Structural basis for Cas9 off-target activity

The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here, we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of noncanonical base-pairing interactions within the guide:off-target heteroduplex. Off-target substrates containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping or multiple noncanonical base pairs rather than RNA bulge formation. Finally, PAM-distal mismatches result in duplex unpairing and induce a conformational change in the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms

A Differential Drug Screen for Compounds That Select Against Antibiotic Resistance

Antibiotics increase the frequency of resistant bacteria by providing them a competitive advantage over sensitive strains. Here, we develop a versatile assay for differential chemical inhibition of competing microbial strains, and use it to identify compounds that preferentially inhibit tetracycline-resistant relative to sensitive bacteria, thus “inverting” selection for resistance. Our assay distinguishes compounds selecting directly against specific resistance mechanisms and compounds whose selection against resistance is based on their physiological interaction with tetracycline and is more general with respect to resistance mechanism. A pilot screen indicates that both types of selection-inverting compounds are secreted by soil microbes, suggesting that nature has evolved a repertoire of chemicals that counteracts antibiotic resistance. Finally, we show that our assay can more generally permit simple, direct screening for drugs based on their differential activity against different strains or targets

Euglena International Network (EIN):Driving euglenoid biotechnology for the benefit of a challenged world

Euglenoids (Euglenida) are unicellular flagellates possessing exceptionally wide geographical and ecological distribution. Euglenoids combine a biotechnological potential with a unique position in the eukaryotic tree of life. In large part these microbes owe this success to diverse genetics including secondary endosymbiosis and likely additional sources of genes. Multiple euglenoid species have translational applications and show great promise in production of biofuels, nutraceuticals, bioremediation, cancer treatments and more exotically as robotics design simulators. An absence of reference genomes currently limits these applications, including development of efficient tools for identification of critical factors in regulation, growth or optimization of metabolic pathways. The Euglena International Network (EIN) seeks to provide a forum to overcome these challenges. EIN has agreed specific goals, mobilized scientists, established a clear roadmap (Grand Challenges), connected academic and industry stakeholders and is currently formulating policy and partnership principles to propel these efforts in a coordinated and efficient manner

Identification of genetic elements in metabolism by high-throughput mouse phenotyping.

Metabolic diseases are a worldwide problem but the underlying genetic factors and their relevance to metabolic disease remain incompletely understood. Genome-wide research is needed to characterize so-far unannotated mammalian metabolic genes. Here, we generate and analyze metabolic phenotypic data of 2016 knockout mouse strains under the aegis of the International Mouse Phenotyping Consortium (IMPC) and find 974 gene knockouts with strong metabolic phenotypes. 429 of those had no previous link to metabolism and 51 genes remain functionally completely unannotated. We compared human orthologues of these uncharacterized genes in five GWAS consortia and indeed 23 candidate genes are associated with metabolic disease. We further identify common regulatory elements in promoters of candidate genes. As each regulatory element is composed of several transcription factor binding sites, our data reveal an extensive metabolic phenotype-associated network of co-regulated genes. Our systematic mouse phenotype analysis thus paves the way for full functional annotation of the genome

Relationship between molecular pathogen detection and clinical disease in febrile children across Europe: a multicentre, prospective observational study

BackgroundThe PERFORM study aimed to understand causes of febrile childhood illness by comparing molecular pathogen detection with current clinical practice.MethodsFebrile children and controls were recruited on presentation to hospital in 9 European countries 2016-2020. Each child was assigned a standardized diagnostic category based on retrospective review of local clinical and microbiological data. Subsequently, centralised molecular tests (CMTs) for 19 respiratory and 27 blood pathogens were performed.FindingsOf 4611 febrile children, 643 (14%) were classified as definite bacterial infection (DB), 491 (11%) as definite viral infection (DV), and 3477 (75%) had uncertain aetiology. 1061 controls without infection were recruited. CMTs detected blood bacteria more frequently in DB than DV cases for N. meningitidis (OR: 3.37, 95% CI: 1.92-5.99), S. pneumoniae (OR: 3.89, 95% CI: 2.07-7.59), Group A streptococcus (OR 2.73, 95% CI 1.13-6.09) and E. coli (OR 2.7, 95% CI 1.02-6.71). Respiratory viruses were more common in febrile children than controls, but only influenza A (OR 0.24, 95% CI 0.11-0.46), influenza B (OR 0.12, 95% CI 0.02-0.37) and RSV (OR 0.16, 95% CI: 0.06-0.36) were less common in DB than DV cases. Of 16 blood viruses, enterovirus (OR 0.43, 95% CI 0.23-0.72) and EBV (OR 0.71, 95% CI 0.56-0.90) were detected less often in DB than DV cases. Combined local diagnostics and CMTs respectively detected blood viruses and respiratory viruses in 360 (56%) and 161 (25%) of DB cases, and virus detection ruled-out bacterial infection poorly, with predictive values of 0.64 and 0.68 respectively.InterpretationMost febrile children cannot be conclusively defined as having bacterial or viral infection when molecular tests supplement conventional approaches. Viruses are detected in most patients with bacterial infections, and the clinical value of individual pathogen detection in determining treatment is low. New approaches are needed to help determine which febrile children require antibiotics.FundingEU Horizon 2020 grant 668303

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Engaging unachieved youths in the football industry

concept of this thesis was made since there have been a lot of players playing football and couldn’t succeed in a higher level due to lack of training or lack of consideration in the football industry along with a lot more. These players have to be given a chance to play and this can be practicable through the scouting team which would be all over the world trying to go to every nook and corner and scout young talents who have the potential and determination to play professional football and with relocate people supporting them with everything needed for their day to day living alongside being a professional football player. There are hundreds and thousands of football clubs which are in the football industry and each and every club has its own motive but try to recruit players who are already trained and successful from other smaller clubs or leagues. The problem is, players do not get enough attention or recognition. There are so many studies that people are not able to play due to lack of financial support and this stays as a problem even till today. Football can be a hard sport to play and at the same time makes it a sport which can lead to a lot of health benefits including less consumption of Illegal drugs and the motive of the club is to take a step on trying to support the parents who are not capable of funding their children or have lack of knowledge on how to make them successful. This thesis shows all the practicable ways and outcomes to come across all these problems which occur in the real life with a practical solution. Everything from food, accommodation to pocket money to be given to the players and in this thesis, everything is explained in detail. This club keenly focuses on recruiting individuals who have been left out due to lack of training or lack of support and recognition who are still desiring to play and commit themselves when given an opportunity. That is why the budget of the whole club is less than half a million just satisfying whatever is absolutely needed for the high level of training and still not letting down the level of professionalism. The whole motive of the club is to not be with a non-profit motive but to keep working for the betterment of the club through sponsors, financiers and investors