Gout is a crystal-associated arthropathy caused by the deposition of monosodium urate crystals and is closely related to purine metabolic disorders and impaired uric acid excretion. It is clinically characterized by hyperuricemia， recurrent joint swelling and pain， and tophus formation. The disease course is divided into three stages： The hyperuricemia stage， acute attack stage， and chronic gouty arthritis stage. Modern medicine has reached a consensus on its pathology， but traditional Chinese medicine （TCM） lacks a systematic stage-specific understanding of gout pathogenesis and its underlying mechanisms， making it difficult to guide precise syndrome differentiation and treatment. By integrating classical TCM theory， clinical practice， and modern medical understanding， and drawing upon descriptions of Bi syndrome caused by endogenous dampness and turbidity in classical texts such as Huangdi Neijing·Ling Shu and Synopsis of the Golden Chamber， our team proposes the pathogenic concept of gout as ''endogenous dampness leading to Bi syndrome'' and the core pathogenesis of ''spleen deficiency with internal retention of dampness-turbidity''. We systematically elucidate the evolution of pathogenesis across different stages and corresponding therapeutic strategies. This study posits that metabolic byproducts such as urate fall under the category of ''endogenous pathogenic dampness-turbidity''. When genetic or dietary factors lead to metabolic abnormalities， it manifests as ''spleen deficiency with impaired transport and transformation''， resulting in ''internal retention of pathogenic dampness-turbidity''. When damp-turbidity stagnates in the blood vessels， serum uric acid levels rise. When it stagnates in the viscera and limbs， monosodium urate crystals deposit in the joints. Triggered by precipitating factors， this leads to gout attacks—the core pathological process of ''endogenous dampness leading to Bi syndrome''. Based on this theory， the stage-specific pathogenic characteristics of gout are proposed： The hyperuricemia stage is characterized by ''spleen deficiency with impaired transport and transformation， internal retention of pathogenic dampness-turbidity''， the acute attack stage is primarily marked by ''dampness-turbidity and static heat obstructing the limbs and joints''， while the chronic stage is defined by ''spleen deficiency with internal retention of pathogenic dampness-turbidity， intermingled with phlegm-stasis binding''. The treatment principle centers on ''strengthening the spleen and draining dampness'' throughout all stages. During the hyperuricemia stage， treatment focuses on ''strengthening the spleen， draining dampness， and eliminating turbidity''. In the acute attack stage， the treatment should "strengthen the spleen， drain dampness， clear heat， eliminate turbidity， alleviate swelling， and relieve pain''. In the chronic stage， the treatments emphasizes to ''strengthen the spleen， drain dampness， transform turbidity， clear heat， resolve phlegm， and activate blood circulation''. This approach has yielded favorable therapeutic outcomes in clinical practice. This theoretical system clarifies the nature of gout as ''spleen deficiency being the root， dampness-turbidity being the secondary manifestation'' and systematically analyzes its pathogenesis evolution process and characteristics. The constructed stage-based treatment protocol has been validated through clinical and basic research， providing systematic theoretical guidance and a practical framework for the precise TCM management of gout， thereby promoting the modernization of TCM pathogenesis theory related to gout.

ObjectiveThis paper aims to observe the effect of Huazhuo Sanjie Chubi prescription （HSCD） on the gouty bone erosion model rats and investigate its action mechanism. MethodsThirty-six two-month-old male SD rats were randomly divided into the blank group with nine rats and the modeling group with 27 rats. The rats in the modeling group were administered hypoxanthine solution at 300 mg·kg^-1·d^-1 and potassium oxonate solution at 250 mg·kg^-1·d^-1， combined with intra-articular injection of 200 μL monosodium urate （MSU） crystal suspension at 25 g·L^-1 into the right ankle joint （joint injection once every three days）， so as to induce the gouty bone erosion model. After four weeks of modeling， three rats were selected from these two groups to validate the model. The modeled 24 rats were randomly divided into the model group， HSCD group （10.35 g·kg^-1·d^-1）， allopurinol group （20 mg·kg^-1·d^-1）， and inhibitor group （LY294002， 10 mg·kg^-1·d^-1）， with six rats per group. Except for the blank group， rats in all other groups continued to receive hypoxanthine solution at 300 mg·kg^-1 and potassium oxonate solution at 250 mg·kg^-1 via gavage concurrently with administration to maintain modeling intervention. The rats in the HSCD group and allopurinol group received administration by gavage at the above doses. The rats in the inhibitor group received an intraperitoneal injection at the above dose. The rats in the blank group and model group received saline （10.35 g·kg^-1·d^-1） by gavage for four consecutive weeks. After administration， ankle joint swelling of the rats in all groups was observed， and the diameters were measured. Bone volume fraction （BV/TV） and bone surface area to bone volume （BS/BV） were observed and quantitatively analyzed by Micro-CT. Histopathological changes in the ankle joint were observed by hematoxylin-eosin （HE） staining and safranin O-fast green staining. The uric acid in the rats' serum was determined by enzyme colorimetry. The levels of inflammatory factors， including tumor necrosis factor-α （TNF-α）， interleukin （IL）-1β， and IL-6 were measured by enzyme-linked immunosorbent assay （ELISA）. The protein expressions of receptor activator of nuclear factor-κB ligand （RANKL） and phosphorylated （p）-phosphatidylinositol-3-kinase （PI3K） in ankle joint tissues of rats were detected by immunofluorescence staining. The mRNA levels of the proteins related to the bone erosion， including RANKL， tartrate-resistant acid phosphatase （TRAP）， cathepsin K （CTSK）， and matrix metalloproteinase-9 （MMP-9） in the ankle joint tissues of rats were determined by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The expressions of the proteins related to the bone erosion， including RANKL， CTSK， TRAP， MMP-9， and the proteins related to the PI3K/protein kinase B （Akt） signaling pathway， including PI3K， Akt， mammalian target of rapamycin （mTOR）， p-PI3K， phosphorylated protein kinase B （p-Akt）， and phosphorylated mammalian target of rapamycin （p-mTOR）， were detected by Western blot. ResultsCompared with the blank group， the modeling group showed marked ankle swelling and increased diameter. Micro-CT revealed an uneven articular surface and honeycomb-like bone erosion in the ankle joint. Quantitative analysis showed decreased BV/TV and increased BS/BV （P<0.05）. HE staining revealed a narrowed joint space， rough and discontinuous cartilage surfaces， reduced and unevenly distributed chondrocytes， partial hypertrophy， and blurred tidemarks， confirming successful model establishment. After four weeks of intervention， compared with those in the blank group， the rats in the model group exhibited severe ankle swelling and increased diameters （P<0.05）. Micro‑CT showed that the ankle joint surface was irregular， and bone erosion exhibited a honeycomb‑like morphology. The microstructural parameters of the bone revealed a decreased BV/TV and an increased BS/BV （P<0.05）. Histopathological examination revealed a narrowed joint space and severe articular cartilage destruction. The levels of uric acid in the serum and inflammatory factors （IL-1β， IL-6， and TNF-α） were increased （P<0.05）. The mRNA and protein levels of the proteins related to bone erosion in joint tissue， including RANKL， CTSK， TRAP， and MMP-9， were upregulated （P<0.05）. The ratios of p-PI3K/PI3K， p-Akt/Akt， and p-mTOR/mTOR in the proteins related to the PI3K/Akt signaling pathway were increased （P<0.05）. RANKL and p-PI3K protein fluorescence intensities were elevated （P<0.05）. Compared with those in the model group， the above indicators in all other administration groups showed varying degrees of improvement. The HSCD group and inhibitor groups exhibited more significant effects in reducing ankle swelling， improving bone erosion， bone microstructure （significant decrease in BV/TV and increase in BS/BV）， and the pathology of cartilage erosion， lowering inflammatory factor levels， downregulating the proteins related to the bone erosion， and suppressing activation of the PI3K/Akt/mTOR pathway （P<0.05）. The uric acid levels in rats' serum were reduced in both HSCD and allopurinol groups （P<0.05）. Compared with those in the HSCD group， the rats in the allopurinol group had larger ankle diameters （P<0.05）， more severe bone erosion and bone microstructure damage （P<0.05）， worse improvement of the pathology of cartilage erosion， higher levels of IL-6 and TNF-α （P<0.05）， elevated expressions of the proteins related to the bone erosion， higher expression ratio of proteins related to the PI3K/Akt signaling pathway （P<0.05）， and higher fluorescence intensities of RANKL and p-PI3K proteins （P<0.05）. The inhibitor group achieved comparable results to the HSCD group in improvements of bone microstructure and the reduction of IL-1β and IL-6， stronger suppression of TNF-α and PI3K/Akt/mTOR signaling pathway activation （P<0.05）， but weaker effects on cartilage repair and serum uric acid reduction （P<0.05）. ConclusionHSCD effectively reduces uric acid levels in serum， suppresses inflammatory factor secretion， and downregulates the expressions of proteins related to bone erosion， including RANKL， CTSK， TRAP， and MMP-9 in the joint tissues. Its action mechanism of improving gouty bone erosion may be associated with inhibition of the PI3K/Akt signaling pathway.

Objective:To observe and analyze the intraocular stability of intraocular lens (IOL) with different haptic designs after cataract surgery by swept-source optical coherence tomography (SS-OCT) and analyze its influencing factors.Methods:A cohort study was conducted.Medical records of 130 consecutive patients (130 eyes) diagnosed with age-related cataract, who underwent phacoemulsification and IOL implantation at The First Affiliated Hospital of Zhengzhou University from August 2021 to August 2022 were collected.According to the type of IOL implanted, the patients were divided into four groups, L-haptic group with 34 cases (34 eyes), modified C-haptic group with 30 cases (30 eyes), plate-haptic group with 36 cases (36 eyes), four-haptic group with 30 cases (30 eyes).Before surgery, the patient's axial length and IOL diopter were measured with an optical biometer, and the angle kappa and angle alpha were measured with corneal topography.At 3 months after surgery, the anterior chamber depth, decentration and tilt of IOL, the space area between IOL and posterior capsule (AREAP) of the four groups were measured using SS-OCT.And anterior chamber depth changes and diopter changes were calculated.This study complied with the Declaration of Helsinki.The study protocol was reviewed and approved by the Medical Ethics Committee of The First Affiliated Hospital of Zhengzhou University (No.2022-KY-1222-002).All subjects were aware of the purpose and significance of this study and voluntarily signed the informed consent form.Results:The average decentration and tilt of the modified C-haptic, L-haptic, plate-haptic, and four-haptic IOL decreased successively at 3 months after surgery, with no statistically significant differences ( H=7.055, F=5.162; all P>0.05).The postoperative anterior chamber depth, the anterior chamber depth change, the diopter change and the AREAP of the modified C-haptic, L-haptic, plate-haptic, and four-haptic IOL decreased successively at 3 months after surgery, and the differences were statistically significant (all P<0.05).Preoperative angle kappa was positively correlated with postoperative 3-month IOL tilt ( r=0.554, P<0.001).Preoperative angle alpha was positively correlated with postoperative 3-month IOL tilt ( r=0.469, P<0.001).The multiple linear regression model constructed with preoperative angle kappa and preoperative angle alpha as independent variables and IOL tilt as dependent variable was statistically significant ( F=39.526, P<0.001, R2=0.384).For every 1 mm increase in preoperative angle kappa, postoperative tilt will increase by 0.438°; for every 1 mm increase in preoperative angle alpha, postoperative tilt will increase by 0.300°. Conclusions:All four IOLs with different haptic designs have good horizontal centering stability.The plate-haptic and the four-haptic IOLs have superior vertical stability compared to L-haptic and modified C-haptic designs.Preoperative angles kappa and alpha can be used as indicators to predict postoperative IOL tilt.

Objectives:To explore whether short-course radiotherapy (SCRT)-based total neoadjuvant therapy (TNT) combined with PD-1 inhibitors could further promote tumor regression and improve the prognosis.Methods:This is a prospective, multicenter, two-arm randomized controlled, seamless phase Ⅱ/Ⅲ trial for proficient mismatch repair or microsatellite stable (pMMR/MSS) locally advanced rectal cancer (LARC). Eligible patients were randomly assigned to the iTNT (TNT+PD-1) group or the TNT group. Patients in the TNT group received SCRT (5 Gy×5) followed by 4 cycles of CAPOX or 6 cycles of mFOLFOX chemotherapy, with the iTNT group receiving SCRT followed by the same regime in combination with 4 cycles of Sintilimab. Total mesorectal excision (TME) surgery or watch and wait (W&W) was performed after neoadjuvant therapy and then 2 cycles of same regimen as before were recommended. The primary endpoints are the complete response (CR) rate for phase Ⅱ trial and 3-year disease-free survival (DFS) for phase Ⅲ trial. A total of 588 patients will be enrolled for the phase Ⅱ/Ⅲ trial. Short-term efficacy and safety data from the initial 100 treated patients were analyzed as planned.Results:From 2022-8-31 to 2023-5-24 the initial 100 patients were enrolled from 10 hospitals in China, 76.0%(76/100) patients were male, and the median age was 61 years (21-74 years). More patients had tumors located in the lower rectum (78.0%, 78/100), staged T3-4 (97.0%, 97/100) and N1-2 (93.0%, 93/100), and about half of the tumors invaded the mesorectal fascia (52.0%, 52/100) and with extramural vascular invasion (51.0%, 51/100). Analyses were performed according to the per-protocal (PP) set. All patients in the iTNT group ( n=52) and the TNT group ( n=48) completed SCRT; The 4-cycle chemotherapy±Sintilimab completion rates were 86.5% and 100.0% in the iTNT and TNT groups, respectively. In the iTNT group, 82.7% (43/52), 11.5% (6/52), and 5.8% (3/52) of the patients received 4, 3, and 2 cycles of PD-1 inhibitor. After TNT, 68 patients underwent radical surgery and 15 patients achieved cCR and adopted W&W. The pathological complete response (pCR) rates were 48.5% (16/33) and 17.1% (6/35) in the iTNT and TNT groups, with CR rates of 50.0% (25/50) and 26.1% (12/46), respectively. The incidence of treatment-related grade 3-4 adverse events was 26.9% (14/52, iTNT group) and 18.8% (9/48, TNT group), with thrombocytopenia and leukopenia being the most common. Among patients receiving immunotherapy, grade 3 immunotherapy-related adverse events occurred in 2 (3.8%, 2/52) patients: one case was pancreatitis, another case was hepatitis combined with myositis and myocarditis. Conclusion:The preliminary results show that SCRT-based TNT combined with PD-1 inhibitors could further improve the CR rate for LARC without unexpected serious adverse events.

Objective:To investigate the association between adiponectin-related copy number variation (CNV) region (CNVR) and gestational diabetes mellitus (GDM).Methods:Pregnant women who had prenatal screening in Anqing Municipal Hospital, Anhui Province, from February 2018 to December 2020 were surveyed for baseline information collection, and blood samples were collected. The outcome information was obtained by post pregnancy follow-up. Latex-enhanced immunoturbidimetry and ASA-CHIA chip were used to detect serum adiponectin levels and CNV of pregnant women, respectively. After genotyping, CNV data were processed with software PennCNV 1.0.5 following standard quality control procedure. CNVR were identified and integrated by using software R 4.3.3. Then the associations between CNVR and adiponectin was evaluated, and gene annotation and over-representation analysis were conducted. The log-binomial regression model was used to adjust relevant covariates and analyze the association between adiponectin-related CNVR and GDM.Results:The detection rate of GDM was 9.54% (176/1 845) in the pregnant women. The genotyping information of 1 840 people (99.73%) passed quality evaluation. A total of 33 878 CNVs were identified, and 1 449 CNVRs were obtained after integration. After the false discovery rate method correction, CNVR_132 (CHR2: 47611743-47635062), CNVR_254 (CHR3: 10182703-10183872), CNVR_691 (CHR7: 150637053-150834539) and CNVR_1101 (CHR14: 104248431-104830620) were correlated with adiponectin levels (all P<0.05). Over- representation analysis showed that the molecular function of ribonucleotide binding [Gene Ontology (GO): 0032553] was significantly enriched based on the GO database. The log-binomial regression model, adjusting age, pre-pregnancy BMI, history of miscarriage, smoking history, and family history of diabetes, indicated that CNVR_132 (CHR2: 47611743-47635062) and CNVR_1101 (CHR14: 104248431-104830620) were not statistically associated with the risk for GDM (both P>0.05). However, CNVR_254 (CHR3: 10182703-10183872, a RR=1.83, 95% CI: 1.15-2.91) and CNVR_691 (CHR7: 150637053-150834539, a RR=1.73, 95% CI: 1.23-2.43) might be associated with an increased risk for GDM (all P<0.05). Conclusion:Adiponectin-related CNVR_254 (CHR3: 10182703-10183872) and CNVR_691 (CHR7: 150637053-150834539) might be risk factors for the incidence of GDM.

Objective:To observe and analyze the intraocular stability of intraocular lens (IOL) with different haptic designs after cataract surgery by swept-source optical coherence tomography (SS-OCT) and analyze its influencing factors.Methods:A cohort study was conducted.Medical records of 130 consecutive patients (130 eyes) diagnosed with age-related cataract, who underwent phacoemulsification and IOL implantation at The First Affiliated Hospital of Zhengzhou University from August 2021 to August 2022 were collected.According to the type of IOL implanted, the patients were divided into four groups, L-haptic group with 34 cases (34 eyes), modified C-haptic group with 30 cases (30 eyes), plate-haptic group with 36 cases (36 eyes), four-haptic group with 30 cases (30 eyes).Before surgery, the patient's axial length and IOL diopter were measured with an optical biometer, and the angle kappa and angle alpha were measured with corneal topography.At 3 months after surgery, the anterior chamber depth, decentration and tilt of IOL, the space area between IOL and posterior capsule (AREAP) of the four groups were measured using SS-OCT.And anterior chamber depth changes and diopter changes were calculated.This study complied with the Declaration of Helsinki.The study protocol was reviewed and approved by the Medical Ethics Committee of The First Affiliated Hospital of Zhengzhou University (No.2022-KY-1222-002).All subjects were aware of the purpose and significance of this study and voluntarily signed the informed consent form.Results:The average decentration and tilt of the modified C-haptic, L-haptic, plate-haptic, and four-haptic IOL decreased successively at 3 months after surgery, with no statistically significant differences ( H=7.055, F=5.162; all P>0.05).The postoperative anterior chamber depth, the anterior chamber depth change, the diopter change and the AREAP of the modified C-haptic, L-haptic, plate-haptic, and four-haptic IOL decreased successively at 3 months after surgery, and the differences were statistically significant (all P<0.05).Preoperative angle kappa was positively correlated with postoperative 3-month IOL tilt ( r=0.554, P<0.001).Preoperative angle alpha was positively correlated with postoperative 3-month IOL tilt ( r=0.469, P<0.001).The multiple linear regression model constructed with preoperative angle kappa and preoperative angle alpha as independent variables and IOL tilt as dependent variable was statistically significant ( F=39.526, P<0.001, R2=0.384).For every 1 mm increase in preoperative angle kappa, postoperative tilt will increase by 0.438°; for every 1 mm increase in preoperative angle alpha, postoperative tilt will increase by 0.300°. Conclusions:All four IOLs with different haptic designs have good horizontal centering stability.The plate-haptic and the four-haptic IOLs have superior vertical stability compared to L-haptic and modified C-haptic designs.Preoperative angles kappa and alpha can be used as indicators to predict postoperative IOL tilt.

Salvia miltiorrhiza (S. miltiorrhiza) represents a crucial component of traditional Chinese medicine, demonstrating effects on blood circulation activation and stasis removal, and has been widely utilized in asthma treatment. This study isolated a novel phenolic acid (S1) from S. miltiorrhiza and investigated its anti-asthmatic activity and underlying mechanisms for the first time. An allergic asthma (AA) model was established using ovalbumin (OVA). The mechanism of S1's effects on AA was investigated using multi-factor joint analysis, flow cytometry, and co-culture systems to facilitate clinical asthma treatment. S1 (10 or 20 mg·kg^-1) was administered daily to mice with OVA-induced AA (OVA-AA) during days 21-25. The study examined airway responsiveness, lung damage, inflammation, and levels of immunoglobulin E (IgE), PGD2, interleukins (IL-4, 5, 10, 13, 17A), tumor necrosis factor α (TNF-α), GM-CSF, CXCL1, CCL11, and mMCP-1. Additionally, mast cell (MC) activation and degranulation were explored, along with T helper type 17 (Th17)/Treg immune cells and TLR4 pathway biomarkers. The antagonistic activity of that specific antagonist of TLR4 (TAK-242) (1 µmol·L^-1), a specific TLR4 blocker, against S1 (10 µmol·L^-1) was examined in co-cultured 16HBE cells and bone marrow-derived cells (BMDCs) or splenic lymphocytes (SLs) induced with LPS (1 µg·mL^-1) to elucidate the TLR4 pathway's mediating role. S1 demonstrated reduced airway responsiveness, lung damage, and inflammation, with downregulation of IgE, PGD2, interleukins, TNF-α, GM-CSF, CXCL1, CCL11, and mMCP-1. It also impeded MC activation and degranulation, upregulated IL-10, and influenced Th17/Treg immune cell transformation following OVA challenge. Furthermore, S1 inhibited the TLR4 pathway in OVA-AA mice, and TLR4 antagonism enhanced S1's positive effects. Analysis using an OVA-AA mouse model demonstrated that S1 alleviates AA clinical symptoms, restores lung function, and inhibits airway response. S1's therapeutic effects occur through regulation of Th17/Treg immune cells and inflammation, attributable at least partially to the TLR4 pathway. This study provides molecular justification for S1 in AA treatment.

ObjectiveTo investigate the whole genomic characteristics and phylogenetic relationships of clinical isolates of enteroaggregative Escherichia coli (EAEC) in diarrhea outpatients in Pudong New Area, Shanghai. MethodsBased on the diarrheal disease surveillance network in Pudong New Area, Shanghai, whole-genome sequencing was performed on a total of 55 EAEC strains isolated from fecal samples of the diarrhea outpatients from January 2015 to December 2019. The genome analyses based on raw sequencing data encompassed genome size, coding genes, dispersed repeat sequences, genomic islands, and protein coding regions, and pan-genome analyses were conducted simultaneously. Contigs sequences assays were performed to analyze molecular characteristics including serotypes, antibiotic resistance genes, and virulence factors. The phylogenetic clusters and multilocus sequence typing (MLST) were identified, and a phylogenetic tree was constructed. ResultsEAEC exhibited an open pan-genome. The predominant serotype of EAEC in diarrhea outpatients in Pudong New Area was O130:H27, and the carriage rate of β-lactam resistance genes was the highest (67.27%, 37/55). A total of 29 virulence factors and 106 virulence genes were identified, phylogenic group B1 was the predominant group, and clonal group CC31 was the dominant clonal group. The strain distribution was highly heterogeneous. ConclusionThe genomic characteristics of EAEC displayed significant strain polymorphism. It is necessary to develop effective strategies for differential diagnosis and improve detection capabilities for infection with EAEC of different serotypes and genotypes.

Objective:To characterize the vaginal flora of pregnant women during the second trimester using full-length 16S rRNA sequencing.Methods:A total of 142 pregnant women were systematically sampled from a pregnancy cohort. Vaginal swabs were collected for full-length 16S rRNA gene sequencing，and bioinformatics analysis was performed to characterize the vaginal microbiota and identify associated influencing factors.Results:Among the 142 pregnant women，the most frequently detected species were Lactobacillus iners（83.10%，118/142）and Lactobacillus crispatus（49.30%，70/142）. The majority of samples（90.85%，129/142）were classified as Lactobacillus-dominant vagitypes，with the Lactobacillus iners vagitype accounting for 48.59%（69/142）and the Lactobacillus crispatus vagitype accounting for 38.73%（55/142）. The vaginal microbiota was clustered into five community state types（CSTs）：Ⅰa，Ⅰb，Ⅲa，Ⅲb，and Ⅳ. The most prevalent CSTs were Lactobacillus iners-dominated CST-Ⅲ（51.41%，73/142）and Lactobacillus crispatus-dominated CST-Ⅰ（24.65%，35/142）. No samples were classified as CST-Ⅱ or CST-Ⅴ. A significant negative correlation was observed between Lactobacill and vaginosis-associated bacteria. Age，alcohol consumption，smoking，and vaginal treatments showed significant associations or trends toward significance with various Alpha diversity indices. Vaginal douching was associated with CST clustering，while obstetric history（primiparity，previous miscarriage history）was associated with vagitype classification. However，no significant associations were identified between maternal baseline characteristics and Beta diversity indices. Conclusions:Full-length 16S rRNA gene sequencing reveals that the vaginal microbiota of pregnant women is dominated by Lactobacillus iners and Lactobacillus crispatus. Maternal age，lifestyle factors such as smoking and alcohol consumption，and obstetric history are significantly associated with variations in vaginal microbiota composition.