METHODS AND USES OF MICROBIOME COMPOSITIONS, COMPONENTS, OR METABOLITES FOR TREATING INSULIN-ASSOCIATED DISEASES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to United States Provisional Patent Application No. 63/407,592 filed September 16, 2022, the entire contents of which are hereby incorporated by reference in their entirety.

BACKGROUND

[0002] Many insulin-associated diseases, disorders, or conditions including, but not limited to, diabetes, obesity, cardiovascular disease, non-alcoholic fatty liver disease, Wolfram syndrome, metabolic syndrome, insulin resistance, diabetic ketoacidosis, hyperosmolar hyperglycemic state, gestational diabetes, diabetic dermadromes, diabetic neuropathy, diabetic foot ulcers, maturity onset diabetes of the young, pancreatogenic diabetes, Polycystic ovary syndrome (PCOS), or Alzheimer’s Disease (AD), can cause degeneration of various cells (e.g. liver cells, pancreatic cells, etc.), and impact physical and/or mental functions. Currently, there are no effective treatments for such diseases and finding new drugs or treatment methods is a priority.

SUMMARY

[0003] The present disclosure provides an insight that compositions (e.g. microbiome compositions) as described herein may be used to treat diseases, disorders, or conditions (e.g. associated with insulin levels (e.g. an insulin-associated disease, disorder, or condition (e.g. diabetes, obesity, cardiovascular disease, non-alcoholic fatty liver disease, Wolfram syndrome, metabolic syndrome, AD, etc.))) in a subject (e.g. a mammal (e.g. human, mice, etc.)). Among other things, the present disclosure describes technologies that can be used to treat, prevent, and/or reduce the risk of a disease, disorder, or condition (e.g. associated with insulin levels). In some embodiments, the present disclosure describes compositions and methods to evaluate the effects of administering such compositions (e.g. microbiome compositions as described herein) to a subject and/or to identify or characterize effects and/or modulation of levels of metabolites or a metabolome in a subject upon administration of such compositions. In some embodiments, the metabolites that may be modulated may be associated with certain diseases, disorders, or conditions. In some embodiments, such technologies can be useful to discern metabolite-level differences in a particular subject (e.g., patient) or population (e.g. before and after administration of disclosed compositions). Accordingly, the present disclosure also provides technologies that can be useful to identify and/or assess the nature and effect of disclosed compositions in specific subjects (e.g., patients) and/or populations and thus provide subject- specific information on how to treat a disease, disorder, or condition (e.g. insulin-associated diseases, disorders, or conditions) in an individual subject or individual population. For example, in some embodiments, technologies provided herein can be useful to identify subject- specific compositions, based on the metabolome in subject-specific samples, and treat and/or prevent a disease, disorder, or condition (e.g. insulin-associated diseases, disorders, or conditions) by administering disclosed compositions (e.g. subject-specific compositions) (e.g. to modulate subject’s metabolome). Thus, technologies described herein may be useful as therapeutics and tools for reducing the risk of certain diseases, disorders, or conditions (e.g. insulin-associated diseases, disorders, or conditions), and for treating and/or preventing such diseases, disorders, or conditions.

[0004] Among other things, the present disclosure provides a method of treating or preventing an insulin-associated disease, disorder, or condition. In some embodiments, a method comprises administering to a subject a composition comprising one or more microbial strains or microbial components. In some embodiments, a method comprises administering to a subject a composition comprising one or more microbial metabolites. In some embodiments, a method comprises administering to a subject a composition comprising: (i) one or more microbial strains or microbial components, or (ii) one or more microbial metabolites. In some embodiments, an insulin-associated disease, disorder, or condition is or comprises diabetes, obesity, cardiovascular disease, non-alcoholic fatty liver disease, Wolfram syndrome, metabolic syndrome, insulin resistance, diabetic ketoacidosis, hyperosmolar hyperglycemic state, gestational diabetes, diabetic dermadromes, diabetic neuropathy, diabetic foot ulcers, maturity onset diabetes of the young, pancreatogenic diabetes, or Polycystic ovary syndrome (PCOS). In some embodiments, an insulin-associated disease, disorder, or condition is or comprises diabetes.

[0005] In some embodiments, a subject has been diagnosed with or is at high risk of developing an insulin-associated disease, disorder, or condition. In some embodiments, a subject is animal. In some embodiments, a subject is a mammal, e.g., a mammal that experiences or is susceptible to a disease, disorder, or condition as described herein. In some embodiments, an animal is a vertebrate, e.g., a mammal, such as a non-human primate, (particularly a higher primate), a sheep, a dog, a rodent (e.g. a mouse or rat), a guinea pig, a goat, a pig, a cat, a rabbit, or a cow. In some embodiments, an animal is a non-mammal animal, such as a chicken, an amphibian, a reptile, or an invertebrate. Tn some embodiments, a subject is a human.

[0006] In some embodiments, a subject is suffering from or susceptible to one or more insulin-associated disease, disorder, or condition as described herein. In some embodiments, a subject displays one or more symptoms of one or more insulin-associated disease, disorder, or condition as described herein. In some embodiments, a subject has been diagnosed with one or more insulin-associated disease, disorder, or condition as described herein. In some embodiments, the subject is receiving or has received certain therapy to diagnose and/or to treat one or more insulin-associated disease, disorder, or condition.

[0007] In some embodiments, one or more microbial strains are from a mammalian microbiome. In some embodiments, one or more microbial strains are from a human microbiome. In some embodiments, a human microbiome is the microbiome of the subject. In some embodiments, a human microbiome is administered to maintain or modulate the microbiome of the subject.

[0008] In some embodiments, one or more microbial components or microbial metabolites are selected from Appendix 1, Appendix 3, or Appendix 4. In some embodiments, metabolites can be from one or more microbial strains. In some embodiments, metabolites can be from a source that is not a microbial strain, e.g., synthetically generated. In some embodiments, one or more microbial metabolites is or comprises a bile acid. In some embodiments, one or more microbial metabolites is or comprises Tauroursodeoxycholic acid. In some embodiments, one or more microbial components or microbial metabolites is Butyrylcamitine, Theobromine, p-Hydroxyphenylpyruvic acid, Propionic acid, Picolinic acid, 2- Hydroxy-4methylvaleric acid, N6-Acetylysine, Urocanic acid, N5-Ethylglutamine, Trigonelline, Stachydrine, Ectoine, 5-Hydroxylysine, Arginine (arg), Cholic acid, 2-(4- Hydroxyphenyl)propionic acid, N-Acetyltryptophan, Hydroxyproline, Argininosuccinic acid, Glutamic acid (Glu), Sarcosine, 5-Methoxyindoleacetic acid, Indole-3-lactic acid, Isovalerylalanine, N-Acetylleucine, 1 -Methylhistidine, N-Acetylephenylalanine, Proline (Pro), or any combination thereof. In some embodiments, one or more microbial components or microbial metabolites is 4-Hydroxyphenylpyruvic, Ectoine, Gramine, N-Acetyl-L-phenylalanine, Nepsilon-Acetyl-L-lysine, Stachydrine, Trigonelline, 3-Ureidopropionic acid, Theobromine, Hippuric acid, Imidazolepropionic acid, NG-Methyl-L-arginine, trans-Urocanic Acid, N-Acetyl- L-leucine, Sarcosine, Isobutyrylcarnitine, b-Hydroxyisovaleric acid, L-Theanine/N5- Ethylglutamine, 5-Hydroxylysine, Phenaceturic acid, betaine, hydroxyproline, Picolinic acid, 2- Aminoadipic acid, Glycerophosphocholine, carnitine, Glycerol 3 -phosphate, Argininosuccinic acid, creatine, Terephthalic acid, Homocitrulline, Mucic acid, Homocysteinesulfinic acid, Trimethyllysine, Spermidine, Glyoxylic acid, XA0013 C6H6O4S, 3-Indoxylsulfuric acid, Nicotinamide, N-Formyl glycine, Ureidoglycolate, N-Methylproline, Glucaric acid, Butyrylcarnitine, Methionine sulfoxide, Carboxymethyllysine, Glycolic acid, Phenaceturic acid, Diethanolamine, Phosphorylcholine, Guanidinosuccinic acid, N-Acetylhistidine, Glyceric acid, S-Methylmethionine, Cysteine glutathione disulfide, Kynurenine, N-Acetylphenylalanine, Threonic acid, Malic acid, 7,8-Dihydrobiopterin, Homovanillic acid, Taurocholic acid, 5- Methoxyindoleacetic acid, butyrate, b-Hydroxyisovaleric acid, 2-Oxoglutaric acid, N- Acetyltryptophan, Thiaproline, Hypotaurine, Cholic acid, Acetoacetic acid, Ethanolamine, Guanidoacetic acid, S-Sulfocysteine, Myristic acid C14:0 XA0027, or any combination thereof.

[0009] In some embodiments, one or more microbial strains are or comprise Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum , Clostridium butyricum, Paenibacillus sp., Veil lone I la sp., Bifidobacterium sp., Bacillus subtilis, Acidaminococcus sp., or a combination thereof. In some embodiments, one or more microbial strains are or comprise Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella sp. , Bifidobacterium sp., or a combination thereof. In some embodiments, one or more microbial strains are or comprise Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus catus, Lactobacillus plantarum, Veillonella atypica, Bifidobacterium breve, or a combination thereof. In some embodiments, one or more microbial strains is or comprises Bacillus subtilis.

[0010] In some embodiments, a composition comprises two or more microbial strains. In some embodiments, a composition comprises five or more microbial strains. In some embodiments, a composition comprises ten or more microbial strains.

[0011] In some embodiments, a composition is administered topically, orally, subcutaneously, intravenously, intramuscularly, intracerebrally, intrathecally, rectally, opthalmically, intravitreally, or suprachoroidally. In some embodiments, a composition is administered orally. In some embodiments, a composition is administered intravenously.

[0012] In some embodiments, a composition is formulated as a syrup, a liquid, a tablet, a troche, a gummy, a capsule, a powder, a gel, a film, an injection, or an eye drop.

[0013] In some embodiments, each microbial strain of the one or more microbial strains is present in the composition at a concentration from 10 ¹ to 10 ¹⁵ CFU. In some embodiments, each microbial strain of the one or more microbial strains is present in the composition at a concentration of at least 10 ⁶ CFU. In some embodiments, each microbial strain of one or more microbial strains in a composition comprises 10 ¹ colony forming units (CFUs) to 10 ²⁰ CFU. In some embodiments, each microbial strain of one or more microbial strains in a composition comprises 10 ¹ colony forming units (CFUs) to 10 ¹⁵ CFU In some embodiments, each microbial strain of one or more microbial strains in a composition comprises 10 ⁶ CFU to 10 ¹⁵ CFUs. In some embodiments, each microbial strain of one or more microbial strains in a composition comprises about 10 ¹ CFU to 10 ⁱ⁵ CFU, or about 10 ² CFU to 10 ⁱ⁴ CFU, or about 10 ³ CFU to 10 ⁱ³ CFU, or about 10 ⁴ CFU to 10 ¹³ CFU, or about 10 ⁵ CFU to 10 ¹² CFU, or about 10 ⁶ CFU to 10 ¹¹ CFU, or about 10 ⁷ CFU to 10 ¹⁰ CFU, or about 10 ⁸ CFU to 10 ⁹ CFU, or about 10 ⁵ CFU to 10 ¹⁰ CFU, or about 10 ⁸ CFU to 10 ¹² CFU. In some embodiments, each microbial strain of one or more microbial strains in a composition comprises at least about 10 ¹ , 5 x 10 ¹ , 10 ² , 5 x 10 ² , 10 ³ , 5 x 10 ³ , 10 ⁴ , 5 x 10 ⁴ , 10 ⁵ , 5 x 10 ⁵ , 10 ⁶ , 5 x 10 ⁶ , 10 ⁷ , 5 x 10 ⁷ , 10 ⁸ , 5 x 10 ⁸ , 10 ⁹ , 5 x 10 ⁹ , 10 ¹⁰ , 5 x 10 ¹⁰ , 10 ¹¹ , 5 x I0 ¹¹ , 10 ¹² , or more CFUs. In some embodiments, each of one or more microbial strains in a composition comprises at most about 10 ¹⁵ , 5 x 10 ¹⁴ , 10 ¹⁴ , 5 x 10 ¹³ , 10 ¹³ , 5 x 10 ¹² , 10 ¹² , 5 x I0 ¹¹ , 10 ¹¹ , 5 x 10 ¹⁰ , 10 ¹⁰ , 5 x 10 ⁹ , 10 ⁹ , 5 x 10 ⁸ , 10 ⁸ , or less CFUs. In some embodiments, each microbial strain of one or more microbial strains in a composition comprises same number of CFUs. In some embodiments, some microbial strains of one or more microbial strains in a composition comprises a different number of CFUs.

[0014] The present disclosure provides, among other things, a composition for treating or a composition for use in treating an insulin-associated disease, disorder, or condition comprising one or more microbial strains, microbial components thereof, or microbial metabolites thereof. In some embodiments, a composition, as described herein, comprises one or more microbial metabolites (e.g. derived from sources other than microbial strains (e.g. synthetically derived), derived from one or more microbial strains), wherein the composition is for treating an insulin- associated disease, disorder, or condition.

[0015] The present disclosure provides a composition comprising one or more microbial strains selected from Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum , Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, Acidaminococcus sp., or a combination thereof. In some embodiments, a composition comprises one or more microbial strains selected from Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella atypica, Bifidobacterium sp., or a combination thereof. In some embodiments, a composition comprises a microbial strain. In some embodiments, a microbial strain is Bacillus subtilis. In some embodiments, a composition comprises at least two microbial strains selected from a group consisting of Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, Acidaminococcus sp., or a combination thereof. In some embodiments, a composition comprises at least two microbial strains selected from a group consisting of Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella atypica, Bifidobacterium sp., or a combination thereof. In some embodiments, a composition comprises at least five microbial strains selected from a group consisting of Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, Acidaminococcus sp., or a combination thereof. In some embodiments, a composition comprises at least five microbial strains selected from a group consisting of Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella atypica, Bifidobacterium sp., or a combination thereof. In some embodiments, a composition comprises or consists of Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, Acidaminococcus sp.. In some embodiments, a composition comprises or consists of Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella atypica, Bifidobacterium sp..

[0016J In some embodiments, a composition, as described herein, comprises one or more microbial metabolites (e.g. derived from sources other than microbial strains (e.g. synthetically derived), derived from one or more microbial strains), wherein the composition is for treating an insulin-associated disease, disorder, or condition.

[0017] In some embodiments, a composition is for topical, oral, subcutaneous, intravenous, intramuscular, intracerebral, intrathecal, rectal, opthalmical, intravitreal, or suprachoroidal administration. In some embodiments, a composition is for oral administration. In some embodiments, a composition is for intravenous administration.

[0018] The present disclosure provides, among other things, a composition for use in treating an insulin-associated disease, disorder, or condition comprising one or more microbial strains or microbial components. In some embodiments, a composition for use in treating an insulin-associated disease, disorder, or condition comprises one or more microbial metabolites.

[0019] The present disclosure provides that a composition as described herein is for use in modulating one or more microbial metabolites in a subject. In some embodiments, a composition is for use in modulating one or more features in a subject. In some embodiments, one or more features is or comprises: (i) level of cell viability; (ii) level or activity of a nucleic acid or protein, or form thereof; (iii) body weight gain; (iv) fat accumulation in liver; (v) lipid accumulation in liver; (vi) blood triglyceride levels; (vii) blood-cholesterol levels; (viii) oxidative stress; or (ix) inflammation.

[0020] In some embodiments, a composition is for use characterizing the ability of one more microbial strains to modulate one or more microbial metabolites in a subject.

[0021] The present disclosure provides that a use of a composition as described herein is for treating or ameliorating a disease, disorder, or condition in a subject, wherein the disease, disorder, or condition is an insulin-associated disease, disorder, or condition associated with one or more microbial metabolites. In some embodiments, a use of a composition as described herein is for treating or ameliorating diabetes.

[0022] The present disclosure provides a method of screening a microbial strain, comprising contacting the microbial strain to a culture comprising pancreatic cells or pancreatic cell lines that model an insulin-associated disease, disorder, or condition, and determining whether the microbial strain altered a feature of the culture, wherein the feature is associated with the insulin-associated disease, disorder, or condition.

[0023] In some embodiments, a step of determining comprises comparing the feature before and after performance of the step of contacting. In some embodiments, a step of determining comprises comparing the feature after the step of contacting with a comparable reference.

[0024] In some embodiments, a comparable reference is a historical reference. In some embodiments, a comparable reference is a negative control reference. In some embodiments, a comparable reference is a positive control reference.

[0025] In some embodiments, a feature is a level of cell viability. In some embodiments, a feature is level or activity of a nucleic acid or protein, or form thereof. In some embodiments, a feature is or comprises weight gain. In some embodiments, a feature is or comprises fat accumulation in liver cells. In some embodiments, a feature is or comprises lipid accumulation in liver cells. In some embodiments, a feature is or comprises triglyceride levels. In some embodiments, a feature is or comprises cholesterol levels. In some embodiments, a feature is or comprises inflammation.

[0026] In some embodiments, a microbial strain may alter one or more features of the culture. In some embodiments, one or more features is associated with the insulin-associated disease, disorder, or condition. In some embodiments, one or more features is or comprises (i) level of cell viability; (ii) level or activity of a nucleic acid or protein, or form thereof; (iii) body weight gain; (iv) fat accumulation in liver; (v) lipid accumulation in liver; (vi) blood triglyceride levels; (vii) blood-cholesterol levels; (viii) oxidative stress; or (ix) inflammation. [0027] The present disclosure provides a method comprising administering to a subject a composition comprising one or more microbial strains or microbial components. In some embodiments, the present disclosure provides a method comprising administering to a subject a composition comprising one or more microbial metabolites. In some embodiments, microbial metabolites can be from one or more microbial strains. In some embodiments, microbial metabolites can be from a source that is not a microbial strain, e g., synthetically generated.

[0028] In some embodiments, a microbial strain, a microbial component, or a microbial metabolite may alter a feature of the subject. In some embodiments, a microbial strain, a microbial component, or a microbial metabolite may alter one or more features of the subject. In some embodiments, feature is a level of cell viability Tn some embodiments, a feature is level or activity of a nucleic acid or protein, or form thereof. In some embodiments, a feature is or comprises body weight gain. In some embodiments, a feature is or comprises fat accumulation in liver. In some embodiments, a feature is or comprises lipid accumulation in liver. In some embodiments, a feature is or comprises triglyceride levels. In some embodiments, a feature is or comprises cholesterol levels. In some embodiments, a feature is or comprises inflammation.

[0029] In some embodiments, a feature is associated with the insulin-associated disease, disorder, or condition.

[0030] The present disclosure provides a method of characterizing a method of characterizing a microbial strain, comprising adding the microbial strain to a culture comprising one or more pancreatic cells or one or more pancreatic cell lines that model an insulin-associated disease, disorder, or condition, and determining whether the microbial strain affects levels of one or more features of the one or more pancreatic cells or pancreatic cell lines, wherein the one or more features are associated with the insulin-associated disease, disorder, or condition.

[0031] The present disclosure provides a method of manufacturing a pharmaceutical treatment comprising characterizing one or more microbial strains, microbial components, or microbial metabolites comprising the steps of adding the one or more microbial strains to a culture comprising one or more pancreatic cells or one or more pancreatic cell lines that model an insulin-associated disease, disorder, or condition, and determining whether the one or more microbial strains affect levels of one or more features of the one or more pancreatic cells or pancreatic cell lines, wherein the one or more features are associated with the insulin-associated disease, disorder, or condition.

[0032] The present disclosure provides a method of manufacturing a pharmaceutical treatment comprising formulating one or more microbial strains or microbial components in a syrup, a liquid, a tablet, a troche, a gummy, a capsule, a powder, a gel, a fdm, an injection, or an eye drop. The present disclosure provides a method of manufacturing a pharmaceutical treatment comprising formulating one or more microbial metabolites in a syrup, a liquid, a tablet, a troche, a gummy, a capsule, a powder, a gel, a fdm, an injection, or an eye drop.

[0033] The present disclosure provides a method of assessing a microbial strain for the ability to affect one or more features of a culture, comprising adding the microbial strain to the culture comprising one or more pancreatic cells or one or more pancreatic cell lines that model an insulin-associated disease, disorder, or condition, and determining whether the microbial strain affects levels of one or more features of the one or more pancreatic cells or pancreatic cell lines, wherein the one or more features are associated with the insulin-associated disease, disorder, or condition.

[0034] In some embodiments, a method further comprises before adding the microbial strain to the culture, determining levels of one or more features of the one or more pancreatic cells or pancreatic cell lines in the culture, after adding the microbial strain to the culture, determining the levels of the same one or more features of the one or more pancreatic cells or pancreatic cell lines in the culture, and comparing the levels of one or more features determined before adding the microbial strain with the levels of one or more features determined after adding the microbial strain.

[0035] In some embodiments, one or more features includes (i) level of cell viability; (ii) level or activity of a nucleic acid or protein, or form thereof; (iii) body weight gain; (iv) fat accumulation in liver; (v) lipid accumulation in liver; (vi) blood triglyceride levels; (vii) blood- cholesterol levels; (viii) oxidative stress; (ix) inflammation; or (x) a combination thereof.

[0036] The present disclosure provides that a composition as described herein is for use in treating or preventing an insulin-associated disease, disorder, or condition, comprising one or more microbial strains or microbial components. The present disclosure further provides that a composition as described herein is for use in treating or preventing an insulin-associated disease, disorder, or condition, comprising one or more microbial metabolites. In some embodiments, one or more microbial components or microbial metabolites of a one or more microbial strains are selected from Appendix 1, Appendix 3, or Appendix 4.

[0037] In some embodiments, a composition as described herein is for use in treating or preventing an insulin-associated disease, disorder, or condition is diabetes.

[0038] The present disclosure provides an injection comprising the composition as described herein.

[0039] The present disclosure provides a food supplement comprising a composition as described herein.

[0040] The present disclosure provides a kit comprising a composition as described herein for use in treating or preventing an insulin-associated disease, disorder, or condition. In some embodiments, a kit comprises a monitoring device. In some embodiments, a monitoring device is a blood-sugar monitor.

[0041] These, and other aspects encompassed by the present disclosure, are described in more detail below and in the claims.

DEFINITIONS

[0042] The scope of the present invention is defined by the claims appended hereto and is not limited by certain embodiments described herein. Those skilled in the art, reading the present specification, will be aware of various modifications that may be equivalent to such described embodiments, or otherwise within the scope of the claims. In general, terms used herein are in accordance with their understood meaning in the art, unless clearly indicated otherwise. Explicit definitions of certain terms are provided below; meanings of these and other terms in particular instances throughout this specification will be clear to those skilled in the art from context.

[0043] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0044] The articles “a” and “an,” as used herein, should be understood to include the plural referents unless clearly indicated to the contrary. Claims or descriptions that include “or” between one or more members of a group are considered satisfied 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 indicated to the contrary or otherwise evident 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. It is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists (e.g, in Markush group or similar format), it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where embodiments or aspects are referred to as “comprising” particular elements, features, etc., certain embodiments or aspects “consist,” or “consist essentially of,” such elements, features, etc. For purposes of simplicity, those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification.

[0045] Administration. As used herein, the term “administration” typically refers to the administration of a composition to a subject or system to achieve delivery of an agent to the subject or system. In some embodiments, the agent is, or is included in, the composition; in some embodiments, the agent is generated through metabolism of the composition or one or more components thereof. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g, by bronchial instillation), buccal, dermal (which may be or comprise, 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, within a specific organ (e. g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In many embodiments provided by the present disclosure, administration is oral administration. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time. Administration of cells can be by any appropriate route that results in delivery to a desired location in a subject where at least a portion of the delivered cells or components of the cells remain viable. A period of viability of cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, i.e., long-term engraftment. In some embodiments, administration comprises delivery of a bacterial extract or preparation comprising one or more bacterial metabolites and/or byproducts but lacking fully viable bacterial cells.

[0046] Analog. As used herein, the term “analog” refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an “analog” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, an analog is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance. In some embodiments, an analog is or can be generated through performance of a synthetic process different from that used to generate the reference substance.

[0047] Approximately . As applied to one or more values of interest, includes to a value that is similar to a stated reference value Tn certain embodiments, the term “approximately” or “about” refers to a range of values that fall within ±10% (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0048] Comparable. As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, subjects, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0049] Conservative: As used herein, refers to instances when describing a conservative amino acid substitution, including a substitution of an amino acid residue by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of interest of a protein, for example, the ability of a receptor to bind to a ligand. Examples of groups of amino acids that have side chains with similar chemical properties include: aliphatic side chains such as glycine (Gly, G), alanine (Ala, A), valine (Vai, V), leucine (Leu, L), and isoleucine (He, 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 (Gin, 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 acids substitution groups include, for example, valine/leucine/isoleucine (Val/Leu/Tle, V/L/T), phenylalanine/tyrosine (Phe/Tyr, FAT), lysine/arginine (Lys/ Arg, K/R), alanine/valine (Ala/Val, A/V), glutamate/aspartate (Glu/Asp, E/D), and asparagine/glutamine (Asn/Gln, N/Q). In some embodiments, a conservative amino acid substitution can be a substitution of any native residue in a protein with alanine, as used in, for example, alanine scanning mutagenesis. In some embodiments, a conservative substitution is made that has a positive value in the PAM250 log- likelihood matrix disclosed in Gonnet, G.H. et al., 1992, Science 256:1443-1445, which is incorporated herein by reference in its entirety. In some embodiments, a substitution is a moderately conservative substitution wherein the substitution has a nonnegative value in the PAM250 log-likelihood matrix.

[0050] Control. As used herein, refers to the art-understood meaning of a “controF being a standard against which results are compared. Typically, controls are used to augment integrity in experiments by isolating variables in order to make a conclusion about such variables. In some embodiments, a control is a reaction or assay that is performed simultaneously with a test reaction or assay to provide a comparator. A “controF also includes a “ control animal ” A “control animaF may have a modification as described herein, a modification that is different as described herein, or no modification (i.e., a wild-type animal). In one experiment, a "test" (i.e., a variable being tested) is applied. In a second experiment, the “control.” the variable being tested is not applied. In some embodiments, a control is a historical control (i.e., of a test or assay performed previously, or an amount or result that is previously known). In some embodiments, a control is or comprises a printed or otherwise saved record. A control may be a positive control or a negative control.

[0051] Determining, measuring, evaluating, assessing, assaying and analyzing Determining, measuring, evaluating, assessing, assaying and analyzing are used interchangeably herein to refer to any form of measurement, and include determining if an element is present or not. These terms include both quantitative and/or qualitative determinations. Assaying may be relative or absolute. “Assaying for the presence of’ can be determining the amount of something present and/or determining whether or not it is present or absent.

[0052] Dosage form. Those skilled in the art will appreciate that the term “dosage form” may 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 quantity of agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen). Those of ordinary skill in the art appreciate that the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.

[0053] Dosing regimen. Those skilled in the art will appreciate that the term “dosing regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population.

[0054] Engineered. In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a cell or organism is considered to be “engineered” if it has been manipulated so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols). As is common practice and is understood by those in the art, progeny of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.

[0055] Excipient. As used herein, refers to an inactive (e.g. , non-therapeutic) agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect. In some embodiments, suitable pharmaceutical excipients may include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. [0056] Functional. As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized. A biological molecule may have two functions (i.e., bifunctional) or many functions (i.e., multifunctional).

[0057] Gene. As used herein, refers to a DNA sequence in a chromosome that codes for a product (e.g., an RNA product and/or a polypeptide product). In some embodiments, a gene includes coding sequence (i.e., sequence that encodes a particular product). In some embodiments, a gene includes non-coding sequence. In some particular embodiments, a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequence. In some embodiments, a gene may include one or more regulatory sequences (e.g., promoters, enhancers, etc.) and/or intron sequences that, for example, may control or impact one or more aspects of gene expression (e.g., cell-type-specific expression, inducible expression, etc.). For the purpose of clarity, we note that, as used in the present disclosure, the term “gene” generally refers to a portion of a nucleic acid that encodes a polypeptide or fragment thereof; the term may optionally encompass regulatory sequences, as will be clear from context to those of ordinary skill in the art. This definition is not intended to exclude application of the term “gene” to non-protein- coding expression units but rather to clarify that, in most cases, the term as used in this document refers to a polypeptide-coding nucleic acid.

[0058] Improve, increase, enhance, inhibit or reduce As used herein, the terms “improve,” “increase,” “enhance,” “inhibit,” “reduce,” or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, a value is statistically significantly difference that a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment. In some embodiments, an appropriate reference is a negative reference; in some embodiments, an appropriate reference is a positive reference. [0059] Isolated. As used herein, refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) designed, produced, prepared, and/or manufactured by the hand of man. In some embodiments, an isolated substance or entity may be enriched; 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 initially associated. In some embodiments, isolated agents are 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 more 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 skilled in the art, a substance may still be considered “enriched”, "isolated" or even "pure", after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g, buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients. Those skilled in the art are aware of a variety of technologies for isolating (e.g, enriching or purifying) substances or agents (e.g, using one or more of fractionation, extraction, precipitation, or other separation).

[0060] Level. As used herein, the term “level” refers to a scale of amount or quantity of a substance (e.g., a metabolite). In some embodiments, a level can be simply the presence or absence of a substance. A level of a substance may be represented in multiple ways or formats. For example, in some embodiments, a level may be represented as a percentage (%), a measure of weight (e.g., mg, pg, ng, etc.), a measure of concentration (e.g., mg/mL, pg/mL, ng/mL, etc.), a measure of volume (e.g., mb, pL, nL, etc.), in % change, etc.

[0061] Metabolite. As used herein, the term “metabolite” refers to a substance (e.g., a small molecule, macromolecule, organic compound, or inorganic compound) made or used during metabolism. Metabolism is generally understood as a process by which a substance (e.g., food, drug, chemical, cell, or tissue) is chemically broken down. In some embodiments, a metabolite is an end product. Tn some embodiments, a metabolite is an intermediate. Exemplary metabolites are provided herein, e.g., in Appendix 1-1, 1-3, and 3. Exemplary metabolic pathways are provided herein, e.g., in Appendix 1-2. In some embodiments, a metabolite may be produced or made by an organism. In some embodiments, a metabolite may be produced or made by a microorganism (e.g. microbial strain). In some embodiments, a microbial metabolite produced or made by a microbial strain. In some embodiments, a metabolite may be produced or made naturally (e.g. by an organism (e.g. microorganism (e.g. microbial strain))). In some embodiments, a metabolite may be produced or made synthetically (e.g. from a source that is not a microbial strain (e.g., synthetically generated)).

[0062] Pharmaceutical composition. As used herein, the term “pharmaceutical composition” refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, a pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those adapted for the following: ophthalmic administration, intravitreal administration, suprachoroidal administration, oral administration, subcutaneous administration, intravenous administration, intramuscular administration, intracerebral administration, intrathecal administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue, capsules, powders, etc. In some embodiments, an active agent may be or comprise a cell or population of cells (e.g., a culture, for example of an Ellagitannin-Enzyme-Synthesizing (EES) microbe); in some embodiments, an active agent may be or comprise an extract or component of a cell or population (e.g., culture) of cells. In some embodiments, an active agent may be or comprise an isolated, purified, or pure compound. In some embodiments, an active agent may have been synthesized in vitro (e.g., via chemical and/or enzymatic synthesis). In some embodiments, an active agent may be or comprise a natural product (whether isolated from its natural source or synthesized in vitro).

[0063] Pharmaceutically acceptable. As used herein, the term "pharmaceutically acceptable" which, for example, may be used in reference to a carrier, diluent, or excipient used to formulate a pharmaceutical composition as disclosed herein, means that the carrier, diluent, or excipient is compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0064] 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 fdler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject (e.g., 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 carboxymethyl cellulose, ethyl cellulose 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; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0065] Prebiotic. As used herein, a “prebiotic” refers to an ingredient that allows or promotes specific changes, both in the composition and/or activity in the gastrointestinal microbiota that may (or may not) confer benefits upon the host. In some embodiments, a prebiotic can include one or more of the following: the prebiotic comprises a pome extract, berry extract and walnut extract.

[0066] Prevention. The term “prevention”, as used herein, refers to a delay of onset, and/or reduction in 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 decrease in the development, 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, when onset of a disease, disorder or condition has been delayed for a predefined period of time.

[0067] Reference. As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control. In some embodiments, a reference is a negative control reference; in some embodiments, a reference is a positive control reference.

[0068] Risk As will be understood from context, “risk” of a disease, disorder, and/or condition refers to a 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 from 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 a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and/or event. In some embodiments a reference sample or group of reference samples are from individuals comparable to a particular individual. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0069] 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, a source of interest is a biological or environmental source. In some embodiments, a source of interest may be or comprise a cell or an organism, such as a microbe, a plant, or an animal (e.g., a human). In some embodiments, a source of interest is or comprises biological tissue or fluid. In some embodiments, a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humour, vomit, plasma, mucous, digestive fluid, stool, and/or combinations or component(s) thereof. In some embodiments, a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and/or a transcellular fluid. In some embodiments, a biological fluid may be or comprise a plant exudate. In some embodiments, a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g, fine needle or tissue biopsy), swab (e.g, oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g, bronchioalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and/or purification of certain components, etc.

[0070] Small molecule As used herein, the term “small molecule” refers to small organic or inorganic molecules of molecular weight below about 3,000 Daltons. In general, small molecules may have a molecular weight of less than 3,000 Daltons (Da). Small molecules can be, e.g., from at least about 100 Da to about 3,000 Da (e.g., between about 100 to about 3,000 Da, about 100 to about 2500 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 1500, about 500 to about 1000, about 300 to about 1000 Da, or about 100 to about 250 Da).

[0071] Subject. As used herein, the term “subject” refers to an individual to which a provided treatment is administered Tn some embodiments, a subject is animal. Tn some embodiments, a subject is a mammal, e.g., a mammal that experiences or is susceptible to a disease, disorder, or condition as described herein. In some embodiments, an animal is a vertebrate, e.g., a mammal, such as a non-human primate, (particularly a higher primate), a sheep, a dog, a rodent (e.g. a mouse or rat), a guinea pig, a goat, a pig, a cat, a rabbit, or a cow. In some embodiments, an animal is a non-mammal animal, such as a chicken, an amphibian, a reptile, or an invertebrate model C. elegans. In some embodiments, a subject is a human. In some embodiments, a subject is suffering from or susceptible to one or more diseases, disorders or conditions as described herein. In some embodiments, a subject displays one or more symptoms of a one or more diseases, disorders or conditions as described herein. In some embodiments, a subject has been diagnosed with one or more diseases, disorders or conditions as described herein. In some embodiments, the subject is receiving or has received certain therapy to diagnose and/or to treat a disease, disorder, or condition. In another embodiment, the subject is an experimental animal or animal substitute as a disease model.

[0072] Substantially : As used herein, refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0073] Therapeutic regimen A “therapeutic regimen”, as that term is used herein, refers to a dosing regimen whose administration across a relevant population may be correlated with a desired or beneficial therapeutic outcome.

[0074] Therapeutically effective amount. As used herein, is meant an amount that produces the desired effect for which it is administered. In some embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of ordinary skill in the art will appreciate that the term "therapeutically effective amount" does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to subjects (e.g., patients) in need of such treatment. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill 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. In some embodiments, a therapeutically effective agent may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.

[0075] Treatment As used herein, the term “treatment” (also “treat” or “treating”) refers to any administration of a therapy that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition. In some embodiments, such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively, or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.

BRIEF DESCRIPTION OF THE DRAWING

[0076] Fig. 1 shows plots of relative values of IDE to Actin for each of the five study groups (Gl, G2, G3, G4, and G5) in TG2576 AD mice. The groups are Gl= wild-type untreated, G2 = transgenic untreated, G3 = transgenic CT 10, G4 = transgenic CTlOx, G5 = transgenic CT 10m. The plots show ANOVA comparison to group G2 (left) and group Gl (right).

[0077] Fig. 2 shows plots of relative values of NSE to Actin for each of the five study groups (Gl, G2, G3, G4, and G5) in TG2576 AD mice. The groups are Gl= wild-type untreated, G2 = transgenic untreated, G3 = transgenic CT 10, G4 = transgenic CTlOx, G5 = transgenic

CT 10m.

[0078] Fig. 3 shows plots of relative values of p-Akt Ser473 to Actin (left) and Aktl to Actin (right) for each of the five study groups (Gl, G2, G3, G4, and G5) in TG2576 AD mice. The groups are Gl= wild-type untreated, G2 = transgenic untreated, G3 = transgenic CT 10, G4 = transgenic CTlOx, G5 = transgenic CT 10m.

[0079] Fig. 4 shows plots of relative values of Insulin Receptor p to Actin for each of the five study groups (Gl, G2, G3, G4, and G5) in TG2576 AD mice. The groups are Gl= wild-type untreated, G2 = transgenic untreated, G3 = transgenic CT 10, G4 = transgenic CTlOx, G5 = transgenic CT 10m.

[0080] Fig. 5 shows plots of relative values of Glut3 to Actin for each of the five study groups (Gl, G2, G3, G4, and G5) in TG2576 AD mice. The groups are Gl= wild-type untreated, G2 = transgenic untreated, G3 = transgenic CT 10, G4 = transgenic CTlOx, G5 = transgenic CT 10m.

[0081] Fig. 6 shows plots of relative values of RBAP48 to Actin for each of the five study groups (Gl, G2, G3, G4, and G5) in TG2576 AD mice. The groups are Gl= wild-type untreated, G2 = transgenic untreated, G3 = transgenic CT 10, G4 = transgenic CTlOx, G5 = transgenic CT 10m.

[0082] Fig. 7 shows plots of relative values of p-4EBPl to Actin for each of the five study groups (Gl, G2, G3, G4, and G5) in TG2576 AD mice. The groups are Gl= wild-type untreated, G2 = transgenic untreated, G3 = transgenic CT 10, G4 = transgenic CTlOx, G5 = transgenic CT 10m.

[0083] Fig. 8 shows plots of relative values of NRF2 to Actin for each of the five study groups (Gl, G2, G3, G4, and G5) in TG2576 AD mice. The groups are Gl= wild-type untreated, G2 = transgenic untreated, G3 = transgenic CT 10, G4 = transgenic CTlOx, G5 = transgenic CT 10m.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0084] Insulin-Associated Diseases, Disorders, and Conditions [0085] Insulin-Associated diseases, disorders, and conditions are an umbrella term for a range of diseases, disorders, and conditions, which primarily arise from varying or unregulated insulin levels in the human body. Insulin is a hormone that helps the body utilize glucose as a source of energy from food. There are several diseased conditions associated with disturbed insulin secretion and utilization by the body. Many insulin-associated diseases, disorders, or conditions including, but not limited to, diabetes, obesity, cardiovascular disease, non-alcoholic fatty liver disease, Wolfram syndrome, metabolic syndrome, insulin resistance, diabetic ketoacidosis, hyperosmolar hyperglycemic state, gestational diabetes, diabetic dermadromes, diabetic neuropathy, diabetic foot ulcers, maturity onset diabetes of the young, pancreatogenic diabetes, Polycystic ovary syndrome (PCOS), or Alzheimer’s Disease (AD), can cause degeneration of various cells (e.g. liver cells, pancreatic cells, etc.), and impact physical and/or mental functions. Some of these are temporary, but others are incurable (e g. chronic) and debilitating conditions that result in progressive degeneration and/or death of cells (e.g. liver cells, pancreatic cells, etc.), which manifest as impaired physical and/or mental functionality are observed.

[0086] Unregulated insulin levels, or insulin resistance, also known as impaired insulin sensitivity, happens when cells in the muscles, fat and liver do not respond as they should to insulin. Muscle, fat and liver cells can respond inappropriately to insulin, which results in inefficient uptake or storage of glucose from the blood. As a result, the pancreas makes more insulin to try to overcome the increasing blood glucose levels. This is called hyperinsulinemia. As long as the pancreas can make enough insulin to overcome the weak response to insulin, blood sugar levels will stay in a healthy range. If the cells become too resistant to insulin, it leads to elevated blood glucose levels (hyperglycemia), which, over time, leads to prediabetes and Type 2 diabetes. In addition to Type 2 diabetes, insulin resistance is associated with several other conditions, including obesity, cardiovascular disease, non-alcoholic fatty liver disease, Wolfram syndrome, metabolic syndrome, Polycystic ovary syndrome (PCOS), and/or other diseases, disorders, or conditions disclosed herein.

[0087] Elevated levels of insulin can also result in weight gain, which in turn makes insulin resistance worse. It is also associated with higher triglyceride levels, hardening of the arteries (atherosclerosis), high blood pressure (hypertension), etc. [0088] Insulin resistance is also the main feature of metabolic syndrome, which is a set of features that link excess fat around the waist and insulin resistance to increased risk of cardiovascular disease, stroke and Type 2 diabetes. Features of metabolic syndrome include elevated blood glucose levels, elevated triglyceride level, low levels of high-density lipoprotein (HDL) cholesterol, and/or high blood pressure.

[0089] Several factors and conditions can cause varying degrees of insulin resistance. It is believed that excess body fat, especially around the belly, and physical inactivity are the two main contributing factors to insulin resistance. Apart from these, insulin resistance may be caused by diet, certain medications, hormonal disorders (e.g. Cushing’s syndrome, Acromegaly, Hypothyroidism, etc ), genetic conditions (e.g. Type A insulin resistance syndrome, Rabson- Mendenhall syndrome, Donohue syndrome, etc.), and other inherited conditions (Myotonic dystrophy, Alstrbm syndrome, Werner syndrome, Inherited lipodystrophy, etc.).

[0090] The present disclosure provides compositions (e.g. microbiome compositions) and methods that inhibit one or more of the events or processes that take place in insulin-associated diseases, disorders, or conditions. The present disclosure is based in part on the discovery that one or more microbial strains or compositions comprising one or more microbial strains are particularly suitable as therapeutic agents for insulin-associated diseases, disorders, or conditions.

[0091] Microbial Preparation(s) and/or Componentls)

[0092] The present disclosure provides systems and methods for assessing, characterizing, and identifying one or more microbial strains of a microbiome. For example, the present disclosure provides systems and methods for assessing, characterizing, and identifying one or more microbial strains of a microbiome that have one or more abilities. Such systems and methods can 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 affect the health of humans, livestock, and/or pets by modulating their respective metabolomes, cell viability, ATP levels, one or more other parameters or features (e.g. of an organ of a subject), or a combination thereof to prevent, treat, or reduce the risk of suffering from a disease, disorder, or condition. For example, technologies described herein may result in modulating the metabolome, improve cell viability, increase ATP levels, modulate one or more other parameters or features (e.g. level or activity of a nucleic acid or protein, or form thereof, body weight gain, fat accumulation in liver, lipid accumulation in liver, blood triglyceride levels, blood-cholesterol levels, oxidative stress, etc.), or a combination thereof of the subject that results in a decrease in production of toxic components and/or components that suggest or are a marker for cellular damage (e.g. hepatic cellular damage, pancreatic cellular damage, neuronal cellular damage (e.g. increased blood levels of neurofilament light protein (NF-L)) in a subject (e.g. in blood of a subject)).

[0093] The present disclosure also provides systems and methods for manufacturing a pharmaceutical composition that comprise assessing, characterizing, and identifying one or more microbial strains of a microbiome.

[0094] In some embodiments, assessing, characterizing, and identifying one or more microbial strains from a microbiome of a snake, lizard, fish, or bird. In some embodiments, assessing, characterizing, and identifying one or more microbial strains from a mammalian microbiome. A mammalian microbiome can be a canine, a feline, an equine, a bovine, an ovine, a caprine, or a porcine microbiome. In some embodiments, a microbiome used in a system or method described herein may prevent or treat a disease or condition.

[0095] A microbiome can be isolated from any system or tissue of an organism that supports microbial growth. For example, a microbiome can be a cutaneous microbiome, an oral microbiome, a nasal microbiome, a gastrointestinal microbiome, a brain microbiome, a pulmonary microbiome, or a urogenital microbiome. A list of exemplary microbial strains found in a gastrointestinal microbiome is included below in Table 1. A person skilled in the art would understand that a microbiome sample can be obtained by various ways known in the art. For example, a cutaneous, oral, nasal, pulmonary, or urogenital microbiome sample could be obtained using a swab or tissue scrapping. In some embodiments, a gastrointestinal microbiome could be sampled from feces. A cutaneous microbiome, an oral microbiome, a nasal microbiome, a gastrointestinal microbiome, a brain microbiome, a pulmonary microbiome, or a urogenital microbiome sample could be obtained via a biopsy. [0096] In some embodiments, a microbiome is a 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 embodiments, a microbiome is a microbiome of an individual that suffers from or is at risk of developing a particular disease, disorder, or condition. In some embodiments, a microbiome is a microbiome of an individual who is known to suffer from a particular disease, disorder, or condition. In some embodiments, a human microbiome is a microbiome of a human with an unknown risk for one or more diseases, disorders, or conditions.

[0097] In some embodiments, a microbiome is a reference microbiome. A reference microbiome can be a microbiome of a healthy individual or an individual who does not suffer from or is not at risk of developing a particular disease, disorder, or condition. Tn some instances, a reference microbiome may be from the same individual as a microbiome to be assessed or characterized, but was obtained at a different time. In some instances, a reference microbiome may be from the same individual as a microbiome to be assessed or characterized, but was obtained from a different system or tissue.

[0098] In some embodiments, an individual microbial strain or a combination of microbial strains may be assessed, characterized, or identified in a different relative amount than such strain or strains are found in a microbiome. For example, the effect of modulation of a cell or organism in response to a single strain may be assessed, characterized, or identified using in vitro methods (e g. mammalian cells) or in vivo methods using mammals (e.g. mice, humans, etc.) as described herein. In some embodiments, for example, the effect of modulation of a cell or organism to treat, prevent, or reduce the risk on a disease, disorder, or condition (e.g. an insulin-associated disease, disorder, or condition as described herein) may be assessed, characterized, or identified using in vitro methods (e.g. mammalian cells) or in vivo methods using mammals (e.g. mice, humans, etc.) as described herein. In some embodiments, for example, the effect of modulation of a cell or organism to treat, prevent, or reduce the risk on a disease, disorder, or condition (e.g. an insulin-associated disease, disorder, or condition as described herein) by modulating one or more metabolites of the cell or organism, one or features or parameters (e.g. cell viability, level or activity of a nucleic acid or protein, or form thereof, body weight gain, fat accumulation in liver, lipid accumulation in liver, blood triglyceride levels, bl ood-chol esterol levels, oxidative stress, etc.) of the cell or organism, or a combination thereof may be assessed, characterized, or identified using in vitro methods (e.g. mammalian cells) or in vivo methods using mammals (e.g. mice, humans, etc.) as described herein. As another example, the effect of modulation (e.g. of levels of one or more metabolites) of a cell or organism to treat, prevent, or reduce the risk on a disease, disorder, or condition, as described herein, in response to two microbial strains may be assessed, characterized, or identified together using methods described herein.

[0099] An extract, component, or compound of a microbial strain may also be assessed, characterized, or identified using methods described herein. In some cases, an extract, component, or compound of a microbial strain that has been determined to treat, prevent, or reduce the risk on a disease, disorder, or condition, as described herein, in an organism (e.g. mammal) may be assessed, characterized, or identified. Assessing, characterizing or identifying an extract, component, or compound of a microbial strain that treats, prevents, or reduces the risk on a disease, disorder, or condition in an organism (e.g. mammal) may provide additional information about potential biomarkers, targets, or protective agents in a microbiome.

[0100] A variety of technologies are known in the art that can be used to prepare extracts of microbial strains, and/or to isolate extracts, components, or compounds therefrom, or to process (e.g., to isolate and/or purify one or more components or compounds from). To give but a few examples, such technologies may include, for example, one or more of organic extraction, vacuum concentration, chromatography, and so on.

[0101] Assessing Biological Impact

[0102] The present disclosure provides the insight that compositions (e.g. microbiome compositions) as described herein can be used to treat, prevent, and/or reduce the risk of a disease, disorder, or condition of an organism (e.g. a mammal (e.g. a human)) by contacting the composition(s) (e.g., feeding the compositions to, administering to) with an organism. In some embodiments, an organism may suffer from or be at risk of suffering from a disease, disorder, or condition (e.g. mammalian disease, disorder, or condition). To determine whether one or more compositions treats, prevents, or reduces the risk of a disease, disorder, or condition (e.g. an insulin-associated disease, disorder, or condition), levels of one or more metabolites can be observed, measured, or assessed in samples that have been contacted with the one or more compositions. For example, levels of the one or more metabolites can be observed, measured, or assessed in samples at different times (e.g. before administration of composition, after administration of composition, during administration of composition, etc.). To determine whether one or more compositions treats, prevents, or reduces the risk of a disease, disorder, or condition (e.g. an insulin-associated disease, disorder, or condition), one or more features or parameters may be observed, measured, or assessed in samples that have been contacted with the one or more compositions. For example, one or more features or parameters may be observed, measured, or assessed in samples at different times (e.g. before administration of composition, after administration of composition, during administration of composition, etc.).

[0103] In some embodiments, methods described herein utilize a first sample and a second sample. Tn some embodiments, a first sample is a reference sample. In some embodiments, a reference sample can be a sample obtained from a subject who is contacted with (e.g., administered or fed) a composition, e.g., CT10 composition, CTlOm composition, CTlOx composition, CT6 composition, or CT6m composition. In some embodiments, a reference sample can be a sample obtained from a subject who is contacted with (e.g., administered or fed) a composition, e.g., CT 10 composition, CTlOx composition, CTIOm composition, CT6 composition, or CT6m composition, at a first time point. In some embodiments, a reference sample can be a sample obtained from a subject prior to being contacted with (e.g., administered or fed) a composition, e.g., CT10 composition, CTlOx composition, CTIOm composition, CT6 composition, or CT6m composition. In some embodiments, a reference sample can be a sample obtained from a healthy individual. In some embodiments, a reference sample can be a sample obtained from an individual who is suffering from or may have a risk for a disease, disorder, or condition (e.g. an insulin-associated disease, disorder, or condition). In some embodiments, a reference sample is a control sample. In some embodiments, a reference sample is a negative control sample. In some embodiments, a reference sample is a positive control sample. In some embodiments, a reference sample may be a historic reference (e.g. value across control samples). In some embodiments, a reference sample may be from a printed publication (e.g. a text book, a journal, etc.).

[0104] In some embodiments, a second sample can be a test sample. In some embodiments, a test sample may be a sample obtained from a subject who is contacted with (e.g., administered or fed) a composition, e g., CT10 composition, CTl Ox composition, CTI Om composition, CT6 composition, or CT6m composition. In some instances, a subject (e.g. patient or population) may be suffering from or at risk of a disease, disorder, or condition (e.g. an insulin-associated disease, disorder, or condition). In some instances, a subject (e.g. patient or population) may have an unknown risk for one or more diseases, disorders, or conditions as described herein. In some embodiments, a test can be a sample obtained from a subject who is contacted with (e.g., administered or fed) a composition, e.g., CT10 composition, CTlOx composition, CTlOm composition, CT6 composition, or CT6m composition, at a second time point.

[0105] In some embodiments, methods described herein comprise comparing one or more metabolite levels (e.g. a metabolome), or one or more parameters or features (e.g. cell viability, level or activity of a nucleic acid or protein, or form thereof, body weight gain, fat accumulation in liver, lipid accumulation in liver, blood triglyceride levels, blood-cholesterol levels, oxidative stress, etc.) obtained from a test sample with one or more metabolite levels (e.g. a metabolome), or one or more parameters or features (e.g. cell viability, level or activity of a nucleic acid or protein, or form thereof, body weight gain, fat accumulation in liver, lipid accumulation in liver, blood triglyceride levels, blood-cholesterol levels, oxidative stress, etc.) obtained from a reference sample. In some embodiments, by comparing one or more metabolite levels, parameters, or features obtained from a test sample with one or more metabolite levels, parameters, or features obtained from a reference sample, a composition described herein can be assessed, characterized or identified as being useful for treating, preventing, or reducing the risk of suffering from a disease, disorder, or condition (e.g. an insulin-associated disease, disorder, or condition) as described herein. In some embodiments, by comparing one or more metabolite levels, parameters, or features obtained from a test sample with one or more metabolite levels, parameters, or features obtained from a reference sample, it can be determined that a composition as disclosed herein increases the severity or incidence of a disease, disorder, or condition phenotype. In some embodiments, by comparing one or more metabolite levels, parameters, or features obtained from a test sample with one or more metabolite levels, parameters, or features obtained from a reference sample, it can be determined that a composition as disclosed herein decreases the severity or incidence of a disease, disorder, or condition phenotype. In some embodiments, by comparing one or more metabolite levels, parameters, or features obtained from a test sample with one or more metabolite levels, parameters, or features obtained from a reference sample, it can be determined that a composition as disclosed herein has no effect on the severity or incidence of a disease, disorder, or condition phenotype. In some embodiments, by comparing one or more metabolite levels, parameters, or features obtained from a test sample with one or more metabolite levels, parameters, or features obtained from a reference sample, it can be determined that a composition as disclosed herein prevents a disease, disorder, or condition phenotype.

[0106] The present disclosure also provides the recognition that compositions and methods provided herein can be used to monitor progression of a disease, disorder, or condition (e.g. an insulin-associated disease, disorder, or condition) in an individual. For example, if metabolite levels, parameters or features (e.g. cell viability, level or activity of a nucleic acid or protein, or form thereof, body weight gain, fat accumulation in liver, lipid accumulation in liver, blood triglyceride levels, blood-cholesterol levels, oxidative stress, etc.) determined to increase the severity of a disease, disorder, or condition decrease in relative amount, it may indicate that the disease, disorder, or condition is being attenuated, e.g., by treatment or immune response.

[0107] The present disclosure also provides the insight that compositions and methods provided herein can be used to tailor treatments (e.g., therapies, nutraceuticals, and/or probiotics) to an individual patient. In some embodiments, compositions and methods provided herein can provide “personalized” therapy. In some cases, metabolite levels, features or parameters (e g. cell viability, level or activity of a nucleic acid or protein, or form thereof, body weight gain, fat accumulation in liver, lipid accumulation in liver, blood triglyceride levels, blood-cholesterol levels, oxidative stress, etc.) within an individual can be assessed, characterized, or identified to determine if they have a disease, disorder, or condition. Based on the results, the individual can be treated with one or more compositions to adjust the metabolite levels (i.e., their metabolome), features or parameters. In some instances, this will affect the disease, disorder, or condition the individual is suffering from or at risk of developing. For example, if an individual is determined to have a relatively low amount of one or more metabolite levels that have been determined to decrease the severity of a disease, disorder, or condition, administration of the one or more compositions that have been determined to decrease the severity of a disease, disorder, or condition to the individual (or an extract, component, or compound thereof) may attenuate the severity of the individual’s disease or condition. [0108] The present disclosure provides the insight that compositions and methods provided herein can be used recursively to treat, prevent, or ameliorate a disease, disorder, or condition. In some embodiments, for example, one or more compositions disclosed herein may be administered (e.g. fed, injected, etc.) to a subject after determining the effect of one or more compositions on subject’s metabolite levels, or after determining the effect of one or more compositions on subject’s features or parameters (e.g. cell viability, level or activity of a nucleic acid or protein, or form thereof, body weight gain, fat accumulation in liver, lipid accumulation in liver, blood triglyceride levels, bl ood-chol esterol levels, oxidative stress, etc.). In some embodiments, a composition may be administered once. In some embodiments, a composition may be administered more than once. In some embodiments, a composition may be administered daily, weekly, biweekly, monthly, bimonthly, etc. In each of these instances, levels of one or more metabolites, or changes in features or parameters may be monitored. In some embodiments, levels of one or more metabolites (e.g. metabolome) or changes in features or parameters may be monitored before administration of a composition. In some embodiments, levels of one or more metabolites (e.g. metabolome) or changes in features or parameters may be monitored after administration of a composition.

[0109] Pharmaceutical Compositions

[0110] Provided herein are compositions comprising individual microbial strains or combinations of microbial strains, metabolites thereof, extracts thereof, or components thereof. In some embodiments, a composition comprises individual microbial strains or combinations of microbial strains from a mammalian microbiome, metabolites thereof, extracts thereof, and/or components thereof, which have been assessed, identified, characterized or assayed using methods as described herein. In some embodiments, a composition provided herein comprises one or more, 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 from a mammalian microbiome, extracts thereof, metabolites thereof, and/or components thereof, which have been assessed, identified, characterized or assayed using methods as described herein.

[OHl] Provided herein are also compositions comprising one or more components or metabolites. In some embodiments, components or metabolites in compositions herein are from a source that is not a microbial strain, e.g., synthetically generated. In some embodiments, components or metabolites in a composition may have been identified from a microbial strain, but are independent from a microbial strain and are not produced by a microbial strain, e.g., they can be synthetically generated.

[0112] In some embodiments, a composition provided herein comprises 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 1 below.

[0113] Table 1: Exemplary Microbial Strains Found in Human Gut Microbiome

[0114] In some embodiments, a composition provided herein comprises Gluconacetobacter house nii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, Acidaminococcus sp.. or a combination thereof. In some embodiments, a composition comprises at least two of, at least three of, at least four of, at least five of, at least six of, at least seven of, at least eight of, at least nine of, or all of Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Clostridium butyr icum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, and Acidaminococcus sp. In some embodiments, for example, a composition comprises all of Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp. , Lactobacillus plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, and Acidaminococcus sp., and may be referred to by different names, including but not limited to, CT 10 composition, CT 10 cocktail, and so forth.

[0115] In some embodiments, a composition provided herein comprises Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella sp., Bifidobacterium sp., or a combination thereof. In some embodiments, a composition comprises at least two of, at least three of, at least four of, at least five of, or all of Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp. , Lactobacillus plantarum, Veillonella sp., and Bifidobacterium sp.. In some embodiments, for example, a composition comprises all of Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella sp., and Bifidobacterium sp. and may be referred to by different names, including but not limited to, CT6 composition, CT6 cocktail, and so forth. In some embodiments, a composition provided herein comprises Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus catus, Lactobacillus plantarum, Veillonella atypica, Bifidobacterium breve, or a combination thereof. In some embodiments, a composition comprises at least two of, at least three of, at least four of, at least five of, or all of Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus catus, Lactobacillus plantarum, Veillonella atypica, and Bifidobacterium breve. In some embodiments, for example, a composition comprises all of Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus catus, Lactobacillus plantarum, Veillonella atypica, and Bifidobacterium breve and may be referred to by different names, including but not limited to, CT6 composition, CT6 cocktail, and so forth.

[0116] Exemplary microbiome compositions (e.g. CT10, CTlOm, CTlOx, CT6, CT6m), as described herein, and their composition details are listed in Tables 2-6 below.

[0117] Table 2; CT10 Composition

[0118] Table 3: CTlOm Composition

[0119] Table 4; CTlOx Composition

[0120J Table 5: CT6 Composition

[0121] Table 6: CT6m Composition

[0122] In some embodiments, a composition provided herein comprises one or more, 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 metabolites. Metabolites which may be assessed, identified, characterized, or assayed and/or comprised in compositions as disclosed herein, include those listed for example in the Appendices submitted herewith (e.g. Appendix 1-1, 1-2, 1-3, 2, 3, or 4).

[0123] In some embodiments, a metabolite may be Butyrylcamitine, Theobromine, p- Hydroxyphenylpyruvic acid, Propionic acid, Picolinic acid, 2-Hydroxy-4methylvaleric acid, N6- Acetylysine, Urocanic acid, N5-Ethylglutamine, Trigonelline, Stachydrine, Ectoine, 5- Hydroxylysine, Arginine (arg), Cholic acid, 2-(4-Hydroxyphenyl)propionic acid, N- Acetyltryptophan, Hydroxyproline, Argininosuccinic acid, Glutamic acid (Glu), Sarcosine, 5- Methoxyindoleacetic acid, Indole-3-lactic acid, Isovalerylalanine, N-Acetylleucine, 1- Methylhistidine, N-Acetylephenylalanine, Proline (Pro), or any combination thereof

[0124] In some embodiments, a metabolite may be 4-Hydroxyphenylpyruvic, Ectoine, Gramine, N-Acetyl-L-phenylalanine, Nepsilon-Acetyl-L-lysine, Stachydrine, Trigonelline, 3- Ureidopropionic acid, Theobromine, Hippuric acid, Imidazolepropionic acid, NG-Methyl-L- arginine, trans-Urocanic Acid, N-Acetyl-L-leucine, Sarcosine, Isobutyrylcarnitine, b- Hydroxyisovaleric acid, L-Theanine/N5-Ethylglutamine, 5-Hydroxylysine, Phenaceturic acid, betaine, hydroxyproline, Picolinic acid, 2-Aminoadipic acid, Glycerophosphocholine, carnitine, Glycerol 3-phosphate, Argininosuccinic acid, creatine, Terephthalic acid, Homocitrulline, Mucic acid, Homocysteinesulfinic acid, Trimethyllysine, Spermidine, Glyoxylic acid, XA0013 C6H6O4S, 3-Tndoxylsulfuric acid, Nicotinamide, N-Formylglycine, Ureidoglycolate, N- Methylproline, Glucaric acid, Butyryl carnitine, Methionine sulfoxide, Carboxymethyllysine, Glycolic acid, Phenaceturic acid, Diethanolamine, Phosphorylcholine, Guanidinosuccinic acid, N-Acetylhistidine, Glyceric acid, S-Methylmethionine, Cysteine glutathione disulfide, Kynurenine, N-Acetylphenylalanine, Threonic acid, Malic acid, 7,8-Dihydrobiopterin, Homovanillic acid, Taurocholic acid, 5-Methoxyindoleacetic acid, butyrate, b-Hydroxyisovaleric acid, 2-Oxoglutaric acid, N-Acetyltry ptophan, Thiaproline, Hypotaurine, Cholic acid, Acetoacetic acid, Ethanolamine, Guanidoacetic acid, S-Sulfocysteine, Myristic acid C14:0 XA0027, or any combination thereof

[0125] In some embodiments, an individual microbial strain or combinations of microbial strains from a mammalian microbiome that have been killed (e.g., heat killed). Alternatively, in some embodiments, an individual microbial strain or combinations of microbial strains from a mammalian microbiome may include cells that are viable or alive.

[0126] In some embodiments, one or more microbial strains comprise a viable or living individual microbial strain or combinations of microbial strains, e.g., from a mammalian microbiome.

[0127] In some embodiments, one or more microbial strains comprise a viable or living individual microbial strain or combinations of microbial strains, e.g., from a mammalian microbiome, as described herein comprises and/or is formulated through use of one or more cell cultures and/or supernatants or pellets thereof, and/or a powder formed therefrom.

[0128] In some embodiments, compositions for use in accordance with the present disclosure are pharmaceutical compositions, e.g., for administration (e.g., topical, oral, subcutaneous, intravenous, intramuscular, intracerebral, intrathecal, rectal (e.g. rectal intubation), opthalmical, intravitreal, or suprachoroidal administration) to a mammal (e.g., a human).

Pharmaceutical compositions typically include an active agent (e.g., individual microbial strains or combinations of microbial strains from a mammalian microbiome, extracts thereof, and/or components thereof), and a pharmaceutically acceptable carrier. Certain exemplary pharmaceutically acceptable carriers include, for instance saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

[0129] In some embodiments, a pharmaceutical composition for use in accordance with the present disclosure may include and/or may be administered in conjunction with, one or more supplementary active compounds; in certain embodiments, such supplementary active agents can 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 in its entirety); prebiotics (non-digestible food ingredients that help support growth of probiotic bacteria, e.g., fructans such as fructooligosaccharides (FOS) and inulins, 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 in its entirety) and combinations thereof.

[0130] In some embodiments, a prebiotic comprises a fructooligosaccharide, an inulin, an isomaltooligosaccharide, a lactilol, a lactosucrose, a lactulose, a soy oligosaccharide, a transgalactooligosaccharide, a xylooligosaccharide, seaweed, or a combination thereof. In some embodiments, a prebiotic comprises seaweed. In some embodiments, a prebiotic comprises a pome extract, berry extract and walnut extract.

[0131] In some embodiments, a probiotic composition can be formulated for oral administration. In some embodiments, a probiotic composition can be a food, a beverage, a feed composition, or a nutritional supplement. In some embodiments, an ellagitannin composition, an enzymatic composition, or both can be a liquid, syrup, tablet, troche, gummy, capsule, powder, gel, or film. In some embodiments, a probiotic composition is an enteric-coated formulation.

[0132] In some embodiments, a probiotic comprises a prebiotic. In some embodiments, a prebiotic comprises a fructooligosaccharide, an inulin, an isomaltooligosaccharide, a lactilol, a lactosucrose, a lactulose, a soy oligosaccharide, a transgalactooligosaccharide, a xylooligosaccharide, seaweed, a pome extract, berry extract and walnut extract, or a combination thereof.

[0133] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include topical, oral, subcutaneous, intravenous, intramuscular, intracerebral, intrathecal, rectal, (e.g. rectal intubation), opthalmical, intravitreal, or suprachoroidal administration. Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21 st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY), which is incorporated in its entirety by reference herein. Oral compositions generally include an inert diluent or an edible carrier (e.g. pharmaceutically acceptable diluent, pharmaceutically acceptable carrier). To give but a few examples, in some embodiments, an oral formulation may be or comprise a syrup, a liquid, a tablet, a troche, a gummy, a capsule, e.g., gelatin capsules, a powder, a gel, a fdm, etc. Similarly, ocular compositions (e.g. for ophthalmic, intravitreal, or suprachoroidal administration) may include an inert diluent or carrier (e.g. pharmaceutically acceptable diluent, pharmaceutically acceptable carrier), various additives such as viscosity enhancers, permeations enhancers, cyclodextrins, etc. Examples of viscosity enhancers include hydroxy methyl cellulose, hydroxy ethyl cellulose, sodium carboxy methyl cellulose, hydroxypropyl methyl cellulose and polyalcohol. Example of permeation enhancers include chelating agents, preservatives, surface active agents, bile salts, Benzalkonium chloride, polyoxyethylene glycol ethers (lauryl, stearyl and oleyl), ethylenediaminetetra acetic acid sodium salt, sodium taurocholate, saponins and cremophor EL, etc. For example, in some embodiments ocular formulations may be or comprise suspensions, emulsions (e.g. water-in-oil or oil-in water), nanocarriers, (e.g. nanoparticles, nanosuspensions, liposomes, nanomicelles, dendrimers, etc.) ointments, gels, eye drops, etc. Cerebral compositions (e.g. for intracerebral or intrathecal administration) may include an inert diluent or carrier, and/or additives. In some embodiments, cerebral compositions are free of preservatives. In some embodiments, cerebral compositions are sterile.

[0134] In some embodiments, pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of a pharmaceutical composition. In some particular embodiments, a pharmaceutical composition can contain, e.g., any one or more of the following inactive 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 com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. In some embodiments, the compositions can be taken as-is or sprinkled onto or mixed into a food or liquid (such as water). In some embodiments, a composition that may be administered to mammals as described herein may be or comprise an ingestible item (e.g., a food or drink) that comprises (e.g., is supplemented) with an individual microbial strain or combinations of microbial strains from a mammalian microbiome, extracts thereof, and/or components thereof.

[0135] In some embodiments, a food can be or comprise one or more of bars, candies, baked goods, cereals, salty snacks, pastas, chocolates, and other solid foods, as well as 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, foods are prepared by a subject by mixing in individual microbial strains or combinations of microbial strains from a mammalian microbiome, extracts thereof, and/or components thereof. [0136] Compositions can be included in a kit, container, pack, or dispenser, together with instructions for administration or for use in a method described herein.

[0137] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, a composition (e.g., a pharmaceutical composition) as described herein may be or comprise one or more cells, tissues, or organisms (e.g., plant or microbe cells, tissues, or organisms) that produce (e.g., have produced, and/or are producing) a relevant compound.

[0138] Those skilled in the art will appreciate that, in some embodiments, technologies for preparing compositions and/or preparations, and/or for preparing (and particularly for preparing pharmaceutical compositions) may include one or more steps of assessing or characterizing a compound, preparation, or composition, e.g., as part of quality control. In some embodiments, if an assayed material does not meet pre-determined specifications for the relevant assessment, it is discarded. In some embodiments, if such assayed material does meet the pre- determined specifications, then it continues to be processed as described herein.

[0139] In some embodiments, a pharmaceutical composition provided herein can promote the colonization of an individual microbial strain or combinations of microbial strains from a mammalian microbiome, particularly microbial strain(s) that have been identified, characterized, or assessed as decreasing the severity or incidence of a mammalian disease, disorder, or condition, in a mammal suffering from or at risk of the mammalian disease, disorder, or condition. In some embodiments, a pharmaceutical composition provided herein can attenuate the colonization of an individual microbial strain or combinations of microbial strains from a mammalian microbiome, particularly microbial strain(s) that have been identified, characterized, or assessed as increasing the severity or incidence of a mammalian disease, disorder, or condition, in a mammal suffering from or at risk of the mammalian disease, disorder, or condition (e.g. an insulin-associated disease, disorder, or condition). In some embodiments, a pharmaceutical composition provided herein can promote the colonization of an individual microbial strain or combinations of microbial strains from a mammalian microbiome, particularly microbial strain(s) that have been identified, characterized, or assessed as not affecting the severity or incidence of the mammalian disease, disorder, or condition but have been identified, characterized, or assessed as being capable of outcompeting one or more microbial strains that have been identified, characterized, or assessed as increasing the severity or incidence of a mammalian disease, disorder or condition, in a mammal suffering from or at risk of the mammalian disease, disorder, or condition.

[0140] In some embodiments, each of the one or more microbial strains in a composition comprises 10 ¹ colony forming units (CFUs) to IO ²⁰ CFU. In some embodiments, each of the one or more microbial strains in a composition comprises 10 ¹ colony forming units (CFUs) to 10 ¹⁵ CFU. In some embodiments, each of the one or more microbial strains in a composition comprises 10 ⁶ CFU to 10 ¹⁵ CFUs. In some embodiments, each of the one or more microbial strains in a composition comprises about 10 ¹ CFU to 10 ¹ ’ CFU, or about 10 ² CFU to 10 ¹⁴ CFU, or about 10 ³ CFU to 10 ¹³ CFU, or about 10 ⁴ CFU to 10 ¹³ CFU, or about 10 ⁵ CFU to 10 ¹² CFU, or about 10 ⁶ CFU to 10 ¹¹ CFU, or about 10 ⁷ CFU to 10 ¹⁰ CFU, or about 10 ⁸ CFU to 10 ⁹ CFU, or about 10 ⁵ CFU to IO ¹⁰ CFU, or about 10 ⁸ CFU to 10 ¹² CFU. In some embodiments, each of the one or more microbial strains in a composition comprises at least about 10 ¹ , 5 x IO ¹ , 10 ² , 5 x 10 ² , 10 ³ , 5 x 10 ³ , 10 ⁴ , 5 x 10 ⁴ , 10 ⁵ , 5 x 10 ⁵ , 10 ⁶ , 5 x 10 ⁶ , 10 ⁷ , 5 x 10 ⁷ , 10 ⁸ , 5 x 10 ⁸ , 10 ⁹ , 5 x 10 ⁹ , 10 ^1(J , 5 x IO ¹⁰ , 10 ¹¹ , 5 x 10 ¹¹ , 10 ¹² , or more CFUs. In some embodiments, each of the one or more microbial strains in a composition comprises at most about IO ¹⁵ , 5 x IO ¹⁴ , IO ¹⁴ , 5 x IO ¹³ , IO ¹³ , 5 x 10 ¹² , 10 ¹² , 5 x IO ¹¹ , 10 ¹¹ , 5 x IO ¹⁰ , IO ¹⁰ , 5 x 10 ⁹ , 10 ⁹ , 5 x 10 ⁸ , 10 ⁸ , or less CFUs. In some embodiments, each of the one or more microbial strains in a composition comprises the same number of CFUs. In some embodiments, some of the one or more microbial strains in a composition comprises a different number of CFUs.

[0141] In some embodiments, a composition comprises a total of 10 ¹ CFU to IO ²⁰ CFUs. In some embodiments, a composition comprises a total of 10 ⁶ CFU to 10 ¹⁵ of CFUs. In some embodiments, a composition can include about 10 ¹ CFU to 10 ^2IJ CFU, or about 10 ⁵ CFU to 10 ¹⁵ CFU, or about 10 ⁵ CFU to 10 ¹² CFU, about 10 ⁵ CFU to IO ¹⁰ CFU, or about 10 ⁸ CFU to 10 ¹² CFU of one or more microbial strains. In some embodiments, a composition can include about 10 ¹ CFU to 10 ¹⁵ CFU, or about 10 ² CFU to 10 ¹⁴ CFU, or about 10 ³ CFU to 10 ¹³ CFU, or about 10 ⁴ CFU to 10 ¹³ CFU, or about 10 ⁵ CFU to 10 ¹² CFU, or about 10 ⁶ CFU to 10 ¹¹ CFU, or about

10 ⁷ CFU to IO ¹⁰ CFU, or about 10 ⁸ CFU to 10 ⁹ CFU, or about 10 ⁵ CFU to IO ¹⁰ CFU, or about

10 ⁸ CFU to 10 ¹² CFU of one or more microbial strains. In some embodiments, a composition can include at least 10 ¹ , 5 x 10 ¹ , 10 ² , 5 x 10 ² , 10 ³ , 5 x 10 ³ , 10 ⁴ , 5 x 10 ⁴ , 10 ⁵ , 5 x 10 ⁵ , 10 ⁶ , 5 x 10 ⁶ , 10 ⁷ , 5 x 10 ⁷ , 10 ⁸ , 5 x 10 ⁸ , 10 ⁹ , 5 x 10 ⁹ , 10 ¹⁰ , 5 x 10 ¹⁰ , 10 ¹¹ , 5 x 10 ¹¹ , 10 ¹² , or more CFUs of one or more microbial strains. In some embodiments, a composition can include at most 10 ¹⁵ , 5 x 10 ¹⁴ , 10 ¹⁴ , 5 X 10 ¹³ , 10 ¹³ , 5 X 10 ¹² , 10 ¹² , 5 X 1 O ¹¹ , 1 O ¹¹ , 5 x 10 ¹⁰ , 10 ¹⁰ , 5 x 10 ⁹ , IO ⁹ , 5 x 10 ⁸ , IO ⁸ , or less CFUs of one or more microbial strains.

[0142] In some embodiments, a pharmaceutical composition is tailored to a specific mammal (e.g., a specific human, e.g., a patient) based on that mammal’s (e.g., human’s) microbiome. In some embodiments, a pharmaceutical composition is specific for a microbiome of an individual mammal (e.g., human). In some embodiments, a pharmaceutical composition is specific for microbiomes of a population of mammals (e.g., humans). Populations of mammals can include, but are not limited to: families, mammals in the same regional location (e.g., neighborhood, city, state, or country), mammals with the same disease or condition, mammals of a particular age or age range, mammals that consume a particular diet (e.g., food, food source, or caloric intake).

[0143] Methods of Treatment

[0144] The present disclosure recognizes that compositions described herein can be useful in the treatment of subjects. Methods provided by the present disclosure include methods for the treatment of certain diseases, disorders and conditions. In some embodiments, relevant diseases, disorders and conditions may be or include an insulin-associated disease, disorder, or condition. In some embodiments, an insulin-associated disease, disorder, or condition may be ALS, AD, PD, or HD.

[0145] Generally, 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 who is in need of, or who has been determined to be in need of, such treatment.

[0146] In some embodiments, methods of treatment provided herein are prophylactic or preventative, e g., may be administered to subjects prior to display of significant symptoms and/or to exposure to a particular expected inducement that is associated with insulin-associated diseases, disorders, or conditions described herein. In some embodiments, methods of treatment provided herein are therapeutic, e g., may be administered to subjects after development of significant symptoms associated with insulin-associated diseases, disorders, or conditions. [0147] In some embodiments, provided methods of treatment are administered to a subject that is a mammal, e.g., a mammal that experiences a disease, disorder, or condition as described herein; in some embodiments, a subject is a human or non-human veterinary subject, e.g., an ape, cat dog, monkey, or pig.

[0148] In many embodiments, treatment involves ameliorating at least one symptom of a disease, disorder, or condition associated with insulin-associated diseases, disorders, or conditions. In some embodiments, a method of treatment can be prophylactic.

[0149] In some embodiments, the methods can include administration of a therapeutically effective amount of compositions disclosed herein before, during (e.g., concurrently with), or after administration of a treatment that is expected to be associated with insulin-associated diseases, disorders, or conditions.

[0150] In some embodiments, subjects who receive treatment as described herein may be receiving and/or may have received other treatment (e.g., pharmacological treatment/therapy, surgical, etc.), for example that may be intended to treat one or more symptoms or features of a disease disorder or condition as described herein (e.g. insulin-associated diseases, disorders, or conditions), so that provided compositions are administered in combination with such other therapy (i.e. treatment) to treat the relevant disease, disorder, or condition.

[0151] In some embodiments, the compositions described herein can be administered in a form containing one or more pharmaceutically acceptable carriers. Suitable carriers have been described previously and vary with the desired form and mode of administration of a composition. For example, pharmaceutically acceptable carriers can include diluents or excipients such as fillers, binders, wetting agents, disintegrators, surface-active agents, glidants, and lubricants. Typically, a carrier may be a solid (including powder), liquid, or any combination thereof. Each carrier is preferably “acceptable” in the sense of being compatible with other ingredients in the composition and not injurious to a subject. A carrier can be biologically acceptable and inert (e.g., it permits the composition to maintain viability of the biological material until delivered to the appropriate site).

[0152] Tablets, pills, capsules, troches and the like can 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 sterotes; 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 flavoring, or other suitable flavorings. These are for purposes of example only and are not intended to be limiting.

[0153] Oral compositions can include an inert diluent or an edible carrier. For purposes of oral therapeutic administration, an active compound can be incorporated with excipients and used in the form of tablets, lozenges, pastilles, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared by combining a composition of the present disclosure with a food. In some embodiments, microbes (e.g. one or more microbial strains) can be formulated in a food item. Some non-limiting examples of food items to be used with the methods and compositions described herein include: popsicles, cheeses, creams, chocolates, milk, meat, drinks, pickled vegetables, kefir, miso, sauerkraut, etc. In other embodiments, food items can be juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish, hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauce, and Chinese soups; soups; dairy products such as milk, dairy beverages, ice creams, and yogurts; fermented products such as fermented soybean pastes, fermented beverages, and pickles; bean products; various confectionery products including biscuits, cookies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; and the like It is preferred that food preparations not require cooking after admixture with microbial strain(s) to avoid killing any microbes. In one embodiment a food used for administration is chilled, for example, iced flavored water. In certain embodiments, the food item is not a potentially allergenic food item (e.g., not soy, wheat, peanut, tree nuts, dairy, eggs, shellfish or fish). Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.

[0154] Ocular formulations (e.g. for ophthalmic, intravitreal, or suprachoroidal administration) can include an inert diluent or a carrier. For purposes of ocular therapeutic administration, an active compound can be incorporated with excipients and used in the form of suspensions, emulsions (e g. water-in-oil or oil-in water), nanocarriers, (e.g. nanoparticles, nanosuspensions, liposomes, nanomicelles, dendrimers, etc.) ointments, gels, eye drops, etc. In some embodiments, administration of such formulations is topical (e.g. eye drops). In some embodiments, administration of such formulations is via injection (e.g. intravitreal, suprachoroidal, etc.).

[0155] Cerebral formulations (e.g. for intracerebral or intrathecal administration) can include an inert diluent or a carrier. For purposes of cerebral therapeutic administration, an active compound can be incorporated with excipients and used in the form of suspensions, emulsions (e.g. water-in-oil or oil-in water), nanocarriers, (e.g. nanoparticles, nanosuspensions, liposomes, nanomicelles, dendrimers, etc.) ointments, gels, etc. In some embodiments, administration of such formulations is topical (e.g. ointments). In some embodiments, administration of such formulations is via injection (e g. intracerebral, intrathecal, etc ).

[0156] In some such embodiments, a composition described herein is administered to a subject according to a dosing regimen that achieves population of the subject’s microbiome with administered cells. In some embodiments, a composition is administered to a subject in a single dose. In some embodiments, a composition is administered to a subject in a plurality of doses. In some embodiments, a dose of a composition is administered to a subject twice a day, daily, weekly, or monthly.

[0157] In some embodiments, each of the one or more microbial strains in a dose comprises 10 ¹ to 10 ¹⁵ colony forming units (CFUs). In some embodiments, each of the one or more microbial strains in a dose comprises 10 ⁶ to 10 ¹⁵ CFUs. In some embodiments, each of the one or more microbial strains in a dose comprises the same number of CFUs. In some embodiments, some of the one or more microbial strains in a dose comprises a different number of CFUs.

[0158] In some embodiments, a dose of one or more microbial strains comprises a total of 10 ⁶ to 10 ¹⁵ CFUs. In some embodiments, a dose of one or more microbial strains comprises a total of 10 ⁷ to 10 ¹⁵ CFUs. In some embodiments, a dose of one or more microbial strains comprises 5-200 billion CFUs. In some embodiments, a dose of one or more microbial strains comprises 5-50 billion CFUs. In some embodiments, a dose of one or more microbial strains comprises 5-20 billion CFUs. In some embodiments, a dose of one or more microbial strains comprises 50-100 billion CFUs. In some embodiments, a dose of one or more microbial strains comprises 100-200 billion CFUs. [0159] In some embodiments, efficacy can be assessed by measuring the degree of oxidative stress of cells in a biological sample prior to and following administration of a composition as described herein. The degree of oxidative stress of cells can be assessed by, for example, measuring the expression of oxidative stress biomarkers, such as reactive oxygen species (ROS) levels, or lipid, protein, and nucleic acid damage levels, or by determining the ratio of oxidized to reduced forms of one or more biomarkers. High levels of oxidative stress can be cytotoxic, so the degree of oxidative stress can be measured by assessing the concentration of intracellular proteins present in the systemic circulation from inflamed or lysed cells (e.g. nerve cells).

EXEMPLIFICATION

[0160] Example 1: Evaluation of efficacy of microbiome compositions in affecting insulin signaling and its upstream molecule IDE in AD mouse model

[0161] This Example provides an evaluation of the efficacy of microbiome compositions, specifically CT 10, CTlOx, and CT 10m compositions, in affecting insulin signaling and its upstream molecule IDE related to A0 clearance and neuroprotection in in vivo mouse model for AD.

[0162] Background: Insulin/IGF pathways support neuronal growth, survival, differentiation, migration, energy metabolism, gene expression, protein synthesis, cytoskeletal assembly, synapse formation, neurotransmitter and memory function, and plasticity (Chesik et al., 2008; de la Monte and Wands, 2005; Gong et al., 2008; Liang et al., 2007). Several AP- degrading enzymes, including neprilysin (NEP), insulin-degrading enzyme (IDE), and endothelin-converting enzyme reduce Ap levels and protect against cognitive impairment in mouse models of AD (Miners et al., 2011). Insulin-degrading enzyme (IDE) is a thiol- metalloprotease that degrades several peptide hormones, including insulin, glucagon, atrial natriuretic peptide (ANP), and IGF-II (Duckworth et al., 1998). IDE has also been identified as a major protease involved in the degradation of Ap peptides (Kurochkin and Goto, 1994; Vekrellis et al., 2000; Farris et al., 2003, 2004). Decreased IDE protein and mRNA levels have been observed in the hippocampus and cortex of AD patients with the apolipoprotein E-e4 (ApoE4) allele (Cook et al, 2003; Zhao et al., 2004).

[0163] Mouse Model; Tg2576 mice that were 9 months old were used for these experiments. This mouse model is one of the most popular transgenic mice models that overexpresses a mutant form of Amyloid Precursor Protein (APP) (isoform 695) with the Swedish mutation (KM670/671NL), resulting in increased levels of Amyloid beta (AP) and ultimately amyloid plaques. Wild type mice were used as a control in all experiments.

[0164] Study; Tg2576 mice were divided into five groups of 15 animals per group, and were provided mock (DPBS) or microbiome composition (CT 10, CT 10m, or CTlOx; composition details listed in Tables 2, 3, and 4) by daily oral gavage for 6 months. The groups were: (i) G1 : Wild type mice treated with DPBS; (ii) G2: Tg2576 transgenic mice treated with DPBS; (iii) G3: Tg2576 transgenic mice treated with CT10 composition; (iv) G4: Tg2576 transgenic mice treated with CTlOx composition; and (v) G5: Tg2576 transgenic mice treated with CT 10m composition. The animals were sacrificed at the end of the study, following which histological analyses were performed.

[0165] Methods; To determine whether CT10, CTlOx, or CTlOm treatment affects insulin signaling and its upstream molecule related to AP clearance and neuroprotection, insulin- degrading enzyme (IDE) protein levels in the brain samples of TG2576, AD mouse model, were checked by western blot. Major parts of cortex, hippocampus, and thalamus of brain lysates were obtained by immersing the tissue in PhosphoSafe buffer (EMD Millipore), and lysing within the Lysing Matrix D-containing tubes (MP Biomedicals). After centrifugation of the samples, the concentration of protein supernatant was determined by the Bradford method using the BioRad Protein assay reagent (Catalog #5000002, Bio-Rad). Total protein lysates (30 pg) were prepared with Sodium Dodecyl Sulfate (SDS) sample buffer, boiled at 70°C for 10 min. The samples were running on custom-made SDS-Polyacryl amide Bis-tris gels (4-12%, Invitrogen) using the MES running buffer followed by transfer onto PVDF membrane (Catalog #IB24001, Invitrogen) using iBlot2 (Invitrogen) system. The membrane was blocked using Intercept® (TBS) Protein-Free Blocking Buffer (Catalog #927-80001, LI-COR) or 5% non-fat dry milk solution for 1 hour and incubated with IDE specific antibody (Catalog #AB9210, Millipore) at 1 :1000 dilution in Intercept® T20 (TBS) Protein-Free Antibody Diluent (Catalog # 927-85001, LI-COR) overnight under gentle shaking conditions at 4°C. The following day, the membrane was thoroughly washed in TBST (0.1% Tween 20) and incubated in Horseradish peroxidase (HRP) conjugated secondary anti-rabbit antibody (Catalog #7074; Cell signaling) at 1 :2000 dilution at room temperature for 1 hour. Densitometric quantification of the immunoblots was performed by GeneTools (Syngene) after visualizing with G:Box Mini (Syngene). Target bands were normalized using their respective P-actin loading control.

[0166] Results; Fig. 1 shows results of this experiment. Each dot in the groups 1, 4, 5 represents pooled brain lysates from two animals. IDE protein level was significantly increased in the brain lysates of TG2576 (G2), CT10 (G3), and CTlOm (G5) groups compared to the control group (Gl), and the average level of IDE is higher in CTlOm (G5) than TG2576 (G2). *p<0.05, **p<0.01. Data represented as Mean ± SEM. Statistical analysis were performed using GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett’s test. This result suggests that CT 10 and CTlOm treatments increase IDE levels and its clearance effect on AP peptides so that it shows neuroprotection.

[0167] Example 2: Evaluation of efficacy of microbiome compositions in affecting insulin signaling and its upstream molecule NSE in AD mouse model

[0168] This Example provides an evaluation of the efficacy of microbiome compositions, specifically CT 10, CTlOx, and CTlOm compositions, in affecting insulin signaling and its upstream molecule NSE related to Akt activation and neuroprotection in in vivo mouse model for AD.

[0169] Background; Insulin/IGF pathways support neuronal growth, survival, differentiation, migration, energy metabolism, gene expression, protein synthesis, cytoskeletal assembly, synapse formation, neurotransmitter and memory function, and plasticity (Chesik et al., 2008; de la Monte and Wands, 2005; Gong et al., 2008; Liang et al., 2007). Neuron-specific enolase (NSE) is a glycolytic isoenzyme, a highly specific marker for central and peripheral neurons and neuroendocrine cells. NSE also expressed in microglia (Hafner et al., 2013; Pislar et al., 2017), and astrocytes, especially in reactive astrocytes (Vinores et al., 1985). NSE levels in serum and CSF have been used as a biomarker in injuries, cancers, and neurodegen erative diseases (Schmidt et al., 2014; Isgro et al., 2015). Elevation of NSE promotes glycolysis, cellular proliferation, activation and migration via the PI3K/AKT and MAPK/ERK pathways.

NSE-mediated activation of PI3K also regulates RhoA kinase, which influences actin cytoskeleton reorganization and induction of neurite outgrowth (Haque et al., 2018).

[0170] Mouse Model: The mouse model described in Example 1 was used in this study.

[0171] Study: Tg2576 mice were divided into five groups of 15 animals per group, and were provided mock (DPBS) or microbiome composition (CT 10, CT 10m, or CTlOx; composition details listed in Tables 2, 3, and 4) by daily oral gavage for 6 months. The groups were: (i) G1 : Wild type mice treated with DPBS; (ii) G2: Tg2576 transgenic mice treated with DPBS; (iii) G3: Tg2576 transgenic mice treated with CT10 composition; (iv) G4: Tg2576 transgenic mice treated with CTlOx composition; and (v) G5: Tg2576 transgenic mice treated with CT 10m composition. The animals were sacrificed at the end of the study, following which histological analyses were performed.

[0172] Methods: To determine whether CT10, CTlOx, or CTlOm treatment affects insulin signaling and its upstream molecule NSE related to Akt activation and neuroprotection, NSE protein levels in the brain samples of TG2576, AD mouse model, were checked by western blot. Major parts of cortex, hippocampus, and thalamus of brain lysates were obtained by immersing the tissue in PhosphoSafe buffer (EMD Millipore), and lysing within the Lysing Matrix D-containing tubes (MP Biomedicals). After centrifugation of the samples, the concentration of protein supernatant was determined by the Bradford method using the BioRad Protein assay reagent (Catalog #5000002, Bio-Rad). Total protein lysates (30 pg) was prepared with SDS sample buffer, boiled at 70°C for 10 min. The samples were run on custom-made SDS- Polyacryl amide Bis-tris gels (4-12%, Invitrogen) using the MES running buffer followed by transfer onto PVDF membrane (Catalog #IB24001, Invitrogen) using iBlot2 (Invitrogen) system. The membrane was blocked using Intercept® (TBS) Protein-Free Blocking Buffer (Catalog #927-80001, Ll-COR) or 5% non-fat dry milk solution for 1 hour and incubated with NSE specific antibody (Catalog #sc-21738, Santa Cruz Biotechnology) at 1 : 1000 dilution in Intercept® T20 (TBS) Protein-Free Antibody Diluent (Catalog # 927-85001, LI-COR) overnight under gentle shaking conditions at 4°C. The following day, the membrane was thoroughly washed in TBST (0.1% Tween 20) and incubated in HRP conjugated secondary anti-rabbit antibody (Catalog #7074; Cell signaling) at 1 :2000 dilution at room temperature for 1 hour. Densitometric quantification of the immunoblots was performed by GeneTools (Syngene) after visualizing with G:Box Mini (Syngene). Target bands were normalized using their respective p- actin loading control.

[0173] Results: Fig. 2 shows results of this experiment. Each dot in the groups 1, 4, 5 represents pooled brain lysates from two animals. NSE protein level was significantly increased in the brain lysates of CT 10 (G3), and CT 10m (G5) groups compared to the TG2576 (G2). **p<0.01, ***p<0 001, Data represented as Mean ± SEM. Statistical analysis were performed using GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett’s test. This result suggests that CT10 and CTlOm treatments increase NSE levels and may trigger Insulin-Akt signaling activation and neuroprotection.

[0174] Example 3: Evaluation of efficacy of microbiome compositions in affecting Akt signaling in AD mouse model

[0175] This Example provides an evaluation of the efficacy of microbiome compositions, specifically CT 10, CTlOx, and CT 10m compositions, in affecting Akt signaling in in vivo mouse model for AD.

[0176] Background: Insulin/IGF pathways support neuronal growth, survival, differentiation, migration, energy metabolism, gene expression, protein synthesis, cytoskeletal assembly, synapse formation, neurotransmitter function, and plasticity (Chesik et al., 2008; de la Monte and Wands, 2005; Gong et al., 2008; Liang et al., 2007). AD is associated with a relative state of insulin resistance in the brain, and impaired insulin/IGF-1 expression and Akt signaling. Insulin receptor are declined and tyrosine kinase activity is reduced (IR desensitization) (Steen E & de la Monte 2005; Frohlich L & Hoyer S. Ann.1999). AD is associated with decreased insulin mediated glucose uptake. Insulin levels are reduced in AD brain and CSF (Frohlich 1998; Craft 1998).

[0177] Mouse Model: The mouse model described in Example 1 was used in this study.

[0178] Study: Tg2576 mice were divided into five groups of 15 animals per group, and were provided mock (DPBS) or microbiome composition (CT 10, CT 10m, or CTlOx; composition details listed in Tables 2, 3, and 4) by daily oral gavage for 6 months. The groups were: (i) G1 : Wild type mice treated with DPBS; (ii) G2: Tg2576 transgenic mice treated with DPBS; (iii) G3: Tg2576 transgenic mice treated with CT10 composition; (iv) G4: Tg2576 transgenic mice treated with CTlOx composition; and (v) G5: Tg2576 transgenic mice treated with CT 10m composition. The animals were sacrificed at the end of the study, following which histological analyses were performed.

[0179] Methods; To determine whether CT10, CTlOx, or CTlOm treatment affects Akt signaling, active form of Akt, p-Akt (Ser473) and total Aktl enzyme protein levels in the brain samples of TG2576 mice were measured by western blot. Major parts of cortex, hippocampus, and thalamus of brain lysates were obtained by immersing the tissue in PhosphoSafe buffer (EMD Millipore), and lysing within the Lysing Matrix D-containing tubes (MP

Biomedicals). After centrifugation of the samples, the concentration of protein supernatant was determined by the Bradford method using the BioRad Protein assay reagent (Catalog #5000002, Bio-Rad). Total protein lysates (30 pg) was prepared with SDS sample buffer, boiled at 70°C for 10 min. The samples were running on custom-made SDS-Polyacryl amide Bis-tris gels (4-12%, Invitrogen) using the MES running buffer followed by transfer onto PVDF membrane (Catalog #IB24001, Invitrogen) using iBlot2 (Invitrogen) system. The membrane was blocked using Intercept® (TBS) Protein-Free Blocking Buffer (Catalog #927-80001, LI-COR) or 5% non-fat dry milk solution for 1 hour and incubated with p-Akt (Ser473), Aktl specific antibodies (Catalog #4060, #4691 Cell Signaling Tech.) at 1 : 1000 dilution in Intercept® T20 (TBS) Protein-Free Antibody Diluent (Catalog #927-85001, LI-COR) overnight under gentle shaking conditions at 4°C. The following day, the membrane was thoroughly washed in TBST (0.1% Tween 20) and incubated in HRP conjugated secondary anti-rabbit antibody (Catalog #7074;

Cell signaling) at 1 :2000 dilution at room temperature for 1 hour. Densitometric quantification of the immunoblots was performed by GeneTools (Syngene) after visualizing with G:Box Mini (Syngene). Target bands were normalized using their respective P-actin loading control.

[0180] Results: Fig. 3 shows results of this experiment. Each dot in the groups 1, 4, 5 represents pooled brain lysates from two animals. p-Akt (Ser473) protein level is significantly decreased in the brain lysates of TG2576 mice compared to the Non-Tg mice. CT10 (G3) or CTlOx (G4), CTlOm (G5) treated mice had significantly restored p-Akt (Ser473) protein levels compared to vehicle-treated TG2576 mice (G2). Total Aktl protein kinase levels in the brain lysates of CT10 (G3) or CTlOx (G4) or CTlOm (G5) treated TG2576 mice were elevated around 120% to that of the levels in control group (Non-Tg, Gl). */?<0.05 and **p<0.01 and ***p<0.001, and ****p<0.0001. Data represented as Mean ± SEM. Statistical analysis were performed using GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett’s test. This result suggests that CT 10, CTlOx, and CT 10m treatments increase and restore insulin/ Akt signaling pathway helping neuronal survival and memory and other brain functions.

[0181] Example 4: Evaluation of efficacy of microbiome compositions in affecting insulin receptor 0 levels in AD mouse model

[0182] This Example provides an evaluation of the efficacy of microbiome compositions, specifically CT10, CTlOx, and CTlOm compositions, in affecting insulin receptor 0 levels in in vivo mouse model for AD.

[0183] Mouse Model: The mouse model described in Example 1 was used in this study.

[0184] Study: Tg2576 mice were divided into five groups of 15 animals per group, and were provided mock (DPBS) or microbiome composition (CT 10, CTlOm, or CTlOx; composition details listed in Tables 2, 3, and 4) by daily oral gavage for 6 months. The groups were: (i) Gl : Wild type mice treated with DPBS; (ii) G2: Tg2576 transgenic mice treated with DPBS; (iii) G3: Tg2576 transgenic mice treated with CT10 composition; (iv) G4: Tg2576 transgenic mice treated with CTlOx composition; and (v) G5: Tg2576 transgenic mice treated with CTlOm composition. The animals were sacrificed at the end of the study, following which histological analyses were performed.

[0185] Methods: To confirm whether CT10, CTlOx, or CTlOm treatment affects insulin signaling, insulin receptor 0 protein levels in the brain samples of TG2576 mice were measured by western blot. Major parts of cortex, hippocampus, and thalamus of brain lysates were obtained by immersing the tissue in PhosphoSafe buffer (EMD Millipore), and lysing within the Lysing Matrix D-containing tubes (MP Biomedicals). After centrifugation of the samples, the concentration of protein supernatant was determined by the Bradford method using the BioRad Protein assay reagent (Catalog #5000002, Bio-Rad). Total protein lysates (30 pg) was prepared with SDS sample buffer, boiled at 70°C for 10 min. The samples were running on custom-made SDS-Polyacryl amide Bis-tris gels (4-12%, Invitrogen) using the MES running buffer followed by transfer onto PVDF membrane (Catalog &IB24001, Invitrogen) using iBlot2 (Invitrogen) system. The membrane was blocked using Intercept® (TBS) Protein-Free Blocking Buffer (Catalog #927-80001, LI-COR) or 5% non-fat dry milk solution for 1 hour and incubated with insulin receptor 0 specific antibody (Catalog #3025, Cell Signaling Tech.) at 1 : 1000 dilution overnight under shaking conditions at 4°C. The following day, the membrane was thoroughly washed in TBST (0.1% Tween 20) and incubated in HRP conjugated secondary anti-rabbit antibody (Catalog # 7074; Cell signaling) at 1:2000 dilution at room temperature for 1 hour. Densitometric quantification of the immunoblots was performed by GeneTools (Syngene) after visualizing with G:Box Mini (Syngene). Target bands were normalized using their respective 0- actin loading control.

[0186] Results; Fig. 4 shows results of this experiment. Each dot in the groups 1, 4, 5 represents pooled brain lysates from two animals. Compared to control group (Non-Tg), the average level of insulin receptor 0 was decreased in TG2576, but not significantly. Insulin receptor 0 protein level was significantly increased in the brain lysates of CT 10m (G5) group compared to the TG2576 (G2). **p<0.01. Data represented as Mean ± SEM. Statistical analysis were performed using GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnetf s test. This result suggests that CTlOm treatment increases and restores insulin/ Akt signaling pathway by increasing of insulin receptor 0 level.

[0187] Example 5; Evaluation of efficacy of microbiome compositions in affecting Glucose transport 3 levels in AD mouse model

[0188] This Example provides an evaluation of the efficacy of microbiome compositions, specifically CT 10, CTlOx, and CT 10m compositions, in affecting Glucose transport 3 levels in in vivo mouse model for AD.

[0189] Mouse Model: The mouse model described in Example 1 was used in this study.

[0190] Study: Tg2576 mice were divided into five groups of 15 animals per group, and were provided mock (DPBS) or microbiome composition (CT 10, CT 10m, or CTlOx; composition details listed in Tables 2, 3, and 4) by daily oral gavage for 6 months. The groups were: (i) G1 : Wild type mice treated with DPBS; (ii) G2: Tg2576 transgenic mice treated with DPBS; (iii) G3: Tg2576 transgenic mice treated with CT10 composition; (iv) G4: Tg2576 transgenic mice treated with CTlOx composition; and (v) G5: Tg2576 transgenic mice treated with CT 10m composition. The animals were sacrificed at the end of the study, following which histological analyses were performed.

[0191] Methods: To confirm whether CT10, CTlOx, or CTlOm treatment affects insulin signaling and its downstream target, glucose transport 3 (Glut3) protein levels in the brain samples of TG2576 mice were measured by western blot. Major parts of cortex, hippocampus, and thalamus of brain lysates were obtained by immersing the tissue in PhosphoSafe buffer (EMD Millipore), and lysing within the Lysing Matrix D-containing tubes (MP Biomedicals). After centrifugation of the samples, the concentration of protein supernatant was determined by the Bradford method using the BioRad Protein assay reagent (Catalog #5000002, Bio-Rad). Total protein lysates (30 pg) was prepared with SDS sample buffer, boiled at 70°C for 10 min. The samples were running on custom-made SDS-Polyacryl amide Bis-tris gels (4-12%, Invitrogen) using the MES running buffer followed by transfer onto PVDF membrane (Catalog #IB24001, Invitrogen) using iBlot2 (Invitrogen) system. The membrane was blocked using Intercept® (TBS) Protein-Free Blocking Buffer (Catalog #927-80001, LI-COR) or 5% non-fat dry milk solution for 1 hour and incubated with glucose transport 3 (Glut3) specific antibody (Catalog #ab 191071, abeam) at 1 : 1000 dilution overnight under shaking conditions at 4°C. The following day, the membrane was thoroughly washed in TBST (0.1% Tween 20) and incubated in HRP conjugated secondary anti-rabbit antibody (Catalog #7074; Cell signaling) at 1 :2000 dilution at room temperature for 1 hour. Densitometric quantification of the immunoblots was performed by GeneTools (Syngene) after visualizing with G:Box Mini (Syngene). Target bands were normalized using their respective P-actin loading control.

[0192] Results: Fig. 5 shows results of this experiment. Each dot in the groups 1, 4, 5 represents pooled brain lysates from two animals. Compared to control group (Non-Tg), the average level of Glut3 was decreased in TG2576 (G2), but not significantly. Glut3 protein level was significantly increased in the brain lysates of CTlOm (G5) group compared to the TG2576 (G2). *p<0.05. Data represented as Mean ± SEM. Statistical analysis were performed using GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett’s test. This result suggests that CT 10m treatment increases and restores insulin/Akt signaling pathway and energy metabolism by increasing of glucose transport 3 (Glut3) protein levels.

[0193] Example 6: Evaluation of efficacy of microbiome compositions in affecting protein RBAP48 levels in AD mouse model

[0194] This Example provides an evaluation of the efficacy of microbiome compositions, specifically CT 10, CTlOx, and CT 10m compositions, in affecting memory function related protein RBAP48 levels in in vivo mouse model for AD.

[0195] Background; Insulin/IGF pathways support neuronal growth, survival, differentiation, migration, energy metabolism, gene expression, protein synthesis, cytoskeletal assembly, synapse formation, neurotransmitter and memory function, and plasticity (Chesik et al., 2008; de la Monte and Wands, 2005; Gong et al., 2008; Liang et al., 2007). RBAP48 is interacting with histones and modify histone acetylation, which is crucial for memory consolidation. The loss of RBAP48 is key to age-related memory decline (Pavlopoulos et al. 2013). RBAP48 Interacts with a complex of CREB Binding Protein and phosphorylated CREB, involved in learning and memory (Zhang et al., 2000).

[0196] Mouse Model: The mouse model described in Example 1 was used in this study.

[0197] Study: Tg2576 mice were divided into five groups of 15 animals per group, and were provided mock (DPBS) or microbiome composition (CT 10, CT 10m, or CTlOx; composition details listed in Tables 2, 3, and 4) by daily oral gavage for 6 months. The groups were: (i) G1 : Wild type mice treated with DPBS; (ii) G2: Tg2576 transgenic mice treated with DPBS; (iii) G3: Tg2576 transgenic mice treated with CT10 composition; (iv) G4: Tg2576 transgenic mice treated with CTlOx composition; and (v) G5: Tg2576 transgenic mice treated with CTlOm composition. The animals were sacrificed at the end of the study, following which histological analyses were performed.

[0198] Methods: To determine whether CT10, CTlOx, or CTlOm treatment affects insulin signaling and its downstream target molecule related to memory function, RBAP48 protein levels in the brain samples of TG2576, AD mouse model, were measured by western blot. Major parts of cortex, hippocampus, and thalamus of brain lysates were obtained by immersing the tissue in PhosphoSafe buffer (EMD Millipore) and lysing within the Lysing Matrix D-containing tubes (MP Biomedicals). After centrifugation of the samples, the concentration of protein supernatant was determined by the Bradford method using the BioRad Protein assay reagent (Catalog #5000002, Bio-Rad). Total protein lysates (30 pg) was prepared with SDS sample buffer, boiled at 70°C for 10 min. The samples were running on custom-made SDS-Polyacryl amide Bis-tris gels (4-12%, Invitrogen) using the MES running buffer followed by transfer onto PVDF membrane (Catalog #IB24001, Invitrogen) using iBlot2 (Invitrogen) system. The membrane was blocked using Intercept® (TBS) Protein-Free Blocking Buffer (Catalog #927-80001, LI-COR) or 5% non-fat dry milk solution for 1 hour and incubated with RBAP48 specific antibody (Catalog #GTX70232, GeneTex) at 1 : 1000 dilution overnight under shaking conditions at 4°C. The following day, the membrane was thoroughly washed in TBST (0.1% Tween 20) and incubated in HRP conjugated secondary anti-mouse antibody (Catalog #7076; Cell signaling) at 1 :2000 dilution at room temperature for 1 hour. Densitometric quantification of the immunoblots was performed by GeneTools (Syngene) after visualizing with G:Box Mini (Syngene). Target bands were normalized using their respective P-actin loading control.

[0199] Results; Fig. 6 shows results of this experiment. Each dot in the groups 1, 4, 5 represents pooled brain lysates from two animals. RBAP48 protein level was significantly increased in the brain lysates of CT10 (G3), CTlOx (G4), and CTlOm (G5) groups compared to the TG2576 (G2). *p<0.05, ****p<0.0001. Data represented as Mean ± SEM. Statistical analysis were performed using GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnetf s test. This result suggests that CT10, CTlOx, CTlOm treatments increase and restore insulin/ Akt signaling pathway and its downstream target of memory function by increasing of RB AP48 protein levels.

[0200] Example 7: Evaluation of efficacy of microbiome compositions in affecting p-4EBPl, Akt and mTOR levels in AD mouse model [0201] This Example provides an evaluation of the efficacy of microbiome compositions, specifically CT10, CTlOx, and CTlOm compositions, in affecting p-4EBPl, Akt and mTOR levels in in vivo mouse model for AD.

[0202] Background: Insulin/IGF pathways support neuronal growth, survival, differentiation, migration, energy metabolism, gene expression, protein synthesis, cytoskeletal assembly, synapse formation, neurotransmitter and memory function, and plasticity (Chesik et al., 2008; de la Monte and Wands, 2005; Gong et al., 2008; Liang et al., 2007).

Hyperphosphorylation of 4EBP1 results in activation of cap-dependent translation, protein synthesis (Pause et al., 1994). Both the PI3 kinase/ Akt pathway and mTOR kinase regulate 4EBP1 activity (Brunn et al., 1997. Gingras et al., 1998). Tg2576 mice showed decreased levels of phospho-4EBP 1 (Thr37/46) in the brain hippocampus. Inhibition of mTOR signaling correlates with impairment in synaptic plasticity in an AD mouse model (Ma et al., 2010).

[0203] Mouse Model: The mouse model described in Example 1 was used in this study.

[0204] Study: Tg2576 mice were divided into five groups of 15 animals per group, and were provided mock (DPBS) or microbiome composition (CT 10, CT 10m, or CTlOx; composition details listed in Tables 2, 3, and 4) by daily oral gavage for 6 months. The groups were: (i) G1 : Wild type mice treated with DPBS; (ii) G2: Tg2576 transgenic mice treated with DPBS; (iii) G3: Tg2576 transgenic mice treated with CT10 composition; (iv) G4: Tg2576 transgenic mice treated with CTlOx composition; and (v) G5: Tg2576 transgenic mice treated with CT 10m composition The animals were sacrificed at the end of the study, following which histological analyses were performed.

[0205] Methods: To determine whether CT10, CTlOx, or CTlOm treatment affects insulin signaling and its downstream target molecule related to protein synthesis and translation, phospho-4EBPl (Thr37/46) protein levels in the brain samples of TG2576, AD mouse model, were measured by western blot. Major parts of cortex, hippocampus, and thalamus of brain lysates were obtained by immersing the tissue in PhosphoSafe buffer (EMD Millipore) and lysing within the Lysing Matrix D-containing tubes (MP Biomedicals). After centrifugation of the samples, the concentration of protein supernatant was determined by the Bradford method using the BioRad Protein assay reagent (Catalog #5000002, Bio-Rad). Total protein lysates (30 pg) was prepared with SDS sample buffer, boiled at 70°C for 10 min. The samples were running on custom-made SDS-Polyacryl amide Bis-tris gels (4-12%, Invitrogen) using the MES running buffer followed by transfer onto PVDF membrane (Catalog #IB24001, Invitrogen) using iBlot2 (Invitrogen) system. The membrane was blocked using Intercept® (TBS) Protein-Free Blocking Buffer (Catalog #927-80001, LI-COR) or 5% non-fat dry milk solution for 1 hour and incubated with phospho-4EBPl (Thr37/46) specific antibody (Catalog #2855, Cell signaling) at 1 :1000 dilution in Intercept® T20 (TBS) Protein-Free Antibody Diluent (Catalog # 927-85001, LI- COR) overnight under gentle shaking conditions at 4°C. The following day, the membrane was thoroughly washed in TBST (0.1% Tween 20) and incubated in HRP conjugated secondary anti- rabbit antibody (Catalog #7074; Cell signaling) at 1 :2000 dilution at room temperature for 1 hour. Densitometric quantification of the immunoblots was performed by GeneTools (Syngene) after visualizing with G:Box Mini (Syngene). Target bands were normalized using their respective 0-actin loading control.

[0206] Results: Fig. 7 shows results of this experiment. Each dot in the groups 1, 4, 5 represents pooled brain lysates from two animals. phospho-4EBPl (Thr37/46) protein level was significantly increased in the brain lysates of CTlOx (G4), and CT 10m (G5) groups compared to the TG2576 (G2). **p<0.01, ****p<0.0001. Data represented as Mean± SEM. Statistical analysis were performed using GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett’s test. This result suggests that CTlOx and CTlOm treatments increase insulin/ Akt signaling pathway and its downstream target of protein synthesis by increasing of phospho-4EBPI (Thr37/46) protein levels.

[0207] Example 8: Evaluation of efficacy of microbiome compositions in affecting antioxidant response regulator NRF2 levels in AD mouse model

[0208] This Example provides an evaluation of the efficacy of microbiome compositions, specifically CT 10, CTlOx, and CT 10m compositions, in affecting antioxidant response regulator NRF2 levels in in vivo mouse model for AD.

[0209] Background: Insulin/IGF pathways support neuronal growth, survival, differentiation, migration, energy metabolism, gene expression, protein synthesis, cytoskeletal assembly, synapse formation, neurotransmitter and memory function, and plasticity (Chesik et al., 2008; de la Monte and Wands, 2005; Gong et al., 2008; Liang et al., 2007). NRF2 (nuclear factor erythroid 2-related factor 2) is the master regulator of the cellular antioxidant response, regulating the expression of over 200 genes that contain antioxidant response elements (AREs). A dietetic herbal medicine (Bungeanum) improves cognitive dysfunction and neurological deficits in an aging mice model via activating PI3K/Akt/NRF2 signaling pathway (Zhao et al., 2020). NRF2 activation through the PI3K/GSK-3 axis protects neuronal cells from Ap-mediated oxidative and metabolic damage (Sotolongo et al., 2020). Activation of NRF2/ARE pathway alleviates the cognitive disfunction in a Tg mouse model of AD through modulation of oxidative stress (Tian et al., 2018).

[0210] Mouse Model; The mouse model described in Example 1 was used in this study.

[0211] Study: Tg2576 mice were divided into five groups of 15 animals per group, and were provided mock (DPBS) or microbiome composition (CT 10, CT 10m, or CTlOx; composition details listed in Tables 2, 3, and 4) by daily oral gavage for 6 months. The groups were: (i) G1 : Wild type mice treated with DPBS; (ii) G2: Tg2576 transgenic mice treated with DPBS; (iii) G3: Tg2576 transgenic mice treated with CT10 composition; (iv) G4: Tg2576 transgenic mice treated with CTlOx composition; and (v) G5: Tg2576 transgenic mice treated with CTlOm composition. The animals were sacrificed at the end of the study, following which histological analyses were performed.

[0212] Methods: To determine whether CT10, CTlOx, or CTlOm treatment affects insulin signaling and its downstream target molecule related to antioxidant response and neuroprotection, NRF2 protein levels in the brain samples of TG2576, AD mouse model, were measured by western blot. Major parts of cortex, hippocampus, and thalamus of brain lysates were obtained by immersing the tissue in PhosphoSafe buffer (EMD Millipore), and lysing within the Lysing Matrix D-containing tubes (MP Biomedicals). After centrifugation of the samples, the concentration of protein supernatant was determined by the Bradford method using the BioRad Protein assay reagent (Catalog #5000002, Bio-Rad). Total protein lysates (30 pg) was prepared with SDS sample buffer, boiled at 70°C for 10 min. The samples were running on custom-made SDS-Polyacryl amide Bis-tris gels (4-12%, Invitrogen) using the MES running buffer followed by transfer onto PVDF membrane (Catalog #IB24001, Invitrogen) using iBlot2 (Invitrogen) system. The membrane was blocked using Intercept® (TBS) Protein-Free Blocking Buffer (Catalog #927-80001, LI-COR) or 5% non-fat dry milk solution for 1 hour and incubated with NRF2 specific antibody (Catalog #12721, Cell signaling) at 1 : 1000 dilution in Intercept® T20 (TBS) Protein-Free Antibody Diluent (Catalog # 927-85001, LI-COR) overnight under gentle shaking conditions at 4°C. The following day, the membrane was thoroughly washed in TBST (0.1% Tween 20) and incubated in HRP conjugated secondary anti-rabbit antibody (Catalog #7074; Cell signaling) at 1 :2000 dilution at room temperature for 1 hour. Densitometric quantification of the immunoblots was performed by GeneTools (Syngene) after visualizing with G:Box Mini (Syngene). Target bands were normalized using their respective 0-actin loading control.

[0213] Results: Fig. 8 shows results of this experiment. Each dot in the groups 1, 4, 5 represents pooled brain lysates from two animals. NRF2 protein level was significantly increased in the brain lysates of CT10 (G3), CTlOx (G4), and CTlOm (G5) groups compared to the TG2576 (G2). *p<0.05, **p<0.01. Data represented as Mean ± SEM. Statistical analysis were performed using GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett’s test. This result suggests that CT 10, CTlOx and CT 10m treatments increase insulin/ Akt signaling pathway and its downstream target of antioxidant response and neuroprotection by increasing of NRF2 protein levels.

OTHER EMBODIMENTS

[0214] It is to be appreciated by those skilled in the art that various alterations, modifications, and improvements to the present disclosure will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of the present disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawing are by way of example only and any invention described in the present disclosure if further described in detail by the claims that follow.

[0215] Those skilled in the art will appreciate typical standards of deviation or error attributable to values obtained in assays or other processes as described herein. The publications, websites and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference in their entireties. [0216] It is to be understood that while embodiments of the invention have been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

EQUIVALENTS

[0217] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Appendix 1-1. Metabolite Abbreviations

Candidates^ Pathway Label t Pathway Index §

1.3 "Diaminopropane DAP Urea cycle relating metaboloism

1 -MethyM-imidazoleacetic acid MIA Urea cycle relating metaboloism

1 “Methylhistamine 1 -Methylhistamine Urea cycle relating metaboloism

1 -Methylnicotinamide 1 -Methvlnicotinamide Metabolism of coenzymes

1 “Pyrroline 5 -carboxy lie acid P5C Urea cycle relating metaboloism

2.3 -Diphosphoglyceric acid Diphosphoglycerate Central carbon metabolism

2,5-Dihydroxybenzoic acid Gensigen Pathway overview

2-Aminoadipic acid 2-Aminoadipic acid Lipid and amino acid metabolism

2'-Deoxyadenosine dAdenosine Nucleotide metabolism

2*-Deoxycytidine dCyt Nucleotide metabolism

2 ’ -De oxy guano s i ne dGuanosine Nucleotide metabolism

2 ’ -Deoxyuri dine dUri Nucleotide metabolism

2 -Hy droxybutyri c acid 2-HBA Lipid and amino acid metabolism

2 -Oxo adipic acid 2 -Oxoadipic acid Lipid and amino acid metabolism

2-Oxobutyric acid 2-Oxobutyric acid Lipid and amino acid metabolism

2-Oxoglutaric acid 2-OG Central carbon metabolism/

Urea cycle relating metaboloism

2 -Oxoisovaleric acid 2-KIV BCAA & aromatic amino acids

2 “Phenyl ethy lam ine Phenylethvl amine BCAA & aromatic amino acids

2 -Phosphoglyceric acid 2-PG Central carbon metabolism

3.3 \ 5 -Triiodothyronine T3 BCAA & aromatic amino acids

3.4-Dihy droxyphenylglycol DHPG Pathway overview

3 ,5 -Diiodotyrosine 3,5-DI-Tvr BCAA & aromatic amino acids

3 - Amino i sobuty ri c ac id 3 -Aminoisobutyric acid BCAA & aromatic amino acids /Nucleotide metabolism

3 ^! -Depliospho CoA Depho spho CoA Metabolism of coenzymes

3 -Hydroxy anthranilic acid 3-OHAA BCAA & aromatic amino acids

3 -Hydroxy butyric acid 3-HBA Central carbon metabolism /

Lipid and amino acid metabolism

3 -Hydroxy kynurenine 3-OHKY BCAA & aromatic amino acids

3-Hydroxypropionic acid b-Lactate BCAA & aromatic amino acids

3-Iodotyrosine MIT BCAA & aromatic amino acids

3-Methoxy-4-hydroxyphenylethyleneglycolMHPG BCAA & aromatic amino acids

3 -Methoxy anthran il i c acid 3~Methoxyanthranilic acid BCAA & aromatic amino acids

3 -Methoxy ty ram i ne 3 -Methoxy tvramine BCAA & aromatic amino acids

3 -Methy 1-2- oxo valeric acid 2K3MVA BCAA & aromatic amino acids

3 -Methyl crotony 1 CoA__divalent 3 -Methyl crotony 1 CoA BCAA & aromatic amino acids

3 -Methyl histidine 3 -Methylhistidine Urea cycle relating metaboloism

3-Phosphoglyceric acid 3-PG Central carbon metabolism / Lipid and amino acid metabolism

3-Ureidopropionic acid 3-Ureidopropionic acid Nucleotide metabolism

4-Acetamidobutanoic acid 4- Acetarn idobutanoic acid Pathway overview

4-Guanidinobutyric acid 4-GBA Urea cycle relating metaboloism

4-Hydroxyphenylacetaldehyde 4-Hydroxyphenylacetaldehyde Pathway overview

4-Methyl-2-oxovaleric acid 2-Oxoleucine BCAA & aromatic amino acids

4 -Methylthio -2-oxobutyri c acid KMTB Lipid and amino acid

4-Pyridoxic acid 4-Pyridoxic acid metabolism

5 , 6-Dimethvlbenzim i dazole Dimethylbenzimidazole Metabolism of coenzymes 5"AminO"4"Oxovaleric acid 5 -ALA* Metabolism of coenzymes

5 -Amino imidazole-4-carboxamide ribotide AICAR Lipid and amino acid metabolism 5'“methylthioadenosine MI Nucleotide metabolism 5'”Deoxy- A

5 -Hydroxy indoleacetic acid 5 -Hydroxy -I A A Urea cycle relating inetaboloism

5 - Hydroxy lysine 5 -Hydroxy lysine BCAA & aromatic amino acids

5 -Hydroxy tryptophan Pretonine Lipid and amino acid metabolism

5 -Methoxy indoleaceti c acid 5-MIAA BCAA & aromatic amino acids

5 -Methoxy tryptamine 5MOT BCAA & aromatic amino acids

5 “Methyltetrahydrofolic acid 5-MTHF BCAA & aromatic amino acids 5 -Oxoproliiie Metabolism of coenzymes

Oxoproline

6-Phosphogluconic acid 6-PG Urea cycle relating metaboloisni

7,8-Dihydrofolic acid Dihydrofolic acid Central carbon metabolism

Acetanilide Acetanilide Metabolism of coenzymes

Acetoacetic acid Acetoacetic acid BCAA & aromatic amino acids Central carbon metabolism /

Lipid and amino acid metabolism

Acetoacetyl CoA divalent AAcCoA Lipid and amino acid metabolism

Acetyl Co A divalent AcCoA Central carbon metabolism /

Lipid and amino acid metabolism / Metabolism of coenzymes

Acetylcholine Acetylcholine Lipid and amino acid metabolism

Adenine Adenine Nucleotide metabolism

Adenosine Adenosine Nucleotide metabolism

Adenyl osuccinic acid Succinyl AMP Nucleotide metabolism ADP ADP Central carbon metabolism /

Nucleotide metabolism

ADP -ribose ADP-Rib Central carbon metabolism /

Metabolism of coenzymes

Adrenaline Adrenaline BCAA & aromatic amino acids

Agmatine Agmatine Urea cycle relating metaboloism

Ala Ala Central carbon metabolism /

Urea cycle relating metaboloism /

Allantoic acid .Allantoic acid BCAA & aromatic amino acids

Pathway overview t Metabolites which have been already known about pathway information were listed up. They included metaboites which were not detected in this study. t Abbreviated names in Pathway Map.

§ Pathway information in the metabolites,

Appendix 1-1. Metabolite Abbreviations

Candidates^ Pathway Label# Pathway Index§

AMP AMP Nucleotide metabolism

Anserine di valent Anserine Urea cycle relating metaboloism

Anthranilic acid Anthranilic acid BCAA & aromatic amino acids

Arg Central carbon metabolism /

Urea cycle relating metaboloism

Argininosuccinic acid Arg Succinate Urea cycle relating metaboloism

Ascorbate 2-glucoside Ascorbate 2-glucoside Metabolism of coenzymes

Ascorbate 2-phosphate Ascorbate 2-phosphate Metabolism of coenzymes

Ascorbate 2-sulfate Ascorbate 2-sulfate Metabolism of coenzymes

Ascorbic acid Ascorbic acid Metabolism of coenzymes

Asn Asn Urea cycle relating metaboloism

Asp Asp Central carbon metabolism /

Urea cycle relating metaboloism /

Nucleotide metabolism

ATP ATP Central carbon metabolism /

Nucleotide metabolism

Betaine Betaine Lipid and amino acid metabolism

Betaine aldehyde +H2O BTL Lipid and amino acid metabolism

Biotin Biotin Metabol ism of coenzymes cAMP cAMP Nucleotide metabolism

Carbamoylphosph ate Carbamoyl-P Urea cycle relating metaboloism

Carnitine Carnitine Lipid and amino acid metabolism

Carnosine Carnosine Urea cycle relating metaboloism

CDP CDP Nucleotide metabolism

CDP-choline CDP-choline Lipid and amino acid metabolism cGMP cGMP Nucleotide metabolism

Cholic acid Cholic acid Lipid and amino acid metabolism

Choline Choline Lipid and amino acid metabolism cis -Aconitic acid cis -Aconitic acid Central carbon metabolism c A-Hy droxy prolin e c A-Hydroxyprol inc Urea cycle relating metaboloism

Citramalic acid Citramalic acid Pathway ovendew

Citric acid Citric acid Central carbon metabolism

Citrulline Citrulline Urea cycle relating metaboloism

CMP CMP Nucleotide metabolism

CMP -N-acetylneuram inate CMP-NeuNAc Co A dival Central carbon metabolism — ent CoA

Central carbon metabolism /

Creatine Creatine Metabolism of coenzymes

Creatinine Creatinine Urea cycle relating metaboloism

CTP CTP Urea cycle relating metaboloism

Cys Cys Nucleotide metabolism

Urea cycle relating metaboloism / Lipid and amino acid metabolism

Cys-Gly Cys-Gly Urea cycle relating metaboloism

Cystathionine Cystathionine Lipid and amino acid metabolism

Cysteamine Cysteamine Lipid and amino acid metabolism

Cysteic acid Cysteic acid Lipid and amino acid metabolism

Cysteinesulfinic acid Cysteinesulfinic acid Lipid and amino acid metabolism

Cystine Cystine Lipid and amino acid metabolism

Cytidine Cytidine Nucleotide metabolism

dADP dADP Nucleotide metabolism dAMP dAMP Nucleotide metabolism dATP dATP Nucleotide metabolism dCDP dCDP Nucleotide metabolism dCMP dCMP Nucleotide metabolism dCTP dCTP Nucleotide metabolism

Deamido-N AD’ ^r Deamido-NAD Metabolism of coenzymes

Desthiobiotiii Desthiobiotin Metabolism of coenzymes dGDP dGDP Nucleotide metabolism dGMP dGMP Nucleotide metabolism dGTP dGTP Nucleotide metabolism

Dihydroorotic acid Dihydroorotic acid Nucleotide metabolism

Dihydrouracil Dihvdrouracil Nucleotide metabolism

Dihydroxyacetone phosphate DIIAP Central carbon metabolism /

Lipid and amino acid metabolism dlMP dlMP Nucleotide metabolism diTP diTP Nucleotide metabolism

DOPA DOPA BCAA & aromatic amino acids

Dopamine Dopamine BCAA & aromatic amino acids dTDP dTDP Nucleotide metabolism dTDP-glucose TDP-Glc Pathway overview dTMP ' dTMP Nucleotide metabolism dTTP dTTP N ucleoti de metabol i sm

dUDP dUDP Nucleotide metabolism dUMP dUMP Nucleotide metabolism dUTP dUTP Nucleotide metabolism

Ergothioneine Ergothioneine Pathway overview

Erythrose 4-phosphate E4P Central carbon metabolism t Metabolites which have been already known about pathway information were listed up.

They included metaboites which were not detected in this study. t Abbreviated names in Pathway Map.

§ Pathway information in the metabolites.

21ppendix 1-L Metabolite Abbreviations

Candidates^ Pathway Label# Pathway lndex§

FAD divalent FAD Metabolism of coenzymes

FMN" FMN Metabolism of coenzymes

Folic acid Folic acid Metabolism of coenzymes

Formylanthranilic acid F ormvl anthranil ate Pathway overview

Fructose 1,6-diphosphate FL6P Central carbon metabolism

Fructose 1 -phosphate D-F1P Central carbon metabolism

Fructose 6-phosphate F6P Central carbon metabolism

Fumaric acid Fumaric acid Central carbon metabolism /

Urea cycle relating metaboloism

GABA GABA Urea cycle relating metaboloism

Galactose 1 -phosphate Gal IP Central carbon metabolism

GDP GDP Nucleotide metabolism

GDP-fucose GDP-fucose Central carbon metabolism

GDP-mannose GDP- Man Central carbon metabolism

Gin Gin Urea cycle relating metaboloism Central carbon metabolism /

Glu Glu Urea cycle relating metaboloism

Glucosamine Glucosamine Central carbon metabolism

Glucosamine 6-phosphate Glc-6P Central carbon metabolism

Glucosaminic acid Glucosaminic acid Central carbon metabolism

Glucose 1 -phosphate G1P Central carbon metabolism

Glucose 6-phosphate G6P Central carbon metabolism

Glucuronic acid Glucuronic acid Central carbon metabolism

Glutaryl CoA divalent GlutaryLCoA Lipid and amino acid metabolism

Glutathione (GSH) GSH Urea cycle relating metaboloism

Glutathione (GSSG) divalent GSSG Urea cycle relating metaboloism

Gly Gly Urea cycle relating metaboloism /

Lipid and amino acid metabolism

Glyceraldehyde 3 -phosphate GAP Central carbon metabolism /

Lipid and amino acid metabolism

Glyceric acid Glyceric acid Central carbon metabolism /

Lipid and amino acid metabolism Glycerol 3 -phosphate G3P Central carbon metabolism /

Lipid and amino acid metabolism

Glycerophosphocholine GPCho Lipid and amino acid metabolism

Glycocholic acid Glycocholic acid Lipid and amino acid metabolism

Glycolic acid Glycolic acid Lipid and amino acid metabolism

Glvoxylic acid Glvoxylic acid Lipid and amino acid metabolism

GMP ’ GMP Nucleotide metabolism

GTP GTP Nucleotide metabolism

Guanidoacetic acid Guanidoacetic acid Urea cycle relating metaboloism

Guanine Guanine Nucleotide metabolism

Guanosine Guanosine Nucleotide metabolism

His His Urea cycle relating metaboloism

Histamine Histamine Urea cycle relating metaboloism

HMG CoA divalent HMG-CoA Lipid and amino acid metabolism

Homocysteine Homocysteine Lipid and amino acid metabolism

Homovanillic acid HVA BCAA& aromatic amino acids

Hydroxyproline Hydroxy prol ine Urea cycle relating metaboloism

Hypotaurine Hypotaurine Lipid and amino acid metabolism

Hypoxanthine Hypoxanthine Nucleotide metabolism

IDP IDP Nucleotide metabolism lie He BCAA& aromatic amino acids

Imidazole-4-acetic acid Imidazole-4-acetic acid Urea cycle relating metaboloism

IMP IMP Nucleotide metabolism

Indole-3 -acetaldehyde Indole acetaldehyde BCAA & aromatic amino acids

Indole-3 -acetic acid Indole-3 -acetic acid BCAA & aromatic amino acids Inosine Inosine Nucleotide metabolism

Isobutyryl Co A divalent Isobutyryl -Co A Lipid and amino acid metabolism / BCAA & aromatic amino acids

Isocitric acid Isocitric acid Central carbon metabolism

ITP ITP Nucleotide metabolism

Kynurenic acid Kynurenic acid BCA A& aromatic amino acids

Kynurenine Kynurenine BCAA& aromatic amino acids

Lactic acid Lactic acid Central carbon metabolism /

Urea cycle relating metaboloism

Leu Leu BCAA & aromatic amino acids

Lvs Lys Lipid and amino acid metabolism

Malic acid Malic acid Central carbon metabolism /

Urea cycle relating metaboloism

Malonyl CoA divalent Malonyl-CoA Central carbon metabolism /

Lipid and amino acid metabolism

Mannose 1 -phosphate Man IP Central carbon metabolism

Mannose 6-phosphate Man6P Central carbon metabolism

Melatonin Melatonin BCAA & aromatic amino acids

Met Met Lipid and amino acid metabolism

Methylmalonic acid Methylmalonic acid Lipid and amino acid metabolism /

BCAA & aromatic amino acid

N ,N -Dimethy Igly cine DMG Lipid and amino acid metabolism

N®,N^,N^"Trimethyllysine Trimethyllysine Lipid and amino acid metabolism

N-Acetylaspartic acid N~Acetylaspartic acid Urea cycle relating metaboloism t Metabolites which have been already known about pathway information were listed up. They included metaboites which were not detected in this study. if Abbreviated names in Pathway Map.

§ Pathway information in the metabolites.

Appendix 1-1. Metabolite Abbreviations

Candidates^ Pathway Laberf Pathway Index »

N - Acety Iglucos amine Gl cN Ac Central carbon metabolism

N -Acety Iglucosamine 1 -phosphate GlcNAc-P Central carbon metabolism

N-Acetvlglucosamine 6-phosphate NAcGlcNP Central carbon metabolism

N-Acetylglutamic acid N-AcGlu Urea cycle relating metaboloism N-Acetylmannosamine ManNAc Central carbon metabolism N-A cetylneuraminic acid NeuNAc Central carbon metabolism N -Ace tvlornithine N- AcOrn Urea cycle relating metaboloism N-Acetvlputrescine N-Acetvlputrescine Urea cycle relating metaboloism NAD ⁺ ' N AD ⁺ " Central carbon metabolism /

Metabolism of coenzymes

NADH NADH Central carbon metabolism /

Metabolism of coenzymes

NADP ⁺ NADP ⁺ Central carbon metabolism /

Metabolism of coenzymes

NADPH div alent NADPH Central carbon metabolism /

Metabolism of coenzymes

N-Carbamoylaspartic acid C arb amoy 1 - A sp Urea cycle relating metaboloism /

Nucleotide metabolism

N -Formyl aspartic acid N-Formyl aspartic acid Urea cycle relating metaboloism Nicotinamide Nicotinamide Metabolism of coenzymes

Nicotinic acid Nicotinic acid Metabolism of coenzymes

N -Methyl serotonin N “Methylserotonin Pathway overview

N -Methyl try ptam ine N -Methyl tryptam ine BCAA & aromatic amino acids

N "Methyltvramine N "Methyltvramine BCAA & aromatic amino acids

KMX NicRN " Metabolism of coenzymes

Noradrenaline Noradrenaline BCAA & aromatic amino acids

N ormetanephrine N ormetanephrine Pathway overview

OAcetyl carnitine ALCAR Lipid and amino acid metabolism o-Aminophenol 2-Aminophenol BCAA & aromatic amino acids o-Hydroxyphenylacetic acid 2-HPAA BCAA & aromatic amino acids O-Phosphoserine 3PSer Lipid and amino acid metabolism

Ornithine Ornithine Urea cycle relating metaboloism Orotic acid Orotic acid Nucleotide metabolism

Orotidine S'-monophosphate Orotidine 5 'P Nucleotide metabolism pl, p4„£)i(ade ₁₁ osine-5 ^t ) AppppA Nucleotide metabolism tetraphosphate divalent

Pantothenic acid Pantothenic acid Metabolism of coenzymes

Phe Phe BCAA & aromatic amino acids

Phenaceturic acid Phen aceturic acid BCAA & aromatic amino acids

Phenylpyruvic acid Phenylpyruvate BCAA & aromatic amino acids

Phosphocreatine Phosphocreatine Urea cycle relating metaboloism

Phosphoenolpyruvic acid PEP Central carbon metabolism

Pho s phory 1 cho line Phosphorvlchol ine Lipid and amino acid metabolism p-Hy droxypheny 1 acetic acid 4-HPAA " BCAA & aromatic amino acids p-Hy droxyph eny Ipyruvi c acid HPP BCAA & aromatic amino acids Phytic acid divalent Phytic acid Pathway overview

Pipecolic acid Pipecolic acid Lipid and amino acid metabolism

Porphobil inogen Porphobil inogen Lipid and amino acid metabolism

Pro Pro Urea cycle relating metaboloism

Propionic acid Propionic acid Lipid and amino acid metabolism /

BCAA & aromatic amino acid

Propionyl CoA divalent Propanoyl-CoA Lipid and amino acid metabolism / BCAA & aromatic amino acids / Nucleotide metabolism

PRPP PRPP Central carbon metabolism /

Nucleotide metabolism Putrescine Putrescine LTrea cycle relating metaboloism

Pyridoxal Pyridoxal Metabolism of coenzymes

Pyridoxal 5 -phosphate PLP Metabolism of coenzymes

Pyridoxamine Pyridoxamine Metabolism of coenzymes

Py ridoxamine 5 '-phosphate Pyridoxamine -P Metabolism of coenzymes

Pyridoxine Pyridoxine Metabolism of coenzymes

Pyruvic acid Pyruvic acid Central carbon metabolism /

Urea cycle relating metaboloism /

Lipid and amino acid metabolism

Quinolinic acid Quinolinic acid BCAA & aromatic amino acids /

Metabolism of coenzymes

Riboflavin Riboflavin Metabolism of coenzymes

Ribose 1 -phosphate RIP Pathway overview

Ribose 5-phosphate R5P Central carbon metabolism /

Metabolism of coenzymes

Ribulose 5-phosphate Ru5P Central carbon metabolism Saccharopine Saccharopine Lipid and amino acid metabolism

S-Adenosylhomocysteine SAHC Lipid and amino acid metabolism

S - Adenosylmethionine SAM Lipid and amino acid metabolism Sarcosine Sarcosine Lipid and amino acid metabolism

Sedoheptulose 7 -phosphate S7P Central carbon metabolism Ser Ser Lipid and amino acid metabolism

Serotonin Serotonin BCA A & aromatic amino acids

S -Lactoy Igl utath ione S -L actoy 1 glutath ione Urea cycle relating metaboloism Spermidine Spermidine Urea cycle relating metaboloism

Spermine Spermine Urea cycle relating metaboloism

Succinic acid Succinic acid Central carbon metabolism /

Urea cycle relating metaboloism

Succinic semialdehyde Succinic semialdehyde Urea cycle relating metaboloism t Metabolites which have been already known about pathway information were listed up. They included metaboites which were not detected in this study.

X Abbreviated names in Pathway Map.

§ Pathway information in the metabolites.

Appendix 1-1. Metabolite Abbreviations

Candidates^ Pathway Labelt Pathway Index §

Succinyl CoA divalent SucCoA Central carbon metabolism

Taurine Taurine Lipid and amino acid metabolism

Taurocholic acid Taurocholic acid Lipid and amino acid metabolism

Taurocyamine Taurocyamine Lipid and amino acid metabolism

Thiamine Thiamine Metabolism of coenzymes

Thiamine diphosphate ThPP Metabolism of coenzymes

Thiamine phosphate TMP Metabolism of coenzymes

Thr Thr Lipid and amino acid' metabolism

Thymidine Thymidine Nucleotide metabolism

Thymine Thymine Nucleotide metabolism

T Trp BCAA & aromatic amino acids

Tr ^r yPptamine Tryptamine BCAA & aromatic amino acids

Tyr Tyr BCAA & aromatic amino acids

Tyramine Tyramine BCAA & aromatic amino acids

UDP UDP Nucleotide metabolism

UDP-glucose UDP-Glc Central carbon metabolism

UDP-glucuronic acid UDP-GlcA Central carbon metabolism

UDP-A'-acetygiucosamine UDP-GlcNAc Central carbon metabolism

UMP UMP Nucleotide metabolism

Uracil Uracil Nucleotide metabolism

Urea. Urea Urea cycle relating metaboloism

Uric acid Uric acid Nucleotide metabolism

Uridine Uridine Nucleotide metabolism

Urocanic acid Urocanic acid Urea cycle relating metaboloism

UTP UTP Nucleotide metabolism

Vai Vai BCAA & aromatic amino acids

Vanillylmandelic acid VMA BCAA & aromatic amino acids Xanthine Xanthine Nucleotide metabolism

Xanthosine Xanthosine Nucleotide metabolism

Xanthurenic acid Xanthurenic acid BCAA & aromatic amino acids

XMP XMP Nucleotide metabolism

XTP XTP Nucleotide metabolism

Xylulose 5 -phosphate X5P Central carbon metabolism p -Ala p-Ala Urea cycle relating metaboloism / Nucleotide metabolism / Metabolism of coenzymes

Y -Butyro betaine Actinine Lipid and amino acid metabolism

Y -Glu-Cys g-Glu-Cys Urea cycle relating metaboloism

Metabolites which have been already known about pathway information were listed up. They included metaboites which were not detected in this study. t Abbreviated names in Pathway Map.

§ Pathway information in the metabolites.

Appendix 1~2. Pathway Abbreviations

Pathway Labetf Candidates"^ Pathway Index§

1 -Methylhistamine 1 “Methylhistamine Urea cycle relating metaboloism

1 -Methvlnicotinamide 1 “Methvlnicotinamide Metabolism of coenzymes

2- Aminoadipic acid 2-Aminoadipic acid Lipid and amino acid metabolism

2-Aminophenol o~ Aminophenol BCAA & aromatic amino acids

2-HBA 2-Hydroxybutyric acid Lipid and amino acid metabolism

2-HPAA o-Hydroxy phenylacetic acid BCAA & aromatic amino acids

2K3MVA. 3-Methyl"2”Oxovaleric acid Lipid and amino acid metabolism

2-KIV 2-Oxoisovaleric acid BCAA & aromatic amino acids

2-OG 2-Oxoglutaric acid Central carbon metabolism / Urea cycle relating metaboloism

2-Oxoadipic acid 2-Oxoadipic acid Lipid and amino acid metabolism

2-Oxobutyric acid 2-Oxo butyric acid Lipid and amino acid metabolism

2-Oxoleucine 4"Methyl-2"Oxovaleri c acid Lipid and amino acid metabolism

2-PG 2-Phosphogly ceric acid Central carbon metabolism

3.5-DLTyr 3.5 -Diiodotyrosine BCAA & aromatic amino acids

3 ~Aminoisobutyric acid 3-Aminoisobutyric acid BCAA & aromatic amino acids /

Nucleotide metabolism

3-HBA 3 -Hydroxy butvne acid Central carbon metabolism /

Lipid and amino acid metabolism

3 -Methoxy anthranil ic acid 3-Methoxyanthranilic acid BCAA & aromatic amino acids

3 ~M ethoxytyramine 3 -Methoxy tyramine BCAA & aromatic amino acids

3 -Methyl crotony 1 -C o A 3 -Methyl crotony 1 -Co A__divalent BCAA & aromatic amino acids

3 -Methvlh i sti d ine 3 -Methylhistidine Urea cycle relating metaboloism

3-OHAA 3 -Hydroxy anthranilic acid BCAA & aromatic amino acids

3-OHKY 3 "Hydroxykynurenine BCAA & aromatic amino acids

3 -PG 3 “Phosphoglyceric acid Central carbon metabolism /

Lipid and amino acid metabolism

3PSer O-Phosphoserine Lipid and amino acid metabolism

3 -Ureidopropionic acid 3 -Ureidopropionic acid Nucleotide metabolism

4-Acetamidobutanoic acid 4-Acetamidobutanoic acid Pathway overview

4-GBA 4-Guanidinobutyric acid Urea cycle relating metaboloism

4-HPAA p-Hydroxyphenylacetic acid BCAA & aromatic amino acids

4 -Hy drox y pheny 1 ace tai dehy de 4-Hydroxyphenylacetaldehyde Pathway overview 4-Pvridoxic acid 4-Pyridoxic acid Metabolism of coenzymes

5 -ALA 5-AminO"4"Oxovaleric acid Lipid and amino acid metabolism

5 -Hydroxy -I AA 5 “Hydroxy indoleacetic aci d BCAA & aromatic amino acids

5 -hydroxyl vsine 5 -hydroxy lysine Lipid and amino acid metabolism

5-MIAA 5-Methoxy indole ace tic acid BCAA & aromatic amino acids

5MOT 5 -Methoxy tryptamine BC A A & aromatic amino acids

5-MHTF 5-Methyltetrahydrofolic acid Metabolism of coenzymes

6~PG 6-Phosphogluconic acid Central carbon metabolism

AAcCoA Acetoacetyl Co A divalent Lipid and amino acid metabolism

AcCoA Acetyl CoA divalent Central carbon metabolism / Lipid and amino acid metabolism / Metabolism of coenzymes

Acetanilide Acetanilide BCAA & aromatic amino acids

Acetoacetic acid Acetoacetic acid Central carbon metabolism /

Lipid and amino acid metabolism

Acetylcholine Acetylcholine Lipid and amino acid metabolism Actin ine y “Butyrobetaine Lipid and amino acid metabolism Adenine Adenine Nucleotide metabolism Adenosine Adenosine Nucleotide metabolism ADP ADP Central carbon metabolism / Nucleotide metabolism

ADP -Rib ADP-ribose Central carbon metabolism /

Metabolism of coenzymes

Adrenaline Adrenaline BCAA & aromatic amino acids Agmatine Agmatine Urea cycle relating metabolosim AICAR 5"Aminoimidazole“4- Nucleotide metabolism carboxamdie ribotide

Ala Ala Central carbon metabolism / Urea cycle relating metabolosim ! BCAA & aromatic amino acids

ALCAR O- Acetyl earn itine Lipid and amino acid metabolism

Allantoic acid Allantoic acid Pathway overview

AMP AMP Nucleotide metabolism

Anserine Anserine dival ent Urea cycle relating metabolosim

Anthranilic acid Anthranilic acid BCAA & aromatic amino acids

AppppA pl, p4~Di(adenosine~5') Nucleotide metabolism tetraphosphate divalent

Arg Arg Central carbon metabolism / Urea cycle relating metabolosim

ArgSuccinate Argininosuccinic acid Urea cycle relating metabolosim

Ascorbate 2 -glucoside Ascorbate 2 -glucoside Metabolism of coenzymes

Ascorbate 2 -phosphate Ascorbate 2 -phosphate Metabolism of coenzymes

Ascorbate 2-sulfate Ascorbate 2-sulfate Metabolism of coenzymes

Ascorbic acid Ascorbic acid Metabolism of coenzymes

Asn Asn Urea cycle relating metabolosim

Asp Asp Central carbon metabolism / Urea cycle relating metabolosim / Nucleotide metabolism

ATP ATP Central carbon metabolism / Nucleotide metabolism

P -Ala P -Ala Central carbon metabolism / Nucleotide metabolism / Metabolism of coenzymes

Betaine Betaine Lipid and amino acid metabolism

Biotin Biotin Metabolism of coenzymes b-Lactate 3 -Hydroxypropionic acid BCAA & aromatic amino acids t Abbreviated names in Pathway Map.

Metabolites which have been already known about pathway information were listed up. They included metaboites which were not detected in this study.

§ Pathway information in the metabolites.

Appendix 1-2. Pathway Abbreviations Pathway Labelt Pathway Index§

Betaine aldehyde_+H2O Lipid and amino acid metabolism cAMP Nucleotide metabolism Carbamoyl- Asp N-Carbamoylaspartic acid Urea cycle relating metaboloism / Nucleotide metabolism

Carbamoyl-P Carbamoyl phosphate Urea cycle relating metaboloism Carnitine Carnitine Lipid and amino acid metabolism Carnosine Carnosine Urea cycle relating metaboloism CDP CDP Nucleotide metabolism

CDP-choline CDP-choline I ipid and amino acid metabolism cGMP cGMP Nucleotide metabolism

Cholic acid Cholic acid Lipid and amino acid metabolism Choline Choline Lipid and amino acid metabolism cA-Aconitic acid cA-Aconitic acid Central carbon metabolism c A-I ly droxy prol ine cA-Hydroxyproline Urea cycle relating metaboloism Citramalic acid Citramalic acid Pathway overview Citric acid Citric acid Central carbon metabolism Citrulline Citrulline Urea cycle relating metaboloism CMP CMP Nucleotide metabolism CMP-NeuNAc CMP-N-acetylneuraminate Central carbon metabolism

CoA CoA divalent Central carbon metabolism /

Metabolism of coenzymes

Creatine Creatine Urea cycle relating metaboloism

Creatinine Creatinine Urea cycle relating metaboloism

CTP CTP Nucleotide metabolism

Cys Urea cycle relating metaboloism / Lipid and amino acid metabolism / Metabolism of coenzymes

Cys-Gly Cys-Gly Urea cycle relating metaboloism Cystathionine Cystathionine Lipid and amino acid metabolism Cysteamine Cysteamine Lipid and amino acid metabolism Cysteic acid Cysteic acid Lipid and amino acid metabolism Cysteinesullinic acid Cysteinesullinic acid Lipid and amino acid metabolism Cystine Cystine Lipid and amino acid metabolism Cytidine Cytidine Nucleotide metabolism dAdenosine 2 ⁸ -Deoxv adeno sine Nucleotide metabolism dADP dADP Nucleotide metabolism dAMP dAMP Nucleotide metabolism DAP 1 ,3 -Diaminopropane Urea cycle relating metaboloism dATP dATP Nucleotide metabolism dCDP dCDP Nucleotide metabolism dCMP dCMP Nucleotide metabolism dCTP dCTP Nucleotide metabolism dCyt 2 ⁸ -Deoxy cy tid ine Nucleotide metabolism

De ami do -N A D Deamido-N AD+ Metabolism of coenzymes Dephospho-CoA 3 '“Dephospho Co A Metabolism of coenzymes Desthiobiotin Desthiobiotin Metabolism of coenzymes D-F1P Fructose 1 -phosphate Central carbon metabolism dGDP dGDP Nucleotide metabolism

dGMP dGMP Nucleotide metabolism dGTP dGTP Nucleotide metabolism dGuanosine 2'-Deoxyguanosine Nucleotide metabolism

DHAP Dihydroxy acetone phosphate Central carbon metabolism / Lipid and amino acid metabolism

DIIPG 3 , 4 -D iliy droxy phenyl gly co 1 Pathway overview

Dihydrofolic acid 7,8-Dihydrofolic acid Metabolism of coenzymes

Dihydroorotic acid Dihydroorotic acid Nucleotide metabolism

Dihydrouracil Dihydrouracil Nucleotide metabolism

Dimethylbenzimidazole 5 , 6~Dimethylbenzimidazole Metaboli sm of coenzymes dIMP dIMP Nucleotide metabolism

Diphosphoglvcerate 23 "Diphosphogly ceric acid Central carbon metabolism diTP diTP Nucleotide metabolism

DMG N,N -Dimethvlgl vcine Lipid and amino acid metabolism

DOPA DOPA BCAA & aromatic amino acids

Dopamine Dopamine BCA A & aromatic amino acids dTDP dTDP Nucleotide metabolism dTMP dTMP Nucleotide metabolism dTTP dTTP Nucleotide metabolism dUDP dUDP Nucleotide metabolism dUMP dUMP Nucleotide metabolism dUri 2 '"Deoxyuridine Nucleotide metabolism

dUTP dUTP Nucleotide metabolism

E4P Erythrose 4-phosphate Central carbon metabolism

Ergothioneine Ergothioneine Pathway overview

F1,6P Fructose 1 ,6-diphosphate Central carbon metabolism

F6P Fructose 6-phospliate Central carbon metabolism _ t Abbreviated names in Pathway Map.

Metabolites which have been already known about pathway information were listed up. They included metaboites which were not detected in this study.

§ Pathway information in the metabolites.

Appendix 1-2. Pathway Abbreviations

Pathway Labelt Candidates^ Pathway lndex§

FAD FAD divalent Metabolism of coenzymes

FMN FMN Metabolism of coenzymes

Folic acid Folic acid Metabolism of coenzymes

Formylanthranilate Formylanthranilic acid Pathway overview

Fumaric acid Fumaric acid Central carbon metabolism /

Urea cycle relating metaboloism G1P Glucose 1 -phosphate Central carbon metabolism

G3P Glycerol 3 -phosphate Central carbon metabolism /

Lipid and amino acid metabolism G6P Glucose 6-phosphate Central carbon metabolism

GABA GABA Urea cycle relating metaboloism

Gal IP Galactose 1 -phosphate Central carbon metabolism

GAP Glyceraldehyde 1 -phosphate Central carbon metabolism /

Lipid and amino acid metabolism

GDP GDP Nucleotide metabolism

GDP-fucose GDP-fucose Central carbon metabolism

GDP -Man GDP-mannose Central carbon metabolism

Gensigen 2,5-Dihydroxybenzoic acid Pathway overview g-Glu-Cys Y -Glu-Cys Urea cycle relating metaboloism

Glc-6p Glucosamine 6-phosphate Central carbon metabolism

GlcNAc N-Acetylglucosamine Central carbon metabolism

GlcNAc-P N-Acetylglucosamine 1 -phosphate Central carbon metabolism

Gin Gin Urea cycle relating metaboloism

Glu Glu Central carbon metabolism /

Urea cycle relating metaboloism

Glucosamine Glucosamine Central carbon metabolism

Glucosaminic acid Glucosaminic acid Central carbon metabolism

Glucuronic acid Glucuronic acid Central carbon metabolism

Glutaryl-CoA Glutaryl-CoA Lipid and amino acid metabolism

Gly Gly Urea cycle relating metaboloism /

Lipid and amino acid metabolism

Glyceric acid Glyceric acid Central carbon metabolism /

Lipid and amino acid metabolism

Glycocholic acid Glycocholic acid Lipid and amino acid metabolism Glycolic acid Glycolic acid Lipid and amino acid metabolism Glyoxylic acid Glyoxylic acid Lipid and amino acid metabolism GMP GMP Nucleotide metabolism GPCho Glycerophosphocholine Lipid and amino acid metabolism GSH Glutathione (GSH) Urea cycle relating metaboloism GSSG Glutathione (GSH) divalent Urea cycle relating metaboloism GTP GTP Nucleotide metabolism Guanidoacetic acid Guanidoacetic acid Urea cycle relating metaboloism Guanine Guanine Nucleotide metabolism Guanosine Guanosine Nucleotide metabolism His His Urea cycle relating metaboloism Histamine Histamine Urea cycle relating metaboloism HMG-CoA HMG CoA divalent Lipid and amino acid metabolism Homocysteine Homocysteine Lipid and amino acid metabolism HPP p-Hydroxyphenylpyruvic acid BCAA & aromatic amino acids HVA Homovanillic acid BCAA & aromatic amino acids Hydroxyproline Hydroxyproline Urea cycle relating metaboloism Hypotaurine Hypotaurine Lipid and amino acid metabolism

Hypoxanthine Hypoxanthine Nucleotide metabolism

IDP IDP Nucleotide metabolism

He He BCAA & aromatic amino acids

Imidazole-4-acetic acid Imidazole-4-acetic acid Urea cycle relating metaboloism

IMP IMP Nucleotide metabolism

Indole-3 -acetic acid Indole-3 -acetic acid BCAA & aromatic amino acids

Indole-3 -acetaldehyde Indole-3 -acetaldehyde BCAA & aromatic amino acids Inosine Inosine Nucleotide metabolism

Isobutyryl -Co A Isobutyryl CoA divalent Lipid and amino acid metabolism /

BCAA & aromatic amino acids Isocitric acid Isocitric acid Central carbon metabolism

ITP ITP Nucleotide metabolism

KMTB 4-Methylthio-2-oxobutyric acid Lipid and amino acid metabolism

Kynurenic acid Kynurenic acid BCAA & aromatic amino acids Kynurenine Kynurenine BCAA & aromatic amino acids Lactic acid Lactic acid Central carbon metabolism /

Urea cycle relating metaboloism

Leu Leu BCAA & aromatic amino acids

Lys Lys Lipid and amino acid metabolism

Malic acid Malic acid Central carbon metabolism /

Urea cycle relating metaboloism

Malonyl -Co A Malonyl CoA divalent Central carbon metabolism /

Lipid and amino acid metabolism

Man IP Mannose 1 -phosphate Central carbon metabolism Man6P Mannose 6-phosphate Central carbon metabolism ManNAc N -Acetylmanno s amine Central carbon metabolism Melatonin Melatonin BCAA & aromatic amino acids Met Met Lipid and amino acid metabolism Abbreviated names in Pathway Map. Metabolites which have been already known about pathway information were listed up. They included metaboites which were not detected in this study.

§ Pathway information in the metabolites.

Appendix 1-2. Pathway Abbreviations

Pathway Labelt Candidates^ Pathway Index§

Methylmalonic acid Methylmalonic acid Lipid and amino acid metabolism / BCAA & aromatic amino acids

MHPG 3 -Methoxy -4 BCAA & aromatic amino acids

-hydroxyphenylethyleneglycol

MIA 1 -Methyl-4-imidazoleacetic acid Urea cycle relating metaboloism

MIT 3~Iodotyrosine BCAA & aromatic amino acids

MTA 5 ’ -Deoxy- 5 ’ -methylthio adenos ine Urea cycle relating metaboloism

N- Acetylaspartic acid A- Acetylaspartic acid Urea cycle relating metaboloism

N - Ace tvlp u ties cine N~ Acetyl putre s cine Urea cycle relating metaboloism

NAcGlcNP yV-Acetylglucosamine 6-phosphate Central carbon metabolism

N-AcGlu A-Acetylglutamic acid Urea cycle relating metaboloism

N- AcOrn A-Acetylomithine Urea cycle relating metaboloism

NAD ⁺ yVAD ⁺ Central carbon metabolism /

Metabolism of coenzymes

NADH NADH Central carbon metabolism /

Metabolism of coenzymes

NADP ⁺ NADP+ Central carbon metabolism /

Metabolism of coenzymes

NADPH NADPH divalent Central carbon metabolism /

Metabolism of coenzymes

NeuNAc A- Acetylneuraminic aci d Central carbon metabolism

N-Formyl aspartic acid AMormylaspartic acid Urea cycle relating metaboloism

Nicotinamide Nicotinamide Metabolism of coenzymes

Nicotinic acid Nicotinic acid Metabolism of coenzymes

NicRN NMN Metabolism of coenzymes

N -Methyl s eroton in ANMethylserotonin Pathway overview

N -Methy Itry ptam i ne N -Me thy 1 try ptam ine BCAA & aromatic amino acids

N "Methyltyramine A^Methyltyramine BCAA & aromatic amino acids

Noradrenaline Noradrenaline BCAA & aromatic amino acids

Normetanephrine Normetanephrine Pathway overview

Ornithine Ornithine Urea cycle relating metaboloism

Orotic acid Orotic acid Nucleotide metabolism

Orotidine5'P Orotidine 5 ^f -monophosphate Nucleotide metabolism

Oxoproline 5 -Oxoproline Urea cycle relating metaboloism

P5C 1 “Pyrroline 5-carboxyIic acid Urea cycle relating metaboloism Pantothenic acid Pantothenic acid Metabolism of coenzymes

PEP Phosphoenolpyruvic acid Central carbon metabolism

Phe Phe BCAA & aromatic amino acids

Phenaceturic acid Phenaceturic acid BCAA & aromatic amino acids

Phenylethylamine 2-Phenylethylamine BCAA & aromatic amino acids

Phenylpyruvate Phenylpyruvic acid BCAA & aromatic amino acids

Phosphocreatine Phosphocreatine Urea cycle relating metaboloism

Phosphoryl choline Phosphorylcholine Lipid and amino acid metabolism

Phytic acid Phytic acid divalent Pathway overview

Pipecolic acid Pipecolic acid I Jpid and amino acid metabolism

PLP Pyridoxal 5 -phosphate Metabolism of coenzymes

Porphobilinogen Porphobilinogen Lipid and amino acid metabolism

Pretonine 5 ~Hy droxy try ptoph an BCAA & aromatic amino acids

Pro Pro Urea cycle relating metaboloism

Phytic acid Phytic acid divalent Pathway overview Pipecoliacid Pipecolic acid Lipid and amino acid metabolism PLP Pyridoxal 5 -phosphate Metabolism of coenzymes Porphobilinogen Porphobilinogen Lipid and amino acid metabolism

Pretonine 5 "Hydroxytryptophan BCAA & aromatic amino acids

Pro Pro Urea cycle relating metaboloism

Propanoyl-CoA Propionyl Co A divalent Lipid and amino acid metabolism / BCAA & aromatic amino acids / Nucleotide metabolism

Propionic acid Propionic acid Lipid and amino acid metabolism / BCAA & aromatic amino acids

PRPP PRPP Central carbon metabolism / Nucleotide metabolism

Putrescine Putrescine Urea cycle relating metaboloism

Pyridoxal Pyridoxal Metabolism of coenzymes

Pyridoxamine Pyridoxamine Metabolism of coenzymes Pyr i doxamine -P Pyridoxamine 5 '“phosphate Metabolism of coenzymes Pyridoxine Pyridoxine Metabolism of coenzymes Pyruvic acid Pyruvic acid Central carbon metabolism / Urea cycle relating metaboloism / Lipid and amino acid metabolism

Quinoliniacid Quinolinic acid BCAA & aromatic amino acids / Metabolism of coenzymes

RIP Ribose 1 -phosphate Pathway overview

R5P Ribose 5 -phosphate Central carbon metabolism / Metabolism of coenzymes

Riboflavin Riboflavin Metabolism of coenzymes

Ru5P Ribulose 5 -phosphate Central carbon metabolism

S7P Sedoheptulose 7-phosphate Central carbon metabolism

Saccharopine Saccharopine Lipid and amino acid metabolism

SAHC S~ Adenosylhomo cy ste ine Lipid and amino acid metabolism

SAM S- Adenosylmethionine Lipid and amino acid metabolism

Sarcosine Sarcosine Lipid and amino acid metabolism

Ser Ser Lipid and amino acid metabolism

Serotonin Serotonin BCAA & aromatic amino acids

S -Lactoy Iglutathi one S~L actoy 1 glutathione Urea cycle relating metaboloism Spermidine Spermidine Urea cycle relating metaboloism

Spermine Spermine Urea cycle relating metaboloism

Succinic acid Succinic acid Central carbon metabolism / Urea cycle relating metaboloism

Succinic semialdehyde Succinic semialdehyde Urea cycle relating metaboloism

Succinyl AMP Adenylosuccinic acid Nucleotide metabolism

+ Abbreviated names in Pathway Map.

" Metabolites which have been already known about pathway information were listed up. They included metaboites which were not detected in this study.

§ Pathway information in the metabolites.

Appendix 1-2. Pathway Abbreviations

Pathway Label t Candidates"^ Pathway Index§ SucCoA Succinyl Co A divalent Central carbon metabolism

T3 3,3 5 -Triiodothyronine BCAA & aromatic amino acids T aurine Taurine Lipid and amino acid metabolism

Taurocholic acid Taurocholic acid Lipid and amino acid metabolism Taurocyamine Taurocy amine Lipid and amino acid metabolism TDP-Glc dTDP-glucose Pathway overview Thiamine Thiamine Metabolism of coenzymes

ThPP Thamine diphosphate Metabolism of coenzymes

Thr Thr Lipid and amino acid metabolism

Thymidine Thymidine Nucleotide metabolism Thymine Thymine Nucleotide metabolism TMP Thamine phosphate Metabolism of coenzymes

Trimethyllysine A ^z ^ ₅ A ^z 6,jV6-Trimethyllysine Lipid and amino acid metabolism

Trp Trp . BCAA & aromatic amino acids

Tryptamine Tryptamine BCAA & aromatic amino acids

Tyr Tyr ' BCAA & aromatic amino acids Tyramine Tyramine BCAA & aromatic amino acids UDP UDP Nucleotide metabolism

UDP-Glc UDP~glucose Central carbon metabolism UDP-GlcA UDP-glucuronic acid Central carbon metabolism UDP-GlcNAc UDP-N -acety glucosamine Central carbon metabolism UMP UMP Nucleotide metabolism Uracil Uracil Nucleotide metabolism Urea Urea Urea cycle relating metaboloism Uric acid Uric acid Nucleotide metabolism Uridine Uridine Nucleotide metabolism

Urocanic acid Urocanic acid Urea cycle relating metaboloism

UTP UTP Nucleotide metabolism

Vai Vai BCAA & aromatic amino acids

VMA Vanillylmandelic acid BCAA & aromatic amino acids

X5P Xylulose 5-phosphate Central carbon metabolism

Xanthine Xanthine Nucleotide metabolism Xanthosine Xanthosine Nucleotide metabolism

Xanthurenic acid Xanthurenic acid BCAA & aromatic amino acids

XMP XMP Nucleotide metabolism

XTP XTP Nucleotide metabolism

£ Abbreviated names in Pathway Map.

Metabolites which have been already known about pathway information were listed up. They included metabolites which were not detected in this study.

§ Pathway information in the metabolites.

Appendix 1-3 Metabolites of Interest

C10H1003S

C32H30O12

C6H10O8 l-Methyl-4-imidazoleacetic acid

1-Methyladenosine

1-Methylhistamine

1-Methy]

3-Methylhistidine

1-Methylhistidine;3-Methylhistidine

1-Methylnicotinamide

1 -Palmitoy l-glycero-3 -phosphocholine- 1

1.2-Dipalmitoyl-glycero-3-phosphoethanolamine-l

1.2-Dipalmitoyl-glycero-3-phosphoethanolamine-2

11-amino-undecanoic acid

15(S)-HETE

17oi-Hydroxyprogesterone

17oi-Hydroxyprogesterone-2

Deoxycorticosterone-2 lH-lmidazole-4-propionic acid lH-lmidazole-4-propionic acid; l-Mcthyl-4-imidazoleacctic acid

2-(beta-D-Glucosyl)-sn-glycerol

2-Aminoadipic acid

2-AminoisobutjTic acid 2-Aminobutyric acid

2-Arachidonoylglycerol

2-Diethylaminoethanol

2-Hydroxy-4-methylvaleric acid

2-Hydroxybutyric acid

2-Hydroxyglutaric acid

2-Hydroxyisobutyric acid

2-Hydroxyva eric acid

2-Keto-glutaramic acid

2-Methylserine

2-Oxoarginine

2-Oxoglutaric acid

2-Oxoisovaleric ack

2-Oxoisovai eric acid;2-Oxovaleric acid

2-Phosphoglyceric acid

2-Phos jphoglyceric acid;3-Phosphoglyceric acid

2-Thiopheneacetic acid

2,3-Diphosphoglyceric acid

2.6-Diaminqpimelic acid

2-Deoxycytidine 107

SUBSTITUTE SHEET ( RULE 26) 2'-Deoxyuridine

20a-Hydroxyprogesterone

21-Deoxycortisol-2

21-Hydroxypregnenolone

3-(4-Hydroxyphenyl)propionic acid

2-(4-Hydroxyphenyl)propionic acid

3-(3-Hydroxyphenyl)propionic acid

3-(4-Hydroxyphenyl)propionic acid;2-(4-Hydroxyphenyl)propionic acid;3-(3-Hydroxyphcnyl)propionic acid;Tropic acid;3-(2-Hydroxyphenyl)propionic acid;m-Ethoxybenzoic acid;3-Phenyllactic acid;p- Methoxyphenylacetic acid

3-cis-Hydroxy-b,e-Caroten-3'-one

3-Guamdinopropanoate

3-Hydrroxy-2-methyl-4-pyrone 3-Hydroxy-3-methylglutaric acid 3-Hydroxy-3 ^l ,4 ^l -didehydro-|3,y-carotene

3-Hydrroxybutyric acicd

3-Hydroxybufyric acid;2-Hydroxybutyric acid;2-Hydroxyisobutyric acid

3-Hydroxybufyric acid;2-Hydroxyisobutyric acid

3-Hydroxyglutaric acid

3-Hydroxytetradecanoic acid-1

3- ndoxylsulfuric acid

3-Mercaptolactic acid

3-Methylhistamine;l-Methylhistamine

3 -Methylhistidine; 1 -Methylhistidine 3-oxocholic acid

3-Phosphoglyceric acid

3-Ureidopropionic acid

3,4-Dihydroxyhydrocinnamic acidiHomovanillic acid;Hydroxyphenyllactic acid -Acetamidobutanoic acid -Guanidinobutyric acid -Hydroxyquinoline -Methyl-2-oxovaleric acic

3-Methyl-2-oxovaleric acic -Oxoh lexanoic acid -Mcthyl-2-oxova eric acid;3-Methyl-2-oxovaleric acid;2-Oxohexanoic acid -Oxopyrrolidine-2-carboxylic acid

5 ■ Amino-4-hy droxynaphthalene- 1 ,3 -disulfonic acid

5-Amino-4-oxovaleric acid

5-Hydroxymdoleacetic acid

5-Hydroxylysine

5-Hydroxypentanoic acid;/A-Hydroxyisovaleric acid;2-Hydroxyvaleric acid

5-Hydroxytryptophan

5-lsopropyl-z-deoxyuridine triphosphate 108

SUBSTITUTE SHEET ( RULE 26) 5-Methoxyindoleacetic acid;lndole-3-lactic acid

5-Methyl-2'-deoxycytidine

5-Oxoproline

5a-Cholestan-3-one-l

5a-Cholestan-3-one _: 2

5u-Pregnane-3, 20-dione

6-Hydroxyhexanoic acid

6-Hydroxyhexanoic acid;2-Hydroxy-4-methylvalenc acid

6-Hydroxynicotinic ack

7-Dehydrocholestero

7-Dehydrocholestero

7-Dehydrocholesterol-2

Desmosterol-2

7-Dehydrocholesterol-3

Desmosterol-3

7-Methoxy-2-methylisoflavone

7-Methylguanine

7-Methylguanine;3-Methylguanine

7.8-Dihydrobiopterin

7.8-Dihydroneopterin

Abietic acid

Abietic acid-1

Abietic acid-3

AC(10:0)

AC(12:0)-l

AC(12:0)-2

AC(12:1)

AC(12:1)-1

AC(12:l)-3

AC(13:1)

AC(13:1)-1

AC(14:0)-l

AC(14:0)-2

AC(14:1)-1

AC(14:l)-2

AC(14:l)-3

AC(14:l)-4

AC(14:2)-1

AC(14:2)-2

AC(14:2)-3

AC(14:3)-1

AC(14:3)-2

109

SUBSTITUTE SHEET ( RULE 26) AC(14:3)-3

AC(14:3)-4

AC(15:O)-1

AC(15:0)-2

AC(16:1)

AC(16:2)-1

AC(16:2)-2

AC(17:0)-l

AC(17:0)-2

AC(17:1)

AC(18:0)

AC(18:1)

AC(18:2)-1

AC(18:2)-2

AC(20:0)

AC(20:l)

AC(22:0)

Acctohydroxamic acidiGly

ADMA;SDMA

ADP

ADP-ribose

AEA(22:6) Ala alaser/glythr Aminoacetone AMP

AMP;dGMP

Anandamide

ANDS(C-SCOPEIS)

Anserine

Arachidic acid

Arachidonic acid

Argininosuccinic acid

Ascorbic acid

Asiatic acid

Asiatic acid-1

Asn

Asp

Asp Asp Pro Ser

Asp Gly His Asp Asp Leu Asn Arg Asp-Pro 110

SUBSTITUTE SHEET ( RULE 26) ATP

Baicalein

Behenic acid

Betaine

Betaine aldehyde_+H20 Betulinic acid Betulinic acid-2

Biopterin

Biotin

Butyrylcarnitine

C'C

C3H8N4O

C4H7NO4

C5H12N2O2

C5H6O7

C6H10O8

C6H11NO2

C6H12N2O3

C7H9N3O2

C8H17NO

C8H18N2O3

C9H18N2O cAMP

Campesterol

Carbachol

Carboxymethyllysine

Carnitine

Carnosine

Camosine;His-Ala;Ala-His Chenodeoxycholic acid Cholesterol

Cholesterol sulfate

Cholic acid

Choline cis- 11-Eicosenoic acid cis-ll,14-Eicosadienoic acid-1 cis-ll,14-Eicosadienoic acid-2 cis-4,7, 10, 13, 16, 19-Docosahexaenoic acid cis-5,8, 11, 14, 17-Eicosapentaenoic acid epa cis-8,11,14-Eicosatrienoic acid cis-Aconitic acid Citric acid

Citrulline

SUBSTITUTE SHEET ( RULE 26) Corosolic acid

Cortexolone Corticosterone 21-Deoxycortisol-l

Cortisol

18-Hydroxycorticosterone Humulone

Creatine

Creatinine

Cratonic acid

CSA;CSA

Cyclodopa glucoside Cyclohexjlamine CysCys Csy AsnAsn Cystathionine

Cysteine glutathione disulfide Cystine Cytidine Cytosine

Daidzein

Dansyl acid(C-SCOPE IS) Deoxy cholic acid Dethiobiotin dGDP;ADP dGTPATP

Diethanolamine

Dimethylaminoethanol DOPA

DPA;DPA

Dyphylline Ectoine Erucic acid

Ethanolamine

Ethanolamine phosphate Ethyl arachidonate Ethyl glucuronide FA 16:1

FAC18:1

FA(12:0)

FA(13:0) FA(14:1) FA(14:l)-2 112

SUBSTITUTE SHEET ( RULE 26) FA(14:2)-1

FA(14:2)-2

FA(14:3)

FA(15:0)

FA(15:0)-l

FA(15:1)

FA(15:l)-2

FA(16:2)-1

FA(16:2)-2

FA(16:3

FA(16:3)-2

FA(17:0)

FA(17.1)

FA(17:2)

FA(17:3)

FA(19:0)

FA(19:0)-l

FA(19:1)

FA(19:2)

FA(20:3)

FA(22:2)

FA(22:3)-1

FA(22:3)-2

FA(22:4)-1

FA(22:4)-2

FA(22:5)-1

FA(22:5)-2

FA(24:0)

FA(24:2)

FA(24:4)

FA(24:5)-1

FA(24:5)-2 Flavanone

Formiminoglutamic acid

Formononetin

Fucosyl tryptophan

Fucosyl-Lysine

Fumaric ack

/A-Ala

JA-Butyrobetaine

/A-Glu-Gly

/A-Glu-Met

/A-Glu-Phc 113

SUBSTITUTE SHEET ( RULE 26) /A-Glu-Ser fA-Glu-Tyr

M-Glu-Val-Gly

GABA

GABA;3-Aminoisobutyric acid

Galactosylhydroxylysine

Galacturonic acid-1

Glucuronic acid-1

Galacturonic acid;Glucuronic acid

Gamma-Glu-Gln

GDP

Genistein

Gin

Glu

Glu;lsoglutamic acid;threo-/A-Methylaspartic acid;N-Methylaspartic acid;N-Acetylserine

Glucaric acid

Gluconic acid

Gluconolactone

Glucosamine

Glucosamine 6-sulfuric acid

Glucose 6-phosphate

Glucosyl-glycerol

Glutaric acid

Methylsuccinic acid

Glutaric acid;Methylsuccinic acid

Glutathione (GSSG) divalent

Gly

Gly Lys

Gly-Ala

Gly-Asp

Gly-Asp;Asp-Gly

Gly-Leu;N-Acetyllysine;Val-Ala;Ala-Val;Leu-Gly

Glyceric acid

Glycerol

Glycerol 3-phosphate

Glycerophosphocholine

Glycitein

Glycochenodeoxycholic acid

Glycocholic acid

Glycodeoxycholic acid

Glyoxylic acid

GMP

SUBSTITUTE SHEET ( RULE 26) Guanidinosuccinic acid

Guanidoacetic acid

Gulonolactone;Gluconolactone

Hccogenin

Heneicosanoic acid

19-Methylarachidic acid

Heptadecanoic acid-1

FA(17:0)-l

Heptadecanoic acid-2

FA(17:0)-2

Heptanoic acid

Hexanoic acid

Hippuric acid

Hippuric acid (benzyl glycine)

His

His Pro Ser Vai ArgTyrThr

His-Asp

Histamine

Homo Arginine

Homo Proline Betaine

Homocamosine

Homocitrulline

Homocysteinesulfmic acid

Homoserine

Homovanillic acid

Hydroxyphenyllactic acid

Hydroxymdole

Hydroxy octanoic acid

Hydroxyprogesterone caproate

Hydroxyproline

Hydroxytetradecanoic acid

Hyodeoxycholic acid

^otaurine 'e le;Leu;A|loisoleucine lle;Leu;/A-Leucine;Alloisoleucine;6-Aminohexanoic acid lmidazole-4-acetic acid

Imidazolelactic acid

IMP ndole-3-acetic acid lndole-3-carboxaldehyde ₁₁₅

SUBSTITUTE SHEET ( RULE 26) lndole-3-lactic acid-1

5-Methoxyindoleacetic acid-1 lndole-3-lactic acid;5-Methoxyindoleacetic acid lndole-3-propionic acid lndole-3-propionic acid (IPA)

Inosine 2',3'-cyclic phosphate clMP sethionic acid sobutyry Icamitine ;Butyrylcamitine socitric acid soglutamic acid lsoliquiritigenin-1 lsoliquiritigenin-2 lsoliquiritigenin-3 lsonicotinamide;Nicotinamide lsovalerylalanine-2

N-Acetylleucine-2 lsovaleiylalanine;N-Acetylleucine

Isovalerylcamitine

Kynurenic acid

Kynurenine

Lactamide

Lactic acid

Lanosterol

Lauric acid

Leu

Leukotriene B4

Linoleic acid

Linolenic acid

Linoleyl ethanolamide

Liquintigenin

Lithocholic acid

Luteolin

Lys

Lys-Asp

Lys-Val

Malic acid

Mannosamine

MCA

Met

Methionine sulfone

Methionine sulfoxide

Methylmalonic acid;Succinic acid 116

SUBSTITUTE SHEET ( RULE 26) Morpholine

Mucic acid

Myristic acid

Myristic acid 14:0

Myristoleic acid

N-( 1 -Deoxy- 1 -fructosyl)valine

N-Acetyl-beta-alanine

N-Acetyl-P-alanine

N-Acctylalamnc

N-Acetylalanine;N-Acetyl-/A-alanine

N-Acetylasparagine

N-Acetylaspartic acid

N-AcetylgaIactosamine;N-AcetylgIucosamine;N-Acctylmannosa mine

N-Acetylglucosamine

N-Acetylglutamic acid

N-Acetylglutamine

N-Acetylglycine

N-Acetylhistidine

N-Acetylleucine

N-Acetyllysine

N-Acctylmcthiomnc

N-Acetylneuraminic acid

N-Acetylomithine

N-Acetylphenylalanine

N-Acetyltryptophan

N-Acetyltyrosine

N-Carbamylglutamate

N-Carboxymethylserine

N-Ethylmaleimide_+H2O

N-Formylaspartic acid

N-Formylglycine

N-Formylmethionine

N-Glycolylneuraminic acid

N-Hvdroxy-L-tiyptophan

N-Methylethanolaminc phosphate

N-Methylproline

N,N-Dimethylglycine

N'-Formylkynurenine

N1 -Acetylspermidine

Nl-Acetylspermidine;N8-Acetylspemudine

N 1 -Methyl-4-pyridone-5 -carboxamide

N1 -Methylguanosine

N5-Ethylglutamine ₁₁₇

SUBSTITUTE SHEET ( RULE 26) N5-Ethylglutamine;N-Acetylomithine N6-Acetyllysine

N6-Methyl-2'-deoxyadenosine N6-Methyllysine N6,N6,N6-mmethyllysine Naringenin Nervonic acid

Nervonyl carnitine Propyl Betaine (Triethylamine) N f-Formylkynurenine Nicotinamide no matches Norophthalmic acid

Norvaline;2-Amino-2-methylbutyric acid;5-Aminovaleric acid;Val Nto-Methylarginine O-Acetylcamitine O-Acetylhomoserine o-Coumaric acid p-Coumaric acid o-Hydroxybcnzoic acid Oleanolic acid Oleic acid

Oleoyl ethanolamide AEA(18:1) Ophthalmic acid Ornithine

Orotidine;Uridine;Pseudouridine p-Hydroxyphenylpyruvic acid )-Hydroxyphenylpyruvic acid;Caffeic acid ’almitic acid Palmitoleic acid Palmitoylcamitine Palmitoylethanolamide Pantothenic acid Penicillamine Penicillamine;Met Pentadecanoic acid Phe Phe Met His Glu Phe PheTrpTrp Phe-Thr Phenaceturic acid Phenol Phenyl Sulfate

Phenylpyruvic acid

Phosphocreatine

Phosphoenolpyruvic acid

Phosphorylcholine

Picolinic acid

Pipecolic acid

Pipecolic acid;N-Methylproline; 1 -Aminocyclopentanecarboxylic acid

Piperidine

Pro

Pro-Gly;Gly-Pro

Progesterone

Proline Betaine

Propionylcamitine

XC0061

Propionylcamitine;XC0061

Prostaglandin El-1

Prostaglandin DI- 1

Prostaglandin El-2

Prostaglandin Dl-2

Putrescine

Pyridoxal

Pyrrolidine

Pyruvic acid

Retinol Vit A

Retinol-2

Riboflavin

Ribose 5-phosphate

Ribulose 5-phosphate

Ribulose 5-phosphate;Ribose l-phosphate;Xylulose 5-phosphate

Ricinoleic acid

Ricinoleic acid 18:1 Hydroxy

Ricinoleic acid-2

Ricinoleic acid-3

S-Acetyldihydrolipoamide (XC0086)

S-Adenosylnomocysteine

S-Adenosylmethionine

S-Carboxymethylcysteine

S-Methylcysteine

S-Methylglutathione

S-Methylmethionine

S-Sulfocysteine Sarcosine

SDMA

Sedoheptulose 7-phosphate

Ser

Ser Ala / Thr gly

Ser GluPro Thr Asp Pro

Serotonin

Sitosterol

Spermidine

Spermine

Sphinganine

Sphingomyelin(dl8:l/16:0)-l

Sphingomyelin(dl8:l/16:0)-2

Sphingomyelin(dl8: 1/18:0)-!

Sphingomyelin(dl8:l/18:0)-2

Sphingosine

Stearic acid

Stearidonic acid

Stearoyl ethanolamide

Stigmasterol-1

Stigmasterol-2

Succinic acid

SulfaguanidinetC-SCOPE IS)

Sulfolithocholylglycine

Taurine

Taurochenodeoxy cholic acid

Taurochohc acid

Taurodeoxycholic acid

Taurolithocholic acid

Tauroursodeoxycholic acid

Terephthalic acid

Theobromine;Aminophylline;Paraxanthine

Thiamine

Thiamine phosphate

Thr

Thr Ala Ala

Thr Asp or Ser Glu threo-3-Hydroxyaspartic acid threo-3-Hydroxyaspartic acid-1 threo-3-Hydroxyaspartic acid-2

Threonic acid

Thymidine

Thyroxine trans-Glutaconic acid trans-Glutaconic acid;ltaconic acid

Tricosanoic acid

Trigonelline

Trilaurin

Trimesic acid;Trimesic acid

Trimethylamine

TrimethylamineN-oxide

Trimethylaminoacetone

Trp

Tyr

UDP-galactose

UDP-glucose

Uracil

Urea

Unc acid

Undine

Uridine;Pseudouridine

Urocanic acid

Ursodeoxycholic acid

Vai

XA0005

XA0008

XA0009

XA0011

XA0017

XA0019

XA0023

XA0026

XA0033

XA0034

XA0037

XA0039

XA0052

Xanthosine

XC0016

XC0039

XC0040

XC0047

XC0049

XC0054:XC0055;/A-Ghi-Gly

XC0056

XC0060 XC0063

XC0064

XC0065

XC0067

XC0070

XC0075

XC0088

XC0094

XCO101

XC0103 ,

XC0107;M-Glu-Gln

XC0114;/A-Gu-His

XC0117

XC0118

XC0119

XC0120

XC0126

XC0133

XC0135

XC0138 XC0139 XC0140 . Zeaxanthin a-Tocophero (/.-Tocophcro acetate 3-Ala 3-Estradiol

17a-Estradiol P-Hydroxyisovaleric acid y-Butyrobetaine y-Glu-Ala u-Asn u-Asp u-Citrulline G n G u Gy His Leu Met Omitine y-Glu-Phe y-Glu-Ser y-Glu-Taurine y-Glu-Thr y-Glu-Trp y-Glu-Tyr y-Glu-Val y-Glu-Val-Gly y-Tocopherol

Appendix 2. Known-Unknown Peaks

The "known-unknown" peaks with out annotation based on the chemical standards are shown in the label _o f "XA — I XC — " in result tables. Among them, several peaks which have been detected from a variety of biological samples are listed in Appendix 2.

Candidate compounds

HMTID Peak ID Mode' - - - mass? PubChem database HMDB database

M90001 XA0001 Anion 107.998

MW XA0002 Anion 111.993 75795

MW XA0003 Anion 125.999 7866

M90004 XA0004 Anion 145.038 440726; 48 HMDB01552

11389478' 125409' 135191

439195;439203; 439204; ’ HMDB00098; HMDB00283;

®05; 439240; 439245; HMDB00366; HMDB00621;

M90005 XA0005 Anion 150.052 ! ? T ^; HMDB00646;HMDB00751;

^; HMDB01644;HMDB03371;

HMDB12194JHMDB1B25

619; 6902

M90006 XA0006 Anion 150,067

M90007 XA0007 Anion 152,014

M90008 XA0008 Amon 154,003 1034; 150865; 440171 wnnnnn V A AAAH A ■ m wii irnr HMDB00152; HMDB00397;

M90009 XA0009 Amon 154,026 19; 3469; 4696; 72 HMDB01856

M90010 XA0010 Anion 155,035 439436; 440231; 440233

M90011 XA0011 Amon 165,019

M90012 XA0012 Amon 167,025 HMDB06462

M90013 XA0013 Amon 173,999 4765; 74426

M90014 XA0014 Anion 174,016 440667; 444212; 4784 HMDB00958; HMDB01264

M90015 XA0015 Anion 174,125

M90016 XA0016 Amon 186,029

M90017 XA0017 Amon 187,121 173; 5282047 HMDB00206; HMDB00446;

M90018 XA0018 Anion 188.115 440139; 92832; 92843; 92907 HMDB00759

102287; 36681; 439290;

HMDB01874;HMDB05971;

M90019 XA0019 Anion 192.027 440165; 440390; 447805; HMDB06511

5318532

M90020 XA0020 Anion 197.036 3082376

146355; 439910; 5206;

M90021 XA0021 Anion 200,008 _545989?

M90022 XA0022 Anion 200,045

M90023 XA0023 Anion 208.021 6812; 8420

M90024 XA0024 Anion 217,104

M90025 XA0025 Anion 224,014

M90026 XA0026 Anion 225,030

M90027 XA0027 Anion 228,208 11005 HMDB00806; HMDB02221

M90028 XA0028 Anion 231,537

M90029 XA0029 Anion 237,030

M90030 XA0030 Anion 238,068 119228; 439706

M90031 XA0031 Anion 240,099

M90032 XA0032 Anion 240,135

M90033 XAOO33 Anion 243.087 53297342; 6175; 6253 HMDB00089

M90034 XA0034 Anion 243,184

M90035 XA0035 Anion 255,988 54675759

M90036 XA0036 Anion 255,988 54675759

M90037 XA0037 Anion 274,014 ions with positive and negative charge are measured in Cation and Anion Mode, respectively edicted mass value was calculated as mono-valent ion. Appendix 2. Known-Unknown Peaks

Candidate compounds

HMT1D Peak ID Mode* - - - mass* PubChem database HMDB database

M90038 XA0038 Anion 274.045 15942876

M90039 XA0039 Anion 287,067

M90040 XA0040 Anion 290,171

M90041 XA0041 Anion 303,540

M90042 XA0042 Anion 309,120

M90043 XA0043 Anion 310,513

11954062; 18172; 5280720; HMDB03871; HMDB04706;

M90044 XA0044 Anion 312.229 5281026; 5283016; 5460412; HMDB06940; HMDB 10201;

6438758; 9548877 HMDB 10208; HMDB 10221

M90045 XA0045 Anion 321,069

M90046 XA0046 Anion 326,526

M90047 XA0047 Anion 333,037

M90048 XA0048 Anion 334,066 HMDB11649

M90049 XA0049 Anion 337.023

M90050 XA0050 Anion 339.073

10267; 105021; 125004; HMDB00968; HMDB01047;

M90051 XA0051 Anion 339.995 128419; 3036654; 439444; HMDB03514; HMDB06234;

440117: 440211; 82400 HMDB06235; HMDB06872

M90052 XA0052 Anion 343.093 10925943

M90053 XAOO53 Anion 353,003

M90054 XA0054 Anion 368,163 12594; 240071 HMDB01032;HMDB02833

M90055 XA0055 Anion 370.006 164735:46906053 M90056 XA0056 Anion 383.052

M90057 XA0057 Anion 397.121

M90058 XA0058 Anion 400,016

M90059 XA0059 Anion 421,027

M90060 XA0060 Anion 422,012

M90061 XA0061 Anion 423.094

M90062 XA0062 Anion 424.036

M90063 XA0063 Anion 425,586

M90064 XA0064 Anion 437.972

M90065 XA0065 Anion 446.060 123727 HMDB01564

M90066 XA0066 Anion 448,141 73607

M90067 XA0067 Anion 495,189

M90068 XA0068 Anion 23724459; 23724466; 439536; HMDB01018;HMDB12301;

46174047 HMDB12303

M90069 XA0069 Anion 536,092

M90070 XA0070 Anion 537,076 165130

M90071 XA0071 Anion 542,274 HMDB10320

M90072 XA0072 Anion 548,129

M90073 XA0073 Anion 633,213 HMDB00825; HMDB06569

M90074 XA0074 Anion 745,093 5884 HMDB00221

M90075 XA0075 Anion 747,024

M90076 XA0076 Anion 767,117 87642 HMDB01423

M90077 XA0077 Anion 785,160 643975 HMDB01248

M90078 XA0078 Anion 841,053

M90079 XC0001 Cation 71,073 443732

M90080 XC0002 Cation 73,053 215; 6228; 67180; 75 111 V117 U u L i J T

M90081 XC0003 Cation 89,083

M90082 XC0004 Cation 89,084 Molecular ions with positive and negative charge are measured in Cation and Anion Mode, respectively Predicted mass value was calculated as mono-valent ion, Appendix 2. Known-Unknown Peaks

Candidate compounds

HMTID PeakID Model - 7 - mw PubChem database HMDS database

M90084 XC0006 Cation 103,073

M«5 XC0007 Cation 108,571

M90086 XC0008 Cation 112,012

M90087 XC00009 Mon 113,053

M90088 XC0010 Cation 114,078 HMDB00323

M90089 XC0011 Cation 115,099

M90090 XC0012 Cation 116,094 439358 HMDB12176

M90091 XC0013 Cation 120,060

M90092 XC0014 Cation 122,586

MOW _11;n „ 194461; 24892813; 3017497;

M90093 XC0015 Cation 125,047 mmuj

M90094 XC0016 Mon 128,058 440769; 440770; 93556 HMDB00079

M90095 XC0017 Cation 129,089 559

M90096 XC0018 Cation 129.594

M90097 XC0019 Cation 130,566

M90098 XC0020 Cation 133.036 5960; 83887 HMDB11753

M90099 XC0021 Cation 133,072

M90100 XC0022 Cation 133.073

M90101 XC0023 Cation 133,073

M90102 XC0024 Cation 133.109

M90103 XC0025 Cation 133.573

M90104 XC0026 Cation 133,574

M90105 XC0027 Cation 142.110

M90106 XC0028 Cation 143,094 115244; 5462194

M90107 XC0029 Cation 143,094 115244; 5462194

M90108 XC0030 Cation 144,569

160603' 18189' 439954' HMDB00730; HMDB00808;

M90109 XC0031 Cation 145,073 V i ’ HMDB01263; HMDB03681;

440077,440805 HMDB12131; HMDB12151

M90110 XC0032 Cation 147,034 440159

M90111 XCOO33 Cation 151,029

M90112 XC0034 Cation 151,576

M90113 XCOO35 Cation 157,109 442645; 4479243

M90114 XC0036 Cation 160,084 439925; 441021 HMDB03459

24906320; 439377; 439389;

M90115 XC0037 Cation 161,068 439943; 440550; 440959;

46173947; 92136

M90116 XC0038 Cation 170,068

M90117 XC0039 Cation 172,047 656724; 782 HMDB01212

M90118 XC0040 Cation 173,079 HMDB04225

M90119 XC0041 Cation 175,028

M90120 XC0042 Cation 175,119

M90121 XC0043 Cation 178,120

M90122 XC0044 Cation 185,104 443003; 443845; 5281740 HMDB06348; HMDB06548

M90123 XC0045 Cation 190,007

M90124 XC0046 Cation 190,057 121396; 441441 HMDB11165

M90125 XC0047 Cation 190,094 439283; 99290 HMDB01370

M90126 XC0048 Cation 190,130

M90127 XC0049 Cation 191.041 27661; 443054; 46173773; olecular ions with positive and negative charge are measured in Cation and Anion Mode, respectively edicted mass value was calculated as mono-valent ion. Appendix 2. Known-Unknown Peaks

, Candidate compounds

HMTID Peak ID Model - - - mass? PubChem database HMDB database

M90128 XC0050 Cation 191059

M90129 XC0051 Cation 193.040

M90130 XC0052 Cation 197,057 440214

M90131 XC0053 Cation 203.125

M90132 XC0054 Cation 204.073 26879 HMDB11162;HMDB11667

M90133 XC0055 Cation 204,074 HMDB11162;HMDB11667

M90134 XC0056 Cation 204,110 128597; 128888; 5799

M90135 XC0057 Cation 204,146

M90136 XC0058 Cation 208,051 5281921; 6763; 6780

M90137 XC0059 Cation 212,115 2479 HMDB11180

M90138 XC0060 Cation 216,073 46173889

M90139 XC0061 Cation 217,130 107738 HMDB00824

M90140 XC0062 Cation 218,089 151284 HMDB03764; HMDB06248

M90141 XC0063 Cation 218,125 193187

M90142 XC0064 Cation 220,069 HMDB11168

M90143 XC0065 Cation 220,083 144; 439280; 442551 HMDB00472

M90144 XC0066 Cation 221,071

M90145 XC0067 Cation 223,104

M90146 XC0068 Cation 225,147

M90147 XC0069 Cation 228,121 441123

M90148 XC0070 Cation 228,146 HMDB11174;HMDB11175

M90149 XC0071 Cation 233,172 HMDB11140

M90150 XC0072 Cation 234,084 HMDB11169

M90151 XC0073 Cation 234,084 HMDB11169 M90152 XC0074 Cation 236.082

HMDB00238; HMDB00468;

M90153 XC0075 Cation 128973; 2380; 439921; 440036;

5460401; 65253 HMDB00633;HMDB00817;

HMDB01195;HMDB02263

M90154 XC0076 Cation 240.146 4845; 49787007

M90155 XC0077 Cation 241.632

M90156 XC0078 Cation 242,175

M90157 XC0079 Cation 245,122

M90158 XC0080 Cation 246,120 HMDB11166;HMDB11172

M90159 XC0081 Cation 246,120 HMDB11166;HMDB11172

M90160 XC0082 Cation 247,081

M90161 XC0083 Cation 247,140 HMDB13127

M90162 XC0084 Cation 248,063 2955 HMDB11163

M90163 XC0085 Cation 248,100

M90164 XC0086 Cation 249,084 1076 HMDB01526;HMDB06878

M90165 XC0087 Cation 253152

M90166 XC0088 Cation 254,038 68134

M90167 XC0089 Cation 254,089 10400039; 9921310

M90168 XC0090 Cation 255,073

M90169 XC0091 Cation 255,074

M90170 XC0092 Cation 256,139

M90171 XC0093 Cation 257,198

M90172 XC0094 Cation 258,084 440569; 65049 HMDB00884;HMDB02331;

HMDB04813

M90173 XC0095 Cation M90174 XC0096 Cation Molecular ions with positive and negative charge are measured in Cation and Anion Mode, respectively Predicted mass value was calculated as mono-valent ion. Appendix 2. Known-Unknown Peaks

Candidate compounds

HMTID Peak ID Model - 7 - mass? PubChem database HMDS database

M90175 XC0097 Cation 261,096

M90176 XC0098 Cation 261,120 181804; 441467; 442866 HMDB13133

M90177 XC0099 Cation 261,131 4098 HMDB02248; HMDB04985;

HMDB04987

M90178 XC0100 Cation 261,131 4098 HMDB02248; HMDB04985;

HMDB04987

M90179 XC0101 Cation 261,156

M90180 XC0102 Cation 262,079 HMDB11164

M90181 XC0103 Cation 265.115 168948

M90182 XC0104 Cation 267,094 107795; 35370; 441037 HMDB00085; HMDB00830

M90183 XC0105 Cation 268.116 439693

M90184 XC0106 Cation 270,095 126220

M90185 XC0107 Cation 275.110 150914; 25137932 HMDB05766; HMDB 11738

M90186 XC0108 Cation 275,135 HMDB1313O

M90187 XC0109 Cation 276D96 69925; 9117; 92865 HMDB11737

M90188 XC0110 Cation 277,564

M90189 XC0111 Cation 278,093

M90190 XC0112 Cation 279,130

M90191 XC0113 Cation 281.110 73317 HMDB04044; HMDB04326;

HMDB06023

M90192 XC0114 Cation 284.110 25447

M90193 XC0115 Cation 128861; 441648; 444150; 6842999

M90194 XC0116 Cation 289,151 HMDB00552 M90195 XC0117 Cation 293.146

M90196 XC0118 Cation 294.105 440002

M90197 XC0119 Cation 294,141

M90198 XC0120 Cation 297,044 HMDB00709

M90199 XC0121 Cation 297,178

M90200 XC0122 Cation 302.137

M90201 XC0123 Cation 305.738

M90202 XC0124 Cation 308.120

M90203 XC0125 Cation 308,120

M90204 XC0126 Cation 309,104 439197; 440038 HMDB00230; HMDB00773

M90205 XC0127 Cation 310,114 HMDB11741

M90206 XC0128 Cation 311,122 HMDB01961; HMDB04824

M90207 XC0129 Cation 319,081

M90208 XC0130 Cation 321,098 115260; 440380

M90209 XC0131 Cation 322,136

M90210 XC0132 Cation 324,152 46174023 HMDB00600

M90211 XC0133 Cation 327,130

M90212 XC0134 Cation 335,132 123826 HMDB00489

M90213 XC0135 Cation 336,164

M90214 XC0136 Cation 337,092 447123; 5360043 HMDB04662

M90215 XC0137 Cation 349,093 11954074; 440596

M90216 XC0138 Cation 366,141

M90217 XC0139 Cation 383,106 23724526 HMDB00912

M90218 XC0140 Cation 387,101

M90219 XC0141 Cation 388,123 50909833

M90220 XC0142 Cation 428,141

M90221 XC0143 Cation 469,136 Molecular ions with positive and negative charge are measured in Cation and Anion Mode, respectively Predicted mass value was calculated as mono-valent ion. Appendix 3. Metabolites Detected

Table 7 Putative Metabolites (1)

ID consists of analysis mode and number. 'C and ^! A' showed cation and anion modes, respectively.

N.D. (Not Detected): The target peak or metabolite was below detection limits.

N.A. (Not Available): The calculation was impossible because of insufficience of the data.

Putative metabolites which were assigned on the basis of m/z and MT in HMT standard compound library.

*1 The ratio is of computed by using averaged detection values. The latter was used as denominator.

I The p-value is computed by Welch’s t-test. (*<0.05, **<0.01, ***<0.001)

The data are sorted by Compound name in ascending order.

Table 7 Putative Metabolites (2)

ID consists of analysis mode and number. 'C and 'A’ showed cation and anion modes, respectively.

N.D. (Not Detected): The target peak or metabolite was below detection limits.

N.A. (Not Available): The calculation was impossible because of insufficience of the data.

Putative metabolites which were assigned on the basis of m/z and MT in HMT standard compound library

The ratio is of computed by using averaged detection values. The latter was used as denominator.

I The p-value is computed by Welch's t-test. (*<0.05, **<0.01, ***<0.001)

The data are sorted by Compound name in ascending order.

Table 7 Putative Metabolites (3)

ID consists of analysis mode and number. 'C and 'A' showed cation and anion modes, respectively.

N.D. (Not Detected): The target peak or metabolite was below detection limits. N.A. (Not Available): The calculation was impossible because of insufficience of the data.

J Putative metabolites which were assigned on the basis of m/z and MT in HMT standard compound library.

The ratio is of computed by using averaged detection values. The latter was used as denominator. h The p-value is computed by Welch's t-test. (*<0.05, **<0.01 , ***<0.001)

The data are sorted by Compound name in ascending order.

Table 7 Putative Metabolites (4)

ID consists of analysis mode and number. 'C and 'A' showed cation and anion modes, respectively.

N.D. (Not Detected): The target peak or metabolite was below detection limits.

N.A. (Not Available): The calculation was impossible because of insufficience of the data. t Putative metabolites which were assigned on the basis of m/z and MT in HMT standard compound library.

The ratio is of computed by using averaged detection values. The latter was used as denominator. i! The p-value is computed by Welch’s t-test. (*<0.05, **<0.01, ***<0.001)

The data are sorted by Compound name in ascending order.

Table 8 Quantitative Estimation of Target Metabolites (1)

ID consists of analysis mode and number. ’C and ’A’ showed cation and anion modes, respectively.

N.D. (Not Detected): The target peak or metabolite was below detection limits.

N.A. (Not Available): The calculation was impossible because of insufficience of the data.

The ratio is of computed by using averaged detection values. The latter was used as denominator. The p-value is computed by Welch's t-test. (*<0.05, **<0.01, ***<0.001)

The data are sorted by Compound name in ascending order.

Table 8 Quantitative Estimation of Target Metabolites (2)

C_0020 Uracil C 0117 Uridine A__0102 UTP C_ 0024 Vai C 0009 P-Ala

ID consists of analysis mode and number. 'C and 'A' showed cation and anion modes, respectively.

N.D. (Not Detected): The target peak or metabolite was below detection limits.

N.A. (Not Available): The calculation was impossible because of msufficience of the data.

Putative metabolites which were assigned on the basis of m/z and MT in HMT standard compound library

The ratio is of computed by using averaged detection values. The latter was used as denominator.

I The p- value is computed by Welch's t-test. (*<0.05, **<0.01 , ***<0.001)

The data are sorted by Compound name in ascending order.

Appendix 4. Metabolites of Interest