Protective Role of Nanoceria-Infused Nanofibrous Scaffold toward Bone Tissue Regeneration with Senescent Cells.

The presence of oxidative stress in bone defects leads to delayed regeneration, especially in the aged population and patients receiving cancer treatment. This delay is attributed to the increased levels of reactive oxygen species (ROS) in these populations due to the accumulation of senescent cells. Tissue-engineered scaffolds are emerging as an alternative method to treat bone defects. In this study, we engineered tissue scaffolds tailored to modulate the adverse effects of oxidative stress and promote bone regeneration. We used polycaprolactone to fabricate nanofibrous mats by using electrospinning. We exploited the ROS-scavenging properties of cerium oxide nanoparticles to alleviate the high oxidative stress microenvironment caused by the presence of senescent cells. We characterized the nanofibers for their physical and mechanical properties and utilized an ionization-radiation-based model to induce senescence in bone cells. We demonstrate that the presence of ceria can modulate ROS levels, thereby reducing the level of senescence and promoting osteogenesis. Overall, this study demonstrates that ceria-infused nanofibrous scaffolds can be used for augmenting the osteogenic activity of senescent progenitor cells, which has important implications for engineering bone tissue scaffolds for patients with low regeneration capabilities.

Comparison of the transcriptome, lipidome, and c-di-GMP production between BCGΔBCG1419c and BCG, with Mincle- and Myd88-dependent induction of proinflammatory cytokines in murine macrophages.

We have previously reported the transcriptomic and lipidomic profile of the first-generation, hygromycin-resistant (HygR) version of the BCGΔBCG1419c vaccine candidate, under biofilm conditions. We recently constructed and characterized the efficacy, safety, whole genome sequence, and proteomic profile of a second-generation version of BCGΔBCG1419c, a strain lacking the BCG1419c gene and devoid of antibiotic markers. Here, we compared the antibiotic-less BCGΔBCG1419c with BCG. We assessed their colonial and ultrastructural morphology, biofilm, c-di-GMP production in vitro, as well as their transcriptomic and lipidomic profiles, including their capacity to activate macrophages via Mincle and Myd88. Our results show that BCGΔBCG1419c colonial and ultrastructural morphology, c-di-GMP, and biofilm production differed from parental BCG, whereas we found no significant changes in its lipidomic profile either in biofilm or planktonic growth conditions. Transcriptomic profiling suggests changes in BCGΔBCG1419c cell wall and showed reduced transcription of some members of the DosR, MtrA, and ArgR regulons. Finally, induction of TNF-α, IL-6 or G-CSF by bone-marrow derived macrophages infected with either BCGΔBCG1419c or BCG required Mincle and Myd88. Our results confirm that some differences already found to occur in HygR BCGΔBCG1419c compared with BCG are maintained in the antibiotic-less version of this vaccine candidate except changes in production of PDIM. Comparison with previous characterizations conducted by OMICs show that some differences observed in BCGΔBCG1419c compared with BCG are maintained whereas others are dependent on the growth condition employed to culture them.

Advanced 3D In Vitro Lung Fibrosis Models: Contemporary Status, Clinical Uptake, and Prospective Outlooks.

Fibrosis has been characterized as a global health problem and ranks as one of the primary causes of organ dysfunction. Currently, there is no cure for pulmonary fibrosis, and limited therapeutic options are available due to an inadequate understanding of the disease pathogenesis. The absence of advanced in vitro models replicating dynamic temporal changes observed in the tissue with the progression of the disease is a significant impediment in the development of novel antifibrotic treatments, which has motivated research on tissue-mimetic three-dimensional (3D) models. In this review, we summarize emerging trends in preparing advanced lung models to recapitulate biochemical and biomechanical processes associated with lung fibrogenesis. We begin by describing the importance of in vivo studies and highlighting the often poor correlation between preclinical research and clinical outcomes and the limitations of conventional cell culture in accurately simulating the 3D tissue microenvironment. Rapid advancement in biomaterials, biofabrication, biomicrofluidics, and related bioengineering techniques are enabling the preparation of in vitro models to reproduce the epithelium structure and operate as reliable drug screening strategies for precise prediction. Improving and understanding these model systems is necessary to find the cross-talks between growing cells and the stage at which myofibroblasts differentiate. These advanced models allow us to utilize the knowledge and identify, characterize, and hand pick medicines beneficial to the human community. The challenges of the current approaches, along with the opportunities for further research with potential for translation in this field, are presented toward developing novel treatments for pulmonary fibrosis.

Cellular Senescence Program is Sensitive to Physical Differences in Polymeric Tissue Scaffolds.

A typical cellular senescence program involves exposing cells to DNA-damaging agents such as ionization radiation or chemotherapeutic drugs, which cause multipronged changes, including increased cell size and volume, the onset of enhanced oxidative stress, and inflammation. In the present study, we examined if the senescence onset decision is sensitive to the design, porosity, and architecture of the substrate. To address this, we generated a library of polymeric scaffolds widely used in tissue engineering of varied stiffness, architecture, and porosity. Using irradiated A549 lung cancer cells, we examined the differences between cellular responses in these 3D scaffold systems and observed that senescence onset is equally diminished. When compared to the two-dimensional (2D) culture formats, there were profound changes in cell size and senescence induction in three-dimensional (3D) scaffolds. We further establish that these observed differences in the senescence state can be attributed to the altered cell spreading and cellular interactions on these substrates. This study elucidates the role of scaffold architecture in the cellular senescence program.

A Platinum(II) Boron-dipyrromethene Complex for Cellular Imaging and Mitochondria-targeted Photodynamic Therapy in Red Light.

Cisplatin-derived platinum(II) complexes [Pt(NH3 )2 (pacac)](NO3 ) (1, DPP-Pt) and [Pt(NH3 )2 (Acac-RB)](NO3 ) (2, Acacplatin-RB), where Hpacac is 1,3-diphenyl-1,3-propanedione and HAcac-RB is a red-light active distyryl-BODIPY-appended acetylacetone ligand, are prepared, characterized and their photodynamic therapy (PDT) activity studied (RB abbreviated for red-light BODIPY). Complex 2 displayed an intense absorption band at λ=652 nm (ϵ=7.3×104  M-1  cm-1 ) and 601 nm (ϵ=3.1×104  M-1  cm-1 ) in 1 : 1 DMSO-DPBS (Dulbecco's Phosphate Buffered Saline). Its emission profile includes a broad maximum at ~673 nm (λex =630 nm). The fluorescence quantum yield (ΦF ) of HAcac-RB and 2 are 0.19 and 0.07, respectively. Dichlorodihydrofluorescein diacetate and 1,3-diphenylisobenzofuran assay of complex 2 indicated photogeneration of singlet oxygen (ΦΔ : 0.36) as reactive oxygen species (ROS). Light irradiation caused only minor extent of ligand release forming chemo-active cisplatin analogue. The complex showed ~70-100 fold enhancement in cytotoxicity on light exposure in A549 lung cancer cells and MDA-MB-231 multidrug resistant breast cancer cells, giving half maximal inhibitory concentration (IC50 ) of 0.9-1.8 µM. Confocal imaging showed its mitochondrial localization and complex 2 exhibited anti-metastasis properties. Immunostaining of ß-tubulin and Annexin V-FITC/propidium iodide staining displayed complex 2 induced photo-selective microtubule rupture and cellular apoptosis, respectively.

Anti-microbial resistance and aging-A design for evolution.

The emergence of resistance to anti-infective agents poses a significant threat to successfully treating infections caused by bacteria. Bacteria acquire random mutations due to exposure to environmental stresses, which may increase their fitness to other selection pressures. Interestingly, for bacteria, the frequency of anti-microbial resistance (AMR) seems to be increasing in tandem with the human lifespan. Based on evidence from previous literature, we speculate that increased levels of free radicals (Reactive Oxygen Species-ROS and Reactive Nitrosative Species-RNS), elevated inflammation, and the altered tissue microenvironment in aged individuals may drive pathogen mutagenesis. If these mutations result in the hyperactivation of efflux pumps or alteration in drug target binding sites, it could confer AMR, thus rendering antibiotic therapy ineffective while leading to the selection of novel drug-resistant variants. This article is categorized under: Immune System Diseases > Genetics/Genomics/Epigenetics Infectious Diseases > Environmental Factors Metabolic Diseases > Environmental Factors.

High throughput screening identifies auranofin and pentamidine as potent compounds that lower IFN-γ-induced Nitric Oxide and inflammatory responses in mice: DSS-induced colitis and Salmonella Typhimurium-induced sepsis.

Interferon-gamma (IFN-γ) is a type II interferon produced primarily by T cells and natural killer cells. IFN-γ induces the expression of inducible nitric oxide synthase (NOS2) to catalyze Nitric Oxide (NO) production in various immune and non-immune cells. Excessive IFN-γ-activated NO production is implicated in several inflammatory diseases, including peritonitis and inflammatory bowel diseases. In this study, we screened the LOPAC®1280 library in vitro on the H6 mouse hepatoma cell line to identify novel non-steroidal small molecule inhibitors of IFN-γ-induced NO production. Compounds with the highest inhibitory activity were validated, which led to identifying the lead compounds: pentamidine, azithromycin, rolipram, and auranofin. Auranofin was the most potent compound determined based on IC50 and goodness of fit analyses. Mechanistic investigations revealed that majority of the lead compounds suppress the IFN-γ-induced transcription of Nos2 without negatively affecting NO-independent processes, such as the IFN-γ-induced transcription of Irf1, Socs1 and MHC class 1 surface expression. However, all four compounds lower IFN-γ-induced reactive oxygen species amounts. In addition, auranofin significantly reduced IFN-γ-mediated NO and IL6 production in resident as well as thioglycolate-elicited peritoneal macrophages (PMs). Finally, in vivo testing of the lead compounds in the pre-clinical DSS-induced ulcerative colitis mice model revealed pentamidine and auranofin to be the most potent and protective lead compounds. Also, pentamidine and auranofin greatly increase the survival of mice in another inflammatory model: Salmonella Typhimurium-induced sepsis. Overall, this study identifies novel anti-inflammatory compounds targeting IFN-γ-induced NO-dependent processes to alleviate two distinct inflammatory models of disease.

Sequestration of histidine kinases by non-cognate response regulators establishes a threshold level of stimulation for bacterial two-component signaling.

Bacterial two-component systems (TCSs) consist of a sensor histidine kinase (HK) that perceives a specific signal, and a cognate response regulator (RR) that modulates the expression of target genes. Positive autoregulation improves TCS sensitivity to stimuli, but may trigger disproportionately large responses to weak signals, compromising bacterial fitness. Here, we combine experiments and mathematical modelling to reveal a general design that prevents such disproportionate responses: phosphorylated HKs (HK~Ps) can be sequestered by non-cognate RRs. We study five TCSs of Mycobacterium tuberculosis and find, for all of them, non-cognate RRs that show higher affinity than cognate RRs for HK~Ps. Indeed, in vitro assays show that HK~Ps preferentially bind higher affinity non-cognate RRs and get sequestered. Mathematical modelling indicates that this sequestration would introduce a 'threshold' stimulus strength for eliciting responses, thereby preventing responses to weak signals. Finally, we construct tunable expression systems in Mycobacterium bovis BCG to show that higher affinity non-cognate RRs suppress responses in vivo.

BCG∆BCG1419c and BCG differ in induction of autophagy, c-di-GMP content, proteome, and progression of lung pathology in Mycobacterium tuberculosis HN878-infected male BALB/c mice.

The efficacy of BCG vaccines against Mycobacterium tuberculosis (Mtb) strains of lineage 2 (Beijing) in preclinical models and humans has been questioned. We have developed BCG∆BCG1419c, by deletion of BCG1419c in BCG Pasteur, which improved control of tuberculosis (TB) in preclinical models. Here, we compared the capacity of BCG and BCG∆BCG1419c to induce autophagy in murine macrophages, modify c-di-GMP content and transcript levels of BCG1416c, encoding the enzyme responsible for c-di-GMP synthesis/degradation, and of BCG1419c, encoding the phosphodiesterase involved in c-di-GMP degradation. Furthermore, we evaluated proteomic differences in vitro and compared protection against TB produced by a low dose of the HN878-Beijing strain at 3- and 6-months post-infection. We found that BCG∆BCG1419c induced more autophagy and produced different levels of c-di-GMP as well as different transcription of BCG1416c with no expression of BCG1419c. BCG∆BCG1419c differentially produced several proteins, including some involved in interaction with host cells. Vaccination with either BCG strain led to control of bacillary burden in lungs and spleen at 3- but not 6-months post-infection, whereas it reduced pneumonic areas compared with unvaccinated controls at 6 months post-infection. Vaccination with BCG∆BCG1419c delayed progression of lung necrosis as this was observed only at 6 months post-infection. Taken together, compared with BCG, BCG∆BCG1419c increased autophagy, presented different levels of c-di-GMP and transcription of BCG1416c in vitro in a growth-phase dependent manner, modified its proteome and delayed progression of lung pathology produced by a highly virulent Beijing strain.

H2S contributed from CSE during cellular senescence suppresses inflammation and nitrosative stress.

Aging involves the time-dependent deterioration of physiological functions attributed to various intracellular and extracellular factors. Cellular senescence is akin to aging and involves alteration in redox homeostasis. This is primarily marked by increased reactive oxygen/nitrogen species (ROS/RNS), inflammatory gene expression, and senescence-associated beta-galactosidase activity, all hallmarks of aging. It is proposed that gasotransmitters which include hydrogen sulfide (H2S), carbon monoxide (CO), and nitric oxide (NO), may affect redox homeostasis during senescence. H2S has been independently shown to induce DNA damage and suppress oxidative stress. While an increase in NO levels during aging is well established, the role of H2S has remained controversial. To understand the role of H2S during aging, we evaluated H2S homeostasis in non-senescent and senescent cells, using a combination of direct measurements with a fluorescent reporter dye (WSP-5) and protein sulfhydration analysis. The free intracellular H2S and total protein sulfhydration levels are high during senescence, concomitant to cystathionine gamma-lyase (CSE) expression induction. Using lentiviral shRNA-mediated expression knockdown, we identified that H2S contributed by CSE alters global gene expression, which regulates key inflammatory processes during cellular senescence. We propose that H2S decreases inflammation during cellular senescence by reducing phosphorylation of IκBα and the p65 subunit of nuclear factor kappa B (NF-κB). H2S was also found to reduce NO levels, a significant source of nitrosative stress during cellular senescence. Overall, we establish H2S as a key gasotransmitter molecule that regulates inflammatory phenotype and nitrosative stress during cellular senescence.

An Evolutionary Paradigm Favoring Cross Talk between Bacterial Two-Component Signaling Systems.

The prevalent paradigm governing bacterial two-component signaling systems (TCSs) is specificity, wherein the histidine kinase (HK) of a TCS exclusively activates its cognate response regulator (RR). Cross talk, where HKs activate noncognate RRs, is considered evolutionarily disadvantageous because it can compromise adaptive responses by leaking signals. Yet cross talk is observed in several bacteria. Here, to resolve this paradox, we propose an alternative paradigm where cross talk can be advantageous. We envisioned programmed environments, wherein signals appear in predefined sequences. In such environments, cross talk that primes bacteria to upcoming signals may improve adaptive responses and confer evolutionary benefits. To test this hypothesis, we employed mathematical modeling of TCS signaling networks and stochastic evolutionary dynamics simulations. We considered the comprehensive set of bacterial phenotypes, comprising thousands of distinct cross talk patterns competing in varied signaling environments. Our simulations predicted that in programmed environments phenotypes with cross talk facilitating priming would outcompete phenotypes without cross talk. In environments where signals appear randomly, bacteria without cross talk would dominate, explaining the specificity widely seen. Additionally, a testable prediction was that the phenotypes selected in programmed environments would display one-way cross talk, ensuring priming to future signals. Interestingly, the cross talk networks we deduced from available data on TCSs of Mycobacterium tuberculosis all displayed one-way cross talk, which was consistent with our predictions. Our study thus identifies potential evolutionary underpinnings of cross talk in bacterial TCSs, suggests a reconciliation of specificity and cross talk, makes testable predictions of the nature of cross talk patterns selected, and has implications for understanding bacterial adaptation and the response to interventions. IMPORTANCE Bacteria use two-component signaling systems (TCSs) to sense and respond to environmental changes. The prevalent paradigm governing TCSs is specificity, where signal flow through TCSs is insulated; leakage to other TCSs is considered evolutionarily disadvantageous. Yet cross talk between TCSs is observed in many bacteria. Here, we present a potential resolution of this paradox. We envision programmed environments, wherein stimuli appear in predefined sequences. Cross talk that primes bacteria to upcoming stimuli could then confer evolutionary benefits. We demonstrate this benefit using mathematical modeling and evolutionary simulations. Interestingly, we found signatures of predicted cross talk patterns in Mycobacterium tuberculosis. Furthermore, specificity was selected in environments where stimuli occurred randomly, thus reconciling specificity and cross talk. Implications follow for understanding bacterial evolution and for interventions.

Culture-Independent Raman Spectroscopic Identification of Bacterial Pathogens from Clinical Samples Using Deep Transfer Learning.

The rapid identification of bacterial pathogens in clinical samples like blood, urine, pus, and sputum is the need of the hour. Conventional bacterial identification methods like culturing and nucleic acid-based amplification have limitations like poor sensitivity, high cost, slow turnaround time, etc. Raman spectroscopy, a label-free and noninvasive technique, has overcome these drawbacks by providing rapid biochemical signatures from a single bacterium. Raman spectroscopy combined with chemometric methods has been used effectively to identify pathogens. However, a robust approach is needed to utilize Raman features for accurate classification while dealing with complex data sets such as spectra obtained from clinical isolates, showing high sample-to-sample heterogeneity. In this study, we have used Raman spectroscopy-based identification of pathogens from clinical isolates using a deep transfer learning approach at the single-cell level resolution. We have used the data-augmentation method to increase the volume of spectra needed for deep-learning analysis. Our ResNet model could specifically extract the spectral features of eight different pathogenic bacterial species with a 99.99% classification accuracy. The robustness of our model was validated on a set of blinded data sets, a mix of cultured and noncultured bacterial isolates of various origins and types. Our proposed ResNet model efficiently identified the pathogens from the blinded data set with high accuracy, providing a robust and rapid bacterial identification platform for clinical microbiology.

How safe are magnetic nanomotors: From cells to animals.

Helical magnetic nanomotors can be actuated using an external magnetic field and have potential applications in drug delivery, colloidal manipulation, and bio-microrheology. Recently, they have been maneuvered in biological environments such as vitreous humour, dentinal tubules, peritoneal fluid, stromal matrix, and blood, which are promising developments for clinical applications. However, their biocompatibility and biodistribution are vital parameters that must be assessed before further use. An extensive quantitative evaluation has been performed for these parameters for the first time through in vitro and in vivo experiments. Investigations of cell death, proliferation, and DNA damage ascertain that the motors are non-toxic. Also, an unbiased transcriptomic analysis affirms that the motors are not genotoxic till 20 motors/ cell. Toxicity studies in mice reveal that the motors show no signs of toxicity up to a dose of 55 mg/ kg body weight. Further, the biodistribution studies show that they remain in the blood circulation after injection and at later stages possibly adhere to the walls of the blood vessel because of adsorption. However, perfusion with physiological saline decreases this adsorption/adhesion. Overall, we demonstrate the biocompatibility of nanomotors in live cellular and organismal systems, and a systemic biodistribution analysis reveals organ-specific retention of motors.

Hybrid Assembly and Annotation of the Genome of the Indian Punica granatum, a Superfood.

The wonder fruit pomegranate (Punica granatum, family Lythraceae) is one of India's economically important fruit crops that can grow in different agro-climatic conditions ranging from tropical to temperate regions. This study reports high-quality de novo draft hybrid genome assembly of diploid Punica cultivar "Bhagwa" and identifies its genomic features. This cultivar is most common among the farmers due to its high sustainability, glossy red color, soft seed, and nutraceutical properties with high market value. The draft genome assembly is about 361.76 Mb (N50 = 40 Mb), ∼9.0 Mb more than the genome size estimated by flow cytometry. The genome is 90.9% complete, and only 26.68% of the genome is occupied by transposable elements and has a relative abundance of 369.93 SSRs/Mb of the genome. A total of 30,803 proteins and their putative functions were predicted. Comparative whole-genome analysis revealed Eucalyptus grandis as the nearest neighbor. KEGG-KASS annotations indicated an abundance of genes involved in the biosynthesis of flavonoids, phenylpropanoids, and secondary metabolites, which are responsible for various medicinal properties of pomegranate, including anticancer, antihyperglycemic, antioxidant, and anti-inflammatory activities. The genome and gene annotations provide new insights into the pharmacological properties of the secondary metabolites synthesized in pomegranate. They will also serve as a valuable resource in mining biosynthetic pathways for key metabolites, novel genes, and variations associated with disease resistance, which can facilitate the breeding of new varieties with high yield and superior quality.

Cell adhesion strength and tractions are mechano-diagnostic features of cellular invasiveness.

The adhesion of cells to substrates occurs via integrin clustering and binding to the actin cytoskeleton. Oncogenes modify anchorage-dependent mechanisms in cells during cancer progression. Fluid shear devices provide a label-free way to characterize cell-substrate interactions and heterogeneities in cell populations. We quantified the critical adhesion strengths of MCF-7, MDAMB-231, A549, HPL1D, HeLa, and NIH3T3 cells using a custom fluid shear device. The detachment response was sigmoidal for each cell type. A549 and MDAMB-231 cells had significantly lower critical adhesion strengths (τ50) than their non-invasive counterparts, HPL1D and MCF-7. Detachment dynamics inversely correlated with cell invasion potentials. A theoretical model, based on τ50 values and the distribution of cell areas on substrates, provided good fits to results from de-adhesion experiments. Quantification of cell tractions, using the Reg-FTTC method on 10 kPa polyacrylamide gels, showed highest values for invasive, MDAMB-231 and A549, cells compared to non-invasive cells. Immunofluorescence studies show differences in vinculin distributions; non-invasive cells have distinct vinculin puncta, whereas invasive cells have more dispersed distributions. The cytoskeleton in non-invasive cells was devoid of well-developed stress fibers, and had thicker cortical actin bundles in the boundary. Fluorescence intensity of actin was significantly lower in invasive cells as compared to non invasive cells. These correlations in adhesion strengths and traction stresses with cell invasiveness may be useful in cancer diagnostics and other pathologies featuring mis-regulation in adhesion.

Mobile Nanobots for Prevention of Root Canal Treatment Failure.

Millions of root canal treatments fail worldwide due to remnant bacteria deep in the dentinal tubules located within the dentine tissue of human teeth. The complex and narrow geometry of the tubules renders current techniques relying on passive diffusion of antibacterial agents ineffective. Here, the potential of actively maneuvered nanobots is investigated to disinfect dentinal tubules, which can be incorporated during a standard root canal procedure. It is demonstrated that magnetically driven nanobots can reach the depths of the tubules not possible with current clinical practices. Subtle alterations of the magnetic drive allow both deep implantations of the nanobots isotopically distributed throughout the dentine and spatially controlled recovery from selected regions, further supported by numerical simulations. Finally, the integration of bactericidal therapeutic modality with the nanobots is demonstrated, thereby validating the tremendous potential of nanobots in dentistry and nanomedicine in general.

Mycobacterium tuberculosis PknK Substrate Profiling Reveals Essential Transcription Terminator Protein Rho and Two-Component Response Regulators PrrA and MtrA as Novel Targets for Phosphorylation.

The Mycobacterium tuberculosis protein kinase K regulates growth adaptation by facilitating mycobacterial survival in response to a variety of in vitro and in vivo stress conditions. Here, we further add that pknK transcription is responsive to carbon and nitrogen starvation signals. The increased survival of an M. tuberculosis ΔpknK mutant strain under carbon- and nitrogen-limiting growth conditions compared to the wild-type (WT) H37Rv suggests an integral role of PknK in regulating growth during metabolic stress. To identify the downstream targets of PknK-mediated signaling, we compared phosphoproteomic and transcription profiles of mycobacterial strains overexpressing WT and phosphorylation-defective PknK. Results implicate PknK as a signaling protein that can regulate several enzymes involved in central metabolism, transcription regulation, and signal transduction. A key finding of this study was the identification of two essential two-component response regulator (RR) proteins, PrrA and MtrA, and Rho transcription terminator, as unique targets for PknK. We confirm that PknK interacts with and phosphorylates PrrA, MtrA, and Rho in vivo. PknK-mediated phosphorylation of MtrA appears to increase binding of the RR to the cognate probe DNA. However, dual phosphorylation of MtrA and PrrA response regulators by PknK and their respective cognate sensor kinases in vitro showed nominal additive effect on the mobility of the protein-DNA complex, suggesting the presence of a potential fine-tuning of the signal transduction pathway which might respond to multiple cues. IMPORTANCE Networks of gene regulation and signaling cascades are fundamental to the pathogenesis of Mycobacterium tuberculosis in adapting to the continuously changing intracellular environment in the host. M. tuberculosis protein kinase K is a transcription regulator that responds to diverse environmental signals and facilitates stress-induced growth adaptation in culture and during infection. This study identifies multiple signaling interactions of PknK and provides evidence that PknK can change the transcriptional landscape during growth transitions by connecting distinctly different signal transduction and regulatory pathways essential for mycobacterial survival.

The ERK-p38MAPK-STAT3 Signalling Axis Regulates iNOS Expression and Salmonella Infection in Senescent Cells.

The cellular changes occurring due to senescence like proliferation arrest, increase in free radical levels, and secretion of pro-inflammatory cytokines have been well studied, but its associated alteration in intracellular signalling networks has been scarcely explored. In this study, we examine the roles of three major kinases viz. p38 MAPK, ERK, and STAT3 in regulating iNOS expression and thereby the levels of the free radical Nitric oxide in senescent cells. Our study revealed that these kinases could differentially regulate iNOS in senescent cells compared to non-senescent cells. Further, we tested the physiological relevance of these alterations with Salmonella infection assays and established an inter-regulatory network between these kinases unique to infected senescent cells. Overall, our findings show how key signalling networks may be rewired in senescent cells rendering them phenotypically different.

A Low-Prevalence Single-Nucleotide Polymorphism in the Sensor Kinase PhoR in Mycobacterium tuberculosis Suppresses Its Autophosphatase Activity and Reduces Pathogenic Fitness: Implications in Evolutionary Selection.

The genome sequencing of Mycobacterium tuberculosis, the causative organism of tuberculosis, has significantly improved our understanding of the mechanisms that drive the establishment of infection and disease progression. Several clinical strains of M. tuberculosis exhibit single-nucleotide polymorphisms (SNPs), the implications of which are only beginning to be understood. Here, we examined the impact of a specific polymorphism in PhoR, the sensor kinase of the PhoPR two-component system. Biochemical analysis revealed reduced autophosphatase/ATPase activity, which led to enhanced downstream gene expression. We complemented M. tuberculosis H37Ra with the wild-type and mutant phoPR genes and characterized the strains in a cell line infection model. We provide an explanation for the low prevalence of the SNP in clinical strains (∼1%), as the mutation causes a survival disadvantage in the host cells. The study provides a rare example of selection of a signaling node under competing evolutionary forces, wherein a biochemically superior mutation aids bacterial adaptation within-host but has low fitness for infection and hence is not selected. Our study highlights the importance of accounting for such SNPs to test therapeutic and co-therapeutic methods to combat TB.