Compositions and methods for characterizing microbiome

Abstract

To provide a system for characterizing microbial strains of a mammalian microbiome and determining whether such microbial strains affect a mammalian disease or disorder.SOLUTION: The present invention provides a system comprising a plurality of C. elegans cultures, wherein each culture comprises a transgenic C. elegans strain that models a mammalian disease or condition. Using the system described herein, it is possible to characterize relationships and / or effects of microbial preparations (e.g., of a microbiome sample) with certain diseases or conditions in target subjects.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 899,718, filed September 12, 2019, and U.S. Provisional Patent Application No. 62 / 988,132, filed March 11, 2020, the entire contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Caenorhabditis elegans is a bacteriovorus nematode that is approximately 1 mm in length and lives in temperate soil environments. Summary of the Invention

[0003] The present disclosure provides insight that C. elegans may provide and / or represent a surprisingly useful system for assessing one or more characteristics of a microbial preparation (e.g., of a microbiome sample). Among other things, the present disclosure describes techniques that may be useful for assessing a microbiome sample to identify or characterize the influence and / or regulation of the microbial strains of such a microbiome sample on a particular disease or condition. In some embodiments, such techniques may be useful for identifying strain-level differences in a particular patient or patient population. Thus, the present disclosure also provides techniques that may be useful for assessing the nature of microbial strains in a patient-specific sample, and thus for providing patient-specific information about how an individual patient's microbiome differentially impacts their health status. For example, in some embodiments, the techniques provided herein may be useful for identifying a disease or condition to which a patient may be susceptible based on the nature of the microbial strains in the patient-specific sample. In some embodiments, the techniques provided herein may be useful for identifying microbial strains in a patient that are beneficial, for example, for protecting the patient from or conferring resistance to a particular disease or condition. Thus, the techniques described herein are useful as diagnostic tools for screening microbiome samples (e.g., human microbiome samples) for disease-modifying agents (e.g., microbial strains that affect a disease or pathology).

[0004] Indeed, in certain embodiments, the transgenic C. elegans whole animal model system is used to identify or screen for microbiome strains (e.g., present in the human microbiome) that may modulate or influence the pathogenesis and / or development of a neurodegenerative disease or condition (e.g., Alzheimer's disease). Upon reviewing this disclosure, one of skill in the art will understand that the technology described herein is applicable not only to uses in connection with neurodegenerative diseases such as Alzheimer's disease, as exemplified, but also to uses in connection with a variety of other diseases or conditions that may be associated with the microbiome, such as, but not limited to, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis, type II diabetes, obesity, hyperglycemia, impaired glucose tolerance, insulin resistance (i.e., hyperinsulinemia, metabolic syndrome, syndrome X), hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia (e.g., dyslipidemia), hypertriglyceridemia, cardiovascular disease, atherosclerosis, peripheral vascular disease, kidney disease, ketoacidosis, thrombotic disorders, nephropathy, diabetic neuropathy, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, glaucoma, sexual dysfunction, dermatopathy, dyspepsia, hypoglycemia, metabolic syndrome, cancer, or edema.

[0005] In some aspects, provided herein are systems comprising a plurality of C. elegans cultures, each culture comprising a transgenic C. elegans strain that models a disease or condition (e.g., a mammalian disease or condition). In some embodiments, such a plurality of C. elegans cultures may comprise at least five or more (e.g., including at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, or more) C. elegans cultures.

[0006] In some embodiments, one or more of the C. elegans cultures in the provided systems comprise a transgenic C. elegans strain that models a disease or condition present in a target subject. In some embodiments, one or more of the C. elegans cultures in the provided systems may comprise a transgenic C. elegans strain that models a mammalian disease or condition (e.g., a human disease or condition). Exemplary human diseases or conditions modeled by C. elegans strains may include neurodegenerative diseases or disorders (e.g., Alzheimer's disease). In some embodiments, the disease or condition (e.g., a human disease or condition) modeled by C. elegans strains may include diseases or conditions associated with an altered or defective HIF-1 pathway (which may include, for example, a cellular stress response). In some embodiments, for example, the disease or condition modeled by C. elegans may be an intraocular neovascular disease or disorder (e.g., diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, or glaucoma). In some embodiments, one or more of the C. elegans cultures in the provided systems may comprise transgenic C. elegans that model a non-human mammalian disease or condition, e.g., a canine, feline, equine, bovine, ovine, caprine, or porcine disease or condition.

[0007] In some embodiments, the transgenic C. elegans strains provided in the systems described herein may include a transgene comprising a characteristic sequence element associated with a target disease or condition (e.g., a mammalian disease or condition). Such a characteristic sequence element associated with a disease or condition (e.g., a mammalian disease or condition) may be or may include a foreign gene (e.g., a mammalian gene), a DNA regulatory element (e.g., a mammalian DNA regulatory element), and / or a mammalian RNA regulatory element. A variety of DNA and RNA regulatory elements are known in the art; thus, one of skill in the art will understand that in some embodiments, a DNA regulatory element associated with a disease or condition (e.g., a mammalian disease or condition) may be or may include an enhancer, promoter, silencer, insulator, locus control region, and combinations thereof. In some embodiments, RNA regulatory elements associated with a disease or condition (e.g., a mammalian disease or condition), such as, for example, untranslated regions, introns, splice sites, and combinations thereof, may be used in accordance with the present disclosure.

[0008] Additionally or alternatively, the transgenic C. elegans strains in the provided systems may contain transgenes, including reporter genes, non-limiting examples of which may include, but are not limited to, fluorescent, phosphorescent, and / or bioluminescent proteins.

[0009] In some embodiments involving C. elegans cultures, at least two or more of such cultures may each comprise a transgenic C. elegans strain that models the same disease or condition (e.g., a mammalian disease or condition). In some such embodiments, at least two or more of such cultures may each comprise a transgenic C. elegans strain that models a different biochemical or molecular pathway associated with the same disease or condition (e.g., a mammalian disease or condition). In some embodiments involving C. elegans cultures, all of such cultures may each comprise the same transgenic C. elegans strain that models the same disease or condition (e.g., a mammalian disease or condition).

[0010] In some embodiments involving C. elegans cultures, at least two or more of such cultures may each comprise a transgenic C. elegans strain that models a different disease or condition (e.g., a mammalian disease or condition).

[0011] The systems described herein can be used to characterize the relationship and / or effect of a microbial preparation (e.g., a microbiome sample) with a particular disease or condition in a target subject. Thus, in some embodiments, one or more C. elegans cultures in such systems comprise microorganisms of a mammalian microbiome (e.g., a human microbiome). In some embodiments, each of such C. elegans cultures may comprise microorganisms of a mammalian microbiome (e.g., a human microbiome). In some embodiments, one or more C. elegans cultures in such systems may comprise microorganisms of a canine, feline, equine, bovine, ovine, caprine, or porcine microbiome. Microbiomes used in accordance with the present disclosure may be obtained or derived from a targeted anatomical site in a mammalian subject. Examples of such microbiomes may include, but are not limited to, a skin microbiome, an oral microbiome, a nasal microbiome, a gastrointestinal microbiome, a brain microbiome, a lung microbiome, and / or a urogenital microbiome.

[0012] In some embodiments, the microorganisms in each C. elegans culture may comprise one or more microbial strains, hi some embodiments, the microorganisms in each culture may comprise a single microbial strain.

[0013] In some embodiments, one or more of the C. elegans cultures may constitute a therapeutic agent or nutritional supplement.

[0014] Also provided herein are methods for characterizing a microbiome using multiple C. elegans cultures. In some embodiments, the methods are for screening an individual's (e.g., mammalian, e.g., human) microbiome to determine whether a microbial strain or combination of microbial strains affects a mammalian disease or disorder. In some embodiments, the methods are for diagnosing an individual (e.g., mammalian, e.g., human) based on one or more microbial strains in the individual's microbiome. In some embodiments, the methods are for monitoring disease or pathological progression in an individual (e.g., mammalian, e.g., human) based on one or more microbial strains in the individual's microbiome.

[0015] In some embodiments described herein, a method is provided that includes adding microorganisms obtained from a mammalian microbiome to each of the C. elegans cultures of the system described herein. In some embodiments, the microorganisms in each C. elegans culture may comprise one or more microbial strains. In some embodiments, the microorganisms in each such culture may comprise a single microbial strain. The microbiome used in the methods described herein may be obtained or derived from a target anatomical site of a mammalian subject. Examples of such microbiomes may include, but are not limited to, a skin microbiome, an oral microbiome, a nasal microbiome, a gastrointestinal microbiome, a brain microbiome, a lung microbiome, and / or a urogenital microbiome.

[0016] In some embodiments, the method may include adding multiple microbial strains of a mammalian microbiome to multiple C. elegans cultures, where a different microbial strain is added to each C. elegans culture, and where each culture contains the same transgenic C. elegans strain, and where the transgenic C. elegans strain is a model for a mammalian disease or condition.

[0017] In some embodiments, one or more of the C. elegans cultures may comprise a therapeutic agent or nutritional supplement. Thus, in some embodiments, the described methods may further comprise adding a therapeutic agent or nutritional supplement to one or more of the C. elegans cultures.

[0018] In some embodiments, the methods described herein may further include determining one or more parameter values of the transgenic C. elegans strain in each of the C. elegans cultures. In some embodiments, such parameter(s) of the transgenic C. elegans are associated with the mammalian disease or pathology modeled by the transgenic C. elegans strain. Exemplary such parameters of the transgenic C. elegans may include the biological function or phenotype and / or level and / or activity of a molecule (e.g., a small molecule, protein, polypeptide, or transcript) associated with the mammalian disease or pathology.

[0019] In some embodiments, the method may further include (a) determining one or more parameter values of a transgenic C. elegans strain in a C. elegans culture before adding the microbial strain to the culture; (b) determining the same one or more parameter values of a transgenic C. elegans strain in the C. elegans culture after adding the microbial strain to the C. elegans culture; and (c) comparing the one or more parameter values determined before adding the microbial strain with the one or more parameter values determined after adding the microbial strain.

[0020] In some embodiments, the techniques described herein can be used to characterize microbial strains in the human biome that are associated with a human disease or condition. Thus, in some such embodiments, the transgenic C. elegans strains involved in the systems and methods described herein model a human disease or condition. An exemplary human disease or condition modeled by the transgenic C. elegans strain is Alzheimer's disease. In some embodiments, the disease or condition (e.g., human disease or condition) modeled by the transgenic C. elegans strain may include a disease or condition associated with an altered or defective HIF-1 pathway (which may include, for example, a cellular stress response). In some embodiments, for example, the disease or condition modeled by the transgenic C. elegans strain may be an intraocular neovascular disease or disorder (e.g., diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, or glaucoma). In some embodiments, the disease or condition that the transgenic C. elegans strain models is diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, or glaucoma.

[0021] Thus, certain aspects described herein relate to techniques for characterizing microbial strains in the human biome. For example, one aspect provides a method comprising: (a) adding a microbial strain to a C. elegans culture, including a transgenic C. elegans strain that models Alzheimer's disease; and (b) determining whether the microbial strain affects one or more parameters of the transgenic C. elegans strain, wherein the one or more parameters are associated with Alzheimer's disease. In some embodiments, the transgenic C. elegans strain may include a transgene encoding human ssApoE4 protein, human Aβ1-42 polypeptide, or human pseudophosphorylated tau protein.

[0022] In some embodiments, the method further comprises: (a) determining one or more parameter values of a transgenic C. elegans strain in a C. elegans culture before adding the microbial strain to the culture; (b) determining the same one or more parameter values of a transgenic C. elegans strain in the culture after adding the microbial strain to the C. elegans culture; and (c) comparing the one or more parameter values determined before adding the microbial strain with the one or more parameter values determined after adding the microbial strain. Exemplary such one or more parameters include, but are not limited to, (i) a level of C. elegans paralysis, (ii) amyloid plaque levels, (iii) tau fibril levels, (iv) neuroinflammation levels, (v) proteasome function levels, and / or (vi) combinations thereof.

[0023] In another aspect, the present disclosure provides a method for characterizing a microbial strain in the human biome associated with an altered or defective HIF-1 pathway in one or more parameters, comprising: (a) adding the microbial strain to a C. elegans culture, including a transgenic C. elegans strain that models a disease or condition associated with an altered or defective HIF-1 pathway; and (b) determining whether the microbial strain affects one or more parameters of the transgenic C. elegans strain. In some embodiments, such a transgenic C. elegans strain may comprise a transgene encoding human prolyl hydroxylase EGLN, a human HIF transcription factor, or a human HIFα protein.

[0024] In some embodiments, such methods further include (a) determining one or more parameter values of the transgenic C. elegans strain in culture before adding the microbial strain to the C. elegans culture; (b) determining the same one or more parameter values of the transgenic C. elegans strain in culture after adding the microbial strain to the C. elegans culture; and (c) comparing the one or more parameter values determined before adding the microbial strain with the one or more parameter values determined after adding the microbial strain.

[0025] Exemplary such one or more parameters include, but are not limited to, (i) the level of neuroinflammation, (ii) the level of proteasome function, (iii) the level of C. elegans egg laying rate, and / or (iv) a combination thereof.

[0026] Also within the scope of this disclosure are transgenic C. elegans strains expressing two or more of: (i) human ssApoE4, (ii) human Aβ1-42, (iii) human pseudophosphorylated tau, and (iv) UbV-GFP proteasome marker.

[0027] The present disclosure also describes, inter alia, the use of the provided transgenic C. elegans strains, systems, and / or methods to screen mammalian microbiomes for microbial strains that affect mammalian diseases or pathologies. Also included within the scope of the present disclosure is the use of the provided C. elegans, systems, and / or methods to characterize the impact that microbial strains in a mammalian microbiome have on mammalian diseases or pathologies. For example, the human microbiome can be screened / characterized in accordance with the present disclosure using the techniques provided herein.

[0028] The present disclosure describes, among other things, compositions comprising one or more microbial strains. In some embodiments, provided herein are compositions comprising one or more microbial strains, extracts thereof, and / or components thereof from a mammalian microbiome assessed, identified, characterized, or assayed using transgenic C. elegans or methods as described herein. In some embodiments, provided herein are compositions comprising two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more microbial strains, extracts thereof, and / or components thereof from a mammalian microbiome assessed, identified, characterized, or assayed using transgenic C. elegans or methods as described herein.

[0029] In some embodiments, provided herein are compositions comprising two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more microbial strains listed in Table 8 below.

[0030] In some embodiments, provided herein are compositions comprising Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., L. plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium, Bacillus subtilis, Acidaminococcus sp., or a combination thereof. In some embodiments, the combination includes at least two of Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., L. plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium, Bacillus subtilis, and Acidaminococcus sp., at least three of them, at least four of them, at least five of them, at least six of them, at least seven of them, at least eight of them, at least nine of them, or all of them.

[0031] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is an ingestible item.

[0032] The present disclosure describes, inter alia, methods that include administering the compositions described herein.

[0033] In some embodiments, a method of treating a disease or condition in a subject comprises administering a composition as described herein to a subject in need thereof. In some embodiments, the disease or condition is a neurodegenerative disease or disorder. In some embodiments, the disease or condition is Alzheimer's disease. In some embodiments, the disease or condition may be associated with an altered or defective HIF-1 pathway. In some embodiments, the disease or condition may be an intraocular neovascular disease or disorder. In some embodiments, the disease or condition is diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, or glaucoma. The present disclosure describes, inter alia, the use of the compositions described herein. In some embodiments, the use of the compositions as described herein is in the treatment of a disease or condition in a subject. In some embodiments, the disease or condition is a neurodegenerative disease or disorder. In some embodiments, the disease or condition is Alzheimer's disease. In some embodiments, the disease or condition may be associated with an altered or defective HIF-1 pathway. In some embodiments, the disease or condition can be an intraocular neovascular disease or disorder, hi some embodiments, the disease or condition is diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, or glaucoma.

[0034] These and other aspects encompassed by the present disclosure are further detailed below and in the claims.

[0035] definition The scope of the present invention is defined by the claims appended hereto, and is not limited by any particular embodiment described herein. Upon reviewing this specification, one of ordinary skill in the art will recognize various modifications that may be equivalent to such described embodiments or that may otherwise be within the scope of the claims. Generally, terms used in this specification follow their understood meanings in the art unless expressly indicated otherwise. Explicit definitions of certain terms are provided below, but the meaning of these and other terms in specific instances throughout this specification will be clear to those of ordinary skill in the art from the context.

[0036] The use of ordinal terms such as "first," "second," "third," etc. in the claims to modify claim elements does not, in itself, imply any priority, precedence, or ordering of one claim element over another claim element, or any chronological order in which acts of a method are performed, but rather is merely used as a label to distinguish one claim element having a particular name from another element having the same name (except for the use of ordinal terms) to distinguish the claim elements.

[0037] As used herein, the articles "a" and "an" should be understood to include plural referents unless a clear indication to the contrary is present. A claim or description including "or" between one or more members of a group is deemed applicable if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless a contrary indication is present or otherwise clear from the context. In some embodiments, exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. In some embodiments, more than one or all group members are present in, employed in, or otherwise relevant to a given product or process. Unless otherwise indicated or unless a contradiction or inconsistency would be apparent to one skilled in the art, the present invention should be understood to encompass all variations, combinations, and permutations of one or more limitations, elements, clauses, descriptive language, etc. from one or more of the enumerated claims, as introduced in another claim (or any other claim, if relevant) dependent on the same base claim. When elements are presented as lists (e.g., in a Markush group or similar format), it should be understood that each subgroup of such elements is also disclosed, and that any element(s) may be removed from the group. Generally, when an embodiment or aspect is referred to as "comprising" certain elements, features, etc., it should be understood that a particular embodiment or aspect "consists of" or "consists essentially of" such elements, features, etc. For purposes of brevity, these embodiments have not been expressly and specifically defined herein in every instance. It should also be understood that any embodiment or aspect may be explicitly excluded from the scope of the claims, regardless of whether the specific exclusion is recited in the specification.

[0038] Administration: As used herein, the term "administration" typically refers to the administration of a composition to a subject or system to achieve delivery of the agent to the subject or system. In some embodiments, the agent is a composition or is contained in a composition. In some embodiments, the agent is produced through metabolism of the composition or one or more of its components. One of skill in the art will recognize various routes that may be utilized for administration to a subject, e.g., a human, under appropriate circumstances. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In certain embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, cutaneous (which may be or include, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc. In many embodiments provided by the present disclosure, administration is oral. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve the application of a number of doses. In some embodiments, administration may involve intermittent (e.g., multiple doses spaced apart) and / or periodic (e.g., individual doses spaced apart by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) over at least a selected period of time. Administration of cells may be by any suitable route that results in delivery to a desired site in a subject, where at least a portion of the delivered cells or cellular components remain viable. The period of cell viability after administration to a subject may be as short as a few hours (e.g., 24 hours), to several days, or even as long as several years (i.e., long-term engraftment). In some embodiments, administration involves delivery of a bacterial extract or preparation that contains one or more metabolic products and / or by-products of the bacteria, but is completely devoid of viable bacterial cells.

[0039] Analog: As used herein, the term "analog" refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an "analog" exhibits significant structural similarity to the reference substance, e.g., sharing a core or consensus structure, while at the same time differing in certain individual ways. In some embodiments, an analog is a substance that can be produced from a reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be produced through the implementation of a synthetic process that is substantially similar to (e.g., shares multiple steps with) the synthetic process that produces the reference substance. In some embodiments, an analog is produced, or can be produced, through the implementation of a synthetic process that is different from the synthetic process used to produce the reference substance.

[0040] Approximately: When applied to one or more values of interest, includes values similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within ±10% (more or less than) of a stated reference value, unless otherwise stated or otherwise clear from the context (except where such number exceeds 100% of possible values).

[0041] Equivalent: As used herein, the term "equivalent" refers to two or more agents, entities, states, sets of conditions, subjects, etc. that may not be identical to one another, but are sufficiently similar to permit a comparison between them, such that one of ordinary skill in the art will understand that conclusions can be reasonably drawn based on the observed differences or similarities. In some embodiments, equivalent sets of conditions, circumstances, individuals, or populations are characterized by multiple substantially identical characteristics and one or a few variable characteristics. One of ordinary skill in the art will understand, in context, the degree of identity required for two or more such agents, entities, states, sets of conditions, etc. to be considered equivalent in any given situation. For example, one of ordinary skill in the art will understand that sets of situations, individuals, or populations are equivalent to one another when they are characterized by a sufficient number and type of substantially identical characteristics to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or by different sets of situations, individuals, or populations are caused by or indicative of variations in the variable characteristics.

[0042] Conservative: As used herein, refers to the case when describing conservative amino acid substitutions, which involve replacing one amino acid residue with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the desired functional properties of a protein, such as the ability of a receptor to bind to a ligand. Examples of groups of amino acids having side chains with similar chemical properties include aliphatic side chains such as glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), and isoleucine (Ile, I); aliphatic-hydroxyl side chains such as serine (Ser, S) and threonine (Thr, T); amide-containing side chains such as asparagine (Asn, N) and glutamine (Gln, Q); aromatic side chains such as phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W); basic side chains such as lysine (Lys, K), arginine (Arg, R), and histidine (His, H); acidic side chains such as aspartic acid (Asp, D) and glutamic acid (Glu, E); and sulfur-containing side chains such as cysteine (Cys, C) and methionine (Met, M). Conservative amino acid substitutions include, for example, valine / leucine / isoleucine (Val / Leu / Ile, V / L / I), phenylalanine / tyrosine (Phe / Tyr, F / Y), lysine / arginine (Lys / Arg, K / R), alanine / valine (Ala / Val, A / V), glutamic acid / aspartic acid (Glu / Asp, E / D), and asparagine / glutamine (Asn / Gln, N / Q). In some embodiments, a conservative amino acid substitution can be the substitution of any naturally occurring residue in a protein with alanine, for example, as used in alanine scanning mutagenesis. In some embodiments, conservative substitutions are made that have a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet, GH et al., 1992, Science 256:1443-1445, which is incorporated herein by reference in its entirety. In some embodiments, the substitution is a moderately conservative substitution, and the substitution has a non-negative value in the PAM250 log-likelihood matrix.

[0043] [Table 1]

[0044] Control: As used herein, refers to the art-understood meaning of "control," a standard against which results are compared. Typically, controls are used to enhance the integrity of an experiment by isolating a variable in order to draw conclusions about that variable. In some embodiments, a control is a reaction or assay performed simultaneously with a test reaction or assay to provide a comparator. "Control" also includes "control animals." A "control animal" may have a modification as described herein, a different modification than those described herein, or no modification (i.e., a wild-type animal). In one experiment, the "test" (i.e., the variable being tested) is administered. In a second experiment, the "control," i.e., the variable being tested, is not administered. In some embodiments, a control is a historical control (i.e., a previously performed test or assay, or of a known amount or result). In some embodiments, a control is or includes a printed or otherwise kept record. A control can be a positive or negative control.

[0045] Determine, measure, evaluate, assess, assay, and analyze: Determine, measure, evaluate, assess, assay, and analyze are used interchangeably herein to refer to any form of measurement, including determining whether an element is present or not. These terms include both quantitative and / or qualitative determinations. Assays can be relative or absolute. "Assaying for the presence of" can be determining the amount of something present and / or determining whether it is present or absent.

[0046] Dosage form: Those of skill in the art will understand that the term "dosage form" can be used to refer to a physically discrete unit of an agent (e.g., a therapeutic agent) for administration to a subject. Typically, each such unit contains a predetermined amount of agent. In some embodiments, such amount is a unit dosage amount (or all of it) appropriate for administration according to a dosing regimen (i.e., a therapeutic dosing regimen) that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population. Those of skill in the art will understand that the total amount of a therapeutic composition or agent to be administered to a particular subject is determined by one or more attending physicians and may involve the administration of multiple dosage forms.

[0047] Dosage regimen: Those skilled in the art will understand that the term "dosage regimen" can refer to a set of unit doses (typically more than one) that are individually administered to a subject, typically spaced apart. In some embodiments, a given medication has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen includes multiple doses, each of which is spaced apart in time from the other doses. In some embodiments, the individual doses are spaced apart by the same length of time, and in some embodiments, a dosing regimen includes multiple doses, each of which is spaced apart by at least two different time periods. In some embodiments, all doses within a dosing regimen are of the same unit dose. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen includes a first dose at a first dose, followed by one or more additional doses at a second dose different from the first dose. In some embodiments, the dosing regimen comprises a first dose at a first dose, followed by one or more additional doses at a second dose that is the same as the first dose. In some embodiments, the dosing regimen correlates with a desired or beneficial outcome when administered across a relevant population.

[0048] Engineered: Generally, the term "engineered" refers to aspects that have been manipulated artificially. For example, a cell or organism is considered "engineered" if it has been manipulated to change its genetic information (e.g., new genetic material not previously present has been introduced, e.g., by transformation, mating, somatic hybridization, transfection, transduction, or other mechanisms, or pre-existing genetic material has been altered or removed, e.g., by substitution or deletion mutations or by breeding protocols). As a matter of convention and as will be understood by those skilled in the art, the progeny of an engineered polynucleotide or cell are typically still referred to as "engineered," even if the actual manipulation was performed on the prior entity.

[0049] Excipient: As used herein, refers to an inert (e.g., non-therapeutic) agent that can be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilization effect. In some embodiments, suitable pharmaceutical excipients can include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, etc.

[0050] Functional: As used herein, a "functional" biomolecule is a biomolecule in a form in which it exhibits a property and / or activity by which it is characterized. A biomolecule may have two functions (i.e., bifunctional) or many functions (i.e., multifunctional).

[0051] Gene: As used herein, refers to a DNA sequence in a chromosome that encodes a product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene comprises coding sequence (i.e., a sequence that encodes a particular product). In some embodiments, a gene comprises non-coding sequence. In certain embodiments, a gene may comprise both coding (e.g., exon) and non-coding (e.g., intron) sequence. In some embodiments, a gene may comprise one or more regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences that, for example, can control or influence one or more aspects of gene expression (e.g., cell-type specific expression, inducible expression, etc.). For clarity, it is noted that as used in this disclosure, the term "gene" generally refers to a portion of a nucleic acid that encodes a polypeptide or fragment thereof. As will be clear from the context to one of skill in the art, the term may optionally encompass regulatory sequences. This definition is not intended to exclude the application of the term "gene" to non-protein-coding expression units, but is intended to clarify that the term as used herein most often refers to a polypeptide-encoding nucleic acid.

[0052] Improve, increase, enhance, inhibit, or reduce: As used herein, the terms "improve," "increase," "enhance," "inhibit," "reduce," or their grammatical equivalents refer to a value that is relative to a baseline or other reference measurement. In some embodiments, the value is a statistically significant difference from the baseline or other reference measurement. In some embodiments, a suitable reference measurement may be or include a measurement in a particular system (e.g., in a single individual) under otherwise equivalent conditions except for (e.g., before and / or after) the presence of a particular agent or treatment, or in the presence of an appropriate equivalent reference agent. In some embodiments, a suitable reference measurement may be or include a measurement in an equivalent system known or expected to respond in a particular manner in the presence of the relevant agent or treatment. In some embodiments, a suitable reference is a negative reference; in some embodiments, a suitable reference is a positive reference.

[0053] Isolated: As used herein, refers to a substance and / or entity that (1) has been separated from at least some of the components with which it was associated when originally produced (whether in nature and / or in an experimental setting) and / or (2) has been designed, produced, prepared, and / or manufactured by human intervention. In some embodiments, an isolated substance or entity may be concentrated, and in some embodiments, an isolated substance or entity may be pure. In some embodiments, isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were originally associated. In some embodiments, an isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those of skill in the art, a substance may still be considered "enriched," "isolated," or even "pure" after being combined with certain other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.). In such embodiments, the isolation or percent purity of a substance is calculated without including such carriers or excipients. Those of skill in the art are aware of various techniques for isolating (e.g., concentrating or purifying) a substance or agent (e.g., using one or more of fractional distillation, extraction, precipitation, or other separations).

[0054] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those intended for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue, capsules, powders, etc. In some embodiments, the active agent may be or include a cell or cell population (e.g., a culture of, for example, an EES microorganism). In some embodiments, the active agent may be or include an extract or component of a cell or cell population (e.g., a culture). In some embodiments, the active agent may be or include an isolated, purified, or pure compound. In some embodiments, the active agent may be synthesized in vitro (e.g., via chemical and / or enzymatic synthesis), hi some embodiments, the active agent may be or include a natural product (whether isolated from its natural source or synthesized in vitro).

[0055] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable," which may be used, for example, in reference to a carrier, diluent, or excipient used to formulate a pharmaceutical composition as disclosed herein, means the carrier, diluent, or excipient is compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0056] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in carrying or transporting a compound of interest from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic, compatible substances used in pharmaceutical formulations.

[0057] Prevention: As used herein, the term "prevention" refers to delaying the onset of, and / or reducing the frequency and / or severity of, one or more symptoms of a particular disease, disorder, or condition. In some embodiments, prevention is assessed on a population basis, such that an agent is considered to "prevent" a particular disease, disorder, or condition if a statistically significant reduction in the occurrence, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition. In some embodiments, prevention may be considered complete, for example, if the onset of the disease, disorder, or condition is delayed for a predetermined period of time.

[0058] Reference: As used herein, describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially contemporaneously with the test or determination of interest. In some embodiments, the reference or control is an existing reference or control, optionally embodied in a tangible medium. Typically, as will be understood by those of skill in the art, a reference or control is determined or characterized under conditions or circumstances comparable to those being assessed. One of skill in the art will understand when there is sufficient similarity to demonstrate the reliability of and / or the validity of a comparison to a particular possible reference or control. In some embodiments, the reference is a negative control reference, and in some embodiments, the reference is a positive control reference.

[0059] Risk: As will be understood from the context, "risk" of a disease, disorder, and / or condition refers to the likelihood that a particular individual will develop the disease, disorder, and / or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or up to 100%. In some embodiments, risk is expressed as a risk relative to the risk associated with a reference sample or group of reference samples. In some embodiments, the reference sample or group of reference samples has a known risk of the disease, disorder, condition, and / or event. In some embodiments, the reference sample or group of reference samples is derived from individuals comparable to the particular individual. In some embodiments, the relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0060] Sample: As used herein, the term "sample" typically refers to an aliquot of material obtained or derived from a source of interest. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may be or include a cell or organism, such as a microorganism, a plant, or an animal (e.g., a human). In some embodiments, the source of interest is or includes a biological tissue or biological fluid. In some embodiments, the biological tissue or fluid may be or include amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, chyme, ejaculate, endolymph, exudate, stool, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural effusion, pus, mucosal secretions, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal fluid, vitreous humor, vomit, and / or combinations or component(s) thereof. In some embodiments, the biological fluid may be or include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph, and / or transcellular fluid. In some embodiments, the biological fluid may be or include plant exudates. In some embodiments, the biological tissue or sample may be obtained by, for example, aspiration, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing, or lavage (e.g., bronchoalveolar epithelial, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from the intended source by any suitable means. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample), for example, filtration using a semipermeable membrane.Such a "processed sample" may include, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques, such as nucleic acid amplification or reverse transcription, isolation and / or purification of certain components, etc.

[0061] Small molecule: As used herein, the term "small molecule" refers to a small organic or inorganic molecule having a molecular weight below about 3,000 Daltons. Generally, a small molecule may have a molecular weight less than 3,000 Daltons (Da). A small molecule can be, for example, at least about 100 Da to about 3,000 Da (e.g., about 100 to about 3,000 Da, about 100 to about 2,500 Da, about 100 to about 2,000 Da, about 100 to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about 1,250 Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1,500, about 500 to about 1,000, about 300 to about 1,000 Da, or about 100 to about 250 Da).

[0062] Subject: As used herein, the term "subject" refers to an individual to whom the provided treatment is applied. In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal, e.g., a mammal experiencing or susceptible to a disease, disorder, or condition as described herein. In some embodiments, the animal is a vertebrate, e.g., a mammal such as a non-human primate (especially a higher primate), sheep, dog, rodent (e.g., a mouse or rat), guinea pig, goat, pig, cat, rabbit, or cow. In some embodiments, the animal is a non-mammal, such as a chicken, amphibian, reptile, or the invertebrate model C. elegans. In some embodiments, the subject is a human. In some embodiments, the patient is suffering from or susceptible to one or more diseases, disorders, or conditions as described herein. In some embodiments, the patient exhibits one or more symptoms of one or more diseases, disorders, or conditions as described herein. In some embodiments, the patient has been diagnosed with one or more diseases, disorders, or conditions as described herein. In some embodiments, the subject is undergoing or has undergone a particular therapy for diagnosing and / or treating a disease, disorder, or condition, hi other embodiments, the subject is an experimental animal or surrogate animal as a disease model.

[0063] Substantially: As used herein, refers to a qualitative state of exhibiting the full or nearly full extent or degree of a desired property or characteristic. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or completeness, or achieve or avoid absolute results. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0064] Therapeutic regimen: "Therapeutic regimen," as that term is used herein, refers to a dosing regimen, the administration of which across a relevant population can be correlated with a desired or beneficial therapeutic outcome.

[0065] Therapeutically effective amount: As used herein, it refers to an amount that produces the desired effect for which it is administered. In some embodiments, the term refers to an amount that, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition according to a therapeutic dosing regimen, is sufficient to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of one or more symptoms of the disease, disorder, and / or condition and / or delays its onset. Those skilled in the art will appreciate that the term "therapeutically effective amount" does not actually require that successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be an amount that, when administered to patients in need of such treatment, provides a specific, desired pharmacological response in a significant number of subjects. In some embodiments, reference to a therapeutically effective amount may be a reference to the amount as measured in one or more specific tissues (e.g., tissues affected by a disease, disorder, or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those skilled in the art will appreciate that in some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and / or administered in a single dose, while in some embodiments, a therapeutically effective agent may be formulated and / or administered in multiple doses, for example, as part of a dosing regimen.

[0066] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to any application of therapy that partially or completely alleviates, improves, relieves, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be treatment of a subject who does not exhibit symptoms of the relevant disease, disorder, and / or condition and / or who exhibits only early signs of the disease, disorder, and / or condition. Alternatively, or additionally, such treatment may be treatment of a subject who exhibits one or more established symptoms of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be treatment of a subject who has been diagnosed with the relevant disease, disorder, and / or condition. In some embodiments, treatment may be treatment of a subject known to have one or more susceptibility factors statistically correlated with an increased risk of developing the relevant disease, disorder, and / or condition. [Brief explanation of the drawings]

[0067] [Figure 1] Data are presented showing evidence that APOE4 protein enhances the Aβ-induced paralysis phenotype in C. elegans. C. elegans animals with the indicated genotypes were administered a standard E. coli OP50 laboratory strain. Animals were monitored every other day from adulthood day 1 until all animals were paralyzed. At least 20 animals were recorded for each assay. Data from three independent trials are presented in the plots. For each data point, the mean ± standard deviation is presented on the graph. [Figure 2]Data are presented showing evidence that expression of Aβ3-42 conjugated to green fluorescent protein (GFP) and human ssAPOE4 significantly increased the number of aggregates. C. elegans animals with the indicated genotypes were administered a standard E. coli OP50 laboratory strain. GFP aggregates in the anterior region of the animals were counted when the animals reached adulthood. At least 17 animals were scored for each assay. For each data point, the mean ± standard deviation is presented in the bar graph. Compared with animals expressing Aβ3-42, animals expressing Aβ3-42 and ssApoE4 had significantly increased GFP aggregates when analyzed using a Student's t-test (P<0.0001). [Figure 3] Data are shown showing evidence that UbV-GFP is stabilized in animals expressing both human ssAPOE4 and human tau352 (PHP). C. elegans animals with the indicated genotypes were administered a standard E. coli OP50 laboratory strain. When the animals reached adulthood, the number of animals expressing GFP in the intestine was counted. At least 30 animals were scored for each assay. Data from three independent trials are presented in bar graphs. For each data point, the mean ± standard deviation is presented on the graph. Compared to animals expressing tau352 (PHP), animals expressing ssAPOE4 and tau352 (PHP) had significantly increased UbV-GFP expression levels when analyzed using a Student's t-test (P < 0.0001). [Figure 4] This figure contains data showing evidence that certain microbial strains from the microbiome increase Aβ-mediated paralysis. C. elegans animals with the indicated genotypes were administered either a standard E. coli OP50 laboratory strain or individual microbiome strains. On adulthood day 4, the number of paralyzed animals was recorded. Data from three independent trials are presented in bar graphs. For each data point, the mean ± standard deviation is presented on the graph. Table 1 contains the raw data, including the number of animals analyzed for each condition. [Figure 5]Data are included to provide evidence that certain microbial strains in the microbiome modulate Aβ3-42::GFP aggregation. C. elegans animals with the indicated genotypes were administered either a standard E. coli op50 laboratory strain or individual microbiome strains. When the animals reached adulthood, the number of GFP aggregates in the anterior region of the animals was counted. GFP aggregates were counted in three animals per condition. For each data point, the mean ± standard deviation is presented on the graph. [Figure 6] Data are included to demonstrate that certain microbial strains altered ATP production. Neuro2A cells were mock-treated or treated with 10 CFU of each bacterium or a combination of all bacteria (CT10) for 16 hours. 2 μM human amyloid beta 1-42 was added to all wells except untreated wells. Cells were incubated for 24 hours, and ATP levels were measured and normalized. ATP percentage was calculated according to the following formula: ATP% = [(normalized luminescence value of sample / normalized luminescence of control) × 100]. [Figure 7] 1 includes a schematic diagram of an exemplary HIF pathway. DETAILED DESCRIPTION OF THE INVENTION

[0068] Provided herein are transgenic C. elegans disease models and methods of using the C. elegans disease models to rapidly identify, assess, or characterize one or more microbial strains from a mammalian microbiome that affect (e.g., enhance or minimize) a phenotype associated with a mammalian disease or condition. In some embodiments, the identification, assessment, or characterization can occur with or without chemical entities and / or biological agents (e.g., antibodies).

[0069] C. elegans is a bacteriovorus nematode that is fed an E. coli diet in the laboratory. C. elegans can provide reliable, valid, and efficient genotypic and phenotypic models for several mammalian diseases and pathologies, including various human diseases and pathologies such as aging, diabetes, neurodegenerative disorders, metabolic diseases, and cancer. The present disclosure provides insights into the use of C. elegans models of mammalian diseases and pathologies to rapidly screen the human microbiome for microbial strains that affect such mammalian diseases and pathologies. Such studies are unlikely to be performed in standard mammalian cell culture assays or animal models. For example, screening millions of microbial species and / or strains individually, or even in combination using, for example, mouse models of human diseases and pathologies, would be economically unfeasible, time-consuming, and laborious. While C. elegans may lack some of the complexity of mammalian systems, C. elegans models share characteristics with mammalian systems, offer a fast response time, and are less expensive to produce and maintain than mammalian model systems. Thus, C. elegans models of mammalian disease and pathology provide a powerful first line of defense for examining the influence that microbial strains in the mammalian biome have on mammalian disease and pathology, and represent a useful tool for prioritizing lead microbial strains that affect specific, sensitive, and conserved therapeutic targets.

[0070] Provided herein is a method for using C. elegans as a tool for rapidly screening the human microbiome for disease-modifying agents. Standard microbiology techniques can be used to culture microbial strains from samples from healthy patients or patients with disease or pathology. These microbial strains can be fed to transgenic C. elegans strains carrying mammalian (e.g., human) disease markers, mammalian (e.g., human) disease gene mutations, or combinations thereof. The microbial strains can be fed to the transgenic C. elegans strains either individually or in combination. In some instances, individual microbial strains or combinations of microbial strains can be combined with chemical entities (e.g., small molecules, e.g., drugs) or biologics (e.g., monoclonal antibodies) and fed to transgenic C. elegans strains carrying mammalian (e.g., human) disease markers, mammalian (e.g., human) disease gene mutations, or combinations thereof.

[0071] The present disclosure recognizes that multiple outcomes can arise from feeding individual microbial strains or combinations of microbial strains in combination with chemical entities (e.g., small molecules, e.g., drugs) or biologics (e.g., monoclonal antibodies) to transgenic C. elegans strains harboring mammalian (e.g., human) disease markers, mammalian (e.g., human) disease gene mutations, or combinations thereof. In the first scenario, the methods described herein can be used to identify, define, assess, and / or detect individual microbial strains or combinations of microbial strains from a mammalian microbiome that increase the phenotypic severity of a mammalian disease or condition in C. elegans. In the second scenario, the methods described herein can be used to identify, define, assess, and / or detect individual microbial strains or combinations of microbial strains from a mammalian microbiome that decrease the phenotypic severity of a mammalian disease or condition in C. elegans. In a third scenario, the methods described herein can be used to identify, define, assess, and / or detect individual microbial strains or combinations of microbial strains from a mammalian microbiome that do not have any phenotypic effects of a mammalian disease or condition in C. elegans. This disclosure recognizes that each of these outcomes provides valuable information.

[0072] For example, individual microbial strains or combinations of microbial strains from a mammalian microbiome that increase the phenotypic severity of a mammalian disease or condition in C. elegans may be potential early diagnostic biomarkers for the mammalian disease or condition. In some embodiments, such individual microbial strains or combinations of microbial strains correlate with a higher incidence or increased severity of the disease or condition in a mammal (e.g., a human). Where such a correlation has not previously been found, the transgenic C. elegans and methods using transgenic C. elegans described herein can be used to rapidly screen and / or evaluate one or more microbial strains in a mammalian microbiome using genetic screening, chemical extraction, or genomic data mining methods to identify potential "toxic" metabolites or components of the microbiome that are responsible for the increased severity or incidence of the disease. These identified microbial strains and / or components of the mammalian microbiome can also be used to develop diagnostic methods. The identification and / or characterization of "toxic" microbial strains or microbiome components can also be used to develop modulators or therapeutic agents that can target the microbial strains, microbiome components, or the biosynthetic pathways that produce them.

[0073] Additionally, the present disclosure recognizes that individual microbial strains or combinations of microbial strains from a mammalian microbiome that reduce the phenotypic severity of a mammalian disease or condition in C. elegans may correlate with disease severity in human patients. In some embodiments, such individual microbial strains or combinations of microbial strains correlate with a lower incidence or reduced severity of the disease or condition in a mammal (e.g., a human). Where such a correlation has not previously been found, the transgenic C. elegans and methods using transgenic C. elegans described herein can be used to rapidly screen and / or evaluate one or more microbial strains in a mammalian microbiome using genetic screening, chemical extraction, or genomic data mining methods to identify potential "beneficial" microbial strains, microbiome components, or metabolites that are involved in reducing disease severity or incidence. These identified microbial strains and / or components of the mammalian microbiome can also be used for the development of diagnostic methods. The identification and / or characterization of "beneficial" microbial strains or microbiome components can also be used as modulators or therapeutic agents for disease.

[0074] In some instances, individual or combinations of microbial strains from a mammalian microbiome can also be combined with chemical entities (e.g., small molecules, e.g., drugs) or biologics (e.g., monoclonal antibodies), or combinations thereof, to feed transgenic C. elegans strains to identify potential biological or signaling pathways or cellular target genes or pathways, which may include, but are not limited to, inflammation, insulin receptor, cell death, mitochondria, endoplasmic reticulum, proteasome, lipogenesis, and detoxification.

[0075] In some instances, individual or combinations of microbial strains from mammalian microbiomes can be combined with chemical entities (e.g., small molecules, e.g., drugs) or biological agents (e.g., monoclonal antibodies), or combinations thereof, to feed C. elegans strains to identify signaling pathways or sets (e.g., comprehensive sets) of target genes or pathways that can modulate or optimize the function of specific organelles relevant to a particular disease. For example, there are multiple pathways or targets that can be modulated to achieve optimal mitochondrial function, which is a key target relevant in several neurodegenerative diseases, including Alzheimer's disease (AD). Biological targets for improving mitochondrial function include biogenesis, bioenergetics, hormesis, and / or repair. The transgenic C. elegans disease models and / or methods using C. elegans disease models described herein can be used to identify individual or combinatorial microbiome species, and / or chemical entities (e.g., small molecules, e.g., drugs) or biologics (e.g., monoclonal antibodies), or combinations thereof, that can modify, improve, or alter mitochondrial biogenesis, bioenergetics, hormesis, or repair, or any combination thereof. Thus, it is possible to identify and combine individual microbial strains or combinations of microbial strains from a mammalian microbiome, and / or chemical entities (e.g., small molecules, e.g., drugs) or biologics (e.g., monoclonal antibodies), or combinations thereof, that can target multiple pathways, e.g., to achieve optimal mitochondrial function.

[0076] C. elegans The free-living nematode C. elegans has been used extensively as a model system. C. elegans is inexpensive to culture, easy to physically manipulate, and has numerous genetic and molecular tools available for research. C. elegans is a simple multicellular organism; adult worms contain approximately 1,000 somatic cells, yet possess a variety of tissue types, including muscle, nerve, and intestinal cells. C. elegans has a short generation time, allowing for rapid experimentation. C. elegans typically progresses from egg to larva to fertile adult within three days at room temperature. A single adult C. elegans can have 300–1,000 offspring, allowing for the use of significant numbers of animals and their subsequent rapid replenishment in a relatively short time. Due to its sexual dimorphism, C. elegans is useful for genetics. Self-fertilizing hermaphrodites can be maintained as homozygous mutants without the need for mating, and males can be used in genetic crosses. C. elegans are transparent throughout all stages of their life cycle, providing the ability to see inside the organism. This allows for the observation of cellular events. It also allows for the use of phosphorescent, luminescent, and fluorescent reporters. RNA-mediated interference (RNAi) can also be used to manipulate protein expression in C. elegans, which may allow for the rapid assessment of gene function. Another advantage of using the C. elegans model system is the ability to freeze and recover the animals, allowing for long-term storage.

[0077] C. elegans can be genetically modified using several techniques to produce transgenic C. elegans strains. The sexual dimorphism of C. elegans allows for relatively easy genetic manipulation according to known procedures. For example, if a strain needs to be propagated, a single hermaphrodite can be used to self-fertilize and produce a population of offspring. Even if an animal becomes infertile due to a mutation, the hermaphrodite can still produce offspring. Another aspect of C. elegans reproduction that makes it a useful genetic tool is the ability of males to mate with hermaphrodites. For example, mating experiments allow genetic markers, such as mutations that cause visible phenotypes, to be placed together with unknown mutations in a single organism to facilitate mapping of the mutations. Hermaphrodites produce only a limited number of sperm and can typically have approximately 300 offspring of their own. Mating increases the number of offspring produced by a single hermaphrodite to approximately 1,000 due to the additional sperm produced by the male. The relatively large number of offspring combined with the short lifespan of C. elegans allows rapid and inexpensive assays to be performed on the animals.

[0078] In addition to genetic modification via reproduction, C. elegans can also be genetically modified via injection of transgenes. Microinjection is an effective method for generating transgenic animals and directly introducing various types of molecules into cells. Regarding DNA transformation, one approach is to inject DNA into the distal arm of the C. elegans gonad. The distal germline of C. elegans contains a central core of cytoplasm shared by many germ cell nuclei. Therefore, DNA injected into the distal arm of the C. elegans gonad can be delivered to many offspring. Direct microinjection into the nucleus of an oocyte can induce chromosomal integration of the transgene, but this technique can be more difficult to implement. C. elegans can also integrate genetic material fed to them.

[0079] C. elegans is relatively simple to culture. It can be cultured in the presence of bacteria, either in liquid culture or on nematode growth medium (NGM) agar plates. It is possible to grow the animals in a chemically defined medium without adding bacteria, which can be useful because the components of the medium can be varied to study the animal's nutritional or other chemical requirements. In some embodiments, C. elegans is grown on agar plates. C. elegans can be grown on nematode growth medium (NGM) agar plates. Bacteria can be spread on NGM plates as a food source for the animals. For example, OP50, a leaky E. coli uracil auxotroph, can be used. OP50 grows slowly and provides nutrients to the animals without overgrowth. Once the animals consume all of the food on the plate, they burrow into the agar and can be maintained on "starvation" plates in an incubator at 15°C for weeks at a time. The animals can be transferred to agar plates with fresh bacteria, either by cutting and removing a small block of agar from the starvation plate with a sterile instrument such as a micropipette tip, by washing the worms off the surface of the plate with sterile water, or by picking one or more individuals onto a fresh plate, resulting in the reappearance of C. elegans. At any point, C. elegans can be cryogenically preserved. C. elegans prefers to grow at 15°C to 25°C, but temperatures can vary depending on the C. elegans strain and the conditions being tested. In some embodiments, C. elegans cultures can be cultured at temperatures of at least 5°C, at least 10°C, at least 15°C, at least 20°C, at least 25°C, at least 30°C, at least 35°C, or at least 40°C. In some embodiments, C. elegans cultures may be cultured at temperatures of at most 65°C, at most 60°C, at most 55°C, at most 50°C, at most 55°C, at most 40°C, at most 35°C, at most 30°C, at most 25°C, or at most 20°C.Standard protocols for C. elegans manipulation and culture are known, as described, for example, in Stiernagle T. Maintenance of C. elegans. Wormbook, ed. The C. elegans Research Community, WormBook. (February 11, 2006), which is incorporated herein by reference.

[0080] Microbial preparation(s) and / or component(s) The present disclosure provides systems and methods for assessing, characterizing, and identifying one or more microbial strains in a microbiome. Such systems and methods may be useful for assessing, characterizing, and identifying one or more microbial strains that affect the health of humans, livestock, and / or pets. In some embodiments, one or more microbial strains from the microbiome of a snake, lizard, fish, or bird are assessed, characterized, and identified. In some embodiments, one or more microbial strains from a mammalian microbiome are assessed, characterized, and identified. The mammalian microbiome may be a canine, feline, equine, bovine, ovine, caprine, or porcine microbiome. Generally, the microbiome used in the systems or methods described herein will correspond to the disease or condition modeled by the transgenic C. elegans used in the system or method. For example, if the transgenic C. elegans models a human disease, a human microbiome will be assessed, characterized, or identified.

[0081] A microbiome can be isolated from any system or tissue of an organism that supports microbial growth. For example, the microbiome can be a skin microbiome, oral microbiome, nasal microbiome, gastrointestinal microbiome, brain microbiome, lung microbiome, or urogenital microbiome. A list of exemplary microbial strains found in the gastrointestinal microbiome is included in Table 8 below. Those skilled in the art will appreciate that microbiome samples can be obtained in various ways known in the art. For example, skin, oral, nasal, lung, or urogenital microbiome samples can be obtained using swabs or tissue scrapes. In some embodiments, gastrointestinal microbiome samples can be collected from stool. Skin, oral, nasal, gastrointestinal, brain, lung, or urogenital microbiome samples can be obtained via biopsy.

[0082] In some embodiments, the microbiome is that of a healthy individual or an individual who does not have or is not at risk for a particular disease or disorder. In some embodiments, the microbiome is that of an individual who has or is at risk for a particular disease or disorder. In some embodiments, the microbiome is that of an individual who is known to have a particular disease or disorder. In some embodiments, the human microbiome is that of a human who is not known to be at risk for one or more diseases or conditions.

[0083] In some embodiments, the microbiome is a reference microbiome. The reference microbiome can be the microbiome of a healthy individual or an individual who does not suffer from or is not at risk of developing a particular disease or disorder. In some cases, the reference microbiome may be derived from the same individual as the microbiome to be assessed or characterized, but obtained at a different time. In some cases, the reference microbiome may be derived from the same individual as the microbiome to be assessed or characterized, but obtained from a different system or tissue.

[0084] In some embodiments, individual microbial strains or combinations of microbial strains may be assessed, characterized, or identified in relative abundances that differ from the relative abundances of such strain(s) found in a microbiome. For example, transgenic C. elegans or methods of using transgenic C. elegans described herein may be used to assess, characterize, or identify a single strain, even if it naturally occurs with other microbial strains in a microbiome. As another example, transgenic C. elegans or methods of using transgenic C. elegans described herein may be used to assess, characterize, or identify two microbial strains together, even if they naturally occur with additional microbial strains in a microbiome.

[0085] The transgenic C. elegans or methods of using transgenic C. elegans described herein may also be used to assess, characterize, or identify extracts, components, or compounds of microbial strains. In some instances, extracts, components, or compounds of microbial strains that have been determined to affect a transgenic C. elegans model of a disease or condition may be assessed, characterized, or identified. Assessing, characterizing, or identifying extracts, components, or compounds of microbial strains that affect a transgenic C. elegans model of a disease or condition may provide additional information about potential biomarkers, targets, or protective factors in the microbiome.

[0086] A variety of techniques are known in the art that can be used to prepare extracts of microbial strains and / or to isolate or process extracts, components, or compounds from microbial strains (e.g., to isolate and / or purify one or more components or compounds therefrom). Such techniques may include, for example, one or more of organic extraction, vacuum concentration, chromatography, etc., to name just a few.

[0087] Biological effect assessment The present disclosure provides insight that C. elegans can be used to identify, characterize, or assess microbial strain(s) of a mammalian microbiome by contacting (e.g., feeding or administering) the microbial strain(s) to transgenic C. elegans that model a mammalian disease or condition. To determine whether a microbial strain or combination of microbial strains affects the transgenic C. elegans that model a mammalian disease or condition, parameters of the transgenic C. elegans can be observed, measured, or assessed in different samples contacted with the microbial strain(s) or combination of microbial strains. To determine whether a microbial strain or combination of microbial strains affects the transgenic C. elegans that model a mammalian disease or condition, various parameters of the transgenic C. elegans can be observed, measured, or assessed. As just a few examples, the behavior (e.g., mating, feeding, food aversion, or movement), genetic variation (e.g., the presence of SNPs, deletions, additions, inversions, or repeats in DNA), transcript levels, protein levels, metabolite levels, lipid levels, carbohydrate levels, protein (e.g., enzyme) activity levels of transgenic C. elegans can be observed, measured, or assessed to determine whether a microbial strain or combination of microbial strains affects transgenic C. elegans that model a mammalian disease or condition.

[0088] In some embodiments, the methods described herein utilize a first sample and a second sample. In some embodiments, the first sample is a reference sample. In some embodiments, the reference sample can be a culture of transgenic C. elegans that has been contacted with (e.g., administered or fed with) OP50, for example. In some embodiments, the reference sample can be a culture of transgenic C. elegans that has been contacted with (e.g., administered or fed with) a microbial strain or combination of microbial strains derived from the microbiome of a healthy individual. In some embodiments, the reference sample can be a culture of transgenic C. elegans that has been contacted with (e.g., administered or fed with) a microbial strain or combination of microbial strains derived from the microbiome of an individual obtained at a first time point.

[0089] In some embodiments, the second sample may be a test sample. In some embodiments, the test sample may be a culture of transgenic C. elegans that has been contacted with (e.g., administered or fed with) an individual or combination of microbial strains from a mammalian microbiome, for example, a human microbiome. In some cases, the human microbiome is the microbiome of a person suffering from or at risk of a disease or pathology. In some cases, the human microbiome is the microbiome of a person who is not known to be at risk for one or more diseases or pathologies. In some embodiments, the test sample may be a culture of transgenic C. elegans that has been contacted with (e.g., administered or fed with) a microbial strain or combination of microbial strains from the microbiome of an individual obtained at a second time point.

[0090] In some embodiments, the methods described herein include comparing one or more parameters obtained from a test sample with one or more parameters obtained from a reference sample. In some embodiments, by comparing one or more parameters obtained from a test sample with one or more parameters obtained from a reference sample, it can be determined that an individual microbial strain or combination of microbial strains from a microbiome increases the severity or incidence of a disease or condition phenotype modeled by cultured transgenic C. elegans. In some embodiments, by comparing one or more parameters obtained from a test sample with one or more parameters obtained from a reference sample, it can be determined that an individual microbial strain or combination of microbial strains from a microbiome decreases the severity or incidence of a disease or condition phenotype modeled by cultured transgenic C. elegans. In some embodiments, by comparing one or more parameters obtained from a test sample with one or more parameters obtained from a reference sample, it can be determined that an individual microbial strain or combination of microbial strains from a microbiome has no effect on the severity or incidence of a disease or condition phenotype modeled by cultured transgenic C. elegans.

[0091] The transgenic C. elegans and methods of using transgenic C. elegans provided herein may be useful in assessing, characterizing, or identifying microbial strains in the microbiome that influence a mammalian disease or condition. The present disclosure also recognizes that the transgenic C. elegans and methods of using transgenic C. elegans provided herein can be used to define and / or characterize microbial signatures associated with a disease or condition. Furthermore, the present disclosure recognizes that the transgenic C. elegans and methods of using transgenic C. elegans provided herein can be used to define and / or characterize microbial signatures associated with one or more characteristics of a disease or condition (e.g., severity, responsiveness to therapy, etc.). For example, if multiple microbial strains are determined to be associated with increased severity of a disease or disorder, e.g., across multiple individuals, the microbial strains, as well as their relative abundance, can be used as a signature to identify individuals at risk of developing increased severity of the disease or disorder. As another example, if multiple microbial strains are determined to be associated with increased severity of a disease or disorder, e.g., in a single individual, at a particular time point (e.g., after withdrawal from treatment), the microbial strains, as well as their relative abundance, can be used as a signature to identify when the individual is at risk of developing increased severity of the disease or disorder.

[0092] The present disclosure also provides the recognition that the transgenic C. elegans and methods of using transgenic C. elegans provided herein can be used to diagnose individuals with a disease or condition. Indeed, microbial signatures associated with a disease or condition determined through use of the transgenic C. elegans and methods of using transgenic C. elegans provided herein can be used to provide an early diagnosis and / or identify individuals as at-risk.

[0093] The present disclosure also recognizes that the transgenic C. elegans and methods of using the transgenic C. elegans provided herein can be used to monitor the progression of a disease or condition in an individual. For example, if the relative abundance within the microbiome of a microbial strain determined to increase the severity of a disease or condition decreases, this may indicate that the disease or condition is being alleviated, e.g., by a treatment or immune response.

[0094] The present disclosure also provides insight that the transgenic C. elegans and methods of using transgenic C. elegans provided herein can be used to tailor treatments (e.g., therapies, nutritional supplements, and / or probiotics) to individual patients. In some embodiments, the transgenic C. elegans and methods of using transgenic C. elegans provided herein can provide "personalized" therapy. In some instances, microbial strains within an individual can be assessed, characterized, or identified to determine whether they have an impact on a disease or disorder. Based on the results, the individual can be treated with one or more microbial strains to adjust the microbial strains (and / or their components or compounds) in their microbiome. In some cases, this will have an impact on the disease or condition that the individual suffers from or is at risk for developing. For example, if an individual is determined to have a relatively low amount of one or more microbial strains determined to reduce the severity of a disease or condition, administering to that individual (or an extract, component, or compound thereof) one or more microbial strains determined to reduce the severity of the disease or condition may alleviate the severity of the disease or condition in that individual.

[0095] Pharmaceutical Compositions Provided herein are compositions comprising individual microbial strains or combinations of microbial strains. In some embodiments, the compositions comprise individual microbial strains or combinations of microbial strains, extracts thereof, and / or components thereof from a mammalian microbiome that have been assessed, identified, characterized, or assayed using transgenic C. elegans or methods as described herein. In some embodiments, provided herein are compositions comprising two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more microbial strains, extracts thereof, and / or components thereof from a mammalian microbiome that have been assessed, identified, characterized, or assayed using transgenic C. elegans or methods as described herein.

[0096] In some embodiments, provided herein are compositions comprising two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more microbial strains listed in Table 8 below.

[0097] In some embodiments, provided herein are compositions comprising Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., L. plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium, Bacillus subtilis, Acidaminococcus sp., or a combination thereof. In some embodiments, the combination includes at least two of Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., L. plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium, Bacillus subtilis, and Acidaminococcus sp., at least three of them, at least four of them, at least five of them, at least six of them, at least seven of them, at least eight of them, at least nine of them, or all of them.

[0098] In some embodiments, the individual or combination of microbial strains from a mammalian microbiome are killed (e.g., heat-killed). Alternatively, in some embodiments, the individual or combination of microbial strains from a mammalian microbiome may comprise viable or living cells.

[0099] In some embodiments, the one or more microbial strains include viable or living individual microbial strains or combinations of microbial strains, for example, from a mammalian microbiome.

[0100] In some embodiments, the one or more microbial strains include viable or living individual microbial strains or combinations of microbial strains, e.g., from a mammalian microbiome, as described herein, and include and / or are formulated through the use of one or more cell cultures and / or supernatants or pellets thereof, and / or powders formed therefrom.

[0101] In some embodiments, a composition for use in accordance with the present disclosure is a pharmaceutical composition, e.g., for administration (e.g., oral administration) to a mammal (e.g., a human). Pharmaceutical compositions typically include an active agent (e.g., an individual microbial strain or combination of microbial strains from a mammalian microbiome, an extract thereof, and / or components thereof) and a pharmaceutically acceptable carrier. Certain exemplary pharmaceutically acceptable carriers include, for example, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration.

[0102] In some embodiments, pharmaceutical compositions for use in accordance with the present disclosure may comprise and / or be administered in combination with one or more supplementary active compounds. In certain embodiments, such co-active agents include ginger, curcumin, probiotics (e.g., probiotic strains of one or more of the following genera: Lactobacillus, Bifidobacterium, Saccharomyces, Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Bacillus, and / or Escherichia coli (see Fijan, Int J Environ Res Public Health. 2014 May;11(5):4745-4767, which is incorporated herein by reference); prebiotics (non-digestible food ingredients that help support the growth of probiotic bacteria, e.g., fructans such as fructooligosaccharides (FOS) and inulin, galactans such as galactooligosaccharides (GOS), dietary fibers such as resistant starch, pectin, beta-glucans, and xylooligosaccharides (Hutkins et al., Curr Opin Biotechnol. 2016 Feb;37:1-7, which is incorporated herein by reference), as well as combinations thereof.

[0103] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. An example of an administration route is oral administration. Methods for formulating suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005, and the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). Oral compositions generally contain an inert diluent or an edible carrier. In some embodiments, oral formulations may be or include syrups, liquids, tablets, lozenges, gummies, capsules (e.g., gelatin capsules), powders, gels, films, etc., to name just a few examples.

[0104] In some embodiments, pharmaceutically compatible binders and / or adjuvant materials may be included as part of the pharmaceutical composition. In certain embodiments, the pharmaceutical composition may contain, for example, any one or more of the following inactive ingredients or compounds of a similar nature: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavor. In some embodiments, the composition can be consumed as is or sprinkled or mixed into food or liquids (such as water). In some embodiments, a composition that can be administered to a mammal as described herein can be or can comprise a ingestible item (e.g., a food or beverage) that contains (e.g., is supplemented with) individual or combinations of microbial strains, extracts thereof, and / or components thereof from the mammalian microbiome.

[0105] In some embodiments, the food product may be or include one or more of bars, candy, baked goods, cereals, savory snacks, pasta, chocolate, and other solid foods, liquid or semi-solid foods including yogurt, soups, and stews, and beverages such as smoothies, shakes, juices, and other carbonated or non-carbonated beverages. In some embodiments, the food product is prepared by the subject by incorporating individual or combinations of microbial strains, extracts thereof, and / or components thereof from a mammalian microbiome.

[0106] The compositions can be included in a kit, container, pack, or dispenser together with instructions for administration or use in the methods described herein.

[0107] Upon review of this disclosure, one of skill in the art will understand that in some embodiments, a composition (e.g., a pharmaceutical composition) as described herein may be or may include one or more cells, tissues, or organisms (e.g., plant or microbial cells, tissues, or organisms) that produce (e.g., have produced and / or are producing) the relevant compound.

[0108] Those skilled in the art will appreciate that in some embodiments, techniques for preparing and / or preparing compositions and / or preparations (and particularly for preparing pharmaceutical compositions) may include one or more steps of assessing or characterizing the compound, preparation, or composition, e.g., as part of quality control. In some embodiments, if assayed material does not meet predetermined specifications for the relevant assessment, it is discarded. In some embodiments, if such assayed material does in fact meet the predetermined specifications, it continues to be processed as described herein.

[0109] In some embodiments, the pharmaceutical compositions provided herein may promote colonization by individual microbial strains or combinations of microbial strains from a mammalian microbiome, particularly microbial strain(s) that have been identified, characterized, or assessed to reduce the severity or incidence of a mammalian disease or condition in a mammal suffering from or at risk for the mammalian disease or condition. In some embodiments, the pharmaceutical compositions provided herein may attenuate colonization by individual microbial strains or combinations of microbial strains from a mammalian microbiome, particularly microbial strain(s) that have been identified, characterized, or assessed to increase the severity or incidence of a mammalian disease or condition in a mammal suffering from or at risk for the mammalian disease or condition. In some embodiments, the pharmaceutical compositions provided herein may promote colonization of individual microbial strains or combinations of microbial strains from the mammalian microbiome, particularly microbial strain(s) that have been identified, characterized, or assessed as being capable of outcompeting one or more microbial strains that do not affect the severity or incidence of a mammalian disease or condition, but that have been identified, characterized, or assessed as increasing the severity or incidence of a mammalian disease or condition in a mammal suffering from or at risk for the mammalian disease or condition.

[0110] In some embodiments, each of the one or more microbial strains in the composition is 10 1 ~10 12 In some embodiments, each of the one or more microbial strains in the composition comprises 10 colony forming units (CFU). 6 ~10 12 In some embodiments, each of the one or more microbial strains in the composition comprises the same number of CFU. In some embodiments, some of the one or more microbial strains in the composition comprise different numbers of CFU.

[0111] In some embodiments, the composition comprises a total of 10 6 ~10 12 Contains CFU.

[0112] In some embodiments, a pharmaceutical composition is tailored to a particular mammal (e.g., a particular human patient) based on the microbiome of that particular mammal (e.g., human). In some embodiments, a pharmaceutical composition is specific to the microbiome of an individual mammal (e.g., human). In some embodiments, a pharmaceutical composition is specific to the microbiome of a population of mammals (e.g., humans). A population of mammals can include, but is not limited to, a family, mammals in the same geographic location (e.g., neighborhood, city, state, or country), mammals with the same disease or condition, mammals of a particular age or age range, or mammals consuming a particular diet (e.g., food, food source, or caloric intake).

[0113] Treatment method The present disclosure recognizes that the compositions described herein may be useful in treating a subject. The methods provided by the present disclosure include methods for treating certain diseases, disorders, and conditions. In some embodiments, the relevant disease, disorder, and condition may be or include a neurodegenerative disease, disorder, or condition. In some embodiments, the neurodegenerative disease, disorder, or condition may be Alzheimer's disease. In some embodiments, the relevant disease, disorder, and condition may be or include an intraocular neovascular disease, disorder, or condition. In some embodiments, the neurodegenerative disease, disorder, or condition may be diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, or glaucoma.

[0114] In general, the methods of treatment provided by the present disclosure involve administering a therapeutically effective amount of a composition as described herein, alone or in combination with other compositions and / or treatments, to a subject in need of, or determined to be in need of, such treatment.

[0115] In some embodiments, the treatment methods provided herein are prophylactic or preventative, e.g., may be administered to a subject prior to the onset of noticeable symptoms and / or prior to exposure to a particular expected trigger associated with a neurodegenerative disease, disorder, or condition. In some embodiments, the treatment methods provided herein are therapeutic, e.g., may be administered to a subject after the onset of noticeable symptoms associated with a neurodegenerative disease, disorder, or condition.

[0116] In some embodiments, provided methods of treatment are administered to a subject that is a mammal, e.g., a mammal experiencing a disease, disorder, or condition as described herein, hi some embodiments, the subject is a human subject or a non-human veterinary subject, e.g., an ape, cat, dog, monkey, or pig.

[0117] In many embodiments, treatment involves ameliorating at least one symptom of the disease, disorder, or condition associated with the neurodegenerative disease, disorder, or condition. In some embodiments, the treatment method may be prophylactic.

[0118] In some embodiments, the methods may involve administration of a therapeutically effective amount of a composition disclosed herein before, during (e.g., simultaneously with), or after administration of a treatment suspected to be associated with a neurodegenerative disease, disorder, or condition.

[0119] In some embodiments, a subject undergoing treatment as described herein may be undergoing and / or may have undergone other treatments (e.g., drug treatments / therapies, surgery, etc.) that may be intended to treat, for example, one or more symptoms or characteristics of a disease, disorder, or condition as described herein (e.g., a neurodegenerative disease, disorder, or condition), whereby the provided compositions are administered in combination with such other therapies (i.e., treatments) to treat the relevant disease, disorder, or condition.

[0120] In some embodiments, the compositions described herein may be administered in a form containing one or more pharmaceutically acceptable carriers. Suitable carriers have been described previously and vary depending on the desired form and mode of administration of the composition. For example, pharmaceutically acceptable carriers may include diluents or excipients such as fillers, binders, wetting agents, disintegrants, surfactants, glidants, and lubricants. Typically, carriers may be solid (including powders), liquid, or any combination thereof. Each carrier is preferably "acceptable" in the sense of being compatible with the other ingredients in the composition and not harmful to the subject. A carrier may be biologically acceptable and inert (e.g., it allows the composition to maintain the viability of the biomaterial until delivery to the appropriate site).

[0121] The tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, primogel, or corn starch; a lubricant such as magnesium stearate or sterote; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, orange flavor, or other suitable flavor, which are intended by way of example only and are not limiting.

[0122] Oral compositions may contain an inert diluent or an edible carrier. For oral therapeutic administration, the active compound may be incorporated with an excipient and used in the form of tablets, lozenges, medicine drops, troches, or capsules, such as gelatin capsules. Oral compositions may also be prepared by combining the compositions of the present disclosure with food. In some embodiments, the microorganisms may be formulated in food products. Some non-limiting examples of food products that can be used with the methods and compositions described herein include popsicles, cheese, cream, chocolate, milk, meat, beverages, spices, kefir, miso, sauerkraut, etc. In other embodiments, the food product may be juice, soft drinks, tea-based drinks, drink preparations, jelly drinks, and functional drinks; alcoholic beverages such as beer; carbohydrate-containing foods such as processed rice foods, noodles, bread, and pasta; fish paste products such as fish, ham, sausage, and fish paste products; retort pouch products such as curry, foods with thick sauces, and Chinese soup; soup; dairy products such as milk, milk drinks, ice cream, and yogurt; fermented products such as miso paste, fermented drinks, and pickles; soy products; various confectionery products including biscuits, cookies, candy, chewing gum, and gummies; cold desserts including jellies, custard puddings, and frozen desserts; instant foods such as instant soups and instant miso soups; and the like. Preferably, the food preparation does not require cooking after mixing with the microbial strain(s) to avoid the destruction of any microorganisms. In one embodiment, the food used for administration is chilled, for example, ice-cold flavored water. In certain embodiments, the foodstuff is not a potentially allergenic foodstuff (e.g., not soy, wheat, peanut, tree nut, dairy, egg, shellfish, or fish). Pharmaceutically compatible binding agents and / or adjuvant materials may be included as part of the composition.

[0123] In some such embodiments, the compositions described herein are administered to a subject according to a dosage regimen that achieves the population of the subject's microbiome with the administered cells.In some embodiments, the compositions are administered to a subject in a single dose.In some embodiments, the compositions are administered to a subject in multiple doses.In some embodiments, the doses of the compositions are administered to a subject twice a day, daily, weekly, or monthly.

[0124] In some embodiments, each of the one or more microbial strains in a dose is 10 1 ~10 12 In some embodiments, each of the one or more microbial strains in a dose comprises 10 colony forming units (CFU). 6 ~10 12 In some embodiments, each of the one or more microbial strains in a dose contains the same number of CFU. In some embodiments, some of the one or more microbial strains in a dose contain different numbers of CFU.

[0125] In some embodiments, a dose of one or more microbial strains comprises a total of 10 6 ~10 12 In some embodiments, a dose of one or more microbial strains comprises a total of 10 CFU. 7 ~10 10 In some embodiments, a single dose of the one or more microbial strains comprises between 5 and 200 billion CFUs. In some embodiments, a single dose of the one or more microbial strains comprises between 5 and 50 billion CFUs. In some embodiments, a single dose of the one or more microbial strains comprises between 5 and 20 billion CFUs. In some embodiments, a single dose of the one or more microbial strains comprises between 50 and 100 billion CFUs. In some embodiments, a single dose of the one or more microbial strains comprises between 100 and 200 billion CFUs. [Example]

[0126] The following examples are provided to illustrate to one of ordinary skill in the art how to make and use the methods and compositions described herein and are not intended to limit the scope of the disclosure.

[0127] Example 1: Materials and Methods Two different constructs for the human APOE4 transgene under the control of the intestinal promoter (pvha-6::apoE4::tbb-2 UTR) were generated by gene synthesis. The constructs were cloned into the KpnI / SalI restriction enzyme sites of the pUC57 plasmid. The human APOE4 protein sequence was codon-optimized for optimal expression in C. elegans. Three synthetic introns were included within the apoE4 sequence in both constructs to avoid gene silencing and for optimal expression in C. elegans. The nucleotide sequences of the introns are included in Table 1 below.

[0128] In one construct, the signal sequence FLP-1 for secretion from C. elegans was included in the apoE4 sequence ("Worm 2"), while in the other construct, no signal sequence was added to the apoE4 sequence ("Worm 1").

[0129] [Table 2]

[0130] Extrachromosomal array strains were constructed by injecting the expression plasmid and co-injection marker (pCFJ90, 2 ng / μl for pmyo-2::mCherry) into either Worm 1 or Worm 2. The sequences used for Worm 1 and Worm 2 are included in Table 2 below. Coding sequences are shown in uppercase and non-coding sequences are shown in lowercase.

[0131] [Table 3-1] [Table 3-2]

[0132] [Table 4]

[0133] Example 2: Exemplary system for characterizing microbial strains that affect parameters associated with Alzheimer's disease Example 2.1: Alzheimer's Disease Alzheimer's disease (AD) is the most common cause of dementia. AD is characterized by the progressive decline of cognitive functions, including memory, language, and cognitive abilities. Senile plaques and intracellular neurofibrillary tangles are generally considered hallmarks of AD pathology. Plaques can contain aggregates of either 40- or 42-amino acid amyloid-β (Aβ) peptides, which can be formed by abnormal processing of amyloid precursor protein (APP) by presenilins (PSEN1 and PSEN2). Soluble Aβ oligomers can also cause synaptic dysfunction, leading to neurodegeneration and cognitive impairment (Mucke, L., and Selkoe, DJ (2012). Neurotoxicity of amyloid β-protein: synaptic and network dysfunction. Cold Spring Harb. Perspect. Med. 2, a006338, which is incorporated herein by reference). Neurofibrillary tangles in AD may contain hyperphosphorylated tau protein, a microtubule-associated protein in neurons (Iqbal, K., et al. (2010). Tau in Alzheimer Disease and Related Tauopathies. Curr. Alzheimer Res. 7, 656-664, which is incorporated herein by reference). In AD, tau may become abnormally hyperphosphorylated and aggregate into fibrils (Grundke-Iqbal, I., et al. (1986). Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology. Proc. Natl. Acad. Sci. USA 83, 4913-4917, which is incorporated herein by reference).Because of the strong evidence for the involvement of Aβ in AD, several monoclonal antibody-based therapeutics have been developed and tested to target amyloid plaques (van Dyck, CH (2018). Anti-Amyloid-β Monoclonal Antibodies for Alzheimer's Disease: Pitfalls and Promise. Biol. Psychiatry 83, 311-319, which is incorporated by reference in its entirety). However, studies in standard mammalian models have shown that interventions that effectively block or clear Aβ accumulation in animal models do not improve cognition in human clinical trials.

[0134] Approximately 5% of AD cases appear to have a genetic cause, while 95% of cases are sporadic or late-onset AD of unknown etiology. Less than 1% of AD cases are caused by genetic mutations in genes including APP, PSEN1, and PSEN2. Although several other genes have been linked to AD, one of the most prominent genetic risk factors for AD is apoE (Lambert, J.-C., et al. (2009). Genome-wide association study identifies variants at CLU and CR1 associated with Alzheimer's disease. Nat. Genet. 41, 1094-1099; Shen, L., and Jia, J. (2016). An Overview of Genome-Wide Association Studies in Alzheimer's Disease. Neurosci. Bull. 32, 183-190 (each of which is incorporated herein by reference)). Apolipoprotein E (APOE), a lipid / cholesterol transporter, is encoded by apoE. In humans, three major protein variants exist, designated APOE2, APOE3, and APOE4, which differ from each other at only two amino acid residues (Mahley, RW (2016). Apolipoprotein E: from cardiovascular disease to neurodegenerative disorders. J. Mol. Med. Berl. Ger. 94, 739-746, which is incorporated herein by reference).Individuals carrying apoe polymorphisms, specifically the apoe4 allele, are significantly more likely to develop not only AD but also early-onset AD compared with individuals carrying either the apoe2 or apoe3 alleles (Roses, AD (1996). Apolipoprotein E alleles as risk factors in Alzheimer's disease. Annu. Rev. Med. 47, 387-400; Strittmatter, WJ, and Roses, AD (1996). Apolipoprotein E and Alzheimer's disease. Annu. Rev. Neurosci. 19, 53-77, each of which is incorporated herein by reference). APOE2 is considered a protective form of APOE, while APOE4 is considered a "toxic" form (Strittmatter and Roses, 1996, which is incorporated herein by reference). APOE4 exacerbates AD-related brain changes, including increased levels of amyloid deposits, brain dysfunction, and neurodegeneration (DiBattista, AM, et al. (2016). Alzheimer's Disease Genetic Risk Factor APOE-ε4 Also Affects Normal Brain Function. Curr. Alzheimer Res. 13, 1200-1207, which is incorporated herein by reference). Despite the importance of APOE4 in AD, the molecular mechanisms by which APOE4 promotes AD pathogenesis remain poorly understood (Kanekiyo, T., et al. (2014). ApoE and Aβ in Alzheimer's disease: accidental encounters or partners? Neuron 81, 740-754, which is incorporated herein by reference in its entirety). APOE4 is thought to contribute to AD pathogenesis through both loss-of-function and gain-of-function mechanisms (DiBattista, 2016; Zepa, L., et al. (2011)).ApoE4-Driven Accumulation of Intraneuronal Oligomerized Aβ42 Following Activation of the Amyloid Cascade In Vivo Is Mediated by a Gain of Function. Int. J. Alzheimers Dis. 2011 (each of which is incorporated herein by reference).

[0135] Earlier studies suggested that APOE isoforms bind to and help clear Aβ (Kim, J., et al. (2009). The role of apolipoprotein E in Alzheimer's disease. Neuron 63, 287-303, which is incorporated herein by reference). Compared to APOE2 and APOE3, APOE4 was suggested to be less efficient at clearing Aβ (Kim, 2009, which is incorporated herein by reference). However, recent studies suggest that APOE competes with Aβ for uptake via the apoE receptor (Verghese, PB, et al. (2013). APOE influences amyloid-β (Aβ) clearance despite minimal APOE / Aβ association in physiological conditions. Proc. Natl. Acad. Sci. USA 110, E1807-1816; Yajima, R., et al. (2015). APOE-isoform-dependent cellular uptake of amyloid-β is mediated by lipoprotein receptor LR11 / SorLA. Biochem. Biophys. Res. Commun. 456, 482-488 (each of which is incorporated herein by reference). While all isoforms were able to compete for binding to the APOE receptor, APOE4-expressing cells were less efficient at clearing Aβ. (Verghese, 2013, which is incorporated herein by reference).

[0136] Although the role of APOE4 in Aβ brain pathology is well documented, its influence on tau pathology has only recently begun to be explored. Using a tauopathy model overexpressing 1N4R human tau containing the P301S mutation, it was shown that ApoE4 exacerbates tau-induced neuroinflammatory and neurodegenerative phenotypes independently of Aβ pathology (Shi, Y., et al. (2017). ApoE4 markedly exacerbates tau-mediated neurodegeneration in a mouse model of tauopathy. Nature 549, 523-527 (the same reference is incorporated herein by reference)). The tau P301S mutation was originally found in a human case with frontotemporal dementia and degeneration (Bugiani, O., et al. (1999). Frontotemporal Dementia and Corticobasal Degeneration in a Family with a P301S Mutation in Tau. J. Neuropathol. Exp. Neurol. 58, 667-677, which is incorporated herein by reference). Furthermore, the tau P301S mutant protein is a more favorable substrate for phosphorylation compared to wild-type tau (Alonso, A. del C., et al. (2004). Promotion of hyperphosphorylation by frontotemporal dementia tau mutations. J. Biol. Chem. 279, 34873-34881, which is incorporated herein by reference). Interestingly, the neurofibrillary tangles found in AD are primarily composed of hyperphosphorylated tau. (Iqbal, 2010, which is incorporated herein by reference).Additionally, the frequency of the APOE4 allele is significantly higher in patients with frontotemporal dementia (Stevens, M., et al. (1997). Apolipoprotein E gene and sporadic frontal lobe dementia. Neurology 48, 1526-1529, which is incorporated herein by reference)), and APOE4 carriers also have increased disease severity (Agosta, F., et al. (2009). Apolipoprotein E ε4 is associated with disease-specific effects on brain atrophy in Alzheimer's disease and frontotemporal dementia. Proc. Natl. Acad. Sci. 106, 2018-2022; Engelborghs, S., et al. (2006). Dose-dependent effect of APOE epsilon4 on behavioral symptoms in frontal lobe dementia. Neurobiol. Aging 27, 285-292 (each of which is incorporated herein by reference). Despite the importance of APOE4 in AD, there is a lack of therapies targeting APOE4 (Michaelson, DM (2014). APOE ε4: The most prevalent yet understudied risk factor for Alzheimer's disease. Alzheimers Dement. J. Alzheimers Assoc. 10, 861-868; Holtzman, DM, et al. (2012). Apolipoprotein E and Apolipoprotein E Receptors: Normal Biology and Roles in Alzheimer Disease. Cold Spring Harb. Perspect. Med. 2 (each of which is incorporated herein by reference)).Furthermore, despite being identified in more than half of all AD patients, ApoE4 carriers are often excluded from clinical trials for AD due to the unpredictability of their response (Qiu, WQ, et al. (2013). Angiotensin converting enzyme inhibitors and the reduced risk of Alzheimer's disease in the absence of apolipoprotein E4 allele. J. Alzheimers Dis. JAD 37, 421-428; Sperling, R., et al. (2012). Amyloid-related imaging abnormalities in patients with Alzheimer's disease treated with bapineuzumab: a retrospective analysis. Lancet Neurol. 11, 241-249; Farlow, MR, et al. (1998). Treatment outcome of tacrine therapy depends on apolipoprotein genotype and gender of the subjects with Alzheimer's disease. Neurology 50, 669-677; Risner, ME, et al. al. (2006). Efficacy of rosiglitazone in a genetically defined population with mild-to-moderate Alzheimer's disease. Pharmacogenomics J. 6, 246-254 (each of which is incorporated herein by reference).

[0137] Interestingly, although APOE4 is expressed in the brain, its expression in peripheral tissues is high, raising the possibility that peripheral APOE4 may contribute to the pathogenesis of AD. Aside from the brain, APOE protein is primarily synthesized in the liver and is involved in lipid transport and cholesterol homeostasis (Safieh, M., et al. (2019). ApoE4: an emerging therapeutic target for Alzheimer's disease. BMC Med. 17, incorporated herein by reference). The liver is the primary site where the body encounters not only nutrients but also small molecules, metabolites, or toxins derived from the gut microbiome through the enterohepatic circulation. Therefore, gut dysbiosis imbalance would have a profound impact on the liver. One possibility is that AD may have an intestinal origin, i.e., "microbiome-derived materials" may leak into the enterohepatic circulation and reach the liver. From the liver, APOE4 may transport these "microbiome-derived materials" to the brain, where they may seed amyloid deposits and / or increase neuroinflammation. Recent studies have suggested that the gut microbiome plays an important role in AD. Significant changes in the microbiome have been observed in human AD patients compared with control populations. However, it is unknown whether these changes are a cause or a consequence of the disease. Although many microbiome components have been associated with either AD susceptibility or pathogenesis, the molecular mechanisms of such interactions remain unknown.

[0138] Example 2.2: APOE4 Augments Aβ-Induced Paralytic Phenotype To test whether microbes regulate AD pathogenesis, we developed transgenic C. elegans strains expressing human APOE4. These transgenic C. elegans strains expressed human APOE4 in the C. elegans intestine under the control of an intestinal promoter with or without a signal sequence (e.g., mbEx2(pvha-6::ssapoe4)) that allows human APOE4 to be secreted from the cell. In C. elegans, the liver is absent, and the intestine performs all functions typically performed by the liver. Animals were challenged with a standard laboratory strain of E. coli OP50. Animals were monitored every other day from adulthood day 1 until all animals were paralyzed. At least 20 animals (as listed in Table 4) were scored for each assay. Data from three independent trials were obtained. For each data point, the mean ± standard deviation is presented in the graph in Figure 1. Transgenic animals expressing human APOE4 with or without the signal sequence did not exhibit any apparent phenotype (Fig. 1).

[0139] Expression of human Aβ1-42 in the muscles of C. elegans has been reported to induce a paralytic phenotype (Link, CD (1995). Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 92, 9368-9372 (incorporated herein by reference)). To determine whether human APOE4 modulates the paralytic phenotype induced by intramuscular human Aβ1-42, the animals listed in Table 4 were analyzed.

[0140] [Table 5]

[0141] Expression of APOE4 with a signal sequence enhanced the dAβ-induced paralysis phenotype. By adult day 8, approximately 40% of animals expressing Aβ were paralyzed, whereas >90% of animals expressing both Aβ and APOE4 with a signal sequence were paralyzed (Fig. 1). However, the paralysis phenotype of animals expressing Aβ and APOE4 without a signal sequence was similar to that of animals expressing Aβ alone (Fig. 1). In contrast, expression of APOE4 alone in the absence of Aβ expression, with or without a signal sequence, did not induce a paralysis phenotype in adult worms (Fig. 1).

[0142] For the remainder of the studies described herein, a line that expressed APOE4 with a signal sequence (referred to as ssApoE4) was analyzed.

[0143] Example 2.3: Aβ3-42 and human ssAPOE4 conjugated to GFP were significantly increased Human Aβ conjugated to GFP in C. elegans muscle 3-42 It has been reported that expression of human ssApoE4 induces Aβ aggregate formation (Link, CD, Fonte, V., Roberts, CM, Hiester, B., Silverman, MA, and Stein, GH (2008). The beta amyloid peptide can act as a modular aggregation domain. Neurobiol. Dis. 32, 420-425 (which is incorporated herein by reference)). To determine whether expression of human ssApoE4 affects Aβ aggregate formation in C. elegans, we analyzed human Aβ conjugated to GFP. 3-42 The animals listed in Table 5 below were challenged with a standard E. coli OP50 laboratory strain.

[0144] [Table 6]

[0145] When animals reached adulthood, GFP aggregates were counted in the anterior part of the animals. At least 17 animals were recorded for each assay. For each data point, the mean ± standard deviation is presented in the graph in Figure 2. Compared with animals expressing Aβ3-42, animals expressing Aβ3-42 and ssApoE4 had significantly increased GFP aggregates when analyzed using a Student's t-test (P < 0.0001).

[0146] Human Aβ conjugated to GFP 3-42 The number of aggregates in the anterior region of animals expressing Aβ conjugated to GFP compared with that of animals expressing Aβ 3-42 The number of aggregates in animals expressing both human ssApoE4 and human ssApoE4 was significantly increased (Figure 2). Interestingly, it has been reported that Aβ deposition in AD patients is higher in APOE4 carriers than in non-carriers (Dorey, E., Chang, N., Liu, QY, Yang, Z., and Zhang, W. (2014). Apolipoprotein E, amyloid-beta, and neuroinflammation in Alzheimer's disease. Neurosci. Bull. 30, 317-330 (the same reference is incorporated herein by reference)).

[0147] Example 2.4: UbV-GFP is stabilized in animals expressing both human ssAPOE4 and human tau352 (PHP) Hyperphosphorylated tau has been reported to be associated with AD. Expression of pseudohyperphosphorylated tau, which mimics the AD-related modifications, has further been reported to induce progressive age-dependent motor impairment in C. elegans (Brandt, R., Gergou, A., Wacker, I., Fath, T., and Hutter, H. (2009). A Caenorhabditis elegans model of tau hyperphosphorylation: induction of developmental defects by transgenic overexpression of Alzheimer's disease-like modified tau. Neurobiol. Aging 30, 22-33, which is incorporated herein by reference).

[0148] To analyze whether ssAPOE4 modulates tau-induced impairment in C. elegans, we generated transgenic strains expressing pseudohyperphosphorylated human tau and human ssAPOE4. Animals of the appropriate genotype were administered standard E. coli OP50 laboratory strains. No apparent differences in locomotion were observed between strains expressing pseudohyperphosphorylated human tau and human ssAPOE4 compared with strains expressing pseudohyperphosphorylated human tau alone (data not shown).

[0149] Proper proteasome function is important for cellular function, and previous studies in the field have shown that proteasome function is impaired in human AD (Bonet-Costa, V., et al. (2016). The Proteasome and Oxidative Stress in Alzheimer's Disease. Antioxid. Redox Signal. 25, 886-901; Upadhya, SC, and Hegde, AN (2007). Role of the ubiquitin proteasome system in Alzheimer's disease. BMC Biochem. 8, S12; Oddo, S. (2008). The ubiquitin-proteasome system in Alzheimer's disease. J. Cell. Mol. Med. 12, 363-373; Zheng, Q., et al. (2016). Dysregulation of Ubiquitin-Proteasome System in Neurodegenerative Diseases. Front. Aging Neurosci. 8 (each of which is incorporated herein by reference). To determine whether ssApoE4 expression affects proteasome function, animals carrying human ssApoE4 and a marker of impaired proteasome function were generated (Table 6). The proteasome dysfunction marker consisted of non-cleavable ubiquitin fused at the N-terminus to GFP (UbV-GFP).

[0150] [Table 7]

[0151] When the animals reached adulthood, the number of animals expressing GFP in the intestine was counted. At least 30 animals were recorded for each assay. Data from three independent trials are presented in (Figure 3). For each data point, the mean ± standard deviation is presented in the graph. Compared with animals expressing tau352(PHP), animals expressing ssAPOE4 and tau352(PHP) had significantly increased UbV-GFP expression levels when analyzed using a Student's t-test (P<0.0001).

[0152] Generally, UbV-GFP undergoes proteasome-dependent degradation, whereas impaired protein homeostasis leads to GFP stabilization (see, e.g., Figure 3). Minimal or absent GFP expression was observed in animals expressing human ssApoE4. However, UbV-GFP was stabilized in animals expressing both human ssApoE4 and human pseudohyperphosphorylated human tau (Figure 3). Expression of pseudohyperphosphorylated human tau by itself did not induce proteasome stress (Figure 3). This result suggested that expression of human ssApoE4 and human pseudohyperphosphorylated human tau induces proteasome stress.

[0153] It has previously been reported that hyperphosphorylated tau is resistant to proteasomal degradation (Poppek, D., Keck, S., Ermak, G., Jung, T., Stolzing, A., Ullrich, O., Davies, K.J.A., and Grune, T. (2006). Phosphorylation inhibits turnover of the tau protein by the proteasome: influence of RCAN1 and oxidative stress. Biochem. J. 400, 511-520, which is incorporated herein by reference), and tau phosphorylation has been reported to regulate proteasome activity (Ren, Q.-G., Liao, X.-M., Chen, X.-Q., Liu, G.-P., and Wang, J.-Z. (2007). Effects of tau phosphorylation on proteasome activity. FEBS Lett. 581, 1521-1528; Johnson, GVW (2006). Tau phosphorylation and proteolysis: insights and perspectives. J. Alzheimers Dis. JAD 9, 243-250 (the same references are incorporated herein by reference). In a C. elegans transgenic line, human pseudohyperphosphorylated human tau was expressed in neurons, while human ssApoE4 was expressed under the control of an intestinal promoter with a signal sequence that allows it to be secreted from the cells. Induction of UbV-GFP was observed primarily in the intestine of the animals (not shown). The intestine is a large and prominent tissue in C. elegans, which may mask the induction of UbV-GFP in other tissues. However, induction of UbV-GFP in the intestine provided an easy visual screen for potential interventions that modify proteasome function.

[0154] Example 2.5: Microbial strain influences Aβ paralysis Animals of the appropriate genotype were challenged with either a standard laboratory strain of E. coli op50 or an individual microbiome strain. On adult day 4, the number of paralyzed animals was recorded. Data from three independent trials are presented. Graphs show the mean ± standard deviation for each data point. See Table 1 below for raw data, including the number of animals analyzed per condition. To facilitate rapid screening of the microbiome for modulators, we generated a novel transgenic C. elegans strain expressing human ssApoE4, human Aβ1-42, human pseudophosphorylated tau, and the UbV-GFP proteasome marker. This transgenic strain can be used not only to identify interventions that suppress paralysis, but also for drugs that improve proteasome function. Furthermore, this model can be used to identify parameters or features of biological pathways that affect (increase or decrease) paralysis. These parameters or features can be small molecules, metabolites, nucleic acids, proteins, lipids, or even microbiome components. Approximately 1,400 individual microbial strains from the human microbiome were administered to animals carrying human ssApoE4, human Aβ1-42, human pseudophosphorylated tau, and the UbV-GFP proteasome marker. The degree of paralysis was observed. A group of microbial populations was found to increase the paralytic phenotype in animals expressing human ssApoE4, human Aβ1-42, human pseudophosphorylated tau, and the UbV-GFP proteasome marker (Table 7, Figure 4). The increase in paralysis was dependent on the presence of ssAPOE4, as administration of many of these bacteria to animals expressing human Aβ1-42, human pseudophosphorylated tau, and the UbV-GFP proteasome marker did not increase the paralytic phenotype (Table 7, Figure 4).

[0155] [Table 8-1] [Table 8-2]

[0156] Example 2.6: Microbial strains modulate Aβ3-42::GFP aggregation Human ssApoE4, human Aβ 1-42 Microbial populations observed to increase paralysis in C. elegans animals expressing tau, human pseudophosphorylated tau, and the UbV-GFP proteasome marker included Porphyromonas gingivalis (Table 1). P. gingivalis has been identified in the brains of AD patients and associated with neurotoxic tau and amyloid deposition (Dominy, SS, et al. (2019). Porphyromonas gingivalis in Alzheimer's disease brains: Evidence for disease causation and treatment with small-molecule inhibitors. Sci. Adv. 5, incorporated herein by reference). Furthermore, P. gingivalis has been reported to increase ubiquitin loading, suggesting disruption of proteasome function (Dominy et al., 2019, incorporated herein by reference). Oral administration of P. gingivalis has previously been shown to be sufficient to induce brain infection and the induction of Aβ deposits (Dominy et al., 2019, which is incorporated herein by reference).

[0157] Animals were administered either a standard E. coli OP50 laboratory strain or individual microbiome strains. GFP aggregates were counted in the anterior region of the animals when they reached adulthood. GFP aggregates were counted in three animals per condition. The graphs show the mean ± standard deviation for each data point.

[0158] Administration of P. gingivalis increased Aβ 3-42 However, Aβ induced GFP aggregates. 3-42 ::GFP aggregates express human ssApoE4 and Aβ 3-42The increase was significantly higher in animals co-expressing ssApoE4 and ::GFP (Figure 5). This result suggested that the ssApoE4 genotype has a detrimental effect on the increased incidence of AD-related symptoms. Therefore, the identification of known microbial populations previously associated with AD confirms the validity of the transgenic human ssApoE4, pseudophosphorylated tau, and Aβ-expressing C. elegans platform assay. The results support the idea that the other microbial populations discovered may be factors influencing AD risk in humans.

[0159] Interestingly, some E. coli isolates exhibited increased Aβ expression, along with increased paralytic phenotypes in an ssAPOE4-dependent manner. 3-42 We found that ::GFP aggregates were induced by the ::GFP aggregates (Table 7, Figures 4 and 5). This is intriguing, at least because C. elegans are fed the standard non-pathogenic E. coli OP50 strain in the laboratory. Previous studies have shown that Gram-negative bacterial molecules, particularly those derived from E. coli, are associated with AD neuropathology (Zhan, X., Stamova, B., Jin, L.-W., DeCarli, C., Phinney, B., and Sharp, FR (2016). Gram-negative bacterial molecules associate with Alzheimer disease pathology. Neurology 87, 2324-2332, which is incorporated herein by reference). It is possible that these factors are not expressed or are weakly expressed in the E. coli OP50 strain, or this could suggest strain-specific differences. The 11 strains of E. coli tested were classified as strains that had either a mild, moderate, or severe effect on paralysis (Table 7, Figure 4) or Aβ 3-42 ::GFP aggregates were phenotyped (Figure 5). These data suggest that strain-specific differences in microbial populations may contribute to the incidence or severity of AD.

[0160] E. fergusonii and E. albertii also showed trends similar to E. coli. Some isolates of E. fergusonii and E. albertii increased the paralysis phenotype, while other strains had either mild or moderate effects (Table 7). This specific effect on paralysis was also observed in isolates of Klebsiella oxytoca, Klebsiella pneumoniae, and Alcaligenes faecalis. While this may be a general trend for other microbial populations as well, this feature may have been overlooked due to the number of strains analyzed. Thus, among other things, the present disclosure teaches that individual strains of specific microorganisms may have differential effects on biological phenotype(s), including particularly disease-related phenotype(s). In some embodiments, the present disclosure provides techniques for identifying and / or characterizing specific strains and / or their components or combinations that can achieve specific effects on biological phenotypes.

[0161] Additionally, microbiome samples from apparently healthy donors were analyzed. It is conceivable that microbiome samples from AD patients may yield a better trend in identifying strains that may have adverse effects. However, this C. elegans characterization system can be used to assess a patient's microbiome for increased or decreased presence of microbial strains associated with, or alternatively affecting, disease. While metagenomic sequencing of patient populations can identify the diversity of microbial species present in a particular patient or patient population, these methods cannot identify differences in strain levels in patient samples compared to healthy populations. The system of the present invention fills this gap in identifying differences in strain levels in patient(s) and / or patient population(s) that may be important for several diseases or conditions, including AD. Thus, this platform may provide a potential early diagnostic disease predictor. Thus, among other things, the present disclosure provides techniques for defining, assessing, and / or detecting microorganisms and / or their components or combinations (i.e., microbial signature(s)) that may be associated with specific disease states. In some embodiments, such microbial signatures can be detected in patient sample(s) and may be useful, for example, to diagnose disease states, monitor the impact of particular therapies with respect to such disease states, etc.

[0162] Example 2.7: Exemplary Microbial Strains That Affect ATP Production Neuro2A cell line was purchased from ATCC and cultured in EMEM medium supplemented with 10% FBS and 1% L-glutamine. Cells were maintained in a 37°C / 5% CO2 incubator. All experiments were performed using only cells from passages 3 to 7. Neuro2A cells (5 × 10 per well) were cultured in EMEM medium supplemented with 10% FBS and 1% L-glutamine. 4Cells) were plated onto 96-well white-walled plates (Corning) and incubated overnight at 37°C / 5% CO2. Each of 10 bacteria or a combination of all bacteria (CT10) were grown in the following media: supplemented Clostridial Broth, peptone yeast extract glucose broth, MRS broth, and tryptic soy broth. Bacteria were grown at 10 in PBS. 8 The cells were resuspended in 10 CFU and stored at -80°C. 8 CFU of each microorganism (referred to as sample) was added to six wells. The total bacterial combination (CT10) was added to six wells at 10 9 CFU (i.e., 10 8 CFU of each bacterium). For control wells, PBS without bacteria was added. After 16 hours of incubation at 37°C / 5% CO2, 2 μM human amyloid-β1 -42 was added to all wells except for the untreated control wells. Cells were incubated at 37°C / 5% CO2 for 24 hours. Cells were washed three times with PBS and 0.05 ml of Promega CellTiter-Glo, and the plate was incubated at room temperature for 1 hour in the dark. Luminescence (representing ATP levels) was measured using a microplate reader (Promega Discoverer, Promega Corp). ATP levels were normalized to protein content measured by a Bradford protein assay kit (ThermoFisher Scientific). 10 μl of sample was added in duplicate to 150 μl of Bradford reagent in a clear 96-well plate, incubated in the dark at room temperature for 5 minutes, and absorbance was measured at 600 nm using a microplate reader (Promega Discoverer, Promega Corp.). Normalized luminescence was calculated by dividing the luminescence value by the OD protein absorbance value. The average value of triplicate wells for each condition was calculated, and ATP% compared to the control was calculated.

[0163] As shown in Figure 6, 2 μM human amyloid beta 1-42Treatment of Neuro2A cells with human amyloid beta ("sham treatment") caused a significant decrease in ATP production. 1-42 Treatment of Neuro2A cells with Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., or Acidaminococcus sp. in the presence of ATP resulted in a significant increase in ATP production compared to mock-treated cells, while the combination of all bacteria together (CT-10) resulted in a further significant increase in ATP production.

[0164] [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7] [Table 9-8]

[0165] Example 3: Exemplary system for characterizing microbial strains that affect the HIF pathway Example 3.1: HIF Pathway The hypoxia-inducible factor (HIF) pathway mediates various metabolic and physiological adaptations to reduced intracellular oxygen levels. Activation of the HIF pathway promotes erythropoiesis and angiogenesis to reduce cellular demand for oxygen. While the HIF pathway is important for cellular stress responses, constitutive activation of HIF-1 leads to angiogenesis in conditions such as diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, and glaucoma. Therefore, developing modulators of the HIF pathway is essential for the treatment of intraocular neovascular diseases.

[0166] The HIF-1 pathway consists of the HIF transcription factor and a negative regulator, the prolyl hydroxylase EGLN. EGLN functions as an oxygen sensor; in the presence of oxygen, it hydroxylates the HIF α-subunit (HIFα). Hydroxylation of HIFα leads to its binding to, among other factors, the von Hippel-Lindau (VHL) E3 ubiquitin ligase, which promotes HIFα degradation (see Figure 7). Under hypoxic conditions, the HIFα protein is stabilized, which promotes the transcriptional activation of genes required for adaptation to low oxygen levels.

[0167] Example 3.2: Constitutively active HIF-1 leads to egg-laying defects In C. elegans, egl-9 encodes the EGLN homolog. In egl-9 loss-of-function (egl-9 lf) C. elegans mutants, HIF-1 (which is a homolog of HIF1α) protein levels are stabilized. Therefore, HIF1 protein is constitutively active, leading to the continuous activation of HIF-1 transcriptional target genes. To identify microorganisms that regulate the HIF-1 pathway, we analyzed egl-9 loss-of-function (egl-9 lf) C. elegans mutants. egl-9 lf mutant C. elegans have constitutively active HIF-1, which leads to egg-laying defects in them. Therefore, they become egg-expanded as adults.

[0168] Example 3.3: Microbial strain affects HIF-1-induced egg-laying defects HIF-1 modulators were identified by screening individual bacterial strains for their ability to suppress the egg-laying defect of egl-9 lf mutants. Wild-type animals lay 8±2 eggs per hour (n=30), while egl-9 lf mutants lay 2±1 eggs / hour (n=30). Each individual microorganism was administered to egl-9 lf C. elegans mutants, and the egg-laying rate for each was measured. In this assay, Gluconacetobacter spp. and Bifidobacterium spp. were found to significantly increase the egg-laying rate of egl-9 lf C. elegans mutants to 11±2 eggs (n=25) and 8±2 eggs (n=29), respectively (see Table 9). This example demonstrates that microbial strains such as those in Table 9 can modulate HIF-1 and the HIF-1 pathway and can be used to ameliorate pathologies and diseases associated with alterations in the HIF-1 pathway.

[0169] [Table 10]

[0170] Other embodiments Those skilled in the art will appreciate that various changes, modifications, and improvements to the present disclosure will readily occur to those skilled in the art. Such changes, modifications, and improvements are intended to be part of this disclosure and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only, and any inventions described in this disclosure are further detailed by the appended claims.

[0171] Those of ordinary skill in the art will understand the typical standard deviation or error attributable to values obtained in assays or other processes such as those described herein. Publications, websites, and other reference materials referred to herein to describe the background of the invention and to provide additional details regarding its practice are hereby incorporated by reference in their entirety. While embodiments of the invention have been described in conjunction with the detailed description, it should be understood that the above description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. A system comprising a plurality of C. elegans cultures, each culture containing a transgenic C. elegans strain that models a mammalian disease or condition.

2. the plurality of C. elegans cultures (a) 5 or more C. elegans cultures; (b) 10 or more C. elegans cultures; (c) 25 or more C. elegans cultures; or (d) 50 or more C. elegans cultures; 2. The system of claim 1, comprising:

3. 3. The system of claim 1 or 2, wherein one or more of the cultures comprises a transgenic C. elegans strain that models a human disease or condition.

4. 4. The system of any one of claims 1 to 3, wherein one or more of the cultures comprises a transgenic C. elegans strain that models Alzheimer's disease.

5. 4. The system of any one of claims 1 to 3, wherein one or more of the cultures comprises a transgenic C. elegans strain that models a disease or condition associated with an altered or defective HIF-1 pathway.

6. 6. The system of claim 5, wherein the disease or condition associated with an altered or defective HIF-1 pathway comprises a cellular stress response.

7. 6. The system of claim 5, wherein the disease or condition associated with an altered or defective HIF-1 pathway is diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, and glaucoma.

8. 6. The system of claim 5, wherein the disease or condition associated with an altered or defective HIF-1 pathway is an intraocular neovascular disease.

9. 3. The system of claim 1 or 2, wherein one or more of the cultures comprises a transgenic C. elegans strain that models a canine, feline, equine, bovine, ovine, caprine, or porcine disease or condition.

10. 10. The system of any one of claims 1 to 9, wherein one or more of the cultures comprises a transgenic C. elegans strain containing a transgene that includes a reporter gene.

11. The system of claim 10 , wherein the reporter gene encodes a fluorescent, phosphorescent, or bioluminescent protein.

12. 12. The system of any one of claims 1-11, wherein one or more of the cultures comprises a transgenic C. elegans strain comprising a transgene comprising a mammalian gene associated with a mammalian disease or condition.

13. 13. The system of any one of claims 1-12, wherein one or more of the cultures comprises a transgenic C. elegans strain comprising a transgene comprising a mammalian DNA regulatory element associated with a mammalian disease or condition.

14. 14. The system of claim 13, wherein the mammalian DNA regulatory element is or comprises an enhancer, promoter, silencer, insulator, locus control region, or a combination thereof.

15. 15. The system of any one of claims 1-14, wherein one or more of the cultures comprises a transgenic C. elegans strain comprising a transgene encoding a mammalian RNA regulatory element associated with a mammalian disease or condition.

16. 16. The system of claim 15, wherein the mammalian RNA regulatory element is or comprises an untranslated region, an intron, a splice site, or a combination thereof.

17. 17. The system of any one of claims 1 to 16, wherein two or more of the cultures comprise transgenic C. elegans strains that model the same mammalian disease or condition.

18. 18. The system of any one of claims 1 to 17, wherein all of said cultures comprise transgenic C. elegans strains that model the same mammalian disease or condition.

19. The system of any one of claims 1 to 18, wherein all of the cultures contain the same transgenic C. elegans strain.

20. 18. The system of any one of claims 1 to 17, wherein two or more of the cultures comprise transgenic C. elegans strains that model different mammalian diseases or pathologies.

21. 21. The system of any one of claims 1 to 20, wherein one or more of the cultures comprises microorganisms of the mammalian microbiome.

22. 22. The system of any one of claims 1 to 21, wherein each of the cultures comprises microorganisms of a mammalian microbiome.

23. 22. The system of any one of claims 1 to 21, wherein one or more of the cultures comprises microorganisms of the human microbiome.

24. 24. The system of claim 23, wherein each of the cultures comprises microorganisms of the human microbiome.

25. 22. The system of any one of claims 1 to 21, wherein one or more of the cultures comprises microorganisms of the canine, feline, equine, bovine, ovine, caprine, or porcine microbiome.

26. 26. The system of any one of claims 21 to 25, wherein the microbiome is a skin microbiome, an oral microbiome, a nasal microbiome, a gastrointestinal microbiome, a brain microbiome, a lung microbiome, a microbiome, or a urogenital microbiome.

27. 27. The system according to any one of claims 21 to 26, wherein the microorganisms in each culture constitute one or more strains of microorganisms.

28. 28. The system of any one of claims 21 to 27, wherein the microorganisms in each culture constitute a single strain of microorganism.

29. The system of any one of claims 1 to 28, wherein one or more of said cultures constitute a therapeutic or nutritional supplement.

30. A method comprising adding microorganisms obtained from a mammalian microbiome to each of the cultures of the system according to any one of claims 1 to 20.

31. 31. The method of claim 30, wherein the microorganisms added to each culture constitute one or more microbial strains.

32. 32. The method of claim 30 or 31, wherein the microorganisms added to each culture constitute a single strain of microorganism.

33. 33. The method of any one of claims 30 to 32, wherein the mammalian microbiome is a skin microbiome, an oral microbiome, a nasal microbiome, a gastrointestinal microbiome, a brain microbiome, a lung microbiome, a microbiome, or a urogenital microbiome.

34. 34. The method of any one of claims 30 to 33, wherein one or more of the cultures constitute a therapeutic or nutritional supplement.

35. 35. The method of any one of claims 30-34, further comprising determining one or more parameters of the transgenic C. elegans strain in each of said cultures, said one or more parameters being related to the mammalian disease or condition modeled by said transgenic C. elegans strain.

36. 36. The method of claim 35, wherein the one or more parameters comprise levels of a small molecule, protein, polypeptide, or transcript.

37. 37. The method of claim 35 or 36, wherein the one or more parameters comprise the level of activity of the transgenic C. elegans strain.

38. adding a plurality of microbial strains of a mammalian microbiome to a plurality of C. elegans cultures; A different microbial strain was added to each C. elegans culture, The method, wherein each culture contains the same transgenic C. elegans strain, said transgenic C. elegans strain being a model for a mammalian disease or condition.

39. 39. The method of claim 38, further comprising determining whether each of the plurality of microbial strains affects one or more parameters of the transgenic C. elegans strain, wherein the one or more parameters are associated with the mammalian disease or condition that the transgenic C. elegans strain models.

40. determining one or more parameter values of the transgenic C. elegans strain in a C. elegans culture prior to adding the microbial strain to the culture; determining the same one or more parameter values of the transgenic C. elegans strain in the culture after adding the microbial strain to the C. elegans culture; comparing the one or more parameter values determined before adding the microbial strain with the one or more parameter values determined after adding the microbial strain; 40. The method of claim 38 or 39, further comprising:

41. 41. The method of claim 39 or 40, wherein the one or more parameters comprise levels of a small molecule, protein, polypeptide, or transcript.

42. 42. The method of any one of claims 39 to 41, wherein the one or more parameters comprise the level of activity of the transgenic C. elegans strain.

43. 43. The method of any one of claims 38 to 42, wherein the mammalian microbiome is a skin microbiome, an oral microbiome, a nasal microbiome, a gastrointestinal microbiome, a brain microbiome, a lung microbiome, a microbiome, or a urogenital microbiome.

44. 44. The method of any one of claims 38-43, wherein the plurality of C. elegans cultures constitute a therapeutic or nutritional supplement.

45. 45. The method of any one of claims 38-44, wherein the transgenic C. elegans strain models a human disease or condition.

46. 46. The method of any one of claims 38-45, wherein the transgenic C. elegans strain is a model for Alzheimer's disease.

47. 46. The method of any one of claims 38-45, wherein one or more of the cultures comprises a transgenic C. elegans strain that models a disease or condition associated with an altered or defective HIF-1 pathway.

48. 48. The method of claim 47, wherein the disease or condition associated with an altered or defective HIF-1 pathway comprises a cellular stress response.

49. 48. The method of claim 47, wherein the disease or condition associated with an altered or defective HIF-1 pathway is diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, or glaucoma.

50. 48. The method of claim 47, wherein the disease or condition associated with an altered or defective HIF-1 pathway is an intraocular neovascular disease.

51. 1. A method for characterizing microbial strains of the human biome, comprising: adding the microbial strain to a C. elegans culture, including a transgenic C. elegans strain that models Alzheimer's disease; and determining whether the microbial strain affects one or more parameters of the transgenic C. elegans strain, wherein the one or more parameters are associated with Alzheimer's disease.

52. determining one or more parameter values of the transgenic C. elegans strain in the culture prior to adding the microbial strain to the C. elegans culture; determining the same one or more parameter values of the transgenic C. elegans strain in the culture after adding the microbial strain to the C. elegans culture; comparing the one or more parameter values determined before adding the microbial strain with the one or more parameter values determined after adding the microbial strain; 52. The method of claim 51, further comprising:

53. 53. The method of claim 51 or 52, wherein the transgenic C. elegans strain comprises a transgene encoding human ssApoE4 protein, human Aβ1-42 polypeptide, or human pseudophosphorylated tau protein.

54. The one or more parameters are: (i) level of C. elegans paralysis; (ii) amyloid plaque levels; (iii) levels of tau fibrils; (iv) the level of neuroinflammation; (v) the level of proteasome function, or (vi) combinations thereof; 54. The method of any one of claims 51 to 53, comprising:

55. 1. A method for characterizing microbial strains of the human biome, comprising: adding the microbial strain to a C. elegans culture, including a transgenic C. elegans strain that models a disease or condition associated with an altered or defective HIF-1 pathway; and determining whether the microbial strain affects one or more parameters of the transgenic C. elegans strain, wherein the one or more parameters are associated with an altered or defective HIF-1 pathway.

56. determining one or more parameter values of the transgenic C. elegans strain in the culture prior to adding the microbial strain to the C. elegans culture; determining the same one or more parameter values of the transgenic C. elegans strain in the culture after adding the microbial strain to the C. elegans culture; comparing the one or more parameter values determined before adding the microbial strain with the one or more parameter values determined after adding the microbial strain; 56. The method of claim 55, further comprising:

57. 57. The method of claim 55 or 56, wherein the transgenic C. elegans strain comprises a transgene encoding the human prolyl hydroxylase EGLN, a human HIF transcription factor, or a human HIFα protein.

58. The one or more parameters are: (i) the level of neuroinflammation; (ii) the level of proteasome function; (iii) the level of C. elegans egg laying rate, or (iv) combinations thereof; 58. The method of any one of claims 55 to 57, comprising:

59. Use of a system according to any one of claims 1 to 20 for screening a mammalian microbiome for microbial strains that affect a mammalian disease or pathology.

60. 60. The use of claim 59, wherein the mammalian microbiome is a human microbiome.

61. 21. Use of the system according to any one of claims 1 to 20 for characterising the influence that microbial strains of a mammalian microbiome have on a mammalian disease or pathology.

62. Transgenic C. elegans strains expressing two or more of: (i) human ssApoE4, (ii) human Aβ1-42, (iii) human pseudophosphorylated tau, and (iv) the UbV-GFP proteasome marker.

63. A composition comprising one or more microbial strains listed in Table 8.

64. 64. The composition of claim 63, comprising two or more microbial strains listed in Table 8.

65. 64. The composition of claim 63, comprising five or more microbial strains listed in Table 8.

66. 64. The composition of claim 63, comprising 10 or more microbial strains listed in Table 8.

67. A composition comprising Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., L. plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, Acidaminococcus sp., or a combination thereof.

68. 68. The composition of claim 67, comprising at least two microbial strains selected from the group consisting of Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., L. plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium, Bacillus subtilis, and Acidaminococcus sp.

69. 68. The composition of claim 67, comprising at least five microbial strains selected from the group consisting of Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., L. plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium, Bacillus subtilis, and Acidaminococcus sp.

70. 68. The composition of claim 67, comprising Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., L. plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium, Bacillus subtilis, and Acidaminococcus sp.

71. The composition of any one of claims 63 to 70, wherein the composition is a pharmaceutical composition.

72. The composition of any one of claims 63 to 70, wherein the composition is an ingestible item.

73. 73. A method of treating a disease or condition in a subject, comprising administering to a subject in need thereof a composition according to any one of claims 63 to 72.

74. 74. The method of claim 73, wherein the disease or condition is a neurodegenerative disease or disorder.

75. 74. The method of claim 73, wherein the disease or condition is Alzheimer's disease.

76. 74. The method of claim 73, wherein the disease or condition is associated with an altered or defective HIF-1 pathway.

77. 74. The method of claim 73, wherein the disease or condition is an intraocular neovascular disease or disorder.

78. 74. The method of claim 73, wherein the disease or condition is diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, or glaucoma.

79. 73. Use of a composition according to any one of claims 63 to 72 in the treatment of a disease or condition in a subject.

80. 80. The use of claim 79, wherein the disease or condition is a neurodegenerative disease or disorder.

81. 80. The use of claim 79, wherein the disease or condition is Alzheimer's disease.

82. 80. The use of claim 79, wherein the disease or condition is associated with an altered or defective HIF-1 pathway.

83. 80. The use of claim 79, wherein the disease or condition is an intraocular neovascular disease or disorder.

84. 80. The use of claim 79, wherein the disease or condition is diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, or glaucoma.

Images