Structural Study of Aavrh.10 Receptor and Antibody Interactions.

Recombinant adeno-associated virus (rAAV) vectors are one of the leading tools for the delivery of therapeutic genes in human gene therapy applications. For a successful transfer of their payload, the AAV vectors have to circumvent potential preexisting neutralizing host antibodies and bind to the receptors of the target cells. Both of these aspects have not been structurally analyzed for AAVrh.10. Here, cryo-electron microscopy and three-dimensional image reconstruction were used to map the binding site of sulfated N-acetyllactosamine (LacNAc; previously shown to bind AAVrh.10) and a series of four monoclonal antibodies (MAbs). LacNAc was found to bind to a pocket located on the side of the 3-fold capsid protrusion that is mostly conserved to AAV9 and equivalent to its galactose-binding site. As a result, AAVrh.10 was also shown to be able to bind to cell surface glycans with terminal galactose. For the antigenic characterization, it was observed that several anti-AAV8 MAbs cross-react with AAVrh.10. The binding sites of these antibodies were mapped to the 3-fold capsid protrusions. Based on these observations, the AAVrh.10 capsid surface was engineered to create variant capsids that escape these antibodies while maintaining infectivity. IMPORTANCE Gene therapy vectors based on adeno-associated virus rhesus isolate 10 (AAVrh.10) have been used in several clinical trials to treat monogenetic diseases. However, compared to other AAV serotypes little is known about receptor binding and antigenicity of the AAVrh.10 capsid. Particularly, preexisting neutralizing antibodies against capsids are an important challenge that can hamper treatment efficiency. This study addresses both topics and identifies critical regions of the AAVrh.10 capsid for receptor and antibody binding. The insights gained were utilized to generate AAVrh.10 variants capable of evading known neutralizing antibodies. The findings of this study could further aid the utilization of AAVrh.10 vectors in clinical trials and help the approval of the subsequent biologics.

Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic or prophylactic. Like other betacoronaviruses, attachment and entry of SARS-CoV-2 is mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in anti-viral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH and 6-O-desulfated enoxaparin with an IC50 of 5.99 {micro}g/L, 1.08 mg/L, 1.77 {micro}g/L, and 5.86 mg/L respectively. The low serum bioavailability of intranasally administered UFH, along with data suggesting that the nasal epithelium is a portal for initial infection and transmission, suggest that intranasal administration of UFH may be an effective and safe prophylactic treatment.

Glycosaminoglycan binding motif at S1/S2 proteolytic cleavage site on spike glycoprotein may facilitate novel coronavirus (SARS-CoV-2) host cell entry

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and continues to spread around the globe at an unprecedented rate. To date, no effective therapeutic is available to fight its associated disease, COVID-19. Our discovery of a novel insertion of glycosaminoglycan (GAG)-binding motif at S1/S2 proteolytic cleavage site (681-686 (PRRARS)) and two other GAG-binding-like motifs within SARS-CoV-2 spike glycoprotein (SGP) led us to hypothesize that host cell surface GAGs might be involved in host cell entry of SARS-CoV-2. Using a surface plasmon resonance direct binding assay, we found that both monomeric and trimeric SARS-CoV-2 spike more tightly bind to immobilized heparin (KD = 40 pM and 73 pM, respectively) than the SARS-CoV and MERS-CoV SGPs (500 nM and 1 nM, respectively). In competitive binding studies, the IC50 of heparin, tri-sulfated non-anticoagulant heparan sulfate, and non-anticoagulant low molecular weight heparin against SARS-CoV-2 SGP binding to immobilized heparin were 0.056 M, 0.12 M, and 26.4 M, respectively. Finally, unbiased computational ligand docking indicates that heparan sulfate interacts with the GAG-binding motif at the S1/S2 site on each monomer interface in the trimeric SARS-CoV-2 SGP, and at another site (453-459 (YRLFRKS)) when the receptor-binding domain is in an open conformation. Our study augments our knowledge in SARS-CoV-2 pathogenesis and advances carbohydrate-based COVID-19 therapeutic development.

Effect of Acupoint Injection at Quyuan (SI13) on the Vibrating Perception Threshold and Its Efficacy in Treating Cervical Spondylosis of Nerve Root Type / 上海针灸杂志

Objective To compare the vibrating perception threshold (VPT) between normal subjects and subjects with cervical spondylosis of nerve root type, and to observe the clinical efficacy of acupoint injection at Quyuan (SI13) in releasing pain in cervical spondylosis of nerve root type.Method Sixty-three patients with cervical spondylosis of nerve root type were recruited and randomized into a treatment group of 35 cases and a control group of 28 cases. The treatment group received acupoint injection at Quyuan, and the control group received acupoint injection at Jiaji (EX-B2) points. The short-form McGill Pain Questionnaire (MPQ) was adopted. From the questionnaire, the Pain Rating Index (PRI), sensory (S), affective (A), and total (T) pain rating indexes, Visual Analogue Scale (VAS), and Present Pain Index (PPI) were used to comprehensively evaluate the pain improvement and to quantify the therapeutic efficacy, and the VPT was also considered. The Clinical Assessment Scale for Cervical Spondylosis (CASCS) by West China Rehabilitation Center was also used to analyze the result in the treatment group.Result After the intervention, the MPQ and VAS scores in the treatment group were significantly lower than those in the control group (P<0.05). The total effective rate of the treatment group was significantly higher than that of the control group (P<0.05). Meanwhile, the VPT was improved after the treatment.Conclusion Acupoint injection at Quyuan can produce a more significant efficacy than at Jiaji (EX-B2) in treating cervical spondylosis of nerve root type.

Simultaneous Determination of the Three Ingredients of Zedoray Turmeric Oil in Ruxiankang Injection by HPLC / 中国药房

OBJECTIVE:To establish a method for the simultaneous determination of curdione,germacrone and furanodiene of zedoray turmeric oil in Ruxiankang injection. METHODS:HPLC was performed on the column of Zorbax SB-C18 with mobile phase of acetonitrile-water (gradient elution) at a flow rate of 1.0 ml/min,detection wavelength was 216 nm,column temperature was 30℃,and the injection volume was 10 μl. RESULTS:The linear range was 60-480 μg/ml for curdione(r=0.999 0),40-320 μg/ml for germacrone(r=0.999 0)and 40-320 μg/ml(r=0.999 0);RSDs of precision,stability and reproducibility tests were lower than 2.0%;recoveries were 95.21%-99.89%(RSD=1.6%,n=6),102.33%-104.89%(RSD=1.0%,n=6)and 97.38%-99.06%(RSD=0.7%,n=6),respectively. CONCLUSIONS:The method is simple and accurate,and can be used for simultaneous contents deter-mination of curdione,germacrone and furanodiene of zedoray turmeric oil in Ruxiankang injection.

Preparation of arbidol hydrochloride multi-layers controlled-released and evaluation of its release rate in vivo and in vitro / 吉林大学学报(医学版)

Objective To study the development of Arbidol hydrochloride(Ahyd) multi-layers controlled-released preparation and evaluate its release rate in order to provide the basis for Ahyd pharmacodynamics reseach.Methods Ahyd multi-layers was used as experiment group and Ahyd single-layer as control,high performance liquid chromatography(HPLC) was used to examine releasing level of two groups for calculating parameters of drug metabolism and bioavailability.Results Compared with Ahyd single-layer,the multi-layers controlled-released preparation had several significant advantages as below:Cmax fell remarkably(P

Development of abdominal breathing training instrument with feedback / 医疗卫生装备

Abdominal breathing is an effective training method for relaxation.It is applicable for resolving sub-health problems in autonomic nervous malfunction and curing some kinds of psychosomatic disorders.In order to improve its training effect,we develop a kind of abdominal breathing training instrument on the basis of the principle of biofeedback.With both features of biofeedback and breathing training apparatus,it may simultaneously feedback the training effect to the trainer during the abdominal breathing training,and the trainer may regulate his breathing mode for desirable effects.In this paper,the instrument is described in such aspects as the circuit design,composition and clinical application.It is an economical,safe and effective instrument,which can be used in Health Promotion Project.

Study of detection of EB virus in peripheral blood of patients with non-hodgkin lymphoma by ramification amplification method / 中华检验医学杂志

Objective To detect EB virus in peripheral blood of patients with non-hodgkin lymphoma (NHL) by ramification amplification method (RAM).To investigate the relativity of NHL with infection of EB virus.Methods RAM was used to detect EB virus with the man-made target gene sequence of promotor of EBER-1. In 120 cases of patients with NHL, EB virus were detected by RAM, and the result of RAM was compared with that of PCR.Raji cells and NB4 cells were used as positive control and negative control respectively.Results RAM could detect 10 molecular target gene of the exponent.76 cases were positive in 120 cases of NHL patients and the positive rate was 63.3%, The result is identical with that of PCR.Conclusion Ramification amplification method is sensitive, convenient and easy to perform.NHL has a high relativity with infection caused by EB virus.

Investigation of Cardiac Failure Therapy by Transplanting Skeletal Satellite Cells / 中国医师杂志

Objective Satellite cells(SCs) transplantation was tried to improve cardiac function in this experiment.Methods A myocaridium infarction was created in 23 male Suzhong pigs by ligating the diagonal ramus,and the alive pigs were divided into the experimental group and the control group.SCs were extracted,purified,cultured by modified Dorfman method,and were transplanted into myocardium by intramyocardial injection.By pathomorphological research,we observed the therapeutic outcomes of heart failure.Results ⑴10 pigs were alive(5 in the control group,and the other 5 in the experimental group).The successful rate of making mould was 43 5%.⑵After cultured in vitro for 3 days,SCs were spindle-shaped,and sticked to walls.SCs growth were stasis and each other fusing into form myotube when we delayed to divede Petri dishs or reduced the concentration of Fetus Bovine Serum(FBS) in the culture medium.⑶In the experimental group,neonate skeletal muscle cells were observed in the infarction regions,which were multi-nuclei near edges.These cells aligned in an accordance direction.Intercalated disks were not found between them.The capillary density in experimental group was much higher than the control group.Conclusions Autograft of SCS can be alive in acceptor,and improve the heart function by altering cardiac compliance and configuration,or by stimulating the blood capillary proliferation.SCs don't form intercalated disks with remnant myocardial cells,so synchro-contraction will not occur between them,but malign arrhythmia are not found.

Changes of serum cardiac troponin levels after percutaneous transluminal coronary angioplasty and its relation to late cardiac events / 介入放射学杂志

Objective To evaluate the possible effect of percutaneous transluminal coronary angioplasty(PTCA) on myocardial injury. Methods Serum cTnI and CK MB levels were measured in 173 patients with coronary artery disease undergone PTCA before and 6, 12, 24, 48 and 72 hours after the procedure. Cardiac events during follow up in these patients were recorded. Results Serum cTnI level was increased after PTCA in 42 patients, remained normal in 84, and was over baseline level before and after the procedure in 47. Serum CK MB level was above baseline before and after the procedure in one patient and increased in 10. Compared with normal cTnI group, elevated cTnI was related to total balloon inflation time, total pressure, number of dilation and stents deployed, contrast medium dose and occurrence of angina during balloon inflation ( P 0.05). Conclusion cTnI was more sensitive and specific than CK MB in identifying minor myocardial injury during PTCA. This injury was related to the intensity of PTCA, but not enough to make worse influence on overall outcome.