ObjectiveTo explore the effect of serum containing Zhenwutang on myocardial mast cell apoptosis induced by angiotensin Ⅱ （AngⅡ） and the mechanism of the correlation between apoptosis and mitochondrial autophagy. MethodsIn this experiment， AngⅡ and serum containing Zhenwutang with different concentrations were used to interfere with H9C2 cardiomyocytes for 24 h， and the survival rate of H9C2 cardiomyocytes was detected by cell counting kit-8 （CCK-8） to screen the optimal concentration for the experiment. Enzyme-linked immunosorbent assay （ELISA） was used to detect the content of B-type natriuretic peptide （BNP） in cell culture supernatant， and immunofluorescence was used to detect the cell surface area to verify the construction of the myocardial mast cell model. Subsequently， the experiment was divided into a blank group （20% blank serum）， a model group （20% blank serum + 5×10^-5 mol·L^-1 AngⅡ）， low-， medium-， and high-dose （5%， 10% and 20%） serum containing Zhenwutang groups， an autophagy inhibitor group （1×10^-4 mol·L^-1 3-MA）， and autophagy inducer group （1×10^-7 mol·L^-1 rapamycin）. The apoptosis level of H9C2 cells and the changes of mitochondrial membrane potential were detected by flow cytometry. The lysosomal probe （Lyso Tracker） and mitochondrial probe （Mito Tracker） co-localization was employed to detect autophagy. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect Caspase-3， Caspase-9， B-cell lymphoma 2 （Bcl-2）， Bcl-2-related X protein （Bax）， and cytochrome C （Cyt C） in apoptosis-related pathways and the relative mRNA expression of ubiquitin ligase （Parkin）， phosphatase and tensin homolog （PTEN）-induced kinase 1 （PINK1）， and p62 protein in mitochondrial autophagy-related pathways. Western blot was used to detect cleaved Caspase-3， cleaved Caspase-9， Bax， Bcl-2， and Cyt C in apoptosis-related pathways， phosphorylated ubiquitin ligase （p-Parkin）， phosphorylated PTEN-induced kinase 1 （p-PINK1）， p62， and Bcl-2 homology domain protein Beclin1 in mitochondrial autophagy-related pathways， and the change of microtubule-associated protein 1 light chain 3 （LC3） Ⅱ/Ⅰ ratio. ResultsCCK-8 showed that when the concentration of AngⅡ was 5×10^-5 mol·L^-1， the cell activity was the lowest， and there was no cytotoxicity. At this concentration， the surface area of cardiomyocytes was significantly increased （P<0.01）， and the content of BNP in the supernatant of culture medium was significantly increased （P<0.05）. Therefore， AngⅡ with a concentration of 5×10^-5 mol·L^-1 was selected for the subsequent modeling of myocardial mast cells. Compared with the blank group， the model group and the autophagy inhibitor 3-MA group had a significantly increased apoptosis rate （P<0.01） and significantly decreased mitochondrial membrane potential （P<0.01）. The results of immunofluorescence co-localization showed that compared with the blank group， the model group had a significantly decreased number of red and green fluorescence spots. The results of Real-time PCR showed that compared with that in the blank group， the relative mRNA expression of Bax， Caspase-3， Caspase-9， Cyt C， and p62 in the model group was significantly up-regulated （P<0.01）， while the relative mRNA expression of Bcl-2， Parkin， and PINK1 was significantly down-regulated （P<0.01）. In addition， the relative protein expression of Bax， cleaved Caspase-3， cleaved Caspase-9， Cyt C， and p62 was significantly up-regulated （P<0.01）. The LC3Ⅱ/Ⅰ was significantly decreased， and the relative protein expression of Bcl-2， p-Parkin， p-PINK1， and Beclin1 was significantly down-regulated （P<0.01）. Compared with the model group， the serum containing Zhenwutang groups and the autophagy inducer group had significantly decreased apoptosis rate （P<0.01）， and the decrease ratio of mitochondrial membrane potential is significantly lowered （P<0.01） in a dose-dependent manner. Additionally， both red and green fluorescence spots became more in these groups. In the 3-MA group， the number of red and green fluorescence spots decreased significantly. The relative mRNA expression of Bax， Caspase-3， Caspase-9， Cyt C， and p62 was significantly down-regulated （P<0.05， P<0.01）， while that of Bcl-2， Parkin， and PINK1 was significantly up-regulated （P<0.01）. In the serum containing Zhenwutang groups， the relative protein expression levels of Bax， cleaved Caspase-3， cleaved Caspase-9， Cyt C， and p62 were significantly down-regulated （P<0.05，P<0.01）. The LC3Ⅱ/Ⅰ was significantly increased， and the relative protein expression levels of Bcl-2， p-Parkin， p-PINK1， and Beclin1 were significantly up-regulated （P<0.01）. ConclusionThe serum containing Zhenwutang can reduce the apoptosis of myocardial mast cells and increase mitochondrial autophagy. This is related to the inhibition of intracellular Bax/Bcl-2/Caspase-3 apoptosis pathway and regulation of Parkin/PINK1 mitochondrial autophagy pathway.

BACKGROUND: The available evidence regarding the benefits of percutaneous coronary intervention (PCI) on patients receiving dialysis with coronary artery disease (CAD) is limited and inconsistent. This study aimed to evaluate the association between PCI and clinical outcomes as compared with medical therapy alone in patients undergoing dialysis with CAD in China. METHODS: This multicenter, retrospective study was conducted in 30 tertiary medical centers across 12 provinces in China from January 2015 to June 2021 to include patients on dialysis with CAD. The primary outcome was major adverse cardiovascular events (MACE), defined as a composite of cardiovascular death, non-fatal myocardial infarction, and non-fatal stroke. Secondary outcomes included all-cause death, the individual components of MACE, and Bleeding Academic Research Consortium criteria types 2, 3, or 5 bleeding. Multivariable Cox proportional hazard models were used to assess the association between PCI and outcomes. Inverse probability of treatment weighting (IPTW) and propensity score matching (PSM) were performed to account for potential between-group differences. RESULTS: Of the 1146 patients on dialysis with significant CAD, 821 (71.6%) underwent PCI. After a median follow-up of 23.0 months, PCI was associated with a 43.0% significantly lower risk for MACE (33.9% [ n  = 278] vs . 43.7% [ n  = 142]; adjusted hazards ratio 0.57, 95% confidence interval 0.45-0.71), along with a slightly increased risk for bleeding outcomes that did not reach statistical significance (11.1% vs . 8.3%; adjusted hazards ratio 1.31, 95% confidence interval, 0.82-2.11). Furthermore, PCI was associated with a significant reduction in all-cause and cardiovascular mortalities. Subgroup analysis did not modify the association of PCI with patient outcomes. These primary findings were consistent across IPTW, PSM, and competing risk analyses. CONCLUSION This study indicated that PCI in patients on dialysis with CAD was significantly associated with lower MACE and mortality when comparing with those with medical therapy alone, albeit with a slightly increased risk for bleeding events that did not reach statistical significance.
Humans ; Percutaneous Coronary Intervention/methods* ; Male ; Female ; Coronary Artery Disease/drug therapy* ; Retrospective Studies ; Renal Dialysis/methods* ; Middle Aged ; Aged ; China ; Proportional Hazards Models ; Treatment Outcome

OBJECTIVE: To construct and validate a predictive model for the therapeutic effect of acupuncture at Tongdu Yangxin prescription (acupoint prescription for promoting the circulation of the governor vessel and nourishing the heart) on insomnia, so as to develop an open-access interactive artificial intelligence (AI)-assisted decision-making platform. METHODS: Clinical data of 139 insomnia patients treated with Tongdu Yangxin acupuncture therapy were included. All the patients had received acupuncture at Baihui (GV20), Yintang (GV24⁺), bilateral Shenmen (HT7), and bilateral Sanyinjiao (SP6); and electric stimulation was attached to Baihui (GV20) and Yintang (GV24⁺), using a continuous wave and a frequency of 2 Hz. The treatment was delivered once every other day, 3 treatments a week, and for 2 consecutive weeks. Patients with Pittsburgh sleep quality index (PSQI) score reduction rate <50% were classified as the "no response group", and those with ≥50% were as the "response group". Outliers were addressed using the 1.5×IQR rule, and missing values were imputed via predictive mean matching. Key features were selected by intersecting the feature importance results from eXtreme Gradient Boosting (XGBoost) and random forest algorithms. After balancing class distribution using the Synthetic Minority Over-sampling Technique (SMOTE), 20% of the data was reserved as a validation set. The remained data underwent the stratified sampling iterations to generate 200 pairs of 3∶1 training-test sets, which was employed for training and internal validation of 8 machine learning algorithms. The optimal algorithm and data partitioning strategy were selected to construct the final model, followed by external validation. The best-performing model was deployed online via Streamlit to create an interactive AI platform. RESULTS: Key predictive features for model construction included insomnia duration, the total PSQI score, PSQI sleep efficiency subscore, the proportion of N1 and N2 sleep stages in total sleep duration, and the maximum pulse rate during sleep. The CatBoost-based model achieved an AUC of 0.92, the average precision of 0.77, and accuracy, average recall, and average F1-score of 0.75 on the test set. On the validation set, it attained an AUC of 0.84, with accuracy, average precision, average recall, and average F1-score all at 0.72, demonstrating robust predictive performance. An interactive AI platform was subsequently developed (https://tdyx-catboost.streamlit.app/). CONCLUSION This study successfully establishes and validates a CatBoost-based efficacy prediction model for Tongdu Yangxin acupuncture therapy in treatment of insomnia. The developed AI platform provides data-driven decision support for acupuncture-based insomnia management.
Humans ; Sleep Initiation and Maintenance Disorders/physiopathology* ; Acupuncture Therapy ; Male ; Acupuncture Points ; Female ; Middle Aged ; Adult ; Artificial Intelligence ; Aged ; Young Adult

Based on the regulation of mitochondrial fatty acid β-oxidation through the PGC1α/PPARα/CPT1A pathway, this study investigated the effect of Jianpi Qinghua Formula on the mitochondrial fatty acid β-oxidation pathway in the livers of mice with metabolic-associated fatty liver disease(MAFLD) induced by a high-fat diet. MAFLD mice were fed a high-fat diet to establish the model, and after successful modeling, the mice were divided into the model group, the Jianpi Qinghua Formula group, and the metformin group, with an additional control group. Each group was treated with the corresponding drug or an equivalent volume of saline via gavage. Body mass and food intake were measured regularly during the experiment. At the end of the experiment, blood lipid levels and liver function-related indices were measured, liver pathological changes were observed, and protein expression levels of PGC1α, PPARα, PPARγ, and CPT1A were detected by Western blot. The results showed that, with no difference in food intake, compared to the model group, the body mass of the Jianpi Qinghua Formula group and the metformin group was reduced, liver weight and liver index decreased, and levels of cholesterol, triglycerides, and low-density lipoprotein cholesterol(LDL-C) were lowered. Additionally, a decrease in alanine aminotransferase(ALT) and aspartate aminotransferase(AST) was observed. Hematoxylin and eosin(HE) staining revealed reduced pathological damage to hepatocytes, while oil red O staining showed improvement in fatty infiltration. The liver disease activity score decreased, and transmission electron microscopy revealed improvement in mitochondrial swelling and restoration of internal cristae. Western blot analysis indicated that Jianpi Qinghua Formula significantly increased the expression of PGC1α, PPARα, and CPT1A proteins in the liver and reduced the expression of PPARγ. These results suggest that the Jianpi Qinghua Formula improves mitochondrial function, promotes fatty acid oxidation, and alleviates the pathological changes of MAFLD. In conclusion, Jianpi Qinghua Formula can improve MAFLD by mediating mitochondrial fatty acid β-oxidation through the PGC1α/PPARα/CPT1A pathway.
Animals ; PPAR alpha/genetics* ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Mice ; Carnitine O-Palmitoyltransferase/genetics* ; Male ; Liver/metabolism* ; Fatty Liver/genetics* ; Humans ; Mice, Inbred C57BL ; Diet, High-Fat/adverse effects*

The structure of intestinal tissue is complex. In vitro simulation of intestinal structure and function is important for studying intestinal development and diseases. Recently, organoids have been successfully constructed and they have come to play an important role in biomedical research. Organoids are miniaturized three-dimensional (3D) organs, derived from stem cells, which mimic the structure, cell types, and physiological functions of an organ, making them robust models for biomedical research. Intestinal organoids are 3D micro-organs derived from intestinal stem cells or pluripotent stem cells that can successfully simulate the complex structure and function of the intestine, thereby providing a valuable platform for intestinal development and disease research. In this article, we review the latest progress in the construction and application of intestinal organoids.
Organoids/cytology* ; Intestines/physiology* ; Humans ; Animals ; Pluripotent Stem Cells

Objective:To investigate the regulatory effect and mechanism of interleukin-22 (IL-22) on the gingival epithelial barrier in the context of periodontal inflammation.Methods:IL-22 knockout (IL-22 KO) mice were constructed, and periodontitis mice models were established through oral gavage with polymicrobial inoculation. DNAs were extracted from the oral plaques of IL-22 KO periodontitis mice group ( n=7) and their wild-type littermates periodontitis group ( n=7) to establish a periodontitis-related oral microbiota database"PD-RiskMicroDB", determining the relationship between changes in oral microbiota and microbial function in two groups using 16S rRNA sequencing results. Gingival epithelial cells (GEC) were cultured by modified trypsinization method, and were stimulated with 100 μg/L IL-22, Porphyromonas gingivalis (Pg) (multiplicity of infection:100), separately or together for 3 and 12 hours. The experimental groups were as follows: control group (no stimulation), IL-22 group, Pg group and Pg+IL-22 group. The expression of barrier protein E-cadherin in each group at 3 h was detected by immunofluorescence, real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting. Fluorescein isothiocyanate-dextran-mediated epithelial cell permeability experiment was conducted to clarify the changes in permeability of GEC in each group at 3 and 12 h. The mRNA expressions of E-cadherin in the gingival epithelium of wild-type littermates periodontitis group and IL-22 KO periodontitis group were detected by RT-qPCR. Fifteen C57BL/6 wild-type mice were randomly divided into control group ( n=5), periodontitis group ( n=5) and periodontitis+IL-22 treatment group ( n=5). RT-qPCR and immunohistochemistry (IHC) staining were used to detect the expression level of E-cadherin in the gingival epithelium of each group. Results:16S rRNA sequencing results showed that the composition of oral microbiota changed in IL-22 KO periodontitis group, of which the abundance of bacterial genera related to periodontal tissue invasion was significantly increased (linear discriminant analysis score: 2.22, P=0.009), compared with wild-type littermates periodontitis group. In vitro cell experiments showed that after Pg infection for 3 hours, the cell connections of GEC in Pg group were interrupted, and the fluorescence intensity of E-cadherin was reduced in Pg group compared with the control group. Meanwhile, the mRNA and protein expression levels of E-cadherin (mRNA: 0.69±0.12; protein: 0.60±0.12) were downregulated compared with the control group [mRNA: 1.00±0.00 ( P=0.043); protein: 1.04±0.08 ( P=0.003)], respectively. The fluorescence intensity of E-cadherin in the Pg+IL-22 group was enhanced compared with Pg group, and expression levels of E-cadherin mRNA (1.16±0.10) and protein (0.98±0.07) in Pg+IL-22 group showed a significant increase compared with Pg group [mRNA: 0.69±0.12 ( P=0.005); protein: 0.60±0.12 ( P=0.007)]. The result of epithelial permeability test showed that there was no statistical difference in epithelial permeability among control group, Pg group, IL-22 group and Pg+IL-22 group with treatment for 3 hours ( F=0.20, P=0.893). While when the treatment time turned to be 12 hours, the epithelial barrier permeability showed a significant increase in Pg group (1.39±0.15) compared with control group (1.00±0.00, P=0.027), and a decrease in Pg+IL-22 group (1.02±0.18) compared with Pg group (1.39±0.15, P=0.034). In vivo, the mRNA expression of E-cadherin in the gingival epithelium of IL-22 KO periodontitis group decreased significantly (0.32±0.21) compared with wild-type littermates periodontitis group (1.01±0.01) ( t=5.70, P=0.005). Moreover, RT-qPCR and IHC staining results showed that the mRNA expression level of E-cadherin (0.40±0.07) and absorbance value of E-cadherin positive expression (0.02±0.00) in gingival epithelial tissue of periodontitis group were both significantly down-regulated compared with control group [mRNA: 1.00±0.00 ( P=0.005); absorbance value of E-cadherin positive expression: 0.04±0.01 ( P=0.006)]. Meanwhile, the mRNA expression level of E-cadherin (1.06±0.24) and the absorbance value of E-cadherin positive expression (0.03±0.01) were both observed increase in periodontitis+IL-22 treatment group compared with periodontitis group ( P=0.003, P=0.039). Conclusions:IL-22 may exert a protective effect on the gingival epithelial barrier in an inflammatory environment by regulating the invasiveness of oral microbiota and the expression of host barrier protein.

Temporomandibular joint (TMJ) diseases are common clinical conditions. The number of patients with TMJ diseases is large, and the etiology, epidemiology, disease spectrum, and treatment of the disease remain controversial and unknown. To understand and master the current situation of the occurrence, development and prevention of TMJ diseases, as well as to identify the patterns in etiology, incidence, drug sensitivity, and prognosis is crucial for alleviating patients′suffering.This will facilitate in-depth medical research, effective disease prevention measures, and the formulation of corresponding health policies. Cohort construction and research has an irreplaceable role in precise disease prevention and significant improvement in diagnosis and treatment levels. Large-scale cohort studies are needed to explore the relationship between potential risk factors and outcomes of TMJ diseases, and to observe disease prognoses through long-term follw-ups. The consensus aims to establish a standard conceptual frame work for a cohort study on patients with TMJ disease while providing ideas for cohort data standards to this condition. TMJ disease cohort data consists of both common data standards applicable to all specific disease cohorts as well as disease-specific data standards. Common data were available for each specific disease cohort. By integrating different cohort research resources, standard problems or study variables can be unified. Long-term follow-up can be performed using consistent definitions and criteria across different projects for better core data collection. It is hoped that this consensus will be facilitate the development cohort studies of TMJ diseases.

Objective:To investigate the correlation between the neoadjuvant rectal (NAR) score and long-term survival in patients with locally advanced rectal cancer who have undergone neoadjuvant chemoradiotherapy.Methods:Clinical and pathological data of 487 patients diagnosed with rectal adenocarcinoma from October 2004 to April 2014 at Sun Yat-sen University Cancer Center who had received neoadjuvant chemoradiotherapy were retrospectively analyzed and the impact of NAR score on prognosis studied. Disease-free-survival (DFS) was calculated by the Kaplan-Meier method and survivals compared using the log-rank test. Cox models were used for univariate and multivariate analyses. Receiver operating characteristic curves were utilized to evaluate the predictive capability of NAR and tumor regression grade scores for the risk of 10-year postoperative recurrence and metastasis. The Delong test was employed to compare the diagnostic performance of the two scores.Results:Of the 487 patients included in the study, 166 were men (34.1%). The median age was 56 years (interquartile range [IQR]: 46–63). All patients completed adequate preoperative chemoradiotherapy and underwent R0 resection.The median interval between the end of chemoradiotherapy and surgery was 51 days (IQR: 44–58). Post-chemoradiotherapy downstaging occurred in 329 patients (67.6%). Tumor regression grades (TRGs) were 1–2 in 246 patients (50.5%) and 3–4 in 241 patients (49.5%). A total of 394 patients (80.9%) received postoperative chemotherapy. NAR scores were <8 in 182 patients (37.4%), 8–16 in 180 (37.0%), and >16 in 125 (25.6%). The median follow-up time was 111.5 months (IQR: 70.7–133.7 months). One hundred and thirteen patients died of rectal cancer, among whom 13 patients developed local recurrence, 88 patients developed distant metastasis, and 12 patients had unknown recurrence patterns. The 10-year DFS and overall survival rate of f the whole group were 68.9% and 71.5% respectively. The 10-year DFS rates for patients with NAR scores <8, 8–16, and >16 were 85.1%, 80.5%, and 66.4%, respectively ( P<0.001). Multivariate analyses revealed that the Dixon operation (HR=0.606, 95%CI: 0.408–0.902, P=0.014), and >16 (HR=2.569, 95%CI: 1.559–4.233, P<0.001) were independent predictors of the 10-year DFS of patients with locally advanced rectal cancer ( P<0.05 for all). In the entire patient cohort, the AUC of the receiver operating characteristic curve for NAR score predicting 10-year recurrence and metastasis was 0.67 (95%CI: 0.62–0.72), whereas the AUC for TRG score was 0.54 (95%CI: 0.49–0.60). The two scores differed significantly in accuracy ( Z=-4.06, P<0.001), the NAR score being a significantly better predictor of risk of 10-year recurrence and metastasis than the TRG score. Conclusion:The NAR score is a reliable predictor of 10-year DFS in patients with locally advanced rectal cancer who have undergone neoadjuvant chemoradiotherapy followed by curative surgery.

Objective:To explore the prognosis efficacy of psycho-cardiological therapy and management on patients with coronary atherosclerosis disease (CAD).Methods:This was a clinical randomized controlled study. This study included inpatients with CAD at the cardiology department in Beijing Anzhen Hospital, Capital Medical University from August 2021 to January 2024. The patients enrolled in this study were asked for basic information, and received measurements for depression, anxiety, sleep quality and living quality by the scales of Patient Health Questionnaire-9 (PHQ-9), General Anxiety Disorder scale-7 (GAD-7), Athens Insomnia Scale (AIS), EuroQol 5-dimension 5-level (EQ-5D-5L) respectively. The patients were randomly grouped into a regular treatment group and a psycho-cardiological treatment group which included WeChat management or antidepressant/antianxiety medical therapy according to the situation. After the patients discharging from hospital for 2, 4, 12, 24, 48, 72, and 96 weeks, professional cardiovascular doctors would follow up by telephone, WeChat, and outpatient department, including scales (2-48 weeks), and cardiac events (2-96 weeks). Kaplan-Meier survival curve and multivariate Cox proportional hazards model were used for analyzing the association between psycho-cardiological treatment and cardiac events.Results:This study recruited a total of 552 patients with CAD, aged 61.0(54.0, 67.0) years, and 379 (68.7%) were male. There were 279(50.5%) in the regular treatment group and 273(49.5%) in the psycho-cardiological treatment group. After treatment for 4, 12 and 48 weeks, the PHQ-9 score in psycho-cardiological was significantly lower than the regular treatment group; After treatment for 12 weeks, the EQ-5D-5L effective value in psycho-cardiological group was higher than the regular treatment group; After treatment for 2, 4, 12, 24 and 48 weeks, the EQ-5D-5L VAS score in psycho-cardiological group was higher than the regular therapy group (all P<0.05). The Kaplan-Meier survival curve showed that, during the different follow-up periods, the rate of cardiac events in psycho-cardiological treatment group was lower than regular treatment group (log-rank P<0.001). The multivariate Cox proportional hazards model adjusted the factor of age, the psycho-cardiological treatment contributed to reducing the cardiac events rate by 80.3% ( HR=0.197, 95% CI: 0.067-0.582, P=0.003). Conclusion:Psycho-cardiological treatment is beneficial for improving psychological stress, living quality, and reducing cardiac events, and helps to improve prognosis and psycho-cardiological rehabilitation in CAD patients.

Objective To identify and conduct antimicrobial susceptibility testing on Vibrio species isolated from blood culture specimens of two patients with bloodstream infection(BSI),analyze the microbiological characteristics of non-O1/non-O139 Vibrio cholerae(NOVC),and provide evidence for the diagnosis,prevention and control of Vibrio cholerae infection.Methods Two Vibrio strains were identified by matrix-assisted laser desorption/ioniza-tion time-of-flight mass spectrometry(MALDI-TOF MS),API bacterial biochemical reaction identification test strip,VITEK 2 Compact identification instrument and 16S rRNA gene sequencing.Serological typing,virulence gene molecular detection,and drug resistance phenotype detection were performed to the Vibrio strains.Results Two strains were identified as Vibrio cholerae,classified as NOVC by serological typing,and were negative for ctxAB virulence gene detection.Antimicrobial susceptibility testing showed that one strain was sensitive to ampici-llin,azi-thromycin,doxycycline and chloramphenicol,while resistant to tetracycline and trimethoprim-sulfametho-xazole.The other strain was sensitive to all tested antimicrobial agents.Conclusion BSI caused by NOVC is rarely reported in China.Accurate identification,typing and drug-resistant phenotype detection of Vibrio cholerae isolated from blood culture specimens are valuable for the diagnosis,treatment,prevention and control of Vibrio cholerae-associated infection.