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BACKGROUND:To investigate the research focus and follow-up research trend of exosomes in the diagnosis and treatment of chronic kidney disease,in order to provide a corresponding reference basis for the future research of exosomes in the diagnosis and treatment of chronic kidney disease,and promote the development of this field. OBJECTIVE:To conduct a bibliometric analysis of relevant studies in each database painstakingly until now for public publication on exosome diagnosis and treatment of chronic kidney disease,to explore the current state and trend of the field in this discipline,and to predict future research directions. METHODS:A computerized search was performed on WanFang,CNKI,CBM,VIP,Web of Science,Cochrane Library,PubMed,and Embase databases from inception to December 2022 for published literature related to the diagnosis and treatment of chronic kidney disease by exosomes.The literature transcripts were screened by NoteExpress for co-occurrence,clustering and mutational analysis among authors,institutions,and keywords through CiteSpace 6.1R4 and VOSviewer software,and the visual knowledge map was plotted. RESULTS AND CONCLUSION:(1)A total of 804 articles,including 133 in Chinese and 671 in English,were included,and the volume of publications climbed year by year with a rapid trend.We included 3 649 literature authors,including 326 Chinese authors and 3 323 English authors,and the field has formed a core team centered on scholars such as Liu Bicheng,Wang Bin,Lyu LinLi,Wang Xiaonan and Wang Haidong,and has formed a stable multicenter collaboration platform among institutions.Research focuses on the three functions of exosomes:carrier,diagnosis and therapy.(2)As a form of extracellular vesicles,exosomes have important mechanisms for carrying,transferring molecular mediators and signal transduction,and have an important role in the physiopathological development of chronic kidney disease,which can provide important health surveillance data for epidemiological studies and clinical decision-making.In recent years,the development of relevant studies on exosome-based diagnosis of chronic kidney disease has expanded dramatically,forming a development layout of collaborative cooperation among multiple institutions worldwide,led by our scientific research institutions.However,at present,the study of the specific function and mechanism of action of exosomes and contents in the disease process has not been fully validated.Their significance for the early diagnosis and prognosis evaluation of chronic kidney disease is not very clear.The intrinsic mechanism of action-related research is still relatively poor.Isolation and purification techniques still need to be improved,and high-quality evidence-based clinical trials with multicenter and large samples have not yet appeared,which still need to be verified by further studies.
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BACKGROUND:The stability of the pelvis is mainly determined by the posterior pelvic ring and the sacroiliac joint.The posterior pelvic ring injury and the dislocation of the sacroiliac joint caused by high energy impacts such as car accidents increase year by year.Surgical treatment is the best method,and there are many kinds of endophytorepair methods in clinical practice,but which treatment method has the best biomechanical properties is still controversial. OBJECTIVE:To compare the biomechanical properties of three kinds of internal implants:anterior double plates,posterior bridging plate and tension nail in the repair of unilateral unstable pelvic posterior ring injury,to provide a reference for the clinical treatment and development of a new pelvic tension screw. METHODS:(1)Finite element simulation:Mimics,Wrap and SolidWorks were used to establish normal pelvic model,unilateral injured pelvis model,and three kinds of internal implant repaired models(anterior double plates,posterior bridging plate and tension nail).Ansys was used to analyze the stress and deformation of the models.(2)Biomechanical test:A total of 15 intact pelvic specimens were randomly grouped into five groups,normal pelvic model,unilateral injured pelvis model,anterior double plates,posterior bridging plate and tension nail groups.The mechanical test was performed using an Instron E10000 testing machine. RESULTS AND CONCLUSION:(1)Simulation:In the normal pelvic model,the average displacement of the sacrum was 0.174 mm,and the maximum stress of the sacral iliac bone was 10.51 MPa,and the stress distribution was uniform.The mean sacral displacement of the unilateral injured pelvis model was 0.267 mm,and the stress concentration of the model was obvious.The mean displacement of the sacrum in the three repaired models was close to that in the normal pelvic model,and the stress distribution of the sacral iliac bone in the tension nail repaired model was uniform.(2)Mechanical test:The stiffness of the normal pelvic model was(226.38±4.18)N/mm,and that of the unilateral unstable pelvic model was the smallest(130.02±2.19)N/mm.The deviation of the normal pelvic model stiffness and the three repaired models'stiffness were all within(±10%),and the repair effect was obvious.(3)The simulation results were in agreement with the experimental results.(4)The biomechanics of the tension nail repaired model was the most similar to that of the normal pelvis,and this method was the best.The repairing stiffness of the anterior double plate was too large,and the stress shielding effect was more significant.The posterior bridging plate repair could not solve the compensatory effect of the normal side soft tissue and had defects.This study provides an optimal basis for clinical surgery.(5)The new type of pelvic tension nail should be improved from the point of view of the tension nail to retain the good biomechanical properties of the tension nail,while adding other advantages,such as being used for the osteoporotic pelvis.
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BACKGROUND:Autophagy and ferroptosis play important roles in the development of chronic kidney disease,but the molecular mechanisms and gene targets related to autophagy and ferroptosis in renal tissue of chronic kidney disease are still unclear. OBJECTIVE:To screen differentially expressed genes in chronic kidney disease-related datasets based on bioinformatics,and to explore potential key biomarkers suitable for screening renal function progression in patients with chronic kidney disease. METHODS:(1)The GSE137570 dataset was obtained from GEO database to screen the differentially expressed genes by Networkanalyst database analysis.Ferroptosis and autophagy related targets were obtained by OMIM,GENECARD,FerrDb and HAMdb databases.The respective data were intersected to obtain autophagy-ferroptosis related differentially expressed genes in chronic kidney disease for parallel enrichment analysis.The STRING website was used to construct the protein-protein interaction network of differentially expressed genes,which was imported into Cytoscape software and analyzed by MCODE and Cytohubba plug-in to screen potential core targets.Enrichment analysis was performed to obtain the functions of these potential core targets.(2)In the in vitro experiment,mouse renal tubular epithelial cells were divided into two groups:the control group received no intervention,while the model group was stimulated with 5 ng/mL transforming growth factor β1 for 24 hours to induce mesenchymal transformation of renal tubular epithelial cells.Flow cytometry was used to measure the levels of reactive oxygen species and changes in mitochondrial membrane potential in the cells.RT-PCR was employed to assess ferroptosis,autophagy-related markers,and the mRNA expression of potential core targets in the cells. RESULTS AND CONCLUSION:After screening the GSE137570 dataset,a total of 480 differentially expressed genes were obtained,including 104 upregulated genes and 376 downregulated genes(log2|(FC)|>1,P<0.05).There were 562 ferroptosis-related targets and 1 266 autophagy-related targets obtained from the OMIM,GENECARD,FerrDb,and HAMdb databases.Intersection of differentially expressed genes with ferroptosis-and autophagy-related targets yielded 15 ferroptosis-related targets and 18 autophagy-related targets,respectively.The enrichment analysis results indicate that ferroptosis-related differentially expressed genes are primarily involved in biological processes such as sulfur amino acid metabolism,neutrophil degranulation,and ferroptosis signaling pathways.Autophagy-related differentially expressed genes are mainly enriched in biological processes such as platelet degranulation,extracellular matrix degradation,and receptor tyrosine kinase signaling.After screened by MCODE and CytoHubba,key genes were identified in the protein-protein interaction network,including CD44,ALB,TIMP1,PLG,CCL2,and DPP4.Immune infiltration analysis results indicate that immune cells such as B cells,CD4+ T cells,NK cells,and monocytes show significant differential expression in renal tissue after chronic kidney disease,and the core targets are also significantly correlated with these immune cells(P<0.05).The results of receiver operator characteristic curve analysis further demonstrate that the pathological progression of chronic kidney disease can be effectively diagnosed by CD44,ALB,TIMP1,PLG,CCL2,and DPP4.Single-cell sequencing results show that,except for PLG,the expression of target genes in the renal tissue of mice in each model group is generally consistent with the results of this experiment.RT-PCR results demonstrate that,for the validation of autophagy and ferroptosis phenotypes,compared with the control group,the model group shows a significant decrease in mRNA expression of LC3B,Nrf2,and SLC7A11(P<0.05),and a significant increase in P62 mRNA expression(P<0.05).Regarding the validation of potential core targets,compared with the control group,the model group exhibits a significant decrease in mRNA expression of ALB and PLG(P<0.05),and a significant increase in TIMP1 and CCL2 mRNA expression(P<0.05).Overall,these findings indicate that,through bioinformatics analysis and experimental validation,CD44,ALB,TIMP1,PLG,and CCL2 are abnormally expressed in the renal tissue of patients with chronic kidney disease,closely correlated with estimated glomerular filtration rate and tubulointerstitial fibrosis,and maybe play a predictive role in the progression of chronic kidney disease.
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Objective:To assess the efficacy and safety of the treat-and-extend (TAE) regimen and pro re nata (PRN) regimen of intravitreal conbercept in polypoidal choroidal vasculopathy (PCV) patients.Methods:A non-randomized controlled study was performed.Ninety-one patients (91 eyes) diagnosed with treatment-na?ve PCV from October 2016 to January 2019 at Department of Ophthalmology, Peking University People's Hospital were enrolled.All the patients received the intravitreal injection of 0.5 mg conbercept.After the initial treatment, the patients were divided into 3+ PRN group and 3+ TAE group according to their willingness.The follow-up time was one year.All the eyes underwent visual acuity test with ETDRS chart, optical coherence tomography (OCT) examination, fundus fluorescein angiography (FFA) and indocyanine green angiography (ICGA). Best corrected visual acuity (BCVA), central retinal thickness (CRT), maximum retinal thickness (MRT), pigment epithelium detachment (PED) height, the number and area of polypoidal lesions, the area of retinal hemorrhage and the area of branching vascular network (BVN) were recorded.Treatment interval and injection frequencies during the one-year follow-up were compared between the two groups.This study adhered to the Declaration of Helsinki.The study protocol was approved by Peking University People's Hospital (No.2020PHB250-01). Written informed consent was obtained from each patient.Results:One-year after treatment, the BCVA improvement in the 3+ PRN group and 3+ TAE group was 5.0(-2.0, 15.0) and 6.0(-1.0, 14.0) letters, respectively, showing no significant difference ( Z=-0.352, P=0.725). No significant differences were found in CRT, MRT and PED height between the two groups ( Z=-0.145, -0.529, -0.985, all at P>0.05). There was no significant difference in polypoidal lesions number, polypoidal lesions area, the number of eyes with different degrees of polyp regression, BVN area and retinal hemorrhage area between the two groups ( Z=-0.502, -0.300, -0.047, -0.265, -1.243, all at P>0.05). After the one-year follow-up, the mean injection frequency of 3+ PRN group was (7.6±0.9) times, which was lower than (8.4±2.0) times of 3+ TAE group, showing a significant difference ( t=2.432, P=0.019). The mean follow-up frequency was (11.3±1.5) times of 3+ PRN group, which was significantly higher than (10.1±1.7) times of 3+ TAE group ( t=3.403, P=0.001). For the 3+ TAE group, 17.1%(6/35) of patients achieved an extension interval of 12 weeks after the first 3 doses, and 48.5%(17/35) of patients achieved an extension interval of 8 weeks or more, with a mean maximum extension interval of (9.5±2.0) weeks.During the follow-up, 10 patients in 3+ PRN group and 8 patients in 3+ TAE group received photodynamic therapy as a rescue treatment. Conclusions:The 3+ PRN and 3+ TAE regimens of intravitreal injection of conbercept combined with photodynamic therapy as a rescue treatment have similar efficacy in visual and anatomical outcomes for PCV patients.3+ TAE regimen has a higher treatment frequency and fewer follow-up visits.
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ObjectiveTo analyze the utilization of outcome indexes and other trial design elements in randomized controlled trials （RCTs） of Chinese medicine for diabetic kidney disease （DKD） and provide a basis for the design of clinical trials and the development of core outcome index sets for Chinese medicine treatment of DKD. MethodSeven medical databases （CNKI， Wanfang Data， VIP， SinoMed， etc.） and two clinical trial registration centers （clinicaltrials.gov and chinadrugtrials.org.cn） were searched for RCTs of Chinese medicine for DKD published in the past 5 years. The included studies were assessed for risk of bias using the Cochrane Handbook for Systematic Reviews of Interventions， and the outcome indexes and other trial design elements were statistically analyzed. ResultNinety-seven RCTs were enrolled， including five trial registration protocols. The overall risk of bias was found to be high in the included studies. Stage Ⅲ DKD （36 studies， 41.38%） and the Qi-Yin deficiency with blood stasis syndrome （16 studies， 26.23%） were the top DKD stage and traditional Chinese medicine （TCM） syndrome， respectively. The treatment duration ranged from 2 weeks to 96 weeks， with 12 weeks being the most common duration （52 studies， 56.52%）. A total of 152 outcome indexes were used in 92 RCTs and five registered trials， with a frequency of 1 040 times. These indexes were classified into eight categories： Laboratory tests （blood）， laboratory tests （urine）， clinical efficacy， TCM syndrome score， quality of life scales， vital signs， other indexes， and other events. The most frequently used outcome indexes were serum creatinine （68 times， 70.10%）， clinical response rate （55 times， 56.70%）， fasting blood glucose （51 times， 52.58%）， blood urea nitrogen （48 times， 49.48%）， total cholesterol （47 times， 48.45%）， and 24-hour urinary protein excretion （43 times， 44.33%）. Safety indexes were used in 56 RCTs and two registered trials， with 53 different indexes and a frequency of 227 times. The most frequently used safety indexes were adverse reactions （49 times， 84.48%）， liver function （28 times， 48.28%）， complete blood count （24 times， 41.38%）， electrocardiogram （17 times， 29.31%）， and urinalysis （14 times， 24.14%）. Ten RCTs and five registered trials reported primary outcome indexes， and 54 RCTs reported clinical response rates. ConclusionThe current design of outcome indexes in RCTs of Chinese medicine for DKD is not standardized. In the future， efforts should be made to develop core outcome index sets that highlight the characteristics of TCM， improve the quality of clinical research， and enhance the applicability of trial results.
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BACKGROUND: To compare widefield swept-source optical coherence tomography angiography (SS-OCTA) with ultra-widefield fundus fluorescein angiography (UWF-FA) for detecting retinal vein occlusion (RVO) lesions. METHODS: Thirty-four eyes of 32 patients with treatment-naïve RVO were enrolled at Peking University People's Hospital from September 2021 to March 2022. Patients were imaged with a UWF-FA (200°) and a widefield SS-OCTA using 24 × 20 mm scan single capture. Quantitative assessments of RVO lesions such as foveal avascular zone (FAZ) area and perimeter, non-perfusion areas (NPA), number of microaneurysms (MAs), capillary changes and collateral vessels were performed. RESULTS: The measurement of FAZ area and perimeter were comparable between SS-OCTA and UWF-FA (0.373 (range, 0.277-0.48) mm2 vs. 0.370 (range, 0.277-0.48) mm2, P = 0.818 and 2.480 (range, 2.011-2.998) vs. 2.330 (range, 2.027-2.807) mm, P = 0.536, respectively). Intraclass correlation coefficients (ICCs) of FAZ area and perimeter between SS-OCTA and UWF-FA was high (0.999, [0.997-0.999] and 0.996 [0.991-0.996], respectively), suggesting good agreement. The mean NPA area was larger on SS-OCTA than that on UWF-FA (89.977 ± 78.805 mm2vs. 87.944 ± 77.444 mm2, P = 0.037). The ICC of NPA area was also high (0.999, [0.999-1.000]). The median of total MA count was less on SS-OCTA than on UWF-FA (7 (range, 0-19) vs.12 (range, 0-23), P < 0.001). Agreement in detecting MAs between SS-OCTA and UWF-FA was found to be good (ICC = 0.920, [0.555-0.974]).The total capillary changes and collateral vessels count were less on UWF-FA than SS-OCTA (11 ± 9 vs 6 ± 7, P < 0.001 and 4 (range, 0-6) vs 0 (range, 0-0), P < 0.001, respectively). Agreement in detecting capillary changes and collateral vessels between OCTA and UWF-FA was found to be fair (ICC = 0.733, [0.081-0.905] and 0.564, [0.039-0.805], respectively). CONCLUSION: Compared with UWF-FA, widefield SS-OCTA was found comparable or even superior in detecting FAZ, NPA, capillary changes and collateral vessels except MAs in RVO. Widefield SS-OCTA may offer a more efficient alternative to FA for diagnosis and monitoring RVO.
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Oclusión de la Vena Retiniana , Tomografía de Coherencia Óptica , Humanos , Angiografía con Fluoresceína/métodos , Tomografía de Coherencia Óptica/métodos , Oclusión de la Vena Retiniana/diagnóstico , Oclusión de la Vena Retiniana/patología , Vasos Retinianos/patología , Fondo de OjoRESUMEN
SARS-CoV-2 variants of concern (VOCs), especially the latest Omicron, have exhibited severe antibody evasion. Broadly neutralizing antibodies with high potency against Omicron are urgently needed for understanding working mechanisms and developing therapeutic agents. In this study, we characterized previously reported F61, which was isolated from convalescent patients infected with prototype SARS-CoV-2, as a broadly neutralizing antibody against all VOCs including Omicron BA.1, BA.1.1, BA.2, BA.3 and BA.4 sublineages by utilizing antigen binding and cell infection assays. We also identified and characterized another broadly neutralizing antibody D2 with epitope distinct from that of F61. More importantly, we showed that a combination of F61 with D2 exhibited synergy in neutralization and protecting mice from SARS-CoV-2 Delta and Omicron BA.1 variants. Cryo-EM structures of the spike-F61 and spike-D2 binary complexes revealed the distinct epitopes of F61 and D2 at atomic level and the structural basis for neutralization. Cryo-EM structure of the Omicron-spike-F61-D2 ternary complex provides further structural insights into the synergy between F61 and D2. These results collectively indicated F61 and F61-D2 cocktail as promising therapeutic antibodies for combating SARS-CoV-2 variants including diverse Omicron sublineages.
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As SARS-CoV-2 Omicron and other variants of concern continue spreading around the world, development of antibodies and vaccines to confer broad and protective activity is a global priority. Here, we report on the identification of a special group of nanobodies from immunized alpaca with exceptional breadth and potency against diverse sarbecoviruses including SARS-CoV-1, Omicron BA.1, and BA.2. Crystal structure analysis of one representative nanobody, 3-2A2-4, revealed a highly conserved epitope between the cryptic and the outer face of the receptor binding domain (RBD). The epitope is readily accessible regardless of RBD in "up" or "down" conformation and distinctive from the receptor ACE2 binding site. Passive delivery of 3-2A2-4 protected K18-hACE2 mice from infection of authentic SARS-CoV-2 Delta and Omicron. This group of nanobodies and the epitope identified should provide invaluable reference for the development of next generation antibody therapies and vaccines against wide varieties of SARS-CoV-2 infection and beyond.
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Since SARS-CoV-2 Omicron variant (B.1.1.529) was reported in November 2021, it has quickly spread to many countries and outcompeted the globally dominant Delta variant in several countries. The Omicron variant contains the largest number of mutations to date, with 32 mutations located at spike (S) glycoprotein, which raised great concern for its enhanced viral fitness and immune escape[1-4]. In this study, we reported the crystal structure of the receptor binding domain (RBD) of Omicron variant S glycoprotein bound to human ACE2 at a resolution of 2.6 [A]. Structural comparison, molecular dynamics simulation and binding free energy calculation collectively identified four key mutations (S477N, G496S, Q498R and N501Y) for the enhanced binding of ACE2 by the Omicron RBD compared to the WT RBD. Representative states of the WT and Omicron RBD-ACE2 systems were identified by Markov State Model, which provides a dynamic explanation for the enhanced binding of Omicron RBD. The effects of the mutations in the RBD for antibody recognition were analyzed, especially for the S371L/S373P/S375F substitutions significantly changing the local conformation of the residing loop to deactivate several class IV neutralizing antibodies.
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Objective:To investigate the clinical features and multimodal imaging features of eyes with perifoveal exudative vascular anomalous complex (PEVAC).Methods:A retrospective case study. From February 2014 to November 2020, 7 eyes of 7 patients with PEVAC diagnosed by ophthalmology examination in Department of Ophthalmologyof Peking University People's Hospital were included in this study. There were 6 males and 1 female. The age was 60.1±9.1 years. All were monocular. The chief complaints of visual deformation and vision loss were 3 and 1 cases, respectively. All patients underwent best corrected visual acuity (BCVA), fundus color photography, optical coherence tomography (OCT), fundus fluorescein angiography (FFA). BCVA examination was performed using the standard logarithmic visual acuity chart, which was converted to logarithm of the minimum angle of resolution (logMAR) visual acuity. OCT angiography (OCTA) and indocyanine green angiography (ICGA) were performed in 4 and 2 eyes, respectively. Three eyes were treated with intravitreal injection of anti-vascular endothelial growth factor (VEGF) combined with local laser photocoagulation. Two eyes were treated with laser photocoagulation alone. The follow-up time was 16.7±19.1 months. During follow-up, relevant examinations were performed with the same equipment and methods as at the initial diagnosis. The multimodal imaging characteristics and treatment response of the affected eyes were observed.Results:The baseline logMAR BCVA was 0.33±0.19 (0.20-0.80). All eyes showed isolated hemangiomatous lesions in the macular fovea with rigid retinal exudation, and 2 adjacent isolated hemangiomatous lesions were observed in 1 eye. FFA and ICGA examination showed that all eyes with macular hemangiomatous lesions showed clear boundary and strong fluorescence in the early stage. No other retinal or choroidal vascular abnormalities were observed. On OCT examination, circular lumen-like structures with strong reflective wall near the fovea were observed in the macular region of all eyes, accompanied by intraretinal cystic lumen. The macular central retinal thickness (CMT) was 326±125 (207-479) μm. In the four eyes examined by OCTA, blood flow signals were observed in the circular lumenoid structures with strong reflective walls adjacent to the fovea. Blood flow signals were observed in the superficial capillary layer (SCP) and deep capillary layer (DCP) of the retina in 3 eyes. SCP showed blood flow signal in 1 eye. In 4 eyes treated with intravitreal injection of anti-VEGF drugs, there was no significant improvement in the intraretinal capsule space after treatment. Subretinal fluid absorption, retinal cystoid edema persisted, and rigid exudation decreased in 1 eye. CMT decreased and BCVA increased in 5 eyes treated with laser photocoagulation or laser photocoagulation alone. At last follow-up, logMAR BCVA was 0.16±0.06 (0.10-0.20) and CMT was 212±34 (154-252) μm. Compared with baseline, the difference of BCVA was statistically significant ( t=2.661, P=0.037). Conclusions:The fundus of PEVAC patients is characterized by solitary or multiple solitary hemangiomatous lesions in the macular fovea. Round lumenoid structures with strong reflective walls, with or without intraretinal cystic lumen, rigid exudate, and subretinal fluid, in which blood flow signals can be seen in OCT.
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Robust severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in nasal turbinate (NT) accounts for high viral transmissibility, yet whether neutralizing IgA antibodies can control it remains unknown. Here, we evaluated receptor binding domain (RBD)-specific monomeric B8-mIgA1 and B8-mIgA2, and dimeric B8-dIgA1 and B8-dIgA2 against intranasal SARS-CoV-2 challenge in Syrian hamsters. These antibodies exhibited comparably potent neutralization against authentic virus by competing with human angiotensin converting enzyme-2 (ACE2) receptor for RBD binding. While reducing viruses in lungs, pre-exposure intranasal B8-dIgA1 or B8-dIgA2 led to 81-fold more infectious viruses and severer damage in NT than placebo. Virus-bound B8-dIgA1 and B8-dIgA2 could engage CD209 as an alternative receptor for entry into ACE2-negative cells and allowed viral cell-to-cell transmission. Cryo-EM revealed B8 as a class II neutralizing antibody binding trimeric RBDs in 3-up or 2-up/1-down conformation. Therefore, RBD-specific neutralizing dIgA engages an unexpected action for enhanced SARS-CoV-2 nasal infection and injury in Syrian hamsters.
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New SARS-CoV-2 variants continue to emerge from the current global pandemic, some of which can replicate faster and with greater transmissibility and pathogenicity. In particular, UK501Y.V1 identified in UK, SA501Y.V2 in South Africa, and BR501Y.V3 in Brazil are raising serious concerns as they spread quickly and contain spike protein mutations that may facilitate escape from current antibody therapies and vaccine protection. Here, we constructed a panel of 28 SARS-CoV-2 pseudoviruses bearing single or combined mutations found in the spike protein of these three variants, as well as additional nine mutations that within or close by the major antigenic sites in the spike protein identified in the GISAID database. These pseudoviruses were tested against a panel of monoclonal antibodies (mAbs), including some approved for emergency use to treat SARS-CoV-2 infection, and convalescent patient plasma collected early in the pandemic. SA501Y.V2 pseudovirus was the most resistant, in magnitude and breadth, against mAbs and convalescent plasma, followed by BR501Y.V3, and then UK501Y.V1. This resistance hierarchy corresponds with Y144del and 242-244del mutations in the N-terminal domain as well as K417N/T, E484K and N501Y mutations in the receptor binding domain (RBD). Crystal structural analysis of RBD carrying triple K417N-E484K-N501Y mutations found in SA501Y.V2 bound with mAb P2C-1F11 revealed a molecular basis for antibody neutralization and escape. SA501Y.V2 and BR501Y.V3 also acquired substantial ability to use mouse and mink ACE2 for entry. Taken together, our results clearly demonstrate major antigenic shifts and potentially broadening the host range of SA501Y.V2 and BR501Y.V3, which pose serious challenges to our current antibody therapies and vaccine protection.
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Neutralizing monoclonal antibodies (nAbs) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represent promising candidates for clinical intervention against coronavirus virus diseases 2019 (COVID-19). We isolated a large number of nAbs from SARS-CoV-2 infected individuals capable of disrupting proper interaction between the receptor binding domain (RBD) of the viral spike (S) protein and the receptor angiotensin converting enzyme 2 (ACE2). In order to understand the mechanism of these nAbs on neutralizing SARS-CoV-2 virus infections, we have performed cryo-EM analysis and here report cryo-EM structures of the ten most potent nAbs in their native full-length IgG or Fab forms bound to the trimeric S protein of SARS-CoV-2. The bivalent binding of the full-length IgG is found to associate with more RBD in the "up" conformation than the monovalent binding of Fab, perhaps contributing to the enhanced neutralizing activity of IgG and triggering more shedding of the S1 subunit from the S protein. Comparison of large number of nAbs identified common and unique structural features associated with their potent neutralizing activities. This work provides structural basis for further understanding the mechanism of nAbs, especially through revealing the bivalent binding and their correlation with more potent neutralization and the shedding of S1 subunit.
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In recognizing the host cellular receptor and mediating fusion of virus and cell membranes, the spike (S) glycoprotein of coronaviruses is the most critical viral protein for cross-species transmission and infection. Here we determined the cryo-EM structures of the spikes from bat (RaTG13) and pangolin (PCoV_GX) coronaviruses, which are closely related to SARS-CoV-2. All three receptor-binding domains (RBDs) of these two spike trimers are in the "down" conformation, indicating they are more prone to adopt this receptor-binding inactive state. However, we found that the PCoV_GX, but not the RaTG13, spike is comparable to the SARS-CoV-2 spike in binding the human ACE2 receptor and supporting pseudovirus cell entry. Through structure and sequence comparisons, we identified critical residues in the RBD that underlie the different activities of the RaTG13 and PCoV_GX/SARS-CoV-2 spikes and propose that N-linked glycans serve as conformational control elements of the RBD. These results collectively indicate that strong RBD-ACE2 binding and efficient RBD conformational sampling are required for the evolution of SARS-CoV-2 to gain highly efficient infection.
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Viruses are the major aetiological agents of acute and chronic severe human diseases that place a tremendous burden on global public health and economy; however, for most viruses, effective prophylactics and therapeutics are lacking, in particular, broad-spectrum antiviral agents. Herein, we identified 2 secreted bacterial lipases from a Chromobacterium bacterium, named Chromobacterium antiviral effector-1 (CbAE-1) and CbAE-2, with a broad-spectrum virucidal activity against dengue virus (DENV), Zika virus (ZIKV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV) and herpes simplex virus (HSV). The CbAEs potently blocked viral infection in the extracellular milieu through their lipase activity. Mechanistic studies showed that this lipase activity directly disrupted the viral envelope structure, thus inactivating infectivity. A mutation of CbAE-1 in its lipase motif fully abrogated the virucidal ability. Furthermore, CbAE-2 presented low toxicity in vivo and in vitro, highlighting its potential as a broad-spectrum antiviral drug.
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The pandemic caused by emerging coronavirus SARS-CoV-2 presents a serious global public health emergency in urgent need of prophylactic and therapeutic interventions. SARS-CoV-2 cellular entry depends on binding between the viral Spike protein receptor-binding domain (RBD) and the angiotensin converting enzyme 2 (ACE2) target cell receptor. Here, we report on the isolation and characterization of 206 RBD-specific monoclonal antibodies (mAbs) derived from single B cells of eight SARS-CoV-2 infected individuals. These mAbs come from diverse families of antibody heavy and light chains without apparent enrichment for particular families in the repertoire. In samples from one patient selected for further analyses, we found coexistence of germline and germline divergent clones. Both clone types demonstrated impressive binding and neutralizing activity against pseudovirus and live SARS-CoV-2. However, the antibody neutralizing potency is determined by competition with ACE2 receptor for RBD binding. Surprisingly, none of the SARS-CoV-2 antibodies nor the infected plasma cross-reacted with RBDs from either SARS-CoV or MERS-CoV although substantial plasma cross-reactivity to the trimeric Spike proteins from SARS-CoV and MERS-CoV was found. These results suggest that antibody response to RBDs is viral species-specific while that cross-recognition target regions outside the RBD. The specificity and neutralizing characteristics of this plasma cross-reactivity requires further investigation. Nevertheless, the diverse and potent neutralizing antibodies identified here are promising candidates for prophylactic and therapeutic SARS-CoV-2 interventions.
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A novel and highly pathogenic coronavirus (2019-nCoV) has caused an outbreak in Wuhan city, Hubei province of China since December 2019, and soon spread nationwide and spilled over to other countries around the world. To better understand the initial step of infection at atomic-level, we determined the crystal structure of the 2019-nCoV spike receptor-binding domain (RBD) bound with the cell receptor ACE2 at 2.45 [A] resolution. The overall ACE2-binding mode of the 2019-nCoV RBD is nearly identical to that of the SARS-CoV RBD, which also utilizes ACE2 as the cell receptor. Structural analysis identified residues in 2019-nCoV RBD critical for ACE2 binding, and majority of which are either highly conserved or shared similar side chain properties with those in the SARS-CoV RBD. Such similarity in structure and sequence strongly argue for a convergent evolution between 2019-nCoV and SARS-CoV RBD for improved binding to ACE2 despite of being segregated in different genetic lineages in the betacoronavirus genus. The epitopes of two SARS-CoV antibodies targeting the RBD are also analyzed with the 2019-nCoV RBD, providing insights into future identification of cross-reactive antibodies.
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Introducing the continuing education of the dental hygienist in eight states of the U.S.,to understand the the category of practice,professional ability,curriculum, teaching methods and teaching evaluation standard. To analyze the continuing education in eight states to provide a reference for constructing a curriculum system that is suitable for China.
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Objective@#To analyze the clinical value of SPECT/CT in diagnosis of skull base bone invasion and clinical decision-making for nasopharyngeal carcinoma (NPC), and to compare their diagnostic value with SPECT/CT, CT, MRI, and MRI combined with SPECT (MRI-SPECT) for skull base bone invasion.@*Methods@#Before treatment, among 348 newly diagnosed NPC patients, CT scan was performed in 186 patients (group A) and the remaining 162 patients received MRI scan (group B). Clinical doctors then made clinical management decisions according to the CT or MRI results. After that, all patients underwent 99Tcm-MDP SPECT/CT examination for nasopharyngeal local tomography, and the results were provided to the clinical doctors to make clinical management decisions again. The changes between the two clinical management decisions were scored according to diagnosis, range of lesion, staging, treatment regimens, and auxiliary examination. The diagnostic value of CT scan, MRI scan, SPECT/CT and MRI-SPECT for skull base bone invasion was then evaluated and compared.@*Results@#In terms of changes in scores of clinical management decisions, the score of group A was 1.387 and group B was 0.951, showing a significant difference between the two groups by Wilcoxon test (Z=6.570, P<0.001). By χ2 test, there were correlations between CT and SPECT/CT (χ2 =98.495, P<0.001), and between MRI and SPECT/CT (χ2 =32.662, P<0.001). The consistency of CT and SPECT/CT (Kappa=0.713) was greater than MRI and SPECT (Kappa=0.449). The sensitivity of CT, MRI, SPECT/CT and MRI-SPECT was 67.1%, 84.5%, 90.8% and 100%, the specificity was 73.3%, 92.3%, 85.6% and 84.6%, and the area under the ROC curve was 0.702, 0.884, 0.882 and 0.923, respectively.@*Conclusions@#SPECT/CT has important impact on clinical management decision for NPC. In the judgement of skull base invasion, the diagnostic value of SPECT/CT is significantly higher than CT and approximately equal to MRI. SPECT/CT should be one of the routine examination methods of nasopharyngeal carcinoma. In addition, in view of its greater diagnostic value, MRI combined with SPECT should be the focus of future imaging studies.
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Objective To investigate the diagnostic efficacy of 13N-NH3 and 18F-FDG PET/CT in the evaluation of untreated gliomas.Methods A total of 45 consecutive patients (29 males,16 females;age range 14-75 years,average age (47.47± 15.64) years) with final diagnosis of glioma from August 2009 to May 2012 were retrospectively analyzed.PET/CT imaging was performed with both 18F-FDG and 13N-NH3.Imaging results were analyzed by tumor-to-gray matter (T/G) ratios.ROC curve analysis was conducted to determine the optimal T/G cutoff values between low-grade and high-grade gliomas.One-way analysis of variance was applied to assess the differential efficacy of the two tracers in different grade of glioma.Results Forty-eight separate lesions were identified (WHO grade Ⅱ,n=16;grade Ⅲ,n=12;and grade Ⅳ,n=20).The sensitivities of 13N-NH3 PET/CT and 18F-FDG PET/CT on predicting high-grade gliomas were 91％(29/32) and 66％ (21/32),respectively.The optimal T/G cutoff values for 18F-FDG and 13N-NH3 were 0.64 and 0.86 with the AUC of 0.910 and 0.943,respectively.The sensitivity and positive predictive value of predicting high-grade gliomas with optimal cutoff values were 84％ (27/32) and 93％ (27/29)for 18F-FDG PET/CT,those for 13N-NH3 PET/CT were 94％ (30/32) and 94％ (30/32),respectively.Conclusion 13N-NH3 PET/CT is superior to 18F-FDG PET/CT not only in separating low-grade gliomas from high-grade ones,but also in the detection of high-grade gliomas for better tumor to normal gray matter contrast.