Objective　To conduct an in-depth study of the molecular biological characteristics and evolutionary trends of H9N2 avian influenza virus (AIV) in live poultry markets in Yunnan Province in 2023, and to provide scientific evidence for the development of control strategies for H9N2 avian influenza in the region. Methods　Environmental samples were collected from live poultry markets in Yunnan Province in 2023 for H9N2 subtype nucleic acid detection. Positive samples were subjected to virus isolation using chicken embryos, and the genome of the 8 isolated strains was amplified, sequenced, and analyzed for genetic characteristics. Results　The eight avian influenza virus (AIV) isolates had the hemagglutinin (HA) cleavage site sequence PSRSSRGLF, which is a non-continuous basic amino acid sequence, consistent with the genetic characteristics of typical low-pathogenicity avian influenza viruses. Mutations Q234L and H191N were observed in the left arm of the HA protein, which enhanced the affinity for α-2,6 sialic acid receptors, suggesting that these viruses may have the potential to infect humans. The neuraminidase (NA) protein exhibited a deletion of three amino acids (TEI) at positions 62–64 in the stalk region, displaying characteristics of high pathogenicity at the molecular level. The increase or absence in potential glycosylation sites were observed in both HA and NA genes. The non-structural protein 1 (NS1) showed no D92E mutation, and had a C-terminal truncation of 13 amino acids, indicating that this virus is of low pathogenicity and poses a lower risk of human transmission. Mutations T37A, R95K, S224N, and K242N in the M1 protein of some isolates increased the risk of infection, while one isolate carried the V27A or S31N mutation in the M2 protein, conferring resistance to M2 ion channel inhibitors. Mutations M317I and S678N were identified in the PB1 protein, which may enhance pathogenicity in mice and increase the potential for mammalian infection. The PB2 protein carried the I292V mutation, which exhibited a stronger infectivity to mammals. Phylogenetic analysis revealed that the HA, NA, and PB2 gene segments belonged to the Y280 lineage, NP and PB1 gene segments were classified under the F98 lineage, the M gene segment of the NH013 isolate belonged to the F98 lineage, while M genes, as well as the NS and PA genes of other isolates belonged to the G1 lineage. Conclusions　These eight AIV isolates exhibited characteristics of low pathogenicity, but simultaneously carry the potential risk of infecting humans. Despite the HA cleavage site and NS1 protein mutations indicating low pathogenicity, the Q234L and H191N mutations in the HA protein enhanced its affinity for human receptors, suggesting the potential for human infection. The TEI deletion in the NA protein, mutations in the M1 protein, and resistance mutations in the M2 protein further increase the risk of human infection. Mutations in the PB1 and PB2 proteins increase the potential for these eight AIV strains to infect humans or mammals.

Background::Limited information exists regarding the impact of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection on psoriasis patients. The objective of this study was to identify clinical factors associated with the prognosis of psoriasis following SARS-CoV-2 infection.Methods::A retrospective, multicenter study was conducted between March and May 2023. Univariable and multivariable logistic regression analyses were employed to identify factors associated with coronavirus disease 2019 (COVID-19)-related psoriasis outcomes. The study included 2371 psoriasis patients from 12 clinical centers, with 2049 of them having been infected with SARS-CoV-2.Results::Among the infected groups, lower exacerbation rates were observed in individuals treated with biologics compared to those receiving traditional systemic or nonsystemic treatments (22.3% [236/1058] vs. 39.8% [92/231] vs. 37.5% [140/373], P <0.001). Psoriasis progression with lesions (adjusted odds ratio [OR] = 8.197, 95% confidence interval [95% CI] = 5.685–11.820, compared to no lesions), hypertension (adjusted OR = 1.582, 95% CI = 1.068–2.343), traditional systemic (adjusted OR = 1.887, 95% CI= 1.263–2.818), and nonsystemic treatment (adjusted OR= 1.602, 95% CI= 1.117–2.297) were found to be associated with exacerbation of psoriasis after SARS-CoV-2 infection, but not biologics (adjusted OR = 0.931, 95% CI = 0.680–1.274, compared to no treatment), according to multivariable logistic regression analysis. Conclusions::A reduced risk of psoriasis exacerbation after SARS-CoV-2 infection was observed with biologics compared to traditional systemic and nonsystemic treatments. Significant risk factors for exacerbation after infection were identified as existing psoriatic lesions and hypertension.

Objective:To investigate the differences in programmed death-ligand 1 (PD-L1) expression and immune cell infiltration characteristics in different molecular subtypes of endometrial cancer.Methods:A retrospective case series study was conducted. Ninety primary treated EC patients who underwent surgery without preoperative neoadjuvant therapy at the Second Hospital of Tianjin Medical University from November 2016 to May 2022 were collected. The surgical paraffin-embedded tissues were selected, and the molecular subtypes of endometrial cancer were classified according to 2020 World Health Organization (WHO) molecular subtypes using POLE gene Sanger sequencing and immunohistochemical staining. The expression of PD-L1, CD3, CD4, CD8, CD68, and CD20 proteins were detected by immunohistochemistry. Stained slides were digitally scanned for quantitative analysis of PD-L1 and immune cell infiltration density. The PD-L1-related scores were evaluated, including tumor cell score (TCS, the percentage of PD-L1 positive tumor cells among total tumor cells ≥1% was TCS positive, <1% was TCS negative), immune cell score (ICS, the percentage of PD-L1 positive tumor-associated lymphocytes and macrophages among total tumor-associated lymphocytes and macrophages ≥1% was ICS positive, <1% was ICS negative) and combined positive score [CPS, PD-L1 positive stained cells (including tumor cells, lymphocytes and macrophages)/total number of viable tumor cells ×100 ≥ 1 was CPS positive, < 1 was CPS negative]. Clinicopathological characteristics, PD-L1 scores and immune cell infiltration densities among different molecular subtypes were analyzed. Kaplan-Meier method was used to plot disease-free survival (DFS) curves for molecular subtypes, PD-L1 scores and immune cell infiltration densities, with subgroup comparisons using log-rank test. Cox proportional hazards models were used for univariate and multivariate analyses of poor DFS in endometrial cancer patients.Results:The median age of 90 patients was 58 years old (range: 33-72 years old); endometrioid carcinoma was present in 78 cases (86.7%), and non-endometrioid carcinoma was present in 12 cases (13.3%). Molecular subtyping identified POLE-mutated subtype in 6 cases (6.7%), mismatch repair deficient (MMRd) subtype in 23 cases (25.6%), p53 abnormal subtype in 14 cases (15.6%), and non-specific molecular profile (NSMP) subtype in 47 cases (52.2%). Significant differences were observed among the 4 molecular subtypes in International Federation of Gynecology and Obstetrics (FIGO) stage, histological grade, morphological subtype, tertiary lymphoid structures, estrogen receptor expression, and progesterone receptor expression (all P < 0.05). Among the 90 cases, 18 cases (20.0%) were positive for TCS, 31 cases (34.4%) were positive for ICS, and 39 cases (43.3%) were positive for CPS. Significant differences were found among the 4 molecular subtypes in PD-L1 + cell density, distribution of patients with ICS positivity, and distribution of patients with CPS positivity (all P < 0.01), but not in distribution of patients with TCS positivity ( P = 0.090); compared to NSMP subtype, the proportions of ICS-positive patients in POLE-mutated and MMRd subtypes were higher, the proportion of CPS-positive patients and PD-L1 + cell density in MMRd and p53 abnormal subtypes were higher, and the differences were statistically significant (all P < 0.05). Significant differences in immune cell densities were observed among the 4 molecular subtypes (all P < 0.01); compared to NSMP subtype, POLE-mutated, MMRd and p53 abnormal subtypes had higher densities of CD3 + and CD8 + cells, MMRd subtype had higher CD4 + cell density, and POLE-mutated and MMRd subtypes had higher CD68 + and CD20 + cell densities (all P < 0.05). The median follow-up was 43 months (range: 7-75 months). Among the molecular subtypes, p53 abnormal patients had the worst DFS, and POLE-mutated patients had the best DFS, and the difference in DFS among the 4 subtypes was statistically significant ( P = 0.046). Grouping according to the median density of immune cells in the entire group, patients with high CD8 + cell density (45 cases) had better DFS than those with low density (45 cases) ( P = 0.010), PD-L1 ICS-positive patients had worse DFS than negative patients ( P = 0.019), and NSMP subtype patients with high CD4 + cell density (24 cases) had better DFS than those with low density (23 cases) ( P < 0.001). There was no statistically significant difference in DFS among patients grouping with other PD-L1 scoring modes and other immune cell infiltration density (all P > 0.05). Cox regression analysis indicated that high CD8 + cell density ( HR = 0.335, 95% CI: 0.113-0.990, P = 0.048) was an independent protective factor for poor DFS in endometrial cancer patients, and high CD4 + cell density was an independent protective factor for poor DFS in NSMP subtype patients ( HR = 0.035, 95% CI: 0.003-0.345, P = 0.004). Conclusions:There are significant differences in PD-L1 expression and immune cell infiltration density among the different molecular subtypes of endometrial cancer, which are correlated with the prognosis of patients, and may provide reference for the selection of immunotherapy strategies and prognosis judgment.

Objective:To explore the correlation of bone marrow polychonal plasma cell proportion (pPC% ) and clinical features in newly diagnosed multiple myeloma (NDMM) patients.Methods:A retrospective analysis of 317 patients with NDMM admitted to Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology from January 2018 to January 2023 was performed. The results of the pPC% in all patients were clear. The relationship between the pPC% and clinical characteristics was analyzed.Results:A total of 317 patients were included, comprising 180 males and 137 females. The median age at diagnosis was 61 (26-91) years, and 55.8% were 60 years or older. The pPC% in the bone marrow of patients with NDMM was different in the DS, International Staging System (ISS), and revised ISS (R-ISS) stages ( P=0.002, 0.010, and 0.049, respectively), whereas no statistical difference in pPC% was observed among patients with different FISH risk stratigrams ( P=0.971). The correlation coefficient between pPC% and hemoglobin (HGB) at the first diagnosis in patients was 0.211 ( P<0.01). The correlation coefficients with serum calcium, serum creatinine, M protein level, and β 2-microglobulin were -0.141, -0.120, -0.181, and -0.207, respectively, and the results of the significance test were P=0.012, 0.033, 0.004, and 0.002, respectively, indicating a negative correlation. Compared with the patients with a pPC% of ≥2.5%, the group of patients with a pPC% of <2.5% had significantly higher levels of light chain, serum calcium, serum creatinine, M protein, and β 2-microglobulin at the initial diagnosis ( P<0.05) ; lower HGB level ( P<0.001) ; and a higher proportion of patients in ISS stage Ⅲ ( P=0.034) . Conclusion:In this study, the pPC% in patients with NDMM was associated with clinical features of good prognosis, including higher HGB, lower serum calcium, serum creatinine, M protein quantity, β 2-microglobulin, light chain involvement, lower proportion of advanced disease (DS stage and ISS stage Ⅲ), and clinical features showing lower tumor burden.

Objective:To establish a nomogram for preoperative differentiating intrahepatic cholangiocarcinoma (ICC) from hepatocellular carcinoma (HCC) based on clinical, ultrasound, and contrast-enhanced ultrasound (CEUS) data.Methods:A retrospective analysis was conducted on ultrasound and CEUS data of 462 patients who underwent hepatectomy in Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School from January 2016 to December 2023, including 262 cases of HCC (56.7%) and 200 cases of ICC (43.3%). The data were randomly divided into training set ( n=324) and validation set ( n=138) in a 7∶3 ratio. Univariate analysis was used to initially screen for variables with statistically significant differences between HCC and ICC groups in the training set, and LASSO regression was performed to select the variables with higher coefficients. Logistic regression analyses were then used to predict independent risk factors for ICC. A nomogram was drawn using R software. The performance of the nomogram was then validated using ROC curve, calibration curve, and decision curve analysis (DCA). Results:Univariate analysis showed that there were significant differences in age, gender, liver cirrhosis, HBsAg (+ ), ALP >185 U/L, CA19-9 >27 kU/L, CA242>10 kU/L, irregular shape, border, cholangiectasis, portal vein tumor thrombus, enhanced pattern in arterial phase, clearance time <60 s, intra-tumoral vein between ICC and HCC groups (all P<0.05). The top 10 features were selected for LASSO regression analysis. Logistic regression analysis revealed that gender, cirrhosis, CA19-9>27 kU/L, CA242>10 kU/L, cholangiectasis, clearance time <60 s, intra-tumoral vein and enhanced pattern in arterial phase were risk factors for ICC (all P<0.05). The area under the ROC curve in the training and validation groups were 0.963 and 0.914, respectively. In the training group, the specificity and sensitivity of the nomogram were 0.926 and 0.917, respectively, and in the validation group, they were 0.875 and 0.871, respectively. The calibration curve showed that the prediction effect of the model was in good agreement with the actual situation. DCA showed that the nomogram could increase the net benefit to the different diagnosis of ICC in patients. Conclusions:The nomogram based on clinical, ultrasound and CEUS features has a good predictive value for preoperative identification of ICC and provides reliable evidence for clinical practice.

Objective:To investigate the relationship between body mass index(BMI) and osteoporosis using Mendelian randomization analysis.Methods:The genetic variation strongly related to BMI was selected as the instrumental variables in the collection data set of the genome-wide association study(GWAS). The MR-Egger regression, weighted median estimator(WME), inverse variance weighted(IVW), simple mode and weighted mode were used for Mendelian randomization(MR) analysis. The causal association between BMI and osteoporosis was evaluated by odds ratio and 95% confidence interval. The MR-APSS method was applied to make the causal inference results based on MR more reliable. The Linkage disequilibrium score regression was applied to evaluate the genetic correlation, and the horizontal pleiotropy test, heterogeneity test, and leave-one-out method were used to evaluate whether the results were reliable, The influence of heterogeneity and horizontal pleiotropy were reduced by the MR-PRESSO outlier test.Results:A total of 421 SNPs were included, with inverse variance-weighted method as the main analysis approach. The calculated OR value and 95% CI were 0.994(95% CI 0.992-0.997), indicating a protective effect of BMI on osteoporosis. The MR-APSS method showed that the effect of BMI on osteoporosis was statistically significant. Linkage disequilibrium score regression demonstrated a genetic correlation between BMI and osteoporosis. MR-Egger regression intercept showed no horizontal pleiotropy of instrumental variables, and the funnel plot showed no bias in instrumental variables. Leave-one-out analysis confirmed robust results. Conclusion:There may be a negative causal relationship between BMI and osteoporosis and BMI is a protective factor for osteoporosis.

Objective: To investigate the mechanism of action of Wuzi Yanzong pill (WYP) in rats with oligoasthenozoospermia (OAZ) via metabolomics and to provide a possible basis for improving this WYP-based treatment. Methods: A rat model of OAZ was established by treating male Sprague–Dawley rats with glucosides from Tripterygium wilfordii Hook. F. Seventy-two rats were randomly divided into six groups: control, L-carnitine (positive control), model, and low-, medium-, and high-dose WYP groups. Rats in the experimental groups were treated with WYP for 4 weeks. At the end of the treatment period, sperm cell quality (density, motility, and viability) was assessed using a semen analysis system, mitochondrial membrane potential (MMP) was assessed using flow cytometry, and testicular injury was assessed using hematoxylin and eosin staining to validate the therapeutic effect of WYP in OAZ. Further, serum metabolomics-based analysis was performed using high-performance liquid chromatography-mass spectrometry to identify differential metabolic pathways and possible mechanisms of action of WYP in OAZ treatment. Results: A rat model of OAZ was considered successfully-established after comparing the quality of spermatozoa in the model group to that in the control group. WYP-M and WYP-H treatments significantly improved sperm cell density, motility, and viability compared with those in the model group (all P < .05). Compared with the model group, both WYP-M and WYP-H treatments increased MMP values (P = .006 and P = .021 respectively), while there was no significant difference in the L-carnitine group. L-carnitine and WYP administration reversed damage to the testes to varying degrees compared with that in the model group. Further, 44 differential metabolites and four metabolic pathways, especially autophagy pathway, related to OAZ were identified via metabolomics. Conclusions WYP improves sperm cell quality and MMP in OAZ primarily via autophagy regulation. These findings can be employed to improve the efficacy of WYP in humans.

Objective:To establish and evaluate a clinical and ultrasound parameters-based nomogram for the preoperative differentiating diagnosis of intrahepatic cholangiocarcinoma (ICC).Methods:A total of 723 patients undergoing hepatectomy in Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University from January 2016 to August 2022 were retrospectively screened. A total of 399 patients with hepatocellular carcinoma (HCC, 198 cases) or ICC (201 cases) were enrolled in this study, including 284 males and 115 females, aged (60.5±10.5) years. Through random sampling using computer-generated random numbers, patients were divided into training ( n=279) and validation groups ( n=120) in a ratio of 7∶3. Univariate and multivariate logistic regression were performed to identify factors differentiating ICC, and a nomogram was established using R software based on independent risk factors for ICC. The accuracy of the nomogram was evaluated by receiver operating characteristic curve and calibration curves. Decision curve analysis was performed to assess the net benefit of the model. Results:Multivariate logistic regression analysis showed that irregular shape, cholangiectasis, female, cirrhosis, carbohydrate antigen 242 >10 U/ml, carbohydrate antigen 125 >30 U/ml and alpha-fetoprotein >10 μg/L were independent differentiating factors for ICC (all P<0.05). A nomogram was constructed based on those factors. The nomogram showed a better discrimination between ICC and HCC. The area under the curve of the training group and the validation group were 0.966 and 0.956, respectively. The calibration curve showed that the prediction effect of the model is in good agreement with the actual situation. Decision curve analysis showed that the nomogram was more effective than diagnosing all patients as either HCC or ICC, which yielded a net benefit at the most reasonable threshold probabilities. Conclusion:The nomogram for the preoperative diagnosis of ICC based on clinical and ultrasound features showed a good diagnostic performance.

Objective:To analyze the non-enterovirus A71 (non-EVA71) and non-coxsackievirus A16 (non-CVA16) enteroviruses causing hand, foot and mouth disease (HFMD) in Kunming and Qujing of Yunnan Province in 2021 by sequencing the VP4/VP2 and VP1 genes and to analyze the phylogenetic characteristics of the VP1 gene of CVA2, aiming to provide reference for the prevention and control of CVA2.Methods:The samples were made and extracted strictly according to the Laboratory Manual for Hand, Foot and Mouth Disease (China Center for Disease Control and Prevention, 2018 Edition). VP4/VP2 junction regions were firstly amplified and sequenced by MD91/OL68-1 primers. These sequences were firstly edited and then "blasted" on the GenBank to determine the virus serotype. To analyze the phylogenetic characteristics of CVA2, the entire VP1 gene sequences were amplified in two segments using enterovirus species A primers. Virus serotype was again confirmed online by "Enterovirus Genotyping Tool Version 0.1". The sequences of the reference virus genotypes/sub-genotypes were downloaded according to the reference. The phylogenetic trees were constructed by Mega5.2 software and the genetic characteristics were analyzed.Results:A total of 749 non-EVA71 and non-CVA16 enteroviruses were detected in the two areas in 2021. Group A enteroviruses were the main pathogens, with CVA16 as the predominant virus, and a small number of group B enteroviruses were reported. Only five strains of CVA2 were detected with a detection rate of 0.67% (5/749), indicating that CVA2 was a rare pathogen for HFMD in the two areas. The sequencing and serotyping results were consistent using the two genomic regions of VP4/VP2 junction region and VP1 region. Phylogenetic analysis showed that three Kunming strains belonged to genotype A, while two Qujing strains belonged to genotype D.Conclusions:The detection rate of CVA2 in Kunming and Qujing was 0.67% in 2021. CVA2 was a rare pathogen for HFMD in the two regions. Phylogenetic analysis showed genotypes A and D spread in Kunming and Qujing, respectively, but had not caused epidemics. To our knowledge, this was the first report of genotype A of CVA2 in China. Strengthening the laboratory surveillance especially molecular epidemiological surveillance is valuable for the monitor and analysis of transmission source for CVA2.

Objective:To understand the characteristics of the local Coronavirus Disease 2019 (COVID-19) epidemic in the border areas of Yunnan province, and to analyze and trace the genome variation of the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2).Methods:The basic information and samples of the first cases of 12 local epidemics in the border areas of Yunnan province from mid-February to April 2022 were collected. The virus genomes, genotypes and mutations were determined by the whole genome sequencing technology, the Pangolin COVID-19 Lineage Assigner and MEGA software. The sources of virus infection were estimated based on the contents of epidemiological investigations.Results:The first cases from 12 local epidemics in the border areas of Yunnan province had diverse occupations, and none of them had ever left the local area. The virus genomes of 12 first cases were Omicron (BA.2) variants and every two of them further belonged to the evolutionary clades BA.2.10 and BA.2.3.2, respectively. The genome-wide nucleotide-level mutation analysis showed that there were 1 or more specific nucleotide mutation sites in 10 first cases of the local epidemics. Combined with the epidemiological investigations, it was suggested that there might be different sources of virus infection in all 12 local epidemics. Further analysis of amino acid mutations revealed that in addition to the representative mutation sites of evolutionary clades, there were also specific amino acid mutation sites (such as S: E1188 D) in the genome sequences between the cases, which also deserved attention. The result of phylogenetic tree analysis were consistent with Pangolin Assigner and mutation analysis.Conclusions:The 12 local epidemics in Yunnan may have different sources of virus infection, which indicates that the COVID-19 epidemic in the border areas of Yunnan province is extremely complicated. Strengthening the frequency of nucleic acid screening and the scope of key populations and monitoring the variation of SARS-CoV-2 are of great significance for the precise prevention and control of the epidemic.