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Key technologies and characteristics of products of anti-SARS-CoV-2 neutralizing antibodies

GE Hua, DIAO Tian-xi

2022, 46(12): 881-890. https://doi.org/10.7644/j.issn.1674-9960.2022.12.001

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The persistent pandemic and large-scale spread of coronavirus disease 2019 （COVID-19） have posed a great challenge to global public health security. Researchers have carried out extensive studies related to vaccines, small molecule antiviral drugs and neutralizing antibodies in order to provide safe and effective means for epidemic prevention and control. Among them, the monoclonal antibody with neutralizing activity has the advantages of a clear mechanism, good specificity, good safety, easy large-scale production, and the potential to be used for prevention and treatment at the same time, so it is one of the most promising therapeutic drugs for COVID-19. Neutralizing antibodies for severe acute respiratory syndrome coronavirus 2 （SARS-CoV-2） are under high-speed development at home and abroad. This paper outlines the product characteristics and key technologies of anti-SARS-CoV-2 neutralizing antibodies in order to provide support for related research.

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An amplicon sequencing assay based on nanopore sequencing technology for detecting the expressions of radiation damage genes

QIAN Kun, LIN Li, HAO Xiao-juan, ZHOU Zhe

2022, 46(12): 891-895. https://doi.org/10.7644/j.issn.1674-9960.2022.12.002

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Objective To establish a method for detecting the expression profile of radiation damage genes based on nanopore sequencing technology. Methods Three sets of 21 primer combinations of radiation damage genes were designed and prepared according to literature. Total RNA was extracted from the peripheral blood of healthy volunteers and reversely transcribed into cDNA. The above radiation damage genes were quantified by digital PCR（dPCR）, and the optimal primer sets were chosen according to the results. Fresh peripheral blood samples of 5 tumor patients before and after primary radiotherapy were collected and the RNA was extracted. Then, RNA reverse transcription and multiplex PCR targeting radiation damage genes were performed. Multiprimers PCR products were sequenced by nanopore sequencing, and peripheral blood RNA was sequenced by the second generation, the consistency of the two sequencing methods on the expression level of radiation damage genes in peripheral blood before and after exposure was analyzed. Results The primer set with the least bias to dPCR results was selected. The amount of data generated in the first, second and third hours after nanopore sequencing had significant consistency （P<0.01）. A comparison of nanopore sequencing and second-generation sequencing data showed that the expression profiles of the 5 patients changes in the same way after radiotherapy. The changes in the expression levels of radiation damage genes obtained by the two methods were significantly consistent （P<0.05）, which verified the reliability of this method. Conclusion A method for detection of expressions of radiation damage genes based on nanopore sequencing technology is established, which can be used for rapid assessment of on-site radiation injuries.

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Bioinformatic prediction of T/B cell epitopes against the Omicron variant

SUN Qi, HE Wei, MA Yue, LI Yuan-yuan, HUANG Zhuan-qing, YANG Sen, YANG Fei, ZHANG Ying, XU Feng-hua

2022, 46(12): 896-900. https://doi.org/10.7644/j.issn.1674-9960.2022.12.003

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Objective To predict antigenic epitopes of the severe acute respiratory syndrome coronavirus 2（SARS-CoV-2） Omicron variant using bioinformatic methods and to provide evidence for the design and development of peptide vaccines. Methods Bioinformatic servers and immunology tools were used to predict cytotoxic T-cell epitopes, helper T-cell epitopes and linear B-cell epitopes of S, M, N and E proteins in the Omicron variant （GenBank：OM095411.1）. The biological properties of candidate epitopes were analyzed based on the immune epitope database （IEDB）, antigenicity prediction （Vaxigen）, allergenicity prediction （AllerTOP） and other databases for epitope analysis. Results Thirty-eight cytotoxic T-cell epitopes, fifteen helper T-cell epitopes and six linear B-cell epitopes with good antigenicity, non-toxicity and non-allergenicity were selected. The candidate epitopes were mainly located in S protein and M protein. Conclusion This method for selecting antigenic epitopes is feasible. The epitopes in our study can be used as candidates for the development of peptide vaccines against the Omicron variant.

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Evaluation of automatic annotation methods for cell types based on single-cell transcriptome datasets

KANG Qi-chuan, YANG Qian, ZHOU Zhe, WANG Sheng-qi

2022, 46(12): 901-908. https://doi.org/10.7644/j.issn.1674-9960.2022.12.004

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Objective To explore suitable automated cell type annotation methods for different application scenarios by collecting single-cell transcriptome datasets and evaluating these methods at both cell and cell cluster levels. Methods Single-cell transcriptome datasets were collected of four kinds of samples, including the cell line, tissue （mice, humans）, and human peripheral blood. Using the F1-score, missed detection rate and running time as performance indicators, the performance of six annotation methods was evaluated that were established by three automated annotation tools （scmap, SingleR, and CelliD）developed based on two resolutions. Results The annotation accuracy of 55 cell subtypes in the whole mouse tissue showed obvious differences. Methodsbased on the resolution of cell annotation generally performed better, especially for the classification of immune-related cell subtypes. Besides, the scmap-cell had the best annotation accuracy in most scenarios, but was significantly slower as the number of cells increased. SingleR-cluster showed the most stable accuracy in case of gene dropout. SingleR had significant speed advantages for annotation with a small-scale reference set. Conclusion These findings can provide reference for automatic annotation of cell types in different scRNA-seq datasets.

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NPM1 promotes cell proliferation,migration and invasion of cholangiocarcinomacell lines by activating the epithelial-mesenchymal transition pathway

ZHANG Zheng, ZENG Ji-feng, LIU Xin-yi, WANG Ya-hui, WANG Cui-ling, ZHOU Gang-qiao

2022, 46(12): 909-917. https://doi.org/10.7644/j.issn.1674-9960.2022.12.005

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Objective To investigate the effects of nucleophosmin 1 （NPM1） on the proliferation and migration of cholangiocarcinoma cell lines Huh28 and RBE, and to explore the potential mechanism. Methods Recombined plasmid containing NPM1 was transiently transfected into Huh28 and RBE cells for overexpression of NPM1. Small interfering RNA （siRNA） was used to knock down the endogenous NPM1 in Huh28 and RBE cells. To verify the effects of overexpression and knockdown of NPM1, NPM1 protein expression levels were detected in Huh28 and RBE by Western blotting assays. CCK-8 assays were used to assess the proliferation of Huh28 and RBE cells. The effects of NPM1 on migration and invasion of Huh28 and RBE cells were verified by Transwell assays.Quantitative real time polymerase chain reaction （qPCR） and Western blotting assays were performed to detect the mRNA and protein expression levels of specific genes in epithelial-mesenchymal transition （EMT）, including cadherin 1（CDH1）, cadherin 2（CDH2）, β-catenin1（CTNNB1） and vimentin（VIM）. Log-rank test was used to reveal the difference in survival between cholangiocarcinoma patients with high and low NPM1 expressions. Results Knockdown of NPM1 reduced the proliferation, migration and invasion of Huh28 and RBE cells, whereas overexpression of NPM1 induced the proliferation, migration and invasion of Huh28 and RBE cells. Overexpression of NPM1 induced EMT, whereas knockdown of NPM1 reduced EMT. NPM1 was up-regulated in cholangiocarcinoma tissues compared with non-tumor liver tissues, and its high expression was marginally associated with poor prognosis in patients with cholangiocarcinoma. Conclusion NPM1 can promote the proliferation, migrationand invasion of cholangiocarcinoma cell lines by activating the EMT pathway. Thus, NPM1 might act as an oncogene in the development of cholangiocarcinoma.

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miR-24-3p regulates IFN-γ/STAT1 pathway to block Th1 cell polarization

MENG Guang-yu, LI Xue, LI Lu, LIU Yuan-lin, WANG Yang, ZHENG Rong-xiu, ZHANG Yi

2022, 46(12): 918-924. https://doi.org/10.7644/j.issn.1674-9960.2022.12.006

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Objective To explore the molecular mechanism by which microRNA-24-3p（miR-24-3p） inhibits type 1 T helper cell（Th1） polarization. Methods Murine naïve CD4⁺ T cells were purified by negative CD4 immuno-magnetic beads isolation kit from murine splenic mononuclear cells before being induced to Th1 cells via mouse IFN-γ. The naïve CD4⁺ T cells transfected with miR-24-3p mimic were cocultured for 72 h and flow cytometry was used to detect the percentage of interferon-γ⁺ （IFN-γ⁺） and CD4⁺ Th1 cells. The expression levels of the key factors, including IFN-γ, Tbx21, STAT1, STAT4 and IL-12Rβ1, which regulated Th1 polarization through JAK-STAT signaling pathway,were examined by qPCR. The protein levels of transcription factor STAT1 and phosphorylated STAT1, which determined Th1 polarization efficiency, were analyzed via Western blotting. Bio-informative analysis was used to predict the targets of miR-24-3p,dual-luciferase assay was used to examine the binding between miR-24-3p and the 3′-UTR of IFN-γ/STAT1. Results Naïve CD4⁺ T cells were obtained using the immune-magnetic beads method. The percentage of CD4⁺ IFN-γ⁺cells in Th1 group was （73.98±3.65）% after IFN-γ induction. Compared with Th1 group, co-transfection of miR-24-3p could reduce the percentage of CD4⁺ IFN-γ⁺cells to （60.61±4.70）% （P=0.0176）. The expression levels of STAT1 （P=0.0002）, Tbx21 （P=0.0056） and STAT4 （P=0.0009）, the key transcription factors determining Th1 cell polarization, were down-regulated after miR-24-3p mimic transfection. Meanwhile, the expressions of IFN-γ （P=0.0003）and IL12Rβ1 （P=0.0038） of JAK-STAT signaling pathway was decreased. Moreover, the protein levels of STAT1（P=0.0113） and activated p-STAT1（P=0.0161） were decreased. Consistent with the bio-informative analysis, the dual-luciferase reporter system showed that miR-24-3p could specifically target to the 3'-UTR of IFN-γ and STAT1,thus inhibiting the expressions of IFN-γ and STAT1. Conclusion MiR-24-3p can specifically down-regulate the expressions of IFN-γ and STAT1, and inhibit Th1 cell polarization by blocking the activation of IFN-γ/STAT1 signaling.

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Synthesis and anti-proliferation on tumor cells of curcumin analogs based on cassumunin A

GUAN Meng-jia, LIU Si-fan, XIA Zi-ming, LI Rui-hong, ZHENG Ya-ping, DONG Jun-xing, LI Bin

2022, 46(12): 925-931. https://doi.org/10.7644/j.issn.1674-9960.2022.12.007

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Objective To design and synthesize a series of cassumunin A derivatives and investigate the anti-proliferation on tumor cells of the target compounds. Methods Cassumunin A derivatives containing amine, bromine and fluorine were synthesized from vanillin and crotyl bromide via substitution reactions, heck reactions and hydroxyl aldol condensation reactions. The inhibitory activities of proliferation on human neuroblastoma cells SH-SY5Y of these compounds were measured by CCK-8 assay. Results Fifteen curcumin analogs were synthesized, fourteen of which were new compounds that had never been reported in literature, and their structures were characterized by ¹H-NMR, ¹³C-NMR. In vitro bioassay tests indicated that compound 5b[IC₅₀=（9.21±0.35）µmol/L] had significantly better anti-proliferation on tumor cells than curcumin [IC₅₀=（26.60±0.64）µmol/L]. Conclusion Introduction of dimethylamine at the hydroxyl neighbor of the benzene ring of cassumunin A contributes to the proliferation-inhibiting effect of the compound on SH-SY5Y cells, while the introduction of trifluoromethyl, bromine, amines of large volumes and other groups hinders the anti-proliferation of the compounds on tumor cells.

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Development of two immunochromatographic strips for clinical paraquat poisoning

LI Nan-nan, XIE Si-zhe, MO Dong-dong, WU Jian-feng, ZHAO Yun-li, XU Yong-nan, XIE Jian-wei

2022, 46(12): 932-938. https://doi.org/10.7644/j.issn.1674-9960.2022.12.008

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Objective To prepare two lateral flow immunochromatography assays （LFIAs） for rapid quantitative detection of paraquat poisoning in trace biological samples （ blood or urine）. Methods LFIAs were developed through hapten preparation, animal immunity, antibody screening and sample pad material optimization. The performance was compared with high performance liquid chromatography-mass spectrometry （HPLC-MS） and surface enhanced Raman spectroscopy （SERS） in terms of requirements of sample pretreatment, sensitivity, detection time and sample applicability. Results The results indicated that the obtained antibody had a good specificity and no significant cross-interference with the structural analogues of paraquat, such as diquat and,4-bipyridine. In addition,the time-resolved fluorescence immunochromatographic assay （TRFICA） had good quantitative ability in some concentration range when coupled with the handheld TRFICA analyzer. The low detection limit in whole blood samples could reach 0.05 ng/ml, and the detection time was less than 10 min. Compared with HPLC-MS and SERS, the developed colloidal gold immunochromatography assay （GICA） and TRFICA had the advantages of low cost, simple operation, fast detection, easy discrimination and high sensitivity. Conclusion The GICA and TRFICA provide a new screening and confirmation method for point-of-care testing and prognosis evaluation of paraquat-poisoned patients.

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Progress in biotechnology projects of DARPA

LIU Mai, ZHANG Yin

2022, 46(12): 939-944. https://doi.org/10.7644/j.issn.1674-9960.2022.12.009

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The development of biotechnology and its integration with artificial intelligence and nanotechnology play a vital role in improving the performance of weapons and equipment and enhancing the combat capability of soldiers. As a defense research institution responsible for improving the combat capability and technological superiorities of the U.S. military, Defense Advanced Research Projects Agency（DARPA） continues to strengthen the deployment and management in the field of biotechnology, and is expected to take biotechnology as a powerful “booster” for the disruptive development of future military technology. This article briefly analyzes the developments of biotechnology projects of DARPA in 2021 so as to provide reference for the development and management of defense biotechnology in China.

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Mental toughness for military personnel：research status and prospects

XU Ke, FENG Zheng-zhi

2022, 46(12): 945-950. https://doi.org/10.7644/j.issn.1674-9960.2022.12.010

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Mental toughness（MT） is not only a core component of mental quality of military personnel,but also a symbol of their mental health. Therefore,the study of MT for military personnel has attracted much attention in recent years. The study of military MT focuses on three areas： ①The concept of military MT is defined in terms of ability. The theoretical basis of MT for military personnel is clarified,and the 4Cs model of MT and a personal construct psychology model of MT are constructed; ②The components of military MT are discussed,and the assessment scale of military MT is established from the perspective of self-evaluation and other evaluation; ③Through quantitative and qualitative empirical investigation,it is found that suicide ideation,anxiety,pain,positive emotion and tough group are the influencing factors of military MT. Such characteristics as the length of military service,age,gender,official ranks,education levels,rearing styles and family structure are revealed. The effect of military MT intervention on physiological function is explored based on psychophysiology and blood biological indexes. This paper also outlines problems with related studies so as to provide reference for the in-depth research of MT for military personnel.

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Research progress in skin vasodilation under cold exposure environments

WANG Ze-jun, WANG Ai-ping, YANG Tian, HUANG Yu-jie, HUANG Qing-yuan

2022, 46(12): 951-954. https://doi.org/10.7644/j.issn.1674-9960.2022.12.011

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When the body is exposed to a cold environment, it will induce the initial contraction of the peripheral blood vessels mediated by the sympathetic nerves, in order to preserve the heat resulting in a decrease in peripheral blood flow and lowering the skin temperature. But what is interesting is that usually 5-10 minutes after cold exposure, the surrounding skin blood flow and temperature will increase, which is known as induced vasodilatation response （CIVD）. CIVD is considered to be an important protective mechanism to prevent local cold injury. So far, it has been studied for nearly a century, but due to differences in research methods, the integration of CIVD literature into a coherent overall framework has been slow. During measurement of CIVD, the lack of standardization for the water temperature, location, depth, time, environmental temperature and individual factors of hand immersion make differences to the research conclusions of CIVD, which makes difficult the determination of the CIVD mechanism. This article summarizes the causes of CIVD and the methodology in research in order to quantify the parameters and influencing factors of CIVD, and reduce variability between and within subjects, improve the repeatability of CIVD, and provide reference for further research on CIVD.

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Corynebacterium glucuronolyticum--an opportunistic pathogenic bacterium that causes genitourinary tract infections in males

ZHANG Wang, YANG Ning, WANG Huan, LIU Ming, ZHANG Tian, BAO Chun-mei

2022, 46(12): 955-960. https://doi.org/10.7644/j.issn.1674-9960.2022.12.012

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There is growing evidence that the pathogenicity of Corynebacterium has been underestimated,and that this group of bacteria can even infect immunocompetent individuals without susceptible factors. Corynebacterium has come to be recognized as important opportunistic pathogenic bacteria. In recent years,there have been successive cases of genitourinary tract infections in males caused by C.glucuronolyticum that not only shows specificity for the genitourinary tract of males,but, as an opportunistic pathogenic bacterium,can cause male non-gonococcal urethritis,chronic bacterial prostatitis,persistent or recurrent cystitis without precipitating factors and other genitourinary tract infectious diseases. This paper reviews literature related to C.glucuronolyticum in terms of biological characteristics,epidemiological characteristics,possible pathogenic mechanisms,clinical significance and related antibacterial drug sensitivity of the bacteriumin order to provide data for the diagnosis and treatment of patients.

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