ObjectiveTo construct a stable monoclonal human embryonic kidney 293 （HEK293） cell line expressing recombinant human coagulation factor Ⅶ （rhFⅦ） and evaluate the expression level and procoagulant bioactivity of rhFⅦ. MethodsThe plasmid pCDNA3.1-EGFP-FⅦ was transfected into HEK293 cells to verify the effectiveness of the transfection system. The plasmid pCDNA3.1-FⅦ was transfected into HEK293 cells， and monoclonal stable transfected cell lines were selected using geneticin （G418）. The transcription of the FⅦ gene was identified by reverse transcription polymerase chain reaction （RT-PCR）. The expression level of rhFⅦ in the supernatant of the monoclonal stable transfected cell line was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. The concentration of rhFⅦ was determined by enzyme-linked immunosorbent assay （ELISA）， and the procoagulant activity of rhFⅦ was measured by human coagulation factor Ⅶ potency assay. ResultsHEK293 cells transfected with pcDNA3.1-EGFP-FⅦ showed green fluorescence， indicating that rhFⅦ was successfully expressed in the supernatant of HEK293 cells after transient transfection with pcDNA3.1-FⅦ. The monoclonal stable transfected cell line was obtained by G418 screening. RT-PCR identified that the FⅦ gene was integrated into the genome of the monoclonal stable transfected cell line. The cell viability was good as detected by Cell Counting Kit-8， and a single band of rhFⅦ was obtained by purification of the cell supernatant. The highest rhFⅦ expression was （1.27±0.09） mg/L， and the highest procoagulant activity was （380.29±13.80）%. ConclusionThe monoclonal HEK293 cell lines which can express rhFⅦ protein efficiently and stably with excellent procoagulant bioactivity is successfully screened.

Objective: To perform transient transfection of recombinant human Factor X(rhFX) into HEK293 cells,to optimize transfection parameters,to develop a high-yield in vitro expression system for rhFX production,and to assess the biological activity of expressed rhFX. Methods: The eukaryotic expression vector pcDNA3. 1-EGFP-FX was constructed by inserting the F10 gene into pcDNA3. 1-EGFP and subsequently transfected into HEK293cells to validate the transfection system efficiency. The recombinant expression vector pcDNA3. 1-FX was generated through ligation of the F10 gene fragment with pcDNA3. 1, followed by liposome-mediated transfection into HEK293 cells. The expression of rhFX in the cell supernatant was analyzed by Western blot and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Transfection conditions were systematically optimized,and rhFX concentration was quantified by enzyme-linked immunosorbent assay(ELISA). The coagulation bioactivity of rhFX was evaluated through prothrombin time(PT) assay and chromogenic substrate method. Results: rhFX was successfully expressed in the supernatant of HEK293 cells. rhFX was successfully expressed in the supernatant of HEK293 cells. The highest expression level of rhFX was achieved on the third day after transfection when the cell density was 80% and the ratio of plasmid DNA to polyethyleneimine(PEI) transfection reagent was 1 ∶ 2.Triplicate ELISA measurements demonstrated a maximum rhFX concentration of 5. 20 ng/mL in the supernatant.The prothrombin time(PT) of rhFX-containing supernatant was significantly shorter(P 50) of etoxaban was determined to be 1. 449 nmol/L using the chromogenic substrate method based on rhFX,which was in the same order of magnitude as the reported 0. 78 nmol/L in the literature. Conclusion HEK293cells successfully express biologically active recombinant human Factor X(rhFX) protein,laying a foundation for advancing the development of rhFX-based therapeutics.

Objective:To investigate whether murine peroxidase reductase-5(mPRDX5)has anti-tumor activity in mice,so as to further confirm the anti-tumor activity and mechanism of recombinant peroxidase reductase-5.Methods:High purity mPRDX5 was obtained by heterologous expression and purification in vitro.Pancreatic cancer Pan02 cells were inoculated subcutaneously on the left axillary back of mice to establish a tumor bearing mouse model.Mice were randomly divided into PBS(solvent control)group,GEM(gemcitabine)50.0 mg/kg group and mPRDX5 10.0 mg/kg group,with 10 mice in each group,and the tumor related indexes were detected in mice.Results:Compared with PBS group,weight of tumor-bearing mice in GEM group was decreased obviously,while weight of mPRDX5 group was increased to a certain extent.Tumor growth was good in PBS group,according to tumor volume,com-pared with PBS group,tumor growth inhibition rates in D7 were 87.07%in GEM group and 52.82%in mPRDX5 10.0 mg/kg group,re-spectively;according to tumor weight,compared with PBS group,GEM group and mPRDX5 10.0 mg/kg group had tumor growth inhibi-tion rates of 95.39%and 48.33%in D7,respectively.Polarization state of macrophages in tumor tissues of mice in PBS group and mPRDX5 group was analyzed,and it was found that compared with PBS group,M1 macrophages expressing CD86 in tumor tissues of mice in mPRDX5 group were significantly increased,while M2 macrophages expressing CD206 were significantly decreased.Conclu-sion:mPRDX5 has significant anti-pancreatic cancer activity in mice,and the activity is exerted by promoting M1-type polarization of macrophages in the tumor microenvironment.

Objective:To investigate the clinical efficacy of butylphthalide combined with hyperbaric oxygen therapy on post-stroke cognitive impairment in patients with acute ischemic stroke.Methods:A total of 90 patients with post-stroke cognitive impairment who were hospitalized within 72 hours of onset in Suining County People's Hospital from December 2019 to November 2020 were included in this study. They were randomly divided into a control group and an observation group ( n = 45/group). The control group was given conventional treatment and the observation group was given butylphthalide combined with hyperbaric oxygen therapy in addition to conventional treatment. The National Institutes of Health Stroke Scale score, Montreal Cognitive Assessment score, and Activities of Daily Living score were compared between the two groups before and after treatment. Results:Before treatment, there were no significant differences in the National Institutes of Health Stroke Scale score, Montreal Cognitive Assessment score, and Activities of Daily Living score between the two groups (all P > 0.05). At 14 days and 1 month after surgery, the National Institutes of Health Stroke Scale scores in the observation group were (4.02 ± 2.18) points and (3.21 ± 2.03) points, which were significantly lower than (5.21 ± 2.24) points and (4.62 ± 2.68) points in the control group ( t =2.55, 2.81, both P < 0.05). At 1 and 3 months after treatment, the Montreal Cognitive Assessment score in the observation group were (19.79 ± 5.67) points and (23.69 ± 2.67) points, which were significantly higher than (16.88 ± 5.12) points and (19.74 ± 2.29) points in the control group ( t = 2.56, 7.53, both P < 0.05). At 1 and 3 months after treatment, Activities of Daily Living scores in the observation group were (54.85 ± 5.69) points and (74.38 ± 4.98) points, which were significantly higher than (46.78 ± 6.24) points and (63.21 ± 5.24) points in the control group ( t = 6.41, 9.76, both P < 0.05). Conclusion:Butylphthalide combined with hyperbaric oxygen therapy for the treatment of post-stroke cognitive impairment in patients with acute ischemic stroke can alleviate neurologic deficits, and improve cognitive function and the ability of daily life.

By combing out the historical development logic of sports and health integration and the current development achievement, the underlying logic of sports and health integration is described systematically in multiple dimensions, and the prominent dilemmas in development are analyzed as follows: lack of residents' health literacy, excessive dependence on medical treatment, low bridging of talent team construction, lack of the guidance of funds centralization, insufficient financial support, imperfect management system and mechanism, and unspecific departmental collaboration.The strategies for solving the problems are as follows: strengthening the integration of sports science and clinical medicine, consolidating the technical integration of sports prescription and clinical standardization process, further improving the business integration of exercise intervention and clinical treatment, and enhancing the industrial integration of sports products and medical treatment.

Objective:To analyze the epidemiological and clinical characteristics of hand-feet-mouth disease (HFMD).Methods:The clinical and pathogenic data of 1 976 cases with HFMD hospitalized in Public Health Clinical Center of Chengdu from August 2018 to September 2019 were retrospective analyzed.Results:Among the 1 976 cases of HFMD, 1 094 cases (55.36%) were one to two years old, and 803 cases (40.64%) lived in the main urban area. The majority were scattered patients (1 344 cases, 68.02%). There were 1 348(72.7%) of 1 854 cases mainly infected by Coxsackie virus 6 (CV-A6). The main distribution of rashes in children infected by CV-A6 were palm/soles, trunks, limbs, oral ulcer and perioral areal, lips and hip. The types of rash were maculopapule, vesicle, and bulla. Fever (1 543 cases, 78.09%) was the main concomitant symptom. Acute tonsillitis (811 cases, 41.04%), myocardial injury (767 cases, 38.82%), and herpetic angina (658 cases, 33.30%) were the most common complications. The incidence of onychomadesis were nine (7.03%) among the 128 patients during follow-up.Conclusions:CV-A6 is the main pathogen of the HFMD prevalence. Children less than two years old are susceptible and the main symptoms of HFMD are rash and fever.

Objective To investigate the characteristics of nosocomial infection of carbapenem resistant En-terobacteriaceae ,then to study the risk factors and prognosis of patients ,as to provide evidence for effective control of CRE hospital infection .Methods A retrospective epidemiology study was conducted of CRE infec-ted patients from January 2016 to June 2017 .This was a 1∶2 matched case-control study of patients with in-fection due to carbapenem resistant Enterobacteriaceae and randomly select 218 patients with non CRE infec-tion in the same hospital as control group ,then the risk factors of CRE infection were investigate .Results A-mong the 109 cases CRE infection patients ,the most common bacteria were Klebsiella pneumoniae ,Enter-obacter cloacae and Escherichia coli .The CRE strains were more sensitive to minocycline than other common antibiotics ,the resistance rate to other antibiotics was more than 40% .Univariate analysis showed that ICU staying time more than 7 d ,using beta lactamase inhibitors and carbapenem antibiotics ,combination therapy and mechanical ventilation were the potential risk factors of CRE nosocomial infection .The non conditional multivariate Logistic regression analysis showed that Check in ICU more than 7 d (OR＝ 4 .981 ,95% CI 2 .451-10 .122 ) ,the use of containing beta lactamase inhibitor antibiotics (OR＝ 3 .718 ,95% CI 2 .162-6 .394) ,use of carbapenem antibiotics (OR＝3 .232 ,95% CI 1 .879-5 .561) ,and mechanical ventilation (OR＝5 .26 ,95% CI 2 .576-10 .742) were independent risk factors of CRE nosocomial infection .The nosocomial in- fection CRE strain was with highly antibiotic resistance rate ,and the average hospitalization time and mortality were significantly higher than those of the control group .Conclusion The carbapenem resistant Enterobacte-riaceae infection had multiple independent risk factors ,strengthening of these independent risk factor control can effectively prevent the spread of CRE isolates infection .

The 'Conclusion' section in the Abstract was published incorrectly in the original publication of the article [1] and is corrected with this erratum as below: "Fetal exposure to the Chinese famine may be associated with an increased risk of hypertension in adulthood in women."

Objective: To explore the function and molecular mechanism of Timeless in promoting hepatocellular carcinoma (HCC) growth. Methods: The expression of Timeless in HCC and paracancer tissues were analyzed by using the public data of HCC. Timeless was overexpressed in MHCC97L cells and silenced in MHCC97H cells, respectively, and the expression of Timeless and its downstream molecules were detected by real-time PCR and western blot. The effects of Timeless on cell glycolysis, oxidative phosphorylation and proliferation were detected by the glucose uptake experiment, lactic acid detection experiment, the extracellular fluid pH detection experiment, cell oxygen consumption test and cell viability assay, respectively. Results: The level of Timeless in HCC tissue was significantly higher than that of paracancer tissue (P<0.05). The relative cellular glucose uptake levels in the groups of Timeless knockdown, including siTimeless-1 and siTimeless-2 group were 0.510±0.119 and 0.508±0.099, respectively, significantly different from that of control group (P<0.05); The relative cellular uptake level of Timeless overexpressed group was 1.953±0.324, significantly different from that of vector transfected group (P<0.05). The relative levels of lactic acid production in the siTimeless-1 and siTimeless-2 group were 0.579±0.096 and 0.550±0.120, respectively, significantly different from that of control group (P<0.05); The relative production level of lactic acid in the Timeless overexpressed group was 1.463±0.179, significantly different that of vector transfected group (P<0.05). The extracellular pH values of siTimeless-1 and siTimeless-2 group were 7.390±0.035 and 7.370±0.060, respectively, significantly different from that of control group (P<0.05); the extracellular pH value of Timeless overexpressed group was 7.130±0.031, significantly different than vector transfected group (P<0.05). Oxygen consumption rate of siTimeless-1 and siTimeless-2 group were 3.686±0.389 and 3.955±0.431, respectively, significantly higher than 1.690±0.297 of control group (P<0.05); Oxygen consumption rate of Timeless overexpressed group was 1.302±0.336, significantly lower than 3.185±0.262 of vector transfected group (P<0.05) Timeless inhibited the expression of p53. The cell glucose uptake, lactic acid production, the pH of extracellular culture medium and cell oxygen consumption of control group were not significantly different from that of Timeless and p53 co-silenced group [(si-Timeless+sip53) group] (P>0.05); the glucose uptake, the production of lactic acid, the pH of the extracellular culture medium and the oxygen consumption of Timeless co-transfected with p53 (Timeless+p53) group were not significantly different from those of vector transfected group (P>0.05). Timeless promoted the proliferation of HCC cells through inhibiting the expression of p53. Conclusion Timeless promotes reprogramming of glucose metabolism and proliferation of HCC cells by inhibiting the p53-dependent signaling pathway.

Objective To evaluate the value of bone marrow imaging by third-generation dual-source dual-energy CT(DSDECT) using virtual noncalcium(VNCa) technique for the assessment of diffuse infiltrative lesions of multiple myeloma(MM). Methods From December 2015 to June 2016,31 patients with plasma disorders at our center were prospectively recruited and received whole-body imaging with third-generation DSDECT and MRI. CT numbers of vertebrae were measured on VNCa images as well as regular CT images. Correlation between VNCa CT numbers and MRI signal intensities of the vertebrae was evaluated. The diagnostic ability of VNCa for MM infiltrative lesions was assessed by ROC analysis,using MRI as the reference standard. Results The mean VNCa CT numbers of vertebrae with MM diffuse infiltration (n=62) were (-13.27±18.96)HU,which were significantly higher than those of non-infiltrated vertebrae[(-63.31±26.75)HU,(n=117)] (Z=-9.731,P=0.000). VNCa CT numbers of vertebrae were negatively correlated with T1WI signal intensity normalized by non-degenerative vertebral discs (r=-0.592,P=0.000). ROC analysis showed the area under the curve of VNCa for the diagnosis of infiltrative lesions was 0.943. With the cut-off value of-37 HU,the sensitivity and specificity of VNCa were 90.32% and 87.18%,respectively. Conclusion Bone marrow imaging by third-generation DSDECT using VNCa technique is a valuable tool for assessing diffuse infiltrative lesions of MM.
Bone Marrow ; diagnostic imaging ; Humans ; Magnetic Resonance Imaging ; Multiple Myeloma ; diagnostic imaging ; ROC Curve ; Sensitivity and Specificity ; Spine ; diagnostic imaging ; Tomography, X-Ray Computed ; methods