Objective:To observe the effect of mirror threshold load training on respiration of persons with respiratory muscle fatigue after a cerebral hemorrhage.Methods:Fifty cerebral hemorrhage patients with respiratory muscle fatigue were randomly divided into an observation group and a control group, each of 25. In addition to routine rehabilitation training, the control group was given threshold load training of the respiratory muscles, while the observation group was provided with mirror threshold load training, twice a day in the morning and afternoon, 5 days a week for 4 weeks. Before the experiment and after 1, 2, 3 and 4 weeks of the treatment, everyone′s maximum inspiratory pressure (MIP) and maximum expiratory pressure (MEP) was recorded. Before and after the 4 weeks forced expiration volume in the first second (FEV1) was measured along with 25% of the forced expiration volume (FEF25), maximum sound time (MPT) and respiration rate (RR).Results:At each time point the MIP and MEP values of both groups were significantly better than those before the treatment. After 4 weeks the average MIP and MEP values of the observation group were significantly better than those of the control group. And after 4 weeks the FEV1, FEF25, MPT and RR values of both groups had also improved significantly, on average. All of the observation group′s averages except MPT were then significantly better than the control group′s averages.Conclusions:Mirror threshold load training of the respiratory muscles can significantly improve the respiration of persons with respiratory muscle fatigue after a cerebral hemorrhage. It is more effective than respiratory muscle threshold load training.

Objective:To analyze the genetic characteristics of the prevalent strains of Varicella-Zoster virus (VZV) in the population of Dali, Yunnan, and to understand its evolutionary status in the population of Dali.Methods:Herpes fluid and 163 sera were collected from 249 patients clinically suspected to have varicella or herpes zoster in the Department of Dermatology of the Second People′s Hospital of Dali city, Yunnan province, China, from 2023 to 2024. The levels of VZV-specific IgG and IgM antibodies in serum were detected using enzyme-linked immunoassay. Viral DNA was extracted from the herpes fluid, and the cycle threshold ( Ct) of the samples was detected using quantitative real-time polymerase chain reaction (qPCR), and some samples with Ct ≦ 22 were selected for sequencing by next-generation sequencing technology (next-generation sequencing). Next-generation sequencing (NGS) was used to obtain 90 whole genome sequences of VZV, and the sequencing result were compared with the sequences of reference strains for multiple sequence comparison and evolutionary analysis. Snapgene was used to translate the nucleotides into amino acids, and the result were compared with the amino acid sequences of the reference strain. Results:Of the 90 VZV whole-genome sequences, one whole-genome sequence was from an adult varicella patient, and the remaining 89 whole-genome sequences were from herpes zoster patients. The serum-specific IgG antibody positivity rate was 99.4%, and the IgM antibody positivity rate was 52.8%. The result of both single nucleotide polymorphism (SNPs) site typing and genome-wide phylogenetic tree analysis showed that 83 of the 90 VZV whole-genome sequences in this study were on the same branch as Clade 2, and 7 VZV whole-genome sequences were on the branch of Clade 9.Conclusions:The main endemic branch in Dali region in 2023-2024 was Clade 2, with the emergence of Clade 9 branch; there were amino acid mutations in the proteins encoded by ORF22 and ORF68 in 83 VZV whole genome sequences of Clade 2 branch, and the mutations did not cause significant changes to the protein structure.

Objective:To analyze the clinical characteristics, diagnosis and prognosis of pregnant women with fetomaternal hemorrhage (FMH) syndrome, and to guide the management of pregnant women with FMH syndrome.Methods:The clinical data of 33 pregnant women with FMH syndrome admitted to Beijing Obstetrics and Gynecology Hospital, Capital Medical University, from January 2010 to December 2024 were collected, and the general information, diagnostic characteristics, treatment and maternal and fetal prognosis were retrospectively analyzed.Results:The incidence of FMH syndrome in our hospital was 1.7/10 000 (33/194 272). The gestational age of onset of FMH syndrome in 33 pregnant women was (35.6±3.1) weeks, 15 cases (45%, 15/33) were full-term delivery and 18 cases (55%, 18/33) were preterm delivery. Decreased fetal movement (51%, 17/33) was the most common initial symptom, followed by abnormal electronic fetal monitoring (33%, 11/33). Thirty-two cases (97%, 32/33) underwent cesarean section, and only one case had spontaneous delivery. Postpartum hemorrhage occurred in 11 cases (33%, 11/33). All the neonates were transferred to neonatal intensive care unit for treatment. Two of them were treated with intrauterine blood transfusion, and the neonates did not receive blood transfusion after birth. The neonatal mortality rate was 6% (2/33), and the remaining 31 cases (94%, 31/33) survived. Complications occurred in 3 premature infants, including 1 case of neonatal neurodevelopmental disorder with cochlear implantation, 1 case of pulmonary artery stenosis, and 1 case of retinopathy of prematurity. Three pregnant women were pregnant again, and none of them had FMH syndrome.Conclusions:Decreased fetal movement or abnormal electronic fetal monitoring in late pregnancy should be alert to the occurrence of FMH syndrome. Early diagnosis and intervention are critical to improve the prognosis of FMH syndrome.

Objective To explore the regulatory role of N-methyl berbamine(N-MB)in intracellular calcium homeostasis in H9c2 car-diomyocytes,and,thereby,clarify the possible mechanism of the myocardial protective effect of N-MB.Methods Binding of N-MB to CaV1.2 channels was simulated using the MOE software,and the binding affinity and binding mode were determined.The hCaV1.2 gene was transfected into HEK293 cells,and the effect of N-MB(30 μmol/L)on the CaV1.2 current was detected using the whole-cell patch clamp technique.In addition,a Fluo 3-AM fluorescent probe was loaded into H9c2 cardiomyocytes,and the effect of N-MB(3,30 μmol/L)on intracellular calcium ion concentration was observed under a laser confocal microscope.The effect of N-MB(3,30 μmol/L)on the expression of Ca2+regulation-related genes Cacna1c,Cacnb2,Ryr2,Serca2a,and Ncx1 in H9c2 cardiomyocytes was examined using real-time quantitative PCR.Results N-MB was predicted to bind to CaV1.2 channels.The binding sites mainly involved Phe1191,Thr1420,and Asn771,and the binding modes were H-donor,pi-pi,and pi-H.N-MB(30 μmol/L)significantly inhibited CaV1.2 currents,with an inhibition rate of 76.09％±7.41％.The fluorescence intensity of intracellular Ca2+level in H9c2 cardiomyocytes was significantly enhanced with N-MB treatment(3,30 μmol/L,P＜0.01).Compared with the control group,differences in the expression of Cacna1c,Serca2a,and Ncx1 in H9c2 cardiomyocytes were not significant after N-MB(3,30 μmol/L)intervention(P＞0.05),whereas the expression of Cacnb2 significantly reduced(P＜0.001)and the expression of Ryr2 significantly increased(P＜0.05).Conclusion N-MB binds to CaV1.2 calcium channels.N-MB may regulate intracellular calcium homeostasis by inhibiting calcium currents by decreasing the gene expression of CaV1.2 calcium channels.Additionally,N-MB may also increase intracellular Ca2+concentration by promoting the expression of Ryr2,which could be the mechanism underlying the myocardial protective effect of N-MB.

Objective To investigate the binding interaction between the calmodulin(CaM)mutant D130V and the IQ motif of the car-diac Cav1.2 channel.Methods The binding of mutant CaM-D130V to the IQ motif was predicted by fold recognition modeling,homology modeling,and protein docking.The plasmid was transformed into Escherichia coli BL-21 sensory cells via heat shock at 42 ℃ to induce the expression of glutathione S-transferase(GST)fusion protein.The protein was extracted by ultrasonic fragmentation and purified using GS-4B beads.PreScission protease was applied to remove the GST.SDS-PAGE was performed to detect the purity of protein.A GST pull-down assay was conducted to detect the interaction between CaM-D130V and IQ motif.Results Protein docking results showed that both CaM-WT and CaM-D130V could bind to the IQ motif of the cardiac Cav1.2 channel,but the binding sites of the mutant CaM-D130V to the IQ motif were reduced,and its binding conformation was changed compared with the CaM-WT,with decreased binding energy(|S|reduced from 48.086 6 kcal/mol to 47.309 5 kcal/mol).The GST pull-down assay indicated that the binding of CaM-D130V to IQ motif significantly decreased(P＜0.01),and the affinity was significantly reduced at 2 mmol/L Ca2+concentration compared with CaM-WT.Conclusion The reduced binding ability of CaM-D130V to the IQ motif of the cardiac Cav1.2 channel may contribute to functional alterations in the channel.These findings provide a theoretical basis for understanding the pathogenesis of CaM mutant-associated cardio-vascular diseases as well as targeted therapies.

Objective:To explore the molecular mechanism by which interferon regulatory factor 1 (IRF1) affects hepatic ischemia-reperfusion injury (HIRI) by regulating the polarization of Kupffer cells.Methods:Twelve male healthy C57BL/6 wild-type mice weighing 20-25 g and aged 6-8 weeks were divided into a sham operation group ( n=6) and a HIRI group ( n=6); Twelve male healthy C57BL/6 IRF1 gene knockout (IRF1 -/-) mice weighing 20-25 g and aged 6-8 weeks were divided into a sham operation IRF1 -/- group ( n=6) and a HIRI IRF1 -/- group ( n=6). The levels of serum alanine transaminase (ALT) and aspartate transaminase (AST) in mice were measured, and hematoxylin-eosin (HE) staining of liver tissues was performed for Suzuki scoring to evaluate liver injury. Fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to evaluate the mRNA levels of IRF1 and tumor necrosis factor α (TNFα) in liver tissues. Flow cytometry and qRT-PCR were used to detect the proportion and functional changes of M1/M2-type Kupffer cells in liver tissues. IRF1 was overexpressed or knocked down in the mononuclear macrophage cell line ANA1, and a co-culture and hypoxia-reoxygenation system with the hepatocyte cell line AML12 was established. Flow cytometry was used to detect the apoptosis of AML12 cells. Results:At 12 hours after hepatic ischemia-reperfusion in wild-type mice, the liver tissue injury was the most severe. Compared with the sham operation group, the levels of serum ALT [(8 073±83) U/L vs. (81±19) U/L, q=13.59] and AST [(11 170±2 890) U/L vs. (412±210) U/L, q=13.77] in the HIRI group were significantly higher, and the differences were statistically significant (both P<0.001). The Suzuki score reached 5-6 points. At 12 hours after hepatic ischemia-reperfusion in IRF1 gene knockout mice, the liver tissue injury was not obvious. There were no significant differences in the levels of serum ALT [668 (514, 2 344) U/L vs. 254 (147, 285) U/L, q=2.52, P=0.348] and AST [1 936 (1 262, 2 003) U/L vs. 628 (423, 759) U/L, q=1.22, P=0.824] between the HIRI IRF1 -/- group and the sham operation IRF1 -/- group. Compared with the HIRI group, the ratio of M1/M2-type Kupffer cells in the liver of the HIRI IRF1 -/- group decreased [(0.958±0.090) vs. (2.788±0.258), q=2.06, P<0.0001], and the mRNA expression of TNFα decreased [(4.363±0.393) vs. (12.900±5.504), q=5.59, P=0.018], and the differences between the two groups were statistically significant. In the co-culture and hypoxia-reoxygenation experiment using ANA1 cells overexpressing IRF1 and AML12 cells, the proportion of AML12 hepatocytes in late apoptosis was higher than that in the control group [(14.05±4.25) vs. (3.15±1.16), t=2.85, P=0.047], and the difference was statistically significant. In contrast, when the expression of IRF1 was knocked down, the proportion of apoptotic AML12 cells decreased [(9.26±3.04) vs. (13.36±4.64), t=2.15, P=0.098], but the difference was not statistically significant. Conclusion:The IRF1 protein can regulate the polarization of Kupffer cells into M1-type macrophages, promote the inflammatory injury of the liver tissue after ischemia-reperfusion, and increase the apoptosis of hepatocytes.

Objective:The impact of bystander CD8 + T cells (CD8 + Tbys) within the tumor microenvironment on the prognosis of early-stage non-small cell lung cancer (NSCLC) remains unclear, particularly concerning their infiltration differences at the invasive margin (IM) and tumor center (TC). Methods:We retrospectively collected postoperative specimens from 173 patients with primary early-stage NSCLC who underwent radical surgery at the Affiliated Tumor Hospital of Shandong First Medical University from 2014 to 2018. Tissue microarrays encompassing IM and TC regions were prepared and subjected to multicolor immunofluorescence staining (CK/CD8/CD103/DAPI). Image processing and phenotype recognition (CD8 + T cells, CK -CD8 +; CD8 + T bys, CK -CD8 +CD103 -) were performed using inFrom software, and automatic quantitative cell density analysis was conducted using R language. Differences in CD8 + T and CD8 + T bys densities at IM and TC were analyzed using the Mann-Whitney U test. The relationship between CD8 + T, CD8 + T bys and clinicopathological features was examined using the Kruskal-Wallis H test. The impact of CD8 + T and CD8 + T bys on recurrence-free survival (RFS) was evaluated using Kaplan-Meier, log-rank, and Cox proportional hazards models. Results:A total of 173 patients with stage ⅠA-ⅡA NSCLC were included, with a recurrence rate of 26.6% (46/173) and a median RFS of 62.3 months (range: 44.7-71.9 months). CD8 + T and CD8 + T bys densities (1/1 000) were significantly higher in the IM region than in the TC region [70(32, 155) vs. 37(18, 88), P＜0.001; 25(11, 46) vs. 18(7, 34), P=0.002]. No significant association was found between CD8 + T, CD8 + T bys and age, gender, smoking history, histological type, or pathological stage (all P＞0.05). Patients with low-density IM CD8 + T cells had lower RFS compared to those with high-density IM CD8 + T cells ( P=0.021; median RFS not reached), Further analysis revealed that patients with low-density IM CD8 + T bys cell had lower RFS compared to those with high-density IM CD8 + T bys ( P=0.047; median RFS not reached), and low-density IM CD8 + T cell was an independent risk factor for postoperative recurrence ( HR=1.836, 95% CI:1.007-3.347, P=0.048). Joint analysis of IM and TC revealed that patients with low IM CD8 + T bys and high TC CD8 + T bys had significantly lower RFS compared to the other three groups ( P=0.006), and this combination was an independent risk factor for postoperative recurrence in early-stage NSCLC ( HR=2.090, 95% CI:1.162-3.760, P=0.014). Conclusions:The spatial distribution of bystander CD8 + T cells within the primary tumor influences the prognosis of patients with early-stage NSCLC. Patients with low-density IM CD8 + T bys and high-density TC CD8 + T bys are more prone to recurrence after radical surgery.

Objective To construct a Ca2+/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ)long-fragment fusion protein plasmid;investigate the expression,extraction,and purification of CaMK Ⅱ;and identify its binding to the Cav1.2 channel.Methods The extracted pGEX-6p-1/CaMK Ⅱ long-fragment plasmid was transformed into Escherichia coli BL21 receptor cells and cultured in a shaking incubator for 12 h.Isopropyl β-D-thiogalactoside was added to promote GST fusion protein expression.Next,the GST-CaMK Ⅱ long frag-ment was isolated and purified with GS-4B using dithiothreitol(DTT)combined with ultrasonic crushing.After treatment with the PreScis-sion protease,the GST label was removed to obtain the CaMK Ⅱ long-fragment protein.The molecular weight and relative purity of the CaMKⅡ long-fragment protein were determined using 15％sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).The concentration of the purified protein was determined using the Bradford method.The binding ability of the CaMK Ⅱ long-fragment pro-tein to the Cav1.2 channel protein was evaluated using the pull-down method combined with Western blotting.Results The sequencing results showed that the CaMK Ⅱ long fragment was successfully constructed.A CaMK Ⅱ long-fragment protein with high purity and con-centration was obtained using DTT combined with ultrasonic crushing.This protein can bind to the CT1 protein of cardiac Cav1.2 calcium channel.Conclusion In this study,we successfully constructed a CaMKⅡ long-fragment plasmid.The CaMKⅡ long-fragment protein was extracted and purified,and was determined to bind to Cav1.2 channel proteins and exhibit biological activity.Collectively,this study provides a basis for further study of the function of CaMK Ⅱ.

Objective:To investigate the effect of interaction between body mass index (BMI) and gender on the survival of advanced gastric cancer after immunotherapy.Methods:The retrospective cohort study was conducted. The clinicopathological data of 317 patients with advanced gastric cancer who were admitted to the Nanfang Hospital, Southern Medical University from November 2019 to October 2023 were collected. There were 205 males and 112 females, aged 56 (range, 21-79)years. All 317 patients were divided into three groups based on BMI of patients, including 58 cases with BMI <18.5 kg/m2 were classified as the low body mass group, 183 cases with BMI 18.5-24.0 kg/m2 were classified as the normal body mass group, and 76 cases with BMI >24.0 kg/m2 were classified as the overweight or obese group. Patients included in the study were treated with a programmed death-ligand 1 (PD-L1) based immunotherapy regimen for 3 cycles based on their specific conditions, and further decision was made whether to undergo radical surgery or continue comprehensive treatment after evaluating the efficacy. Observation indicators: (1) clinicopathological characteristics of patients; (2) follow-up and mortality status; (3) analysis of factors affecting survival of patients with advanced gastric cancer after immunotherapy. Comparison of measurement data with normal distribution among groups was conducted using the ANOVA. Comparison of measurement data with skewed distribution among groups was conducted using the Kruskal-Wallis H test. Comparison of count data among groups was conducted using the chi-square test. The Cox proportional hazard model was used for univariate and multivariate analyses. Nonlinear trend was analyzed using the restricted cubic spline (RCS) curve, and trend and correction graphs were created using the rcssci package (v1.0). Results:(1) Clinicopathological characteristics of patients. There was no significant difference in gender, age, Borrmann classification, Lauren classification, combined positive score of PD-L1, expression of human epidermal growth factor receptor 2, Epstein-Barr virus infection, carcino-embryonic antigen, CA19-9, CA72-4, alpha-fetoprotein, conversion surgery among the 3 groups of patients ( P>0.05), and there was a significant difference in mismatch repair combined with micro-satellite stability among the 3 groups of patients ( P<0.05). (2) Follow-up and mortality status. Of the 317 patients, 316 cases completed follow-up and 1 case in the overweight or obese group was lost to follow-up. The follow-up time of the 316 cases was 13.8(range, 0.9-48.2)months. During the follow-up, the number of death in the low body mass group, normal body mass group and overweight or obese group were 27, 70 and 31, respectively. (3) Analysis of factors affecting survival of patients with advanced gastric cancer after immunotherapy. Results of multivariate analysis showed that gender and BMI were independent factors affecting survival of patients with advanced gastric cancer after immunotherapy ( hazard ratio=0.066, 0.922, 95% confidence interval as 0.005-0.846, 0.855-0.994, P<0.05). Results of further analysis showed that the interaction between BMI and gender was an independent factor affecting survival of patients with advanced gastric cancer after immuno-therapy ( hazard ratio=1.152, 95% confidence interval as 1.024-1.296, P<0.05). Results of Cox regre-ssion analysis based on different gender showed that took patients of the normal body mass group as a reference, the male patients of the low body mass group had a significantly increased risk of death, showing a significant statistically difference ( hazard ratio=1.809, 95% confidence interval as 1.037-3.155, P<0.05). Results of RCS curve analysis showed that there was a non-linear correlation between BMI and survival of patients with advanced gastric cancer after immunotherapy ( P<0.05). Results of corrected RCS curve analysis using the rcssci package showed that there was a U-shaped relationship between BMI and survival of patients with advanced gastric cancer after immuno-therapy ( P<0.05), with the optimal cut-off value of BMI as 22.2 kg/m 2. Results of RCS curve analysis based on different gender showed that there was a U-shaped relationship between BMI and survival of male patients with advanced gastric cancer after immunotherapy ( P<0.05), with the optimal cut-off value of BMI as 22.7 kg/m 2. Conclusions:Gender, BMI and the interaction between BMI and gender are independent factors affecting survival of patients with advanced gastric cancer after immuno-therapy. There is a U-shaped relationship between BMI and survival of patients, with the optimal cut-off value of BMI as 22.2 kg/m 2, and there is a U-shaped relationship between BMI and survival of male patients, with the optimal cut-off value of BMI as 22.7 kg/m 2.

To assess the cross-contamination risk and genetic evolution ofStaphylococcus aureus(S.aureus)at various stages in a swine slaughterhouse,and to provide a reference for risk evalua-tion and control measures of S.aureus contamination during swine slaughtering,we conducted whole-genome sequencing on 31 isolates of S.aureus collected and preserved from the slaughter-house.Bioinformatics analyses were performed to investigate the genomic characteristics and genet-ic relationships of these isolates.Results revealed cross-contamination across different slaughter-house stages,predominantly with ST398-t1451 strains.Additionally,highly virulent ST9-t899 strains and ST398-t11 strains with numerous resistance genes were detected at various stages.The strains predominantly carried virulence genes such as hlgA,hlgB,and hlgC,with varying num-bers of resistance genes.Notably,two strains carried the optrA gene and three strains carried the cfr gene;the presence of the optrA gene is relatively rare among S.aureus.These genes confer re-sistance to novel synthetic antibiotics such as oxazolidinones and florfenicol and have the potential for horizontal gene transfer,increasing the risk of dissemination both within and beyond the slaughterhouse.Importantly,the study also detected a LA-MRSA-ST9 strain in water samples from the slaughterhouse,which could potentially infect humans.This strain exhibits zoonotic char-acteristics,highlighting the need for stricter protective measures for slaughterhouse workers to mitigate occupational exposure and infection risks.