HK1225077A1 - Customized skin care products and personal care products based on the analysis of skin flora - Google Patents

Description

Customized skin care and personal care products based on analysis of skin flora

Cross Reference to Related Applications

The present application claims the benefit of priority from U.S. series No. 61/947,316 filed on 3/3 mesh 2014 and U.S. series No. 61/836,589 filed on 6/18/2013, each of which is incorporated herein by reference in its entirety, according to 35u.s.c. § 119 (e).

Background

Technical Field

The present invention is based generally on skin care products and more particularly on methods of analyzing the skin and subcutaneous tissue flora and their associated metabolite sets to develop or identify personalized skin care products.

Background information

About 100 trillion microorganisms live in and on the human body, greatly exceeding about 10 trillion individual cells of the human body. These generally harmless viruses, bacteria and fungi are called commensal or mutualistic organisms. Symbiotic and mutualistic organisms help to maintain our health in many ways: they help us digest food and obtain nutrients such as vitamins B and K, stimulate the immune system to develop and prevent, for example, colonization by pathogenic bacterial pathogens by competing with them. In summary all microorganisms living in and on the body-symbiotic, mutualistic and pathogenic-are called microbiome and their balance and related metabolome are closely related to the health status of the individual and vice versa.

Next Generation Sequencing (NGS) has established the opportunity to quickly and accurately identify and characterize the microbiome inhabiting the skin and subcutaneous tissue, which in turn has created the opportunity to create customized or personalized skin care products and personal care products that maintain a healthy microbiome or shift the profile (profile) towards a healthy balance or profile by mixing a mixture of symbiotic and/or mutualistic organisms that are specifically generated to create a healthy profile (heatyprofile). The flora also interacts in a synergistic manner with the host immune system, further extending its health benefits. The relevant metabolome of an individual can also be characterized by mass spectrometry-based systems or using genome-based metabolome modeling and flow balance analysis and used to generate a profile of a healthy metabolome. Deficiencies in any beneficial metabolites may also be compensated for.

Currently, the only way to treat skin disorders is by antibiotics that greatly affect the microbiome (including commensal and mutualistic bacteria), or by attenuating the body's immune response and mitigating the epidermal cell's response to pathogens, but may not address the underlying cause of skin disorders (if it is caused by an imbalance or absence of commensal or mutualistic microorganisms, an overabundance of opportunistic or pathogenic bacteria, or a lack of essential or beneficial metabolites).

Summary of The Invention

The present invention relates generally to the development of customized skin care products and personal care products for human and animal use, and more particularly, but not by way of limitation, to the development of personal care products based on a preliminary assessment of the flora of bacteria residing in the skin and subcutaneous tissue. The present disclosure relates to methods and systems for analyzing skin and subcutaneous tissue flora, such as microbiome and its associated metabolite panel, comparing the resulting skin and subcutaneous tissue flora and metabolite panel profiles to health profiles (expressed as the number and diversity of flora falling within a range determined from a set of healthy skin types), and then customizing skin care and personal care products that will increase the flora and its associated metabolite panel resident in the skin and subcutaneous tissue of a test subject or replicate the healthy flora profile on the test subject's flora profile.

Accordingly, in one aspect, the present invention provides a method of characterizing the microbiome of skin or subcutaneous tissue of a subject. The method comprises the following steps: a) obtaining a sample comprising a plurality of microorganisms from the skin or subcutaneous tissue of a subject; and b) analyzing and classifying the plurality of microorganisms of (a) to characterize the microbiome of the subject, thereby characterizing the microbiome of the subject. In some embodiments, the method further comprises comparing the microbiome of the subject to a reference microbiome or generating a microbiome profile of the subject, or identifying a disease or disorder that the subject has, or is at risk of developing, or providing a personalized treatment regimen for the subject. In various embodiments, the reference microbiome is classified as having a healthy profile, and similarity between the microbiome of the subject and the reference microbiome identifies the microbiome of the subject as having a healthy profile. Alternatively, the reference microbiome is classified as having, or at risk of having, a disease or disorder, and the similarity between the subject's microbiome and the reference microbiome identifies the subject's microbiome as having, or at risk of having, the disease or disorder.

In another aspect, the invention provides methods of characterizing microbiome of skin or subcutaneous tissue of a plurality of subjects. The method comprises the following steps: a) obtaining a plurality of samples from skin or subcutaneous tissue of a plurality of subjects, each sample comprising a plurality of microorganisms; and b) analyzing and classifying the plurality of microorganisms of each of the samples of (a) to identify a microbiome for each of the plurality of samples, thereby characterizing the microbiome of the plurality of subjects. In embodiments, the method further comprises clustering the results of (b) to identify individual clusters in the plurality of samples. In some embodiments, each individual cohort displays a particular phenotype or profile. In some embodiments, each individual cohort comprises samples with similar microbiomes, samples from subjects with a common skin disease or disorder, or samples from subjects with similar metabolite profiles.

In another aspect, the invention provides a method of diagnosing a disease or disorder in a subject. The method comprises the following steps: a) obtaining a sample comprising a plurality of microorganisms from the skin or subcutaneous tissue of a subject; b) analyzing and classifying the plurality of microorganisms of (a) to identify a microbiome of the subject; and c) comparing the microbiome of the subject to a reference microbiome representing a microbiome of a subject having, or at risk of having, a disease or disorder, wherein a similarity between the microbiome of the subject and the reference microbiome indicates that the subject is at risk of, or has, the disease or disorder, thereby diagnosing the disease or disorder in the subject. In embodiments, the method further comprises providing the subject with a personalized treatment regimen. In some embodiments, the method further comprises formulating a customized therapeutic formulation and administering the formulation to the subject.

In another aspect, the invention provides a method of formulating a customized therapeutic formulation for a subject having, or at risk of having, a disease or condition. The method comprises the following steps: a) obtaining a sample comprising a plurality of microorganisms from the skin or subcutaneous tissue of a subject; b) analyzing and classifying the plurality of microorganisms of (a) to identify a microbiome of the subject; c) comparing the subject's microbiome to a reference microbiome representative of a microbiome of a subject having, or at risk of having, a disease or disorder, wherein a similarity between the subject's microbiome and the reference microbiome indicates that the subject is at risk of having, or has, the disease or disorder; and d) formulating the customized therapeutic preparation based on (c).

In another aspect, the invention provides a therapeutic formulation formulated by the method of the invention.

Brief Description of Drawings

FIG. 1 is a high level summary of experimental and analytical channels used to analyze skin and subcutaneous tissue flora profiles. Informative regions of the microbial genome of a mixed population collected from skin or subcutaneous tissue samples are amplified using universal primer sequences designed to capture the greatest diversity of various bacterial species. The amplified regions were uniquely indexed to allow for multiple processing of samples from various sources (nucleic acid harvesting and preparation). Amplified regions from different sources can be combined and sequenced using Paired End (PE) mode on NGS platforms, or alternatively, can be analyzed based on Sanger sequencing, mass spectrometry, quantitative PCR, immunofluorescence, in situ hybridization, or microbial stain-based platforms. The raw output of the identification platform is analyzed using a proprietary analysis pipeline that assigns raw reads to species from different taxonomic groups. Similar workflows can be used to map metabolites associated with any given sample.

Figure 2 shows the collection of multiple samples from a healthy cohort of individuals and the processing of the samples through our pipeline as described in figure 1 and their use to establish a common profile for healthy populations, capturing the populations of metabolites that constitute the dominant species or their associated groups. The health profile is used as a reference to compare any affected cohort, population or individual.

Figure 3 shows that multiple samples from a cohort of individuals with one characteristic skin disorder will be collected and their skin flora and their associated metabolites profiled as previously described. The seed metadata profile is used to identify characteristic microorganisms or metabolites that are responsible for or associated with the skin disorder. Any abnormalities in the composition of the skin and subcutaneous tissue flora or metabolome of a new customer (shown by the question mark in the figure) can be detected even at an early stage compared to the previously identified health profile, and can be repaired or repaired with customized or personalized skin care products that divert the affected profile towards a healthy balance produced by mixing a mixture of commensal organisms or metabolites specifically expected to establish the health profile.

Detailed Description

It is now well known that about 100 trillion microorganisms live in and on the human body, much in excess of about 10 trillion individual cells. These normally harmless viruses, bacteria and fungi are said to be symbiotic (harmless to their host) or mutualistic (provide benefits). Symbiotic and mutualistic organisms help maintain our health in many ways: they help us digest food and obtain nutrients such as vitamins B and K, stimulate our immune system to develop and prevent, for example, colonization by pathogenic bacterial pathogens by competing with them. In summary, all microorganisms living in and on the body-symbiotic, mutualistic and pathogenic-are called the microbiome of the body, and their balance and related metabolome are closely related to the health status of the individual, and vice versa.

Currently, the main method of treating skin diseases is to alleviate the response of epidermal cells to pathogens by greatly changing the microbiome of an individual or by attenuating the body's immune response, but there is no anti-inflammatory agent that addresses the underlying cause of skin diseases. Embodiments of the present invention relate to a combination of experimental and computerized workflows that allow for the characterization of the skin microbial flora and its associated metabolome with the aim of first evaluating the skin of an individual to determine whether their skin disease is caused by an imbalance or absence of commensal or mutualistic microorganisms or their associated metabolome.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods and/or steps of the type described herein, which will become apparent to those skilled in the art upon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

The present invention relates generally to the development of customized skin care products and personal care products for human and animal use and, more particularly, but not by way of limitation, the development of personal care products based on a preliminary assessment of the flora of bacteria residing in the skin and subcutaneous tissue. The present disclosure relates to methods and compositions for analyzing skin and subcutaneous tissue flora and their associated metabolome, comparing the resulting profile of skin and subcutaneous tissue flora and metabolome to a health profile (expressed as the number and diversity of flora falling within the range determined from a set of healthy skin types), and then customizing skin care and personal care products that will increase the flora and its associated metabolome residing in the skin and subcutaneous tissue of a test subject or replicate the healthy flora profile on the test subject's flora profile.

Personalized skin test results may be the basis for the development of personalized skin care products and personal care products that are tailored to maintain a healthy skin microbiome and metabolome, or to shift the profile towards a healthy balance or profile by adding one or more commensal and/or complementary microorganisms and/or substrates that facilitate the growth of commensal and mutualistic organisms on the skin. The exact composition of the skin care mixture can be determined after comparing the resulting profile of skin and subcutaneous tissue flora and metabolome of any individual to the health profile, and then customizing the skin care and personal care products that best convert the subject's skin and subcutaneous tissue flora and metabolome to the health profile. The optimal flora and the set of substrates and metabolites may act synergistically with the immune system of the host and contribute to health from this perspective.

As used herein, the term "subject" refers to any individual or patient on whom the subject methods are performed. Typically, the subject is a human, although as will be understood by those skilled in the art, the subject may be an animal. Thus other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals (including cows, horses, goats, sheep, pigs, etc.) and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.

Accordingly, in one aspect, the invention provides a method of characterizing a microbiome from skin or subcutaneous tissue of a subject. The method comprises the following steps: a) obtaining a sample comprising a plurality of microorganisms from the skin or subcutaneous tissue of a subject; and b) analyzing and classifying the plurality of microorganisms of (a) to characterize the microbiome of the subject, thereby characterizing the microbiome of the subject.

As used herein, the terms "sample" and "biological sample" refer to any sample suitable for the methods provided by the present invention. The sample of cells may be any sample, including, for example, a skin or subcutaneous tissue sample obtained by a non-invasive technique, such as peel-off tape, scraping, swabbing, or a more invasive technique such as a biopsy of the subject. In one embodiment, the term "sample" refers to any preparation derived from the skin or subcutaneous tissue of a subject. For example, cell samples obtained using the non-invasive methods described herein can be used to isolate nucleic acid molecules or proteins for use in the methods of the invention. Samples for use in the present invention may be obtained from an area of skin that is shown to exhibit a disease or condition (which is suspected to be the result of a disease or a pathological or physiological state such as psoriasis or dermatitis), or a surrounding margin or tissue. As used herein, "peripheral margin" or "surrounding tissue" refers to tissue of a subject that is adjacent to skin that is shown to exhibit a disease or disorder, but otherwise appears normal.

In some embodiments, the method further comprises comparing the subject's microorganisms to a reference microorganism or generating a microbiome profile of the subject, or identifying a disease or disorder that the subject has, or is at risk of developing, or providing a personalized treatment regimen to the subject. In various embodiments, the reference microbiome is classified as having a healthy profile, and similarity between the microbiome of the subject and the reference microbiome identifies the microbiome of the subject as having a healthy profile. Alternatively, the reference microorganism is classified as having, or at risk of having, a disease or disorder, and the similarity between the subject's microbiome and the reference microbiome identifies the subject's microbiome as having, or at risk of having, the disease or disorder.

As used herein, a "health profile" refers to a microbiome associated with a sample from a subject determined to have a healthy microbiome, e.g., no disease or disorder or risk thereof. Thus, in one embodiment, the reference microbiome is a microbiome of a cell sample obtained from a healthy individual who does not have a skin disorder or a particular undesirable phenotype.

The term health profile is the number and colony diversity that falls within the range determined from a set of healthy skin types. The term healthy skin refers to skin without skin disorders, diseases or conditions including, but not limited to, inflammation, rash, dermatitis, atopic dermatitis, eczema, psoriasis, dandruff, acne, cellulitis, rosacea, warts, seborrheic keratosis, actinic keratosis, tinea versicolor, viral eruptions, shingles, tinea, and cancers such as basal cell carcinoma, squamous cell carcinoma, and melanoma.

In addition, as used herein, "disease" or "disorder" means generally any skin-related disease, such as, but not limited to, inflammation, rash, dermatitis, atopic dermatitis, eczema, psoriasis, dandruff, acne, cellulitis, rosacea, warts, seborrheic keratosis, actinic keratosis, tinea versicolor, viral eruptions, shingles, tinea, and cancers such as basal cell carcinoma, squamous cell carcinoma, and melanoma.

As used herein, the term "cancer" includes any malignancy, including but not limited to: carcinomas, melanomas and sarcomas. Cancer results from uncontrolled and/or abnormal division of cells, which subsequently invade and destroy surrounding tissues. As used herein, "proliferation" and "hyperplasia" refer to cells undergoing mitosis. As used herein, "metastasis" refers to the spread of a malignant tumor distal to its primary site. Cancer cells can metastasize through the bloodstream, through the lymphatic system, through body cavities, or any combination thereof. As provided herein, the term "cancer cell" includes a cell that has any of the cancerous conditions provided herein. The term "cancer" refers to a malignant new growth consisting of epithelial cells that tend to infiltrate the surrounding tissues and cause metastases. The term "melanoma" refers to a malignant tumor of melanocytes found primarily in the skin but also in the intestine and eye. "melanocytes" refers to cells located in the bottom basement membrane of the epidermis of the skin and the middle layer of the eye. Thus, "melanoma metastasis" refers to the spread of melanoma cells to regional lymph nodes and/or distant organs (e.g., liver, brain, breast, prostate, etc.).

The term "skin" or "subcutaneous tissue" refers to the outer protective layer of the body, which is composed of the epidermis (including the stratum corneum) and the underlying dermis, and is understood to include sweat and sebaceous glands, as well as structures and nails due to hair follicles. Throughout this application, the adjectives "cutaneous" and "subcutaneous" may be used, and should be understood to refer generally to the properties of skin, depending on the context in which they are used. The epidermis of human skin comprises several different layers of skin tissue. The bottom layer is the basal layer, which is composed of columnar cells. The overlayer is a spinous layer, which is composed of polyhedral cells. Cells pushed up from the spinous layer are flattened and keratohyalin granules are synthesized to form a layer of the granular layer. As these cells move outward, they lose the nucleus and the keratohyalin granules fuse and mix with the tension fibrils. This forms a transparent layer which is a transparent layer. The cells of the stratum lucidum are closely packed. As the cells move up from the stratum lucidum, they are squeezed into many layers of opaque scales. These cells are all flat cell remnants that have become completely filled with keratin and have lost all other internal structures, including the nucleus. These scales constitute the outer layer of the epidermis and the stratum corneum. At the bottom of the stratum corneum, the cells are tightly packed and strongly adhere to each other, but higher up in the stratum corneum they become loosely packed and eventually flake off the surface.

As discussed further herein, next generation sequencing or NGS (a powerful DNA sequencing technique that allows for rapid and accurate sequencing of cells or organisms) now enables the evaluation of complex bacterial communities (a good example of which is microbiome). The present invention contemplates the identification of the habitat population of each individual to be performed on such Next Generation Sequencing (NGS) platforms. Such platforms allow rapid and accurate identification and analysis of the profile of the microbiome inhabiting the skin with sufficiently high sensitivity and specificity and relatively short turnaround times and scalable throughput. Alternatively, individual profiles can be characterized using Sanger sequencing, mass spectrometry, quantitative PCR, immunofluorescence, in situ hybridization, or a microbial stain-based platform. Similarly, analysis of the profile of a microbiome or metabolome can be performed by mass spectrometry based systems or using genomics-based metabolome modeling and flux balance analysis. All of the above assays can be performed on samples collected directly from an individual without any proliferation steps. Thus, minimal bias is introduced for the identification of mixtures of culturable and non-culturable microorganisms or their relevant metabolome.

By utilizing the high throughput capability of NGS or other microbial identification methods such as mass spectrometry or Sanger sequencing, hundreds of microbes and their related microbiome and metabolome in the subcutaneous tissue of an individual can be identified simultaneously and the resulting signature profiles compared to the health signature profiles from a database of skin and subcutaneous tissue signature profiles. Independent of the platform utilized to analyze the profile, the above-described platform can be provided as a test to any customer, and the output can be used to identify which commensal, pathogenic or mutualistic microorganisms or their related metabolites are depleted or overabundant on the skin and subcutaneous tissues of the subject as compared to the healthy profile.

In addition, the subject's flora and metabolite panel is compared to a previously compiled database of different skin conditions to see if he or she is likely to develop any of these skin conditions in the future. Based on the customized or personalized test results, a customized or personalized skin care or personal care mixture for the individual may be formulated by mixing a mixture of commensal and mutualistic microorganisms or their related metabolites depleted in the individual's flora or metabolome with or without essential substrates and nutrients that facilitate the proliferation of commensal and mutualistic organisms. The customized or personalized skin care or personal care product is specifically produced in a manner that establishes an optimal profile (by maintaining a healthy microbiome or biasing a sub-optimal profile towards a healthy balance). In addition, the synergy between the optimal microbial flora and its associated metabolome and the host's immune system will further contribute to skin hygiene and health.

Thus, the methods and platforms described herein can utilize analysis of nucleic acid molecules, such as sequencing nucleic acid molecules. Sequencing methods may include whole genome sequencing, next generation sequencing, Sanger sequencing, 16SrDNA sequencing and 16SrRNA sequencing. Further, such methods and platforms described herein can utilize mass spectrometry, quantitative PCR, immunofluorescence, in situ hybridization, microbial stain-based platforms, or combinations thereof.

In embodiments, the input to the identification platform may be any nucleic acid, including single-stranded or double-stranded DNA, RNA, cDNA, miRNA, mtDNA. The nucleic acid can be of any length, as short as an oligonucleotide of about 5bp to as long as megabases or even longer. The term "nucleic acid molecule" as used herein refers to single, double or triple stranded DNA, RNA and any chemical modifications thereof. Virtually any modification of the nucleic acid is contemplated. A "nucleic acid molecule" can have almost any length, 10, 20, 30, 40, 50, 60, 75, 100, 125, 150, 175, 200, 225, 230, 275, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 30,000, 40,000, 50,000, 75,000, 100,000, 150,000, 200,000, 500,000, 1,000,000, 1,500,000, 2,000,000, 5,000,000 or even more bases in length, up to a full length chromosomal DNA molecule. For methods of analyzing gene expression, the nucleic acid isolated from the sample is typically RNA.

Micro RNA (mirna) is a small single-stranded RNA molecule of length on average 22 nucleotides, which is involved in the regulation of mRNA expression in different species, including humans (reviewed in Bartel 2004). The first report of miRNA was found in the lin-4 gene of C.elegans (Lee, Feinbaum et al 1993). Since then, hundreds of mirnas have been found in drosophila, plants, and mammals. mirnas regulate gene expression by binding to the 3' -untranslated region of mRNA and catalyzing i) cleavage of mRNA, or 2) inhibition of translation. The regulation of gene expression by miRNAs is central to many biological processes such as cell development, differentiation, communication, and apoptosis (Reinhart, Slack et al 2000; Baehreche 2003; Brennecke, Hipfner et al 2003; Chen, Li et al 2004). Recently, mirnas have been shown to be active during mouse epithelial embryogenesis and to play an important role in skin morphogenesis (Yi, O' Carroll et al 2006).

In view of the role of mirnas in gene expression, it is clear that mirnas will affect, if not be completely specific, the relative amount of mRNA in a particular cell type, thereby determining the expression profile of a particular gene (i.e., a particular mRNA population) in different cell types. Furthermore, the specific distribution of a particular miRNA in a cell may also be unique among different cell types. Thus, determination of the miRNA profile of a tissue can be used as a tool for analyzing the expression profile of an actual mRNA population in that tissue. Thus, the detection of miRNA levels and/or miRNA mutations is useful for disease detection, diagnosis, prognosis or treatment-related decisions (i.e., indicating a response before or after a treatment regimen has been initiated) or for the purpose of characterizing a particular disease in a subject.

As used herein, the term "protein" refers to at least two covalently linked amino acids, which include proteins, polypeptides, oligopeptides, and peptides. Proteins can be composed of naturally occurring amino acids and peptide bonds or synthetic peptidomimetic structures. Thus, as used herein, "amino acid" or "peptide residue" refers to both naturally occurring and synthetic amino acids. For example, homophenylalanine, citrulline, and norleucine are considered amino acids for the purposes of the present invention. "amino acid" also includes imino acid residues such as proline and hydroxyproline. The side chain may be present in either the (R) or (S) configuration.

A "probe" or "probe nucleic acid molecule" is a nucleic acid molecule that is at least partially single stranded and is at least partially complementary, or at least partially substantially complementary, to a target sequence. The probe may be RNA, DNA or a combination of RNA and DNA. Also within the scope of the invention are probe nucleic acid molecules having a nucleic acid comprising a backbone sugar that is not ribose or deoxyribose. The probe nucleic acid may also be a peptide nucleic acid. The probe may comprise a linkage to anti-nucleolytic activity or a detectable label, and may be operably linked to other moieties, such as peptides.

A single-stranded nucleic acid molecule is "complementary" to another single-stranded nucleic acid molecule when it can base pair (hybridize) with all or part of the other nucleic acid molecule to form a double helix (double-stranded nucleic acid molecule) based on the ability of guanine (G) to base pair with cytosine (C) and adenine (a) to base pair with thymine (T) or uridine (U). For example, the nucleotide sequence 5 '-TATAC-3' is complementary to the nucleotide sequence 5 '-GTATA-3'.

As used herein, "hybridization" refers to the process by which a strand of nucleic acid joins with a complementary strand through base pairing. The hybridization reaction may be sensitive and selective, so that a particular target sequence may even be identified in a sample where it is present at low concentrations. In the in vitro case, suitably stringent conditions may be defined by, for example, the concentration of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art. Specifically, stringency can be increased by decreasing the salt concentration, increasing the formamide concentration, or increasing the hybridization temperature. For example, hybridization under high stringency conditions can occur in about 50% formamide at about 37 ℃ to 42 ℃. Hybridization can occur under reduced stringency conditions (at about 30 ℃ to 35 ℃ in about 35% to 25% formamide). Specifically, hybridization can occur under high stringency conditions (at 42 ℃ in 50% formamide, 5 XSSPE, 0.3% SDS, and 200mg/ml sheared and denatured salmon sperm DNA). Hybridization can occur under reduced stringency conditions as described above, but at a reduced temperature of 35 ℃ in 35% formamide. The temperature range corresponding to a particular stringency level can be further narrowed by calculating the ratio of purines to pyrimidines in the target nucleic acid and adjusting the temperature accordingly. Variations on the above ranges and conditions are well known in the art.

As used herein, the term "skin flora" or "microbiome" refers to microorganisms, including bacteria, viruses, and fungi, that inhabit the skin or subcutaneous tissue of a subject.

As used herein, the term microbial, microbe or microorganism (microorganissm) refers to any microscopic organism, including prokaryotes or eukaryotes, bacteria, archaea, fungi, viruses, or protists (unicellular or multicellular).

As used herein, the term "ameliorating" or "treating" means that clinical signs and/or symptoms associated with cancer or melanoma are reduced as a result of the ongoing behavior. The signs or symptoms to be monitored are characteristic of a particular cancer or melanoma and are well known to the skilled clinician, as are methods for monitoring the signs and conditions. Thus, a "treatment regimen" refers to the plan or course of any system for treating a disease or cancer in a subject.

In embodiments, nucleic acid molecules may also be isolated by lysing cells and cellular material collected from a skin sample using a number of methods well known to those skilled in the art. For example, a number of commercial products that can be used to isolate polynucleotides can be used, including, but not limited to, RNeasy^TM(Qiagen, Valencia, CA) and TriReagent^TM(molecular research laboratory, Inc, Cincinnati, OH). The isolated polynucleotides may then be tested and assayed for a particular nucleic acid sequence, including a polynucleotide encoding a cytokine. Methods for recovering target nucleic acid molecules within a nucleic acid sample are well known in the art and can include microarray analysis.

As discussed further herein, nucleic acid molecules can be analyzed in a number of methods known in the art that can aid in the determination of microbiome and/or metabolome associated with the skin of an individual. For example, the presence of a nucleic acid molecule can be detected by DNA-DNA or DNA-RNA hybridization or amplification using a probe or fragment of the particular nucleic acid molecule. Nucleic acid amplification-based assays include the use of oligonucleotides or oligomers based on the nucleic acid sequence to detect transformants containing a particular DNA or RNA.

In another embodiment, an antibody that specifically binds to an expression product of a nucleic acid molecule of the invention can be used to characterize a skin lesion in a subject. The antibodies may be used modified or unmodified, and may be labeled by covalently or non-covalently linking them with a reporter molecule.

A variety of labels and conjugation techniques are known to those skilled in the art and can be used for a variety of nucleic acid and amino acid assays. Methods for generating labeled hybridization or PCR probes for detecting sequences associated with the nucleic acid molecules of tables 1-6 include oligonucleotide labeling, nick translation, end labeling, or PCR amplification using labeled nucleotides. Alternatively, the nucleic acid molecule or any fragment thereof may be cloned into a vector for the production of mRNA probes. Such vectors are known in the art, are commercially available, and can be used for in vitro synthesis of RNA probes by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These methods can be performed using various commercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.); Promega (Madison Wis.) and U.S. Biochemical Corp., Cleveland, Ohio). Suitable reporter molecules or labels that can be used to facilitate detection include radionuclides, enzymes, fluorescence, chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

PCR systems typically use two amplicon-specific fluorescent hybridization probes that specifically bind to sites within the amplicon and two amplicon primers. The probe may comprise one or more fluorescent label moieties. For example, the probe may be labeled with two fluorescent dyes: 1) 6-carboxy-Fluorescein (FAM) as a reporter at the 5 'end, and 2) 6-carboxy-tetramethyl-rhodamine (TAMRA) as a quencher at the 3' end. When amplification occurs, the 5 '-3' exonuclease activity of the taq dna polymerase cleaves the reporter from the probe during the extension phase, releasing it from the quencher. The resulting increase in fluorescence emission of the reporter dye, which is representative of the number of DNA fragments produced, is monitored during PCR. In situ PCR can be used for direct localization and visualization of target nucleic acid molecules, and can further be used to correlate expression with histopathological findings.

Methods for generating specific hybridization probes for the nucleic acid molecules of the invention include cloning the nucleic acid sequence into a vector for generating mRNA probes. Such vectors are known in the art, are commercially available, and can be used for the synthesis of RNA probes in vitro by addition of an appropriate RNA polymerase and appropriate labeled nucleotides. The hybridization probes may be labeled with a variety of reporter groups, e.g., radionuclides such as 32P or 35S, or enzymatic labels such as alkaline phosphatase coupled to the probe via an avidin/biotin coupling system, or the like.

The term "skin care" or "personal care" refers to skin care products, including, but not limited to, cleansing products, shampoos, conditioners, toners or creams, topical ointments and gels, and topical (e.g., under-the-eye) gels, all of which can be formulated to contain ingredients (with or without symbiotic or mutualistic organisms, or mixtures of symbiotic or mutualistic organisms, in an active or dormant state) specifically designed to bias microbial populations toward a healthy profile. Such skin care products may also include therapeutic agents, vitamins, antioxidants, minerals, skin conditioners, polymers, excipients, surfactants, probiotics or fractions thereof, microorganisms or products from cultures thereof, such bacteria, fungi, and the like (live, dormant, or inactivated).

By "skin commensal microorganism" is meant prokaryotes and eukaryotes that can colonize (i.e., live and reproduce on human skin) or inhabit human skin transiently in vitro, ex vivo, and/or in vivo. Exemplary skin commensal microorganisms include, but are not limited to, alpha-Proteobacteria (Alphaproteobacteria), beta-Proteobacteria (Betaproteobacteria), gamma-Proteobacteria (Gamma Proteobacteria), Propionibacterium (Propionibacterium), Corynebacterium (Corynebacterium), Actinomycetes (Actinobacillus), Clostridium (Clostridium), Lactobacillus (Lactobacilliales), Staphylococcus (Staphylococcus), Bacillus (Bacillus), Micrococcus (Micrococcus), Streptococcus (Streptococcus), Bacteroides (Bacteroides), Flavobacterium (Flavobacterium), Enterococcus (Enterococcus), Pseudomonas (Pseudomonas), Marasmyces (Malassezia), Malassezia, Debaromyces (Debaryomyces) and Cryptococcus (Cryptococcus).

Propionibacterium acnes (p.acnes) is a commensal, non-spore-forming, rod-shaped gram-positive bacterium found in various locations on the human body, including areas of the skin, oral cavity, urinary tract, and large intestine. Propionibacterium acnes can consume skin oil and produce byproducts, such as short chain fatty acids and propionic acid, which are known to help maintain a healthy skin barrier. Propionibacteria such as propionibacterium acnes also produce bacteriocins and bacteriocin-like compounds (e.g., propionibacterin P1G-1, jenserin G, propionibacterin SM1, SM2T1, and acnecin) that are inhibitory to undesirable lactic acid producing bacteria, gram negative bacteria, yeasts, and molds. In embodiments, a subject having skin identified as having propionibacterium acnes can be treated with a personal care product designed to inhibit the growth and proliferation of propionibacterium acnes.

In embodiments, an individual's skin profile is translated into personalized skinniq by capturing the diversity of the skin flora and the ridges that help formulate personal care products^TMIndex, which is an overall snapshot of skin health. The main factors contributing to uplift are probiotic balance, mutual benefit and ratio of symbiotic microorganisms to (opportunistic) pathogens. However, the bumps may also contain other factors that may positively affect the health of the skin. These factors may include the presence of genes, gene products or proteins of key biosynthetic microorganisms responsible for the promotion and maintenance of healthy host skin. All of these factors will contribute to the overall health of the skin by, but not limited to, reducing skin inflammation, reducing the relative amount of pathogens, and biosynthesis of provitamins, antimicrobial peptides, vitamins, and fatty acids. By SkinIQ^TMThe combination of the exponentially represented diversity and elevations can also be a predictive measure of skin health. For example, the dominance of a certain subspecies of propionibacterium acnes may be strongly correlated with the risk of acne outbreaks. Similarly, SkinIQ^TMOther skin diseases may be predicted including, but not limited to, eczema, psoriasis, atopic dermatitis and rosacea flare-up.

The SkinIQ^TMThe index is defined in terms of a skin health measurement system that compares any individual profile to a "common health profile" from a database of skin profiles (microbiome and/or metabolome) and places each profile in the context of a healthy population. A common health profile is defined for each bacterial species separately. Data from healthy populations is used to define the range in which any given bacterial species is expected to be found in healthy individuals. All these ranges define references for future comparisons. Skin health test system (the SkinHealthMeasurementSystem)^TMAlso as usable for diggingA powerful tool for the discovery of a rich set of novel microorganisms that can be used in skin care formulations to positively affect different skin diseases including, but not limited to, acne, atopic dermatitis, psoriasis and eczema. As such, it can be used to mine higher levels of interaction between different bacterial species, with potential therapeutic implications.

Thus, the present invention contemplates generating a reference database containing a number of reference projection profiles generated from skin samples of subjects having known conditions (such as normal or healthy skin and various skin disease conditions). The personal profile may be compared to a reference database containing reference profiles. A subject is diagnosed as having a particular disease state if the subject's profile best matches the profile of that disease state in the database. Various computer systems and software may be used to perform the analysis method of the present invention and will be apparent to those skilled in the art. Exemplary software programs include, but are not limited to, Cluster & TreeView (Stanford, URLs: rana. lbl. gov or micro. org), GeneCluster (MIT/Whitehead institute, URL: MPR/GeneCluster. html), ArrayExplorer (SpotFireInc, URL: spot. com/Products/Scicomp. asp # SAE), and GeneSpring (Silicongenetics Inc, URL: signatures. com/Products/GeneSpring/index. html) (see also U.S. Pat. No. 6,203,987 for computer systems and software, incorporated herein by reference).

In embodiments, the present invention provides methods of characterizing skin and/or subcutaneous tissue, the methods comprising collecting a sample from a subject containing a population of skin or subcutaneous tissue. Healthy individuals with a healthy flora of skin and subcutaneous tissue may be collected using a wipe, scrape, swab, strip of paper or any other effective collection method of microorganisms. The profiles of the collected samples were analyzed on NGS, Sanger sequencing, mass spectrometry, quantitative PCR, immunofluorescence, in situ hybridization, or a microbial stain-based platform. For sequencing-based platforms, this can be done using whole genome sequencing methods, or by targeted applications (a prominent example of which is 16SrDNA sequencing). All of the above-described identification methods can be performed on samples collected directly from an individual without any proliferation steps. Thus, minimal bias is introduced to the identification of mixtures of culturable and non-culturable microorganisms. A dedicated analysis algorithm can be used to identify the species composition of each individual. A consensus health profile can be constructed from the health cohort. The health profile may be updated in real time as more samples are collected over time. The health profile will be used as a reference for comparing all individual samples, i.e. the profiles. Examples of bacteria identified belong to any phylum, including Actinomycetes, Firmicutes, Proteobacteria, Bacteroides. It generally includes species of common species such as propionibacterium (Propionibacterium), Staphylococcus (Staphylocci), Corynebacterium (Corynebacterium) and Acinetobacter (Aceniobacter).

In embodiments, the present invention provides a platform or method for characterizing the skin and subcutaneous tissue microbial flora of individuals with skin disorders. Skin and subcutaneous tissue flora of healthy individuals can be collected using a wipe, a swab, a paper strip, or any other effective collection of microorganisms. The profiles of the harvested samples can be analyzed on NGS, Sanger sequencing, mass spectrometry, quantitative PCR, immunofluorescence, in situ hybridization, or a microbial stain-based platform. For sequencing-based platforms, this can be done using whole genome sequencing methods, or by the target application (whose prominent example is 16SrDNA sequencing). All of the above-described identification methods can be performed on samples collected directly from an individual without any proliferation steps. Thus, minimal bias is introduced to the identification of mixtures of culturable and non-culturable microorganisms. A profile of the individual skin and subcutaneous tissue flora characteristics of each individual can be generated. Individuals based on their phenotypic characteristics may likewise be placed under a particular skin disorder classification. Such clustering efforts will help identify biologically significant patterns characteristic of each cluster. The microbial composition of the affected group is significantly different from the health profile. The species of microorganism associated with any given skin disorder can be used as an early diagnostic marker for individuals who do not develop a visible skin disorder but who may be susceptible to such a skin disorder. Examples of bacteria identified belong to any phylum, including Actinomyces, firmicutes, Proteobacteria, Bacteroides. It usually includes common species such as propionibacterium, staphylococcus, corynebacterium and acinetobacter species. Damaged skin may affect the composition of the flora or may cause nonpathogenic bacteria to become pathogenic.

In embodiments, the present invention provides a platform or method for characterizing a common healthy skin and subcutaneous tissue metabolite profile. The profile of the metabolome associated with the skin and subcutaneous tissue flora can also be analyzed by mass spectrometry based systems or using genomics based metabolome modeling and flux balance analysis. The extraction may be performed on samples collected by using a wipe, swab, strip of tissue, or any other effective collection of microorganisms. Alternatively, these metabolites and biochemicals, particularly extracellular metabolites and biochemicals, can be isolated directly from any individual without undergoing any collection of cells. The entire metabolite panel can be characterized or the characterization focused on only a subset of metabolites, which are known, or can be shown to be of great significance in a particular disease pathology. All of the above identification methods can be performed on samples collected directly from an individual without any proliferation steps. Thus, minimal deviation in population composition is introduced. Dedicated analysis algorithms can be used to identify the metabolite composition of each individual's skin flora. A consensus health profile can be constructed from the health cohort. The health profile may be updated in real time as more samples are collected over time. The health profile will be used as a reference for comparing all individual samples, i.e. the profiles.

In embodiments, the present invention provides a platform or method for characterizing the skin and subcutaneous tissue microbial flora of individuals with skin disorders. The metabolite composition of the skin and subcutaneous tissue microbial flora of individuals considered to be most appropriate with skin disorders can be analyzed by mass spectrometry based systems or using genomics based metabolome modeling and flux balance analysis. The extraction may be performed on samples collected by using a wipe, swab, strip of tissue, or any other effective collection of microorganisms. Alternatively, these metabolites and biochemicals, particularly extracellular metabolites and biochemicals, can be isolated directly from any individual without undergoing any collection of cells. The entire metabolite panel may be characterized or the characterization focused on only a subset of metabolites, which are known or may be shown to be of great significance. All of the above identification methods can be performed on samples collected directly from an individual without any proliferation steps. Thus, minimal deviation in population composition is introduced. An individual profile reflecting the metabolite composition of the skin and subcutaneous tissue flora may be generated for each individual. Likewise, individuals may be placed under specific categories of skin disorders based on their phenotypic characteristics. Such clustering efforts will help identify biologically significant patterns that are characteristic of each cluster. The metabolite composition of the affected cohort differed significantly from the healthy profile. Metabolites associated with any given skin disorder can be used as an early diagnostic marker for individuals who have not developed a visible skin disorder but who may be susceptible to the skin disorder.

In embodiments, the platform or methods described herein may be provided as a test for analyzing the profile of the skin flora and their associated metabolome of any individual (healthy or with a skin disorder). Such tests can sensitively characterize the predominant skin flora and metabolites of any individual. A customized or personalized assessment of the flora of any individual can be made and the identified skin and subcutaneous tissue flora and metabolites can be compared to a health profile and also to an affected skin profile. A customized or personalized report can be generated that will detail the species composition of the individual's skin and subcutaneous flora and also its associated metabolites. Such reports would list beneficial and commensal species that are depleted or over-represented in each individual. It will also include a list of beneficial or undesirable metabolites that are depleted or over-represented in each individual. This can be useful in the formulation of customized or personalized skin care or personal care products. Alternatively, the test may be applied to evaluate the performance of other skin care and personal care, therapies,or to assess any disruption of normal skin flora or metabolites. Tests can be performed before, during and after any skin treatment to monitor the efficacy of the treatment regimen on the skin flora or its related metabolites. The test is also useful for the early diagnosis of skin diseases associated with characteristic microbial signatures or their concomitant metabolites. The sensitivity of the tests may allow for their phenotype_{Outbreak of hair}Early diagnosis of such skin diseases has previously been carried out. In one aspect, the present invention provides a method for producing a customized or personalized skin care or personal care product formulated for a particular individual. The customized or personalized products contain one or more beneficial or commensal microorganisms or a group of chemicals and metabolites that can be depleted in any given individual. Conventional applications of such skin care and personal care products should shift the sub-optimal profile towards a healthy balance. The skin care product may be applied after the existing flora is cleared with a special lotion that will increase the colonization efficiency of the skin care product microorganisms or their constituent metabolites. Any customized or personalized skin care or personal care product may contain one or more microorganisms (culturable or non-culturable). The customized or personalized products can optionally be substrates and nutrients that facilitate the establishment or proliferation of commensal or mutualistic organisms and/or the inhibition of pathogenic organisms. These chemicals and metabolites can be synthesized in vitro or purified from microorganisms.

Although the present invention has been described with reference to the above embodiments, it is to be understood that modifications and variations are included within the spirit and scope of the present invention. Accordingly, the invention is not limited except as by the following claims.

Claims (47)

1. A method of characterizing a microbiome of skin or subcutaneous tissue of a subject, the method comprising:

a) obtaining a sample comprising a plurality of microorganisms from the skin or subcutaneous tissue of the subject; and

b) analyzing and classifying the plurality of microorganisms of (a) to characterize the microbiome of the subject, thereby characterizing the microbiome of the subject.

2. The method of claim 1, further comprising comparing the subject's microbiome to a reference microbiome.

3. The method of claim 1, further comprising generating a microbiome profile of the subject.

4. The method of claim 2, further comprising identifying a disease or disorder that the subject has, or is at risk of developing.

5. The method of claim 4, further comprising providing the subject with a personalized treatment regimen.

6. The method of claim 5, further comprising formulating and administering a customized therapeutic formulation to the subject.

7. The method of claim 2, wherein the reference microbiome is classified as having a healthy profile and the similarity between the subject's microbiome and the reference microbiome identifies the subject's microbiome as having a healthy profile.

8. The method of claim 2, wherein the reference microorganism is classified as having, or at risk of having, a disease or disorder, and the similarity between the subject's microbiome and the reference microbiome identifies the subject's microbiome as having, or at risk of having, the disease or disorder.

9. The method of claim 1, wherein the sample is obtained from the skin or subcutaneous tissue by scraping, swabbing, biopsy, or taping.

10. The method of claim 9, wherein the tape is an adhesive tape.

11. The method of claim 1, wherein the plurality of microorganisms comprise bacteria, fungi, or a combination thereof.

12. The method of claim 11, wherein the microorganism is a bacterium selected from the group consisting of alpha-proteobacteria, beta-proteobacteria, gamma-proteobacteria, propionibacterium, corynebacterium, actinomycetales, clostridiales, lactococca, staphylococcus, bacillus, micrococcus, streptococcus, bacteroides, flavobacteriales, firmicutes, enterococcus, pseudomonas, malassezia, Maydida, debaryomyces, and cryptococcus.

13. The method of claim 11, wherein the microorganism is a bacterium of the genus propionibacterium, staphylococcus, corynebacterium, or acinetobacter.

14. The method of claim 13, wherein the bacterium is propionibacterium acnes.

15. The method of claim 1, wherein said analyzing and classifying comprises sequencing nucleic acids.

16. The method of claim 15, wherein the sequencing is selected from whole genome sequencing, next generation sequencing, Sanger sequencing, 16SrDNA sequencing, and 16SrRNA sequencing.

17. The method of claim 1, wherein the analyzing and classifying comprises mass spectrometry, quantitative PCR, immunofluorescence, in situ hybridization, a microbial stain-based platform, or a combination thereof.

18. A method of characterizing a microbiome of skin or subcutaneous tissue of a plurality of subjects, the method comprising:

a) obtaining a plurality of samples from skin or subcutaneous tissue of the plurality of subjects, each sample comprising a plurality of microorganisms; and

b) analyzing and classifying the plurality of microorganisms of each of the samples of (a) to identify a microbiome for each of the plurality of samples, thereby characterizing the microbiome of the plurality of subjects.

19. The method of claim 18, further comprising clustering the results of (b) to identify individual cohorts of the plurality of samples.

20. The method of claim 19, wherein each population of individuals exhibits a particular phenotype or profile.

21. The method of claim 20, wherein each individual cohort comprises samples with a similar microbiome.

22. The method of claim 20, wherein an individual cohort comprises samples from subjects having a common skin disease or disorder.

23. The method of claim 20, wherein an individual cohort comprises samples from subjects with similar metabolite profiles.

24. The method of claim 19, wherein each sample is obtained from the skin or subcutaneous tissue by scraping, swabbing, biopsy, or taping.

25. The method of claim 24, wherein the tape is an adhesive tape.

26. The method of claim 19, wherein the plurality of microorganisms comprises bacteria, fungi, or a combination thereof.

27. The method of claim 26, wherein the microorganism is a bacterium selected from the group consisting of alpha-proteobacteria, beta-proteobacteria, gamma-proteobacteria, propionibacterium, corynebacterium, actinomycetales, clostridiales, lactococca, staphylococcus, bacillus, micrococcus, streptococcus, bacteroides, flavobacteriales, firmicutes, enterococcus, pseudomonas, malassezia, Maydida, debaryomyces, and cryptococcus.

28. The method of claim 26, wherein the microorganism is a bacterium of the genus propionibacterium, staphylococcus, corynebacterium, or acinetobacter.

29. The method of claim 28, wherein the bacterium is propionibacterium acnes.

30. The method of claim 19, wherein said analyzing and classifying comprises sequencing nucleic acids.

31. The method of claim 30, wherein the sequencing is selected from whole genome sequencing, next generation sequencing, Sanger sequencing, 16SrDNA sequencing, and 16SrRNA sequencing.

32. The method of claim 19, wherein the analyzing and classifying comprises mass spectrometry, quantitative PCR, immunofluorescence, in situ hybridization, a microbial stain-based platform, or a combination thereof.

33. A method of diagnosing a disease or disorder in a subject, the method comprising:

a) obtaining a sample comprising a plurality of microorganisms from the skin or subcutaneous tissue of the subject;

b) analyzing and classifying the plurality of microorganisms of (a) to identify a microbiome of the subject; and

c) comparing the subject's microbiome to a reference microbiome representing a microbiome of a subject having, or at risk of having, a disease or disorder, wherein a similarity between the subject's microbiome and the reference microbiome indicates that the subject is at risk of having, or is at risk of having, the disease or disorder, thereby diagnosing the disease or disorder in the subject.

34. The method of claim 33, further comprising generating a microbiome profile of the subject.

35. The method of claim 33, further comprising providing the subject with a personalized treatment regimen.

36. The method of claim 36, further comprising formulating a customized therapeutic formulation and administering the therapeutic formulation to the subject.

37. The method of claim 33, wherein the sample is obtained from the skin or subcutaneous tissue by scraping, swabbing, biopsy, or taping.

38. The method of claim 38, wherein the tape is an adhesive tape.

39. The method of claim 33, wherein the plurality of microorganisms comprises bacteria, fungi, or a combination thereof.

40. The method of claim 40, wherein the microorganism is a bacterium selected from the group consisting of alpha-Proteobacteria, beta-Proteobacteria, gamma-Proteobacteria, Propionibacterium, Corynebacterium, Actinomycetes, Clostridium, Lactobacillus, Staphylococcus, Bacillus, Micrococcus, Streptococcus, Bacteroides, Flavobacterium, firmicutes, enterococcus, Pseudomonas, Malassezia, Maydida, Debaryomyces, and Cryptococcus.

41. The method of claim 40, wherein the microorganism is a bacterium of the genus Propionibacterium, Staphylococcus, Corynebacterium, or Acinetobacter.

42. The method of claim 42, wherein the bacterium is Propionibacterium acnes.

43. The method of claim 33, wherein said analyzing and classifying comprises sequencing nucleic acids.

44. The method of claim 44, wherein the sequencing is selected from whole genome sequencing, next generation sequencing, Sanger sequencing, 16SrDNA sequencing and 16SrRNA sequencing.

45. The method of claim 33, wherein the analyzing and classifying comprises mass spectrometry, quantitative PCR, immunofluorescence, in situ hybridization, a microbial stain-based platform, or a combination thereof.

46. A method of formulating a customized therapeutic formulation for a subject having or at risk of having a disease or disorder, the method comprising:

a) obtaining a sample comprising a plurality of microorganisms from the skin or subcutaneous tissue of the subject;

b) analyzing and classifying the plurality of microorganisms of (a) to identify a microbiome of the subject; and

c) comparing the subject's microbiome to a reference microbiome representative of a microbiome of a subject having or at risk of having the disease or disorder, wherein a similarity between the subject's microbiome and the reference microbiome indicates that the subject is at risk of having the disease or disorder, or has the disease or disorder, and

d) formulating the customized therapeutic preparation based on (c), thereby formulating the customized therapeutic preparation.

47. A therapeutic preparation formulated by the method of claim 47.