NU-6027 Inhibits Growth of Mycobacterium tuberculosis by Targeting Protein Kinase D and Protein Kinase G.

Tuberculosis (TB) is a global health concern, and this situation has further worsened due to the emergence of drug-resistant strains and the failure of BCG vaccine to impart protection. There is an imperative need to develop highly sensitive, specific diagnostic tools, novel therapeutics, and vaccines for the eradication of TB. In the present study, a chemical screen of a pharmacologically active compound library was performed to identify antimycobacterial compounds. The phenotypic screen identified a few novel small-molecule inhibitors, including NU-6027, a known CDK-2 inhibitor. We demonstrate that NU-6027 inhibits Mycobacterium bovis BCG growth in vitro and also displayed cross-reactivity with Mycobacterium tuberculosis protein kinase D (PknD) and protein kinase G (PknG). Comparative structural and sequence analysis along with docking simulation suggest that the unique binding site stereochemistry of PknG and PknD accommodates NU-6027 more favorably than other M. tuberculosis Ser/Thr protein kinases. Further, we also show that NU-6027 treatment induces the expression of proapoptotic genes in macrophages. Finally, we demonstrate that NU-6027 inhibits M. tuberculosis growth in both macrophage and mouse tissues. Taken together, these results indicate that NU-6027 can be optimized further for the development of antimycobacterial agents.

Estrogen-regulated expression of cyp19a1a and cyp19a1b genes in swim-up fry of Labeo rohita.

P450 aromatase is the terminal enzyme in the steroidogenic pathway and catalyzes the conversion of androgens to estrogens. The expression of cyp19a1 genes in brain and gonad of Indian major carp, Labeo rohita swim-up fry was measured by quantitative real-time polymerase chain-reaction. Results demonstrated that cyp19a1b and cyp19a1a predominate in brain and gonad respectively. Treatment of fry with an aromatase inhibitor fadrozole for 6days attenuated brain cyp19a1b expression, but not cyp19a1a of gonad. Fadrozole also attenuated brain aromatase activity. Treatment with 17ß-estradiol (E2) for 6days resulted in up-regulation of brain cyp19a1b transcripts in a dose- and time-dependent manner, but not cyp19a1a. Whole-body concentration of vitellogenin also increased in response to E2. Altogether, these results indicate L. rohita swim-up fry can be used to detect environmental estrogens either using vitellogenin induction or cyp19a1b gene expression.

Effects of phenol on ovarian P450arom gene expression and aromatase activity in vivo and antioxidant metabolism in common carp Cyprinus carpio.

Ovarian cyp19a mRNA expression and P450 aromatase activity were measured in vivo in common carp Cyprinus carpio exposed to phenol for 96 h. Production of reactive oxygen species (ROS) and parameters of antioxidant defense system in serum ovary and liver of this fish after long-term phenol exposure were also studied. In vivo exposure of fish to sublethal dose of phenol for 96 h caused marked attenuation of ovarian cyp19a1a gene expression and P450 aromatase activity. Production of ROS like hydrogen peroxide and hydroxyl radicals in serum, liver and ovary in fish exposed to phenol for 15 days elevated significantly from day 1 to day 7 with no further significant increase thereafter compared to their respective control values. Total superoxide dismutase (SOD) and catalase activities in serum and ovary decreased gradually and significantly from day 1 to day 4, which then increased significantly for the rest of the exposure days. Liver SOD activity seemed to be distinctly responsive to phenol. SOD activity in liver of phenol-exposed fish started to increase gradually from day 1 to 4 with no further increase thereafter. Catalase activities in all the tissues showed significant inhibition up to day 4 which then increased gradually and significantly up to day 15 of phenol exposure compared to their respective control values. From our results, it appears that sublethal dose of phenol has the endocrine disruptive potential and effect is mediated via inhibition of ovarian P450arom gene expression and aromatase activity in vivo. Sublethal dose of phenol also caused oxidative stress, and antioxidant systems are very much effective to prevent the damages caused by the generation of ROS.

G-protein coupled estrogen receptor (GPER) inhibits final oocyte maturation in common carp, Cyprinus carpio.

GPR-30, now named as GPER (G protein-coupled estrogen receptor) was first identified as an orphan receptor and subsequently shown to be required for estrogen-mediated signaling in certain cancer cells. Later studies demonstrated that GPER has the characteristics of a high affinity estrogen membrane receptor on Atlantic croaker and zebra fish oocytes and mediates estrogen inhibition of oocyte maturation in these two distantly related teleost. To determine the broad application of these findings to other teleost, expression of GPER mRNA and its involvement in 17ß-estradiol mediated inhibition of oocyte maturation in other cyprinid, Cyprinus carpio was investigated. Carp oocytes at pre-vitellogenic, late-vitellogenic and post-vitellogenic stages of development contained GPER mRNA and its transcribed protein with a maximum at late-vitellogenic oocytes. Ovarian follicular cells did not express GPER mRNA. Carp oocytes GPER mRNA was essentially identical to that found in other perciformes and cyprinid fish oocytes. Both spontaneous and 17,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P)-induced oocyte maturation in carp was significantly decreased when they were incubated with either E2, or GPER agonist G-1. On the other hand spontaneous oocyte maturation was significantly increased when carp ovarian follicles were incubated with an aromatase inhibitor, fadrozole, GPER antagonist, G-15 and enzymatic removal of the ovarian follicle cell layers. This increase in oocyte maturation was partially reversed by co-treatment with E2. Consistent with previous findings with human and fish GPR30, E2 treatment in carp oocytes caused increase in cAMP production and simultaneously decrease in oocyte maturation, which was inhibited by the addition of 17,20ß-P. The results suggest that E2 and GPER play a critical role in regulating re-entry in to meiotic cell cycle in carp oocytes.

Doxorubicin induced epigenetic regulation of dendritic cell maturation in association with T cell activation facilitates tumor protective immune response in non-small cell lung cancer (NSCLC).

BACKGROUND: NSCLC is one of the leading causes of death and is often diagnosed at late stages with no alternative therapeutic approach. DCs are professional antigen-presenting cells and DC-based immunotherapy has been under the spotlight for its anti-cancer properties. Epigenetic modifications including DNA methylation and histone modification in DCs play a crucial role in regulating their functions such as maturation and activation,innate immune responses, T cell priming, antigen presentation, and cytokine production. In the current study, we investigated the anti-cancer properties of Doxorubicin at a noncytotoxic concentration that could be extrapolated as an epigenetic regulator for DC maturation to elicit anti-tumor activity. METHODOLOGIES: PBMCs from normal and NSCLC blood samples were isolated and treated with growth factors. DCs were matured with low dose Doxorubicin and the DC maturation markers were checked by using flow-cytometry. Further, ELISA was performed and low dose Doxorubicin-induced DCs were pulsed with LCA (Lung Cancer Antigen) and primed with CD4 +T helper (Th) cells for cytotoxicity assessment. Further, epigenetic markers of T: DC conjugation were immunofluorescently visualized under a microscope. ChIP-qPCR and Invitro assays such as histone methylation, DNA methylation, and m6A methylation were performed to study the epigenetic changes under low dose Dox treatment. IL-12 neutralization assay was performed to check for the IL-12 dependency of DCs and their effect under Dox at low dose treatment. This was further followed by a Western Blotting analysis for histone and non-histone proteins. RESULTS: Low dose Doxorubicin induces epigenetic changes in DCs to elicit an anti-tumor response in NSCLC through the generation of CTLs with a concomitant increase in the extracellular secretions of anti-inflammatory cytokines. We also found that low dosage of Doxorubicin matured DCs when pulsed with LCA and primed with CD4 +T helper cells, secrete IFN-γ which is important in orchestrating adaptive immunity by activating CD8 + cytotoxic T-lymphocytes. Also, the secretions of IL-12 help us infer that protective immunity is also induced via Th1 response which triggered selectively the translocation of PKCθ to immunological synapse in between DC and Th. Further, methylation and acetylation markers H3K4me3 and H3K14Ac respectively upregulated whereas levels of STAT5, NFkB, NOTCH1, and DNAPKcs were downregulated. DNA and RNA methylation assays then lead to confirmations about the epigenetic changes caused by low dose Dox treatment. DNA methylation was reduced which resulted in the activation of tumor suppressor gene p53 and Th1-associated transcription factor TBX21. On the other hand, both absolute and relative RNA methylation quantification increased in the presence of Dox at a low dose. CONCLUSION: From this study, we understand that non-cytotoxic concentration of Doxorubicin increases the Ag-presenting ability of DCs via an IL-12-dependent mechanism and causes epigenetic modifications in NSCLC.

Cannabis effectiveness on immunologic potency of pulmonary contagion.

Respiratory illnesses and its repercussions are becoming more prevalent worldwide. It is necessary to research both innovative treatment and preventative techniques. Millions of confirmed cases and fatalities from the COVID-19 epidemic occurred over the previous two years. According to the review research, cannabinoids are a class of medicines that should be considered for the treatment of respiratory conditions. Cannabinoids and inhibitors of endocannabinoid degradation have illustrated advantageous anti-inflammatory, asthma, pulmonary fibrosis, and pulmonary artery hypotension in numerous studies (in vitro and in vivo). It has been also noted that CB2 receptors on macrophages and T-helper cells may be particularly triggered to lower inflammation in COVID-19 patients. Since the majority of lung tissue contains cannabinoid receptors, cannabis can be an effective medical tool for treating COVID-19 as well as pulmonary infections. Notably, CB2 and CB1 receptors play a major role in immune system modulation and anti-inflammatory activities. In this review, we put forth the idea that cannabis might be helpful in treating pulmonary contagion brought on by viral integration, such as that caused by SARS-CoV-2, haemophilus influenza type b, Streptococcus pneumoniae, influenza virus, and respiratory syncytial virus. Also, a detailed overview of CB receptors, intricate mechanisms, is highlighted connecting link with COVID-19 viral structural modifications along with molecular basis of CB receptors in diminishing viral load in pulmonary disorders supported through evident literature studies. Further, futuristic evaluations on cannabis potency through novel formulation development focusing on in vivo/in vitro systems can produce promising results.

Effect of cadmium chloride on secretion of 17ß-estradiol by the ovarian follicles of common carp, Cyprinus carpio.

Cadmium (Cd(2+)) is a common environmental pollutant present in wastes associated with mining, smelting and electroplating. It is a major constituent of the tobacco smoke. Exposure of this heavy metal has been linked to wide range of detrimental effects on mammalian reproduction particularly on ovarian steroidogenesis. Low doses of Cd(2+) are reported to stimulate ovarian luteal progesterone synthesis whereas high doses inhibited it. Cd(2+) exposure is also reported to inhibit gonadal function in fish. In the present study the effects of cadmium chloride (CdCl(2)) on the secretion of gonadotropin-induced 17ß-estradiol was examined in female common carp Cyprinus carpio. Vitellogenic stage fish were exposed to physiological safe dose of CdCl(2) for 0, 24, 48 and 96 h and serum and ovarian 17ß-estradiol levels were estimated. In the in vitro experiments, vitellogenic follicles were incubated with CdCl(2) and a dose- and time-dependent effects on steroid production were estimated induced by LH. Exposure of fish with CdCl(2) gradually attenuated serum and ovarian 17ß-estradiol levels with increasing time and maximum inhibition was noticed after 96 h. Administration of CdCl(2) to the incubations significantly inhibited LH-induced release of 17ß-estradiol in vitro. To clarify the mechanism of attenuated production of 17ß-estradiol, in vitro effects of CdCl(2) on LH induced P450 aromatase activity (conversion of testosterone to 17ß-estradiol) and cytochrome P450arom gene expression in carp ovarian follicles were evaluated. Results show that LH-stimulated P450 aromatase activity and P450arom gene expression in ovarian follicles were significantly inhibited by CdCl(2). The present study further demonstrated that LH-induced stimulation of ovarian steroidogenic factor-1 (SF-1) which activates aromatase enzyme, is strongly inhibited by cadmium chloride treatment.

Effect of phenol on ovarian secretion of 17ß-estradiol in common carp Cyprinus carpio.

Phenol is a common substance present in many industrial wastewaters and in nonspecific pesticides. Due to its solubility and volatility phenol is often found in marine and freshwater environment. It is lipophilic compound and has a high potential for accumulating along the trophic chain. Phenol thus is not only a threat to natural environment but also to human health. The effects of phenol on the secretion of 17ß-estradiol were examined in female common carp Cyprinus carpio. Vitellogenic stage fish were exposed to physiological safe dose of phenol for 0, 24, 48 and 96 h. In the in vitro experiments, vitellogenic follicles were incubated with phenol and dose- and time-course effects on leuteinising hormone (LH) induced steroid production were examined. Exposure of fish with phenol gradually attenuated serum and ovarian 17ß-estradiol levels with increasing time and maximum inhibition was noticed after 96 h. Administration of phenol significantly inhibited LH-induced secretion of 17ß-estradiol by the ovarian follicles in vitro. To clarify the mechanism of attenuated production of 17ß-estradiol in phenol-treated follicles, stimulated by LH, in vitro effect phenol and LH on aromatase activity (conversion of testosterone to 17ß-estradiol) and cytochrome P450arom gene expression in carp ovarian follicles were investigated. Physiological safe dose of phenol significantly inhibited LH-stimulated aromatase activity and P450arom gene expression in ovarian follicles. The present study further demonstrated that LH-induced activation of ovarian steroidogenic factor-1 (SF-1) is strongly inhibited by phenol treatment. These results suggest that physiological safe dose of phenol as endocrine disruption (ED) potential and the effect can be mediated via several cellular pathways including the inhibition of SF-1 activity, aromatase activity and P450arom gene expression.

A Comparative Prospective Study Between Conventional Chemo-Radiotherapy and Pure Accelerated Radiotherapy With Concurrent Chemotherapy for the Treatment of Locally Advanced Head and Neck Cancer.

BACKGROUND: The established standard treatment for locally advanced head and neck squamous cell carcinoma is concurrent chemoradiotherapy, but the optimum radiotherapy schedule for best disease control and acceptable toxicity is still evolving. Tumor control probability decreases with each day's prolongation of treatment time. Shortening the overall treatment time of radiation by pure accelerated radiotherapy may be a good option. MATERIAL AND METHODS: One hundred and sixty-five patients with histopathologically proven squamous cell carcinoma of the head and neck were included in the study and were assigned into two groups from January 2017 to June 2019. The total dose of 70 Gy was given, 2 Gy/fraction/day. Treatment was given five days a week (conventional radiotherapy) and six days a week (pure accelerated radiotherapy). Both groups received weekly concurrent injections of cisplatin. RESULTS:  The stage (p=0.006) and fractionation of radiation (p=0.018) were the independent factors affecting disease-free survival (DFS). There was a statistically significant difference (p=0.019) in the recurrence of patients in different fractionation schedules. The median DFS was 39 months with a 95% CI of 31.44 - 46.55. One- and three-year DFS was 51% and 8.5% respectively in the five fractions/week schedule arm while 54.5% and 9.5% respectively in the six fractions/week schedule group. CONCLUSION: Pure accelerated radiotherapy is more efficacious in terms of disease control with comparable mildly increased acute side effects.

Total and soluble oxalate content of some Indian spices.

Spices, such as cinnamon, cloves, cardamom, garlic, ginger, cumin, coriander and turmeric are used all over the world as flavouring and colouring ingredients in Indian foods. Previous studies have shown that spices contain variable amounts of total oxalates but there are few reports of soluble oxalate contents. In this study, the total, soluble and insoluble oxalate contents of ten different spices commonly used in Indian cuisine were measured. Total oxalate content ranged from 194 (nutmeg) to 4,014 (green cardamom) mg/100 g DM, while the soluble oxalate contents ranged from 41 (nutmeg) to 3,977 (green cardamom) mg/100 g DM. Overall, the percentage of soluble oxalate content of the spices ranged from 4.7 to 99.1% of the total oxalate content which suggests that some spices present no risk to people liable to kidney stone formation, while other spices can supply significant amounts of soluble oxalates and therefore should be used in moderation.

Clinical relevance and therapeutic aspects of professional antigen-presenting cells in lung cancer.

Lung cancer stays the preeminent cause of death worldwide. Despite recent advancements in chemotherapy, radiotherapy, and immunotherapy, the survival rate for people with advanced stages of the disease is still appalling. Moreover, there is a severe lack of reliable prognoses and indicators for classification in newly developed immunotherapies. A better understanding of immune cells is necessary to harness immune response mechanisms for therapeutic effects. Professional antigen-presenting cells are responsible for determining the fate of the immune response through the antigen processing and presentation pathway (APP). The most professional antigen-presenting cells (APC) include the dendritic cells (DC), macrophages, and B cells, which present antigens to the T-helper cells. Dendritic cells are significantly explored as a tool for immunotherapy owing to their precise ability to provoke and alter T-cell responses. Moreover, the role of tumor-associated macrophages (TAMs), an abundant leukocyte in lung cancer, is also a potential target for adjuvant anti-cancer therapies. In this review, we summarize the recent advances in our understanding of the various types of immunotherapy mapped out via professional antigen-presenting cells in lung cancer.

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.