Optogenetic regulation of site-specific subtelomeric DNA methylation

Telomere length homeostasis, critical for chromosomal integrity and genome stability, is controlled by intricate molecular regulatory machinery that includes epigenetic modifications. Here, we examine site-specific and spatiotemporal alteration of the subtelomeric methylation of CpG islands using optogenetic tools to understand the epigenetic regulatory mechanisms of telomere length maintenance. Human DNA methyltransferase3A (DNMT3A) were assembled selectively at chromosome ends by fusion to cryptochrome 2 protein (CRY2) and its interacting complement, the basic helix loop helix protein-1 (CIB1). CIB1 was fused to the telomere-associated protein telomere repeat binding factor-1 (TRF1), which localized the protein complex DNMT3A-CRY2 at telomeric regions upon excitation by blue-light monitored by single-molecule fluorescence analyses. Increased methylation was achieved selectively at subtelomeric CpG sites on the six examined chromosome ends specifically after blue-light activation, which resulted in progressive increase in telomere length over three generations of HeLa cell replications. The modular design of the fusion constructs presented here allows for the selective substitution of other chromatin modifying enzymes and for loci-specific targeting to regulate the epigenetic pathways at telomeres and other selected genomic regions of interest

Cerebral Venous Thrombosis and Acute Pulmonary Embolism following Varicella Infection

Varicella infection is caused by varicella-zoster virus (VZV) and commonly presents as a self-limiting skin manifestation in children. VZV also causes cerebral arterial vasculopathy and antibody-mediated hypercoagulable states leading to thrombotic complications in children, although there are very few such reports in adults. Postulated causal factors include vasculitis, direct endothelial damage, or acquired protein S deficiency secondary to molecular mimicry. These induced autoantibodies to protein S could lead to acquired protein S deficiency and produce a hypercoagulable state causing venous sinus thrombosis. Here we report the case of a 26-year-old man who presented with cortical venous sinus thrombosis and acute pulmonary embolism following varicella infection. Both conditions responded to anticoagulation treatment

Identification of SARS-CoV-2 inhibitors targeting Mpro and PLpro using in-cell-protease assay

SARS-CoV-2 proteases Mpro and PLpro are promising targets for antiviral drug development. In this study, we present an antiviral screening strategy involving a novel in-cell protease assay, antiviral and biochemical activity assessments, as well as structural determinations for rapid identification of protease inhibitors with low cytotoxicity. We identified eight compounds with anti-SARS-CoV-2 activity from a library of 64 repurposed drugs and modeled at protease active sites by in silico docking. We demonstrate that Sitagliptin and Daclatasvir inhibit PLpro, and MG-101, Lycorine HCl, and Nelfinavir mesylate inhibit Mpro of SARS-CoV-2. The X-ray crystal structure of Mpro in complex with MG-101 shows a covalent bond formation between the inhibitor and the active site Cys145 residue indicating its mechanism of inhibition is by blocking the substrate binding at the active site. Thus, we provide methods for rapid and effective screening and development of inhibitors for blocking virus polyprotein processing as SARS-CoV-2 antivirals. Additionally, we show that the combined inhibition of Mpro and PLpro is more effective in inhibiting SARS-CoV-2 and the delta variant

A Chemical Strategy for Intracellular Arming of an Endogenous Broad-Spectrum Antiviral Nucleotide

The naturally occurring nucleotide 3′-deoxy-3′,4′-didehydro-cytidine-5′-triphosphate (ddhCTP) was recently found to exert potent and broad-spectrum antiviral activity. However, nucleoside 5′-triphosphates in general are not cell-permeable, which precludes the direct use of ddhCTP as a therapeutic. To harness the therapeutic potential of this endogenous antiviral nucleotide, we synthesized phosphoramidate prodrug HLB-0532247 (1) and found it to result in dramatically elevated levels of ddhCTP in cells. We compared 1 and 3′-deoxy-3′,4′-didehydro-cytidine (ddhC) and found that 1 more effectively reduces titers of Zika and West Nile viruses in cell culture with minimal nonspecific toxicity to host cells. We conclude that 1 is a promising antiviral agent based on a novel strategy of facilitating elevated levels of the endogenous ddhCTP antiviral nucleotide

Structure-Function Studies of DNA Binding Domain of Response Regulator KdpE Reveals Equal Affinity Interactions at DNA Half-Sites

Expression of KdpFABC, a K+ pump that restores osmotic balance, is controlled by binding of the response regulator KdpE to a specific DNA sequence (kdpFABCBS) via the winged helix-turn-helix type DNA binding domain (KdpEDBD). Exploration of E. coli KdpEDBD and kdpFABCBS interaction resulted in the identification of two conserved, AT-rich 6 bp direct repeats that form half-sites. Despite binding to these half-sites, KdpEDBD was incapable of promoting gene expression in vivo. Structure-function studies guided by our 2.5 Å X-ray structure of KdpEDBD revealed the importance of residues R193 and R200 in the α-8 DNA recognition helix and T215 in the wing region for DNA binding. Mutation of these residues renders KdpE incapable of inducing expression of the kdpFABC operon. Detailed biophysical analysis of interactions using analytical ultracentrifugation revealed a 2∶1 stoichiometry of protein to DNA with dissociation constants of 200±100 and 350±100 nM at half-sites. Inactivation of one half-site does not influence binding at the other, indicating that KdpEDBD binds independently to the half-sites with approximately equal affinity and no discernable cooperativity. To our knowledge, these data are the first to describe in quantitative terms the binding at half-sites under equilibrium conditions for a member of the ubiquitous OmpR/PhoB family of proteins

Warranty Cost Analysis And Optimization To Finalize Asuitable Warranty Period For An Organization

Since the beginning of industrialization, reliability and warranty concerns have taken top priority. It's mostly because the warranty failures costs a lot of money for the manufacturer (Glickman and Berger 1976). The turnover and profitability of the business are significantly impacted by the warranty cost. The goal of manufacturers is to extend the product warranty period not only to improve customer perception and satisfaction, but also to improve product development and production methods to reduce the warranty cost.The advancement in technology over the last decade has been tremendous. This rise in the use of digital technology has not only led to making the competition fierce among the industrial companies but also increase customer expectations towards products and services. Among the different choices, there has been an immense increase in the demand for product warranty and services related to warranty. The warranty period is one of the crucial elements in the decision-making process for consumers. Customers typically choose the product with the best warranty when choosing between numerous options with comparable features and specifications. All these factors have led to a stronger belief among the manufacturers to have a good warranty period and services related to warranty. In order to remain competitive in the market there is a need for the manufacturers to arrive at the best warranty policy in this competitive worldThis thesis study concerns using an existing failure data set from a manufacturing company based out of Sweden to • Develop an optimization model that would help the organization choose the optimal warranty period / policy based on current warranty costs and based on competitors’ warranty period or time frame.The above analysis would thus, help to answer the following research questionHow can the organization arrive at the optimal warranty period (i.e., free replacement warranty and pro rata warranty) and based on its current warranty failure rate and warranty cost

Warranty Cost Analysis And Optimization To Finalize Asuitable Warranty Period For An Organization

Since the beginning of industrialization, reliability and warranty concerns have taken top priority. It's mostly because the warranty failures costs a lot of money for the manufacturer (Glickman and Berger 1976). The turnover and profitability of the business are significantly impacted by the warranty cost. The goal of manufacturers is to extend the product warranty period not only to improve customer perception and satisfaction, but also to improve product development and production methods to reduce the warranty cost.The advancement in technology over the last decade has been tremendous. This rise in the use of digital technology has not only led to making the competition fierce among the industrial companies but also increase customer expectations towards products and services. Among the different choices, there has been an immense increase in the demand for product warranty and services related to warranty. The warranty period is one of the crucial elements in the decision-making process for consumers. Customers typically choose the product with the best warranty when choosing between numerous options with comparable features and specifications. All these factors have led to a stronger belief among the manufacturers to have a good warranty period and services related to warranty. In order to remain competitive in the market there is a need for the manufacturers to arrive at the best warranty policy in this competitive worldThis thesis study concerns using an existing failure data set from a manufacturing company based out of Sweden to • Develop an optimization model that would help the organization choose the optimal warranty period / policy based on current warranty costs and based on competitors’ warranty period or time frame.The above analysis would thus, help to answer the following research questionHow can the organization arrive at the optimal warranty period (i.e., free replacement warranty and pro rata warranty) and based on its current warranty failure rate and warranty cost