Research progress on the mechanism of gut microbiota participating in diabetes nephropathy / 药学实践与服务

With the increasing prevalence of diabetes, the prevention and treatment of diabetes nephropathy have become a worldwide problem. The molecular mechanism of the occurrence and development of diabetes nephropathy is still unclear, but many studies in recent years have shown that gut microbiota plays an important role in the progress on diabetes nephropathy. The research progress on the mechanism of gut microbiota participating in diabetes nephropathy was reviewed in this article.

Construction and validation of a mouse model with systemic overexpression of human METRNL gene / 药学实践与服务

Objective To generate mice with whole-body overexpression of human METRNL gene. Methods Based on Cre-loxP system, Dppa3-Cre mice were mated with Rosa26-LSL-METRNL knock-in mice（R26-LSL-METRNL+/-）to generate R26-L-METRNL+/- mice. The genotypes of the offsprings were identified, and tissues of the blood, heart, liver, spleen, lung, kidney, brain, white adipose and muscle were collected. The expression of human METRNL gene in mice was investigated by quantitative real-time PCR, western blot and enzyme linked immunosorbent assay. Results Compared with wild type control mice, human METRNL in R26-L-METRNL+/- mice significantly expressed at both mRNA and protein levels in tissues, with abundant METRNL protein in blood. Conclusion The mouse model overexpressing human METRNL gene（R26-L-METRNL+/- mouse）was successfully constructed.

The increasement of blood METRNL protein by insulin sensitizer rosiglitazone / 药学实践与服务

Objective To investigate the effect of insulin sensitizer rosiglitazone on blood METRNL levels. Methods After fed with high fat diet (HFD) for 3 months, obese mice were treated with rosiglitazone for 1 month. Glucose tolerance was tested with glucose tolerance test (GTT), and METRNL levels in blood were measured by ELISA. Real time fluorescence quantitative PCR was used to detect the expression of METRNL in various tissues such as muscle, liver, white fat, brown fat, brain, spleen and kidney, as well as the expression of mitochondrial proteins in brown adipose tissue. Results Glucose tolerance of animals fed a high-fat diet was improved in rosiglitazone group, and blood METRNL levels were also increased significantly in this group. Rosiglitazone treatment increased the expression of METRNL in brown fat and kidney tissue. There was no effect on METRNL expression in muscle, liver, white fat, brain and spleen. Rosiglitazone increased the expression of mitochondrial-associated proteins in brown adipose tissue. Conclusion The insulin sensitizer rosiglitazone might increase the serum METRNL level by increasing the METRNL expression in brown fat and kidney tissue, suggesting that METRNL may be involved in the therapeutic effect of rosiglitazone on diabetes.

Anti-metastatic Pharmacology Based on Tumor Microenvironment Regulation： A Review / 中国实验方剂学杂志

Tumor metastasis is the major cause of death for tumor patients and the key bottleneck of clinical treatment. In recent years， basic and clinical studies have recognized that tumor microenvironment （TME） is highly correlated with tumor metastasis， which provides hope for anti-metastatic drug development and clinical treatment. At present， the mainstream studies on TME represented by immune checkpoint inhibitors （ICIs） mainly focus on the rectification of immune function of T cells and B cells. However， a large number of studies have shown that the significance of other members of TME for tumor metastasis cannot be ignored， which greatly reflects the progress of anti-metastatic research based on TME regulation. This review focused on tumor metastasis， summarized the mechanism of action of non-T and non-B immune cells ［tumor-associated macrophages （TAMs） and tumor-associated neutrophils （TANs）］ and non-immune members ［vascular endothelial cells （ECs）， tumor-associated fibroblasts （CAFs）， and blood platelet］ in the process of tumor metastasis in TME based on the literature over the recent five years， and explored their key value in the treatment of metastasis. At the treatment level， this review focused on the perspective of the integration of frontier and traditional methods and took the functional homeostasis remodeling of TME as the entry point to summarize the activity and mechanism of traditional Chinese medicine （TCM） regulation of non-T and non-B immune cells and non-immune members and highlight its advantages and characteristics in clinical intervention of metastasis. This review helps to break through the limitations of over-reliance on T and B immune cells in anti-metastatic research， make the research rely on a wider range of cell groups， explore the potential value of TME in anti-metastatic drug intervention， and enrich the idea and strategy of understanding the anti-metastatic pharmacological activity. The review is also expected to provide a broader vision for the research and development of new anti-metastatic drugs.

Expert consensus on cryoablation therapy of oral mucosal melanoma / 实用口腔医学杂志

Cryoablation therapy with explicit anti-tumor mechanisms and histopathological manifestations has a long history.A large number of clinical practice has shown that cryoablation therapy is safe and effective,making it an ideal tumor treatment method in theory.Previously,its efficacy and clinical application were constrained by the limitations of refrigerants and refrigeration equipment.With the development of the new generation of cryoablation equipment represented by argon helium knives,significant progress has been made in refrigeration efficien-cy,ablation range,and precise temperature measurement,greatly promoting the progression of tumor cryoablation technology.This consensus systematically summarizes the mechanism of cryoablation technology,indications for oral mucosal melanoma(OMM)cryotherapy,clinical treatment process,adverse reactions and management,cryotherapy combination therapy,etc.,aiming to provide reference for carrying out the standardized cryoablation therapy of OMM.

Clinical study on the application of guide catheter window guidance technology in intracranial artery stent angioplasty / 中国医师杂志

Objective:To explore the application value of guide tube fenestration and drainage technology in intracranial artery stenting surgery.Methods:A total of 120 patients with symptomatic intracranial atherosclerotic stenosis admitted to the Xingtai Third Hospital from January 2021 to December 2022 were selected and randomly divided into an observation group ( n=60) and a control group ( n=60). Both groups were treated with intracranial artery stenting, with the observation group receiving guidance catheterization and window opening technique during the surgery. Two groups of surgeries were observed and compared: the degree of vascular stenosis before and after surgery, the Montreal Cognitive Assessment Scale (MoCA) scores before and one month after surgery, intraoperative complications, and prognosis. Results:The surgical time and catheter placement time in the observation group were (110.20±23.32)minutes and (11.32±2.01)minutes, respectively, which were significantly shorter than those in the control group (all P<0.05). The stent placement rate and operation success rate in the observation group were 95.00% and 96.67%, respectively, significantly higher than those in the control group (all P<0.05). The degree of postoperative vascular stenosis in the observation group was (32.29±7.11)%, significantly milder than that in the control group [(44.43±8.15)%, P<0.05]. One month after surgery, the MoCA scores of both groups improved significantly compared to before surgery (all P<0.05), and there was no statistically significant difference between the groups ( P>0.05). There was no statistically significant difference in the incidence of collateral circulation occlusion and vascular rupture between the two groups (all P>0.05). During the follow-up period, there was no statistically significant difference in the incidence of restenosis, intracranial hemorrhage, and recurrent ischemic stroke between the observation group and the control group (all P>0.05); The good prognosis rate of the observation group was 73.33%, significantly higher than that of the control group (53.33%, P<0.05). Conclusions:The application of guided catheter fenestration technique in intracranial artery stenting has good value, which is beneficial for improving the stent placement rate and operation success rate, and improving the degree of vascular stenosis.

Therapeutic effect and mechanism of Eucommia lignans on diabetes encephalopathy rats / 中华老年心脑血管病杂志

Objective To explore the therapeutic effect of lignans extracted from Eucommia Ulmoides Oliver on diabetes encephalopathy(DE)rats and the effects on nuclear factor E2 related factor 2(Nrf2)/heme oxygenase-1(HO-1)signaling pathway.Methods A rat model of DE was established,and 48 successfully established DE rats were randomly divided into model group,and low-and high-dose lignans groups(200 and 400 mg/kg),and metformin(200 mg/kg)group,with 12 rats in each group.Another 12 healthy rats served as control group.Each group of rats was giv-en corresponding drug intervention once a day for 6 consecutive weeks.Cognitive and memory abilities,serum levels of IL-6,TNF-α,MDA and SOD,and mRNA and protein levels of Nrf2 and HO-1 in the hippocampus were measured in all groups.Pathological changes of the hippocampus were observed after HE staining.Results Compared with the control group,significantly longer escape latency and increased serum levels of IL-6,TNF-α and MDA,while lower times of crossing original platform,shorter residence duration of original platform quadrant,decreased serum SOD level,and reduced mRNA and protein levels of Nrf2 and HO-1 in the hippocampus were observed in the model group(all P＜0.05).Low-and high-dose lignans treatment reversed all above indica-tors when compared with model group(P＜0.05).What's more,the effects of high-dose lignans treatment were more significant than low-dose treatment in escape latency,serum levels of IL-6,TNF-α and MDA,times through the original platform,residence duration of the original platform quadrant,serum SOD level,and mRNA Nrf2 and HO-1 in the hippocampus(6.07±0.70 vs 4.76± 0.52,30.96±2.96 s vs 23.49±2.60 s,77.02±6.51 U/ml vs 61.71±6.75 U/ml,0.69±0.06 vs 0.49± 0.05,0.74±0.07 vs 0.57±0.06,P＜0.05).Conclusion Eucommia lignans can improve the cogni-tive ability,enhance the antioxidant capacity,inhibit inflammatory response and protect the hippo-campus of DE rats,and the mechanism may be related to the activation of Nrf2/HO-1 signaling pathway.

Analysis and comparison of major plant databases in China and ASEAN countries / 国际中医中药杂志

At present, countries around the world are paying greater attention to the protection of medicinal plants and traditional medicinal knowledge resources, and are looking for various ways to protect medicinal plants. Many countries have established their own databases to save the medicinal plant information resources. This paper focuses on the introduction of medicinal plant databases in six countries including Malaysia, Philippines, and Singapore, and compares their basic information. It is difficult to achieve integration and sharing among these databases. It brings certain difficulties to the use of researchers in related fields. It is suggested that the construction of a multinational common medicinal plant database should be included in the "Belt and Road Initiative" to systematically organize massive information, enhance exchanges between countries on traditional medicinal plants, and achieve medicinal plant information sharing, and the establishment of a shared database will reduce optimization and maintenance to a certain extent or renewal work, laying the foundation for the protection, development and sustainable use of traditional medicinal plant resources.

Literature Survey on Edible Medicinal Plants in Lancang-Mekong Countries / 中国实验方剂学杂志

Lancang-Mekong countries refer to the six countries that the Lancang-Mekong River flows through， including China， Laos， Myanmar， Thailand， Cambodia， and Vietnam. These countries are geographically adjacent with similar cultures and have long history of traditional medicine and high plant diversity. Since ancient times， medicinal plants have been introduced into China from the countries along the river， including a variety of medicinal plants with edible and healthcare values， which is an important way for the transnational circulation of medicinal resources. This paper briefly described the history and application of edible medicinal plants in the six Lancang-Mekong countries and summarized more than 150 edible medicinal plants from the other five countries except China. These 150 medicinal plants belong to 66 families such as Labiatae， and 12 species of them are used as edible medicinal plants in all the six countries. Further， we collected the information of these edible medicinal plants， including the origins， efficacy， indications， medicinal edible parts， edible values， and the traditional application of these plants in China. Some valuable edible medicinal plants in the other five countries are considered to have a promising prospect of application in China and may be introduced to China as new medicinal resources. These efforts will be conducive to the cooperation in traditional medicine among Lancang-Mekong countries.

Endothelial METRNL determines circulating METRNL level and maintains endothelial function against atherosclerosis

METRNL is a recently identified secreted protein with emerging functions. This study is to find major cellular source of circulating METRNL and to determine METRNL novel function. Here, we show METRNL is abundant in human and mouse vascular endothelium and released by endothelial cells using endoplasmic reticulum-Golgi apparatus pathway. By creating endothelial cell-specific Metrnl knockout mice, combined with bone marrow transplantation to produce bone marrow-specific deletion of Metrnl, we demonstrate that most of circulating METRNL (approximately 75%) originates from the endothelial cells. Both endothelial and circulating METRNL decrease in atherosclerosis mice and patients. By generating endothelial cell-specific Metrnl knockout in apolipoprotein E-deficient mice, combined with bone marrow-specific deletion of Metrnl in apolipoprotein E-deficient mice, we further demonstrate that endothelial METRNL deficiency accelerates atherosclerosis. Mechanically, endothelial METRNL deficiency causes vascular endothelial dysfunction including vasodilation impairment via reducing eNOS phosphorylation at Ser1177 and inflammation activation via enhancing NFκB pathway, which promotes the susceptibility of atherosclerosis. Exogenous METRNL rescues METRNL deficiency induced endothelial dysfunction. These findings reveal that METRNL is a new endothelial substance not only determining the circulating METRNL level but also regulating endothelial function for vascular health and disease. METRNL is a therapeutic target against endothelial dysfunction and atherosclerosis.

First Report of Rhizoctonia solani AG-4 HG-III Causing Sheath Blight on Foxtail Millet in China.

Foxtail millet (Setaria italica) is an important grain and forage crop. This crop is widely grown in Northern China (Yang et al.2020). In Aug 2021, foxtail millet variety of Jigu42 showing lodging were found in Baoding China with the incidence of 30% and irregular brown lesions were found in sheaths and leaves of infected plants. The center of the lesions was kraurotic and pale, and the edges were gray-brown or dark brown. Twelve samples with typical lesions were collected from the surveyed field to isolate the pathogen. The infected samples were cut into square pieces of about 3 to 5 mm and were immersed into NaOCl (1%) for 1 min followed by washing with sterile water for three times. Then all sterilized tissues were inoculated on potato dextrose agar (PDA) plates and incubated at 25℃. After 3 days, fresh mycelial tips grown from the tissues were transferred to new plates for purification and incubated in the dark at 25°C for 4-5 days until the hyphae covered the whole plates. The colonies of 15 isolates on PDA medium showed similar colonial characteristics, which were fluffy and white initially, gradually turned light brown, and no sclerotia was observed even at 20 days later. Micro-examination revealed that all isolates showed the identical morphological features as Rhizoctonia sp. (Sneh et al. 1991), which contained the septate and right-angled branching hyphae with slight constriction at the base of mycelial branches, and three to seven nuclei per cell (Yang et al. 2013). Total genomic DNA was extracted from 5-day-old cultures, and the internal transcribed spacer (ITS) region of rDNA was amplified with ITS1 and ITS4 as the primers (Garibaldi et al. 2019). The sequencing results showed that the nucleotide sequences of 15 amplicons were identical and shared 100% identity with the corresponding fragments of R. solani AG-4 HG-III from sugar beet (GenBank accession No. MH172666 and MH172663) in Blastn search. The sequencing size of ITS in this study was 3 bp shorter than that of sugar beet, with a length of 722, because the base 'T' in the beginning and 'GA' in the end of the sequences did not detected in our study. Phylogenetic tree of 16 isolates of different AG4 subgroups was created by the software MEGA 7.0 through the NJ method, and the showed that the isolates were clustered to the clade of AG-4 HG-III group. The sequences of three isolates were deposited in GenBank under the accession No. ON810364, ON810365 and ON810366. For pathogenicity test, 5 mm diameters plate of the 5-day-old fungus which cultured on PDA were inoculated to the sheath of 10 foxtail millet plants grown in pots at 5- or 6-leaf stage. Then, the inoculated plants were placed into a growth chamber, and the inoculated sheaths were covered with wet cotton ball for 2 days to keep humidity, while sterile water was inoculated as the control. All plants were cultivated at 26°C with 14 h light and 10 h dark for 14 days. The experiment was repeated for three times. As the result, the same lesions observed in the field appeared on the inoculated plants at 10-14 days post inoculation, whereas the mock was healthy. The pathogen was re-isolated from the infected samples. The morphological characteristics and the nucleotide sequences of ITSs were same as that of the original isolates. All in above, the pathogen cusing sheath blight on foxtail millet was identified as R. solani AG-4 HG-III. To our knowledge, this is the first report of R. solani AG-4 HG-III causing sheath blight on S. italica in China. This finding expands the host range known for R. solani AG-4 HG-III and will be helpful for developing effective control strategies of foxtail millet sheath blight.

Application of rapamycin eluting stent in popliteal artery acute plaque events: a report of four cases / 中国综合临床

Short segment lesions in the middle and upper segment of popliteal artery caused by acute plaque events can lead to moderate and severe limb ischemia, but there is little professional attention and research reports. The effect of rapamycin eluting stent in the treatment of short segment popliteal artery lesions was reviewed. The characteristics, operation methods and follow-up results of 4 cases were analyzed to summarize the operation experience and evaluate the treatment effect. In 4 cases, the operation time was short, the operation was smooth, and the technical success rate was 100%. The blood flow of the stent was good, no complications such as displacement, rupture and vascular injury were found, and the blood supply of the lower limbs was improved satisfactorily. Rapamycin eluting stent implantation can achieve good medium and long-term results in the treatment of specific middle and upper popliteal artery lesions.

Study on optimization and stability of Artemisia annua pollen-induced allergic rhinitis model in mice / 中华微生物学和免疫学杂志

Objective:To optimize the challenge scheme for establishing a stable mouse model of Artemisia annua pollen-induced allergic rhinitis. Methods:BALB/c mice were subcutaneously injected with 0.1 ml allergen extract containing 20 μg/ml Art a1 from Artemisia pollen on 1 d, 4 d and 7 d. One week after the sensitization, these mice were divided into three groups and intranasally challenged with Artemisia annua pollen allergen extract containing 500 μg/ml Art a1 for 7 (7 d group), 10 (10 d group) and 14 (14 d group) consecutive days, respectively. The first challenge was followed by another 7 days of challenge every four weeks. Blank control group was set up through sensitizing and challenging BALB/c mice with normal saline. Behavioral changes and nasal pathological changes were observed. The changes in humoral and cellular responses were also detected. After the first challenge cycle was decided, the challenge frequency was further optimized. Results:After the first challenge, the allergic symptoms of mice in 10 d group were significantly severe than those in 7 d and 14 d groups, and the levels of serum specific IgE antibody in 10 d and 14 d groups were significantly higher than that in 7 d group. After the second challenge, the mice in the three model groups still had obvious allergic symptoms as compared with the blank control group. There were obvious pathological changes in the nose, including epithelial cell proliferation, turbinate enlargement and inflammatory cell increase. Moreover, the level of serum specific IgE antibody increased significantly and the proliferation of antigen-specific IL-4 and IL-6 lymphocytes was significantly up-regulated, especially in 10 d and 14 d groups. The frequency of challenge had a great impact on the stability of the allergic model. The allergic symptoms of sensitized mice challenged every two weeks were significantly severe than those of mice challenged every four weeks and the level of serum antigen-specific antibody was also higher.Conclusions:This study optimized the first challenge cycle and challenge frequency for establishing a mouse model of Artemisia annua pollen-induced allergic rhinitis, which provided reference for the establishment of drug efficacy evaluation system for desensitization therapy.

Investigation of medication adherence for superficial mycoses among students in a college based on Morisky Medication Adherence Scale / 药学实践杂志

Objective To investigate the medication adherence of military academy students with superficial mycoses. Methods A 8-item Morisky medication adherence scale (MMAS) was modified into 7-item scale to evaluate the compliance of antifungal drugs in the sick students. The reliability and validity of the scale were analyzed. Results A total of 243 questionnaires were collected, of which 242 were valid questionnaires. 90.08% of the students took topical medication and 8.68% were treated both with topical and systematic combination. High, medium and low medication adherence rates as assessed by the modified MMAS were 9.09%, 23.97% and 66.94%, respectively. The reliability analysis showed that the internal consistency coefficient (Cronbach’s α ) was 0.781，and the adjusted Cronbach’s α was 0.790, indicating the high reliability of the scale. The KMO value was 0.798, and the Bartlett’s spherical test value was 440.866, P=0.000. One factor was extracted by exploratory factor analysis. The factor loadings of the items were all above 0.5. Therefore, the high convergent validity was good. Conclusion The modified MMAS has good reliability and validity and is applicable for the evaluation of medication compliance for superficial mycoses. In this study, the military students have a low level of medication adherence for superficial mycoses. Effective measures should be taken to help students strengthen their daily medication management and improve compliance.

Shear wave elastography to evaluate the protective effect of testicular torsion fascial compartment decompression treatment on spermatogenesis / 中华超声影像学杂志

Objective:To investigate the feasibility of shear wave elastography (SWE) to evaluate the protective effect of testicular compartment decompression on spermatogenesis after testicular torsion reduction.Methods:Thirty-two rabbits were randomly divided into 4 groups: control group (S group), testicular torsion simple reduction group (I group), and testicular torsion reduction+ compartment decompression group (T group: T1 group, T2 group), 8 rabbits per group. After the complete testicular torsion model was established in the I and T groups, the I group was simply reperfused, and the T group was reperfused before decompression of the compartment. Rabbits in each group were kept for 30 days after successful modeling. Each group of rabbits underwent testicular fascia intracompartment pressure measurement and SWE examination before operation, after successful complete torsion modeling, after reperfusion and 30 days later. After the experiment, the surgical side testicles were taken for pathological examination.Results:After testicular torsion, the pressure of testicular fascia and the average Young′s modulus (Emean) of testicular tissue in each experimental group increased (all P<0.05), and further increased with the extension of torsion time (all P<0.05). After reperfusion, the testicular fascial compartment pressure and testicular tissue Emean value in group I further increased (all P<0.05), while the testicular fascial compartment pressure and testicular tissue Emean value in group T decreased (all P<0.05). Thirty days later, testicular fascial compartment pressure and testicular tissue Emean value in group I were higher than those in group T (all P<0.05), while Johnsen′s score of testicular tissue was lower than that in group T ( P<0.05), and testicular tissue apoptosis index and malondialdehyde content were higher than those in group T Group T (all P<0.05). Conclusions:Decompression of the testicular compartment has a protective effect on spermatogenesis after testicular torsion reduction. SWE can indirectly evaluate the severity of testicular compartment syndrome after testicular torsion and reduction, and the protective effect of compartment decompression on spermatogenesis.

Analyses of low-dose chemotherapy effect and prognostic factors in advanced gastrointestinal cancer patients with weak physical condition / 肿瘤研究与临床

Objective:To investigate the clinical therapeutic effect of low-dose chemotherapy in advanced gastrointestinal cancer patients with weak physical condition, and to evaluate the related factors affecting the prognosis.Methods:The clinical data of 118 advanced gastrointestinal cancer patients with weak physical condition who were admitted to Xiamen Branch, Zhongshan Hospital, Fudan University from March 2018 to May 2019 were retrospectively analyzed. Kaplan-Meier method was used to make survival analysis in patients stratified by different factors, and log-rank was used to test. Multiple factor analysis of prognosis was performed by using Cox regression model. The association of clinicopathological factors with the prognosis was evaluated.Results:Among 118 patients, 16 (13.6%) cases were esophageal cancer, 41 (34.7%) cases were gastric cancer, 48 (40.7%) cases were colorectal cancer, 13 (11.0%) cases were pancreatic cancer. The overall survival (OS) rates of 6 months and 1-year were 44.1% and 10.2%, respectively. Survival analysis showed that patients with hemoglobin ≥100 g/L, serum albumin > 40 g/L and normal gastrointestinal function had better OS (all P < 0.05), while patients with physical status score of 3 scores and pain had worse OS (all P < 0.05). Cox multivariate analysis showed that after adjusting age and gender, hemoglobin level ( HR = 0.314, 95% CI 0.241-0.977, P = 0.001), pain ( HR = 2.016, 95% CI 1.697-7.038, P = 0.008) and gastrointestinal function ( HR = 1.751, 95% CI 1.607-6.080, P = 0.036) were independent influencing factors of OS in advanced gastrointestinal cancer patients with weak physical condition after receiving low-dose chemotherapy. Conclusions:Low-dose chemotherapy can still benefit advanced gastrointestinal cancer patients with weak physical condition. Hemoglobin level, pain and gastrointestinal function are independent prognostic factors for these patients.

Progress of programmed death ligand 2 in immunotherapy of solid tumors / 肿瘤研究与临床

With the breakthrough of immune checkpoint blocking therapy in the clinical treatment of a variety of malignant solid tumors, tumor immunotherapy has opened a new era. However, clinical practice has proved that the response rate of this therapy is low. Seeking for the key factors limiting its response rate has become a research hotspot in this field. Programmed death ligand 2 (PD-L2) is the second important ligand binding to programmed death 1 (PD-1) after programmed death ligand 1 (PD-L1). The encoded protein can bind to PD-1 and then inhibit immune cells. This paper reviews the biological characteristics of PD-L2, its mechanism in immune regulation and its research progress in solid tumor immunotherapy to provide more theoretical support for solid tumor immunotherapy.

Virus-free and live-cell visualizing SARS-CoV-2 cell entry for studies of neutralizing antibodies and compound inhibitors

The ongoing COVID-19 pandemic, caused by SARS-CoV-2 infection, has resulted in hundreds of thousands of deaths. Cellular entry of SARS-CoV-2, which is mediated by the viral spike protein and host ACE2 receptor, is an essential target for the development of vaccines, therapeutic antibodies, and drugs. Using a mammalian cell expression system, we generated a recombinant fluorescent protein (Gamillus)-fused SARS-CoV-2 spike trimer (STG) to probe the viral entry process. In ACE2-expressing cells, we found that the STG probe has excellent performance in the live-cell visualization of receptor binding, cellular uptake, and intracellular trafficking of SARS-CoV-2 under virus-free conditions. The new system allows quantitative analyses of the inhibition potentials and detailed influence of COVID-19-convalescent human plasmas, neutralizing antibodies and compounds, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. This approach may also be adapted to develop a viral entry visualization system for other viruses.

Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells

The global pandemic of Coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable in vitro neutralization assay is very important for the development of neutralizing antibodies, vaccines and other inhibitors. In this study, G protein-deficient vesicular stomatitis virus (VSVdG) bearing full-length and truncated spike (S) protein of SARS-CoV-2 were evaluated. The virus packaging efficiency of VSV-SARS-CoV-2-Sdel18 (S with C-terminal 18 amino acid truncation) is much higher than VSV-SARS-CoV-2-S. A neutralization assay for antibody screening and serum neutralizing titer quantification was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and human angiotensin-converting enzyme 2 (ACE2) overexpressed BHK21 cell (BHK21-hACE2). The experimental results can be obtained by automatically counting EGFP positive cell number at 12 hours after infection, making the assay convenient and high-throughput. The serum neutralizing titer of COVID-19 convalescent patients measured by VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with live SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting receptor binding domain (RBD) of SARS-CoV-2-S were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.

Development and Multicenter Performance Evaluation of The First Fully Automated SARS-CoV-2 IgM and IgG Immunoassays

BACKGROUNDThe outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread globally. The laboratory diagnosis of SARS-CoV-2 infection has relied on nucleic acid tests. However, there are many limitations of nucleic acid tests, including low throughput and high rates of false negatives. More sensitive and accurate tests to effectively identify infected patients are needed. METHODSThis study has developed fully automated chemiluminescent immunoassays (CLIA) to determine IgM and IgG antibodies to SARS-CoV-2 in human serum. The assay performance has been evaluated at 10 hospitals. Clinical specificity was evaluated by measuring 972 hospitalized patients with diseases other than COVID-19, and 586 donors of a normal population. Clinical sensitivity was assessed on 503 confirmed cases of SARS-CoV-2 by RT-PCR and 52 suspected cases. RESULTSThe assays demonstrated satisfied assay precision with coefficient of variation (CV) of less than 4.45%. Inactivation of specimen does not affect assay measurement. SARS-CoV-2 IgM shows clinical specificity of 97.33% and 99.49% for hospitalized patients and normal population respectively. SARS-CoV-2 IgG shows clinical specificity of 97.43% and 99.15% for the hospitalized patients and the normal population respectively. SARS-CoV-2 IgM and IgG show clinical sensitivity of 85.88% and 96.62% respectively for confirmed SARS-Cov-2 infection with RT-PCR, of 73.08% and 86.54% respectively for suspected cases. CONCLUSIONSwe have developed fully automated immunoassays for detecting SARS-CoV-2 IgM and IgG antibodies in human serum. The assays demonstrated high clinical specificity and sensitivity, and add great value to nucleic acid testing in fighting against the global pandemic of the SARS-CoV-2 infection.