Targeting S6K/NFκB/SQSTM1/Polθ signaling to suppress radiation resistance in prostate cancer.

In this study we have identified POLθ-S6K-p62 as a novel druggable regulator of radiation response in prostate cancer. Despite significant advances in delivery, radiotherapy continues to negatively affect treatment outcomes and quality of life due to resistance and late toxic effects to the surrounding normal tissues such as bladder and rectum. It is essential to develop new and effective strategies to achieve better control of tumor. We found that ribosomal protein S6K (RPS6KB1) is elevated in human prostate tumors, and contributes to resistance to radiation. As a downstream effector of mTOR signaling, S6K is known to be involved in growth regulation. However, the impact of S6K signaling on radiation response has not been fully explored. Here we show that loss of S6K led to formation of smaller tumors with less metastatic ability in mice. Mechanistically we found that S6K depletion reduced NFκB and SQSTM1 (p62) reporter activity and DNA polymerase θ (POLθ) that is involved in alternate end-joining repair. We further show that the natural compound berberine interacts with S6K in a in a hitherto unreported novel mode and that pharmacological inhibition of S6K with berberine reduces Polθ and downregulates p62 transcriptional activity via NFκB. Loss of S6K or pre-treatment with berberine improved response to radiation in prostate cancer cells and prevented radiation-mediated resurgence of PSA in animals implanted with prostate cancer cells. Notably, silencing POLQ in S6K overexpressing cells enhanced response to radiation suggesting S6K sensitizes prostate cancer cells to radiation via POLQ. Additionally, inhibition of autophagy with CQ potentiated growth inhibition induced by berberine plus radiation. These observations suggest that pharmacological inhibition of S6K with berberine not only downregulates NFκB/p62 signaling to disrupt autophagic flux but also decreases Polθ. Therefore, combination treatment with radiation and berberine inhibits autophagy and alternate end-joining DNA repair, two processes associated with radioresistance leading to increased radiation sensitivity.

DNA Aptamer-Based Staining and Fluorescence Microscopy for Rapid Detection of Cyclospora Cayetanensis Oocysts.

There are no commercial antibodies for detection of Cyclospora cayetanensis, only a relatively slow polymerase chain reaction (PCR) test developed by the U.S. Food and Drug Administration (FDA). However, DNA aptamers have recently been developed by our group against known proteins and whole oocysts of C. cayetanensis and shown to specifically detect the oocysts when attached on their 5' ends to red-emitting fluorophores and used as probes for fluorescence microscopy. Aptamers developed against recombinant wall protein 2 and TA4 antigen-like protein as well as whole oocysts specifically stained C. cayetanensis oocysts while exhibiting little, if any, staining of numerous other waterborne parasite species. Interestingly, the aptamers stained both exterior cell wall moieties and internal structures, suggesting that the aptamers penetrate the oocysts even without added detergents.

Novel recombinant proteins and peptides from Clonorchis sinensis and Opisthorchis viverrini for liver fluke exposure ELISA.

Human serum samples from individuals living in Vietnam and Taiwan suspected of past Clonorchis sinensis or Opisthorchis viverrini infection were screened using several novel peptides and recombinant liver fluke proteins to determine if any consistent patterns could be discerned and used as the basis for future liver fluke ELISA development. Absorbance values at 405 nm were compared to those of pooled unexposed normal human serum and analyzed for statistical significance. The data exhibited some interesting patterns consistent with egg antigen sequestration in the gut possibly leading to lower serum antibody levels and potential regional exposure differences between Vietnamese and Taiwanese subjects. In particular, antibodies against Cathepsin B and B2 peptides, as well as a partial Cahedrin Domain peptide may be elevated in some Taiwanese serum samples while antibodies against recombinant Clonorchis egg protein and Hepatocellular Carcinoma Peptide Antigen 59 may be elevated in some samples from both Taiwan and Vietnam. The data appear to suggest that some of the novel recombinant protein and peptide antigens selected and tested herein warrant further study with larger sample sizes as possible targets for detecting anti-liver fluke antibodies by ELISA from humans suspected of liver fluke infections.

Alpha-thiol deoxynucleotide triphosphates (S-dNTPs) as radioprotective agents: A novel approach.

In this study, the ability of a mixture of four different alpha-thiol deoxynucleotide triphosphates (S-dNTPs) each at a concentration of 10µM when incorporated into the genomic DNA of proliferating human HL-60 and Mono-Mac-6 (MM-6) cells in vitro to provide protection from 2, 5, and 10 Gy of gamma radiation was investigated. Incorporation of the four different S-dNTPs into nuclear DNA at 10 µM concentration for five days was validated by agarose gel electrophoretic band shift analysis. S-dNTP-treated genomic DNA reacted with BODIPY-iodoacetamide demonstrated a band shift to higher molecular weight to confirm the presence of sulfur moieties in the resultant phosphorothioate DNA backbones. No overt signs of toxicity or obvious morphologic cellular differentiation were noted in the presence of 10 µM S-dNTPs even after 8 days in culture. Significantly reduced radiation-induced persistent DNA damage measured at 24 and 48 h post-exposure by γ-H2AX histone phosphorylation using FACS analysis in S-dNTP incorporated HL-60 and MM6 cells indicated protection against radiation-induced direct and indirect DNA damage. Statistically significant protection by S-dNTPs was noted at the cellular level by CellEvent™ Caspase-3/7 assay, which assess the extent of apoptotic events, and by trypan blue dye exclusion to assed cell viability. The results appear to support an innocuous antioxidant thiol radioprotective effect built into genomic DNA backbones as the last line of defense against ionizing radiation and free radical-induced DNA damage.

MMP-9 reinforces radiation-induced delayed invasion and metastasis of neuroblastoma cells through second-signaling positive feedback with NFκB via both ERK and IKK activation.

Neuroblastoma (NB) progression is branded with hematogenous metastasis and frequent relapses. Despite intensive multimodal clinical therapy, outcomes for patients with progressive disease remain poor, with negligible long-term survival. Therefore, understanding the acquired molecular rearrangements in NB cells with therapy pressure and developing improved therapeutic strategies is a critical need to improve the outcomes for high-risk NB patients. We investigated the rearrangement of MMP9 in NB with therapy pressure, and unveiled the signaling that facilitates NB evolution. Radiation-treatment (RT) significantly increased MMP9 expression/activity, and the induced enzyme activity was persistently maintained across NB cell lines. Furthermore, RT-triggered NFκB transcriptional activity and this RT-induced NFκB were required/adequate for MMP9 maintenance. RT-triggered NFκB-dependent MMP9 actuated a second-signaling feedback to NFκB, facilitating a NFκB-MMP9-NFκB positive feedback cycle (PFC). Critically, MMP9-NFκB feedback is mediated by MMP9-dependent activation of IKKß and ERK phosphotransferase activity. Beyond its tumor invasion/metastasis function, PFC-dependent MMP9 lessens RT-induced apoptosis and favors survival pathway through the activation of NFκB signaling. In addition, PFC-dependent MMP9 regulates 19 critical molecular determinants that play a pivotal role in tumor evolution. Interestingly, seven of 19 genes possess NFκB-binding sites, demonstrating that MMP9 regulates these molecules by activating NFκB. Collectively, these data suggest that RT-triggered NFκB-dependent MMP9 actuates feedback to NFκB though IKKß- and ERK1/2-dependent IκBα phosphorylation. This RT-triggered PFC prompts MMP9-dependent survival advantage, tumor growth, and dissemination. Targeting therapy-pressure-driven PFC and/or selective inhibition of MMP9 maintenance could serve as promising therapeutic strategies for treatment of progressive NB.

Surgical Challenges in the Management of Post COVID-19 Midface Mucormycosis (PCoMM): An Institutional Protocol.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has posed another serious threat, mucormycosis infection, affecting the maxilla and orbitocerebral region. This condition has not spared world population from its merciless claws. This article addresses the challenges faced by the maxillofacial surgeons in setting the protocols from preoperative diagnosis, surgical management to postoperative care, including short-term and long-term rehabilitation. To manage this relentlessly progressing condition, a multispecialty team approach is to be activated in diagnosing, managing, and rehabilitating the patients. PURPOSE: The purpose of this clinical study is to document and analyze the clinical and demographic data, presentation of the lesion, the diagnostic methods followed for early clinical detection, and management of post COVID-19 midface mucormycosis. The article also discusses postoperative medical management and prosthetic rehabilitation. RESULTS: Most of the mucormycosis cases reporting to our center were treated and recovered patients of Severe Acute Respiratory Syndrome Coronavirus 2 infection. Thirty-four (n=34) case were operated for post COVID-19 midface mucormycosis between October 2020 and December 2021. Male to Female ratio is 1:42. The average age of the patients was 57.5 years. Maximum patients were in fifth and sixth decade of life. Maxilla was the involved bone. Treatment was primarily surgical debridement to extended or radical maxillectomy. All patients were treated with Liposomal Amphotericin B and tab posaconazole for 3 to 4 weeks depending upon the age, weight, and physiological state of the patients to attain an optimal cumulative load. Three patients succumbed to illness postoperatively (n=3, 1.02%). Average duration of hospital stay was 47 days. The average review period was 5.1 months.

Chemoprevention of neuroblastoma: progress and promise beyond uncertainties.

Neuroblastoma is the most common extracranial solid tumor in children and comprises one-tenth of all childhood cancer deaths. The current clinical therapy for this deadly disease is multimodal, involving an induction phase with alternating regimens of high-dose chemotherapeutic drugs and load reduction surgery; a consolidation phase with more intensive chemotherapy, radiotherapy, and stem cell transplant; and a maintenance phase with immunotherapy and immune-activating cytokine treatment. Despite such intensive treatment, children with neuroblastoma have unacceptable life quality and survival, warranting preventive measures to regulate the cellular functions that orchestrate tumor progression, therapy resistance, metastasis, and tumor relapse/recurrence. Globally, active efforts are underway to identify novel chemopreventive agents, define their mechanism(s) of action, and assess their clinical benefit. Some chemoprevention strategies (e.g., retinoids, difluoromethylornithine) have already been adopted clinically as part of maintenance phase therapy. Several agents are in the pipeline, while many others are in preclinical characterization. Here we review the classes of chemopreventive agents investigated for neuroblastoma, including cellular events targeted, mode(s) of action, and the level of development. Our review: (i) highlights the pressing need for new and improved chemopreventive strategies for progressive neuroblastoma; (ii) lists the emerging classes of chemopreventive agents for neuroblastoma; and (iii) recognizes the relevance of targeting dynamically evolving hallmark functions of tumor evolution (e.g., survival, differentiation, lineage transformation). With recent gains in the understanding of tumor evolution processes and preclinical and clinical efforts, it is our strong opinion that effective chemopreventive strategies for aggressive neuroblastoma are a near reality.

Differential activation of Ca2+ influx channels modulate stem cell potency, their proliferation/viability and tissue regeneration.

Stem cells have indefinite self-renewable capability; however, factors that modulate their pluripotency/function are not fully identified. Here we show that store-dependent Ca2+ entry is essential for modulating the function of bone marrow-derived mesenchymal stem cells (MSCs). Increasing external Ca2+ modulated cell cycle progression that was critical for MSCs survival. Additionally, Ca2+ was critical for stem proliferation, its differentiation, and maintaining stem cell potential. Ca2+ channel characterization, including gene silencing, showed two distinct Ca2+ entry channels (through Orai1/TRPC1 or via Orai3) that differentially regulate the proliferation and viability of MSCs. Importantly, NFκB translocation, but not JNK/ERK into the nucleus, was observed upon store depletion, which was blocked by the addition of Ca2+ channel inhibitors. Radiation lead to a decrease in saliva secretion, decrease in acinar cell number, and enlarged ducts were observed, which were restored by the transplantation of stem cells that were propagated in higher Ca2+. Finally radiation showed a decrese in TRPC1 expression along with a decrese in AQP5, which was again restored upon MSC tranplantation. Together these results suggest that Ca2+ entry is essential for stem cell function that could be critical for regenerative medicine.

Impact of Diabetes and Low Body Mass Index on Tuberculosis Treatment Outcomes.

BACKGROUND: Diabetes was identified as a tuberculosis (TB) risk factor mostly in retrospective studies with limited assessments of metabolic variables. The prospective Effects of Diabetes on Tuberculosis Severity study compared adults with pulmonary TB in Chennai, India, who were classified as having either diabetes or a normal glucose tolerance at enrollment. METHODS: Baseline TB severity, sputum conversion, and treatment outcomes (cure, failure, death, or loss to follow-up) were compared between groups with respect to glycemic status and body mass index (BMI). RESULTS: The cohort of 389 participants included 256 with diabetes and 133 with a normal glucose tolerance. Low BMIs (<18.5 kg/m2) were present in 99 (74.4%) of nondiabetic participants and 85 (33.2%) of those with diabetes. Among participants with normal or high BMIs, rates of cure, treatment failure, or death did not vary by glycemic status. Participants with low BMIs had the highest radiographic severity of disease, the longest time to sputum culture conversion, and the highest rates of treatment failure and death. Among participants with low BMIs, poorly controlled diabetes (glycohemoglobin [HbA1c] ≥8.0%) was unexpectedly associated with better TB treatment outcomes. A high visceral adiposity index was associated with adverse outcomes and, despite an overall correlation with HbA1c, was elevated in some low-BMI individuals with normal glucose tolerance. CONCLUSIONS: In this South Indian cohort, a low BMI was significantly associated with an increased risk for adverse TB treatment outcomes, while comorbid, poorly controlled diabetes lessened that risk. A high visceral adiposity index, either with or without dysglycemia, might reflect a novel TB susceptibility mechanism linked to adipose tissue dysfunction.

Heterogeneity in the cytokine profile of tuberculosis - diabetes co-morbidity.

Tuberculosis - diabetes (TB-DM) co-morbidity is characterized by heterogeneity in clinical and biochemical parameters between newly diagnosed diabetic individuals with TB (TB-NDM) and known diabetic individuals at incident TB (TB-KDM). However, the immunological profile underlying this heterogeneity is not explored. To identify the cytokine profiles in TB-NDM and TB-KDM individuals, we examined the plasma cytokine levels as well as TB-antigen stimulated levels of pro-inflammatory cytokines. TB-KDM individuals exhibit significantly higher levels of IFNγ, IL-2, TNFα, IL-17A, IL-1α, IL-1ß and IL-6 in comparison to TB-NDM, TB alone and DM alone individuals. TB-NDM individuals are characterized by significantly lower levels of blood glucose and glycated hemoglobin in comparison to TB-KDM with both groups exhibiting a significant lowering of glycated hemoglobin levels at 6  months of anti-tuberculosis therapy (ATT). TB-NDM individuals are characterized by significantly diminished - unstimulated levels of IFNγ, IL-2, TNFα, IL-17A, IL-1α, IL-1ß and IL-12 at pre-treatment, of IFNγ, IL-2 and IL-1α at 2  months of ATT and IL-2 at post-treatment in comparison to TB-KDM. TB-NDM individuals are also characterized by significantly diminished TB-antigen stimulated levels of IFNγ, IL-2, TNFα, IL-17A, IL-17F, IL-1α, IL-1ß and/or IL-6 at pre-treatment and at 2  months of ATT and IFNγ, IL-2, IL-1α and IL-1ß at post-treatment. In addition, TB-NDM individuals are characterized by significantly diminished mitogen - stimulated levels of IL-17F and IL-6 at pre-treatment and IL-6 alone at 6 months of ATT. Therefore, our data reveal considerable heterogeneity in the immunological underpinnings of TB-DM co-morbidity. Our data also suggest that TB-NDM exhibits a characteristic profile, which is both biochemically and immunologically distinct from TB-KDM.

Plasma chemokines are biomarkers of disease severity, higher bacterial burden and delayed sputum culture conversion in pulmonary tuberculosis.

Plasma cytokines are biomarkers of disease extent and mycobacterial burden in pulmonary tuberculosis (PTB). Whether chemokines can perform the same role in PTB is not known. We examined the plasma levels of chemokines in individuals with PTB, latent TB (LTB) or healthy controls (HC) and their association with disease severity and mycobacterial burdens in PTB. We also examined the chemokines in PTB individuals at the end of anti-tuberculous chemotherapy (ATT). PTB individuals exhibited significantly higher levels of CCL1, CCL3, CXCL1, CXCL2, CXCL9 and CXCL10 in comparison to LTB and/or HC individuals. PTB individuals with bilateral or cavitary disease displayed significantly elevated levels of CCL1, CCL3, CXCL1, CXCL10 and CXCL11 compared to those with unilateral or non-cavitary disease and also exhibited a significant positive relationship with bacterial burdens. In addition, PTB individuals with slower culture conversion displayed significantly elevated levels of CCL1, CCL3, CXCL1 and CXCL9 at the time of PTB diagnosis and prior to ATT. Finally, the chemokines were significantly reduced following successful ATT. Our data demonstrate that PTB is associated with elevated levels of chemokines, which are partially reversed followed chemotherapy. Our data demonstrate that chemokines are markers of disease severity, predicting increased bacterial burden and delayed culture conversion in PTB.

Fucoidan from marine brown algae attenuates pancreatic cancer progression by regulating p53 - NFκB crosstalk.

Poor pancreatic cancer (PC) prognosis has been attributed to its resistance to apoptosis and propensity for early systemic dissemination. Existing therapeutic strategies are often circumvented by the molecular crosstalk between cell-signalling pathways. p53 is mutated in more than 50% of PC and NFκB is constitutively activated in therapy-resistant residual disease; these mutations and activations account for the avoidance of cell death and metastasis. Recently, we demonstrated the anti-PC potential of fucoidan extract from marine brown alga, Turbinaria conoides (J. Agardh) Kützing (Sargassaceae). In this study, we aimed to characterize the active fractions of fucoidan extract to identify their select anti-PC efficacy, and to define the mechanism(s) involved. Five fractions of fucoidan isolated by ion exchange chromatography were tested for their potential in genetically diverse human PC cell lines. All fractions exerted significant dose-dependent and time-dependent regulation of cell survival. Fucoidans induced apoptosis, activated caspase -3, -8 and -9, and cleaved Poly ADP ribose polymerase (PARP). Pathway-specific transcriptional analysis recognized inhibition of 57 and 38 nuclear factor κB (NFκB) pathway molecules with fucoidan-F5 in MiaPaCa-2 and Panc-1 cells, respectively. In addition, fucoidan-F5 inhibited both the constitutive and Tumor necrosis factor-α (TNFα)-mediated NFκB DNA-binding activity in PC cells. Upregulation of cytoplasmic IκB levels and significant reduction of NFκB-dependent luciferase activity further substantiate the inhibitory potential of seaweed fucoidans on NFκB. Moreover, fucoidan(s) treatment increased cellular p53 in PC cells and reverted NFκB forced-expression-related p53 reduction. The results suggest that fucoidan regulates PC progression and that fucoidans may target p53-NFκB crosstalk and dictate apoptosis in PC cells.

Targeting acquired oncogenic burden in resilient pancreatic cancer: a novel benefit from marine polyphenols.

The upsurge of marine-derived therapeutics for cancer treatment is evident, with many drugs in clinical use and in clinical trials. Seaweeds harbor large amounts of polyphenols and their anti-cancer benefit is linear to their anti-oxidant activity. Our studies identified three superlative anti-cancer seaweed polyphenol drug candidates (SW-PD). We investigated the acquisition of oncogenic burden in radiation-resilient pancreatic cancer (PC) that could drive tumor relapse, and elucidated the efficacy of SW-PD candidates as adjuvants in genetically diverse in vitro systems and a mouse model of radiation-residual disease. QPCR profiling of 88 oncogenes in therapy-resilient PC cells identified a 'shared' activation of 40 oncogenes. SW-PD pretreatment inflicted a significant mitigation of acquired (shared) oncogenic burden, in addition to drug- and cell-line-specific repression signatures. Tissue microarray with IHC of radiation-residual tumors in mice signified acquired cellular localization of key oncoproteins and other critical architects. Conversely, SW-PD treatment inhibited the acquisition of these critical drivers of tumor genesis, dissemination, and evolution. Heightened death of resilient PC cells with SW-PD treatment validated the translation aspects. The results defined the acquisition of oncogenic burden in resilient PC and demonstrated that the marine polyphenols effectively target the acquired oncogenic burden and could serve as adjuvant(s) for PC treatment.

De novo regulation of RD3 synthesis in residual neuroblastoma cells after intensive multi-modal clinical therapy harmonizes disease evolution.

Most high-risk neuroblastomas that initially respond to therapy will ultimately relapse. Currently, no curative treatment is available. Acquired genetic/molecular rearrangement in therapy-resistant cells contributes to tumor relapse. Recently, we identified significant RD3 loss in progressive disease (PD) and defined its association with advanced disease-stage and poor clinical outcomes. Here, we investigated whether RD3 loss is an acquired process in cells that survive intensive multi-modal clinical therapy (IMCT) and its significance in disease evolution. RD3 status (mRNA, protein) during diagnosis (Dx) and PD after IMCT was investigated in NB patient cohort (n = 106), stage-4 NB cell lines (n = 15) with known treatment status and validated with independent data from another set of 15 cell-lines. Loss of RD3 in metastatic disease was examined using a mouse model of PD and metastatic-site-derived aggressive cells (MSDACs) ex vivo. RD3 silencing/expression assessed changes in metastatic state. Influence of RD3 loss in therapy resistance was examined through independent in vitro and in vivo studies. A significant loss of RD3 mRNA and protein was observed in resistant cells derived from patients with PD after IMCT. This is true to the effect within and between patients. Results from the mouse model identified significant transcriptional/translational loss of RD3 in metastatic tumors and MSDACs. RD3 re-expression in MSDACs and silencing RD3 in parental cells defined the functional relevance of RD3-loss in PD pathogenesis. Analysis of independent studies with salvage therapeutic agents affirmed RD3 loss in surviving resistant cells and residual tumors. The profound reductions in RD3 transcription indicate the de novo regulation of RD3 synthesis in resistant cells after IMCT. Defining RD3 loss in PD and the benefit of targeted reinforcement could improve salvage therapy for progressive neuroblastoma.

Persistent inflammation during anti-tuberculosis treatment with diabetes comorbidity.

Diabetes mellitus (DM) increases risk for pulmonary tuberculosis (TB) and adverse treatment outcomes. Systemic hyper-inflammation is characteristic in people with TB and concurrent DM (TBDM) at baseline, but the impact of TB treatment on this pattern has not been determined. We measured 17 plasma cytokines and growth factors in longitudinal cohorts of Indian and Brazilian pulmonary TB patients with or without DM. Principal component analysis revealed virtually complete separation of TBDM from TB individuals in both cohorts at baseline, with hyper-inflammation in TBDM that continued through treatment completion at six months. By one year after treatment completion, there was substantial convergence of mediator levels between groups within the India cohort. Non-resolving systemic inflammation in TBDM comorbidity could reflect delayed lesion sterilization or non-resolving sterile inflammation. Either mechanism portends unfavorable long-term outcomes including risk for recurrent TB and for damaging immune pathology.

Identification and Characterization of Genetic Determinants of Isoniazid and Rifampicin Resistance in Mycobacterium tuberculosis in Southern India.

Drug-resistant tuberculosis (TB), one of the leading causes of death worldwide, arises mainly from spontaneous mutations in the genome of Mycobacterium tuberculosis. There is an urgent need to understand the mechanisms by which the mutations confer resistance in order to identify new drug targets and to design new drugs. Previous studies have reported numerous mutations that confer resistance to anti-TB drugs, but there has been little systematic analysis to understand their genetic background and the potential impacts on the drug target stability and/or interactions. Here, we report the analysis of whole-genome sequence data for 98 clinical M. tuberculosis isolates from a city in southern India. The collection was screened for phenotypic resistance and sequenced to mine the genetic mutations conferring resistance to isoniazid and rifampicin. The most frequent mutation among isoniazid and rifampicin isolates was S315T in katG and S450L in rpoB respectively. The impacts of mutations on protein stability, protein-protein interactions and protein-ligand interactions were analysed using both statistical and machine-learning approaches. Drug-resistant mutations were predicted not only to target active sites in an orthosteric manner, but also to act through allosteric mechanisms arising from distant sites, sometimes at the protein-protein interface.

Endothelial cell-specific overexpression of endothelial nitric oxide synthase in Ins2Akita mice exacerbates diabetic nephropathy.

Previous studies demonstrated that global deficiency of eNOS in diabetic mice exacerbated renal lesions and that overexpression of eNOS may protect against tissue injury. Our study revealed for the first time overexpression of eNOS leads to disease progression rather than protection. Transgenic mice selectively expressing eNOS in endothelial cells (eNOSTg) were cross bred with Ins2Akita type-1 (AK) diabetic mice to generate eNOS overexpressing eNOSTg/AK mice. Wild type, eNOSTg, AK and eNOSTg/AK mice were assessed for kidney function and blood glucose levels. Remarkably, overexpressing eNOSTg mice showed evidence of unpredicted glomerular injury with segmental mesangiolysis and occasional microaneurysms. Notably, in eNOSTg/AK mice overexpression of eNOS led to increased glomerular/endothelial injury that was associated with increased superoxide levels and renal dysfunction. Results indicate for the first time that overexpressing eNOS in endothelial cells cannot ameliorate diabetic lesions, but paradoxically leads to progression of nephropathy likely due to eNOS uncoupling and superoxide upsurge. This novel finding has a significant impact on current therapeutic strategies to improve endothelial function and prevent progression of diabetic renal disease. Further, the eNOSTg/AK model developed in this study has significant translational potentials for elucidating the underlying mechanism implicated in the deflected function of eNOS in diabetic nephropathy.

Elevated circulating levels of monocyte activation markers among tuberculosis patients with diabetes co-morbidity.

Alteration in the frequency of monocyte subsets is a hallmark of tuberculosis-diabetes co-morbidity (TB-DM). To study this association, we examined the plasma levels of sCD14, sCD163, C-reactive protein (CRP) and soluble tissue factor (sTF) in individuals with TB-DM, TB or diabetes mellitus (DM), and in healthy controls (HC). Circulating levels of sCD14, sCD163 and sTF were significantly increased in TB-DM and DM compared with TB and HC; however, CRP was significantly increased in TB-DM and TB compared with DM and HC. During longitudinal follow up, sCD14, CRP and sTF levels remained significantly increased in TB-DM compared with TB from baseline (pre-treatment), during treatment (2nd month) and at the completion (6th month) of anti-TB treatment (ATT), whereas sCD163 was significantly higher in TB-DM compared with TB only at baseline. Moreover, the levels of sCD14 and sCD163 were significantly higher in TB-DM individuals with bilateral and cavitary disease and exhibited a significant positive relationship with bacterial burden. Levels of sCD14, sCD163 and CRP exhibited a positive relationship with HbA1c levels. Within the TB-DM group, those with known diabetes before incident TB (KDM) exhibited significantly higher levels of sCD14 and sCD163 compared with individuals with newly diagnosed DM with TB (NDM). Finally, KDM individuals on metformin treatment exhibited significantly lower levels of sCD14, sCD163 and CRP compared with those on non-metformin-containing regimens. Our data demonstrate that systemic monocyte activation marker levels reflect baseline disease severity and extent in TB-DM, differentiate KDM from NDM and are modulated by ATT and metformin therapy.

Heterozygous Tsc2 (Tsc2+/-) mouse model to study induced renal cancer in response to ionizing radiation at low doses.

Kidneys are one of the main dose-limiting organs in radiotherapeutic procedures of lower abdomen. Likewise, the threat of exposure of radiosensitive organs such as kidneys in warfare or radiation accidents among military personal or due to terrorist activities in general public is of increasing concern. These events warrant the need for appropriate animal models to study the acute and chronic effects of low- and high-dose rate radiation exposures. In this study, for the first time, we validated Tsc2+/- mouse model to study whether radiation accelerates carcinogenesis in kidneys. Tsc2+/- mice at increasing age groups at 8 and 10 months were exposed to repeated doses of gamma radiation (0.4 Gy × 5) and assessed for aggravated kidney tumor formation at 2 months post-irradiation. Animals from irradiated group showed a significant increase in numbers of bilateral, multifocal tumors compared with mock-irradiated animals. Intra-glomerular reactive oxygen species (ROS) levels measured by dihydroethidium florescence showed significant increases in ROS production in irradiated Tsc2+/- mice compared with non-irradiated animals. Similarly, selective hematological parameters and glomerular filtration rate were further reduced significantly in irradiated Tsc2+/- mice. Tsc2 protein, tuberin in irradiated mice, however, remains at the same reduced levels as that of the mock-irradiated heterozygous Tsc2 mice. The results indicate that radiation alters kidney homeostatic function and influences high spontaneous incidence of renal cell carcinoma in this rodent model. Repurposing of Tsc2+/- mice model will, therefore, provide a unique opportunity to study acute and delayed effects of radiation in the development of kidney cancers.

Association of Nitric Oxide Synthase2 gene polymorphisms with leprosy reactions in northern Indian population.

The pathogen Mycobacterium leprae causes leprosy that affects mainly skin and nerves. Polymorphisms of certain genes are substantiated to be associated with the susceptibility/resistance to leprosy. The present investigation addressed the association of Nitric Oxide Synthase2 gene polymorphisms and leprosy in a population from northern part of India. A total of 323 leprosy cases and 288 healthy controls were genotyped for four NOS2 promoter variants (rs1800482, rs2779249, rs8078340 and rs2301369) using FRET technology in Real Time PCR. None of these SNPs in promoter sites was associated with susceptibility/resistance to leprosy. NOS2 rs1800482 was found to be monomorphic with GG genotype. However, NOS2-1026T allele was observed to be in higher frequency with leprosy cases (BL and LL) who were not suffering from any reactional episodes compared to cases with ENL reaction {OR=0.30, 95% CI (0.10-0.86), p=0.024}. NOS2-1026GT genotype was more prevalent in cases without reaction (BT, BB and BL) compared to RR reactional patients {OR=0.38, 95% CI (0.17-0.86), p=0.02}. Although haplotype analysis revealed that no haplotype was associated with leprosy susceptibility/resistance with statistical significance, GTG haplotype was noted to be more frequent in healthy controls. These SNPs are observed to be in linkage disequilibrium. Although, these SNPs are not likely to influence leprosy vulnerability, -1026G>T SNP was indicated to have noteworthy role in leprosy reactions.