Molecular mechanisms and modulation of Endothelial Progenitor Cell function in South Asian men

South Asian ethnicity has long been considered a contributory factor in the development of atherosclerosis and cardiovascular disease. Endothelial dysfunction is considered to be the precursor of atherosclerosis, and South Asian ethnicity has been linked to endothelial progenitor cell dysfunction and impaired endothelium dependent vasodilatation. Endothelial progenitor cell (EPC) dysfunction may contribute to ineffective endogenous endothelial repair, although the exact mechanisms remain unclear. This project aimed to validate existing data, and identify signalling pathways and molecular mechanisms that may be involved in EPC dysfunction in healthy South Asian men when compared to matched white European controls. In addition, the study was designed to assess the impact of South Asian ethnicity on EPC function in vivo, and whether modifying the signalling pathways linked with EPC dysfunction improved EPC function. Early outgrowth EPC numbers were significantly lower in South Asian men. EPC numbers were significantly increased in both South Asian and white European groups by treating EPCs with Simvastatin in vitro. Functional parameters such as migration to VEGF, adhesion to fibronectin and endothelial tubule formation induced by EPC conditioned medium were also impaired in South Asian EPCs. Some aspects of functional impairment were improved by Simvastatin. Late outgrowth EPCs derived from South Asian men demonstrated reduced proliferation and increased senescence. Western blot analysis showed reduced basal Akt and endothelial Nitric Oxide Synthase (eNOS) expression in South Asian late EPCs. Vascular repair of injured conduit vessel following transfusion of South Asian late EPCs was significantly impaired - this was rescued by increasing Akt activity using Lentiviral delivery of constitutively active Akt. Abnormal phosphotidyl-inositol-3-kinase/Akt signalling may therefore contribute to EPC dysfunction in South Asian men. These data provide novel insights into mechanisms underlying EPC dysfunction which may contribute to increased atherosclerosis burden in people of South Asian origin. Future research avenues have been identified, which may guide development of novel strategies in preventing and treating cardiovascular disease

PerSort Facilitates Characterization and Elimination of Persister Subpopulation in Mycobacteria.

Mycobacterium tuberculosis (MTB) generates phenotypic diversity to persist and survive the harsh conditions encountered during infection. MTB avoids immune effectors and antibacterial killing by entering into distinct physiological states. The surviving cells, persisters, are a major barrier to the timely and relapse-free treatment of tuberculosis (TB). We present for the first time, PerSort, a method to isolate and characterize persisters in the absence of antibiotic or other pressure. We demonstrate the value of PerSort to isolate translationally dormant cells that preexisted in small numbers within Mycobacterium species cultures growing under optimal conditions but that dramatically increased in proportion under stress conditions. The translationally dormant subpopulation exhibited multidrug tolerance and regrowth properties consistent with those of persister cells. Furthermore, PerSort enabled single-cell transcriptional profiling that provided evidence that the translationally dormant persisters were generated through a variety of mechanisms, including vapC30, mazF, and relA/spoT overexpression. Finally, we demonstrate that notwithstanding the varied mechanisms by which the persister cells were generated, they converge on a similar low-oxygen metabolic state that was reversed through activation of respiration to rapidly eliminate persisters fostered under host-relevant stress conditions. We conclude that PerSort provides a new tool to study MTB persisters, enabling targeted strategies to improve and shorten the treatment of TB.IMPORTANCE Mycobacterium tuberculosis (MTB) persists and survives antibiotic treatments by generating phenotypically heterogeneous drug-tolerant subpopulations. The surviving cells, persisters, are a major barrier to the relapse-free treatment of tuberculosis (TB), which is already killing \u3e1.8 million people every year and becoming deadlier with the emergence of multidrug-resistant strains. This study describes PerSort, a cell sorting method to isolate and characterize, without antibiotic treatment, translationally dormant persisters that preexist in small numbers within Mycobacterium cultures. Characterization of this subpopulation has discovered multiple mechanisms by which mycobacterial persisters emerge and unveiled the physiological basis for their dormant and multidrug-tolerant physiological state. This analysis has discovered that activating oxygen respiratory physiology using l-cysteine eliminates preexisting persister subpopulations, potentiating rapid antibiotic killing of mycobacteria under host-relevant stress. PerSort serves as a new tool to study MTB persisters for enabling targeted strategies to improve and shorten the treatment of TB

Heavy and moderate interval exercise training alters low-flow-mediated constriction but does not increase circulating progenitor cells in healthy humans.

Moderate-intensity endurance exercise training improves vascular endothelial vasomotor function; however, the impact of high-intensity exercise training has been equivocal. Thus, the effect of the physiological stress of the exercise remains poorly understood. Furthermore, enhanced vascular repair mediated by circulating progenitor cells may also be improved. To address whether the physiological stress of exercise training is an important factor contributing to these adaptations, 20 healthy participants trained for 6 weeks. Training involved either moderate (MSIT; n = 9) or heavy metabolic stress (HSIT; n = 11) interval exercise training programmes matched for total work and duration of exercise. Before and after training, flow-mediated dilatation, low-flow-mediated constriction and total vessel reactivity were measured at the brachial artery using Doppler ultrasound. Circulating progenitor cells (CD34(+), CD133(+) and CD309/KDR(+)) were measured by flow cytometry (means ± SD). Relative (MSIT pre- 5.5 ± 3.4 versus post-training 6.6 ± 2.5%; HSIT pre- 6.6 ± 4.1 versus post-training 7.0 ± 3.4%, P = 0.33) and normalized (P = 0.16) flow-mediated dilatation did not increase with either training programme. However, low-flow-mediated constriction was greater after training in both groups (MSIT pre- -0.5 ± 3.2 versus post-training -1.9 ± 3.1%; HSIT pre- -1.0 ± 1.7 versus post-training -2.9 ± 3.0%, P = 0.04) and contributed to greater total vessel reactivity (MSIT pre- 7.4 ± 3.3 versus post-training 10.1 ± 3.7%; HSIT pre- 10.9 ± 5.9 versus post-training 12.7 ± 6.2%, P = 0.01). Peak reactive hyperaemia and the area under the shear rate curve were not different between groups, either before or after training. Although circulating progenitor cell numbers increased following heavy-intensity interval exercise training, variability was great amongst participants [MSIT pre- 16 ± 18 versus post-training 14 ± 12 cells (ml whole blood)(-1); HSIT pre- 8 ± 6 versus post-training 19 ± 23 cells (ml whole blood)(-1), P = 0.50]. Overall, vasoconstrictor function may be augmented by moderate- and heavy-intensity interval exercise training in young adults. However, circulating progenitor cell numbers were not increased, suggesting that these cells are not likely to be upregulated as a result of training

Transcriptome signature of cell viability predicts drug response and drug interaction in

There is an urgent need for new drug regimens to rapidly cure tuberculosis. Here, we report the development of drug response assayer (DRonA) and MLSynergy, algorithms to perform rapid drug response assays and predict response of Mycobacterium tuberculosis (Mtb) to drug combinations. Using a transcriptome signature for cell viability, DRonA detects Mtb killing by diverse mechanisms in broth culture, macrophage infection, and patient sputum, providing an efficient and more sensitive alternative to time- and resource-intensive bacteriologic assays. Further, MLSynergy builds on DRonA to predict synergistic and antagonistic multidrug combinations using transcriptomes of Mtb treated with single drugs. Together, DRonA and MLSynergy represent a generalizable framework for rapid monitoring of drug effects in host-relevant contexts and accelerate the discovery of efficacious high-order drug combinations

Intricate Genetic Programs Controlling Dormancy in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB) displays the remarkable ability to transition in and out of dormancy, a hallmark of the pathogen\u27s capacity to evade the immune system and exploit susceptible individuals. Uncovering the gene regulatory programs that underlie the phenotypic shifts in MTB during disease latency and reactivation has posed a challenge. We develop an experimental system to precisely control dissolved oxygen levels in MTB cultures in order to capture the transcriptional events that unfold as MTB transitions into and out of hypoxia-induced dormancy. Using a comprehensive genome-wide transcription factor binding map and insights from network topology analysis, we identify regulatory circuits that deterministically drive sequential transitions across six transcriptionally and functionally distinct states encompassing more than three-fifths of the MTB genome. The architecture of the genetic programs explains the transcriptional dynamics underlying synchronous entry of cells into a dormant state that is primed to infect the host upon encountering favorable conditions

Disrupting the ArcA Regulatory Network Amplifies the Fitness Cost of Tetracycline Resistance in Escherichia coli.

There is an urgent need for strategies to discover secondary drugs to prevent or disrupt antimicrobial resistance (AMR), which is causing \u3e700,000 deaths annually. Here, we demonstrate that tetracycline-resistant (TetR) Escherichia coli undergoes global transcriptional and metabolic remodeling, including downregulation of tricarboxylic acid cycle and disruption of redox homeostasis, to support consumption of the proton motive force for tetracycline efflux. Using a pooled genome-wide library of single-gene deletion strains, at least 308 genes, including four transcriptional regulators identified by our network analysis, were confirmed as essential for restoring the fitness of TetR E. coli during treatment with tetracycline. Targeted knockout of ArcA, identified by network analysis as a master regulator of this new compensatory physiological state, significantly compromised fitness of TetR E. coli during tetracycline treatment. A drug, sertraline, which generated a similar metabolome profile as the arcA knockout strain, also resensitized TetR E. coli to tetracycline. We discovered that the potentiating effect of sertraline was eliminated upon knocking out arcA, demonstrating that the mechanism of potential synergy was through action of sertraline on the tetracycline-induced ArcA network in the TetR strain. Our findings demonstrate that therapies that target mechanistic drivers of compensatory physiological states could resensitize AMR pathogens to lost antibiotics. IMPORTANCE Antimicrobial resistance (AMR) is projected to be the cause of \u3e10 million deaths annually by 2050. While efforts to find new potent antibiotics are effective, they are expensive and outpaced by the rate at which new resistant strains emerge. There is desperate need for a rational approach to accelerate the discovery of drugs and drug combinations that effectively clear AMR pathogens and even prevent the emergence of new resistant strains. Using tetracycline-resistant (TetR) Escherichia coli, we demonstrate that gaining resistance is accompanied by loss of fitness, which is restored by compensatory physiological changes. We demonstrate that transcriptional regulators of the compensatory physiologic state are promising drug targets because their disruption increases the susceptibility of TetR E. coli to tetracycline. Thus, we describe a generalizable systems biology approach to identify new vulnerabilities within AMR strains to rationally accelerate the discovery of therapeutics that extend the life span of existing antibiotics

MtrA modulates Mycobacterium tuberculosis cell division in host microenvironments to mediate intrinsic resistance and drug tolerance

Summary: The success of Mycobacterium tuberculosis (Mtb) is largely attributed to its ability to physiologically adapt and withstand diverse localized stresses within host microenvironments. Here, we present a data-driven model (EGRIN 2.0) that captures the dynamic interplay of environmental cues and genome-encoded regulatory programs in Mtb. Analysis of EGRIN 2.0 shows how modulation of the MtrAB two-component signaling system tunes Mtb growth in response to related host microenvironmental cues. Disruption of MtrAB by tunable CRISPR interference confirms that the signaling system regulates multiple peptidoglycan hydrolases, among other targets, that are important for cell division. Further, MtrA decreases the effectiveness of antibiotics by mechanisms of both intrinsic resistance and drug tolerance. Together, the model-enabled dissection of complex MtrA regulation highlights its importance as a drug target and illustrates how EGRIN 2.0 facilitates discovery and mechanistic characterization of Mtb adaptation to specific host microenvironments within the host

MtrA modulates Mycobacterium tuberculosis cell division in host microenvironments to mediate intrinsic resistance and drug tolerance.

The success of Mycobacterium tuberculosis (Mtb) is largely attributed to its ability to physiologically adapt and withstand diverse localized stresses within host microenvironments. Here, we present a data-driven model (EGRIN 2.0) that captures the dynamic interplay of environmental cues and genome-encoded regulatory programs in Mtb. Analysis of EGRIN 2.0 shows how modulation of the MtrAB two-component signaling system tunes Mtb growth in response to related host microenvironmental cues. Disruption of MtrAB by tunable CRISPR interference confirms that the signaling system regulates multiple peptidoglycan hydrolases, among other targets, that are important for cell division. Further, MtrA decreases the effectiveness of antibiotics by mechanisms of both intrinsic resistance and drug tolerance. Together, the model-enabled dissection of complex MtrA regulation highlights its importance as a drug target and illustrates how EGRIN 2.0 facilitates discovery and mechanistic characterization of Mtb adaptation to specific host microenvironments within the host