Lactobacillus acidophilus, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, and Lactobacillus delbrueckii) intake. The results obtained showed an increase in the number of members of the phylum Actinobacteriota in the group taking nutritional supplements, while the number of phylum Bacteroidota decreased in comparison with the control group. There has also been an increase in potentially beneficial groups: Bifidobacterium, Blautia, Eubacterium, Anaerostipes, Lactococcus, Lachnospiraceae ND3007, Streptococcus, Escherichia-Shigella, and Lachnoclostridium. Along with this, a decrease in the genera was demonstrated: Faecalibacterium, Pseudobutyrivibrio, Subdoligranulum, Faecalibacterium, Clostridium sensu stricto 1 and 2, Catenibacterium, Megasphaera, Phascolarctobacterium, and the Oscillospiraceae NK4A214 group, which contribute to the development of various metabolic disorders. Modulation of the gut microbiota by lactic acid bacteria may be one of the ways to treat obesity.Obesity is a problem of modern health care that causes the occurrence of many concomitant diseases: arterial hypertension, diabetes mellitus, non-alcoholic fatty liver disease, and cardiovascular diseases. New strategies for the treatment and prevention of obesity are being developed that are based on using probiotics for modulation of the gut microbiota. Our study aimed to evaluate the bacterial composition of the gut of obese patients before and after two weeks of lactic acid bacteria ( The gut microbiome strongly contributes to the overall health of the host including the risk of developing obesity. Although our knowledge about the composition of the human microbiome is evolving quite rapidly, there are many questions about the metabolic mechanisms and functionality of the human microbiome. It is known that probiotics have a positive effect in the context of various diseases, particularly those associated with gastroenterology . A deepeThe prevalence of obesity worldwide continues to rise . Even thSince weight loss drugs often have specific toxic side effects, it is necessary to look for more moderate and effective substitutes. Some research suggests that the gut microbiota is directly linked to obesity and taking probiotics may have a positive impact on weight loss ,10.Lactic acid bacteria are often used as probiotics. Many species of lactic acid bacteria can boost nutrient absorption, improve the functioning of the gastrointestinal tract, lower serum cholesterol levels, prevent obesity, inhibit the growth of putrefactive bacteria in the intestine, and improve immune function ,12,13,14Lactobacillus acidophilus is one of the main commercially important species of lactic acid bacteria used as a probiotic [L. acidophilus promotes nutrient absorption, has a beneficial effect on digestive functions, and prevents diarrhea [robiotic . L. aciddiarrhea .L. plantarum may benefit human health [L. plantarum can lower cholesterol levels in adults with hypercholesterolemia, potentially reducing the risk of coronary heart disease [L. plantarum often exhibits the broadest ability to inhibit pathogen growth among the Lactobacilli species [There is growing evidence that n health . For exa disease . Additio species ,20.L. fermentum is one of the most promising probiotics. It is often used as a typical reference species in comparative investigations with different probiotics due to its health benefits [L. fermentum in various diseases such as intestinal inflammation [benefits . There aammation , respiraammation , and hepammation .Despite the reported beneficial properties of these lactic acid bacteria species, supporting studies are needed to further understand the role of these probiotics in modulating gut microbial populations.It is possible that restoring the gut microbiome to a healthy state could improve the conditions associated with obesity. However, the role of probiotics in the prevention and treatment of obesity has not yet been fully explored.The purpose of this work was to study the effect of probiotic intake on the composition of the gut microbiome in obese people.At the beginning of the study, each patient completed a questionnaire that included questions about age, nutrition, physical activity, a history of infectious and non-infectious pathologies, taking medications including hormonal and antibacterial drugs (for the last 6 months), and there were clarifications on anthropometric indicators ) and waist circumference (OT). Based on the results of the survey, eight patients were selected, who were subsequently included in the group with diagnosed obesity.At the beginning of the study, each of the eight patients had a fecal sampling, approximately 20 ± 5 g. Then, each patient consumed 55 g of the supplement, which consisted of lactic acid bacteria, with food for two weeks. During this period, patients did not change their usual diet in any way. At the end of the 2-week supplementation, approximately 20 ± 5 g of feces were again collected from each patient for microbiome profiling.The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Board of Voronezh State University . Before the beginning of the study, all patients gave their written consent to the use of anonymized personal data for scientific purposes.Lactobacillus delbrueckii subsp. bulgaricus VSUET15; Lactiplantibacillus plantarum VSUET13; Lactobacillus acidophilus VSUET12; and Limosilactobacillus fermentum VSUET14 for further placement in the Microbiological Museum of the Voronezh State University of Engineering Technologies.Probiotic bacteria were obtained from LLC “White Lily” . After purification, the bacterial cultures received the following numbers: Streptococcus thermophilus was added. The activation of microbial cells of the concentrate occurred due to mixing and further 4 h exposure at a constant temperature of 37 ± 1 °C, cyclically stirring (shaking) for a uniform distribution of bacterial cells in the mixture. Then, the produced concentrate was immediately added to pasteurized  and chilled (37–42 °C) skimmed milk with constant stirring. The density of microorganisms in the finished product was adjusted to 108 CFU/mL. Subsequently, the cultures were kept at a temperature of −u °C for no more than three days. The viable cells of lactic acid bacteria were accounted on MRS agar.For subsequent work, the bacteria were added to 100 mL of sterilized skimmed milk. To improve the fermentation process in the mixture of bacteria, A commercial Quick-DNA Fungal/Bacterial Microprep Kit  was used to obtain a DNA product from a biomaterial.16S rRNA gene.Analysis of the bacterial diversity of the intestinal microbiome by sequencing based on the Ion Torrent PGM platform was carried out for the V3 hypervariable region of the For further PCR analysis, two universal primers 337F and 518R  for further preparation of the sequencing libraries. This is a necessary step for preparing high-quality sequencing libraries.For the preparation of sequencing libraries, a commercial NEBNext Fast DNA Kit  was used in the work according to the method provided by the manufacturer. In further work, the resulting library was barcoded with a commercial NEXTflex Kit . Then, another cleaning of the finished libraries was carried out with AMPureXP magnetic particles . The Ion Torrent PGM platform was used for sequencing. Libraries were loaded onto an Ion 318™ Chip v2 BC using the standard protocols of the Ion PGM Hi-Q View OT2 Kit for further enrichment using emulsion PCR and the Ion PGM Hi-Q View Sequencing Kit for post-processing and loading libraries onto the chip as well as for running the instrument itself .The obtained analysis results were processed using the R programming language in the R Studio environment .At the initial stage, BAM files were generated for each sample, which were later modified into the FastQ format based on the fileexporterwin plugin.To exclude low-quality reads from the subsequent analysis, we used the maximum expected error threshold, all reads for which values of less than 1.0 were eliminated (DADA2 package). In addition, the reads were truncated to an optimized total size, followed by demultiplexing. After all the manipulations, the reads were dereplicated and, as a result, all identical reads were combined into unique sequences. Then, based on the UNOISE2 algorithm, we formed operational taxonomic units (OTUs).https://www.arb-silva.de/, accessed on 14 March 2022). The taxonomy was determined relative to the peak limit of identity with variant amplicon sequences corresponding to 100%. All raw sequences obtained from the analyses for each sample are available in the BioProject repository (Bio Project:PRJNA862929).Generic identification was carried out using version 132 of the SILVA database . The statistical significance of differences in the microbiome profiles of patients was achieved using the Mann–Whitney U test. The average abundance of individual taxa was expressed as the mean ± standard error of the mean (SEM). The intra-sample alpha diversity score was expressed using Shannon’s test.In total, 128,390 unique reads were obtained during sequencing, corresponding to 103 bacterial genera .We analyzed the microbiome composition at the phylum level for the study groups .The phylum Firmicutes dominated in both cases. Before taking lactic acid bacteria, its abundance was 0.569 ± 0.072; after taking it, it decreased to 0.498 ± 0.048. The phylum Actinobacteriota was the next most numerous both before and after taking the lactic acid bacteria supplement. In the control group, its abundance was 0.272 ± 0.025, while in the experimental group, it was 0.436 ± 0.036. The abundance of the phylum Bacteroidota before taking the supplement was 0.155 ± 0.078; after, it was—0.061 ± 0.020. Proteobacteria were next, with an abundance of 0.003 ± 0.001 and 0.005 ± 0.001, respectively. The number of Verrucomicrobiota before taking lactic acid bacteria was 0.0008 ± 0.001; after, it was — 0.0002 ± 0.001. The Desulfobacterota phylum was the least numerous; in both groups, its abundance was less than 0.005.We found statistically significant differences before and after the intake of lactic acid bacteria for the two phyla .The abundance of the phylum Actinobacteriota increased significantly after supplementation with lactic acid bacteria, while the abundance of Bacteroidota decreased.Bacterial genera whose abundance was less than 0.005 were grouped into “Other Genus”. We analyzed the differences among all identified genera of bacteria for each study group. Twenty-two of the most common bacterial genera were identified for the group of obese patients participating in a study evaluating the effects of a nutritional supplement containing lactic acid bacteria on the gut microbiome composition .Faecalibacterium with a population of 0.294 ± 0.05 was also the most common genus. The next largest genera were Prevotella—0.203 ± 0.072; Bifidobacterium—0.084 ± 0.019; Subdoligranulum—0.062 ± 0.014; Blautia—0.037 ± 0.011; Bacteroides—0.035 ± 0.006; Roseburia—0.032 ± 0.006; Catenibacterium—0.031 ± 0.005; Fusicatenibacter—0.024 ± 0.006; Collinsella—0.022 ± 0.006; Clostridium sensu stricto 2—0.022 ± 0.006; Phascolarctobacterium—0.020 ± 0.005; Holdemanella—0.019 ± 0.005; Pseudobutyrivibrio—0.017 ± 0.001; Megasphaera—0.013 ± 0.002. The average abundance of other genus in the group of patients with obesity was less than 0.001.We detected a different microbiome landscape in the group of patients with obesity. Bifidobacterium—0.346 ± 0.070 after a two-week intake of lactic acid bacteria in obese patients. The next largest genera were Blautia—0.142 ± 0.033; Streptococcus—0.132 ± 0.014; Collinsella—0.088 ± 0.038; Faecalibacterium—0.046 ± 0.009; Bacteroides—0.034 ± 0.013; Fusicatenibacter—0.028 ± 0.004; Eubacterium hallii group—0.026 ± 0.004; Prevotella—0.024 ± 0.007; Roseburia—0.021 ± 0.005; Anaerostipes—0.014 ± 0.003; Subdoligranulum—0.012 ± 0.006; Ruminococcus—0.011 ± 0.005. The number of other bacterial genera was less than 0.010.We observed a microbiome landscape dominated by the genus We also evaluated the alpha diversity before and after lactic acid bacteriaintake by obesity group of patients .p < 0.001).The alpha diversity index before taking the lactic acid bacteria was 2.540 ± 0.031, while after taking it decreased to 2.351 ± 0.035 , Blautia—0.142 ± 0.033 vs. 0.037 ± 0.011 (p < 0.01), Streptococcus—0.132 ± 0.014 vs. 0.004 ± 0.000 (p < 0.01), Eubacterium hallii group—0.026 ± 0.005 vs. 0.003 ± 0.001 (p < 0.01), Anaerostipes—0.013 ± 0.003 vs. 0.002 ± 0.001 (p < 0.01), Dialister—0.004 ± 0.001 vs. 0.000 ± 0.000 (p < 0.05), Lactococcus—0.002 ± 0.001 vs. 0.000 ± 0.000 (p < 0.05), Escherichia-Shigella—0.003 ± 0.001 vs. 0.000 ± 0.000 (p < 0.05), Lachnospiraceae ND3007 group—0.005 ± 0.001 vs. 0.001 ± 0.000 (p < 0.05), Erysipelotrichaceae UCG-003—0.002 ± 0.000 vs. 0.001 ± 0.000 (p < 0.05), and Lachnoclostridium—0.005 ± 0.002 vs. 0.001 ± 0.000 compared with the pre-supplement microbiome control obesity group, respectively.After taking lactic acid bacteria, we observed a statistically significant increase in the genus Faecalibacterium—0.046 ± 0.009 vs. 0.294 ± 0.051 (p < 0.01), Prevotella—0.024 ± 0.007 vs. 0.203 ± 0.072 (p < 0.05), Subdoligranulum—0.012 ± 0.006 vs. 0.062 ± 0.014 (p < 0.01), Holdemanella—0.000 ± 0.000 vs. 0.019 ± 0.005 (p < 0.01), Oscillospiraceae UCG-002—0.001 ± 0.000 vs. 0.004 ± 0.000 (p < 0.01), Alistipes—0.001 ± 0.000 vs. 0.004 ± 0.001 (p < 0.05), Pseudobutyrivibrio—0.006 ± 0.002 vs. 0.017 ± 0.001 (p < 0.05), Ruminococcaceae CAG-352—0.001 ± 0.000 vs. 0.003 ± 0.000 (p < 0.01), Clostridium sensu stricto 2—0.001 ± 0.000 vs. 0.022 ± 0.006 (p < 0.01), Catenibacterium—0.000 ± 0.000 vs. 0.031 ± 0.005 (p < 0.01), Parabacteroides—0.001 ± 0.000 vs. 0.007 ± 0.001 (p < 0.01), Megasphaera—0.002 ± 0.001 vs. 0.013 ± 0.002 (p < 0.01), Phascolarctobacterium—0.000 ± 0.000 vs. 0.020 ± 0.005 (p < 0.01), Christensenellaceae R-7 group—0.000 ± 0.000 vs. 0.003 ± 0.000 (p < 0.01), Monoglobus—0.000 ± 0.000 vs. 0.001 ± 0.000 (p < 0.05), Solobacterium—0.000 ± 0.000 vs. 0.006 ± 0.001 (p < 0.01), Oscillospiraceae NK4A214 group—0.000 ± 0.000 vs. 0.001 ± 0.000 (p < 0.05), Oscillospiraceae UCG-005—0.000 ± 0.000 vs. 0.001 ± 0.000 (p < 0.01), and Clostridium sensu stricto 1—0.000 ± 0.000 vs. 0.001 ± 0.000 (p < 0.05) after taking the lactic acid bacteria compared to the obesity control, respectively.At the same time, we observed a decrease in the number for the genus The phylum Actinobacteriota, whose increase we observed after the ingestion of lactic acid bacteria, includes members that can have a beneficial effect on intestinal health. It is also known that there is an inverse correlation of the number of this phylum with indicators characteristic of irritable bowel syndrome ,26.After 14 days of lactic acid bacteria intake, the number of the Bacteroidota phyla decreased. Members of this type are the main colonizers of the gastrointestinal tract. Many of these have the ability to produce butyrate, which is involved in maintaining gut health . HoweverBifidobacterium, Blautia, Eubacterium hallii group, Anaerostipes, Lactococcus, and Lachnospiraceae ND3007 group. In contrast, the number of representatives of some other beneficial genus  decreased after probiotics in obese patients.In our study, we observed changes in the abundance of some potentially beneficial bacteria, particularly the genera Bifidobacterium are commensal microorganisms found in the human gastrointestinal tract and are traditionally considered beneficial to human health [Bifidobacterium species were involved in the reduction of body weight and fat mass in Sprague-Dawley rats fed a high-fat diet [Bifidobacterium spp. potentially have anti-obesity properties.Members of the genus n health . A studyfat diet . This suBlautia are well-known as butyrate-producing bacteria. This metabolite may explain the beneficial role of these bacteria in glucose metabolism and inflammation associated with obesity [Members of the genus  obesity ,33.Eubacterium hallii group is a butyrate-producing bacterium [Roseburia) that produce butyrate from monosaccharides, E. hallii can produce butyrate from lactate acetate in a low pH environment [E. hallii improved the insulin sensitivity in severely resistant db/db mice insulin. In addition, E. hallii has been shown to contribute to changes in SCFA production and bile acid composition. The ability to exert these effects suggests that E. hallii is capable of beneficially affecting insulin sensitivity [acterium . Unlike ironment . In a stsitivity .Anaerostipes is the most efficient consumer of lactate in the human colon [Anaerostipes.an colon . It was an colon , and canan colon . These dLactococcus is a species of lactic acid bacteria that produce lactic acid as a result of the fermentation of carbohydrates [Lactococcus are widely used as starter bacteria in the production of cheese and other fermented dairy products. However, there have been several studies showing the possibility of the presence of strains of Lactococcus in the flora of the gastrointestinal tract of humans or animals [Lactococcus lactis and Lactococcus garvieae, which are able to produce antimicrobial agents [Lactococcus including Lactococcus lactis and Lactococcus petauri have probiotic properties [Lactococcus.The genus hydrates . Many sp animals ,41. Theyl agents . Some spoperties ,44. All Lachnospiraceae ND3007 group are putative producers of short-chain fatty acids (SCFAs) [Lachnospiraceae ND3007 group has been associated with a decrease in the blood glucose levels in rats with diabetes [Members of the  (SCFAs) . It has diabetes .Subdoligranulum are SCFA producers and are conditionally positive for the body [Subdoligranulum is positively associated with overall metabolic health including lower body mass index and fat mass in adult humans [It is known that bacteria of the genus the body . It has t humans .Holdemanella is considered to be a potential health contributor [Holdemanella with the consumption of fermented dairy products, carbohydrates, and fiber was previously reported [The genus tributor . A positreported .Parabacteroides are saccharolytic bacteria and produce major fermentation end products such as acetic acid and succinic acid. According to numerous studies, the relative abundance of Parabacteroides has been negatively associated with BMI [Parabacteroides goldsteinii and Parabacteroides distasonis are considered as promising probiotics that may alleviate obesity and metabolic dysfunctions associated with obesity [Members of the genus with BMI ,51,52,53 obesity . HoweverClostridium sensu stricto 2 and Solobacterium. Furthermore, an increase in the relative abundance of the potentially pathogenic genus Streptococcus was observed.As a result of taking probiotics, we observed a decrease in some pathogenic bacteria such as Clostridium sensu stricto 2 was identified in human feces and clinical specimens such as blood, peritoneal and pleural fluid as well as lung biopsies from lung infections [Previously, the bacterial genus fections . Membersfections . Thus, tSolobacterium is a Gram-positive obligate anaerobic genus in the family Erysipelotrichidae with one known species. This genus is part of the salivary microbiome, but is also found in small amounts in the colon [he colon ,58, whiche colon ,60Streptococcus species are considered pathogenic, but Streptococcus thermophilus, for example, is a probiotic [S. thermophilus was in the composition of the final product containing lactic acid bacteria . S. thermophilus is known to ferment lactose and sucrose, and can also metabolize fructose monosaccharide [Most robiotic . S. therccharide . High frccharide .Dialister and Lachnoclostridium. Furthermore, some bacteria with a role associated with obesity decreased including Faecalibacterium, Alistipes, Prevotella, Catenibacterium, Megasphaera, Phascolarctobacterium, Christensenellaceae R-7 group, Monoglobus, and the Oscillospiraceae NK4A214 group.In our study, we found an increase in obesity-associated bacteria such as Dialister bacteria was lower in the overweight group compared to the lean group [Dialister play some role in the development of obesity, but more detailed studies of these bacteria are needed to understand the exact mechanisms.A study by Yanrong Lv et al. showed that the abundance of an group . In our Lachnoclostridium bacteria led to lower levels of circulating acetate, which ultimately led to increased visceral fat. However, the study only looked at women and also only measured the changes in acetate, not all SCFAs [Lachnoclostridium increased in obese patients after taking probiotics. This suggests that more research is needed on the role of Lachnoclostridium bacteria in obesity.In a study by Ana Nogal et al., increased levels of ll SCFAs . We foun16S rRNA gene sequencing) with obesity or type 2 diabetes mellitus showed that the reduced activity of Faecalibacterium, Alistipes is significant in relation to obesity [Studies using the analysis of the association of individual microbial genera , where a significant difference was demonstrated between the obese and control group [Prevotella in people with obesity [Prevotella may be useful on a low-calorie diet, but in turn can lead to obesity on a fairly high-calorie diet. The results of studies demonstrate the association of the abundance of the Prevotella enterotype with a long-term diet that included a high intake of soluble carbohydrates and simple sugars and a low intake of proteins, amino acids, and saturated fats [ol group . Hu H.J. obesity . It has  obesity . The revted fats ,72.Catenibacterium were significantly higher in obese participants, while the bacterial genus Alistipes was reduced [The study by Martínez-Cuesta et al. (2021) noted that bacteria of the genus  reduced .Megasphaera is a genus of Gram-negative, non-motile, coccoid, opportunistic bacteria [bacteria . It is abacteria . Howeverbacteria . A direcbacteria ,77,78.Phascolarctobacterium is a relatively numerous genus of bacteria in the human gastrointestinal tract. A study in obese rats showed that an increase in Phascolarctobacterium was positively correlated with an increase in body weight, fat mass, plasma leptin, triglycerides, and glucose tolerance [olerance . It is aolerance . In addiolerance .Christensenellaceae R-7 group genus are saccharolytic, and they can ferment carbohydrates with the accumulation of a mixture of SCFAs [Christensenellaceae R-7 group and body weight and visceral fat content [Christensenellaceae R-7 is believed to be reduced in patients suffering from intestinal diseases, we noted a reduction in this genus with a probiotic mixture of lactic acid bacteria.Most members of the of SCFAs . Studies content ,83,84. AMonoglobus is one of the main specialized taxa involved in the fermentation of pectin and mannan in the human colon [Monoglobus in the effect of metabolic disorders. It is known to be inversely correlated with BMI as well as with the development of ulcerative colitis [The genus an colon ,86. Ther colitis ,88.Oscillospiraceae NK4A214 group genus has a direct correlation with obesity, regardless of the cohort studied, and therefore is a contender for one of the prognostic genera for obesity [According to the literature data, the abundance of the  obesity .The role of some bacteria, the number of which changed in our study, is still poorly understood.Escherichia and Shigella are two closely related genera that share bioenergetic mechanisms that allow them to fill a specific niche in the gut microbiome ecosystem [Escherichia-Shigella includes four Escherichia species, four Shigella species, and several lineages currently unclassified as “Escherichia cryptic clades” [Escherichia-Shigella with a wide range of functions. Thus, this does not allow us to draw clear conclusions about the role played by the increase in the relative abundance of bacteria of the genus Escherichia-Shigella in the case of obesity after the ingestion of lactic acid bacteria.cosystem . The gen clades” . All of  clades” ,93,94. IOscillospiraceae UCG-002 was demonstrated after two weeks of lactic acid supplementation. Bacteria of the genus Oscillospiraceae UCG-002 are known to significantly contribute to aspartate degradation and glycine degradation [In our study, a reduction in bacteria belonging to the genus radation .Ruminococcaceae CAG-352 also showed a decrease after two weeks of lactic acid bacteria supplementation. The genus Ruminococcaceae CAG-352 is known to be Gram-positive bacteria [The bacterial genus bacteria . We did Oscillospiraceae UCG-005, we observed a decrease in abundance after lactic acid bacteria supplementation, but the relationship of this genus with any metabolic pathologies in humans is poorly understood. Further research is needed to shed light on the contribution of this genus to gut health.For the genus Clostridium sensu stricto 1 to human gut health is poorly understood. There are data that have found a direct correlation of Clostridium sensu stricto 1 with acute pancreatitis in rats [Clostridium sensu stricto 1 may be responsible for the visceral hypersensitivity seen in irritable bowel syndrome [The contribution of  in rats  and with in rats . It is asyndrome . FurtherErysipelotrichaceae UCG-003, so it is difficult to say exactly what role members of this genus play.Little is known about the role of bacteria of the genus L. acidophilus VSUET12, L. plantarum VSUET13, L. fermentum VSUET14, and Lactobacillus delbrueckii subsp bulgaricus VSUET15, which was taken by obese people, had a significant effect on changing the gut microbiome landscape. According to the results of the study, we observed at the phylum level, a decrease in Bacteroidota and an increase in Actinobacteriota, both of which belong to the main phylum that form the core of the microbiome. At the generic level, a 14-day intake of the mixture of lactic acid bacteria led to the enrichment of the potentially beneficial groups Bifidobacterium, Blautia, E. halli group, Anaerostipes, Lactococcus, and Lachnospiraceae ND3007. We also observed an increase in the genera Streptococcus, Escherichia-Shigella, and Lachnoclostridium, which contributed to gut health and is highly member-dependent.The studied mixture of lactic acid bacteria, consisting of strains of Faecalibacterium, Pseudobutyrivibrio, Subdoligranulum, Faecalibacterium, Clostridium sensu stricto 1 and 2, Catenibacterium, Megasphaera, Phascolarctobacterium, Oscillospiraceae NK4A214 group, which can be associated with the development of various metabolic disorders as well as inflammatory bowel diseases. At the same time, the abundance of beneficial genera such as Subdoligranulum, Holdemanella, Alistipes, Parabacteroides, Christensenellaceae R-7, Monoglobus, and Solobacterium also decreased. The decrease in the genera Prevotella, Oscillospiraceae UCG-002, and UCG-005, Ruminococcaceae CAG-352 cannot be interpreted unambiguously. The effect of some of them on the host organism depends on the specific species and strain, while others are poorly understood.We also found a decrease in the genera Thus, based on the data obtained, we cannot unambiguously conclude on the beneficial effect of the mixture of the studied lactic acid bacteria on the gut of obese people. Positive microbiome changes were observed against the background of the intake, but along with these, there were changes in the gut microbiota that could not be interpreted as positive.


The separation was carried out on an ACQUITY UPLC®HSS T3 column , using methanol and 5 mmol/L ammonium acetate water as the mobile phase. The three compounds were quantified using the multiple reaction monitoring mode with the electrospray ion source in both the positive and negative mode. Liquid-liquid extraction was applied to the plasma sample preparation. Bifendate was selected as the internal standard. The intra-day and inter-day precision and the accuracy of the method were all within receivable ranges. The lower limit of quantification of ziyuglycoside I, 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-d-glucopyranosyl ester, and pomolic acid were 6.50, 5.75, and 2.63 ng/mL, respectively. The extraction recoveries of analytes in rat plasma ranged from 83 to 94%. The three components could be rapidly absorbed into the blood  both in the single-administration group or S. officinalis extract group, but the first peak of PA occurred at 0.5 h and the second peak at 4–5 h in the S. officinalis extract. Three compounds were eliminated relatively slowly . The research was to establish a rapid, sensible, and sensitive UHPLC–MS/MS method using the multi-ion mode for multi-channel simultaneous mensuration pharmacokinetics parameters of three compounds in rats after oral administration of S. officinalis extract. This study found, for the first time, differences in the pharmacokinetic parameters of the three compounds in the monomer compounds and S. officinalis extract administration, which preliminarily revealed the transformation and metabolism of the three compounds in vivo.A selective and rapid ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was established and validated for the determination of ziyuglycoside I, 3 Sanguisorba officinalis L. , belonging to the Rosaceae family and Sanguisorba genus, has detoxification, analgesic + can acquire strong and steady signals. To improve the sensitivity and enhance the response, the negative mode of [M − H]− at m/z 471.2 was more appropriate for PA. To obtain a good resolution, an ACQUITY UPLC®HSS T3  column was adopted to separate the three analytes. In summary, two scanning time intervals with positive and negative modes were adopted: positive (ESI+: 0–5.0 min) and negative (ESI−: 5.0–6.5 min) ion modes. The main reason for segmental detection of the three compounds was to protect the capillary. Finally, the separating conditions were set with methanol (mobile phase A) and 5 mM ammonium acetate (mobile phase B) as the mobile phase at a flow rate of 0.3 mL/min.Both the negative and positive ion modes were checked by different mobile phases. Both The gradient elution program was as follows: 0–4.5 min, 70–90% B; 4.5–6.0 min, 90% B; 6.0–6.3 min, 90–70% B; 6.3–6.5 min, 70% B. The total running time was 6.5 min, and the injection volume was 10 μL.S. officinalis extract, as listed in The selectivity was appraised by comparing the chromatograms of blank plasma, a plasma sample spiked with LLOQ analytes and IS, and a plasma sample from rats following single oral administration of monomers and Y = 1.522X + 1.390 × 10−1 for ZGI, Y = 4.734X + 3.194 × 10−2 for DGE, Y = 0.685X + 6.882 × 10−3 for PA, where X means the plasma concentration of analytes and Y represents the peak area ratio of analytes to IS. As shown in r > 0.9960. The results showed that the compounds were within the linearity ranges. The LLOQ of ZGI, DGE, and PA were 6.50, 5.75, and 2.63 ng/mL, respectively.The typical equations of the calibration curves were RSD) of the three analytes were not more than 13%, and the accuracies (RE) for the three analytes ranged from −0.71% to 4.7%. The precision and accuracy were in line with the relevant provisions of the biological sample analysis guidelines.The precision and accuracies of the three analytes in rat plasma at four levels are listed in The extraction recoveries and matrix effects of the three components in rat plasma are listed in The stability of rat plasma under different storage conditions was determined. The results  showed tThe blank rat plasma sample was immediately analyzed after the injection of the highest concentration calibration standard sample. The interference in the blank rat plasma sample was 6.0% at LLOQ (<20%) and 0.85% of the response for IS (<5%), which suggested that no obvious carryover effect was found under the described conditions.S. officinalis extract in rats . Based on the body surface area calculations of people and the animals and equivalent dose conversion calculations, the dosage of the rats was 0.55 g/kg. The mean plasma concentration–time curves of compounds are listed in t1/2), maximum plasma concentration (Cmax), time to reach Cmax (Tmax), and area under concentration–time curve (AUC0→t and AUC0→∞) were calculated as shown in The UHPLC–MS/MS method was successfully used in the pharmacokinetic studies of the three analytes after single dose administration of ZGI, DGE, PA, and officinalis extract. The Tmax of ZGI after consuming the extract was similar to that of after consuming pure ZGI. In contrast, the Tmax of DGE after consuming extract is higher than that of the pure DGE, suggesting that DGE in extract needs a longer time for absorption. The Tmax values of ZGI, DGE, and PA were 1.4 ± 0.20, 1.6 ± 0.20, and 4.2 ± 0.41 h in the S. officinalis extract, and 1.4 ± 0.20, 0.92 ± 0.20, 0.92 ± 0.20 h for the ZGI, DGE, and PA groups, respectively. The absorbance velocity of the ZGI, DGE, and PA was rapid; they reached Cmax between 0.92 and 2.00 h after single dose administration of ZGI, DGE, PA, and S. officinalis extract, but the first peak of PA appeared at 0.5 h and the second peak reached Cmax at 4.2 h after administration of S. officinalis extract, compared with PA group. The Cmax of ZGI, DGE, and PA in the S. officinalis extract was 1091 ± 1.1 × 102, 359 ± 51, and 703 ± 96 ng/mL, respectively. The Cmax of ZGI, DGE, and PA of the monomer groups was 678 ± 47, 166 ± 21, and 262 ± 31 ng/mL. Moreover, the AUC0→t of the three compounds was 2877 ± 1.1 × 102, 1200 ± 76, and 3315 ± 89 ng·h/mL in the S. officinalis extract, and 1716 ± 3.1 × 102, 394 ± 33, and 1021 ± 55 ng·h/mL from the ZGI, DGE, and PA groups, respectively. The AUC0→∞ of the three compounds in the S. officinalis extract was 3098 ± 1.2 × 102, 1314 ± 1.5 × 102, and 4026 ± 2.1 × 102 ng·h/mL, and the AUC0→∞ of the three compounds in the monomer groups was 2166 ± 3.3 × 102, 551 ± 47, and 1183 ± 92 ng·h/mL. The increasing Cmax, AUC0→t, and AUC0→∞ showed the better absorption of the three compounds in the S. officinalis extract. The mechanism of the difference in pharmacokinetics between the monomer groups and S. officinalis extract remains unclear. It may be inferred that some ingredients in the S. officinalis extract may enhance the absorption of the three analytes. Furthermore, the t1/2 values of ZGI, DGE, and PA were 9.3 ± 2.4, 7.3 ± 2.7, and 11 ± 1.4 h in the S. officinalis extract. The t1/2 values of ZGI, DGE, and PA were 11 ± 0.95, 11 ± 0.79, and 8.8 ± 1.0 h in the monomer groups. It was revealed that ZGI and DGE in the S. officinalis extract were more easily eliminated and metabolized than in the monomer groups. In addition, from these analysis results, we could conclude that PA has a different absorption compared with ZGI and DGE. The t1/2 of PA after consuming the extract is higher than that of the pure PA, suggesting that PA in extract needs a longer time for elimination. The Tmax of PA was at 4.2 h, which was longer than the other two compounds. However, the content of PA in S. officinalis was much lower than that of ZGI, yet the AUC0→t and AUC0→∞ of PA were greater than those of ZGI and DGE. The reason for these results was probably that ZGI and DGE metabolized PA in vivo.Notably, as S. officinalis in the rat. Therefore, this research established a rapid, sensible, and sensitive UHPLC–MS/MS method for simultaneous determination and investigation of three compounds in rats after oral administration of S. officinalis extract. Meanwhile, the rats were given the three monomers; respectively, it was found that the three monomers have a transformational relationship and affect their pharmacokinetic parameters, to explore the process of the transformation of the three components in the rat.Few previous studies indicated that a method was developed to simultaneously determine ZGI, DGE, and PA from For rapid, sensible, and simultaneous determination of three compounds, a UHPLC–MS/MS method was optimized. First, different proportions of mobile phase systems were tested, and ammonium acetate (5 mM) water was opted for, to enhance the peak strength of the three analytes. To realize a high extraction yield and weaken the matrix effect, LLE was chosen as the sample extraction method. As a result of the three compounds with similar polarities, ethyl acetate was the best option for extraction efficiency and repeatability. In addition, it is worth mentioning that to improve the sensitivity and accuracy, the mass spectrometer adopts multi-channel positive and negative ion mode to simultaneously detect a variety of compounds, which greatly optimizes the UHPLC–MS/MS method.t1/2. The t1/2 of both ZGI and DGE monomers were lower than those of S. officinalis extract. The reason may be that the presence of ZGI and DGE in the extract may be converted into PA in vivo, increasing the amount of PA and reducing the elimination rate of PA. In contrast, the conversion of ZGI and DGE in S. officinalis extracts reduces the amount in the body, so the elimination rate of these two compounds was improved. From the Tmax, the absorption rates of the three compounds can be reflected. It is worth noting that the DGE in S. officinalis extract was absorbed for a longer time, but the absorption amount was higher than that of the pure compound. The interactions between compounds of herbal extracts in vivo may affect the absorption of target compounds [The elimination rate of the three compounds can be reflected from the ompounds  The reasIn addition, according to the data in S. officinalis extract. The pharmacokinetic and transformation characteristics of the three compounds in S. officinalis were initially revealed in vivo. However, the relevant mechanism of its specific metabolic behaviors needs further study.In this work, a rapid, sensible, and sensitive UHPLC–MS/MS method was established with the multi-ion mode, multi-channel simultaneous mensuration, and further applied to the pharmacokinetic studies of three compounds in rats after oral administration of Sanguisorba officinalis L., which was purchased from the Anguo Traditional Chinese Medicine Market of Hebei, was appraised by Professor Zhenyue Wang of Heilongjiang University of Chinese Medicine.The ZGI, DGE, and PA, with purity of more than 98%, were refined in our laboratory (identified by NMR and MS) . Bifendan = 6).The experimental protocol was permitted by the Animal Ethics Committee of Harbin Medical University  and conformed to the principles for the Care and Use of Laboratory Animals. Twenty-four male Sprague-Dawley rats (weighting 200 ± 20 g), which were provided by the Laboratory Animal Center at Harbin Medical University , were used for the pharmacokinetic study. The animals were maintained in a SPF-grade room on a 12 h light/dark cycle with a controlled temperature conditions (21 ± 2 °C) and relative humidity (50 ± 5%) before the experiment. During the adaptation process, all rats were provided with standard food and purified water each morning and evening. Each rat fasted for 12 h before giving the drug and was randomly divided into one of four groups  solution two times at 85 °C, 1 h each time, and was then filtrated. The combined filtrate was evaporated into steam, and the residue was dissolved in water to obtain the concentration of the S. officinalis extract equivalent to 0.025 g/mL.After smashing the dried root of +: 0.0–5.0 min) and negative (ESI−: 5.0–6.5 min) ion modes with MRM. The transitions were m/z 784.5→437.4 for ZGI, m/z 652.5→455.4 for DGE, m/z 471.2→453.2 for PA, and m/z 418.9→342.8 for bifendate (IS). The mobile phases were as follows: solvent A was 5 mM ammonium acetate water, and solvent B was methanol, which was delivered at a flow rate of 0.3 mL/min. The gradient elution program was as follows: 0–4.5 min, 70 to 90% B; 4.5–6.0 min, 90% B; 6.0–6.3 min, 90–70% B; 6.3–6.5 min, 70% B. The sample injection volume was 10 μL.The analytes were evaluated on an Agilent series 1290 UHPLC instrument combined with an Agilent Technologies 6430 mass spectrometer. The eluant was surveyed by applying a triple quadrupole tandem mass spectrometer equipped with an ESI source and operated in positive (ESI2) flow rate, 11 L/min; drying gas temperature, 300 °C; high purity nitrogen (N2) was atomized as the nebulizing gas; capillary voltage, 4000 V. The mass parameters for the three analytes and IS are listed in The conditions for MS analysis were as below: drying gas . Standard solutions were prepared by compatible dilutions of the stock solutions with methanol . The IS solution (520 ng/mL) was prepared by diluting stock solution of methanol. Calibration standards were prepared by spiking each working stock solution at seven concentrations of 6.5, 13.0, 26.0, 130.0, 260.0, 520.0, and 2600 ng/mL for ZGI; 5.8, 11.5, 23.0, 115.0, 230.0, 460.0, and 2300 ng/mL for DGE; and 2.6, 5.3, 10.5, 52.5, 105.0, 210.0, and 1050 ng/mL for PA. Quality control (QC) samples were prepared at 13.0, 130.0, and 2080 ng/mL for ZGI, 11.5, 115.0, and 1840 ng/mL for DGE, and 5.3, 52.5, and 840 ng/mL for PA. All solutions were immediately stored at 4 °C.2 blowing at 40 °C. After stirring for 4 min at 3800 rpm, the residue was reassembled with 100 μL methanol, then mixed by vortexing for 120 s and filtered by a 0.22 μm organic membrane. Finally, 10 μL sample solution was injected into the UHPLC–MS/MS system [The supernatant was separated by adding 10 μL IS (520 ng/mL) solution into 100 μL plasma sample, vortexing for 30 s, and mixing with 3 mL ethyl acetate by being vortex-mixed for 60 s. After centrifuging, the supernatant was dried by Nhttps://www.fda.gov/media/70858/download (accessed on 21 May 2018) [The method was appraised with the FDA guidelines, ay 2018) .S. officinalis extract.Selectivity evaluation was conducted by comparing chromatograms of blank plasma samples from six individual rats, corresponding blank plasma spiked with LLOQ of the three analytes and IS, and the plasma samples from the rats after oral administration of the three analytes and x2) least square linear regression, using standard plasma samples. The lower limit of quantification (LLOQ) was the lowest analytical concentration of the calibration curve, for which the signal-to-noise ratio was >10.The calibration curves were constructed by plotting the peak area ratio versus the concentration of the three analytes and IS with a weighted  and relative error (RE), respectively. The RSD values should not exceed 15% for the QC samples, except for the LLOQ, which should not exceed 20%. The RE values should be within ±15% of the nominal values for the QC samples, except for the LLOQ, which should be within ±20% of the nominal value.The extraction recovery of analytes was determined by comparing the peak areas of the three analytes from the QC samples with those obtained from blank plasma samples with the three analytes spiked into the post-extraction supernatant at three QC levels in six replicates. The matrix effect was evaluated by comparing the peak areas of analytes spiked after plasma extraction into the post-extraction supernatants at three QC levels in six replicates. The extraction recovery and matrix effects of IS were also determined at one concentration. The RSD values should be within ±15% of the nominal values for the QC samples and IS, except for the LLOQ, which should be within ±20% of the nominal value.The stability of the three compounds in rat plasma, including freeze and thaw stability (three freeze-thaw cycles at −20 °C), long-term stability (storage for 2 weeks at −80 °C), room temperature stability (storage for 4 h at ambient temperature), and post-preparation stability (storage for 12 h after sample preparation at 4 °C), was tested at two QC levels with six replicates at each level. All stability testing QC samples were determined using the calibration curve of freshly prepared standard samples.The carryover effect was assessed by injecting a blank rat plasma sample after calibration of the standard sample at the upper limit of quantification. Carryover in a blank rat plasma sample should not be greater than 20% of response at the LLOQ for samples and 5% of response for IS.S. officinalis extract , ZGI , DGE , and PA  was administrated to the rats. The S. officinalis extract was dissolved in water. A single dose of 0.55 g/kg of S. officinalis extract was administrated to the rats. Blood was obtained from the retinal venous plexus at 0, 0.083, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, 12.0, and 24.0 h after dosing. The plasma was immediately separated by centrifugation at 8000 rpm for 4 min at −4 °C.A single dose of the Cmax) and the time to attain it (Tmax) were observed directly from the measured data. The elimination rate constant (Ke) was calculated by linear regression of the terminal points in a semi-log plot of the plasma concentration against time. The area under plasma concentration–time curve (AUC0→t) to the last measurable plasma concentration (Ct) was estimated using the linear trapezoidal rule.The maximum concentration  . All resS. officinalis in rat plasma. This is the first report of a pharmacokinetic study of these three triterpenes together in vivo following the oral administration of ZGI, DGE, PA, and S. officinalis extract. This paper may be useful for further studies on the metabolism and absorption process of S. officinalis extract in vivo, and may also be beneficial for the application of this TCM in clinical therapy.This study demonstrated that a simple, rapid, and sensitive LC–MS/MS method was successfully developed for simultaneous quantification of the three representative components from ZGI, DGE, PA, and


To evaluate pelvic floor muscle strength using surface electromyography and risk factors for pelvic floor muscle strength in the early postpartum period.This retrospective study included 21,302 participants who visited FujianMaternity and Child Health Hospital from September 2019 to February 2022. All participantswere assessed bymedical professionals for general information and surface electromyography.Univariate analysis indicated that age was inversely related to tonic and endurance contractions. In contrast, all the other variables, including education level, body mass index, neonatal weight, and number of fetuses, had a positive impact on rapid, tonic, and endurance contractions. Likewise, parity was also positively associated with rapid contractions. In addition, compared with vaginal delivery, cesarean section delivery had a protective effect on the amplitude of the three types of contractions. Stepwise regression analysis showed that both age and neonatal weight had a negative linear relationship with the amplitude of rapid, tonic and endurance contractions. In contrast, the amplitude of rapid, tonic and endurance contractions significantly increased as body mass index, parity (≤ 3), education level and gestational weight gain (endurance contractions only) increased. Participants with cesarean section delivery showed positive effects on rapid, tonic, and endurance contractions compared to participants with vaginal delivery.r pelvic floor muscle strength; in contrast, body mass index, parity (≤ 3) and gestational weight gain had a positive relationship with pelvic floor muscle strength.We found that age, neonatal weight, vaginal delivery, episiotomy, and forceps delivery were risk factors fo The pelvic floor is composed of three layers of muscles combined with ligaments and fascia that act as a sling to support the bladder, reproductive organs, and rectum , 2. The Therefore, it is important to assess PFM strength and determine the factors that may affect it. The aim of this study was to evaluate PFM strength using surface electromyography (EMG) and risk factors for PFM strength in the early postpartum period.This retrospective study included 21,302 participants who visited Fujian Maternity and Child Health Hospital from September 2019 to February 2022. All participants were assessed by medical professionals for general information and surface EMG. The inclusion criteria were as follows: participants who were 40 days to 6 months postpartum and who could tolerate a gynecological examination [In China, the assessment of EMG has been routinely used to evaluate pelvic floor conditions for years . A humanAll statistical analyses were performed using SPSS software version 26.0. Univariable analysis for categorical and continuous parameters was performed with chi square tests and t tests, respectively. The greater the absolute value of the standardized regression coefficient (β), the greater the influence of the corresponding independent variable on the dependent variable. Stepwise regression analysis was used to assess the relationship between the independent and dependent variables. For all tests, a two-tailed p value < 0.05 was considered statistically significant.A total of 4511 participants were excluded, and 21,302 participants were included in this analysis. The mean age, height, weight, body mass index (BMI), gestational weight gain (GWG), and neonatal weight (NW) were 30.43 ± 4.035 years, 160.37 ± 5.240 cm, 59.78 ± 8.084 kg, 23.23 ± 2.834 kg, 12.88 ± 4.651 kg, 3.27 ± 0.516 kg respectively. There were 9066 (42.6%) participants who were younger than 29 years, 11,809 (55.4%) who were aged 30–39 years, and 427 (2.0%) who were 40–49 years. A total of 590 (2.8%) participants had a BMI less than 18.5, 13,002 (61.0%) had a BMI from 18.5–23.9, 6508 (30.6%) had a BMI from 24–27.9, and 1202 (5.6%) had a BMI from more than 28. A total of 13,211 (62.0%) participants had a parity of one, 7348 (34.5%) had a parity of two, 698 (3.3%) had a parity of three, and 45 (0.2%) had a parity more than three. A total of 3834 (18.0%) participants received less than 12 years of education, and 17,468 (82.0%) received more than 12 years. A total of 20,086 (94.3%) of the infants weighed less than 4 kg, and 1216 (5.7%) weighed more than 4 kg. A total of 20,860 (97.9%) participants had single births and 442 (2.1%) had twin or triplet births. A total of 7664 (36.0%) participants had a cesarean section (CS), 10,481 (49.2%) had a noninstrumental vaginal delivery (NIVD), 2600 (12.2%) had an episiotomy (EP), and 557 (2.6%) had a forceps delivery (FD). The baseline demographic features are summarized in Table P < 0.001, and P < 0.001, respectively). In contrast, all the other variables, including education level, BMI, NW, and NOF, had a positive impact on rapid contractions, tonic contractions, and endurance contractions . Likewise, parity was also associated with rapid contractions (P < 0.001), and the average strength significantly increased as the number of parities increased. In addition, CS delivery also had a protective effect on PFM strength, including the three types of contractions, compared with NIVD , EP , and FD   Table .Table 2CP < 0.001; β = -0.107, P < 0.001; β = -0.109, P < 0.001, respectively; β = -0.034, P < 0.001; β = -0.015, P < 0.05; β = -0.020, P < 0.01, respectively). Secundiparas showed a positive effect on rapid, tonic, and endurance contractions compared with primiparas . All factors showed even positive values for tertiparas . In contrast, BMI, education level and GWG (endurance contractions only) also showed a positive linear relationship with three types of contractions . Participants with CS delivery showed a positive effect on rapid, tonic, and endurance contractions compared with participants with NIVD , EP , and FD .  , 34. UniThe literature on the association between parity and the risk of PFDs indicates that multiparas are more likely to develop PFDs –43. UnliSome studies have shown a significant relationship between educational level and PFM strength , 48. LikAs NW increases, the possibility of PFDs also increases. Previous studies have shown that excessive NW might harm PFM strength and was an independent risk factor for PFDs , 50. StePrevious studies reported that vaginal delivery increased the risk of PFM dysfunction compared with cesarean delivery , 54. LimIn this study, we found that age, NW, NIVD, EP, and FD were risk factors for PFM strength. Although BMI, parity (≤ 3) and GWG had a positive relationship with PFM strength, this is likely due to body’s adaptation ability and self-repair ability, rather than the benefits of weight gain or parity.Only female participants were included; the number of multiparas (≥ 3) was too small to observe the changes in PFM strength when parity continued to grow, the assessment of surface EMG alone cannot reflect the overall function of the pelvic floor, and we lacked assessments of pelvic floor associated scales to assess the participants’ clinical symptoms, which made it difficult to relate our results to the clinic.


The TCGA molecular groups of endometrial carcinoma are “POLE‐mutated” (POLEmut), “microsatellite‐instable/mismatch repair‐deficient” (MSI/MMRd), “TP53‐mutated/p53‐abnormal” (TP53mut/p53abn), and “no specific molecular profile” (NSMP).Prognostic assessment of the TCGA groups in uterine carcinosarcoma (UCS).Systematic review from January 2000 to January 2021.Studies assessing the TCGA groups in UCS.P < 0.050.Progression‐free survival (PFS) and overall survival (OS) were assessed by Kaplan–Meier and Cox analyses (reference: TP53mut/p53abn group) and compared with endometrioid and serous carcinomas , with a significant P < 0.001) but similar OS (P = 0.788), whereas NSMP UCS showed similar PFS (P = 0.936) and OS (P = 0.240). Compared with their endometrioid/serous counterparts, NSMP and TP53mut/p53abn UCS showed significantly worse PFS (P < 0.001 and P = 0.004) and OS (P < 0.001 and P < 0.001), while MSI/MMRd UCS showed similar PFS (P = 0.595) but significantly worse OS (P < 0.001). The POLEmut group showed neither recurrences nor deaths in both the UCS and the endometrioid/serous carcinoma cohorts.Five studies with 263 UCS were included. Compared with TP53mut/p53abn UCS, MSI/MMRd UCS showed significantly better PFS (POLEmut UCS show excellent prognosis, whereas TP53mut/p53abn and NSMP UCS show a prognosis even worse than that of TP53mut/p53abn endometrioid/serous carcinomas. The prognosis of MSI/MMRd UCS remains to be defined. POLEmut UCS show the same excellent prognosis as POLEmut endometrioid carcinomas, while TP53mut/p53abn and NSMP UCS show a prognosis even worse than TP53mut/p53abn endometrioid/serous carcinomas. The epithelial component reflects the endometrial carcinoma histotypes, such as serous (most common), endometrioid, clear cells, mixed, and undifferentiated. The stromal component may be “non‐otherwise specified” or reflect the typical uterine sarcomas; in these cases, the stromal component is defined as “homologous”. If a differentiation towards extrauterine mesenchymal tissues is present, the stromal component is defined as “heterologous”.The classification of UCS has changed over time. Indeed, UCS had previously been listed among the “mixed Müllerian tumors” and was lumped together with uterine sarcomas in terms of staging and treatment.Among UCS, the vast majority (>70%) fall into the TP53mut/p53abn group, which is consistent with its aggressive behavior.2a priori, based on previous studies.Methods of this study were defined 2.1Four electronic databases  were searched from January 2000 to January 2021 for all studies assessing the TCGA molecular signatures in UCS. We adopted the following word combination:  AND  AND (TCGA OR ProMisE OR ultramutated OR POLE OR hypermutated OR mismatch OR microsatellite OR copy number OR TP53 OR p53). We also assessed the reference lists of eligible studies. We included all studies that assessed the TCGA groups and survival outcomes in UCS. Given the rarity of POLEmut UCS,2.2PICO2.3The QUADAS‐22.4P value less than 0.05 was considered significant. Statistical Package for Social Science (SPSS) 18.0 package  was used for the analyses.PFS and OS for each TCGA group in UCS were represented graphically by using Kaplan–Meier curves. Cox regression survival analysis was used to calculate hazard ratio (HR) for recurrence and death in the TCGA group of UCS. Two analyses were performed: in the first analysis, HR was calculated in each TCGA group of UCA by using the TP53mut/p53abn group as reference; in the second analysis, each TCGA group of UCS was compared with the TCGA groups in the original TCGA series published in 2013, which included 232 patients with endometrial carcinoma . A 33.1POLE sequencing. The MSI/MMRd group was assessed by MMR immunohistochemistry in three studies and by MSI testing in two studies. The TP53mut/p53abn group was assessed by p53 immunohistochemistry in two studies, by TP53 sequencing in two studies and by both methods in the remaining study. Characteristics of the included studies are reported in Table Five studies3.2POLE mutations not assessed). For the “reference standard” domain, all studies were considered at low risk of bias. For the “flow” domain, the risk of bias was considered low for four studies and unclear for one study (only part of the cases was assessed by MMR immunohistochemistry). Authors’ judgments are reported in Figure For the patient selection domain, three studies were considered at low risk of bias and two studies at unclear risk (period of enrollment not reported). For the index test domain, three studies were considered at low risk of bias and two studies at unclear risk (3.3P < 0.001), but no significant difference was found between NSMP and TP53mut/p53abn cases . Compared with the correspondent groups in the 2013 TCGA cohorts, MSI/MMRd UCS showed similar PFS , while NSMP and TP53mut/p53abn UCS showed significantly worse PFS, with HR of 5.31  and 2.18 , respectively  and 1.51 , respectively. Compared with the correspondent groups in the 2013 TCGA cohorts, MSI/MMRd, NSMP, and TP53mut/p53abn UCS showed significantly worse OS, with HR of 5.90 , 22.02 , and 3.51 , respectively  of the same group in the 2013 TCGA cohort. In the OS analysis, MSI/MMRd UCS showed a very poor prognosis similar to that of TP53mut/p53abn and NSMP UCS and worse than any 2013 TCGA group. By assessing the possible causes for such discrepancy, we noted that most MSI/MMRd UCS in the OS analysis derived from a study with an unusually high prevalence of the MSI/MMRd signature.A limitation of our results is the low number of patients in the groups other than TP53mut/p53abn, especially in the POLEmut group and in the OS analysis. Furthermore, we had no sufficient data to perform a multivariate survival analysis, because not all studies reported individual clinical data. Finally, we could not review histologic and immunohistochemical findings to confirm the diagnosis of UCS.In conclusion, the TCGA classification significantly stratifies OS and PFS in UCS. POLEmut UCS show an excellent prognosis similar to that of POLEmut endometrioid carcinomas, supporting their inclusion in the same low‐risk category for management purposes. TP53mut/p53abn and NSMP UCS show a very poor prognosis, seemingly even worse than that of serous carcinomas. Whether MSI/MMRd UCS should be considered intermediate‐risk like MSI/MMRd carcinomas remains to be defined; for this purpose, a screening with SWI/SNF protein immunohistochemistry would be useful to exclude highly aggressive dedifferentiated carcinomas, which may mimic UCS and confound the results.The authors have no conflicts of interest.The study was conceived by AT, AR, DR, DA, AS, RS, and GFZ and the protocol was developed by AT, AR, DR, DA, GA, GS, AM, AS, RS, GFZ. Electronic search was oerfirned by GS and NDA; study selection by MV and GA; data extraction by AM and DR; risk of bias assessment by PC and FI; and data analysis by AT and AR. Disagreements were resolved by AT, AR, DR, DA, GA, MV, GS, NDA, PC, FI, AM, AS, RS, and GFZ. Interpretation was by AT, AR, DR, DA, PC, FI, AM, AS, RS, and GFZ. The first draft was written by DR, DA, GA, MV, GS, NDA, and FI and was revised by AT, AR, AS, RS, PC, AM, and GFZ. The study was supervised by AT, AR, AM, AS, RS, and GFZ.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Supplementary MaterialClick here for additional data file.


The height of the temporal iris increased to 0.45 ± 0.31 mm at 0 s (p < 0.001) and returned to −0.06 ± 0.18 mm at 0.2 s. The height of the nasal iris at 0 s in the ISF group was significantly lower, and that of the temporal iris was significantly higher than the control . Iris fluttering can act as a check valve for aqueous and vitreous humor movements and can be quantified by consecutive AS-OCT images. Large amplitude iris fluttering in eyes with intrascleral fixation is important because it can lead to a reverse pupillary block.Saccadic eye movements induce movements of the aqueous and vitreous humor and iris fluttering. To evaluate iris fluttering during eye movements, anterior segment optical coherence tomography (AS-OCT) was used in 29 eyes with pars plana vitrectomy (PPV) and intrascleral fixation of an intraocular lens (ISF group) and 15 eyes with PPV and an IOL implantation into lens capsular bag (control group). The height of the iris from the iris plane (the line between the anterior chamber angles) was compared every 0.2 s after the eye had moved from a temporal to the primary position (time 0). The height of the nasal iris in the ISF group decreased to −0.68 ± 0.43 mm at 0 s ( Intrascleral fixation or ciliary suturing of an intraocular lens (IOLs) has been used on eyes without adequate support by the lens capsule ,5,6,7,8.The eye is filled with fluid, and saccadic eye movements and body motions cause movement of the intraocular fluids, which generates shear stress on the ocular tissues in contact with the fluids . The movThe anterior segment optical coherence tomographic (AS-OCT) devices were designed to record cross-sectional images of the anterior segment of the eye ,19. The We present a dynamic analysis with swept-source AS-OCT, which allowed us to evaluate and quantify the degree of iris fluttering produced by eye movements in eyes with an intrasclerally fixed IOL. The purpose of this study was to determine whether iris flutter immediately after a saccadic eye movement can be detected and quantified. Another purpose was to determine whether the movement of the iris can lead to a pupillary capture by the IOL after intrascleral fixation.This single-center, observational study was approved by the Institutional Review Committee of the Kyorin University School of Medicine (1507). It adheres to the tenets of the Declaration of Helsinki. All of the patients received a detailed explanation of the surgical and ophthalmic procedures, and all signed an informed consent form. All of the patients consented to our review of their medical records and their anonymized use in medical publications.The findings in 29 eyes of 29 patients after pars plana vitrectomy (PPV) and intrascleral fixation of an IOL (ISF group) were compared to that in 15 eyes of 15 patients after PPV combined with cataract surgery with implantation of an IOL in the lens capsular bag (control group). In the ISF group, the surgery was performed on 21 eyes with IOL dislocation, 4 eyes with lens dislocation, and 4 eyes with aphakia. In the control group, there were 6 eyes with an epiretinal membrane, 6 eyes with a macular hole, 1 eye each with a lamellar macular hole, proliferative diabetic retinopathy, and rhegmatogenous retinal detachment. The eyes with a posterior synechia, impending reverse pupillary block, and intraoperative complications, including posterior capsule rupture during cataract surgery in the control group, were excluded. The age, sex, laterality, axial length, and anterior chamber depth (ACD) were compared between the ISF and the control groups and with and without peripheral iridectomy (PI) in the ISF group.The axial length was measured with the OA2000 Optical Biometer . Swept-source AS-OCT  with a scan rate of 50,000 A-scan per second was used. The ACD was measured from the corneal endothelium to the anterior surface of the IOL along the visual axis with caliper software integrated into the AS-OCT device.Cross-sectional images of the anterior segment, including the iris, were obtained by swept-source AS-OCT (CASIA2). Consecutive images were recorded with the movie mode from both groups one month after the surgery. For this, the patient’s head was fixed to the head holders of the AS-OCT device, and the patient was instructed to fixate a target located 30 degrees laterally from the primary visual axis for 1 s and then move the eye back to the primary position and to hold this eye position for 3 s a,b. ThisThe iris height was defined as the distance to the anterior surface of the middle sector of the iris from the iris plane, which is a reference line connecting the anterior chamber angles c and mea® Vision System  under local anesthesia. The surgery of the ISF group was for a dislocated or subluxated crystalline lens or an IOL, or aphakia. For these eyes, the dislocated lens was lifted by suction of a vitreous cutter and then phacoemulsified or extracted. A dislocated acrylic or silicone IOL was cut into 2 or 3 pieces by scissors and then extracted through a transconjunctival single plane corneoscleral incision. A residual posterior hyaloid cortex was made more visible by intravitreal injection of triamcinolone acetonide , and the residual posterior hyaloid cortex was removed by suction with a vitreous cutter if it was present. The PPV and all other surgical procedures were performed by two surgeons (TK and MI) with 27-gauge instruments of the Constellation®, Alcon Laboratories, Fort Worth, TX, USA).The intrascleral fixation was performed as described by Yamane et al. ,8. The lThe control group underwent PPV combined with cataract surgery and implantation of an IOL in the lens capsular bag before the PPV. Posterior vitreous detachment, membrane peeling, internal limiting peeling with the aid of brilliant blue G, endo-photocoagulation, tamponade by air, or 20% sulfur hexafluoride (SF6) were performed as needed.The significance of the differences in the baseline characteristics and the degree and time course of the iris fluttering between the 2 groups was determined by the Wilcoxon signed rank tests, Mann–Whitney U tests, or Fisher’s exact probability tests. All statistical analyses were performed using SPSS .The differences in age, sex distribution, and axial length between the ISF and the control groups were not significant . HoweverAt the time when the AS-OCT was used to evaluate iris fluttering, one month after the implantation of the IOL, no intravitreal gas bubbles were seen in any of the eyes that had undergone a gas tamponade in both groups. p < 0.001, Wilcoxon signed-rank test, p < 0.01 was taken to be significant with Bonferroni correction, p = 0.207), to −0.24 ± 0.22 mm at 0.4 s (p = 0.163), to −0.22 ± 0.17 mm at 0.6 s (p = 0.396), and to −0.21 ± 0.17 mm at 0.8 s (p = 0.977) while the eye remained in the primary position. The height of the nasal iris in the control group decreased from the baseline at −0.06 ± 0.17 mm to −0.11 ± 0.19 mm at 0 s (p = 0.058) and returned to −0.05 ± 0.16 mm at 0.2 s (p = 0.813), to -0.08 ± 0.17 mm at 0.4 s (p = 0.041), to −0.06 ± 0.17 mm at 0.6 s (p = 0.475), and to −0.06 ± 0.16 mm at 0.8 s (p = 0.762) but were not significant (p < 0.01 was taken to be significant with Bonferroni correction). After the eye moved from the lateral to the primary position, the height of the nasal sector of the iris in the ISF group decreased significantly from the baseline at −0.20 ± 0.16 mm to −0.88 ± 0.40 mm at 0 s , 0.4 s (p = 0.931), 0.6 s (p = 0.832), and 0.8 s (p = 0.89). The amount of height change of the nasal iris from the baseline was only significant at 0 s in the ISF group, and this amount of height change of the nasal iris at 0 s was not significant group with age , ACD (p = 0.446), and axial length (p = 0.365). The amount of height change of the nasal iris from the baseline was not significant at 0 s in the control group, and this height change at 0 s was also not significant with age (p = 0.745), ACD (p = 0.788), and axial length (p = 0.176). The amount of height change of the nasal iris from the baseline was significantly greater at 0 s in the ISF group (−0.68 ± 0.43 mm) than in the control group , and then returned to −0.29 ± 0.22 mm at 0.2 s (p = 0.071), to −0.26 ± 0.18 mm at 0.4 s (p = 0.124), to −0.25 ± 0.15 mm at 0.6 s (p = 0.008), and to −0.23 ± 0.14 mm at 0.8 s (p = 0.483). The height of the temporal iris in the control group decreased from the baseline at −0.08 ± 0.16 mm to −0.05 ± 0.19 mm at 0 s (p = 0.091) and returned to −0.07 ± 0.16 mm at 0.2 s (p = 0.682), to −0.08 ± 0.14 mm at 0.4 s (p = 0.694), to −0.07 ± 0.15 mm at 0.6 s (p = 0.027), and to −0.08 ± 0.15 mm at 0.8 s (p = 0.079) but were not significant (p < 0.01 was taken to be significant with Bonferroni correction). The direction of the changes in the height of the temporal sector of the iris was in the opposite direction from that of the nasal sector. Thus, the height of the temporal iris in the eyes of the ISF group increased significantly from the baseline (−0.23 ± 0.13 mm) to 0.22 ± 0.32 mm at 0 s (p < 0.001) in the ISF group at 0 s than that in the control group (0.03 ± 0.06 mm). However, the amount of height change of the temporal iris was not significantly different between these 2 groups at 0.2 s (p = 0.084), 0.4 s (p = 0.244), 0.6 s (p = 0.019), and 0.8 s (p = 0.089). The amount of height change of the temporal iris from the baseline was only significant at 0 s in the ISF group, and this amount of height change of the temporal iris at 0 s was not significant with age , ACD (p = 0.482), and axial length (p = 0.082). The amount of height change of the temporal iris from the baseline was not significant at 0 s in the control group, and this change at 0 s was significant with axial length (p = 0.009) but not with age (p = 0.086), and ACD (p = 0.889).The amount of change in the height of the temporal iris from the baseline was significantly greater  at 0 s and returned to −0.32 ± 0.28 mm at 0.2 s (p = 0.131), −0.29 ± 0.23 mm at 0.4 s (p = 0.117), −0.25 ± 0.17 mm at 0.6 s (p = 0.67), and −0.24 ± 0.16 mm at 0.8 s (p = 0.981). The height of the nasal sector of the iris in eyes without PI decreased significantly to −0.88 ± 0.42 mm at 0 s from the baseline . Then, the height of the nasal iris returned to −0.16 ± 0.30 mm at 0.2 s (p = 1.0), −0.13 ± 0.17 mm at 0.4 s (p = 0.859), −0.15 ± 0.17 mm at 0.6 s (p = 0.362), and −0.13 ± 0.17 mm at 0.8 s . The height of the temporal iris then decreased significantly to −0.35 ± 0.22 mm at 0.2 s (p = 0.025), −0.32 ± 0.18 mm at 0.4 s (p = 0.017), −0.29 ± 0.15 mm at 0.6 s (p = 0.015), and returned to −0.24 ± 0.16 mm at 0.8 s  at 0 s. The height of the temporal iris then returned to −0.15 ± 0.14 mm at 0.2 s (p = 0.858), −0.14 ± 0.11 mm at 0.4 s (p = 0.477), −0.16 ± 0.12 mm at 0.6 s (p = 0.574), and −0.13 ± 0.17 mm at 0.8 s (p = 0.249).The height of the temporal sector of the iris in the eyes with PI increased to 0.20 ± 0.26 mm at 0 s from the baseline , 0.2 s (p = 0.444), 0.4 s (p = 0.417), 0.6 s (p = 0.764), and 0.8 s (p = 0.594). The amount of height change of the temporal iris in eyes with PI was not significantly different from that without PI at 0 s (p = 0.627), 0.2 s (p = 0.153), 0.4 s (p = 0.069), 0.6 s (p = 0.062), and 0.8 s (p = 0.417). The height change of the nasal iris from the baseline in eyes with PI was not significantly different from that of eyes without PI at 0 s . On the other hand, even if the temporal sector of the iris is lifted due to the inflow of vitreous humor into the anterior chamber, the pupil margin does not contact the IOLs. Thus, the flow of the vitreous humor into the anterior chamber on the temporal side is enhanced more than the outflow of aqueous humor on the nasal side as the valve is opened. These movements change the iris height, which then returns to the baseline level after 0.2 s. However, the heights of both the nasal and temporal sectors of the iris remain lower than the baseline and gradually return to the baseline level after 0.4 s. The decreased height of the iris indicates the process of recovery from the influx of vitreous humor into the anterior chamber.A reverse pupillary block occurs when either side of the iris is pushed posteriorly during eye movements and the pupillary margin does not stay on the anterior surface of IOL but is pushed behind the IOL by the flow of aqueous humor. A PI was thought to be able to prevent a reverse pupillary block ,27,28. IThe results indicated that the PI did not reduce iris fluttering due to eye movements; however, it is assumed the PI prevented a reverse pupillary block by blocking a homeostatic backward movement of the iris. We believe that a simulation of fluid dynamics can be reproduced using a computational model. However, in vivo and live evaluations by AS-OCT are probably more important because the conditions can vary in each case.Our study has several limitations. First, the number of patients was too few to perform meaningful statistical analyses. In addition, the types of patients in the two groups varied because of their retrospective design. Second, the AS-OCT analyses were performed one month after the surgery and lacked data for longer postoperative follow-up times. Third, we evaluated the iris height with AS-OCT, and we did not evaluate the intracameral flow directly.In conclusion, iris fluttering can be detected and quantified by examining consecutive swept-source AS-OCT images. Iris fluttering can act as a check valve for aqueous and vitreous humor movements. The greater amount of iris fluttering by eye movements in eyes after intrascleral fixation of the IOL may cause reverse pupillary block because of the absence of a lens capsular support behind the iris. A PI does not reduce the fluttering of the iris caused by eye movements, but it stabilizes the iris height to prevent a pupillary block.


Bifidobacteria is an important microbe that inhabits the human gut. It is capable of metabolizing complex compounds in the human diet. Albiflorin, an antidepressant natural product from Radix Paeoniae Alba in China, is difficult to absorb after oral administration, and its metabolism has been proven to be closely related to the gut microbiota. In this study, we demonstrated in vitro that several Bifidobacteria species were able to convert albiflorin to benzoic acid, and four esterases  from Bifidobacterium breve and Bifidobacterium longum were found through genome mining and modeled by SWISS-MODEL. B2 and B3 presented the strongest albiflorin metabolism ability. The optimal conditions, including temperature, buffer, and pH, for the conversion of albiflorin by the four esterases were investigated. Furthermore, the effect of esterase on the metabolism of albiflorin in vivo was confirmed by transplanting bacteria containing esterase B2. This study demonstrated the vital role of esterases from Bifidobacteria in the metabolism of natural compounds containing ester bonds, which could contribute to the development of new enzymes, microbial evolution, and probiotic adjuvant compounds for treatment. The esterase in Bifidobacteria may be one of the key metabolic enzymes in the metabolic process  and can hardly cross the blood–brain barrier. The level of albiflorin in the blood and brain is considerably low after oral administration . Previou process . Similaroyao Wan . Esterasoyao Wan . In contoyao Wan .Bifidobacteria species to hydrolyze albiflorin into benzoic acid and to identify the enzymes involved in this reaction. Hydrolysis seems to be a characteristic phenomenon of Bifidobacteria. Four Bifidobacteria strains found in the human intestine were chosen for the conversion of albiflorin. We detected the activity of esterases in Bifidobacterium breve and B. longum, which can hydrolyze albiflorin. By genome mining and modeling with SWISS-MODEL, four esterases were found, and they all have the core domain of esterase, but the surrounding regions are very different. The functional characterization of the four esterases, which were expressed in Escherichia coli, was performed and compared under different conditions. Further, we proved the important role of esterase B2 in affecting the metabolism of albiflorin and improving the concentration of benzoic acid in plasma by pharmacokinetic study in vivo with bacteria transplantation. This demonstrated the possible role of Bifidobacteria in albiflorin metabolism, which revealed new prospects for the development of novel enzyme preparations and probiotics specifically designed for the enhancement of the bioconversion of traditional Chinese medicinal (TCM) chemicals into biologically active compounds.This study aimed to explore the ability of probiotic Albiflorin, benzoic acid, propranolol, anaerobic medium, and MRS broth medium were all purchased from Solarbio . The purity of the compounds was higher than 98% (HPLC). Albiflorin and benzoic acid were dissolved to prepare a 0.5 mmol/L stock solution in water. HPLC-grade ammonia, acetonitrile, and methanol were obtained from Fisher Scientific .Bifidobacteria strains  were provided by the ATCC Biological Resource Center. The Bifidobacteria strains were cultured in MRS medium at 37°C under anaerobic conditions in N2 gas. The E. coli strains DH5α and BL21 (DE3) were used to clone the pET-28a plasmid and express the esterase protein, respectively. The E. coli strain was grown in Luria-Broth (LB) medium  at 37°C. If necessary, 10–15 μg/ml kanamycin (km) was added to the medium.The bacterial strains and plasmids employed in this study are listed in 18 column . The gradient elution started with 80% mobile phase A (water) and decreased to 50% mobile phase A over 5 min at a flow rate of 0.4 ml/min. Then, it was maintained at 50% for 2 min and returned to 80% mobile phase A over 1 min, where it was maintained for 10 min. Mobile phase B consisted of methanol. The column temperature and autosampler temperature were set at 30°C and 4°C, respectively. The mass spectrometer was run in multiple reaction monitoring (MRM) mode: 503.00 → 341.05 (m/z) for albiflorin (+), 121.10 → 77.10 (m/z) for benzoic acid (−), and 260.20 → 116.10 (m/z) for propranolol (−). The sample processing method was as follows: 100 μl of sample was added to 300 μl of methanol containing 50 ng/ml of internal standard (propranolol). Then, the mixture was vortexed for 30 s and centrifuged at 12,000 rpm for 10 min. The supernatant was prepared for quantitative analysis by LC-MS/MS, and the injection volume was 1 μl.The concentrations of albiflorin and benzoic acid were determined using an HPLC-MS/MS 8050 system from Shimadzu Corporation . The substance to be tested was separated in liquid phase with an Alltima CSprague-Dawley (SD) male rats weighing 200 ± 20 g and ICR mice weighting 18 ± 22 g were provided by the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences . The animals were placed in a cage with free access to chow and water. The temperature of the cage was kept at approximately 22°C with 50% humidity and a 12-h day/night cycle. Rats were fasted for 12 h before the experiment but had free access to water. This study was conducted in accordance with institutional and ethics guidelines approved by the Laboratory Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College.2 atmosphere) at 37°C for 1 h. After preincubation, 10 μl of albiflorin was transferred into 990 μl of the culture. The cultures were inactivated by high temperature and high pressure at 121°C as a negative control. The final concentrations of albiflorin in the incubation system were 0.05 mM and 0.5 mM. The cultures were incubated at 37°C for 0, 12, or 24 h. The method was performed in accordance with the previously described procedure  with an LC/MSn-IT-TOF system  in both positive and negative modes. Mass spectra were acquired in the range of m/z 100–1,000 for MS1. The MSn data were collected in automatic mode. An elution gradient was employed at a flow rate of 0.4 ml/min, with water as mobile phase A and methanol as mobile phase B, by using the following program: 0.10 min (90% A and 10% B), 3.00 min (60% A and 40% B), 7.00 min (40% A and 60% B), 9.00 min (10% A and 90% B), 9.01 min (90% A and 10% B), and 12.00 min (controller stop).Albiflorin and its metabolite were analyzed by an Alltima CBifidobacteria, B. breve ATCC15700, B. longum ATCC15697, B. animalis ATCC27673, and B. adolescentis ATCC15703 were cultured to convert albiflorin in vitro. After resuscitation, the strains were cultured overnight in MRS medium at 37°C under anaerobic conditions (N2 atmosphere), and the bacterial concentration was uniformly maintained at 3 × 106 cells/ml. The cultures were incubated at 37°C for 0, 12, and 24 h, and the final concentration of albiflorin in the incubation system was 0.05 mM. The contents of albiflorin and its metabolite benzoic acid were determined and analyzed by LC-MS/MS.Four strains of B. breve and B. longum for genes labeled “carboxylesterase” and “esterase.” By searching for genes annotated as “carboxylesterase” in the genome of B. breve ATCC15700 (NZ_AP012324.1), we found three carboxylesterase genes, NZ_AP012324.1: 1013236–1014057, NZ_AP012324.1: 506173–507018, and NZ_AP012324.1: 506173–507018. The amino acid sequences can be accessed as WP_003829196.1 , WP_003828396.1 , and WP_003828023.1 . By searching for the gene annotated as “esterase” in the genome of B. longum ATCC15697 (NC_011593.1), we found an esterase gene, NC_011593.1: c2783438-2782680, and its protein sequence can be found in KAB6720564.1 . Detailed information on each protein was obtained from the NCBI database, as shown in The NCBI databasehttp://swissmodel.expasy.org/workspace/. We used the automatic modeling mode and applied the protein sequences of four esterases, which were available in GenBank. The data obtained from the homology model were visualized using DeepView ver. 4.0.1 (The Swiss Institute of Bioinformatics).3-D structural modeling of the esterase was conducted with SWISS-MODEL,B. breve and B. longum, extraction was carried out according to the instructions of the bacterial genome extraction kit . Using the genomic DNA of B. breve and B. longum as templates, Primer Premier 5.0 was used to design primers based on the four esterase genes  and the vector pET-28a. All of the primers  are listed in B. breve and B. longum as templates, the four esterase genes were amplified by PCR and then purified along with the PCR-amplified vector DNA using the Gel Recovery Purification Kit . A Gibson Assembly Kit  was used to insert the four esterase DNA fragments into the vector pET-28a to construct the recombinant vectors. Escherichia coli DH5α cells were transformed by the heat shock method and used for plasmid propagation to obtain the recombinant plasmids pET-28a-b2, pET-28a-b3, pET-28a-b4, and pET-28a-bl. All fragments were validated by Sanger 3730 sequencing.To obtain the genomic DNA of b2, pET-28a-b3, pET-28a-b4, and pET-28a-bl were transformed into E. coli BL21 (DE3) cells. Cells were grown in liquid LB media supplemented with kanamycin (40 μg/ml). Cultures were maintained at 37°C until the OD600 nm reached 0.6. Then, the temperature was lowered to 16°C, and protein expression was induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 20 h. The bacterial cells were harvested by centrifugation , concentrated 10-fold, and suspended in approximately 10 ml of lysis buffer (10 mM PBS buffer or 25 mM Tris-HCl buffer). After ultrasonic lysis, the supernatant and precipitate were prepared for SDS-PAGE analysis, with the empty pET-28a plasmid in E. coli BL21 cells as a control. The B2 and B3 proteins were purified using the PrepEase His-tag protein purification kit .The recombinant plasmids pET-28a-Escherichia coli cells with B2, B3, B4, and BL and the cell extract supernatant were used for the bioconversion of albiflorin to characterize B2, B3, B4, and BL. Escherichia coli BL21 (DE3) transformed with the empty pET-28a vector was used as a negative control. The final concentration of albiflorin in the incubation system was 0.05 mM. The cultures were incubated at 37°C for 0, 12, and 24 h. Albiflorin and its metabolite benzoic acid were detected by LC-MS/MS. The enzymatic reaction conditions were as follows: temperatures, 30°C and 37°C; buffers, PBS buffer and Tris-HCl buffer; and pH, 6.0, 7.0, and 8.0. The LC-MS/MS conditions refer to the above experimental methods.n = 6/group) were orally administered E. coli (1 × 1010 CFU), the E. coli with esterase B2 (1 × 1010 CFU), and B. breve (1 × 1010 CFU) once a day for 3 days, respectively. Another six ICR mice were orally administered an equivalent volume of saline. Pharmacokinetic evaluations were performed 3 days after the final administration.Three groups of ICR mice , the four groups of mice were fasted overnight with free access to water. Blood samples were collected before and at 5, 10, 20, 30, 45, 60, 90, 120, 180, 240, 360, and 480 min after drug treatment.t-test. Data are expressed as the mean ± standard deviation, and p values less than 0.05 were considered statistically significant.Statistical analyses were performed with GraphPad Prism Version 5  using two-way ANOVA and Student’s n-IT-TOF was used for identifying albiflorin and BA. The mass spectrum of albiflorin is shown in m/z of the [M + Na]+ peak was 503, the m/z of the secondary fragments were 201, 307, and 341, and the tertiary ion fragment had an m/z of 175. The mass spectrum of benzoic acid is shown in + peak of benzoic acid had an m/z of 122. Moreover, different concentrations of albiflorin (0.05 and 0.5 mM) were cultured with the intestinal bacterial culture and inactivated intestinal bacterial culture at 37°C for 12 and 24 h, and the concentrations of albiflorin and benzoic acid were determined after incubation. As shown in Intestinal bacteria from SD rats were collected for anaerobic incubation in the presence of albiflorin, and the concentrations of albiflorin in the culture solution were 0.05 and 0.5 mM, respectively, with the inactivated intestinal bacteria culture functioning as a negative control. The structure of albiflorin  includesBifidobacteria strains  found among gut microbes to metabolize albiflorin under anaerobic conditions over a period of 24 h was examined. The cells were maintained at a consistent concentration with 3 × 106 cells/ml in the presence of 0.05 mM albiflorin. Samples were removed at 0, 12, and 24 h for LC-MS/MS detection. As shown in B. breve, B. longum, and B. adolescentis showed significant metabolism of albiflorin at 12 and 24 h, while B. longum had the strongest ability to metabolize albiflorin and resulted in a high conversion rate at 24 h of 18.1%, followed by the rates of B. breve (16.8%) and B. adolescentis (1.5%). Accordingly, benzoic acid was produced differently by the tested Bifidobacteria strains, resulting in 7.14 μg/ml for B. breve, 5.29 μg/ml for B. longum, and 4.96 μg/ml for B. adolescentis. According to the above results, B. breve and B. longum have a strong albiflorin metabolism ability, and we propose that both play an important role in albiflorin metabolism in the gut microbiota.The capability of four common B. breve ATCC15700 (NZ_AP012324.1), three carboxylesterase genes were found, including NZ_AP012324.1: 1013236–1014057, NZ_AP012324.1: 506173–507018, and NZ_AP012324.1: 506173–507018. The corresponding amino acid sequences are WP_003829196.1 , WP_003828396.1 , and WP_003828023.1 . By searching for the genes annotated as “esterase” in the genome of B. longum ATCC15697 (NC_011593.1), an esterase gene was found, which was named NC_011593.1:c2783438-2,782,680, and its protein identifier is KAB6720564.1 . Detailed information on each protein was obtained from the NCBI database, and the ProtParam tool by ExPASyBy searching for genes annotated as “carboxylesterase” in the genome of We found that the four enzymes, B2, B3, B4, and BL, belong to three superfamilies. As shown in B. animalis subsp. lactis WC 0432 exhibits hydrolytic activity against chlorogenic acid  for Bifidobacterium esterase expression, we first verified the absence of homologous genes in its genome by analyzing its genome, and the E. coli cells with the empty vector pET-28a were incapable of metabolizing any albiflorin (data not shown). Several primer pairs designed based on the B2, B3, B4, and BL coding sequences of B. breve ATCC15700 and B. longum ATCC15697 were used to clone the four genes  was transformed with the above expression vectors bearing four esterase genes. SDS-PAGE showed that the expression of the four esterases occurred at high levels in IPTG-induced recombinant cells  expressing the four esterases were obtained. After being cultured overnight with IPTG, the four E. coli strains were individually used to investigate albiflorin metabolism at 37°C for 24 h. The cells of each strain were controlled at a consistent concentration of OD600 nm = 2.0 in the presence of 0.05 mM albiflorin. Samples were removed at 0, 12, and 24 h for LC-MS/MS analysis. As shown in E. coli EB2 had a significant effect on albiflorin metabolism, the level of which was reduced by 31.9% at 24 h compared with that at 0 h. Moreover, the production of benzoic acid at 24 h was increased by 5.37-fold compared with that at 0 h. The concentration of benzoic acid reached 23.9 μg/ml. However, EB3, EB4, and EBL had no effect on albiflorin metabolism, and no significant increase was detected in the production of benzoic acid. This result showed the metabolic ability of the four esterases expressed by E. coli cells in suspension culture, and the E. coli strain expressing esterase B2 showed the greatest potential for albiflorin metabolism.Based on the above results, four E. coli cells expressing the four esterases were collected and resuspended in PBS buffer (pH = 7) and sonicated to obtain the B2, B3, B4, and BL proteins. The supernatants were mixed with 0.05 mM albiflorin and incubated at 37°C. Samples were taken at 0, 12, and 24 h for the detection of albiflorin and its metabolite benzoic acid. As shown in Furthermore, the Furthermore, the effects of different temperatures, buffers, and pH on the ability of the four enzymes to convert albiflorin were investigated. First, as shown in Then, the effect of the reaction conditions of the PBS buffer and Tris-HCl buffer on the conversion of albiflorin by the four enzymes was investigated. As shown in Finally, the effect of different pH buffer reaction conditions on the conversion of albiflorin by the four enzymes was investigated. As shown in E. coli 1 × 1010 CFU/day, E. coli with Esterase B2 1 × 1010 CFU/day, B. breve 1 × 1010 CFU/day), ICR mice were given a single dose of albiflorin (7 mg/kg) to investigate the differences in albiflorin and benzoic acid concentrations over time in the four groups ((0– t) of the four groups were 5,965.590 ± 2,004.089, 5,452.317 ± 1,045.409, 4,254.94 ± 1,591.654, and 4,355.606 ± 934.475 μg/L h, respectively. The Cmax of the four groups were 44.064 ± 24.12, 45 ± 10.607, 21.61 ± 5.363, and 26.528 ± 11.105 μg/L, respectively. Similar pharmacokinetic parameters were observed in the NS group and the E. coli group. The transplantation of E. coli had little effect on the drug metabolism of albiflorin. The administration of E. coli containing esterase B2 obviously affected the absorption and metabolism of albiflorin in vivo, while the administration of B. brevis containing esterase B2 also affected the metabolism of albiflorin in vivo to a certain extent. At the same time, the plasma concentration-time profiles of albiflorin metabolites benzoic acid were determined, the results showed that the benzoic acid concentration of B2 group obviously greater than the NS group, the B. breve group could also increase the benzoic acid concentration in plasma, and the benzoic acid concentration in plasma of E. coli group and NS group is similar. In vivo experiments showed that supplement of esterase B2 could affect the metabolism of albiflorin in mice and significantly increased the concentration of benzoic acid, the metabolite of albiflorin in plasma. Esterase B2 is an important enzyme for the conversion of albiflorin to benzoic acid.After 3 days of oral administration of different bacteria , and it contains abundant enzyme resources, such as α-glucosidase, β-glucosidase, hydrolase, and esterase  compared to non-depressed controls in observational studies, and depressive symptoms were improved compared to controls in interventional studies with probiotics , which is generally known as carboxylesterase, belongs to the class of hydrolytic enzymes, the function of which is to hydrolyze carboxylic acid esters : a serine, a glutamate/aspartate, and a histidine. The catalytic mechanism often involves a nucleophilic attack on a carbonyl carbon atom   the National Natural Science Foundation of China (nos. 81973290 and 82173888), Beijing Natural Sciences Fund Key Projects (no. 7181007), the Beijing Key Laboratory of Non-Clinical Drug Metabolism and PK/PD study (Z141102004414062), and the Youth Science Foundation Project from National Natural Science Foundation of China (no. 81803613).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.