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Recently, the gut microbiota has been found to be associated with many diseases, such as inflammatory bowel disease, depression, Parkinson's disease, cancer, metabolic syndrome, and cardiovascular disease (CVD). Among various gut microbiota-derived metabolites (GMs), short-chain fatty acids (SCFAs), bile acids (BAs), and tryptophan (TRP) metabolites are the most frequently discussed metabolites. LC-MS/MS shows advantages in quantifying the levels of metabolites with good sensitivity and selectivity; however, the poor ionization efficiency and polar characteristics of SCFAs make their analysis challenging, especially when analyzing plasma samples with low SCFA concentrations. Moreover, without characteristic fragment ions for unconjugated BAs and different detection ion modes for TRP metabolites and BAs, GM analysis is complex and time-consuming. To overcome these problems, we developed a derivatization method combined with LC-MS/MS to enhance the sensitivity and LC retention of GMs. Through derivatization with 3-nitrophenylhydrazine (3-NPH), 7 SCFAs, 9 bile acids, and 6 tryptophan metabolites can be simultaneously analyzed via separation within 14 min on a reversed-phase C18 column. For accurate quantification, 13C6-3NPH-labeled standards were used as one-to-one internal standards. This derivatization approach was optimized and then validated. We further applied this method to investigate the targeted GM profile in patients with CVD. The results showed a significant reduction in plasma butyrate levels in CVD patients compared with healthy controls, suggesting its potentially protective role in CVD. In summary, this work provides a sensitive and effective LC-MS/MS method for simultaneously quantifying gut microbiota-related metabolites in human plasma, which could benefit various future gut microbiota-related studies.

Human gut microbiota is a dynamic and variable system that can change over time and in response to different diets and treatments. There is currently no doubt that gut microbiota can provide interesting therapeutic opportunities, since it can metabolize biologically active molecules, drugs, and their precursors, and control their bioavailability. Moreover, it can produce both beneficial and dangerous metabolites that influence host's health. In this review, we summarize the current knowledge on the involvement of gut microbiota in two chronic human pathologies that represent the greatest challenges of modern medicine: atherosclerosis and cancer. Interesting parallels are observed between the mechanisms and possible treatment approaches of these pathologies. Some of the common effects of therapeutic agents targeting both pathologies, such as anti-inflammatory activity, are partially mediated by the gut microbiota. We will discuss the effects of common drugs (metformin, statins and aspirin) and various nutraceuticals on gut microbiota and outline the pathways of microbial involvement in mediating the pleiotropic beneficial effects of these agents in atherosclerosis and cancer.

Background/purpose Appropriate storage of fecal samples is a critical step for the unbiased analysis of microbial communities in metagenomic studies. Rapid freezing at −80 °C is usually considered to be best practice, but this approach is challenging. DNA stabilizing kits may provide a more convenient method to preserve and store clinical samples. We evaluated the reliability of two collection kits (Stratec stool collection tube with stabilizer, #1038111200 and OMNIgene.GUT OMR-200) on preserving fecal microbiota. Methods Samples were collected from two locations of the fecal specimen, in four healthy volunteers. The samples were sub-aliquoted and stored in a −80 °C freezer, in Stratec and OMNIgene.GUT (incubation at ambient temperature for 0, 3, or 7 days). The fecal microbial composition was assessed by 16S rRNA sequencing. Results We found that alpha diversity was not significantly affected by storage conditions. Samples stored in DNA stabilizers were still representative of the original microbial community after 7 days at ambient temperature. Individual differences were found to have a greater contribution to the differences in microbial community composition than storage conditions or sampling location. Samples subjected to stabilizers displayed microbial community shifts compared with immediately frozen samples. A linear discriminant analysis effect size (LEfSe) analysis showed that the relative abundances of Faecalibacterium were significantly higher in samples stored in Stratec kits. Conclusion Our study reveals that both Stratec and OMNIgene.GUT kits provide good microbiome preservation for up to 7 days in ambient temperature and would represent good options for fecal sample collection in large scale, population-based studies.

The progression of metabolic dysfunction associated fatty liver disease (MAFLD) leads to steatohepatitis, liver fibrosis and hepatocellular carcinoma. Thus far, there have been no FDA-approved medications for MAFLD. Bariatric surgery (BS) has been found to improve insulin resistance, steatohepatitis and liver fibrosis but is not recommended for treating MAFLD due to its invasiveness. Recent studies suggest the improved glucose metabolism after BS is a result of, at least partly, alterations to the gut microbiota and its associated metabolites, including short chain fatty acids and bile acids. It makes sense the improved steatohepatitis and fibrosis after BS are also induced by the gut microbiota that involves in host metabolic modulation, for example, through altering bile acids composition. Given that the gut–liver axis is a path that may harbor unexplored mechanisms behind MAFLD, we review current literatures about disentangling the metabolic benefits of MAFLD after BS, with a focus on gut microbiota. Some useful research tools including the rodent BS model, the multiomics approach, and the human microbiota associated (HMA) mice are presented and discussed. We believe, by taking advantage of these modern translational tools, researchers will uncover microbiota related pathways to serve as potential therapeutic targets for treating MAFLD.

The emergence of the novel coronavirus in December 2019 in China marked the beginning of a pandemic that impacted healthcare systems and economic life all over the world. The virus primarily targets the respiratory system causing severe acute respiratory syndrome (SARS) in some patients, and therefore received the name of SARS-CoV-2. The pathogen stands out among other coronaviruses by its rapid transmission from human to human, with the majority of infected individuals being asymptomatic or presenting with only minor illness, therefore facilitating the pathogen spread. At the same time, people from the risk groups, such as the elderly, patients suffering from chronic diseases, or obese individuals, have increased chances of developing a severe or even fatal disease. The search for risk factors explaining this phenomenon continues. In this review, we focus on the known mechanisms of SARS-CoV-2 infection affecting the functioning of the immune system and discuss potential risk factors responsible for the severe disease course. Oxidative stress is one of such factors, which plays a prominent role in innate immunity activity, and recent research has revealed its tight involvement in SARS-CoV-2 infection. We discuss these recent findings and the development of excessive inflammation and cytokine storm observed during SARS-CoV-2 infection. Finally, we consider potential use of antioxidant drugs for alleviating the severe symptoms in affected patients.

Atherosclerosis is a major cause of disease-related mortality around the globe. The main characteristic of the disease is an accumulation of plaque on the arterial wall and subsequent erosion or rupture of some plaques. Atherosclerosis often leads to cardiovascular disease and such acute complications as myocardial infarction or ischemic stroke due to thrombus formation. Most recent advances in atherosclerotic research state that the modifications of low-density lipoprotein (LDL) are one of the most significant stages in the disease initiation, and among these modifications desialylation is of particular interest. Sialic acids are widely expressed on all types of cells of many organisms and participate in numerous biological processes. Regarding atherosclerosis, sialidases that are responsible for the regulation of the sialic component of different molecules, are probably one of the most crucial enzymatic families. Sufficient sialylation of vascular endothelium defines its susceptibility to an atherogenic plaque formation. Moreover, the desialylation of LDL provokes an accumulation of cholesterol and lipids in the arterial walls. According to the multiple involvements of sialic acids and related enzymes, sialidases, in the initiation and development of atherosclerosis, the deeper understanding of their exact role, as well as cellular and molecular mechanisms, will allow creating more targeted and effective therapeutic and diagnostic approaches.

Even though advancement in medicine has contributed to the control of many diseases to date, cancer therapy continues to pose several challenges. Hepatocellular carcinoma (HCC) etiology is multifactorial. Recently, non-alcoholic fatty liver disease (NAFLD) has been considered as an important risk factor of HCC. NAFLD can be divided into non-alcoholic simple fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) based on histopathological features. Recently, studies have indicated that the gut microbiota is associated with NAFLD and HCC. Therefore, in this review, we have discussed the effects of gut microbiota-related mechanisms, including dysbiosis and gut barrier function, and gut microbiota-derived metabolites on NAFLD and HCC pathogenesis and the potential therapeutic strategies for NAFLD and HCC. With a better understanding of the gut microbiota composition and function, new and improved diagnostic, prognostic, and therapeutic strategies for common liver diseases can be developed.

There is an important task of current medicine to identify mechanisms and new markers of subclinical atherosclerosis in order to develop early targets for the diagnosis and treatment of this disease, since it causes such widespread diseases as myocardial infarction, stroke, sudden death, and other common reasons of disability and mortality in developed countries. In recent years, studies of the human microbiome in different fields of medicine have become increasingly popular; there is evidence from numerous studies of the significant contribution of microbiome in different steps of atherogenesis. This review attempted to determine the current status of the databases PubMed and Scopus (until May, 2020) to highlight current ideas on the potential role of microbiome and its metabolites in atherosclerosis development, its mechanisms of action in lipids metabolism, endothelial dysfunction, inflammatory pathways, and mitochondrial dysfunction. Results of clinical studies elucidating the relationship of microbiome with subclinical atherosclerosis and cardiovascular disease considered in this article demonstrate strong association of microbiome composition and its metabolites with atherosclerosis and cardiovascular disease. Data on microbiome impact in atherogenesis open a wide perspective to develop new diagnostic and therapeutic approaches, but further comprehensive studies are necessary.

The capability of gut microbiota in degrading foods and drugs administered orally can result in diversified efficacies and toxicity interpersonally and cause significant impact on human health. Production of atherogenic trimethylamine N-oxide (TMAO) from carnitine is a gut microbiota-directed pathway and varies widely among individuals. Here, we demonstrated a personalized TMAO formation and carnitine bioavailability from carnitine supplements by differentiating individual TMAO productivities with a recently developed oral carnitine challenge test (OCCT). By exploring gut microbiome in subjects characterized by TMAO producer phenotypes, we identified 39 operational taxonomy units that were highly correlated to TMAO productivity, including Emergencia timonensis, which has been recently discovered to convert γ-butyrobetaine to TMA in vitro. A microbiome-based random forest classifier was therefore constructed to predict the TMAO producer phenotype (AUROC = 0.81) which was then validated with an external cohort (AUROC = 0.80). A novel bacterium called Ihubacter massiliensis was also discovered to be a key microbe for TMA/TMAO production by using an OCCT-based humanized gnotobiotic mice model. Simply combining the presence of E. timonensis and I. massiliensis could account for 43% of high TMAO producers with 97% specificity. Collectively, this human gut microbiota phenotype-directed approach offers potential for developing precision medicine and provides insights into translational research.

Immunoglobulins are the potent effector proteins of the humoral immune response. In the course of evolution, immunoglobulins have formed extremely diverse types of molecular structures with antigen-recognizing, antigen-binding, and effector functions embedded in a single molecule. Polysaccharide moiety of immunoglobulins plays the essential role in immunoglobulin functioning. There is growing evidence that the carbohydrate composition of immunoglobulin-linked glycans, and especially their terminal sialic acid residues, provide a key effect on the effector functions of immunoglobulins. Possibly, sialylation of Fc glycan is a common mechanism of IgG anti-inflammatory action in vivo. Thus, the post-translational modification (glycosylation) of immunoglobulins opens up significant possibilities in the diagnosis of both immunological and inflammatory disorders and in their therapies. This review is focused on the analysis of glycosylation of immunoglobulins, which can be a promising addition to improve existing strategies for the diagnosis and treatment of various immuno-inflammatory diseases.

Atherosclerosis with associated cardiovascular diseases remains one of the main causes of disability and death worldwide, requiring development of new solutions for prevention and treatment. Macrophages are the key effectors of a series of events involved in atherogenesis, such as inflammation, plaque formation, and changes in lipid metabolism. Some of these events were shown to be associated with mitochondrial dysfunction and excessive mitochondrial DNA (mtDNA) damage. Moreover, macrophages represent a promising target for novel therapeutic approaches that are based on the expression of various receptors and nanoparticle uptake. Lipid‐based gene delivery to mitochondria is considered to be an interesting strategy for mtDNA damage correction. To date, several nanocarriers and their modifications have been developed that demonstrate high transfection efficiency and low cytotoxicity. This review discusses the possibilities of lipid‐based gene delivery to macrophage mitochondria for atherosclerosis therapy.

ObjectiveThe gut microbiota-derived metabolite, trimethylamine N-oxide (TMAO) plays an important role in cardiovascular disease (CVD). The fasting plasma TMAO was shown as a prognostic indicator of CVD incident in patients and raised the interest of intervention targeting gut microbiota. Here we develop a clinically applicable method called oral carnitine challenge test (OCCT) for TMAO-related therapeutic drug efforts assessment and personalising dietary guidance.DesignA pharmacokinetic study was performed to verify the design of OCCT protocol. The OCCT was conducted in 23 vegetarians and 34 omnivores to validate gut microbiota TMAO production capacity. The OCCT survey was integrated with gut microbiome, host genotypes, dietary records and serum biochemistry. A humanised gnotobiotic mice study was performed for translational validation.ResultsThe OCCT showed better efficacy than fasting plasma TMAO to identify TMAO producer phenotype. The omnivores exhibited a 10-fold higher OR to be high TMAO producer than vegetarians. The TMAO-associated taxa found by OCCT in this study were consistent with previous animal studies. The TMAO producer phenotypes were also reproduced in humanised gnotobiotic mice model. Besides, we found the faecal CntA gene was not associated with TMAO production; therefore, other key relevant microbial genes might be involved. Finally, we demonstrated the urine TMAO exhibited a strong positive correlation with plasma TMAO (r=0.92, pConclusionThe OCCT can be used to identify TMAO-producer phenotype of gut microbiota and may serve as a personal guidance in CVD prevention and treatment.Trial registration numberNCT02838732; Results.

The gut microbiota has attracted a great deal of interest in recent years due to its association with many diseases. Short-chain fatty acids (SCFAs), the end products of dietary fiber fermentation by the intestinal microbiota, are among the most frequently discussed gut metabolites. As the sample handling method greatly affects the integrity of data, this study investigated the most important parameters that affect the bias of SCFA comparisons in human fecal studies. An accurate gas chromatography-mass spectrometry (GC-MS) method was first established and validated for quantifying six SCFAs, including acetic, propionic, butyric, isobutyric, isovaleric, and valeric acids. To remove interfering species, we used butanol to extract SCFAs from acidified fecal suspensions. The validated quantification method was then applied to evaluate fecal sample handling protocols. We found that lyophilization of fecal samples can not only minimize bias due to the water content but also provide better stability of SCFAs. Six SCFAs were stable and that their recoveries were higher than 90% after lyophilization. Lyophilization of a large fecal sample is extremely time-consuming, and 1 g of fecal sample is suggested for lyophilization to minimize sampling bias. The interindividual difference was significantly higher than the intra-individual difference when using 1 g of fecal sample to study SCFAs. Finally, an effective protocol from sample collection to GC-MS analysis was proposed. As SCFAs have been shown to play an important role in health maintenance and disease development, the proposed protocol is anticipated to be applicable to clinical studies to delineate the biological functions of each SCFA.

Inflammation is the key for the initiation and progression of atherosclerosis. Accumulating evidence has revealed that an altered gut microbiome (dysbiosis) triggers both local and systemic inflammation to cause chronic inflammatory diseases, including atherosclerosis. There have been some microbiome-relevant pro-inflammatory mechanisms proposed to link the relationships between dysbiosis and atherosclerosis such as gut permeability disruption, trigger of innate immunity from lipopolysaccharide (LPS), and generation of proatherogenic metabolites, such as trimethylamine N-oxide (TMAO). Meanwhile, immune responses, such as inflammasome activation and cytokine production, could reshape both composition and function of the microbiota. In fact, the immune system delicately modulates the interplay between microbiota and atherogenesis. Recent clinical trials have suggested the potential of immunomodulation as a treatment strategy of atherosclerosis. Here in this review, we present current knowledge regarding to the roles of microbiota in contributing atherosclerotic pathogenesis and highlight translational perspectives by discussing the mutual interplay between microbiota and immune system on atherogenesis.

Chronic obstructive pulmonary disease (COPD) is a multifactorial disease, in which systemic inflammation plays a key role. This 6-month randomized double-blinded placebo-controlled study evaluates the possible effect of natural preparation Inflaminat on clinical symptoms of COPD, indicators of respiratory function, and exacerbation frequency in 60 patients with moderate severity of COPD. Inflaminat is a combination of natural ingredients black elder (Sambucus nigra L.) berries, violet (Viola tricolor L.) herb, and calendula (Calendula officinalis L.) flowers. The preparation has been previously demonstrated to possess anticytokine and anti-inflammatory effects in experimental studies. In present study, COPD dynamics were evaluated by means of BCSS (Breathlessness, Cough, and Sputum Scale) and spirometry tests. It was shown that 6-months Inflaminat administration led to significant decrease of BCSS points from 3.0 &plusmn, 0.6 to 1.9 &plusmn, 0.7, (p = 0.002) as well as significant increase of FEV1 from 66 &plusmn, 18% to 73 &plusmn, 17%, (p = 0.042), there were no beneficial dynamics in placebo group. Side effects associated with preparation administration were not identified. The results of the study suggest that Inflaminat may be employed in treatment of patients with moderate severity of COPD, since it has a positive effect on COPD symptoms according BCSS and indicators of respiratory function FEV1.

Atherosclerosis can be regarded as chronic inflammatory disease affecting the arterial wall. Despite the recent progress in studying the pathogenesis of atherosclerosis, some of the pathogenic mechanisms remain to be fully understood. Among these mechanisms is oxidative stress, which is closely linked to foam cells formation and other key events in atherosclerosis development. Two groups of enzymes are involved in the emergence of oxidative stress: Pro-oxidant (including NADPH oxidases, xanthine oxidases, and endothelial nitric oxide synthase) and antioxidant (such as superoxide dismutase, catalases, and thioredoxins). Pro-oxidant enzymes in normal conditions produce moderate concentrations of reactive oxidant species that play an important role in cell functioning and can be fully utilized by antioxidant enzymes. Under pathological conditions, activities of both pro-oxidant and antioxidant enzymes can be modified by numerous factors that can be relevant for developing novel therapies. Recent studies have explored potential therapeutic properties of antioxidant molecules that are capable to eliminate oxidative damage. However, the results of these studies remain controversial. Other perspective approach is to inhibit the activity of pro-oxidant enzymes and thus to slow down the progression of atherosclerosis. In this review we summarized the current knowledge on oxidative stress in atherosclerosis and potential antioxidant approaches. We discuss several important antioxidant molecules of plant origin that appear to be promising for treatment of atherosclerosis.

This randomized double-blinded, placebo-controlled clinical trial evaluated the progression of intima-media thickness of common carotid artery (cIMT) and the effect of phytoestrogen therapy on atherosclerosis development in early and late postmenopausal women. The 2-year cIMT progression was evaluated in 315 early postmenopausal women aged 40−55 years and in 231 late postmenopausal women aged 60−69 years free of cardiovascular disease. B-mode ultrasound was done at baseline and after 12 and 24 months of follow-up. The study revealed no significant changes in the rate of cIMT progression in 315 early postmenopausal women. By contrast, a statistically significant difference in the rate of atherosclerosis development was observed in late postmenopausal women treated with phytoestrogens compared to placebo (p = 0.008). The rate of cIMT progression in the placebo group was 0.019 mm/year led to a significant increase of cIMT during the observation period (p = 0.012), while the rate of cIMT progression in phytoestrogen late postmenopausal recipients was 0.011 mm/year, and total change did not reach statistical significance during the follow-up period (p = 0.101). These results suggest that late postmenopausal women can be a suitable cohort for trials assessing the anti-atherosclerosis effects of phytoestrogen preparations. In particular, the beneficial effect of phytoestrogens on cIMT progression was demonstrated in late postmenopausal women.

Atherosclerosis is associated with acute cardiovascular conditions, such as ischemic heart disease, myocardial infarction, and stroke, and is the leading cause of morbidity and mortality worldwide. Our understanding of atherosclerosis and the processes triggering its initiation is constantly improving, and, during the last few decades, many pathological processes related to this disease have been investigated in detail. For example, atherosclerosis has been considered to be a chronic inflammation triggered by the injury of the arterial wall. However, recent works showed that atherogenesis is a more complex process involving not only the immune system, but also resident cells of the vessel wall, genetic factors, altered hemodynamics, and changes in lipid metabolism. In this review, we focus on foam cells that are crucial for atherosclerosis lesion formation. It has been demonstrated that the formation of foam cells is induced by modified low-density lipoprotein (LDL). The beneficial effects of the majority of therapeutic strategies with generalized action, such as the use of anti-inflammatory drugs or antioxidants, were not confirmed by clinical studies. However, the experimental therapies targeting certain stages of atherosclerosis, among which are lipid accumulation, were shown to be more effective. This emphasizes the relevance of future detailed investigation of atherogenesis and the importance of new therapies development.

We thank Arduini et al 1 for the feedback. The importance of identifying trimethylanine N-oxide (TMAO)-producer phenotype was challenged in doubt about the significance of TMAO in cardiovascular disease (CVD).1 2 The reason we regard TMAO as a potential harmful rather than a beneficial or bystander molecule for CVD is based on a careful literature review from both affirmative and negative sides.3 4 We appreciated the opportunity to extend our discussions about the role of TMAO in CVD. The letter mentioned a study conducted by Collins et al 5 that TMAO was inversely correlated to atherosclerosis in mice and concluded TMAO as protective for atherosclerosis. In this study, the plasma TMAO only reached 0.2ppm (2.66 µM) for the carnitine group that is far from the estimated pathological TMAO level (>10 µM). Besides, the negative …

Although great interest has been displayed by researchers in the contribution of gut microbiota to human health, there is still no standard protocol with consensus to guarantee the sample quality of metagenomic analysis. Here we reviewed existing methodology studies and present suggestions for optimizing research pipeline from fecal sample collection to DNA extraction. First, we discuss strategies of clinical metadata collection as common confounders for microbiome research. Second, we propose general principles for freshly collected fecal sample and its storage and share a DIY stool collection kit protocol based on the manual procedure of Human Microbiome Project (HMP). Third, we provide a useful information of collection kit with DNA stabilization buffers and compare their pros and cons for multi-omic study. Fourth, we offer technical strategies as well as information of novel tools for sample aliquoting before long-term storage. Fifth, we discuss the substantial impact of different DNA extraction protocols on technical variations of metagenomic analysis. And lastly, we point out the limitation of current methods and the unmet needs for better quality control of metagenomic analysis. We hope the information provided here will help investigators in this exciting field to advance their studies while avoiding experimental artifacts. Keywords: Gut microbiome, Stool collection, Sample processing, Storage condition, Standardization

This study investigated the liver-protective effects of allicin, an active compound in fresh garlic, against alcoholic fatty liver disease (AFLD) and liver inflammation. Its effects were investigated in an AFLD model in male C57BL/6 mice, which were fed Lieber-DeCarli liquid diet containing ethanol. Allicin (5 and 20 mg/kg bw/day) was orally administered daily in the AFLD mice for 4 weeks. The results indicate that allicin promotes hepatoprotection by significantly reducing aspartate transaminase (AST) and alanine transaminase (ALT) levels (p0.05) in the plasma, which are key indicators of liver damage. Allicin reduced fat accumulation, increased glutathione and catalase levels, and decreased microsomal protein cytochrome P450 2E1 (CYP2E1) expression (p0.05) in the livers of the AFLD mice. Furthermore, allicin supplementation significantly decreased the levels of proinflammatory tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 and suppressed the expression of sterol regulatory element-binding protein-1 (SREBP-1) (p0.05). Additionally, it improved the hepatic alcohol dehydrogenase (ADH) activity (p0.05). Collectively, these findings demonstrate that allicin attenuates liver oxidative stress and inflammation.

Supplemental Digital Content is available in the text, Neutralizing anti-interferon-γ autoantibody (nAIGA)-associated immunodeficiency is an emerging medical issue worldwide. In the present study, we describe and discuss the clinical features and outcomes of patients with nAIGAs and disseminated infections by nontuberculous mycobacteria (dNTM). We thoroughly reviewed the medical records of all patients. Microorganisms and nAIGAs were identified using previously described methods with modifications. All data were calculated and analyzed using SPSS software. Among 46 adult patients with dNTM infections, we identified 45 cases (97.8%) with nAIGAs. The average patient age was 58.6 years, and there was no sex predominance. Cervical lymphadenitis (81.8%) was the most common clinical manifestation. Endocrine disorder was the leading comorbidity (7 cases). Malignancies were found in 4 patients, and all of the malignancies originated from the T-cell/macrophage lineage. More than half of the identifiable isolates were slow-growing NTMs. Twenty-eight (62.2%) and 18 (40.0%) patients had a history of zoster and salmonellosis, respectively. A high proportion of patients with recurrent episodes of NTM infection or a history of zoster and dNTM infection had initial nAIGA titers ≥10–5 dilution (P