Methods and compositions for identifying fecal protein bioprofiles and treating inflammatory and neurological disorders

Abstract

The present inventive concept provides a subject's fecal protein bioprofile and methods for establishing the same. Also provided are methods of determining a subject's risk for developing an inflammatory disorder, a neurological disorder, a neurodegenerative disorder, or a disorder associated with aging or monitoring the same. The inventive concept further provides kits for use in the methods described herein.

Description

Attorney Docket No.190412-00026WO METHODS AND COMPOSITIONS FOR IDENTIFYING FECAL PROTEIN BIOPROFILES AND TREATING INFLAMMATORY AND NEUROLOGICAL DISORDERS Cross-Reference to Priority Application

[0001] This application claims the benefit of and priority to U.S. Provisional Application No.63 / 621,679, filed January 17, 2024, the content of which is hereby incorporated herein by reference in its entirety. Reservation of Copyright

[0002] A portion of the disclosure of this patent document contains material, which is subject to copyright protection. The copyright owner, East Carolina University, Greenville, North Carolina, a constituent institution of the University of North Carolina, has no objection to the reproduction by anyone of the patent document or the patent disclosure, as it appears in U.S. Patent and Trademark Office patent files or records, but otherwise reserves all copyright rights whatsoever. Field

[0003] The present inventive concept is directed to methods and compositions of matter for identifying fecal protein bioprofiles in healthy and disease states and methods and compositions for treating and / or managing inflammatory disorders; neurological disorders; neurodegenerative disorders; including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, motor neuron disorder and multiple sclerosis; and disorders and diseases associated with aging. Background

[0004] An estimated 1 in 100 Americans, or ~3.4 million people, have some form of inflammatory bowel disease (IBD); environmental factors- including pollution, stress, and ultra-processed foods- as well as genetic factors and an aging population are further contributing to the increasing prevalence of IBD with nearly 56,000 cases diagnosed each year. In addition to those with IBD where gut healthAttorney Docket No.190412-00026WO is important to curtailing disease associated gut inflammation, maintaining gut health in healthy individuals is important to both physical and mental well-being, in contrast, poor gut health leads to chronic gut inflammation. If left unchecked, chronic gut inflammation can contribute to obesity and insulin resistance, cause widespread intestinal tissue damage, and potentially increase a person’s risk for developing neurogenerative diseases later in life. Similar patterns of tissue damage and increased incidences of neurodegenerative diseases are also seen in individuals with IBD.

[0005] According to the National Institute of Neurological Disorders and Stroke, each year ~50 million Americans are affected with neurological diseases. Over the next 20 years, the number of individuals diagnosed with a neurological disease is expected to more than double. Currently, the diagnosis of neurodegenerative disease onset is limited to single biomarkers that are often poorly correlated to clinical disease manifestations (Hor, J. W., Lim, S.-Y., Khor, E. S., Chong, K. K., Song, S. L., Ibrahim, N. M., Teh, C. S. J., Chong, C. W., Hilmi, I. N., and Tan, A. H. (2021) Fecal Calprotectin in Parkinson’s Disease and Multiple System Atrophy. JMD 15, 106–114; Rao, B. B., Bhattacharya, A., and Lichtenstein, G. R. (2020) Precision Medicine: Predicting Disease Course in Patients with Inflammatory Bowel Disease. Curr. Treat. Options Gastroenterol.18, 574–588).

[0006] Further, not only are socioeconomically disadvantaged individuals at a higher risk for developing neurodegenerative disease, current diagnostic approaches can be cost prohibitive (Fotenos, A. F., Mintun, M. A., Snyder, A. Z., Morris, J. C., and Buckner, R. L. (2008) Brain Volume Decline in Aging: Evidence for a Relation Between Socioeconomic Status, Preclinical Alzheimer Disease, and Re- serve. Arch. Neurol.65, 113–120; Ullah, I., Ali, S., Rahman, H. U., Ali, S., Ullah, Z., and Ali, F. (2022) Socioeconomic Study on the Prevalence and Pattern of Various Neurological Disorders in Urban and Rural Areas. Sch. J. Appl. Méd. Sci.10, 409–416).

[0007] In addition to humans, companion animals, farm animals, and wildlife also suffer from neurological disease including Bovine spongiform encephalopathy (BSE), Vestibular syndrome, and Chronic Wasting Disease (CWD) (https: / / pubmed.ncbi.nlm.nih.gov / 15259778 / ANDAttorney Docket No.190412-00026WO https: / / pubmed.ncbi.nlm.nih.gov / 21598099 / AND https: / / pubmed.ncbi.nlm.nih.gov / 19942058 / ). In certain cases such neurological diseases are caused by infections that can spread between people and animals, and therefore reinforcing the need for early testing in animal populations to prevent disease spread (https: / / pubmed.ncbi.nlm.nih.gov / 34271977 / AND https: / / pubmed.ncbi.nlm.nih.gov / 35760448 / AND https: / / pubmed.ncbi.nlm.nih.gov / 29913297 / ).

[0008] Therefore, there is an imperative need for a cost-effective bioassay that is performed without having to visit a doctor’s office and provides early risk detection for the development of inflammatory and neurodegenerative diseases and disorders associated with aging prior to the onset of symptoms.

[0009] The present inventive concept overcomes previous shortcomings in the art by providing a non-invasive, accessible, and robust screening tool for identifying and monitoring issues with gut health, inflammation, neurological disorders, and aging using a subject’s fecal protein bioprofile, or fingerprint, in both healthy subjects and those affected with the disorders described herein. Summary

[0010] Aspects of the present inventive concept provide methods of establishing a fecal protein bioprofile including extracting proteins found in a subject’s fecal matter; determining a relative abundance of the proteins; categorizing the proteins with respect to their biological pathway; and generating a unique fecal protein bioprofile.

[0011] Aspects of the inventive concept further include methods of determining a subject’s risk for developing an inflammatory disorder, a neurological disorder, a neurodegenerative disorder, or a disorder associated with aging, the methods include using a subject’s fecal protein bioprofile to determine a risk assessment score based upon an analysis of protein pathway enrichment scores.

[0012] In other aspects, methods of monitoring or managing the status of disease progression in a subject suffering from an inflammatory disorder, a neurological disorder, a neurodegenerative disorder, or a disorder associated with aging, the methodAttorney Docket No.190412-00026WO including using a subject’s fecal protein bioprofile to determine the progression of the disease based upon an analysis of protein pathway enrichment scores.

[0013] Aspects of the inventive concept further provide recommending and / or administering treatment modalities and / or therapeutic agents to the subject identified as being in need of treatment for alleviation of a condition described herein or prevention of the same.

[0014] In another aspect, provided is a kit for determining a subject’s risk for developing an inflammatory disorder, a neurological disorder, or a disorder associated with aging or monitoring or managing the same, the kit including a container to collect a subject’s fecal sample and optional instructions for using the kit. Brief Description of the Drawings

[0015] FIG.1 is a graphical depiction of the general workflow for fecal protein bioprofile analysis. (1) Fecal samples are collected. (2) Host proteomes are separated from the proteomes of other organisms. Proteins identified in a host fecal samples are measured. (3) The identified proteins and abundance of the same are used to construct fecal protein bioprofiles. Detailed Description

[0016] The present inventive concept will now be described with reference to the following embodiments. As is apparent by these descriptions, the inventive concept can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the inventive concept to those skilled in the art. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. In addition, numerous variations and additions to the embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention.Attorney Docket No.190412-00026WO

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the inventive concept belongs. The terminology used in the description of the inventive concept herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the inventive concept.

[0018] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0019] The present inventive concept focuses on measuring proteins from fecal samples. To aid in these measurements, animal data have been correlated to human ortholog proteins. Embodiments of the inventive concept not only provide details on the overall risk for an individual for developing a neurological disease, but also the specific type of neurological disease that the individual may be at risk of developing. A market in need for the benefits provided by embodiments of the inventive concept is related to Parkinson's disease (PD). Exemplary embodiments of the inventive concept may be provided in the form of a test kit that may be performed in a clinical setting, or alternatively, sent or taken to the individual patient's house. The test performed with the kit can be requested by the clinician or directly by the patient. Once the sample is collected / provided, it is sent back / delivered to a commercial lab for testing via a proteomics-based platform. Once an analysis of the sample is performed, the patient is provided with access to their bioprofile. The bioprofile may be in the form of a digital dashboard bioprofile that can track changes in the subject’s bioprofile and risk scores over time and can be accessed by telehealth professionals for counseling. This feature helps to keep a patient further engaged with their health. This model aligns with the estimated growth in the number of patients with neurological diseases.

[0020] The present inventive concept is based on the unexpected discovery of clusters of host proteins in fecal samples that are differentially expressed in wildtype versus animal models of neurological conditions such as multiple sclerosis and Parkinson’s disease. The present inventive concept provides both details on the person’s overall risk of neurological disease development and other diseases as well as the specific type of neurological disease for which they are at risk of developing. This technology represents a huge disruption, particularly, into how currentAttorney Docket No.190412-00026WO neurological diseases are diagnosed, especially by those people who do not always have access to a physician or other health care professional, or laboratory testing.

[0021] Embodiments of the inventive concept leverage analysis of host proteins in fecal samples to provide an early, accurate, and non-invasive method for detecting neurological disorders. By identifying unique biomarkers associated with the gut-brain axis, this solution empowers the target customers to be diagnosed at earlier stages, enabling timely intervention and personalized treatment strategies. With its simplicity, affordability, and ability to enhance patient outcomes, this groundbreaking approach can address a critical unmet need for accessible and reliable diagnostics.

[0022] This technology identifies clusters of host proteins in fecal samples that are differentially abundant in animal models of various neurological conditions.

[0023] Embodiments of the present inventive concept include methods of establishing a fecal protein bioprofile, the methods include extracting proteins found in a subject’s fecal matter; determining a relative abundance of the proteins; categorizing the proteins with respect to their biological pathway; and generating a unique fecal protein bioprofile.

[0024] The extracted proteins, include a diverse array of proteins involved in various biological processes. The term protein refers to all conformational states, post- transcriptionally or post-translationally modified forms including, but not limited to, phosphorylated, deamidated, acylated, zymogen, or cleaved forms of the parent protein. Exemplary proteins, include, but are not limited to, 4-aminobutyrate aminotransferase (mitochondrial), ATP-binding cassette sub-family A member 2, Long-chain specific acyl-CoA dehydrogenase (mitochondrial), Medium-chain specific acyl-CoA dehydrogenase (mitochondrial), Very long-chain specific acyl-CoA dehydrogenase (mitochondrial), Angiotensin-converting enzyme, Angiotensin- converting enzyme 2, Aconitate hydratase (mitochondrial), Peroxisomal acyl-coenzyme A oxidase 1, Actin (cytoplasmic 1), Actin (alpha cardiac muscle 1), Alpha-actinin-1, Alpha-actinin-4, Aminoacylase-1, N-acyl-aromatic-L-amino acid amidohydrolase (carboxylate-forming), Adenosine deaminase, Alcohol dehydrogenase 1A, N(4)-(beta- N-acetylglucosaminyl)-L-asparaginase, Anterior gradient protein 2 homolog, Adenosylhomocysteinase, Adenylate kinase 2, Aldo-keto reductase family 1 memberAttorney Docket No.190412-00026WO A1, Aldo-keto reductase family 1 member B1, Albumin Aldehyde dehydrogenase 1A1, Aldehyde dehydrogenase (mitochondrial), Fructose-bisphosphate aldolase A, Alpha- amylase 1A, Pancreatic alpha-amylase, Aminopeptidase N, Annexin A2, Annexin A4, Amiloride-sensitive amine oxidase [copper-containing], Acylamino-acid-releasing enzyme, Rho GDP-dissociation inhibitor 1, Arylsulfatase A, Arylsulfatase B, Neutral ceramidase, Potassium-transporting ATPase alpha chain 2, Bleomycin hydrolase 3'(2'),5'-bisphosphate nucleotidase 1, Complement C3, Calmodulin 1-5 isoforms, Calreticulin, Adenylyl cyclase-associated protein 1, Carbonic anhydrase isoforms 1-7, Cobalamin binding intrinsic factor, Carbonyl reductase, CD177 antigen, Cadherin-1, Cadherin-17 Cadherin-related family member 2 Cadherin-related family member 3 Carcinoembryonic antigen-related cell adhesion molecule 1 Carcinoembryonic antigen- related cell adhesion molecule 18 Chymotrypsin-like elastase family member 1 Chymotrypsin-like elastase family member 2A Chymotrypsin-like elastase family member 3B Liver carboxylesterase 1 Carboxylesterase 3 Carboxylesterase 5A Carboxylesterase 4A Acidic mammalian chitinase Chitinase-3-like protein 2 Calcium and integrin-binding protein 1 Creatine kinase U-type, mitochondrial Calcium-activated chloride channel regulator 1 Calcium-activated chloride channel regulator 2 Calcium- activated chloride channel regulator 4 Beta-Ala-His dipeptidase Ceruloplasmin Carboxypeptidase A1 Carboxypeptidase A2 Carboxypeptidase M Carboxypeptidase Q Protein CREG1 Peroxisomal carnitine O-octanoyltransferase C-reactive protein Lambda-crystallin homolog Citrate synthase, mitochondrial Cystatin-C Cystatin-A Cystatin-B Cystathionine gamma-lyase Catenin alpha-1 Chymotrypsinogen B1 Cathepsin B Procathepsin H Procathepsin L Cathepsin S Cubilin CUB and zona pellucida-like domain-containing protein 1 Coxsackievirus and adenovirus receptor Cytochrome b5 Cytochrome b5 type B Cytochrome c Acyl-CoA-binding protein D- dopachrome decarboxylase 2,4-dienoyl-CoA reductase [(3E)-enoyl-CoA-producing], mitochondrial Dihydrolipoyl dehydrogenase, mitochondrial Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial Deleted in malignant brain tumors 1 protein Deoxyribonuclease-1 Deoxyribonuclease-2-alpha Aspartyl aminopeptidase Dipeptidase 1 Dipeptidyl peptidase 4 Dipeptidyl peptidase 2 Dihydropyrimidinase-related protein 1 Desmocollin-Attorney Docket No.190412-00026WO 3 Desmoglein-2 Enoyl-CoA delta isomerase 1, mitochondrial Elongation factor 2 EF- hand domain-containing protein D2 Alpha-enolase Beta-enolase Glutamyl aminopeptidase Ectonucleotide pyrophosphatase / phosphodiesterase family member 3 Ectonucleotide pyrophosphatase / phosphodiesterase family member 7 Epithelial cell adhesion molecule Epidermal growth factor receptor kinase substrate 8-like protein 1 Epidermal growth factor receptor kinase substrate 8-like protein 3 Endoplasmic reticulum aminopeptidase 2 Extended synaptotagmin-1 and -2 Persulfide dioxygenase ETHE1, mitochondrial Ezrin Fatty acid-binding protein, liver Fatty acid-binding protein, intestinal Gastrotropin Acylpyruvase FAHD1, mitochondrial Protein FAM151A Protein FAM3D Farnesyl pyrophosphate synthase Fibrinogen alpha chain Filamin-B Ferritin heavy chain Ferritin light chain Galactose mutarotase N-acetylgalactosamine-6- sulfatase Neutral alpha-glucosidase AB Glyceraldehyde-3-phosphate dehydrogenase Vitamin D-binding protein Guanine deaminase Rab GDP dissociation inhibitor alpha, beta and gamma isoforms Gamma-glutamyl hydrolase Glutathione hydrolase 1 proenzyme PDZ domain-containing protein GIPC2 Lactoylglutathione lyase Glyoxalase domain-containing protein 5 GDP-mannose 4,6 dehydratase N-acetylglucosamine-6- sulfatase Aspartate aminotransferase, cytoplasmic Aspartate aminotransferase, mitochondrial Pancreatic secretory granule membrane major glycoprotein GP2 Cell surface A33 antigen Glucose-6-phosphate isomerase Glutathione peroxidase 1 Glutathione peroxidase 7 Gelsolin Glutathione reductase, mitochondrial Glutathione synthetase Glutathione S-transferase A4 Glutathione S-transferase Mu 1 Glutathione S-transferase Mu 2 Glutathione S-transferase P Guanylyl cyclase C Beta- glucuronidase HLA class II histocompatibility antigen, DR alpha chain HLA class II histocompatibility antigen, DO beta chain HLA class II histocompatibility antigen, DQ beta 2 chain Histone H2A type 1-H Histone H2B type 1-B HLA class I histocompatibility antigen, B alpha chain Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial Hydroxyacylglutathione hydrolase, mitochondrial 2-Hydroxyacid oxidase 2 Hemoglobin subunit alpha Hemoglobin subunit beta Beta-hexosaminidase subunit beta Haptoglobin Hemopexin Heat shock 70 kDa protein 1B Heat shock protein beta-1 Endoplasmic reticulum chaperone BiP Heat shock cognate 71 kDa protein Intestinal-type alkaline phosphatase Isocitrate dehydrogenase [NADP] cytoplasmic Isocitrate dehydrogenaseAttorney Docket No.190412-00026WO [NADP], mitochondrial Ras GTPase-activating-like protein IQGAP1 Inositol-3- phosphate synthase 1 Integrin alpha-2 Integrin alpha-3 Integrin beta-1 Intelectin-1 Immunoglobulin J chain kallikrein 1 Keratin, type II cytoskeletal 1 Keratin, type I cytoskeletal 10 Keratin, type I cytoskeletal 14 Keratin, type I cytoskeletal 18 Keratin, type I cytoskeletal 19 Keratin, type II cytoskeletal 2 epidermal Keratin, type I cytoskeletal 20 Keratin, type II cytoskeletal 6A Keratin, type II cytoskeletal 6B Keratin, type II cytoskeletal 8 Lysosome-associated membrane glycoprotein isofroms 1-3 Cytosol aminopeptidase Lipocalin isoforms 1-3 L-lactate dehydrogenase A chain Protein LEG1 homolog Galectin-3 Galectin-3-binding protein Galectin-4 Prelamin-A / C Leukotriene A-4 hydrolase Latexin Lysozyme C Lysosomal alpha-mannosidase Mesencephalic astrocyte-derived neurotrophic factor S-adenosylmethionine synthase isoform type-2 Mannose-binding protein C Malate dehydrogenase, cytoplasmic Malate dehydrogenase, mitochondrial NAD-dependent malic enzyme, mitochondrial Meprin A subunit alpha Meprin A subunit beta Macrophage migration inhibitory factor Neprilysin Putative mucosal pentraxin homolog Mucin-13 Mucin-2 Mucin-4 Major vault protein Myosin regulatory light chain 12B Myosin light polypeptide 6 Aminopeptidase NAALADL1 NAD(P)H-hydrate epimerase Nicastrin CD177 antigen NHL repeat- containing protein 3 Omega-amidase NIT2 NME / NM23 nucleoside diphosphate kinase 1 Nucleoside diphosphate kinase B NPC1-like intracellular cholesterol transporter 1 Probable aminopeptidase NPEPL1 Puromycin-sensitive aminopeptidase nucleobindin 1 Ornithine aminotransferase, mitochondrial Oncomodulin-1 Protein disulfide- isomerase Protein disulfide-isomerase A3 Protein disulfide-isomerase A6 Phosphatidylethanolamine-binding protein 1 Xaa-Pro dipeptidase Profilin -1 and -2 Phosphoglycerate mutase 1 Phosphoglycerate kinase 1 Peptidoglycan recognition protein 1 Phosphopentomutase Glycerol-3-phosphate phosphatase Polymeric immunoglobulin receptor Prolactin-inducible protein Pyruvate kinase PKLR Phospholipase B1, membrane-associated Phospholipase B-like 1 Plastin-1 Plexin-B2 Pancreatic triacylglycerol lipase Pancreatic lipase-related protein 2 Purine nucleoside phosphorylase Inorganic pyrophosphatase Peptidyl-prolyl cis-trans isomerase A Peptidyl-prolyl cis-trans isomerase B Lysosomal Pro-X carboxypeptidase Peroxiredoxin-1 Peroxiredoxin-2 Thioredoxin-dependent peroxide reductase,Attorney Docket No.190412-00026WO mitochondrial Peroxiredoxin-4 Peroxiredoxin-5, mitochondrial Peroxiredoxin-6 Prolyl endopeptidase Prominin-1 Trypsin-2 Trypsin-3 Prosaposin Proteasome subunit alpha type-1 Proteasome subunit alpha type-3 Proteasome subunit alpha type-5 Proteasome subunit alpha type-6 proteasome subunit alpha 7 Proteasome subunit beta type-1 Proteasome subunit beta type-6 Phosphotriesterase-related protein 6-pyruvoyl tetrahydrobiopterin synthase Pyrroline-5-carboxylate reductase 3 Pregnancy zone protein Sulfhydryl oxidase 1 Ras-related protein Rab-1A Ras-related protein Rab-7a Ras-related protein Ral-A Lithostathine-1-beta Lithostathine-1-alpha Protein S100-A13 Protein S100-A8 (Calrprotectin) Protein S100-A9 (Calprotectin) Protein S100-G Scinderin Methanethiol oxidase Alpha-1-antitrypsin Alpha-1-antitrypsin-related protein Kallistatin Alpha-1-antichymotrypsin Plasma serine protease inhibitor Leukocyte elastase inhibitor Serpin B6 Antithrombin-III Alpha-2-antiplasmin Plasma protease C1 inhibitor Neuroserpin Serpin I2 Sialate O-acetylesterase Solute carrier family 15 member 1 Sodium- and chloride-dependent neutral and basic amino acid transporter B(0+) Acid sphingomyelinase-like phosphodiesterase 3a Acid sphingomyelinase-like phosphodiesterase 3b Superoxide dismutase [Cu-Zn] Superoxide dismutase [Mn], mitochondrial Spectrin alpha chain, non-erythrocytic 1 Spectrin beta chain, non- erythrocytic 1 Suppressor of tumorigenicity 14 protein homolog Sushi domain- containing protein 2 Transgelin-2 Transaldolase Protein-glutamine gamma- glutamyltransferase E Thimet oligopeptidase Transketolase Enteropeptidase Triosephosphate isomerase Thiamin pyrophosphokinase 1 Tropomyosin alpha-3 chain Trehalase Serotransferrin Tyrpsin-2 Trypsin-3 Serine protease 1 Transthyretin Thioredoxin Thioredoxin domain-containing protein 17 Thioredoxin reductase 1, cytoplasmic Cytochrome b-c1 complex subunit Rieske, mitochondrial Harmonin vinculin Villin-1 Pantetheinase WD repeat-containing protein 1 Xanthine dehydrogenase / oxidase Xaa-Pro aminopeptidase 214-3-3 protein epsilon 14-3-3 protein zeta / delta Zymogen granule membrane protein 16 Zinc finger ZZ-type and EF- hand domain-containing protein 1 Immunoglobulin kappa variable regions 3-20 Immunoglobulin kappa variable regions 3-15 Immunoglobulin kappa variable regions 3- 11 Immunoglobulin kappa variable regions 3-12 Immunoglobulin kappa constant Immunoglobulin kappa variable 1-39 Immunoglobulin kappa variable 4-1Attorney Docket No.190412-00026WO Immunoglobulin kappa variable 1-5 Immunoglobulin kappa variable 1-33 Immunoglobulin lambda variable 1-51 Immunoglobulin lambda variable 1-44 Immunoglobulin lambda variable 1-47 Immunoglobulin lambda variable 2-11 Immunoglobulin lambda variable 6-57 Immunoglobulin lambda variable 2-14 Immunoglobulin lambda constant 2 Immunoglobulin lambda constant 3 Immunoglobulin heavy constant alpha 2 Immunoglobulin heavy constant alpha 1 Immunoglobulin heavy variable 3-23 Immunoglobulin heavy variable 2-70 Immunoglobulin heavy variable 3-30 Immunoglobulin heavy variable 1-69 Immunoglobulin heavy variable 3-7 Immunoglobulin heavy variable 3-33 Immunoglobulin heavy variable 1-2 Immunoglobulin heavy constant gamma 1 Immunoglobulin heavy constant gamma 3 Immunoglobulin heavy constant gamma 4 Immunoglobulin heavy constant gamma 2 Immunoglobulin heavy constant gamma 1 Immunoglobulin kappa constant Immunoglobulin lambda constant 3, and Ester hydrolase C11orf54.

[0025] The proteins may be extracted from a subject’s fecal sample and separated from bacterial, viral, and fungal proteomes.

[0026] The proteins may be extracted from a subject’s fecal sample and proteins of human origin may be isolated from viral, bacterial, and fungal proteomes via methods like filtration and sonication. Isolated proteins may be then further purified and processed depending on the technology used for identification.

[0027] In particular embodiments, the biological pathway is determined to be activated or suppressed. Activated pathways may include biological pathways including proteins with higher measured protein abundances than predicted. In contrast, suppressed pathways may include biological pathways including proteins with lower measured protein abundances compared to predicted levels. Notably, the protein may be associated with more than one biological pathway. The biological pathway is associated with an immune response, an inflammatory response, a neurological disorder, wound healing, aging, protein homeostasis, oxidative stress, autophagy, mitochondrial homeostasis, axonal transport, protein seeding and propagation, protein unfolding response, apoptosis, protein degradation, and combinations thereof. In some embodiments, the biological pathway isAttorney Docket No.190412-00026WO associated with Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis, multiple sclerosis, motor neuron disorder, inflammatory bowel disease, ulcerative colitis, Crohn’s disease, or aging.

[0028] According to some embodiments, the fecal protein bioprofile is generated based on gender-related assessments of relative protein abundance and / or protein categorization of biological pathways. The fecal protein bioprofile may also be generated based on age-related assessments of relative protein abundance and / or protein categorization of biological pathways. The fecal protein bioprofile may also be generated based on a combination of gender and age-related assessments.

[0029] Embodiments of the present inventive concept further provide methods of determining a subject’s risk for developing an inflammatory disorder, a neurological disorder, a neurodegenerative disorder, or a disorder associated with aging. The method may employ using the fecal protein bioprofile described herein to determine a risk assessment score based upon an analysis of protein pathway enrichment scores.

[0030] Inflammatory disorders are conditions characterized by inflammation, involving, generally, the immune system, blood vessels, and various signaling molecules. Examples of inflammatory disorders include, but are not limited to, rheumatoid arthritis, inflammatory bowel disease or syndrome, psoriasis, systemic lupus erythematosus, ankylosing spondylitis, gout, asthma, multiple sclerosis and Type 1 diabetes.

[0031] Neurological disorders include medical conditions that affect the nervous system. Non-limiting neurological disorders include, but are not limited to, epilepsy, migraine, stroke, multiple sclerosis (MS), Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, peripheral neuropathy, traumatic brain injury (TBI), and cerebral palsy.

[0032] Neurodegenerative disorders are generally diseases characterized by the progressive degeneration or death of nerve cells associated with the nervous system. Examples of neurodegenerative disorders include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple system atrophy (MSA), frontotemporal dementia (FTD), Creutzfeldt-JakobAttorney Docket No.190412-00026WO disease (CJD), progressive supranuclear palsy (PSP), peripheral neuropathy, and spinocerebellar ataxias.

[0033] Disorders associated with aging include, but are not limited to atherosclerosis, cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension, hearing loss, muscle weakness, cell senescence, urologic changes, dementia, mobility disorders, frailty, continence, depression, memory, lung function, genetic aging disease, vision loss, collagen loss, and Alzheimer's disease. In some embodiments, a subject’s risk for Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, dementia, amyotrophic lateral sclerosis, motor neuron disorder, inflammatory bowel disease, ulcerative colitis, and / or Crohn’s disease is determined.

[0034] Disorders associated with animals that can produce inflammation and / or neurological impacts include, but are not limited to Campylobacteriosis, Tularemia, Yersiniosis, Encephalitis, Yellow Fever, Zika, Chikungunya, Dengue, Rabies, Coxiella burnetii infection, Psittacosis, Giardia, Hantavirus Infection, Plague, Cryptosporidiosis, Brucellosis, Epilepsy, Meningitis, Degeneration, Vestibular Disease, Dementia, Feline Hyperesthesia Syndrome, Hydrocephalus, Wobbler’s Syndrome, Polyneuropathy, Degenerative lumbosacral stenosis, West Nile Virus, Herpes Myeloencephalopathy, Protozoal Myeloencephalitis, Chronic wasting disease (CWD), influenza, Cerebellar Hypoplasia, Polioencephalomalacia (PEM) syn Cerebrocortical necrosis (CCN), Listeriosis, Heavy Metal Poisoning, Basillar Empyema, Bovine Spongiform Encephalopathy (BSE), Poxvirus, Herpesvirus, Viral Infections, Bacterial Infections, Mycobacterium Infections, Myelin Disorders, Dysautonomia, Paralysis, Neoplasia, Paraneoplastic Disorders, Scrapie, Equine Arboviral Encephalomyelitis, Louping III, Pesudorabies, Teschovirus Encephalomyelitis, Sporadi Bovine Encephalomyelitis, Equine Protozoal Myeloencephalitis, and Tick Paralysis.

[0035] Embodiments of the present inventive concept also allow early risk detection assessment of a subject’s risk for developing an inflammatory disorder, a neurological disorder, a neurodegenerative disorder, or a disorder associated with aging prior to the onset of symptoms. Further, methods of monitoring the status of disease progression in a subject suffering from an inflammatory disorder, a neurological disorder, a neurodegenerative disorder, or a disorder associated with aging are also provided. TheAttorney Docket No.190412-00026WO methods include using the fecal protein bioprofile of the present inventive concept to determine the progression of the disease based upon an analysis of the protein modulations and / or biological pathway modulations in the subject. Protein and / or biological modulation in the subject is assessed using a protein-abundance weighted enrichment score where the measured protein abundances in a subject’s fecal sample is used as a mathematical weighting factor when generating a subject’s bioprofiles. The subject’s weighted bioprofiles are compared against those predicted to be found in a healthy subject or those with an inflammatory disorder, a neurological disorder, a neurodegenerative disorder, or a disorder associated with aging using multivariate statistical analysis (Smaga, L. (2020) A note on repeated measures analysis for functional data. AStA Adv. Stat. Anal.104, 117–139, Rao, B. B., Bhattacharya, A., and Lichtenstein, G. R. (2020) Precision Medicine: Predicting Disease Course in Patients with Inflammatory Bowel Disease. Curr. Treat. Options Gastroenterol.18, 574–588). The extent of contribution of these quantitative differences to the overall risk assessment score is based on the identity of the pathway, whether it is activated or suppressed, and the quantitative (amount?) difference determined in the analysis.

[0036] The overall score related to a subject’s gut health, risk for developing an inflammatory disorder, a neurological disorder, a neurodegenerative disorder, or a disorder associated with aging prior to the onset of symptoms, or disease progression may include the following or combinations thereof: the quantitative comparisons of activated and suppressed biological pathways from protein-weighted fecal bioprofile analysis to those with the disease and healthy subject’s, the qualitative assessments of a subject’s fecal bioprofile, and additional factors including, but not limited to age, gender, and medication and diet history.

[0037] Embodiments of the present inventive concept provide kits for determining a subject’s risk for developing an inflammatory disorder, a neurological disorder, a neurodegenerative disorder, or a disorder associated with aging. The kits may also be used to monitor disease states and / or their progression thereof. The kits include a container to collect a fecal sample and optional instructions for using the kit. In some embodiments, the kits further include a preservative, buffer, and / or an antimicrobial agent. According to some embodiments, the kits are used in a medical setting such asAttorney Docket No.190412-00026WO a clinic or hospital. In other embodiments, the kits are an at-home risk determination kit or disease state monitoring kit.

[0038] In particular embodiments, a kit as described herein may be sent to a subject’s residence for use at home. After collection, the sample is sent to a laboratory for testing via technology such as mass spectrometry or on a proteomics- based-based platform, and analysis is performed. A scorecard describing the subject’s risk assessment or disease state indication can be delivered to the subject or representative thereof through U.S. postal mail, electronic mail, text messaging, patient portal, etc.

[0039] For example, an exemplary test can be requested by a clinician or directly by the patient. Once the sample is provided, it is sent back to a commercial lab for testing via a proteomics-based platform. Once the analysis is performed, the patient may be provided with access to their own digital dashboard bioprofile that can track changes in their bioprofile and risk scores over time and have access to telehealth professionals for counseling.

[0040] Upon receiving the risk assessment or indication of disease state, appropriate treatments, preventive care, lifestyle modification recommendations, physical and / or mental therapy can be recommended and / or administered to the subject. Definitions

[0041] As used herein, "a" or "an" or "the" can mean one or more than one. Also as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0042] Furthermore, the term "about," as used herein when referring to a measurable value such as an amount of a compound or agent of the inventive concept, dose, time, temperature, and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0043] The terms "comprise," "comprises" and "comprising" as used herein, specify the presence of the stated features, integers, steps, operations, elements, and / orAttorney Docket No.190412-00026WO components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0044] As used herein, the transitional phrase "consisting essentially of" means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term "consisting essentially of" when used in a claim of the inventive concept is not intended to be interpreted to be equivalent to "comprising."

[0045] Nonetheless, in some embodiments, "comprise" may also encompass, and, in some embodiments, may refer to the expressions "consist essentially of" and / or "consist of." Thus, the expression "comprise" can also refer to and encompass embodiments, wherein that which is claimed "comprises" specifically listed elements does not include further elements, as well as embodiments wherein that which is claimed "comprises" specifically listed elements may and / or does encompass further elements, or encompass further elements that do not materially affect the basic and novel characteristic(s) of that which is claimed. For example, that which is claimed, such as a method, kit, system, etc. "comprising" specifically listed elements also encompasses, for example, a method, kit, system, etc. "consisting of," i.e., wherein that which is claimed does not include further elements, and, for example, a method, kit, system, etc. "consisting essentially of," i.e., wherein that which is claimed may include further elements that do not materially affect the basic and novel characteristic(s) of that which is claimed.

[0046] Notably, aspects of the present inventive concept may comprise, consist essentially of, or consist of the elements as described herein. For example, any method may comprise, consist essentially of, or consist of the steps recited.

[0047] The terms "management" or “managing” as used herein refer to the ability to affect a method, process, state of being, disorder or the like. The effect may be that of prevention, treatment, modulation, or inactivity. “Monitoring” refers to observing the point of interest over a period of time.

[0048] By the terms "treat," "treating" or "treatment of," it is intended that the severity of the disorder or the symptoms of the disorder are reduced, or the disorder is partiallyAttorney Docket No.190412-00026WO or entirely eliminated, as compared to that which would occur in the absence of treatment. Treatment does not require the achievement of a complete cure of the disorder.

[0049] By the terms "preventing" or "prevention," it is intended that the inventive methods eliminate or reduce the incidence or onset of the disorder, as compared to that which would occur in the absence of the measure taken. Alternatively stated, the present methods slow, delay, control, or decrease the likelihood or probability of the disorder in the subject, as compared to that which would occur in the absence of the measure taken.

[0050] A "therapeutically effective" or "effective" amount is intended to designate a dose that causes a relief of symptoms of a disease or disorder as noted through clinical testing and evaluation, patient observation, and / or the like. "Effective amount" or "effective" can further designate a dose that causes a detectable change in biological or chemical activity. The detectable changes may be detected and / or further quantified by one skilled in the art for the relevant mechanism or process. Moreover, "effective amount" or "effective" can designate an amount that maintains a desired physiological state, i.e., reduces or prevents significant decline and / or promotes improvement in the condition of interest. As is generally understood in the art, the dosage will vary depending on the administration routes, symptoms and body weight of the patient but also depending upon the compound being administered.

[0051] A "subject" as used herein can be a human subject and can include, but is not limited to, a patient. The subject may be male, female or any gender or gender identity, and may be of any race or ethnicity, including, but not limited to, Caucasian, African- American, African, Asian, Hispanic, Native American, etc., and combinations thereof. The subject may be of any age, including newborn, neonate, infant, child, juvenile, adolescent, adult, and geriatric. In some embodiments, the subject is over 30, 40, 50, 60, 70, 80 or 90 years of age. A subject can also include an animal subject, including mammalian subjects such as canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g., rats and mice), lagomorphs, primates (including non-human primates), etc., avian subjects (e.g., fowl, parrots, raptors etc.), and the like, for prevention and treatment purposes as well as veterinary medicine and / orAttorney Docket No.190412-00026WO pharmaceutical drug development purposes. A subject of the inventive concept can be believed to be at risk for an inflammatory disorder, a neurological disorder, a neurodegenerative disorder, or a disorder associated with aging, or can be experiencing one or more of these disorders.

[0052] EXAMPLE 1: Summary of Proteins Identified and Protein Modulations by Disease Type

[0053] We measured the abundances of host proteins from fecal samples in several rodent models of neurological disorders, including multiple sclerosis and Parkinson’s disease. Host proteins in fecal samples are present in much lower abundance than microbe proteins, however we have a method to deplete the amount of microbe proteins in the fecal samples. Host proteins are measured reliably and reproducibly from non-preserved samples and are consistent across control and disease models (~400 proteins, >95% confidence ID, Tables 1-11). Importantly, these data show specific protein abundances increase or decrease depending on the disease model indicating the use of host proteins isolated from fecal samples as an indicator of neurological disorders is feasible. The following tables summarize the proteins identified and protein modulations by disease type.

[0054] Dopamine D3- Receptor Knockout Model. All 217 proteins identified from male rodents are provided in Table 1. The Dopamine D3-Receptor Knockout model mimics aging associated changes in hypertension and cardiovascular changes, (Johnson, T.L., Tulis, D.A., Keeler, B.E., Virag, J.A., Lust, R.M., Clemens, S. (2013) The Dopamine D3 Receptor Knockout Mouse Mimics Aging-Related Changes in Autonomic Function and Cardiac Fibrosis. Plos One, 8, ee74116) and is also a model for restless leg syndrome, a chronic, neurological disorder linked with dopamine level imbalances (Meneely, S., Dinkins, M-L, Kassai, M., Lyu, S., Lin, C-T., Brewer, K., Li, Y., Clemens, S. (2018) Differential Dopamine D1 and D3 Receptor Modulation and Expression in the Spinal Cord of Two Mouse Models of Restless Legs Syndrome. Front. Behav. Neurosci., 12, 1-14).Attorney Docket No.190412-00026WO

[0055] Table 1. Dopamine D3-Receptor Knockout Model. Protein Protein ID Entry Name GENE DESCRIPTION Isoform 2 of 4- aminobut rate 1 id- edc-Attorney Docket No.190412-00026WO Protein Protein ID Entry Name GENE DESCRIPTION sp|P31809- related cell adhesion 4|CEAM1 MOUSE molecule 1 - eeea e e e ke1Attorney Docket No.190412-00026WO Protein Protein ID Entry Name GENE DESCRIPTION sp|Q60997-2|DMBT1_MOUSE sp|Q60997-3|DMBT1 MOUSE Isoform 2 of Deleted in -2 di diAttorney Docket No.190412-00026WO Protein Protein ID Entry Name GENE DESCRIPTION H-2 class II sp|P14483|HB2A MOUSE P14483 HB2A MOUSE H2-Ab1 histocompatibility antigen A a 96lin-3 1lnAttorney Docket No.190412-00026WO Protein Protein ID Entry Name GENE DESCRIPTION sp|Q9CPY7-2|AMPL_MOUSE Q9CPY7-2 AMPL_MOUSE Lap3Isoform 2 of CytosolaminopeptidaseC L1 e,a b a e e-eAttorney Docket No.190412-00026WO Protein Protein ID Entry Name GENE DESCRIPTION sp|P17742|PPIA_MOUSE P17742 PPIA_MOUSE PpiaPeptidyl-prolyl cis-transisomerase Aseoree-],-Attorney Docket No.190412-00026WO Protein Protein ID Entry Name GENE DESCRIPTION sp|P97435|ENTK_MOUSE P97435 ENTK_MOUSE Tmprss15 Enteropeptidase -p pp Receptor Knockout Model.

[0057] Table 2. Protein Modulation in D3-Receptor Knockout Model. Proteins Enhanced (114) Proteins suppressed (34) Abat AceAttorney Docket No.190412-00026WO Proteins Enhanced (114) Proteins suppressed (34) Ces1c IapAttorney Docket No.190412-00026WO Proteins Enhanced (114) Proteins suppressed (34) Gstm2Attorney Docket No.190412-00026WO Proteins Enhanced (114) Proteins suppressed (34) Qsox1

[0058] DSS-Induced Colitis Model. All 149 proteins identified from male rodents are in Table 3. Administration of dextran sulfate (DSS) is proposed to induce inflammatory bowel disease-like pathologies in rodents (Chassing, B., Aitken, J.D., Malleshappa, M. Vijay-Kumar, M. (2015) Dextran Sulfate Sodium (DSS)-Induced Colitis in Mice. Curr. Protoc. Immunol.104, Unit-15.25.).

[0059] Table 3. DSS-Induced Colitis Model. Protein Protein ID Entry Name Gene DescriptionAttorney Docket No.190412-00026WO Protein Protein ID Entry Name Gene Description sp|P97429|ANXA4_MOUSE P97429 ANXA4_MOUSE Anxa4 Annexin A4 5 g elelAttorney Docket No.190412-00026WO Protein Protein ID Entry Name Gene Description sp|Q60997-2|DMBT1_MOUSE sp|Q60997- 1 as as al ine e 2Attorney Docket No.190412-00026WO Protein Protein ID Entry Name Gene Description sp|P10853|H2B1F_MOUSE sp|P10854|H2B1M MOUSE, n se ic aAttorney Docket No.190412-00026WO Protein Protein ID Entry Name Gene Description sp|P04939|MUP3_MOUSE P04939 MUP3_MOUSE Mup3 Major urinary protein 3 orAttorney Docket No.190412-00026WO Protein Protein ID Entry Name Gene Description sp|P58242|ASM3B_MOUSE P58242 ASM3B_MOUSE Smpdl3bAcid sphingomyelinase-likephosphodiesterase 3bylInduced Colitis model.

[0061] Table 4. Protein modulation in DSS-Induced Colitis Model. Proteins Enhanced (29) Proteins suppressed (11)Attorney Docket No.190412-00026WO Proteins Enhanced (29) Proteins suppressed (11) Hp

[0062] Experimena auommune encep aomye s mo el for multiple sclerosis. All 152 proteins identified in male rodents in Table 5. The EAE experimental model is the most common rodent model used for multiple sclerosis. (Constantinescu, C.S, Farooqi, N., O’Brien, K., Gran, B. (2011) Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Br. J. Pharmacol.164. 1079-1106).

[0063] Table 5. EAE model for multiple sclerosis. Protein Entry Name Gene DescriptionAttorney Docket No.190412-00026WO Protein Entry Name Gene Description P00920 CAH2_MOUSE Ca2 Carbonic anhydrase 2 fAttorney Docket No.190412-00026WO Protein Entry Name Gene Description Q9DCM0 ETHE1_MOUSE Ethe1 Persulfide dioxygenase ETHE1, mitochondrialAttorney Docket No.190412-00026WO Protein Entry Name Gene Description P17897 LYZ1_MOUSE Lyz1 Lysozyme C-1Attorney Docket No.190412-00026WO Protein Entry Name Gene Description Q9D7P9 SPB12_MOUSE Serpinb12 Serpin B12 1

[0064] Table 6 shows the proteins that were enhanced or suppressed in the EAE model for multiple sclerosis.

[0065] Table 6. Protein modulation in EAE model for Multiple Sclerosis. Proteins Enhanced (74) Proteins suppressed (1)Attorney Docket No.190412-00026WO Proteins Enhanced (74) Proteins suppressed (1) Zzef1Attorney Docket No.190412-00026WO Proteins Enhanced (74) Proteins suppressed (1) Pepd

[0066] A53T-model. All 391 proteins identified both in male and female rodents are in Table 7. The A53T-model is a transgenic rodent model where the human alpha- Synuclein A53T-varient is constituently expressed leading to disease pathologies consistent with Parkinson’s disease (Lee, M.K, Stirling, W., Xu, Y., Qui, D., Mandir, A.S., Dawson, T.M., Copeland, N.G., Jenkins. N.A., Price, D.L. (2002) Human α- synuclein-harboring familial Parkinson's disease-linked Ala-53 → Thr mutation causes neurodegenerative disease with α-synuclein aggregation in transgenic mice. PNAS, 99, 8968-8973).Attorney Docket No.190412-00026WO

[0067] Table 7. A53T Model for Parkinson’s Disease. Protein Protein Entry Name Gene Description ID P619222GABT MOUSE P619222 GABT MOUSE Ab t Isoform 2 of 4-aminobutyrateB1 l L- gAttorney Docket No.190412-00026WO Protein Protein Entry Name Gene Description ID sp|Q9JHE3|ASAH2 MOUSE Q9JHE3 ASAH2 MOUSE Asah2 Neutral ceramidase einily eing elAttorney Docket No.190412-00026WO Protein Protein Entry Name Gene Description ID s|Q9QX15|CA3A1 MOUSE Q9QX15 CA3A1 MOUSE Clca3a1Calcium-activated chloride channelelAttorney Docket No.190412-00026WO Protein Protein Entry Name Gene Description ID sp|Q9ET22|DPP2 MOUSE Q9ET22 DPP2 MOUSE Dpp7 Dipeptidyl peptidase 2 ininas as al ial e taAttorney Docket No.190412-00026WO Protein Protein Entry Name Gene Description ID s|Q9Z0L8-2|GGH MOUSE Q9Z0L8-2 GGH MOUSE G hIsoform II of Gamma-glutamyle 2 eneaAttorney Docket No.190412-00026WO Protein Protein Entry Name Gene Description ID sp|Q91X72|HEMO MOUSE Q91X72 HEMO MOUSE Hpx Hemopexin in A se ,ynAttorney Docket No.190412-00026WO Protein Protein Entry Name Gene Description ID sp|Q9D312|K1C20 MOUSE Q9D312 K1C20 MOUSE Krt20 Keratin type I cytoskeletal 20 75 n ic aAttorney Docket No.190412-00026WO Protein Protein Entry Name Gene Description ID sp|Q8JZM8|MUC4 MOUSE Q8JZM8 MUC4 MOUSE Muc4 Mucin-4 1 g 1Attorney Docket No.190412-00026WO Protein Protein Entry Name Gene Description ID sp|Q8CHP8|PGP MOUSE Q8CHP8 PGP MOUSE Pgp Glycerol-3-phosphate phosphatase or g M 2 eese l 3Attorney Docket No.190412-00026WO Protein Protein Entry Name Gene Description ID sp|P51150|RAB7A MOUSE P51150 RAB7A MOUSE Rab7a Ras-related protein Rab-7a 20Attorney Docket No.190412-00026WO Protein Protein Entry Name Gene Description ID Protein-glutamine gamma-glutamyle

[0068] Table 8 shows the proteins that were enhanced or suppressed in females in the A53T model for Parkinson’s disease.

[0069] Table 8. Protein modulation in females for A53T model of Parkinson’s disease. Enhanced Female (63) Suppressed Female (18)Attorney Docket No.190412-00026WO Enhanced Female (63) Suppressed Female (18) Ces1c Hspa1lAttorney Docket No.190412-00026WO Enhanced Female (63) Suppressed Female (18) Ppib

[0070] Table9 shows the proteins that were enhanced or suppressed in males in the A53T model for Parkinson’s disease.

[0071] Table 9. Protein modulation in males for A53T model of Parkinson’s disease. Enhanced Male (15) Suppressed Male (64) Al A lAttorney Docket No.190412-00026WO Enhanced Male (15) Suppressed Male (64) S100a13 Ca1Attorney Docket No.190412-00026WO Enhanced Male (15) Suppressed Male (64) Prdx1

[0072] Table 1p ppressed in the A53T model for Parkinson’s disease in a separate analysis account for gender.

[0073] Table 10. Combined protein modulation in males and females for A53T model of Parkinson’s disease. Combined M / F Enhanced (74) Combined M / F Suppressed (75)Attorney Docket No.190412-00026WO Combined M / F Enhanced (74) Combined M / F Suppressed (75) Dpep1 Ces1fAttorney Docket No.190412-00026WO Combined M / F Enhanced (74) Combined M / F Suppressed (75) Psma3 Prcp

[0074] The proens cy , y a, e , , rcp, an r x were suppressed in males and enhanced in females. The protein S100a13 was enhanced in males and suppressed in females. The proteins Alb, Ces1c, Ggt1, Klk1, and Tf were enhanced in males and females. Lastly, the proteins Ace, Agr2, Clca4aq, Hspa1l, and Pdia3 were suppressed in both males and females

[0075] Table 11 shows the proteins that were enhanced or suppressed in the A53T model for Parkinson’s disease in a combined analysis not accounting for gender.

[0076] Table 11. Combined analysis of protein modulation not accounting for gender for A53T model of Parkinson’s disease. Enhanced Proteins (19) Suppressed Proteins (35)Attorney Docket No.190412-00026WO Enhanced Proteins (19) Suppressed Proteins (35) Gusb Clca4a

[0077] EXAMPLE 2. Summary of Fecal Bioprofiles by Disease Type.

[0078] We constructed fecal bioprofiles in rodent models for several disease types using the proteins identified in EXAMPLE 1. Importantly, when compared to age- matched controls, diseased animals have bioprofiles enriched in biological pathways associated with the disease, and abundances and enrichments corresponded with the severity of the neurological disorders. Tables 12-15 summarize the fecal bioprofiles determined for the disease types in EXAMPLE 1. Pathways denoted in bold overlap with each disease type, however, either magnitude or enrichment / suppression is different.Attorney Docket No.190412-00026WO

[0079] Table 10 shows the biological pathways enhanced or suppressed in a non- protein abundance weighted fecal bioprofile for the D3-receptor knockout model. FDR is the false discovery rate (<0.05).

[0080] Table 12. Fecal bioprofile for D3-receptor knockout model. Fold- Enriched Biological Pathways Enriched p-value FDR

[0081] Table 13 shows the biological pathways enhanced in a non-protein abundance weighted fecal bioprofile for the experimental autoimmuneAttorney Docket No.190412-00026WO encephalomyelitis (EAE) model for multiple sclerosis. FDR is the false discovery rate (<0.05).

[0082] Table 13. Fecal bioprofile for experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis. Enriched Biological Pathways Fold-Enriched p-value FDR protein targeting to lysosome involved in hi h100 0.000104 0.0348[a e s ows e oogca pa ways en ance n a non-proen abundance weighted fecal bioprofile for female rodents in the A53T model for Parkinson’s disease. FDR is the false discovery rate (<0.05).

[0084] Table 14. Fecal bioprofile for female rodents in A53T model for Parkinson’s disease. Enriched Biological Pathways Fold-Enriched p-value FDR 01 22 18 96 93 12 96 32 86 16 91 66Attorney Docket No.190412-00026WO

[0085] Table 15 shows the biological pathways enhanced and suppressed in a non- protein abundance weighted fecal bioprofile for male rodents in the A53T model for Parkinson’s disease. FDR is the false discovery rate (<0.05).

[0086] Table 15. Fecal bioprofile for male rodents in the A53T model for Parkinson’s disease. Enhanced Biological Pathways Fold-Enriched p-value FDR proteolysis 9.69 6.06E-08 0.00095

[0087] EXAMPLE 3. Parkinsons (A53T) model pathways - p<0.05, z-score weighted protein abundance pathway enrichment and suppression scores.

[0088] Weighted protein abundance pathway enrichment scores (z-score, p<0.05) for biological pathways activated in female PD (A53T model) are shown in Table 16.

[0089] Table 16. Pathways activated in female PD (A53T Model). Pathway Score p-value FDR Function / RoleAttorney Docket No.190412-00026WO Pathway Score p-value FDR Function / Role deoxyribonucleosideo pa ways were o serve o e suppresse n emae mo e . Weighted protein abundance pathway enrichment scores in male PD (A53T model) are shown in Table 17, and protein abundance scores for biological pathways suppressed in male PD (A53T model) are shown in Table 18.

[0091] Table 17. Pathways activated in male PD (A53T model) Pathway Score p-value FDR Function / Role

[0092] Table 18. Pathways suppressed in male PD (A53T model) Pathway Score p-value FDR Function / RoleAttorney Docket No.190412-00026WO Pathway Score p-value FDR Function / Role positive regulation of protein folding -45.27 9.22E-05 2.73E-02 Protein homeostasis. es ess eg syn rome mo e pa ways p<0.05, z-score weighted protein abundance pathway enrichment scores.

[0094] Weighted protein abundance pathway enrichment scores (z-score, p<0.05) for biological pathways activated in RLS (D3KO model) are shown in Table 19.

[0095] Table 19 Pathways enriched in RLS model (D3KO). Pathway Score p-value FDR Function / RoleAttorney Docket No.190412-00026WO Pathway Score p-value FDR Function / Role homocysteine metabolic process 20.37 3.88E-05 0.00879 Oxidative stressroen a un ance pa way epe on scores or oogca pa ways suppressed in RLS (D3KO model) are shown in Table 20.

[0097] Table 20. Pathways suppressed in RLS model (D3KO). Pathway Score p-value FDR Function / Role

[0098] References: (1) Hor, J. W., Lim, S.Y., Khor, E. S., Chong, K. K., Song, S. L., Ibrahim, N. M., Teh, C. S. J., Chong, C. W., Hilmi, I. N., and Tan, A. H. (2021) Fecal Calprotectin in Parkinson’s Disease and Multiple System Atrophy. JMD 15, 106–114.Attorney Docket No.190412-00026WO (2) Rao, B. B., Bhattacharya, A., and Lichtenstein, G. R. (2020) Precision Medicine: Predicting Disease Course in Patients with Inflammatory Bowel Disease. Curr. Treat. Options Gastroenterol.18, 574–588. (3) Fotenos, A. F., Mintun, M. A., Snyder, A. Z., Morris, J. C., and Buckner, R. L. (2008) Brain Volume Decline in Aging: Evidence for a Relation Between Socioeconomic Status, Preclinical Alzheimer Disease, and Reserve. Arch. Neurol. 65, 113–120. (4) Ullah, I., Ali, S., Rahman, H. U., Ali, S., Ullah, Z., and Ali, F. (2022) Socioeconomic Study on the Prevalence and Pattern of Various Neurological Disorders in Urban and Rural Areas. Sch. J. Appl. Méd. Sci.10, 409–416. (5) Konings, B., Villatoro, L., Eynde, J. V. den, Barahona, G., Burns, R., McKnight, M., Hui, K., Yenokyan, G., Tack, J., and Pasricha, P. J. (2023) Gastrointestinal syndromes preceding a diagnosis of Parkinson’s disease: testing Braak’s hypothesis using a nationwide database for comparison with Alzheimer’s disease and cerebrovascular diseases. Gut gutjnl 2023-329685. (6) Kim, T.K., Bae, E.J., Jung, B. C., Choi, M., Shin, S. J., Park, S. J., Kim, J. T., Jung, M. K., Ulusoy, A., Song, M.-Y., Lee, J. S., Lee, H.J., Monte, D. A. D., and Lee, S.J. (2022) Inflammation promotes synucleinopathy propagation. Exp Mol Med 54, 2148– 2161. (7) Bairamian, D., Sha, S., Rolhion, N., Sokol, H., Dorothée, G., Lemere, C. A., and Krantic, S. (2022) Microbiota in neuroinflammation and synaptic dysfunction: a focus on Alzheimer’s disease. Mol. Neurodegener.17, 19. (8) Iskandar, H. N., and Ciorba, M. A. (2012) Biomarkers in inflammatory bowel disease: current practices and recent advances. Transl. Res.159, 313–325. (9) Mendoza, J. L., and Abreu, M. T. (2009) Biological markers in inflammatory bowel disease: Practical consideration for clinicians. Gastroentérologie Clin. Biol.33, S158– S173. (10) Sandor, C., Millin, S., Dahl, A., Schalkamp, A.K., Lawton, M., Hubbard, L., Rahman, N., Williams, N., Ben- Shlomo, Y., Grosset, D. G., Hu, M. T., Marchini, J., and Webber, C. (2022) Universal clinical Parkinson’s disease axes identify a major influence of neuroinflammation. Genome Med 14, 129. (11) Villar-Piqué, A., Schmitz, M., Candelise, N., Ventura, S., Llorens, F., and Zerr, I. (2018) Molecular and Clinical Aspects of Protein Aggregation Assays in Neurodegenerative Diseases. Mol Neurobiol 55, 7588–7605. (12) Pagan, F. L. (2012) Improving outcomes through early diagnosis of Parkinson’s disease. Am. J. Manag. care 18, S176-82. (13) Chen, J.H., Chan, L., Chung, C.C., Bamodu, O. A., and Hong, C.T. (2021) Blood Neurofilament Light Chain in Parkinson’s Disease: Comparability between Parkinson’s Progression Markers Initiative (PPMI) and Asian Cohorts. J. Clin. Med. 10, 5085.Attorney Docket No.190412-00026WO (14) Chen, L., Reynolds, C., David, R., and Brewer, A. P. (2020) Development of an Index Score for Intestinal Inflammation-Associated Dysbiosis Using Real-World Stool Test Results. Dig. Dis. Sci.65, 1111–1124. (15) Whelan, S. A., Hendricks, N., Dwight, Z. L., Fu, Q., Moradian, A., Eyk, J. E. V., and Mockus, S. M. (2023) Assessment of a 60-Biomarker Health Surveillance Panel (HSP) on Whole Blood from Remote Sampling Devices by Targeted LC / MRM-MS and Discovery DIA-MS Analysis. Anal. Chem.95, 11007–11018. (16) Smaga, L. (2020) A note on repeated measures analysis for functional data. AStA Adv. Stat. Anal.104, 117–139. (17) Johnson, T.L., Tulis, D.A., Keeler, B.E., Virag, J.A., Lust, R.M., Clemens, S. (2013) Plos One, 8, ee74116. (18) Meneely, S., Dinkins, M-L, Kassai, M., Lyu, S., Lin, C-T., Brewer, K., Li, Y., Clemens, S. (2018) Differential Dopamine D1 and D3 Receptor Modulation and Expression in the Spinal Cord of Two Mouse Models of Restless Legs Syndrome. Front. Behav. Neurosci., 12, 1-14 (19) Chassing, B., Aitken, J.D., Malleshappa, M. Vijay-Kumar, M. (2015) Dextran Sulfate Sodium (DSS)-Induced Colitis in Mice. Curr. Protoc. Immunol.104, Unit-15.25. (20) Constantinescu, C.S, Farooqi, N., O’Brien, K., Gran, B. (2011) Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Br. J. Pharmacol.164.1079-1106. (21) Lee, M.K, Stirling, W., Xu, Y., Qui, D., Mandir, A.S., Dawson, T.M., Copeland, N.G., Jenkins. N.A., Price, D.L. (2002) Human α-synuclein-harboring familial Parkinson's disease-linked Ala-53 → Thr mutation causes neurodegenerative disease with α-synuclein aggregation in transgenic mice. PNAS, 99, 8968-8973

[0099] The foregoing is illustrative of the present inventive concept and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein. All publications, patent applications, patents, patent publications, and any other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

Claims

Attorney Docket No.190412-00026WO THAT WHICH IS CLAIMED:

1. A method of establishing a fecal protein bioprofile, comprising: (a) determining a relative abundance of proteins extracted from a subject’s fecal matter; (b) categorizing the proteins with respect to their biological pathway; and (c) generating a subject’s unique fecal protein bioprofile.

2. The method of claim 1, wherein the extracted proteins are separated from bacterial, viral, and fungal proteomes.

3. The method of claim 1 or 2, wherein the biological pathway is determined to be activated or suppressed.

4. The method of claim 1, wherein the proteins are associated with at least one biological pathway.

5. The method of claim 1, wherein the biological pathway is associated with an immune response, an immune response, an inflammatory response, a neurological disorder, wound healing, aging, protein homeostasis, oxidative stress, autophagy, mitochondrial homeostasis, axonal transport, protein seeding and propagation, protein unfolding response, apoptosis, protein degradation, and combinations thereof.

6. The method of claim 1, wherein the biological pathway is associated with Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, dementia, amyotrophic lateral sclerosis, motor neuron disorder, inflammatory bowel disease, ulcerative colitis, Crohn’s disease, or aging.Attorney Docket No.190412-00026WO 7. The method of claim 1 or 6, wherein the fecal protein bioprofile is associated with or generated based on gender-related assessments of relative protein abundance and / or protein categorization of biological pathways.

8. The method of claim 1 or 6, wherein the fecal protein bioprofile is associated with or generated based on age-related assessments of relative protein abundance and / or protein categorization of biological pathways.

9. The method of claim 1, wherein the subject is an asymptomatic or healthy subject.

10. The method of claim 1, wherein the subject is afflicted with an inflammatory disorder, a neurological disorder, a neurodegenerative disorder, or a disorder associated with aging.

11. A method of determining a subject’s risk for developing an inflammatory disorder, a neurological disorder, a neurodegenerative disorder, or a disorder associated with aging, the method comprising using the fecal protein bioprofile of claim 1 to determine a risk assessment score based upon an analysis of protein pathway or protein-weighted biological pathway enrichment scores.

12. The method of claim 11, wherein a subject’s risk for Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, dementia, amyotrophic lateral sclerosis, motor neuron disorder, inflammatory bowel disease, ulcerative colitis, and / or Crohn’s disease is determined.

13. The method of claim 11, wherein the subject’s risk for developing an inflammatory disorder, a neurological disorder, a neurodegenerative disorder, or a disorder associated with aging is an early risk detection prior to the onset of symptoms.Attorney Docket No.190412-00026WO 14. A method of monitoring the status of disease progression in a subject suffering from an inflammatory disorder, a neurological disorder, a neurodegenerative disorder, or a disorder associated with aging, the method comprising using the fecal protein bioprofile of claim 1 to determine the progression of the disease based upon an analysis of protein pathway or protein-weighted biological pathway enrichment scores.

15. A kit for determining a subject’s risk for developing an inflammatory disorder, a neurological disorder, a neurodegenerative disorder, or a disorder associated with aging, the kit comprising a container to collect a fecal sample and optional instructions for using the kit.

16. The kit of claim 15, further comprising a preservative, buffer, and / or an antimicrobial agent.

17. The kit of claim 15, wherein the kit is an at-home risk determination kit or disease state monitoring kit.

18. A fecal bioprofile derived according to the method of claim 1.

19. A method of establishing a fecal protein bioprofile as described substantially herein.

20. The methods and kits as described substantially herein.

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