The Mycobacterial Efflux Pump EfpA Can Induce High Drug Tolerance to Many Antituberculosis Drugs, Including Moxifloxacin, in Mycobacterium smegmatis.

Active efflux of drugs across the membrane is a major survival strategy of bacteria against many drugs. In this work, we characterize an efflux pump, EfpA, from the major facilitator superfamily, that is highly conserved among both slow-growing and fast-growing Mycobacterium species and has been found to be upregulated in many clinical isolates of Mycobacterium tuberculosis. The gene encoding EfpA from Mycobacterium smegmatis was overexpressed under the control of both a constitutive and an inducible promoter. The expression of the efpA gene under the control of both promoters resulted in >32-fold-increased drug tolerance of M. smegmatis cells to many first-line (rifampicin, isoniazid, and streptomycin) and second-line (amikacin) antituberculosis drugs. Notably, the drug tolerance of M. smegmatis cells to moxifloxacin increased by more than 180-fold when efpA was overexpressed. The increase in MICs correlated with the decreased uptake of drugs, including norfloxacin, moxifloxacin, and ethidium bromide, and the high MIC could be reversed in the presence of an efflux pump inhibitor. A correlation was observed between the MICs of drugs and the efflux pump expression level, suggesting that the latter could be modulated by varying the expression level of the efflux pump. The expression of high levels of efpA did not impact the fitness of the cells when supplemented with glucose. The efpA gene is conserved across both pathogenic and nonpathogenic mycobacteria. The efpA gene from Mycobacterium bovis BCG/M. tuberculosis, which is 80% identical to efpA from M. smegmatis, also led to decreased antimicrobial efficacy of many drugs, although the fold change was lower. When overexpressed in M. bovis BCG, 8-fold-higher drug tolerance to moxifloxacin was observed. This is the first report of an efflux pump from Mycobacterium species that leads to higher drug tolerance to moxifloxacin, a promising new drug for the treatment of tuberculosis.

Ethanol in Combination with Oxidative Stress Significantly Impacts Mycobacterial Physiology.

Here, we investigate the mycobacterial response to the combined stress of an organic oxidant (cumene hydroperoxide [CHP]) and a solvent (ethanol). To understand the interaction between the two stressors, we treated Mycobacterium smegmatis cells to a range of ethanol concentrations (2.5% to 10% [vol/vol]) in combination with a subinhibitory concentration of 1 mM CHP. It was observed that the presence of CHP increases the efficacy of ethanol in inducing rapid cell death. The data further suggest that ethanol reacts with the alkoxy radicals to produce ethanol-derived peroxides. These radicals induce significant membrane damage and lead to cell lysis. The ethanol-derived radicals were primarily recognized by the cells as organic radicals, as was evident by the differential upregulation of the ohr-ohrR genes that function in cells treated with the combination of ethanol and CHP. The role of organic peroxide reductase, Ohr, was further confirmed by the significantly higher sensitivity of the deletion mutant to CHP and the combined stress treatment of CHP and ethanol. Moreover, we also observed the sigma factor σB to be important for the cells treated with ethanol alone as well as the aforementioned combination. A ΔsigB mutant strain had significantly higher susceptibility to the stress conditions. This finding was correlated with the σB-dependent transcriptional regulation of ohr and ohrR In summary, our data indicate that the combination of low levels of ethanol and organic peroxides induce ethanol-derived organic radicals that lead to significant oxidative stress on the cells in a concentration-dependent manner.IMPORTANCE Bacterial response to a combination of stresses can be unexpected and very different compared with that of an individual stress treatment. This study explores the physiological and transcriptional response of mycobacteria in response to the combinatorial treatment of an oxidant with the commonly used solvent ethanol. The presence of a subinhibitory concentration of organic peroxide increases the effectiveness of ethanol by inducing reactive peroxides that destroy the membrane integrity of cells in a significantly short time span. Our work elucidates a mechanism of targeting the complex mycobacterial membrane, which is its primary source of intrinsic resistance. Furthermore, it also demonstrates the importance of exploring the effect of various stress conditions on inducing bacterial clearance.

Identification of Disease-relevant, Sex-based Proteomic Differences in iPSC-derived Vascular Smooth Muscle.

The prevalence of cardiovascular disease varies with sex, and the impact of intrinsic sex-based differences on vasculature is not well understood. Animal models can provide important insight into some aspects of human biology, however not all discoveries in animal systems translate well to humans. To explore the impact of chromosomal sex on proteomic phenotypes, we used iPSC-derived vascular smooth muscle cells from healthy donors of both sexes to identify sex-based proteomic differences and their possible effects on cardiovascular pathophysiology. Our analysis confirmed that differentiated cells have a proteomic profile more similar to healthy primary aortic smooth muscle than iPSCs. We also identified sex-based differences in iPSC- derived vascular smooth muscle in pathways related to ATP binding, glycogen metabolic process, and cadherin binding as well as multiple proteins relevant to cardiovascular pathophysiology and disease. Additionally, we explored the role of autosomal and sex chromosomes in protein regulation, identifying that proteins on autosomal chromosomes also show sex-based regulation that may affect the protein expression of proteins from autosomal chromosomes. This work supports the biological relevance of iPSC-derived vascular smooth muscle cells as a model for disease, and further exploration of the pathways identified here can lead to the discovery of sex-specific pharmacological targets for cardiovascular disease. Significance: In this work, we have differentiated 4 male and 4 female iPSC lines into vascular smooth muscle cells, giving us the ability to identify statistically-significant sex-specific proteomic markers that are relevant to cardiovascular disease risk (such as PCK2, MTOR, IGFBP2, PTGR2, and SULTE1).

Influence of PEGylation on WS2 Nanosheets and Its Application in Photothermal Therapy.

Photothermal therapy (PTT) is an alternative cancer therapy with minimal side effects and higher efficiency and selectivity. In this study, WS2 nanosheets were developed by ultrasonic exfoliation with different ratios of polyethylene glycol (PEG), and their effects on physicochemical properties were studied. The utilization of PEG during sonication significantly influenced the size and thickness of the resulting WS2 nanosheet layers, which was confirmed through scanning electron microscopy, atomic force microscopy, and dynamic light scattering analyses. PEG functionalization also improved the dispersibility of WS2 in aqueous solution by making its surface hydrophilic, which resulted in better biocompatibility. The intrinsic near-infrared absorbance of the nanosheets positions them as valuable agents for PTT. The study further explores the efficacy of these nanosheets as photothermal agents in the ablation of MDAMB-231 breast cancer cells. Although the use of PEG to demonstrate exfoliation and biocompatibility for WS2 has been reported previously, the effect of PEGylation on various physicochemical properties has not been studied in-depth until now. This study paves the way for the use of highly versatile PEG across a range of 2D material systems.

The Extent of Antimicrobial Resistance Due to Efflux Pump Regulation.

A key mechanism driving antimicrobial resistance (AMR) stems from the ability of bacteria to up-regulate efflux pumps upon exposure to drugs. The resistance gained by this up-regulation is pliable because of the tight regulation of efflux pump levels. This leads to temporary enhancement in survivability of bacteria due to higher efflux pump levels in the presence of antibiotics, which can be reversed when the cells are no longer exposed to the drug. Knowledge of the extent of resistance thus gained would inform intervention strategies aimed at mitigating AMR. Here, we combine mathematical modeling and experiments to quantify the maximum extent of resistance that efflux pump up-regulation can confer via phenotypic induction in the presence of drugs and genotypic abrogation of regulation. Our model describes the dynamics of drug transport in and out of cells coupled with the associated regulation of efflux pump levels and predicts the increase in the minimum inhibitory concentration (MIC) of drugs due to such regulation. To test the model, we measured the uptake and efflux as well as the MIC of the compound ethidium bromide (EtBr), a substrate of the efflux pump LfrA, in wild-type Mycobacterium smegmatis mc2155, as well as in two laboratory-generated strains. Our model captured the observed EtBr levels and MIC fold-changes quantitatively. Further, the model identified key parameters associated with the resulting resistance, variations in which could underlie the extent to which such resistance arises across different drug-bacteria combinations, potentially offering tunable handles to optimize interventions aimed at minimizing AMR.

Asporin, an extracellular matrix protein, is a beneficial regulator of cardiac remodeling.

Heart failure is accompanied by adverse cardiac remodeling involving extracellular matrix (ECM). Cardiac ECM acts as a major reservoir for many proteins including growth factors, cytokines, collagens, and proteoglycans. Activated fibroblasts during cardiac injury can alter the composition and activity of these ECM proteins. Through unbiased analysis of a microarray dataset of human heart tissue comparing normal hearts (n = 135) to hearts with ischemic cardiomyopathy (n = 94), we identified Asporin (ASPN) as the top differentially regulated gene (DEG) in ischemic cardiomyopathy; its gene-ontology terms relate closely to fibrosis and cell death. ASPN is a Class I small leucine repeat protein member implicated in cancer, osteoarthritis, and periodontal ligament mineralization. However, its role in cardiac remodeling is still unknown. Here, we initially confirmed our big dataset analysis through cells, mice, and clinical atrial biopsy samples to demonstrate increased Aspn expression after pressure overload or cardiac ischemia/reperfusion injury. We tested the hypothesis that Aspn, being a TGFß1 inhibitor, can attenuate fibrosis in mouse models of cardiac injury. We found that Aspn is released by cardiac fibroblasts and attenuates TGFß signaling. Moreover, Aspn-/- mice displayed increased fibrosis and decreased cardiac function after pressure overload by transverse aortic constriction (TAC) in mice. In addition, Aspn protected cardiomyocytes from hypoxia/reoxygenation-induced cell death and regulated mitochondrial bioenergetics in cardiomyocytes. Increased infarct size after ischemia/reperfusion injury in Aspn-/- mice confirmed Aspn's contribution to cardiomyocyte viability. Echocardiography revealed greater reduction in left ventricular systolic function post-I/R in the Aspn-/- animals compared to wild type. Furthermore, we developed an ASPN-mimic peptide using molecular modeling and docking which when administered to mice prevented TAC-induced fibrosis and preserved heart function. The peptide also reduced infarct size after I/R in mice, demonstrating the translational potential of ASPN-based therapy. Thus, we establish the role of ASPN as a critical ECM molecule that regulates cardiac remodeling to preserve heart function.

Publisher Correction: Ramifications of Atmospheric Humidity on Monsoon Depressions over the Indian Subcontinent.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Ramifications of Atmospheric Humidity on Monsoon Depressions over the Indian Subcontinent.

In this study, a comprehensive investigation is carried out to examine the sensitivity of tropospheric relative humidity (RH) on monsoon depressions (MDs) under a changing climate regime through surrogate climate change approach over the Indian region. Composite analysis of four MDs show a persistent warming (RH2+) and cooling (RH2-) throughout the troposphere in the sensitivity experiments. In-depth analysis of a MD over the Arabian Sea (AS) exhibits sustained warming for RH2+, which is accredited to 2.6% increase in stratiform clouds accounting for 13% increment in heating, whereas 5% increment in convective clouds hardly contribute to total heating. Frozen hydrometeors (graupel and snow) are speculated to be the major contributors to this heating. Stratiform clouds showed greater sensitivity to RH perturbations in the lower troposphere (1000-750 hPa), albeit very less sensitivity for convective clouds, both in the lower and mid-troposphere (700-500 hPa). Precipitation is enhanced in a moist situation (RH2+) owing to positive feedbacks induced by moisture influx and precipitation efficiency, while negative feedbacks suppressed precipitation in a dry troposphere (RH2-). In a nutshell, it is inferred that under moist (dry) situations, it is highly likely that intense (weak) MDs will occur in the near future over the Indian region.