This study aims to construct a recombinant vaccine containing the tetanus toxin C frag-ment(HC)with a built-in adjuvant of metallothionein 3(MT-3)and evaluate its immune effect in mice.The gene sequences of MT-3 and HC were fused via a linker to create the pET30a-MT-3-HC recombinant plasmid,which was then transformed into E.coli BL21(DE3)competent cells.The re-combinant plasmid was confirmed through double enzyme digestion.The M3C recombinant protein expression was induced,identified by SDS-PAGE and Western blot,and purified using Ni-NTA af-finity chromatography.Five groups of vaccines,including PBS,HC,TT(tetanus toxoid),M3C,and M3C+CpG(composite adjuvant),were administered to mice via intramuscular injection at 7-day intervals for three immunizations.Blood samples were periodically collected from the tail vein.ELISA was used to measure changes in specific IgG antibody titers in the serum,and on day 28,antibody subtypes(IgG1,IgG2a,IgG2b,IgG3,and IgM)and cytokine levels(IL-4,IFN-γ)were also measured.The results demonstrated that the pET30a-MT-3-HC recombinant plasmid was cor-rectly constructed,and the M3C recombinant protein was highly expressed in the supernatant fol-lowing ultrasonic disruption of the induced bacterial culture,with a single band observed post-puri-fication.ELISA results showed that the titer of specific IgG antibodies in the M3C+CpG group peaked at 3.54×105 14 days after the third immunization,which was 141 times,70 times,and 6.6 times higher than that in HC,TT,and TT groups,respectively.Antibody subtype analysis revealed significantly higher specific antibody subtype titers in the M3C and M3C+CpG groups compared to the PBS,HC,and TT groups(P＜0.05),with the M3 C group showing an IgG1/IgG2a ratio greater than 1,and the M3C+CpG group having an IgG1/IgG2a ratio of approximately 1.Serum concentrations of IFN-γ and IL-4 in the M3C and M3C+CpG groups were also significantly higher than those in the other experimental groups(P＜0.05).These results showed that the recombinant antigen containing MT-3 built-in adjuvant and tetanus toxin C fragment was successfully expressed and showed strong immunogenicity,which laid the experimental foundation for the development of this recombinant vaccine.

Objective The study explored the psychological help-seeking experiences of adolescents with mood disorder,so as to provide references for healthcare professionals to carry out relevant interventions to facilitate adolescents'early access to mental health services.Methods A qualitative research was adopted,and 14 adolescents with mood disorder admitted to a tertiary hospital in Lanzhou City from December 2023 to March 2024 were interviewed by semi-structured interviews.The data was analyzed by Colaizzi seven-step method.Results 3 themes were extracted,including long and tortuous help-seeking journey(maladaptive coping,continuous exploration of informal help-seeking behavior,formal help-seeking for crisis-triggered or needs-driven),the psychological experience intertwining struggles and expectations(disappointment at not being taken seriously,helplessness at not being understood,expectation of more understanding and support),barriers to help-seeking(lack of knowledge,difficulties with self-disclosure,lack of trust,negative attitudes encountered when seeking informal help,negative experiences during formal help-seeking).Conclusion Adolescents with mood disorder experienced a long and tortuous journey while seeking help,faced with numerous barriers along the process,and desired to be understood and supported.Healthcare professionals should collaborate with families,schools,and other resources,to improve adolescents'mental health literacy and establish a robust social support system,so as to provide reliable support for adolescents with mood disorder in accessing professional mental health services.

Background Irritable bowel syndrome with diarrhea(IBS-D)is a common gastrointestinal dysfunction disease,but currently,single Western medicine treatments have significant side effects and are prone to recurrence.Probiotics and traditional Chinese medicine have certain therapeutic effects on IBS-D,but the exact treatment mechanism is still unclear,and there is limited research on the combination of the two.Objective To investigate the effects of Chongkang Capsules combined with Bifidobacterium on gut microbiota,mucosal barrier function,and neurotransmitter levels in patients with IBS-D and comorbid anxiety and depression,and to analyze its therapeutic mechanisms.Methods A total of 150 patients with irritable bowel syndrome with diarrhea(IBS-D)and anxiety and depression who were treated in the gastroenterology department of our hospital from January 2021 to June 2022,were selected as the study subjects.A randomized controlled trial was designed with three treatment groups:50 patients in the observation group received Chongkang Capsules combined with Bifidobacterium for 4 weeks;50 patients in the positive control group received Rifaximin combined with Bifidobacterium for 4 weeks;and 50 patients in the blank control group received Bifidobacterium alone for 4 weeks(to exclude interference from the bacterial species in the Bifidobacterium capsules in intestinal flora detection).The changes in IBS-D symptom severity,stool consistency,anxiety and depression scores(HAM-A/HAM-D),intestinal flora(lactobacilli,Bifidobacterium,bacteroides,enterobacteria),intestinal barrier markers(serum D-lactic acid,DAO),inflammatory factors(IL-6,IL-8,TNF-α),intestinal tight junction proteins(Occludin,ZO-1),neurotransmitters(5-HT),and recurrence rates were compared among the three groups before and after treatment.Results Symptom improvement:the improvement in IBS-SSS scores,stool classification,and HAM-A/HAM-D scores in the observation and positive control groups was significantly better than that in the blank control group(all P<0.001),but there was no significant difference between the two groups(P>0.05).Flora regulation:after treatment,the observation and positive control groups showed a significant increase in beneficial bacteria(lactobacilli and Bifidobacterium)and a significant decrease in pathogenic bacteria(bacteroides and enterobacteria)compared to the blank control group(all P<0.001).The improvement in flora persisted up to 6 months after treatment(P<0.001).Barrier and inflammation:the reduction in serum D-lactic acid,DAO,and IL-6/IL-8/TNF-α levels was significantly greater in the observation and positive control groups than in the blank control group(all P<0.001).The expression of intestinal barrier proteins(Occludin and ZO-1)was significantly upregulated(P<0.001).Neurotransmitters:the increase in serum 5-HT levels were significantly higher in the two groups than in the blank control group(P<0.001).Recurrence rate:the 3-month recurrence rates in the observation group(8.0%)and positive control group(10.0%)were significantly lower than that in the blank control group(30.0%).The 6-month recurrence rates(14.0%vs.16.0%vs.44.0%)also showed significant differences(all P<0.05).Conclusion Chongkang Capsules combined with Bifidobacterium can effectively alleviate clinical symptoms in patients with IBS-D and comorbid anxiety and depression.The mechanism may be related to regulating intestinal flora balance,repairing the intestinal mucosal barrier,suppressing inflammatory responses,and increasing 5-HT levels.Additionally,the low recurrence rate suggests its clinical application value.

Objective:To investigate the incidence of shoulder work-related musculoskeletal disorders (WMSDs) among occupational population in China, and to explore their intrinsic association with personal and work-related factors.Methods:In April 2024, 73497 valid questionnaires of the Chinese version of the Musculoskeletal Disorders Electronic Questionnaire were retrospectively analyzed from June 2018 to December 2023 in 22 provinces and 29 key industries in China, and the general information, occurrence of WMSDs and related risk factors of key occupational populations in different regions in China were collected. By using Chi-square test and confirmatory factor analysis, the relationship between shoulder fatigue and pain in key occupational groups and individual factors, work type, work posture and work organization was discussed, and the internal relationship was analyzed based on structural equation model.Results:Higher incidence of shoulder fatigue and pain were associated with female, lack of physical exercise, uncomfortable working posture and neck leaning forward ( P<0.05). Structural equation model analysis showed that work type, work posture and work organization were strongly correlated ( r=0.58, 0.55). Work organization and work type were strongly correlated with shoulder fatigue ( r=0.65) and moderately correlated with shoulder fatigue ( r=0.21). Shoulder fatigue was moderately associated with shoulder pain ( r=0.40). Individual factors, work type, work posture and shoulder fatigue could directly affect shoulder pain ( OR=0.07, -0.09, 0.17 and 0.40), and work type and work posture could also indirectly affect shoulder pain through shoulder fatigue ( OR=0.08, 0.03). Work organization only indirectly affected shoulder pain through shoulder fatigue ( OR=0.26) . Conclusion:The main influencing factor of shoulder pain is shoulder fatigue, followed by work posture and individual factors. Structural equation model can better reflect the complex relationship between work type, work posture and work organization and shoulder WMSDs. Improving work posture and work organization may be an effective way to control the influence of shoulder fatigue on shoulder pain.

Objective:To explore the structural equation model to explore the levels of work-related musculoskeletal disorders (WMSDs) and various risk factors in the feet and ankle of China's occupational population, providing scientific basis for for preventing WMSDs in feet and ankles.Methods:Data of 73497 national occupational epidemiological cases were selected from June 2018 to December 2023 used the Chinese version of the Electronic Questionnaire on Musculoskeletal Disorders. The adverse ergonomic factors and their source classification standard and confirmatory factor analysis were used to investigate foot and ankle WMSDs and their related risk factors (including individual factors, work organization, work posture, work type, fatigue, etc.) in key occupational groups in China, and structural equation model hypothesis, fitting, verification, and path and intermediary effect analysis were carried out. The model fit evaluation indexes included Chi-square specific degrees of freedom ( χ2/ df), gauge fit index (NFI), Tucker Lewis index (TLI), goodness of Fit index (GFI), adjusted Goodness of Fit index (AGFI) and approximate root mean square error (RMSEA) . Results:A total of 73497 occupational workers were surveyed, with local muscle fatigue and WMSDs incidence rates in the feet and ankles being 17.17% and 12.06%, respectively. The fitting index of the adjusted structural equation model basically meets the standard (GFI=1, AGFI=1, RMESA=0.042, NFI=0.716, TLI=0.663). The top three factors affecting feet and ankle WMSDs are feet and ankle muscle fatigue, work type, and work organization, with standardized path coefficients of 0.221, 0.105, and 0.095, respectively. The top two factors affecting feet and ankle muscle fatigue are work organization and work type, with standardized path coefficients of 0.548 and 0.383, respectively. Feet and ankle muscle fatigue, work type, work organization, and work posture have a direct effect on feet and ankle WMSDs, with effect values of 0.221, 0.105, 0.095, and 0.077, respectively. The organization and type of work can also have indirect effects through feet and ankle muscle fatigue, with effect values of 0.121 and 0.084, respectively.Conclusion:Feet and ankle muscle fatigue has a direct impact on WMSDs, and plays a mediating role between ankle and ankle WMSDs caused by work organization and work type. Feet and ankle muscle fatigue is an important pathway leading to feet and ankle WMSDs. It is recommended that employers and managers detect job fatigue early and take corresponding prevention and intervention measures, which can play a key role in preventing feet and ankle WMSDs.

Objective:To explore the structural relationship between WMSDs in the upper limbs and various risk factors in the occupational population in China, based on a large sample epidemiological survey and structural equation analysis, and to establish a structural equation model, so as to lay a foundation for the prevention and control of such diseases.Methods:The Chinese version of the Musculoskeletal Disorders Electronic Questionnaire was used to conduct a nationwide survey on the prevalence of WMSDs in the upper extremity. Six factors related to WMSDs in the upper extremity were extracted by the classification standard of adverse ergonomic factors and their source and confirmatory factor analysis, including work organization, work type, upper extremity work posture, individual factors, upper extremity fatigue and upper extremity WMSDs. The structural equation analysis was carried out and the structural equation model was established.Results:The incidence of WMSDs and fatigue in the upper limbs was 24.44% and 43.76%, respectively. The adjusted structural equation model fitting indicators were generally up to the standard (GFI=1.000, AGFI=1.000, RMSEA=0.043, NFI=0.808, TLI=0.784) . The four exogenous latent variables of work organization, work type, upper limb work posture and individual factors were correlated. There was a strong positive correlation between job type and upper limb work posture ( r=0.865) , a moderate positive correlation between work organization and job type and upper limb work posture ( r=0.570, 0.490) , and a weak negative correlation between individual factors and the other three exogenous latent variables. Upper limb work posture and individual factors had direct effects on upper limb WMSDs, and the effect coefficients were 0.10 and 0.06, respectively. Upper limb fatigue played a mediating role between work organization, work type, upper limb work posture and upper limb WMSDs. The effect coefficient was 0.46, and the composition ratios of indirect effects were 100.0%, 100.0%, and 38.3%, respectively. The direct path effect of upper limb work posture, individual factors and upper limb WMSDs was weaker than the mediating path through upper limb fatigue. Conclusion:When carrying out the prevention and control of upper limbWMSDs, it is necessary to comprehensively consider the pathogenesis path of upper limb muscle fatigue and upper limb WMSDs caused by work organization, work type, and upper limb work posture, so as to provide theoretical reference for improving the prevention and control level of such diseases.

Background Irritable bowel syndrome with diarrhea(IBS-D)is a common gastrointestinal dysfunction disease,but currently,single Western medicine treatments have significant side effects and are prone to recurrence.Probiotics and traditional Chinese medicine have certain therapeutic effects on IBS-D,but the exact treatment mechanism is still unclear,and there is limited research on the combination of the two.Objective To investigate the effects of Chongkang Capsules combined with Bifidobacterium on gut microbiota,mucosal barrier function,and neurotransmitter levels in patients with IBS-D and comorbid anxiety and depression,and to analyze its therapeutic mechanisms.Methods A total of 150 patients with irritable bowel syndrome with diarrhea(IBS-D)and anxiety and depression who were treated in the gastroenterology department of our hospital from January 2021 to June 2022,were selected as the study subjects.A randomized controlled trial was designed with three treatment groups:50 patients in the observation group received Chongkang Capsules combined with Bifidobacterium for 4 weeks;50 patients in the positive control group received Rifaximin combined with Bifidobacterium for 4 weeks;and 50 patients in the blank control group received Bifidobacterium alone for 4 weeks(to exclude interference from the bacterial species in the Bifidobacterium capsules in intestinal flora detection).The changes in IBS-D symptom severity,stool consistency,anxiety and depression scores(HAM-A/HAM-D),intestinal flora(lactobacilli,Bifidobacterium,bacteroides,enterobacteria),intestinal barrier markers(serum D-lactic acid,DAO),inflammatory factors(IL-6,IL-8,TNF-α),intestinal tight junction proteins(Occludin,ZO-1),neurotransmitters(5-HT),and recurrence rates were compared among the three groups before and after treatment.Results Symptom improvement:the improvement in IBS-SSS scores,stool classification,and HAM-A/HAM-D scores in the observation and positive control groups was significantly better than that in the blank control group(all P<0.001),but there was no significant difference between the two groups(P>0.05).Flora regulation:after treatment,the observation and positive control groups showed a significant increase in beneficial bacteria(lactobacilli and Bifidobacterium)and a significant decrease in pathogenic bacteria(bacteroides and enterobacteria)compared to the blank control group(all P<0.001).The improvement in flora persisted up to 6 months after treatment(P<0.001).Barrier and inflammation:the reduction in serum D-lactic acid,DAO,and IL-6/IL-8/TNF-α levels was significantly greater in the observation and positive control groups than in the blank control group(all P<0.001).The expression of intestinal barrier proteins(Occludin and ZO-1)was significantly upregulated(P<0.001).Neurotransmitters:the increase in serum 5-HT levels were significantly higher in the two groups than in the blank control group(P<0.001).Recurrence rate:the 3-month recurrence rates in the observation group(8.0%)and positive control group(10.0%)were significantly lower than that in the blank control group(30.0%).The 6-month recurrence rates(14.0%vs.16.0%vs.44.0%)also showed significant differences(all P<0.05).Conclusion Chongkang Capsules combined with Bifidobacterium can effectively alleviate clinical symptoms in patients with IBS-D and comorbid anxiety and depression.The mechanism may be related to regulating intestinal flora balance,repairing the intestinal mucosal barrier,suppressing inflammatory responses,and increasing 5-HT levels.Additionally,the low recurrence rate suggests its clinical application value.

Objective To explore the effects of hypoxia on spermatid differentiation and stability of sperm flagellar microtubule,and investigate the underlying molecular mechanisms in order to clarify the potential adverse effects of hypoxia on male reproductive function.Methods Forty-eight 8-week-old healthy male SD rats(weighing 300～399 g)were subjected in this study.The experiments included ① an oxygen concentration gradient experiment(n=6):21％oxygen was regarded as normoxia(control),and 13.5％,11.8％,and 10.4％ oxygen were used to simulate hypoxic environments at altitudes of 3 500,4 500 and 5 500 m,respectively,for a continuous exposure of 2 months;② a time gradient experiment(n=6):the rats were exposed to 10.4％ oxygen for 0,0.5,1,and 2 months,respectively.Flow cytometry was employed to isolate round spermatids,and the following methods were employed to measure relevant indicators:① RNA sequencing to analyze gene expression profile changes related to impaired spermatogenesis and abnormal flagellar structure under hypoxic stress;②Western blotting to detect the expression levels of key proteins CEP290,RING 1A,and H2AK119ub;③ fluorescence recovery after photobleaching(FRAP)to monitor microtubule assembly dynamics and assess the immediate impact of hypoxia on microtubule stability.Results In the oxygen concentration gradient experiment,after 2 months of exposure to 10.4％ oxygen,the proportions of spermatogonia,secondary spermatocytes,and round spermatids in rat seminiferous tubules were significantly increased(P＜0.05),reaching 1.33±0.04,1.06±0.01 and 1.60±0.02 times higher,respectively than that of the 21％ normoxia group.Conversely,the proportions of primary spermatocytes and elongated spermatids were obviously decreased(P＜0.05),taking 0.89±0.01 and 0.88±0.000 2 times respectively when compared with that of the 21％ normoxia group,in a oxygen concentration-depended manner.In the time gradient experiment,after 0.5 months of exposure to 10.4％oxygen,the proportions of spermatogonia,secondary spermatocytes,and round spermatids began to increase(P＜0.05),reaching 1.11±0.03,1.04±0.01 and 1.29±0.003 times higher,respectively than that of the 0-month control group.The proportions of primary spermatocytes and elongated spermatids started to significantly decrease(P＜0.05)after 1 month of exposure,only 0.94±0.03 and 0.95±0.008 times,respectively than that of the 0-month control group.After 2 months of exposure to 10.4％ oxygen,the rate of sperm tail abnormalities in the epididymis of rats was significantly increased(P＜0.05),rising from(12.1±1.7)％ in the 21％ normoxia group to(30.8±3.7)％.In G2 spermatocytes exposed to 1％ hypoxia for 24 h,FRAP revealed a decrease in microtubule assembly rate and enhanced microtubule dynamic instability,with the maximum fluorescence recovery value decreasing from 0.37±0.02 in the normoxia group to 0.29±0.01.The results of RNA sequencing showed that under hypoxic condition,the transcription level of the key cilium basal body molecule CEP290 was increased,with an upregulation of 1.81±0.11 times than that of the 21％ normoxia group.In contrast,the expression levels of PRC1 complex members RING 1A,RING 1B,CBX2,PHC1,and PCGF1 were decreased,to 0.74±0.02,0.73±0.01,0.78±0.02,0.71±0.01 and 0.86±0.03 times of that of the 21％ normoxia group,respectively.Western blotting indicated that the protein level of CEP290 was up-regulated in the hypoxia group,while that of RING 1A was down-regulated.ChIP-qPCR experiments showed that the binding of RING 1A and its product H2AK119ub to the CEP290 promoter were significantly decreased(P＜0.000 1),with binding strengths of 0.38±0.02 and 0.52±0.06 times of that of the 21％ normoxia group,respectively.In siRING 1A-treated G2 cells,the binding of H2AK119ub to the CEP290 promoter was significantly decreased(P＜0.000 1),with a binding strength of 0.74±0.06 times of that of the control group,while CEP290 mRNA level was significantly increased(P＜0.000 1),with an up-regulation of 3.35±0.37 times.Conclusion Hypoxic environment impair sperm flagellar microtubule stability via the RING 1A-H2AK119ub-CEP290 signaling axis,which affects spermatid differentiation and leads to spermatogenic dysfunction.

BACKGROUND: Non-invasive computed tomography angiography (CTA)-based fractional flow reserve (CT-FFR) could become a gatekeeper to invasive coronary angiography. Deep learning (DL)-based CT-FFR has shown promise when compared to invasive FFR. To evaluate the performance of a DL-based CT-FFR technique, DeepVessel FFR (DVFFR). METHODS: This retrospective study was designed for iScheMia Assessment based on a Retrospective, single-center Trial of CT-FFR (SMART). Patients suspected of stable coronary artery disease (CAD) and undergoing both CTA and invasive FFR examinations were consecutively selected from the Beijing Anzhen Hospital between January 1, 2016 to December 30, 2018. FFR obtained during invasive coronary angiography was used as the reference standard. DVFFR was calculated blindly using a DL-based CT-FFR approach that utilized the complete tree structure of the coronary arteries. RESULTS: Three hundred and thirty nine patients (60.5 ±10.0 years and 209 men) and 414 vessels with direct invasive FFR were included in the analysis. At per-vessel level, sensitivity, specificity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) of DVFFR were 94.7%, 88.6%, 90.8%, 82.7%, and 96.7%, respectively. The area under the receiver operating characteristics curve (AUC) was 0.95 for DVFFR and 0.56 for CTA-based assessment with a significant difference (P < 0.0001). At patient level, sensitivity, specificity, accuracy, PPV and NPV of DVFFR were 93.8%, 88.0%, 90.3%, 83.0%, and 95.8%, respectively. The computation for DVFFR was fast with the average time of 22.5 ± 1.9 s. CONCLUSIONS The results demonstrate that DVFFR was able to evaluate lesion hemodynamic significance accurately and effectively with improved diagnostic performance over CTA alone. Coronary artery disease (CAD) is a critical disease in which coronary artery luminal narrowing may result in myocardial ischemia. Early and effective assessment of myocardial ischemia is essential for optimal treatment planning so as to improve the quality of life and reduce medical costs.