WO2019081551A1

WO2019081551A1 - Process for performing a chemotaxis assay on a porous wet surface

Info

Publication number: WO2019081551A1
Application number: PCT/EP2018/079098
Authority: WO
Inventors: Michael RAYNER; Kentaro Kawata; Marianne Dohmen; Barbara GERTEN; Michael Dietze
Original assignee: Merck Patent GmbH
Current assignee: Merck Patent GmbH
Priority date: 2017-10-26
Filing date: 2018-10-24
Publication date: 2019-05-02
Anticipated expiration: 2020-04-26

Abstract

The present invention relates to a process for performing a nematode chemotaxis assay. Instead of using agar the assay is performed on a three- dimensional porous wet surface.

Description

Process for performing a chemotaxis assay on a porous wet surface

The present invention relates to a process for performing a nematodes chemotaxis assay. Instead of using agar the assay is performed on a three- dimensional porous wet surface.

Background of the invention

Chemotaxis is the movement of an organism in response to a chemical stimulus. Somatic cells, bacteria, and other single-cell or multicellular organisms like nematodes direct their movements according to certain chemicals in their environment. Positive chemotaxis occurs if the movement is towards a higher concentration of the chemical in question. The chemical in question is thus an attractant. Negative chemotaxis occurs if the movement is in the opposite direction. In this case the chemical in question is a repellant.

Nematodes are one type of multicellular organism which is known to show chemotaxis. The nematodes or roundworms constitute the phylum

Nematoda. They are a diverse animal phylum inhabiting a broad range of environments. Nematode species can be difficult to distinguish, and although over 25,000 have been described, of which more than half are parasitic, the total number of nematode species has been estimated to be about 1 million. Nematodes are classified along with insects and have tubular digestive systems with openings at both ends.

The nematode Caenorhabditis elegans, also called C. elegans, has a highly developed chemosensory system that enables it to detect a wide variety of volatile (olfactory) and water-soluble (gustatory) cues associated with food, danger, or other animals. Much of its nervous system and more than 5% of its genes are devoted to the recognition of environmental chemicals. C. elegans uses chemosensation to find food, avoid noxious conditions, develop appropriately, and mate. It senses chemicals with chemosensory neurons that penetrate the cuticle to expose their sensory cilia to the environment.

There are two main types of chemotaxis assays using nematodes. One type is an assay in which the nematodes move through a three-dimensional gel like an agarose gel. This type of assay is e.g. performed with Root-knot nematodes (RKNs) which are destructive pathogens of agricultural crops and are attracted to host roots.

Another type of chemotaxis assay uses the ability of nematodes to move on a surface. Nematodes are applied onto the surface of an agar plate. If they are attracted or repelled by an olfactory and optionally also a gustatory cue they move on the surface of the plate - either towards the cue or away from it. With such an assay olfactory cues can be detected.

This assay is for example described for the detection of cancer (US

2017016906). This assay as well as other surface chemotaxis assays can be very valuable. But their industrial applicability is limited as the

nematodes need to have a suitable environment and the assay set up is consequently complicated. It would be favorable if the complexity of the assay set up could be reduced.

Brief description of the Invention

It was found that one aspect of the assay set up for chemotaxis assays for detecting olfactory cues can be greatly reduced in complexity. At present the nematodes are put on agar plates for the assay as it is known that they can move on the surface of the agar. It was found that instead of agar any porous, contiguous, wettable support can be used for the assay. As long as the support has a surface that provides enough liquid to the nematode and is three-dimensionally connected it is suitable. This avoids the use of agar which typically needs to be freshly prepared.

The present invention is thus directed to a method for performing a nematode chemotaxis assay for detecting olfactory cues whereby the nematodes are put on the surface of a non-agar porous, contiguous wet support and typically after application onto said surface also move along said surface while being repelled or attracted by an olfactory cue.

In a preferred embodiment, the support is a natural, non-agar gel, a non- natural gel, a filter or a membrane.

In a very preferred embodiment, the support is made of or comprises gellan gum or carrageenan.

In another very preferred embodiment, the support is a filter, especially a membrane, like a cellulose-based filter membrane.

In another embodiment, the support is wetted with an aqueous liquid prior to application of the nematodes by putting it on a wetted sponge.

In another embodiment, the assay is performed by

a) Providing a porous, wet non-agar support

b) Applying the nematodes and the one or more samples to the

support, typically to one or more certain positions on the surface of the support.

c) Incubating the support

d) Detecting the nematodes on the surface of the support during or after the incubation of step c).

Typically, in step d) the position of the nematodes on the support is detected. In a preferred embodiment the nematode is C. elegans.

In another preferred embodiment the movement of the nematodes is induced by an olfactory cue resulting from the sample.

Description of the invention

Figure 1 shows a general format that can be used in a chemotaxis assay according to the invention. It is also the format used in the Examples. The flat support 1 is in the base part of a box 2 (the cover is not shown but closed during performance of the assay). The nematodes are applied to the middle of the support indicated with an N and a dotted line which divides the support into two halves, one half called "+", the other half called The sample is applied close to the edges on one half (the "+" half) of the support. Suitable positions for application of the sample, in this case two identical samples, are indicated with S. If the nematodes are attracted by the olfactory cue of the sample they will move towards the sample and into the "+" - half of the support in which the sample is positioned (left of the dotted line in Figure 1 ). Nematodes that are repelled will move away from the sample towards the other half of the support - the "-" half. If the nematodes do not react to the sample they will spread equally over both halves of the support. The same type of assay can of course be performed with other formats that e.g. have another shape, another position of the sample or the roundworms etc. Further details about the Figures can be found in the Examples.

The gelling fraction in agar is the polysaccharide agarose which forms the supporting structure in the cell walls of certain species of algae, and which is released on treatments like boiling. These algae are known as

agarophytes and belong to the Rhodophyta (red algae) phylum. Agar is actually the resulting mixture of two components: the linear polysaccharide agarose, and a heterogeneous mixture of generally smaller, generally more charged molecules called agaropectin. Non-agar means that the support is not made of agar, more precisely it does not comprise more than 25% by weight of agarose and/or agaropectin, preferably not more than 10%. Most preferred the support does not comprise any agarose and/or agaropectin.

Carrageenans are large, highly flexible molecules that curl forming helical structures. This gives them the ability to form a variety of different gels at room temperature. They are widely used in the food and other industries as thickening and stabilizing agents. Carrageenans are also extracted from seaweeds like agar but are different from agar.

All carrageenans are high-molecular-weight polysaccharides made up of repeating galactose units and 3,6 anhydrogalactose (3,6-AG), both sulfated and nonsulfated. The units are joined by alternating a-1 ,3 and β-1 ,4 glycosidic linkages.

There are three main commercial classes of carrageenan:

• Kappa forms strong, rigid gels in the presence of potassium ions; it reacts with dairy proteins.

• lota forms soft gels in the presence of calcium ions.

• Lambda does not gel, and is used to thicken dairy products.

The primary differences that influence the properties of kappa, iota, and lambda carrageenan are the number and position of the ester sulfate groups on the repeating galactose units. Higher levels of ester sulfate lower the solubility temperature of the carrageenan and produce lower strength gels, or contribute to gel inhibition (lambda carrageenan).

Gellan gum is a water-soluble anionic polysaccharide produced by the bacterium Sphingomonas elodea. The repeating unit of the polymer is a tetrasaccharide, which consists of two residues of D-glucose and one of each residues of L-rhamnose and D-glucuronic acid. Gellan gum is commercially available as GELRITE ®, Nanogel-TC,"Gelrich" Grovgel, AppliedGel or Phytagel. Nanocellulose is a very clear gel. It can be made of cellulose nanofibers, also called microfibrillated cellulose, or nanocrystalline cellulose or bacterial nanocellulose. Gelatine is a mixture of peptides and proteins produced by partial hydrolysis of collagen typically extracted from the skin, bones, and connective tissues of animals such as domesticated cattle, chicken, pigs, and fish. Nata de coco (also marketed as "coconut gel") is a gel produced by the fermentation of coconut water. It gels through the production of microbial cellulose by Acetobacter xylinum.

A support is a three-dimensional porous network that has a surface with pores not larger than the width of the worm in diameter. It can be a gel or a solid three-dimensional body like a filter or a membrane. To be suitable for the present invention, the support needs to be contiguous. That means it comprises a three-dimensional, interconnected, cross-linked network. It is not made by paste or slurry of particles which can change their position and flow within the paste or slurry. The support according to the invention comprises a three-dimensional network, e.g. made by chemically linked molecules or particles. Though the support might still be flexible like a gel, the support is preferably made of one piece. Wet or wettable support means that at least the surface of the support is or can be put in a state where its surface if lightly pressed with a dry piece of thin paper tissue would visibly wet said filter paper. The aqueous liquid which wets the filter paper can e.g. be present as a film at least on the surface of the support, in the form of droplets at least on the surface of the support, in the pores of the support and/or like in a gel. Preferably, the support is like a gel or a sponge which is soaked with liquid and provides part of the liquid to the surface. Also in case of the support being a filter the support itself may behave as its own water reservoir without the need for a further reservoir.

A gel is a solid or semi-solid jelly-like material that can have properties ranging from soft and weak to hard and tough. Gels are defined as a substantially dilute cross-linked system, which exhibit no or minimum flow when in the steady-state. By weight, gels are mostly liquid, yet they behave like solids due to a three-dimensional cross-linked network within the liquid. It is the crosslinking within the fluid that gives a gel its structure (hardness) and contributes to the adhesive stick. In this way gels are a dispersion of molecules of a liquid within a solid in which liquid particles are dispersed in the solid medium. Gels consist of a solid three-dimensional network that spans the volume of a liquid medium and ensnares it through surface tension effects. This internal network structure may result from physical bonds (physical gels) or chemical bonds (chemical gels), as well as crystallites or other junctions that remain intact within the extending fluid. The fluid that is used as an extender in the application according to the present invention is water or an aqueous liquid so that so-called hydrogels are formed.

A sample is any substance or mixture that shall be tested. A sample can be a pure chemical compound or a mixture of compounds. Preferred samples are food samples, environmental samples and especially body samples. Body samples are blood, tissue samples and urine. The type of sample might depend on the purpose of the assay. If a cancer test is performed the sample is typically a body sample. If the response of a nematode to a certain compound or mixture of compounds shall be evaluated, the sample is typically said compound or a mixture assumed to comprise said compound. The sample can be applied to the support in any format, like e.g. in dry, liquid or gas format. Up to now agar was regarded as the standard medium and support for performing a surface chemotaxis assay with nematodes in which an olfactory cue induces movement of the nematodes. It has been found that it is not necessary to use agar for this application. It is sufficient to provide a wet support on which the nematodes can move. The support according to the present invention is wet, planar (which means planar at least compared to the size of the worms) and porous whereby the pores should be in a range so that the nematodes can move along the surface of the support without being distracted by the pores and that the pores should be at least as big such that water can nevertheless flow through the said pores.

Preferably the pores have a diameter below the width of the worm, preferably below one fifth of the width. Suitable pore sizes are for example between one five hundredth and one tenth of the width of the worm.

Consequently, the pores have typically a diameter below 50 μιτι, more preferably below 10 μιτι.

In one embodiment, the support is a non-agar gel. The gel can be a natural or a synthetic, non-natural gel.

Examples of natural gels are carrageenan, gellan gum, nanocellulose, gelatine and nata de coco.

Examples of synthetic, non-natural gels are polyacrylamide,

polyvinylalcohol, polyvinyl pyrrol idon and cellulose ethers. The texture of the gel is such that the nematodes can move on the surface of the gel without sinking into the gel. A person skilled in the art is able to prepare the gels so that they have a suitable texture.

In another embodiment the support is a porous three-dimensional body which keeps its shape also in dry state (in contrast to a gel). The three- dimensional body has at least one planar surface. Also the pores of this body are preferably below the width of the worm, as discussed in detail above. The porous body can be made of any material, like metals, inorganic materials like metal oxides, e.g. silica; preferably it is made of organic polymers like polyolefins, polysulfones, polyesters, polyvinyls, polyacryls, silicones, cellulose and cellulose derivatives, PTFE

(polytetrafluorethylene) or paper.

Examples of suitable porous three-dimensional bodies are filters, especially membranes, e.g. cellulose based filters or filter membranes like cellulose paper filters as well as membranes or filters made of or comprising cellulose nitrate and/or cellulose acetate like cellulose acetate/cellulose nitrate mixed esters. Suitable supports are e.g. cellulose based Whatman® filters or filter membranes.

The support typically has a length and width between 1 cm and 20 cm. Its depth typically ranges between 1 mm and several centimetres, preferably, between 1 mm and 10 mm. Typically the support is a flat sheet with square or round shape.

In one embodiment the support is coloured other than white, e.g. red or black, so that the nematodes contrast against the support. The support may also comprise fluorescent or phosphorescent components which, e.g. upon inducement, improve the visualisation and detection of the nematodes.

An aqueous solution or an aqueous liquid is a liquid based on water or any aqueous liquid like a liquid growth medium or buffers. The aqueous solution might comprise up to 20% of a water soluble organic solvent like ethanol. But preferably the aqueous solution does not contain any organic solvent(s). A preferred aqueous solution according to the present invention is water, a buffer comprising salts of phosphate, sodium and/or potassium together with calcium and/or magnesium and may contain a surface active agent(s) like a protein/peptide and/or a detergent-like materials such as Triton-X derivatives or a liquid NGM (nematode growth medium) medium. A suitable NGM medium comprises for example around 3.0 g/L sodium chloride, around 2.5 g/L peptone and 0.005 g/L cholesterol. Such buffers and solutions are physiologically compatible with the worms and shall have a composition so that the nematodes are minimally adversely perturbed.

For performing the assay according to the present invention, a wet porous, contiguous, non-agar support is provided. If it is in form of a gel, a wetting step is typically not necessary as the gel already comprises enough aqueous solution. If the support is not wet, yet, it is wetted by contacting it with an aqueous solution. This can be done by partly or completely immersing the support in the aqueous liquid, pouring or spraying liquid over the support or any other suitable means. Afterwards, typically, excess aqueous solution is removed by letting the water drip off, optionally by slight shaking of the support.

In one embodiment, the support is wetted by placing it onto a wet sponge. A sponge is any medium which is able to provide liquid to the support when it is contacted with the support. A sponge might be a gel, a wet slurry of particles, or a porous three-dimensional cellulose based or polymer network. When using a sponge to wet the support, typically, the wet sponge is put in a box, the support, in this case preferably a flat porous sheet, like a filter paper or a membrane, is placed onto the sponge so that the support is completely wetted. By amending the type of support, its thickness and the amount of water with which it is wetted, the amount of water that is present on the surface of the support can be amended to have an amount that is optimal for the nematodes to dwell and move. By using a sponge to wet the support, the amount of water that is available in and onto the support can be modulated and then kept very precisely.

It is also possible to put the dry support on the dry sponge and wet them afterwards by applying a certain amount of an aqueous solution. The support is preferably placed in a box for performing the assay, either before or after the optional wetting step. The box can be any closable vessel. Typically the box has a base and a cover. During execution of the assay, as the assay is based on the nematodes detecting an odour, the box should be closed as otherwise the odour gradient which forms could be disturbed by external influences.

Preferably, the box is made of transparent plastic material. Very preferred, the box is a petri dish or similar to a petri dish.

Inoculation of the support with the sample or samples to be tested and the nematodes can be performed by either applying the sample and afterwards the nematodes or the other way round. In one embodiment, the nematodes are first put onto the surface of the support. Depending on the test format this can be in a certain area of the surface, e.g. in the centre of the support surface or along a line on the support or it can be randomly over the whole surface of the support. A person skilled in the art knows how to apply the nematodes. Typically, they are applied by applying a liquid comprising the nematodes and if necessary distributing them in the envisaged or required area. Excess liquid can be removed e.g. by soaking it up with a filter paper. Afterwards the one or more samples are applied. Their position on the surface of the support is also depending on the assay format.

In one format, the nematodes are put in the centre of the support surface and the typically two or more samples are put close to the edge of the support whereby they have the greatest possible distance to each other. In another embodiment, the nematodes are applied on a line dividing the surface in two halves of ideally the same size. The typically two samples are put close to the edge of the surface with the same distance to line where the nematodes are applied.

In another embodiment, the one or more samples are applied prior to or simultaneously to applying the nematodes. The nematodes used in this assay can be any nematode, either wild type or mutant. The nematode should of course be sensitive to olfactory cues. Preferably, the nematode is C. elegans. The number of nematodes to be applied onto the surface depends on the assay format. It may vary from one to 5 nematodes to up to more than 100 - 500 nematodes.

After application of the nematodes and the one or more samples, the box is preferably closed to ensure uniform conditions and the support is incubated under suitable conditions for a certain time.

Typically, the incubation is performed for a time between 1 minute and 5 hours, preferably between 15 minutes and 1 hour.

A suitable temperature is typically between 10 and 30 °C, preferably around 20 to 25 °C.

Further details about suitable nematodes like C. elegans, growing and culturing C. elegans can be found in US 2017016906 .especially in paragraphs [0099] to [0101 ].

In one embodiment, an anaesthetic or poison is additionally positioned on certain spots on the surface of the support, preferably in close proximity, preferably contacted to the sample spots. By this, the worms who have come close to the sample spot are anesthetized or killed and are hindered from moving any further and from potentially spreading the cue of the sample on their bodies to other parts of the plate. A suitable compound can be any substance or mixture of substances that hinders the worms from moving any further after contact with the compound. An example of a suitable compound is sodium azide. During the incubation of the nematodes on the surface they can move over the whole surface of the support. Depending on the effect the one or more samples have on the nematodes they might be directed to certain areas of the support surface. It has been found that all porous, contiguous, wet supports are suitable. The nematodes do not need the agar but only a wet, preferably planar surface. A person skilled in the art can choose the type of support e.g. based on its price, availability, mode of handling etc. One aspect can also be its visual appearance. Gellan gum for example is colourless and transparent. Compared to agar, it is much easier to see the nematodes on the gel.

During and/or after incubating the nematodes for a defined time, the assay is analysed. This can be done visually directly by eye or with using magnifying lenses, by taking a photo which can be analysed or by any other means that is suitable to detect the position of the nematodes on the support surface.

Unexpectedly, the nematodes do not show any negative reaction when using them for olfactory attractant repellent detection on the surfaces according to the invention. Instead of using expensive pre-made agar plates or preparing them freshly, one is free to use another support that is suitable for the assay to be performed. This can either be another gel or even just a wetted filter paper. It is even possible to adjust the properties of the support to further test requirements e.g. detectability of the nematodes by choosing a certain colour or other optic property.

The present invention is further illustrated by the following examples, however, without being restricted thereto.

The entire disclosure of all applications, patents, and publications cited above and below, especially of the European patent application EP

17198633.4, filed on October 26, 2017, are hereby incorporated by reference. Examples

1 . Breeding of Nematodes (C. elegans)

Five or six wild-type adult nematodes N2 were placed on a petri dish (containing an NGM medium, on which Escherichia coli had been dispersed), and they were then cultured at 20 degrees centigrade for 4 days. Approximately 300 to 500 next-generation nematodes were allowed to grow up to adult worms.

2. Preparation of the nematodes

To prepare the nematodes for use in an assay according to the invention 1 ml of wash buffer (M9 buffer containing 0.2% gelatin) was applied onto a nematode-breeding plate (as generated in Example 1 ), and floating nematodes, together with the buffer, were then recovered in a tube. When the thus recovered nematodes were left for a while, they sedimented to the bottom and the supernatant was discarded. Thereafter, 1 ml of wash buffer was placed into the tube, and the procedure was repeated. This washing operation was repeated three times to remove Escherichia coli and small worms.

3. Chemotaxis assay (general format)

The assay is performed in a 9-cm petri dish comprising the support, a schematic view of the assay format is shown in Figure 1 , and 0.5 micro I each of sodium azide (NaN3) is then placed on each of four spots on the wet, porous surface in the petri dish. The four spots are the two spots marked with "S" where also the sample is applied to as well as two spots with an identical position on the other half of the plate (mirrored).

1 μΙ of sample is placed on the "S" marks on the support on two adjacent spot positions directly onto the sodium azide spots (see Figure 1 ). Subsequently, approximately 100 nematodes are placed on the center line of the support (dotted line N in Figure 1 ), and the nematodes are allowed to move for 30 minutes. The temperature is set at 23 degrees centigrade plus or minus 1 degrees centigrade

Thirty minutes later, the number of nematodes on the + half and the number of nematodes on the - half are counted. Chemotaxis indices are calculated according to US 2017/016906.

All analyses are carried out two to five times. The results shown are the mean values resulting from the analyses.

4. Performance of chemotaxis assay

The chemotaxis assay according to Example 3 is performed with the known C. elegans attractant IAA (isoamyl alcohol - at a concentration of 10^"4 or 10^~ ³ (v/v) in pure ethanol). The control spots receive the same volume, 1 micro liter, of pure ethanol without attractant or repellant.

The supports used are 10 mL of a 2% dilution of the following hydrocolloids each containing M9 buffer:

a) Agar

b) Gellan gum (Gelrite®)

c) Carrageenan

d) Agar, as above to which cellulose nitrate filters (Whatman ® 10-400212) have been placed on the surface (note the filters were previously briefly wetted by contact with M9 buffer)

e) Polyacrylamide Gel, Invitrogen NuPAGE ® Tris Acetate Mini Gel NP 0323PK2. The gel was removed from its packaging and carefully placed in a beaker with 50 mL M9 buffer without Gelatine, then occasionally swirled to allow diffusion out of the chemicals in the gel for 30 mins. Then the solution was decanted and new M9 buffer added and the process repeated. Then the gel was removed and carefully cut to fit a 9cm Petri dish. Then the gel was dried using a Kim wipe to remove excess fluid. Then the gel was dried in the air until the surface was almost dry. Then the test was performed as usual,

f) Gelatine,„cook gelatine" purchased from Morinaga & Co Ltd., Japan.

Solid Gelatine was dissolved in water at a temperature of 80°C.

The results of these assays are shown in Figure 2/Table 1 . All of the responses are clearly positive compared to a neutral response of zero.

Claims

1 A method for performing a nematode chemotaxis assay whereby the nematodes are put of the surface of a non-agar porous, contiguous, wet support.

2. Method according to claim 1 , whereby the support is a natural, non-agar gel, a non-natural gel, a filter or a membrane.

3. Method according to claim 1 or 2, whereby the support comprises gellan gum or carrageenan.

4. Method according to claim 1 or 2 whereby the support is a filter

membrane.

5. Method according to one or more of claims 1 to 4, whereby the support is wetted with an aqueous solution prior to application of the nematodes

6. Method according to claim 5, whereby the support is wetted by putting onto a wet sponge.

7. Method according to one or more of claims 1 to 6, whereby the assay is performed by

a) Providing a porous, wet non-agar support

b) Applying the nematodes and the one or more samples to the surface of the support

c) Incubating the support

d) Detecting the position of the nematodes on the surface of the

support.

8. Method according to one or more of claims 1 to 7, whereby the

nematode is C. elegans.

9. Method according to one or more of claims 1 to 8, whereby the movement of the nematodes is induced by an olfactory cue coming from the sample.

10. Method according to one or more of claims 1 to 9, whereby the support is wetted with an aqueous solution comprising phosphate and sodium and/or potassium together with calcium and/or magnesium ions prior to application of the nematodes.

1 1 . Method according to one or more of claims 1 to 10, whereby in step c) the support is incubated in a closed box.