Genome-wide CRISPR screens identify PKMYT1 as a therapeutic target in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with an overall 5-year survival rate of <12% due to the lack of effective treatments. Novel treatment strategies are urgently needed. Here, PKMYT1 is identified through genome-wide CRISPR screens as a non-mutant, genetic vulnerability of PDAC. Higher PKMYT1 expression levels indicate poor prognosis in PDAC patients. PKMYT1 ablation inhibits tumor growth and proliferation in vitro and in vivo by regulating cell cycle progression and inducing apoptosis. Moreover, pharmacological inhibition of PKMYT1 shows efficacy in multiple PDAC cell models and effectively induces tumor regression without overt toxicity in PDAC cell line-derived xenograft and in more clinically relevant patient-derived xenograft models. Mechanistically, in addition to its canonical function of phosphorylating CDK1, PKMYT1 functions as an oncogene to promote PDAC tumorigenesis by regulating PLK1 expression and phosphorylation. Finally, TP53 function and PRKDC activation are shown to modulate the sensitivity to PKMYT1 inhibition. These results define PKMYT1 dependency in PDAC and identify potential therapeutic strategies for clinical translation.

RUNX1 targeting AKT3 promotes alveolar hypercoagulation and fibrinolytic inhibition in LPS induced ARDS.

BACKGROUND: Alveolar hypercoagulation and fibrinolytic inhibition are mainly responsible for massive alveolar fibrin deposition, which are closely related with refractory hypoxemia in acute respiratory distress syndrome (ARDS). Our previous study testified runt-related transcription factor (RUNX1) participated in the regulation of this pathophysiology in this syndrome, but the mechanism is unknown. We speculate that screening the downstream genes associated with RUNX1 will presumably help uncover the mechanism of RUNX1. METHODS: Genes associated with RUNX1 were screened by CHIP-seq, among which the target gene was verified by Dual Luciferase experiment. Then the efficacy of the target gene on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS was explored in vivo as well as in vitro. Finally, whether the regulatory effects of RUNX1 on alveolar hypercoagulation and fibrinolytic in ARDS would be related with the screened target gene was also sufficiently explored. RESULTS: Among these screened genes, AKT3 was verified to be the direct target gene of RUNX1. Results showed that AKT3 was highly expressed either in lung tissues of LPS-induced rat ARDS or in LPS-treated alveolar epithelia cell type II (AECII). Tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) were increasingly expressed both in lung tissues of ARDS and in LPS-induced AECII, which were all significantly attenuated by down-regulation of AKT3. Inhibition of AKT3 gene obviously ameliorated the LPS-induced lung injury as well as the collagen I expression in ARDS. RUNX1 overexpression not only promoted the expressions of TF, PAI-1, but also boosted AKT3 expression in vitro. More importantly, the efficacy of RUNX1 on TF, PAI-1 were all effectively reversed by down-regulation of AKT3 gene. CONCLUSION: AKT3 is an important target gene of RUNX1, through which RUNX1 exerted its regulatory role on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS. RUNX1/ATK3 signaling axis is expected to be a new target for the exploration of ARDS genesis and treatment.

Tumor cell-intrinsic SETD2 inactivation sensitizes cancer cells to immune checkpoint blockade through the NR2F1-STAT1 pathway.

BACKGROUND: Cancer immunotherapies can induce durable tumor regression, but most patients do not respond. SETD2 mutation has been linked to the efficacy of immune checkpoint inhibitors (ICIs) immunotherapy. The functional importance of the SETD2 inactivation and how to modulate immunotherapy response remains unclear. METHODS: To explore the function of SETD2 in immunotherapy, knockout and subsequent functional experiments were conducted. Bulk RNA-seq, ATAC-seq, Chip-seq and single-cell RNA-seq were performed to dissect the mechanism and explore the immune microenvironment of mouse tumor. Flow cytometry was used to assess cell surface antigen and intratumoral T cell levels. RESULTS: We comprehensively determine the effect of SETD2 inactivation in ICIs therapy and elucidate the mechanistic impact on tumor immunity. Murine syngeneic tumors harboring Setd2 inactivation are sensitive to ICIs. By bulk and single-cell RNA-seq, we further reveal that SETD2 inactivation reprograms intratumoral immune cells and inflames the tumor microenvironment, which is characterized by high infiltration of T cells and enhanced antigen presentation to activate CD8+ T cell-mediated killing. Mechanistically, via an integrated multiomics analysis using ATAC-seq, ChIP-seq and RNA-seq, we demonstrate that SETD2 inactivation reduces NR2F1 transcription by impairing H3K36me3 deposition and chromatin accessibility, which activates the STAT1 signaling pathway to promote chemokines and programmed cell death protein-1 (PD-1) expression and enhance antigen presentation. All these regulatory mechanisms synergistically promote the effects of anti-programmed cell death ligand 1 immunotherapy in Setd2-knockout syngeneic mouse models. The SETD2-NR2F1-STAT1 regulatory axis is conserved in human and murine cancers. Finally, cancer patients harboring SETD2 mutations who received ICIs show increased durable clinical benefits and survival. CONCLUSIONS: These findings provide novel insights into the biology of SETD2 inactivation regulation and reveal a new potential therapeutic biomarker for ICIs immunotherapy in various refractory cancers.

HUB genes transcriptionally regulate lipid metabolism in alveolar type II cells under LPS stimulation.

Objective: Alveolar type II (ATII) cells produce pulmonary surfactant (PS) essential for maintaining lung function. The aberration or depletion of PS can cause alveolar collapse, a hallmark of acute respiratory distress syndrome (ARDS). However, the intricacies underlying these changes remain unclear. This study aimed to elucidate the mechanisms underlying PS perturbations in ATII cells using transcriptional RNA-seq, offering insights into the pathogenesis of ARDS. Methods: ATII cells were identified using immunofluorescence targeting surface-active protein C. We used 24-h lipopolysaccharide (LPS)-induced ATII cells as an ARDS cell model. The efficacy of the injury model was gauged by detecting the presence of tumour necrosis factor-α and interleukin-6. RNA-seq analysis was performed to investigate the dynamics of PS deviation in unaltered and LPS-exposed ATII cells. Results: Whole-transcriptome sequencing revealed that LPS-stimulated ATII cells showed significantly increased transcription of genes, including Lss, Nsdhl, Hmgcs1, Mvd, Cyp51, Idi1, Acss2, Insig1, and Hsd17b7, which play key roles in regulating cholesterol biosynthesis. We further verified gene levels using real-time quantitative PCR, and the results showed that the mRNA expression of these genes increased, which was consistent with the RNA-seq results. Conclusion: Our study revealed pivotal transcriptional shifts in ATII cells after LPS exposure, particularly in nine key lipid and cholesterol metabolism genes. This altered expression might disrupt the lipid balance, ultimately affecting PS function. This finding deepens our understanding of the aetiology of ARDS and may lead to new therapeutic directions.

[The fliL gene significantly affects the motility and sporulation abilities of Clostridioides difficile].

Flagella are the main motility structure of Clostridioides difficile that affects the adhesion, colonization, and virulence of C. difficile in the human gastrointestinal tract. The FliL protein is a single transmembrane protein bound to the flagellar matrix. This study aimed to investigate the effect of the FliL encoding gene flagellar basal body-associated FliL family protein (fliL) on the phenotype of C. difficile. The fliL gene deletion mutant (ΔfliL) and its corresponding complementary strains (: : fliL) were constructed using allele-coupled exchange (ACE) and the standard molecular clone method. The differences in physiological properties such as growth profile, antibiotic sensitivity, pH resistance, motility, and spore production ability between the mutant and wild-type strains (CD630) were investigated. The ΔfliL mutant and the : : fliL complementary strain were successfully constructed. After comparing the phenotypes of strains CD630, ΔfliL, and : : fliL, the results showed that the growth rate and maximum biomass of ΔfliL mutant decreased than that of CD630. The ΔfliL mutant showed increased sensitivity to amoxicillin, ampicillin, and norfloxacin. Its sensitivity to kanamycin and tetracycline antibiotics decreased, and the antibiotic sensitivity partially returned to the level of CD630 strain in the : : fliL strain. Moreover, the motility was significantly reduced in the ΔfliL mutant. Interestingly, the motility of the : : fliL strain significantly increased even when compared to that of the CD630 strain. Furthermore, the pH tolerance of the ΔfliL mutant significantly increased or decreased at pH 5 or 9, respectively. Finally, the sporulation ability of ΔfliL mutant reduced considerably compared to the CD630 strain and recovered in the : : fliL strain. We conclude that the deletion of the fliL gene significantly reduced the swimming motility of C. difficile, suggesting that the fliL gene is essential for the motility of C. difficile. The fliL gene deletion significantly reduced spore production, cell growth rate, tolerance to different antibiotics, acidity, and alkalinity environments of C. difficile. These physiological characteristics are closely related to the survival advantage in the host intestine, which is correlated with its pathogenicity. Thus, we suggested that the function of the fliL gene is closely related to its motility, colonization, environmental tolerance, and spore production ability, which consequently affects the pathogenicity of C. difficile.

Binding domain peptide ameliorates alveolar hypercoagulation and fibrinolytic inhibition in mice with lipopolysaccharide-induced acute respiratory distress syndrome Via NF-κB signaling pathway.

BACKGROUND: Alveolar hypercoagulation and fibrinolytic inhibition are shown to be associated with refractory hypoxemia in acute respiratory distress syndrome (ARDS), and the NF-κB pathway is involved in this process. The purpose of this study is to explore the role of NEMO-binding domain peptide (NBDP) in alleviating alveolar hypercoagulation and fibrinolytic inhibition induced by lipopolysaccharide (LPS) in ARDS mice and its related mechanisms. MATERIALS AND METHODS: ARDS was induced by inhalation of LPS (mg/L) in adult male BALB/c mice. Mice were treated with intratracheal inhalation of NBDP or saline aerosol at increased concentrations 30 minutes before LPS administration. Six hours after LPS treatment, bronchoalveolar lavage fluids (BALF) were collected and then all mice were euthanized. In addition, coagulation and fibrinolysis associated factors in lung tissues and BALF were detected, and the activation of NF-κB signaling pathway was observed. RESULTS: NBDP pretreatment dose-dependently inhibited the expression of tissue factor (TF) and plasminogen activator inhibitor (PAI) 1 in lung tissues, reduced the secretions of TF, PAI-1, thrombin-antithrombin (TAT) complex, and promoted activated protein C (APC) secretion in BALF induced by LPS. LPS-induced high expression of pulmonary procollagen peptide type lll (PIIIP) was also reduced in a dose-dependent manner under NBDP pretreatment. Western blotting showed that NBDP pretreatment significantly attenuated LPS-induced activation of IKKα/ß, Iκα and NF-κB p65. NBDP pretreatment also inhibited the DNA binding activity of p65 induced by LPS. We also noticed that NBDP protected mice against LPS-induced lung injury in a dose-dependent manner. CONCLUSIONS: The experimental findings demonstrate that through inhibiting the NF-κB signaling pathway, NBDP dose-dependently ameliorates LPS-induced alveolar hypercoagulation and fibrinolytic inhibition, which is expected to be a new therapeutic target to correct the abnormalities of alveolar coagulation and fibrinolytic pathways in ARDS.

The Efficacy and Safety of Early Renal Replacement Therapy in Critically Ill Patients With Acute Kidney Injury: A Meta-Analysis With Trial Sequential Analysis of Randomized Controlled Trials.

The efficacy and safety of early renal replacement therapy (eRRT) for critically ill patients with acute kidney injury (AKI) remain controversial. Therefore, the purpose of our study was to perform an up-to-date meta-analysis with the trial sequential analysis (TSA) of randomized controlled trials (RCTs) to evaluate the therapeutic effect of eRRT on patients in an intensive care unit (ICU). We extensively searched MEDLINE, EMBASE, LILACS, the Cochrane Central Register of Controlled Trials and ClinicalTrials.gov, Gray Literature Report, and Bielefeld Academic Search Engine (BASE), and conducted an updated search on December 27, 2021. The included studies were RCTs, which compared the efficacy and safety of eRRT and delayed renal replacement therapy (dRRT) on critically ill patients with AKI. We adopted TSA and sensitivity analysis to strengthen the robustness of the results. About 12 RCTs with a total of 5,423 participants were included. Patients receiving eRRT and dRRT had the similar rate of all-cause mortality at day 28 (38.7% vs. 38.9%) [risk ratio (RR), 1.00; 95%CI, 0.93-1.07, p = 0.93, I 2 = 0%, p = 0.93]. A sensitivity and subgroup analysis produced similar results for the primary outcome. TSA showed that the required information size was 5,034, and the cumulative Z-curve crossed trial sequential monitoring boundaries for futility. Patients receiving eRRT had a higher rate of renal replacement therapy (RRT) (RR, 1.50, 95% CI: 1.28-1.76, p < 0.00001, I 2 = 96%), and experienced more adverse events comparing to those receiving dRRT (RR: 1.41, 95% CI: 1.22-1.63, p < 0.0001, heterogeneity not applied). The most remarkable and important experimental finding is that, to our knowledge, the current meta-analysis included the largest sample size from the RCTs, which were published in the past 10 years to date, show that eRRT had no significant survival benefit for ill patients with AKI compared with dRRT and TSA indicating that no more studies were needed to confirm it. Trial Registration: INPLASY, INPLASY2020120030. Registered 04 December 2020.

The cwp66 Gene Affects Cell Adhesion, Stress Tolerance, and Antibiotic Resistance in Clostridioides difficile.

Clostridioides difficile is a Gram-positive, spore-forming anaerobic bacteria that is one of the leading causes of antibiotic-associated diarrhea. The cell wall protein 66 gene (cwp66) encodes a cell wall protein, which is the second major cell surface antigen of C. difficile. Although immunological approaches, such as antibodies and purified recombinant proteins, have been implemented to study the role of Cwp66 in cell adhesion, no deletion mutant of the cwp66 gene has yet been characterized. We constructed a cwp66 gene deletion mutant using Clustered Regularly Interspaced Short Palindromic Repeats Cpf1 (CRISPR-Cpf1) system. The phenotypic and transcriptomic changes of the Δcwp66 mutant compared with the wild-type (WT) strain were studied. The deletion of the cwp66 gene led to the decrease of cell adhesive capacity, cell motility, and stresses tolerance (to Triton X-100, acidic environment, and oxidative stress). Interestingly, the Δcwp66 mutant is more sensitive than the WT strain to clindamycin, ampicillin, and erythromycin but more resistant than the latter to vancomycin and metronidazole. Moreover, mannitol utilization capability in the Δcwp66 mutant was lost. Comparative transcriptomic analyses indicated that (i) 22.90-fold upregulation of cwpV gene and unable to express gpr gene were prominent in the Δcwp66 mutant; (ii) the cwp66 gene was involved in vancomycin resistance of C. difficile by influencing the expression of d-Alanine-d-Alanine ligase; and (iii) the mannose/fructose/sorbose IIC and IID components were upregulated in Δcwp66 mutant. The present work deepens our understanding of the contribution of the cwp66 gene to cell adhesion, stress tolerance, antibiotic resistance, and mannitol transportation of C. difficile. IMPORTANCE The cell wall protein 66 gene (cwp66) encodes a cell wall protein, which is the second major cell surface antigen of C. difficile. Although immunological approaches, such as antibodies and purified recombinant proteins, have been implemented to study the role of Cwp66 in cell adhesion, no deletion mutant of the cwp66 gene has yet been characterized. The current study provides direct evidence that the cwp66 gene serves as a major adhesion in C. difficile, and also suggested that deletion of the cwp66 gene led to the decrease of cell adhesive capacity, cell motility, and stresses tolerance (to Triton X-100, acidic environment, and oxidative stress). Interestingly, the antibiotic resistance and carbon source utilization profiles of the Δcwp66 mutant were significantly changed. These phenotypes were detrimental to the survival and pathogenesis of C. difficile in the human gut and may shed light on preventing C. difficile infection.

Prognostic significance of steroid response in pediatric acute lymphoblastic leukemia: The CCCG-ALL-2015 study.

Introduction: Whether steroid response is an independent risk factor for acute lymphoblastic leukemia (ALL) is controversial. This study aimed to investigate the relationship between response to dexamethasone and prognosis in children with ALL. Methods: We analyzed the data of 5,161 children with ALL who received treatment in accordance with the Chinese Children's Cancer Group ALL-2015 protocol between January 1, 2015, and December 31, 2018, in China. All patients received dexamethasone for 4 days as upfront window therapy. Based on the peripheral lymphoblast count on day 5, these patients were classified into the dexamethasone good response (DGR) and dexamethasone poor response (DPR) groups. A peripheral lymphoblast count ≥1× 109/L indicated poor response to dexamethasone. Results: The age, white blood cell counts, prevalence of the BCR/ABL1 and TCF3/PBX1 fusion genes, and rates of recurrence in the central nervous system were higher in the DPR than in the DGR group (P<0.001). Compared to the DPR group, the DGR group had a lower recurrence rate (18.6% vs. 11%) and higher 6-year event-free survival (73% vs. 83%) and overall survival (86% vs. 92%) rates; nevertheless, subgroup analysis only showed significant difference in the intermediate-risk group (P<0.001). Discussion: Response to dexamethasone was associated with an early treatment response in our study. In the intermediate-risk group, dexamethasone response added a prognostic value in addition to minimal residual disease, which may direct early intervention to reduce the relapse rate.

Andrographolide sulfonate attenuates alveolar hypercoagulation and fibrinolytic inhibition partly via NF-κB pathway in LPS-induced acute respiratory distress syndrome in mice.

BACKGROUND: Alveolar hypercoagulation and fibrinolytic inhibition are important characteristics during acute respiratory distress syndrome (ARDS), and NF-κB p65 signaling pathway is involved to regulate these pathophysiologies. We hypothesize that targeting NF-κB signal pathway could ameliorate alveolar hypercoagulation and fibrinolyitc inhibition, thus attenuating lung injury in ARDS. PURPOSE: We explore the efficacy and the potential mechanism of andrographolide sulfonate (Andro-S) on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS in mice. METHODS: ARDS was made by lipopolysaccharide (LPS) inhalation in C57BLmice. Andrographolide sulfonate (2.5, 5 and 10 mg/kg) was intraperitoneally given to the mice (once a day for three consecutive days) before LPS administration. NEMO binding domain peptide (NBD), an inhibitor of NF-κB, was used as the positive control and it replaced Andro-S in mice of NBD group. Mice in normal control received saline instead of LPS. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected for analysis of alveolar coagulation, fibrinolytic inhibition as well as of pulmonary inflammatory response after 8 h of LPS inhalation. NF-κB signal pathway in lung tissue was simultaneously determined. RESULTS: Andro-S dose-dependently inhibited tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 expressions either in mRNA or in protein in lung tissue of ARDS mice, and it also decreased the concentrations of TF, PAI-1, thrombin-antithrombin complex (TAT), procollagen peptide type Ⅲ (PⅢP) while promoting the production of activated protein C (APC) in BALF. Meanwhile, Andro-S effectively inhibited inflammatory response (interleukin 1ß and myeloperoxidase) induced by LPS. LPS stimulation dramatically activated NF-κB signal pathway, indicated by increased expressions of phosphorylation of p65 (p-p65), p-IKKα/ß and p-IκBα and the higher p65-DNA binding activity, which were all dose-dependently reversed by Andro-S. Andro-S and NBD presented similar efficacies. CONCLUSIONS: Andro-S treatment improves alveolar hypercoagulation and fibrinolytic inhibition and attenuates pulmonary inflammation in LPS-induced ARDS in mice partly through NF-κB pathway inactivation. The drug is expected to be an effective choice for ARDS.

Triptolide dose-dependently improves LPS-induced alveolar hypercoagulation and fibrinolysis inhibition through NF-κB inactivation in ARDS mice.

BACKGROUND: Alveolar hypercoagulation and fibrinolysis inhibition were associated with the refractory hypoxemia and the high mortality in patient with acute respiratory distress syndrome (ARDS), and NF-κB pathway was confirmed to contribute to the process. Triptolide (TP) significantly inhibited NF-κB pathway and thus depressed accessive inflammatory response in ARDS. We speculate that TP could improve alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS via NF-κB inactivation. PURPOSE: The aim of this experiment was to explore the efficacy and potential mechanism of TP on alveolar hypercoagulation and fibrinolysis inhibition in LPS-induced ARDS in mice. METHODS: 50 µl of LPS (5 mg/ml) was inhalationally given to C57BL/6 mice to set up ARDS model. Male mice were randomly accepted with LPS, LPS + TP (1 µg/kg, 10 µg/kg, 50 µg/kg respectively), or with NEMO Binding domain peptide (NBD), an inhibitor of NF-κB. TP (1 µg/kg, 10 µg/kg, 50 µg/kg) were intraperitoneally injected or 10 µg/50 µl of NBD solution were inhaled 30 min before LPS inhalation. A same volume of normal saline (NS) substituted for TP in mice in control. The endpoint of experiment was at 8 hours after LPS stimulation. Pulmonary tissues were taken for hematoxylin-eosin (HE) staining, wet / dry ratio and for lung injury scores (LIS). Tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 in lung tissue were detected by Western-blotting and by quantitative Real-time PCR(qPCR) respectively. Concentrations of TF, PAI-1, thrombin-antithrombin complex (TAT), procollagen peptide type Ⅲ (PⅢP) and activated protein C (APC) in bronchoalveolar lavage fluid (BALF) were measured by ELISA. NF-κB activation and p65-DNA binding activity in pulmonary tissue were simultaneously determined. RESULTS: LPS stimulation resulted in pulmonary edema, neutrophils infiltration, obvious alveolar collapse, interstitial congestion, with high LIS, which were all dose-dependently ameliorated by Triptolide. LPS also dramatically promoted the expressions of TF and PAI-1 either in mRNA or in protein in lung tissue, and significantly stimulated the secretions of TF, PAI-1, TAT, PⅢP but inhibited APC production in BALF, which were all reversed by triptolide treatment in dose-dependent manner. TP dose-dependently inhibited the activation of NF-κB pathway induced by LPS, indicated by the changes of phosphorylations of p65 (p-p65), p-IKKα/ß and p-IκBα, and weakened p65-DNA binding activity. TP and NBD had same efficacies either on alveolar hypercoagulation and fibrinolysis inhibition or on NF-κB signalling pathway in ARDS mice. CONCLUSIONS: TP dose-dependently improves alveolar hypercoagulation and fibrinolysis inhibition in ARDS mice through inhibiting NF-κB signaling pathway. Our data demonstrate that TP is expected to be an effective selection in ARDS.

[Impact and mechanism of NEMO binding domain peptide on pulmonary inflammation and apoptosis of lung tissues in mice with acute respiratory distress syndrome].

OBJECTIVE: To investigate the effect of NEMO binding domain peptide (NBDP) on lung inflammation and apoptosis in mice with acute respiratory distress syndrome (ARDS) and its mechanism. METHODS: Thirty-six male BALB/c mice were divided into normal saline (NS) control group, ARDS model group, NBDP negative control group and 6, 12 and 18 µg NBDP pretreatment group by random number table method, with 6 mice in each group. ARDS mouse model was reproduced by aerosol inhalation lipopolysaccharide (LPS) 50 µL. An equivalent among of NS was inhaled in NS control group. The mice in NBDP negative control group were inhaled the materials similar to the non-functional NBDP 30 minutes before the aerosol inhalation LPS; 6, 12 and 18 µg of NBDP 50 µL were respectively inhaled in NBDP pretreatment groups. After inhalation of LPS for 6 hours, mice were sacrificed to get lung tissue and observe the degree of pathological injury and edema. Western blotting was used to detect the phosphorylation of nuclear factor-κB (NF-κB) pathway related proteins [NF-κB inhibitor (IκB) kinaseα/ß(IKKα/ß), IκBα and NF-κB p65; p-IKKα/ß, p-IκBα, p-p65] and the expression of caspase-3 in lung tissue. The bronchoalveolar lavage fluid (BALF) was collected and the levels of inflammatory markers such as myeloperoxidase (MPO), interleukins (IL-1ß, IL-8), and tumor necrosis factor-α (TNF-α) were detected by enzyme linked immunosorbent assay (ELISA). RESULTS: ARDS model group had severe edema and hemorrhage, alveolar structure destruction, pulmonary hemorrhage and hyaline membrane formation etc. under light microscope, consistent with the pathological characteristics of ARDS lung tissue, suggesting that the ARDS model was successfully reproduced. ELISA showed that MPO, IL-1ß, IL-8 and TNF-α levels of BALF in ARDS model group were obviously higher than those in NS control group. There were no significant differences in the above inflammatory indicators between NBDP negative control group and ARDS model group. The levels of MPO, IL-1ß, IL-8 and TNF-α in NBDP pretreatment groups were significantly lower than those in ARDS model group in a dose-dependent manner, especially in 18 µg NBDP, the differences were statistically significant as compared with ARDS model group [MPO (ng/L): 393.32±19.35 vs. 985.87±101.50, IL-1ß (ng/L): 43.05±5.11 vs. 97.68±10.88, IL-8 (ng/L): 84.64±2.32 vs. 204.00±17.37, TNF-α (ng/L): 229.13±17.03 vs. 546.73±62.72, all P < 0.05]. Western blotting showed that p-IKKα/ß, p-IκBα, p-p65 and caspase-3 protein expressions in ARDS model group were significantly higher than those in NS control group. There was no significant difference in above NF-κB pathway and apoptosis-related protein expression between the NBDP negative control group and ARDS model group. The p-IKKα/ß, p-IκBα, p-p65 and caspase-3 protein expression in NBDP pretreatment groups were significantly lower than those in ARDS model group in a dose-dependent manner, especially in 18 µg NBDP, the differences were statistically significant as compared with ARDS model group [p-IKKα/ß protein (p-IKKα/ß/ß-actin): 0.15±0.02 vs. 0.42±0.04, p-IκBα protein (p-IκBα/ß-actin): 0.10±0.01 vs. 0.93±0.30, p-p65 protein (p-p65/ß-actin): 0.22±0.05 vs. 1.37±0.21, all P < 0.05]. CONCLUSIONS: NBDP can inhibit inflammatory response and apoptosis in ARDS lung tissue in a dose-dependent manner, and its mechanism is associated with interference NF-κB signaling pathway transduction.

[Effects of andrographolide on the expression of procoagulant and fibrinolytic inhibition factors in rat type II alveolar epithelial cells stimulated by lipopolysaccharide].

OBJECTIVE: To determine the effect of andrographolide (AD) on the expression of procoagulant and fibrinolytic inhibitory factors in rat type II alveolar epithelial cells (AEC II) stimulated by lipopolysaccharide (LPS). METHODS: The AEC II cells RLE-6TN in the logarithmic growth phase were divided into 5 groups: the normal control (NC) group, the LPS group, and the 6.25, 12.5, and 25 mg/L AD groups (AD 6.25 group, AD 12.5 group, AD 25 group). The NC group was cultured with RPMI 1640 conventional medium. In the LPS group, 5 mg/L LPS was added to the RPMI 1640 conventional medium for stimulation. Cells in the AD groups were treated with 6.25, 12.5, and 25 mg/L AD in advance for 1 hour and then given LPS to stimulate the culture. The cells and cell culture supernatant were collected 24 hours after LPS stimulation. The protein and mRNA expressions of tissue factor (TF), tissue factor pathway inhibitor (TFPI), and plasminogen activator inhibition-1 (PAI-1) in cells were detected by Western blotting and real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). The levels of procollagen III peptide (PIIIP), thrombin-antithrombin complex (TAT), antithrombin III (AT-III) and activated protein C (APC) in the cell supernatant were detected by enzyme linked immunosorbent assay (ELISA). RESULTS: Compared with the NC group, the protein and mRNA expressions of TF and PAI-1 in the LPS group were significantly increased, and the protein and mRNA expressions of TFPI were significantly reduced. At the same time, the levels of PIIIP and TAT in the cell supernatant were significantly increased, the levels of AT-III, APC were significantly reduced. Compared with the LPS group, the protein and mRNA expressions of TF and PAI-1 in AD 6.25 group, AD 12.5 group, AD 25 group were significantly reduced [TF/GAPDH: 0.86±0.08, 0.45±0.04, 0.44±0.04 vs. 1.32±0.10, TF mRNA (2-ΔΔCt): 2.59±0.25, 2.27±0.05, 1.95±0.04 vs. 4.60±0.26, PAI-1/GAPDH: 2.11±0.07, 1.45±0.04, 0.86±0.09 vs. 2.56±0.09, PAI-1 mRNA (2-ΔΔCt): 3.50±0.22, 2.23±0.29, 1.84±0.09 vs. 6.60±0.27, all P < 0.05], while the protein and mRNA expressions of TFPI were significantly increased [TFPI/GAPDH: 0.78±0.05, 0.81±0.03, 0.84±0.07 vs. 0.36±0.02, TFPI mRNA (2-ΔΔCt): 0.46±0.09, 0.69±0.07, 0.91±0.08 vs. 0.44±0.06, all P < 0.05]. Also the levels of PIIIP and TAT in the cell supernatant were significantly reduced, and the levels of AT-III and APC were significantly increased [PIIIP (µg/L): 13.59±0.23, 12.66±0.23, 10.59±0.30 vs. 15.82±0.29, TAT (ng/L): 211.57±6.41, 205.69±4.04, 200.56±9.85 vs. 288.67±9.84, AT-III (µg/L): 102.95±3.86, 123.92±2.63, 128.67±1.67 vs. 92.93±3.36, APC (µg/L): 1 188.95±14.99, 1 366.12±39.93, 1 451.15±29.69 vs. 1 145.55±21.07, all P < 0.05]. With the increase of the dose of AD, the above-mentioned promotion and inhibition effects became more obvious. In the AD 25 group, TF, PAI-1 protein and mRNA expressions decreased, TFPI mRNA expression increased, PIIIP level in the supernatant decreased and AT-III, APC levels increased compared with AD 6.25 group, the difference was statistically significant, and the decrease of PAI-1 protein expression and PIIIP level in the supernatant were also statistically significant compared with AD 12.5 group. CONCLUSIONS: Andrographolide in the dose range of 6.25-25 mg/L can dose-dependently inhibit the expression and secretion of procoagulant and fibrinolytic inhibitor-related factors in AEC II cells RLE-6TN stimulated by LPS, and promote the secretion of anticoagulant factors. 25 mg/L has the most obvious effect.

BAY11-7082 inhibits the expression of tissue factor and plasminogen activator inhibitor-1 in type-II alveolar epithelial cells following TNF-α stimulation via the NF-κB pathway.

Pulmonary inflammation strongly promotes alveolar hypercoagulation and fibrinolytic inhibition. NF-κB signaling regulates the expression of molecules associated with coagulation and fibrinolytic inhibition in type-II alveolar epithelial cells (AECII) stimulated by lipopolysaccharide. However, whether TNF-α-induced alveolar hypercoagulation and fibrinolysis inhibition is also associated with the NF-κB pathway remains to be determined. The aim of the present study was to determine whether BAY11-7082, an inhibitor of the NF-κB pathway, inhibits the expressions of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) in AECⅡ in response to TNF-α. Rat AECII were treated with BAY11-7082 for 24 h and stimulated with TNF-α for 1 h. The expression of TF and PAI-1 were determined using western blotting and reverse transcription-quantitative PCR. The concentrations of TF and PAI-1 in culture supernatant were also measured by ELISA. Moreover, levels of NF-κB p65 (p65), phosphorylated (p)-p65 (p-p65), inhibitor of NF-κB α (IκBα) and p-IκBα were also evaluated. Immunofluorescence was used to detect p65 levels in cell nuclei. TNF-α significantly promoted TF and PAI-1 expression either at the mRNA or protein level in AECII cells. Concentrations of TF and PAI-1 in supernatant also significantly increased upon TNF-α stimulation. Furthermore, TNF-α upregulated the levels of p-IκBα, p65, and p-p65 in the cytoplasm. Immunofluorescence analysis indicated that TNF-α increased p65 translocation from the cytoplasm to the nucleus. However, AECII pre-treated with BAY11-7082 expressed lower levels of TF and PAI-1 following TNF-α treatment. Levels of p-IκBα, p65 and p-p65 in the cytoplasm also decreased, and translocation of p65 from cytoplasm into the nucleus was inhibited by BAY11-7082 pretreatment. These findings suggest that BAY11-7082 improves the hypercoagulation and fibrinolytic inhibition induced by TNF-α in alveolar epithelial cells via the NF-κB signaling pathway. BAY11-7082 might represent a therapeutic option for alveolar hypercoagulation and fibrinolytic inhibition in acute respiratory distress syndrome.

[Berberine dose-dependently inhibits the expression of procoagulant and fibrinolytic inhibitory factors in lipopolysaccharide-induced rat type II alveolar epithelial cells].

OBJECTIVE: To observe the effects of berberine on procoagulant and fibrinolytic inhibitory factors produced by rat type II alveolar epithelial cell (AEC II) induced by lipopolysaccharide (LPS). METHODS: AEC II cells (RLE-6TN cells) were cultured in vitro, and the cells in logarithmic growth phase were collected. The cytotoxicity text of berberine was detected by cell counting kit-8 (CCK-8) to determine the drug concentration range according to inhibition concentration of half cells (IC50). The RLE-6TN cells were divided into five groups, the cells in blank control group were cultured in DMEM; the cells in LPS group were stimulated with 5 mg/L LPS; and the cells in berberine pretreatment groups were pretreated with 20, 50 and 80 µmol/L berberine for 1 hour, and then were co-cultured with 5 mg/L LPS. The cells were collected after LPS induced for 24 hours. The protein and mRNA expression levels of tissue factor (TF), tissue factor pathway inhibitor (TFPI) and plasminogen activator inhibitor-1 (PAI-1) in the cells were detected by Western blotting and real-time fluorescence quantification reverse transcription-polymerase chain reaction (RT-qPCR). The levels of activated protein C (APC), precollagen III peptide (PIIIP), thrombin-antithrombin complex (TAT) and antithrombin III (AT III) in the cell supernatant were measured by enzyme linked immunosorbent assay (ELISA). RESULTS: According to the inhibition rate curve, the IC50 of berberine on RLE-6TN cells was 81.16 µmol/L. Therefore, 20, 50 and 80 µmol/L were selected as the intervention concentration of berberine. Compared with the blank control group, the expression and secretion of procoagulant and fibrinolytic inhibitory factors were abnormal in RLE-6TN cells after LPS induced for 24 hours. The protein and mRNA expression levels of TF and PAI-1 in the LPS group were significantly increased, but the protein and mRNA expression levels of TFPI were significantly decreased. Meanwhile, the levels of APC and AT III in the cell supernatant were significantly decreased, while the levels of PIIIP and TAT were significantly increased. After pretreatment with berberine, the abnormal expression and secretion of procoagulant and fibrinolytic inhibitory factors induced by LPS were corrected in a dose-dependent manner, especially in 80 µmol/L. Compared with the LPS group, the protein and mRNA expression levels of TF and PAI-1 in the berberine 80 µmol/L group were significantly decreased [TF protein (TF/GAPDH): 0.45±0.02 vs. 0.55±0.03, TF mRNA (2-ΔΔCt): 0.39±0.08 vs. 1.48±0.11, PAI-1 protein (PAI-1/GAPDH): 0.37±0.02 vs. 0.64±0.04, PAI-1 mRNA (2-ΔΔCt): 1.14±0.29 vs. 4.18±0.44, all P < 0.01] and those of TFPI were significantly increased [TFPI protein (TFPI/GAPDH): 0.53±0.02 vs. 0.45±0.02, TFPI mRNA (2-ΔΔCt): 0.94±0.08 vs. 0.40±0.05, both P < 0.01]. Meanwhile, the levels of APC and AT III in the cell supernatant were significantly increased [APC (µg/L): 1 358.5±26.0 vs. 994.2±23.1, AT III (µg/L): 118.0±7.4 vs. 84.4±2.7, both P < 0.01], while those of PIIIP and TAT were significantly decreased [PIIIP (µg/L): 11.2±0.4 vs. 18.6±0.9, TAT (ng/L): 222.1±2.8 vs. 287.6±7.0, both P < 0.01]. CONCLUSIONS: Berberine could inhibit the LPS-induced expressions of procoagulant and fibrinolytic inhibitory factors in rat AEC II cells and promote the expressions of anticoagulant factors in a dose-dependent manner. Berberine may be a new therapeutic target for alveolar hypercoagulability and fibrinolysis inhibition in acute respiratory distress syndrome (ARDS).

Variation of Gut Microbiome in Free-Ranging Female Tibetan Macaques (Macaca thibetana) across Different Reproductive States.

The gut microbiome is expected to adapt to the varying energetic and nutritional pressures in females of different reproductive states. Changes in the gut microbiome may lead to varying nutrient utilizing efficiency in pregnant and lactating female primates. In this study, we examined variation in the gut bacterial community composition of wild female Tibetan macaques (Macaca thibetana) across different reproductive states (cycling, pregnancy and lactation). Fecal samples (n = 25) were collected from ten adult females harvested across different reproductive states. Gut microbial community composition and potential functions were assessed using 16 S rRNA gene sequences. We found significant changes in gut bacterial taxonomic composition, structure and their potential functions in different reproductive states of our study species. In particular, the relative abundance of Proteobacteria increased significantly during pregnancy and lactation. In addition, the relative abundance of Succinivibrionaceae and Succinivibrio (Succinivibrionaceae) were overrepresented in pregnant females, whereas Bifidobacteriaceae and Bifidobacterium (Bifidobacteriaceae) were overrepresented in lactating females. Furthermore, the relative abundance of predicted functional genes of several metabolic pathways related to host's energy and nutrition, such as metabolism of carbohydrates, cofactors and vitamins, glycans and other amino acids, were enriched in pregnancy and lactation. Our findings suggest that changes in the gut microbiome may play an important role in meeting the energetic needs of pregnant and lactating Tibetan macaques. Future studies of the "microbial reproductive ecology" of primates that incorporate food availability, reproductive seasonality, female reproductive physiology and gut inflammation are warranted.

Emodin improves alveolar hypercoagulation and inhibits pulmonary inflammation in LPS-provoked ARDS in mice via NF-κB inactivation.

BACKGROUND: Alveolar hypercoagulation and pulmonary inflammation are important characteristics and they regulate each other in acute respiratory distress syndrome (ARDS). NF-κB pathway has been confirmed to be involved in regulation of this crosstalk. Emodin, a traditional Chinese herb, shows potent inhibitory effect on NF-κB pathway, but whether it is effective in alveolar hypercoagulation and pulmonary inflammation in ARDS remains to be elucidated. PURPOSE: The aim of this experiment was to evaluate the efficacy of emodin on LPS-provoked alveolar hypercoagulation and excessive pulmonary inflammation in ARDS, and its potential mechanism. METHODS: Mice ARDS was set up through LPS (40 µl, 4 mg/ml) inhalation. Male mice were randomly received with BPS, LPS only, LPS+ emodin (5 mg/kg, 10 mg/kg, 20 mg/kg, respectively) and BAY65-1942, an inhibitor of IKKß. After 48 h of LPS stimulation, pulmonary pathological injury, expressions of Tissue factor (TF), plasminogen activator inhibitor (PAI)-1, activated protein C (APC), collagen Ⅰ, collagen III, interleukin (IL) 8, IL-1ß and tumor necrosis factor (TNF)-α in lung tissues, as well as concentrations of antithrombin III (AT III), procollagen peptide type III (PIIIP), soluble thrombomodulin (sTM), thrombin antithrombin complex (TAT), myeloperoxidase (MPO) and the percentage of inflammatory cells in bronchoalveolar lavage fluid (BALF) were all determined. NF-κB pathway activation as well as NF-κB DNA binding activity in pulmonary tissue were simultaneously checked. RESULTS: LPS stimulation resulted in obvious lung injury, excessive inflammatory cells infiltration, which all were dose-dependently ameliorated by emodin. Expressions of TF, PAI-1, collagen Ⅰ and collagen III as well as IL-8, IL-1ß and TNF-α in pulmonary tissue were all elevated while APC decreased under LPS provocation, which were all reversed by emodin treatment in dose-dependent manner. LPS promoted the secretions of PIIIP, sTM, TAT and inhibited AT III production in BALF, and resulted in high levels of MPO and the percentage of inflammatory cells in BALF, all of which were significantly and dose-dependently attenuated while AT III production was increased by emodin. Meanwhile, emodin effectively inhibited NF-κB pathway activation and attenuated p65 DNA binding activity induced by LPS inhalation. Emodin and BAY-65-1942 had similar impacts in this experiment. CONCLUSIONS: Emodin improves alveolar hypercoagulation and fibrinolytic inhibition and depresses excessive pulmonary inflammation in ARDS mice in dose-dependent manner via NF-κB inactivation. Our data demonstrate that emodin is expected to be an effective drug in alveolar hypercoagulation and pulmonary inflammation in ARDS.

[A temperature-inducible Targetron system for efficient gene inactivation in Escherichia coli].

To construct TeI3c/4c-based and temperature-inducible gene inactivation system (Thermotargetron) and to apply it to gene inactivation of mesophilic bacteria. The subunit of flagellum (fliC) and C4 dicarboxylate orotate:H⁺ symporter (dctA) genes were chosen as targets in the genome of Escherichia coli HMS174 (DE3) strain. According to recognition roles of TeI3c/4c intron, the fliC489a, fliC828s, fliC1038s and dctA2a sites were chosen as target sites. Gene-targeting plasmids were constructed based on pHK-TT1A by using overlap PCR method and transformed into HMS174 cells. An aliquot mid-log phase cultures of the transformants were shocked at 48 °C and plated on LB plate (containing chloramphenicol). Afterwards, gene mutants were screened by using colony PCR and DNA sequencing. After the mutants were obtained, the phenotypes of ΔfliC and ΔdctA gene mutants were characterized by using agar puncture and carbon metabolism experiments. Colony PCR and sequencing results show that TeI3c/4c intron was inserted in the designed sites of fliC and dctA genes. The gene-targeting efficiency of Thermotargetron system was 100%. Phenotype verification experiments of the mutants demonstrated that the cell motility of all ΔfliC mutants was damaged and the malate assimilation ability of ΔdctA mutant was deprived comparing to wild-type HMS174 strain. In our study, a temperature-inducible and high-efficiency gene inactivation system was established for mesophilic bacteria. This system could achieve high efficiency and precise gene inactivation by modulation of the incubation duration of the transformants at 48 °C.

Perinatal listeriosis patients treated at a maternity hospital in Beijing, China, from 2013-2018.

BACKGROUND: Listeriosis is a rare but severe foodborne infectious disease. Perinatal listeriosis is often associated with septicemia, central nervous system (CNS) infection, and serious adverse pregnancy outcomes (miscarriage and neonate death). Here we report the characteristics and outcomes of perinatal listeriosis cases treated over 6 years at Beijing Obstetrics and Gynecology Hospital (BOGH), the largest maternity hospital in China. METHODS: We retrospectively reviewed the records of laboratory-confirmed, pregnancy-associated listeriosis cases treated from January 1, 2013 to December 31, 2018. The clinical manifestations, laboratory results, perinatal complications and outcomes (post-natal follow-up of 6 months) were investigated. RESULTS: In BOGH, 12 perinatal listeriosis cases were diagnosed based on Listeria monocytogenes positive culture, including 10 single pregnancies and 2 twin pregnancies. The corresponding incidence of pregnancy-associated listeriosis was 13.7/100,000 deliveries. Among those cases, four pregnant women and four newborns had septicemia, and two of the neonates with septicemia also suffered CNS infection. All the maternal patients recovered. Two inevitable miscarriages and four fetal stillbirths occurred. Of the eight delivered newborns, six survived, and two died within 2 days from birth. None of the survivors had neurological sequelae during a 6-month follow-up. The overall feto-neonatal fatality rate was 57.1%; notably, this rate was 100% for infections occurring during the second trimester of pregnancy and only 14.3% for those occurring in the third trimester. CONCLUSIONS: Perinatal listeriosis is associated with high feto-neonatal mortality, and thus, a public health concern. Additional large-scale studies are needed to strengthen the epidemiological understanding of listeriosis in China.