Biology subjects

Klein, R.

Publications and source records attributed to Klein, R..

6 recordsLinked to original sources

bioRxiv2018

In Silico Identification and In Vitro Validation of Novel KPC-2 β-lactamase Inhibitors

Bacterial resistance has become a worldwide concern, particularly after the emergence of resistant strains overproducing carbapenemases. Among these, the KPC-2 carbapenemase represents a significant clinical challenge, being characterized by a broad substrate spectrum that includes aminothiazoleoxime and cephalosporins such as cefotaxime. Moreover, strains harboring KPC-type {beta}-lactamases are often reported as resistant to available {beta}-lactamase inhibitors (clavulanic acid, tazobactam and sulbactam). Therefore, the identification of novel non {beta}-lactam KPC-2 inhibitors is strongly necessary to maintain treatment options. This study explored novel, non-covalent inhibitors active against KPC-2, as putative hit candidates. We performed a structure-based in silico screening of commercially available compounds for non-{beta}-lactam KPC-2 inhibitors. Thirty-two commercially available high-scoring, fragment-like hits were selected for in vitro validation and their activity and mechanism of action vs the target was experimentally evaluated using recombinant KPC-2. N-(3-(1H-tetrazol-5-yl)phenyl)-3-fluorobenzamide (11a), in light of its ligand efficiency (LE = 0.28 kcal/mol/non-hydrogen atom) and chemistry, was selected as hit to be directed to chemical optimization to improve potency vs the enzyme and explore structural requirement for inhibition in KPC-2 binding site. Further, the compounds were evaluated against clinical strains overexpressing KPC-2 and the most promising compound reduced the MIC of the {beta}-lactam antibiotic meropenem by four fold.

biochemistry↗

bioRxiv2018

Cortical circuit alterations precede disease onset in Huntington’s disease mice

AbstractHuntingtons disease (HD) is a devastating hereditary movement disorder, characterized by degeneration of neurons in the striatum and cortex. Studies in human patients and mouse HD models suggest that disturbances of neuronal function in the neocortex play an important role in the disease onset and progression. However, the precise nature and time course of cortical alterations in HD have remained elusive. Here, we use chronic in vivo two-photon calcium imaging to monitor the activity of single neurons in layer 2/3 of the primary motor cortex in awake, behaving R6/2 transgenic HD mice and wildtype littermates. R6/2 mice show age-dependent changes in neuronal activity with a clear increase in activity at the age of 8.5 weeks, preceding the onset of motor and neurological symptoms. Furthermore, quantitative proteomics demonstrate a pronounced downregulation of synaptic proteins in the cortex, and histological analyses in R6/2 mice and HD patient samples reveal reduced inputs from parvalbumin-positive interneurons onto layer 2/3 pyramidal cells. Thus, our study provides a time-resolved description as well as mechanistic details of cortical circuit dysfunction in HD.\n\nSignificance statementFuntional alterations in the cortex are believed to play an important role in the pathogenesis of Huntingtons disease (HD). However, studies monitoring cortical activity in HD models in vivo at a single-cell resultion are still lacking. We have used chronic two-photon imaging to investigate changes in the activity of single neurons in the primary motor cortex of awake presymptomatic HD mice. We show that neuronal activity increases before the mice develop disease symptoms. Our histological analyses in mice and in human HD autopsy cases furthermore demonstrate a loss inhibitory synaptic terminals from parvalbimun-positive interneurons, revealing a potential mechanism of cortical circuit impairment in HD.

neuroscience↗

bioRxiv2018

Validation of Prostate Cancer Risk Variants by CRISPR/Cas9 Mediated Genome Editing

GWAS have identified numerous SNPs associated with prostate cancer risk. One such SNP is rs10993994. It is located in the MSMB promoter, associates with MSMB encoded {beta}-microseminoprotein prostate secretion levels, and is associated with mRNA expression changes in MSMB and the adjacent gene NCOA4. In addition, our previous work showed a second SNP, rs7098889, is in LD with rs10993994 and associated with MSMB expression independent of rs10993994. Here, we generate a series of clones with single alleles removed by double guide RNA (gRNA) mediated CRISPR/Cas9 deletions, through which we demonstrate that each of these SNPs independently and greatly alters MSMB expression in an allele-specific manner. We further show that these SNPs have no substantial effect on the expression of NCOA4. These data demonstrate that a single SNP can have a large effect on gene expression and illustrate the importance of functional validation to deconvolute observed correlations. The method we have developed is generally applicable to test any SNP for which a relevant heterozygous cell line is available.\n\nAuthor summaryIn pursuing the underlying biological mechanism of prostate cancer pathogenesis, scientists utilized the existence of common single nucleotide polymorphisms (SNPs) in human genome as genetic markers to perform large scale genome wide association studies (GWAS) and have so far identified more than a hundred prostate cancer risk variants. Such variants provide an unbiased and systematic new venue to study the disease mechanism, and the next big challenge is to translate these genetic associations to the causal role of altered gene function in oncogenesis. The majority of these variants are waiting to be studied and lots of them may act in oncogenesis through gene expression regulation. To prove the concept, we took rs10993994 and its linked rs7098889 as an example and engineered single cell clones by allelic-specific CRISPR/Cas9 deletion to separate the effect of each allele. We observed that a single nucleotide difference would lead to surprisingly high level of MSMB gene expression change in a gene specific and tissue specific manner. Our study strongly supports the notion that differential level of gene expression caused by risk variants and their associated genetic locus play a major role in oncogenesis and also highlights the importance of studying the function of MSMB encoded {beta}-MSP in prostate cancer pathogenesis.

genetics↗

bioRxiv2017

High-depth whole genome sequencing of a large population-specific reference panel: Enhancing sensitivity, accuracy, and imputation

BackgroundWhile increasingly large reference panels for genome-wide imputation have been recently made available, the degree to which imputation accuracy can be enhanced by population-specific reference panels remains an open question. In the present study, we sequenced at full-depth ([≥]30x) a moderately large (n=738) cohort of samples drawn from the Ashkenazi Jewish population across two platforms (Illumina X Ten and Complete Genomics, Inc.). We developed and refined a series of quality control steps to optimize sensitivity, specificity, and comprehensiveness of variant calls in the reference panel, and then tested the accuracy of imputation against target cohorts drawn from the same population.\n\nResultsFor samples sequenced on the Illumina X Ten platform, quality thresholds were identified that permitted highly accurate calling of single nucleotide variants across 94% of the genome. The Complete Genomics, Inc. platform was more conservative (fewer variants called) compared to the Illumina platform, but also demonstrated relatively greater numbers of false positives that needed to be filtered. Quality control procedures also permitted detection of novel genome reads that are not mapped to current reference or alternate assemblies. After stringent quality control, the population-specific reference panel produced more accurate and comprehensive imputation results relative to publicly available, large cosmopolitan reference panels. The population-specific reference panel also permitted enhanced filtering of clinically irrelevant variants from personal genomes.\n\nConclusionsOur primary results demonstrate enhanced accuracy of a population-specific imputation panel relative to cosmopolitan panels, especially in the range of infrequent (<5% non-reference allele frequency) and rare (<1% non-reference allele frequency) variants that may be most critical to further progress in mapping of complex phenotypes.

genomics↗

bioRxiv2017

A Strategy for Large-Scale Systematic Pan-Cancer Germline Rare Variation Analysis

Traditionally, genetic studies in cancer are focused on somatic mutations found in tumors and absent from the normal tissue. Identification of shared attributes in germline variation could aid discrimination of high-risk from likely benign mutations and narrow the search space for new cancer predisposing genes. Extraordinary progress made in analysis of common variation with GWAS methodology does not provide sufficient resolution to understand rare variation. To fulfil missing classification for rare germline variation we assembled datasets of whole exome sequences from >2,000 patients with different types of cancers: breast cancer, colon cancer and cutaneous and ocular melanomas matched to more than 7,000 non-cancer controls and analyzed germline variation in known cancer predisposing genes to identify common properties of disease associated mutations and new candidate cancer susceptibility genes. Lists of all cancer predisposing genes were divided into subclasses according to the mode of inheritance of the related cancer syndrome or contribution to known major cancer pathways. Out of all subclasses only genes linked to dominant syndromes presented significant rare germline variants enrichment in cases. Separate analysis of protein-truncating and missense variation in this subclass of genes confirmed significant prevalence of protein-truncating variants in cases only in loss-of-function tolerant genes (pLI<0.1), while ultra-rare missense mutations were significantly overrepresented in cases only in constrained genes (pLI>0.9). Taken together, our findings provide insights into the distribution and types of mutations underlying inherited cancer predisposition.

genetics↗

bioRxiv2017

Central Amygdala Circuits Modulate Food Consumption Through A Positive Valence Mechanism

The complex behaviors underlying the pursuit and consumption of rewards are integral to an organisms survival. The hypothalamus and mesolimbic dopamine system are key mediators of these behaviors, yet regulation of appetitive and consummatory behaviors outside of these regions is not well understood. The central nucleus of the amygdala (CeA) is implicated in feeding and reward behavior, but the specific neural players and circuit mechanisms that positively regulate these behaviors remain unclear. Here, we define the neuronal mechanisms by which the CeA promotes consumption of food. We show, using in vivo activity manipulations and Ca2+ imaging, that CeA GABAergic neurons expressing the serotonin receptor 2a (Htr2a) modulate food consumption in multiple contexts, promote positive reinforcement and are active in vivo during eating. We demonstrate using electrophysiology, anatomical tracing methods and in vivo optogenetics that both intra-CeA and long-range circuit mechanisms underlie these functional effects. Finally, we show that CeAHtr2a neurons are poised to regulate food consumption through inputs from feeding-relevant brain regions. Our study highlights a mechanism by which defined CeA neural circuits positively regulate food consumption.

neuroscience↗