This work was aimed at predicting and verifying B-cell epitopes of SARS-CoV-2 E protein through bioinformatics methods,and clarifying the dominant B cell epitopes with mouse polyclonal antibody serum prepared through SARS-CoV-2 re-combinant E protein immunization and human positive serum vaccinated with inactivated SARS-CoV-2 vaccine.The structural and B-cell linear epitopes of SARS-CoV-2 E protein were predicted with SOPMA,Expasy,SWISS-MODEL,IEDB database,and Bepid-2.0 software.Candidate epitopes were expressed as GST-tagged recombinant protein fragments in an E.coli sys-tem,and their immunoreactivity with mouse and human poly-clonal positive serum against SARS-CoV-2 E protein was de-tected by western blotting and indirect ELISA,respectively.The epitope prediction results showed that E protein contained linear B cell epitopes Ser6-Val14 and Tyr57-Pro71,and the conformational epitopes of Glu8-Val12,Leu39-Tyr59,and Ser60-Leu65.The GST tagged recombinant E protein fragments of E1 and E3,containing Ser6-Val14 and Tyr57-Pro71 epitopes,respectively,as well as E2 without an epitope sequence as a control,were expressed in an E.coli expression system and successfully purified with an Ni-NTA column.Western blotting and indirect ELISA analysis indicated that all mouse and human SARS-CoV-2 positive sera positively reacted with only E1 and E3 proteins,but negatively reacted with E2 protein,thus indicating that the corresponding epitope prediction with Ser6-Val14 and Tyr57-Pro71 was correct.This study successfully predicted and preliminarily identified two linear B cell epitopes of SARS-CoV-2 E protein,thus providing a reference for the preparation of new coronavirus vaccines and the analysis of immune response characteristics.

Outpatient humanistic care refered to providing a full process of caring medical services to outpatients. In order to standardize the human caring services for outpatients in medical institutions, promote the comprehensive service level of outpatient services, and improve the patient′s medical experience, Chinese Association for Life Care issued the group standard of Management Norms for Human caring of Outpatients in April 2023. This standard clarified the relevant terms and definitions of human caring for outpatients, specified the basic requirements for human caring, the humanistic quality and care responsibilities of outpatient staff, the outpatient care environment and facilities, the outpatient care process and measures, and quality management. It designed standardized and personalized full process care service norms, providing references for medical institutions at all levels to promote the development of human caring for outpatients.

Objective To explore the causal association between gut microbes and non-alcoholic fatty liver disease(NAFLD)by Mendelian randomisation analysis.Methods Genetic instrumental variables for gut microbiota were identified from a gene-wide association study of 18 340 participants,and summary statistics for NAFLD were ob-tained from the FinnGen database,which provided data on 894 NAFLD cases and 217 898 controls using the IVW method as the primary analysis.In order to test the robustness of the results,MR-Egger method,WM method,Simple Mode method,Weighted Mode method were used for Mendelian randomisation analysis,and heterogeneity test,sensitivity analysis,and multiplicity analysis were performed.Results class Gammaproteobacteria IVW re-sults showed(OR=0.621,95％CI=0.412～0.934,P=0.022);family Enterobacteriaceae IVW results showed(OR=1.481,95％CI=1.069～2.053,P=0.018);genus Lachnospiraceae IVW results showed(OR=1.405,95％CI=1.036～1.904,P=0.029);genus Prevotella7 IVW results showed(OR=0.834,95％CI=0.714～0.974,P=0.021);genus Prevotella9 IVW results showed(OR=1.251,95％CI=1.025～1.527,P=0.027);order Desulfovibrionales IVW results showed(OR=0.714,95％CI=0.519～0.982,P=0.038);or-der Enterobacteriales IVW results showed(OR=1.481,95％CI=1.069～2.053,P=0.018).And there was no heterogeneity in the heterogeneity test,and the sensitivity analyses all showed robustness and no pleiotropy was found.Conclusion This study implicates class Gammaproteobacteria,family Enterobacteriaceae,genus Lachno-spiraceae,genus Prevotella7,genus Prevotella9,order Desulfovibrionales,order Enterobacteriales seven species of gut microorganisms have a causal relationship with NAFLD.

Exhaled ammonia(NH3)is an essential noninvasive biomarker for disease diagnosis.In this study,an acetone-modifier positive photoionization ion mobility spectrometry(AM-PIMS)method was developed for accurate qualitative and quantitative analysis of exhaled NH3 with high selectivity and sensitivity.Acetone was introduced into the drift tube along with the drift gas as a modifier,and the characteristic NH3 product ion peak of(C3H6O)4NH4+(K0=1.45 cm2/V·s)was obtained through the ion-molecule reaction with acetone reactant ions(C3H6O)2H+(K0=1.87 cm2/V·s),which significantly increased the peak-to-peak resolution and improved the accuracy of exhaled NH3 qualitative identification.Moreover,the interference of high humidity and the memory effect of NH3 molecules were significantly reduced via online dilution and purging sampling,thus realizing breath-by-breath measurement.As a result,a wide quantitative range of 5.87-140.92 μmol/L with a response time of 40 ms was achieved,and the exhaled NH3 profile could be synchronized with the concentration curve of exhaled CO2.Finally,the analytical capacity of AM-PIMS was demonstrated by measuring the exhaled NH3 of healthy subjects,demon-strating its great potential for clinical disease diagnosis.

Objective:To compare the differences in the prevalence of mild micro-hepatic encephalopathy (MHE) among patients with cirrhosis by using the psychometric hepatic encephalopathy score (PHES) and the Stroop smartphone application (Encephal App) test.Methods:This prospective, multi-center, real-world study was initiated by the National Clinical Medical Research Center for Infectious Diseases and the Portal Hypertension Alliance and registered with International ClinicalTrials.gov (NCT05140837). 354 cases of cirrhosis were enrolled in 19 hospitals across the country. PHES (including digital connection tests A and B, digital symbol tests, trajectory drawing tests, and serial management tests) and the Stroop test were conducted in all of them. PHES was differentiated using standard diagnostic criteria established by the two studies in China and South Korea. The Stroop test was evaluated based on the criteria of the research and development team. The impact of different diagnostic standards or methods on the incidence of MHE in patients with cirrhosis was analyzed. Data between groups were differentiated using the t-test, Mann-Whitney U test, and χ2 test. A kappa test was used to compare the consistency between groups. Results:After PHES, the prevalence of MHE among 354 cases of cirrhosis was 78.53% and 15.25%, respectively, based on Chinese research standards and Korean research normal value standards. However, the prevalence of MHE was 56.78% based on the Stroop test, and the differences in pairwise comparisons among the three groups were statistically significant (kappa = -0.064, P < 0.001). Stratified analysis revealed that the MHE prevalence in three groups of patients with Child-Pugh classes A, B, and C was 74.14%, 83.33%, and 88.24%, respectively, according to the normal value standards of Chinese researchers, while the MHE prevalence rates in three groups of patients with Child-Pugh classes A, B, and C were 8.29%, 23.53%, and 38.24%, respectively, according to the normal value standards of Korean researchers. Furthermore, the prevalence rates of MHE in the three groups of patients with Child-Pugh grades A, B, and C were 52.68%, 58.82%, and 73.53%, respectively, according to the Stroop test standard. However, among the results of each diagnostic standard, the prevalence of MHE showed an increasing trend with an increasing Child-Pugh grade. Further comparison demonstrated that the scores obtained by the number connection test A and the number symbol test were consistent according to the normal value standards of the two studies in China and South Korea ( Z = -0.982, -1.702; P = 0.326, 0.089), while the other three sub-tests had significant differences ( P < 0.001). Conclusion:The prevalence rate of MHE in the cirrhotic population is high, but the prevalence of MHE obtained by using different diagnostic criteria or methods varies greatly. Therefore, in line with the current changes in demographics and disease spectrum, it is necessary to enroll a larger sample size of a healthy population as a control. Moreover, the establishment of more reliable diagnostic scoring criteria will serve as a basis for obtaining accurate MHE incidence and formulating diagnosis and treatment strategies in cirrhotic populations.

To report a case of Noonan-like syndrome with loose anagen hair, and to detect gene mutations in the family. A 3-year-old female patient presented with a special facial appearance, short stature, deep palmar and plantar creases, and dark skin. The scalp hair was sparse and thin, dermoscopy showed black spot sign, single-hair follicular units, hair shafts varying in the diameter, upright hair and new vellus hair, and constriction of hair shafts and nodular hair were occasionally observed. Peripheral blood samples were collected from the proband and her parents, and the genomic DNA was extracted for whole-exome sequencing. A heterozygous missense mutation c.4A>G was identified in exon 2 of the SHOC2 gene in the proband, resulting in the substitution of serine by glycine at amino acid position 2 (p.S2G) . The mutation was not identified in either of her parents. Combined with the clinical phenotype and gene mutation, the diagnosis of Noonan-like syndrome with loose anagen hair was confirmed.

Objective:To investigate the diagnostic accuracy of serological indicators and evaluate the diagnostic value of a new established combined serological model on identifying the minimal hepatic encephalopathy (MHE) in patients with compensated cirrhosis.Methods:This prospective multicenter study enrolled 263 compensated cirrhotic patients from 23 hospitals in 15 provinces, autonomous regions and municipalities of China between October 2021 and August 2022. Clinical data and laboratory test results were collected, and the model for end-stage liver disease (MELD) score was calculated. Ammonia level was corrected to the upper limit of normal (AMM-ULN) by the baseline blood ammonia measurements/upper limit of the normal reference value. MHE was diagnosed by combined abnormal number connection test-A and abnormal digit symbol test as suggested by Guidelines on the management of hepatic encephalopathy in cirrhosis. The patients were randomly divided (7∶3) into training set ( n=185) and validation set ( n=78) based on caret package of R language. Logistic regression was used to establish a combined model of MHE diagnosis. The diagnostic performance was evaluated by the area under the curve (AUC) of receiver operating characteristic curve, Hosmer-Lemeshow test and calibration curve. The internal verification was carried out by the Bootstrap method ( n=200). AUC comparisons were achieved using the Delong test. Results:In the training set, prevalence of MHE was 37.8% (70/185). There were statistically significant differences in AMM-ULN, albumin, platelet, alkaline phosphatase, international normalized ratio, MELD score and education between non-MHE group and MHE group (all P<0.05). Multivariate Logistic regression analysis showed that AMM-ULN [odds ratio ( OR)=1.78, 95% confidence interval ( CI) 1.05-3.14, P=0.038] and MELD score ( OR=1.11, 95% CI 1.04-1.20, P=0.002) were independent risk factors for MHE, and the AUC for predicting MHE were 0.663, 0.625, respectively. Compared with the use of blood AMM-ULN and MELD score alone, the AUC of the combined model of AMM-ULN, MELD score and education exhibited better predictive performance in determining the presence of MHE was 0.755, the specificity and sensitivity was 85.2% and 55.7%, respectively. Hosmer-Lemeshow test and calibration curve showed that the model had good calibration ( P=0.733). The AUC for internal validation of the combined model for diagnosing MHE was 0.752. In the validation set, the AUC of the combined model for diagnosing MHE was 0.794, and Hosmer-Lemeshow test showed good calibration ( P=0.841). Conclusion:Use of the combined model including AMM-ULN, MELD score and education could improve the predictive efficiency of MHE among patients with compensated cirrhosis.

The bacterial ATP-competitive GyrB/ParE subunits of type II topoisomerase are important anti-bacterial targets to treat super drug-resistant bacterial infections. Herein we discovered novel pyrrolamide-type GyrB/ParE inhibitors based on the structural modifications of the candidate AZD5099 that was withdrawn from the clinical trials due to safety liabilities such as mitochondrial toxicity. The hydroxyisopropyl pyridazine compound 28 had a significant inhibitory effect on Gyrase (GyrB, IC₅₀ = 49 nmol/L) and a modest inhibitory effect on Topo IV (ParE, IC₅₀ = 1.513 μmol/L) of Staphylococcus aureus. It also had significant antibacterial activities on susceptible and resistant Gram-positive bacteria with a minimum inhibitory concentration (MIC) of less than 0.03 μg/mL, which showed a time-dependent bactericidal effect and low frequencies of spontaneous resistance against S. aureus. Compound 28 had better protective effects than the positive control drugs such as DS-2969 ( 5) and AZD5099 ( 6) in mouse models of sepsis induced by methicillin-resistant Staphylococcus aureus (MRSA) infection. It also showed better bactericidal activities than clinically used vancomycin in the mouse thigh MRSA infection models. Moreover, compound 28 has much lower mitochondrial toxicity than AZD5099 ( 6) as well as excellent therapeutic indexes and pharmacokinetic properties. At present, compound 28 has been evaluated as a pre-clinical drug candidate for the treatment of drug-resistant Gram-positive bacterial infection. On the other hand, compound 28 also has good inhibitory activities against stubborn Gram-negative bacteria such as Escherichia coli (MIC = 1 μg/mL), which is comparable with the most potent pyrrolamide-type GyrB/ParE inhibitors reported recently. In addition, the structure-activity relationships of the compounds were also studied.

Objective:To investigate the application value of the anatomically oriented "six steps with six windows" approach in laparoscopic radical total gastrectomy.Methods:The retros-pective cohort study was conducted. The clinicopathological data of 121 patients who underwent laparoscopic radical total gastrectomy in Henan Provincial People′s Hospital from January 2019 to February 2022 were collected. There were 53 males and 68 females, aged (68±12)years. Of the 121 patients, 72 patients using the traditional approach in the surgery were divided into the control group, and 49 patients using the "six steps with six windows" approach in the surgery were divided into the experiment group. Observation indicators: (1) surgical situations; (2) postoperative situations. Measurement data with normal distribution were represented as Mean± SD, and comparison between groups was conducted using the t test. Count data were described as absolute numbers or percen-tages, and comparison between groups was conducted using the chi-square test. Results:(1) Surgical situations. All 121 patients underwent laparoscopic radical total gastrectomy successfully, without laparotomy conversion. The operation time, volume of intraoperative blood loss, number of lymph nodes dissected were (250±50)minutes, (150±34)mL, 41±6 in the control group, versus (180±16)minutes, (55±13)mL, 51±5 in the experiment group, showing significant differences in the above indicators between the two groups ( t=11.04, 21.47, 9.42, P<0.05). (2) Postoperative situations. The times of postoperative analgesic injection, time to postoperative ?rst ?atus, duration of postoperative hospital stay, postoperative pathological staging (stage Ⅰ, stage Ⅱ, stage Ⅲ), incidence of postoperative complication were 3.4±1.6, (4.0±1.4)days, (13.1±2.0)days, 9, 32, 31, 15.3%(11/72) in the control group, versus 3.5±1.7, (4.1±1.5)days, (13.1±1.7)days, 6, 25, 18, 16.3%(8/49), showing no significant difference in the above indicators between the two groups ( t=0.35, 0.18, 0.03, Z=0.55, χ2=0.02, P>0.05). There was no perioperative death in both groups of patients. Conclusion:Appli-cation of the anatomically oriented "six steps with six windows" approach in laparoscopic radical total gastrectomy is safe and feasible.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis and insulin resistance and there are currently no approved drugs for its treatment. Hyperactivation of mTOR complex 1 (mTORC1) and subsequent impairment of the transcription factor EB (TFEB)-mediated autophagy-lysosomal pathway (ALP) are implicated in the development of NAFLD. Accordingly, agents that augment hepatic TFEB transcriptional activity may have therapeutic potential against NAFLD. The objective of this study was to investigate the effects of nuciferine, a major active component from lotus leaf, on NAFLD and its underlying mechanism of action. Here we show that nuciferine activated ALP and alleviated steatosis, insulin resistance in the livers of NAFLD mice and palmitic acid-challenged hepatocytes in a TFEB-dependent manner. Mechanistic investigation revealed that nuciferine interacts with the Ragulator subunit hepatitis B X-interacting protein and impairs the interaction of the Ragulator complex with Rag GTPases, thereby suppressing lysosomal localization and activity of mTORC1, which activates TFEB-mediated ALP and further ameliorates hepatic steatosis and insulin resistance. Our present results indicate that nuciferine may be a potential agent for treating NAFLD and that regulation of the mTORC1-TFEB-ALP axis could represent a novel pharmacological strategy to combat NAFLD.