Dietary Dihydroquercetin Alleviated Colitis via the Short-Chain Fatty Acids/miR-10a-5p/PI3K-Akt Signaling Pathway.

Gut microbiota provides an important insight into clarifying the mechanism of active substances with low bioavailability, but its specific action mechanism varied case by case and remained unclear. Dihydroquercetin (DHQ) is a bioactive flavonoid with low bioavailability, which showed beneficial effects on colitis alleviation and gut microbiota modulation. Herein, we aimed to explore the microbiota-dependent anticolitis mechanism of DHQ in sight of gut microbiota metabolites and their interactions with microRNAs (miRNAs). Dietary supplementation of DHQ alleviated dextran sulfate sodium-induced colitis phenotypes and improved gut microbiota dysbiosis. Fecal microbiota transplantation further revealed that the anticolitis activity of DHQ was mediated by gut microbiota. To clarify how the modulated gut microbiota alleviated colitis in mice, the tandem analyses of the microbiome and targeted metabolome were performed, and altered profiles of metabolite short-chain fatty acids (SCFAs) and bile acids and their producers were observed in DHQ-treated mice. In addition, SCFA treatment showed anticolitis activity compared to that of bile acids, along with the specific inhibition on the phosphoinositide-3-kinase (PI3K)-protein kinase B (Akt) pathway. Subsequently, the colonic miRNA profile of mice receiving SCFA treatment was sequenced, and a differentially expressed miR-10a-5p was identified. Both prediction analysis and dual-luciferase reporter assay indicated that miR-10a-5p directly bind to the 3'-untranslated regions of gene pik3ca, inhibit the PI3K-Akt pathway activation, and lead to colitis alleviation. Together, we proposed that gut microbiota mediated the anticolitis activity of DHQ through the SCFAs/miR-10a-5p/PI3K-Akt axis, and it provided a novel insight into clarifying the microbiota-dependent mechanism via the interaction between metabolites and miRNAs.

Limosilactobacillus reuteri RE225 alleviates gout by modulating the TLR4/MyD88/NF-κB inflammatory pathway and the Nrf2/HO-1 oxidative stress pathway, and by regulating gut microbiota.

BACKGROUND: Gout poses a significant health threat. The use of Lactobacillus from the gut microbiota is one potential remedy. However, the intricate molecular mechanisms governing the impact of Lactobacillus on gout remain largely uncharted. In this study, a strain of Limosilactobacillus reuteri RE225 was separated from the gut of mice and colitis was treated with polypeptide intervention. RESULTS: Limosilactobacillus reuteri RE225 reduced foot tumefaction markedly in mice with gout and extended the pain threshold time in their feet. It also improved the health of gut microbiota. Intervention with L. reuteri RE225 also suppressed the TLR4/MyD88/NF-κB and nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathways in the mice, reduced the levels of pro-inflammatory cytokines - interleukin 1ß (IL-1ß), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) - and increased the level of the anti-inflammatory cytokine interleukin 10 (IL-10), thereby mitigating inflammation. CONCLUSION: This study provides a theoretical basis for the comprehensive development of Limosilactobacillus reuteri and new ideas for the non-pharmacological treatment of gout. © 2024 Society of Chemical Industry.

Functional Analysis of Stress Resistance of Bacillus cereus SCL10 Strain Based on Whole-Genome Sequencing.

A Gram-positive, rod-shaped, aerobic, motile, and spore-forming bacterium, designated SCL10, was isolated from Acaudina molpadioides exposure to Co-60 radiation. In this study, whole-genome sequencing was performed to identify the strain as Bacillus cereus and functional characterization, with a focus on stress resistance. The genome of the B. cereus SCL10 strain was sequenced and assembled, revealing a size of 4,979,182 bp and 5167 coding genes. The genes involved in biological functions were annotated by using the GO, COG, KEGG, NR, and Swiss-Prot databases. The results showed that genes related to alkyl hydroperoxide reductase (ahpC, ahpF), DNA-binding proteins from starved cells (dps), spore and biofilm formation (spoVG, spo0A, gerP), cold shock-like protein (cspC, cspE), ATP-dependent chaperone (clpB), and photolyase, small, acid-soluble spore protein (SASP) and DNA repair protein (recA, radD) could explain the stress resistance. These findings suggest that antioxidant activity, sporulation, biofilm formation, and DNA protection may be considered as the main resistance mechanisms under exposure to radiation in the B. cereus SCL10 strain.

Fluorescence resonance energy transfer in atomically precise metal nanoclusters by cocrystallization-induced spatial confinement.

Understanding the fluorescence resonance energy transfer (FRET) of metal nanoparticles at the atomic level has long been a challenge due to the lack of accurate systems with definite distance and orientation of molecules. Here we present the realization of achieving FRET between two atomically precise copper nanoclusters through cocrystallization-induced spatial confinement. In this study, we demonstrate the establishment of FRET in a cocrystallized Cu8(p-MBT)8(PPh3)4@Cu10(p-MBT)10(PPh3)4 system by exploiting the overlapping spectra between the excitation of the Cu10(p-MBT)10(PPh3)4 cluster and the emission of the Cu8(p-MBT)8(PPh3)4 cluster, combined with accurate control over the confined space between the two nanoclusters. Density functional theory is employed to provide deeper insights into the role of the distance and dipole orientations of molecules to illustrate the FRET procedure between two cluster molecules at the electronic structure level.

Tuna Dark Muscle Feeding Improved the Meat Quality of Holland Mini-Piglets and Modulated the Gut Microbiota.

Pork is one of the most widely produced and consumed meats in the world, and it is also an important source of animal protein. The continuous rise in feed prices has forced the pig industry to consider adding cost-effective alternative feed to pig diets. In this study, we aimed to explore the beneficial effects of tuna dark muscle as a nutritional supplement on the growth performance, serum lipids and antioxidant levels of Holland mini-piglets, as well as on the odor and volatile substances of pork and the gut microbiota. Two-month-old male mini-piglets (n = 24) were fed a control diet or supplemented with either 2% (LD) or 4% (HD) tuna dark muscle for 8 weeks. The use of tuna dark muscle at low and high dosages significantly increased the average daily weight gain, but it showed no significant effect on organ indices or blood lipids. In addition, dark muscle treatment significantly increased the antioxidant capacity, characterized by increased SOD and GSH-Px activities, and it decreased the content of MDA in serum. Moreover, tuna dark muscle feeding shifted the odor of rib muscle and tendon meat away from that of the control group, while similar odor patterns were observed in the longissimus dorsi muscle. Among these volatile substances, hexanal, nonanal, and heptanal increased in response to dietary tuna dark muscle and were regarded as indispensable contributors to the feeding. Furthermore, dietary tuna dark muscle modulated the gut microbiota of the piglets, increasing the abundance of beneficial bacteria such as butyric acid-producing bacteria, and reduced the abundance of harmful bacteria. The feeding strategy reported in this study not only reduces the production costs of pork but also utilizes tuna processing by-products in an environmentally friendly way.

Whole genome sequencing analysis of Limosilactobacillus reuteri from the intestinal tract of mice recovering from ulcerative colitis and preliminary study on anti-inflammatory effects of its derived peptides.

Limosilactobacillus reuteri is an indigenous inhabitant of the animal gut known for its probiotic effects on the host. In our previous study, a large number of L. reuteri strains were isolated from the gastrointestinal tract of mice recovering from ulcerative colitis, from which we randomly selected L. reuteri RE225 for whole genome sequencing to explore its probiotic properties. The results of next-generation sequencing and third-generation single molecule sequencing showed that L. reuteri RE225 contained many genes encoding functional proteins associated with adhesion, anti-inflammatory and pathogen inhibition. And compared to other L. reuteri strains in NCBI, L. reuteri RE225 has unique gene families with probiotic functions. In order to further explore the probiotic effect of the L. reuteri RE225, the derived peptides were identified by LC-MS/MS, and the peptides with tumor necrosis factor-α binding ability were screened by reverse molecular docking and microscale thermophoresis. Finally, cell experiments demonstrated the anti-inflammatory ability of the peptides. Western blotting and qPCR analyses confirmed that the selected peptides might alleviate LPS-induced inflammation in NCM460 cells by inhibiting JAK2/STAT3 pathway activation.

Rapid and simultaneous multiepitope antigen-based detection of Enterococcus by microscale thermophoresis and immunomagnetic separation.

Background: Generally, enterococci bacteria cause nosocomial infections and are major indicators of bacterial contamination in marine bathing beach. However, a method for the rapid and simultaneous detection of multiple pathogenic enterococci has not been developed on account of the wide variety of pathogenic enterococci and their existence in complex matrices. Methods: Immunoinformatics tools were used to design a multi-epitope antigen for the detection of various pathogenic enterococci by using the sequence of dltD gene on enterococci lipoteichoic acid (LTA) surface, which is associated with toxicological effects. The multi-epitopes included enterococci such as Enterococcus faecalis, E. gallinarum, E. raffinosus, E. durans, E. faecium, E. hirae, E. thailandicus, E. casseliflavus, E. avium, E. mundtii, E. lactis, E. solitarius, E. pseudoavium, and E. malodoratum. Microscale thermophoresis (MST) and western blot were carried out to detect the affinity between multi-epitope antigens and antibodies and between multi-epitope antibodies and bacteria. Furthermore, the detection of pathogenic enterococci was carried out by using immunomagnetic beads (IMBs) and immune chromatographic test strip (ICTS). Results: The multi-epitope antibody had a satisfactory affinity to the antigen and enterococci. IMBs and ICTS were detected with a minimum of 101 CFU/mL and showed incompatibility for Vibrio parahemolyticus, V. vulnifcus, V. harveyi, V. anguillarum, and Edwardsiella tarda. Implication: The present study demonstrated that the multi-epitope antigens exhibited excellent specificity and sensitivity, making them highly suitable for efficient on-site screening of enterococci bacteria in marine bathing beaches.

Analysis of antimicrobial biological activity of a marine Bacillus velezensis NDB.

A new strain of Bacillus velezensis NDB was isolated from Xiangshan Harbor and antibacterial test revealed antibacterial activity of this strain against 12 major pathogenic bacteria. The whole genome of the bacterium was sequenced and found to consist of a 4,214,838 bp circular chromosome and a 7410 bp circular plasmid. Furthermore, it was predicted by AntiSMASH and BAGEL4 to have 12 clusters of secondary metabolism genes for the synthesis of the inhibitors, fengycin, bacillomycin, macrolactin H, bacillaene, and difficidin, and there were also five clusters encoding potentially novel antimicrobial substances, as well as three bacteriocin biosynthesis gene clusters of amylocyclicin, ComX1, and LCI. qRT-PCR revealed significant up-regulation of antimicrobial secondary metabolite synthesis genes after 24 h of antagonism with pathogenic bacteria. Furthermore, MALDI-TOF mass spectrometry revealed that it can secrete surfactin non-ribosomal peptide synthase and polyketide synthase to exert antibacterial effects. GC-MS was used to analyze methanol extract of B. velezensis NDB, a total of 68 compounds were identified and these metabolites include 16 amino acids, 17 acids, 3 amines, 11 sugars, 11 alcohols, 1 ester, and 9 other compounds which can inhibit pathogenic bacteria by initiating the antibiotic secretion pathway. A comparative genomic analysis of gene families showed that the specificity of B. velezensis NDB was mainly reflected in environmental adaptability. Overall, this research on B. velezensis NDB provides the basis for elucidating its biocontrol effect and promotes its future application as a probiotic.

A strategy to boost xanthine oxidase and angiotensin converting enzyme inhibitory activities of peptides via molecular docking and module substitution.

Molecular docking and activity evaluation screened the dipeptide module GP with low xanthine oxidase (XOD) inhibitory activity and modules KE and KN with high activity, and identified them as low- and high-contribution modules, respectively. We hypothesized the substitution of low-contribution modules in peptides with high contributions would boost their XOD inhibitory activity. In the XOD inhibitory peptide GPAGPR, substitution of GP with both KE and KN led to enhanced affinity between the peptides and XOD. They also increased XOD inhibitory activity (26.4% and 10.3%) and decreased cellular uric acid concentrations (28.0% and 10.4%). RNA sequencing indicated that these improvements were attributable to the inhibition of uric acid biosynthesis. In addition, module substitution increased the angiotensin-converting enzyme inhibitory activity of GILRP and GAAGGAF by 84.8% and 76.5%. This study revealed that module substitution is a feasible strategy to boost peptide activity, and provided information for the optimization of hydrolysate preparation conditions.

Gallic acid ameliorates colitis by trapping deleterious metabolite ammonia and improving gut microbiota dysbiosis.

Gut microbiota dysbiosis is causally related to inflammatory bowel disease (IBD), and increased levels of the gut metabolite ammonia have been proposed to contribute to IBD development. In this study, we aimed to clarify the anti-colitis mechanism of gallic acid (GA) based on its ability to trap the deleterious metabolite ammonia and improve gut microbiota. Aminated product was detected in the fecal samples of mice after oral gavage of gallic acid (GA) and identified as 4-amino-substituted gallic acid (4-NH2-GA), thus confirming the ability of GA to trap ammonia in vivo. Then, we compared the beneficial effects of GA and 4-NH2-GA on dextran sulfate sodium (DSS)-induced colitis mouse and found that both compounds managed to alleviate colitis phenotypes, indicating ammonia trapping had no adverse effect on the original anti-colitis activity of GA. In addition, both GA and 4-NH2-GA improved the gut microbiota dysbiosis induced by DSS, and fecal microbiota transplantation was subsequently performed, which further revealed that the gut microbiota mediated the anti-colitis activity of both GA and 4-NH2-GA. In summary, this study clarified that GA alleviated colitis by targeting both the symptoms and root causes: it directly reduced the deleterious metabolite ammonia by forming aminated metabolites without compromising the original anti-colitis activity, and it also improved gut microbiota dysbiosis, which in turn contributed to the alleviation of colitis. Since the GA structure is presented in various polyphenols as a common building block, the novel anti-colitis mechanism obtained from GA may also apply to other complex polyphenols.IMPORTANCEThe dysbiosis of the gut microbiota and its metabolism directly cause the emergence of IBD. In this study, we aimed to clarify the anti-colitis mechanism of GA in sight of gut microbiota and its metabolite ammonia. We discovered that GA directly captured and reduced the harmful metabolite ammonia in vivo to produce the aminated metabolite 4-NH2-GA, while the amination of GA had no adverse effect on its initial anti-colitis activity. In addition, both GA and its aminated metabolite improved the gut microbiota in colitis mice, and the modified gut microbiota, in turn, helped to relieve colitis. Since the GA structure is presented in diverse polyphenols as a common building block, the novel anti-colitis mechanism targeting the symptoms and root causes might also apply to other complex polyphenols.

Comparison of Lactiplantibacillus plantarum isolates from the gut of mice supplemented with different types of nutrients: a genomic and metabolomic study.

Many studies have focused on the influence of dietary supplements on gut microbiota composition, but limited research have reported their effects on specific bacterial species in the gut. Lactiplantibacillus plantarum is one of the most widely studied probiotics, with a wide range of sources and good environmental adaptability. In this study, in order to elucidate the adaptation strategies of L. plantarum to the gut of mice supplemented with carbohydrates, peptides and minerals, whole genome resequencing and intracellular metabolites detection were performed, and high-frequency mutant genes and differential metabolites were screened. The results suggested different types of dietary supplements do have different effects on L. plantarum from the gut of mice. Additionally, KEGG annotation unveiled that the effects of these dietary supplements on the gene level of L. plantarum primarily pertained to environmental information processing, while the differential metabolites were predominantly associated with metabolism. This study provided new perspectives on the adaptive mechanism of L. plantarum in response to the host's gut environment, suggesting that the diversity of the genome and metabolome of L. plantarum was correlated with dietary supplements. Furthermore, this study offered useful guidance in the effective utilization of dietary supplements.

Disabling spidroin N-terminal homologs' reverse reaction unveils why its intermolecular disulfide bonds have not evolved for 380 million years.

Spiders, ubiquitous predators known for their powerful silks, rely on spidroins that self-assemble from high-concentration solutions stored in silk glands, which are mediated by the NT and CT domains. CT homodimers containing intermolecular disulfide bonds enhance silk performance, promoting spider survival and reproduction. However, no NT capable of forming such disulfide bonds has been identified. Our study reveals that NT homodimers with sulfur substitution can form under alkaline conditions, shedding light on why spiders have not evolved intermolecular disulfide bonds in the NT module during their 380 million years of evolution. This discovery significantly advances our comprehension of spider evolution and silk spinning mechanisms, while also providing novel insights into protein storage, assembly, as well as the mechanisms and therapeutic strategies for neurodegenerative diseases associated with protein aggregation.

Structural and Biochemical Characterization of Silver/Copper Binding by Dendrorhynchus zhejiangensis Ferritin.

Ferritin with a highly symmetrical cage-like structure is not only key in the reversible storage of iron in efficient ferroxidase activity; it also provides unique coordination environments for the conjugation of heavy metal ions other than those associated with iron. However, research regarding the effect of these bound heavy metal ions on ferritin is scarce. In the present study, we prepared a marine invertebrate ferritin from Dendrorhynchus zhejiangensis (DzFer) and found that it could withstand extreme pH fluctuation. We then demonstrated its capacity to interact with Ag+ or Cu2+ ions using various biochemical and spectroscopic methods and X-ray crystallography. Structural and biochemical analyses revealed that both Ag+ and Cu2+ were able to bind to the DzFer cage via metal-coordination bonds and that their binding sites were mainly located inside the three-fold channel of DzFer. Furthermore, Ag+ was shown to have a higher selectivity for sulfur-containing amino acid residues and appeared to bind preferentially at the ferroxidase site of DzFer as compared with Cu2+. Thus, it is far more likely to inhibit the ferroxidase activity of DzFer. The results provide new insights into the effect of heavy metal ions on the iron-binding capacity of a marine invertebrate ferritin.

Zinc-Catalyzed Asymmetric Cascade Michael/Acyl Transfer Reaction between α-Hydroxy Aryl Ketones and Enynones.

We described herein a neoteric enantioselective cascade Michael/acyl transfer reaction of enynones and α-hydroxy aryl ketones catalyzed by dinuclear zinc cooperative catalysis. A series of structurally diverse chiral 1,5-dicarbonyl compounds were synthesized in good yields with excellent stereoselectivities. This strategy features broad substrate scope, high atom economy, as well as enynones as efficient electrophilic acyl transfer reagents in asymmetric cascade reactions for the first time.

Analyzing the mechanism by which oyster peptides target IL-2 in melanoma cell apoptosis based on RNA-seq and m6A-seq.

Melanoma is a kind of skin cancer with high malignancy and strong proliferation and invasion abilities. Chemotherapy drugs in the clinic have the disadvantages of high price and high toxicity. Peptides are natural active ingredients that have many functions and are safe and effective. Previous studies have shown that oysters are rich in protein and have antitumor effects. In this study, a high-throughput strategy combined with MALDI TOF/TOF-MS and molecular docking was developed to screen peptides with antitumor functions from oyster hydrolysate. Three dominant peptides were predicted to have similar functions to IL-2 via molecular docking. Then, the activity of the peptides was confirmed in B16 cells, and we found that the three peptides increased the apoptosis of B16 cells. Furthermore, via RNA-seq and m6A-seq of B16 cells treated with the peptides, we found that ILADSAPR downregulates the expression of Pcna, Tlr4, and Ncbp2 and upregulates the expression of Bax, Bad, Pak4, Rasa2, Cct6, and Gbp2. ILADSAPR inhibited B16 cell proliferation and promoted cell apoptosis by regulating the expression of these genes. In addition, the result of metabolic pathway analysis also proved this point. This study provides a preliminary reference for antitumor research on oyster peptides.

Spatial distribution of gut microbiota in mice during the occurrence and remission of hyperuricemia.

BACKGROUND: Previous studies have shown that anserine can alleviate hyperuricemia by changing the fecal microbiota of hyperuricemic mice. TOPIC: However, the fecal microbiota could not fully represent the distribution of the whole gut microbiota. Knowing the spatial distribution of the gastrointestinal tract microbiota is therefore important for understanding its action in the occurrence and remission of hyperuricemia. METHODS: This study provides a comprehensive map of the most common bacterial communities that colonize different parts of the mouse gastrointestinal tract (stomach, duodenum, ileum, cecum, and colon) using a modern methodological approach. RESULTS: The stomach, colon, and cecum showed the greatest richness and diversity in bacterial species. Three clusters of bacterial populations were observed along the digestive system: (1) in the stomach, (2) in the duodenum and ileum, and (3) in the colon and cecum. A high purine solution changed the composition and abundance of the digestive tract microbiota, and anserine relieved hyperuricemia by restoring the homeostasis of the digestive tract microbiota, especially improving the abundance of probiotics in the digestive tract. IMPLICATION: This could be the starting point for further research on the regulation of hyperuricemia by gut microbiota with the ultimate goal of promoting health and welfare. © 2022 Society of Chemical Industry.

Comparison of the gut microbiota and metabolism in different regions of Red Swamp Crayfish (Procambarus clarkii).

Background: The gut microbiota is very important for maintaining the homeostasis and health of crustaceans. Many factors affect the gut microbiota of crustaceans, one of which is temperature. However, it is currently unclear how temperature affects the gut microbiota and metabolites of Procambarus clarkii. Methods: Using metagenomic sequencing and gas chromatography-mass spectrometry (GC-MS) techniques, the gut microbiota and metabolites of P. clarkii from Hubei (HB), Jiangsu (JS), Shandong (SD), and Zhejiang (ZJ) in China were investigated. Results: Under the impact of temperature, the gut microbiota and metabolites of P. clarkii exhibit a specific trend of change. The primary pathogenic bacteria affecting P. clarkii are Citrobacter, Enterobacterium, and Aeromonas, which are affected by temperature. Two metabolites, namely, sugars and amino acids, are regulated by temperature. Implication: This study demonstrated that the gut microbiota and gut metabolites of P. clarkii were considerably affected by temperature. It provides a theoretical basis for the systematic study of P. clarkii and provides a basis for a healthy culture of P. clarkii.

Structural and Functional Insights into the Roles of Potential Metal-Binding Sites in Apostichopus japonicus Ferritin.

Ferritin is widely acknowledged as a conservative iron storage protein found in almost all living kingdoms. Apostichopus japonicus (Selenka) is among the oldest echinoderm fauna and has unique regenerative potential, but the catalytic mechanism of iron oxidation in A. japonicus ferritin (AjFER) remains elusive. We previously identified several potential metal-binding sites at the ferroxidase center, the three- and four-fold channels in AjFER. Herein, we prepared AjFER, AjFER-E25A/E60A/E105A, AjFER-D129A/E132A, and AjFER-E168A mutants, investigated their structures, and functionally characterized these ferritins with respect to Fe2+ uptake using X-ray techniques together with biochemical analytical methods. A crystallographic model of the AjFER-D129A/E132A mutant, which was solved to a resolution of 1.98 Å, suggested that the substitutions had a significant influence on the quaternary structure of the three-fold channel compared to that of AjFER. The structures of these ferritins in solution were determined based on the molecular envelopes of AjFER and its variants by small-angle X-ray scattering, and the structures were almost consistent with the characteristics of well-folded and globular-shaped proteins. Comparative biochemical analyses indicated that site-directed mutagenesis of metal-binding sites in AjFER presented relatively low rates of iron oxidation and thermostability, as well as weak iron-binding affinity, suggesting that these potential metal-binding sites play critical roles in the catalytic activity of ferritin. These findings provide profound insight into the structure-function relationships related to marine invertebrate ferritins.

Gut microbiome and metabolome analyses reveal the protective effect of special high-docosahexaenoic acid tuna oil on d-galactose-induced aging in mice.

Aging is closely related to altered gut function and its microbiome composition. To elucidate the mechanisms involved in the preventive effect of special high-docosahexaenoic acid tuna oil (HDTO) on senescence, the effects of different doses of HDTO on the gut microbiome and metabolome of d-galactose-induced aging mice were studied. Deferribacteres and Tenericutes and uridine might be used as indicator bacteria and characteristic metabolites to identify aging, respectively. HDTO markedly improved the impaired memory and antioxidant abilities induced by d-galactose. At the phylum level, the abundance of Firmicutes and Tenericutes was significantly increased upon d-galactose induction, while that of Bacteroidetes, Proteobacteria, and Deferribacteres was significantly decreased. At the genus level, the variation mainly presented as an increase in the abundance of the Firmicutes genera Ligilactobacillus, Lactobacillus, and Erysipelothrix, the decrease in the abundance of the Bacteroidetes genera Bacteroides and Alistipes, the Firmicutes genus Dielma, and the Deferribacteres genus Mucispirillum. HDTO supplementation reversed the alterations in the intestinal flora by promoting the proliferation of beneficial flora during the aging process; the metabolic pathways, such as glycine-serine-threonine metabolism, valine-leucine-isoleucine biosynthesis, and some metabolic pathways involved in uridine, were also partially restored. Furthermore, the correlation analysis illustrated an obvious correlation between gut microbiota, its metabolites, and aging-related indices. Moreover, it is worth noting that the metabolic regulation by dietary intervention varied with different HDTO doses and did not present a simple additive effect; indeed, each dose showed a unique modulation mechanism.

Prediction of the generalization of myasthenia gravis with purely ocular symptoms at onset: a multivariable model development and validation.

Background: About half of myasthenia gravis (MG) patients with purely ocular symptoms at onset progress to generalized myasthenia gravis (gMG). Objectives: To develop and validate a model to predict the generalization of MG at 6 months after disease onset in patients with ocular-onset myasthenia gravis (OoMG). Methods: Data of patients with OoMG were retrospectively collected from two tertiary hospitals in Germany and China. An accelerated failure time model was developed using the backward elimination method based on the German cohort to predict the generalization of OoMG. The model was then externally validated in the Chinese cohort, and its performance was assessed using Harrell's C-index and calibration plots. Results: Four hundred and seventy-seven patients (275 from Germany and 202 from China) were eligible for inclusion. One hundred and three (37.5%) patients in the German cohort progressed from OoMG to gMG with a median follow-up time of 69 (32-116) months. The median time to generalization was 29 (16-71) months. The estimated cumulative probability of generalization was 30.5% [95% CI (confidence interval), 24.3-36.2%) at 5 years after disease onset. The final model, which was represented as a nomogram, included five clinical variables: sex, titer of anti-AChR antibody, status of anti-MuSK antibody, age at disease onset and the presence of other autoimmune disease. External validation of the model using the bootstrap showed a C-index of 0.670 (95% CI, 0.602-0.738). Calibration curves revealed moderate agreement of predicted and observed outcomes. Conclusion: The nomogram is a good predictor for generalization in patients with OoMG that can be used to inform of the individual generalization risk, which might improve the clinical decision-making.