In the ongoing arms race between bacteria and bacteriophages,bacteriophages have evolved anti-CRISPR proteins to counteract bacterial CRISPR-Cas systems.Recently,AcrⅡA25.1 and AcrⅡA32 have been found to effectively ...In the ongoing arms race between bacteria and bacteriophages,bacteriophages have evolved anti-CRISPR proteins to counteract bacterial CRISPR-Cas systems.Recently,AcrⅡA25.1 and AcrⅡA32 have been found to effectively inhibit the activity of Spy Cas9 both in bacterial and human cells.However,their molecular mechanisms remain elusive.Here,we report the cryo-electron microscopy structures of ternary complexes formed by AcrⅡA25.1 and AcrⅡA32 bound to Spy Cas9-sg RNA.Using structural analysis and biochemical experiments,we revealed that AcrⅡA25.1 and AcrⅡA32 recognize a novel,previously-unidentified anti-CRISPR binding site on Spy Cas9.We found that both AcrⅡA25.1 and AcrⅡA32 directly interact with the WED domain,where they spatially obstruct conformational changes of the WED and PI domains,thereby inhibiting Spy Cas9 from recognizing protospacer adjacent motif(PAM)and unwinding double-stranded DNA.In addition,they may inhibit nuclease activity by blocking the dynamic conformational changes of the Spy Cas9 surveillance complex.In summary,our data elucidate the inhibition mechanisms of two new anti-CRISPR proteins,provide new strategies for the modulation of Spy Cas9 activity,and expand our understanding of the diversity of anti-CRISPR protein inhibition mechanisms.展开更多
The purpose of this study was to evaluate the storage conditions as a way to increase the safety of fermented sausage, with the specific objectives to investigate the effect of storage time, pH value, water activity a...The purpose of this study was to evaluate the storage conditions as a way to increase the safety of fermented sausage, with the specific objectives to investigate the effect of storage time, pH value, water activity and microbiological status （Lactobacillus spp and Enterobacteriaceae） on the levels of Yersinia enterocolitica in fermented sausages. For this experiment, meat was divided into four equal portions of 10 kg each. The first portion was control group （C1 group） without addition of Y. enterocolitica and starter culture. The second portion （CII group） was inoculated starter, the third portion （EI group） was inoculated with I1. enterocolitica and the fourth portion （Eli group） was inoculated Y.. enterocolitica and starter culture. Sampling was performed by randomly selecting two links of each sausage preparation at the 0, 3rd, 6th, 9th, 15th, 25th and 35th day of drying. Results show that Y. enterocolitica and Enterobacteriaceae were not detected after 25 d of storage. The results indicate that practice utilizing starter culture is satisfactory to reduction of pathogen if present. Thus, inclusion with starter cultures in a maturation period may increase the safety of fermented sausages.展开更多
基金supported by the National Key Research and Development Program of China(2023YFF1000200)the National Natural Science Foundation of China(U21A20276)+1 种基金the Tencent Foundation through the XPLORER PRIZE and the New Cornerstone Science Foundation to Z.H.,Heilongjiang Touyan Team(HITTY-20190034 to Z.H.)Natural Science Foundation of Heilongjiang Province of China(YQ2023C032 to Y.Z.)。
文摘In the ongoing arms race between bacteria and bacteriophages,bacteriophages have evolved anti-CRISPR proteins to counteract bacterial CRISPR-Cas systems.Recently,AcrⅡA25.1 and AcrⅡA32 have been found to effectively inhibit the activity of Spy Cas9 both in bacterial and human cells.However,their molecular mechanisms remain elusive.Here,we report the cryo-electron microscopy structures of ternary complexes formed by AcrⅡA25.1 and AcrⅡA32 bound to Spy Cas9-sg RNA.Using structural analysis and biochemical experiments,we revealed that AcrⅡA25.1 and AcrⅡA32 recognize a novel,previously-unidentified anti-CRISPR binding site on Spy Cas9.We found that both AcrⅡA25.1 and AcrⅡA32 directly interact with the WED domain,where they spatially obstruct conformational changes of the WED and PI domains,thereby inhibiting Spy Cas9 from recognizing protospacer adjacent motif(PAM)and unwinding double-stranded DNA.In addition,they may inhibit nuclease activity by blocking the dynamic conformational changes of the Spy Cas9 surveillance complex.In summary,our data elucidate the inhibition mechanisms of two new anti-CRISPR proteins,provide new strategies for the modulation of Spy Cas9 activity,and expand our understanding of the diversity of anti-CRISPR protein inhibition mechanisms.
文摘The purpose of this study was to evaluate the storage conditions as a way to increase the safety of fermented sausage, with the specific objectives to investigate the effect of storage time, pH value, water activity and microbiological status （Lactobacillus spp and Enterobacteriaceae） on the levels of Yersinia enterocolitica in fermented sausages. For this experiment, meat was divided into four equal portions of 10 kg each. The first portion was control group （C1 group） without addition of Y. enterocolitica and starter culture. The second portion （CII group） was inoculated starter, the third portion （EI group） was inoculated with I1. enterocolitica and the fourth portion （Eli group） was inoculated Y.. enterocolitica and starter culture. Sampling was performed by randomly selecting two links of each sausage preparation at the 0, 3rd, 6th, 9th, 15th, 25th and 35th day of drying. Results show that Y. enterocolitica and Enterobacteriaceae were not detected after 25 d of storage. The results indicate that practice utilizing starter culture is satisfactory to reduction of pathogen if present. Thus, inclusion with starter cultures in a maturation period may increase the safety of fermented sausages.
文摘目的:观察重楼复方(Chonglou Fufang,CLFF)对小鼠H22肝癌的抑制作用及其免疫调节作用。方法:建立小鼠肝癌H22实体瘤模型,将小鼠随机分为6组:空白对照组、CLFF高、中、低剂量组、环磷酰胺(Cyclo-phosphamide,CTX)组、复方斑蝥(Fufang Banmao,FFBM)组。各组连续灌胃给药10d。于末次给药后24h,断颈处死小鼠,剥离瘤体,无菌条件下取脾脏,分别计算抑瘤率、脾指数,并采用MTT显色法检测药物对细胞毒性T淋巴细胞(Cytotoxic T Lymphocyte,CTL)的细胞不良反应。结果:CLFF显著抑制肿瘤生长,高、中、低剂量组抑瘤率分别为54.84%、54.38%、37.27%,与空白对照组比较具有显著性差异(P<0.01);CLFF高、中、低剂量组脾指数分别为(7.20±3.07)mg/g、(7.23±2.34)mg/g、(10.89±3.91)mg/g,各剂量组与环磷酰胺组比较具有显著性差异(P<0.01),中低剂量组与空白对照组比较具有统计学意义(P<0.05);CLFF高中低剂量组对小鼠的CTL细胞的杀伤率分别为51.76%、44.18%、22.74%,高剂量组与空白对照组及CTX组比较,P<0.05,具有统计学意义。结论:CLFF具有显著抑制肿瘤作用,其抑瘤机制可能与其调节机体的免疫功能有关。
