Myristoylation is required for human immunodeficiency virus type 1 Gag-Gag multimerization in mammalian cells

The Gag protein of human immunodeficiency virus type 1 directs the virion assembly process. Gag proteins must extensively multimerize during the formation of the spherical immature virion shell. In vitro, virus-like particles can be generated from Gag proteins that lack the N-terminal myristic acid modification or the nucleocapsid (NC) protein. The precise requirements for Gag-Gag multimerization under conditions present in mammalian cells, however, have not been fully elucidated. In this study, a Gag-Gag multimerization assay measuring fluorescence resonance energy transfer was employed to define the Gag domains that are essential for homomultimerization. Three essential components were identified: protein-protein interactions contributed by residues within both the N- and C-terminal domains of capsid (CA), basic residues in NC, and the presence of myristic acid. The requirement of myristic acid for multimerization was reproduced using the heterologous myristoylation sequence from v-src. Only when a leucine zipper dimerization motif was placed in the position of NC was a nonmyristoylated Gag protein able to multimerize. These results support a three-component model for Gag-Gag multimerization that includes membrane interactions mediated by the myristoylated N terminus of Gag, protein-protein interactions between CA domains, and NC-RNA interactions.     

Manufacturing a chimpanzee adenovirus-vectored SARS-CoV-2 vaccine to meet global needs

Manufacturing has been the key factor limiting rollout of vaccination during the COVID-19 pandemic, requiring rapid development and large-scale implementation of novel manufacturing technologies. ChAdOx1 nCoV-19 (AZD1222, Vaxzevria) is an efficacious vaccine against SARS-CoV-2, based upon an adenovirus vector. We describe the development of a process for the production of this vaccine and others based upon the same platform, including novel features to facilitate very large-scale production. We discuss the process economics and the “distributed manufacturing” approach we have taken to provide the vaccine at globally-relevant scale and with international security of supply. Together, these approaches have enabled the largest viral vector manufacturing campaign to date, providing a substantial proportion of global COVID-19 vaccine supply at low cost.     

MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes

Characterization of dynamic DNA methylomes in diverse phylogenetic groups has attracted growing interest for a better understanding of the evolution of DNA methylation as well as its function and biological significance in eukaryotes. Sequencing-based methods are promising in fulfilling this task. However, none of the currently available methods offers the ‘perfect solution’, and they have limitations that prevent their application in the less studied phylogenetic groups. The recently discovered Mrr-like enzymes are appealing for new method development, owing to their ability to collect 32-bp methylated DNA fragments from the whole genome for high-throughput sequencing. Here, we have developed a simple and scalable DNA methylation profiling method (called MethylRAD) using Mrr-like enzymes. MethylRAD allows for de novo (reference-free) methylation analysis, extremely low DNA input (e.g. 1 ng) and adjustment of tag density, all of which are still unattainable for most widely used methylation profiling methods such as RRBS and MeDIP. We performed extensive analyses to validate the power and accuracy of our method in both model (plant Arabidopsis thaliana) and non-model (scallop Patinopecten yessoensis) species. We further demonstrated its great utility in identification of a gene (LPCAT1) that is potentially crucial for carotenoid accumulation in scallop adductor muscle. MethylRAD has several advantages over existing tools and fills a void in the current epigenomic toolkit by providing a universal tool that can be used for diverse research applications, e.g. from model to non-model species, from ordinary to precious samples and from small to large genomes, but at an affordable cost.     

Evaluating the Impact of Test-and-Treat on the HIV Epidemic among MSM in China Using a Mathematical Model

Background

Various studies have modeled the impact of test-and-treat policies on the HIV epidemics worldwide. However, few modeling studies have taken into account China’s context. To understand the potential effect of test-and-treat on the HIV epidemic among men who have sex with men (MSM) in China, we developed a mathematical model to evaluate the impact of the strategy.

Method

Based on the natural history of the CD4 count of people living with HIV and AIDS (PLWHA), we constructed a dynamic compartmental model of HIV transmission among Chinese MSM to project the number of HIV new infections and prevalence over 10 years. We predicted the annual number of HIV new infections and the total number of MSM living with HIV and AIDS (based on Beijing data) between 2010 and 2022 under the following conditions: (1) current practice (testing rate of 50% and ART coverage of 39%); (2) both testing rate and ART coverage increasing to 70% in 2013; (3) both testing rate and ART coverage increasing to 90% in 2013; and (4) both testing rate and ART coverage increasing gradually every year until 90% since 2013.

Results

Based on our model, if the HIV test-and-treat policy was implemented among Chinese MSM, the total number of HIV new infections over 10 years (2013-2022) would be reduced by 50.6-70.9% compared with the current policy. When ART coverage for PLWHA increased to 58% since 2013, the ‘turning point’ would occur on the curve of HIV new infections by 2015. A 25% reduction in annual number of HIV new infections by 2015 might be achieved if the testing rate increased from 50% to 70% and treatment coverage for PLWHA increased to 55% since 2013.

Conclusion

Implementation of the test-and-treat strategy may significantly reduce HIV new infections among MSM in China. Great efforts need to be made to scale up HIV testing rate and ART coverage among Chinese MSM.     

Prevalence and Impact on Stroke in Patients Receiving Maintenance Hemodialysis versus Peritoneal Dialysis: A Prospective Observational Study

Background

Patients undergoing maintenance dialysis are at increased risk of stroke, however, less is known about the prevalence and impact on stroke in the patients.

Methods

In this prospective cohort study, 590 patients undergoing hemodialysis (HD; n = 285) or peritoneal dialysis (PD; n = 305) from January 1, 2008 to December 31, 2012 were recruited. Baseline demographic, clinical, and laboratory data were collected. Timeline incidence data were analyzed using a Poisson model. The Cox proportional hazards regression assessed adjusted differences in stroke risk, a multivariate analysis was also performed.

Results

62 strokes occurred during 1258 total patient-years of follow-up. Stroke occurred at a rate of 49.2/1,000 patient-years with a predominance in HD patients compared with PD patients (74.0 vs. 31.8/1,000 patient-years). The cumulative hazard of developing stroke was significantly higher in HD patients (hazard ratio [HR], 1.75; 95% confidence interval [CI], 1.15–3.62; p = 0.046) after adjusting for potential confounders. HD patients had an increased risk of ischemic stroke (HR, 2.62; 95% CI, 1.56–4.58; p = 0.002). The risk of hemorrhagic stroke was not significantly different between PD and HD patients. On multivariate Cox analysis, risk factors of stroke in both HD and PD patients were older age, diabetes, and cardiovascular disease. Other independent risk factors of stroke were lower albumin-corrected calcium in HD patients and higher triglycerides in PD patients.

Conclusions

Patients undergoing PD were less likely to develop ischemic stroke than those undergoing HD. Comprehensive control of diabetes, cardiovascular disease, calcium-phosphorus metabolism, and triglyceride levels may be useful preventive strategies for stroke in dialysis patients.     

Unwinding forward and sliding back: an intermittent unwinding mode of the BLM helicase

There are lines of evidence that the Bloom syndrome helicase, BLM, catalyzes regression of stalled replication forks and disrupts displacement loops (D-loops) formed during homologous recombination (HR). Here we constructed a forked DNA with a 3′ single-stranded gap and a 5′ double-stranded handle to partly mimic a stalled DNA fork and used magnetic tweezers to study BLM-catalyzed unwinding of the forked DNA. We have directly observed that the BLM helicase may slide on the opposite strand for some distance after duplex unwinding at different forces. For DNA construct with a long hairpin, progressive unwinding of the hairpin is frequently interrupted by strand switching and backward sliding of the enzyme. Quantitative study of the uninterrupted unwinding length (time) has revealed a two-state-transition mechanism for strand-switching during the unwinding process. Mutational studies revealed that the RQC domain plays an important role in stabilizing the helicase/DNA interaction during both DNA unwinding and backward sliding of BLM. Especially, Lys1125 in the RQC domain, a highly conserved amino acid among RecQ helicases, may be involved in the backward sliding activity. We have also directly observed the in vitro pathway that BLM disrupts the mimic stalled replication fork. These results may shed new light on the mechanisms for BLM in DNA repair and homologous recombination.     

Defective chromatin architectures in embryonic stem cells derived from somatic cell nuclear transfer impair their differentiation potentials

Nuclear transfer embryonic stem cells (ntESCs) hold enormous promise for individual-specific regenerative medicine. However, the chromatin states of ntESCs remain poorly characterized. In this study, we employed ATAC-seq and Hi-C techniques to explore the chromatin accessibility and three-dimensional (3D) genome organization of ntESCs. The results show that the chromatin accessibility and genome structures of somatic cells are re-arranged to ESC-like states overall in ntESCs, including compartments, topologically associating domains (TADs) and chromatin loops. However, compared to fertilized ESCs (fESCs), ntESCs show some abnormal openness and structures that have not been reprogrammed completely, which impair the differentiation potential of ntESCs. The histone modification H3K9me3 may be involved in abnormal structures in ntESCs, including incorrect compartment switches and incomplete TAD rebuilding. Moreover, ntESCs and iPSCs show high similarity in 3D genome structures, while a few differences are detected due to different somatic cell origins and reprogramming mechanisms. Through systematic analyses, our study provides a global view of chromatin accessibility and 3D genome organization in ntESCs, which can further facilitate the understanding of the similarities and differences between ntESCs and fESCs.Subject terms: Embryonic stem cells, Reprogramming, Stem-cell differentiation     

Knockdown of ubiquitin-like modifier-activating enzyme 2 promotes apoptosis of clear cell renal cell carcinoma cells

Small ubiquitin-related modifier (SUMO) proteins are involved in the development of tumors. Ubiquitin-like modifier-activating enzyme 2 (UBA2) is an important member of the SUMO modification system; however, its role in clear cell renal cell carcinoma (ccRCC) is unclear. Therefore, we investigated the expression and function of UBA2 in ccRCC. Both mRNA and protein expression levels of UBA2 were found to be higher in ccRCC than in normal renal tissues and significantly related to the tumor size, Fuhrman grade, and tumor stage. UBA2 knockdown inhibited ccRCC cell growth, promoted apoptosis in vitro and in vivo, and decreased the abundance of a p53 mutant, c-Myc, and key enzymes of the SUMO modification system. Meanwhile, overexpression of UBA2 had the opposite effects. Overexpression of the p53 mutant or c-Myc alleviated the effects of UBA2 knockdown on ccRCC cell proliferation and apoptosis. In conclusion, targeting UBA2 may have a therapeutic potential against ccRCC.Subject terms: Oncogenes, Oncogenesis     

鸟类对新疆农区的危害评估和预防探讨

长期以来,鸟类对人类造成的危害一直是一个世界性的问题,涉及到多个行业,造成经济损失,且难以防控。本文旨在摸清对新疆农业造成损失的鸟类种类及危害程度,阐述鸟类出没规律,进而提出科学有效的防治措施。2019至2021年,基于样线法、问卷法、田间观测与取样法、防鸟粘网计数、物种鉴定与胃容物解剖等,在新疆的墨玉、乌什、拜城和昌吉大范围布设观测点,对农田、果园、水库等典型生境进行调查,得出现有害鸟种类以及受害的类型、损失程度、预防措施、经济补偿等,最后完成鸟害评估。共记录到对农业有害鸟类10目19科40属49种(类),已超出了新疆鸟类种数的10%。它们危害作物包括玉米、小麦、水稻、葡萄、红枣、油葵等,其中以冬小麦(52.9%)和酿酒葡萄(14.4%)损失较大。除此以外,也会对收获中的土豆、西瓜、甜瓜、核桃、草莓、枸杞等构成危害。对于整个调查区域而言,鸟类光顾或平均取食的概率约为28.6%,呈由北向南递减的规律(R²=0.893,P<0.05)。抽样分析,估计一些地方收成减产或损失达26.6%。即使如此,当地农民却较少采取预防措施,仅有18.0%的农户采取了防范措施(如大喇叭、粘鸟网、稻草人、驱鸟剂等)。目前,保险理赔与政府补贴微乎其微,低于9.53%或无。最后,笔者探讨了国内外驱鸟与粮食安全措施,包括生物防治、化学驱鸟和物理恐吓等。--些方法会伤及无辜种类,如一些猛禽和食虫鸟类。未来鸟类与人类的冲突将会愈演愈烈,同时折射出野生动物生存与人类发展之间的尴尬处境。其实,有效地防止鸟害,也是为了更好地保护鸟类。