Whole genome enrichment approach for rapid detection of Mycobacterium tuberculosis and drug resistance-associated mutations from direct sputum sequencing.

Tuberculosis is the leading cause of death among infectious diseases worldwide. Detection of Mycobacterium tuberculosis (Mtb), using routine culture-based methods is time consuming resulting in delayed diagnosis and poor treatment outcomes. Currently available molecular tests provide faster diagnosis but are able to screen only limited hot-spot mutations. Whole genome sequencing from direct sputum offers a potential solution, however, due to the presence of other microbes and host DNA its use in diagnostic testing remains challenging. In this study, we present a targeted Mtb-enrichment assay for lineage-4 coupled with an improved analysis pipeline that uses 1657 bacterial taxa as background for reducing non-Mtb genome from sputum DNA. This method drastically improved the recovery of Mtb DNA from sputum (Mtb alignment increased from 3% to >65%) as compared to non-enrichment-based sequencing. We obtained >99% Mtb genome coverage as compared to 49% in non-enriched sputum sequencing. We were able to identify Mtb positive samples from controls with 100% accuracy using Mpt64 gene coverage. Our method not only achieved 100% sensitivity to resistance variants profiled by line probe assay (LPA), but also outperformed LPA in determining drug resistance based on phenotypic drug susceptibility tests for 6 anti-tuberculosis drugs (accuracy of 97.7% and 92.8% by enriched WGS and LPA, respectively).

Cortisol regulates immune and metabolic processes in murine adipocytes and macrophages through HTR2c and HTR5a serotonin receptors.

Epidemiological studies implicate stress as an important factor contributing to the increasing prevalence of metabolic disorders. Studies have correlated visceral obesity and atherosclerosis with hyper-cortisolemia, a sequela of chronic psychological stress in humans and animals. Although several hormonal markers of stress have been associated with various metabolic disorders, the mechanism by which these hormones alter metabolic functions have not been established. We used an in vitro model system, culturing 3T3-L1 pre-adipocytes and RAW 264.7 macrophages in the presence or absence of cortisol, to analyze cell signaling pathways mediating changes in metabolic functions. Our analysis revealed that cortisol up-regulated the expression and function of two serotonin (S) receptors, HTR2c and HTR5a. HTR2c and HTR5a were also directly involved in mediating cortisol enhanced adipogenesis when pre-adipocytes were cultured alone or in the presence of macrophages. Finally, cortisol treatment of pre-adipocytes co-cultured with macrophages enhanced adipogenesis in both macrophages and pre-adipocytes.

Role of murine macrophage in temporal regulation of cortisol- and serotonin-induced adipogenesis in pre-adipocytes when grown together.

Regulation of adipogenesis, the root cause for obesity, is very poorly understood. However, studies have presented evidence of immuno-metabolic regulation of adipose tissue during periods of chronic psychological stress, leading to adverse conditions related to stress manifestation, including visceral obesity and atherosclerosis. Despite pronounced association of hormonal markers of stress with dys-regulated metabolic states, the contributing signalling events are yet to be established. It is apparent that to understand contributing signalling events we need a model. Although an in vivo model is preferred, it is difficult to establish. The current report, therefore, presents an in vitro model system for the simulation of adipose tissue in a chronic stress micro-environment by growing pre-adipocytes with macrophages in the presence and absence of stress hormones. In this report, effects of cortisol and serotonin on the kinetics of immune and metabolic changes in adipocytes and macrophage (alone and co-cultured) was studied through whole genome transcriptome profiling. A transition from pro- to anti-inflammatory response in the immune profile of pre-adipocytes, with increasing time in co-culture with macrophages, was observed. This transition was reversed by stress hormones cortisol and/or serotonin.

A Computational Approach Identifies Immunogenic Features of Prognosis in Human Cancers.

A large number of tumor intrinsic and extrinsic factors determine long-term survival in human cancers. In this study, we stratified 9120 tumors from 33 cancers with respect to their immune cell content and identified immunogenomic features associated with long-term survival. Our analysis demonstrates that tumors infiltrated by CD8+ T cells expressing higher levels of activation marker (PD1hi) along with TCR signaling genes and cytolytic T cell markers (IL2hi/TNF-αhi/IFN-γhi/GZMA-Bhi) extend survival, whereas survival benefit was absent for tumors infiltrated by anergic and hyperexhausted CD8+ T cells characterized by high expression of CTLA-4, TIM3, LAG3, and genes linked to PI3K signaling pathway. The computational approach of using robust and highly specific gene expression signatures to deconvolute the tumor microenvironment has important clinical applications, such as selecting patients who will benefit from checkpoint inhibitor treatment.

Estimation of psychological stress in humans: a combination of theory and practice.

Stress has long been known to increase susceptibility to health disorders. In 2009, American Psychological Association further established association of stress to serious health problems. However, a quantitative and accurate way to evaluate and estimate stress status of individuals is still a big challenge. It has been shown, in large animal models using cattle, that psychological stress can be quantified as well as disease susceptibility could be predicted through biomarker discovery. Taking cue from those studies, we have evaluated and estimated psychological stress level of individuals theoretically and validated experimentally. Various biomarkers have also been identified which can be associated to psychological stress to predict stress status of unknown individuals.

Effects of psychological stress on innate immunity and metabolism in humans: a systematic analysis.

Stress is perhaps easiest to conceptualize as a process which allows an organism to accommodate for the demands of its environment such that it can adapt to the prevailing set of conditions. Psychological stress is an important component with the potential to affect physiology adversely as has become evident from various studies in the area. Although these studies have established numerous effects of psychological stress on physiology, a global strategy for the correlation of these effects has yet to begin. Our comparative and systematic analysis of the published literature has unraveled certain interesting molecular mechanisms as clues to account for some of the observed effects of psychological stress on human physiology. In this study, we attempt to understand initial phase of the physiological response to psychological stress by analyzing interactions between innate immunity and metabolism at systems level by analyzing the data available in the literature. In light of our gene association-networks and enrichment analysis we have identified candidate genes and molecular systems which might have some associative role in affecting psychological stress response system or even producing some of the observed terminal effects (such as the associated physiological disorders). In addition to the already accepted role of psychological stress as a perturbation that can disrupt physiological homeostasis, we speculate that it is potentially capable of causing deviation of certain biological processes from their basal level activity after which they can return back to their basal tones once the effects of stress diminish. Based on the derived inferences of our comparative analysis, we have proposed a probabilistic mechanism for how psychological stress could affect physiology such that these adaptive deviations are sometimes not able to bounce back to their original basal tones, and thus increase physiological susceptibility to metabolic and immune imbalance.

Psychological stressors as interventions: good out of the evil.

Stress in general can be defined as a state of threatened balance, equilibrium or harmony that tends to disturb the homeostasis of the body. Stress can be of many kinds viz. psychological, physiological, social, emotional, and nutritional. Albeit the distinct kinds of stress stated in the aforementioned stress list, it is hard to bring out a clear distinction between them since each stress may precede or succeed the manifestation of any other. The studies discussed in the review elucidate effects of psychological stressors (PS) on diseases such as cancer, AIDS, epidermal abnormalities, obesity, and various inflammatory diseases like colonic inflammations, Coronary Artery Disease (CAD), Coronary Heart Disease (CHD), asthma. From these studies, further attempt was made to establish the basic mechanisms which come into play during a stressor stimulus and consequently modulate the physiology of the body. In this review we have highlighted effects of PS on diseases while simultaneously building on the modes of operation of PS to alter physiology and its further implications in developing potential psychotherapeutic methods for disease treatment.