The Chinese hamster ovary (CHO) cell is the most representative mammalian cell protein expression system, and it is widely used in recombinant protein, vaccine and other biopharmaceutical fields. However, due to its vulnerability to environmental factors, apoptosis, and metabolic inhibitors, CHO cells demonstrate poor robustness, and thus the integrated viable cell density and unit cell productivity are largely limited. To improve the robustness and foreign protein expression efficiency of CHO cells, we employed CRISPR/Cas9 to knock out the apoptosis genes Bax and Bak and the lactate dehydrogenase gene LDHa, thereby blocking apoptosis and lactic acid metabolism pathways. The results of apoptosis and single cell viability detection showed that the number of apoptotic cells in the knockout cell lines Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- was reduced by 22.51%, 37.73%, and 64.12%, respectively, compared with the wild-type cell line CHO-K1, which indicated that the anti-apoptotic ability was significantly improved. After staurosporine treatment, the single cell viability of Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- cells was increased by 30.8%, 22%, and 41.1%, respectively. After treatment with puromycin, the single cell viability of Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- cells was increased by 26.7%, 30.7%, and 38.8%, respectively. To further investigate the production performance of cells obtained after blocking apoptosis and lactic acid metabolism pathways, we induced transient expression of human tissue plasminogen activator (tPA) in these cells. The results showed that the secretion of tPA in Bax^-/-, Bax-Bak^-/-, and LDHa-Bax-Bak^-/- cells was 11.12%, 46.18%, and 63.13%, respectively, higher than that in wild-type CHO-K1 cells. The expression of intracellular tPA was increased by 35.65%, 130%, and 192.15%. In conclusion, blocking apoptosis and lactic acid metabolism pathways simultaneously can improve cell robustness and productivity, with the performance better than blocking the apoptosis pathway alone. The above results indicated that the constructed cell lines were expected to be the delivery carriers of protein drugs such as medicinal peptides, and better used for the treatment of diseases.
CHO Cells ; Cricetulus ; Animals ; Apoptosis/genetics* ; Lactic Acid/metabolism* ; Recombinant Proteins/biosynthesis* ; L-Lactate Dehydrogenase/genetics* ; bcl-2-Associated X Protein/genetics* ; bcl-2 Homologous Antagonist-Killer Protein/genetics* ; Cricetinae ; CRISPR-Cas Systems ; Staurosporine/pharmacology*

Objective To analyze the composition characteristics and biological functions of tumor cell subpopulations in glioblastoma(GBM)through multi-transcriptomics sequencing technology,and explore the hypoxia characteristics and spatial localization features of the mesenchymal-like(MES-like)tumor cell subpopulation in GBM and the influence on malignant biological behaviors.Methods Multi-transcriptomics sequencing data,including single-cell RNA sequencing(scRNA-seq)data(18 patients),bulk RNA sequencing(bulk RNA-seq)and spatial transcriptomics(ST)data of GBM,were employed to define cell subpopulations in GBM,and Gene Ontology(GO)and Gene Set Enrichment Analysis(GSEA)were utilized to analyze their functions.The proportions and locations of cell subpopulations in bulk RNA-seq data were evaluated with BayesPrism deconvolution.Immunofluorescence assay was conducted for verification on 12 paraffin samples of GBM from patients who visited the neurosurgical department of our hospital from 2015 to 2023 and met the pathological diagnostic criteria for GBM(10 males and 2 females,at an average age of 53.50 years and a median age of 54.50 years).pySCENIC was applied to predict specific transcription factors of tumor cell subpopulations.Results Tumor cells in GBM were highly heterogeneous,and could be mainly divided into 4 subpopulations:astrocyte-like(AC-like),neural progenitor-like(NPC-like),oligodendrocyte progenitor-like(OPC-like)and MES-like.Differential gene analysis found that the MES-like tumor cells highly expressed vascular endothelial growth factor A(VEGFA),adrenomedullin(ADM),N-myc downstream regulated 1(NDRG1),insulin like growth factor binding protein 5(IGFBP5),and A-kinase anchoring protein 12(AKAP12)(P<0.001).pySCENIC transcription factor prediction found that the high-active transcription factors of the MES-like tumor cells were AT-rich interaction domain 3A(ARID3A),FOS like 2,AP-1 transcription factor subunit(FOSL2),endothelial PAS domain protein 1(EPAS1),CCAAT enhancer binding protein delta(CEBPD),and CCAAT enhancer binding protein beta(CEBPB)(P<0.05).GO and GSEA enrichment analyses found that the MES-like tumor cells were enriched in hypoxia-related pathways,especially the pathway of cell responses to hypoxia levels(NES=2.437,P<0.001).BayesPrism deconvolution showed that the MES-like tumor cells mainly existed in PAN(Pseudopalisading cells around necrosis)and perinecrotic zone.Immunofluorescence assay confirmed CD44+(CD44 antigen)MES-like tumor cells were mainly located in hypoxia areas with highly expression of hypoxia inducible factor 1 subunit alpha(HIF1α)(P<0.01).Multivariate Cox regression analysis indicated that the MES-like tumor cells were significantly correlated with the adverse prognosis of GBM patients(HR=1.71,95%CI:1.38～2.11,P<0.001).Conclusion Tumor cells in GBM are of highly heterogeneity.They could be mainly divided into 4 subpopulations:AC-like,NPC-like,OPC-like and MES-like.MES-like tumor cells,mainly locating in PAN and perinecrotic zone,are characterized by hypoxia,which can promote the malignant progression of GBM.

Objective:To investigate the curative effects of Shugan Hewei Zhitong Recipe on chronic gastritis complicated by Helicobacter pylori infection. Methods:Eighty-seven patients with chronic gastritis complicated by Helicobacter pylori infection who received treatment in the Huguosi Hospital of Traditional Chinese Medicine, Beijing University of Chinese Medicine, China from July 2018 to June 2020 were included in this study. They were randomly assigned to undergo treatment with either conventional Western medicine ( n = 43, control group) or Shugan Hewei Zhitong Recipe based on conventional Western medicine ( n = 44, observation group) for 2 weeks. Before and after treatment, traditional Chinese medicine syndrome scores were compared between the two groups. Curative effects of Shugan Hewei Zhitong Recipe on chronic gastritis complicated by Helicobacter pylori infection were evaluated. Results:Total response rate in the observation group was significantly higher than that in the control group [91% (40/44) vs. 72% (31/43), χ2 = 5.13, P = 0.024]. In each group, the traditional Chinese medicine symptom score post-treatment was significantly lower than the traditional Chinese medicine symptom score pre-treatment (both P < 0.001). After treatment, the traditional Chinese medicine score of the observation group was significantly lower than that of the control group ( P < 0.05). There were no adverse reactions in each group. Conclusion:Shugan Hewei Zhitong Recipe is safe and effective on chronic gastritis complicated by Helicobacter pylori infection, which is worthy of clinical promotion.

OBJECTIVE To compare the dosage forms and specifications of over-the-counter drug （OTC）（chemical drugs ） for children at home and abroad ，and to provide reference for the addition of new dosage forms and specifications of OTC for children in China . METHODS Data analysis was used to comb the active ingredients of OTC single -ingredient preparation for children in China . The similarities and differences of the dosage forms and specifications of OTC for children with the same active ingredients among China and 8th edition of WHO Model List of Essential Medicines for Children （WHO EMLc ）and US/UK/EU （this article refers specifically to EU countries ）/Japan were analyzed by comparative analysis . RESULTS There were 72 active ingredients of OTC single -ingredient preparation for children in China ，corresponding to 34 dosage forms and 216 specifications； 39 same active ingredients of OTC for children were retrieved in WHO EMLc and US/UK/EU/Japan ，corresponding to 38 dosage forms and 258 specifications. Among OTC for children corresponding to 39 active ingredients ，there were 10 unique dosage forms in China ，and 16 unique dosage forms in WHO EMLc and US/UK/EU/Japan ，of which some dosage forms have advantages for children（such as chewing gums ，gels for external use ，spray for oral liquid ，etc.），were included in the latter while not included in China . There were 107 unique specifications in China ，and 214 unique specifications in WHO EMLc and US/UK/EU/Japan ，of which the division of applicable age groups for specifications was more detailed . In addition ，the dosage forms and specifications corresponding to a few active ingredients （such as ibuprofen ，cetirizine hydrochloride ）were not included in OTC for children in China， while included in UK/EU . CONCLUSIONS The dosage forms and specifications of OTC for children in WHO EMLc and US/UK/EU/Japan are generally more abundant and E-mail：zjfyouyou@163.com flexible than in China . Relevant departments in China should learn from the advanced experience of WHO and foreign countries and increase the research and development of dosage forms and specifications of OTC for children .

Objective:To summarize our experience in prevention of COVID-19 infection in emergency and confined operations during the first 3 weeks after Spring Festival in 2020.Methods:From February 3rd to 23rd, 2020, 151 patients were admitted to Department of Orthopaedic Trauma, Beijing Jishuitan Hospital for emergency and confined operations. In this cohort, 125 patients were admitted to ordinary wards. They were 70 males and 55 females with an age of 51.1 years ± 14.9 years. Of them, 2 were subjected to emergency operation and 123 to confined operation. The mean time from injury to operation was 9.9 days ± 6.1 days. There were 26 cases in the senile wards, 7 males and 19 females with an age of 80.8 years ± 7.0 years all of whom underwent confined operations. The mean time from injury to operation was 8.4 days ± 6.3 days. The protocols for emergency diagnosis, admission, emergency and confined operations, postoperative rehabilitation and management of suspects with COVID-19 during the epidemic of COVID-19 were optimized according to Diagnosis and Treatment Protocols for Novel Coronavirus Pneumonia (Trial version 5), emergency responding pre-plans of our hospital, and our experience in Enhanced Recovery After Surgery (ERAS) as well.Results:The patients in the ordinary wards had a hospital stay of 6.8 days ± 4.6 days while those in the senile wards 5.1 days ± 2.0 days. Abnormal temperature (≥37.3 ℃) was observed perioperatively in 17 cases in the ordinary wards. It was absorption fever in all and appeared in 4 cases upon admission. Fever appeared in 11 patients in the senile wards and upon admission in 3 of them. One senile patient who had been diagnosed of normal pneumonia returned to normal temperature and remained stable conditions after antibiotic therapy. The other patients were free of complications related to COVID-19 during their hospital stay.Conclusion:The first-line medical staff working at emergency department, wards and surgical theaters must heighten their vigilance against COVID-19 infection and rigorously follow protocols for prevention of COVID-19 infection in their daily clinical practice.

Objective To investigate the needs and service of rehabilitation status of the people with intellectual disability, to support rehabilitation service for them. Methods A total of 167,872 cases with intellectual disability from the Administration Data of 2017 Basic Service Status and Needs of People with Disabilities in Henan Province were analyzed and rehabilitation needs included medical service and support, assistive productions, and rehabilitation training. Results 56.4% of the people with intellectual disability reported their rehabilitation needs. The rehabilitation needs were different with their ages, levels of disability and household registration (χ2 > 976.42, P < 0.001). Only 19.0% of the people with intellectual disability accessed rehabilitation services, and the influential factors included demographic variables, family economic status, transportation, knowledge and information, etc. Conclusion There are huge needs of rehabilitation services for the people with intellectual disability needed to be tailored. It is necessary to increase the supply of rehabilitation services using approach of precise rehabilitation, and improve the quality of rehabilitation services.

Objective:To study the acute toxicity of Momordica Cochinchinensis in mice, observe and evaluate the acute toxicity of Momordica Cochinchinensis herbs in order to provide basis for the safe and reasonable clinical medication. Methods: According to the classic experiment methods, the acute toxicity of Momordica Cochinchinensis in mice was compared with different administration meth-ods, and the LD50 was calculated by the Bliss method with the continuous administration and observation for 7 d. At the end of the tri-al, the pathologic changes of the relevant organs were also observed. Results:The LD50 of Momordica Cochinchinensis in mice with oral administration was 4. 03g·kg-1 , and the 95% confidence interval was 3. 29-4. 92 g·kg-1 . The LD50 of Momordica Cochinchinensis in mice with intraperitoneal injection was 146. 17 mg·kg-1 , and the 95% confidence interval was 118. 92-177. 31 mg·kg-1 . Conclu-sion:The Momordica Cochinchinensis extract is poisonous, which is consistent with the literatures and pharmacopoeia. By comparing the acute toxicity with different administration methods of Momordica Cochinchinensis in mice, it is beneficial to the evaluation of acute toxicity of Momordica Cochinchinensis to provide basis for the reasonable medication in clinics.

Objective To research the unmet needs and services of rehabilitation for people with physical disabilities in rural areas in Henan, China. Methods The data of 2016 Basic Service Status and Unmet Needs for People with Disabilities in Henan, China were ana-lyzed. Results A total of 1,028,598 people with physical disabilities from all 19 cities had been surveyed, accounting for 61.7% male. In terms of rehabilitation needs, there were 19,348 (1.9%) responsed surgery, 202,855 (19.7%) responsed medical treatment, 197,592 (19.2%) responsed functional training, 360,754 (35.1%) responsed assistive products, and 158,012 (15.4%) responsed nursing, there were significant differences in the number of people who needed rehabilitation among different disability levels (χ2>18.080, P<0.001). For rehabilitation ser-vice, there were 6061 (0.6%) reported surgery, 93,905 (9.1%) reported medicine, 62,967 (6.1%) reported function training, 81,437 (7.9%) reported assistive products, and 34,595 (3.4%) reported nursing, there were significant differences in the number of people who got rehabili-tation among different disability levels (χ2>15.920, P<0.01). For the people without rehabilitation services, there were 165,172 (20.3%) per-son-times lack of understanding of rehabilitation policy, 151,448 (18.6%) reported lack of rehabilitation financial support, 20,369 (2.5%) re-ported not to access rehabilitation institutions, 540,971 (66.5%) reported lack of rehabilitation professionals or services, there were signifi-cant differences in the number of people who did not get rehabilitation services among different disability levels (χ2>19.182, P<0.001). Con-clusion The unmet needs and services of rehabilitation for people with physical disabilities in rural areas in Henan were explicated. It recom-mended to train rehabilitation professionals and improve the service delivery capacity, to raise awareness, increase investment in rehabilita-tion services and provide reasonable accommodation for people with disabilities.

Objective To depermine oupcome of papienps wiph non-ST elevapion acupe coronart stndromes (NSTEACS) preaped wiph FFR-guided versus CAG-guided sprapegt. Methods From Jult 1. 2014 po Jult 30. 2015 in Beijing Anzhen Hospipal, papienps admipped for NSTEACS were reprospecpivelt analtsed wiph a 10-monph follow-up. 142 cases on CAG were furpher assessed wiph FFR ( phe FFR group). Papienps were mapched as 1 : 2 wiph NSTEACS who had moderape lesions shown on CAG in phe same period were enrolled (CAG group, n = 284). End poinps were deaph, nonfapal mtocardial infarcpion (MI), pargep vessel revascularizapion ( TVR), and procedure cosps. Major adverse cardiac evenps ( MACE) were defined as deaph, nonfapal MI, and TVR. Results Fifpt-pwo papienps (36. 6% ) in phe FFR group had FFR less phan 0. 80 underwenp percupaneous coronart inpervenpion (PCI) while 133 papienps (46. 8% ) in phe CAG group received PCI (P =0. 037). Papienps preaped wiph FFR-guided sprapegt had significanplt lower rape of nonfapal MI (2. 2% vs. 4. 5% , P =0. 040) and TVR (5. 9% vs. 11. 7% , P = 0. 046). No spapispical difference was observed in morpalipt (0. 7% vs. 1. 1% , P = 0. 682) and MACE (8. 8% vs. 14. 4% , P = 0. 085). Topal financial cosp was less in phe FFR group (P = 0. 033). Conclusions FFR-guided sprapegt for papienps wiph NSTEACS resulps in less rape of PCI,lower cosp and bepper clinical oupcomes when compared wiph an angio-guided sprapegt.

Objective To study the correlation between the level of CaMKIV and essential hypertension (EH) in Shihezi community. Methods One hundred and forty-six patients with EH and 142 with normal blood pressure are enrolled from 15 communities in shihezi. We collected the clinical data, including blood pressure, body mass index, waist circumference, blood lipids. The serum level of CaMKIV was detected using an enzyme linked immunosorbent assay. Results The serum level of CaMKIV in the EH group was significantly lower than that in the normal blood pressure group (P < 0.05). The serum CaMKIV was negatively correlated with systolic BP and diastolic BP (r = -0.304, -0.452, all P < 0.05), with more obvious correlation with patients whose diastolic blood pressure was over 100 mmHg (r = -0.571, P < 0.05). Conclusions The serum level of CaMKIV is negatively correlated with EH, which is an independent factor of EH.