Wilson disease (WD) is one of the most prevalent genetic diseases with an estimated global carrier frequency of 1 in 90 and a prevalence of 1 in 30,000. The disease owes its genesis to Kinnier Wilson who described the disease, and is caused by accumulation of Copper (Cu) in various organs including the liver, central nervous system, cornea, kidney, joints and cardiac muscle which contribute to the characteristic clinical features of WD. A number of studies have reported genetic variants in the ATP7B gene from diverse ethnic and geographical origins. The recent advent of next-generation sequencing approaches has also enabled the discovery of a large number of novel variants in the gene associated with the disease. Previous attempts have been made to compile the knowledgebase and spectrum of genetic variants from across the multitude of publications, but have been limited by the utility due to the significant differences in approaches used to qualify pathogenicity of variants in each of the publications. The recent formulation of guidelines and algorithms for assessment of the pathogenicity of variants jointly put forward by the American College of Medical Genetics and the Association of Molecular Pathologists (ACMG &) has provided a framework for evidence based and systematic assessment of pathogenicity of variants. In this paper, we describe a comprehensive resource of genetic variants in ATP7B gene manually curated from literature and data resources and systematically annotated using the ACMG & AMP guidelines for assessing pathogenicity. The resource therefore serves as a central point for clinicians and geneticists working on WD and to the best of our knowledge is the most comprehensive and only clinically annotated resource for WD. The resource is available at URL http://clingen.igib.res.in/WilsonGen/. We compiled a total of 3662 genetic variants from publications and databases associated with WD. Of these variants compiled, a total of 1458 were found to be unique entries. This is the largest WD database comprising 656 pathogenic/likely pathogenic variants reported classified according to ACMG & AMP guidelines. We also mapped all the pathogenic variants corresponding to ATP7B protein from literature and other databases. In addition, geographical origin and distribution of ATP7B pathogenic variants reported are also mapped in the database.
Background Atrocious use of antibiotics has led to the emergence of MDR in uropathogenic Escherichia coli (UPEC) that poses a serious challenge in the management of urinary tract infections (UTIs). The WHO has described antibiotic resistance in uropathogens as a key pressure point in the burgeoning global antimicrobial resistance crisis. Given this grim situation, we are exploring phage-encoded lysins as plausible alternatives. Objectives Our study involved an in silico strategy for the discovery and characterization of lysin sequences (seq) targeting E. coli cell wall and evaluating the bactericidal activity of these recombinant lysins using in-vitro assays. Methods Novel lysin sequences were searched by BLAST homology and by screening E. coli prophages in the database (using PHASTER). Lysozyme-like domain was observed in 9 out of 16 lysins. Their characterization depicted modular or globular structure. Based on the physicochemical properties, 7 out of 16 lysins were selected for cloning, expression, and purification as recombinant proteins for evaluating the bactericidal activity. Results Among the several lysins screened, lysin seq 5 demonstrated the highest activity using in vitro assays. Using static biofilm assay, lysin seq 5 (180 μg) showed efficient reduction (>50%) in the biofilm formed by ATCC UPEC 700928 strain. As per turbidity reduction method, lysin seq 5 (50 μg) showed 37% drop in OD600nm on UPEC 700928 strain after 3 h of incubation at 37°C. Using spot on lawn assay, lysin seq 5 (20 μg) exhibited lytic activity (zone of inhibition) on five drug-resistant clinical UTI isolates, which were pretreated with an outer membrane permeabilizer (OMP), viz., EDTA (0.3 mM). Conclusions Lysin seq 5 exhibited antibiofilm activity against UPEC 700928 strain as well as lytic activity against drug-resistant clinical UTI isolates. Screening of additional drug-resistant clinical isolates from UTI patients is underway.
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