Biochemical maturation of human central nervous system myelin.

—A developmental study of the lipid and protein composition of human CNS myelin was undertaken. The relative concentrations of the major lipid classes, cholesterol, glycolipids and phospholipids exhibited little change except for a modest decrease in the concentration of the phospholipids. In contrast to the total phospholipids, marked variations in the relative concentrations of individual phospholipids were found. Sphingomyelin increased over two-fold, and phosphatidyl choline decreased to almost half its original concentration. While the concentration of total myelin protein remained constant during maturation, variations in the concentrations of individual proteins were observed. Basic protein constituted 8·5 per cent of the total myelin proteins in the newborn brain and increased to about 30 per cent of the protein in the older ages. The concentrations of proteolipid protein and DM-20 seemed to increase with age, while the relative amounts of high molecular weight proteins decreased. The presence of myelin basic protein in newborn human brain was confirmed by electrophoretic studies involving several different polyacrylamide gel systems and by immunodiffusion experiments which showed a reaction of identity between a constituent present in the fraction containing the presumptive myelin basic protein and authentic myelin basic protein isolated from adult human brain.     

Moths complement bumblebee pollination of red clover: a case for day-and-night insect surveillance

Recent decades have seen a surge in awareness about insect pollinator declines. Social bees receive the most attention, but most flower-visiting species are lesser known, non-bee insects. Nocturnal flower visitors, e.g. moths, are especially difficult to observe and largely ignored in pollination studies. Clearly, achieving balanced monitoring of all pollinator taxa represents a major scientific challenge. Here, we use time-lapse cameras for season-wide, day-and-night pollinator surveillance of Trifolium pratense (L.; red clover) in an alpine grassland. We reveal the first evidence to suggest that moths, mainly Noctua pronuba (L.; large yellow underwing), pollinate this important wildflower and forage crop, providing 34% of visits (bumblebees: 61%). This is a remarkable finding; moths have received no recognition throughout a century of T. pratense pollinator research. We conclude that despite a non-negligible frequency and duration of nocturnal flower visits, nocturnal pollinators of T. pratense have been systematically overlooked. We further show how the relationship between visitation and seed set may only become clear after accounting for moth visits. As such, population trends in moths, as well as bees, could profoundly affect T. pratense seed yield. Ultimately, camera surveillance gives fair representation to non-bee pollinators and lays a foundation for automated monitoring of species interactions in future.     

An ecological niche model to predict the geographic distribution of Haemagogus janthinomys,Dyar, 1921 a yellow fever and Mayaro virus vector,in South America

Yellow fever virus (YFV) has a long history of impacting human health in South America. Mayaro virus (MAYV) is an emerging arbovirus of public health concern in the Neotropics and its full impact is yet unknown. Both YFV and MAYV are primarily maintained via a sylvatic transmission cycle but can be opportunistically transmitted to humans by the bites of infected forest dwelling Haemagogus janthinomys Dyar, 1921. To better understand the potential risk of YFV and MAYV transmission to humans, a more detailed understanding of this vector species’ distribution is critical. This study compiled a comprehensive database of 177 unique Hg. janthinomys collection sites retrieved from the published literature, digitized museum specimens and publicly accessible mosquito surveillance data. Covariate analysis was performed to optimize a selection of environmental (topographic and bioclimatic) variables associated with predicting habitat suitability, and species distributions modelled across South America using a maximum entropy (MaxEnt) approach. Our results indicate that suitable habitat for Hg. janthinomys can be found across forested regions of South America including the Atlantic forests and interior Amazon.     

体外重构STUB1介导α-1抗胰蛋白酶突变体Z蛋白泛素化修饰反应体系

α-1抗胰蛋白酶Z型突变体蛋白(α-1 antitrypsin Z-mutant protein, ATZ)是引发α-1抗胰蛋白酶缺陷症(α-1 antitrypsin deficiency, AATD)的主要原因,研究ATZ蛋白的泛素化修饰和降解对于治疗AATD具有重要意义。STUB1是一种重要的E3泛素连接酶,参与调节多种蛋白质的泛素化修饰。然而,STUB1是否参与ATZ的泛素化修饰尚未明确。本研究首先将ATZ和STUB1的编码基因克隆到pET28a质粒,构建了这2个蛋白的表达质粒。随后,将重组质粒转入大肠杆菌表达系统,在优化诱导条件实现了重组蛋白的异源表达。通过金属螯合亲和层析技术纯化得到目的蛋白,并通过蛋白质谱分析验证了其氨基酸序列的准确性。利用纯化的ATZ和STUB1重组蛋白,构建了一个体外泛素化修饰反应体系。实验结果显示,在ATP、E1泛素激活酶和E2泛素结合酶的协同作用下,STUB1成功催化了ATZ的泛素化修饰。本研究提供了一种体外获得Z型突变体ATZ纯化蛋白的方法,并确认了STUB1介导ATZ的泛素化修饰功能,推进了对α-1抗胰蛋白酶Z型突变体蛋白在细胞内降解过程的调控机制的理解。     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.     

Mismatch discrimination and sequence bias during end-joining by DNA ligases

DNA ligases, critical enzymes for in vivo genome maintenance and modern molecular biology, catalyze the joining of adjacent 3′-OH and 5′-phosphorylated ends in DNA. To determine whether DNA annealing equilibria or properties intrinsic to the DNA ligase enzyme impact end-joining ligation outcomes, we used a highly multiplexed, sequencing-based assay to profile mismatch discrimination and sequence bias for several ligases capable of efficient end-joining. Our data reveal a spectrum of fidelity and bias, influenced by both the strength of overhang annealing as well as sequence preferences and mismatch tolerances that vary both in degree and kind between ligases. For example, while T7 DNA ligase shows a strong preference for ligating high GC sequences, other ligases show little GC-dependent bias, with human DNA Ligase 3 showing almost none. Similarly, mismatch tolerance varies widely among ligases, and while all ligases tested were most permissive of G:T mismatches, some ligases also tolerated bulkier purine:purine mismatches. These comprehensive fidelity and bias profiles provide insight into the biology of end-joining reactions and highlight the importance of ligase choice in application design.     

TBX2 acts as a potent transcriptional silencer of tumour suppressor genes through interaction with the CoREST complex to sustain the proliferation of breast cancers

Chromosome 17q23 amplification occurs in 20% of primary breast tumours and is associated with poor outcome. The TBX2 gene is located on 17q23 and is often over-expressed in this breast tumour subset. TBX2 is an anti-senescence gene, promoting cell growth and survival through repression of Tumour Suppressor Genes (TSGs), such as NDRG1 and CST6. Previously we found that TBX2 cooperates with the PRC2 complex to repress several TSGs, and that PRC2 inhibition restored NDRG1 expression to impede cellular proliferation. Here, we now identify CoREST proteins, LSD1 and ZNF217, as novel interactors of TBX2. Genetic or pharmacological targeting of CoREST emulated TBX2 loss, inducing NDRG1 expression and abolishing breast cancer growth in vitro and in vivo. Furthermore, we uncover that TBX2/CoREST targeting of NDRG1 is achieved by recruitment of TBX2 to the NDRG1 promoter by Sp1, the abolishment of which resulted in NDRG1 upregulation and diminished cancer cell proliferation. Through ChIP-seq we reveal that 30% of TBX2-bound promoters are shared with ZNF217 and identify novel targets repressed by TBX2/CoREST; of these targets a lncRNA, LINC00111, behaves as a negative regulator of cell proliferation. Overall, these data indicate that inhibition of CoREST proteins represents a promising therapeutic intervention for TBX2-addicted breast tumours.     

Expression of the recombinant antibacterial peptide sarcotoxin IA in Escherichia coli cells

Sarcotoxin IA is an antibacterial peptide that is secreted by a meat-fly Sarcophaga peregrina larva in response to a hypodermic injury or bacterial infection. This peptide is highly toxic against a broad spectrum of both Gram-positive and Gram-negative bacteria and lethal to microbes even at nanomolar concentrations. However, research needs as well as its potential use in medicine require substantial amounts of highly purified sarcotoxin. Because heterologous expression systems proved to be inefficient due to sarcotoxin sensitivity to intracellular proteases, here we propose the biosynthesis of sarcotoxin precursors in Escherichia coli cells that are highly sensitive to the mature peptide. To optimize its biosynthesis, sarcotoxin was translationally fused with proteins highly expressed in E. coli. A fusion partner and the position of sarcotoxin in the chimeric polypeptide were crucial for protecting the sarcotoxin portion of the fusion protein from proteolysis. Released after chemical cleavage of the fusion protein and purified to homogeneity, sarcotoxin displayed antibacterial activity comparable to that previously reported for the natural peptide.     

Microbial light-activatable proton pumps as neuronal inhibitors to functionally dissect neuronal networks in C. elegans

Essentially any behavior in simple and complex animals depends on neuronal network function. Currently, the best-defined system to study neuronal circuits is the nematode Caenorhabditis elegans, as the connectivity of its 302 neurons is exactly known. Individual neurons can be activated by photostimulation of Channelrhodopsin-2 (ChR2) using blue light, allowing to directly probe the importance of a particular neuron for the respective behavioral output of the network under study. In analogy, other excitable cells can be inhibited by expressing Halorhodopsin from Natronomonas pharaonis (NpHR) and subsequent illumination with yellow light. However, inhibiting C. elegans neurons using NpHR is difficult. Recently, proton pumps from various sources were established as valuable alternative hyperpolarizers. Here we show that archaerhodopsin-3 (Arch) from Halorubrum sodomense and a proton pump from the fungus Leptosphaeria maculans (Mac) can be utilized to effectively inhibit excitable cells in C. elegans. Arch is the most powerful hyperpolarizer when illuminated with yellow or green light while the action spectrum of Mac is more blue-shifted, as analyzed by light-evoked behaviors and electrophysiology. This allows these tools to be combined in various ways with ChR2 to analyze different subsets of neurons within a circuit. We exemplify this by means of the polymodal aversive sensory ASH neurons, and the downstream command interneurons to which ASH neurons signal to trigger a reversal followed by a directional turn. Photostimulating ASH and subsequently inhibiting command interneurons using two-color illumination of different body segments, allows investigating temporal aspects of signaling downstream of ASH.