Tas2R activation relaxes airway smooth muscle by release of Gαt targeting on AChR signaling.

Both chronic obstructive pulmonary disease (COPD) and asthma are severe respiratory diseases. Bitter receptor-mediated bronchodilation is a potential therapy for asthma, but the mechanism underlying the agonistic relaxation of airway smooth muscle (ASM) is not well defined. By exploring the ASM relaxation mechanism of bitter substances, we observed that pretreatment with the bitter substances nearly abolished the methacholine (MCh)-induced increase in the ASM cell (ASMC) calcium concentration, thereby suppressing the calcium-induced contraction release. The ASM relaxation was significantly inhibited by simultaneous deletion of three Gαt proteins, suggesting an interaction between Tas2R and AChR signaling cascades in the relaxation process. Biochemically, the Gαt released by Tas2R activation complexes with AChR and blocks the Gαq cycling of AChR signal transduction. More importantly, a bitter substance, kudinoside A, not only attenuates airway constriction but also significantly inhibits pulmonary inflammation and tissue remodeling in COPD rats, indicating its modulation of additional Gαq-associated pathological processes. Thus, our results suggest that Tas2R activation may be an ideal strategy for halting multiple pathological processes of COPD.

Faecalibacterium prausnitzii as a potential Antiatherosclerotic microbe.

BACKGROUND: The gut microbiota plays a crucial role in coronary artery disease (CAD) development, but limited attention has been given to the role of the microbiota in preventing this disease. This study aimed to identify key biomarkers using metagenomics and untargeted metabolomics and verify their associations with atherosclerosis. METHODS: A total of 371 participants, including individuals with various CAD types and CAD-free controls, were enrolled. Subsequently, significant markers were identified in the stool samples through gut metagenomic sequencing and untargeted metabolomics. In vivo and in vitro experiments were performed to investigate the mechanisms underlying the association between these markers and atherosclerosis. RESULTS: Faecal omics sequencing revealed that individuals with a substantial presence of Faecalibacterium prausnitzii had the lowest incidence of CAD across diverse CAD groups and control subjects. A random forest model confirmed the significant relationship between F. prausnitzii and CAD incidence. Notably, F. prausnitzii emerged as a robust, independent CAD predictor. Furthermore, our findings indicated the potential of the gut microbiota and gut metabolites to predict CAD occurrence and progression, potentially impacting amino acid and vitamin metabolism. F. prausnitzii mitigated inflammation and exhibited an antiatherosclerotic effect on ApoE-/- mice after gavage. This effect was attributed to reduced intestinal LPS synthesis and reinforced mechanical and mucosal barriers, leading to decreased plasma LPS levels and an antiatherosclerotic outcome. CONCLUSIONS: Sequencing of the samples revealed a previously unknown link between specific gut microbiota and atherosclerosis. Treatment with F. prausnitzii may help prevent CAD by inhibiting atherosclerosis.

Quality markers of Dajianzhong decoction based on multicomponent qualitative and quantitative analysis combined with network pharmacology and chemometric analysis.

INTRODUCTION: Dajianzhong decoction (DJZD), a classic famous prescription, has a long history of medicinal application. Modern studies have demonstrated its clinical utility in the treatment of postoperative ileus (POI). But none of the current quality evaluation methods for this compound is associated with efficacy. OBJECTIVES: This study aimed to identify the quality markers (Q-Markers) connected to the treatment of POI in DJZD. METHODOLOGY: Ultra-performance liquid chromatography quadrupole Exactive Orbitrap mass spectrometry (UPLC-Q-Exactive Orbitrap-MS) was used to identify the main constituents in DJZD. Based on the qualitative results obtained by fingerprinting, chemical pattern recognition (CPR) was used to analyse the key components affecting the quality and finally to establish the network of the active ingredients in DJZD with POI. RESULTS: A total of 64 chemical components were detected. After fingerprint analysis, 13 common peaks were identified. The fingerprint similarity of 15 batches of samples ranged from 0.860 to 1.000. CPR analysis was able to categorically classify 15 batches of DJZD into two groups. And gingerenone A, methyl-6-gingerdiol, 6-gingerol, and hydroxy-ß-sanshool contributed to their grouping. Twelve common components interact with the therapeutic targets for treating POI. In addition, the mechanism of this prescription for treating POI may be related to the jurisdiction of the neurological system, the immunological system, and the inflammatory response. CONCLUSIONS: This integrated approach can accurately assess and forecast the quality of DJZD, presume the Q-Markers of DJZD for POI, and lay the foundation for studying the theoretical underpinnings and exploring the mechanism of DJZD in the treatment of POI.

The thymus regulates skeletal muscle regeneration by directly promoting satellite cell expansion.

The thymus is the central immune organ, but it is known to progressively degenerate with age. As thymus degeneration is paralleled by the wasting of aging skeletal muscle, we speculated that the thymus may play a role in muscle wasting. Here, using thymectomized mice, we show that the thymus is necessary for skeletal muscle regeneration, a process tightly associated with muscle aging. Compared to control mice, the thymectomized mice displayed comparable growth of muscle mass, but decreased muscle regeneration in response to injury, as evidenced by small and sparse regenerative myofibers along with inhibited expression of regeneration-associated genes myh3, myod, and myogenin. Using paired box 7 (Pax7)-immunofluorescence staining and 5-Bromo-2'-deoxyuridine-incorporation assay, we determined that the decreased regeneration capacity was caused by a limited satellite cell pool. Interestingly, the conditioned culture medium of isolated thymocytes had a potent capacity to directly stimulate satellite cell expansion in vitro. These expanded cells were enriched in subpopulations of quiescent satellite cells (Pax7highMyoDlowEdUpos) and activated satellite cells (Pax7highMyoDhighEdUpos), which were efficiently incorporated into the regenerative myofibers. We thus propose that the thymus plays an essential role in muscle regeneration by directly promoting satellite cell expansion and may function profoundly in the muscle aging process.

Two new halogenated metabolites from the red alga Laurencia sp.

Two new halogenated metabolites, laurenhalogens A (1) and B (2), along with four known ones (3-6), were isolated from the red alga Laurencia sp. The structures of 1 and 2 were determined by the means of UV, IR, MS, NMR and X-ray diffraction analysis. In addition, the antibacterial activities of 1-6 were also evaluated.

Three New Butenolides from the Green Alga Caulerpa racemosa var. turbinata.

Three new butenolides, caulerpalide A and a pair of enantiomers, (+)-caulerpalide B and (-)-caulerpalide B, together with seven known compounds, have been isolated from the green alga Caulerpa racemosa var. turbinata. All these structures were determined by spectroscopic techniques. The absolute configurations of caulerpalide A, (+)-caulerpalide B and (-)-caulerpalide B were elucidated by the method of ECD calculation. This is the first separation of butenolides from the algae of genus Caulerpa. Additionally, the antibacterial activities of the nine isolated compounds were also evaluated.

Berberine inhibits fluphenazine-induced up-regulation of CDR1 in Candida albicans.

Over-expression of the Candida drug resistance gene CDR1 is a common mechanism generating azole-resistant Candida albicans in clinical isolates. CDR1 is transcriptionally activated through the binding of the transcription factor Tac1p to the cis-acting drug-responsive element (DRE) in its promoter. We previously demonstrated that the combination of fluconazole (FLC) and berberine (BBR) produced significant synergy when used against FLC-resistant C. albicans in vitro. In this study, we found that BBR inhibited both the up-regulation of CDR1 mRNA and the transport function of Cdr1p induced by fluphenazine (FNZ). Further, electrophoretic mobility shift assays suggested that the transcription activation complex of protein-DRE was disrupted by BBR, and electrospray ionization mass spectrometry analysis showed that BBR bound to the DRE of CDR1. Thus we propose that BBR inhibits the FNZ-induced transcriptional activation of CDR1 in C. albicans by blocking transcription factor binding to the DRE of CDR1. These results contribute to our understanding of the mechanism of synergistic effect of BBR and FLC.

Exogenous 24-Epibrassinolide Enhanced Drought Tolerance and Promoted BRASSINOSTEROID-INSENSITIVE2 Expression of Quinoa.

Brassinosteroids (BRs) are involved in the regulation of biotic and abiotic stresses in plants. The molecular mechanisms of BRs that alleviate the drought stress in quinoa have rarely been reported. Here, quinoa seedlings were treated with 24-epibrassinolide (EBR) and we transiently transferred CqBIN2 to the quinoa seedlings' leaves using VIGS technology to analyze the molecular mechanism of the BR mitigation drought stress. The results showed that EBR treatment significantly increased the root growth parameters, the antioxidant enzyme activities, and the osmolyte content, resulting in a decrease in the H2O2, O2∙-, and malondialdehyde content in quinoa. A transcriptome analysis identified 8124, 2761, and 5448 differentially expressed genes (DEGs) among CK and Drought, CK and EBR + Drought, and Drought and EBR + Drought groups. WGCNA divided these DEGs into 19 modules in which these characterized genes collectively contributed significantly to drought stress. In addition, the EBR application also up-regulated the transcript levels of CqBIN2 and proline biosynthesis genes. Silenced CqBIN2 by VIGS could reduce the drought tolerance, survival rate, and proline content in quinoa seedlings. These findings not only revealed that exogenous BRs enhance drought tolerance, but also provided insight into the novel functions of CqBIN2 involved in regulating drought tolerance in plants.

Gallbladder dysfunction caused by MYPT1 ablation triggers cholestasis-induced hepatic fibrosis in mice.

BACKGROUND: The incidence of gallbladder diseases is as high as 20%, but whether gallbladder diseases contribute to hepatic disorders remains unknown. METHODS: Here, we established an animal model of gallbladder dysfunction and assessed the role of a diseased gallbladder in cholestasis-induced hepatic fibrosis (CIHF). RESULTS: Mice with smooth muscle-specific deletion of Mypt1, the gene encoding the main regulatory subunit of myosin light chain phosphatase (myosin phosphatase target subunit 1 [MYPT1]), had apparent dysfunction of gallbladder motility. This dysfunction was evidenced by abnormal contractile responses, namely, inhibited cholecystokinin 8-mediated contraction and nitric oxide-resistant relaxation. As a consequence, the gallbladder displayed impaired bile filling and biliary tract dilation comparable to the alterations in CIHF. Interestingly, the mutant animals also displayed CIHF features, including necrotic loci by the age of 1 month and subsequently exhibited progressive fibrosis and hyperplastic/dilated bile ducts. This pathological progression was similar to the phenotypes of the animal model with bile duct ligation and patients with CIHF. The characteristic biomarker of CIHF, serum alkaline phosphatase activity, was also elevated in the mice. Moreover, we observed that the myosin phosphatase target subunit 1 protein level was able to be regulated by several reagents, including lipopolysaccharide, exemplifying the risk factors for gallbladder dysfunction and hence CIHF. CONCLUSIONS: We propose that gallbladder dysfunction caused by myosin phosphatase target subunit 1 ablation is sufficient to induce CIHF in mice, resulting in impairment of the bile transport system.

Rate pressure product as a novel predictor of long-term adverse outcomes in patients after percutaneous coronary intervention: a retrospective cohort study.

OBJECTIVES: Previous studies have suggested that heart rate and blood pressure play important roles in the development of adverse outcomes in patients with coronary artery disease (CAD) who underwent percutaneous coronary intervention (PCI). However, the relationship between the rate pressure product (RPP) and long-term outcomes has rarely been investigated. This study investigated the effects of RPP on the clinical outcomes of patients with CAD who underwent PCI. DESIGN: In this study, a total of 6015 patients with CAD were enrolled. All patients were from the CORFCHD-PCI (Clinical Outcomes and Risk Factors of Patients with Coronary Heart Disease after PCI) Study. They were divided into two groups according to RPP (RPP <10 269, n=4018 and RPP ≥10 269, n=1997). In addition, the median follow-up time was 32 months. PARTICIPANTS: Data was obtained from 6050 patients with CAD who underwent PCI at the First Affiliated Hospital of Xinjiang Medical University from January 2008 to December 2016. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary endpoint was long-term mortality, including all-cause mortality (ACM) and cardiac mortality (CM). The secondary endpoints were major adverse cardiovascular events (MACEs) and major adverse cardiovascular and cerebrovascular events (MACCEs). RESULTS: We found that there were significant differences between the two groups in the incidence of ACM, CM, MACCEs and MACEs (all p<0.05). Among the patients with CAD having ACM, CM, MACCEs and MACEs, the mean survival time of the low-value group was significantly higher than that of the high-value group. Multivariate Cox regression analyses showed that RPP was an independent predictor for ACM (HR=1.605, 95% CI: 1.215-2.120, p=0.001), CM (HR=1.733, 95% CI: 1.267-2.369, p=0.001), MACCEs (HR=1.271, 95% CI: 1.063-1.518, p=0.008) and MACEs (HR=1.315, 95% CI: 1.092-1.584, p=0.004) in patients with stable CAD. On the other hand, there was no significant correlation between the RPP and the adverse outcomes in patients with acute coronary syndrome. CONCLUSION: In summary, RPP is an independent predictor of long-term prognosis in patients with CAD who underwent PCI. A higher baseline RPP before PCI increased the risk of adverse outcomes. Compared with heart rate and blood pressure alone, RPP has a higher predictive value for adverse clinical outcomes.

A Recurrence Predictive Model for Node-negative Esophageal Squamous Cell Carcinoma After Upfront Esophagectomy.

The prognosis for pathologically node-negative (pN0) esophageal squamous cell carcinoma (ESCC) with surgery alone remains poor. We aimed to develop a model for a more precise prediction of recurrence, which will allow personalized management for pN0 ESCC after upfront complete resection. Clinical and pathological records of patients with completely resected pT1-3N0M0 ESCC were retrospectively analyzed between January 2014 and December 2019. A nomogram for the prediction of recurrence was established based on the Cox regression analysis and evaluated by C-index, AUC, and calibration curves. The model was further validated using bootstrap resampling and k-fold cross-validation and compared with the 8th edition of the AJCC TNM staging system using Td-ROC, NRI, IDI, and DCA. Two-hundred-and seventy cases were included in this study. The median follow-up was 45 months. Distant and/or loco-regional recurrences were noted in 89 (33.0%) patients. The predictive model revealed pT-category, differentiation, perineural invasion, examined lymph nodes (ELN), and prognostic nutritional index (PNI) as independent risk factors for recurrence, with a c-index of 0.725 in the bootstrapping cohort. Td-ROC, NRI, and IDI showed a better predictive ability than the AJCC 8th TNM staging system. Based on this model, patients in the low-risk group had a significantly lower recurrence incidence than those in the high-risk group (p < .001). The predictive model developed in this study may facilitate the precise prediction of recurrences for pN0 ESCC after upfront surgery. Stratifying management of those patients might bring significantly better survival benefits.

ALB-dNLR Score Predicts Mortality in Coronary Artery Disease Patients After Percutaneous Coronary Intervention.

Background: The influence of the albumin/derived neutrophil and lymphocyte ratio (ALB-dNLR) on the outcomes of patients with coronary artery disease (CAD) after percutaneous coronary intervention (PCI) is not known. Here, we aimed to determine the association between the ALB-dNLR score and post-PCI CAD patient outcomes. Methods: A total of 6,050 patients from the First Affiliated Hospital of Xinjiang Medical University were enrolled between January 2008 and December 2016. These patients were divided into three groups according to their ALB-dNLR scores (0 points, n = 1,121; 1 point, n = 3,119; 2 points, n = 1,810). Mortality after PCI [all-cause (ACM) and cardiac (CM)] was taken as the primary endpoint. The prognostic value of the ALB-dNLR score was determined with the Cox proportional hazard model after adjustment for covariates. Results: The ACM and CM rates differed among participants in the three groups (P = 0.007 and P = 0.034, respectively). Multivariate Cox analysis showed that the ALB-dNLR score independently predicted both ACM [1 point vs. 0 points, HR = 1.249 (95% CI: 0.79-1.774), P = 0.215; 2 points vs. 0 points, HR = 1.777 (95% CI: 1.239-2.549), P = 0.002] and CM [1 point vs. 0 points, HR = 1.294 (95% CI: 0.871-1.922), P = 0.202; 2 points vs. 0 points, HR = 1.782 (95% CI: 1.185-1.782), P = 0.027]. We also found that among male patients in the three groups, both ACM and CM rates differed (P = 0.006 and P = 0.017, respectively). Multivariate Cox analysis showed that the ALB-dNLR score independently predicted both ACM [1 point vs. 0 points, HR = 1.237 (95% CI: 0.806-0.330), P = 0.330; 2 points vs. 0 points, HR = 1.790 (95% CI: 1.159-2.764), P = 0.009] and CM [1 point vs. 0 points HR = 1.472 (95% CI: 0.892-2.430), P = 0.130; 2 points vs. 0 points, HR = 1.792 (95% CI: 1.182-3.289), P = 0.009]. Conclusion: The ALB-dNLR score is a credible predictor for mortality in patients with CAD who have undergone PCI.

A Shigella species variant is causally linked to intractable functional constipation.

Intractable functional constipation (IFC) is the most severe form of constipation, but its etiology has long been unknown. We hypothesized that IFC is caused by refractory infection by a pathogenic bacterium. Here, we isolated from patients with IFC a Shigella species - peristaltic contraction-inhibiting bacterium (PIB) - that significantly inhibited peristaltic contraction of the colon by production of docosapentenoic acid (DPA). PIB colonized mice for at least 6 months. Oral administration of PIB was sufficient to induce constipation, which was reversed by PIB-specific phages. A mutated PIB with reduced DPA was incapable of inhibiting colonic function and inducing constipation, suggesting that DPA produced by PIB was the key mediator of the genesis of constipation. PIBs were detected in stools of 56% (38 of 68) of the IFC patients, but not in those of non-IFC or healthy individuals (0 of 180). DPA levels in stools were elevated in 44.12% (30 of 68) of the IFC patients but none of the healthy volunteers (0 of 97). Our results suggest that Shigella sp. PIB may be the critical causative pathogen for IFC, and detection of fecal PIB plus DPA may be a reliable method for IFC diagnosis and classification.

Selenoprotein W gene expression in the gastrointestinal tract of chicken is affected by dietary selenium.

Selenoprotein W (SelW) and selenium (Se) plays important roles in gastrointestinal function and that SelW expression in the gastrointestinal system of mammals is sensitive to Se levels. However, little is known about the pattern of SelW expression in the bird gastrointestinal tract. To investigate the distribution of SelW and effects of dietary Se levels on the SelW mRNA expression in the gastrointestinal tract tissues of birds, 1-day-old male chickens were fed either a commercial diet or a Se-supplemented diet containing 1.0, 2.0, 3.0 or 5.0 mg/kg sodium selenite for 90 days. The gastrointestinal tract tissues (tongue, esophagus, crop, proventriculus, gizzard, duodenum, small intestine, cecum and rectum) were collected and examined for Se content and mRNA levels of SelW. The mRNA expression of SelW was detected in all tissues. The greatest increase in SelW mRNA levels was observed in the gizzard, whereas Se content was highest in the duodenum and small intestine. A significant increase in SelW mRNA levels was observed in the gastrointestinal tract tissues of chickens fed the diets containing 1-3 mg/kg sodium selenite while decreased SelW mRNA levels were observed in the esophagus, crop, proventriculus, gizzard, duodenum and cecum in chickens fed the diet containing 5 mg/kg sodium selenite. These data indicate that SelW is widely expressed in the gastrointestinal tract tissues of birds and the transcription of the SelW gene is very sensitive to dietary Se.

A novel polyamide SL-A92 as a potential fungal resistance blocker: synthesis and bioactivities in Candida albicans.

AIM: To synthesize a novel polyamide SL-A92 and evaluate its bioactivity against drug resistance in Candida albicans. METHODS: SL-A92 was synthesized using N-hydroxybenzotriazole (HOBT)/N,N'-dicyclohexylcarbodiimide (DCC) in solution phase. Its antifungal activities and effects on strain growth were tested using the micro-broth dilution method and growth curves, respectively. Induced drug resistance in the C. albicans collection strain SC5314 was obtained by incubation with fluconazole (12 microg/mL) for 21 passages. Meanwhile, incubations with SL-A92 plus fluconazole were also carried out in SC5314 strains, and the MIC(80)s were used to evaluate the inhibitory effects of SL-A92 on drug resistance during the induction process. Real time RT-PCR was performed to investigate the CDR1 and CDR2 mRNA levels in induced SC5314 strains. RESULTS: SC5314 strain induced by the combination of fluconazole and SL-A92 (200 microg/mL) did not develop drug resistance. On day 24, the CDR1 and CDR2 mRNA levels in SC5314 strain co-treated with fluconazole and SL-A92 relative to fluconazole alone were 26% and 24%, respectively, and on day 30 the CDR1 and CDR2 mRNA levels were 43% and 31%, respectively. CONCLUSION: SL-A92 can block the development of drug resistance during the fluconazole induction process, which partially results from the down-regulation of CDR1 and CDR2.

Hepcidin Gene Cloning and Expression Pattern in Turbot (Scophthalmus maximus) after Vibrio. anguillarum Infection.

BACKGROUND: Antimicrobial peptides play crucial roles in organisms as the first line of defense against invading pathogens. OBJECTIVE: To isolate the hepcidin (hepc1) gene from the liver of turbot (Scophthalmus maximus) challenged with Vibrio anguillarum (GenBank accession number: AM113708), characterize it, and assess its expression level in various tissues. MATERIALS AND METHODS: The DNA sequence of hepcidin from S. maximus was determined from the total RNA extracted and reverse transcribed from this fish. The expression levels of tissue-specific hepcidin transcripts were determined using reverse-transcriptase polymerase chain reactions. RESULTS: Hepcidin levels increased in the livers, head kidneys and spleens of the fish. The transcriptional increase was especially noticeable in the liver after bacterial infection commencement. The presence of hepcidin and interleukin-beta (IL-1ß) in blood leukocytes was compared at the transcription level and hepcidin transcripts were detected earlier than IL-1ß transcripts after infection, indicating that hepcidin might serve as the first line of defense to kill bacteria and may also play a more direct and effective role than that of IL-1ß during the initial stage of the innate immune response when turbot are exposed to bacteria invasion. CONCLUSIONS: Hepcidin might serve as the first line of defense to kill bacteria and may also play a more direct and effective role than that of IL-1ß during the initial stage of the innate immune response when turbot are exposed to bacteria invasion.

UV-cured nanocomposite coating for surface charging mitigation and breakdown strength enhancement: exploring the combination of surface topographical structure and perfluorooctyl chain.

A facile method using a nanocomposite coating is proposed to suppress surface charge accumulation and enhance the surface breakdown strength of polymeric insulating materials like epoxy resin, by covering a thin 1,1,2,2-tetrahydroperfluorodecyltrimethoxysilane modified alumina (Al2O3) flake/UV curable resin nanocomposite coating. Due to the peculiar characteristics of perfluorooctyl chains at the microscale and the intricate topographical structure of morphology at the mesoscale, the coating exhibits enhanced water/oil repellence, surface charge accumulation resistance, and flashover withstanding capability. It is found that increasing the content of modified Al2O3 is conducive to decreasing the surface free energy of the specimens, rendering them superhydrophobic. Experimental tests in air show that the presence of nanoparticles generates numerous carrier traps at the surface layer, contributing to a much faster charge decay rate. Furthermore, impulse flashover voltage tests under vacuum show a >100% improvement of surface electrical strength. Further experimental results reveal that lower secondary electron emission yield remarkably alleviates the surface charging phenomenon, thus relieving electric field distortion caused by hetero-charges. We envision that such a multifunctional strategy for surface discharge mitigation is efficient, adaptable and easy to scale up, and thereby exhibits great prospects for applications in electronics and electrical power systems.

[Transition of Particulate Pollutant in the Parcel-based Catchment of Sponge City].

Most sponge city constructions in China are carried out in urban parcel-based catchments, and the quality and quantity of surface runoff can be controlled by several low impact development (LID) facilities. However, there are few reports on the generation and control of urban diffuse pollution. In this study, two areas with different hardening rates were compared to analyze the load conditions during the accumulation-wash-off-transport process of particulate pollutants. The results showed that the road surface in the catchment was the main underlying surface that the particulate pollutants contributed to. The road dust accumulation in the medium hardening rate (61.1%) and high hardening rate (73.6%) plots accounted for 88.4% (2.22-12.51 g ·m-2) and 90.1% (4.99-33.43 g ·m-2) of the catchment area unit, respectively. The contribution to the suspended solids (SS) load of runoff was 91.7% (0.97-7.34 g ·m-2) and 90.5% (0.92-18.77 g ·m-2), respectively. The SS load of road runoff accounted for approximately 95.2% and 83.1%, respectively. The pollution load (SS) after treatment by the LID facilities was approximately 24.0% and 40.2% of the surface runoff, respectively. The particle size distribution of road dust during the accumulation and wash-off processes was>150 µm, while that in surface and output runoff was <50 µm. With the increase in the impervious area, the distribution of finer particles (<105 µm) in the process of accumulation and wash-off increased (24.4%, 106.4%), while the distribution of particles <50 µm in road runoff decreased (12.4%). The particle size distribution of the accumulated, washed dust, and the rain runoff on the roof were roughly similar to those on the road. However, compared to the particle size distribution of road dust, in the accumulation and wash-off processes, the coarser particles (>1000 µm) of the medium hardening rate plot and the particles of size 250-450 µm and <45 µm of the high hardening rate plot increased significantly (>1000 µm: 58.1%, 108.5%; 250-450 µm: 72.9%, 41.8%; <45 µm: 59.2%, 64.8%). The results revealed the entire distribution process (accumulation-wash-off-transport) of particulate pollutants and the effect of LID facilities on the total SS pollution load of the catchment, which can provide an important reference for the scientific assessment of the project performance of LID installation in urban parcel-based catchments.

[Influence of Pervious/Impervious Pattern on Runoff and Pollution in Parcel-based Catchment].

Determining the influence of pervious/impervious underlying surface pattern (composition, position, proportion, etc.) on the generation capacity of surface runoff and pollution in the urban parcel-based catchment could aid in alleviating the urban waterlogging and control non-point source pollution significantly. Landscape metrics were used to analyze the relationship between the characteristics of pervious/impervious underlying surface pattern and total runoff (Q) and the cumulative load of dissolved pollutants (Ld) and particulate pollutants (Lp). The results showed that: ① For the metrics of fragmentation, the patch density (PD) was positively correlated with Q and Ld. and largest patch index (LPI) was negatively correlated with them. Especially, the PD exhibited a significantly positive correlation with Ld(r=0.59, P<0.05, calculated in COD). However, the LPI exhibited a significantly negative correlation with Ld (r=-0.60, P<0.05, calculated in COD). ② For the metrics of complexity, landscape shape index (LSI) was positively correlated with Q and Lp, and mean shape index (MSI) was negatively correlated with them. Especially, the LSI exhibited a significantly positive correlation with Lp (r=0.61, P<0.05, calculated in TP) significantly. However, the MSI exhibited a significantly negative correlation with Lp (r=-0.62, P<0.01, calculated in TP) significantly. ③ For the metrics of vergence, the split index (SPLIT) was positively correlated with Q and Ld, and the cohesion index (COHESION) was negatively correlated with them. The COHESION exhibited a significantly negative correlation with Ld(r=-0.59, P<0.05, calculated in COD), whereas the SPLIT exhibited a significantly positive correlation with Ld (r=0.6, P<0.05, calculated in COD). ④ In the planning on the distribution and pattern of pervious surface under small-scale catchment, it is suggested that the scattered small-regular patches should be transformed to large-irregular patches or patch group. The relationship of the fragmentation, complexity, and vergence of pervious/impervious surface, with the runoff generation, and pollution output in parcel-based catchment was analyzed, which provided a new method for rainfall runoff and pollution control by considering rational allocation of LID facilities in terms of its pattern characteristics (area, distance, shape, etc.).

Quantification of Proliferative and Dead Cells in Enteroids.

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. Epithelial barrier function requires the integrity of intestinal epithelial cells. While epithelial cell proliferation maintains a continuous layer of cells that forms a barrier, epithelial damage leads to barrier dysfunction. As a result, luminal contents can across the intestinal barrier via an unrestricted pathway. Dysfunction of intestinal barrier has been associated with many intestinal diseases, such as inflammatory bowel disease. Isolated mouse intestinal crypts can be cultured and maintained as crypt-villus-like structures, which are termed intestinal organoids or "enteroids". Enteroids are ideal to study the proliferation and cell death of intestinal epithelial cells in vitro. In this protocol, we describe a simple method to quantify the number of proliferative and dead cells in cultured enteroids. 5-ethynyl-2'-deoxyuridine (EdU) and propidium iodide are used to label proliferating and dead cells in enteroids, and the proportion of proliferating and dead cells are then analyzed by flow cytometry. This is a useful tool to test the effects of drug treatment on intestinal epithelial cell proliferation and cell survival.