Integrated Bioinformatics Analysis for Revealing CBL is a Potential Diagnosing Biomarker and Related Immune Infiltration in Parkinson's Disease.

Purpose: There is growing evidence that the immune system plays an important role in the progression of Parkinson's disease, the second most common neurodegenerative disorder. This study aims to address the comprehensive understanding of the immunopathogenesis of Parkinson's disease and explore new inflammatory biomarkers. Patients and Methods: In this study, Immune-related differential expressed genes (DEIRGs) were obtained from GEO database and Immport database. The hub gene was screened in DEIRGs using LASSO regression and random forest algorithm, and the mRNA expression of the identified hub gene was validated using clinical blood samples. Results: We obtained a total of 157 DEIRGs that played an important role in the immune response. The results of immune cell infiltration analysis showed that the degree of memory B cells infiltration was higher in PD patients, while the degree of Monocytes, resting mast cells and M0 macrophages infiltration was lower (p<0.05). A total of 8 hub genes were screened by machine learning methods, and RT-PCR results showed that the expression level of CBL gene in PD was significantly increased (p<0.05). Conclusion: Our findings suggest that CBL is a new potential diagnostic biomarker for PD and that abnormal immune cell infiltration may influence PD development.

Classification and hemodynamic characteristics of delayed intracerebral hemorrhage following stent-assisted coil embolism in unruptured intracranial aneurysms.

Background and objective: Stent-assisted coil (SAC) embolization is a commonly used endovascular treatment for unruptured intracranial aneurysms (UIAs) but can be associated with symptomatic delayed intracerebral hemorrhage (DICH). Our study aimed to investigate the hemodynamic risk factors contributing to DICH following SAC embolization and to establish a classification for DICH predicated on hemodynamic profiles. Methods: This retrospective study included patients with UIAs located in the internal carotid artery (ICA) treated with SAC embolization at our institution from January 2021 to January 2022. We focused on eight patients who developed postoperative DICH and matched them with sixteen control patients without DICH. Using computational fluid dynamics, we evaluated the hemodynamic changes in distal arteries [terminal ICA, the anterior cerebral artery (ACA), and middle cerebral artery (MCA)] pre-and post-embolization. We distinguished DICH-related arteries from unrelated ones (ACA or MCA) and compared their hemodynamic alterations. An imbalance index, quantifying the differential in flow velocity changes between ACA and MCA post-embolization, was employed to gauge the flow distribution in distal arteries was used to assess distal arterial flow distribution. Results: We identified two types of DICH based on postoperative flow alterations. In type 1, there was a significant lower in the mean velocity increase rate of the DICH-related artery compared to the unrelated artery (-47.25 ± 3.88% vs. 42.85 ± 3.03%; p < 0.001), whereas, in type 2, there was a notable higher (110.58 ± 9.42% vs. 17.60 ± 4.69%; p < 0.001). Both DICH types demonstrated a higher imbalance index than the control group, suggesting an association between altered distal arterial blood flow distribution and DICH occurrence. Conclusion: DICH in SAC-treated UIAs can manifest as either a lower (type 1) or higher (type 2) in the rate of velocity in DICH-related arteries. An imbalance in distal arterial blood flow distribution appears to be a significant factor in DICH development.

Calcium sensing receptor: A promising therapeutic target in pulmonary hypertension.

Pulmonary hypertension (PH) is characterized by elevation of pulmonary arterial pressure and pulmonary vascular resistance. The increased pulmonary arterial pressure and pulmonary vascular resistance due to sustained pulmonary vasoconstriction and pulmonary vascular remodeling can lead to right heart failure and eventual death. A rise in intracellular Ca2+ concentration ([Ca2+]i) and enhanced pulmonary arterial smooth muscle cells (PASMCs) proliferation contribute to pulmonary vasoconstriction and pulmonary vascular remodeling. Recent studies demonstrated that extracellular calcium sensing receptor (CaSR) as a G-protein coupled receptor participates in [Ca2+]i increase induced by hypoxia in the experimental animals of PH and in PH patients. Pharmacological blockade or gene knockout of CaSR significantly attenuates the development of PH. This review will aim to discuss and update the pathogenicity of CaSR attributed to onset and progression in PH.

Diagnostic performance of microRNA-29a in active pulmonary tuberculosis: a systematic review and meta-analysis.

BACKGROUND: In recent years, more and more studies have shown that microRNA-29a (miRNA-29a) can be used as a potential biomarker for active tuberculosis, but the results of these studies are not consistent. OBJECTIVE: To comprehensively evaluate the value of miRNA-29a in the diagnosis of active tuberculosis by meta-analysis. METHODS: The databases of CNKI, WanFang, PubMed, The Cochrane Library, Web of Science and EMBASE were searched for relevant studies. Studies were screened strictly according to inclusion and exclusion criteria. QUADAS-2 scale was used to evaluate the quality of the included studies. Data were extracted and analyzed by Meta-DiSc 1.4 and Stata 16.0 software. RESULTS: 13 articles were included, including a total of 1598 subjects, including 872 active tuberculosis patients and 726 controls. The combined sensitivity and specificity of miRNA-29a in the diagnosis of active tuberculosis were 78 % and 76 %, respectively, and the area under the overall summary receiver operating characteristic curve was 0.8564. CONCLUSION: miRNA-29a can be used as a biomarker for the diagnosis of active tuberculosis.

How single-cell techniques help us look into lung cancer heterogeneity and immunotherapy.

Lung cancer patients tend to have strong intratumoral and intertumoral heterogeneity and complex tumor microenvironment, which are major contributors to the efficacy of and drug resistance to immunotherapy. From a new perspective, single-cell techniques offer an innovative way to look at the intricate cellular interactions between tumors and the immune system and help us gain insights into lung cancer and its response to immunotherapy. This article reviews the application of single-cell techniques in lung cancer, with focuses directed on the heterogeneity of lung cancer and the efficacy of immunotherapy. This review provides both theoretical and experimental information for the future development of immunotherapy and personalized treatment for the management of lung cancer.

Rational design of an influenza-COVID-19 chimeric protective vaccine with HA-stalk and S-RBD.

Highly contagious respiratory illnesses like influenza and COVID-19 pose serious risks to public health. A two-in-one vaccine would be ideal to avoid multiple vaccinations for these diseases. Here, we generated a chimeric receptor binding domain of the spike protein (S-RBD) and hemagglutinin (HA)-stalk-based vaccine for both SARS-CoV-2 and influenza viruses. The S-RBD from SARS-CoV-2 Delta was fused to the headless HA from H1N1 (H1Delta), creating a chimera that forms trimers in solution. The cryo-electron microscopy structure of the chimeric protein complexed with the RBD-targeting CB6 and the HA-stalk-targeting CR9114 antibodies shows that the trimeric protein is stable and accessible for neutralizing antibody binding. Immunization with the vaccine elicited high and long-lasting neutralizing antibodies and effectively protected mice against the challenges of lethal H1N1 or heterosubtypic H5N8, as well as the SARS-CoV-2 Delta or Omicron BA.2 variants. Overall, this study offers a two-in-one universal vaccine design to combat infections caused by both SARS-CoV-2 variants of concern and influenza viruses.

Diagnostic performance of microRNA-29a in active pulmonary tuberculosis: a systematic review and meta-analysis

Abstract Background: In recent years, more and more studies have shown that microRNA-29a (miRNA-29a) can be used as a potential biomarker for active tuberculosis, but the results of these studies are not consistent. Objective: To comprehensively evaluate the value of miRNA-29a in the diagnosis of active tuberculosis by meta-analysis. Methods: The databases of CNKI, WanFang, PubMed, The Cochrane Library, Web of Science and EMBASE were searched for relevant studies. Studies were screened strictly according to inclusion and exclusion criteria. QUA-DAS-2 scale was used to evaluate the quality of the included studies. Data were extracted and analyzed by Meta-DiSc 1.4 and Stata 16.0 software. Results: 13 articles were included, including a total of 1598 subjects, including 872 active tuberculosis patients and 726 controls. The combined sensitivity and specificity of miRNA-29a in the diagnosis of active tuberculosis were 78 % and 76 %, respectively, and the area under the overall summary receiver operating characteristic curve was 0.8564. Conclusion: miRNA-29a can be used as a biomarker for the diagnosis of active tuberculosis.

Vaginal microecological imbalance and expression of serum inflammatory factors in pregnant women with group B streptococcus infection and pregnancy outcome.

The objective of this study was to explore the correlation between vaginal microecological imbalance and the expression of related inflammatory factors in pregnant women with group B streptococcus (GBS) infection and pregnancy outcomes. For this purpose, 100 GBS-positive pregnant women were recruited as the experimental group, and 100 GBS-negative pregnant women were recruited as the controls. The balance of vaginal microecology of pregnant women in different groups was compared. Results showed that the probability of vaginal microecological imbalance in the experimental group was much higher than against the controls. Fasting venous blood was drawn from the pregnant women in two groups. After centrifugation, the expression levels of interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and interleukin-1ß (IL-1ß) in serum were detected. It was found that the expression levels of IL-6, IL-1ß, and TNF-α in the experimental group were higher than against the controls. After delivery, it suggested that the incidence of premature delivery, neonatal infection, premature rupture of membranes, and other adverse childbirth in the experimental group was much higher in contrast to the controls, up to 87%. In conclusion, GBS infection can increase the incidence of vaginal microecological imbalance and the expression of serum inflammatory factors in pregnant women, and it can greatly raise the incidence of adverse pregnancy outcomes.

Protective efficacy of a universal influenza mRNA vaccine against the challenge of H1 and H5 influenza A viruses in mice.

Current influenza vaccines need to be updated annually owing to constant antigenic drift in the globular head of the viral surface hemagglutinin (HA) glycoprotein. The immunogenic subdominant stem domain of HA is highly conserved and can be recognized by antibodies capable of binding multiple HA subtypes. Therefore, the HA stem antigen is a promising target for the design of universal influenza vaccines. On the basis of an established lipid nanoparticle-encapsulated mRNA vaccine platform, we designed and developed a novel universal influenza mRNA vaccine (mHAs) encoding the HA stem antigen of the influenza A (H1N1) virus. We tested the efficacy of the mHAs vaccine using a mouse model. The vaccine induced robust humoral and specific cellular immune responses against the stem region of HA. Importantly, two doses of the mHAs vaccine fully protected mice from lethal challenges of the heterologous H1N1 and heterosubtypic H5N8 influenza viruses. Vaccinated mice had less pathological lung damage and lower viral titers than control mice. These results suggest that an mRNA vaccine using the conserved stem region of HA may provide effective protection against seasonal and other possible influenza variants.

Clinical and laboratory features in health care volunteers with inactivated SARS-CoV-2 vaccination.

Background/aim: To better optimize the inactivated vaccine-induced immune response and improve vaccine protection efficiency, a preliminary study was conducted on the influencing factors of producing neutralizing antibody (NAb) titers against the inactivated coronavirus disease 2019 (COVID-19) vaccine. Materials and methods: A total of 91 health care volunteers were enrolled from the Immunology Division of the Laboratory Department of Chongqing General Hospital from February to March 2021. The study had a cross-sectional design. All of the volunteers were scheduled to receive a complete dose regimen of the inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine and the vaccination interval between 2 doses was 14 days. Clinical and laboratory features were collected for further analysis. Results: The NAb titers gradually increased after COVID-19 vaccination, and 72.53% (n = 66) of the volunteers had NAbs after the second dose. Eight variables, including CD16+CD56+ NK cell level before the first dose (HR = 0.94, p = 0.02), CD16+CD56+ NK cell level after the second dose (HR = 0.94, p = 0.03), interleukin (IL)-2 level before the first dose (HR = 2.09, p = 0.05), mean corpuscular volume (HR = 0.86, p = 0.02), serum urea level (HR = 0.69, p = 0.05), increment of CD19+ B cells (HR = 0.86, p = 0.03), increment of CD4+/CD8+ T cells (HR = 0.21, p = 0.03), and increment of the IL-6 level (HR = 0.75, p = 0.04) demonstrated a correlation with the NAb titers after COVID-19 vaccination. In the multivariate logistical regression analysis, the serum urea level (HR = 2.32, P = 0.03) and increment of CD19+ B cells (HR = 1.96, p = 0.03) were positively correlated with the NAb titers. The principal component analysis effectively distinguished the response after COVID-19 vaccination. The Pearson correlation analysis indicated that the CD19+ B cell level (r = 0.23, p < 0.001) and IL-2 (r = 0.24, p < 0.001) and IL-6 levels (r = 0.22, p < 0.001) were weakly positively correlated with the concentration of NAbs. Conclusion: The NAbs titers of the inactivated vaccines were positively correlated with the ratio of CD19+ B cell, IL-6, and IL-2 levels in the serum, which provide clinical guidance for inactivated SARS-CoV-2 vaccines.

Machine learning for identifying benign and malignant of thyroid tumors: A retrospective study of 2,423 patients.

Thyroid tumors, one of the common tumors in the endocrine system, while the discrimination between benign and malignant thyroid tumors remains insufficient. The aim of this study is to construct a diagnostic model of benign and malignant thyroid tumors, in order to provide an emerging auxiliary diagnostic method for patients with thyroid tumors. The patients were selected from the Chongqing General Hospital (Chongqing, China) from July 2020 to September 2021. And peripheral blood, BRAFV600E gene, and demographic indicators were selected, including sex, age, BRAFV600E gene, lymphocyte count (Lymph#), neutrophil count (Neu#), neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), red blood cell distribution width (RDW), platelets count (PLT), red blood cell distribution width-coefficient of variation (RDW-CV), alkaline phosphatase (ALP), and parathyroid hormone (PTH). First, feature selection was executed by univariate analysis combined with least absolute shrinkage and selection operator (LASSO) analysis. Afterward, we used machine learning algorithms to establish three types of models. The first model contains all predictors, the second model contains indicators after feature selection, and the third model contains patient peripheral blood indicators. The four machine learning algorithms include extreme gradient boosting (XGBoost), random forest (RF), light gradient boosting machine (LightGBM), and adaptive boosting (AdaBoost) which were used to build predictive models. A grid search algorithm was used to find the optimal parameters of the machine learning algorithms. A series of indicators, such as the area under the curve (AUC), were intended to determine the model performance. A total of 2,042 patients met the criteria and were enrolled in this study, and 12 variables were included. Sex, age, Lymph#, PLR, RDW, and BRAFV600E were identified as statistically significant indicators by univariate and LASSO analysis. Among the model we constructed, RF, XGBoost, LightGBM and AdaBoost with the AUC of 0.874 (95% CI, 0.841-0.906), 0.868 (95% CI, 0.834-0.901), 0.861 (95% CI, 0.826-0.895), and 0.837 (95% CI, 0.802-0.873) in the first model. With the AUC of 0.853 (95% CI, 0.818-0.888), 0.853 (95% CI, 0.818-0.889), 0.837 (95% CI, 0.800-0.873), and 0.832 (95% CI, 0.797-0.867) in the second model. With the AUC of 0.698 (95% CI, 0.651-0.745), 0.688 (95% CI, 0.639-0.736), 0.693 (95% CI, 0.645-0.741), and 0.666 (95% CI, 0.618-0.714) in the third model. Compared with the existing models, our study proposes a model incorporating novel biomarkers which could be a powerful and promising tool for predicting benign and malignant thyroid tumors.

Serum peptidome profiles immune response of COVID-19 Vaccine administration.

Background: Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused significant loss of life and property. In response to the serious pandemic, recently developed vaccines against SARS-CoV-2 have been administrated to the public. Nevertheless, the research on human immunization response against COVID-19 vaccines is insufficient. Although much information associated with vaccine efficacy, safety and immunogenicity has been reported by pharmaceutical companies based on laboratory studies and clinical trials, vaccine evaluation needs to be extended further to better understand the effect of COVID-19 vaccines on human beings. Methods: We performed a comparative peptidome analysis on serum samples from 95 participants collected at four time points before and after receiving CoronaVac. The collected serum samples were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to profile the serum peptides, and also subjected to humoral and cellular immune response analyses to obtain typical immunogenicity information. Results: Significant difference in serum peptidome profiles by MALDI-TOF MS was observed after vaccination. By supervised statistical analysis, a total of 13 serum MALDI-TOF MS feature peaks were obtained on day 28 and day 42 of vaccination. The feature peaks were identified as component C1q receptor, CD59 glycoprotein, mannose-binding protein C, platelet basic protein, CD99 antigen, Leucine-rich alpha-2-glycoprotein, integral membrane protein 2B, platelet factor 4 and hemoglobin subunits. Combining with immunogenicity analysis, the study provided evidence for the humoral and cellular immune responses activated by CoronaVac. Furthermore, we found that it is possible to distinguish neutralizing antibody (NAbs)-positive from NAbs-negative individuals after complete vaccination using the serum peptidome profiles by MALDI-TOF MS together with machine learning methods, including random forest (RF), partial least squares-discriminant analysis (PLS-DA), linear support vector machine (SVM) and logistic regression (LR). Conclusions: The study shows the promise of MALDI-TOF MS-based serum peptidome analysis for the assessment of immune responses activated by COVID-19 vaccination, and discovered a panel of serum peptides biomarkers for COVID-19 vaccination and for NAbs generation. The method developed in this study can help not only in the development of new vaccines, but also in the post-marketing evaluation of developed vaccines.

Longitudinal Serum Proteome Characterization of COVID-19 Patients With Different Severities Revealed Potential Therapeutic Strategies.

The COVID-19 pandemic caused by SARS-CoV-2 is exerting huge pressure on global healthcare. Understanding of the molecular pathophysiological alterations in COVID-19 patients with different severities during disease is important for effective treatment. In this study, we performed proteomic profiling of 181 serum samples collected at multiple time points from 79 COVID-19 patients with different severity levels (asymptomatic, mild, moderate, and severe/critical) and 27 serum samples from non-COVID-19 control individuals. Dysregulation of immune response and metabolic reprogramming was found in severe/critical COVID-19 patients compared with non-severe/critical patients, whereas asymptomatic patients presented an effective immune response compared with symptomatic COVID-19 patients. Interestingly, the moderate COVID-19 patients were mainly grouped into two distinct clusters using hierarchical cluster analysis, which demonstrates the molecular pathophysiological heterogeneity in COVID-19 patients. Analysis of protein-level alterations during disease progression revealed that proteins involved in complement activation, the coagulation cascade and cholesterol metabolism were restored at the convalescence stage, but the levels of some proteins, such as anti-angiogenesis protein PLGLB1, would not recovered. The higher serum level of PLGLB1 in COVID-19 patients than in control groups was further confirmed by parallel reaction monitoring (PRM). These findings expand our understanding of the pathogenesis and progression of COVID-19 and provide insight into the discovery of potential therapeutic targets and serum biomarkers worth further validation.

Janus wireframe DNA cube-based 3D nanomachine for rapid and stable fluorescence detection of exosomal microRNA.

Exosomal microRNAs (miRNAs) have been demonstrated to be credible biomarkers for the diagnosis and monitoring of tumors. Nevertheless, developing simple, rapid, and stable biosensing strategies that are capable of accurately detecting exosomal miRNAs remains a challenge. Herein, an accelerated and biostable three-dimensional (3D) nanomachine based on Janus wireframe DNA cube was constructed for sensitive fluorescence measurement of exosomal miRNA. The Janus wireframe DNA cube could propel target exosomal miRNA-21 rapid movement on its surface by catalytic hairpin assembly (CHA), releasing a massive fluorescence signal. Benefiting from the Janus wireframe DNA cube, the developed 3D nanomachine exhibited significantly improved reaction rate and enhanced biostability in complex biofluids compared to conventional CHA. As a result, this fluorescence biosensing strategy achieved rapid, stable, and single-step detection of exosomal miRNA-21 with the detection limit down to the picomole level. Therefore, this work offers a brief sensing tool for nucleic acid biomarkers detection, which has great application potential in tumor diagnosis.

Mutation of TonB-Dependent Receptor Encoding Gene MCR_0492 Potentially Associates with Macrolides Resistance in Moraxella catarrhalis Isolates.

Introduction: Moraxella catarrhalis, which is an opportunistic pathogen and is one of the three major pathogens of community-acquired pneumonia, causes a variety of infections in clinic. In recent years, the isolation rate of Moraxella catarrhalis has gradually increased. In China, due to the clinical empirical use of antibiotics, the resistance rate of Moraxella catarrhalis isolated from children to ß-lactam antibiotics has reached 99%. The non-susceptible rate of Moraxella catarrhalis to macrolide antibiotics has also increased significantly. Methods: Two isolates of Moraxella catarrhalis (R17123922_R and R18013231_R) were isolated from in-patients and were confirmed to be resistant to macrolide antibiotics using the standard disk diffusion and broth microdilution method recommended by CLSI. Whole-genome sequencing (WGS) analysis was performed in these two resistant strains. Results: A total of 696 SNVs (single nucleotide variations), and 79 indels (Insertion and Deletion) were found in R17123922_R and R18013231_R. These SNVs and indels were distributed evenly in the genome, and no centralized distribution occurred. Moreover, two isolates did not harbor any previously reported mutations in the 23S rRNA and ribosomal proteins. Conclusion: A novel indel in the MCR_0492 gene encoding TonB-dependent receptor protein was identified, and we speculated that TonB-dependent protein receptor may play an important role in macrolide resistance of Moraxella catarrhalis.

Highly sensitive fluorescence multiplexed miRNAs biosensors for accurate clinically diagnosis lung cancer disease using LNA-modified DNA probe and DSN enzyme.

With the emergence of microRNAs as key biomarkers for disease diagnosis such as lung cancer, various techniques have been settled for their detection. However, these current methods require different amplification steps since numerous challenges for detecting circulating miRNAs are attributable to their intrinsic properties accounting for tiny sizes, high sequence similarity, and low abundance. Duplex specific nuclease (DSN)-based microRNA amplification has recently gained interest in biosensing applications thanks to its catalytic activity based on target recycling. In this context, we designed a highly selective, sensitive, and multiplexed fluorescence-based biosensor combining DSN enzyme and magnetic beads to detect three distinct microRNAs, including microRNA-21, microRNA-210, and microRNA-486-5p. By exploiting the above approach, we were able to detect as low as 98 aM, 120 aM, and 300 aM of mir-21, miR-210, and miR-486-5p, respectively. Furthermore, this recommended strategy displays a high selectivity toward an entirely matched target than the off-target. These results are ascribed to the potent DSN enzyme activity and to the locked nucleic acid (LNA)-modified DNA probe that boosted the hetero-duplex probe/target stability. Lastly, our proposed method was applied to detect microRNAs in the serum samples and displayed a high efficacy to discriminate between healthy controls and lung cancer patients. Furthermore, the analytical accuracy of the proposed strategy was validated with the computed tomography (CT) technique of the chest. Thus based on these findings, this strategy could open new directions for detecting microRNAs associated with several diseases.

Pressure vessel-oriented visual inspection method based on deep learning.

The detection of surface parameters of pressure vessel welds guarantees safe operation. To address the problems of low efficiency and poor accuracy of traditional manual inspection methods, a method for welding morphological parameters combined with vision and structured light is proposed in this study. First, a feature point extraction algorithm for weld parameters based on deep convolution was proposed. An accurate extraction method of weld image feature point coordinates was designed based on the combination of the loss function via seam undercut feature recognition and weld feature point extraction network structure. Second, a training data enhancement method based on the third-order non-uniform rational B-spline (NURBS) curve was proposed to reduce the amount of data collection for training. Finally, a pressure vessel measurement device was designed, and the feature point extraction performance of the deep network and common feature point extraction networks, DeepLabCut and HR-net, proposed in this study were compared to analyze the theoretical accuracy of the surface parameter measurement. The results indicated that the theoretical accuracy of the parameter measurements was within 0.065 mm.

Endogenous health risks, poverty traps, and the roles of health insurance in poverty alleviation.

BACKGROUND: Family education investment is a key factor in reducing intergenerational transmission of poverty. At the price of higher health risk, the poor may overdraw their bodies to earn more money to invest in education. This study investigates the effect of physical overdraft, health risks and health insurance on poverty and economic growth. METHODS: This paper proposes an economic development model of endogenous health risks and poverty by setting up a physical overdraft decision. Furthermore, we introduce mutual health insurance mechanism to analyze its poverty alleviation effects. RESULTS: First, this study shows that health risks weaken the economy and are among the leading causes of poverty. Second, mutual health insurance can alleviate, but not completely eliminate, the negative impact of health risks on the economy. Third, appropriate health insurance arrangements can lift some or even all poor households out of poverty. CONCLUSION: Health risks have a significant effect on poverty. Furthermore, health insurance mechanisms have the advantages of transferring health risks, reducing poverty and improving health equity.

Insertion Mutation of MSMEG_0392 Play an Important Role in Resistance of M. smegmatis to Mycobacteriophage SWU1.

PURPOSE: Phage is a new choice for the treatment of multi-drug-resistant bacteria, and phage resistance is also an issue of concern. SWU1 is a mycobacteriophage, and the mechanism of its resistance remain poorly understood. METHODS: The mutant strains which were stably resistant to SWU1 were screened by transposon mutation library. The stage of phage resistance was observed by transmission electron microscope (TEM). The insertion site of transposon was identified by thermal asymmetric interlaced PCR (TAIL-PCR). The possible relationship between insertion site and phage resistance was verified by gene knockout technique. The fatty acid composition of bacterial cell wall was analyzed by Gas Chromatography-Mass Spectrometer (GC-MS). Through the amplification and sequencing of target genes and gene complement techniques to find the mechanism of SWU1 resistance. RESULTS: The transposon mutant M12 which was stably resistant to mycobacteriophage SWU1 was successfully screened. It was confirmed that resistance occurred in the adsorption stage of bacteriophage. It was verified that the insertion site of the transposon was located in the MSMEG_3705 gene, but after knocking out the gene in the wild type M. smegmatis mc2 155, the resistance of the knockout strain to SWU1 was not observed. Through the amplification and sequencing of the target gene MSMEG_0392, it was found that there was an adenine insertion mutation at position 817. After complementing MSMEG_0392 in M12, it was found that M12 returned to sensitivity to SWU1. CONCLUSION: We confirmed that the resistance of M12 to SWU1 was related to the functional inactivation of MSMEG_0392 and this phenomenon may be caused by the change of cell wall of M. smegmatis.

External Quality Assessment for Severe Acute Respiratory Syndrome Coronavirus 2 RNA Detection in Chongqing, China.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a huge threat to public health. Therefore, clinical laboratories must have the ability to detect SARS-CoV-2 RNA. With the enhanced detection in Chongqing, many laboratories rapidly implemented assays for the molecular detection of SARS-CoV-2 based on real-time reverse transcription polymerase chain reaction (rRT-PCR) assays. This study aimed to improve the detection capabilities of clinical laboratories by evaluating their performance for SARS-CoV-2 RNA detection through the external quality assessment (EQA) programs of 2020 in Chongqing to contribute to the prevention of this epidemic. METHODS: The EQA panels consist of eight positive samples with concentrations within 2.7 - 5.0 log10 copies/mL quantified by digital PCR and two negative samples with other human coronaviruses clinically validated by four commercial assays. All 21 samples from four rounds were distributed to the participating laboratories through cold-chain transportation. Depending on the results from each sample, laboratories were asked to use one or two assays to detect SARS-CoV-2 RNA. Test results and raw data were also required. All data were evaluated, and the testing performance of commercial assays was compared. For the rounds, all laboratories used commercial assays. RESULTS: Four rounds of EQA programs were performed, and the percent agreements of participants were 97.5% (39/40), 97.5% (39/40), 98.9% (88/89), 100.0% (131/131). Only three false negative results and one false positive result were obtained. Statistical significance in the Ct values of the ORF region and N region of SARS-CoV-2-RNA was found by using one-step, one-step concentration, and magnetic bead methods (p < 0.05). The Ct values of the ORF region of SARS-CoV-2-RNA in P5 and P6 were significantly different in the different batches of reagent A (p < 0.05). The ORF region of SARS-CoV-2-RNA was not detected in a batch of reagent B. CONCLUSIONS: The majority of laboratories in Chongqing have reliable diagnostic ability for SARS-CoV-2 detection. Our data emphasized the importance of EQA for monitoring the performance of clinical laboratories. However, clinical laboratories must first effectively evaluate the performance of reagents prior to their use.