AAV8 transduction capacity is reduced by prior exposure to endosome-like pH conditions

Adeno-associated virus(AAV) is an essential instrument in the neuroscientist's toolkit, which allows delivery of DNA to provide labeling with fluorescent proteins or genetic instructions to regulate gene expression. In the field of neural regeneration, the transduction of neurons enables the observation and regulation of axon growth and regeneration, and in the future will likely be a mechanism for delivering molecular therapies to promote sprouting and regeneration after central nervous system injury. Traditional formulations of AAV preparations permit efficient viral transduction under physiologic conditions, but an improved understanding of the mechanistic limitations of AAV transduction may facilitate production of more resilient AAV strains for investigative and therapeutic purposes. We studied AAV transduction in the context of prior exposure of AAV serotype 8(AAV8) to environmental p H within the range encountered during endosomal endocytosis(p H 7.4 to p H 4.4), during which low p H-triggered structural and autoproteolytic changes to the viral capsid are believed to be necessary for endosome escape and virus uncoating. Due to the fundamental nature of these processes, we hypothesized that premature exposure of AAV8 particles to acidic p H would decrease viral transduction of HT1080 cells in vitro, as measured by fluorescent reporter gene expression using high-content imaging analysis. We found that increasingly acidic incubation conditions were associated with concomitant reductions in transduction efficiency, and that quantitative levels of reporter gene expression in transduced cells were similarly decreased. The biggest decrease in transduction occurred between p H 7.4 and p H 6.4, suggesting the possible co-occurrence of a p H-associated event and viral inactivation within that range. Taken together, these findings indicate that exposure of AAV8 to acidic p H for as little as 1 hour is deleterious to transduction ability. Future studies are necessary to understand the p H-associated causative mechanisms involved. This study was approved by the University of Miami Institutional Animal Care and Use Committee, USA(Protocol #18-108-LF) on July 12, 2018.     

Independent folding of autolytic fragments of thermolysin and their domain-like properties

High molecular weight autolysis products from thermolysin have been isolated and identified. The primary fragments correspond to residues 1 to 187–204 (21kD) and residues 187–204 to 316 (12kD), respectively. The fragments are both capable of independent refolding upon removal of denaturant. On the basis of these results, we suggest that the first step in the unfolding pathway of thermolysin involves unfolding of an interdomain region and domain separation. Bound calcium ions at sites 1, 2 and 4 play a major role in protecting the protein against both autolysis and unfolding, probably by stabilizing the interdomain region and enhancing domain-domain interactions.     

Antifungal drug resistance mechanisms

The present view focuses on the possibility that cationic antimicrobial peptides (CAMPs) might, in addition to their killing effects due to permeabilization of microbial membranes, also function similarly to β-lactam antibiotics to activate nascent autolytic wall enzymes, leading to bacteriolysis. Since the massive release of microbial cell wall components is a major cause of postinfectious sequelae, the in vivo process of bacteriolysis must be controlled. Due to the emergence of antibiotic resistance in pathogenic bacteria, CAMPs might be useful as an alternative to antibiotics. However, they should be used with caution, since they might also function as a ‘double-edged sword’ by injuring both the bacteria and host.     

Study on Autolytic Mechanism and Self-Healing Properties of Autolytic Clinker Microsphere in Alkaline Environment

In this study, the autolytic clinker microsphere with clinker as core and polyvinyl pyrrolidone (PVP) as coating film was prepared. Pretreatment of clinker with silane coupling agent was firstly processed during the preparation. To investigate the autolytic mechanism, the microstructures of the autolytic clinker microsphere at different curing ages were observed using environmental scanning electron microscopy (ESEM), equipped with an energy dispersive spectrometer (EDS). The autolytic stages were also identified based on the microstructural evolution. The influence of pretreatment degree on autolytic behavior was also studied by measurements of micro-morphology and isothermal calorimetry. Experimental results indicated that the compressive strength recovery of specimens was increased by 15–19% due to the addition of autolytic clinker microspheres. The recovery of compressive strength was also improved with the increase of pH value. The improvements in compressive strength recovery of specimens with microspheres were in the range of 15–19%, 15–31%, 25–36%, and 29–50% with the pH value of 7, 8, 10, and 12, respectively. It was also found that inner damage of cement-based matrix had greater recovery when pre-cracked specimens were cured in alkaline environments.     

家兔心肌自溶的酶组织化学研究

作者应用酶组织化学方法对家兔离体心肌自溶的早期改变进行了研究。自溶30～60min,PAS,PPr和分枝酶呈阴性;SDH,GDH,NADPHD,ATPase和CCO对自溶的抵抗力较强,240～480min,活性略有降低;MAO,ACP和ANAE活性在自溶480min时无明显变化。作者认为SDH,GDH,NADPHD,ATPasc和CCO可用于急性心肌缺血的早期诊断。     

小檗碱对MRSA自溶酶系统作用的研究

目的探讨小檗碱与β内酰胺类抗生素对耐甲氧西林金葡菌(MRSA)的协同抗菌作用机制。方法采用蛋白印迹试验(Western-blot)测定小檗碱对MRSA的青霉素结合蛋白(PBP)2a表达的影响,TritonX-100诱导的自溶试验及自溶酶谱分析测定小檗碱对MRSA自溶酶系统的影响。结果小檗碱对MRSA所产PBP2a的表达无影响,但能显著诱导MRSA的自溶酶系统。结论小檗碱对自溶酶系统的激活作用可能是小檗碱增强β内酰胺类抗生素对MRSA抗菌作用的机制之一。本研究为今后小檗碱用于临床抗MRSA治疗提供了科学的理论基础。     

家兔心肌自溶的超微结构变化

作者对离体家兔心肌自溶的超微结构进行了观察。结果表明糖原颗粒减少,核和线粒体均出现变化、线粒体表现肿胀,基质密度降低,基质内出现絮状致密体。随自溶时间延长,絮状致密体体积增大,数量增多。上述变化有助于死亡时间的判定。     

一种改良的金黄色葡萄球菌自溶酶谱分析方法

目的建立金黄色葡萄球菌自溶酶谱的研究方法。方法用SDS抽提金黄色葡萄球菌的自溶酶粗提蛋白。将同样菌体热杀灭后用SDS抽提其细胞壁粗提物作为自溶酶的底物,以SDS-PAGE电泳分析金黄色葡萄球菌的自溶酶谱。结果凝胶在黑色背景下可见每条泳带上有20多条大小不一的黑色条带,如用1%亚甲基蓝染色,则可见凝胶背景为蓝色,自溶酶条带呈透明状。结论该方法可用于研究金黄色葡萄球菌在不同因素影响下的自溶酶谱及其活性,有助于研究抗菌药物对金黄色葡萄球菌自溶酶系统的作用。