Objective To investigate the relationship between the expression of keratin 15(KRT15)and keratin 18(KRT18)proteins in colorectal cancer tissue and their clinicopathological features and prognosis.Methods A total of 97 patients with colorectal cancer who underwent surgical treatment in a hospital from June 2018 to June 2019 were selected as the study objects.Immunohistochemistry was used to detect the ex-pression of KRT15 and KRT18 protein in colorectal cancer tissues and adjacent tissues,and the differences of KRT15 and KRT18 protein expression in colorectal cancer patients with different clinicopathological features were compared.The patients with colorectal cancer were followed up for 3 years after discharge,and their o-verall survival(OS)during the follow-up period was analyzed.Kaplan-Meier survival curve and Log-rank test were used to analyze the difference in OS rate among colorectal cancer patients with different KRT15 and KRT18 protein expression.Univariate and multivariate COX proportional regression analysis was performed to analyze the factors affecting the prognosis of patients with colorectal cancer.Results The positive expres-sion rates of KRT15 and KRT18 protein in colorectal cancer tissues were higher than those in adjacent tis-sues,and the difference was statistically significant(P＜0.05).The positive expression rates of KRT15 and KRT18 protein in colorectal cancer tissues of patients with low differentiation,TNM Ⅲ stage,perineural inva-sion and preoperative carcinoembryonic antigen(CEA)level ≥5 ng/mL were higher than those of patients with medium-high differentiation,TNM Ⅰ-Ⅱ stage,without perineural invasion and preoperative CEA level＜5 ng/mL,the difference was statistically significant(P＜0.05).The 3-year OS rates of colorectal cancer patients with positive expression of KRT15 and KRT18 protein were 64.29％and 60.00％respectively,which were lower than those of patients with negative expression of KRT15 and KRT18 protein(83.64％and 85.96％respec-tively),and the difference was statistically significant(x2=6.497,7.987,P＜0.05).Multivariate COX pro-portional regression analysis showed that TNM stage Ⅲ,positive expression of KRT15 protein and positive expression of KRT18 protein were risk factors affecting the survival of patients with colorectal cancer(P＜0.05).Conclusion The expression of KRT15 and KRT18 protein in colorectal cancer tissues can provide ref-erence for prognosis assessment of patients with colorectal cancer.

The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (M^pro) and papain like protease (PL^pro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851M^pro inhibitors and 205 PL^pro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both M^pro and PL^pro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of M^pro inhibitors and over 20% of PL^pro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 M^pro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.
Humans ; Antiviral Agents/chemistry* ; COVID-19 ; COVID-19 Drug Treatment ; High-Throughput Screening Assays ; Molecular Docking Simulation ; Protease Inhibitors/chemistry* ; SARS-CoV-2/enzymology* ; Viral Nonstructural Proteins

Objective To analyze the chemical compounds of Shenqi Dihuang decoction by the ultraperformance liquid chromatography coupled with linear quadrupole ion trap-orbitrap mass spectrometry (UPLC-LTQ-Orbitrap-MS). Methods Warters ACQUITY UPLC HSS T3 (2.1 mm ×100 mm, 1.8 μm) was used as chromatographic column with mobile phase: 0.1% formic acid water (A)-0.1% formic acid acetonitrile (B) with gradient elution, and flow rate was 0.3 ml/min. Electrospray ion source (ESI) and an electrostatic field orbital ion trap mass analyzer were adopted, which was used to collect mass spectrometry fragment information with positive and negative ion modes, by comparing with the relative retention time of the reference substance. In addition, the fragment information of the mass spectrum was used to identify the compounds. The accurate identification of the chemical components in Shenqi Dihuang decoction was confirmed with literature. Results The study found that UPLC-LTQ-Orbitrap-MS technology could be used to identify 62 chemical components, including 13 aromatic acids, 9 flavonoids, 8 saponins, and 5 aromatic amines, 3 keto acids, 2 phenols, 1 aromatic quinone and other ingredients in Shenqi Dihuang decoction. Conclusion The identification analysis method in this study was efficient and accurate, which could be applied to the identification and analysis of chemical components in Shenqi Dihuang decoction and provided the important experimental data for the research on the material basis and mechanism.

The gut microbiota of intensive care unit (ICU) patients displays extreme dysbiosis asso-ciated with increased susceptibility to organ failure, sepsis, and septic shock. However, such dysbio-sis is difficult to characterize owing to the high dimensional complexity of the gut microbiota. We tested whether the concept of enterotype can be applied to the gut microbiota of ICU patients to describe the dysbiosis. We collected 131 fecal samples from 64 ICU patients diagnosed with sepsis or septic shock and performed 16S rRNA gene sequencing to dissect their gut microbiota compo-sitions. During the development of sepsis or septic shock and during various medical treatments, the ICU patients always exhibited two dysbiotic microbiota patterns, or ICU-enterotypes, which could not be explained by host properties such as age, sex, and body mass index, or external stressors such as infection site and antibiotic use. ICU-enterotype I (ICU E1) comprised predominantly Bac-teroides and an unclassified genus of Enterobacteriaceae, while ICU-enterotype Ⅱ(ICU E2) com-prised predominantly Enterococcus. Among more critically ill patients with Acute Physiology and Chronic Health Evaluation Ⅱ(APACHE Ⅱ) scores > 18, septic shock was more likely to occur with ICU E1 (P = 0.041). Additionally, ICU E1 was correlated with high serum lactate levels (P = 0.007). Therefore, different patterns of dysbiosis were correlated with different clinicaloutcomes, suggesting that ICU-enterotypes should be diagnosed as independent clinical indices. Thus, the microbial-based human index classifier we propose is precise and effective for timely mon-itoring of ICU-enterotypes of individual patients. This work is a first step toward precision medicine for septic patients based on their gut microbiota profiles.

In 2011, the term "enterotype" first appeared to the general public in Nature, which refers to stratification of human gut microbiota. However, with more studies on enterotypes conducted nowadays, doubts about the existence and robustness of enterotypes have also emerged. Here we reviewed current opinions about enterotypes from both conceptual and analytical points of view. We firstly illustrated the definition of the enterotype and various factors influencing enterotypes, such as diet, administration of antibiotics, and age. Then we summarized lines of evidence that pose the concept against the enterotype, and described the current methods for enterotype analysis. Finally, we showed that the concept of enterotype has been extended to other ecological niches. Based on current studies on enterotypes, it has been clear that more studies with larger sample sizes are needed to characterize the enterotypes. Improved computational methods are also required to build sophisticated models, reflecting the dynamics and resilience of enterotypes.
Adult ; Age Factors ; Aged ; Anti-Bacterial Agents ; pharmacology ; Child ; Diet ; Gastrointestinal Microbiome ; drug effects ; Humans ; Infant

Objective: To investigate the delayed effect of liver injury and metabolism of dimethylformamide (DMF) after high exposures in rats. Methods: A total of 12 rats were randomly divided into four groups and 3 rats were in each group. Rats in 1d DMF+2 d delayed group were dosed for 1 day and rested for 2 days, and sacrificed at the 4th day. Rats in 3 d DMF group were dosed for 3 days and sacrificed at the 4th day. Rats in 3 d DMF+3 d delayed group were dosed for 3 days and rested for 3 days, and sacrificed at the 7th day. Rats in control group were administrated with water for 3 days, sacrificed at the 7th day. The administrated dose was 1 000 mg/kg (body weight·d) DMF by oral. The daily observation and body weight were recorded during the study period. After the experiment, the blood biochemistry, including alanine aminotransferase (ALT) , aspartate aminotransferase (AST) , lactic dehydrogenase (LDH) , alkaline phosphatase (ALP) , total bilirubin (TBIL) etc. were detected. Liver weight, kidney weight, liver/body ratio, kidney/body ratio and pathologic examination of liver and kidney were investigated. The concentrations of hemoglobin-adduct (NMHb) were detected. Results: During the period of 1~3 d, body weight growth rate of rats in each treated group had no significant difference with control rats. In the 4~6 th day of the period, rats in group 3 became thinner than before, and the body weight was negative growth (-4.22±3.29 g/d) and significant lower than that of control rats (10.33±3.21 g/d, F=30.07, P<0.05) . AST and LDH levels of 3 d DMF group were significant higher than control group (P<0.05) . Liver/body ratio in 3 d DMF+3 d delayed group were significant higher than control group (P<0.05) . The gross inspection showed 1 rat and 3 rats were observed liver injury in 3 d DMF group and 3 d DMF+3 d delayed group, respectively. Histopathological lesions of 1d DMF+2 d delayed group, 3 d DMF group and 3 d DMF+3 d delayed group were mainly spotty necrosis, focal necrosis and large necrosis of liver cells, respectively. Only NMHb level of control group was undetectable. NMHb levels in 3 d DMF+3 d delayed group were significantly higher than 3 d DMF group (F=135.46, P<0.05) . Conclusion The DMF-induced liver injury and DMF metabolism may be delayed after high DMF exposures in rats.

Human 5-lipoxygenase (5-LOX) is a well-validated drug target and its inhibitors are potential drugs for treating leukotriene-related disorders. Our previous work on structural optimization of the hit compound 2 from our in-house collection identified two lead compounds, 3a and 3b, exhibiting a potent inhibitory profile against 5-LOX with IC50 values less than 1 µmol/L in cell-based assays. Here, we further optimized these compounds to prepare a class of novel pyrazole derivatives by opening the fused-ring system. Several new compounds exhibited more potent inhibitory activity than the lead compounds against 5-LOX. In particular, compound 4e not only suppressed lipopolysaccharide-induced inflammation in brain inflammatory cells and protected neurons from oxidative toxicity, but also significantly decreased infarct damage in a mouse model of cerebral ischemia. Molecular docking analysis further confirmed the consistency of our theoretical results and experimental data. In conclusion, the excellent in vitro and in vivo inhibitory activities of these compounds against 5-LOX suggested that these novel chemical structures have a promising therapeutic potential to treat leukotriene-related disorders.

Objective: To explore the protective mechanism of resvertrol (RSV) suppressing the rapid electrical stimulation incurred oxidative stress injury in neonatal rats cardiomyocytes. Methods: The neonatal rats cardiac ifbroblasts and myocytes were isolated by double enzyme digestion and differential adhesion method, and the cardiomyocytes were randomly divided into 5 groups:①Control (CTR) group,②Rapid electrical stimulation (RES) group,③RES+APO group, cells were pretreated with NADPH oxidase inhibitor APO,④RES+RSV group,⑤RES+AIP group, cells were pretreated with CaMKII inhibitor AIP. In order to conifrm whether RSV protection was via MsrA-OX-CaMKⅡpathway, the cells were further divided into another 3 groups:①DMSO control group,②RSV+DMSO group,③RES+ RSV+DMSO group. The best dose of RSV was measured with Kit-8 by cardiomyocytes surviving condition, the optimal electrical stimulation time was detected with ELISA by Ang II level in conditioned medium. The level of reactive oxygen species (ROS) in cardiomyocytes was detected by flow cytometry, the protein expressions of MsrA, Nox4, Nox2, P22phox, OX-CaMK II and apoptosis related cleaved caspase-3 were observed by Western blot analysis.Results:①Compared with CTR group, RES group showed increased AngII secretion with increased protein expressions of Nox4, Nox2, P22phox, OX-CaMK II and cleaved caspase-3.②Compared with RES group, the RES+APO, RES+RSV, RES+AIP groups had decreased ROS level, the ROS was even lower in RES+RSV group.③Compared with RES group, the RES+APO, RES+RSV groups presented decreased protein expressions of Nox4, Nox2, P22phox, OX-CaMK II and cleaved caspase-3, while RES+AIP group only had decreased Nox2, OX-CaMK II and cleaved caspase-3.④Compared with DMSO control group, RES+ RSV+DMSO group had the lower level of cleaved caspase-3 expression. Conclusion: RSV has protective effect on rapid electrical stimulation incurred oxidative stress injury in neonatal rats cardiomyocytes, which might be via NADPH oxidase with the increased MsrA expression .

Objective: To explore the effect of resveratrol (Res) on angiogenesis of human umbilical vein endothelial cells (HUVEC) with the possible mechanisms in vitro.
Methods: The HUVECs were cultured in 6 groups.①Control group, HUVEC were cultured with high glucose in DMEM,②Res group, the cell were cultured with Res at different concentrations,③Res with PI3K blocker LY294002 (Res+Blocker 1) group, ④Blocker 1 group, HUVEC were cultured with LY294002 alone, ⑤Res with eNOS blocker L-NAME (Res +Blocker 2) group and ⑥Blocker 2 group. The effect of Res on HUVEC proliferation was detected by CCK-8 kit, the protein expressions of p-Akt, p-eNOS were examined by Westin blot analysis, nitric oxide (NO) level was measured by nitrate reduction method and the endothelial cell migration was assayed by transwell chamber method.
Results: ① Compared with Control group, HUVEC proliferation increased in Res (1, 5μmol/L ) group, P<0.01, the proliferation in Res (5μmol/L) group was higher than those in Res (0.2, 10, 20μmol/L) group, P<0.01, while Res (20 μmol/L) group could inhibit the proliferation P<0.01. ②Compared with Control group, Res (5μmol/L) group had the higher protein expressions of p-Akt, p-eNOS, P<0.05-0.01, higher NO level, P<0.05.③Compared with Res group, Res+Blocker 1 group had lower expressions of p-Akt, p-eNOS, P<0.01, lower NO, P<0.05; the expressions of p-Akt, p-eNOS and NO level were similar between Res+Blocker 1 group and Blocker 1 group, all P>0.05.④Compared with Control group, the cell migration and tubing formation were higher in Res (5μmol/L) group, P<0.01;compared with Res group, the cell migration and tubing formation were lower in Res+Block2 group, P<0.01.
Conclusion: Res could up-regulate NO level via activating PI3-K/Akt/eNOS signaling and therefore, improving the proliferation, migration and tubing formation of HUVEC in vitro.