Immunogenicity of mucosal COVID-19 vaccine candidates based on the highly attenuated vesicular stomatitis virus vector (VSVMT) in golden syrian hamster

COVID-19 is caused by coronavirus SARS-CoV-2. Current systemic vaccines generally provide limited protection against viral replication and shedding within the airway. Recombinant VSV (rVSV) is an effective vector which inducing potent and comprehensive immunities. Currently, there are two clinical trials investigating COVID-19 vaccines based on VSV vectors. These vaccines were developed with spike protein of WA1 which administrated intramuscularly. Although intranasal route is ideal for activating mucosal immunity with VSV vector, safety is of concern. Thus, a highly attenuated rVSV with three amino acids mutations in matrix protein (VSVMT) was developed to construct safe mucosal vaccines against multiple SARS-CoV-2 variants of concern. It demonstrated that spike protein mutant lacking 21 amino acids in its cytoplasmic domain could rescue rVSV efficiently. VSVMT indicated improved safeness compared with wild-type VSV as the vector encoding SARS-CoV-2 spike protein. With a single-dosed intranasal inoculation of rVSVΔGMT-SΔ21, potent SARS-CoV-2 specific neutralization antibodies could be stimulated in animals, particularly in term of mucosal and cellular immunity. Strikingly, the chimeric VSV encoding SΔ21 of Delta-variant can induce more potent immune responses compared with those encoding SΔ21 of Omicron- or WA1-strain. VSVMT is a promising platform to develop a mucosal vaccine for countering COVID-19.

Long-term passaging of replication competent pseudo-typed SARS-CoV-2 reveals the antiviral breadth of monoclonal and bispecific antibody cocktails

The continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants poses challenges to the effectiveness of neutralizing antibodies. Rational design of antibody cocktails is a realizable approach addressing viral immune evasion. However, evaluating the breadth of antibody cocktails is essential for understanding the development potential. Here, based on a replication competent vesicular stomatitis virus model that incorporates the spike of SARS-CoV-2 (VSV-SARS-CoV-2), we evaluated the breadth of a number of antibody cocktails consisting of monoclonal antibodies and bispecific antibodies by long-term passaging the virus in the presence of the cocktails. Results from over two-month passaging of the virus showed that 9E12+10D4+2G1 and 7B9-9D11+2G1 from these cocktails were highly resistant to random mutation, and there was no breakthrough after 30 rounds of passaging. As a control, antibody REGN10933 was broken through in the third passage. Next generation sequencing was performed and several critical mutations related to viral evasion were identified. These mutations caused a decrease in neutralization efficiency, but the reduced replication rate and ACE2 susceptibility of the mutant virus suggested that they might not have the potential to become epidemic strains. The 9E12+10D4+2G1 and 7B9-9D11+2G1 cocktails that picked from the VSV-SARS-CoV-2 system efficiently neutralized all current variants of concern and variants of interest including the most recent variants Delta and Omicron, as well as SARS-CoV-1. Our results highlight the feasibility of using the VSV-SARS-CoV-2 system to develop SARS-CoV-2 antibody cocktails and provide a reference for the clinical selection of therapeutic strategies to address the mutational escape of SARS-CoV-2.

Efficient Neutralization of SARS-CoV-2 Omicron and Other VOCs by a Broad Spectrum Antibody 8G3

Numerous mutations in the spike protein of SARS-CoV-2 B.1.1.529 Omicron variant pose a crisis for antibody-based immunotherapies. The efficacy of emergency use authorized (EUA) antibodies that developed in early SARS-CoV-2 pandemic seems to be in flounder. We tested the Omicron neutralization efficacy of an early B cell antibody repertoire as well as several EUA antibodies in pseudovirus and authentic virus systems. More than half of the antibodies in the repertoire that showed good activity against WA1/2020 previously had completely lost neutralizing activity against Omicron, while antibody 8G3 displayed non-regressive activity. EUA antibodies Etesevimab, Casirivimab, Imdevimab and Bamlanivimab were entirely desensitized by Omicron. Only Sotrovimab targeting the non-ACE2 overlap epitope showed a dramatic decrease activity. Antibody 8G3 efficiently neutralized Omicron in pseudovirus and authentic virus systems. The in vivo results showed that Omicron virus was less virulent than the WA1/2020 strain, but still caused deterioration of health and even death in mice. Treatment with 8G3 quickly cleared virus load of mice. Antibody 8G3 also showed excellent activity against other variants of concern (VOCs), especially more efficient against authentic Delta plus virus. Collectively, our results suggest that neutralizing antibodies with breadth remains broad neutralizing activity in tackling SARS-CoV-2 infection despite the universal evasion from EUA antibodies by Omicron variant.

Comprehensive Evaluation of ACE2-Fc Combination with Neutralization Antibody on Broad Protection against SARS-CoV-2 and Its Variants

Emerging SARS-CoV-2 variants are threatening the efficacy of antibody therapies. Combination treatments including ACE2-Fc have been developed to overcome the evasion of neutralizing antibodies (NAbs) in individual cases. Here we conducted a comprehensive evaluation of this strategy by combining ACE2-Fc with NAbs of diverse epitopes on the RBD. NAb+ACE2-Fc combinations efficiently neutralized HIV-based pseudovirus carrying the spike protein of the Delta or Omicron variants, achieving a balance between efficacy and breadth. In an antibody escape assay using replication-competent VSV-SARS-CoV-2-S, all the combinations had no escape after fifteen passages. By comparison, all the NAbs without combo with ACE2-Fc had escaped within six passages. Further, the VSV-S variants escaped from NAbs were neutralized by ACE2-Fc, revealing the mechanism of NAb+ACE2-Fc combinations survived after fifteen passages. We finally examined ACE2-Fc neutralization against pseudovirus variants that were resistant to the therapeutic antibodies currently in clinic. Our results suggest ACE2-Fc is a universal combination partner to combat SARS-CoV-2 variants including Delta and Omicron.

Broad ultra-potent neutralization of SARS-CoV-2 variants by monoclonal antibodies specific to the tip of RBD

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) continue to wreak havoc across the globe. Higher transmissibility and immunologic resistance of VOCs bring unprecedented challenges to epidemic extinguishment. Here we describe a monoclonal antibody, 2G1, that neutralizes all current VOCs and has surprising tolerance to mutations adjacent to or within its interaction epitope. Cryo-electron microscopy structure showed that 2G1 bound to the tip of receptor binding domain (RBD) of spike protein with small contact interface but strong hydrophobic effect, which resulted in nanomolar to sub-nanomolar affinities to spike proteins. The epitope of 2G1 on RBD partially overlaps with ACE2 interface, which gives 2G1 ability to block interaction between RBD and ACE2. The narrow binding epitope but high affinity bestow outstanding therapeutic efficacy upon 2G1 that neutralized VOCs with sub-nanomolar IC50 in vitro. In SARS-CoV-2 and Beta- and Delta-variant-challenged transgenic mice and rhesus macaque models, 2G1 protected animals from clinical illness and eliminated viral burden, without serious impact to animal safety. Mutagenesis experiments suggest that 2G1 could be potentially capable of dealing with emerging SARS-CoV-2 variants in future. This report characterized the therapeutic antibodies specific to the tip of spike against SARS-CoV-2 variants and highlights the potential clinical applications as well as for developing vaccine and cocktail therapy.

Characterization of SHATTERPROOF Homoeologs and CRISPR-Cas9-Mediated Genome Editing Enhances Pod-Shattering Resistance in Brassica napus L.

Brassica napus is the most important oil crop plant for edible oil and renewable energy source worldwide. Yield loss caused by pod shattering is a main problem during B. napus harvest. In this study, six BnSHP1 and two BnSHP2 homoeologs were targeted by the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) genome editing system and multiple SHP1 and SHP2 mutated lines were identified for evaluating the contribution for pod-shattering resistance. Our data suggest that BnSHP1A09 is probably a promising homoeolog for controlling lignin contents at dehiscence zone. Simultaneous mutation of BnSHP1A09/C04-B/A04 and BnSHP2A05/C04-A exhibited reduced lignified layer and separation layer adjacent to valves and replum. The pod-shattering resistance index (SRI) subsequently increased to 0.31 in five homoeolog mutation lines compared with the wild type (SRI = 0.036), which provide the theoretical basis for breeding of commercial pod-shattering resistance variety.

CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Since it was first recognized in bacteria and archaea as a mechanism for innate viral immunity in the early 2010s, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) has rapidly been developed into a robust, multifunctional genome editing tool with many uses. Following the discovery of the initial CRISPR/Cas-based system, the technology has been advanced to facilitate a multitude of different functions. These include development as a base editor, prime editor, epigenetic editor, and CRISPR interference (CRISPRi) and CRISPR activator (CRISPRa) gene regulators. It can also be used for chromatin and RNA targeting and imaging. Its applications have proved revolutionary across numerous biological fields, especially in biomedical and agricultural improvement. As a diagnostic tool, CRISPR has been developed to aid the detection and screening of both human and plant diseases, and has even been applied during the current coronavirus disease 2019 (COVID-19) pandemic. CRISPR/Cas is also being trialed as a new form of gene therapy for treating various human diseases, including cancers, and has aided drug development. In terms of agricultural breeding, precise targeting of biological pathways via CRISPR/Cas has been key to regulating molecular biosynthesis and allowing modification of proteins, starch, oil, and other functional components for crop improvement. Adding to this, CRISPR/Cas has been shown capable of significantly enhancing both plant tolerance to environmental stresses and overall crop yield via the targeting of various agronomically important gene regulators. Looking to the future, increasing the efficiency and precision of CRISPR/Cas delivery systems and limiting off-target activity are two major challenges for wider application of the technology. This review provides an in-depth overview of current CRISPR development, including the advantages and disadvantages of the technology, recent applications, and future considerations.

Investigation and analysis on the pressure and stressors of pediatric medical staff of a children′s hospital / 中华医院管理杂志

Objective:To provide effective measures and suggestions for alleviating the pressure on pediatric medical workers and improve hospital management system, by means of investigating the working behavior, attitude and stress status of pediatric medical staff, and analyzing the influencing factors.Methods:From March to October 2017, case studies and questionnaires were used to comprehensively analyze the sources of work stress and stress of pediatric medical staff. A total of 375 valid samples were included. Inter-group comparison was conducted through correlation analysis and independent sample t test. Results:From such aspects as the psychological contract, organizational fairness, organizational transparency, age, and seniority, the main sources of pressure for pediatric medical workers were analyzed, and it was found that organizational fairness, organizational transparency, and seniority had a greater impact on the pressure. Perceived stress was negatively correlated with organizational equity( r=-0.13, P<0.05), negatively correlated with organizational transparency( r=-0.11, P<0.05), and positively correlated with years of employment( r=0.11, P<0.05). The perceived degree of organizational fairness of the high pressure personnel( M=4.71)was lower than that of the low pressure personnel( M=5.07), the perceived transparency of the high pressure personnel( M=4.47)was lower than that of the low pressure personnel( M=4.71), and the working years of the high pressure personnel( M=11.35)were higher than that of the low pressure personnel( M=9.18). Conclusions:Pediatric medical staff were under great pressure at work. It was suggested to improve the management concept and system of the hospital, and work positively to cope with the pressure so as to enhance staff satisfaction and sense of belongings.

Safety study on infusion of PICC intraconazole injection / 中国实用护理杂志

Objective To study the safety of transfusion of itraconazole through PICC and to evaluate the effect of different amount of blood transfusion before and after the infusion. Methods Patients were recruited from January 1, 2014, until December 31, 2015, in the Hematology hematopoietic stem cell transplantation ward. Thiry-two patients were recruited in the control group. Ninety patients wererecruited in the experience group. They were randomly assigned to three groups with 30 cases each, extracting different amounts of itraconazole before infusion, back phlebotomize in group A, B, C respectively was 10.0, 0.5, 1.0 ml. Comparing the phlebitis and obstruction after ten days from the transfusion day on. Results Catheter obstruction was not observed in any case. There was a significant difference between control group (21.9%,7/32) and observation group (0) regarding the incidence of phlebitis (χ2=21.157,P 0.05). Conclusions Drawing a small amount of blood volume before itraconazole injection through PICC can effectively avoid the drug-induced catheterobstruction. What′s more, transfusion through PICC can significantly reduce the incidence of phlebitis compared with peripheral infusion.

Nursing of a pediatric patient with serious skin problems at the puncturing site of peripherally inserted central catheter after methotrexate use / 中华临床营养杂志

Objective To summarize the nursing experience of a child with serious skin problems at the puncturing site of peripherally inserted central catheter (PICC) for methotrexate (MTX) infusion.MethodsA pediatric patient from the Hematology Ward of Beijing University People''s Hospital was found to be with serious skin problems at the puncturing site of PICC for MTX infusion.Local irradiation with ultraviolet, disinfection of local skin with iodine volts and 0.9％ sodium chloride injection, coverage of the broken skin with alginate dressing, and fixation of PICC with sterile gauzes were applied.Results After 22 days of nursing, the pain and itching at the broken skin around PICC disappeared.The skin returned normal, without exudates and prolapse of catheter.Conclusions After MTX use via PICC, the children can easily become irritable due to local symptoms and their own feelings after skin allergy.Meanwhile, prolonged treatment and nursing may make the child cry and increase the stress of parents.Caregivers needs to explain the related factors of allergy to the children and their families, and give the correct nursing intervention, so as to improve local allergy and facilitate the retention of PICC.

Caenorhabditis elegans: A Model System for Anti-Cancer Drug Discovery and Therapeutic Target Identification

The nematode Caenorhabditis elegans (C. elegans) offers a unique opportunity for biological and basic medical researches due to its genetic tractability and well-defined developmental lineage. It also provides an exceptional model for genetic, molecular, and cellular analysis of human disease-related genes. Recently, C. elegans has been used as an ideal model for the identification and functional analysis of drugs (or small-molecules) in vivo. In this review, we describe conserved oncogenic signaling pathways (Wnt, Notch, and Ras) and their potential roles in the development of cancer stem cells. During C. elegans germline development, these signaling pathways regulate multiple cellular processes such as germline stem cell niche specification, germline stem cell maintenance, and germ cell fate specification. Therefore, the aberrant regulations of these signaling pathways can cause either loss of germline stem cells or overproliferation of a specific cell type, resulting in sterility. This sterility phenotype allows us to identify drugs that can modulate the oncogenic signaling pathways directly or indirectly through a high-throughput screening. Current in vivo or in vitro screening methods are largely focused on the specific core signaling components. However, this phenotype-based screening will identify drugs that possibly target upstream or downstream of core signaling pathways as well as exclude toxic effects. Although phenotype-based drug screening is ideal, the identification of drug targets is a major challenge. We here introduce a new technique, called Drug Affinity Responsive Target Stability (DARTS). This innovative method is able to identify the target of the identified drug. Importantly, signaling pathways and their regulators in C. elegans are highly conserved in most vertebrates, including humans. Therefore, C. elegans will provide a great opportunity to identify therapeutic drugs and their targets, as well as to understand mechanisms underlying the formation of cancer.

Analysis of basic research in endocrine and metabolic projects granted by National Natural Science Foundation of China / 中华内分泌代谢杂志

ObjectiveTo investigate the effect of National Natural Science Foundation ( NSFC ) on the progress of the discipline of endocrine and metabolic research from 1987 to 2010.MethodsThe data regarding the NSFC allocated to endocrine and metabolic research from 1987 to 2010 were collected.Total expenses and numbers of the majority of programs,unit distribution,times of funding and the situation of completed program finished in recent two years were provided.ResultsFrom 1987 to 2010,a total of 731 projects and 178 398 thousands Yuan expenses of NSFC were allocated to endocrine and metabolic research.The detailed allocations are as follows:general program ( n =462 ),Young Scientists Fund ( n =187 ),regional fund ( n =28 ),Key Program ( n =9 ),National Science Fund for Distinguished Young Scholars ( n =5 ),Joint Research Fund for Overseas Chinese Young Scholars( n =2 ),Fund for Creative Research Groups ( n =1 ),International ( regional )joint research program ( n =11 ),and the others ( n =26 ).Taking the projects ( n =102 ) completed in 2009 and 2010 as an example,279 papers were published in Science Citation Index ( SCI ) included journals and 236 papers were published in Chinese journals.During the time of the projects completed,8 post-doctoral students,169 students for PhD degree,and 227 students for Master degree have been graduated.ConclusionOver the past 25 years,the continuously increased funding of NSFC on endowrine and metabolic research has led to substantial achievement.The grants of talent training and research program have increased dramatically,and the units of funding increased yearly.Talent training and subject-specific development have increased greatly.

The relationship between hyperuricaemia and clinic pathology of IgA nephropathy / 中华内科杂志

objective To analyze the correlation between the level of serum uric acid and the clinical and pathological features of IgA nephropathy.Methods Totally 148 patients diagnosed as IgA nephropathy by renal biopsy in our hospital from January 2007 to December 2010 were divided into hyperuricaemic group(41 cases)and non-hyperuricaemic group(107 cases)according to the level of serum uric acid.The clinical parameters and renal pathology grade were compared.Results There were significant differences between hyperuricaemic group and non-hyperuricaemic group in the incidences of hypertension(63.4%vs 38.3%),disease duration[(18.90±10.12)months vs(9.46±3.91)months]and body mass index[(22.81±3.60)kg/m2vs(15.32±2.54)kg/m2](all P0.05).The blood urea nitrogen(BUN)[(8.93±4.28)mmol/L vs (5.21±2.18)mmol/L],creatinine(Cr)[(155.96±107.72)μmol/L vs(79.52±40.01)μmol/L],serum triglycerides[(2.11±1.06)mmoVL vs(1.86±1.20)mmol/L]and 24-hour urine protein amount [(4328.16±1434.25)mg/24 h vs(2885.10±1388.15)mg/24 h]were significantly different between the two groups(all P<0.05).The percentage of Lee's grade I+Ⅱin hyperuricaemic group was 12.2%,and IV+V grade was 39.0%,while percentage of Lee's grade I+Ⅱin non-hyperuricaemic group was 25.2%,and IV+V grade was 16.9%(P<0.05).Tubulointerstitial lesions(TIL)gradeⅢ+IV was more in hyperuricaemic group,which was 68.3%,while TIL grade II was more in non-hyperuricaemic group,which was 76.6%.Renal artery damage grade II+Ⅲ was more in hyperuricaemic group.which was 73.2%,while renal artery damage grade 0+1 was more in non-hyperuricaemic group,which was 69.2%.Conclusion The level of serum uric acid was related with 24-hour urine protein amount,blood pressure and kidney function in IgA nephropathy,and Lee's grade,TIL grade and renal artery damage grade were severe in hyperuricaemic group.

Urotensin Ⅱ aggravated β-glycerophosphate-stimulated calcification in cultured rat vascular smooth muscle cells / 中国药理学通报

Aim To investigate the effect of urotensin Ⅱ on vascular calcification.Methods Calcified VSMCs of rat in vitro were induced by β-glycerophosphate.Cellular calcium content,ALP activities,~(45)Ca accumulation and osteocalcin content were measured.Results Compared with those of control group,calcium content,ALP activities,~(45)Ca uptake and osteocalcin in calcified VSMCs increased greatly(P<0.01).Calcium content,ALP activities,~(45)Ca uptake and osteocalcin of calcified VSMCs stimulated by urotensin Ⅱ (10~(-10)、10~(-9) and 10~(-8) mol·L~(-1))were greatly increased in a concentration-dependent manner as compared with those of calcified group(P<0.01).Conclusion UrotensinⅡ aggravates the calcification of VSMCs induced by β-glycerophosphate.

Urotensin Ⅱ aggravated ?-glycerophosphate-stimulated calcification in cultured rat vascular smooth muscle cells / 中国药理学通报

Aim To investigate the effect of urotensin Ⅱ on vascular calcification.Methods Calcified VSMCs of rat in vitro were induced by ?-glycerophosphate.Cellular calcium content,ALP activities,45Ca accumulation and osteocalcin content were measured.Results Compared with those of control group,calcium content,ALP activities,45Ca uptake and osteocalcin in calcified VSMCs increased greatly(P

Role of N-terminal pro-brain natriuretic peptide in differentiating heart failure in the patients with acute dyspnea / 中国实用内科杂志

Objective To study the differential diagnostic value of serum N-terminal pro-brain natriuretic peptide(NT-proBNP)in the patients with acute dyspnea.Methods Sixty-two patients in Medical Department of the People's Hospital of Huangpu were included in this study.Patients were divided into two groups:dyspneic patients with heart failure and without heart failure.Thirty healthy cases were selected as the control group.The concentration of serum NT-proBNP,LVEF was determined in all cases.We analyzed the relationship between NT-proBNP and LVEF.Furthermore we analyzed the relationship between NT-proBNP and the NYHA Class.Results LVEF of the control group,dyspneic patients without heart failure and with heart failure was(60.82?5.53)%,(55.92?5.62)% and(40.25?4.85)%.It had significant difference in the control group,dyspneic patients without heart failure and with heart failure(P

Changes of heme oxygenase-carbon monoxide system in vascular calcification in rats / 中国病理生理杂志

AIM: To investigate the change of heme oxygenase (HO)-carbon monoxide (CO)-cyclic guanosine monophosphate (cGMP) pathway in vascular calcification, to clarify the cellular and molecular mechanimsm in vascular calcification.METHODS: Vascular calcification model was established in rats by using vitamin D 3 and nicotine. The relative content of HO-1 mRNA, immunochemistry (IH) for HO-1, HO activity, HbCO formation and content of cGMP in aorta were measured. RESULTS: Compared to those of control rats, the HO-1 mRNA level in vessels of rats in VDN group(vascular calcification group) were decreased by 34.9% ( P

Changes in L-arginine/nitric oxide pathway of the aorta in septic shock rats / 中国病理生理杂志

AIM: To observe the change of nitric oxide (NO) generation system in the vascular adventitia, media and intima in septic shock rats. METHODS: The septic shock model was made in rats by caecal ligation and puncture. The intima, media and adventitia of the rat aorta were separated. NO production (NO - 2) , nitric oxide synthase(NOS) activity and L-arginine (L-Arg) transport were measured, separately. Inducible NOS (iNOS) distribution was detected by immunohistochemistry. RESULTS: Both in early and late stage of septic shock, NO - 2 from the intima was decreased by 66.1% and 78.9%( P

Taurine attenuated ?-glycerophosphate-stimulated calcification in cultured rat vascular smooth muscle cells / 中国病理生理杂志

AIM: To investigate the effect of taurine on calcification of vascular smooth muscle cells (VSMCs).METHODS: Calcified VSMCs of rat in vitro were induced by ?-glycerophosphate. Cellular calcium content,alkaline phosphatase(ALP) activities and accumulation were measured. DNA synthesis were evaluated by -thymidine (-TdR) incorporation. RESULTS: Calcium content,ALP activities and uptake of calcified VSMCs stimulated by taurine (5-20 mmol/L) were greatly decreased in a concentration-dependent manner as compared with calcified group ( P

Alterations of nitric oxide synthase in cardiac sarcoplasmic reticulum from rats with myocardial calcification / 中国病理生理杂志

AIM: To study alterations of nitric oxide synthase (NOS) in cardiac sarcop lasmic reticulum from rats with myocardial calcification, and to explore the mec hanism of inhibition of SR function in the rats with myocardial calcification. METHODS: The myocardial calcification rat models were prepared by vit amin D3 plus nicotine for 2 weeks and 6 weeks. Cardiac SR was separated by centrifugating. T he nitric oxide (NO) production, NOS activity and NOS protein expression in the SR were perfor med. RESULTS: Compared with control, myocardial calcium content in t he 6 weeks i ncreased by 408%(P