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  • Original articles
    Establishment of a rapid detection method for the mutation site of the SARS-CoV-2 based on RAA-CRISPR
    NIU Meng-wei, LI Hao, YANG Lan, DONG Xue, HAN Yao, XIA Xue-shan, SUN Yan-song
    2023, 47(3): 161-168. https://doi.org/10.7644/j.issn.1674-9960.2023.03.001
    Abstract ( ) Download PDF ( )   Knowledge map   Save
    Objective To establish a rapid and on-site detection method for SRAS-CoV-2 mutation sites using reverse transcription recombinase aided amplification（RT-RAA） and clustered regularly interspaced short palindromic repeats （CRISPR）nucleic acid detection technology combined with easy-readout and sensitive enhanced（ “ERASE”）CRISPR strip. Methods Based on sequence analysis of SRAS-CoV-2 HV69-70del mutation sites, RT-RAA primers for highly efficient amplification of HV69-70del mutation nucleic acid and crRNA （CRISPR RNA） for specific recognition of the HV69-70del site were designed and screened. The target nucleic acid was amplified by RT-RAA technology, the products were detected by the CRISPR-Cas13a system, and the results were interpreted by the “ERASE” strip. Results The detection sensitivity of this method was 1 copy/µl and the HV69-70del mutation site could be specifically identified within 1 h without reliance on fluorescence detection equipment. Then, this method did not cross-react with the other 7 pathogen nucleic acids. Conclusion Based on RT-RAA technology and “ERASE” CRISPR strip technology, a rapid detection method has been established for the HV69-70del mutation site of SRAS-CoV-2——RAA-CRISPR, which does not rely on fluorescence detection equipment and is a new method for rapid detection and identification of SRAS-CoV-2 variants.
  • Original articles
    Efficiency detection of Lyz2Cre mediated yellow fluorescent protein labeling of myeloid immune cells in different parts of tumor-bearing mice
    ZHANG Jing-ru, TANG Li
    2023, 47(3): 169-174. https://doi.org/10.7644/j.issn.1674-9960.2023.03.002
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    Objective To evaluate the specificity and efficiency of Lyz2Cre mediated yellow fluorescent protein（YFP）labeling of myeloid immune cells in different parts of bodies of mice. Methods Lyz2Cre mice were mated with Rosa26RYFP mice,and double-positive offspring mice were screened via PCR genotype identification. Once the mice became adults,a subcutaneous tumor transplantation model was established. After 14 days,immune cells from peripheral blood,bone marrow and tumors were isolated. Flow cytometry analysis was performed of YFP labeling efficiency in myeloid immune cells at different sites. Results The percentage of monocytes that expressed YFP was（36.87±2.03）% in peripheral blood,compared with（78.46±0.84）% for neutrophils,（15.13±1.17）% for monocytes and （69.62±1.75）% for neutrophils in bone marrow,（32.89±1.89）% for monocytes and （65.56±4.17）% for neutrophils in tumors. The proportion of tumor-associated macrophages expressing YFP was（40.80±3.89）%. Conclusion Lyz2Cre mediated YFP might be not specific and efficient for tracking a particular type of myeloid immune cells. Lyz2Cre gene conditional knockout mice as tumor myeloid macrophages have some defects.
  • Original articles
    Site-specific integration efficiency of CHO-K1 cells improved by DNA ligase Ⅳ inhibitor SCR7
    WANG Chuan-jie, SUN Zhao-lin, SHEN Bei-fen, WANG Jing, FENG Jian-nan
    2023, 47(3): 175-181. https://doi.org/10.7644/j.issn.1674-9960.2023.03.003
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    Objective To find out whether SCR7,a small molecule inhibitor of DNA ligase Ⅳ （Lig4）,can improve the integration efficiency of exogenous genes at the ROSA26 safe harbor mediated by CRISPR/Cas9 in Chinese hamster ovary（CHO）-K1 cells. Methods A specific pX330-sgRNA-ROSA26 plasmid targeting the ROSA26 site in CHO-K1 cells was constructed,so was a homologous targeting vector carrying GFP gene without any promoter. Then,the appropriate conditions under which SCR7 worked in CHO cells were explored.The effect of SCR7 on site-specific integration efficiency of CHO cells was detected by flow cytometry, and the cell apoptosis induced by SCR7 was also evaluated. Results A GFP reporting system targeting ROSA26 was constructed, which could be used to analyze the efficiency of site-specific integration mediated by CRISPR/Cas9. The appropriate dose and time of SCR7 were screened out, and there was no obvious cytotoxicity on CHO-K1 cells within this dose range. It was confirmed that SCR7 could improve the integration efficiency of CHO-K1 cells at ROSA26 site. Conclusion SCR7,as a small-molecular inhibitor of Lig4, could dramatically increase the site-specific integration efficiency in CHO-K1 cells. It may be used for the construction of recombinant CHO cell lines with high expressions of exogenous genes in the future.
  • Original articles
    Identification of ubiquitin ligase activity and ubiquitin ligation mode of enteropathogenic Escherichia coli effector E2348C-1040
    HUANG Yan-yu, LI Zhan, CHEN Fang-hong, LI Tao, WANG Hui
    2023, 47(3): 182-187. https://doi.org/10.7644/j.issn.1674-9960.2023.03.004
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    Objective To identify the ubiquitin ligase activity of enteropathogenic Escherichia coli （EPEC）effector E2348C-1040, and detect the ubiquitin chain type catalyzed by the effector. Methods The E2348C-1040 gene was cloned into pET-22b（+） and transformed into E.coli BL21（DE3）, which was used to express and purify E2348C-1040 protein. The ubiquitin ligase activity was identified by in vitro ubiquitination assay. The ubiquitin mutants and Western blotting assay were used to detect the ubiquitin chain binding site. Results The prokaryotic expression plasmid pET-22b（+）-E2348C-1040 was constructed. The purity of the obtained E2348C-1040 protein was more than 85%, and the concentration was about 0.5 mg/ml. The results of in vitro ubiquitination assay showed that E2348C-1040 could catalyze the formation of lysine-independent ubiquitin chain. Then, an eukaryotic expression plasmid pcDNA3.1（+）-E2348C-1040 was constructed and used to transfect HEK-293T cells. The results showed that E2348C-1040 could catalyze the formation of methionine 1 （M1）-linked linear ubiquitin chain in host cells. Conclusion The EPEC effector E2348C-1040 is an E3 ubiquitin ligase, which can catalyze ubiquitin to form M1 linked linear ubiquitin chain. This discovery will provide clues to the mechanism of effector E2348C-1040 in host cells.
  • Original articles
    Osteogenic differentiation of adipose derived-stem cells in type 1 diabetes mellitus
    XU Ting-ting, LIU Wei-jiang, WU Chu-shan, LIU Yuan-lin, WANG Yang, ZHENG Rong-xiu, ZHANG Yi
    2023, 47(3): 188-195. https://doi.org/10.7644/j.issn.1674-9960.2023.03.005
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    Objective To investigate the osteogenic differentiation capacity of adipose derived-stem cells （ADSCs） in mice with type 1 diabetes mellitus （T1DM）. Methods Ten C57BL/6 mice were randomly divided into two groups. Streptozotocin （STZ） was administrated to C57BL/6 mice through peritoneal injection to induce a T1DM model （n=5）, while the normal control group （NC group, n=5） was given the same volume of sodium citrate buffer injection. After one week, the random blood glucose of the mice was monitored and measured for 3 consecutive days. ADSCs were isolated and cultured from groin and gonad fat in two groups. Cell characteristics were identified by cell morphology, immunophenotype, and induced differentiation assay. The key transcription factors in adipogenic and osteogenic differentiation were measured by real-time quantitative PCR （qPCR） and Western blotting. To study the difference in the osteogenic differentiation ability between the two groups, the third passage ADSCs （P3） in the two groups were used for osteogenic differentiation assay in vitro. Alkaline phosphatase （ALP） staining was performed to measure the osteogenic capacity at 3, 5 and 7 d, respectively, and the expression of osteogenic differentiation related genes were detected by qPCR. Results In the T1DM group,the random blood glucose of the mice was found to be ≥16.7 mmol/L for 3 consecutive days. Typical symptoms of diabetes,including polydipsia,polyphagia,polyuria and slowed weight growth occurred in T1DM mice induced by STZ. Cultured cells from murine adipose tissue in the T1DM group and NC group were spindle-shaped and showed adherent vortex growth. Flow cytometry results showed that CD29 and CD90 were highly expressed, but the expressions of CD11b, CD34, CD45 and MHC-Ⅱ were lower or not expressed. Moreover, these cells presented adipogenic and osteogenic differentiation ability. These results demonstrated that ADSCs from T1DM mice and normal mice had been isolated. In vitro osteogenic differentiation assay results showed that the ALP activities in T1DM-ADSCs were decreased at 3, 5 and 7 d, compared with NC-ADSCs. Furthermore, the mRNA expression levels of RUNX2, OCN, ALP and BMP2 in T1DM-ADSCs were significantly reduced. Western blotting results revealed that the RUNX2 expression was decreased, while CEBPα was increased in T1DM-ADSCs. Conclusion ADSCs isolated from T1DM mice display weak osteogenic differentiation ability.
  • Original articles
    Roles of pyroptosis-related genes in immunotherapy and prognosis in hepatocellular carcinoma
    LI Hao, WANG Zi-hao, LU Yi-ming
    2023, 47(3): 196-204. https://doi.org/10.7644/j.issn.1674-9960.2023.03.006
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    Objective To investigate the role of pyroptosis-related genes （PRGs） in prognosis and immune-targeted therapy in hepatocellular carcinoma （HCC）. Methods PRGs that were key to HCC were identified using bioinformatics methods before being subjected to prognostic value analysis, enrichment analysis, and immune signature analysis. Then, based on these key PRGs, patients with HCC were divided into two subtypes （C1/C2） via non-negative matrix factorization （NMF）. The clinical features, immune cell infiltration, immune checkpoints and mutations were also analyzed. Finally, a drug-gene interaction network was established to identify potential compounds targeting HCC-related PRGs. Results A total of 16 key PRGs were differentially expressed between HCC and normal tissues, and 5 genes were significantly correlated with prognosis in HCC. These 16 key PRGs were closely related to the immune microenvironment. Based on these key PRGs, it was found that the enrichment pathways, immune cell infiltration, immune checkpoints and mutations between the two clusters were significantly different, so was the prognosis. Conclusion Pyroptosis may provide new clues to better understanding the pathogenesis of HCC. The expression of PRGs is closely related to the immune microenvironment and prognosis of HCC, among which IL-6 and IL-1β may be regarded as promising potential therapeutic targets.
  • Original articles
    Immunohistochemical detection technology for polyubiquitination signals based on the tandem hybrid ubiquitin-binding domain probe
    HUANG Shuai, XIAO Wei-di, LI Yan-chang, XU Ping
    2023, 47(3): 205-209. https://doi.org/10.7644/j.issn.1674-9960.2023.03.007
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    Objective To improve the sensitivity of the tandem hybrid ubiquitin-binding domain （ThUBD） probe for detecting polyubiquitination signals in formaldehyde-fixed paraffin-embedded （FFPE） sections. Methods Buffer solutions with different salt ions and pH values were used for antigen retrieval via microwave. The polyubiquitination signals in FFPE sections were detected by the ThUBD probe with or without any reducing agent of a high concentration. Results Addition of 50 mmol/L DTT in pH 9.5 EDTA buffer solutions enhanced polyubiquitination signals in FFPE sections. Conclusion pH 9.5 EDTA buffer solutions plus 50 mmol/L DTT can facilitate the exposure and structure recovery of polyubiquitin chains, and significantly improve the sensitivity of the ThUBD probe for immunohistochemical detection.
  • Original articles
    yEmRFP-based bimolecular fluorescence complementary technology in yeast
    MI Qing-kun, SHANG Li-min, JIN Chao-zhi, YUAN Yan-zhi, WANG Jian
    2023, 47(3): 210-216. https://doi.org/10.7644/j.issn.1674-9960.2023.03.008
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    Objective To establish a bimolecular fluorescence complementary technology based on a red fluorescent protein,yEmRFP, for detection of protein-protein interactions in yeast cells. Methods The N-terminal fragment （1-159 aa, RN159） and C-terminal fragment （160-236 aa, RC160） of yEmRFP were fused with bJun and bFos or bFosΔZip, respectively, to construct recombinant plasmids via molecular cloning technology. Among them, bJun and bFos were the basic leucine zipper domains （bZIP） of transcription factors Jun and Fos, and there was no interaction between bFosΔZip and bJun, which could be used as a negative control. After the recombinant plasmids were transformed into yeast cells, the transformed yeast colonies were screened in auxotrophic medium, and the fluorescence signals of recombinant yEmRFP protein in yeast were detected by fluorescence microscopy and flow cytometry. Thus, it was confirmed that this method was effective for investigating protein-protein interactions. Results The recombinant plasmids of RN159 and RC160 fused with bJun and bFos or bFosΔZip were constructed. The interactions between bJun and bFos reassembled yEmRFP into complete fluorescent protein in yeast cells and strong red fluorescence signals were detected by fluorescence microscopy.About 25% of the 20 000 yeast cells counted by the phycoerythrin （PE） channel of flow cytometry had red fluorescence signals. No fluorescence signal was detected in the negative control group,and no red fluorescentce signal was clearly detected in the same PE channel of the flow cytometry, thus indicating that this system had no self-activation. Conclusion In this study, a yEmRFP-based yeast bimolecular fluorescence complementation technology has been developed, which can be used for direct and rapid detection and screening of protein-protein interactions.
  • Original articles
    Management of and research progress in Combat Casualty Care in the U.S. Armed Forces
    LIU Wei, LOU Tie-zhu, LI Li-juan
    2023, 47(3): 217-222. https://doi.org/10.7644/j.issn.1674-9960.2023.03.009
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    In the future, all-domain operations will pose new challenges to Combat Casualty Care. To deal with these challenges, the U.S. military has continued to innovate the model and concept related to health services,impacting the new style of Combat Casualty Care. This article summarized the U.S. military's progress in Combat Casualty Care management and research in 2021, analyzed the measures taken by the U.S. military, including the continuous promotion of the health-information system reform, updating the new edition of Combat Casualty Care Guidelines, and organizing advanced training on Prolonged Casualty Care by focusing on the layout of a number of prospective projects as well as the new technologies and equipment for Combat Casualty Care in order to provide reference for the development of Combat Casualty Care here in China.
  • Original articles
    Changes of contents of hemoglobin among soldiers trained on the plateau
    LI Shan-shan, SHI Bao-lin, LIU Xiao-yan, BAI Jun-bei, DONG Cheng, HAN Ju-qiang
    2023, 47(3): 223-227. https://doi.org/10.7644/j.issn.1674-9960.2023.03.010
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    Objective To investigate the way in which hemoglobin（Hb） contents change in soldiers under training on the plateau. Methods Two independent cohorts were selected in this study. Peripheral vein blood was aseptically taken to test the blood routine, liver function, renal function, main electrolytes and other basic biochemical indexes. Results Hb content was one of the most sensitive markers in soldiers trained on the plateau. Hb contents quickly increased significantly （173.43±20.73）g/L in those soldiers within 2 weeks of training on the plateau. After those soldiers returned to the plains, Hb contents returned to normal （148.97±13.12）g/L within 2 weeks. Furthermore, a significant time-effect correlation was found between Hb contents and duration of training time on the plateau within one year. However, after over one year of training on the plateau, this time-effect correlation disappeared and Hb contents of the soldiers remained at an abnormally high level. Additionally, Hb contents changed independently of the basic biochemical indexes. Conclusions A Hb content is one of the most sensitive indicators to affect the physiological function of soldiers during training on the plateau, which can contribute to precise medical support at a high altitude.
    • Reviews
  • Reviews
    Advances in cardiovascular physiological effects of +Gz acceleration
    ZHANG Xiao-xue, YANG Ming-hao
    2023, 47(3): 228-231. https://doi.org/10.7644/j.issn.1674-9960.2023.03.011
    Abstract ( ) Download PDF ( )   Knowledge map   Save
    The development of high-performance flighter aircraft requires better anti-G ability of aircrew. Under the influence of continuous positive acceleration, aircrew can suffer from visual change or even loss of consciousness, which poses huge threat to flight safety. In recent years, some achievements have been made in the physiological research of sustained positive acceleration, especially the research on G-induced loss of consciousness （G-LOC） and the cardiovascular physiological mechanism of sustained positive acceleration. This paper summarizes the research progress in the occurrence of G-LOC, cardiovascular compensatory response and arrhythmia, It is expected to provide reference for subsequent exploration of the mechanism of sustained positive acceleration and the selection of ways to protect against anti-G.
  • Reviews
    Research progress of Internet of things in the field of medical treatment
    CHEN Jing, ZHU Mei-lin, LIN Rong, XU Bao-ling, ZHANG Wei-qin, SU Hong-hong, SHEN Xian-ying
    2023, 47(3): 232-235. https://doi.org/10.7644/j.issn.1674-9960.2023.03.012
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    With the development of Internet of Things information technology, informatization in modern health care has become a very important sphere of study. This paper analyzes the applications of Internet of Things in the field of war-time medical treatment, and summarizes the latest research progress at home and abroad in early treatment and medical evacuation.
  • Reviews
    IgSF8 and its post-translational modifications
    ZHANG Ke, LI Xu, WANG Yan
    2023, 47(3): 236-239. https://doi.org/10.7644/j.issn.1674-9960.2023.03.013
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    Immunoglobulin superfamily 8（IgSF8） is one member of the IgSF,and intracellular short-tail motif of IgSF8 molecule has a strong correlation with many biological effects. Palmitoylation and glycosylation,two of the main post-translational modification forms of IgSF8 and CD9,may play a fundamental role in the formation of heteropolymers and their biological functions. In recent years,a number of studies have shown that IgSF8 is of great significance in many biological processes,such as cell proliferation,cell migration and development of the nervous system. This article focuses on the research progress in IgSF8 molecular characteristics and post-translational modification,and proposes future areas of study on IgSF8.
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