A single upstream mutation of whiB7 underlies amikacin and clarithromycin resistance in Mycobacterium abscessus.

Aims: We aimed to investigate the molecular mechanisms underlying the survival of Mycobacterium abscessus when faced with antibiotic combination therapy. By conducting evolution experiments and whole-genome sequencing (WGS), we sought to identify genetic variants associated with stress response mechanisms, with a particular focus on drug survival and resistance.

Methods And Results: We conducted evolution experiments on M.

in Non-Cystic Fibrosis Bronchiectasis: Prevalence and Genomic Basis of High Inoculum β-Lactam Resistance.

The pathobiology of in non-cystic fibrosis bronchiectasis (nCFB) is poorly defined. When present at high density or "inoculum," some methicillin-sensitive (MSSA) can inefficiently degrade antistaphylococcal β-lactam antibiotics via BlaZ penicillinases (termed the "inoculum effect" [IE]). Given the high burden of organisms in bronchiectatic airways, this is particularly relevant.

Identification of biologic agents to neutralize the bicomponent leukocidins of .

A key aspect underlying the severity of infections caused by is the abundance of virulence factors that the pathogen uses to thwart critical components of the human immune response. One such mechanism involves the destruction of host immune cells by cytolytic toxins secreted by , including five bicomponent leukocidins: PVL, HlgAB, HlgCB, LukED, and LukAB. Purified leukocidins can lyse immune cells ex vivo, and systemic injections of purified LukED or HlgAB can acutely kill mice.

Staphylococcus aureus Strain Newman D2C Contains Mutations in Major Regulatory Pathways That Cripple Its Pathogenesis.

is a major human pathogen that imposes a great burden on the health care system. In the development of antistaphylococcal modalities intended to reduce the burden of staphylococcal disease, it is imperative to select appropriate models of strains when assessing the efficacy of novel agents. Here, using whole-genome sequencing, we reveal that the commonly used strain Newman D2C from the American Type Culture Collection (ATCC) contains mutations that render the strain essentially avirulent.

Antibody-Based Biologics and Their Promise to Combat Staphylococcus aureus Infections.

The growing incidence of serious infections mediated by methicillin-resistant Staphylococcus aureus (MRSA) strains poses a significant risk to public health. This risk is exacerbated by a prolonged void in the discovery and development of truly novel antibiotics and the absence of a vaccine. These gaps have created renewed interest in the use of biologics in the prevention and treatment of serious staphylococcal infections.

Antisense RNA amplification for target assessment of total mRNA from a single cell.

This protocol describes how to amplify mRNA isolated from a single cell and then analyze its gene expression profile using polymerase chain reaction (PCR). Single-cell analysis is advantageous over studies of cell populations because it allows identification of a range of normal physiological states expressed by different cells of the same cell type without the confounding effects of averaging that result from measuring physiological states of cell populations. This is especially important when addressing questions of physiology in tissues, which comprises many different cell types.

Divergence of RNA localization between rat and mouse neurons reveals the potential for rapid brain evolution.

Background: Neurons display a highly polarized architecture. Their ability to modify their features under intracellular and extracellular stimuli, known as synaptic plasticity, is a key component of the neurochemical basis of learning and memory. A key feature of synaptic plasticity involves the delivery of mRNAs to distinct sub-cellular domains where they are locally translated.

Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue.

Transcriptome profiling of single cells resident in their natural microenvironment depends upon RNA capture methods that are both noninvasive and spatially precise. We engineered a transcriptome in vivo analysis (TIVA) tag, which upon photoactivation enables mRNA capture from single cells in live tissue. Using the TIVA tag in combination with RNA sequencing (RNA-seq), we analyzed transcriptome variance among single neurons in culture and in mouse and human tissue in vivo.

Serotonergic neuron regulation informed by in vivo single-cell transcriptomics.

Despite the recognized importance of the dorsal raphe (DR) serotonergic (5-HT) nuclei in the pathophysiology of depression and anxiety, the molecular components/putative drug targets expressed by these neurons are poorly characterized. Utilizing the promoter of an ETS domain transcription factor that is a stable marker of 5-HT neurons (Pet-1) to drive 5-HT neuronal expression of YFP, we identified 5-HT neurons in live acute slices. We isolated RNA from single 5-HT neurons in the ventromedial and lateral wings of the DR and performed single-cell RNA-Seq analysis identifying >500 G-protein coupled receptors (GPCRs) including receptors for classical transmitters, lipid signals, and peptides as well as dozens of orphan-GPCRs.

Cytoplasmic intron retention, function, splicing, and the sentinel RNA hypothesis.

Cytoplasmic splicing represents a newly emerging level of transcriptional regulation adding to the molecular diversity of mammalian cells. As examples of this noncanonical form of transcript processing are discovered, the evidence of its importance to normal cellular function grows. Work from a number of groups using a variety of cell types is steadily identifying a large number of transcripts (and soon to be even larger as genome-wide analyses of retained introns across a number of cellular phenotypes are currently underway) that undergo some level of regulated endogenous extranuclear splicing as part of their normal biosynthetic pathway.

Subcellular RNA sequencing reveals broad presence of cytoplasmic intron-sequence retaining transcripts in mouse and rat neurons.

Recent findings have revealed the complexity of the transcriptional landscape in mammalian cells. One recently described class of novel transcripts are the Cytoplasmic Intron-sequence Retaining Transcripts (CIRTs), hypothesized to confer post-transcriptional regulatory function. For instance, the neuronal CIRT KCNMA1i16 contributes to the firing properties of hippocampal neurons.

Pharmacological characterization of GSK573719 (umeclidinium): a novel, long-acting, inhaled antagonist of the muscarinic cholinergic receptors for treatment of pulmonary diseases.

Activation of muscarinic subtype 3 (M3) muscarinic cholinergic receptors (mAChRs) increases airway tone, whereas its blockade improves lung function and quality of life in patients with pulmonary diseases. The present study evaluated the pharmacological properties of a novel mAChR antagonist, GSK573719 (4-[hydroxy(diphenyl)methyl]-1-{2-[(phenylmethyl)oxy]ethyl}-1-azoniabicyclo[2.2.

Tyrosine urea muscarinic acetylcholine receptor antagonists: achiral quaternary ammonium groups.

Tyrosine ureas had been identified as potent muscarinic receptor antagonists with promising in vivo activity. Controlling the stereochemistry of the chiral quaternary ammonium center had proved to be a serious issue for this series, however. Herein we describe the preparation and SAR of tyrosine urea antagonists containing achiral quaternary ammonium centers.

Drug targets: single-cell transcriptomics hastens unbiased discovery.

Drug discovery in neuro- and psychopharmacology is lagging, and the most commonly mentioned cause is the scarcity of drug targets. Using NextGen 'sequencing based single-cell transcriptomics' (SBSCT), several hundred different receptors and channels can be identified in individual neurons, and the functional gene product can subsequently be validated. The use of single-cell transcriptome data to reveal the entire receptor repertoire is crucial, as the copy numbers of mRNAs encoding receptors are low, and when cells are pooled dilution of rare mRNAs leads to loss of signal.

Cytoplasmic intron sequence-retaining transcripts can be dendritically targeted via ID element retrotransposons.

RNA precursors give rise to mRNA after splicing of intronic sequences traditionally thought to occur in the nucleus. Here, we show that intron sequences are retained in a number of dendritically-targeted mRNAs, by using microarray and Illumina sequencing of isolated dendritic mRNA as well as in situ hybridization. Many of the retained introns contain ID elements, a class of SINE retrotransposon.

mRNA for the EAAC1 subtype of glutamate transporter is present in neuronal dendrites in vitro and dramatically increases in vivo after a seizure.

The neuronal Na(+)-dependent glutamate transporter, excitatory amino acid carrier 1 (EAAC1, also called EAAT3), has been implicated in the control of synaptic spillover of glutamate, synaptic plasticity, and the import of cysteine for neuronal synthesis of glutathione. EAAC1 protein is observed in both perisynaptic regions of the synapse and in neuronal cell bodies. Although amino acid residues in the carboxyl terminal tail have been implicated in the dendritic targeting of EAAC1 protein, it is not known if mRNA for EAAC1 may also be targeted to dendrites.

Intron retention facilitates splice variant diversity in calcium-activated big potassium channel populations.

We report that the stress axis-regulated exon (STREX)-containing calcium-activated big potassium (BKCa) channel splice variant expression and physiology are regulated in part by cytoplasmic splicing and intron retention. NextGen sequencing of the mRNA complement of pooled hippocampal dendrite samples found intron 17a (i17a), the intron immediately preceding STREX, in the BKCa mRNA. Further molecular analyses of i17a revealed that the majority of i17a-containing BKCa channel mRNAs associate with STREX.

Melanocyte-like cells in the heart and pulmonary veins contribute to atrial arrhythmia triggers.

Atrial fibrillation is the most common clinical cardiac arrhythmia. It is often initiated by ectopic beats arising from the pulmonary veins and atrium, but the source and mechanism of these beats remains unclear. The melanin synthesis enzyme dopachrome tautomerase (DCT) is involved in intracellular calcium and reactive species regulation in melanocytes.

The reduced folate carrier (SLC19A1) c.80G>A polymorphism is associated with red cell folate concentrations among women.

Low folate status may be a consequence of suboptimal intake, transport or cellular utilization of folate and, together with elevated homocysteine, is a recognized risk factor or marker for several human pathologies. As folate transport across cell membranes is mediated in part by the reduced folate carrier (RFC1), variants within SLC19A1, the gene that encodes RFC1, may influence disease risk via an effect on folate and/or homocysteine levels. The present study was undertaken to assess the association between the SLC19A1 c.

Discovery of (3-endo)-3-(2-cyano-2,2-diphenylethyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octane bromide as an efficacious inhaled muscarinic acetylcholine receptor antagonist for the treatment of COPD.

Design and syntheses of a novel series of muscarinic antagonists are reported. These efforts have culminated in the discovery of (3-endo)-3-(2-cyano-2,2-diphenylethyl)-8,8-dimethyl-8-azoniabicyclo[3.2.

Cytoplasmic BK(Ca) channel intron-containing mRNAs contribute to the intrinsic excitability of hippocampal neurons.

High single-channel conductance K+ channels, which respond jointly to membrane depolarization and micromolar concentrations of intracellular Ca2+ ions, arise from extensive cell-specific alternative splicing of pore-forming alpha-subunit mRNAs. Here, we report the discovery of an endogenous BK(Ca) channel alpha-subunit intron-containing mRNA in the cytoplasm of hippocampal neurons. This partially processed mRNA, which comprises approximately 10% of the total BK(Ca) channel alpha-subunit mRNAs, is distributed in a gradient throughout the somatodendritic space.

HIV-1 Viral protein-r (Vpr) protects against lethal superantigen challenge while maintaining homeostatic T cell levels in vivo.

The HIV-1 accessory protein Vpr exhibits many interesting features related to macrophage and T cell biology. As a viral protein or as a soluble molecule it can suppress immune cell activation and cytokine production in vitro in part by targeted inhibition of NF-kappaB. In this regard we sought to test its effects in vivo on an NF-kappaB-dependent immune pathway.

Nonpeptidic urotensin-II receptor antagonists I: in vitro pharmacological characterization of SB-706375.

1. SB-706375 potently inhibited [(125)I]hU-II binding to both mammalian recombinant and 'native' UT receptors (K(i) 4.7+/-1.

Cloning and pharmacological characterization of the cat urotensin-II receptor (UT).

Urotensin-II (U-II), acting through its G-protein-coupled receptor, UT, is a possible contributor to hypertension. Variable functional responses to U-II, both within and between species studied to date, complicate the characterization of UT antagonists. In the cat, however, U-II causes systemic hypertension and constricts arterial segments isolated from several vascular beds.