IJMMS_2024v14n2

International Journal of Molecular Medical Science 2024, Vol.14 http://medscipublisher.com/index.php/ijmms © 2024 MedSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved.

International Journal of Molecular Medical Science 2024, Vol.14 http://medscipublisher.com/index.php/ijmms © 2024 MedSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. MedSci Publisher, operated by Sophia Publishing Group (SPG), is an international Open Access publishing platform that publishes scientific journals in the field of life science. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher. Publisher MedSci Publisher Editedby Editorial Team of International Journal of Molecular Medical Science Email: edit@ijmms.medscipublisher.com Website: http://medscipublisher.com/index.php/ijmms Address: 11388 Stevenston Hwy, PO Box 96016, Richmond, V7A 5J5, British Columbia Canada International Journal of Molecular Medical Science (ISSN 1927-6656) is an open access, peer reviewed journal published online by MedSci Publisher. The journal publishes scientific articles like original research articles, case reports, review articles, editorials, short communications and correspondence of the high quality pertinent to all aspects of human biology, pathophysiology and molecular medical science, including genomics, transcriptomics, proteomics, metabolomics of disease therapy, clinical pharmacology, clinical biochemistry, vaccines, immunology, microbiology, epidemiology, aging, cancer biology, infectious diseases, neurological diseases and myopathies, stem cells and regenerative medicine, vascular and cardiovascular biology, as well as the important implications for human health and clinical practice research. All the articles published in International Journal of Molecular Medical Science are Open Access, and are distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MedSci Publisher uses CrossCheck service to identify academic plagiarism through the world’s leading plagiarism prevention tool, iParadigms, and to protect the original authors’ copyrights.

International Journal of Molecular Medical Science (online), 2024, Vol. 14, No. 2 ISSN 1927-6656 http://medscipublisher.com/index.php/ijmms © 2024 MedSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. Latest Content Innate Defense Role of Extracellular Vesicles: The Critical Role of Phosphatidylserine in Combating Apoptotic Mimicry Viruses ManmanLi International Journal of Molecular Medical Science, 2024, Vol. 15, No. 2, 100-105 Unraveling the Gut-Brain Axis: The Potential of Engineered Synthetic Microbial Communities in Modulating Neurotransmitter Production and Mental Health Jingqiang Wang International Journal of Molecular Medical Science, 2024, Vol. 15, No. 2, 106-122 The Application and Challenges of Mesenchymal Stem Cells' Immunomodulation in the Treatment of Autoimmune Diseases CaixinLi International Journal of Molecular Medical Science, 2024, Vol. 15, No. 2, 123-131 Epigenetic Biomarkers in Patients with Hypertensive Heart Disease Jianli Zhong International Journal of Molecular Medical Science, 2024, Vol. 15, No. 2, 132-143 Advancements in Insulin Therapy for Type 1 Diabetes Guangying Zong, Guangman Xu International Journal of Molecular Medical Science, 2024, Vol. 15, No. 2, 144-152

International Journal of Molecular Medical Science, 2024, Vol.14, No.2, 100-105 http://medscipublisher.com/index.php/ijmms 100 Scientific Commentary Open Access Innate Defense Role of Extracellular Vesicles: The Critical Role of Phosphatidylserine in Combating Apoptotic Mimicry Viruses ManmanLi Hainan Institute of Tropical Agricultural Resources, Sanya, 572024, Hainan, China Corresponding email: lmm314.editor@gmail.com International Journal of Molecular Medical Science, 2024, Vol.14, No.2 doi: 10.5376/ijmms.2024.14.0013 Received: 27 Mar., 2024 Accepted: 18 Apr., 2024 Published: 29 Apr., 2024 Copyright © 2024 Li, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Li M.M., 2024, Innate defense role of extracellular vesicles: the critical role of phosphatidylserine in combating apoptotic mimicry viruses, International Journal of Molecular Medical Science, 14(2): 100-105 (doi: 10.5376/ijmms.2024.14.0013) The paper titled "Phosphatidylserine-exposing extracellular vesicles in body fluids are an innate defence against apoptotic mimicry viral pathogens" was published in the journal "Nature Microbiology" on March 25, 2024. Authored by Rüdiger Groß, Hanna Reßin, and Janis A. Müller, the research was conducted at the Institute of Molecular Virology at the University Medical Center Ulm, Germany, and the Institute of Virology at Philipps University of Marburg, Germany. The study reveals that extracellular vesicles (EVs) in body fluids combat viral infections by exposing phosphatidylserine (PS), which disrupts viral attachment and entry, thus serving an antiviral function. The results indicate that these EVs can effectively inhibit several viruses, including Dengue, West Nile, Chikungunya, Ebola, and Vesicular stomatitis viruses. However, they show lower inhibitory effects on SARS-CoV-2, HIV-1, Hepatitis C, and Herpes viruses due to these pathogens utilizing different receptor mechanisms for cell infection. 1 Interpretation of Experimental Data Researchers collected critical data through lipidomic analysis, flow cytometry, and viral infection experiments. The experimental data indicate that the exposure of phosphatidylserine is crucial to the antiviral properties of extracellular vesicles (EVs). Additionally, the study shows that liposomes containing synthetically produced phosphatidylserine can also inhibit viral infections. Figure 1 illustrates the role of phosphatidylserine (PS) in Zika virus (ZIKV) and other viral infections. Panel a shows the process where phosphatidylserine decarboxylase (PSD) converts PS in the viral envelope into phosphatidylethanolamine (PE). Panel b indicates that after PSD treatment, the infection rates of ZIKV, HIV-1, HSV-1, and HSV-2 significantly decrease. Panels c and d explore the effects of liposomes with different PS contents on inhibiting ZIKV infection, revealing that the inhibitory effect on infection significantly increases with higher PS content. The data reveal the crucial role of PS in the viral infection process, establishing it as a potential antiviral target. Figure 2 investigates the effects of liposomes containing phosphatidylserine (PS) in inhibiting Zika virus (ZIKV) infection. Panel a shows that adding PS liposomes either prior to or simultaneously with the virus significantly inhibits ZIKV infection. Panel b demonstrates that PS liposomes exhibit a concentration-dependent inhibitory effect across different viral inoculation doses (MOI). Panel c indicates that individual headgroup small molecules do not possess significant antiviral activity. Panels d and e reveal that liposomes with a higher PS content have stronger antiviral effects. Panels f and g, using fluorescence quantification, show that PS liposomes can reduce viral attachment and increase the attachment of the liposomes themselves. Panel h, through fluorescence microscopy images, visually displays the competitive inhibitory effect of the liposomes on the virus. Figure 3 explores the levels and characteristics of phosphatidylserine (PS) in extracellular vesicles (EVs) across different body fluids. Panel a describes the workflow for purifying EVs using Tangential Flow Filtration (TFF) and BE-SEC. Panel b confirms the enrichment of characteristic EV proteins in different samples through Western

International Journal of Molecular Medical Science, 2024, Vol.14, No.2, 100-105 http://medscipublisher.com/index.php/ijmms 101 Blot analysis. Panel c shows the content of major phospholipids in EVs from various body fluids, finding that semen and urine have the highest proportions of PS. Panels d-f indicate that EVs from semen and urine have significantly higher contents of PS and PE compared to those from blood. Panel g reveals that the PS+PE/PC ratios are higher in semen, saliva, and urine. Panel h uses flow cytometry to detect subpopulations of EVs exposing PS. Panel i displays the proportion of PS and TSPN positive vesicles in semen EVs. The results suggest significant fluid-specific differences in the lipid composition and PS exposure of EVs. Figure 4 investigates how extracellular vesicles (EVs) rich in phosphatidylserine (PS) interfere with the viral infection mechanisms. Panel a shows that EVs from semen and other body fluids more effectively inhibit Zika virus (ZIKV) infection compared to control liposomes. Panels b-c, using fluorescence microscopy, confirm that semen EVs effectively reduce viral attachment. Panels d-e reveal that PS-rich EVs block ZIKV infection by binding to the Axl receptor. Panels f-i present "shaving" experiments conducted after treating EVs with phospholipase D, finding that removing PS exposure significantly reduces the antiviral activity of EVs, while replenishing PS restores this capability. This study highlights the critical role of PS in the antiviral mechanisms of EVs. Figure 1 PS exposure in the viral envelope of ZIKV is essential for infection

International Journal of Molecular Medical Science, 2024, Vol.14, No.2, 100-105 http://medscipublisher.com/index.php/ijmms 102 Figure 2 PS-containing vesicles interfere with virion attachment Figure 5 evaluates the inhibitory effects of semen extracellular vesicles (EVs) on Zika virus (ZIKV) infection in reproductive tract cells and tissues. Panel a shows that both semen EVs and seminal plasma (SP) effectively inhibit ZIKV infection in primary human foreskin fibroblasts, while control liposomes do not show significant inhibitory effects. Panel b uses immunofluorescence imaging to display the infection status under different treatment conditions. Panel c further assesses the inhibitory effect of semen EVs and SP on ZIKV in ex vivo vaginal tissue blocks from two donors, finding that semen EVs significantly reduce viral genome levels, confirming their potential application in preventing the spread of ZIKV. Figure 6 evaluates the inhibitory effects of semen extracellular vesicles (EVs) and liposomes containing phosphatidylserine (PS) or phosphatidylcholine (PC) against various viruses. Panel a shows that semen EVs and PS liposomes significantly inhibit viruses that infect via apoptotic mimicry mechanisms (such as ZIKV, DENV, WNV, CHIKV, EBOV, and VSV), while PC liposomes show no significant inhibitory effect. Panel b demonstrates that for viruses that rely on non-lipid receptors (such as SARS-CoV-2, HIV-1, HSV-1, HSV-2, HCMV, and HCV), the inhibitory effects of semen EVs and PS liposomes are weaker. The results indicate that semen EVs and PS liposomes have specific antiviral activity against viruses that employ apoptotic mimicry.

International Journal of Molecular Medical Science, 2024, Vol.14, No.2, 100-105 http://medscipublisher.com/index.php/ijmms 103 Figure 3 EVs from body fluids expose PS at varying levels 2 Insight of Research Findings Extracellular vesicles (EVs) enriched with phosphatidylserine (PS) significantly reduce viral attachment by competing with viruses for cellular receptors. There are variations in the PS content of EVs from different sources. Data charts illustrate the effectiveness of PS-enriched extracellular vesicles and liposomes in preventing viral infections. 3 Evaluation of the Research This study validates the theory of extracellular vesicles (EVs) as an innate antiviral mechanism. Experiments have shown that EVs exposing more phosphatidylserine (PS) are most effective at blocking viruses that utilize apoptotic mimicry mechanisms. Additionally, the research also demonstrates the differences in antiviral activity among various types of vesicles and liposomes. 4 Concluding Remarks The conclusion drawn from the research is that extracellular vesicles (EVs) exposing phosphatidylserine (PS) as a defense mechanism in body fluids may be a potentially effective means of combating specific viruses. Synthetic liposomes that mimic this mechanism have the potential to be used in developing new antiviral therapies.

International Journal of Molecular Medical Science, 2024, Vol.14, No.2, 100-105 http://medscipublisher.com/index.php/ijmms 104 Figure 4 PS-rich EVs from body fluids interfere with viral apoptotic mimicry 5 Access the Full Text Groß, R., Reßin, H., von Maltitz, P. et al. Phosphatidylserine-exposing extracellular vesicles in body fluids are an innate defence against apoptotic mimicry viral pathogens. Nat Microbiol 9, 905–921 (2024). https://doi.org/10.1038/s41564-024-01637-6. Acknowledgement Thanks to the open access policy of "Nature Microbiology," readers can freely share this valuable research. Thanks to the excellent research work of the von Maltitz, P. (corresponding author) research team, providing a very good case study for the research community. If the reviewers' understanding and perspectives on the research data, results, and evaluations differ from the authors' intentions, I sincerely apologize.

International Journal of Molecular Medical Science, 2024, Vol.14, No.2, 100-105 http://medscipublisher.com/index.php/ijmms 105 Figure 5 Semen EVs inhibit ZIKV infection of primary cells and tissues from the anogenital tract Figure 6 EVs specifically inhibit viral pathogens depending on apoptotic mimicry Disclaimer/Publisher's Note The statements, opinions, and data contained in all publications are solely those of the individual authors and contributors and do not represent the views of the publishing house and/or its editors. The publisher and/or its editors disclaim all responsibility for any harm or damage to persons or property that may result from the application of ideas, methods, instructions, or products discussed in the content. Publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

International Journal of Molecular Medical Science, 2024, Vol.14, No.2, 106-122 http://medscipublisher.com/index.php/ijmms 106 Research Perspective Open Access Unraveling the Gut-Brain Axis: The Potential of Engineered Synthetic Microbial Communities in Modulating Neurotransmitter Production and Mental Health Jingqiang Wang Institute of Life Science, Jiyang College of Zhejiang A&F University, Zhuji, 311800, Zhejiang, China Corresponding email: jingqiang.wang@gmail.com International Journal of Molecular Medical Science, 2024, Vol.14, No.2 doi: 10.5376/ijmms.2024.14.0014 Received: 18 Mar., 2024 Accepted: 22 Apr., 2024 Published: 05 May, 2024 Copyright © 2024 Wang, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Wang J.Q., 2024, Unraveling the gut-brain axis: the potential of engineered synthetic microbial communities in modulating neurotransmitter production and mental health, International Journal of Molecular Medical Science, 14(2): 106-122 (doi: 10.5376/ijmms.2024.14.0014) Abstract This study delves into the intricate communication network between the gastrointestinal tract and the central nervous system, known as the gut-brain axis, and its significant impact on mental health. The study focuses on the potential of engineered synthetic microbial consortia (SynComs) in regulating neurotransmitter production and promoting mental well-being. By examining recent advancements in synthetic biology and multi-omics technologies, it highlights the substantial improvements in SynComs' ability to precisely target neurochemical pathways. The integration potential of personalized SynComs in the treatment of mental health disorders is emphasized, offering a promising alternative to traditional therapies. Additionally, the study discusses the challenges related to technology, safety, ethics, and regulation, providing a comprehensive overview of the current state and future prospects of SynComs in clinical applications. The importance of interdisciplinary collaboration in advancing SynCom research is underscored, calling for continued efforts to fully realize its therapeutic potential. This study demonstrates the transformative potential of SynComs in the field of mental health, providing a theoretical foundation for innovative and personalized therapeutic strategies. Keywords Gut-brain axis; Neurotransmitter modulation; Synthetic microbial communities; Mental health; Synthetic biology 1 Introduction The gut-brain axis, a complex bidirectional communication network between the gastrointestinal tract and the central nervous system, has garnered significant interest for its role in regulating mental health. Emerging research has highlighted how gut microbiota can influence brain function through various pathways, including immune modulation, hormone secretion, and direct microbial metabolite production. Understanding this intricate connection is crucial for developing novel therapeutic strategies for mental health disorders (Huang and Wu, 2021). The gut-brain axis (GBA) involves multiple pathways, including neural, hormonal, and immune mechanisms, which facilitate the interaction between gut microbiota and brain function (Petra et al., 2015; Cryan et al., 2019; Socała et al., 2021). The gut microbiota, comprising trillions of microorganisms, plays a crucial role in maintaining homeostasis and influencing various physiological processes, including neural development, neurotransmission, and behavior (Martin et al., 2018; Cryan et al., 2019; Socała et al., 2021). Emerging evidence suggests that alterations in the gut microbiota composition can significantly impact mental health, contributing to the pathogenesis and progression of neuropsychiatric and neurological disorders such as depression, anxiety, schizophrenia, autism spectrum disorders, and Parkinson's disease (Huang and Wu, 2021; Margolis et al., 2021; Socała et al., 2021). Synthetic microbial communities (SynComs) are engineered consortia of microorganisms designed to perform specific functions within a host environment. These communities offer a promising approach to modulate the gut microbiota and, consequently, the gut-brain axis. By precisely controlling the composition and metabolic activities of SynComs, researchers aim to influence neurotransmitter production and other biochemical pathways that affect brain function and mental health (Liu et al., 2015). SynComs can be tailored to produce specific metabolites, such as short-chain fatty acids, neurotransmitters, and other signaling molecules, which can interact with the central nervous system through various routes, including the vagus nerve, immune system, and endocrine pathways (Petra

International Journal of Molecular Medical Science, 2024, Vol.14, No.2, 106-122 http://medscipublisher.com/index.php/ijmms 107 et al., 2015; Wiley et al., 2017; Martin et al., 2018). This targeted modulation holds potential for developing novel therapeutic strategies for mental health disorders. This study article aims to deeply explore the potential of engineered synthetic microbial communities (SynComs) in regulating neurotransmitter production and improving mental health. By comprehensively analyzing existing research on the gut-brain axis and the impact of the gut microbiota on neuropsychiatric disorders, this article reveals how SynCom interventions can influence brain function and behavior. Key findings from preclinical and clinical studies are highlighted, mechanisms of gut-brain communication are discussed, and gaps in the current knowledge base are identified to guide future research. This study emphasizes the importance of SynComs as an innovative and promising therapeutic approach for the prevention and treatment of mental health issues, providing a theoretical foundation for future research and clinical practice. 2 The Gut-Brain Axis: A Complex Communication Network 2.1 Definition and components of the Gut-Brain axis The gut-brain axis (GBA) is a bidirectional communication network that links the gastrointestinal (GI) tract and the central nervous system (CNS). This axis encompasses various components, including the enteric nervous system (ENS), the autonomic nervous system (ANS), the hypothalamic-pituitary-adrenal (HPA) axis, and the gut microbiota (Martin et al., 2018; Cryan et al., 2019; Hattori and Yamashiro, 2021). The ENS, often referred to as the "second brain", resides within the intestinal wall and communicates with the brain via the vagus nerve and other neural pathways (Hattori and Yamashiro, 2021). The ANS, comprising sympathetic and parasympathetic branches, modulates gut motility, secretion, and permeability, thereby influencing the gut microbiota (Martin et al., 2018). The HPA axis plays a crucial role in stress responses, linking the gut and brain through hormonal signaling (Hattori and Yamashiro, 2021). Collectively, these components form a complex network that maintains homeostasis and influences both gut and brain functions. 2.2 Mechanisms of communication between the gut and the brain The communication between the gut and the brain occurs through multiple mechanisms, including neural, endocrine, and immune pathways (Figure 1). The vagus nerve is a primary neural conduit for gut-brain communication. It transmits signals from the gut to the brain and vice versa, playing a crucial role in regulating gut motility, secretion, and immune responses. The ENS also communicates directly with the CNS through intrinsic primary afferent neurons that detect changes in the gut environment (Bistoletti et al., 2020). The research of Ma et al. (2019) shows the interaction between the gut microbiota and the gut-brain axis through various mechanisms, affecting the health of the central nervous system. Short-chain fatty acids regulate immune cells, promoting anti-inflammatory responses; microglial cell maturation is impaired under germ-free conditions, affecting neuroprotection; specific probiotics promote hippocampal neurogenesis; blood-brain barrier permeability increases under germ-free conditions but can be restored to normal function by microbial colonization or short-chain fatty acids; the vagus nerve directly and indirectly influences brain function by transmitting neural signals and metabolites. These mechanisms highlight the crucial role of gut microbiota in maintaining and regulating central nervous system health. The HPA axis mediates the endocrine communication between the gut and the brain. In response to stress, the hypothalamus releases corticotropin-releasing hormone (CRH), which stimulates the pituitary gland to secrete adrenocorticotropic hormone (ACTH). ACTH then prompts the adrenal glands to release cortisol, a hormone that influences gut permeability and immune function. Additionally, gut hormones such as ghrelin, peptide YY, and glucagon-like peptide-1 (GLP-1) can signal the brain to regulate appetite and metabolism (Makris et al., 2021). The immune system is a key player in the gut-brain axis. Gut-associated lymphoid tissue (GALT) monitors and responds to pathogens and other antigens in the gut. Immune cells release cytokines that can influence brain function and behavior. Chronic inflammation in the gut can lead to increased permeability of the blood-brain barrier, allowing immune signals to affect the brain directly. This interaction is bidirectional, as brain inflammation can also impact gut health and microbiota composition (Fung, 2020).

International Journal of Molecular Medical Science, 2024, Vol.14, No.2, 106-122 http://medscipublisher.com/index.php/ijmms 108 Figure 1 The complex interactions between the gut microbiota and the gut-brain axis (Adapted from Ma et al., 2019) Image caption: A shows that short-chain fatty acids (SCFAs) promote the generation of regulatory T cells (Tregs) by modulating the Foxp3 promoter, while inhibiting the formation of pro-inflammatory T cells; B explains that under germ-free conditions or with antibiotic use, the maturation of microglia is inhibited, affecting the anti-inflammatory effects in the nervous system; C indicates that specific probiotics can promote neurogenesis in the hippocampus; D demonstrates that under germ-free conditions, the permeability of the blood-brain barrier (BBB) increases, and the colonization of microbiota or the use of SCFAs can restore its normal permeability; E emphasizes that the vagus nerve interacts with the gut microbiota directly and indirectly, influencing brain function (Adapted from Ma et al., 2019) Additionally, microbial metabolites such as short-chain fatty acids (SCFAs), tryptophan metabolites, and peptidoglycans can modulate these communication pathways. These mechanisms collectively ensure a dynamic and responsive interaction between the gut and the brain. 2.3 Role of the gut microbiota in modulating these communication pathways The gut microbiota, comprising trillions of microorganisms, plays a pivotal role in modulating the communication pathways of the GBA. The microbiota influences neural communication by interacting with the ENS and modulating vagal signaling (Cryan et al., 2019; Kuwahara et al., 2020). It also affects endocrine pathways by influencing the release of hormones such as serotonin, which is predominantly produced in the gut (Gao et al., 2019; Margolis et al., 2021). The immune system is another critical interface, with the gut microbiota modulating immune responses and maintaining the integrity of the intestinal barrier (Wang and Wang, 2016; Hattori and Yamashiro, 2021). Dysbiosis, or an imbalance in the gut microbiota, has been linked to various neurological and psychiatric disorders, including anxiety, depression, and autism (Wiley et al., 2017; Cryan et al., 2019). Probiotics, prebiotics, and other microbial-based interventions are being explored as potential therapeutic strategies to restore balance in the GBA and improve mental health outcomes (Wiley et al., 2017; Suganya and Koo, 2020). The intricate interplay between the gut microbiota and the GBA underscores the importance of maintaining a healthy and diverse microbial community for optimal brain function and mental health. 3 Neurotransmitter Production in the Gut 3.1 Overview of neurotransmitters produced in the gut The gut is a significant site for the production of various neurotransmitters, including serotonin, dopamine, and gamma-aminobutyric acid (GABA). These neurotransmitters play critical roles in maintaining gastrointestinal (GI) homeostasis and facilitating communication along the gut-brain axis. Serotonin, for instance, is predominantly produced in the gut, with approximately 90% of the body's total serotonin synthesized by enterochromaffin cells in the gastrointestinal tract (Strandwitz, 2018; Liu and Huang, 2019). Dopamine, another critical neurotransmitter, is also produced in the gut, albeit in smaller quantities compared to the brain (Strandwitz, 2018; Bhatia et al., 2023). GABA, which plays a crucial role in inhibiting neural activity, is synthesized by certain gut bacteria, contributing to the overall pool of this neurotransmitter in the body (Strandwitz, 2018; Bhatia et al., 2023).

International Journal of Molecular Medical Science, 2024, Vol.14, No.2, 106-122 http://medscipublisher.com/index.php/ijmms 109 3.2 Microbial involvement in neurotransmitter synthesis and regulation The gut microbiota plays a pivotal role in the synthesis and regulation of neurotransmitters. Various gut bacteria are capable of producing neurotransmitters directly. For example, certain strains of Lactobacillus and Bifidobacterium can produce GABA, while other bacteria can synthesize serotonin and dopamine (Strandwitz, 2018; Liu and Huang, 2019; Bhatia et al., 2023). Additionally, the gut microbiota can influence the availability of precursors for neurotransmitter synthesis. The metabolism of aromatic amino acids (AAAs) by gut bacteria can affect the levels of tryptophan and tyrosine, which are precursors for serotonin and dopamine, respectively (Gao et al., 2018; Gao et al., 2019). This microbial modulation of neurotransmitter precursors can subsequently impact neurotransmitter levels in the brain (Gao et al., 2018; Gao et al., 2019). 3.3 Impact of Gut-Derived neurotransmitters on brain function and mental health Gut-derived neurotransmitters have a profound impact on brain function and mental health. The gut-brain axis facilitates bidirectional communication between the gut and the brain, allowing gut-derived neurotransmitters to influence central nervous system activities. For instance, alterations in gut microbiota composition have been linked to changes in brain neurotransmitter levels, which can affect mood and behavior (Chen et al., 2021; Huang and Wu, 2021; Socała et al., 2021). Studies have shown that Alterations in gut serotonin levels have been linked to conditions like irritable bowel syndrome (IBS) and depression. Serotonergic pathways in the gut can influence central serotonergic activity, impacting mood and behavior (Kumar et al., 2020). Increased GABA production in the gut, particularly under conditions like hepatic encephalopathy, can affect brain inhibition and lead to altered neural activity (Altaib et al., 2021). Moreover, interventions targeting the gut microbiota, such as the use of probiotics, prebiotics, or antibiotics, have been shown to alter neurotransmitter levels and improve symptoms of mental health disorders (Dinan and Cryan, 2017; Strandwitz, 2018; Huang and Wu, 2021). 4 Engineering Synthetic Microbial Communities (SynComs) for Neurotransmitter Modulation 4.1 Definition and principles of SynComs Synthetic microbial communities (SynComs) are engineered consortia of microorganisms designed to perform specific functions that natural microbial communities may not efficiently achieve. These communities are constructed using principles of synthetic biology, which involves the design and assembly of genetic components to create new biological systems or reprogram existing ones. SynComs can be tailored to produce desired metabolites, including neurotransmitters, by incorporating specific microbial strains with known metabolic capabilities (Petra et al., 2015; Dinan and Cryan, 2017; Huang and Wu, 2021). 4.2 Techniques for engineering SynComs to enhance neurotransmitter production Several techniques are employed to engineer SynComs for enhanced neurotransmitter production such as serotonin, dopamine, and gamma-aminobutyric acid (GABA): 1) Genetic Engineering: This involves the insertion, deletion, or modification of genes within microbial genomes to enhance their ability to produce specific neurotransmitters. For example, genes responsible for the production of serotonin, dopamine, and gamma-aminobutyric acid (GABA) can be inserted into microbial genomes to boost their production (Strandwitz, 2018; Baj et al., 2019; Huang and Wu, 2021). 2) Synthetic Biology: This broad field includes techniques such as the design of synthetic gene circuits and metabolic pathways that can control and optimize microbial behavior. Synthetic biology approaches enable the creation of complex genetic networks that regulate neurotransmitter production in response to environmental signals or internal cellular states (Kang et al., 2020). 3) Metabolic Engineering: This involves the optimization of metabolic pathways within microorganisms to increase the yield of neurotransmitters. By redirecting metabolic fluxes and eliminating competing pathways, the production of target neurotransmitters can be maximized (Dinan and Cryan, 2017; Margolis et al., 2021; Socała et al., 2021).

International Journal of Molecular Medical Science, 2024, Vol.14, No.2, 106-122 http://medscipublisher.com/index.php/ijmms 110 4) Quorum Sensing: By engineering quorum sensing systems, SynComs can coordinate their behavior and maintain stable population dynamics. This technique allows microbial communities to modulate neurotransmitter production collectively, based on cell density and environmental conditions (Scott and Hasty, 2016). 5) Mathematical Modeling and Computational Tools: Predictive models and computational tools are used to design and optimize SynComs. These models help in understanding the complex interactions within microbial consortia and guide the engineering of stable and functional communities (Zomorrodi and Segrè, 2016). 4.3 Advantages of using engineered SynComs over natural microbial communities Engineered SynComs offer several advantages over natural microbial communities: 1) Predictability and Control: SynComs are designed to have predictable behaviors and functions, reducing the variability often seen in natural communities. This predictability is crucial for therapeutic applications where consistent outcomes are necessary (Karkaria et al., 2021). 2) Targeted Functionality: SynComs can be engineered to perform specific functions, such as the production of particular neurotransmitters, which might not be achievable with natural microbial communities. This targeted functionality allows for precise interventions in the gut-brain axis (Kang et al., 2020). 3) Stability: Engineered communities can be designed to be more stable and resilient to environmental changes compared to natural communities. This stability ensures that the desired functions are maintained over time and under varying conditions (Scott and Hasty, 2016). 4) Scalability and Reproducibility: SynComs can be scaled up and reproduced consistently, which is advantageous for clinical and industrial applications. This scalability ensures that large populations can be managed and utilized effectively for therapeutic purposes (Zomorrodi and Segrè, 2016). 5) Safety: Engineered SynComs can be designed to minimize the risk of pathogenicity and unwanted side effects. By using non-pathogenic strains and incorporating safety switches, the potential for adverse effects can be reduced (Dinan and Cryan, 2017; Strandwitz, 2018; Huang and Wu, 2021). 5 Applications of SynComs in Mental Health 5.1 SynComs for anxiety and depression management Synthetic microbial communities (SynComs) have shown potential in managing anxiety and depression by modulating the gut-brain axis. The gut microbiome plays a crucial role in the production of neurotransmitters such as serotonin and gamma-aminobutyric acid (GABA), which are essential for mood regulation. Studies have demonstrated that specific microbial strains can influence these neurotransmitter levels, thereby alleviating symptoms of anxiety and depression. For instance, a study investigating the use of Microbial Ecosystem Therapeutic-2 (MET-2), which comprises 40 strains of bacteria, showed promising results in treating major depressive disorder and generalized anxiety disorder. Participants who received MET-2 capsules daily for eight weeks exhibited significant improvements in depression and anxiety symptoms, as measured by the Montgomery-Asberg Depression Rating Scale (MADRS) and the Generalized Anxiety Disorder 7-item scale (GAD-7). Furthermore, studies have linked specific gut microbial compositions to mental health profiles. For example, reduced diversity of gut microbiota, particularly a decrease in Fusicatenibacter saccharivorans, has been associated with increased anxiety and depression symptoms. This finding suggests that enhancing the abundance of beneficial microbes could alleviate these symptoms. 5.2 Role of SynComs in neurodevelopmental and neurodegenerative disorders The application of SynComs in neurodevelopmental and neurodegenerative disorders is an emerging area of research. Neurodevelopmental disorders such as autism and attention deficit hyperactivity disorder (ADHD) are often associated with cognitive and emotional regulation impairments. SynComs could potentially improve these

International Journal of Molecular Medical Science, 2024, Vol.14, No.2, 106-122 http://medscipublisher.com/index.php/ijmms 111 conditions by enhancing cognitive control processes such as working memory, inhibition, and shifting, which are linked to better emotion regulation and reduced internalizing/externalizing symptoms (Tajik-Parvinchi et al., 2021). In neurodegenerative disorders like schizophrenia, targeting neural synchrony deficits has been shown to improve cognitive function. For example, normalizing aberrant neural synchrony in a schizophrenia-related model improved cognitive control and reduced hyperlocomotion, indicating that SynComs could be engineered to achieve similar outcomes (Lee et al., 2014). Altered gut microbiota has been observed in patients with generalized anxiety disorder (GAD) and major depressive disorder (MDD), with significant differences in microbial richness and diversity compared to healthy controls. This dysbiosis can affect neurodevelopment and contribute to neurodegenerative processes (Jiang et al., 2018).By introducing SynComs designed to restore healthy microbial balance, it may be possible to mitigate the progression of these disorders. For instance, the use of probiotics such as Lactobacillus plantarum has been shown to relieve symptoms of anxiety and depression, potentially through the modulation of gut microbiota and neuroactive metabolites (Zhu et al., 2023). 5.3 Potential for SynComs to enhance cognitive function and mood SynComs hold significant potential for enhancing cognitive function and mood by influencing the gut-brain axis. The gut microbiome's role in cognitive processes is well-documented, with specific microbial strains being linked to improved cognitive performance and mood regulation. For example, probiotic supplementation has been linked to increased levels of beneficial gut bacteria such as Bifidobacterium and Fecalibacterium, which are associated with improved mood and cognitive performance. Moreover, the application of SynComs could complement existing cognitive-behavioral therapies by providing a biological basis for mood enhancement and cognitive improvement. The studies have shown that specific gut bacteria can influence the synthesis and metabolism of neurotransmitters like gamma-aminobutyric acid (GABA), serotonin, and dopamine. By leveraging SynComs to enhance the presence of these beneficial microbes, it may be possible to develop targeted therapies for cognitive enhancement and mood regulation (Chung et al., 2019).This is particularly relevant in the context of telepsychology interventions, which have been effective in treating anxiety and depression through various delivery methods. By integrating SynComs with these interventions, it may be possible to achieve more robust and sustained improvements in mental health. The potential applications of SynComs in mental health are vast, ranging from managing anxiety and depression to addressing neurodevelopmental and neurodegenerative disorders and enhancing cognitive function and mood. The integration of SynComs with existing therapeutic approaches could pave the way for more effective and personalized treatments for mental health conditions. 6 Mechanistic Insights and Biological Pathways 6.1 Molecular mechanisms through which SynComs modulate neurotransmitter levels The gut-brain axis (GBA) is a complex communication network that involves various molecular mechanisms through which synthetic microbial communities (SynComs) can modulate neurotransmitter levels. One of the primary pathways involves the metabolism of tryptophan, an essential amino acid that serves as a precursor to serotonin, a key neurotransmitter. Gut microbiota can influence tryptophan metabolism, thereby affecting serotonin synthesis and other neuroactive metabolites such as kynurenine and indole (O'Mahony et al., 2015; Gao et al., 2019; Gheorghe et al., 2019). Additionally, gut bacteria are capable of producing and consuming a range of neurotransmitters, including dopamine, norepinephrine, and gamma-aminobutyric acid (GABA), which can directly impact host physiology (Strandwitz, 2018). Another significant mechanism is the modulation of glutamatergic signaling. Glutamate, a crucial neurotransmitter, is involved in various brain functions, including stress response, mood, and behavior. Alterations in glutamatergic transmission due to microbial activity can contribute to the pathogenesis of both gut and brain disorders (Margolis et al., 2021). Furthermore, the gut microbiota can produce short-chain fatty acids

International Journal of Molecular Medical Science, 2024, Vol.14, No.2, 106-122 http://medscipublisher.com/index.php/ijmms 112 (SCFAs) like butyrate, which have been shown to influence neurotransmitter synthesis and release (Wijdeveld et al., 2020). 6.2 Interaction between SynComs and host cells in the gut The interaction between SynComs and host cells in the gut is multifaceted, involving direct and indirect pathways. Direct interactions include the production of neuroactive compounds by gut bacteria, which can interact with host receptors and influence neurotransmitter levels. For instance, certain gut bacteria can produce serotonin and other neurotransmitters that interact with the enteric nervous system and subsequently affect the central nervous system (Strandwitz, 2018; Margolis et al., 2021). Indirect interactions involve the modulation of the host's immune system. Gut microbiota can influence the production of cytokines and other immune mediators, which can affect the hypothalamic-pituitary-adrenal (HPA) axis and subsequently alter neurotransmitter levels (Petra et al., 2015). Additionally, gut bacteria can affect the integrity of the intestinal barrier, leading to changes in the permeability of the gut and the subsequent translocation of microbial metabolites that can influence brain function (Guo et al., 2021). 6.3 Influence of diet, environment, and lifestyle on SynCom efficacy The efficacy of SynComs in modulating neurotransmitter production and mental health is significantly influenced by diet, environment, and lifestyle (Figure 2). A diet high in sugar and fat has been shown to disrupt gut microbiota composition, leading to changes in neurotransmitter metabolism and brain function (Guo et al., 2021). Conversely, diets rich in fiber and prebiotics can promote the growth of beneficial gut bacteria that produce SCFAs and other neuroactive compounds, thereby enhancing SynCom efficacy (Wijdeveld et al., 2020). Figure 2 Factors affecting the gut microbiota profile (Adopted from Long-Smith et al., 2020)

International Journal of Molecular Medical Science, 2024, Vol.14, No.2, 106-122 http://medscipublisher.com/index.php/ijmms 113 Figure 2 demonstrates the various factors influencing the composition and diversity of the gut microbiota. These factors include geographic location, host genetics, exercise, stress, antibiotic use, age, diet, and mode of delivery. Geographic location and host genetics establish the foundational characteristics of an individual's gut microbiota; exercise helps enhance microbial diversity; stress may disrupt microbial balance. Antibiotic use significantly reduces microbiota diversity, impacting gut health. The gut microbiota varies considerably across different ages, with distinct microbial structures present at each life stage from infancy to old age. Diet directly affects the composition of gut microbiota, while the mode of delivery (such as vaginal birth or cesarean section) plays a crucial role in the early establishment of the microbiota. These factors collectively determine the health and function of an individual's gut microbiota. Foster et al. (2017) highlighted that diet is a critical factor influencing the gut-brain axis. Microorganisms communicate with the brain through pathways such as the vagus nerve, gut hormone signaling, the immune system, tryptophan metabolism, and short-chain fatty acids. Alterations in the early gut microbiome can have profound impacts on later health, including stress-related physiological and behavioral changes. The study also explored the role of the microbiome in stress-related diseases such as anxiety, depression, and irritable bowel syndrome, proposing that psychobiotics might serve as an intervention to improve mental health. The Table 1 demonstrates the effects of targeting the gut microbiota on depression and anxiety in both clinical and preclinical studies. Clinical evidence indicates that the prebiotic B-GOS and various probiotics such as Lactobacillus and Bifidobacterium have significant effects on improving cognitive processing, mood, and reducing psychological stress, while also decreasing physiological stress responses like cortisol levels. Preclinical studies further support these findings, showing improvements in depression-like and anxiety-like behaviors in animal models treated with prebiotics and probiotics, along with corresponding physiological changes such as reduced corticosterone and pro-inflammatory cytokine levels. These results suggest that the potential role of the gut microbiota in mental health warrants further exploration. Environmental factors such as stress and exposure to antibiotics can also impact gut microbiota composition and function, thereby influencing the effectiveness of SynComs. For example, stress can alter the gut microbiota and increase gut permeability, leading to changes in neurotransmitter levels and brain function (Petra et al., 2015). Similarly, antibiotic use can disrupt gut microbiota composition, affecting the production of neurotransmitters and other neuroactive compounds (Gao et al., 2019). Lifestyle factors, including physical activity and sleep patterns, also play a crucial role in modulating gut microbiota and SynCom efficacy. Regular physical activity has been shown to promote a healthy gut microbiota composition, which can enhance the production of beneficial neurotransmitters (Wijdeveld et al., 2020). Adequate sleep is essential for maintaining gut microbiota balance and optimal neurotransmitter production, thereby supporting mental health (Gao et al., 2019). 7 Clinical Studies and Performance Evaluation 7.1 Overview of preclinical and clinical trials involving SynComs for mental health The exploration of synthetic microbial communities (SynComs) in modulating mental health through the gut-brain axis has gained significant traction in recent years. Preclinical studies have demonstrated the potential of SynComs in altering neurotransmitter levels and influencing behavior. For instance, germ-free rodent models and those subjected to antibiotic treatments have shown that the absence or alteration of gut microbiota can significantly impact anxiety and depression-like behaviors, suggesting a critical role of gut microbes in mental health (Huang and Wu, 2021; Socała et al., 2021). Clinical trials, although still in their nascent stages, have begun to explore the efficacy of SynComs in human subjects. These studies aim to translate the promising results observed in animal models to human applications, focusing on conditions such as depression, anxiety, and irritable bowel syndrome (IBS) (Foster et al., 2017; Martin et al., 2018; Iannone et al., 2019).

International Journal of Molecular Medical Science, 2024, Vol.14, No.2, 106-122 http://medscipublisher.com/index.php/ijmms 114 Table 1 Clinical and preclinical evidence for the antidepressant and anxiolytic properties associated with targeting the gut microbiota (Adopted from Foster et al., 2017) Behavioural outcomes Physiological outcomes Clinical evidence B-GOS Increased cognitive processing of positive versus negative attentional vigilance Reduced cortisol awakening response Lactobacillus casei strain Shirota Reduced anxiety scores in patients with chronic fatigue syndrome Increased numbers of Lactobacillus and Bifidobacterium in faecal samples Improved mood in individuals with a low mood prior to taking the probiotic NA Probiotic formulation: Lactobacillus helveticus and Bifidobacterium longum Reduced psychological distress as measured by the HADS Reduced 24-h UFC levels Multispecies probiotic formulation: Lactobacillus and Bifidobacterium species Reduced cognitive processing of sad mood; decreased aggressive feelings and rumination NA Preclinical evidence Prebiotic- FOS and GOS Antidepressant and anxiolytic-like effects in adult mice. Reversed the behavioural effects of chronic psychosocial stress in mice. Increased BDNF, NR1 and NR2A mRNA, and protein expression in the dentate gyrus and frontal cortex. Reduced acute and chronic stress-induced corticosterone release. Modified specific gene expression in the hippocampus and hypothalamus. Reduced chronic stress-induced elevations in pro-inflammatory cytokines levels. Prebiotic- 3′Sialyllactose and 6'sialyllactose Anxiolytic effect in mice exposed to SDR Prevented SDR-mediated reduction in the number of immature neurons Prebiotic- GOS & polydextrose with lactoferrin (Lf) and milk fat globule membrane Reduced immobility time of maternally separated rats in a forced swimtest Improves NREM Sleep, Enhance REM Sleep Rebound and Attenuate the Stress-Induced Decrease in Diurnal Temperature. Attenuated exaggerated IL-6 response in maternally separated rats following concanavalin A stimulation. Bifidobacterium breve Improved depressive and anxiety-related behaviours in mice No effect upon circulating corticosterone Bifidobacterium longum Anxiolytic effect in step-down inhibitory avoidance Anxiolytic effect mediated via the vagus nerve Lactobacillus plantarum PS128 Reduced immobility time and increased sucrose preference in ELS mice Decreased basal and stress-induced circulating corticosterone levels; attenuated circulating TNF-α and IL-6 levels while increasing IL-10 levels in ELS mice Lactobacillus rhamnosus Reduced immobility time in the forced swim test. Decreased stress-induced anxiety-like behaviour. Decreased stress-induced circulating corticosterone secretion and altered central GABA receptor subunit expression. Attenuated chronic stress-related activation of dendritic cells while increasing IL-10 + regulatory T cells. Lactobacillus fermentum NS9 Reduced ampicillin-induced anxiety behaviour Decreased ampicillin-induced corticosterone secretion and increased hippocampal mineralocorticoid receptor and NMDA receptor levels. Butyric acid Reduced immobility time in Flinders sensitive line rats exposed to a forced swimtest Increased BDNF expression within the prefrontal cortex Behavioural outcomes Physiological outcomes Note: BDNF (brain-derived neurotrophic factor), ELS (early life stress–exposed), FOS (fructo-oligosaccharide), GABA (γ-aminobutyric acid), GOS (galacto-oligosaccharide), HADS (Hospital Anxiety and Depression Scale), IL (interleukin), mRNA (messenger RNA), NA (not assessed), NMDA (N-methyl-d-aspartate), SDR (social disruption stress), TNF (tumour necrosis factor), UFC (urinary free cortisol), NR (NMDA Receptor) (Adopted from Foster et al., 2017)

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