WO2024238373A1 - Cross-reference to related applications - Google Patents

Abstract

The present disclosure describes a method and system for quantifying satiety signal(s) upon exposure to one or more stimuli. Provided method(s) are essentially characterized as high-throughput. Provided method(s) quantify satiety signal(s), enabling conclusion(s) regarding feature(s) of stimuli.

Description

ASSESSING SATIETY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/466,226, filed May 12, 2023; U.S. Provisional Patent Application No. 63/517,303, filed August 2, 2023; and U.S. Provisional Patent Application No. 63/617,760, filed January 4, 2024; the title of each of which is “Assessing Satiety” and the content of each of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 5, 2024, is named “2024-05-06 VK8.xml” and is 7.1 kilobytes in size.

TECHNICAL FIELD

[0003] The present disclosure is generally related to methods and/or approaches and/or devices and/or systems and/or technologies within the food and/or beverage and/or nutraceutical industries.

BACKGROUND

[0004] The health benefits of formulated meals, formulated foods, formulated beverages, formulated supplements, and/or their nutritional constituents to mammals, such as humans, have been studied for several decades. However, in many cases, challenges associated with identifying and/or optimizing (e.g., synergistic nutrients and/or food components that enable and/or facilitate and/or provide benefits (e.g., satiety) from foods and/or health and wellness products and/or supplements and/or nutrients and/or their constituents remain unsolved. As such, meals and/or foods and/or beverages and/or supplements and/or their nutritional constituents have yet to realize their full potential in controlling the downstream benefits (e.g., satiety, energy acquisition, etc.) of nutrients.

SUMMARY

[0005] The present disclosure provides technologies related to assessment of satiety, including for example, providing a quantitative assessment of satiety in a system (e.g., determining a quantitative satiety state) and/or providing assessment of satiety-modulating character of one or more agents of interest (e.g., one or more stimuli). Among other things, the present disclosure appreciates that it is both valuable and feasible to perform assessments relating to satiety. The present disclosure specifically appreciates that it is valuable to quantify satiety, and provides technologies that can achieve such quantification. Additionally, the present disclosure specifically appreciates that it is valuable to provide assessments of satietymodulating character, e.g., of particular stimuli of interest. In some embodiments, provided technologies for achieving quantitative assessment of satiety permit assessment of satietymodulating character of stimuli of interest.

[0006] In some embodiments, the present disclosure provides specific insights and technologies that achieve quantitative assessment of satiety in a system (e.g., in a cellular system, for example a system that is or comprises living cells, such as a complex cell composition, comprising two or more cells types and/or one or more structural features, e.g., that may be representative of a tissue or organ; in some embodiments cells are mammalian cells, e.g., human cells.

[0007] Among other things, the present disclosure provides insights that analysis of a plurality of satiety signals, particularly in high throughput and/or over time, and furthermore in a representative context, provides remarkable advantages and contributions in achieving quantitative assessment of satiety. Among other things, such advantages and contributions may facilitate, or even make possible, assessments of satiety-modulating character of stimuli as described herein.

[0008] The present disclosure furthermore provides teachings relating to particular technologies that it demonstrates can be specifically useful for assessing (e g., quantifying) satiety as described herein. For example, the present disclosure teaches that technologies that utilize one or more of transgenic organisms (e.g., transgenic microbes such as transgenic yeast), probiotic quorum sensing, multiplexed specific binder agents (e.g., aptamers, antibody agents such as scFvs, etc), cAMP/Ca sensor systems, etc. are specifically useful to achieve assessment (e g., quantification) of satiety in a system and/or of satiety-modulating character of a stimulus applied to such system.

[0009] In some embodiments, the present disclosure provides specific insights and technologies relating to assessment of satiety-modulating character of stimuli of interest, e.g., of nutrient compositions (e.g., of food products), including for example of complex samples of such compositions and, furthermore including of engineered nutrient compositions (e.g., formulated meals, formulated foods, formulated beverages, formulated supplements, etc.).

[0010] In some embodiments, certain provided technologies are characterized by ability to achieve high-throughput assessments. In some embodiments, certain provide technologies are characterized by ability to assess complex stimuli (e.g., nutrient compositions such as food products).

[0011] The present disclosure provides a variety of particular insights and technologies related to satiety assessment, and in particular relating to use of cellular systems (e.g., systems that are or comprise living cells) to achieve relevant assessments. Among other things, in some embodiments, the present disclosure provides an insight that complex cellular systems (e.g., comprising two or more distinct cell types and/or one or more structural features (e g., three- dimensional arrangements of cells, apical/basolateral polarity, or other components) may be particularly desirable for and/or important to satiety-related assessments - e.g., to assessment of satiety-modulating character. Indeed, the present disclosure provides a particular insight that primary cell samples, and/or specifically of organotypic models (e.g., that utilize cellular sample(s) obtained from living subjects and particularly from human subjects) may be particularly desirable for use, and/or may provide particular benefits when used, in satiety-related assessments.

[0012] Without wishing to be bound by any particular theory, the present disclosure proposes that advantages of such complex cellular systems and/or primary cell systems, and particularly of organotypic models (e.g., that utilize primary samples) include, for example, ability to detect a readout of interest (e.g., impact on satiety state) independent of which or how many biological pathway(s) may be impacted. Furthermore, the present disclosure provides technologies that permit high-throughput assessments, including with such complex cellular systems and/or primary cell systems, and particularly of organotypic models (e.g., that utilize primary samples). Still further, the present disclosure provides technologies that permit assessments, including high-throughput assessments, of a variety of stimuli, including of complex stimuli (e.g., including complex materials such as complex nutrient compositions including engineered foods (e.g., formulated meals, formulated foods, formulated beverages, formulated supplements, etc.), real foods, and/or crude preparations obtained or derived from either), including utilizing complex cellular systems and/or primary cell systems, and particularly of organotypic models (e g., that utilize primary samples).

[0013] Among other things, the present disclosure teaches that analysis (e.g., detection and in many embodiments quantitative evaluation) of one or more, including specifically of a plurality, of particular satiety signals can achieve quantitative assessment of satiety in a system such as a cellular system (e.g., a complex cellular system, including specifically an organotypic model system). Furthermore, the present disclosure teaches that, in some embodiments, valuable assessments (e.g., quantitative assessments) may include performing such analysis over time - e.g., at multiple points in time and/or continuously over a period of time. The present disclosure provides technologies that can achieve such quantitative assessment of satiety, including over time and/or in the presence of one or more stimuli that may modulate satiety.

[0014] In some embodiments, the present disclosure provides technologies that involve determination of presence and/or level of one or more, and in many embodiments, of a plurality of satiety signals (e.g., a plurality of distinct satiety signals) in a cellular system such as a complex cellular system (e.g., comprising a plurality of cell types and/or one or more structural features; in some embodiments, such complex cellular systems may be or comprise a tissue, an organ or an organotypic model, e.g., that may be or comprise cells such as cells that are human cells and/or cells of a primary sample). In many embodiments, such provided technologies achieve quantitative assessment of satiety in the system. Indeed, without wishing to be bound by any particular theory, the present disclosure proposes that quantitative analysis of a plurality of satiety signals, particularly when assessed over time, provides a particularly useful quantitative assessment of satiety in such system.

[0015] In some embodiments, the present disclosure provides methods comprising steps of (a) contacting a cell population with a stimulus of interest for a period of time; and (b) quantifying a change in the cell population that occurs during or after the period of time, which change comprises an increase or decrease in one or more satiety signals produced by cells of the population and is indicative of a change in satiety state of cells in the population, so that satietymodulating character of the stimulus is determined. In some embodiments, the relevant cell population is or comprises cells of enteroendocrine and/or neuroendocrine origin. In some such embodiments, the cell population is or comprises human cells and/or is a complex population in that it comprises at least two cell types and/or is an organotypic model, e.g., that comprises or was derived from a primary cell sample obtained from a human subject.

[0016] In some embodiments, provided technologies involve a step of quantifying that comprises (a) receiving one or more signals indicative of the change; and (b) converting the received one or more signals into one or more readouts by way of a signal adaptor. In some embodiments, a satiety signal comprises one or more extracellular agents. In some embodiments, one or more satiety signals are one or more first messenger(s), second messenger(s), nucleic acid(s), reactive species, receptor agonist(s), receptor antagonist(s), receptor activator(s), receptor inhibitor(s), and/or combinations thereof. In some embodiments, one or more satiety signals is or comprises one or more satiety hormones.

[0017] In some embodiments, the present disclosure provides an agent that is characterized as a satiety modulator when assessed as described herein. In some embodiments, a provided such agent is incorporated into a nutritional composition.

[0018] In some embodiments, the present disclosure provides a method of manufacturing a nutritional composition by incorporating an agent that is characterized as a satiety modulator when assessed as described herein into a nutritional composition.

[0019] In some embodiments, the present disclosure provides technologies for assessing an agent, or a nutritional composition that includes it, as described herein. [0020] Among other things, the present disclosure provides technologies for assessing (e.g., quantifying) one or more feature(s) of a biological system(s)-for example, that may be indicative of satiety or a change therein. In some embodiments, provided technologies utilize or otherwise relate to biological system(s) comprising or undergoing biological signaling. In certain embodiments, provided technologies involve assessing (e.g., quantifying one or more aspects of biological signaling, for example of satiety signaling). In some embodiments, provided technologies for quantifying satiety signaling quantify one or more changes in features (e.g., chemical, physical, and/or electrical properties) of a biological system (e.g., that is or comprises living cells) and, in many embodiments includes cells of two or more different cell types and/or one or more structural features characteristic of organ organization in response to (e.g., correlating with) a change(s) in environment (e.g., presence or absence of a stimulus as described herein), metabolic state(s), and/or energy storage need(s).

[0021] In some embodiments, provided method(s) of assessing satiety (e.g., quantifying satiety signaling and/or otherwise assessing satiety-modulating character) comprise or may comprise one or more step(s). Typically, such step(s) occur or may occur sequentially; those skilled in the art, reading the present disclosure will appreciate those when provided technologies require steps be performed in a particular order, and when alternate orders may be utilized.

[0022] In certain embodiments, provided technologies may comprise a step of exposing one or more cells or cellular systems (e.g., combinations of cell types, tissues, organs, organotypic models, organisms) to one or more stimuli and assessing (e.g., quantifying) one or more aspects of satiety signaling. In some embodiments, provided technologies include or utilize a system for adapting a satiety signal or set of signals into a readout (e.g., a quantified assessment). In some embodiments, such a system for adapting a signal is referred to herein as a “signal adapter”. In some embodiments, output from a signal adapter permits determination of a conclusion, e.g., regarding impact (of a stimulus of interest) on satiety, for example by correlating such impact (e.g., satiety-modulating character) of the stimulus with the readout.

One advantage of certain technologies provided herein is their ability to relate one or more easily controlled input(s) too difficult to control output(s).

[0023] In some embodiments, provided technologies incorporate or utilize one or more systems, components, or methodologies familiar to those skilled in the art. For example, certain embodiments may utilize, for example, assays ranging from cell-free binding assays to in vivo dosing and biomarker quantification.

[0024] In certain aspects, the present disclosure provides new applications for certain technologies developed for and/or utilized in other contexts. For example, the present disclosure provides an insight that certain technological strategies that have been developed and/or employed in assessment of pharmacological interventions for particular disease states can usefully be applied to the important question of assessing satiety (e.g., impact thereon). In fact, the present disclosure identifies the source of a problem with many current efforts to evaluate satiety in that they tend to rely upon subjective or observational rating(s) by subject(s) who receive or are exposed to a particular stimulus. The present disclosure appreciates that such strategies provide only limited success in assessing ability of stimuli of interest to modulate (e.g., to increase, decrease, extend, shorten, etc.) satiety. As a result, identification or characterization of stimuli to increase and/or decrease satiety remains difficult.

[0025] The present disclosure appreciates that satiety in humans may involve the arcuate nucleus. Without wishing to be bound by any particular theory, it is contemplated that neurons comprising the arcuate nucleus are or may be central regulators of satiety. Without wishing to be bound by any particular theory, it is contemplated that such neurons are or may be spatially distinct from target(s) of their intended regulation in the gut. The present disclosure appreciates that such an arrangement would present particular challenges for the study of satiety, and particular for quantifying satiety signal(s) in response to easily controlled stimuli. Moreover, it is contemplated that one or more satiety-controlling neurons within the arcuate nucleus respond or may respond to endocrine signal(s) mediated by afferent vagal nerve(s). The present disclosure observes that certain recent data (see, for example, Heise et al., Diabetes Care 46:998, March 1, 2023 (correlates systemic circulation of arcuate nucleus ligand(s) to reduction in appetite, and notes that such correlation supports a link between endocrine hormone(s) and appetite.

Conventional strategies for controlling appetite primarily focus on optimizing insulin secretion; prior to the present disclosure, the value of assessment (e.g., quantification) of satiety signal(s) in response to easily controlled stimuli has not been well recognized, and technologies capable of achieving such assessment (e g., quantification) have not previously been provided. [0026] The present disclosure also observes that conventional contexts for assessing (e.g., quantifying) biological signaling tend to focus on specific signal(s) implicated in one or more disease state(s). Even when such disease states (e.g., obesity) may be considered related in at least some aspect to satiety, existing analyses fail to assess or determine satiety-modulating character and therefore cannot achieve benefits of technologies provided herein. As already noted, in many embodiments, provided technologies achieve assessment (e.g., high throughput assessment and/or assessment in relevant context(s) such as cellular systems and specifically including complex cellular systems such as complex cellular compositions, e.g., organotypic models) of satiety-modulating character by, for example, permitting quantification of satiety signal(s) in response to easily controlled stimuli.

[0027] In certain embodiments, provided herein are method(s) of quantifying satiety signaling by, for example, providing one or more stimuli of interest to a system that is or comprises one or more first living cell(s), where satiety modulation by such one or more stimuli generates one or more signals amenable to conversion by a signal adapter, contacting the signal adapter with the one or more signals so that the one or more signals are converted to readout(s) indicative of the satiety modulation (e.g., in light of correlation between the one or more stimuli and the readout reflects correlation between the one or more stimuli and the generated signal(s) indicative of the satiety modulation).

[0028] Among other things, the present disclosure provides an insight that technologies as described herein may be particularly useful or effective at assessing satiety (e.g., determining satiety-modulating character of one or more stimuli) when one or more of the following is employed: cells of enteroendocrine and/or neuroendocrine origin or utilized, and/or complex cellular systems (e.g., comprising two or more cell types and/or one or more structural features characteristic of an organ of interest, such as for example may be present in an organotypic model) are utilized, satiety signal(s) are monitored in real time (e.g., at multiple time points), and/or multiple stimuli are assessed high throughput. Still further, the present disclosure provides an insight that provided technologies permit satiety assessments (e.g., quantification of satiety response(s) to environmental cue(s). The present disclosure thus provides various objective and quantitative insights relating to impact(s) of consumed food on regulation of consumption, for example. [0029] In some embodiments, the present disclosure provides technologies characterized by one or more features or advantages including, for example (but not limited to): a) provided technologies permit assessment of a wide range of stimuli - e.g., of any chemical, physical, and/or electrical type or class, specifically including complex materials (e.g., engineered foods such as formulated meals, formulated foods, formulated beverages, formulated supplements; food products such as real foods; and/or crude or otherwise complex extracts or samples thereof); b) provided technologies permit assessment of stimuli provided non-invasively (e.g., through use of model systems such as cell cultures, cultured tissues or organs, organotypic models, etc.); c) provided technologies permit assessment of stimuli over time and/or under controlled circumstances; in some embodiments, one or more aspects such as timing, frequency, amount, form, combination, etc. of exposure to stimuli is/are controlled; d) provided technologies can be applied to a variety of different cell types that may or may not have previously been known to participate in satiety signaling; e) included among cell types that can be assessed by provided technologies are healthy and/or primary cell(s), including healthy and/or primary human cells; f) receiving satiety signal(s) is or may be achieved by harvesting sample(s) from first living cell(s) and/or capturing sample(s) in the presence of first living cell(s); g) received satiety signal(s) are or may be monitored in real time; h) a variety of signal adapter(s) technologies can be utilized, many of which comprise second living cell(s) compatible with monolayer(s), tissue(s), organotypic model(s), organ(s), and/or organism(s); i) provided technologies can identify and/or characterize stimuli not previously known to have satiety-modulating (e.g., inducing and/or reducing) character or capability; j) provided technologies permit comparison of multiple stimuli for relative effect(s) on satiety; k) provided technologies can provide conclusion(s) quantifying one or more health attributes of living cell(s); and l) provided technologies can identify and/or characterize satiety signaling pathway(s) and/or features thereof, including activity in cells, cell types or other contexts (e.g., cell combinations, tissues, organs, etc.) not previously appreciated to participate in satiety signaling.

[0030] The present disclosure provides various insights relating to challenges associated with conventional strategies for quantifying biological signaling utilizing monolayer cell(s) in that they often are limited to assessment of chemical stimuli whereas the present disclosure provides technologies that (and/or provides an insight that certain available technologies) are compatible with a variety of different types of stimuli, including for example with chemical, physical and/or electrical stimuli.

[0031] The present disclosure further provides an insight that the source of a problem with many conventional approaches to assessing aspects of satiety is that they require or at least utilize full organisms and, in some cases, administer stimuli intravenously. Among other things, the present disclosure teaches that organismal assessments are not required; valuable information can be obtained with cellular systems such as cell cultures, cultured tissues or organs, and/or organotypic model systems. Moreover, the present disclosure appreciates that intravenous administration is invasive causing pain, discomfort, and/or damage. Still further, intravenous administration risks irrelevant assessments, as satiety-impacting stimuli (e.g., foods or other nutritional compositions) are typically preferably ingested (e.g., received orally). Particularly advantageous features of certain embodiments of provided technologies include their ability to assess stimuli of a various different types (e.g., chemical, physical, and/or electrical stimuli), in a variety of contexts and via various modes or routes of exposure. For example, in some particular embodiments, stimuli are or may be provided as an added component to media, as an aerosol, and/or as a food and/or beverage, for example. [0032] The present disclosure further provides an insight that one limitation of strategies for assessing satiety by intravenous administration of stimuli is that such intravenous administration is typically in a single bolus dose, or ad libitum. Without wishing to be bound by any particular theory, the present disclosure observes that naturally encountered satiety stimuli occur or may occur for intermittent durations and with varied frequency. One advantage of certain embodiments of provided technologies is that they permit assessments of relevant stimuli according to various regiments - e.g., for varied duration(s) and/or with varied frequency.

[0033] The present disclosure yet further provides an insight that one limitation of various available or potentially contemplated strategies for assessing satiety is that they may rely upon cell type(s) or contexts that are poorly representative of in vivo satiety signaling. Without wishing to be bound by any particular theory, the present disclosure suggests that quantification of satiety signaling in cancerous endocrine line(s) and/or modified epithelial cell(s) may be of particularly low fidelity to in vivo signaling pathway(s). Certain provided technologies for assessing (e.g., quantifying) satiety are particularly advantageous as primary cells, or even primary-sample-derived organotypic model(s) are or may be utilized.

[0034] The present disclosure yet further provides an insight that one limitation of various available or potentially contemplated strategies for assessing satiety is that they may rely upon lysis of one or more first living cell(s) to achieve readout(s). Without wishing to be bound by any particular theory, it is contemplated that destruction of one or more first living cell(s) is labor intensive, reducing method throughput. In some embodiments, provided technologies for assessing (e.g., quantifying) satiety are particularly advantageous in that they achieve signal capture in the presence of first living cell(s) (e.g., in that they do not require and/or do not employ, cell lysis).

[0035] The present disclosure yet further provides an insight that one limitation of various available or potentially contemplated strategies for assessing satiety is that they may rely upon generating readout(s) characterized as endpoint readout(s). Without wishing to be bound by any particular theory, it is contemplated that satiety signaling is highly heterogeneous and time dependent may be and difficult to capture through endpoint readout(s). In some embodiments, provided technologies for assessing (e.g., quantifying) satiety signaling are particularly advantageous in that they provide one or more real time readout(s) correlating to satiety signal(s).

[0036] The present disclosure yet further provides an insight that one limitation of various available or potentially contemplated strategies for assessing satiety is that they may rely upon invasive collection of blood plasma and/or genetic information with low signal to noise ratio. One advantage of certain embodiments of provided technologies is that they may utilize genetically-encoded second cell(s) compatible with satiety signal quantification using monolayer(s), organotypic model(s), organoid(s), and/or organism(s). In certain provided embodiments, signal adapter(s) characterized as second living cell(s) provide or may provide continuous real-time monitoring of one or more satiety signal(s).

[0037] Without wishing to be bound by any particular theory, it is contemplated that limitations in existing method(s) of quantifying satiety signal(s) limit or may limit conclusion(s) to monitoring the health of one or more living cell(s). Without wishing to be bound by any particular theory, it is contemplated that high-throughput and/or endpoint method(s) pose or may pose the greatest hurdle towards the utility of existing method(s) of quantifying satiety signaling. In some embodiments, provided technologies for assessing (e.g., quantifying) satiety are particularly advantageous by enabling the quantitative comparison of satiety signaling resulting from several stimuli. In some embodiments, provided technologies for assessing (e.g., quantifying) satiety are particularly advantageous by enabling the identification of previously unidentified stimuli resulting in satiety signal(s). In some embodiments, provided technologies for assessing (e.g., quantifying) satiety are particularly advantageous by enabling the identification of previously unidentified modulator(s) enhancing known satiety signaling. In some embodiments, provided technologies for assessing (e.g., quantifying) satiety are particularly advantageous by enabling the identification of previously unidentified signal pathway(s) comprising satiety signaling.

[0038] The present disclosure yet further provides embodiments of provided technologies that can be characterized by the following various features.

[0039] In one aspect, the present disclosure is directed to method(s) comprising the steps of providing a system that is or comprises living cells; and detecting in the system presence or level of at least one satiety signal, wherein such detection is performed continuously or at a plurality of time points over a period of time or in a high throughput format, so that the detecting achieves quantitative assessment of satiety in the system. In some embodiments, the step of detecting comprises contacting the system with a signal adaptor comprising a set of binding agents, each of which binds specifically to a satiety signal that is a chemical agent or entity present in the system; and determining binding of each of the binding agents to its target satiety signal. In some embodiments, each binding agent is or comprises a polypeptide or a nucleic acid. In some embodiments, each polypeptide binding agent is or comprises an antibody agent. In some embodiments, each nucleic acid binding agent is or comprises an aptamer.

[0040] In another aspect, the present disclosure is directed to method(s) comprising the steps of contacting a cell population with a stimulus of interest for a period of time; and quantifying a change in the cell population that occurs during or after the period of time, which change comprises an increase or decrease in one or more satiety signals produced by cells of the population and is indicative of a change in satiety state of cells in the population, so that satietymodulating character of the stimulus is determined. In some embodiments, the cell population is or comprises cells of enteroendocrine and/or neuroendocrine origin. In some embodiments, the cell population is or comprises human cells. In some embodiments, the cell population is a complex population in that it comprises at least two cell types. In some embodiments, the cell population is cultured in the upper apical chamber of a transwell plate. In some embodiments, the cell population is an organotypic model. In some embodiments, the organotypic model comprises or was derived from a primary cell sample obtained from a human subject.

[0041] In some embodiments, the step of quantifying comprises receiving one or more signals indicative of the change; and converting the received one or more signals into one or more readouts by way of one or more signal adaptors. In some embodiments, the one or more satiety signals comprise one or more extracellular agents. In some embodiments, the one or more satiety signals are one or more first messenger(s), second messenger(s), nucleic acid(s), reactive species, receptor agonist(s), receptor antagonist(s), receptor activator(s), receptor inhibitor(s), and/or combinations thereof. In some embodiments, one of the one or more satiety signals is a second messenger. In some embodiments, the second messenger is selected from calcium and cAMP. In some embodiments, the one or more satiety signals is or comprises one or more satiety hormones. In some embodiments, wherein the one or more signal adaptors is a genetically encoded sensor. In some embodiments, the one or more signal adaptors is selected from a genetically encoded calcium indicator (GECI) and a genetically encoded fluorescent indicator (GEFI). In some embodiments, the one or more signal adaptors is TWITCH-NR or gFLAMP. In some embodiments, a first signal of the one or more signals is converted by a first signal adaptor of the one or more signal adaptors, and a second signal of the one or more signals is converted by a second signal adaptor of the one or more signal adaptors. In some embodiments, the presence or level of at least one satiety signal is detected within 1 second, 5 seconds 30 seconds, 1 minute, 5 minutes, 7 minutes, 10 minutes, 30 minutes, 45 minutes, 1 hour, or 2 hours of contacting the cell population with the stimulus of interest. In some embodiments, the presence or level of at least one satiety signal is detected in less than 2 hours. In some embodiments, the presence or level of at least one satiety signal is detected in less than 30 minutes.

[0042] In some embodiments, an agent is characterized as a satiety modulator when assessed according to methods of the present disclosure. In some embodiments, a method of manufacturing a nutritional composition incorporates an agent into a food. In some embodiments, a nutritional composition comprising an agent. In some embodiments, a method of characterizing a nutritional composition comprises using it as a stimulus in an assessment as described herein.

[0043] In some embodiments, the living cells comprise Caco-2 cells, NCI-H716 cells, or Caco-2 cells co-cultured with NCI-H716 cells. In some embodiments, the one or more satiety hormones comprises peptide YY (PYY), glucagon, glucagon-like peptide-1 (GLP-1), glucagon- like peptide-2 (GLP-2), gastric inhibitory polypeptide (GIP), or any combination thereof. In some embodiments, the stimulus of interest induces a physicochemical change in one or more membrane protein(s). In some embodiments, the one or more membrane proteins comprise a g- protein coupled receptor (GPCR) comprising at least one of GPR40, GPR70, and GPR120. In some embodiments, the one or more membrane proteins comprise TGR5, GPR17, GPR40, GPR119, GPR120, SGLT-1, SGLT-2, CaSR, or any combination thereof.

[0044] In another aspect, the present disclosure is directed to a system for screening satiety modulators comprising active cells comprising a plurality of endocrine cells (for example, enteroendocrine cells and/or neuroendocrine cells) and/or a plurality of intestinal endothelial cells; at least one satiety screening sensor comprising at least one recombinant satiety adapter protein expressed in the active cells, the at least one satiety adapter protein comprising at least one fluorescent protein; at least one nutrient for dosing the active cells; means for exciting the at least one fluorescent protein; and means for reading and/or detecting fluorescence of the at least one fluorescent protein; wherein dosing of the active cells with the at least one nutrient alters a level of fluorescence of the at least one fluorescent protein.

[0045] In another aspect, the present disclosure is directed to a method for screening satiety modulators comprising providing active cells comprising a plurality of enteroendocrine cells and/or a plurality of intestinal endothelial cells; providing at least one satiety screening sensor comprising at least one recombinant satiety adapter protein expressed in the active cells, the at least one satiety adapter protein comprising at least one fluorescent protein; dosing (or stimulating) the active cells with at least one nutrient; exciting the at least one fluorescent protein; and detecting fluorescence of the at least one fluorescent protein; wherein dosing of the active cells with the at least one nutrient alters a level of fluorescence of the at least one fluorescent protein.

[0046] In some embodiments, upon dosing (for example, within 1 second, 5 seconds 30 seconds, 1 minute, 5 minutes, 7 minutes, 10 minutes, 30 minutes, 45 minutes, 1 hour, or 2 hours) with the at least one nutrient, the plurality of enteroendocrine cells and/or the plurality of intestinal endothelial cells propagate at least one signal (i.e., a satiety signal) via one or more signal transduction pathways to the at least one satiety adapter protein. In some embodiments, means for exciting the at least one fluorescent protein and/or means for reading and/or detecting the at least one fluorescent protein comprises a flow cytometer. In some embodiments, wherein the flow cytometer comprises a laser configured to excite the active cells in a range from about 300 nm to about 700 nm (for example, in a range from about 300 nm to about 600 nm, or from about 300 nm to about 500 nm, or from about 350 nm to about 470 nm, or at about 355 nm and/or at about 460 nm). In some embodiments, the at least one satiety adapter protein comprises at least one of TWITCH-NR and gFLAMP. In some embodiments, the at least one satiety adapter protein is excited by a secondary messenger. In some embodiments, the secondary messenger is selected from cAMP and calcium. In some embodiments, the at least one fluorescent protein comprises at least one of a green fluorescent protein and a red fluorescent protein. In some embodiments, the at least one fluorescent protein comprises both a green fluorescent protein and a red fluorescent protein. In some embodiments, the green fluorescent protein is circularly permutated. In some embodiments, the green fluorescent protein comprises mNEON. In some embodiments, the red fluorescent protein comprises mScarlet.

[0047] In some embodiments, the at least one satiety adapter protein comprises a nuclear export sequence comprising the amino acid sequence of SEQ. ID NO: 7. In some embodiments, the at least one satiety adapter protein comprises at least a portion of calcium-binding protein troponin C. In some embodiments, the active cells comprise enteroendocrine cells comprising receptors disposed on the cell surface for propagation of satiety signaling resulting from the nutrient dosing. In some embodiments, the at least one nutrient comprises at least one of a- linoleic acid, quercetin, glucose, and oleic acid. In some embodiments, the at least one nutrient comprises at least two of a-linoleic acid, quercetin, glucose, and oleic acid.

[0048] In some embodiments, the system and/or method further comprises a well plate (for example, a 96-well plate, 384-well plate, etc.) wherein the active cells and the at least one satiety adapter protein are aliquoted such that multiple wells of the well plate contain the active cells and the at least one satiety adapter protein, wherein the multiple wells contain the at least one nutrient, and wherein at least one well of the multiple wells contains a nutrient that is different than at least one other well of the multiple wells. In some embodiments, at least one well of the multiple wells contains more than one nutrient. In some embodiments, the level of fluorescence of the at least one fluorescent protein is altered such that an increase in a range from about 0.5% to about 1.5% (for example, from about 0.9% to about 1.3%) is observed over a fluorescence level corresponding to an unstimulated protein. In some embodiments, the at least one nutrient comprises at least one of a caloric nutrient and an acaloric nutrient. In some embodiments, the at least one nutrient comprises a caloric nutrient and an acaloric nutrient. In some embodiments, the at least one nutrient comprises an acaloric nutrient comprising at least one of diindolylmethane (DIIM), quercetin, and piperine.

[0049] In some embodiments, the system and/or method further comprises at least one of linoleic acid (LA), alpha-linoleic acid (ALA), disodium inosinate plus disodium guanylate (I + G), and digested whey protein isolate (dWPI). In some embodiments, each of the caloric nutrient and the acaloric nutrient alters a level of fluorescence of the at least one fluorescent protein.

INCORPORATION BY REFERENCE

[0050] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWING

[0051] The present disclosure includes a Drawing comprised of Figures as described below:

[0052] FIG. 1 shows, in a non-limiting example, quantification of secreted satiety signal(s) from one or more enteroendocrine cell(s) utilizing protein signal adapter(s). (A) Schematic depicting provision of stimuli to first living cell(s), collection of satiety signal(s) shown as black squares, circle, and triangle, and conversion of signal(s) to readout(s) (stars) by immunoassay. (B) Calibration curve and quantification of samples generated for exemplary satiety signal(s) (e.g., GLP-1) using immunoassay signal adapter(s).

[0053] FIG. 2 shows, in a non-limiting example, quantification of secreted satiety signal(s) from one or more enteroendocrine cell(s) utilizing a second living cell (e.g., yeast) as a protein signal adapter. (A) Signal adapter comprising a transgene GPCR specific for single satiety signal (e.g., GLP-1) in quenched FRET pair with cognate Ga subunit; (B) Signal adapter comprising soluble cAMP and/or Ca2+ FRET binding protein(s) to detect GPCR binding in real time; (C) Signal adapter comprising direct quantification of transcripts generated in response to stimuli; (D) Signal adapter comprising quantification of secreted satiety signal(s) using protein immunoassay (s) and/or aptamers.

[0054] FIG. 3 shows, in a non-limiting example, a schematic of a phage display process to identify satiety signal-binding receptor(s) and/or protein(s). [0055] FIGs. 4A-4C shows, in a non4imiting example, quantification of secreted satiety signal(s) from one or more enteroendocrine cell(s) utilizing a second living cell (e.g., probiotic) as a signal adapter. (A) Schematic of engineered probiotic comprising hormone-sensing quorum sensors linked to a genetically-encoded secreted readout; (B) Theoretical data showing selection and refinement of engineered quorum sensing proteins over repeated panning steps; (C) Theoretical data depicting increase in readout(s) in response to increasing satiety signal(s).

[0056] FIGs. 5A-5C shows, in a non-limiting example, quantification of secreted satiety signal(s) from one or more enteroendocrine cell(s) utilizing a multiplexed peptide(s) and/or multiplexed aptamers as signal adapters. (A) First cell(s) secreting satiety signal(s) (grey squares) binding to either multiplexed protein(s) or aptamers, said protein(s) or aptamers conjugated to fluorescent dye(s) that emit photon(s) only in the presence of satiety signal(s); (B) Theoretical data of readout(s) generated from multiplexed signal adapter(s) binding to satiety signal(s); (C) Theoretical aptamer sequences to optimize binding affinity to one or more signal adapter(s).

[0057] FIG. 6 shows, in a non-limiting example, transformation of HEK cells to express libraries and scale up production of peptide binding partners towards key satiety signal(s).

[0058] FIGs. 7A-7B shows, in a non-limiting example, dosing of NCI-H716 enteroendocrine cells with various chemical stimuli and quantifying resulting satiety signal(s) via protein signal adapter(s). NCI-H716 cells, grown in complete F-12 media at 37 °C and 5% CO2, were seeded on 96-well plates and serum starved overnight prior to dosing of either vehicle (black), glucose (dark grey squares), Quercetin (grey triangles), Oleic Acid (light grey hexagon), or Oleic Acid and Quercetin (black diamonds with dashed lines) in the presence of 50 pM Sitagliptin. Glucose, Quercetin, and Oleic Acid were dosed in serum-free F-12 media at 500 mM, 100 pM, and 100 pM, respectively. Concentrations match post-prandial luminal concentrations and/or prior literature doses to NCI-H716 cells. Cells were incubated for either 0, 5, 30, 120, or 240 minutes and media was collected for subsequent analysis. Samples were analyzed using (A) commercial GLP-1 ELISA kits (Sigma- Aldrich) or (B) GIP ELISA kits (Raybiotech) at t = 0, 5, 30, 120, and 240 minutes. Concentrations of GLP-1 are reported in picomolar units, while concentrations of GIP are reported in picograms per milliliter. Data is shown as average of 3 replicates +/- standard deviation. [0059] FIGs. 8A-8D show, in a non-limiting example, widefield fluorescence microscopy images of NCI-H716 cells co-cultured with Caco-2 cells stained with DAPI nuclear stain (FIG. 8 A), ZO-1 tight-j unction complex protein (FIG. 8B), L-cell specific G-protein coupled receptor (GPR40) (FIG. 8C). FIG. 8D shows an overlaid composite image of FIGs. 8A-8C. The inset scale bar indicates 20 pm.

[0060] FIG. 8E shows, in a non-limiting example, a bar graph of the nutrient receptor gene expression determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Data are shown as means +/- standard deviation relative to expression levels of a GAPDH control.

[0061] FIGs. 9A-9D show, in a non-limiting example, widefield fluorescence microscopy images of NCI-H716 cells co-cultured with Caco-2 cells stained with DAPI nuclear stain (FIG. 9A), ZO-1 tight-j unction complex protein (FIG. 9B), umami taste receptor T1R1 (GPR70) (FIG. 9C). FIG. 9D shows an overlaid composite image of FIGs. 9A-9C. The inset scale bar indicates 20 pm.

[0062] FIG. 10A-10F show, in a non-limiting example, flow cytometry plots of nutrient receptor surface expression of NCI-H716 cells co-cultured with Caco-2 cells. FIG. 10A shows a side-scatter area vs. forward scatter area density plot of voltage resolved NCI-H716 cells within the Caco-2/NCI-H716 co-culture system. FIG. 10B shows a live/dead cell discrimination gating strategy of Invitrogen® Fixable Live/Dead violet-stained cells. FIG. 10C is a histogram showing the staining of NCI-H716 cells co-cultured with Caco-2 cells for GPR40 as compared to unstained control cells. FIG. 10D is a histogram showing the staining of NCI-H716 cells co- cultured with Caco-2 cells for GPR120 as compared to unstained control cells. FIG. 10E is a histogram showing the staining of NCI-H716 cells co-cultured with Caco-2 cells for GPR70 as compared to unstained control cells. FIG. 1 OF is a histogram showing the overlaid plots of FIGs. 10C-10E.

[0063] FIG. 11 shows, in a non-limiting example, a bar graph of satiety hormone gene expression of NCI-H716 cells, Caco-2 cells, NCI-H716-Caco-2 co-cultures as determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Data are shown as means +/- standard deviation relative to expression levels of a GAPDH control. [0064] FIG. 12A-12D show, in a non-limiting example, graphs of expression of various cellular nutrient receptors that may be used for identifying or characterizing stimuli with satiety modulating characteristics. FIG 12A shows a graph of relative nutrient receptor gene expression (compared to GAPDH) in NCI-H716 cells as determined by quantitative polymerase chain reaction (qPCR). FIG 12B shows a graph of relative nutrient receptor gene expression (compared to GAPDH) in a NCI-H716 and Caco-2 co-culture plated in the upper apical compartment of a Transwell plate as determined by qPCR. FIG 12C shows flow cytometry histograms of nutrient receptor expression on NCI-H716 cells. FIG 12D shows a bar graph of relative gene expression of PYY, GIP, and GCG satiety hormone peptides in NCI-H716 or Caco- 2 monocultures or NCI-H716 and Caco-2 co-cultures plated in the upper apical compartment of a Transwell plate as determined by qPCR.

[0065] FIG. 13 is a schematic table describing exemplary nutrient receptors and their corresponding known or predicted nutrient stimuli.

[0066] FIG. 14 shows, in a non-limiting example, a bar graph of GIP satiety hormone peptide secretion from NCI-H716 cells simulated with various nutrients and nutrient combinations as determined by ELISA.

[0067] FIG. 15 shows, in a non-limiting example, a bar graph of GLP-1 satiety hormone peptide secretion from NCI-H716 cells simulated with various nutrients and nutrient combinations as determined by ELISA.

[0068] FIG. 16A-16B show, in a non-limiting example, the relative fluorescence (as measured by flow cytometry) of unstimulated or stimulated NCI-H716 cells transfected with plasmids encoding TWITCH-NR or gFLAMP. FIG. 16A shows a bar graph of the relative fluorescence of the intracellular calcium responsive protein, TWITCH-NR, in NCLH716 cells transfected with a plasmid encoding TWITCH-NR and unstimulated or stimulated with calcium and ionomycin. FIG. 16B shows a bar graph of the relative fluorescence of the intracellular cAMP -responsive protein, gFLAMP, in NCI-H716 cells transfected with a plasmid encoding gFLAMP and unstimulated or stimulated with forskolin.

[0069] FIG. 17 shows, in a non-limiting example, the relative fluorescence (as measured by flow cytometry) of NCI-H716 cells transfected with a plasmid encoding TWITCH-NR and stimulated with various nutrients, calcium and ionomycin positive control, or unstimulated negative control.

[0070] FIG. 18 shows, in a non-limiting example, the relative fluorescence (as measured by flow cytometry) of NCI-H716 cells transfected with a plasmid encoding gFLAMP and stimulated with various nutrients, or forskolin or unstimulated positive and negative controls, respectively.

[0071] FIG. 19 shows, in a non-limiting example, the relative fluorescence (as measured by flow cytometry) of NCI-H716 cells transfected with a plasmid encoding gFLAMP and stimulated with various concentrations of quercetin, or forskolin or unstimulated positive and negative controls, respectively.

[0072] FIG. 20 shows, in a non-limiting example, normalized percent fluorescence increases (as measured by flow cytometry) of NCI-H716 cells transfected with a plasmid encoding TWITCH-NR and stimulated with various concentrations of quercetin, calcium and ionomycin positive control, or unstimulated negative control.

[0073] FIG. 21A-21B show, in a non-limiting example, fluorescence signals (as measured by flow cytometry) of NCI-H716 cells transfected with plasmids encoding TWITCH- NR or gFLAMP and stimulated with various concentrations of single nutrients or nutrient combinations. FIG. 21 A shows normalized percent flourescence increases (as measured by flow cytometry) of NCLH716 cells transfected with a plasmid encoding TWITCH-NR and stimulated with various concentrations and/or combinations of nutrients, calcium and ionomycin positive control, or unstimulated negative control. FIG. 21B shows relative flourescence (as measured by flow cytometry) of NCI-H716 cells transfected with a plasmid encoding gFLAMP and stimulated with various concentrations and/or combinations of nutrients, or forskolin or unstimulated positive and negative controls, respectively.

[0074] FIG. 22, panels A-C show, in a non-limiting example, plots of satiety hormone secretion and intracellular calcium release after treatment with a panel of caloric and acaloric nutrients in enteroendocrine cells modified to recombinantly express TWITCH-NR. FIG. 22, panel A shows a line graph of GLP-1 secretion by NCI-H716 cells transfected with a plasmid encoding TWITCH-NR and stimulated with 13 caloric or acaloric nutrients or nutrient combinations over a 30-hour period. FIG. 22, panel B shows a bar graph of a intracellular calcium release of the cells of FIG. 22, panel A stimulated with 13 caloric or acaloric nutrients or nutrient combinations over a 30-hour period. FIG. 22, panel C shows a plot of the correlation between the GLP-1 release data of FIG. 22, panel A and the intracellular calcium release data of FIG. 22, panel B.

[0075] FIG. 23 shows, in a non-limiting example, a workflow schematic for a high- throughput satiety modulator screen.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0076] Section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

A. Certain Terminology

[0077] Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood to which the claimed subject matter belongs. In the event that there are a plurality of definitions for terms herein, those in this section prevail.

[0078] It is to be understood that the general description and the detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, use of the term "including" as well as other forms, such as "include", "includes," and "included," is not limiting.

[0079] Unless the context requires otherwise, throughout the specification and claims which follow, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense, that is, as "including, but not limited to." Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed invention. [0080] Definition of standard chemistry terms may be found in reference works, including but not limited to, Carey and Sundberg "Advanced Organic Chemistry 4th Ed." Vols. A (2000) and B (2001), Plenum Press, New York.

[0081] As used herein, the symbol "<" means less than or fewer than. As used herein, the symbol ">" means more than or greater than.

[0082] About. As used herein, the term "about" or "approximately" means within 10%, preferably within 10%, and more preferably within 5% of a given value or range.

[0083] Administration: As used herein, the term “administration” typically refers to the administration (e.g., of a composition or treatment) to a subject or system (e.g., that is or comprises one or more cells, tissues, organisms, etc), for example to achieve delivery of an agent that is, is included in, or is otherwise delivered or generated by, such composition or treatment.

[0084] Affinity: As is known in the art, “affinity” is a measure of the tightness with which two or more binding partners associate with one another. Those skilled in the art are aware of a variety of assays that can be used to assess affinity, and will furthermore be aware of appropriate controls for such assays. In some embodiments, affinity is assessed in a quantitative assay. In some embodiments, affinity is assessed over a plurality of concentrations (e.g., of one binding partner at a time). In some embodiments, affinity is assessed in the presence of one or more potential competitor entities (e.g., that might be present in a relevant - e g., physiological - setting). In some embodiments, affinity is assessed relative to a reference (e.g., that has a known affinity above a particular threshold [a “positive control” reference] or that has a known affinity below a particular threshold [a “negative control” reference”]. In some embodiments, affinity may be assessed relative to a contemporaneous reference; in some embodiments, affinity may be assessed relative to a historical reference. Typically, when affinity is assessed relative to a reference, it is assessed under comparable conditions.

[0085] Agent: In general, the term “agent”, as used herein, is used to refer to an entity (e g., for example, a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc., or complex, combination, mixture or system [e.g., cell, tissue, organism] thereof), or phenomenon (e.g., heat, electric current or field, magnetic force or field, etc.). [0086] Ambient: The term "ambient", as used herein, refers to a typical indoor (e.g., climate-controlled) temperature, usually within a range of about 18 °C to about 32 °C, and/or typical indoor (e.g., climate-controlled) humidity, usually within a range of about 30% to 50%. In some embodiments, ambient temperature is within a range of about 20 °C to about 30 °C.

[0087] Analog: As used herein, the term “analog” refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an “analog” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete way(s). In some embodiments, an analog is a substance that can be generated from the reference substance, e g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance. In some embodiments, an analog is or can be generated through performance of a synthetic process different from that used to generate the reference substance.

[0088] Aptamer The term “aptamer”, as used herein, refers to one or more polynucleotide(s) characterized by strong and/or specific binding to a particular ligand of interest. Those skilled in the art will appreciate that an aptamer is typically designed or selected to interact with one or a set of potential ligand(s) preferentially relative to other potential ligand(s) present in a relevant system. Those skilled in the art will further appreciate that, in some embodiments, an aptamer may include one or more nucleic acid analogs (e.g., sugar and/or base analogs) and/or one or more inter-residue linkages that is not a phosphodiester bond. To give but a few examples, in some embodiments, an aptamer is or may be comprised of double stranded and/or single stranded nucleic acids. In some embodiments, an aptamer for use in accordance with the present disclosure characterized by a < 1 pM binding constant with respect to one or more target ligands (e.g., a singular ligand). In certain embodiments, an aptamer may be detectably labeled (e.g., associated with a label such as a fluorophore). In some embodiments, an aptamer may adopt a hairpin structure. In some embodiments, an aptamer may be associated with (e.g., covalently linked to) each of a signal generator (e.g., a fluorophore) and a quencher, and may adopt distinguishable states in which the signal generator and quencher are either separated from one another (so that signal from the signal generator is detectable) or in proximity with one another (so that signal from the signal generator is not detectable); in some such embodiments, an aptamer adopts such distinguishable states when bound to vs not bound to its target ligand. In certain embodiments, an aptamer is utilized in accordance with the present disclosure in conjunction with a small molecule fluorescent dye to which the aptamer binds with lower affinity than it does with its target ligand. In some embodiments, an aptamer is utilized in accordance with the present disclosure in conjunction with covalently-attached hairpin-quenched dyes; in some embodiments, binding between such an aptamer and its target ligand displaces the hairpin structure.

[0089] Binding. It will be understood that the term “binding”, as used herein, typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts - including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell). Binding between two entities may be considered “specific” if, under the conditions assessed, the relevant entities are more likely to associate with one another than with other available binding partners.

[0090] Comparable. As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0091] Culture. The term “culture”, when used as a verb, herein, refers to growth (e.g., proliferation) and/or maintenance of one or more living cells or system(s). To give but a few embodiments, in some instances, the term “culture” refers to expansion of monolayer cell(s) within a nutrient-rich aqueous medium. In some embodiments, the term “culture” refers to maintenance of a structured cellular material, such as a tissue or sample thereof, or an organotypic model(s), e.g., within a balanced salt solution and/or for a designated period of time. In some embodiments, the term “culture” refers to maintenance of an organism, such as a mammalian organism, which may for example be a human or a domesticated animal, for example by feeding and/or hydrating.

[0092] Detectable entity . The term “detectable entity” as used herein refers to an element, molecule, functional group, compound, fragment or moiety that is detectable. In some embodiments, a detectable entity is provided or utilized alone. In some embodiments, a detectable entity is provided and/or utilized in association with (e.g., joined to) another agent. Examples of detectable entities include, but are not limited to: various ligands, radionuclides (e.g., 3H, 14C, 18F, 19F, 32P, 35S, 1351, 1251, 1231, 64Cu, 187Re, Ulin, 90Y, 99mTc, 177Lu, 89Zr etc.), fluorescent dyes (for specific exemplary fluorescent dyes, see below), chemiluminescent agents (such as, for example, acridinium esters, stabilized dioxetanes, and the like), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductors nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes (for specific examples of enzymes, see below), colorimetric labels (such as, for example, dyes, colloidal gold, and the like), biotin, dioxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available.

[0093] Engineered. In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and/or when a particular residue in a polynucleotide is non-naturally occurring and/or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature. For example, in some embodiments described and/or utilized herein, an engineered polynucleotide comprises a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with the second coding sequence. Comparably, a polypeptide may be considered to be “engineered” if encoded by or expressed from an engineered polynucleotide, and/or if produced other than natural expression in a cell. Analogously, a cell or organism is considered to be “engineered” if it has been subjected to a manipulation, so that its genetic, epigenetic, and/or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or comprises a genetic manipulation, so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols). In some embodiments, an engineered cell is one that has been manipulated so that it contains and/or expresses a particular agent of interest (e.g., a protein, a nucleic acid, and/or a particular form thereof) in an altered amount and/or according to altered timing relative to such an appropriate reference cell. As is common practice and is understood by those in the art, progeny of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.

[0094] Food Product. The term “food product”, as used herein, refers to an edible material (e.g., solid, liquid, gel, etc.) that can be ingested, swallowed, chewed, or otherwise consumed by a person or animal without material risk to the person or animal and/in many embodiments, which providing one or more nutritional attributes (e.g., one or more benefits, including specifically health benefits such as survival benefits) resulting from release (e.g., controlled release), absorption, spatial access, concentration, and/or residence time of one or more nutrients. In some embodiments, a food product can be or comprise agricultural seed, dry powders, supplements, solid foods, beverages and/or drinks, etc. In some embodiments, a “food product” may generally refer to a food and/or beverage product. In some embodiments, a “food product” may generally refer to an edible object that is intended to confer a benefit (e.g., health, energy, nutrition, performance, well-being) on one or more animal(s). In some embodiments, a food product is a substantially pure preparation of a single component (e.g., ingredient), or a small number of components (e.g., ingredients). In some embodiments, a food product is a combination of multiple components (e.g., ingredients). In some embodiments, a food product is a complex mixture of multiple components (ingredients). In some embodiments, a food product is a structured combination (e.g., comprising two or more discrete portions of different chemical and/or physical arrangement) of two or more components (e.g., ingredients). Indeed, as described herein, one advantage of certain embodiments of provided technologies is their usefulness to assess satiety character of food products, and in particular of complex food products.

[0095] Genetic Information. The term “genetic information”, as used herein, refers to information encoded in, or relating to expression or processing of, nucleic acids in living cell(s). For example, in some embodiments, genetic information refers to or is embodied in one or more of the DNA (e.g., genes) of living cell(s), the epigenetic state of DNAin living cell(s), the transcription state of or transcripts (i.e., RNA) in living cell(s), the splicing (e.g., mRNA) of such RNA in living cells, the cytosolic (e.g., miRNA) silencing of sRNA in living cells, and/or translation of RNA into protein in living cell(s). In some embodiments, genetic information is endogenous to a living cell in which it is present. In some embodiments, a living cell is or has been (or is progeny of a cell that has been) engineered to contain particular genetic information.

[0096] Immobilized. The term “immobilized”, as used herein, refers to the attachment of one or more microscopic (e.g., nano-sized) molecule(s) to one or more macroscopic (e.g., visible to the eye) surfaces using at least one covalent, ionic, dipolar, hydrogen bonding, and/or hydrophobic interaction.

[0097] “ Improved f ' increased," or 'reduced As used herein, these terms, or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with an agent of interest may be “improved” relative to that obtained with a comparable reference agent. Alternatively or additionally, in some embodiments, an assessed value achieved in a subject or system of interest may be “improved” relative to that obtained in the same subject or system under different conditions (e.g., prior to or after an event such as administration of an agent of interest), or in a different, comparable subject (e.g., in a comparable subject or system that differs from the subject or system of interest in presence of one or more indicators of a particular disease, disorder or condition of interest, or in prior exposure to a condition or agent, etc.). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and/or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and/or prevalence of difference that is required or sufficient to achieve such statistical significance.

[0098] Internucleotidic linkage. As used herein, the phrase “internucleotidic linkage” refers generally to a linkage linking nucleoside units of a polynucleotide. In some embodiments, an internucleotidic linkage is a phosphodi ester linkage, as is extensively found in naturally occurring DNA and RNA molecules (natural phosphate linkage (-OP(=O)(OH)O-), which as appreciated by those skilled in the art may exist as a salt form). In some embodiments, an internucleotidic linkage is a modified internucleotidic linkage (not a natural phosphate linkage). In some embodiments, an internucleotidic linkage is a “modified internucleotidic linkage” wherein at least one oxygen atom or -OH of a phosphodiester linkage is replaced by a different organic or inorganic moiety. In some embodiments, such an organic or inorganic moiety is selected from =S, =Se, =NR’, -SR’, -SeR’, -N(R’)2, B(R’)s, -S-, -Se-, and -N(R’)-, wherein each R’ is independently as defined and described in the present disclosure. In some embodiments, an internucleotidic linkage is a phosphotriester linkage, phosphorothioate linkage (or phosphorothioate diester linkage, -OP(=O)(SH)O-, which as appreciated by those skilled in the art may exist as a salt form), or phosphorothioate triester linkage. In some embodiments, a modified internucleotidic linkage is a phosphorothioate linkage. In some embodiments, an internucleotidic linkage is one of, e.g., PNA (peptide nucleic acid) or PMO (phosphorodiamidate Morpholino oligomer) linkage. In some embodiments, a modified internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, a modified internucleotidic linkage is a neutral internucleotidic linkage (e.g., nOOl in certain provided oligonucleotides). It is understood by a person of ordinary skill in the art that an internucleotidic linkage may exist as an anion or cation at a given pH due to the existence of acid or base moieties in the linkage. [0099] Nucleobase. The term “nucleobase” refers to the parts of nucleic acids that are involved in the hydrogen-bonding that binds one nucleic acid strand or sequence element to another complementary strand or sequence element in a sequence specific manner. The most common naturally-occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, nucleobases are modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, nucleobases are methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, a nucleobase comprises a heteroaryl ring wherein a ring atom is nitrogen, and when in a nucleoside, the nitrogen is bonded to a sugar moiety. In some embodiments, a nucleobase comprises a heterocyclic ring wherein a ring atom is nitrogen, and when in a nucleoside, the nitrogen is bonded to a sugar moiety. In some embodiments, a nucleobase is a “modified nucleobase,” e.g., a nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, a modified nucleobase is substituted A, T, C, G or U. In some embodiments, a modified nucleobase is a substituted tautomer of A, T, C, G, or U. In some embodiments, a modified nucleobases is methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, a modified nucleobase mimics the spatial arrangement, electronic properties, or some other physicochemical property of the nucleobase and retains the property of hydrogen-bonding that binds one nucleic acid strand to another in a sequence specific manner. In some embodiments, a modified nucleobase can pair with all of the five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting the melting behavior, recognition by intracellular enzymes or activity of the oligonucleotide duplex. In some embodiments, as will be understood by those skilled in the art, the term “nucleobase” encompasses structural analogs used in lieu of natural or naturally-occurring nucleotides, such as modified nucleobases and nucleobase analogs. In some embodiments, a nucleobase is optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U. In some embodiments, a “nucleobase” refers to a nucleobase unit in an oligonucleotide or a nucleic acid (e.g., A, T, C, G or U as in an oligonucleotide or a nucleic acid).

[0100] Nucleoside. The term “nucleoside” refers to a moiety wherein a nucleobase or a modified nucleobase is covalently bound to a sugar or a modified sugar. In some embodiments, a nucleoside is a natural nucleoside, e.g., adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, or deoxycytidine. In some embodiments, a nucleoside is a modified nucleoside, e.g., a substituted natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. In some embodiments, a nucleoside is a modified nucleoside, e.g., a substituted tautomer of a natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. In some embodiments, a “nucleoside” refers to a nucleoside unit in an oligonucleotide or a nucleic acid.

[0101] Nucleotide. The term “nucleotide” as used herein refers to a monomeric unit of a polynucleotide that consists of a nucleobase, a sugar, and one or more intemucleotidic linkages (e.g., phosphate linkages in natural DNA and RNA). The naturally occurring bases [guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and uracil (U)] are derivatives of purine or pyrimidine, though it should be understood that naturally and non-naturally occurring base analogs are also included. The naturally occurring sugar is the pentose (five-carbon sugar) deoxyribose (which forms DNA) or ribose (which forms RNA), though it should be understood that, in various embodiments, as will be clear to those skilled in the art, naturally and non- naturally occurring sugar analogs are included. Nucleotides are linked via intemucleotidic linkages to form nucleic acids, or polynucleotides. Various intemucleotidic linkages are known in the art (such as, though not limited to, phosphate, phosphorothioates, boranophosphates and the like). Artificial nucleic acids include PNAs (peptide nucleic acids), phosphotriesters, phosphorothionates, H-phosphonates, phosphoramidates, boranophosphates, methylphosphonates, phosphonoacetates, thiophosphonoacetates and other variants of the phosphate backbone of native nucleic acids. In some embodiments, a natural nucleotide comprises a naturally occurring base, sugar and intemucleotidic linkage. As used herein, the term “nucleotide” also encompasses structural analogs used in lieu of natural or naturally- occurring nucleotides, such as modified nucleotides and nucleotide analogs. In some embodiments, a “nucleotide” refers to a nucleotide unit in a polynucleotide.

[0102] Nutrient. The term “nutrient” is used herein to refer to an agent, entity, event or condition that imparts nourishment, e.g., as may be required for growth and/or maintenance of life, for living cell(s) or systems (e.g., tissues, organs, organoids, organisms, etc). In some embodiments, a nutrient is or comprises an amino acid or polypeptide, a nucleotide or nucleic acid, a lipid, a carbohydrate, a vitamin, a mineral, etc., or a combination thereof. Often, a nutrient is provided to a cell, e.g., by virtue of being present in an environment to which the cell is exposed or in which the cell is present. In some embodiments of the present disclosure, such an environment may be or comprise a culture well; in some embodiments of the present disclosure, such an environment may be or comprise an intestinal lumen.

[0103] Organotypic. The term “organotypic”, is used herein to describe a collection of cells characterized by resemblance to one or more organ(s). In some embodiments, an “organotypic” collection of cells is referred to herein as an “organotypic model” of the relevant organ. In many embodiments, an “organotypic” collection of cells (e.g., an “organotypic model”) includes two or more different cell types that are typically found in the relevant organ. Furthermore, in many embodiments, an “organotypic” collection of cells (e.g., an “organotypic model”) is characterized by spatial organization or other structure analogous to or otherwise reasonably representative of a spatial organization or other structure present in the relevant organ. In some embodiments, an “organotypic” collection of cells (e.g., an “organotypic model”) is characterized by one or more functional characteristic not associated with the cell types included in the model when those cell types are cultured individually (e.g., separately from one another and/or otherwise not in the context of the organotypic model). In one particular embodiment, an intestinal organotypic model may be comprised of enterocytes, Paneth cells, goblet cells, and L cells, and optionally may be characterized, for example, by a stratified epithelium oriented apically towards an air interface and basolaterally towards a liquid interface and/or by barrier function (e g., excluding solutes from apical to basolateral transport).

[0104] Pathway . The term “pathway”, as used herein, refers to a sequence of chemical, physical, and/or electrical interactions that occurs with a living cell. Typically, activity of a pathway results in a change of state of the cell. In some embodiments, activity of a pathway results in production of one or more new molecules. In some embodiments, activity of a pathway transduces a signal from outside the cell; in some such embodiments, such signal represents intracellular communication. Those skilled in the art will be familiar with a variety of biological pathways (e.g., signaling pathways that operate in living cells (e.g., in mammalian cells and/or in cells of neuroendocrine and/or enteroendocrine origin and/or in relevant organs such as the intestine). [0105] Pharmaceutical Device. The term “pharmaceutical device”, as used herein, refers to one or more device(s) comprising one or more active pharmaceutical ingredients for the treatment of one or more disease(s). For example, one or more pharmaceutical device(s) is or may be a tablet, a pill, a capsule, a gel cap, a topical ointment, a lozenge, a sublingual patch, a nasal spray, a nebulized solution, an elixir, a syrup, an oil, a subcutaneous injection, a dermal patch, an intravenous injection, and/or an intramuscular injection.

[0106] Piezoelectric. The term “piezoelectric”, as used herein, refers to one or more process(es) by which translating force (e.g., pressure) is converted into one or more electrical properties. Typically, as defined herein, piezoelectric refers to one or more material(s) (e.g., metals, minerals, ceramics, nucleic acids, certain proteins) characterized by a change in current upon application of pressure. In some instances, piezoelectric refers to piezoresistive, characterized by a change in the resistance to current upon application of pressure.

[0107] Polynucleotide. The term “polynucleotide”, as used herein, refers to a nucleic acid polymer, e.g., comprising at least three residues. In some embodiments, a polynucleotide may be single stranded. In some embodiments, a polynucleotide may be partially or wholly double stranded. In some embodiments, a polynulecotide is or comprises ribonucleotides (RNA); in some embodiments a polynucleotide is or comprises deoxyribonucleotides (DNA). In some embodiments, a polynucleotide may comprise a one or more RNA residues, one or more DNA residues and/or one or more residue analogs. Alternatively or additionally, in some embodiments, a polynucleotide may include one or more modified internucleotidic linkages. In some embodiments, a polynucleotide may include one or more natural RNA or DNA residues, one or more RNA or DNA residues derived from N-glycosides or C-glycosides of nucleobases and/or modified nucleobases; one or more RNA or DNA residues derived from sugars and/or modified sugars; and/or one or more phosphate bridges and/or modified internucleotidic linkages. The term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified internucleotidic linkages. Particular exemplary polynucleotides include nucleic acids containing ribose moieties, nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. [0108] Quenched. The term “quenched”, as used herein, refers to the optoelectronic properties of one or more fluorescent, luminescent, and/or phosphorescent chemical(s). For example, in some instances, one or more quenched first chemical(s) refers or may refer to the spatial proximity of a second chemical, wherein orbital(s) comprising said second chemical exhibit or may exhibit a lower band gap than the excited state orbital(s) of said first chemical(s). Thus, excitation of first chemical(s) by one or more photon(s) and/or electron(s) channels electron(s) towards the second chemical thereby preventing fluorescence, luminescence, and/or phosphorescence. Typically, as provided herein, quenching is or may be reversed upon spatial dissociation of said first chemical from said second chemical.

[0109] Reference. As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.

[0110] Sample. As used herein, the term “sample” typically refers to an aliquot of material obtained or derived from a source of interest, as described herein. In some embodiments, a source of interest is a biological or environmental source. In some embodiments, a source of interest may be or comprise a cell or an organism, such as an animal or a human. In some embodiments, a source of interest may be or comprise an in vitro source such as a cell culture (e.g., a medium thereof). In some embodiments, a source of interest is or comprises biological tissue or fluid. In some embodiments, a biological tissue or fluid may be or comprise neuroendocrine and/or enteroendocrine cells. In some embodiments, a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e g., bronchoalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and/or purification of certain components, etc. In some embodiments, a sample may be a “crude” sample in that it has been subjected to relatively little processing and/or is complex in that it includes components of relatively varied chemical classes.

[0111] Satiety The term “satiety”, as used herein, refers to a state of sufficient energy consumption experienced by one or more cell(s), tissues, organotypic model(s), organ(s), and/or organism(s). In some instances, satiety may be characterized by production and/or release (e.g., secretion) of one or more satiety signals (e.g., one or more agent(s), entity(ies), event(s) or condition(s) whose presence (e.g., occurrence and/or frequency), level (e.g., intensity), form, and/or activity is correlated with or otherwise indicative of a state of satiety). In some instances, a satiety signal may be or comprise a chemical signal (e.g., an agent or entity); in some instances, a satiety signal may be or comprise a physical signal and/or an electrical signal (e.g., a force or discharge or event). In some instances, satiety is or may refer to a change in the expression profde(s) of one or more metabolic protein(s). In some instances, satiety is or may refer to a feeling of fullness in one or more organism(s). In some instances, satiety is or may refer to a feeling of contentment in one or more organism(s).

[0112] Secretion : The term “secretion”, as used herein, refers to the export of one or more chemical entity(ies) from the interior of one or more cell(s), tissues, organotypic model(s), organ(s), and/or organism(s) to the exterior. In many embodiments, secretion may occur in response to a particular signal or stimulus. To give one particular example, in some embodiments, GLP-1 secretion is or may refer to the production and export of GLP-1 from one or more L-cells. To give a different example, in some embodiments, secretion is or may refer to the production and export of saliva from the salivary glands.

[0113] Signal. The term “signal”, as used herein, refers to a agent, entity, event or condition that conveys information, e.g., to a cell or cellular system. In some embodiments, a signal is or may be provided to a cell or cellular system(s) endogenously. In some embodiments, a signal is or may be provided to a cell or cellular system(s) exogenously.

[0114] Stimulus The term “stimulus”, as used herein, refers to an agent, entity, event or condition that, when applied exogenously to a cell or cellular system, acts as a signal. In some embodiments, a stimulus may be or comprise a chemical, physical, and/or electrical entity. In some embodiments, a stimulus is an agent of interest whose satiety-modulating character is assessed as described herein. In some embodiments, a stimulus is or comprises one or more nutrients. In some embodiments, a stimulus is or comprises a food product. In some embodiments, a stimulus is or comprises a food or a crude extract thereof. In some embodiments, a stimulus is or comprises an engineered nutritional material.

[0115] Transfection: The term “transfection”, as used herein, refers to the introduction of exogenous genetic material (e.g., DNA and/or RNA) into a recipient cell. In many embodiments, transfection is or may be achieved through complexation of nucleic acids with or within lipids (e.g., in liposomes, micelles, lipid nanoparticles, etc), carbohydrates, polymers, viral capsids or virus-like particles, etc., which are contacted with recipient cells. In some cases, transfection may utilize or be achieved through nutrient deprivation. In some cases, transfection is or may be achieved through transient introduction of membrane pores under a high electric voltage.

B. Overview

[0116] Disclosed herein, among other things, in some embodiments, are provided technologies for assessing (e.g., quantifying) satiety by measuring certain signal(s) (e.g., signal level(s)).

[0117] As provided herein, quantification of satiety signaling refers to the assignment of a numerical (e.g., discrete, continuous, decimal, whole) value to satiety signaling involving one or more first living cell(s). In certain embodiments, such one or more first living cell(s) generate or may generate one or more satiety signal(s) for intercellular and/or intracellular communication. In certain embodiments, one or more satiety signal(s) represents or may represent the presence of nutrients in surrounding environment(s). In certain embodiments, one or more satiety signal(s) represents or may represent change(s) in metabolic priority. In certain embodiments, one or more satiety signal(s) represents or may represent change(s) in energy storage need(s). As provided herein, quantification of satiety signaling relies or may rely upon the measurement of level(s) of one or more signal(s). In certain embodiments, one or more satiety signal(s) generated by one or more living cell(s) is or may be chemical, physical, and/or electrical signal(s). In certain embodiments, said chemical, physical, and/or electrical signal(s) are or may be challenging to directly observe and/or quantitate. Provided herein are method(s) of converting said chemical, physical, and/or electrical signal(s) to essentially electrical signal(s) to enable quantitation. As provided herein, quantification of satiety signal(s) in response to one or more stimuli enable or may enable determination of one or more conclusion(s) regarding feature(s) of stimuli and/or feature(s) of one or more first living cell(s).

[0118] Disclosed herein, among other things, are method(s) of quantifying satiety signaling by measuring signal level(s). As provided herein, said technologies for assessing (e.g., quantifying) satiety are or may be comprised of a step of providing stimuli to one or more first living cell(s) for a predetermined period of time, a step of receiving one or more signal(s) generated by one or more first living cell(s), a step of converting one or more signal(s) to readout(s) by a signal adapter, and a step of determining a conclusion with the method based on correlating at least one feature(s) of stimuli towards one or more readout(s). In certain embodiment s) said method(s) of quantifying satiety signal(s) are or may be utilized to assign numerical value(s) to one or more stimuli. In certain embodiment(s) said method(s) of quantifying satiety signal(s) are or may be utilized to assign numerical value(s) to one or more first living cell(s).

[0119] In certain embodiments of the present disclosure, provided technologies for assessing (e.g., quantifying) satiety comprise a step of providing stimuli to one or more first living cell(s) for a predetermined period of time. In certain embodiments, a step of providing stimuli to one or more first living cell(s) is or may be characterized by application. In certain embodiments, application is or may comprise modification of one or more extracellular environment(s) by the addition, subtraction, replacement, and/or conversion of said extracellular matrices. In certain embodiments, a step of providing stimuli to one or more first living cell(s) is or may be characterized by one or more stimuli. In certain embodiments, one or more stimuli is or may be characterized as chemical, physical, and/or electrical stimuli. For example, in some instances, one or more chemical stimuli is or may be nutrient(s), pharmacological intervention(s), and/or digested food product(s). In certain embodiments, a step of providing stimuli to one or more first living cell(s) is or may be characterized by one or more cell(s). In certain embodiments, one or more cell(s) is or may be characterized as monolayer cell(s), suspension cell(s), tissue(s), organotypic model(s), organoid(s), organ(s), and/or organism(s). For example, in some instances, one or more cell(s) is or may be a monolayer of cancerous enteroendocrine cell(s), a differentiated intestinal model generated from gavage of human patient(s), and/or excised human ileum(s). In certain embodiments, a step of providing stimuli to one or more first living cell(s) is or may be characterized by time. In certain embodiments, one or more predetermined duration(s) is or may be characterized as a period of continuous exposure to stimuli, frequency of exposure to stimuli, and/or delay required to stimulate one or more first living cell(s).

[0120] In certain embodiments of the present disclosure, provided technologies for assessing (e.g., quantifying) satiety comprise a step of receiving one or more signal(s) generated by one or more first living cell(s). In certain embodiments, a step of receiving one or more signal(s) generated by one or more first living cell(s) is or may be characterized by method(s) of receiving. In certain embodiments, one or more method(s) of receiving is or may be characterized as collection (e.g., harvesting) of acellular and/or cellular sample(s). In certain embodiments, one or more method(s) of receiving is or may be capture of satiety signal(s) by signal adapter(s) spatially adjacent to one or more first living cell(s). In certain embodiments, a step of receiving one or more signal(s) generated by one or more first living cell(s) is or may be characterized by said satiety signal(s). In certain embodiments, one or more satiety signal(s) is or may be characterized as first messengers, second messengers, genetic information, and/or receptor expression, as provided herein.

[0121] In certain embodiments of the present disclosure, provided technologies for assessing (e g., quantifying) satiety comprise a step of converting one or more signal(s) to readout(s) by a signal adapter. In certain embodiments, a step of converting one or more signal(s) to readout(s) by a signal adapter is or may be characterized by suitable signal adapter(s). In certain embodiments, one or more signal adapter(s) exhibits chemical, physical, and/or electrical change(s) in the presence of satiety signal(s). In certain embodiments, said chemical, physical, and/or electrical change(s) are converted to one or more electrical signal(s). In certain embodiments, one or more signal adapter(s) is or may comprise second living cell(s) and/or is or may be acellular. In certain embodiments, one or more signal adapter(s) comprise one or more carbohydrate(s), protein(s), lipid(s), nucleic acid(s), metal(s), and/or ion(s) exhibiting change(s) in the presence of specific signal(s). In some embodiments, change(s) in one or more carbohydrate(s), protein(s), lipid(s), nucleic acid(s), metal(s), and/or ion(s) results or may result in increased photon emission, photon absorption, electron emission, and/or electron absorption. In certain embodiments, said increases in photon emission, fluorescence emission, photon absorption, electron emission, and/or electron absorption are or may be quantified by one or more laboratory instrument(s).

[0122] In certain embodiments of the present disclosure, provided technologies for assessing (e.g., quantifying) satiety comprise a step of determining a conclusion with the method based on correlating at least one feature(s) of stimuli towards one or more readout(s). In certain embodiments, a step of determining a conclusion with the method based on correlating at least one feature(s) of stimuli towards one or more readout(s) is or may be characterized by drawn conclusion(s). In some cases, one or more conclusion(s) is or may be characterized as a ranking of satiety signaling in response to stimuli, an identification of unknown stimuli capable of modulating (e.g., inducing) satiety signal(s), a quantification of health of living system(s), and/or an identification of signaling pathway(s).

[0123] Disclosed herein, among other things, are technologies for assessing (e.g., quantifying) satiety further characterized as high-throughput, non-invasive, integrative, and/or applicable to human enteroendocrine and/or neuroendocrine signaling. In certain embodiments, provided technologies for assessing (e.g., quantifying) satiety are or may be performed on multiwell plates (e.g., 24-well, 48-well, 96-well, and/or 384-well plates) thus enabling quantification of at least 100, at least 200, at least 300, at least 400, and/or at least 500 unique sample(s) in a 2- hour period, for example. In certain embodiments, provided technologies for assessing (e.g., quantifying) satiety are or may be performed using representative model(s) of human patient(s) and/or introduced within a pharmaceutical device to human patient(s), for example. In certain embodiments, provided technologies for assessing (e.g., quantifying) satiety quantify or may quantify response(s) of several distinct satiety signal(s) in response to a single stimulus. In certain embodiments, provided method(s) of satiety signaling quantify or may quantify the response of a single satiety signal in response to several distinct stimuli. In certain embodiments, provided method(s) of quantifying satiety signal(s) are or may utilize cell(s), tissue(s), organotypic model(s), organoid(s), and/or organ(s) from primary human source(s) expected to generate satiety signal(s) equivalent to those of enteroendocrine and/or neuroendocrine signaling in vivo.

[0124] In certain embodiments, the present disclosure is of particular novelty in providing method(s) of quantifying intercellular and/or intracellular communication comprising satiety in response to food. Methods of quantifying intercellular and/or intracellular communication known to those skilled in the art rely or may rely upon knowledge of single signaling pathway(s) implicated in disease state(s). As a result, methods of quantifying intercellular and/or intracellular communication known to those skilled in the art identify or may identify signaling in response to pharmacological intervention(s). Moreover, methods of quantifying intercellular and/or intracellular communication known to those skilled in the art identify or may identify single stimuli, and/or single signal(s). Method(s) of quantifying intercellular and/or intracellular communication as provided herein simultaneously quantify or may quantify several signal(s). Moreover, method(s) of quantifying intercellular and/or intracellular communication as provided herein do not rely upon the presentation of one or more disease state(s).

[0125] In certain embodiments, the present disclosure is of particular novelty in providing method(s) of quantifying satiety signal(s) characterized as high-throughput. Methods of quantifying intercellular and/or intracellular communication known to those skilled in the art comprise or may comprise endpoint assays and/or microscope assays requiring at least 4 hours, at least 5 hours, at least 6 hours, and/or at least 8 hours to quantify at least 100 samples. Moreover, methods of quantifying intercellular and/or intracellular communication known to those skilled in the art require or may require several concurrent assays to quantify changes in one or more signal(s) over time. Methods of quantifying satiety signal(s) as provided herein are or may be essentially characterized as quantifying at least 10 samples, at least 20 samples, at least 30 samples, at least 40 samples, at least 50 samples, at least 60 samples, at least 70 samples, at least 80 samples, at least 90 samples , at least 100 samples, at least 150 samples, at least 200 samples, at least 250 samples at least 500 samples, at least 750 samples, or at least 1000 samples within a 30 minute, within a 1-hour, or within a 2-hour period. In some embodiments, methods of quantifying satiety signal(s) as provided herein are or may be characterized as real-time.

[0126] In some embodiments, methods of quantifying satiety signal(s) as provided herein are or may be characterized by the measurement of a satiety signal within about 1 minute to 2 hours of addition of one or more satiety modulating agents to one or more living cells. For example, in some embodiments, methods of quantifying satiety signal(s) as provided herein are or may be characterized by the measurement of a satiety signal within about 1 minute to 2 hours, about 15 minutes to 2 hours, about 30 minutes to 2 hours, about 45 minutes to 2 hours, about 1 hour to 2 hours, about 1.25 hours to 2 hours, about 1.5 hours to 2 hours, about 1.75 hours to 2 hours, about 1 minute to 1.75 hours, about 15 minutes to 1.75 hours, about 30 minutes to 1.75 hours, about 45 minutes to 1.75 hours, about 1 hour to 1.75 hours, about 1.25 hours to 1.75 hours, about 1.5 hours to 1.75 hours, about 1 minute to 1.5 hours, about 15 minutes to 1.5 hours, about 30 minutes to 1.5 hours, about 45 minutes to 1.5 hours, about 1 hour to 1.5 hours, about 1.25 hours to 1.5 hours, about 1 minute to 1.25 hours, about 15 minutes to 1.25 hours, about 30 minutes to 1.25 hours, about 45 minutes to 1.25 hours, about 1 hour to 1.25 hours, about 1 minute to 1 hour, about 15 minutes to 1 hour, about 30 minutes to 1 hour, about 45 minutes to 1 hour, about 1 minute to 45 minutes, about 15 minutes to 45 minutes, about 30 minutes to 45 minutes, about 1 minute to 30 minutes, about 15 minutes to 30 minutes, or about 1 minute to 15 minutes of addition of one or more satiety modulating agents to one or more living cells.

[0127] In certain embodiments, the present disclosure provides method(s) of quantifying satiety signal(s) characterized as uninformed. Methods of quantifying intercellular and/or intracellular communication known to those skilled in the art comprise or may comprise assays for known stimuli and/or known targets. As a result, methods of quantifying intercellular and/or intracellular communication known to those skilled in the art are unable to quickly identify novel stimuli. Method(s) of quantifying satiety signal(s), as provided herein, are or may be free from knowledge of underlying satiety signaling pathway(s) instead relying upon rapid quantification of satiety marker(s).

[0128] In certain embodiments, the present disclosure provides method(s) of quantifying satiety signal(s) characterized as biorelevant. Methods of quantifying intercellular and/or intracellular communication known to those skilled in the art comprise or may comprise assays utilizing model cell lines (e.g., HEK) failing to recapitulate in vivo satiety signal(s). Method(s) of quantifying satiety signal(s), as provided herein, are or may be performed on one or more primary cell(s) (e.g., monolayer cell(s), tissue(s), organotypic model(s), organoid(s), organ(s), and/or organism(s)) thus providing an accurate representation of satiety signaling in vivo. In some embodiments, method(s) of quantifying satiety signal(s), as provided herein, are or may be performed on one or more cell line(s) that have been modified (e.g., modified to express a recombinant protein) and/or cultured in a manner (e.g., in cell mixtures, on basement matrices, and/or Transwell systems) to provide a more accurate representation of satiety signaling in vivo than cell line monocultures alone.

C. Assessing satiety (e.g., quantifying satiety signaling)

[0129] Among other things, the present disclosure provides one or more method(s) of assessing (e.g., quantifying) satiety (e.g., by quantifying satiety signaling). In some instances, quantifying satiety signaling involves assignment of a numeric value, as described herein, to satiety signaling (and/or to a satiety state). In some embodiments, satiety signaling is characterized by a physical, chemical, and/or electrical change in a living system upon exposure to one or more physical, chemical, and/or electrical stimuli. For example, in some instances, one or more living systems is or may be characterized by the provision (e.g., secretion) of one or more chemical entities upon exposure to one or more chemical, physical, and/or electrical stimuli. In some cases, one or more chemical, physical, and/or electrical change(s) provided by one or more living systems upon exposure to said one or more stimuli is or may be further characterized as signaling. In some instances, one or more signal(s) provided by one or more living system(s) essentially mediate intercellular communication, as provided herein. In some instances, one or more signal(s) provided by one or more living system(s) essentially mediate intracellular communication, as provided herein.

[0130] Without wishing to be bound by any particular theory, it is contemplated that production (e.g., secretion) of one or more chemical entities (e.g., hormones) by one or more living system(s) upon exposure to one or more physical, chemical, and/or electrical stimuli indicates the health and/or functionality, or relevant characteristic or state thereof (e.g., satiety state) of said living system(s). Without wishing to be bound by any particular theory, it is contemplated that production (e.g., secretion) of one or more chemical entities (e.g., hormones) by one or more living system(s) upon exposure to one or more physical, chemical, and/or electrical stimuli is or may be compared to that of reference living system(s) under exposure to an identical physical, chemical, and/or electrical stimulus. Without wishing to be bound by any particular theory, it is contemplated that the assignment of a numerical value towards one or more secretion(s) performed by one or more living system(s) enables the facile comparison of health and/or functionality of said living system(s), or relevant characteristic or state thereof (e.g., satiety state).

[0131] Without wishing to be bound by any particular theory, it is contemplated that production (e.g., emission) of one or more fluorescent or luminescent signal by one or more living system(s) upon exposure to one or more physical, chemical, and/or electrical stimuli indicates the health and/or functionality, or relevant characteristic or state thereof (e.g., satiety state) of said living system(s). Without wishing to be bound by any particular theory, it is contemplated that production (e.g., emission) of one or more fluorescent or luminescent signal by one or more living system(s) upon exposure to one or more physical, chemical, and/or electrical stimuli is or may be compared to that of reference living system(s) under exposure to an identical physical, chemical, and/or electrical stimulus. Without wishing to be bound by any particular theory, it is contemplated that the assignment of a numerical value towards one or more emission(s) performed by one or more living system(s) enables the facile comparison of health and/or functionality of said living system(s), or relevant characteristic or state thereof (e.g., satiety state).

[0132] Without wishing to be bound by any particular theory, it is contemplated that the provision of one or more physical responses (e.g., physical reorganization) by one or more living system(s) upon exposure to one or more physical, chemical, and/or electrical stimuli indicates the health and/or functionality of said living system(s), or relevant characteristic or state thereof (e.g., satiety state). Without wishing to be bound by any particular theory, it is contemplated that the provision of one or more physical responses (e.g., physical reorganization) by one or more living system(s) upon exposure to one or more physical, chemical, and/or electrical stimuli is or may be compared to that of reference living system(s) under exposure to an identical or comparable physical, chemical, and/or electrical stimulus. Without wishing to be bound by any particular theory, it is contemplated that the assignment of a numerical value towards one or more physical response(s) performed by one or more living system(s) enables the facile comparison of health and/or functionality of said living system(s), or relevant characteristic or state thereof (e g., satiety state).

[0133] Without wishing to be bound by any particular theory, it is contemplated that the provision of one or more electrical responses (e.g., change in membrane potential) by one or more living system(s) upon exposure to one or more physical, chemical, and/or electrical stimuli indicates the health and/or functionality of said living system(s), or relevant characteristic or state thereof (e.g., satiety state). Without wishing to be bound by any particular theory, it is contemplated that the provision of one or more electrical responses (e.g., change in membrane potential) by one or more living system(s) upon exposure to one or more physical, chemical, and/or electrical stimuli is or may be compared to that of reference living system(s) under exposure to an identical physical, chemical, and/or electrical stimulus. Without wishing to be bound by any particular theory, it is contemplated that the assignment of a numerical value towards one or more electrical response(s) performed by one or more living system(s) enables the facile comparison of health and/or functionality of said living system(s), or relevant characteristic or state thereof (e.g., satiety state).

[0134] Among other things, the present disclosure provides one or more technologies for assessing (e.g., quantifying) satiety. In certain embodiments of the present invention, quantifying satiety signaling involves assignment of a numeric value, as described herein, to chemical, physical, and/or electrical change(s) in one or more living system(s). In some instances, one or more assigned numerical value(s) is or may describe the amount of one or more secreted chemical entities from one or more living system(s). In some instances, one or more assigned numerical value(s) is or may describe the force exerted by one or more living system(s) upon one or more sensor(s). In some instances, one or more assigned numerical value(s) is or may describe the membrane potential of one or more living system(s). In some embodiments, the chemical, physical, and/or electrical response(s) provided by one or more living system(s) in response to one or more chemical, physical, and/or electrical stimuli are or may be quantified absolutely. In some embodiments, a chemical, physical, and/or electrical response(s) provided by one or more living system(s) in response to one or more chemical, physical, and/or electrical stimuli are or may be quantified referentially. For example, in some cases, quantification of chemical, physical, and/or electrical response(s) provided by one or more living system(s) is a change occurring in response to one or more chemical, physical, and/or electrical stimuli. For example, in some cases, quantification of chemical, physical, and/or electrical response(s) provided by one or more living system(s) is a change occurring in reference to a separate living system(s).

[0135] Without wishing to be bound by any particular theory, provided methods of quantifying satiety is or may be advantageous for assigning a numerical value towards the intercellular and/or intracellular communication (e.g., signaling) comprising the response of one or more living system(s) to satiety. Without wishing to be bound by any particular theory, provided technologies for assessing (e.g., quantifying) satiety are or may be advantageous for assigning a numerical value to changes in satiety experienced by one or more living system(s). Without wishing to be bound by any particular theory, provided technologies for assessing (e.g., quantifying) satiety are or may be advantageous for assigning a relative numerical value between the satiety signaling of several distinct living system(s). Without wishing to be bound by any particular theory, provided technologies for assessing (e.g., quantifying) satiety are or may be particularly advantageous for identifying (e.g., previously unknown) chemical, physical, and/or electrical stimuli resulting in satiety signaling in one or more living system(s). Without wishing to be bound by any particular theory, provided technologies for assessing (e.g., quantifying) satiety are or may be particularly advantageous for identifying (e.g., previously unknown) chemical, physical, and/or electrical stimuli resulting in hunger signaling in one or more living system(s). In some instances, without wishing to be bound by any particular theory, the provided technologies for assessing (e g., quantifying) satiety are or may be particularly advantageous for comparing (e.g., ranking) the response elicited by one or several chemical, physical, and/or electrical stimuli in one or more living system(s).

[0136] For example, in some instances, provided technologies for assessing (e.g., quantifying) satiety are or may be useful for predicting the satiety signaling, as provided herein, of one or more living system(s) by means of distinct living system(s). The present disclosure appreciates that assessing (e.g., quantifying) satiety are particularly challenging in living system(s), particularly for example in healthy human(s). In certain embodiments, the present disclosure provides technologies that can achieve assessment (e.g., quantification) of satiety in healthy human(s). For example, the present disclosure provides a particular insight that use of organotypic model(s) as described herein may permit assessment (e.g., quantification) of satiety relevant to healthy humans.

[0137] Yet another advantage of certain technologies provided by the present disclosure is that they are not reliant upon the collection and assay of human plasma. It is contemplated that such collection and assay of human plasma causes or may cause pain, discomfort, nausea, and/or trauma upon the subject(s), thus reducing willingness to participate in sample collection and/or interfering with sufficient sample collection. In certain embodiments, provided technologies for assessing (e.g., quantifying) satiety sufficient to predict satiety signaling in healthy human(s) utilize or may utilize living system(s) incapable of pain, discomfort, nausea, and/or trauma. Additionally, or alternatively, in some embodiments, provided technologies for assessing (e.g., quantifying) satiety sufficient to predict satiety signaling in healthy human(s) utilize or may utilize signal readouts, as provided herein, that do not impose pain, discomfort, nausea, and/or trauma upon the subject(s). For example, in some instances, one or more technologies for assessing (e.g., quantifying) satiety sufficient to predict satiety signaling in healthy human(s) are or may be characterized by interspecies signaling within the ileum of the subject(s).

[0138] Yet another advantage of various embodiments of technologies provided herein is that they permit high throughput assessments. In some embodiments, an assessment may be characterized as low throughput when the number of distinct response(s) quantified in at least one of about 1 minute, about 30 minutes, about 1 hour, about 4 hours, about 8 hours, about 12 hours, and/or about 24 hours. For example, an assessment may be considered to be low throughput if it quantifies fewer than 10 satiety signaling response(s) in 1 hour. Low throughput technologies typically fail to enable the identification of unknown chemical, physical, and/or electrical stimuli eliciting satiety signaling in one or more living system(s).

[0139] In certain embodiments, provided technologies for assessing (e.g., quantifying) satiety are essentially characterized as high throughput, as provided herein. In some instances, provided method(s) characterized as high throughput are or may be further characterized by the number of distinct response(s) quantified in at least one of about 1 minute, about 30 minutes, about 1 hour, about 4 hours, about 8 hours, about 12 hours, and/or about 24 hours. For example, in one instance, one or more method(s) characterized as high throughput is or may be characterized as quantifying at least 90 satiety signaling response(s) in 1 hour.

[0140] Yet a further advantage provided by certain embodiments of the present disclosure is that provided technologies are not limited to quantifying a single chemical, physical, and/or electrical change in one or more living system(s) in response to a single chemical, physical, and/or electrical stimulus. Without wishing to be bound by any particular theory, it is contemplated that a single stimulus can often affect several chemical, physical, and/or electrical changes a living system, combinations of which comprise satiety signaling. Without wishing to be bound by any particular theory, it is contemplated that several stimuli together may sometimes affect a single chemical, physical, and/or electrical change a living system, comprising satiety signaling. In certain embodiments, provided technologies for assessing (e.g., quantifying) satiety, characterized as high throughput, enable quantification of satiety signaling from combinations of stimuli and/or impacting or generating a plurality of signals.

[0141] In certain embodiments, provided technologies for assessing (e.g., quantifying) satiety comprise a step of providing one or more stimuli to one or more first living cell(s) for a period of time. In certain embodiments, one or more stimuli are or may be further characterized as chemical, physical, and/or electrical stimuli originating external to the cell membrane of one or more first living cell(s). Without wishing to be bound by any particular theory, it is contemplated that satiety signal(s) generated by one or more living system(s) enable intracellular and/or intercellular communication of environmental (e.g., external) conditions. Without wishing to be bound by any particular theory, it is contemplated that satiety signal(s) generated by one or more living system(s) rely or may rely upon sensing protein(s) (e.g., receptor(s)) present in the cell membrane. As provided herein, one or more stimuli are or may be characterized as present in buffer(s), cell culture media, serum, intestinal lumen, mucus, and/or water. In certain embodiments, one or more first living cell(s) generating one or more satiety signal(s) is or may be characterized as neuroendocrine and/or enteroendocrine. For example, in some instances, one or more first living cell(s) characterized as neuroendocrine and/or enteroendocrine is or may be further characterized as buccal, gastric, pancreatic, hepatic, intestinal, neuronal, and/or bacterial in origin. In some embodiments, one or more first living cell(s) is essentially characterized as living. In certain embodiments, one or more first living cell(s) is or may be further characterized by metabolic activity, intracellular trafficking, and/or expression of sensing protein(s) (e.g., receptor(s)). In certain embodiments, one or more stimuli are or may be provided to one or more first living cell(s) for a predetermined period of time. Without wishing to be bound by any particular theory, incubation of one or more chemical, physical, and/or electrical stimuli in the presence of one or more first living cell(s) for a predetermined period of time is contemplated to provide sufficient time for the stimuli to interact with said first living cell(s). Without wishing to be bound by any particular theory, incubation of one or more chemical, physical, and/or electrical stimuli in the presence of one or more first living cell(s) for a predetermined period of time is contemplated to provide sufficient time for said first living cell(s) to generate one or more signaling response(s).

[0142] In certain embodiments, provided technologies for assessing (e.g., quantifying) satiety comprise a step of receiving one or more signal(s) generated by one or more first living cell(s). In certain embodiments, one or more signal(s) generated by one or more first living cell(s) is or may be characterized as chemical, physical, and/or electrical change(s) originating from said one or more living cell(s). For example, in some instances, one or more chemical change(s) characterized as a signal is or may be secretion of GLP-1, gastric inhibitory protein (GIP), or a combiantion thereof. For example, in some instances, one or more physical change(s) characterized as a signal is or may be tensile stress. For example, in some instances, one or more electrical change(s) characterized as a signal is or may be membrane depolarization. In certain embodiments, provided technologies for assessing (e.g., quantifying) satiety involve receiving one or more signal(s) generated by one or more first living cell(s). As provided herein, receiving one or more si nal(s) refers or may refer to capturing one or more chemical, physical, and/or electrical change(s). In some instances, capturing one or more chemical change(s) is or may refer to collection of cell culture media, a receptor-ligand interaction, a binding (e.g., complexation), collection of serum, and/or harvesting of one or more cell(s). In some instances, receiving one or more physical change(s) is or may refer to collection of cell force data by a piezoelectric sensor. In some instances, receiving one or more electrical change(s) is or may refer to collection of membrane potential by protein conformational changes and/or patch-clamp recording. In certain embodiments, capture of one or more signal(s) requires the separation of one or more first living cell(s) from collected signal(s). In certain embodiments, capture of one or more signal(s) occurs in the presence of one or more first living cell(s). In certain embodiments, provided technologies for assessing (e.g., quantifying) satiety involve receiving one single signal from one or more first living cell(s). In certain embodiments, provided technologies for assessing (e.g., quantifying) satiety involve receiving several independent signals from one or more first living cell(s).

[0143] In certain embodiments, provided technologies for assessing (e.g., quantifying) satiety comprise a step of converting one or more signal(s) to readout(s) by a signal adapter. In certain embodiments, one or more chemical, physical, and/or electrical signal(s) comprising one or more signal(s) is or may be received by capture (e.g., collection). In certain embodiments, one or more signal(s) received by capture are or may be further purified before conversion to readout(s) by signal adapter. In certain embodiments, one or more signal(s) received by capture are or may be used without further purification before conversion to readout(s) by signal adapter. In certain embodiments, one or more signal(s) is or may be converted to one or more readout(s) by a signal adapter. In some embodiments, one or more signal adapter(s) is or may be a molecular binding event, a piezoelectric sensor, and/or electrical measurement device. In certain embodiments, one or more signal adapter(s) is essentially characterized as converting one or more chemical, physical, and/or electrical signal(s) to at least one of: light (e.g., fluorescence, phosphorescence, luminescence), color (e.g., new covalent bonds), heat (e.g., molecular motion), electrical current (e.g., movement of electrons), and/or sound (e.g., molecular motion). In certain embodiments, one or more signal adapter(s) convert one or more chemical, physical, and/or electrical signal(s) to a numerical (e.g., discrete and/or continuous) readout. In certain embodiments, provided technologies for assessing (e.g., quantifying) satiety are or may essentially comprise a detector for at least one of light, color, heat, electrical current, and/or sound thus converting said signal(s) to a quantifiable electrical current. In certain embodiments, a signal adapter is or may comprise, for example, one or more carbohydrate(s), lipid(s), protein(s), nucleic acid(s), receptor(s), enzyme(s), antibody(s), bacterium(a), small molecule(s), or a fluorescent dye(s).

[0144] In certain embodiments, provided technologies for assessing (e.g., quantifying) satiety comprise a step of determining a conclusion with the method(s) based on correlating at least one feature(s) of stimuli to one or more readout(s). In certain embodiments, determining one or more conclusion(s) is or may comprise ranking of one or more stimuli by quantity and/or magnitude of one or more readout(s). In certain embodiments, determining one or more conclusion(s) is or may comprise identifying new stimuli by quantity and/or magnitude of one or more readout(s). In some embodiments, one or more conclusion(s) is determined from readout(s) following the provision of one known stimuli in combination with several unknown stimuli to one or more first living cell(s). In certain embodiments, determining one or more conclusion(s) is or may comprise identifying and/or quantifying the health of one or more living system(s). In certain embodiments, determining one or more conclusion(s) is or may comprise identifying and/or quantifying the signaling pathway(s), as provided herein, of one or more living system(s). In some embodiments, one or more conclusion(s) is determined comparing readout(s) obtained between first living cell(s) characterized as monolayer cell(s), organoids(s), and/or organism(s). In some embodiments, one or more conclusion(s) determined using readout(s) obtained from one or more first living cell(s) identifies and/or quantifies one or more unknown satiety signaling pathway(s).

(i) Providing one or more stimuli

[0145] As provided herein, provided technologies for assessing (e.g., quantifying) satiety comprise a step of providing one or more stimuli to one or more first living cell(s) for a period of time (e.g., a predetermined amount of time). In some instances, provision of one or more stimuli to one or more first living cell(s) occurs or may occur once (e.g., one single time). In some instances, provision of one or more stimuli to one or more first living cell(s) occurs or may occur several times (e.g., repeatedly, regularly, etc.). In some instances, provision of one or more stimuli is characterized as providing a single chemical, physical, and/or electrical stimulus. In some instances, provision of one or more stimuli is characterized as providing several chemical, physical, and/or electrical stimuli.

[0146] In certain embodiments, a step of providing one or more stimuli to one or more first living cell(s) is or may be particularly useful for inducing a chemical, physical, and/or electrical response in one or more first living cell(s). In certain embodiments, one or more stimuli are known to induce a certain chemical, physical, and/or electrical response in one or more first living cell(s). In certain embodiments, one or more stimuli are not known to induce a certain chemical, physical, and/or electrical response in one or more first living cell(s). In certain embodiments, provided technologies for assessing (e.g., quantifying) satiety are essentially characterized as high throughput. Without wishing to be bound by any particular theory, it is contemplated that high throughput method(s) are or may facilitate inducing chemical, physical, and/or electrical response(s) in one or more first living cell(s) from one or more unknown stimuli.

[0147] In certain embodiments, one or more stimuli is or may be applied to one or more first living cell(s) to induce chemical, physical, and/or electrical response(s). In some cases, application of one or more stimuli to one or more first living cell(s) occurs in vitro. In some cases, the application of one or more stimuli to one or more first living cell(s) occurs in vivo. In some cases, the application of one or more stimuli to one or more first living cell(s) occurs ex vivo. In some cases, the application of one or more stimuli introduces new stimuli to one or more first living cell(s). In some cases, the application of one or more stimuli removes existing stimuli to one or more first living cell(s).

[0148] In certain embodiments, provided technologies for assessing (e.g., quantifying) satiety comprise a step of providing one or more essentially extracellular stimuli to one or more first living cell(s). Without wishing to be bound by any particular theory, one or more extracellular stimuli are or may be indicative or predictive of satiety signaling in one or more healthy living organism(s). Without wishing to be bound by any particular theory, satiety signaling in one or more healthy living system(s) is or may comprise inter- and/or intra-cellular communication regarding extracellular chemical, physical, and/or electrical stimuli. In certain embodiments, one or more extracellular stimuli interact or may interact (e.g., receptor-ligand interaction) with one or more membrane proteins comprising one or more first living cell(s). In certain embodiments, one or more extracellular stimuli interact or may interact (e.g., receptorligand interaction) with one or more nucleic acids comprising one or more first living cell(s). In certain embodiments, one or more extracellular stimuli interact or may interact (e.g., receptorligand interaction) with one or more lipids comprising one or more first living cell(s).

[0149] In certain embodiments, provided technologies for assessing (e.g., quantifying) satiety comprise a step of providing one or more extracellular stimuli to one or more first living cell(s). As provided herein, in many embodiments, utilized cells are or comprise cells that are neuroendocrine and/or enteroendocrine in origin. In some embodiments, one or more first living cell(s) are further characterized by receptor expression. In some embodiments, one or more first living cell(s) are further characterized by one or more chemical, physical, and/or electrical response(s) to extracellular stimuli. In certain embodiments, one or more first living cell(s) are or may be further characterized as buccal, gastric, pancreatic, hepatic, intestinal, neuronal, and/or bacterial in origin.

[0150] In certain embodiments, provided technologies for assessing (e.g., quantifying) satiety comprise a step of providing one or more extracellular stimuli for a period of time (e.g., a predetermined period of time). Without wishing to be bound by any particular theory, it is contemplated that one or more signal(s) generated by one or more living cell(s) upon exposure to extracellular stimuli rely or may rely upon one or more binding interaction(s). In certain embodiments of the present disclosure, the period of time in which one or more extracellular stimuli are provided to one or more living cell(s) controls the signal(s) generated by one or more first living cell(s). In certain embodiments of the present disclosure, the period of time in which one or more stimuli are provided to one or more living cell(s) is independent of the signal(s) generated by one or more first living cell(s). In certain embodiments of the present disclosure, the period of time in which one or more stimuli are provided to one or more living cell(s) refers, or may refer to, a period of time before receiving one or more signal(s). In certain embodiments of the present disclosure, the period of time in which one or more stimuli are provided to one or more living cell(s) refers, or may refer to, a period of time before repeated provision of one or more stimuli.

(it) Application of stimuli [0151] In certain embodiments of the present disclosure, technologies for assessing (e.g., quantifying) satiety comprising a step of applying one or more stimuli are disclosed. In certain embodiments, application of one or more stimuli provides or may provide for the interaction of one or more stimuli with one or more first living cell(s). Without wishing to be bound by any particular theory, it is contemplated that quantification of satiety signaling is or may be accomplished through certain methods of application, as provided herein.

[0152] In certain embodiments of the present disclosure, technologies for assessing (e.g., quantifying) satiety comprise stimulation of one or more first living cell(s). In certain embodiments of the present disclosure, extracellular stimuli are or are present in natural extracellular media of one or more living cell(s). In certain embodiments, one or more extracellular stimuli are or may be provided as a gas, a liquid, a solid, through shear, through pressure, through tension, through voltage, through current, and/or combinations thereof.

[0153] In certain embodiments, application of one or more stimuli utilizes or may utilize one or more delivery device(s). For example, in certain embodiments, application of one or more stimuli utilizes or may utilize nanoparticle(s), microparticle(s), protein aggregate(s), polymer(s), hydrogel(s), aerosol(s), pipette(s), microinjector(s), syringe(s), fluidic device(s), electrode(s), or any combination(s) thereof.

[0154] In certain aspects of the present disclosure, one or more living cell(s), further characterized as monolayer culture(s), suspension culture(s), tissue(s), or organ(s) may be cultivated immersed in one or more culture media. In certain embodiments, one or more culture media is or may comprise one or more component(s). For example, one or more culture media comprise or may comprise sugars, salts, animal-derived serum, amino acid(s), ion(s), or combination(s) thereof. In some embodiments, one or more culture media comprise or may comprise those component s) necessary for growth and/or maintenance of one or more living cell(s), further characterized as basal culture media. In some embodiments, addition of one or more chemical, physical, and/or electrical stimuli to one or more basal culture media comprises or may comprise provision of stimuli. In some embodiments, replacement of one or more basal media with culture media comprising differing component(s) comprises or may comprise provision of stimuli. [0155] In some embodiments, provided technologies assess satiety in tissue sample(s). In some embodiments, a tissue sample is or comprises a biopsy, a seeded gavage, portion(s) of one or more organ(s), an organ, and/or combinations thereof. In some embodiments of the present disclosure, a tissue sample(s) is or may be cultivated at an air-liquid interface, and/or a liquidliquid interface. In some embodiments, said air-liquid interface comprises one or more tissue sample(s) wherein the apical surface is or may be directly exposed to air and the basolateral surface is or may be directly exposed to liquid. In some embodiments, an air-liquid interface comprises one or more tissue sample(s) wherein the apical surface is or may be directly exposed to liquid and the basolateral surface is or may be directly exposed to liquid. In certain embodiments, a liquid interface(s) is or may be comprised of culture media, as provided herein.

[0156] Technologies provided herein, in some embodiments, comprise a step of applying one or more stimuli to one or more living cell(s) characterized as tissue sample(s). In some embodiments, application of one or more stimuli to tissue sample(s) is or may comprise aerosolization of one or more stimuli, addition to culture media, replacement and/or alteration of culture media, application of mechanical force(s), application of an electric current, and/or combinations thereof. Without wishing to be bound by any particular theory, it is contemplated that application of stimuli to one or more tissue sample(s) is particularly advantageous so as to maintain spatial arrangement s), physiological response(s), and heterogeneous profile(s) of tissue(s) found in healthy intact organ(s) and/or organism(s).

[0157] In some embodiments, provided technologies assess satiety in a human being. In certain embodiments, a step of providing stimuli in one or more first living cell(s), thus, may comprise intravenous injection(s), subcutaneous injection(s), intraperitoneal injection(s), retro- orbital injection(s), intrathecal inj ection(s), and/or oral gavage of solid(s), liquid(s), or gas(es).

[0158] In some embodiments, a step of applying one or more stimuli to one or more first living cell(s) comprises or may comprise one or more formulated food and/or beverage product(s). For example, in some instances, one or more chemical stimuli is or may comprise one or more nutrient(s) comprising one or more food(s) and/or beverage(s). In some instances, said one or more nutrient(s) is or may be encapsulated and/or spatially distributed so as to restrict release. In some instances, said one or more nutrient(s) is or may be encapsulated and/or spatially distributed so as to restrict receptor-ligand interaction(s). In some embodiments food and/or beverage product(s) are or may be further characterized as non-formulated, formulated, mashed, an intact product, blended, fermented, digested, and/or any combination(s) thereof. Without wishing to be bound by any particular theory, it is contemplated that controlled spatial arrangement(s) of one or multiple stimuli component(s) may alter the rate of receptor-binding, rate of uptake, extent of formulation stability, extent of formulation degradation, and/or combination(s) thereof.

[0159] As disclosed herein, in certain embodiments, formulated is further characterized as exhibiting a controlled spatial arrangement of one or multiple stimuli component(s). It is known to those skilled in the art that controlled spatial arrangement(s) of one or multiple stimuli component(s) may alter the rate of receptor-binding, rate of uptake, extent of formulation stability, extent of formulation degradation, and/or any combination(s) thereof.

(Hi) Stimuli

[0160] The present invention pertains, in part, to technologies for assessing (e.g., quantifying) satiety sin one or more first living cell(s) as a function of stimuli. Without wishing to be bound by any particular theory, it is contemplated that native (e.g., natural) neuroendocrine and/or enteroendocrine signaling is propagated in response to one or more environmental stimuli. In some cases, said one or more first living cell(s) may be further characterized as a single layer of one type of cell. For example, in some instances, one or more living cell(s) may be further characterized as a cell monolayer. In some cases, said one or more first living cell(s) may be further characterized as tissue sample(s). For example, in some instances, one or more living cell(s) may be further characterized as an organotypic model. In some cases, said one or more first living cell(s) may further comprise a living organism. For example, one or more living cell(s) may be further characterized as a healthy human.

[0161] In some embodiments, one or more extracellular stimuli is a chemical, physical, and/or electrical stimulus originating external to the cell membrane of one or more first living cell(s). In some embodiments, one or more stimuli are generated by one performing provided method(s) of quantifying satiety signaling. In some embodiments, one or more extracellular stimuli are generated by one first living cell(s). [0162] In certain embodiments, one or more stimuli is or may be characterized as interacting with membrane-bound receptors comprising one or more first living cell(s). In certain embodiments, an interaction with membrane-bound receptor(s) is or may be characterized as a receptor-ligand interaction, a conformational change, and/or a chemical reaction. In certain embodiments, one or more stimuli is or may be characterized as interacting with cytosolic proteins comprising one or more first living cell(s). In certain embodiments, such interaction with cytosolic proteins is or may be characterized as a receptor-ligand interaction, a conformational change, and/or a chemical reaction. In certain embodiments, one or more stimuli is or may be characterized as interacting with nucleic acid(s) comprising one or more first living cell(s). In certain embodiments, such interaction with nucleic acid(s) is or may be characterized as a receptor-ligand interaction, a conformational change, and/or a chemical reaction.

[0163] In certain embodiments, one or more stimuli is or may comprise one or more stimuli applied to the apical and/or basolateral surfaces of one or more cell culture monolayer(s) and/or tissue sample(s). In certain embodiments, one or more stimuli is or may comprise one or more stimuli applied enterally, parenterally, and/or topically in one or more organism(s).

(a) Chemical stimulus

[0164] In some instances, one or more chemical stimuli, as provided herein, is or may be a chemical entity capable of a receptor-ligand interaction with one or more first living cell(s). In some instances, one or more chemical stimuli, as provided herein, is or may be a chemical entity capable of modulating (e.g., inducing) satiety signaling in one or more first living cell(s).

[0165] In some embodiments, one or more chemical stimuli characterized as one or more chemical entities capable of modulating (e.g., inducing) satiety signaling in one or more first living cell(s) is or may be further characterized as carbohydrate(s), ion(s), lipid(s), caloric nutrient(s), acaloric nutrient(s), peptide(s), nucleic acid(s), polymer(s), surfactant(s), and/or any combination thereof. In some embodiments, addition of said chemical entity(ies) to one or more first living cell(s) is characterized as a stimulus. In some embodiments, removal of said chemical entity(ies) from one or more first living cell(s) is characterized as a stimulus. [0166] In some embodiments, one or more chemical stimuli are or may be capable of modulating (e.g., inducing) satiety signaling in one or more first living cell(s) by receptor-ligand interaction(s). In certain embodiments, one or more receptor-ligand interaction(s) is or may be further characterized as an agonist, antagonist, orthosteric activator, allosteric activator, orthosteric inhibitor, allosteric inhibitor, partial agonist, partial antagonist, partial allosteric activator, partial orthosteric inhibitor, partial allosteric inhibitor, and/or combinations thereof.

[0167] In some embodiments, one or more stimuli is or may be the act of replacing culture media, adding culture media, removing culture media, increasing chemical entity concentration(s), decreasing chemical entity concentration concentration s), or any combination thereof.

[0168] In some embodiments, a chemical stimulus is or may be further characterized as a carbohydrate. In some embodiments, one or more carbohydrate(s) is or may be D-glucose, D- galactose, D-fructose, maltose, D-xylose, D-mannose, sucrose, isomaltulose, trehalose, D- psicose, tagatose, lactose, lactulose, arabinose, amylopectin, dextran, pectin, amylose, inulin, locust bean gum, maltodextrin, xanthan gum, gum arabic, karaya gum, ghatti gum, guar gum, sodium carboxymethylcellulose, sodium alginate, sodium hyaluronate, calcium alginate, agarose, chitosan, chitin, carrageenan, chondroitin sulfate, hydroxypropyl methylcellulose, methyl cellulose, modified cellulose gum, ethyl cellulose, hydroxyethylcellulose, com starch, cellulose triacetate, cellulose acetate butyrate, cellulose, cellulose acetate propionate, cellulose acetate succinate, cellulose acetate phthalate, and/or hydroxypropyl methylcellulose acetate succinate.

[0169] In some embodiments, a chemical stimulus is or may be further characterized as a ketone. In some embodiments, one or more ketone(s) may be a ketone ester. In some embodiments, one or more ketone(s) is or may comprise acetoacetate, beta-hydroxybutyrate, deltaG® (“Delta G”), and acetone. In some embodiments, one or more ketone(s) is or may comprise acetoacetate, beta-hydroxybutyrate, and acetone.

[0170] In some embodiments, a chemical stimulus is or may be further characterized as an ion. In some embodiments, one or more ion(s) are characterized as comprising one or more of sodium ion(s), potassium ion(s), magnesium ion(s), calcium ion(s), chloride ion(s), phosphate ion(s), bicarbonate ions, and/or any combination of these. [0171] In some embodiments, a chemical stimulus is or may be further characterized as a lipid. In some embodiments, one or more lipid(s) is or may be characterized as acid(s), amide(s), monoglyceride(s), diglyceride(s), ether(s), diether(s), and/or triglyceride(s). For example, in some embodiments, one or more lipid(s) is or may comprise linoleic acid, a-linoleic acid (ALA), palmitic acid, arachidonic acid, myristic acid, lauric acid, eicosapentaenoic acid, stearic acid, myristic acid, lauric acid, decanoic acid, octanoic acid, palmitoleic acid, caprylic acid, arachidic acid, caproic acid, butyric acid, erucic acid, gamma-linoleic acid, omega-6 fatty acid, lignoceric acid, behenic acid, elaidic acid, dihomo-y-linolenic acid, stearic acid, omega-3 fatty acids, oleic acid, docosahexaenoic acid, capric acid, butyric acid, lauric acid, behenic acid, lignoceric acid, lauroleic acid, caproleic acid, vaccenic acid, gadoleic acid, brassidic acid, nervonic acid, columbic acid, stearidonic acid, mead acid, docosapentaenoic acid, docosahexaenoic acid, or any combinations or derivative(s) thereof.

[0172] In some embodiments, a chemical stimulus is or may be further characterized as acaloric nutrient(s). In some embodiments, one or more acaloric nutrient(s) is or may comprise quercetin, kaempferol, myricetin, fisetin, rutin, isorhamnetin, naringenin, silybin, eriodictyol, apigenin, chrysin, delphinidin, betanin, cyanidin chloride, neohesperidin, epigall ocatechin, diosmetin, baicalein, genistein, oleuropein, amarogentin, genipin, aucubin, catalpol, olivetol, cannabidiol, tetrahydrocannabinol, daidzein, pelargonidin, tangeritin, luteolin, wogonin, epicatechin, catechin, theaflavin, resveratrol, cholesterol, sitosterol, oryzanol, diindolylmethane, piperine, vitamin(s) A, vitamin(s) B, vitamin(s) C, vitamin(s) D, vitamin(s) E, vitamin(s) K, and/or hydroxytyrosol.

[0173] In some embodiments, a chemical stimulus is or may be a protein(s). In some embodiment(s), one or more protein(s) may be further characterized as comprising one or more amino acid(s). In certain embodiments, one or more protein(s) may be further characterized as naturally derived. In certain embodiments, one or more protein(s) may be further characterized as semi-synthetic. In certain embodiments, one or more protein(s) may be further characterized as fully synthetic. In certain embodiments, one or more protein(s) may be further characterized as possessing a molecular weight of at least 10 Da, at least 100 Da, at least 1000 Da, at least 10000 Da, and/or at least 100000 Da. In certain embodiments, one or more chemical stimuli characterized as protein(s) is or may comprise disodium guanylate, disodium inosinate, L- glycine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamine, L- glutamic acid, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L- proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, amylase, avenin, calcium caseinate, cellulase, collagen, corn protein isolate, fibroin, gelatin, ghrelin, leptin, cholecystokinin, amylin, peptide yy, GLP-1, GLP-2, GIP, insulin, glutanin, kefirin, lipase, milk protein concentrate, oat protein isolate, pea protein isolate, protease, rice protein isolate, sodium caseinate, soy protein isolate, wheat protein isolate, whey protein isolate (digested or undigested), and/or zein.

[0174] In some embodiments, a chemical stimulus may be a surfactant(s). In some embodiments one or more surfactant(s) is or may comprise sorbitan laurate, sorbitan palmitate, sorbitan stearate, sorbitan oleate, sorbitan trioleate, polyoxyethylene sorbitan laurate, polyoxyethylene sorbitan palmitate, polyoxyethylene sorbitan stearate, polyoxyethylene trioleate, brij 30, brij 35, sodium oleate, potassium oleate, polyethylene glycol, sodium lauryl sulfate, sodium dodecyl sulfate, or any derivative(s) thereof.

[0175] In some instances, a chemical stimulus may be a single chemical entity. In some cases, a chemical stimulus may comprise a plurality of chemical entities. In some embodiments, a chemical stimulus forms a covalent bond, an ionic bond, a hydrogen bond, a dipolar interaction, a Van der Waals interaction, and/or combinations thereof with at least one or more carbohydrate(s), lipid(s), protein(s), and/or nucleic acid(s) comprising one or more first living cell(s). In some embodiments, characterized chemical stimulus participates in receptor-ligand interaction(s), as provided herein.

[0176] In certain specific embodiment(s), chemical stimuli are or may be pharmacological ligands; for example falling under the general categories of analgesics, antacids, antianxiety, antiarrhythmics, antibacterials, anticoagulants, thrombolytics, anticonvulsants, antidepressants, antidiarrheals, antiemetics, antifungals, antihistamines, antihypertensives, antiinflammatories, antineoplastics, antipsychotics, antipyretics, antivirals, barbiturates, betablockers, bronchodilators, corticosteroids, cytotoxics, decongestants, diuretics, expectorants, hormones, hypoglycemics, immunosuppressives, laxatives, muscle relaxants, sedatives, sex hormones, tranquilizers, and/or vitamins, or any combination(s) thereof. [0177] In certain embodiments, a virus may be a chemical stimulus. In certain embodiments, one or more chemical stimuli characterized as virus(es) encode or may encode genetic information producing one or more random peptides on the surface of encoded viral capsid. In certain embodiments, one or more encoded virus(es) displaying random peptide(s) are or may be added to one or more first living cell(s) in order to generate one or more satiety signal(s). Without wishing to be bound by any particular theory, it is contemplated that one or more viral-encoded random peptide(s) exhibit or may exhibit particular binding affinity towards one or more receptor(s) comprising one or more first living cell(s), thereby modulating (e g., inducing) satiety signaling.

[0178] In some embodiments, one or more receptor-ligand interaction(s) is further characterized by having a binding constant, a binding affinity, an effective concentration, an inhibitory concentration, or any combinations thereof. In certain embodiments, the concentration of one or more chemical stimuli further characterized as a ligand is or may be quantified by one or more of the following method(s), immunoassay, scintillation proximity assay, radiolabeling, high performance liquid chromatography, mass spectroscopy, fluorimetry, luminometry, or any combination(s) thereof.

(b) Physical stimulus

[0179] In certain embodiments of the present disclosure, a physical stimulus is characterized as providing one or more forces towards one or more first living cell(s). In certain embodiments, provided stimuli characterized as one or more force(s) towards one or more first living cell(s) induce or may induce formation of one or more covalent bond(s), ionic bond(s), hydrogen bond(s), dipolar interaction(s), Van der Waals interaction(s), conformational change(s) and/or combinations thereof. In certain embodiments, provided stimuli characterized as one or more force(s) towards one or more first living cell(s) break or may break one or more covalent bond(s), ionic bond(s), hydrogen bond(s), dipolar interaction(s), Van der Waals interaction(s), conformational change(s) and/or combinations thereof.

[0180] In some embodiments, one or more stimuli characterized as physical stimuli are or may be further characterized by one or more force(s). For example, in some embodiments, one or more stimuli characterized as physical stimuli is or may be characterized as the application of a pressure, a tension, a compression, and/or a shear towards one or more first living cell(s). In certain embodiments, one or more stimuli characterized as physical stimuli is or may be characterized as disrupting membrane protein(s), tight junction(s), focal adhesion(s), actin(s), and/or extracellular matrices.

[0181] In some embodiments, one or more stimuli characterized as physical stimuli are or may be further characterized by the magnitude and/or frequency of one or more force(s). For example, in some embodiments, one or more stimuli characterized as physical stimuli are or may be characterized by the application of a compression and/or tension of at least 10 qN at least 50 qN, at least 100 qN. For example, in some embodiments, one or more stimuli characterized as physical stimuli are or may be characterized by the application of a pressure and/or shear of at least 0.01 Pa, at least 0.05 Pa, at least 0.07 Pa, at least 0.15 Pa, at least 0.30 Pa, at least 0.50 Pa, at least 0.80 Pa, at least 1.0 Pa. For example, in some embodiments, one or more stimuli characterized as physical stimuli are or may be characterized by application of a compression and/or tension with a frequency of at least 10 Hz, at least 25 Hz, at least 50 Hz, at least 100 Hz, at least 150 Hz, at least 200 Hz, at least 300 Hz, at least 500 Hz, at least 800 Hz, at least 1000 Hz. For example, in some embodiments, one or more stimuli characterized as physical stimuli are or may be characterized by application of a pressure and/or shear with a frequency of at least 10 Hz, at least 25 Hz, at least 50 Hz, at least 100 Hz, at least 150 Hz, at least 200 Hz, at least 300 Hz, at least 500 Hz, at least 800 Hz, at least 1000 Hz.

[0182] In some embodiments, one or more physical stimuli are applied to one or more organisms; in some embodiments, such application modulates (e.g., initiates, enhances, reduces, or terminates) satiety signaling in such one or more organism. In some embodiments, one or more physical stimuli characterized as consumption provides or may provide one or more physical stimuli towards one or more first living cell(s). For example, in some instances, one or more physical stimuli further characterized as eating, over-eating, chewing, over-chewing, swallowing, over-swallowing, peristalsis, drinking, over-drinking, and/or digesting is or may comprise stimuli. (c) Electrical Stimulus

[0183] In some instances, one or more electrical stimuli is characterized as one or more electromagnetic (e.g., electrical) excitation(s) towards one or more first living cell(s). In certain embodiments, provided stimuli characterized as one or more electromagnetic excitation(s) towards one or more first living cell(s) induce or may induce formation of one or more covalent bond(s), ionic bond(s), hydrogen bond(s), dipolar interaction(s), Van der Waals interaction(s), conformational change(s) and/or combinations thereof. In certain embodiments, provided stimuli characterized as one or more electromagnetic excitation(s) towards one or more first living cell(s) break or may break one or more covalent bond(s), ionic bond(s), hydrogen bond(s), dipolar interaction(s), Van der Waals interaction(s), conformational change(s) and/or combinations thereof.

[0184] For example, in some embodiments, one or more stimuli characterized as electrical stimuli is or may be characterized as the application of an electric current, an electromagnetic voltage, an electrical resistance, an electrical inductance, and/or light (e.g., photons) towards one or more first living cell(s). For example, in some embodiments, one or more stimuli characterized as electrical stimuli is or may be characterized as the removal of an electric current, an electromagnetic voltage, an electrical resistance, an electrical inductance, and/or light (e.g., photons) from one or more first living cell(s).

[0185] Without wishing to be bound by any particular theory, it is contemplated that satiety signaling is or may be essentially comprised of combination(s) of chemical, electrical, and/or physical response(s) to one or more stimuli. In particular, it is contemplated that satiety signaling is or may be essentially mediated by changes in electrical membrane potential(s). Without wishing to be bound by any particular theory, it is thus contemplated that providing one or more stimuli further characterized as electrical stimuli towards one or more cell(s) results or may result in satiety signaling.

[0186] In some embodiments, one or more stimuli characterized as electrical stimuli is or may be further characterized by membrane potential(s). In certain embodiments, one or more electrical stimuli is or may be a magnitude of change in membrane potential(s) by at least 5, at least 10, at least 20, at least 40, and/or at least 70 mV. In certain embodiments, one or more electrical stimuli is or may be a duration of change in membrane potential(s) of at least 1 ms, at least 5 ms, at least 10 ms, at least 50 ms, at least 100 ms, at least 500 ms, and/or at least 1 s. In some embodiments, one or more electrical stimuli is or may be characterized as combination(s) of changes in magnitude of membrane potential(s) and duration(s).

(Hi) Living cell(s)

[0187] In certain embodiments one or more living cell(s) is or may comprise one or more class(es) within the animal, plant, fungal, protist, and/or monera kingdoms. In certain embodiments, one or more living cell(s) is or may be further characterized by metabolic activity, intracellular trafficking, and/or expression of sensing protein(s) (e.g., receptor(s)).

[0188] In some embodiments, one or more living cell(s), as utilized in accordance with the present disclosure, is or may be further characterized by metabolic activity. In some cases, metabolic activity is or may be quantification of cellular respiration and/or quantification of enzymatic activity. For example, in some instances, one or more cell(s) is or may be characterized as living by measuring at least one of oxygen consumption, Alamar Blue, esterase activity, Live-Dead assay, NADPH oxidoreductase activity, MTT assay, and/or MTS assay.

[0189] In some embodiments, one or more living cell(s) as provided herein, is or may be further characterized by intracellular trafficking. In some cases, intracellular trafficking is or may be active transport of one or more protein(s) comprising one or more living cell(s). For example, in some instances, one or more cell(s) is or may be characterized as living by measuring the secretion of one or more protein(s) in response to one or more stimuli. For example, in some instances, one or more cell(s) is or may be characterized as living by measuring secreted ghrelin, leptin, cholecystokinin, amylin, peptide yy, GLP-1, GLP-2, GIP, or insulin.

[0190] In some embodiments, one or more living cell(s) as provided herein, is or may be further characterized by expression of one or more membrane receptor(s). For example, in some instances, one or more membrane receptor(s) is or may be characterized by receptor-ligand interactions with one or more chemical stimuli. For example, in some instances, one or more cell(s) is or may be characterized as living by measuring cell surface expression of GPRC6A, CaSR, TasteR, GPR93, FFAR2, FFAR3, FFAR1, FFAR4, GPR40, GPR70, GPR119, CB1 , GLP- 1R, GLP-2R, GIPR, IR, Y2R, Y4R, Y1R,

[0191] TGR5, GPR17, SGLT-1, SGLT-2, GPR120, or any combination thereof. In some embodiments, one or more cell(s) is or may be characterized as living by measuring gene expression (e.g., by quantitative PCR (qPCR)) of GPRC6A, CaSR, TasteR, GPR93, FFAR2, FFAR3, FFAR1, FFAR4, GPR40, GPR70, GPR119, CB1, GLP-1R, GLP-2R, GIPR, IR, Y2R, Y4R, Y1R, TGR5, GPR17, SGLT-1, SGLT-2, GPR120, or any combination thereof.

[0192] In some embodiments, one or more cell(s) is or may be characterized as responsive to satiety modulating agents by measuring cell surface expression of TGR5, GPR17, GPR40, GPR119, GPR120, SGLT-1, SGLT-2, CaSR, or any combination thereof. In some embodiments, one or more cell(s) is or may be characterized as responsive to satiety modulating agents by measuring gene expression (e.g., by qPCR) of TGR5, GPR17, GPR40, GPR119, GPR120, SGLT-1, SGLT-2, CaSR, or any combination thereof.

[0193] In many embodiments, desirable living cell(s) is or may comprise one or more cell(s) further characterized as neuroendocrine and/or enteroendocrine. In some embodiments, provided method(s) quantify signaling of the neuroendocrine and/or enteroendocrine system; including but not limited to the pituitary gland, the parathyroid gland(s), the adrenal gland(s), the stomach and/or surrounding tissue(s), the duodenum and/or surrounding tissue(s), the ileum and/or surrounding tissue(s), the jejunum and/or surrounding tissue(s), the colon and/or surrounding tissue(s), the vagus nerve(s), afferent nerve(s), sensory neuron(s), and/or tissue vasculature system(s).

[0194] In some embodiments, provided method(s) quantify one or multiple signals from cell(s) derived from cancerous origins. In some embodiments, provided method(s) quantify one or multiple signals from cell(s) derived from non-cancerous origins. In some embodiments, provided method(s) quantify one or multiple signals from neuroendocrine and/or enteroendocrine cell(s) derived from primary neuroendocrine and/or enteroendocrine tissue sample(s), including but not limited to samples harvested from organ biopsy, organoid structures, organ-on-a-chip technologies, stem-cell derived tissue(s), or other iteration(s) thereof. In some embodiments, provided method(s) quantify signal(s) generated from cell(s) or tissues cultured in, for example, adherent cultures on plastic surfaces, suspension culture(s), organoid(s), co-culture(s), transwell(s), glass surfaces, bioreactor(s), and/or combinations thereof.

[0195] In some embodiments, provided method(s) quantify one or multiple signals from cell(s) derived from the endocrine organs, as provided herein. In some embodiments, the method quantifies one or multiple signals from cell(s) further characterized as immortalized cells derived from STC-1, NCI-H716, GluTag, HT29-MTX, Caco2, TC7, IPEC-2, IPEC-J2, IEC-6, IEC-18, HIEC-6, SK-CO-1, SNU-C1, LoVo, CT26.CL25, HCT-15, SW-620, SW-480, HCT-8, SNU-5, or any variant(s) thereof. In some embodiments, the method quantifies one or multiple signals from cell co-cultures including cells selected from, but not limited to STC-1, NCI-H716, GluTag, HT29-MTX, Caco2, TC7, IPEC-2, IPEC-J2, IEC-6, IEC-18, HIEC-6, SK-CO-1, SNU-C1, LoVo, CT26.CL25, HCT-15, SW-620, SW-480, HCT-8, SNU-5, and any variant(s) thereof. For example, co-cultures may include 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cells selected from, but not limited to STC-1, NCI-H716, GluTag, HT29-MTX, Caco2, TC7, IPEC-2, IPEC-J2, IEC-6, IEC- 18, HIEC-6, SK-CO-1, SNU-C1, LoVo, CT26.CL25, HCT-15, SW-620, SW-480, HCT-8, SNU-5 and any variant(s) thereof. In some embodiments, the method quantifies one or multiple signals from a cell co-culture including NCLH716 and Caco2 cells.

[0196] In some embodiments, the method quantifies one or multiple signals form cells in monoculture or co-culture cultured in matrices, and/or compartmentalized culture systems (e.g., transwell cultures) that promote or achieve cellular polarization, and/or three dimensional structures that mimic in vivo physiology.

[0197] In some embodiments, provided method(s) quantify one or multiple signals from cell(s) derived from the endocrine organs, as provided herein. In some embodiments, the method quantifies one or multiple signals from cell(s) further characterized as immortalized cells derived from HeLA, GH3, PC12, MDA-MB-231, MDA-MB-453, MCF-7, T-47D, U2OS, HUVEC, HEK-293T, GOT1, P-STS, BON-1, QGP-1, HBMC, PC-12, Capan-2, Pane 10.05, CFPAC-1, Pane 05.04, AsPC-1, PSN-1, SW 1990, HPAC, WRO, FTC133, BCPAP, TPC1, KI, 8505C, Neu41 or any combination(s) or transformation(s) thereof. (iv) Period of time

[0198] In some embodiments, provided methods of quantifying satiety signaling comprise a step of providing one or more stimuli to one or more first living cell(s) for a period of time (e.g., a predetermined period of time). In certain embodiments, one or more signal(s) is or may be generated by one or more first living cell(s) in response to such one or more stimuli. In certain embodiments, the magnitude of one or more signal(s) is or may depend on the duration of exposure to such one or more stimuli. In certain embodiments, the frequency of one or more signal(s) is or may depend on the duration of exposure to such one or more stimuli. In certain embodiments, the magnitude and/or frequency of one or more signal(s) is or may depend on the frequency of exposure to such one or more stimuli.

[0199] Without wishing to be bound by any particular theory, it is contemplated that distinct satiety signal(s) are released from one or more first living cell(s) in a time dependent manner. For example, in some instances, one or more satiety signal(s) characterized as a secreted protein is or may be characterized by secretion over 30 minutes. For example, in some instances, one or more satiety signal(s) characterized as calcium release is or may be characterized by release over 5 minutes. Moreover, various conventional technologies for assessing satiety fail to relate stimuli induced satiety signaling as a function of time. In some embodiments, one or multiple satiety signals are or may be quantified following at least about 1 minute, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 12 hours, about 24 hours, and/or about 48 hours exposure to one or more stimuli, as provided herein.

1. Satiety signal(s)

[0200] In certain embodiments of the present disclosure, provided technologies for assessing (e.g., quantifying) satiety involve receiving signal(s) generated by one or more first living cell(s). In certain embodiments, one or more satiety signal(s) generated by one or more first living cell(s) comprise or may comprise one or more receptor(s), primary messenger(s), secondary messenger(s), transcription factor(s), and/or nucleic acid(s). Without wishing to be bound by any particular theory, it is contemplated that receiving one or more satiety signal(s), in many embodiments, involves or relates to signal transduction (e.g., intercellular and/or intracellular signaling) carried out by one or more first living cell(s).

[0201] In certain embodiments, provided technologies quantify or may quantify increases in single signal quantity as a result of a single input stimulus. For example, in some embodiments, provided technologies quantify or may quantify signal level(s) after a single signal of chemical, physical, electrical, pharmacological, biological, or environmental stimulus. In certain embodiments, provided technologies quantify or may quantify increases in single signal quantity as a result of multiple input signal(s). For example, provided technologies quantify or may quantify signal level(s) after a combination of chemical, physical, electrical, pharmacological, biological, or environmental stimuli.

[0202] In certain embodiments, provided technologies quantify or may quantify decreases in single signal quantity as a result of a single input stimulus. For example, provided method(s) quantify or may quantify signal level(s) after a single signal of chemical, physical, electrical, pharmacological, biological, or environmental stimulus. In certain embodiments, provided method(s) quantify or may quantify decreases in single signal quantity as a result of multiple input signal(s). For example, in some embodiments, provided technologies quantify or may quantify signal level(s) after a combination of chemical, physical, electrical, pharmacological, biological, or environmental stimuli.

[0203] In certain embodiments, receiving one or more signal(s) from one or more first living cell(s) is or may be useful for collecting (e.g., harvesting) one or more satiety signal(s). For example, in certain embodiments, receiving one or more satiety signal(s) facilitates (e.g., enables) measuring the quantity of one or more satiety signal(s) by one or more signal transducer(s).

(i) Receiving one or more signal(s)

[0204] In certain embodiments of the present disclosure, provided technologies involve receiving a signal generated by one or more first living cell(s). Without wishing to be bound by any particular theory, it is contemplated that receiving such a generated signal comprises or may comprise collecting sample(s) from one or more first living cell(s). In some embodiments, collecting is further characterized as lysing, pipetting, sampling, centrifuging, harvesting serum, biopsying, resecting, freezing, fixing, or any combinations thereof. In certain embodiments, collecting is further characterized as a binding (e.g., complexation), a piezoelectric recording, and/or an electrical recording.

[0205] In some embodiments, receiving a signal generated by one or more first living cell(s) comprises or may comprise destruction of one or more first living cell(s). In certain embodiments, such destruction of one or more first living cell(s) is or may be further characterized as lysing. In certain embodiments, lysing refers or may refer to the disintegration of the cell, tissue, or organ membrane. In some embodiments, disintegration is or may be mediated by detergent(s), organic solvent(s), fixative(s), preservative(s), salt(s), and/ or enzyme(s).

[0206] In some embodiments, receiving a signal generated by one or more first living cell(s) comprises or may comprise removing a portion of the surrounding environment comprising one or more first living cell(s). In certain embodiments, such receiving of one or more signal(s) is or may be further characterized as sampling. In some embodiments, one or more collected sample(s) is or may be further characterized as a volume, mass, density, concentration, pressure, force, voltage, temperature, and/or rate. In some embodiments, one or more collected sample(s) is or may be portions of living cell(s) comprising larger tissue(s), organ(s), and/or organism(s). In certain embodiments, one or more portions of said living cell(s) is or may be further characterized as a biopsy and/or resection.

[0207] In some embodiments, provided technologies involve receiving one or more portion(s) of surrounding environment(s) of neuroendocrine, enteroendocrine, endocrine and/or nervous system cells.

[0208] In certain embodiments, provided technologies utilize one or more sample(s) that comprise one or more culture media. In some instances, for example, one or more culture media is or may be further characterized as water, phosphate buffered saline solution, simulated intestinal fluid, simulated gastric fluid, simulated tear fluid, simulated urine, HEPES buffered saline solution, Dulbecco’s Modified Eagle Medium, Hank’s balanced salt solution, biological intestinal fluid, biological gastric fluid, plasma, saliva, urine, feces, sweat, tear fluid, and/or Kreb’s buffer.

[0209] In some embodiments, provided technologies involve receiving one or more signal(s); in some such embodiments, one or more signal(s) are or may be received by sampling, as provided herein. In some embodiments, one or more signal(s) received by sampling may comprise a volume of at least 1 qL, at least 10 qL, at least 50 pL, at least 100 pL, at least 500 qL, at least 1 mL, at least 5 mL, at least 10 mL, at least 20 mL, at least 50 mL, at least 100 mL, and/or at least 1 Liter. In some embodiments, one or more received signal(s) are or may be stored in a separate collection vessel further characterized as a microcentrifuge tube, a conical tube, a beaker, a graduated cylinder, a bottle, and/or combination(s) thereof.

[0210] Among other things, in some embodiments, provided technologies quantify satiety signal(s) collected from one or first living cell(s) further characterized as one or more organ(s) as described herein. Without wishing to be bound by any particular theory, it is contemplated that cells(s) are or may be physiologically relevant models for quantifying satiety signal(s) in response to one or more stimuli. In some embodiments, enteroendocrine and/or neuroendocrine cell(s) are further characterized as the mouth, stomach, small intestine, large intestine, liver, pancreas, gallbladder, hypothalamus, pituitary gland, thyroid, adrenal gland, kidney, and/or heart. In some embodiments, receiving one or more signal(s) comprises harvesting, as provided herein, one or more organ(s) further characterized as lethal and/or non- lethal. In some embodiments, lethal organ harvesting is further characterized as collection of an organ otherwise required for the organism to survive; for example, removal of a liver from a murine or porcine model specifically for quantifying satiety signals. In some embodiments, non- lethal organ harvesting is comprised of organ donation, removal of a whole organ not required for survival (i.e., gallbladder), in vitro growth of organs, and/or 3D bioprinting of organ(s).

[0211] In some embodiments, provided technologies comprise a step of receiving one or more signal(s) generated by one or more first living cell(s), which may, for example be received as one or more binding interact! on(s). For example, in some embodiments, one or more si nal(s) received as one or more binding interaction(s) comprises or may comprise a receptor-ligand interaction, a organometallic interaction, a nucleic acid/protein interaction, and/or a nucleic acid/small molecule interaction. For example, in some instances, one or more signal(s) received as one or more binding interaction(s) is or may be a peptide-gold interaction, a peptide-RNA interaction, and/or a peptide-receptor interaction.

(ii) Satiety signaling

[0212] Without wishing to be bound by any particular theory, it is contemplated that signal transduction pathways dictate or may dictate the health and survival of living cell(s). Without wishing to be bound by any particular theory, it is contemplated that one or more signal transduction pathway(s) comprise satiety signaling. In some embodiments, signal transduction is further characterized as the process by which living cell(s) responds to one or more stimuli, as provided herein.

[0213] Without wishing to be bound by any particular theory, it is contemplated that one or more components of satiety signaling is a receptor, a ligand, a primary messenger, a secondary messenger, a transcription factor, and/or a nucleic acid. In some embodiments, provided technologies quantify or may quantify one or multiple components of satiety signaling, for example, a receptor, a ligand, a primary messenger, a secondary messenger, a transcription factor, and/or a nucleic acid in the presence of one or more nutrient(s). In some embodiments, provided technologies quantify or may quantify one or multiple components of biological signaling, for example, a receptor, a ligand, a primary messenger, a secondary messenger, a nucleic acid, and/or a transcription factor in the absence of one or more nutrient(s).

[0214] In some embodiments, quantifying signaling is or comprises quantification of one or more primary messenger(s), as provided herein. Without wishing to be bound by any particular theory, it is contemplated that quantification of one or more primary messenger(s) relates or may relate to the activation of one or more signal transduction pathway(s). In some embodiments, one or more primary messenger(s) are further characterized as hormone(s), ligand(s), neurotransmitter(s), growth factor(s), cytokine(s), and/or chemokine(s).

[0215] In some embodiments, provided technologies involve quantification of one or more second messenger(s) (alternately referred to as secondary messenger(s)), as provided herein. Without wishing to be bound by any particular theory, it is contemplated that quantification of one or more second messenger(s) relates or may relate to the activation of one or more signal transduction pathway(s). In some embodiments, one or more second messenger(s) are further characterized as cyclic nucleotide(s), lipid messenger(s), ion(s), gas(es), or free radical(s). In some embodiments, one or more second messenger(s) are further characterized as intracellular calcium (Ca²⁺), and/or cAMP.

[0216] In some embodiments, provided technologies involve quantification of one or more nucleic acid(s), as provided herein. Without wishing to be bound by any particular theory, it is contemplated that quantification of one or more nucleic acid(s) relates or may relate to the activation of one or more signal transduction pathway(s). In some embodiments, one or more nucleic acid(s) are further characterized as DNA and/or RNA.

[0217] In some embodiments, provided technologies involve quantification of one or more receptor(s), as provided herein. Without wishing to be bound by any particular theory, it is contemplated that quantification of one or more receptor(s) relates or may relate to the activation of one or more signal transduction pathway(s). In some embodiments, one or more receptor(s) are further characterized as nutrient-sensing receptor(s).

(Hi) First messenger(s)

[0218] In certain embodiments, one or more satiety signal(s) generated by one or more first living cell(s) is or may be characterized as first messenger(s). In some cases, one or more first messenger(s) mediates intercellular and/or intracellular communication, for example, between the neuroendocrine, enteroendocrine, endocrine, and/or central nervous system(s). In certain embodiments, one or more satiety signal(s) characterized as first messenger(s) is or may be an amino acid, a peptide, a protein, a neurotransmitter, a cytokine, a growth factor, a hormone, and/or a transcription factor. Without wishing to be bound by any particular theory, it is contemplated that quantifying one or more satiety signal(s) characterized as first messenger(s) is sufficient to quantify satiety signaling in one or more first living cell(s).

[0219] In certain embodiments, one or more satiety signal(s) characterized as one or more first messenger(s) is or may be further characterized as a peptide, a protein, and/or a hormone. One or more first messenger(s), thus, is or may be leptin, GLP-1, GLP-2, motilin, gastrin, insulin, amylin, spexin, glucagon, ghrelin, peptide yy, cholecystokinin, GIP, endorphin peptides, their pre hormones, their isoforms, their degradation products, and/or their full length and/or spliced transcripts.

[0220] In some embodiments, one or more satiety signal/s) characterized as one or more first messenger(s) is or may be further characterized as neurotransmitter(s). One or more neurotransmitter(s), as provided herein, mediate intercellular and/or intracellular communication between and/or interfacing with one or more neuron(s). One or more first messenger(s) further characterized as one or more neurotransmitter/s), thus, is or may be serotonin, histamine, anandamide, dopamine, glutamate, oleoyl ethanol ami de, palmitoylethanolamide, linolenoyl ethanolamide, gamma aminobutyric acid, acetylcholine, epinephrine, norepinephrine, and/or glycine.

[0221] In some embodiments, one or more satiety signal/s) characterized as one or more first messenger(s) is or may be further characterized as growth factor(s). One or more growth factor(s), as provided herein, mediate intercellular and/or intracellular communication between and/or interfacing with cell(s). For example, in some instances, one or more growth factor(s) is or may be characterized as activating the transcription, translation, and/or expression of one or several gene(s). In some cases, one or more growth factor/ s) is or may be further characterized as cytokine(s) and/or chemokine(s). One or more first messenger(s) further characterized as one or more growth factor/ s), thus, is or may be AM, Ang, Autocrine motility factor, BMPs, CNTF, LIF, M-CSF, G-CSF, GM-CSF, EGF, Ephrin, EPO, FGF, FBS, GDNF, Neurturin, Persephin, Artemin, GDF9, HGF, HDGF, Insulin, IGF-1, IGF -2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, KGF, MSF, HGFLP, GDF-8, NRG1, NRG2, (NRG3, NRG4, Neurotrophin, BDNF, NGF, NT-3, NT-4, PGF, PDGF, RNLS, TCGF, TPO, TGF-a, TGF-0, TNF-a, VEGF, and/or WNT.

[0222] In some embodiments, one or more satiety signal/s) characterized as one or more first messenger/ s) is or may be further characterized as transcription factor/ s). One or more transcription factor/ s), as provided herein, mediate intercellular and/or intracellular communication between and/or interfacing with cell/s). For example, in some instances, one or more transcription factor/ s) is or may be characterized as activating the transcription, translation, and/or expression of one or several gene/s). One or more first messenger/ s) further characterized as one or more transcription factor/s), thus, is or may be NR3A1, NR3A2, NR3C4, NR3B3, NR1C1, NR1C13, NR1A1, NR1A2, NR1I12, NR2B1, NR2B2, NR2B3, any nuclear receptor homodimer formed from the listed receptors, any nuclear receptor heterodimer formed from the listed receptors, KLF4, ATF3, CEBPA, Myc, CTCF, TBP, ASCL1, ELK1, bZIP domains, CEBPB, E2F4, ETV4, HNF4A, SOX2, ARID3A, ATF1, CEBPD, BCL11A, BACH1, F0XA2, BCL11B, IRF3, ETS2, CICR, EPAC2A, CREB, NFkb, MEF2, HSF01, SPF, any factors within the same genetic families, and/or any combinations thereof.

(iv) Second messenger(s)

[0223] In certain embodiments, one or more satiety signal(s) generated by one or more first living cell(s) is or may be characterized as second messenger(s). In some cases, one or more second messenger(s) mediates intercellular and/or intracellular communication, for example, between the neuroendocrine, enteroendocrine, endocrine, and/or central nervous system(s). In certain embodiments, one or more satiety signal(s) characterized as second messenger(s) is or may be nucleotide, a lipid, a gas, a protein, and/or an ion. Without wishing to be bound by any particular theory, it is contemplated that quantifying one or more satiety signal(s) characterized as second messenger(s) is sufficient to quantify satiety signaling in one or more first living cell(s).

[0224] In certain embodiments, the quantity of one or more second messenger(s) is or may relate to the quantity of one or more first messenger(s) comprising one or more satiety signal(s). Without wishing to be bound by any particular theory, it is contemplated that the quantification of one or more second messenger(s) is or may be more accessible than that of one or more first messenger(s). Thus, in certain embodiments, quantification of one or more second messenger(s) is or may be characterized as quantification of satiety signal(s).

[0225] Among other things, the present disclosure provides technologies for quantification of second messengers in a satiety pathway. In some aspects, quantification of second messengers is advantageous to determine the propagation of signal transduction pathways in response to one or more stimuli. In some embodiments, second messenger(s) may be further characterized as cyclic nucleotide(s), protein(s), lipid messenger(s), ion(s), gas(es), or free radical(s). In certain embodiments, one or more second messenger(s) is or may be characterized as PI3K, cAMP, Epac2, PKB/Akt, PKA, MEK,ERK, TCF1, beta-arrestin, PLC, ATF4, ATP, cGMP, calcium ions, potassium ions, sodium ions, magnesium ions, phosphate ions, chloride ions, bicarbonate ions, sulfate ions, any genetic isoforms or variants, and/or any combination(s) thereof.

[0226] In some embodiments, quantification of second messengers in a satiety pathway includes quantification of intracellular calcium ions, cAMP, or a combination thereof. In some embodiments, quantification of intracellular calcium ions is achieved by one or more genetically encoded calcium indicators (GECIs). See, e.g., Zhang et al., Comm. Bio. 4, 924 (2021); Salgado-Almario et al., Int. J. Mol. Sci. 2020, 21(18), 6610. GECIs can effectively report calcium activity in a cell via a fluorescence response to calcium concentration changes. In some embodiments, a GECI is a single-fluorophore GECI. In some embodiments, a GECI is a fluorescence resonance energy transfer (FRET) GECI. In some embodiments, quantification of intracellular calcium ions is achieved by the recombinant expression of TWITCH-NR in one or more first living cell(s). See, e.g., Wang et al., Nat. Commun. 2022, 13:5363; and Liu et al., Front. Pharmacol. 13-2022. In some embodiments, quantification of intracellular cAMP is achieved by one or more genetically encoded fluorescent indicators (GEFIs). In some embodiments, a GEFI is a single fluorescent protein GEFI. In some embodiments, a GEFI is a FRET GEFI. In some embodiments, quantification of cAMP is achieved by the recombinant expression of gFLAMP in one or more first living cell(s).

(v) Genetic information

[0227] In certain embodiments, one or more satiety signal(s) generated by one or more first living cell(s) is or may be characterized as genetic information. In some cases, one or more genetic information mediates intercellular and/or intracellular communication, for example, between the neuroendocrine, enteroendocrine, endocrine, and/or central nervous system(s). In certain embodiments, one or more satiety signal(s) characterized as genetic information is or may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and/or protein expression profile(s). Without wishing to be bound by any particular theory, it is contemplated that quantifying one or more satiety signal(s) characterized as genetic information is sufficient to quantify satiety signaling in one or more first living cell(s). [0228] In some embodiments, one or more satiety signal(s) generated by one or more first living cell(s) is or may be characterized as messenger RNA (mRNA) encoding leptin, GLP- 1, GLP-2, motilin, gastrin, insulin, amylin, spexin, glucagon, ghrelin, peptide yy, cholecystokinin, GIP, endorphin, or any combination thereof.

[0229] In some embodiments, provided technologies for assessing (e.g., quantifying) satiety are characterized as quantifying change(s) in the genetic material of one or more cell(s), tissue(s), organ(s), and/or organism(s). In some embodiments, said change(s) in genetic material comprise transformation(s), modification(s), spatial arrangement(s), structural arrangement(s), mutation(s), damage(s), and/or any diseases impacting characteristics of DNA, RNA, and/or protein expression(s). In some embodiments, provided technologies quantify changes in genetic material(s) after a single stimulus, at a single time point. In some embodiments, provided method(s) quantify changes in genetic material(s) after multiple stimuli, at a single time point. In some embodiments, provided method(s) quantify changes in genetic material(s) after a single stimulus, at time(s) including but not limited to about 1 minute after stimulus, about 30 minutes after stimulus, about 1 hour after stimulus, about 4 hours after stimulus, about 8 hours after stimulus, about 12 hours after stimulus, about 24 hours after stimulus, and/or about 48 hours after stimulus. In some embodiments, provided method(s) quantify changes in genetic material(s) after multiple stimuli, at time(s) including but not limited to about 1 minute after stimuli, about 30 minutes after stimuli, about 1 hour after stimuli, about 4 hours after stimuli, about 8 hours after stimuli, about 12 hours after stimuli, about 24 hours after stimuli, and/or about 48 hours after stimuli.

(vi) Cellular receptor(s)

[0230] In certain embodiments, one or more receptor(s) will be quantified by the method in response to stimuli as described herein. In some instances, quantifying receptor(s) in response stimuli may be advantageous for assessing stimuli-specific receptor expression changes. In some embodiments, receptors are classified as proteins that propagate biological signaling. In some embodiments, receptors are quantified by the method in response to one or more nutrients. For example, one or more receptor(s) quantified by the method is or may be: GPRC6A, CaSR, TasteR, GPR93, FFAR2, FFAR3, FFAR1, FFAR4, GPR40, GPR70, GPR119, CB1, GLP-1R, GLP-2R, GIPR, IR, Y2R, Y4R, Y1R, TGR5, GPR17, SGLT-1 , SGLT-2, GPR120, or any combination thereof .

[0231] In some cases, provided technologies will involve quantifying gene expression level of a single receptor in response to one stimulus as described herein. In some cases, the method will quantify the gene expression level of a single receptor in response to multiple stimuli as described herein. In some cases, the method will quantify the gene expression level(s) of any and all combination(s) of the receptors listed in response to one stimulus as described herein. In some cases, the method will quantify the gene expression level(s) of any and all combination(s) of the receptors listed in response to multiple stimuli as described herein.

[0232] In some embodiments, provided technologies quantify protein expression level of a single receptor in response to one stimulus as described herein. In some cases, the method will quantify the protein expression level of a single receptor in response to multiple stimuli as described herein. In some cases, the method will quantify the protein expression level(s) of any and all combination(s) of the receptors listed in response to one stimulus as described herein. In some cases, the method will quantify the protein expression level(s) of any and all combination(s) of the receptors listed in response to multiple stimuli as described herein.

2. Converting signals

[0233] The present invention pertains, in part, to technologies for quantifying signaling in one or more first living cell(s). Without wishing to be bound by any particular theory, it is contemplated that quantification of one or multiple satiety signal(s) is or may be advantageous assigning numerical value(s) to satiety signaling. In some embodiments, one or more received satiety signal(s) generated by one or more first living cell(s) is essentially characterized as chemical, physical, and/or electrical change(s). Without wishing to be bound by any particular theory, it is contemplated that quantification of one or more chemical, physical, and/or electrical change(s) in one or more first living cell(s) requires or may require one or more electrical signal(s).

[0234] In certain embodiments of the present disclosure, one or more chemical, physical, and/or electrical change(s) in one or more first living cell(s) are or may be converted to one or more electrical signal(s) (e g., electromagnetic interaction(s)) by one or more signal adapter(s). In certain embodiments, one or more signal adapter(s) is essentially characterized by a chemical, physical, and/or electrical change, in response to one or more chemical, physical, and/or electrical change(s) in one or more first living cell(s). Moreover, in certain embodiments, said chemical, physical, and/or electrical change(s) exhibited by one or more signal adapter(s) are or may be essentially converted to one or more electromagnetic interaction(s). As provided herein, one or more electromagnetic interaction(s) is or may be the emission of photon(s), the absorbance of photon(s), the emission of electron(s), and/or absorbance of electron(s), Moreover, in certain embodiments, said chemical, physical, and/or electrical change(s) exhibited by one or more signal adapter(s) converted to electromagnetic interaction(s) are or may be measured by one or more laboratory instrument(s), further comprising one or more readout(s).

[0235] In certain embodiments, one or more signal adapter(s) is or may comprise one or more protein(s), carbohydrate(s), lipid(s), nucleic acid(s), small molecule(s), metal(s), and/or polymer(s). In certain embodiments, one or more signal adapter(s), thus comprises or may comprise one or more component(s) characterized as exhibiting one or more chemical, physical, and/or electrical change(s) in the presence of one or more satiety signal(s). For example, in some instances, one or more signal adapter(s) forms or may form a covalent bond, an ionic bond, a hydrogen bond, a dipolar interaction, and/or a Van der Waals interaction with one or more satiety signal(s). For example, in some instances, one or more signal adapter(s) is or may be characterized by a change in physical structure and/or spatial arrangement in the presence of one or more satiety signal(s). For example, in some instance(s) one or more signal adapter(s) is or may be characterized by a change in electrical properties in the presence of one or more satiety signal(s).

[0236] In certain embodiments, one or more technologies for quantifying one or more satiety signal(s) generated by one or more first living cell(s) utilizes or may utilize one or more signal adapter(s). In certain embodiments, one or more satiety signal(s) are or may be received in the presence of one or more first living cell(s), as provided herein. In certain embodiments, one or more satiety signal(s) are or may be received in the absence of one or more first living cell(s). In certain embodiments, received satiety signal(s) interact or may interact with one or more signal adapter(s) in the presence of one or more first living cell(s). In certain embodiments, received satiety signal(s) interact or may interact with one or more signal adapter(s) in the absence of one or more first living cell(s).

[0237] In certain embodiments, one or more technologies for quantifying one or more satiety signal(s) generated by one or more first living cell(s) utilizes or may utilize one or more signal adapter(s). In certain embodiments, one or more signal adapter(s) is or may be characterized as a binding moiety, a piezoelectric sensor, and/or an electrode. In some cases, one or more protein(s) comprise one or more binding moieties, piezoelectric sensor(s), and/or electrode(s). In some cases, one or more metal(s) and/or metal ion(s) comprise one or more binding moieties, piezoelectric sensor(s), and/or electrode(s). In some cases, one or more nucleic acids comprise one or more binding moieties, piezoelectric sensor(s), and/or electrode(s). In some cases, one or more second living cell(s) comprise one or more binding moieties, piezoelectric sensor(s), and/or electrode(s).

[0238] In certain embodiments, one or more technologies for quantifying one or more satiety signal(s) generated by one or more first living cell(s) utilizes or may utilize one or more signal adapter(s). In certain embodiments, one or more signal adapter(s) is or may be characterized as a second living cell(s). In certain embodiments, one or more second living cell(s) comprise one or more binding moieties, piezoelectric sensor(s), and/or electrode(s). In certain embodiments, the genetic information comprising one or more second living cell(s) is altered so as to enable one or more binding moieties, piezoelectric sensor(s), and/or electrode(s). In certain embodiments, the genetic information comprising one or more second living cell(s) is altered so as to induce one or more chemical, physical, and/or electrical change(s) in the presence of one or more satiety signal(s). In certain embodiments, the genetic information comprising one or more second living cell(s) is altered so as to encode one or more virus(es) comprising the genetic information necessary to generate one or more randomly generated peptide(s) on a capsid surface.

[0239] In certain embodiments, one or more signal adapter(s) exhibit one or more chemical, physical, and/or electrical change(s) converted to electromagnetic interaction(s). In certain embodiments, one or more electromagnetic interaction(s) are or may be measured by one or more laboratory instrument(s), further comprising one or more readout(s). In some aspects of the present invention, one or more laboratory instrument(s) are or may be one or more plate readers, flow cytometers, spectrophotometer, liquid chromatograph, mass spectrometer, mass cytometers, microscopes, microcontrollers, electrophoretic chambers, sequencers, whole body imaging devices, magnetic resonance imagers, and/or any iteration or combination(s) thereof.

[0240] In certain embodiments, one or more computational method(s) further comprise one or more electromagnetic interaction(s) measured by one or more laboratory instrument s). In some cases, one or more computational method(s) are or may be molecular modeling, molecular docking, protein modeling, protein docking, x-ray crystallography modeling, homology modeling, thread recognition reconstruction, fold recognition reconstruction, sequence alignment, ab initio modeling, and/or combinations thereof.

[0241] In certain embodiments, one signal adapter(s) recognizes or may recognize one feature of received satiety signal(s). In certain embodiments, one adapter(s) recognizes or may recognize several features of received satiety signal(s). In certain embodiments, several signal adapter(s) together recognize one or more features of received satiety signal(s).

(i) Second engineered living cell

[0242] In some embodiments, provided technologies involve a step of converting one or more received satiety signal(s) to one or more readout(s). In some embodiments, a step of converting one or more received satiety signal(s) to one or more readout(s) comprises or may comprise one or more signal adapter(s). In some embodiments, one or more signal adapter(s) is or may comprise one or more second living cell(s). In some cases, one or more second living cell(s) is characterized as receiving one or more chemical, physical, and/or electrical signal(s) generated by one or more first living cell(s) upon exposure to one or more stimuli. In certain embodiments, one or second living cell(s) is characterized as comprising one or more binding moieties, piezoelectric sensor(s), and/or electrode(s). In certain embodiments, one or more second living cell(s) is essentially characterized as generating one or more electromagnetic interaction(s) upon exposure to one or more satiety signal(s).

[0243] In certain embodiments, one or more signal adapter(s) is or may be characterized as one or more second living cell(s). In certain embodiments one or more second living cell(s) is or may comprise one or more class(es) within the animal, plant, fungal, protist, and/or monera kingdoms. One or more second living cell(s) is or may be further characterized as prokaryotic and/or eukaryotic. In certain embodiments, one or more second living cell(s) is or may be further characterized as a bacterium, a yeast, a fungus, a probiotic, a symbiote, a live biotherapeutic product, and/or any combinations thereof.

[0244] In certain embodiments, one or more signal adapter(s) is or may be characterized as one or more second living cell(s). In certain embodiments, one or more second living cell(s) is or are characterized as comprising one or more binding moieties, piezoelectric sensor(s), and/or electrode(s). In certain embodiments, said one or more binding moieties, piezoelectric sensor(s), and/or electrode(s) are or may be naturally expressed in one or more second living cell(s). In certain embodiments, said one or more binding moieties, piezoelectric sensor(s), and/or electrode(s) are or may be unnaturally expressed in one or more second living cell(s). For example, in some embodiments, said one or more binding moieties, piezoelectric sensor(s), and/or electrode(s) are or may be expressed using genetic material introduced in one or more second living cell(s). In certain embodiments, said introduced genetic material is or may be identified by iterative selection process(es) relying on affinity to one or more satiety signal(s). In some cases, one or more binding moieties, piezoelectric sensor(s) and/or electrode(s) expressed in one or more second living cell(s) is retained within (e.g., as a component of) said living cell(s). In some cases, one or more binding moieties, piezoelectric sensor(s) and/or electrode(s) expressed in one or more second living cell(s) is secreted from said living cell(s).

[0245] In certain embodiments, one or more signal adapter(s) is or may be characterized as one or more second living cell(s). In certain embodiments, one or more second living cell(s) is or are characterized as generating one or more electromagnetic interaction(s) upon exposure to one or more satiety signal(s). In certain embodiments, component(s) generating one or more electromagnetic interaction(s) upon exposure to one or more satiety signal(s) are or may be naturally expressed in one or more second living cell(s). In certain embodiments, component(s) generating one or more electromagnetic interaction(s) upon exposure to one or more satiety signal(s) are or may be unnaturally expressed in one or more second living cell(s). For example, in some embodiments, said component(s) generating one or more electromagnetic interaction(s) upon exposure to one or more satiety signal(s) are or may be expressed using genetic material introduced in one or more second living cell(s). In some cases, one or more component(s) generating one or more electromagnetic interaction(s) upon exposure to one or more satiety signal(s) expressed in one or more second living cell(s) is retained within (e.g., as a component of) said living cell(s). In some cases, one or more component(s) generating one or more electromagnetic interaction(s) upon exposure to one or more satiety signal(s) expressed in one or more second living cell(s) is secreted from said living cell(s). In certain embodiments, one or more component(s) generating one or more electromagnetic interaction(s) is or may be characterized as the interaction of one or more chemical, physical, and/or electrical entities with one or more component s) comprising and/or secreted from one or more second living cell(s).

[0246] In certain embodiments, one or more signal adapter(s) is or may be characterized as one or more second living cell(s) comprising one or more binding moi eties, piezoelectric sensor(s), and/or electrode(s). In certain embodiments, one or more binding moieties is or may be one or more carbohydrate(s), protein(s), nucleic acid(s), and/or lipid(s). In certain embodiments, one or more piezoelectric sensor(s) is or may be one or more carbohydrate(s), protein(s), nucleic acid(s), and/or lipid(s). In certain embodiments, one or more electrode(s) is or may be one or more carbohydrate(s), protein(s), nucleic acid(s), and/or lipid(s). In certain embodiments, one or more binding moieties, piezoelectric sensor(s), and/or electrode(s) is further characterized as a protein. For example, in some instances, one or more binding moieties is or may be further characterized as a quorum sensing protein, a G-protein coupled receptor, a receptor tyrosine kinase, an ion channel, a rhodopsin, a calcium -binding protein, Piezo 1, Piezo2, an enzyme, a cyclic AMP binding protein, a cyclic GMP binding protein, a primary messenger, a second/secondary messenger, a transcription factor, a chemokine, associated genetic and/or interkingdom variants, and/or any combinations thereof. In certain embodiments, one or more binding moieties, piezoelectric sensor(s), and/or electrode(s) is further characterized as a nucleic acid. For example, in some instances, one or more binding moieties is or may be further characterized as an aptamer.

[0247] In certain embodiments, one or more signal adapter(s) is or may be characterized as one or more second living cell(s) comprising one or more component(s) generating one or more electromagnetic interaction(s). In certain embodiments, one or more component(s) generating one or more electromagnetic interaction(s) is or may be one or more carbohydrate(s), protein(s), nucleic acid(s), and/or lipid(s). In certain embodiments, one or more component(s) generating one or more electromagnetic interaction(s) is or may be a protein further characterized as a fluorescent protein, a luminescent protein, a phosphorescent protein, a fluorescence-quenching protein, a non-fluorescent protein bound to a quenched fluorescent substrate, an enzyme, associated genetic variants, and/or any combinations thereof. For example, in some instances, one or more component(s) generating one or more electromagnetic interaction(s) is or may be mCherry, EGFP, EYFP, Fura-2-AM, Epac-S-H188, luciferases, galactosidases, GCaMP6f, antibiotic resistance proteins, Cre recombinases, Zinc-Finger proteins, CRISPR-associated proteins, and/or combinations thereof. In certain embodiments, one or more component(s) generating one or more electromagnetic interaction(s) characterized as a nucleic acid is or may be further characterized as an aptamer. For example, in some instances, one or more component(s) generating one or more electromagnetic interaction(s) is or may be an aptamer-dye hybrid, and/or a FRET (e g., BRET)-quenched aptamer.

[0248] In certain embodiments, one or more binding moieties, piezoelectric sensor(s), electrode(s), and/or component(s) generating one or more electromagnetic interaction(s) is or may be introduced as genetic material to one or more second living cell(s). For example, in some instances, one or more binding moieties, piezoelectric sensor(s), electrode(s), and/or component s) generating one or more electromagnetic interaction(s) is or may be introduced using viral gene transfection, non-viral gene transfection, polymer-based gene transfection, and/or enzymatic gene transfection. In some embodiments, one or more method(s) of viral gene transfection further comprises or may comprise the use of adenovirus, retrovirus, lentivirus, adeno-associated virus, and/or oncoretrovirus. In certain embodiments, one or more second living cell(s) receive or may receive genetic information in one or more native culture environment(s). In certain embodiments, one or more second living cell(s) receives or may receive genetic information in a temporary culture environment and re-introduced into one or more native culture environment(s).

[0249] In certain embodiments, one or more signal adapter(s) is or may be characterized as one or more second living cell(s). In certain embodiments, one or more signal adapter(s) characterized as one or more second living cell(s) is or may be further characterized as one or more first living cell(s). For example, in certain embodiments, one or more second living cell(s) generates or may generate one or more satiety signal(s). In some instances, one or more satiety signal(s) recognized by one or more signal adapter(s) comprising one or more second living cell(s) is characterized as intercellular. In some instances, one or more satiety signal(s) recognized by one or more signal adapter(s) comprising one or more second living cell(s) is characterized as intracellular.

[0250] In certain embodiments, one or more second living cell(s) is or may be cultured in the presence of one or more first living cell(s). For example, one or more second living cell(s) is or may comprise one or more cell culture dish(es), transwell dish(es), organoid(s), organ(s), and/or organism(s). In certain embodiments, one or more second living cell(s) is or may be a subset of one or more first living cell(s). In certain embodiments, one or more second living cell(s) is or may be ingested as a pharmaceutical device by one or more organism(s) subsequently cultured in the cecum and/or ascending colon of said one or more organism(s). In certain embodiments, one or more second living cell(s) is or may be cultured separately from one or more first living cell(s) receiving one or more previously collected satiety signal(s).

[0251] In certain embodiments, one or more signal adapter(s) further characterized as one or more second living cell(s) is or may comprise at least 10¹ cells, at least 10² cells, at least 10³ cells, at least 10⁴ cells, at least 10⁵ cells, at least 10⁶ cells, at least 10⁷ cells, at least 10⁸ cells, and/or at least 10⁹ cells.

(ii) Acellular signal adapter(s)

[0252] In some embodiments, one or more signal adapter(s) is or may comprise one or more acellular signal adapter(s). As provided herein, one or more acellular signal adapter(s) comprises or may comprise signal adapter(s) in the spatiotemporal absence of one or more second living cell(s). Alternatively, or additionally, one or more acellular signal adapter(s) comprises or may comprise signal adapter(s) of synthetic origin and subsequently purified. Alternatively, or additionally, one or more acellular signal adapter(s) comprises or may comprise signal adapter(s) of biological origin and subsequently purified to remove associated cell(s).

[0253] In certain embodiments, one or more received satiety signal(s) are or may be converted to one or more readout(s) by acellular signal adapter(s). As provided herein, one or more acellular signal adapter(s) comprises or may comprise one or more binding moieties, piezoelectric sensor(s), and/or electrode(s). In certain embodiments, one or more acellular signal adapter(s) is or may be utilized in the presence of one or more first living cell(s) generating one or more satiety signal(s) thus received. In certain embodiments, one or more acellular signal adapter(s) is or may be utilized with one or more received satiety signal(s) in the absence of one or more first living cell(s). For example, in some embodiments, one or more acellular signal adapter(s) is or may be one or more carbohydrate(s), lipid(s), protein(s), nucleic acid(s), small molecule(s), metal(s), and/or ion(s).

[0254] In certain embodiments, one or more acellular signal adapter(s) recognizing one or more satiety signal(s) is or may be further characterized as water soluble. In certain embodiments, one or more acellular signal adapter(s) recognizing one or more satiety signal(s) is or may be further characterized as water insoluble. In certain embodiments, one or more acellular signal adapter(s) is or may be further characterized as immobilized on a plastic substrate. For example, in some instances, one or more acellular signal adapter(s) is or may be immobilized on one or more plastic bead(s), one or more plastic microwell plate(s), one or more plastic dish(es), and/or one or more plastic microfluidic device(s). In certain embodiments, one or more acellular signal adapter(s) is or may be further characterized as immobilized on a metal substrate. For example, in some instances, one or more acellular signal adapter(s) is or may be immobilized on one or more magnetic bead(s), one or more quantum dots(s), barium titanate surface(s), potassium sodium niobate surface(s), gold surface(s), metallic microwell plate(s), metallic dish(es), and/or metallic microfluidic device(s). In certain embodiments, one or more acellular signal adapter(s) is or may be further characterized as immobilized on a lipid substrate. For example, in some instances, one or more acellular signal adapter(s) is or may be immobilized within and/or on one or more lipid bilayers, liposomes, water-in-oil-in-water emulsions, lipid nanoparticles, and/or solid lipid microparticles.

[0255] In certain embodiments, one or more acellular signal adapter(s) is or may comprise one or more protein(s). In certain embodiments, said protein(s) recognizing one or more satiety signal(s) comprise or may comprise one or more binding moiety(ies), piezoelectric sensor(s), and/or electrode(s). In some cases, said one or more protein(s) are or may be further characterized as an antibody, a quorum sensing protein, a G-protein coupled receptor, a receptor tyrosine kinase, an ion channel, an enzyme, a rhodopsin, a calcium-binding protein, a cyclic AMP binding protein, a cyclic GMP binding protein, a primary messenger, a second messenger, a transcription factor, a chemokine, associated genetic and/or inter-kingdom variants, and/or any combinations thereof. In certain embodiments, one or more signal adapter(s) further characterized as an antibody is or may be further characterized as enzyme-linked. In some cases, one or more acellular signal adapter(s) is or may be characterized as an enzyme-linked immunosorbent assay (e.g., ELISA). In certain embodiments, one or more signal adapter(s) characterized as a protein is or may be further characterized by spatial rearrangement upon binding to one or more satiety signal(s). In certain embodiments, one or more signal adapter(s) characterized as a protein is or may be characterized as a soluble antibody. For example, one or more signal adapter(s) is or may be characterized as a nanobody, a scFv, and/or a camelid antibody.

[0256] In certain embodiments, one or more acellular signal adapter(s) is or may comprise one or more nucleic acid(s). In certain embodiments, said nucleic acid(s) recognizing one or more satiety signal(s) comprise or may comprise one or more binding moiety(ies), piezoelectric sensor(s), and/or electrode(s). In some cases, said one or more nucleic acid(s) are or may be further characterized as an aptamer. In certain embodiments, one or more acellular signal adapter(s) characterized as a nucleic acid is or may be further characterized by a spatial rearrangement upon binding to one or more satiety signal(s).

[0257] In certain embodiments, one or more acellular signal adapter(s) is or may comprise one or more metal(s). In certain embodiments, said metal(s) recognizing one or more satiety signal(s) comprise or may comprise one or more binding moiety(ies), piezoelectric sensor(s), and/or electrode(s).For example, in some instances, one or more acellular signal adapter(s) is or may comprise one or more magnetic bead(s), one or more quantum dots(s), barium titanate surface(s), potassium sodium niobate surface(s), gold surface(s), metallic microwell plate(s), metallic dish(es), and/or metallic microfluidic device(s).

[0258] In certain embodiments, one or more signal adapter(s) is or may be characterized as one or more acellular signal adapter(s) comprising one or more component s) generating one or more electromagnetic interaction(s). In certain embodiments, one or more component s) generating one or more electromagnetic interaction(s) is or may be one or more carbohydrate(s), protein(s), nucleic acid(s), metal(s), ion(s), and/or lipid(s). In certain embodiments, one or more component(s) generating one or more electromagnetic interact! on(s) is or may be a protein further characterized as a fluorescent protein, a luminescent protein, a phosphorescent protein, a fluorescence-quenching protein, a non-fluorescent protein bound to a quenched fluorescent substrate, an enzyme, associated genetic variants, and/or any combinations thereof. For example, in some instances, one or more component(s) generating one or more electromagnetic interaction(s) is or may be mCherry, EGFP, EYFP, Fura-2-AM, Epac-S-H188, luciferases, galactosidases, antibiotic resistance proteins, Cre recombinases, Zinc-Finger proteins, CRISPR- associated proteins, horseradish peroxidase, alkaline phosphatase, and/or combinations thereof.

[0259] In certain embodiments, one or more signal adapter(s) is or may be characterized as one or more acellular signal adapter(s) comprising one or more component(s) generating one or more electromagnetic interact! on(s). In certain embodiments, one or more component(s) generating one or more electromagnetic interaction(s) is or may be one or more carbohydrate(s), protein(s), nucleic acid(s), metal(s), ion(s), and/or lipid(s). In certain embodiments, one or more component(s) generating one or more electromagnetic interaction(s) is or may be a metal further characterized as a quantum dot, as plasmon resonant, a conductive metal, a photoelectric material, a semiconductive metal, a piezoelectric metal, and/or any combinations thereof. For example, in some instances, one or more component(s) generating one or more electromagnetic interaction(s) is or may be indium phosphide, zinc sulfide, copper indium sulfide, cadmium selenide, silver, platinum, gold, and/or any combinations thereof.

[0260] In certain embodiments, one or more signal adapter(s) is or may be characterized as one or more acellular signal adapter(s) comprising one or more component(s) generating one or more electromagnetic interaction(s). In certain embodiments, one or more component(s) generating one or more electromagnetic interaction(s) is or may be one or more carbohydrate(s), protein(s), nucleic acid(s), metal(s), ion(s), and/or lipid(s). In certain embodiments, one or more component(s) generating one or more electromagnetic interaction(s) is or may be ion(s) further characterized as a patch clamp recording, voltage-sensitive dyes, and/or any combinations thereof. For example, in some instances, one or more component s) generating one or more electromagnetic interaction(s) is or may be a current clamp sensor(s), Di-4-ANEPPS, and/or any combinations thereof. [0261] In certain embodiments, one or more acellular signal adapter(s) characterized as generating one or more electromagnetic interaction(s) is or may be further characterized by an increase in emission of photon(s), the absorbance of photon(s), the emission of electron(s), and/or absorbance of electron(s) in the presence of one or more satiety signal(s). In certain embodiments, one or more acellular signal adapter(s) characterized as generating one or more electromagnetic interaction(s) is or may be further characterized by a decrease in emission of photon(s), the absorbance of photon(s), the emission of electron(s), and/or absorbance of electron(s) in the presence of one or more satiety signal(s). For example, in some instances, the presence of one or more satiety signal(s) induces or may induce a structural change in one or more acellular signal adapter(s) further characterized as a protein, thus enabling measurable Forster Resonance Energy Transfer (e.g., FRET). For example, in some instances, the presence of one or more satiety signal(s) induces or may induce a change in the electrical conductivity (e g., electrical impedance) of one or more acellular signal adapter(s) further characterized as a metal, thus enabling an increase in measurable current. For example, in some instances, the presence of one or more satiety signal(s) induces or may induce a change in the optical clarity (e g., light scattering) of one or more acellular signal adapter(s) further characterized as a metal, thus enabling an increase in measurable current. For example, in some instances, the presence of one or more satiety signal(s) displaces a quenched fluorescent substrate from one or more protein(s), nucleic acid(s), and/or metal(s).

[0262] In certain embodiments, one or more signal adapter(s) further characterized as one or more acellular signal adapter(s) is or may comprise at least 10-16, at least 10-14, at least 10- 12, at least 10-10, at least 10-8, at least 10-6, at least 10-4, at least 10-2, at least 100, at least 102, at least 104, and/or at least 106 moles.

(Hi) Genetic satiety signal(s)

[0263] In certain embodiments, one or more received satiety signal(s) in accordance with the present disclosure is or may comprise genetic information, as provided herein. For example, in some embodiments, one or more received satiety signal(s) is or may comprise DNA, RNA, and/or any combinations thereof. In certain embodiments, DNA and/or RNA received from one or more first living cell(s) is or may be indicative of satiety signal(s) in response to provided stimuli. Without wishing to be bound by any particular theory, it is contemplated that the identity and/or quantity of genetic information comprising one or more received satiety signal(s) is or may be used to quantify satiety signaling.

[0264] In some embodiments, one or more satiety signal(s) comprising genetic information is or may be converted to one or more readout(s) by one or more signal adapter(s). For example, in some cases, the quantity of one or more RNAtranscript(s) is or may be measured by in situ hybridization. As provided herein, one or more signal adapter(s) characterized as converting genetic information to one or more readout(s) essentially comprise(s) DNA, RNA, peptide nucleic acids, morpholinos, and/or phosphorothioate nucleic acids complementary to (e.g., hybridizing with) said genetic information. In certain embodiments, one or more hybrid(s) comprising genetic information are or may be identified and/or quantified by one or more protein(s) and/or nucleic acid(s). In certain embodiments, one or more protein(s) recognizes and/or binds to one or more hybrids and subsequently generates, or may generate, one or more electromagnetic interact! on(s). In certain embodiments, one or more nucleic acid(s) recognizes and/or binds to one or more hybrids and subsequently generates, or may generate one or more electromagnetic interaction(s).

[0265] In certain embodiments, one or more received satiety signal(s) characterized as genetic information comprises or may comprise RNA. In some cases, signal adapter(s) comprising DNA, RNA, peptide nucleic acids, morpholinos, and/or phosphorothioate nucleic acids are incubated in the presence of DNA polymerase, reverse transcriptase, and one or more RNA generated by one or more first living cell(s). For example, in some embodiments, signal adapter(s) may comprise hairpin DNA further comprising a quenched FRET pair. Alternatively, or additionally, in some embodiments, signal adapter(s) may comprise a double-stranded DNA- binding dye.

3. Readouts

[0266] Among other things, the present disclosure provides technologies for quantification of satiety signal(s). In some embodiments, provided technologies provide correlating at least one feature(s) of stimuli with one or more readout(s), e.g., and determining a conclusion based on such correlating. Without wishing to be bound by any particular theory, it is contemplated that a step of determining one or more conclusion(s) from the provided method(s) of quantifying satiety is or may be particularly useful for describing one or more satiety response(s). For example, it is contemplated that one or more readout(s) is or may be indicative of the response(s) of one or more living system(s) to stimuli. In some instances, one or more readout(s) is or may be indicative of the health and/or performance of one or more living system(s). Without wishing to be bound by any particular theory, it is contemplated that a step of determining one or more conclusion(s) from the provided method(s) of quantifying satiety is or may be particularly advantageous for identifying stimuli to maximize satiety response(s) from one or more first living cell(s). Without wishing to be bound by any particular theory, it is contemplated that a step of determining one or more conclusion(s) from the provided method(s) of quantifying satiety is or may be particularly advantageous for identifying stimuli to minimize satiety response(s) from one or more first living cell(s).

[0267] In certain embodiments of provided technologies, one or more conclusion(s) is or may comprise ranking of one or more stimuli by quantity and/or magnitude of one or more readout(s). Without wishing to be bound by any particular theory, it is contemplated that the quantity and/or magnitude of one or more readout(s) is or may be related to the quantity and/or magnitude of satiety signal(s) in one or more first living cell(s). In some embodiments, one or more conclusion(s) is determined comparing readout(s) prior to and following the provision of one stimulus to one or more first living cell(s). In some embodiments, one or more conclusion(s) is determined comparing readout(s) following the provision of several stimuli to one or more first living cell(s).

[0268] In certain embodiments, one or more conclusion(s) achieved by technologies of the present disclosure is or may comprise identifying new stimuli by quantity and/or magnitude of one or more readout(s). Without wishing to be bound by any particular theory, it is contemplated that the quantity and/or magnitude of one or more readout(s) is or may be related to the quantity and/or magnitude of satiety signal(s) in one or more first living cell(s). In some embodiments, one or more conclusion(s) is determined comparing readout(s) following the provision of several unknown stimuli to one or more first living cell(s) to readout(s) following the provision of a known stimulus to one or more first living cell(s). In some embodiments, one or more conclusion(s) is determined from readout(s) following the provision of one known stimuli in combination with several unknown stimuli to one or more first living cell(s).

[0269] In certain embodiments, one or more conclusion(s) achieved by technologies provided by the present disclosure is or may comprise identifying and/or quantifying the health of one or more living system(s). Without wishing to be bound by any particular theory, it is contemplated that the quantity and/or magnitude of one or more readout(s) is or may be related to the quantity and/or magnitude of satiety signal(s) in one or more first living cell(s). In some embodiments, one or more conclusion(s) is determined comparing readout(s) between one or more first living cell(s) known to be healthy and one or more first living cell(s) of unknown health following the provision of several known stimuli. In some embodiments, one or more conclusion(s) is determined comparing readout(s) between one or more first living cell(s) known to be diseased and one or more first living cell(s) of unknown health following the provision of several known stimuli.

[0270] In certain embodiments, one or more conclusion(s) achieved by technologies provided by the present disclosure is or may comprise identifying and/or quantifying the signaling pathway(s), as provided herein, of one or more living system(s). Without wishing to be bound by any particular theory, it is contemplated that the quantity and/or magnitude of one or more readout(s) is or may be related to the signaling pathway(s) in one or more first living cell(s). In some embodiments, one or more conclusion(s) is determined comparing readout(s) obtained between first living cell(s) comprising several species. In some embodiments, one or more conclusion(s) is determined comparing readout(s) obtained between first living cell(s) characterized as monolayer cell(s), organoids(s), and/or organism(s). In some embodiments, one or more conclusion(s) determined using readout(s) obtained from one or more first living cell(s) identifies and/or quantifies one or more unknown satiety signaling pathway(s).

[0271] In certain embodiments, one or more feature(s) of stimuli is or may be correlated towards one or more readout(s). In certain embodiments, one or more correlated readout(s) is or may be a change in one or more electrical signal(s), as provided herein, thus indicating an increase in the quantity and/or identities of one or more satiety signal(s). In certain embodiments, one or more correlated readout(s) is or may be a change in one or more electrical signal(s), as provided herein, thus indicating a decrease in the quantity and/or identities of one or more satiety signal(s). In certain embodiments, one or more conclusion(s) is or may be drawn from one correlated readout(s) comprising changes in one electrical signal. In certain embodiments, one or more conclusion(s) is or may be drawn from multiple correlated readout(s) comprising changes in multiple electrical signal(s). In some cases, change(s) in one electrical signal(s) are or may be understood to comprise increases and/or decrease in multiple satiety signal(s). In some cases, one or more conclusion(s) are or may be drawn when single readout(s) essentially indicate change(s) in single satiety signal(s).

[0272] In some embodiments, one or more conclusion(s) achieved by technologies provided by the present disclosure is or may comprise selecting, screening, predicting, ranking, scoring, and/or correlating one or more nutrient(s) by identity and/or quantity of one or more satiety signal(s).

(i) Ranking stimuli

[0273] In some embodiments, provided technologies permit and/or achieve ranking of one or more stimuli. Without wishing to be bound by any particular theory, it is contemplated that a ranking of one or more stimuli is or may be particularly advantageous for comparing the satiety induced by several independent food product(s).

[0274] In certain embodiments, one or more readout(s) is or may be correlated to classification(s) of stimuli to conclude upon those classification(s) yielding one or more desirable readout(s). For example, in some instances, one or more feature(s) grouping stimuli is or may be their classification as chemical, physical, and/or electrical stimuli. For example, in some instances, one or more feature(s) grouping stimuli further characterized as chemical stimuli is or may be a classification as a carbohydrate, a protein, a lipid, a nucleic acid, a metal, an ion, and/or combinations thereof. For example, in some instances, one or more feature(s) grouping stimuli further characterized as fatty acids is or may be a classification as a long chain unsaturated fatty acid, medium chain unsaturated fatty acid, medium chain saturated fatty acid, and/or combinations thereof. In some instances, one or more classification(s) of stimuli are or may be utilized to infer and/or predict readout(s) resulting from untested stimuli exhibiting similar classification(s). [0275] In certain embodiments, one or more readout(s) is or may be classified by magnitude and/or frequency, thus providing a grouping (e.g., classification) of one or more stimuli. For example, in some instances, one or more feature(s) grouping stimuli is or may be a maximization of one or more readout(s). For example, in some instances, one or more feature(s) grouping stimuli is or may be a minimization of one or more readout(s). In certain embodiments, one or more stimuli are or may be ranked by comparing the magnitude and/or frequency of one or more readout(s) resulting from exposing one or more first living cell(s) to said stimuli. In some instances, one or more magnitude(s) and/or frequency(ies) of one or more readout(s) are or may be utilized to infer and/or predict classification(s) of one or more stimuli.

(ii) Characterizing stimuli (and combinations thereof) not known to modulate satiety

[0276] In some embodiments, provided technologies permit and/or achieve identification and/or characterization of stimuli resulting in one or more particular readout(s). Without wishing to be bound by any particular theory, it is contemplated that an identification or characterization of one or more satiety modulating (e g., inducing) stimuli is or may be particularly advantageous for identifying chemical, physical, and/or electrical entities to reduce over-eating. Without wishing to be bound by any particular theory, it is contemplated that an identification or characterization of one or more satiety reducing stimuli is or may be particularly advantageous for improving the appeal of one or more consumer food product(s).

[0277] In certain embodiments, one or more readout(s) is or may be classified by magnitude and/or frequency correlated to changes in one or more satiety signal(s). In certain embodiments, one or more stimuli provided to one or more first living cell(s) is or are characterized by unknown readout(s). In certain embodiments, one or more stimuli provided to one or more first living cell(s) is or are characterized by known readout(s). In certain embodiments, readout(s) generated by one or more unknown stimuli are or may be compared to readout(s) generated by one or more known stimuli to determine one or more conclusion(s). For example, in some instances, one or more known stimuli comprise or may comprise GLP-1, GIP, PYY, TUG-891, Danuglipron, oleoylethanolamide, or any combinations thereof. In certain embodiment s), one or more unknown stimuli providing readout(s) matching and/or exceeding those of known stimuli are or may be further characterized as one or more satiety modulator(s). In certain embodiments, one or more unknown stimuli comprise or may comprise stimuli further classified as nutrient(s). In certain embodiments, one or more unknown stimuli comprise or may comprise stimuli further classified as pharmacological intervention(s). In certain embodiments, provided method(s) identify or may identify component(s) of diet(s) maximizing and/or minimizing satiety signal(s).

[0278] In certain embodiments, one or more known stimuli provided to one or more first living cell(s) is or are characterized by readout(s) and/or by signaling pathway (s). In certain embodiments, one or more stimuli known to independently afford negligible change in one or more readout(s) enhances or may enhance readout(s) of said known stimuli. In certain embodiments, correlating change(s) in one or more readout(s) due to unknown combination(s) of one known stimulus and several additional stimuli identifies or may identify improved satiety signaling. In certain embodiments, combination(s) of one or more unknown stimuli with known stimuli resulting in readout(s) indicative of increased satiety enable conclusion(s) that said unknown stimuli participate or may participate in allosteric modulation, pathway synergism, and/or competitive inhibition.

(Hi) Conclusions regarding health of living system(s)

[0279] In some embodiments, provided technologies permit and/or achieve conclusion(s) relating to quantification and/or identification of health of one or more living system(s). Without wishing to be bound by any particular theory, it is contemplated that quantifying and/or identifying the health of one or more living system(s) is or may be particularly advantageous for determining if a nutritional and/or pharmacological intervention is required.

[0280] In certain embodiments, one or more known stimuli provided to one or more first living cell(s) is or are characterized by known readout(s) and/or by known signaling pathway(s). In some cases, known readout(s) and/or known signaling pathway (s) derive or may derive from cell(s) characterized as healthy (e.g., free from disease). In certain embodiments, one or more known readout(s) and/or known signaling pathway(s) generated by one or more healthy first living cell(s) is or may be reproducible upon exposure to one or more known stimuli. In certain embodiments, readout(s) and/or signaling pathway(s) generated by one or more first living cell(s) are or may be compared to readout(s) and/or signaling pathway(s) generated by one or more healthy living cell(s) to said known stimuli. In certain embodiments, one or more deviation(s) in readout(s) and/or signaling pathway(s) generated by one or more first living cell(s) is or may be indicative of poor health. For example, in some instances, satiety signal(s) generated from one or more first living cell(s) characterized as a biopsy from diabetic patient(s) are or may be compared to satiety signal(s) generated from one or more first living cell(s) characterized as a biopsy from non-diabetic patient(s).

(iv) Cross-species or cell-organization-level pathway characterization

[0281] In some embodiments, provided technologies utilize and/or achieve comparing and/or contrasting satiety signal(s) generated between different species and/or between component(s) and/or subcomponent(s) of larger organism(s). Without wishing to be bound by any particular theory, it is contemplated that comparing and/or contrasting satiety signal(s) between one or more living system(s) is or may be particularly advantageous for determining translatability of one or more nutritional and/or pharmacological intervention(s).

[0282] In certain embodiments, one or more stimuli provided to one or more first living cell(s) is or are characterized by readout(s) and/or by signaling pathway(s). For example, in some cases, readout(s) and/or signaling pathway(s) derive or may derive from interaction(s) with nearby cell(s), geometric orientation(s), species, oxygen level(s), pH, and/or combinations of these factors. In some cases, one or more conclusion(s) for one or more stimuli determined in one species are valid in another, distinct species. In some cases, one or more conclusion(s) for one or more stimuli determined in one species are invalid in another, distinct species. In certain embodiments, conclusion(s) for readout(s) indicative of satiety signal(s) in monolayer cell(s) are valid in organism(s). In certain embodiments, conclusion(s) for readout(s) indicative of satiety signal(s) in monolayer cell(s) are invalid in organism(s). In certain embodiments, provided method(s) of quantifying satiety signaling, essentially characterized as high-throughput, enable or may enable comparison of satiety signal(s) in first living cell(s) characterized as distinct species and/or distinct cell organization(s). [0283] In certain embodiments, one or more known readout(s) originating from one or more known stimuli is or may be essentially characterized by increased and/or decreased satiety. In some cases, one or more signaling pathway(s) responsible for changes in one or more readout(s) are unknown. In certain embodiments, one or more signal adapter(s), as provided herein, enable or may enable the identification of specific satiety signal(s) (e.g., chemical, physical, and/or electrical signal(s)) generated by one or more first living cell(s) by knowledge of said readout(s). For example, in some instances, one or more readout(s) in response to a known stimulus characterized as known increased secretion of GLP-1, further characterized as increased satiety, includes one or more readout(s) characterized as unknown increased secretion of PYY. In this aforementioned exemplary embodiment, a new signaling pathway deriving from said known stimulus is or may be identified.

D. Exemplary Applications

[0284] Among other things, the present disclosure provides technologies for identifying and/or characterizing stimuli with satiety modulating character.

[0285] In some embodiments, provided technologies are useful to identify and/or characterize stimuli that may be usefully utilized as and/or incorporated into nutritional compositions.

[0286] In some embodiments, the present disclosure provides nutritional compositions that are or comprise one or more agents that, when utilized as a stimulus as described herein, triggers or enhances satiety, particularly, for example, when assessed in a complex cellular system such as a system comprising two or more different cell types and/or including one or more structural features characteristic of a relevant organ; in some particular embodiments, such agents induce or enhance satiety in one or more organotypic systems and/or one or more cellular systems (e.g., complex cellular systems) that are or comprise cells (e g., mammalian cells) of neuroendocrine and/or enteroendocrine origin.

[0287] Alternatively or additionally, in some embodiments the present disclosure provides technologies for producing and/or characterization of such nutritional compositions. EXEMPLIFICATION

[0288] The following examples are intended to illustrate but not limit the disclosed embodiments. The following examples are useful to confirm aspects of the disclosure described above and to exemplify certain embodiments of the disclosure.

[0289] These non-limiting examples demonstrate particular features and advantages of provided technologies - e.g., of provided technologies for assessing (e.g., quantifying) satiety.

[0290] Among other things, provided technologies for assessing (e.g., quantifying) satiety may be characterized by significant improvements, including, for example, any chemical, physical, and/or electrical stimuli are or may be provided to one or more first living cell(s), said chemical, physical, and/or electrical stimuli are or may be provided non-invasively, said chemical, physical, and/or electrical stimuli are provided with control over both time and frequency of exposure, said chemical, physical, and/or electrical stimuli are provided to any cell type known or unknown to participate in satiety signaling, said cell type(s) are or may be healthy and/or primary cell(s) within human(s), receiving satiety signal(s) is or may be achieved by harvesting sample(s) from first living cell(s) and/or capturing sample(s) in the presence of first living cell(s), received satiety signal(s) are or may be monitored in real time, signal adapter(s) comprise second living cell(s) compatible with monolayer(s), tissue(s), organotypic model(s), organ(s), and/or organism(s), conclusion(s) identifying new stimuli for inducing and/or reducing satiety, conclusion(s) comparing stimuli for relative effect(s) on satiety, conclusion(s) quantifying the health of living cell(s), and, conclusion(s) identifying satiety signaling pathway (s).

[0291] Certain exemplary embodiments, as provided herein, describe or may describe one or more step(s) of disclosed method(s). For example, in some instances, provided example(s) describe a step of providing stimuli to one or more first living cell(s). For example, in some instances, provided example(s) describe a step of receiving one or more signal(s). For example, in some instances, provided example(s) describe a step of converting one or more satiety signal(s) to a readout by signal adapter(s). For example, in some instances, provided example(s) describe a step of providing stimuli to one or more first living cell(s). For example, in some instances, provided example(s) describe a step of drawing one or more conclusion(s). A. Example 1: Converting satiety signal(s) to readout(s) via proteins

[0292] The present Example describes a particular embodiment of a provided technology for assessing (e.g., quantifying) satiety. As exemplified, a system is provided in which satiety state of certain cells (e.g., of a population of mammalian cells and/or of cells of neuroendocrine and/or enteroendocrine origin) can be determined, for example through detection (e.g., quantification) of one or more (and in this particular Example, of a plurality of) satiety signal(s). In some embodiments, detection (e.g., quantification) of such satiety signal(s) constitutes determination of a satiety state. Provided technologies, including as exemplified in the present Example, permit detection (e.g., quantification) of such satiety signal(s) (e.g., permit determination of satiety state), and furthermore permit assessment (e.g., quantitation) of impact(s) of one or more stimuli that may be applied to the system on such satiety signal(s) and/or satiety state. In some embodiments, for example, provided such technologies permit characterization of such one or more stimuli as “satiety modulating” if, for example, presence, level, activity or form of the one or more stimuli correlates with a change in one or more relevant satiety signals and/or otherwise with a change in satiety state of the system exposed to the system. In some particular embodiments, provided such technologies permit characterization of such one or more stimuli as “satiety inducing” (e.g., for example, presence, level, activity or form the one or more stimuli correlates with increase in (e.g., increased level, frequency, intensity, etc.) of one or more relevant satiety signal(s) and/or otherwise with increase in satiety state in the system exposed to the one or more stimuli), and/or as “satiety reducing” (e.g., if, for example, presence, level, activity or form the one or more stimuli correlates with decrease in (e.g., decreased level, frequency, intensity, etc.) of one or more relevant satiety signal(s) and/or otherwise with decrease in satiety state in the system exposed to the one or more stimuli). In some embodiments, such modulation (e.g., increase or decrease) may be assessed relative to absence of the one or more stimuli; alternatively or additionally, in some embodiments, such modulation (e.g., increase or decrease) may be assessed relative to a reference stimulus (or stimuli) known to have a particular impact (e.g., a positive reference known to achieve satiety or a negative reference known not to). [0293] In this particular example, as illustrated in Figure 1(A), relevant satiety signals are satiety hormones generated by living cell(s) (e.g., by cells of neuroendocrine and/or enteroendocrine origin). Exemplary cells that can be utilized in an assessment as depicted in Figure 1 include, but are not limited to, NCI-H716 cells (which are human lymphoblast cells; specifically a colorectal ademocarcinoma cell line), Caco-2 cells (which are a human colorectal adenocarcinoma cells line understood to be useful as a model of intestinal epithelial barrier and reported to differentiate spontaneously into a heterogeneous mixture of intestinal epithelial cells), STC-1 cells (which are intestinal secretin tumor cells described as having features of native intestinal enteroendocrine cells, particularly including secretion of gastrointestinal hormones in repose to stimuli such as food components), and IPEC-J2 cells (which are porcine intestinal enterocytes isolated from the jejunum of a neonatal unsuckled piglet; these cells are neither transformed nor tumorigenic). In certain particular embodiments, primary cells are utilized. Moreover, in various embodiments, complex cell populations and/or tissue models are utilized. In some embodiments, a three-dimensional tissue model, such as for example the MATTEK epilntestinal 3D human tissue model, is utilized.

[0294] In the particular embodiment of a provided assessment that is depicted in Figure 1, released hormone(s) of interest (i.e., satiety hormones) are detected (e.g., quantified) using an enzyme-linked immunosorbent assay (Figure 1A). Thus, the release (and/or released level) of one or more hormone(s) is a biological event that can be considered to be a “signal” generated by the cells, and the ELISA assay can be considered to be a “signal adapter” that converts the biological event (i.e., the “signal”) into a detectable (e g., quantifiable) readout (as depicted in Figure 1, quantification of a label, e.g., a fluorescent label, on an antibody). Those skilled in the art are familiar with a variety of ELIS A formats - e.g., direct, indirect, sandwich, and competitive (see Figure 1 A) useful in accordance with this embodiment of the present invention.

[0295] The present disclosure teaches that ELISA assays performed, e.g., on cells of neuroendocrine and/or enteroendocrine origin, e.g., on mammalian cells of neuroendocrine and/or enteroendocrine origin, and specifically, in some embodiments, on primary cells and/or on complex cell mixtures or structures, may usefully be employed to assess satiety -modulating character of a stimulus or stimuli of interest. Among other things, the present disclosure provides an insight that such assays may usefully be implemented in high-throughput format - for example using plates or other culture systems with multiple wells (e.g., commercially available culture well plates, which are available, for example, with 24 wells, 48 wells, 96 wells, 384 wells, 1536 wells or more). Moreover, the present disclosure provides an insight that such assays can be useful to assess satiety character of isolated compounds or of complex materials (e g., combinations of components, which may be provided, for example, as structured materials or mixtures, whole foods or crude extracts or multi-component samples thereof, etc.).

[0296] Figure IB presents results of exemplary satiety assessments utilizing ELISA assays. To generate the results of Figure IB, a black-walled 96-well immunosorbent plate was coated with an anti-GLP-1 capture monoclonal antibody at a concentration of 1-10 ug per mL. Plates were subsequently washed with a buffer (1 mM PBS, Tween-20, sodium azide) 3 times before addition of samples. 100 uL of assay standard(s), quality control(s), and recombinant GLP-1 was loaded into each well (n=2). The assay standard(s), quality control(s), and recombinant GLP-1 were resuspended and diluted in assay buffer comprising 0.05 M PBS, pH 6.8, comprising protease inhibitors, Tween 20 0.08% (w/v), sodium azide, and 1% (w/v) bovine serum albumin. The assay standards comprised purified GLP-1 at concentrations of 2 pM, 5 pM, 10 pM, 20 pM, 50 pM, and 100 pM. Recombinant GLP-1 was diluted to concentrations of 200 pM, 50 pM, 25 pM, 10 pM and 1 pM. Quality control samples comprised purified GLP-1 at ranges of 5.6-12 pM for QC1, and 31-65 pM for QC2. Samples derived from NCLH716 cells were incubated at 4 °C overnight. Following incubation with the samples, plates were washed 5 times with a buffer (1 mM PBS, Tween 20, sodium azide). A GLP-1 HRP-conjugated detection antibody was added and allowed to incubate for 1 hour at room temperature. Plates were again washed 5 times with a buffer (1 mM PBS, Tween 20, sodium azide), and 0.05 mg/mL of 4- methylumbelliferyl phosphate substrate was added to each well for 30 minutes in the dark. After incubation, a stop solution consisting of 2M sulfuric acid was added to each well. Fluorescence was read at excitation/emission wavelengths of 355/460 nm. In this exemplary, non-limiting embodiment, assay standard(s) were observed to maintain a linear relationship with an R-squared value of > 0.99 (Figure IB). The recombinant peptide standard curve(s) also maintained a linear relationship with an R-squared value of > 0.90. Additionally, incorporation of potential assay interference did not alter the detection sensitivity of the assay (Figure IB). B. Example 2: Converting satiety signal(s) to readout(s) via yeast second living cell(s)

[0297] The present Example describes an embodiment of provided technologies for assessing (e.g., quantifying) satiety, e.g., as described in Example 1 except that, in this Example, one or more signal(s) generated by one or more first living cell(s) is converted to readout(s) utilizing one or more second living cell(s).

[0298] In this non-limiting example, further characterized as a genetically-encoded realtime living hormone sensor, one or more strains of yeast (ex. Saccharomyces cerevisiae) comprise one or more second living cell(s) (Figure 2). Optically dense samples comprising approximately 2E7 yeast cells/mL are used for transformation(s). 1 m of carrier DNA (linearized plasmid or PCR products) is denatured for 5 minutes at 100 °C. The yeast are washed and centrifuged 3 times in sterile water with a final resuspension volume of 1 mL. The cell pellets are then transferred to a microcentrifuge tube and spun at > 10,000 xg. 100 uL of cells are added into individual tubes per each transformation. For lithium acetate transformations, 50% polyethylene glycol 3350, 1.0 M of lithium acetate, are added to 100 uL of cells alongside carrier DNA (2 mg/mL) or plasmid DNA (100 ug/mL). The solution is then incubated at 42 degrees C for 40 minutes, followed by centrifugation at 13,000xg for 30 seconds in a microcentrifuge. Yeast with antibiotic resistance are grown in appropriate YPAD media.

[0299] Readout A: For generation of GPCR reporter(s), plasmid constructs of GLP-1R, GIPR, and Y4R are genetically fused to mTangerine, mOrange, and mPlum, respectively with a flexible glycine-serine linker inserted between transmembrane helices 5-6. Cognate Ga protein(s) are genetically fused to a 27 amino acid quenching peptide derived from influenza M2. Without wishing to be bound by any particular theory, this peptide is contemplated to quench fluorescence of mTangerine, mOrange, and/or mPlum when in close proximity. In this nonlimiting embodiment, inactive fluorophore-engineered GPCR(s) remain non-fluorescent, while activation, and subsequent Ga protein release, initiates quencher relocation and removes quenching of fluorescent protein(s). Genetic constructs are PCR amplified and transformed into yeast. To confirm presence of fluorescent protein(s) and GPCRs, yeast are lysed and western blots are performed against each protein and GPCR, respectively. To assess theoretical Readout A as depicted in Figure 2A, yeast are incubated with recombinant GLP-1, GIP, and PYY, respectively, and fluorescence was quantified over time with a plate-reader with excitation/emission ranges at 548/562 nm, 568/585 nm, and 590/649 nm, respectively.

[0300] Readout B. To quantify the cyclic adenosine monophosphate level(s) (cAMP), transcripts for CAMPER are amplified via PCR and transformed into yeast via the lithium acetate method described herein. Presence of the CAMPER construct is verified by flow cytometry and widefield microscopy. To monitor levels of cAMP, ranges of glucose from 0-100 mM are dosed to the yeast and fluorescence quantified in the excitation emission range of 488/507 nm. This exemplary, non-limiting depiction of the yeast engineering is shown stepwise in Figure 2B, denoted as theoretical readout B.

[0301] Readout C: To monitor levels of transcription factors, the coding sequence of luciferase is fused to the insulin gene promoter (INS) to monitor levels of GLP-1 response. The INS promoter region also contains a CRE octamer motif (TGACGTCA) which, without wishing to be bound by any particular theory, promotes cAMP/PKA signaling pathway component binding. Relevant portions of the plasmid construct are amplified via PCR and transformed into yeast via the lithium acetate method described herein. Reporter expression is assessed with doses of recombinant GLP-1 at varying concentrations, followed by addition of luciferin substrate. Luminescence is measured in a white-walled 96-well plate at the emission wavelength 560 nm. This exemplary, non-limiting depiction of the yeast engineering is shown stepwise in Figure 2C, denoted as theoretical readout C.

[0302] Readout D: In order to further monitor the stages at which the yeast are responding to extracellular hormone concentrations, the supernatant of stimulated yeast is collected and centrifuged at > 10,000 xg to remove any residual cells. The supernatant is then incubated with anti -m Tangerine, anti-mOrange, anti-mPlum, anti-GFP, and anti-luciferase magnetic beads and separated from solution. The beads are then run on a flow cytometer, and fluorescence values are compared against reference bead-protein conjugate concentrations to determine the amount of protein that had been secreted (Figure 2D). Similar steps are performed with fixed and permeabilized whole yeast samples to determine the extent of signal activation. C. Example 3: Converting satiety signal(s) to readout(s) via probiotic second living cell(s)

[0303] The present Example describes an embodiment of provided technologies for assessing (e.g., quantifying) satiety, e.g., as described in Example 2 except that, in this Example, one or more signal(s) generated by one or more first living cell(s) is converted to readout(s) utilizing a genetically-encoded real-time living hormone sensor in which one or more strains of the bacterium Bacillus derived from Bacillus subtilis and Bacillus coagulans comprise one or more second living cell(s). Bacillus strains are grown in liquid competence medium supplemented with 0.5% glucose, as well as 1-histidine, 1-leucine, and 1-methionine all at a concentration of 50 ug/mL. To uncover GLP-1, GIP, PYY or other hormones capable of binding modified quorum sensing receptors, phagemid vector libraries are generated and cloned into the vector pG8SAET or a functional equivalent. An example phage display protocol is visualized in Figure 3. Escherichia coli strain ER2738 are transformed with the modified receptor pG8SAET plasmid library via electroporation. Transformed strains are plated on agar plates containing Xgal and IPTG. Without wishing to be bound by any particular theory, the ER2738 strain contains a lacZa gene, which allows the phage plaques to appear blue. Blue plaques are inoculated into LB medium and allowed to grow until the OD600 reached approximately 0.5. Phage titering is performed via serial dilutions on appropriate agar plates. Phages are serially diluted and incubated at room temperature for 5 minutes prior to addition of 200 uL of unmodified ER2738. Target recombinant peptide(s) (GLP-1, GIP, PYY, etc.) are modified with an N-terminal biotin moiety, and incubated at concentration ranging from 10-100 ug/mL on a neutravidin-coated plate. Coating is conducted in 0.1 M NaHCO3 at pH 8.6 overnight at 4 degrees Celsius. Serial dilutions of the phage library are added to each well and plates are incubated for 1 hour at room temperature. Non-binding phages are discarded and plates are washed 10 times with TBST. Bound phages are eluted using a solution of recombinant GLP-1 (ranging from 0.1-lmM) in TBS, and incubated for 1 hour at room temperature. Eluates are pipetted off and incubated with non-infected ER2738 for 4 hours at 37 degrees Celsius. Cultures are then centrifuged for 10 minutes at 12,000 g and the supernatant is harvested. The upper 80% of the supernatant is incubated at a 1 :6 dilution with 20% PEG/2.5M NaCl and incubated at 4 degrees C overnight. Phage precipitates are spun at 12,000 g for 15 minutes and the supernatant is discarded. Pellets are washed in TBS and precipitated a second time, and phages are re-inoculated and steps are repeated 5 times to generate binding candidates. After each display round, phage DNA is harvested and sequenced by Sanger sequencing and next generation sequencing. High affinity clones are introduced into B. coagulans via plasmid transformation, and immunoassays against the hormone of interest are performed as described herein to generate theoretical binding curve(s) as shown in Figure 4A. Theoretical sequence diversity of binders decreases significantly as sequencing rounds progress as represented in Figure 4B. The transfected modified receptors on the bacterium also display different but similar levels of secreted readout as theoretically depicted in Figure 4C.

D. Example 4: Converting satiety signal(s) to readout(s) via multiplexed scFv and/or aptamer signal adapters

[0304] The present Example describes an embodiment of provided technologies for assessing (e.g., quantifying) satiety, e.g., as described in Example 1 except that, in this Example, one or more signal(s) generated by one or more first living cell(s) is converted to readout(s) a multiplexed hormone-binding assay, one or more scFvs and/or aptamers comprise one or more signal adapter(s). First, multiple hormone binding molecules are generated as depicted in Figure 5A. scFv: Protein-based binding molecules, primarily antibody fragment(s) such as scFvs, are designed and assessed via a modified phage display technique described herein. The modification(s) to the phage display protocol consisted of the target molecule (GLP-1, GIP, PYY, etc ). High affinity clones are sequenced and adapted for mammalian protein expression to generate scFvs specific for each hormone. High affinity sequences are codon-optimized for mammalian cell expression, a histidine tag was added to the c-terminus, and gene fragments are cloned into a lentiviral transfection vector (PLVX or functional equivalent), and example protocol of which is shown in Figure 6. Expression vectors are combined with envelope and packaging vectors and dosed to HEK-293T producing cells. Viruses are titered and CHO cells are transfected at varying MOIs (1-100 virions/cell) prior to antibiotic selection. Surviving cells are assessed for protein expression via western blot and RT-qPCR of the transfected transcript. Cell supernatants are harvested and scFvs isolated via immobilized metal ion affinity chromatography to capture the poly-histidine tag on the c-terminus of the scFv. ScFvs are eluted off of the column by addition of varying concentrations of imidazole-containing elution buffer.

Concentrations of the scFv are quantified via immunoassay as described herein.

[0305] Exemplary engineered scFvs are used to generate multiplexed protein binding hormone detection readout(s). First, cells are plated in a 24-well cell culture plate until they reach 80% confluency. Cells are dosed with a range of stimuli for 2-4 hours, depending on the cell type. Without wishing to be bound to by any particular theory, cells are also dosed with known stimuli for each hormone under investigation (GLP-1 - glucose, PYY - vomitoxin, and GIP - a-methyl d glucopyranoside). Cell supernatants are collected and centrifuged at 2,000 g to remove cell debris and protein aggregates. After sample collection, fixed concentrations of scFvs for each hormone are added into the supernatant and incubated with rotation at 4 degrees C overnight. After sample incubation, supernatants are coated on a black-walled 96-well immunosorbent plate, and each peptide is further detected with a fluorophore conjugated detection antibody as described herein. The assay is capable of detecting multiple hormones in the same sample through the use of the scFvs, and detection antibodies raised in different species with spectrally distinct fluorophores (Stokes shift >50 nanometers). Theoretical binding curves for multiple exemplary scFvs are proposed in Figure 5A.

[0306] An exemplary, non-limiting procedure to generate a library of aptamers utilizes a modified protocol of systematic evolution of ligands by exponential enrichment (SELEX) in combination with fluorescent enhancement determination. In brief, SELEX is performed with a library of > 1012 RNA sequences targeted against hormones of interest (GLP-1, GIP, PYY, etc). RNA libraries are incubated with the target peptide, washed, amplified, sequenced, and the process was conducted 10 times, with each iteration decreasing the overall RNA load given to the target. At the end of the 10th round of sequencing, sequences are analyzed via nextgeneration sequencing and bioinformatics techniques to uncover the optimal binding sequences. After 10 rounds of “traditional” SELEX, light up aptamers are optimized with small-molecule binding partners after transfection into E. coli and fluorescence activated cell sorting. In addition to light up aptamer pairs, aptamer quenched fluorophores are also screened simultaneously to determine small molecules that fluoresce once outside of the bound aptamer. After binding sequences and binding partners were found, 2D and 3D structural assessments of each are performed to determine flexibility, stability, free-energy, and aptamer binding characteristics. To determine binding affinity and fluorescent enhancement, the following formula was used;

F([apt]) = F' [(F_D + [apt] + [fluorophore])

— (([apt] — [fluorophore])² + K_D(K_D + 2 [apt] + 2[fluorophore]))]/2 wherein [apt] and [fluorophore] are the RNA aptamer and initial fluorophore concentrations, respectively, and F' is the molar maximum fluorescence (i.e., F' = Fmax/[fluorophore]). Ideal binding partners have lower binding affinities than those observed for the hormones of interest. In one exemplary embodiment of aptamer signal adapter(s) to determine the quantity of hormone secreted hormones from a cell supernatant, Aptamer A is designed to release a quenched fluorophore after specific hormone binding as shown in Figure 5A. Prior to aptamer incubation, a cocktail of RNAse and protease inhibitors are added to the media to prevent aptamer or protein degradation. Fixed concentrations of aptamer are pre-incubated with a 10% molecular equivalent amount of fluorophore (aptamer in 10-fold excess to prevent non-specific fluorescence). Aptamer-fluorophore conjugates are then added to cell culture media, and fluorophore release was quantified over time in a kinetic fluorescence plate reader. In another exemplary embodiment of aptamer signal adapter(s), Aptamer B is designed to have two fluorescent molecules that are quenched within close proximity to one another. Structural changes after hormone binding allow the fluorophores to light up. The fluorophores chosen for this purpose are chemically added to the aptamer, with the 5’ end of the aptamer containing fluorescein amidites (FAM), and the 3’ end of the aptamer containing the black-hole quencher 1 (BHQ1). Prior to aptamer addition into collected cell culture media, RNAse and protease inhibitors are added to prevent aptamer degradation. After addition to the media, the mitigation of fluorescent quenching is quantified over time with a kinetic fluorescence plate reader. In another exemplary embodiment of aptamer signal adapter(s), Aptamer C is designed in a similar fashion to Aptamer B, wherein the FAM and BHQ1 are quenched only when the hormone is in the bound state to the aptamer. Within these assays, the decrease in fluorescence over time is measured using a kinetic fluorescence plate reader. For each aptamer and hormone binding partner, multiple sequences are bound at varying affinities for each portion. Representative sequence alignments are shown in Figure 5C, wherein the sequences depicted are randomly generated. E. Example 5: quantitative readout(s) of satiety signaling over time in response to one or more chemical stimuli.

[0307] The present example describes an embodiment of provided technologies for assessing (e.g., quantifying) satiety. In the following non-limiting example, one or more first living cell(s), characterized as neuroendocrine and/or enteroendocrine, are exposed to chemical stimuli for a prolonged duration resulting in the production of one or more satiety signal(s). Said satiety signal(s) are then converted to a quantitative readout by means of one or more protein(s), as provided in Example 1. In this non-limiting example, a combination of the quantified magnitude and duration of production of one or more satiety signal(s) is utilized to determine a conclusion regarding applied chemical stimuli.

[0308] In this particular example, NCI-H716 cells (which are human lymphoblast cells; specifically a colorectal adenocarcinoma cell line), cultured in complete F-12 media to 80% confluency at 37 °C, 5% CO2, were seeded onto 96-well plates and subsequently starved of serum for 16 hours prior to dosing. Then, the serum-depleted F-12 media was replaced with serum-depleted F-12 media further comprising one or more chemical stimuli in the presence of 50 pM Sitagliptin (which is a small molecule inhibitor of DPP-IV added to mitigate degradation of satiety signal(s)). In this non-limiting example, experimental chemical stimuli included media alone (vehicle control), media with 500 mM glucose, media with 100 pM oleic acid, media with 100 pM quercetin, and/or media with 100 pM oleic acid and 100 pM quercetin. Concentrations of chemical stimuli were chosen to either mimic physiological concentrations (e.g., for glucose) or match concentrations used in prior studies with NCI-H716 cells (e.g., for oleic acid and quercetin). Cells were incubated in the presence of chemical stimuli with media collection at 0, 5, 30, 120, and 240 minutes, and the collected satiety signal(s) were converted to a readout using protein adapter(s). In this non-limiting example, commercial GLP-1 ELISA (from Sigma- Aldrich) and commercial GIP ELISA (from Raybiotech) kits were used to convert satiety signal(s) into quantitative readout(s). As shown in Figure 7A, the NCI-H716 cells responded strongly to chemical stimuli comprising oleic acid and oleic acid with quercetin, with nearly 3- and 8-fold increase in secretion of GLP-1 satiety signals, respectively, after 2 hours of incubation. Importantly, while quercetin alone did not increase secretion of GLP-1, the combination of quercetin and oleic acid demonstrated synergy. Similarly, chemical stimuli comprising oleic acid yielded substantial increases in secretion of GIP (Figure 7B), however, at significantly earlier time points (~5 minutes) than GLP-1. This non-limiting example highlights the unexpected advantage of high-throughput assessment of satiety signaling by demonstrating that increasing the frequency of readout sampling allows for the capture of significant signaling events that would have otherwise gone undetected.

F. Example 6: Measuring quantitative readout(s) of satiety signaling over time in response to one or more chemical stimuli.

[0309] The present example describes an embodiment of provided technologies for assessing (e.g., quantifying) satiety. In the following non-limiting example, one or more first living cell(s), is exposed to chemical stimuli for a prolonged duration resulting in the production of one or more satiety signal(s). Said satiety signal(s) are then converted to a quantitative readout by means of one or more protein(s), as provided in Example 1. In this non-limiting example, a combination of the quantified magnitude and duration of production of one or more satiety signal(s) is utilized to determine a conclusion regarding applied chemical stimuli. In this particular example, the one or more satiety signal(s) include, but are not limited to, signaling events downstream of nutrient receptors (e.g., GPR40, GPR120, and GPR70) and gene expression of one or more satiety hormones (e.g., glucagon, gastric inhibitory polypeptide, and peptide YY) in NCI-H716/Caco-2 co-cultures. In this particular example, signaling events downstream of nutrient receptors include modified expression, activation/phosphorylation, and/or flux of one or more second messenger(s) including, but not limited to, PI3K, cAMP, Epac2, PKB/Akt, PKA, MEK, ERK, TCF1, beta-arrestin, PLC, ATF4, ATP, cGMP, calcium ions, potassium ions, sodium ions, magnesium ions, phosphate ions, chloride ions, bicarbonate ions, sulfate ions, any genetic isoforms or variants, and/or any combination(s) thereof.

NCI-H716/Caco-2 co-cultures

[0310] Caco-2 cells were obtained from Sigma-Aldrich (Cat# 12-35-22-00) and NCI- H716 cells were obtained from ATCC (Cat# CCL-251). All cells were maintained in 37° cell culture incubators with 5% CO2. Caco-2 and NCI-H716 culture media was composed of Phenol- free Dulbecco’s minimum essential medium (Gibco) supplemented with 20% fetal bovine serum for Caco-2 cells or 10% fetal bovine serum for NCI-H716 cells (Genessee Scientific), 2.5 mM L- glutamine (Gibco), 10 mM HEPES (Coming), 1% penicillin-streptomycin (Genessee Scientific), and 1% non-essential amino acids for Caco-2 (Gibco). Cells were grown until confluency and detached using 0.05% Trypsin (Corning). Detached cells were spun at 500 x g for 5 minutes, resuspended in 1 mL of growth medium and counted with trypan blue exclusion using a Countessa (Life Technologies) automated cell counter.

[0311] To establish a co-culture, 50,000 NCLH716 cells and 200,000 Caco-2 cells were seeded into the upper chamber of 12-well transwell plates. Cells were polarized for 21 days with media changes on days 2. 4, 8, 12, 16, and 18.

Fluorescence Microscopy

[0312] On day 21, cells were detached with trypsin and centrifuged at 500 xg for 10 minutes then seeded onto 8-well chamber slides and allowed to adhere. Media was removed and cells were washed 3X with sterile phosphate buffered-saline prior to a 10 minute fixation using pre-warmed 4% paraformaldehyde. Cells were then washed 3X, and permeabilized with 0.5% Triton X-100 for 10 minutes at room temperature. Cells were washed 3X with PBS and blocked for 2 hours at room temperature with a 10% fetal-bovine serum (FBS) solution in PBS. Cells were again washed and stained with a primary antibody raised against ZO-1, GPR40 (Abeam Cat# ab236285), or GPR70 (Bioss BS-8612R) for 1 hour at room temperature as recommended by the manufacturer. Cells were then washed 3X in PBS and incubated with an Alexa Fluor conjugated secondary antibody (Invitrogen cat#A-11012) for 30 minutes at room temperature. Cells were then washed and mounted using Fluoromount ™ with DAPI overnight prior to imaging on the EVOS M7000 microscope (Thermofisher).

[0313] As shown in FIG. 8C and FIG. 8D, cells in the co-culture system express surface expression of GPR40. Similarly, as shown in FIG. 9C and FIG. 9D, cells in the co-culture system express GPR70. Accordingly, these data indicate that cells in this co-culture model will be responsive to various nutrient stimuli and elicit one or more quantifiable satiety signal(s) downstream of these nutrient receptors.

Flow Cytometry

[0314] On day 21, cells were detached with trypsin and centrifuged at 500 xg for 10 minutes. Cells were washed with Phosphate buffered saline 3X prior to live-dead staining. Cells were stained with Invitrogen ® Fixable Live/Dead violet stain for 30 minutes following the manufacturer’s instructions. Cells were then washed 3X with PBS and divided into 4 separate 1.5 mL centrifuge tubes. Cells were then stained with anti-GPR40 (Abeam), anti-GPR120 (Invitrogen), and anti-GPR19 (Bioss) at a concentration of 5 pg/mL for 1 hour at 37 °C with rotation. Cells were then washed 3X with PBS, and secondary antibodies (Alexa-Fluor 488 for GPR120 and Alexa-fluor 594 for GPR40 and GPR19) were diluted 1 : 1000 in 5% FBS and stained for 30 minutes at 37 °C. Cells were then washed 3X and kept on ice in the dark until analysis on an Attune CytPix Flow cytometer.

[0315] As shown in FIGs. 10C-10F, cells in the co-culture system express surface expression of GPR40 (FIG. 10C and FIG. 10F), GPR120 (FIG. 10D and FIG. 10F), and GPR70 (FIG. 10E and FIG. 1 OF). These data further indicate that cells in this co-culture model will be responsive to various nutrient stimuli and elicit one or more quantifiable satiety signal(s) downstream of these nutrient receptors.

Quantitative PCR

[0316] Select wells of the 12-well transwell plate co-cultures were used for RNA isolation and subsequent quantitative PCR (qPCR). Cells were lysed with 1 mL of Trizol (Invitrogen) for 5 minutes at room temperature. Cell lysates were then mixed gently with 0.2 mL of chloroform, and centrifuged at >15,000 x G for 30 minutes at 4 °C. The upper aqueous phase was then transferred to 1 mL of isopropanol and incubated at -20°C for 10 minutes. RNA was pelleted by centrifugation >15,000 x G for 30 minutes at 4 °C. RNA pellets were washed twice with 1 mL of 70% ethanol and centrifuged for 15 minutes at >15,000 x G at 4°C after each wash. RNA pellets were air dried for 5-10 minutes or until no ethanol droplets were visible. RNA pellets were resuspended in 50 pL of nuclease-free water and concentrations assessed on a Nanodrop One™ (Thermofisher). RNA was converted into cDNA using the NEB Protoscript First strand cDNA synthesis kit (New England Biolabs), following the manufacturer’s specifications. In brief, 50 ng of RNA was loaded into the cDNA synthesis reaction alongside manufacturer-provided random primer, oligo dT mixes, reaction buffer, and reverse-transcriptase. Controls without enzyme were included on initial runs to ensure kit functionality. After cDNA synthesis, 1 uL of transcribed cDNA was added to a 96-well semi-skirted PCR plate (Thermofisher). Custom PCR primers for the transcripts of G protein-coupled bile acid receptor 1 (TGR5), G-protein coupled receptor 17 (GPR17), free fatty acid receptor 1 (GPR40), G-protein coupled receptor 119 (GPR119), free fatty acid receptor 4 (GPR120), SLC5A1 solute carrier family 5 member 1 (SLGT1), SLC5A2 solute carrier family 5 member 2 (SGLT2), calcium sensing receptor (CASR), peptide YY (PYY), gastric inhibitory polypeptide (GIP), glucagon (GCG), and the house-keeping gene Glyceraldehyde 3 -phosphate dehydrogenase (GAPDH), were ordered and synthesized by Integrated DNA technologies for use in qPCR reactions. Master mixes of gene-specific primers, nuclease-free water, and SYBR green supermix (BioRad) were generated so that the following reaction volumes were added per well: 0.2 uL forward primer, 0.2 uL reverse primer, 3.6 uL nuclease-free water, and 5 uL SYBR. PCR plates were run using a Quantstudio 7 RT-PCR machine.

[0317] As shown in FIG. 8E, cells in the co-culture system express detectable levels of nutrient receptor gene transcripts. These data provide further support that cells in this co-culture model will be responsive to various nutrient stimuli and elicit one or more quantifiable satiety signal(s) downstream of these nutrient receptors.

[0318] As shown in FIG. 11, cells in the co-culture system express detectable levels of glucagon (GCG), gastric inhibitory polypeptide (GIP), and peptide YY (PYY) gene transcripts. These data indicate that co-culture of NCI-H716 cells and Caco-2 cells does not significantly change the levels of gene expression of these satiety hormones as compared to NCI-H716 cells or Caco-2 cells monocultures. These baseline data further suggest that NCI-H716 cells and Caco-2 cells may be used to quantify modulations in the gene expression of these satiety hormones, and others, in response to various nutrient stimuli.

[0319] As shown in FIG. 14, differential nutrient stimulation of enteroendocrine cells results in the release of signal peptides, or hormones. In this figure, the signal peptide is gastric inhibitory polypeptide (GIP). The quantity of signal peptides (e.g., hormones) secreted after single and multi -nutrient stimulation of enteroendocrine cells can be quantified with an antibody signal adapter. In FIG. 14, cells were cultured in 10% fetal bovine serum (FBS, Genessee Scientific), Dubecco’s Modified Eagle Medium (DMEM -GIBCO), 2 mM L-glutamine (GIBCO), and passaged after reaching confluency. NCI-H716 cells were expanded until approximately 80% confluent in a T-75 cell-culture flask. Matrigel (Corning) was thawed overnight on ice prior to use. Pipette tips and plates were kept cold to prevent polymerization prior to coating. 12-well transwell assay plates were coated with 0.5 uL of Matrigel per plate, and plates were cured at 37 degrees C for one hour prior to seeding cells. Approximately 50,000 NCI-H716 cells were seeded per well in the Matrigel-coated plates. Cells were serum starved for 1 hour prior to conducting the experiment. Dosing solutions were prepared in secretion buffer containing 4.5 mM KC1, 138 mM NaCl, 4.2 mM NaHCO3, 1.2 mM NaH2PO4, 2.6 mM CaC12, 1.2 mM MgC12, 10 mM HEPES at pH 7.4, and 50 uM sitagliptin as a peptidase inhibitor. Dosing solutions contained glucose, oleic acid, quercetin, or combinations thereof at combinations including but not limited to 10 uM - 10 mM. Cells were given the dosing solution in the upper compartment of the transwell, with samples harvested for ELISA at 4 hours post-dose.

[0320] ELISA Assay: A clear-walled 96-well immunosorbent plate was coated with anti- GIP (Raybiotech) capture monoclonal antibody at a concentration 0.1 - 1000 pg/mL, respectively. Plates were subsequently washed with a buffer (1 mM PBS, Tween-20, sodium azide) 3 times before addition of samples. 100 uL of assay standard(s), quality control(s), and biotinylated-GIP was loaded into each well (n=2). The assay standard(s) and quality control(s) were resuspended and diluted in assay buffer comprising 0.05 M PBS, pH 6.8, comprising protease inhibitors, Tween 20 0.08% (w/v), sodium azide, and 1% (w/v) bovine serum albumin. The assay standards comprised biotinylated GIP at concentrations of 1000 pg/mL, 100 pg/mL, 10 pg/mL, 1 pg/mL, and 0.1 pg/mL, respectively. Samples derived from NCI-H716 cells were incubated at 4 °C overnight. Following incubation with the samples, plates were washed 5 times with a buffer (1 mM PBS, Tween 20, sodium azide). A GIP-1 HRP-conjugated detection antibody was added and allowed to incubate for 1 hour at room temperature. Plates were again washed 5 times with a buffer (1 mM PBS, Tween 20, sodium azide), and 0.05 mg/mL of 4- methylumbelliferyl phosphate substrate was added to each well for 30 minutes in the dark. After incubation, a stop solution consisting of 2M sulfuric acid was added to each well. Fluorescence was read at excitation/emission wavelengths of 355/460 nm. Data are shown as mean +/- standard deviation, statistical significance was performed using a one-way ANOVA with multiple comparisons against the untreated group with * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0321] As shown in FIG. 15, differential nutrient stimulation of enteroendocrine cells results in the release of signal peptides, or hormones. In this figure, the signal peptide is glucagon-like peptide 1 (GLP-1). The quantity of signal peptides (e.g., hormones) secreted after single and multi -nutrient stimulation of enteroendocrine cells can be quantified with an antibody

Ill signal adapter. In FIG. 15, cells were cultured in 10% fetal bovine serum (FBS, Genessee Scientific), Dubecco’s Modified Eagle Medium (DMEM -GIBCO), 2 mM L-glutamine (GIBCO), and passaged after reaching confluency. NCI-H716 cells were expanded until approximately 80% confluent in a T-75 cell-culture flask. Matrigel (Corning) was thawed overnight on ice prior to use. Pipette tips and plates were kept cold to prevent polymerization prior to coating. 12-well transwell assay plates were coated with 0.5 uL of Matrigel per plate, and plates were cured at 37 degrees C for one hour prior to seeding cells. Approximately 50,000 NCI-H716 cells were seeded per well in the Matrigel-coated plates. Cells were serum starved for 1 hour prior to conducting the experiment. Dosing solutions were prepared in secretion buffer containing 4.5 mM KC1, 138 mM NaCl, 4.2 mM NaHCO3, 1.2 mM NaH2PO4, 2.6 mM CaC12, 1.2 mM MgCL2, 10 mM HEPES at pH 7.4, and 50 uM sitagliptin as a peptidase inhibitor. Dosing solutions contained glucose, oleic acid, quercetin, or combinations thereof at combinations including but not limited to 10 uM - 10 mM. Cells were given the dosing solution in the upper compartment of the transwell, with samples harvested for ELISA at 4 hours postdose.

[0322] ELISA Assay: A black-walled 96-well immunosorbent plate was coated with an anti-GLP-1 (Millipore Sigma) capture monoclonal antibody at a concentration of 1-10 ug per mL. Plates were subsequently washed with a buffer (1 mM PBS, Tween-20, sodium azide) 3 times before addition of samples. 100 uL of assay standard(s), quality control(s), and recombinant GLP-1 was loaded into each well (n=2). The assay standard(s), quality control(s), and recombinant GLP-1 were resuspended and diluted in assay buffer comprising 0.05 M PBS, pH 6.8, comprising protease inhibitors, Tween 20 0.08% (w/v), sodium azide, and 1% (w/v) bovine serum albumin. The assay standards comprised purified GLP-1 at concentrations of 2 pM, 5 pM, 10 pM, 20 pM, 50 pM, and 100 pM. Recombinant GLP-1 was diluted to concentrations of 200 pM, 50 pM, 25 pM, 10 pM and 1 pM. Quality control samples comprised purified GLP-1 at ranges of 5.6-12 pM for QC1, and 31-65 pM for QC2. Samples derived from NCI-H716 cells were incubated at 4 °C overnight. Following incubation with the samples, plates were washed 5 times with a buffer (1 mM PBS, Tween 20, sodium azide). A GLP-1 HRP- conjugated detection antibody was added and allowed to incubate for 1 hour at room temperature. Plates were again washed 5 times with a buffer (1 mM PBS, Tween 20, sodium azide), and 0.05 mg/mL of 4-methylumbelliferyl phosphate substrate was added to each well for 30 minutes in the dark. After incubation, a stop solution consisting of 2M sulfuric acid was added to each well. Fluorescence was read at excitation/emission wavelengths of 355/460 nm. Data is shown as mean +/- standard deviation, statistical significance was performed using a one-way ANOVA with multiple comparisons against the untreated group with * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

G. Example 7: Characterization of Nutrient-Responsive Cell Culture Systems

[0323] The present example describes an embodiment of provided technologies for assessing (e.g., quantifying) satiety. In the following non-limiting example, one or more first living cell(s) is exposed to chemical stimuli (e.g., one or more nutrient(s)), which produces a satiety signal that is read by a signal adapter to yield a quantifiable signal correlating to satiety. Specific nutrient sensing receptors expressing in a living cell(s) can receive one or more chemical stimuli in complex culture systems (e.g., a transwell system) as shown in FIGS. 12A- 12D.

Receptor Gene Expression Analysis

[0324] Certain cellular compositions are required for identifying and/or characterizing stimuli with satiety modulating characteristics. As shown in FIG. 12A, for example, nutrient receptor expression is required for receiving one or more chemical stimuli. An analysis of receptor gene expression relative to GAPDH was performed in NCI-H716 cells. Cells were cultured in 10% fetal bovine serum (FBS, Genessee Scientific), Dubecco’s Modified Eagle Medium (DMEM -GIBCO), 2 mM L-glutamine (GIBCO), and passaged after reaching confluency. NCI-H716 cells were expanded until approximately 80% confluent in a T-75 cellculture flask. On the day of isolation, cells were centrifuged for 5 minutes at 200 times the speed of gravity. Cells were then lysed with 4 mL of Trizol (Invitrogen) for 5 minutes at room temperature per T-75 flask. Cell lysates were then mixed gently with 0.8 mL of chloroform, and centrifuged at >15,000xG for 30 minutes at 4°C. The upper aqueous phase was then transferred to 1 mL of isopropanol and incubated at -20°C for 10 minutes. RNA was pelleted by centrifugation >15,000xG for 30 minutes at 4°C. RNA pellets were washed twice with 1 mL of 70% ethanol and centrifuged for 15 minutes at >15,000xG at 4°C after each wash. Pellets were then air dried for 5-10 minutes or until no ethanol droplets were visible. RNA was resuspended in 50 uL of nuclease-free water and concentrations assessed on a Nanodrop One™ (Thermofisher). RNA was then converted into cDNA using the NEB Protoscript First strand cDNA synthesis kit (New England Biolabs), following manufacturer’s specifications. Specifically, 50 ng of RNA was loaded into the cDNA synthesis reaction alongside manufacturer-provided random primer, oligo dT mixes, reaction buffer, and reverse-transcriptase. Controls without enzyme were included on initial runs to ensure kit functionality. After cDNA synthesis, 1 uL of transcribed cDNA was added to a 96-well semi-skirted PCR plate (Thermofisher). Custom PCR primers for the transcripts of Bile acid membrane receptor (TGR5), G-protein coupled receptor 17 (GPR17), G-protein coupled receptor 40 (GPR40), G-protein coupled receptor 119 (GPR119), G-protein coupled receptor 120 (GRP 120), Sodium-glucose transporter protein 1 (SGLT1), Sodium-glucose transporter protein 2 (SGLT2), Calcium-sensing receptor (CASR) were ordered and synthesized by Integrated DNA technologies for use in qPCR reactions. Master mixes of gene-specific primers, nuclease-free water, and SYBR green supermix (BioRad) were generated so that the following reaction volumes were added per well: 0.2 uL forward primer, 0.2 uL reverse primer, 3.6 uL nuclease-free water, and 5 uL SYBR. PCR plates were run using a Quantstudio 7 RT-PCR machine (Thermofisher) with the following cycling steps; activation and denaturation 45 seconds at 95°C, annealing + extension 30 seconds at 60 °C (repeated 40X), hold temperature at 4 °C. Cycle threshold (CT) values were exported and analyzed using the delta-delta CT method compared against the housekeeping genes as described previously. Data is shown as mean +/- standard deviation at n=3.

[0325] Nutrient receptor expression, shown relative to GAPDH in complex-culture (e.g., co-culture) systems show receipt of one or more chemical stimuli is possible in conditions that mimic physiological conditions. As shown in FIG. 12B, NCI-H716 cells were cultured as described above for FIG. 12Aprior to RNA Isolation. For co-culture assays, Caco-2 cells were obtained from Sigma-Aldrich (Cat# 12-35-22-00). All cells were maintained in 37° cell culture incubators with 5% CO2. Caco-2 culture media was composed of Phenol-free Dulbecco’s minimum essential medium (Gibco) supplemented with 20% fetal bovine serum (Genessee Scientific), 2.5 mM L-glutamine (Gibco), 10 mM HEPES (Corning), 1% penicillin-streptomycin (Genesee Scientific), and 1% non-essential amino acids (Gibco). Cells were grown until confluency and detached using 0.05% Trypsin (Corning). Detached cells were spun at 500xg for 5 minutes, resuspended in 1 mL of growth medium and counted with trypan blue exclusion using a Countessa (Life Technologies) automated cell counter. Approximately 80,000 Caco-2 cells and 10,000 NCI-H716 cells were then plated together into the upper apical compartment of a 1.1 cm² 12-well transwell assay system (Coming). Then 1 mL of growth medium was added to the lower basal compartment, and 0.5 mL of medium was added to the upper apical compartment. On the day of isolation, cells were washed IX in sterile PBS. Cells were then lysed with 0.5 mL of Trizol (Invitrogen) for 5 minutes at room temperature per well of the 12-well transwell plate. Cell lysates were then mixed gently with 0.4 mL of chloroform, and centrifuged at >15,000xG for 30 minutes at 4°C. The upper aqueous phase was then transferred to 1 mL of isopropanol and incubated at -20°C for 10 minutes. RNA was pelleted by centrifugation >15,000xG for 30 minutes at 4°C. RNA pellets were washed twice with 1 mL of 70% ethanol and centrifuged for 15 minutes at >15,000xG at 4°C after each wash. Pellets were then air dried for 5-10 minutes or until no ethanol droplets were visible. RNA was resuspended in 50 uL of nuclease-free water and concentrations assessed on a Nanodrop One™ (Thermofisher). RNA was then converted into cDNA using the NEB Protoscript First strand cDNA synthesis kit (New England Biolabs), following manufacturer’s specifications. To summarize, 50 ng of RNA was loaded into the cDNA synthesis reaction alongside manufacturer-provided random primer, oligo dT mixes, reaction buffer, and reverse-transcriptase. Controls without enzyme were included on initial runs to ensure kit functionality. After cDNA synthesis, 1 uL of transcribed cDNA was added to a 96- well semi-skirted PCR plate (Thermofisher). Custom PCR primers for the transcripts of Bile acid membrane receptor (TGR5), G-protein coupled receptor 17 (GPR17), G-protein coupled receptor 40 (GPR40), G-protein coupled receptor 119 (GPR119), G-protein coupled receptor 120 (GRP 120), Sodium-glucose transporter protein 1 (SGLT1), Sodium-glucose transporter protein 2 (SGLT2), Calcium-sensing receptor (CASR) were ordered and synthesized by Integrated DNA technologies for use in qPCR reactions. Master mixes of gene-specific primers, nuclease-free water, and SYBR green supermix (BioRad) were generated so that the following reaction volumes were added per well: 0.2 uL forward primer, 0.2 uL reverse primer, 3.6 uL nuclease-free water, and 5 uL SYBR. PCR plates were run using a Quantstudio 7 RT-PCR machine

(Therm ofisher) with the following cycling steps; activation and denaturation 45 seconds at 95°C, annealing + extension 30 seconds at 60 °C (repeated 40X), hold temperature at 4 °C. Cycle threshold (CT) values were exported and analyzed using the delta-delta CT method compared against the housekeeping genes as described previously. Data are shown as mean +/- standard deviation at n=3.

[0326] As shown in FIG. 12C, a flow cytometry graph depicts cells’ nutrient receptor membrane expression for extracellular ligand-binding interactions important for satiety signaling. In FIG. 12C, cells were cultured as described above for FIG. 12A. On the day of analysis, cells were harvested and centrifuged for 5 minutes at 200 times the speed of gravity. Cells were washed in 5 mL of sterile phosphate buffered saline three times. After the third wash, cells were resuspended in 1 milliliter of Hank’s balanced salt solution (HBSS) containing 1 uL of LIVE/DEAD™ Fixable blue dead cell stain kit and incubated for 30 minutes at 37 degrees Celsius. After incubation, cells were fixed for 10 minutes at room temperature with 4% paraformaldehyde. Cells were then washed 3 times with PBS, and blocked for 2 hours at room temperature with buffer containing 5% fetal bovine serum in PBS. After blocking, cells were washed 3 times with PBS and incubated with primary human antibodies against the nutrient receptors GPR40, GPR70, and GPR120. The antibody staining solution contained an individual antibody concentration of 2 ug/mL, 5% fetal bovine serum, and 2 mM ethylenediamine tetraacetic acid (EDTA). Cells were divided into 4 groups, (1) unstained, (2) GPR40, (3) GPR70, and (4) GPR120, and diluted 1 : 1 in antibody staining solution. Cells were incubated for 2 hours at room temperature with their respective antibodies. After 2 hours, cells were centrifuged at 500xg and washed with PBS 3X prior to addition of appropriate anti-primary secondary antibodies tagged to Alexa-Fluor 488™ or Alexa-Fluor 568™. Cells were incubated for 30 minutes with the secondary antibody at 37 degrees Celsius. Cells were centrifuged and washed 5X with sterile PBS prior to analysis on the Attune NxT flow cytometer.

[0327] Baseline hormone characterization for screening of satiety modulators in simple and complex culture conditions relative to GAPDH are required for functional analysis of satiety signaling. As shown in FIG. 12D, NCI-H716 cells were cultured as described above for FIG.

12A prior to RNA Isolation. For co-culture assays, Caco-2 cells were obtained from Sigma- Aldrich (Cat# 12-35-22-00). All cells were maintained in 37° cell culture incubators with 5% CO2. Caco-2 culture media was composed of Phenol-free Dulbecco’s minimum essential medium (Gibco) supplemented with 20% fetal bovine serum (Genessee Scientific), 2.5 mM L- glutamine (Gibco), 10 mM HEPES (Coming), 1% penicillin-streptomycin (Genesee Scientific), and 1% non-essential amino acids (Gibco). Cells were grown until confluency and detached using 0.05% Trypsin (Corning). Detached cells were spun at 500xg for 5 minutes, resuspended in 1 mL of growth medium and counted with trypan blue exclusion using a Countessa (Life Technologies) automated cell counter. Approximately 80,000 Caco-2 cells and 10,000 NCLH716 cells were then plated together into the upper apical compartment of a 1.1 cm² 12-well transwell assay system (Corning). 1 mL of growth medium was added to the lower basal compartment, and 0.5 mL of medium was added to the upper apical compartment. On the day of isolation, cells were washed IX in sterile PBS. Cells were then lysed with 0.5 mL of Trizol (Invitrogen) for 5 minutes at room temperature per well of the 12-well transwell plate. Cell lysates were then mixed gently with 0.4 mL of chloroform, and centrifuged at >15,000xG for 30 minutes at 4°C. The upper aqueous phase was then transferred to 1 mL of isopropanol and incubated at -20°C for 10 minutes. RNA was pelleted by centrifugation >15,000xG for 30 minutes at 4°C. RNA pellets were washed twice with 1 mL of 70% ethanol and centrifuged for 15 minutes at >15,000xG at 4°C after each wash. Pellets were then air dried for 5-10 minutes or until no ethanol droplets were visible. RNA was resuspended in 50 uL of nuclease-free water and concentrations assessed on a Nanodrop One™ (Thermofisher). RNA was then converted into cDNA using the NEB Protoscript First strand cDNA synthesis kit (New England Biolabs), following manufacturer’s specifications. Particularly, 50 ng of RNA was loaded into the cDNA synthesis reaction alongside manufacturer-provided random primer, oligo dT mixes, reaction buffer, and reversetranscriptase. Controls without enzyme were included on initial runs to ensure kit functionality. After cDNA synthesis, 1 uL of transcribed cDNA was added to a 96-well semi-skirted PCR plate (Thermofisher). Custom PCR primers for Peptide YY (pyy), Gastric inhibitory polypeptide (gip), and the GLP-1 precursor transcript glucagon (gcg), were ordered from IDT technologies. Master mixes of gene-specific primers, nuclease-free water, and SYBR green supermix (BioRad) were generated so that the following reaction volumes were added per well: 0.2 uL forward primer, 0.2 uL reverse primer, 3.6 uL nuclease-free water, and 5 uL SYBR. PCR plates were run using a Quantstudio 7 RT-PCR machine (Thermofisher) with the following cycling steps; activation and denaturation 45 seconds at 95°C, annealing + extension 30 seconds at 60 °C (repeated 40X), hold temperature at 4 °C. Cycle threshold (CT) values were exported and analyzed using the deltadelta CT method compared against the housekeeping genes as described previously. Data are shown as mean +/- standard deviation at n=3.

[0328] As shown in FIG. 13, certain nutrient stimuli are known or predicted stimuli for various receptors expressed in cells. FIG. 13 depicts a table of 8 receptors of interest and any knowns or predicted classes of nutrient stimuli. FIGS. 12A-13 show that these receptors have characteristic expression profdes in monoculture and co-culture cell systems. Further, these data show characteristic satiety protein gene expression in enteroendocrine cells and in conditions similar to physiological conditions.

H. Example 8: Nutrient-Responsive Cell Culture Systems for Quantifying Satiety Signaling

[0329] Certain signal adapters may be employed to assess satiety signals via measurement of a second messenger. As shown in FIG. 16A, the introduction of a calcium binding protein TWITCH-NR into enteroendocrine cells quantifies a second messenger, calcium, after a signal is received. The readout of this quantification can take under thirty minutes as compared to other processes (e.g., an ELISA assay) that take upwards of 30 hours.

[0330] TWITCH-NR is comprised of (1) a portion of the calcium-binding protein troponin C, (2) the green fluorescent protein mNEON, (3) the red fluorescent protein mScarlet, and (4) the nuclear export sequence MLQNELALKLAGLDINKTG (i.e., SEQ. ID NO 7). To prepare TWITCH-NR containing genetically engineered enteroendocrine cells, a TWITCH-NR gene was first designed. Gblocks with this design were custom ordered from IDT. In silica molecular cloning and reading frame validation was performed using Snapgene. This gene fragment was designed to be inserted into a lentiviral backbone (Addgene #51024) and used for stable gene integration. The plasmid backbone was transformed into Escherichia coli (E. call) strain DH5a (Thermofisher) and grown overnight on Luria-Bertani (LB) agar plates with 50 ug/mL ampicillin. Single colonies were picked and grown overnight at 37 degrees Celsius with agitation in liquid LB-Ampicillin medium. Bacteria were spun at 3000 times gravity for 15 minutes at room temperature. Pellets underwent DNA isolation using the GeneJet Plasmid mini prep kit (Thermofisher) following manufacturer's instructions. The isolated plasmid was then quantified using a NanoDrop One (Thermofisher), and a minimum of 4 micrograms was digested overnight using the restriction enzymes Xhol (NEB) and BsrGI (NEB), Cutsmart™ buffer, and a volume of nuclease free water up to 50 uL. Digests were conducted overnight at 37 degrees C. In tandem with plasmid digests, the gene fragment containing TWITCH-NR was digested with the same enzyme and buffer mixture. Digested plasmid backbones were run on a 1% agarose gel for 40 minutes at 120V. Gels were stained using GelRed nucleic acid stain 10000X (Sigma- Aldridge) for 30 minutes at room temperature, and visualized on a GelBright ™ gel imager (Thermofisher). Digested vector bands were excised, and digested TWITCH NR were both purified using the Wizard gel and PCR purification kit (Promega). The purified DNA backbone and TWITCH-NR insert were ligated together overnight at 16 degrees C using T4 DNA ligase (NEB) at varying ratios as calculated by NEB ligation calculator. Ligated plasmids were then transformed into E.coli DH5a chemically competent cells using the manufacturer’s instructions. Transformed bacteria were grown in LB for 2 hours at 37 degrees C before being streaked onto LB-Amp Agar plates. Plates were grown overnight at 37 degrees Celsius, and successful colonies were grown in liquid LB-Amp overnight. DNA was harvested from the bacteria as described above and digested with Xhol and BsrGI for 4 hours at 37 degrees Celsius. Clones with the gene fragment were then sent for Sanger DNA sequencing using custom primers. Positive clones were grown in large batches of liquid LB-Amp media overnight at 37 degrees C and DNA was isolated using the ZymoPure Midi Prep (II) (Fisher Scientific).

[0331] To engineer NCI-H716 cells with TWITCH-NR, HEK-293T were first cultured in 10% fetal bovine serum (FBS, Genessee Scientific), Dubecco’s Modified Eagle Medium (DMEM -GIBCO), 2 mM L-glutamine (GIBCO), and passaged after reaching confluency. Cells were plated in a 6-well plate and allowed to reach 80% confluency. Seven micrograms of prepped plasmid containing the TWITCH NR gene were resuspended in sterile nuclease free water and incubated with the Lenti-X Packaging Single Shots (Clontech) for 10 minutes as per manufacturer's protocol. The transfection reagent and plasmid were then added to the HEK-293T cells. Lentiviral media was harvested at 48 and 72 hours post transfection and tittered using the Lenti-X GloStix (Clontech). In tandem with the viral harvest, NCLH716 cells were plated to be approximately 80% confluent in Matrigel™ (Corning) coated 24-well culture plates. Virus was added to the cells at multiplicities of infection (MOI) ranging from 1-100, where MOI is calculated as (Virus concentration in particles/mL)/Cell number. Viral media was removed after 48 hours of transduction, and cells were switched into standard media. After 48 hours, NCI-H716 cells were switched into a media containing 5 ug/mL of puromycin. Cells underwent puromycin selection for 2-weeks, with media interchanged every other day. Positive colonies were expanded and used for subsequent experiments.

[0332] TWITCH-NR NCI cells were grown to approximately 80% confhiency in a T-75 tissue culture flask. Cells were detached using 0.05% Trypsin (Corning) for five minutes at 37 degrees C. Detached cells were spun down at 500 times gravity for 5 minutes and washed 3X in sterile Hank’s balanced salt solution (HBSS). After the third wash, cells were resuspended in 1 milliliter of Hank’s balanced salt solution (HBSS) containing 1 uL of LIVE/DEAD™ Fixable blue dead cell stain kit and incubated for 30 minutes at 37 degrees Celsius. Cells were divided into two groups, unstimulated and stimulated. Stimulated cells were then dosed with 10 uM of ionomycin (Cayman) and 5 mM of CaCl for 5 minutes before being placed on ice to halt further stimulation. Cells were subsequently run on an Attune NxT flow cytometer (Thermofisher) and the fluorescence of the mScarlet protein under the mNEON excitation was quantified. Data are shown as the normalized relative fluorescence units (RFU) of 3 biological replicates, with a minimum of 10,000 live-cell events per sample. Data are shown as mean +/- standard deviation.

[0333] As shown in FIG. 16B, the introduction of a cyclic adenosine monophosphate (cAMP) binding protein gFLAMP into enteroendocrine cells quantifies cAMP levels after a signal is received. The gene gFLAMP is comprised of (1) a portion of the bacterial cAMP channel MlotiKl, (2) a circularly permutated green fluorescent protein. To design the gFLAMP gene, gblocks with this design were custom ordered from IDT. In silico molecular cloning and reading frame validation was performed using Snapgene. This gene fragment was designed to be inserted into a lenti viral backbone (Addgene #51024) and used for stable gene integration. The plasmid backbone was transformed into Escherichia coli (E. coli) strain DH5a (Thermofisher) and grown overnight on Luria-Bertani (LB) agar plates with 50 ug/mL ampicillin. Single colonies were picked and grown overnight at 37 degrees Celsius with agitation in liquid LB- Ampicillin medium. Bacteria were spun at 3000 times gravity for 15 minutes at room temperature. Pellets underwent DNA isolation using the GeneJet Plasmid mini prep kit (Therm ofisher) following manufacturer's instructions. The isolated plasmid was then quantified using a NanoDrop One (Thermofisher), and a minimum of 4 micrograms was digested overnight using the restriction enzymes Xhol (NEB) and BsrGI (NEB), Cutsmart™ buffer, and a volume of nuclease free water up to 50 uL. Digests were conducted overnight at 37 degrees C. In tandem with plasmid digests, the gene fragment containing gFLAMP was digested with the same enzyme and buffer mixture. Digested plasmid backbones were run on a 1% agarose gel for 40 minutes at 120V. Gels were stained using GelRed nucleic acid stain 10000X (Sigma-Aldridge) for 30 minutes at room temperature, and visualized on a GelBright ™ gel imager (Thermofisher). Digested vector bands were excised, and digested TWITCH NR were both purified using the Wizard gel and PCR purification kit (Promega). The purified DNA backbone and gFLAMP insert were ligated together overnight at 16 degrees C using T4 DNA ligase (NEB) at varying ratios as calculated by NEB ligation calculator. Ligated plasmids were then transformed in to E.coli DH5a chemically competent cells using the manufacturer’s instructions. Transformed bacteria were grown in LB for 2 hours at 37 degrees C before being streaked onto LB-Amp Agar plates. Plates were grown overnight at 37 degrees Celsius, and successful colonies were grown in liquid LB-Amp overnight. DNA was harvested from the bacteria as described above and digested with Xhol and BsrGI for 4 hours at 37 degrees Celsius. Clones with the gene fragment were then sent for Sanger DNA sequencing using custom primers. Positive clones were grown in large batches of liquid LB-Amp media overnight at 37 degrees C and DNA was isolated using the ZymoPure Midi Prep (II) (Fisher Scientific).

[0334] To engineer NCLH716 cells with gFLAMP, NCLH716 cells were grown to confluency in T-75 cell-culture flasks as described above. Prepared plasmids were diluted to a concentration of 100 nanograms/microliter prior to Lipofectamine 3000 transfection. Plasmid DNA containing the cAMP sensor was first diluted in 750 uL of Opti-Mem (Gibco) medium with 50 uL of P3000 reagent (Thermofisher). The plasmid and P3000 preparation were then combined with 59.2 uL of Lipofectamine 3000 reagent in 750 uL of Opti-mem as a recommended scale up by the manufacturer. Plasmid-complexes were incubated for 15 minutes at room temperature prior to adding to the NCLH716 basal cell culture medium. Complexes were incubated on the cells for 16 hours at 37 degrees Celsius with 5% Carbon Dioxide before being removed from the cells. Cell media was replaced, and the transfected cells were analyzed 48 hours posttransfection. [0335] The gFLAMP NCI cells were grown to approximately 80% confluency in a T-75 tissue culture flask. Cells were detached using 0.05% Trypsin (Coming) for five minutes at 37 degrees C. Detached cells were spun down at 500 times gravity for 5 minutes and washed 3X in sterile Hank’s balanced salt solution (HBSS). After the third wash, cells were resuspended in 1 milliliter of Hank’s balanced salt solution (HBSS) containing 1 uL of LIVE/DEAD™ Fixable blue dead cell stain kit and incubated for 30 minutes at 37 degrees Celsius. Cells were divided into two groups, unstimulated and stimulated. Stimulated cells were then dosed with 60 uM of forskolin for 5 minutes before being placed on ice to halt further stimulation. Cells were subsequently run on an Attune NxT flow cytometer (Thermofisher) and the fluorescence of the cpGFP was quantified. Data are shown as the normalized relative fluorescence units (RFU) of 3 biological replicates, with a minimum of 10,000 live-cell events per sample. Data are shown as mean +/- standard deviation.

[0336] Enteroendocrine cells containing the satiety adapter protein TWITCH-NR are able to receive one or more stimuli that is followed by a binding interaction of a second messenger (e.g., calcium) and generate a quantifiable readout. As shown in FIG. 17, the levels of various chemical stimuli are measurable in TWITCH-NR containing cells as a result of a binding event between TWITCH-NR and calcium ions after the enteroendocrine cells are exposed to the different pharmacological or nutrient stimuli. Dosing of a nutrient leads to the production of a satiety signal (e.g., intracellular calcium), which leads to a quantifiable signal (e.g., fluorescence) by the signal adapter within 5 minutes of dosing.

[0337] In FIG. 17, TWITCH-NR NCI cells were grown to approximately 80% confluency in a T-75 tissue culture flask. Cells were detached using 0.05% Trypsin (Corning) for five minutes at 37 degrees C. Detached cells were spun down at 500 times gravity for 5 minutes and washed 3X in sterile Hank’s balanced salt solution (HBSS). After the third wash, cells were resuspended in 1 milliliter of Hank’s balanced salt solution (HBSS) containing 1 uL of LIVE/DEAD™ Fixable blue dead cell stain kit and incubated for 30 minutes at 37 degrees Celsius. Cells were divided into 5 groups, unstimulated, calcium +ionomycin, glucose, a-linoleic acid, and quercetin. Stimulated cells were then dosed with non-limiting concentration(s) of 10 uM of ionomycin (Cayman) and 5 mM of CaCl, 10 mM glucose, 10 uM a-linoleic acid, and 10 uM quercetin for 5 minutes before being placed on ice to halt further stimulation. Cells were subsequently run on an Attune NxT flow cytometer (Thermofisher) and the fluorescence of the mScarlet protein under the mNEON excitation was quantified. Data are shown as the normalized relative fluorescence units (RFU) of 3 biological replicates, with a minimum of 10,000 live-cell events per sample. Data are shown as mean +/- standard deviation.

[0338] Enteroendocrine cells containing the satiety adapter protein gFLAMP are able to receive one or more stimuli that is followed by a binding interaction of a second messenger (e.g., cAMP) and generate a quantifiable readout. As shown in FIG. 18, the levels of various chemical stimuli are measurable in gFLAMP containing cells as a result of a binding event between gFLAMP and cAMP after the enteroendocrine cells are exposed to the different pharmacological or nutrient stimuli. Dosing of a nutrient leads to the production of a satiety signal (e.g., intracellular cAMP), which leads to a quantifiable signal (e.g., fluorescence) by the signal adapter within 5 minutes of dosing.

[0339] In FIG. 18, gFLAMP NCI cells were grown to approximately 80% confluency in a T-75 tissue culture flask. Cells were detached using 0.05% Trypsin (Coming) for five minutes at 37 degrees C. Detached cells were spun down at 500 times gravity for 5 minutes and washed 3X in sterile Hank’s balanced salt solution (HBSS). After the third wash, cells were resuspended in 1 milliliter of Hank’s balanced salt solution (HBSS) containing 1 uL of LIVE/DEAD™ Fixable blue dead cell stain kit and incubated for 30 minutes at 37 degrees Celsius. Cells were divided into 5 groups, unstimulated, forskolin, glucose, a-linoleic acid, and quercetin. Stimulated cells were then dosed with a non-limiting concentration of 60 uM of forskolin, 10 mM glucose, 10 uM a-linoleic acid, and 10 uM quercetin for 5 minutes before being placed on ice to halt further stimulation. Cells were subsequently run on an Attune NxT flow cytometer (Thermofisher) and the fluorescence of the cpGFP was quantified. Data are shown as the normalized relative fluorescence units (RFU) of 3 biological replicates, with a minimum of 10,000 live-cell events per sample. Data are shown as mean +/- standard deviation.

[0340] As shown in FIG. 19, a binding event between the satiety adapter protein gFLAMP and cAMP after enteroendocrine cells are exposed to different concentrations of an identical chemical stimuli results in a correlated readout of satiety signaling. An escalated dose of a nutrient correlates to a stronger satiety response in the gFLAMP containing enteroendocrine cells. [0341] In FIG. 19, gFLAMP NCI cells were grown to approximately 80% confluency in a T-75 tissue culture flask. Cells were detached using 0.05% Trypsin (Corning) for five minutes at 37 degrees C. Detached cells were spun down at 500 times gravity for 5 minutes and washed 3X in sterile Hank’s balanced salt solution (HBSS). After the third wash, cells were resuspended in 1 milliliter of Hank’s balanced salt solution (HBSS) containing 1 uL of LIVE/DEAD™ Fixable blue dead cell stain kit and incubated for 30 minutes at 37 degrees Celsius. Cells were divided into 4 groups and dosed as follows; unstimulated, forskolin, Quercetin 1 uM (Low), Quercetin 3 uM (Medium), Quercetin 10 uM (High) for 10 minutes prior to fixation with 4% paraformaldehyde. Cells were subsequently washed 3X with sterile PBS and run on an Attune NxT Flow cytometer (Thermofisher). The fluorescence of the cpGFP was quantified. Data are shown as the normalized relative fluorescence units (RFU) of 3 biological replicates, with a minimum of 10,000 live-cell events per sample. Data shown as mean +/- standard deviation.

[0342] As shown in FIG. 20, secondary satiety messengers (e.g., calcium) increase in abundance after increasing the stimulation level of genetically-engineered enteroendocrine cells, as measurable by TWITCH-NR within 5 minutes of dosing.

[0343] In FIG. 20, TWITCH NCI cells were grown to approximately 80% confluency in a T-75 tissue culture flask. Cells were detached using 0.05% Trypsin (Coming) for five minutes at 37 degrees C. Detached cells were spun down at 500 times gravity for 5 minutes and washed 3X in sterile Hank’s balanced salt solution (HBSS). After the third wash, cells were resuspended in 1 milliliter of Hank’s balanced salt solution (HBSS) containing 1 uL of LIVE/DEAD™ Fixable blue dead cell stain kit and incubated for 30 minutes at 37 degrees Celsius. Cells were divided into 4 groups and dosed as follows; unstimulated, Calcium + ionomycin, Quercetin 1 uM (Low), Quercetin 3 uM (Medium), Quercetin 10 uM (High) for 10 minutes prior to fixation with 4% paraformaldehyde. Cells were subsequently washed 3X with sterile PBS and run on an Attune NxT Flow cytometer (Thermofisher). The fluorescence of the cpGFP and mScarlet was quantified. F/F0 (% increase) was calculated as the mScarlet signal prior to stimulation (F0) and after stimulation (F) *100. Data are shown as the normalized relative fluorescence units (RFU) of 3 biological replicates, with a minimum of 10,000 live-cell events per sample. Data shown as mean +/- standard deviation. [0344] Screening for satiety modulators can yield differential secondary messenger responses to the same single and multi -nutrient stimuli within genetically engineered NCI-H716 cells. As shown in FIG. 21 A, TWITCH-NR NCI cells were grown to approximately 80% confluency in a T-75 tissue culture flask. Cells were detached using 0.05% Trypsin (Corning) for five minutes at 37 degrees C. Detached cells were spun down at 500 times gravity for 5 minutes and washed 3X in sterile Hank’s balanced salt solution (HBSS). After the third wash, cells were resuspended in 1 milliliter of Hank’s balanced salt solution (HBSS) containing 1 uL of LIVE/DEAD™ Fixable blue dead cell stain kit and incubated for 30 minutes at 37 degrees Celsius. Cells were divided into 4 groups and dosed as follows; unstimulated, Calcium + lonomycin, Quercetin 1 uM, Quercetin 1 uM + a-linoleic acid 1 uM, Quercetin 1 uM + a-linoleic acid 10 uM for 10 minutes prior to fixation with 4% paraformaldehyde. Cells were subsequently washed 3X with sterile PBS and run on an Attune NxT Flow cytometer (Thermofisher). The fluorescence of the cpGFP and mScarlet was quantified. F/F0 (% increase) was calculated as the mScarlet signal prior to stimulation (F0) and after stimulation (F) *100. Data are shown as the normalized relative fluorescence units (RFU) of 3 biological replicates, with a minimum of 10,000 live-cell events per sample. Data are shown as mean +/- standard deviation.

[0345] As shown in FIG. 2 IB, gFLAMP NCI cells were grown to approximately 80% confluency in a T-75 tissue culture flask. Cells were detached using 0.05% Trypsin (Corning) for five minutes at 37 degrees C. Detached cells were spun down at 500 times gravity for 5 minutes and washed 3X in sterile Hank’s balanced salt solution (HBSS). After the third wash, cells were resuspended in 1 milliter of Hank’s balanced salt solution (HBSS) containing 1 uL of LIVE/DEAD™ Fixable blue dead cell stain kit and incubated for 30 minutes at 37 degrees Celsius. Cells were divided into 4 groups and dosed as follows; ; unstimulated, forskolin, Quercetin 1 uM, Quercetin 1 uM + a-linoleic acid 1 uM, Quercetin 1 uM + a-linoleic acid 10 uM for 10 minutes prior to fixation with 4% paraformaldehyde. Cells were subsequently washed 3X with sterile PBS and run on an Attune NxT Flow cytometer (Thermofisher). The fluorescence of the cpGFP was quantified. Data are shown as the normalized relative fluorescence units (RFU) of 3 biological replicates, with a minimum of 10,000 live-cell events per sample. Data shown as mean +/- standard deviation. [0346] These data demonstrate that a satiety response may be measured depending on the specific nutrient that is triggering the secondary messenger increase. In FIG. 21A, for example, a combination of 1 pM quercetin and 1 pM linoleic acid produced a statistically significant response measured in the TWITCH-NRNCI cells. In contrast, in FIG. 21B, the combination of 1 pM quercetin and 1 pM linoleic acid produced a similar response to a combination of 1 pM quercetin and 10 pM linoleic acid in the gFLAMP NCI cells. These data further demonstrate that satiety response stemming from stimulation via novel combinations of nutrients can lead to satiety responses that enable the identification of nutrients providing for maximal satiety.

[0347] As shown in FIG. 22A-22C, quantification of satiety hormones may be performed after rapid screening of satiety modulating compounds in genetically engineered enteroendocrine cells.

[0348] In FIG. 22A, a plot of ELISA data over 30 hours demonstrates that differential caloric and acaloric stimulation of genetically engineered enteroendocrine cells results in the release of GLP-1. These data can also be seen in the following table.

TABLE 1. ELISA Data from FIG. 22A.

[0349] Cells utilized in the ELISA assay were cultured in 10% fetal bovine serum (FBS, Genessee Scientific), Dubecco’s Modified Eagle Medium (DMEM -GIBCO), 2 mM L-glutamine (GIBCO), and passaged after reaching confluency. NCI-H716 cells were expanded until approximately 80% confluent in a T-75 cell-culture flask. Matrigel (Corning) was thawed overnight on ice prior to use. Pipette tips and plates were kept cold to prevent polymerization prior to coating. 12-well transwell assay plates were coated with 0.5 uL of Matrigel per plate, and plates were cured at 37 degrees C for one hour prior to seeding cells. Approximately 50,000 NCI-H716 cells were seeded per well in the Matrigel-coated plates. Cells were serum starved for 1 hour prior to conducting the experiment. Dosing solutions were prepared in fed state simulated intestinal fluid (FeSSIF). Dosing solutions were prepared at the following concentrations, Positive control - Calcium 5 mM + ionomycin 10 uM, Glucose - 55 mM, alpha-linoleic acid (ALA) - 10 uM, digested whey protein isolate (dWPI) - 20 mM, quercetin - 10 uM, Delta G - 20 mg/mL, I+G umami flavor - 10 mg/mL, Diindolylmethane (DIIM) - 10 mM, Piperine - 100 uM. Combinations of given nutrients were maintained at these concentrations. Peptide aliquots were taken from the receiver compartment containing phosphate-buff ered saline (PBS) + 50 uM sitagliptin at T=0, 1 hour, and 3 hours post-dosing.

[0350] ELISA Assay: A black-walled 96-well immunosorbent plate was coated with an anti-GLP-1 (Millipore Sigma) capture monoclonal antibody at a concentration of 1-10 ug per mL. Plates were subsequently washed with a buffer (1 mM PBS, Tween-20, sodium azide) 3 times before addition of samples. 100 uL of assay standard(s), quality control(s), and recombinant GLP-1 was loaded into each well (n=2). The assay standard(s), quality control(s), and recombinant GLP-1 were resuspended and diluted in assay buffer comprising 0.05 M PBS, pH 6.8, comprising protease inhibitors, Tween 20 0.08% (w/v), sodium azide, and 1% (w/v) bovine serum albumin. The assay standards comprised purified GLP-1 at concentrations of 2 pM, 5 pM, 10 pM, 20 pM, 50 pM, and 100 pM. Recombinant GLP-1 was diluted to concentrations of 200 pM, 50 pM, 25 pM, 10 pM and 1 pM. Samples derived from NCI-H716 cells were incubated at 4 °C overnight. Following incubation with the samples, plates were washed 5 times with a buffer (1 mM PBS, Tween 20, sodium azide). A GLP-1 HRP-conjugated detection antibody was added and allowed to incubate for 1 hour at room temperature. Plates were again washed 5 times with a buffer (1 mM PBS, Tween 20, sodium azide), and 0.05 mg/mL of 4-methylumbelliferyl phosphate substrate was added to each well for 30 minutes in the dark. After incubation, a stop solution consisting of 2M sulfuric acid was added to each well. Fluorescence was read at excitation/emission wavelengths of 355/460 nm. Data are shown as mean +/- standard deviation, statistical significance was performed using a one-way ANOVA with multiple comparisons against the untreated group with * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0351] FIG. 22B shows the satiety sensor readout (e.g., calcium concentration) after about 1 hour from the same cells as described above for FIG. 22A. These data can also be seen in the table below.

TABLE 2: Satiety Secondary Messenger Data from FIG. 22B.

[0352] FIG. 22C depicts a plot of the correlation of FIG. 22A and 22B relative to the positive control (calcium and ionomycin), with a value of 1 on either axis indicating the positive control. These data suggest that GLP-1 can be secreted from multiple different cellular pathways. For example, the outlined group “a” as depicted in FIG. 22C showed GLP-1 secretion and no calcium response, whereas the outlined group “b” showed a modest level of calcium response and low GLP-1 secretion, and the outlined group “c” showed both high calcium and high GLP-1 responses. These data may further suggest that rapid detection of satiety signals with TWITCH- NR NCI cells may correlate with high GLP-1 secretion (group “c”), but other sensors may provide high correlation with other groups (e.g., groups “a” and “b”).

[0353] Nonetheless, by performing the assay using a system comprising only ONE signal adapter as discussed above with reference to FIG. 22B, 4 nutrients and nutrient combinations have been identified as providing a satiety response, validated by secretion of GLP-1.

Additionally, the readout of TWITCH-NR can be used to identify nutrient combinations providing for GLP-1 secretion. Further, these data show that data related to satiety signaling can be procured in efficient time frames (e g., 30 minutes - 2 hours) with embodiments of the present disclosure, as compared to other methods in the art (e.g., greater than 2 hours). The signals produced in enteroendocrine cells utilizing embodiments of the present disclosure are quantifiable in response to various nutrient signals and satiety modulators. Further, these signals are dose-responsive in quantifiable levels.

[0354] In a non-limiting example. FIG. 23 describes an embodiment of provided technologies for assessing (e.g., quantifying) satiety in a high-throughput screening system. In the following non-limiting example, as shown in FIG. 23, the process is depicted in five phases, (1) preparation and dosing of compounds to satiety sensors that may be set up on, for example, transwell plates comprising a differentiated cell layer with several cells types (one of which is enteroendocrine cells with several genetically encoded signal adapters for satiety signals), (2) quantification of satiety readouts produced by signal adapters, for example via microscopy, (3) analysis of satiety modulator response from each signal adapter, (4) correlating the multiplexed readouts to satiety, and (5) iteratively improving nutrients or formulations that modulate satiety. Satiety modulating compounds will be dissolved in buffers including but not limited to phosphate-buffered saline, Hank’s balanced salt-solution (HBSS), fasted state simulated intestinal fluid, fasted state simulated gastric fluid, fed state simulated intestinal fluid, fed state simulated gastric fluid, simulated saliva fluid, or any other biorelevant buffer that is compatible with the compound(s) or organisms comprising the satiety sensor. These potential satiety modulating compounds will be dosed at varying concentrations for time points including but not limited to 0-24 hours. The compounds may be dosed in non-limited examples of well-plates, transwell systems, microfluidic systems, or bioreactors. After dosing, the readouts of the satiety sensors will be quantified using combinations of imaging and computational algorithms that feed into an analysis pipeline. The analysis pipeline may or may not be automated, machine-learning based, or utilize artificial intelligence. Once readouts from one or multiple sensor(s) is quantified and analyzed, findings will be coded into a decision tree wherein compounds that induce satiety will be annotated and compounds or combinations that increase or decrease satiety readouts will be iteratively re-tested either alone or in combination with other modulating compounds.

EXEMPLARY EMBODIMENTS

[0355] Certain exemplary embodiments of provided technologies are enumerated here:

1.) A method of quantifying satiety signaling by measuring signal level(s), the method comprising a step of providing stimuli to one or more first living cell(s) for a predetermined period of time, a step of receiving one or more resulting satiety signal(s), a step of converting one or more signal(s) to readout(s) by a signal adapter, and a step of determining a conclusion with the method based on correlating at least one feature(s) of stimuli towards one or more readout(s).

2) The method of embodiment 1, considered as high-throughput.

3) The method of embodiments 1-2, wherein a 24-well, 48-well, 96-well, and/or 384- well microplate comprises one or more first living cell(s).

4) The method of embodiments 1-3, wherein at least 24, at least 48, at least 96 and/or at least 384 distinct readouts are measured in 2 hours. 5) The method of embodiments 1-4, wherein quantifying refers to assignment of a numerical, percentage, decimal, and/or binary value to said readout(s).

6) The method of embodiments 1-5, wherein a satiety signal comprises one or more first messenger(s), second messenger(s), nucleic acid(s), reactive species, receptor agonist(s), receptor antagonist(s), receptor activator(s), receptor inhibitor(s), and/or combinations thereof.

7) The method of embodiments 1-6, wherein one or more first messenger(s) comprises a small molecule, a peptide, a carbohydrate, a lipid, an electric potential, a gas, and/or combinations thereof.

8) The method of embodiments 1-6, wherein one or more second messenger(s) comprises a small molecule, a peptide, a carbohydrate, a lipid, an electric potential, a gas, and/or combinations thereof.

9) The method of embodiments 1-6, wherein one or more small molecule(s) is or comprises dopamine, 5-hydroxytryptamine, epinephrine, norepinephrine, acetylcholine, glutamate, histamine, cyclic adenosine monophosphate, or cyclic guanosine monophosphate.

10) The method of embodiments 1-9, wherein one or more peptide(s) is or comprises ghrelin, leptin, cholecystokinin, amylin, peptide yy, GLP-1, GLP-2, GIP, or insulin.

11) The method of embodiments 1-9, wherein one or more lipid(s) is or comprises arachidonic acid, anandamide, oleoylethanolamide, lysophosphatidylcholine, or 2- arachidonylglycerol.

12) The method of embodiments 1-11, wherein said one or more satiety signal(s) result by exposing said first living cell(s) to one or more stimuli for a predetermined period of time.

13) The method of embodiments 1-12, wherein said first living cell comprise any class in the group(s) of the animal, plant, fungal, protist, or monera kingdoms. 14) The method of embodiments 1-13, wherein said first living cell(s) is an organism, a cell, a tissue, a monolayer culture, a transwell culture, an organotypic model, and/or an organ.

15) The method of embodiments 1-14, wherein the first living cell(s) are analyzed in the group(s) comprised of the level(s) of single-cell(s), tissue culture plate(s), suspension culture(s), bioreactor(s), whole organism(s), and any combination(s) thereof.

16) The method of embodiments 1-15, wherein said first living cell(s) are characterized as neuroendocrine and/or enteroendocrine in origin.

17) The method of embodiments 1-16, wherein said satiety signal(s) is or may comprise ghrelin, leptin, cholecystokinin, amylin, melanocortin, peptide yy, GLP-1, GLP- 2, GIP, insulin, cyclic adenosine monophosphate, cyclic guanosine monophosphate, P- arrestin, calcium, nitric oxide, anandamide, or oleoylethanolamide.

18) The method of embodiments 1-17, wherein said stimuli induces a physicochemical change in one or more membrane protein(s).

19) The method of embodiments 1-18, wherein said stimulus is a chemical, physical, and/or electrical stimulus.

20) The method of embodiments 1-19, wherein said chemical stimulus is a nutrient.

21) The method of embodiments 1-20, wherein said nutrient is of plant, animal, fungal, or bacterial origin.

22) The method of embodiments 1-19, wherein said chemical stimulus is a pharmacological treatment.

23) The method of embodiment 1-22, wherein said pharmacological treatment falls under the general category of analgesics, antacids, antianxiety, antiarrhythmics, antibacterials, anticoagulants, thrombolytics, anticonvulsants, antidepressants, antidiabetics, antidiarrheals, antiemetics, antifungals, antihistamines, antihypertensives, anti-inflammatories, antineoplastics, antiobesity, antipsychotics, antipyretics, antivirals, barbiturates, beta-blockers, bronchodilators, corticosteroids, cytotoxics, decongestants, diuretics, expectorants, hormones, hypoglycemics, immunosuppressives, laxatives, muscle relaxants, sedatives, sex hormones, tranquilizers, and/or vitamins, or any combination(s) thereof.

24) The method of embodiments 1-19, wherein said physical stimulus is a change in pressure, tension, compression, and/or shear.

25) The method of embodiment 1-24, wherein said membrane protein(s) are constitutively expressed and/or engineered into one or more first living cell(s).

26) The method of embodiments 1-25, wherein said membrane protein(s) comprise cell surface receptor(s), transmembrane receptor(s), gap junction(s), tight junction(s), ion channel receptor(s), g-protein coupled receptor(s) (GPCRs), receptor tyrosine kinases (RTKs), nuclear receptor(s), cytosolic receptor(s), or any isoform(s) thereof.

27) The method of embodiments 1-26, wherein receiving a sample comprises harvesting cell(s), harvesting organism(s), harvesting organ(s), harvesting biological fluid(s), harvesting genetic material(s), harvesting cell culture media, harvesting secreted hormone(s), or any combination(s) thereof.

28) The method of embodiments 1-27, wherein said secreted hormone(s) comprise ghrelin, leptin, cholecystokinin, amylin, peptide yy, melanocortin, GLP-1, GLP-2, GIP, insulin, cyclic adenosine monophosphate, cyclic guanosine monophosphate, P-arrestin, calcium, nitric oxide, anandamide, or oleoylethanolamide.

29) The method of embodiments 1-27, wherein said secreted genetic material(s) comprise nuclear DNA, genomic DNA, plasmid DNA, histone(s), cytosolic DNA, secreted DNA, nuclear RNA, messenger RNA (mRNA), transfer RNA (tRNA), microRNA (miRNA), long-non coding RNA (IncRNA), small nucleolar ma (snoRNA), ribosomal rna (rRNA), secreted RNA, and any iteration(s) or transformation(s) thereof. 30) The method of embodiments 1-29, wherein said readout(s) utilize a signal adapter characterized as an antibody, an aptamer, a second engineered living cell, a fluorescent protein, an enzyme, a quantum dot, a nucleic acid, a short chain variable fragment peptide, a small molecule, or combinations thereof.

31) The method of embodiments 1-30, wherein said second engineered living cell is an organism, a cell, a tissue, a monolayer culture, a transwell culture, an organotypic model, and/or an organ.

32) The method of embodiments 1-31, wherein said second engineered living cell comprise any class in the group(s) of the animal, plant, fungal, protist, or monera kingdoms.

33) The method of embodiments 1-32, wherein said second engineered living cell is further characterized as a probiotic.

34) The method of embodiments 1-33, wherein at least one component of the genome or phenome of said probiotic is not found in wild type probiotic.

35) The method of embodiments 1-30, wherein said signal adapters(s) are immobilized on a plastic and/or metallic surface.

36) The method of embodiment 1-30, wherein said signal adapter(s) are freely soluble in aqueous buffer.

37) The method of embodiments 1-36, wherein said signal adapter(s) emit light upon either direct or indirect excitation by light.

38) The method of embodiments 1-37, wherein said signal adapter(s) effect a chemical reaction (e.g., formation or cleavage of a covalent bond, ionic bond, hydrogen bonding, Van der Waals, and/or dipolar interaction) generating at least one of light, color, heat, electric current, and/or sound. 39) The method of embodiments 1-37, wherein said signal adapter(s) effect a chemical reaction (e.g., formation or cleavage of a covalent bond, ionic bond, hydrogen bonding, Van der Waals, and/or dipolar interaction) decreasing at least one of light, color, heat, electric current, and/or sound.

40) The method of embodiments 1-39, wherein said readout(s) are measured as total signal(s), signal(s) change over time, signal(s) relative to assay, signal(s) relative to other assay measurement(s), and combinations thereof.

41) The method of embodiments 1-40, wherein said concentration of one or more hormone(s) is further characterized as molecular weight per unit gram, molecular weight per unit mole, molarity, gram(s) per unit volume, and/or any transformation(s) or iterations thereof.

42) The method of embodiments 1-41, wherein the conclusion is based on one or more multiple measurement(s).

43) The method of embodiments 1-41, wherein the conclusion is based on incomplete measurement(s).

44) The method of embodiments 1-43, wherein the conclusion is based on calculation(s) of synergy, antagonism, additivity, potentiation, relative synergy, relative antagonism, relative potentiation, non-zero effects, and combinations thereof.

45) The method of embodiments 1-44, wherein a predetermined period of time is at least about 1 minute, at least about 5 minutes, at least about 30 minutes, at least about 1 hour, at least about 4 hours, at least about 12 hours, at least about 24 hours, at least about 3 days, at least about 7 days, at least about 30 days, at least about 90 days, at least about 1 year, at least about 5 years, and/or at least about 20 years.

46) The method of embodiments 1-45, wherein said method is used to quantify satiety signaling in response to chemical, physical, and/or electrical stimuli 47) The method of embodiments 1-46, wherein said chemical, physical, and/or electrical stimuli are known to stimulate satiety signaling in one or more first living cell(s).

48) The method of embodiments 1-46, wherein said chemical, physical, and/or electrical stimuli are unknown to stimulate satiety signaling in one or more first living cell(s).

SEQUENCES

SEQ ID NO: 1 atcgatcgat cgatcgatcg atcgatcgat cgatcgatcg atcgatcgat cgatcgatcg

SEQ ID NO: 2 atcgatcgat cgatcgatcg atcgatcgat cgatgtctca gatgatcgat cgatcgatcg

SEQ ID NO: 3 atcgatcgat cgatcgatcg atcgatcgat cggtgtctca gatgatcaaa agatcgatcg

SEQ ID NO: 4 atcgatcgat cgatcgatcg atcgatcgat cggtgtctca gcgcggatca agatcgatcg

SEQ ID NO: 5 atcgatcgat cgatcgatcg atcgatcgat cggtgatatc agcgcggatc agatcgatcg

SEQ ID NO: 6 atcgatcgat cgatcgatcg atcgatcgat cggtgatgtc agacaggatc agatcgatcg

SEQ ID NO: 7

MLQNELALKL AGLDINKTG

Claims

CLAIMS What is claimed is:

1. A method comprising steps of: providing a system that is or comprises living cells; and detecting in the system presence or level of at least one satiety signal, wherein such detection is performed continuously or at a plurality of time points over a period of time or in a high throughput format, so that the detecting achieves quantitative assessment of satiety in the system.

2. The method of claim 1, wherein the step of detecting comprises: contacting the system with a signal adaptor comprising a set of binding agents, each of which binds specifically to a satiety signal that is a chemical agent or entity present in the system; and determining binding of each of the binding agents to its target satiety signal.

3. The method of claim 2, wherein each binding agent is or comprises a polypeptide or a nucleic acid.

4. The method of claim 3, wherein each polypeptide binding agent is or comprises an antibody agent.

5. The method of claim 3 or claim 4, wherein each nucleic acid binding agent is or comprises an aptamer.

6. A method comprising steps of: contacting a cell population with a stimulus of interest for a period of time; and quantifying a change in the cell population that occurs during or after the period of time, which change comprises an increase or decrease in one or more satiety signals produced by cells of the population and is indicative of a change in satiety state of cells in the population, so that satiety-modulating character of the stimulus is determined.

7. The method of claim 6, wherein the cell population is or comprises cells of enteroendocrine and/or neuroendocrine origin.

8. The method of claim 6 or claim 7, wherein the cell population is or comprises human cells.

9. The method of any one of claims 6-8, wherein the cell population is a complex population in that it comprises at least two cell types.

10. The method of any one of claims 6-9, wherein the cell population is cultured in the upper apical chamber of a transwell plate.

11. The method of claim 9 wherein the cell population is an organotypic model.

12. The method of claim 11, wherein the organotypic model comprises or was derived from a primary cell sample obtained from a human subject.

13. The method of claim 6 wherein the step of quantifying comprises: receiving one or more signals indicative of the change; and converting the received one or more signals into one or more readouts by way of one or more signal adaptors.

14. The method of claim 6, wherein the one or more satiety signals comprise one or more extracellular agents.

15. The method of claim 6 or claim 14, wherein the one or more satiety signals are one or more first messenger(s), second messenger(s), nucleic acid(s), reactive species, receptor agonist(s), receptor antagonist s), receptor activator(s), receptor inhibitor(s), and/or combinations thereof.

16. The method of claim 15, wherein one of the one or more satiety signals is a second messenger.

17. The method of claim 16, wherein the second messenger is selected from calcium and cAMP.

18. The method of claim 6 or claim 14, wherein the one or more satiety signals is or comprises one or more satiety hormones.

19. The method of claim 13, wherein the one or more signal adaptors is a genetically encoded sensor.

20. The method of claim 19, wherein the one or more signal adaptors is selected from a genetically encoded calcium indicator (GECI) and a genetically encoded fluorescent indicator (GEFI).

21. The method of claim 19, wherein the one or more signal adaptors is TWITCH-NR or gFLAMP.

22. The method of claim 13, wherein a first signal of the one or more signals is converted by a first signal adaptor of the one or more signal adaptors, and a second signal of the one or more signals is converted by a second signal adaptor of the one or more signal adaptors.

23. The method of claim 6, wherein the presence or level of at least one satiety signal is detected within 1 second, 5 seconds 30 seconds, 1 minute, 5 minutes, 7 minutes, 10 minutes, 30 minutes, 45 minutes, 1 hour, or 2 hours of contacting the cell population with thestimulus of interest.

24. The method of claim 6, wherein the presence or level of at least one satiety signal is detected in less than 2 hours.

25. The method of claim 6, wherein the presence or level of at least one satiety signal is detected in less than 30 minutes.

26. An agent that is characterized as a satiety modulator when assessed according to the method of claim 6.

27. A method of manufacturing a nutritional composition by incorporating the agent of claim 12 into a food.

28. A nutritional composition comprising an agent of claim 26.

29. A method of characterizing a nutritional composition of claim 26 by using it as a stimulus in an assessment as described herein.

30. The method of claim 1, wherein the living cells comprise Caco-2 cells, NCI-H716 cells, or Caco-2 cells co-cultured with NCI-H716 cells.

31. The method of claim 18, wherein the one or more satiety hormones comprises peptide YY (PYY), glucagon, glucagon-like peptide- 1 (GLP-1), glucagon-like peptide-2 (GLP- 2), gastric inhibitory polypeptide (GIP), or any combination thereof.

32. The method of claim 6, wherein the stimulus of interest induces a physicochemical change in one or more membrane protein(s).

33. The method of claim 32, wherein the one or more membrane proteins comprise a g- protein coupled receptor (GPCR) comprising at least one of GPR40, GPR70, and GPR120.

34. The method of claim 32, wherein the one or more membrane proteins comprise

TGR5, GPR17, GPR40, GPR119, GPR120, SGLT-1, SGLT-2, CaSR, or any combination thereof.

35. A system for screening satiety modulators comprising: active cells comprising a plurality of endocrine cells (for example, enteroendocrine cells and/or neuroendocrine cells) and/or a plurality of intestinal endothelial cells; at least one satiety screening sensor comprising at least one recombinant satiety adapter protein expressed in the active cells, the at least one satiety adapter protein comprising at least one fluorescent protein; at least one nutrient for dosing the active cells; means for exciting the at least one fluorescent protein; and means for reading and/or detecting fluorescence of the at least one fluorescent protein; wherein dosing of the active cells with the at least one nutrient alters a level of fluorescence of the at least one fluorescent protein.

36. A method for screening satiety modulators comprising: providing active cells comprising a plurality of enteroendocrine cells and/or a plurality of intestinal endothelial cells; providing at least one satiety screening sensor comprising at least one recombinant satiety adapter protein expressed in the active cells, the at least one satiety adapter protein comprising at least one fluorescent protein; dosing (or stimulating) the active cells with at least one nutrient; exciting the at least one fluorescent protein; and detecting fluorescence of the at least one fluorescent protein; wherein dosing of the active cells with the at least one nutrient alters a level of fluorescence of the at least one fluorescent protein.

37. The system of claim 35 and/or the method of claim 36, wherein, upon dosing (for example, within 1 second, 5 seconds 30 seconds, 1 minute, 5 minutes, 7 minutes, 10 minutes, 30 minutes, 45 minutes, 1 hour, or 2 hours) with the at least one nutrient, the plurality of enteroendocrine cells and/or the plurality of intestinal endothelial cells propagate at least one signal (i.e., a satiety signal) via one or more signal transduction pathways to the at least one satiety adapter protein.

38. The system and/or method of claims 35-37, wherein means for exciting the at least one fluorescent protein and/or means for reading and/or detecting the at least one fluorescent protein comprises a flow cytometer.

39. The system and/or method of claim 38, wherein the flow cytometer comprises a laser configured to excite the active cells in a range from about 300 nm to about 700 nm (for example, in a range from about 300 nm to about 600 nm, or from about 300 nm to about 500 nm, or from about 350 nm to about 470 nm, or at about 355 nm and/or at about 460 nm).

40. The system and/or method of claims 35-39, wherein the at least one satiety adapter protein comprises at least one of TWITCH-NR and gFLAMP.

41. The system and/or method of claims 35-40, wherein the at least one satiety adapter protein is excited by a secondary messenger.

42. The system and/or method of claim 41, wherein the secondary messenger is selected from cAMP and calcium.

43. The system and/or method of claims 35-42, wherein the at least one fluorescent protein comprises at least one of a green fluorescent protein and a red fluorescent protein.

44. The system and/or method of claims 35-43, wherein the at least one fluorescent protein comprises both a green fluorescent protein and a red fluorescent protein.

45. The system and/or method of claims 36-44, wherein the green fluorescent protein is circularly permutated.

46. The system and/or method of claims 35-45, wherein the green fluorescent protein comprises mNEON.

47. The system and/or method of claims 35-46, wherein the red fluorescent protein comprises mScarlet.

48. The system and/or method of claims 35-47, wherein the at least one satiety adapter protein comprises a nuclear export sequence comprising the amino acid sequence of SEQ. ID NO: 7.

49. The system and/or method of claims 35-48, wherein the at least one satiety adapter protein comprises at least a portion of calcium-binding protein troponin C.

50. The system and/or method of claims 35-49, wherein the active cells comprise enteroendocrine cells comprising receptors disposed on the cell surface for propagation of satiety signaling resulting from the nutrient dosing.

51. The system and/or method of claims 35-50, wherein the at least one nutrient comprises at least one of a-linoleic acid, quercetin, glucose, and oleic acid.

52. The system and/or method of claims 35-51, wherein the at least one nutrient comprises at least two of a-linoleic acid, quercetin, glucose, and oleic acid.

53. The system and/or method of claims 35-52, further comprising a well plate (for example, a 96-well plate, 384-well plate, etc.) wherein the active cells and the at least one satiety adapter protein are aliquoted such that multiple wells of the well plate contain the active cells and the at least one satiety adapter protein, wherein the multiple wells contain the at least one nutrient, and wherein at least one well of the multiple wells contains a nutrient that is different than at least one other well of the multiple wells.

54. The system and/or method of claims 35-53, wherein at least one well of the multiple wells contains more than one nutrient.

55. The system and/or method of claims 35-54, wherein the level of fluorescence of the at least one fluorescent protein is altered such that an increase in a range from about 0.5% to about 1.5% (for example, from about 0.9% to about 1.3%) is observed over a fluorescence level corresponding to an unstimulated protein.

56. The system and/or method of claims 35-55, wherein the at least one nutrient comprises at least one of a caloric nutrient and an acaloric nutrient.

57. The system and/or method of claims 35-56, wherein the at least one nutrient comprises a caloric nutrient and an acaloric nutrient.

58. The system and/or method of claims 35-57, wherein the at least one nutrient comprises an acaloric nutrient comprising at least one of diindolylmethane (DIIM), quercetin, and piperine.

59. The system and/or method of claim 58, further comprising at least one of linoleic acid (LA), alpha-linoleic acid (ALA), disodium inosinate plus disodium guanylate (I + G), and digested whey protein isolate (dWPI).

60. The system of claim 57, wherein each of the caloric nutrient and the acaloric nutrient alters a level of fluorescence of the at least one fluorescent protein.