Objective:In the present study,Bac-to-Bac baculovirus expression system was used to obtain recombinant human Cosmc extracellular domain protein,which can lay the foundation for the research about the structure and function of Cosmc protein in vitro,and simultaneously provide ideas for the research of O-glycosylation and related diseases. Methods: The Cosmc extracellular domain ( Cosmc-ED) gene was cloned into a transfer vector pFastBac1 to form the recombinant donor plasmid pFastBac1-Cosmc ED, which was transformed into competent cells DH10Bac. By using blue-white selection and PCR analysis,we could obtain recombinant shuttle vector rBacmid-Cosmc ED. Then, the recombinant gene DNA of rBacmid-Cosmc ED was used to transfect Sf-9 mediated by cationic lipid formulation,and the recombinant baculovirus bacmid was obtained,which was further used to infect the serum-free cell Sf-9 to express Cosmc-ED in the supernatant. Then the protein of interest was detected by SDS-PAGE and Western blot and purified with Ni-NTA affinity column. Results:SDS-PAGE and Western blot analysis showed a specific band about 33 kD,consistent with the interest protein. Mass spectrometry results further prove that the protein was Cosmc extracellular domain protein. Conclusion: Human Cosmc-ED protein can be successfully expressed in Sf-9 insect cells and laid basis for subsequent studies.

Objective To explore the effect of comprehensive intervention on anxiety in patients of acute myocardial infarction (AMI) undergoing intra-aortic balloon pump (IABP). Methods 50 patients with AMI undergoing IABP with the score of Hamilton Anxiety Rating Scale (HAMA) more than 14 points, were divided into conventional intervention group (n=25) and comprehensive intervention group (n=25). The conventional intervention group received all the conventional nursing measures including cognitive behavioral intervention, and the comprehensive intervention group received propofol intravenous pumping in addition with Ramsay sedation at II-III level. The vital signs, HAMA scores, major cardiovascular events, and vascular complications were recorded before and the 1st-5th days after intervention. Results The HAMA scores, systolic blood pressure, diastolic blood pressure and mean pressure decreased in most of the time points after intervention in the comprehensive intervention group (P<0.05). And there was no complication such as low blood pressure, respiratory depression. The incidence rates of cardiac arrhythmias, puncture hematoma/bleeding and catheter displacement were lower in the comprehensive intervention group than in the conventional intervention group (P<0.05). Conclusion Comprehensive intervention can improve the symptoms of anxiety in the patients with AMI undergoing IABP, and reduce the incidence of arrhythmia, vascular complications and catheter displacement.

Objective To investigate the effect of intermedin (IMD) on renal ischemia/ reperfusion (I/R) injury after the up-regulation of IMD. Methods A total of 24 healthy Wistar male rats were randomly divided into four groups,sham-operated group,I/R group,IMD gene transfection +I/R group and empty plamid +I/R group.All the animals were killed at the end of 24 h of reperfusion.Histological changes and renal function were estimated.The expression and site of IMD were determined by Immunohistochemistry method,semi-quantitative RT-PCR and Western blotting.The protein expressions of endothelin 1 (ET-1),tumor necrosis factor αt (TNF-α) were detected by Western blotting. Results Compared with sham-operated group,tubulointerstitial pathological injury was significant aggravated in I/R group (7.6±2.3) and empty plamid +I/R group (7.0±1.8),and such injury was improved in IMD+I/R group (1.5±0.8) (P＜0.05).Compared with I/R group and empty plamid +I/R group,the renal dysfunction of IMD +I/R group was obviously lessened [BUN:(7.73±1.03) mmol/L vs (10.13±2.14) mmol/L,(9.77±1.92) mmol/L; Scr:(58.50±3.27) μmol/L vs (80.33±7.15) μmol/L, (75.67±7.58) μmol/L,all P＜0.05].IMD expression was weak in the plasma of tubulointerstitial cells in sham-operated group,and was up-regulated in I/R group. Compared with I/R group, immunohistochemical IMD expression increased obviously (262.03±67.89 vs 175.57±48.06,P＜0.01).The mRNA expression of IMD in IMD+I/R group was up-regulated significantly by 60.7％,66.1％ and the protein expression of IMD in IMD+I/R group increased significantly by 51.4％,55.9％ as compared to I/R and empty plasmid +I/R group.Meanwhile,the protein expressions of ET-1 and TNF-αt in IMD+I/R group were obviously lower compared with those in I/R group (ET-1:0.08±0.02 vs 0.17±0.02; TNF-α:0.21±0.04 vs 0.35± 0.02,all P＜0.05). Conclusion IMD gene transfected into kidneys of rats prior to I/R surgery can attenuate the over-expressions of both ET-1 and TNF-o in I/R injured rat kidneys as well as the damages to the structure and function of the kidneys.

Objective To assess the relationship between serum vitamin D and autoimmune thyroid disease.Methods Subjects included total 520 persons receiving regular health examination,and serum calcium,phosphorus,parathyroid hormone (PTH),thyroid peroxidase autoantibody (TPOAb) and 25-dihydroxy vitamin Ds was measured.The incidence of 25-dihydroxy vitamin D3 deficiency (≤30 μ g/L)was observed.The relationship between 25-dihydroxy vitamin D3 deficiency and autoimmune thyroid disease was analyzed.Results The serum 25-dihydroxy vitamin D3 of all the subjects was (24.47 ± 7.21) μ g/L,and the incidence of 25-dihydroxy vitamin D3 deficiency (≤30 μg/L) was 61.15％ (318/520),and the positive rate of TPOAb was 21.54％ (112/520).The proportion of TPOAb ＞ 50 kU/L or ＞ 100 kU/L in subjects with 25-dihydroxy vitamin D3≤30 μ g/L was higher than that in subjects with 25-dihydroxy vitamin D3 ＞ 30 μg/L [25.79％(82/318) vs.19.80％(40/202) and 9.43％(30/318) vs.4.46％(9/202)],and there was significant difference (P ＜ 0.05).The relationship between 25-dihydroxy vitamin D3 and TPOAb was assessed and showed significant inverse correlation (r =-0.13,P ＜0.05).Conclusions Vitamin D deficiency is very common in the population,and autoimmune thyroid disease is related with vitamin D deficiency,which may has impact on the body's immune regulation.Specific mechanism and whether vitamin D supplementation can intervene and treat autoimmune thyroid disease need further study.

Objective To construct eukaryotic expression vector encoding rat IMD gene and deliver it into rat renal tissue via ultrasound-mircobubbles. Methods IMD gene was inserted into pCDNA3.1 ( + )between Hind Ⅲ and EcoRI enzyme sites. The recombinant plasmid designated as IMD-pCDNA 3.1 wasconfirmed by restrictive enzyme digestion and sequencing. Eighteen male Wistar rats were randomized into 3groups, which were treated with no transfection, empty vector transfection and IMD transfection, respectively, in renal tissue via ultrasound-microbubbles. RT-PCR and Western blotting were applied to detect the expression level of IMD. Results Enzyme- digestion and sequencing data showed that IMD-pCDNA 3.1 was correctly constructed. The differences in ALT, AST, BUN and SCr were not significant; No obvious damage in the glomerular, tubular and interstitial was observed in all the treated groups;Compared with non-transfection group and empty vector-transfection group, IMD mRNA and protein expression in IMD transgenic renal tissue were significantly increased. Conclusion IMD-pCDNA 3.1 expression vector was successfully constructed and well expressed in rat kidney.

An electrolyte cathode atmospheric glow discharge atomic emission spectroscopy(ELCAD-AES) has been developed to determine the hardness of water. By the standard curves of Mg and Ca, the concentration curves of Mg and Ca measurement were obtained. The limits of detection of Mg and Ca were 0.2 and 0.8 mg/L, respectively. The precision and recovery were experimentally elucidated, which indicated that the homemade ELCAD-AES system has a good performance. Finally, practical water samples such as tap water, boiled tap water, ground water, boiled ground water and lake water were analyzed by the method. The results were in agreement with those by the EDTA titration. This demonstrates that ELCAD-AES has the capacity for on-line determination of water hardness.

Objective To investigate the effect of α-crystallin on the proliferation of rat retinal microglia after optic nerve injury. Methods The effect of α-crystallin on number and proliferation of microglia were analyzed by MTT assay.After the rat model with optic nerve injury was established,α-crystallin was iniected into vitreous cavity and the microglia cell number were counted and compared by retinal fiat counting and immunofluorescence labeling in different groups. Results The proliferation and activation of microglia cells could be stimulated by LPS at 10-6g/L to 10-2g/L.α-crystallin at 10-4g/L and 10-6g/L could inhibit proliferation and activation of microglia cells.Compared to BSA iniection group,α-crystallin could inhibit more significantly the number of microglia cells 1-3 weeks after injury (P＜0.05). Conclusions α-crystallin can inhibit proliferation and activation of retinal microglia and alleviate overphagocytosis and secondary damage of retinal microglia to retinal ganglion cells(RGCs),which may be another mechanism that α-crystallin contributes to indirect protection of RGCs.

Objective To test the effect of an APP in the adherence of inhalation medicines for children with asthma,improve the rate of inhalation medicines compliance,self management ability and the quality of life of children with asthma.Methods The research use self-control study.We use Medication Adherence Report Scale for Asthma (MARS-A) to test the adherence of inhalation medicines for children with asthma in outpatient department.After 2 months using the APP,the children with asthma may be re-tested by the MARS-A to compare the differences in medication adherence,asthma knowledge,use method and pulmonary function,etc.Finally,data analysis using paired t test.Results After using the APP,the inhalation medicines adherence rate is increased from 25.78 ％ to 68.75 ％,the awareness rate of patients and their caregivers are increased from 25 ％ to 65 ％,and the APP usage rate is 42.18％,there was significant difference (P＜0.01).Conclusion The APP can effectively improve the adherence of inhalation drugs,standardized medication management,guarantee treatment effect and improve the quality of life of children with asthma.It could also reducing medical costs and improving work efficiency and service quality,thus deserves promotion.

Objective:To investigate the diagnostic value of metagenomic next-generation sequencing (mNGS) in AIDS patients complicated with Pneumocystis jirovecii ( P. jirovecii) infection. Methods:This is a retrospective study. From January 2019 to June 2021, the respiratory tract and other body fluid samples of 236 cases of AIDS co-infected patients diagnosed in the AIDS Department of Changsha First Hospital were collected, along with corresponding medical histories. Traditional etiological hexamine silver staining and serum 1,3-β-D glucan (BDG) were performed simultaneously with mNGS detection, and Fisher′s exact test was used to analyze the results and compare the diagnostic performances of mNGS with those of hexamine silver staining and serum G test.Results:A total of 236 cases of AIDS patients with pulmonary infection were collected and tested. Seventy-seven cases were clinically diagnosed with Pneumocystis jiroveci pneumonia and 159 cases with non- Pneumocystis jiroveci pneumonia. Among the 236 AIDS patients with pulmonary infection, mNGS detected 77 [32.63%(77/236)] positive cases of Pneumocystis jiroveci, while hexamine silver staining detected 10[4.24%(10/236)] and serum BDG detected 146 [61.86% (146/236). Based on these clinical diagnostic results, the sensitivity of mNGS detection was 100% (77/77) for the 77 patients with Pneumocystis pneumoniae, significantly higher than that of silver hexamine staining [12.99% (10/77), P=0.046] and serum BDG [58.44% (45/77), P=0.038]. The mNGS showed good specificity, which was the same as that of hexamine silver staining [100% (159/159)] and significantly higher than that of serum BDG [36.48% (58/159), P=0.026]. With therapeutic clinical diagnosis as the reference method, the accuracy of mNGS detection was 100% (236/236). Conclusions:This study evaluated the diagnostic value of mNGS detection in AIDS patients with Pneumocystis jirovecii infection. The results showed that the sensitivity and specificity of mNGS detection were high, and it had exceptional clinical application value in the pathogenic detection of infectious diseases.

Objective:To explore the efficacy of haploidentical hematopoietic stem cell transplantation (haplo-HSC) combined with tpCB in the treatment of children with bone marrow failure syndromes (BMFs).Methods:The clinical chara-cteristics of 78 BMFs pediatric patients, including inherited BMFs (4 cases) and acquired BMFs (74 cases) under-went haplo-HSC combined with the third-party cord blood (tpCB) in Chinese People′s Liberation Army General Hospital-Sixth Medical Center between July 2012 and July 2019 and were retrospectively analyzed, with 41 males and 37 females.Among them, 73 cases experienced first transplantation and 5 cases accepted second transplantation, with the median age of 5.6 years.The conditioning regimen was based on Busulfan, with 74 acquired BMFs cases using non-myeloablative and the remaining 4 cases using myeloablative.The prophylaxis of acute graft versus host disease (aGVHD) includes Cyclosporine, Mycophenolate mofetil (MMF) and Methotrexate.All patients received bone marrow from haploid donor and tpCB on day 1 and peripheral stem cell from haploid donor on day 2.The median dose of the total donor nucleated cells was 12.19×10 8/kg of recipient weight and CD 34+ cell dose was 6.13×10 6/kg of recipient weight. Results:The median time of granulocytes over 0.5×10 9/L and platelets over 20×10 9/L were + 13 d and + 17 d, respectively.All patients displayed complete donor-type chimerism at + 30 d. No primary graft failure occurred in any patient and second graft failure occurred in two cases.The incidence rate of grade ⅡtoⅣ and grade Ⅲ to Ⅳ aGVHD were 39.0% and 13.9%, respectively.The incidence of chronic GVHD with limited type and extensive type were 7.8% (95% CI: 7.1%-8.5%) and 2.6% (95% CI: 2.1%-3.1%), respectively.the median follow-up was 1 550 days, and 76 patients survived with free disease.The rate of transplant related mortality was 2.8%, and both of the estimated 5-year overall survival and failure-free survival rate were 97.2%(95% CI: 96.8%-97.6%). Conclusions:Haplo-HSC and umbilical cord blood can quickly provide hematopoietic stem cells.The results of haplo-HSC combined with the tpCB in pediatric patients with life-threatening BMFs are promising.