Tetrahydropyrazolo[1,5-a]Pyrimidine-3-Carboxamide and N-Benzyl-6′,7′-Dihydrospiro[Piperidine-4,4′-Thieno[3,2-c]Pyran] analogues with bactericidal efficacy against Mycobacterium tuberculosis targeting MmpL3

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-ca​rboxamide(THPP) and N-benzyl-6′,7′-dihydrospiro[piperidine-4,​4′-thieno[3,2-c]pyran](Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This ‘genetic phenotype’ was further confirmed by a ‘chemical phenotype’, whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice

Lentiviral Hematopoietic Stem Cell Gene Therapy Benefits Metachromatic Leukodystrophy

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A Meta-analysis of Multiple Myeloma Risk Regions in African and European Ancestry Populations Identifies Putatively Functional Loci

Genome-wide association studies (GWAS) in European populations have identified genetic risk variants associated with multiple myeloma (MM)

Improving the efficiency and effectiveness of an industrial SARS-CoV-2 diagnostic facility.

On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Large Impact of Low Concentration Oxidized LDL on Angiogenic Potential of Human Endothelial Cells: A Microarray Study

<div><p>Oxidized LDL (ox-LDL) is a key factor in atherogenesis. It is taken up by endothelial cells primarily by ox-LDL receptor-1 (LOX-1). To elucidate transcriptional responses, we performed microarray analysis on human coronary artery endothelial cells (HCAECs) exposed to small physiologic concentration of ox-LDL- 5 µg/ml for 2 and 12 hours. At 12 hours, cultures treated with ox-LDL exhibited broad shifts in transcriptional activity involving almost 1500 genes (>1.5 fold difference, p<0.05). Resulting transcriptome was enriched for genes associated with cell adhesion (p<0.002), angiogenesis (p<0.0002) and migration (p<0.006). Quantitative PCR analysis revealed that LOX-1 expression in HCAECs is at least an order of magnitude greater than the expression of other major ox-LDL specific receptors CD36 and MSR1. In keeping with the data on LOX-1 expression, pre-treatment of HCAECs with LOX-1 neutralizing antibody resulted in across-the-board inhibition of cellular response to ox-LDL. Ox-LDL upregulated a number of pro-angiogenic genes including multiple receptors, ligands and transcription factors and altered the expression of a number of genes implicated in both stimulation and inhibition of apoptosis. From a functional standpoint, physiologic concentrations of ox-LDL stimulated tube formation and inhibited susceptibility to apoptosis in HCAECs. In addition, ox-LDL exposure resulted in upregulation of miR-1974, miR-1978 and miR-21 accompanied with significant over-presentation of their target genes in the downregulated portion of ox-LDL transcriptome. Our observations indicate that ox-LDL at physiologic concentrations induces broad transcriptional responses which are mediated by LOX-1, and are, in part, shaped by ox-LDL-dependent miRNAs. We also suggest that angiogenic effects of ox-LDL are partially based on upregulation of several receptors that render cells hypersensitive to angiogenic stimuli.</p> </div

Ox-LDL signaling is partially mediated by miR-21.

<p>(<b>A</b>) - qPCR analysis of miR-21 expression in response to ox-LDL (left) and (<b>B</b>) -microarray expression of corresponding validated target genes; (<b>C</b>) - qPCR analysis of miR-221 expression in response to ox-LDL and (<b>D</b>) - microarray expression of corresponding validated target genes. (<b>E</b>) – the percentage of target genes for miR-1974, miR-1978 and miR-21 in downregulated and upregulated portions of ox-LDL induced transcriptome.</p

Ox-LDL transcriptome and its dependence on LOX-1 in endothelial cells.

<p>(<b>A</b>) – basal expression of scavenger receptors in HCAECs evaluated by qPCR. (<b>B</b>) – ox-LDL induced expression of scavenger receptors in HCAECs by qPCR. (<b>C</b>) - Differentially expressed genes (1.5-fold, p<0.05) in HCAECs exposed to 5 µg/ml ox-LDL for 12 hrs. Blue lines represent genes up- or down-regulated more than 2-fold. Red triangles represent expression of these genes in cultures pre-treated with LOX-1 neutralizing antibody (TS92, 10 mg/ml) before ox-LDL exposure.</p

Schematics of pro-angiogenic signaling of ox-LDL based on microarray data partially validated by qPCR.

<p>Color coding: Shades of green – upregulation. Rred-inhibition; Grey –no change; Pro-angiogenic action of ox-LDL appears to be most consistently associated with VEGF and Notch pathways. Downstream target genes of VEGF also show corresponding shifts in expression patterns.</p

Effects of LOX-1 abrogation on choroid angiogenesis.

<p>Eyes of 8 week old wild type (C57BL) and LOX-1 KO (C57BL background) mice were subjected to laser photocoagulation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047421#s4" target="_blank">Materials and Methods</a>). Choroid angiogenesis was visualized in RPE-choroid-sclera flat mounts from animals perfused with 1 ml of PBS containing 50 mg/ml FITC-dextran after 7 days. Mounts were examined under a ZEISS LSM 510 laser confocal microscope and images of laser spots were captured. The images represent 3D reconstruction of the choroid neovascularization complex.</p