Glycosylation modification is an important post-translational modification of proteins, which participates in regulating protein half-life, biological activity, and immunogenicity, thereby affecting their functions. Glycoproteins expressed in Pichia pastoris predominantly carry high-mannose type glycans, primarily composed of mannose residues, which starkly contrasts with the complex-type glycans synthesized by mammalian cells. This study aims to transform the high mannose glycosylation modification of P. pastoris into a hybrid glycosylation modification similar to that of mammalian cells through genetic engineering technology. We introduced the mannosidase Ⅰ gene (MDSⅠ) from Trichoderma viride and the human β-1,2-N-acetylglucosaminyltransferase I gene (GnTⅠ) into a previously constructed P. pastoris strain (∆och1) capable of producing Man₈GlcNAc₂ glycans. To precisely regulate the expression of MDSⅠ and GnTⅠ, we designed various promoter combinations, including the strong inducible AOX promoter and the constitutive GAP promoter. The receptor-binding domain (RBD, residues 377-588) of the spike protein from the Middle East respiratory syndrome coronavirus (MERS-CoV) was selected as the reporter protein for this investigation (MERS-RBD). The N-glycosylation profile of MERS-RBD was systematically analyzed using PNGase F digestion coupled with mass spectrometry. The results showed that after the knockout of och1 and the introduction of MDSⅠ and GnTⅠ genes with different promoter combinations, P. pastoris strains capable of producing GlcNAcMan₅GlcNAc₂ glycans were successfully generated. When the AOX promoter was used to control the MDSⅠ gene and the GAP promoter was used to control the GnTⅠ gene, the engineered strain exhibited the highest proportion of hybrid-type GlcNAcMan₅GlcNAc₂ glycans, which accounted for 68.38% of the total N-glycosylation. In conclusion, we successfully engineered a P. pastoris strain capable of synthesizing hybrid-type GlcNAcMan₅GlcNAc₂ glycans, establishing a foundation for subsequent research on the biosynthesis of complex-type N-glycans in P. pastoris.
Glycosylation ; Glycoproteins/genetics* ; Polysaccharides/metabolism* ; N-Acetylglucosaminyltransferases/metabolism* ; Pichia/metabolism* ; Humans ; Mannosidases/metabolism* ; Genetic Engineering ; Trichoderma/genetics* ; Recombinant Proteins/genetics* ; Saccharomycetales

Objective By analyzing the characteristics of hidden poisoning homicide cases,provide scientific references for grass-roots forensic examination and toxicological screening and provide direction for the efficient discovery of hidden or missed cases of poisoning homicide in non-natural death cases.Methods Poisoning incidents in Hunan Province from 2018 to 2023 were retrospectively analyzed and compiled using Origin and EXCEL software.Results There were a total of 149 poisoning homicide cases,with 15 occurring in urban areas(accounting for 10.1%)and 134 in rural areas(accounting for 89.9%).Out of the 46 cases that led to casualties,rodenticide poisoning was the most prevalent(accounting for 52.2%),followed by cyanide(4.3%),nitrite(4.3%),thallium(2.2%),and other forms of poisoning.These incidents were primarily concentrated in urban areas.The main way of poisoning was to mix poisons into drinking water(accounting for 28.3%),meals(26.1%),and dark or heavy seasoning(8.7%).There was no significant disparity in the gender distribution of suspects,although the number of male victims was notably higher than that of female victims.The suspects were predominantly between the ages of 19 and 45,while the victims were mostly between the ages of 46 and 60.Among the 103 cases that did not result in injury or death,99%occurred in rural areas,with more than 95%of the involved poisons included pungent pesticides or herbicides,while rodenticides accounted for the least(1.9%).Conclusion There are obvious urban-rural differences in the case occurrence areas and types of involved poisons in poisoning homicide cases.Therefore,it is imperative to focus on both urban and rural areas during the investigation and evidence collection process in suspected poisoning cases.

Objective:To summarize the concept, theoretical basis, evaluation tools and mechanisms, influencing factors, and intervention measures of kinesiophobia.Methods:The literature on kinesiophobia in patients undergoing total knee replacement was electronically searched on databases such as China National Knowledge Infrastructure, China Biology Medicine disc, WanFang Data, PubMed, CINAHL, Web of Science, Embase, Scopus, PsycINFO, Cochrane Library. The search period was from database establishment to June 24, 2023. This study extracted and analyzed data from the included literature.Results:A total of 32 articles were included. The Tampa Scale for Kinesiophobia was a widely used tool for evaluating kinesiophobia. The influencing factors of kinesiophobia were demographic and disease factors, body motor function, and psychological and social factors. The intervention measures for kinesiophobia mainly included cognitive behavioral intervention, pain health education, exercise, art video or music intervention, multidisciplinary collaborative intervention, and so on.Conclusions:The concept and theoretical basis of kinesiophobia are not yet complete. It is necessary to revise and improve the theoretical model and assessment tool for kinesiophobia and construct an intervention program for kinesiophobia in combination with the concept of rapid rehabilitation.

Objective:To systematically evaluate the risk factors for perioperative malnutrition in aged fracture patients.Methods:A comprehensive search of databases including CNKI, WanFang, VIP, SinoMed, Web of Science, Embase, PubMed, and CINAHL was conducted to identify relevant studies on risk factors for perioperative malnutrition in aged fracture patients from inception to May 10, 2023. Additional grey literature was identified by snowball sampling and manual searches. Two postgraduate students trained in evidence-based nursing independently screened the literature, extracted data, and assessed the methodological quality of the studies. Meta-analysis was performed using RevMan 5.4 software.Results:Fourteen original studies were included, covering 46 risk factors for malnutrition. Meta-analysis of factors reported in at least two studies indicated that age≥ 70 years, low body mass index, low hemoglobin, preoperative hypoalbuminemia, female, physical inactivity, non-home residence, low self-care ability, intertrochanteric fracture, chronic obstructive pulmonary disease, chronic gastritis, dementia/cognitive decline, cancer, stroke, chronic heart failure, multiple chronic diseases, high Charlson Comorbidity Index, weight loss, depression, American Society of Anesthesiologists classification≥Ⅲ, taking multiple medications≥ 4 types, and longer hospital stays all increased the incidence of perioperative malnutrition in aged fracture patients ( P< 0.05) . Conclusions:The incidence of perioperative malnutrition in the aged fracture population is high and influenced by multiple factors. This highlights the need for healthcare providers to conduct dynamic and precise nutritional screenings for high-risk patients and to implement personalized perioperative nutritional management to prevent malnutrition and promote rapid recovery in aged fracture patients.

Objective To systematically retrieve relevant research on the application of exercise intervention dur-ing hematopoietic stem cell transplantation for hospitalized children at home and abroad,to generalize and summa-rize the content of exercise intervention,the effect of intervention,and safety monitoring,with the purpose of provid-ing reference bases for the clinical application of exercise intervention in the future.Methods In accordance with the scoping review framework,we systematically retrieved databases including Web of Science,PubMed,Scopus,Cochrane Library,Embase,CINAHL,CNKI,VIP Database,Wanfang Data and China Biomedical Literature Database,with a retrieval timeframe from database establishment to May 19,2023.Based on the criteria for inclusion and ex-clusion of literature,we screened,summarized,extracted,and analyzed the literature.Results A total of 17 pieces of literature were included,including 5 randomized controlled trials,9 quasi-experimental studies,and 3 mixed-method studies.There were 4 categories of exercise including aerobic,anti-gravity,resistance,and stretching;most of the ex-ercises were of low to moderate intensity;the exercise interventions had a positive impact on muscle strength,quali-ty of life,cardiorespiratory function,body composition,and hematological indices;the safety monitoring consisted of red blood cell,hemoglobin,and platelet counts,temperature,joint and muscle pains,as well as falls,and syringe shedding.Conclusion The application of exercise intervention is safe and feasible,with a positive effect on chil-dren with hematopoietic stem cell transplantation in muscle strength,quality of life.Medical staff should combine with the clinical situation to formulate a reasonable and unified exercise program and to carry out scientific and standardized exercise management.

Objective:To evaluate the efficacy and safety of hypomethylating agents (HMA) in patients with myelodysplastic syndromes (MDS) .Methods:A total of 409 MDS patients from 45 hospitals in Zhejiang province who received at least four consecutive cycles of HMA monotherapy as initial therapy were enrolled to evaluate the efficacy and safety of HMA. Mann-Whitney U or Chi-square tests were used to compare the differences in the clinical data. Logistic regression and Cox regression were used to analyze the factors affecting efficacy and survival. Kaplan-Meier was used for survival analysis. Results:Patients received HMA treatment for a median of 6 cycles (range, 4-25 cycles) . The complete remission (CR) rate was 33.98% and the overall response rate (ORR) was 77.02%. Multivariate analysis revealed that complex karyotype ( P=0.02, OR=0.39, 95% CI 0.18-0.84) was an independent favorable factor for CR rate. TP53 mutation ( P=0.02, OR=0.22, 95% CI 0.06-0.77) was a predictive factor for a higher ORR. The median OS for the HMA-treated patients was 25.67 (95% CI 21.14-30.19) months. HMA response ( P=0.036, HR=0.47, 95% CI 0.23-0.95) was an independent favorable prognostic factor, whereas complex karyotype ( P=0.024, HR=2.14, 95% CI 1.10-4.15) , leukemia transformation ( P<0.001, HR=2.839, 95% CI 1.64-4.92) , and TP53 mutation ( P=0.012, HR=2.19, 95% CI 1.19-4.07) were independent adverse prognostic factors. There was no significant difference in efficacy and survival between the reduced and standard doses of HMA. The CR rate and ORR of MDS patients treated with decitabine and azacitidine were not significantly different. The median OS of patients treated with decitabine was longer compared with that of patients treated with azacitidine (29.53 months vs 20.17 months, P=0.007) . The incidence of bone marrow suppression and pneumonia in the decitabine group was higher compared with that in the azacitidine group. Conclusion:Continuous and regular use of appropriate doses of hypomethylating agents may benefit MDS patients to the greatest extent if it is tolerated.

Objective:To investigate the current situation of medical equipment allocation in 103 tertiary public hospitals,analyze and compare the proportion of medical equipment in different regional hospitals in our country,and provide basis for scientific and reasonable allocation of medical equipment.Methods:A questionnaire was designed by literature research and expert consultation,and an electronic questionnaire was released through the public account of"China Medical Equipment"and the platform of"Good Medical Workers"to analyze the correlation between the number of medical equipment configuration,the proportion of domestic products,the region where the hospital was located and the number of hospital beds.Results:A total of 103 valid questionnaires were collected,involving 103 tertiary public hospitals in East China,North China,Central China,South China,Southwest,Northwest and Northeast China,of which 18%were in North China,37%were in East China and 9%were in South China.In North China,South China and East China,the average number of medical equipment in the tertiary public hospitals in the region was 9785 units per hospital,9245 units per hospital and 8153 units per hospital,and the number of the expensive equipment was relatively small,which had no correlation with the number of beds.There were statistically significant differences between North China and East China in the number of beds,the number of pieces of equipment per 100 beds and the amount of equipment per 100 beds(t=-2.582,2.939,4.653,P＜0.05).The number of life support equipment configuration was significantly positively correlated with the number of beds,and the domestic share,especially the domestic share of life support equipment,increased.Conclusion:The analysis of medical equipment allocation in public hospitals in China can provide data support for hospitals to better adapt to the adjustment of the new expensive medical equipment catalogue,provide reference for the demonstration and promotion of large medical equipment allocation and localization in tertiary hospitals,and provide reference for relevant health management departments.

Objective:The degree of involvement of extraocular muscles varies across different regions of retrobulbar tissue in patients with thyroid eye disease, but the mechanism is unclear. This study aims to explore the relationship between differential expression of thyroid-stimulating hormone receptor(TSHR) in different parts of the extraocular muscles and the varying degrees of muscle involvement.Methods:The medial, lateral, superior, and inferior rectus muscle were separated from the retrobulbar tissue of rats, and the expression level of TSHR in four extraocular muscles was detected by immunofluorescence and qPCR. Extraocular muscle tissue of patients with strabismus was collected to detect the expression of TSHR and the cell types expressed by fluorescence.Results:The results of qPCR showed that the expression of TSHR in the medial rectus muscle was significantly higher than that in the lateral, superior, and inferior rectus muscle(medial rectus vs lateral rectus, P=0.012; medial rectus vs superior rectus, P=0.015; medial rectus vs inferior rectus, P=0.013), but there was no difference in insulin-like growth factor 1(IGF-1R) expression. Immunofluorescence showed that TSHR was co-expressed with PAX7, a molecular marker of muscle satellite cells, and the expression level in the medial rectus muscle of rats and humans was significantly higher than those in the other three extraocular muscles. Conclusion:The high specific expression of TSHR in the satellite cells of the medial rectus muscle may be the reason why the medial rectus muscle is most susceptible to involvement in thyroid eye disease.

Objective To establish a universally applicable logistic risk prediction model for diabetes mellitus type 2(T2DM)in the middle-aged and elderly populations based on the results of a Meta-analysis,and to validate and confirm the efficacy of the model using the follow-up data of medical check-ups of National Basic Public Health Service.Methods Cohort studies evaluating T2DM risks were identified in Chinese and English databases.The logistic model utilized Meta-combined effect values such as the odds ratio(OR)to derive β,the partial regression coefficient,of the logistic model.The Meta-combined incidence rate of T2DM was used to obtain the parameter α of the logistic model.Validation of the predictive performance of the model was conducted with the follow-up data of medical checkups of National Basic Public Health Service.The follow-up data came from a community health center in Chengdu and were collected between 2017 and 2022 from 7 602 individuals who did not have T2DM at their baseline medical checkups done at the community health center.This community health center was located in an urban-rural fringe area with a large population of middle-aged and elderly people.Results A total of 40 cohort studies were included and 10 items covered in the medical checkups of National Basic Public Health Service were identified in the Meta-analysis as statistically significant risk factors for T2DM,including age,central obesity,smoking,physical inactivity,impaired fasting glucose,a reduced level of high-density lipoprotein cholesterol(HDL-C),hypertension,body mass index(BMI),triglyceride glucose(TYG)index,and a family history of diabetes,with the OR values and 95% confidence interval(CI)being 1.04(1.03,1.05),1.55(1.29,1.88),1.36(1.11,1.66),1.26(1.07,1.49),3.93(2.94,5.24),1.14(1.06,1.23),1.47(1.34,1.61),1.11(1.05,1.18),2.15(1.75,2.62),and 1.66(1.55,1.78),respectively,and the combined β values being 0.039,0.438,0.307,0.231,1.369,0.131,0.385,0.104,0.765,and 0.507,respectively.A total of 37 studies reported the incidence rate,with the combined incidence being 0.08(0.07,0.09)and the parameter α being-2.442 for the logistic model.The logistic risk prediction model constructed based on Meta-analysis was externally validated with the data of 7 602 individuals who had medical checkups and were followed up for at least once.External validation results showed that the predictive model had an area under curve(AUC)of 0.794(0.771,0.816),accuracy of 74.5%,sensitivity of 71.0%,and specificity of 74.7% in the 7 602 individuals.Conclusion The T2DM risk prediction model based on Meta-analysis has good predictive performance and can be used as a practical tool for T2DM risk prediction in middle-aged and elderly populations.