Human Gut Microbiota for Diagnosis and Treatment of Depression.

Nowadays, depressive disorder is spreading rapidly all over the world. Therefore, attention to the studies of the pathogenesis of the disease in order to find novel ways of early diagnosis and treatment is increasing among the scientific and medical communities. Special attention is drawn to a biomarker and therapeutic strategy through the microbiota-gut-brain axis. It is known that the symbiotic interactions between the gut microbes and the host can affect mental health. The review analyzes the mechanisms and ways of action of the gut microbiota on the pathophysiology of depression. The possibility of using knowledge about the taxonomic composition and metabolic profile of the microbiota of patients with depression to select gene compositions (metagenomic signature) as biomarkers of the disease is evaluated. The use of in silico technologies (machine learning) for the diagnosis of depression based on the biomarkers of the gut microbiota is given. Alternative approaches to the treatment of depression are being considered by balancing the microbial composition through dietary modifications and the use of additives, namely probiotics, postbiotics (including vesicles) and prebiotics as psychobiotics, and fecal transplantation. The bacterium Faecalibacterium prausnitzii is under consideration as a promising new-generation probiotic and auxiliary diagnostic biomarker of depression. The analysis conducted in this review may be useful for clinical practice and pharmacology.

Sweet-Tasting Natural Proteins Brazzein and Monellin: Safe Sugar Substitutes for the Food Industry.

This article presents the results of a comprehensive toxicity assessment of brazzein and monellin, yeast-produced recombinant sweet-tasting proteins. Excessive sugar consumption is one of the leading dietary and nutritional problems in the world, resulting in health complications such as obesity, high blood pressure, and cardiovascular disease. Although artificial small-molecule sweeteners widely replace sugar in food, their safety and long-term health effects remain debatable. Many sweet-tasting proteins, including thaumatin, miraculin, pentadin, curculin, mabinlin, brazzein, and monellin have been found in tropical plants. These proteins, such as brazzein and monellin, are thousands-fold sweeter than sucrose. Multiple reports have presented preparations of recombinant sweet-tasting proteins. A thorough and comprehensive assessment of their toxicity and safety is necessary to introduce and apply sweet-tasting proteins in the food industry. We experimentally assessed acute, subchronic, and chronic toxicity effects, as well as allergenic and mutagenic properties of recombinant brazzein and monellin. Our study was performed on three mammalian species (mice, rats, and guinea pigs). Assessment of animals' physiological, biochemical, hematological, morphological, and behavioral indices allows us to assert that monellin and brazzein are safe and nontoxic for the mammalian organism, which opens vast opportunities for their application in the food industry as sugar alternatives.

Unveiling the Connection between Microbiota and Depressive Disorder through Machine Learning.

In the last few years, investigation of the gut-brain axis and the connection between the gut microbiota and the human nervous system and mental health has become one of the most popular topics. Correlations between the taxonomic and functional changes in gut microbiota and major depressive disorder have been shown in several studies. Machine learning provides a promising approach to analyze large-scale metagenomic data and identify biomarkers associated with depression. In this work, machine learning algorithms, such as random forest, elastic net, and You Only Look Once (YOLO), were utilized to detect significant features in microbiome samples and classify individuals based on their disorder status. The analysis was conducted on metagenomic data obtained during the study of gut microbiota of healthy people and patients with major depressive disorder. The YOLO method showed the greatest effectiveness in the analysis of the metagenomic samples and confirmed the experimental results on the critical importance of a reduction in the amount of Faecalibacterium prausnitzii for the manifestation of depression. These findings could contribute to a better understanding of the role of the gut microbiota in major depressive disorder and potentially lead the way for novel diagnostic and therapeutic strategies.

Oxidative Stress Response of Probiotic Strain Bifidobacterium longum subsp. longum GT15.

Bifidobacterium is a predominant and important genus in the bacterial population of the human gut microbiota. Despite the increasing number of studies on the beneficial functionality of bifidobacteria for human health, knowledge about their antioxidant potential is still insufficient. Several in vivo and in vitro studies of Bifidobacterium strains and their cellular components have shown good antioxidant capacity that provided a certain protection of their own and the host's cells. Our work presents the data of transcriptomic, proteomic, and metabolomic analyses of the growing and stationary culture of the probiotic strain B. longum subsp. longum GT15 after exposure to hydrogen peroxide for 2 h and oxygen for 2 and 4 h. The results of the analysis of the sequenced genome of B. longum GT15 showed the presence of 16 gene-encoding proteins with known antioxidant functions. The results of the full transcriptomic analysis demonstrated a more than two-fold increase of levels of transcripts for eleven genes, encoding proteins with antioxidant functions. Proteomic data analysis showed an increased level of more than two times for glutaredoxin and thioredoxin after the exposure to oxygen, which indicates that the thioredoxin-dependent antioxidant system may be the major redox homeostasis system in B. longum bacteria. We also found that the levels of proteins presumably involved in global stress, amino acid metabolism, nucleotide and carbohydrate metabolism, and transport had significantly increased in response to oxidative stress. The metabolic fingerprint analysis also showed good discrimination between cells responding to oxidative stress and the untreated controls. Our results provide a greater understanding of the mechanism of oxidative stress response in B. longum and the factors that contribute to its survival in functional food products.

Multi-level analysis of the gut-brain axis shows autism spectrum disorder-associated molecular and microbial profiles.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by heterogeneous cognitive, behavioral and communication impairments. Disruption of the gut-brain axis (GBA) has been implicated in ASD although with limited reproducibility across studies. In this study, we developed a Bayesian differential ranking algorithm to identify ASD-associated molecular and taxa profiles across 10 cross-sectional microbiome datasets and 15 other datasets, including dietary patterns, metabolomics, cytokine profiles and human brain gene expression profiles. We found a functional architecture along the GBA that correlates with heterogeneity of ASD phenotypes, and it is characterized by ASD-associated amino acid, carbohydrate and lipid profiles predominantly encoded by microbial species in the genera Prevotella, Bifidobacterium, Desulfovibrio and Bacteroides and correlates with brain gene expression changes, restrictive dietary patterns and pro-inflammatory cytokine profiles. The functional architecture revealed in age-matched and sex-matched cohorts is not present in sibling-matched cohorts. We also show a strong association between temporal changes in microbiome composition and ASD phenotypes. In summary, we propose a framework to leverage multi-omic datasets from well-defined cohorts and investigate how the GBA influences ASD.

Macrophages from naked mole-rat possess distinct immunometabolic signatures upon polarization.

The naked mole-rat (NMR) is a unique long-lived rodent which is highly resistant to age-associated disorders and cancer. The immune system of NMR possesses a distinct cellular composition with the prevalence of myeloid cells. Thus, the detailed phenotypical and functional assessment of NMR myeloid cell compartment may uncover novel mechanisms of immunoregulation and healthy aging. In this study gene expression signatures, reactive nitrogen species and cytokine production, as well as metabolic activity of classically (M1) and alternatively (M2) activated NMR bone marrow-derived macrophages (BMDM) were examined. Polarization of NMR macrophages under pro-inflammatory conditions led to expected M1 phenotype characterized by increased pro-inflammatory gene expression, cytokine production and aerobic glycolysis, but paralleled by reduced production of nitric oxide (NO). Under systemic LPS-induced inflammatory conditions NO production also was not detected in NMR blood monocytes. Altogether, our results indicate that NMR macrophages are capable of transcriptional and metabolic reprogramming under polarizing stimuli, however, NMR M1 possesses species-specific signatures as compared to murine M1, implicating distinct adaptations in NMR immune system.

Kidney-Related Function of Mitochondrial Protein Mitoregulin.

A small protein, Mitoregulin (Mtln), localizes in mitochondria and contributes to oxidative phosphorylation and fatty acid metabolism. Mtln knockout mice develop obesity on a high-fat diet, demonstrating elevated cardiolipin damage and suboptimal creatine kinase oligomerization in muscle tissue. Kidneys heavily depend on the oxidative phosphorylation in mitochondria. Here we report kidney-related phenotypes in aged Mtln knockout mice. Similar to Mtln knockout mice muscle mitochondria, those of the kidney demonstrate a decreased respiratory complex I activity and excessive cardiolipin damage. Aged male mice carrying Mtln knockout demonstrated an increased frequency of renal proximal tubules' degeneration. At the same time, a decreased glomerular filtration rate has been more frequently detected in aged female mice devoid of Mtln. An amount of Mtln partner protein, Cyb5r3, is drastically decreased in the kidneys of Mtln knockout mice.

Mitoregulin Contributes to Creatine Shuttling and Cardiolipin Protection in Mice Muscle.

Small peptides compose a large share of the mitochondrial proteome. Mitoregulin (Mtln) is a mitochondrial peptide known to contribute to the respiratory complex I functioning and other processes in mitochondria. In our previous studies, we demonstrated that Mtln knockout mice develop obesity and accumulate triglycerides and other oxidation substrates in serum, concomitant with an exhaustion of tricarboxylic acids cycle intermediates. Here we examined the functional role of Mtln in skeletal muscles, one of the major energy consuming tissues. We observed reduced muscle strength for Mtln knockout mice. Decrease of the mitochondrial cardiolipin and concomitant increase in monolysocardiolipin concentration upon Mtln inactivation is likely to be a consequence of imbalance between oxidative damage and remodeling of cardiolipin. It is accompanied by the mitochondrial creatine kinase octamer dissociation and suboptimal respiratory chain performance in Mtln knockout mice.

Mitochondrial peptide Mtln contributes to oxidative metabolism in mice.

Mitoregulin (Mtln) is a recently identified 56 amino acid long mitochondrial peptide conserved in vertebrates. Mtln is known to enhance function of respiratory complex I, which is likely mediated by modulation of lipid composition. To address an influence of Mtln gene on the metabolism we created knockout mice deficient in Mtln gene. In line with accumulation of triglycerides observed earlier on a model of Mtln knockout cell lines, we observed Mtln KO mice to develop obesity on a high fat diet. An increased weight gain could be attributed to enhanced fat accumulation according to the magnetic resonance live imaging. In addition, Mtln KO mice demonstrate elevated serum triglycerides and other oxidation substrates accompanied by an exhaustion of tricarboxylic acids cycle intermediates, suggesting suboptimal oxidation of respiration substrates by mitochondria lacking Mtln.

A New Mouse Strain with a Mutation in the NFE2L2 (NRF2) Gene.

Transcription factor NRF2 is involved in inflammatory reactions, maintenance of redox balance, metabolism of xenobiotics, and is of particular interest for studying aging. In the present work, the CRISPR/Cas9 genome editing technology was used to generate the NRF2ΔNeh2 mice containing a substitution of eight amino acid residues at the N-terminus of the NRF2 protein, upstream of the functional Neh2 domain, which ensures binding of NRF2 to its inhibitor KEAP1. Heterozygote NRF2wt/ΔNeh2 mice gave birth to homozygous mice with lower than expected frequency, accompanied by their increased embryonic lethality and visual signs of anemia. Mouse embryonic fibroblasts (MEFs) from the NRF2ΔNeh2/ΔNeh2 homozygotes showed impaired resistance to oxidative stress compared to the wild-type MEFs. The tissues of homozygous NRF2ΔNeh2/ΔNeh2 animals had a decreased expression of the NRF2 target genes: NAD(P)H:Quinone oxidoreductase-1 (Nqo1); aldehyde oxidase-1 (Aox1); glutathione-S-transferase A4 (Gsta4); while relative mRNA levels of the monocyte chemoattractant protein 1 (Ccl2), vascular cell adhesion molecule 1 (Vcam1), and chemokine Cxcl8 was increased. Thus, the resulting mutation in the Nfe2l2 gene coding for NRF2, partially impaired function of this transcription factor, expanding our insights into the functional role of the unstructured N-terminus of NRF2. The obtained NRF2ΔNeh2 mouse line can be used as a model object for studying various pathologies associated with oxidative stress and inflammation.

Alterations of the Composition and Neurometabolic Profile of Human Gut Microbiota in Major Depressive Disorder.

Major depressive disorder (MDD) is among the most prevalent mental disorders worldwide. Factors causing the pathogenesis of MDD include gut microbiota (GM), which interacts with the host through the gut-brain axis. In previous studies of GM in MDD patients, 16S rRNA sequencing was used, which provided information about composition but not about function. In our study, we analyzed whole metagenome sequencing data to assess changes in both the composition and functional profile of GM. We looked at the GM of 36 MDD patients, compared with that of 38 healthy volunteers. Comparative taxonomic analysis showed decreased abundances of Faecalibacterium prausnitzii, Roseburia hominis, and Roseburia intestinalis, and elevated abundances of Escherichia coli and Ruthenibacterium lactatiformans in the GM of MDD patients. We observed decreased levels of bacterial genes encoding key enzymes involved in the production of arginine, asparagine, glutamate, glutamine, melatonin, acetic, butyric and conjugated linoleic acids, and spermidine in MDD patients. These genes produced signature pairs with Faecalibacterium prausntizii and correlated with decreased levels of this species in the GM of MDD patients. These results show the potential impact of the identified biomarker bacteria and their metabolites on the pathogenesis of MDD, and should be confirmed in future metabolomic studies.

Unique Features of the Tissue Structure in the Naked Mole Rat (Heterocephalus glaber): Hypertrophy of the Endoplasmic Reticulum and Spatial Mitochondrial Rearrangements in Hepatocytes.

The reason for the exceptional longevity of the naked mole rat (Heterocephalus glaber) remains a mystery to researchers. We assumed that evolutionarily, H. glaber acquired the ability to quickly stabilize the functioning of mitochondria and endoplasmic reticulum (ER) to adjust metabolism to external challenges. To test this, a comparison of the hepatic mitochondria and ER of H. glaber and C57BL/6 mice was done. Electron microscopy showed that 2-months-old mice have more developed rough ER (RER) than smooth ER (SER), occupying ~17 and 2.5% of the hepatocytic area correspondingly, and these values do not change with age. On the other hand, in 1-week-old H. glaber, RER occupies only 13% constantly decreasing with age, while SER occupies 35% in a 1-week-old animal, constantly rising with age. The different localization of mitochondria in H. glaber and mouse hepatocytes was confirmed by confocal and electron microscopy: while in H. glaber, mitochondria were mainly clustered around the nucleus and on the periphery of the cell, in mouse hepatocytes they were evenly distributed throughout the cell. We suggest that the noted structural and spatial features of ER and mitochondria in H. glaber reflect adaptive rearrangements aimed at greater tolerance of the cellular system to challenges, primarily hypoxia and endogenous and exogenous toxins. Different mechanisms of adaptive changes including an activated hepatic detoxification system as a hormetic response, are discussed considering the specific metabolic features of the naked mole rat.

Mitochondrial rRNA Methylation by Mettl15 Contributes to the Exercise and Learning Capability in Mice.

Mitochondrial translation is a unique relic of the symbiotic origin of the organelle. Alterations of its components cause a number of severe human diseases. Hereby we report a study of mice devoid of Mettl15 mitochondrial 12S rRNA methyltransferase, responsible for the formation of m4C839 residue (human numbering). Homozygous Mettl15-/- mice appeared to be viable in contrast to other mitochondrial rRNA methyltransferase knockouts reported earlier. The phenotype of Mettl15-/- mice is much milder than that of other mutants of mitochondrial translation apparatus. In agreement with the results obtained earlier for cell cultures with an inactivated Mettl15 gene, we observed accumulation of the RbfA factor, normally associated with the precursor of the 28S subunit, in the 55S mitochondrial ribosome fraction of knockout mice. A lack of Mettl15 leads to a lower blood glucose level after physical exercise relative to that of the wild-type mice. Mettl15-/- mice demonstrated suboptimal muscle performance and lower levels of Cox3 protein synthesized by mitoribosomes in the oxidative soleus muscles. Additionally, we detected decreased learning capabilities in the Mettl15-/- knockout mice in the tests with both positive and negative reinforcement. Such properties make Mettl15-/- knockout mice a suitable model for mild mitochondriopathies.

CRISPR-Cas Systems in Gut Microbiome of Children with Autism Spectrum Disorders.

The human gut microbiome is associated with various diseases, including autism spectrum disorders (ASD). Variations of the taxonomical composition in the gut microbiome of children with ASD have been observed repeatedly. However, features and parameters of the microbiome CRISPR-Cas systems in ASD have not been investigated yet. Here, we demonstrate such an analysis in order to describe the overall changes in the microbiome CRISPR-Cas systems during ASD as well as to reveal their potential to be used in diagnostics and therapy. For the systems identification, we used a combination of the publicly available tools suited for completed genomes with subsequent filtrations. In the considered data, the microbiomes of children with ASD contained fewer arrays per Gb of assembly than the control group, but the arrays included more spacers on average. CRISPR arrays from the microbiomes of children with ASD differed from the control group neither in the fractions of spacers with protospacers from known genomes, nor in the sets of known bacteriophages providing protospacers. Almost all bacterial protospacers of the gut microbiome systems for both children with ASD and the healthy ones were located in prophage islands, leaving no room for the systems to participate in the interspecies competition.

Comparative Analysis of Genome Editors Efficiency on a Model of Mice Zygotes Microinjection.

Genome editing is an indispensable tool for functional genomics. The caveat of the genome-editing pipeline is a prevalence of error-prone non-homologous end joining over homologous recombination, while only the latter is suitable to introduce particularly desired genetic variants. To overcome this problem, a toolbox of genome engineering was appended by a variety of improved instruments. In this work, we compared the efficiency of a number of recently suggested improved systems for genome editing applied to the same genome regions on a murine zygote model via microinjection. As a result, we observed that homologous recombination utilizing an ssDNA template following sgRNA directed Cas9 cleavage is still the method of choice for the creation of animals with precise genome alterations.

Biomarkers and Utility of the Antioxidant Potential of Probiotic Lactobacilli and Bifidobacteria as Representatives of the Human Gut Microbiota.

Lactobacilli and bifidobacteria are an important part of human gut microbiota. Among numerous benefits, their antioxidant properties are attracting more and more attention. Multiple in vivo and in vitro studies have demonstrated that lactobacilli and bifidobacteria, along with their cellular components, possess excellent antioxidant capacity, which provides a certain degree of protection to the human body against diseases associated with oxidative stress. Recently, lactobacilli and bifidobacteria have begun to be considered as a new source of natural antioxidants. This review summarizes the current state of research on various antioxidant properties of lactobacilli and bifidobacteria. Special emphasis is given to the mechanisms of antioxidant activity of these bacteria in the human gut microbiota, which involve bacterial cell components and metabolites. This review is also dedicated to the genes involved in the antioxidant properties of lactobacilli and bifidobacteria strains as indicators of their antioxidant potential in human gut microbiota. Identification of the antioxidant biomarkers of the gut microbiota is of great importance both for creating diagnostic systems for assessing oxidative stress and for choosing strategies aimed at restoring the normal functioning of the microbiota and, through it, restoring human health. In this review, the practical application of probiotic strains with proven antioxidant properties to prevent oxidative stress is also considered.

Proarrhythmic atrial ectopy associated with heart sympathetic innervation dysfunctions is specific for murine B6CBAF1 hybrid strain.

A lot of animal models are developed with aim to advance in atrial fibrillation (AF) understanding. The hybrid B6CBAF1 mice are used extensively as a background to create manifestation of various diseases, however, their atrial electrophysiology, autonomic sympathetic innervation of the heart and potential for AF investigation is poorly characterized. In the present study we used ECG and microelectrode recordings from multicellular atrial preparations to reveal attributes of atrial electrical activity in B6CBAF1. Also, experiments with a fluorescent false monoamine neurotransmitter and glyoxylic acid-based staining were carried out to characterize functionally and morphologically catecholaminergic innervation of the B6CBAF1 atria. Atrial myocardium of B6CBAF1 is highly prone to ectopic automaticity and exhibits abnormal spontaneous action potential accompanied by multiple postdepolarizations that result in proarrhythmic triggered activity unlike two parental C57Bl/6 and CBA strains. In vivo experiments revealed that B6CBAF1 hybrids are more susceptible to the norepinephrine induced AF. Also, sympathetic nerve terminals are partially dysfunctional in B6CBAF1 revealing lower ability to accumulate and release neurotransmitters unlike two parental strains. The analysis of the heart rate variability revealed suppressed sympathetic component of the autonomic heart control in B6CBAF1. The organization of sympathetic innervation is very similar morphologically in all three murine strains however the abundance of non-bifurcated catecholamine-positive fibers in B6CBAF1 was increased. These results suggest that B6CBAF1 mice exhibit enhanced intrinsic atrial proarrhythmicity, while the abnormalities of sympathetic neurotransmitter cycling probably underlie disturbed autonomic heart control.

Surprisingly long survival of premature conclusions about naked mole-rat biology.

Naked mole-rats express many unusual traits for such a small rodent. Their morphology, social behaviour, physiology, and ageing have been well studied over the past half-century. Many early findings and speculations about this subterranean species persist in the literature, although some have been repeatedly questioned or refuted. While the popularity of this species as a natural-history curiosity, and oversimplified story-telling in science journalism, might have fuelled the perpetuation of such misconceptions, an accurate understanding of their biology is especially important for this new biomedical model organism. We review 28 of these persistent myths about naked mole-rat sensory abilities, ecophysiology, social behaviour, development and ageing, and where possible we explain how these misunderstandings came about.

Bacterial Metabolites of Human Gut Microbiota Correlating with Depression.

Depression is a global threat to mental health that affects around 264 million people worldwide. Despite the considerable evolution in our understanding of the pathophysiology of depression, no reliable biomarkers that have contributed to objective diagnoses and clinical therapy currently exist. The discovery of the microbiota-gut-brain axis induced scientists to study the role of gut microbiota (GM) in the pathogenesis of depression. Over the last decade, many of studies were conducted in this field. The productions of metabolites and compounds with neuroactive and immunomodulatory properties among mechanisms such as the mediating effects of the GM on the brain, have been identified. This comprehensive review was focused on low molecular weight compounds implicated in depression as potential products of the GM. The other possible mechanisms of GM involvement in depression were presented, as well as changes in the composition of the microbiota of patients with depression. In conclusion, the therapeutic potential of functional foods and psychobiotics in relieving depression were considered. The described biomarkers associated with GM could potentially enhance the diagnostic criteria for depressive disorders in clinical practice and represent a potential future diagnostic tool based on metagenomic technologies for assessing the development of depressive disorders.

Mild depolarization of the inner mitochondrial membrane is a crucial component of an anti-aging program.

The mitochondria of various tissues from mice, naked mole rats (NMRs), and bats possess two mechanistically similar systems to prevent the generation of mitochondrial reactive oxygen species (mROS): hexokinases I and II and creatine kinase bound to mitochondrial membranes. Both systems operate in a manner such that one of the kinase substrates (mitochondrial ATP) is electrophoretically transported by the ATP/ADP antiporter to the catalytic site of bound hexokinase or bound creatine kinase without ATP dilution in the cytosol. One of the kinase reaction products, ADP, is transported back to the mitochondrial matrix via the antiporter, again through an electrophoretic process without cytosol dilution. The system in question continuously supports H+-ATP synthase with ADP until glucose or creatine is available. Under these conditions, the membrane potential, ∆ψ, is maintained at a lower than maximal level (i.e., mild depolarization of mitochondria). This ∆ψ decrease is sufficient to completely inhibit mROS generation. In 2.5-y-old mice, mild depolarization disappears in the skeletal muscles, diaphragm, heart, spleen, and brain and partially in the lung and kidney. This age-dependent decrease in the levels of bound kinases is not observed in NMRs and bats for many years. As a result, ROS-mediated protein damage, which is substantial during the aging of short-lived mice, is stabilized at low levels during the aging of long-lived NMRs and bats. It is suggested that this mitochondrial mild depolarization is a crucial component of the mitochondrial anti-aging system.