Previous studies have revealed the microbial metabolism of dietary choline in the gut, leading to its conversion into trimethylamine (TMA). Polymethoxyflavones (PMFs), exemplified by tangeretin, have shown efficacy in mitigating choline-induced cardiovascular inflammation. However, the specific mechanism by which these compounds exert their effects, particularly in modulating the gut microbiota, remains uncertain. This investigation focused on tangeretin, a representative PMFs, to explore its influence on the gut microbiota and the choline-TMA conversion process. Experimental results showed that tangeretin treatment significantly attenuated the population of CutC-active bacteria, particularly Clostridiaceae and Lactobacillus, induced by choline chloride in rat models. This inhibition led to a decreased efficiency in choline conversion to TMA, thereby ameliorating cardiovascular inflammation resulting from prolonged choline consumption. In conclusion, tangeretin\'s preventive effect against cardiovascular inflammation is intricately linked to its targeted modulation of TMA-producing bacterial activity.

BACKGROUND: Trimethylamine N-oxide (TMAO), a metabolite produced by intestinal microbiota through metabolizing phosphatidylcholine, choline, l-carnitine and betaine in the diet, has been implicated in the pathogenesis of atherosclerosis (AS). Concurrently, dietary polyphenols have garnered attention for their potential to ameliorate obesity, diabetes and atherosclerosis primarily by modulating the intestinal microbial structure. Hickory (Carya cathayensis) nut, a polyphenol-rich food product favored for its palatability, emerges as a candidate for exploration.
OBJECTIVE: The relationship between polyphenol of hickory nut and atherosclerosis prevention will be firstly clarified, providing theoretical basis for the discovery of natural products counteracting TMAO-induced AS process in hickory nut.
METHODS: Employing Enzyme-linked Immunosorbent Assay (ELISA) and histological examination of aortic samples, the effects of total polyphenol extract on obesity index, inflammatory index and pathological changes of atherosclerosis in C57BL/6 J mice fed with high-fat and high choline diet were evaluated. Further, the composition, abundance, and function of mouse gut microbiota were analyzed through 16srDNA sequencing. Concurrently, the levels of TMAO and the expression of key enzymes (CutC and FMO3) involved in its synthesis are quantified using ELISA, Western Blot and Real-Time Quantitative PCR (RT-qPCR). Additionally, targeted metabolomic profiling of the hickory nut polyphenol extract was conducted, accompanied by molecular docking simulations to predict interactions between candidate polyphenols and the CutC/FMO3 using Autodock Vina. Finally, the docking prediction were verified by microscale thermophoresis (MST) .
RESULTS: Polyphenol extracts of hickory nut improved the index of obesity and inflammation, and alleviated the pathological changes of atherosclerosis in C57BL/6 J mice fed with high-fat and high-choline diet. Meanwhile, these polyphenol extracts also changed the composition and function of intestinal microbiota, and increased the abundance of microorganisms in mice. Notably, the abundance of intestinal microbiota endowed with CutC gene was significantly reduced, coherent with expression of CutC catalyzing TMA production. Moreover, polyphenol extracts also decreased the expression of FMO3 in the liver, contributing to the reduction of TMAO levels in serum. Furthermore, metabonomic profile analysis of these polyphenol extracts identified 647 kinds of polyphenols. Molecular docking predication further demonstrated that Casuariin and Cinnamtannin B2 had the most potential inhibition on the enzymatic activities of CutC or FMO3, respectively. Notably, MST analysis corroborated the potential for direct interaction between CutC enzyme and available polyphenols such as Corilagin, (-)-Gallocatechin gallate and Epigallocatechin gallate.
CONCLUSIONS: Hickory polyphenol extract can mitigate HFD-induced AS by regulating intestinal microflora in murine models. In addition, TMA-FMO3-TMAO pathway may play a key role in this process. This research unveils, for the inaugural time, the complex interaction between hickory nut-derived polyphenols and gut microbial, providing novel insights into the role of dietary polyphenols in AS prevention.

UNASSIGNED: Previous studies have indicated that diarrhea with kidney-yang deficiency syndrome leads to a disorder of small intestine contents and mucosal microbiota. However, the relationship of TMA-lyase (CutC) activity and TMAO with diarrhea with kidney-yang deficiency syndrome remains unexplored. Therefore, this study explores the relationship between cecal microbiota and choline TMA-lyase (CutC) activity, as well as the correlation between trimethylamine oxide (TMAO), inflammatory index, and CutC activity.
UNASSIGNED: Twenty SPF-grade male KM mice were randomly divided into the normal group (CN) and the diarrhea model group (CD). Diarrhea mouse models were established by adenine combined with Folium sennae administration. CutC activity, TMAO, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) levels were detected, and the cecal content microbiota was sequenced.
UNASSIGNED: After 14 days, diarrhea occurred in the CD group. Compared with the CN group, there was no significant change in the activity of CutC in the small intestine of the CD group, while the activity of CutC in the cecum was significantly increased, and the levels of TMAO, IL-6, and TNF-α showed a significant increase. The Chao1 index, Observed_species index, Shannon index, and Simpson index all exhibited a decreasing trend. The main changes at the bacterial genus level were Alistipes, Enterorhabdus, Desulfovibrio, Bacteroides, Candidatus_Saccharimonas, and [Ruminococcus]_torques_group. The results of LEfSe analysis, random forest analysis and ROC curve analysis revealed Paludicola, Blautia, Negativibacillus, Paraprevotella, Harryflintia, Candidatus_Soleaferrea, Anaerotruncus, Oscillibacter, Colidextribacter, [Ruminococcus]_torques_group, and Bacteroides as characteristic bacteria in the CD group. Correlation analysis showed a significant negative correlation between cecal CutC activity and Ligilactobacillus, and a significant positive correlation with Negativibacillus and Paludicola. The level of TMAO was significantly positively correlated with CutC activity and IL-6.
UNASSIGNED: Diarrhea with kidney-yang deficiency syndrome significantly affects the physiological status, digestive enzyme activity, CutC activity, TMAO levels, and inflammatory response in mice. Additionally, there are changes in the composition and function of cecal microbiota, indicating an important impact of diarrhea with kidney-yang deficiency syndrome on the host intestinal microbiota balance. The occurrence of diarrhea with kidney-yang deficiency syndrome may be associated with dysbiosis of intestinal microbiota, increased CutC activity, elevated TMAO levels, and heightened inflammatory factor levels.

Gut microbiota metabolism can have profound effects on human health. Choline, a quaternary amine (QA) highly abundant in our diet, is canonically cleaved by a glycyl radical enzyme, choline trimethylamine lyase (CutC), and its SAM-dependent radical activator, CutD. CutC cleaves choline to form trimethylamine (TMA) and acetaldehyde. TMA is oxidized to TMAO by FMO3 in the liver, which plays a role in causing atherosclerosis. We hypothesized that alternative pathways for choline degradation occur within gut microbes and that certain gut microbiota can anaerobically respire or ferment QAs, such as choline. Based on this prediction we established QA-supplemented enrichment cultures using fecal material from healthy volunteers as the inocula. We have isolated, from a choline-supplemented enrichment of a human fecal sample, a strain of Citrobacter amalonaticus, that we have designated CJ25. This strain is capable of anaerobically utilizing choline as its sole carbon and energy source. Its genome does not contain the cutCD genes or genes encoding any COG5598 methyltransferases. We have confirmed the degradation of choline and production of acetate by the organism during growth of the strain. However, we used multiple analytical methods to confirm that no TMA accumulated in the medium during growth. Hence, strain CJ25 is a unique bacterium that degrades choline without the production of the proatherogenic metabolite TMA.

In the current study, the gut microbiota of patients with and without coronary heart disease was compared and the relationship between gut microbiota distribution, intending to reveal the role of gut microbiota in the coronary atherosclerosis process, was investigated.This study included 50 patients diagnosed with coronary heart disease (CHD) who received conventional coronary angiography or computed tomography angiography and 50 patients with CHD at Changshu No. 2 People\'s Hospital, Suzhou, China, from May 2020 to January 2021. Trimethylamine N-oxide (TMAO) level was tested and feces were collected, the DNA of the gut microbiota was extracted, and the distribution by 16SrRNA gene sequencing was obtained from the two groups of patients.Plasma TMAO concentrations were significantly higher in patients with CHD (P < 0.001). In the CHD group, 22 patients with multivessel disease had a higher level of TMAO compared with the 28 patients who had the single-vessel disease (P < 0.001). No difference in the gut microbiota diversity was noted between the two groups (P < 0.001). Patients with CHD had a significantly lower proportion of Bacteroidetes phyla and more proportion of Epsilonbacteraeota phyla. At the genus level, patients with CHD had an increased abundance of Enterococcus, whereas healthy controls had significantly higher levels of Streptococcus. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2 analysis found that, in the KEGG ORTHOLOGY, the level of choline trimethylamine-lyase gene expression correlated with TMAO production was higher in the fecal microbiome of the CHD group (P < 0.05).Gut microbiota and its product were expected to become a diagnostic marker and a new target for preventing CHD.

Clinical studies have demonstrated associations between circulating levels of the gut-microbiota-derived metabolite trimethylamine-N-oxide (TMAO) and stroke incident risk. However, a causal role of gut microbes in stroke has not yet been demonstrated. Herein we show that gut microbes, through dietary choline and TMAO generation, directly impact cerebral infarct size and adverse outcomes following stroke. Fecal microbial transplantation from low- versus high-TMAO-producing human subjects into germ-free mice shows that both TMAO generation and stroke severity are transmissible traits. Furthermore, employing multiple murine stroke models and transplantation of defined microbial communities with genetically engineered human commensals into germ-free mice, we demonstrate that the microbial cutC gene (an enzymatic source of choline-to-TMA transformation) is sufficient to transmit TMA/TMAO production, heighten cerebral infarct size, and lead to functional impairment. We thus reveal that gut microbiota in general, specifically the metaorganismal TMAO pathway, directly contributes to stroke severity.

A substantial proportion of patients with common variable immunodeficiency (CVID) have inflammatory and autoimmune complications of unknown etiology. We have previously shown that systemic inflammation in CVID correlates with their gut microbial dysbiosis. The gut microbiota dependent metabolite trimethylamine N-oxide (TMAO) has been linked to several metabolic and inflammatory disorders, but has hitherto not been investigated in relation to CVID. We hypothesized that TMAO is involved in systemic inflammation in CVID. To explore this, we measured plasma concentrations of TMAO, inflammatory markers, and lipopolysaccharide (LPS) in 104 CVID patients and 30 controls. Gut microbiota profiles and the bacterial genes CutC and CntA, which encode enzymes that can convert dietary metabolites to trimethylamine in the colon, were examined in fecal samples from 40 CVID patients and 86 controls. Furthermore, a food frequency questionnaire and the effect of oral antibiotic rifaximin on plasma TMAO concentrations were explored in these 40 patients. We found CVID patients to have higher plasma concentrations of TMAO than controls (TMAO 5.0 [2.9-8.6] vs. 3.2 [2.2-6.3], p = 0.022, median with IQR). The TMAO concentration correlated positively with tumor necrosis factor (p = 0.008, rho = 0.26), interleukin-12 (p = 0.012, rho = 0.25) and LPS (p = 0.034, rho = 0.21). Dietary intake of meat (p = 0.678), fish (p = 0.715), egg (p = 0.138), dairy products (p = 0.284), and fiber (p = 0.767) did not significantly impact on the TMAO concentrations in plasma, nor did a 2-week course of the oral antibiotic rifaximin (p = 0.975). However, plasma TMAO concentrations correlated positively with gut microbial abundance of Gammaproteobacteria (p = 0.021, rho = 0.36). Bacterial gene CntA was present in significantly more CVID samples (75%) than controls (53%), p = 0.020, potentially related to the increased abundance of Gammaproteobacteria in these samples. The current study demonstrates that elevated TMAO concentrations are associated with systemic inflammation and increased gut microbial abundance of Gammaproteobacteria in CVID patients, suggesting that TMAO could be a link between gut microbial dysbiosis and systemic inflammation. Gut microbiota composition could thus be a potential therapeutic target to reduce systemic inflammation in CVID.

A copper-mediated synthesis of diaryl sulfides utilizing Cu(I)-thiophene-2-carboxylate (CuTC) is described. We demonstrate the use of CuTC as a soluble, non-basic catalyst in the coupling of aryl iodides and aryl thiols in the synthesis of synthetically advanced diaryl sulfides. This method allows for the successful coupling of challenging substrates including ortho-substituted and heteroaryl iodides and thiols. Additionally, most of the aryl iodide substrates used here contain the privileged piperazine scaffold bound to a pyrimidine, pyridine, or phenyl ring and thus this method allows for the elaboration of complex piperazine scaffolds into molecules of biological interest. The method described here enables the incorporation of late-stage structural diversity into diaryl sulfides containing the piperazine ring, thus enhancing the number and nature of derivatives available for SAR investigation.

In enteric bacteria, the transcription factor σ(E) maintains membrane homeostasis by inducing synthesis of proteins involved in membrane repair and two small regulatory RNAs (sRNAs) that down-regulate synthesis of abundant membrane porins. Here, we describe the discovery of a third σ(E)-dependent sRNA, MicL (mRNA-interfering complementary RNA regulator of Lpp), transcribed from a promoter located within the coding sequence of the cutC gene. MicL is synthesized as a 308-nucleotide (nt) primary transcript that is processed to an 80-nt form. Both forms possess features typical of Hfq-binding sRNAs but surprisingly target only a single mRNA, which encodes the outer membrane lipoprotein Lpp, the most abundant protein of the cell. We show that the copper sensitivity phenotype previously ascribed to inactivation of the cutC gene is actually derived from the loss of MicL and elevated Lpp levels. This observation raises the possibility that other phenotypes currently attributed to protein defects are due to deficiencies in unappreciated regulatory RNAs. We also report that σ(E) activity is sensitive to Lpp abundance and that MicL and Lpp comprise a new σ(E) regulatory loop that opposes membrane stress. Together MicA, RybB, and MicL allow σ(E) to repress the synthesis of all abundant outer membrane proteins in response to stress.