Identification of Sodium- and Chloride-Sensitive Sites in the Slack Channel.

The Slack channel (KCNT1, Slo2.2) is a sodium-activated and chloride-activated potassium channel that regulates heart rate and maintains the normal excitability of the nervous system. Despite intense interest in the sodium gating mechanism, a comprehensive investigation to identify the sodium-sensitive and chloride-sensitive sites has been missing. In the present study, we identified two potential sodium-binding sites in the C-terminal domain of the rat Slack channel by conducting electrophysical recordings and systematic mutagenesis of cytosolic acidic residues in the rat Slack channel C terminus. In particular, by taking advantage of the M335A mutant, which results in the opening of the Slack channel in the absence of cytosolic sodium, we found that among the 92 screened negatively charged amino acids, E373 mutants could completely remove sodium sensitivity of the Slack channel. In contrast, several other mutants showed dramatic decreases in sodium sensitivity but did not abolish it altogether. Furthermore, molecular dynamics (MD) simulations performed at the hundreds of nanoseconds timescale revealed one or two sodium ions at the E373 position or an acidic pocket composed of several negatively charged residues. Moreover, the MD simulations predicted possible chloride interaction sites. By screening predicted positively charged residues, we identified R379 as a chloride interaction site. Thus, we conclude that the E373 site and the D863/E865 pocket are two potential sodium-sensitive sites, while R379 is a chloride interaction site in the Slack channel.SIGNIFICANCE STATEMENT The research presented here identified two distinct sodium and one chloride interaction sites located in the intracellular C-terminal domain of the Slack (Slo2.2, KCNT1) channel. Identification of the sites responsible for the sodium and chloride activation of the Slack channel sets its gating property apart from other potassium channels in the BK channel family. This finding sets the stage for future functional and pharmacological studies of this channel.

The neuropeptide GsMTx4 inhibits a mechanosensitive BK channel through the voltage-dependent modification specific to mechano-gating.

The cardiac mechanosensitive BK (Slo1) channels are gated by Ca2+, voltage, and membrane stretch. The neuropeptide GsMTx4 is a selective inhibitor of mechanosensitive (MS) channels. It has been reported to suppress stretch-induced cardiac fibrillation in the heart, but the mechanism underlying the specificity and even the targeting channel(s) in the heart remain elusive. Here, we report that GsMTx4 inhibits a stretch-activated BK channel (SAKcaC) in the heart through a modulation specific to mechano-gating. We show that membrane stretching increases while GsMTx4 decreases the open probability (Po) of SAKcaC. These effects were mostly abolished by the deletion of the STREX axis-regulated (STREX) exon located between RCK1 and RCK2 domains in BK channels. Single-channel kinetics analysis revealed that membrane stretch activates SAKcaC by prolonging the open-time duration (τO) and shortening the closed-time constant (τC). In contrast, GsMTx4 reversed the effects of membrane stretch, suggesting that GsMTx4 inhibits SAKcaC activity by interfering with mechano-gating of the channel. Moreover, GsMTx4 exerted stronger efficacy on SAKcaC under membrane-hyperpolarized/resting conditions. Molecular dynamics simulation study revealed that GsMTx4 appeared to have the ability to penetrate deeply within the bilayer, thus generating strong membrane deformation under the hyperpolarizing/resting conditions. Immunostaining results indicate that BK variants containing STREX are also expressed in mouse ventricular cardiomyocytes. Our results provide common mechanisms of peptide actions on MS channels and may give clues to therapeutic suppression of cardiac arrhythmias caused by excitatory currents through MS channels under hyper-mechanical stress in the heart.

No intrauterine vertical transmission in pregnancy with COVID-19: A case report.

The coronavirus disease 2019 (COVID-19) has been a worldwide pandemic diseases, nearly 400,000 people died at now. The data of status of pregnant women and neonates after infection of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) is limited. We report a case of pregnant woman in her third trimester with critical COVID-19, and amniotic fluid, umbilical cord blood, placenta, and neonatal gastric fluid were retained during cesarean section. The SARS-COV-2 nucleic acid test results of these specimens were negative. There is no evidence of intrauterine vertical transmission during delivery in the third trimester, but the data are limited and need to be further explored.

Diagnostic Value of Non-Real-Time Radial Probe Endobronchial Ultrasound (RP-EBUS) Guided Positioning Method for Peripheral Pulmonary Lesions.

BACKGROUND The aim of this study was to analyze the diagnostic value of thin bronchoscopy lung biopsy for peripheral pulmonary lesions under non-real-time guidance of radial ultrasound (RP-EBUS). MATERIAL AND METHODS We used a retrospective analysis of ultrasound images of 165 patients with peripheral pulmonary disease admitted to Suzhou Municipal Hospital Affiliated to Nanjing Medical University from February 2016 to December 2018 who were given RP-EBUS examination. Ultrasound images were obtained for all patients. There were 76 patients treated using traditional positioning method as the control group; 89 patients were treated by probe combined with bronchoscopy positioning method as the research group where the biopsy of the lesion along the path of the ultrasound probe was taken. The positive rate of the 2 methods was observed, and the factors affecting the quality of ultra-thin bronchoscopy under RP-EBUS non-real-time guidance were analyzed. RESULTS The detection rate of the study group was 77.64%, which was significantly higher than that in control group, which was 63.16% (χ²=5.238, P<0.05). The number of biopsies in the study group was 6±1.25, which was significantly lower than that of the control group which was 9±1.87 (t=4.116, P<0.05). The diagnostic positive rate of the RP-EBUS probe was significantly higher than that of the RP-EBUS probe (χ²=5.081, P<0.05). CONCLUSIONS The diagnostic positive rate of RP-EBUS non-real-time guided subtotal bronchoscopy lung biopsy for peripheral lung disease using probe combined with bronchoscopy positioning method was higher than the traditional positioning method, and the number of biopsies in the study group was significantly lower than that in the control group, which was related to the size, location, whether the probe was wrapped, or the characteristics of the ultrasound image.

miR-4326 promotes lung cancer cell proliferation through targeting tumor suppressor APC2.

microRNAs have been reported to play vital role in lung cancer proliferation and metastasis; the role of miR-4326 in tumor progression has not been studied. Here, we studied the effect of miR-4326 on lung cancer cell proliferation; we found that miR-4326 was significantly upregulated in lung cancer tissues determined using TCGA dataset and clinical specimens, meanwhile it was also upregulated in lung cancer cells. Overexpression of miR-4326 promoted lung cancer cell proliferation analyzed by MTT, soft agar growth, and BrdU incorporation assay, while miR-4326 knockdown suppressed lung cancer cell proliferation. We found miR-4326 targets tumor suppressor adenomatous polyposis coli 2 (APC2), which is a negative regulator of Wnt pathway, by binding to the 3'UTR of APC2. Wnt pathway could increase Cyclin D1 and c-MYC expression, we also found that miR-4326 could increase their expression, suggesting that APC2 was the target of miR-4326. Moreover, double knockdown of APC2 and miR-4326 promoted lung cancer cell proliferation, confirming that miR-4326 promoted lung cancer cell proliferation by inhibiting APC2.

Combined systemic immune-inflammatory index and prognostic nutritional index predict outcomes in advanced non-small cell lung cancer patients receiving platinum-doublet chemotherapy.

Background: Systemic immune-inflammatory index (SII) and prognostic nutritional index (PNI) could evaluate the therapeutic efficacy and prognosis in different tumors. However, no studies investigated the SII-PNI score to predict outcomes in non-small cell lung cancer (NSCLC) patients treated with platinum-doublet chemotherapy. The aim of this study was to investigate the SII-PNI score in predicting outcomes in non-small cell lung cancer (NSCLC) patients treated with platinum-doublet chemotherapy. Materials and methods: Our study retrospectively analyzed clinical data from 124 patients with advanced NSCLC receiving platinum-doublet chemotherapy. The SII and PNI were calculated based on peripheral blood cell counts and serum albumin, and the optimal cut-off values were determined using receiver operating characteristic (ROC). All patients were divided into three groups according to the SII-PNI score. The association between the SII-PNI score and the clinicopathological characteristics of the patients was examined. The Kaplan-Meier and Cox regression models were used to assess progression-free survival (PFS)and overall survival (OS). Results: There was no significant correlation between SII, PNI at baseline and chemotherapy response in patients with advanced NSCLC (p>0.05). However, after receiving 4 cycles of platinum-doublet chemotherapy, the SII of the SD group (p=0.0369) and PD group (p=0.0286) was significantly higher than that of the PR group. At the same time, the PNI of the SD group (p=0.0112) and the PD group (p=0.0007) was significantly lower than that of the PR group. The PFS of patients with SII-PNI scores of 0, 1, and 2 were 12.0, 7.0, and 5.0 months, and the OS of patients with SII-PNI scores of 0, 1, and 2 were 34.0, 17.0, and 10.5 months, respectively. There was statistical significance among the three groups (all p <0.001). Multivariate analyses showed that the chemotherapy response of progressive disease (PD) (HR, 3.508; 95% CI, 1.546-7.960; p=0.003) and SII-PNI score of 2 (HR, 4.732; 95% CI, 2.561-8.743; p < 0.001) were independently associated with a shorter OS. The uses of targeted drugs (HR, 0.543; 95% CI, 0.329-0.898; p=0.017) and immune checkpoint inhibitors (HR, 0.218; 95% CI, 0.081-0.584; p=0.002) were protective factors for OS in patients with NSCLC. Conclusion: Compared with baseline parameters, the correlation between SII, PNI after 4 cycles of chemotherapy and the chemotherapy effect was more significant. The SII-PNI score after 4 cycles of chemotherapy is an effective prognostic biomarker for advanced NSCLC patients treated with platinum-doublet chemotherapy. Patients with a higher SII-PNI score had a worse prognosis.

Prognostic factors of survival in patients with non-small cell lung cancer: a competing risk model using the SEER database.

Background: To explore the prognostic factors of survival in non-small cell lung cancer (NSCLC) patients using the competing risk analysis. Methods: This was a retrospective cohort study. NSCLC patients with complete data were selected from the Surveillance, Epidemiology, and End Results (SEER) database between 2010 and 2015. Outcomes were censored, cancer-specific mortality in NSCLC, and other-cause mortality. Gray's test was used in univariable analysis, and a multivariable Fine-Gray competing risk model with backward elimination was used to explore the prognostic factors of survival. The screened variables were incorporated into a random survival forest (RSF) model for the prediction of 1-, 3-, and 5-year survival in patients with NSCLC. Receiver operator characteristic (ROC) curves, calibration curves, the value of area under the curve (AUC), and decision curve analysis (DCA) were used to evaluate the performance. Results: Totally 1,251 eligible patients were included, 678 (54.20%) patients were cancer-specific mortality, and 128 (10.23%) patients were other-cause mortality. The median follow-up time was 26 months. Age, primary site, N stage, M stage, surgery type, tumor size, and lymph nodes (LNs) count were included in the multivariable Fine-Gray model for further analysis (P<0.05). The six most important features (surgery type, tumor size, M stage, LNs count, N stage, and primary site) were included in the competing risk analysis using the RSF model. The value of AUC for predicting 1-, 3-, and 5-year survival in the testing set were 0.796, 0.804, and 0.792, respectively. Calibration curves were well-fitted. DCA curves showed that the RSF model had similar or greater clinical net benefits in survival compared with the 8th edition the American Joint Committee on Cancer (AJCC) staging. The good performance of the RSF model under different surgery types, T, N, and M stages. Conclusions: We conducted a competing risk analysis using the RSF model for predicting the 1-, 3-, and 5-year survival of NSCLC. We generated a web calculator (https://github.com/YingChen19/Prognostic-factors-of-long-term-survival-of-non-small-cell-lung-cancer), which could provide a convenient assessment and could help improve the prognosis and survival of NSCLC.

Epidemic characteristics of Mycoplasma pneumoniae infection: a retrospective analysis of a single center in Suzhou from 2014 to 2020.

Background: Mycoplasma pneumoniae (M. Pneumoniae) is a common pathogen of respiratory tract infections, but there is still a lack of detailed investigation on the large sample of M. Pneumoniae infection in the all age population. And patients with severe M. Pneumoniae pneumonia (SMPP) still have a certain risk of death. How to identify the clinical characteristics and population of patients with SMPP as soon as possible is still an urgent problem in clinical practice. Methods: Demographic characteristics, patient clinical information, and laboratory data of 81,131 patients with respiratory tract infections (RTIs) in the Affiliated Suzhou Hospital of Nanjing Medical University from 2014 to 2020 were retrospectively collected from all patient records. The serum particle agglutination (PA) test was used to determine M. Pneumoniae infection by detecting specific antibodies. The white blood cell count, the proportion of neutrophils and lymphocytes, C-reactive protein (CRP) and lactate dehydrogenase (LDH) levels between children and adults with SMPP were compared by Student's t-test; other clinical features were analyzed by χ2 test or Fisher's exact test. Results: A total of 81,131 patients with RTIs were included, and 21,582 (26.60%) M. Pneumoniae immunoglobulin M (IgM)-positive patients were detected. From 2014 to 2020, the annual proportions of M. Pneumoniae RTIs were 23.60%, 28.18%, 38.08%, 27.05%, 23.44%, 25.26%, and 18.33%, respectively. In terms of seasonal distribution, April-June and September-November were the peak seasons of M. Pneumoniae infection each year. Children and women have a high proportion of M. Pneumoniae infection. The peak age of M. Pneumoniae infection was between 4 and 14 years old. There were 301 cases of SMPP, including 281 children and 20 adults (8 cases of pregnant women). Children and pregnant women accounted for a high proportion of SMPP. Children with SMPP had more extrapulmonary symptoms, multilobar infiltrates, and increased CRP and LDH levels compared with adults. Conclusions: M. Pneumoniae infection has seasonal, sex, and age distribution trends. Children and pregnant women accounted for a high proportion of SMPP. Extrapulmonary symptoms, multilobar infiltrates, and increased CRP and LDH levels may be helpful to identify SMPP in children than in adults.

Overexpression of B7-H4 in tumor infiltrated dendritic cells.

DCs infiltrated tumors appears to be phenotypically and functionally defective. B7-H4 was highlighted for its inhibitory role in T cell responses. In this study, we showed that B7-H4 was moderately expressed in imDCs, and up-regulated by IL-10, and TNF-α could counteract the up-regulatory effects of IL-10 on expression of B7-H4 in DCs in vitro. Furthermore, tumor infiltrated DCs expressed B7-H4 at high levels. Blockade of B7-H4 expressed in DCs highly resulted in enhanced T cell proliferation and IFN-γ production significantly. Otherwise, the high level of IL-10 and TNF-α was both detected in the tumor, which suggested that TNF-α can not antagonize the effects of IL-10 on expression of B7-H4 in DCs in vivo. These data indicate that tumor environment may condition local DCs to become dysfunctional in the phenotype, and that the high expression of B7-H4 may contribute to the tumor infiltrated DCs to mediate immune invasion.

Metagenomic next-generation sequencing of radial ultrasound bronchoscopy-guided "cocktail" specimens as an efficient method for the diagnosis of focal pulmonary infections: a randomised study.

BACKGROUND: To investigate the value of metagenomic next-generation sequencing (mNGS) in the diagnosis of focal pulmonary infections by using radial ultrasound bronchoscopic "cocktail" specimens. METHODS: From March 2019 to May 2020, 90 patients with focal pulmonary infections [locatable by a radial probe endobronchial ultrasound (RP-EBUS)] treated by the Department of Respiratory and Critical Care Medicine at the Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital (Suzhou, China) were randomly allocated to the "cocktail" group, transbronchial brushing (TBBr) group or bronchoalveolar lavage fluid (BALF) group; 30 patients were assigned to each group. Using the extracted material, the diagnostic efficacy of the mNGS test for pathogen detection was compared across the three groups, and the effect of an antibiotic treatment on detection rate was assessed. RESULTS: The sensitivity of mNGS analysis in cocktail group, TBBr group and BALF group was 90% (27/30), 66.7 (20/30) and 50% (15/30), respectively. The analysis of the sensitivity of the positive test, the detection rate of multiple pathogens, and the effect of antibiotics on the detection rate by mMGS indicated that the "cocktail" group was significantly higher number of samples positive for pathogens than the TBBr group (P<0.05), and the TBBr group was significantly higher number of samples positive for pathogens than the BALF group (P<0.05). CONCLUSIONS: "Cocktail" specimens had high sensitivity in the identification of focal pathogens, and the use of antibiotics had little effect on the results of the mNGS analysis. These clinically valuable results can be used in the diagnosis and treatment of patients with focal pulmonary infections.

The regulation of CPNE1 ubiquitination by the NEDD4L is involved in the pathogenesis of non-small cell lung cancer.

Our previous studies revealed that oncogene CPNE1 is positively correlated with the occurrence, TNM stage, lymph node metastasis, and distant metastasis of non-small-cell lung cancer (NSCLC), and it could be regulated by micro RNAs. But no direct role of post-translational modification of CPNE1 in NSCLC has been reported. This study confirms that CPNE1 is degraded by two pathways: the ubiquitin-proteasome pathway and the autophagy-lysosome pathway. CPNE1 binds with the ubiquitin molecule via its K157 residue. Moreover, we determined that the ubiquitin ligase NEDD4L can mediate the ubiquitination of CPNE1 and promote its degradation. In addition, we find that NEDD4L knockdown promotes the proliferation and metastasis of NSCLC cells by regulating CPNE1 in vitro and vivo. This study aims to further investigate the mechanism of CPNE1 ubiquitination in the occurrence and development of NSCLC and provide a new potential target for NSCLC treatment.

Regulatory Effect of General Anesthetics on Activity of Potassium Channels.

General anesthesia is an unconscious state induced by anesthetics for surgery. The molecular targets and cellular mechanisms of general anesthetics in the mammalian nervous system have been investigated during past decades. In recent years, K+ channels have been identified as important targets of both volatile and intravenous anesthetics. This review covers achievements that have been made both on the regulatory effect of general anesthetics on the activity of K+ channels and their underlying mechanisms. Advances in research on the modulation of K+ channels by general anesthetics are summarized and categorized according to four large K+ channel families based on their amino-acid sequence homology. In addition, research achievements on the roles of K+ channels in general anesthesia in vivo, especially with regard to studies using mice with K+ channel knockout, are particularly emphasized.

Zoledronic acid inhibits infiltration of tumor-associated macrophages and angiogenesis following transcatheter arterial chemoembolization in rat hepatocellular carcinoma models.

Hepatic transcatheter arterial chemoembolization (TACE), a minimally invasive procedure to block the blood supply of tumors and release of cytotoxic agents, is preferentially applied to patients with hepatocellular carcinoma (HCC) who are not able to receive radical treatments. However, the long-term effects of TACE are unsatisfactory, as the microenvironment following procedure stimulates tumor angiogenesis, which promotes recurrence and metastasis of residual tumors. Tumor associated macrophages (TAMs) have been revealed to stimulate tumor growth and angiogenesis associated with poor prognosis in HCC. The present study focused on the changes in TAMs following TACE, and explored the effects of TACE in combination with the TAM inhibitor zoledronic acid (ZA) in rat HCC models. Orthotropic HCC rats were divided into three groups: Sham TACE, TACE alone and TACE combined with ZA treatment. At 7 or 14 days following TACE, tumor growth was evaluated by magnetic resonance imaging (MRI). Infiltration of TAMs was assessed by histological analysis and flow cytometry. Tumor angiogenesis was measured as the mean vessel density, and initial slope was calculated from dynamic contrast enhancement MRI. Local and systemic levels of vascular endothelial growth factor (VEGF) were determined by western blotting or an ELISA, respectively. The results revealed that TACE inhibited tumor growth at 7 days following the procedure, but this inhibition was attenuated at 14 days following the procedure compared with the sham TACE control. If combined with ZA treatment, TACE exhibited a stable inhibition effect on tumor growth until the end of observation. Investigation of the underlying mechanisms demonstrated that TACE combined with ZA treatment inhibited infiltration of F4/80 positive TAMs and tumor angiogenesis compared with the TACE alone group at 14 days following the procedure. Additionally, the combination treatment significantly inhibited secretion of VEGF in the present models. In conclusion, ZA treatment enhanced the effects of TACE through inhibiting TAM infiltration and tumor angiogenesis in rat HCC models.

miR-937 contributes to the lung cancer cell proliferation by targeting INPP4B.

Lung cancer is the leading cause of cancer death worldwide, microRNAs play critical role in the initiation and development of lung cancer. Here, we used MTT assay, colony formation assay, soft agar growth assay and BrdU incorporation assay to investigate miR-937's role in lung cancer. We found that miR-937 was upregulated in lung cancer tissues and cells. Overexpression of miR-937 in A549 promoted anchorage -dependent and -independent growth, whereas knockdown of miR-937 reduced this effect. Meanwhile, we also found miR-937 overexpression increased CCND1 and c-Myc levels in both mRNA and protein levels, knockdown of miR-937 reduced this effect, confirming miR-937 promoted cell proliferation. Mechanism analyses found polyphosphate 4-phosphatase type II (INPP4B) was the target of miR-937, miR-937 directly bound to the 3'UTR of INPP4B, knockdown of INPP4B in A549 with miR-937 inhibitor promoted anchorage -dependent and -independent growth, suggesting miR-937 contributed to cell proliferation of lung cancer by inhibiting INPP4B, it might be a valuable target for lung cancer therapy.