Overcoming heat shock protein inhibition at critical temperature vital for survival in Solanum tuberosum L. in vivo condition

Heat stress proteins (HSPs) and related cognates are candidates mediating and preventing cellular damage  from heat-stress, but their expression can be inhibited midway. The time-based occurrence pattern for heat mediated inhibition underlying HSPs expression at 41.5°C and revival subsequent stress was studied in vivo for four Solanum tuberosum L. cultivars viz. Kufri Pukhraj, Kufri Jyoti, Kufri Chandramukhi and Kufri Ashoka. Our  results show that the inhibition process is a functional variance of time and genetic variability characterized by differential down-regulation of housekeeping proteins (HKPs) of about 55.7 and 43.5 KD in some cultivars and  complete inhibition of a prominent 19.9 KD HKP in Kufri Jyoti at all stressed time. Furthermore, the results  strongly suggest HSPs inhibition process bridges the gap between normal proteome and spur expression  maxima for stress proteome and may last for about 1 h for cultivars that effectively eludes the process  upgrading their thermotolerance in vivo.Key words: Solanum tuberosum L., heat-mediated inhibition, heat shock proteins, housekeeping proteins

Differential Targeting of c-Maf, Bach-1, and Elmo-1 by microRNA-143 and microRNA-365 Promotes the Intracellular Growth of Mycobacterium tuberculosis in Alternatively IL-4/IL-13 Activated Macrophages

Mycobacterium tuberculosis (Mtb) can subvert the host defense by skewing macrophage activation toward a less microbicidal alternative activated state to avoid classical effector killing functions. Investigating the molecular basis of this evasion mechanism could uncover potential candidates for host directed therapy against tuberculosis (TB). A limited number of miRNAs have recently been shown to regulate host-mycobacterial interactions. Here, we performed time course kinetics experiments on bone marrow-derived macrophages (BMDMs) and human monocyte-derived macrophages (MDMs) alternatively activated with IL-4, IL-13, or a combination of IL-4/IL-13, followed by infection with Mtb clinical Beijing strain HN878. MiR-143 and miR-365 were highly induced in Mtb-infected M(IL-4/IL-13) BMDMs and MDMs. Knockdown of miR-143 and miR-365 using antagomiRs decreased the intracellular growth of Mtb HN878, reduced the production of IL-6 and CCL5 and promoted the apoptotic death of Mtb HN878-infected M(IL-4/IL-13) BMDMs. Computational target prediction identified c-Maf, Bach-1 and Elmo-1 as potential targets for both miR-143 and miR-365. Functional validation using luciferase assay, RNA-pulldown assay and Western blotting revealed that c-Maf and Bach-1 are directly targeted by miR-143 while c-Maf, Bach-1, and Elmo-1 are direct targets of miR-365. Knockdown of c-Maf using GapmeRs promoted intracellular Mtb growth when compared to control treated M(IL-4/IL-13) macrophages. Meanwhile, the blocking of Bach-1 had no effect and blocking Elmo-1 resulted in decreased Mtb growth. Combination treatment of M(IL-4/IL-13) macrophages with miR-143 mimics or miR-365 mimics and c-Maf, Bach-1, or Elmo-1 gene-specific GapmeRs restored Mtb growth in miR-143 mimic-treated groups and enhanced Mtb growth in miR-365 mimics-treated groups, thus suggesting the Mtb growth-promoting activities of miR-143 and miR-365 are mediated at least partially through interaction with c-Maf, Bach-1, and Elmo-1. We further show that knockdown of miR-143 and miR-365 in M(IL-4/IL-13) BMDMs decreased the expression of HO-1 and IL-10 which are known targets of Bach-1 and c-Maf, respectively, with Mtb growth-promoting activities in macrophages. Altogether, our work reports a host detrimental role of miR-143 and miR-365 during Mtb infection and highlights for the first time the role and miRNA-mediated regulation of c-Maf, Bach-1, and Elmo-1 in Mtb-infected M(IL-4/IL-13) macrophages

Differential Targeting of c-Maf, Bach-1, and Elmo-1 by microRNA-143 and microRNA-365 Promotes the Intracellular Growth of Mycobacterium tuberculosis in Alternatively IL-4/IL-13 Activated Macrophages.

Mycobacterium tuberculosis (Mtb) can subvert the host defense by skewing macrophage activation toward a less microbicidal alternative activated state to avoid classical effector killing functions. Investigating the molecular basis of this evasion mechanism could uncover potential candidates for host directed therapy against tuberculosis (TB). A limited number of miRNAs have recently been shown to regulate host-mycobacterial interactions. Here, we performed time course kinetics experiments on bone marrow-derived macrophages (BMDMs) and human monocyte-derived macrophages (MDMs) alternatively activated with IL-4, IL-13, or a combination of IL-4/IL-13, followed by infection with Mtb clinical Beijing strain HN878. MiR-143 and miR-365 were highly induced in Mtb-infected M(IL-4/IL-13) BMDMs and MDMs. Knockdown of miR-143 and miR-365 using antagomiRs decreased the intracellular growth of Mtb HN878, reduced the production of IL-6 and CCL5 and promoted the apoptotic death of Mtb HN878-infected M(IL-4/IL-13) BMDMs. Computational target prediction identified c-Maf, Bach-1 and Elmo-1 as potential targets for both miR-143 and miR-365. Functional validation using luciferase assay, RNA-pulldown assay and Western blotting revealed that c-Maf and Bach-1 are directly targeted by miR-143 while c-Maf, Bach-1, and Elmo-1 are direct targets of miR-365. Knockdown of c-Maf using GapmeRs promoted intracellular Mtb growth when compared to control treated M(IL-4/IL-13) macrophages. Meanwhile, the blocking of Bach-1 had no effect and blocking Elmo-1 resulted in decreased Mtb growth. Combination treatment of M(IL-4/IL-13) macrophages with miR-143 mimics or miR-365 mimics and c-Maf, Bach-1, or Elmo-1 gene-specific GapmeRs restored Mtb growth in miR-143 mimic-treated groups and enhanced Mtb growth in miR-365 mimics-treated groups, thus suggesting the Mtb growth-promoting activities of miR-143 and miR-365 are mediated at least partially through interaction with c-Maf, Bach-1, and Elmo-1. We further show that knockdown of miR-143 and miR-365 in M(IL-4/IL-13) BMDMs decreased the expression of HO-1 and IL-10 which are known targets of Bach-1 and c-Maf, respectively, with Mtb growth-promoting activities in macrophages. Altogether, our work reports a host detrimental role of miR-143 and miR-365 during Mtb infection and highlights for the first time the role and miRNA-mediated regulation of c-Maf, Bach-1, and Elmo-1 in Mtb-infected M(IL-4/IL-13) macrophages

An overview of natural products that modulate the expression of non-coding RNAs involved in oxidative stress and inflammation-associated disorders

Oxidative stress is a state in which oxidants are produced in excess in the body’s tissues and cells, resulting in a biological imbalance amid the generation of reactive oxygen and nitrogen species (RONS) from redox reactions. In case of insufficient antioxidants to balance, the immune system triggers signaling cascades to mount inflammatory responses. Oxidative stress can have deleterious effects on major macromolecules such as lipids, proteins, and nucleic acids, hence, Oxidative stress and inflammation are among the multiple factors contributing to the etiology of several disorders such as diabetes, cancers, and cardiovascular diseases. Non-coding RNAs (ncRNAs) which were once referred to as dark matter have been found to function as key regulators of gene expression through different mechanisms. They have dynamic roles in the onset and development of inflammatory and oxidative stress-related diseases, therefore, are potential targets for the control of those diseases. One way of controlling those diseases is through the use of natural products, a rich source of antioxidants that have drawn attention with several studies showing their involvement in combating chronic diseases given their enormous gains, low side effects, and toxicity. In this review, we highlighted the natural products that have been reported to target ncRNAs as mediators of their biological effects on oxidative stress and several inflammation-associated disorders. Those natural products include Baicalein, Tanshinone IIA, Geniposide, Carvacrol/Thymol, Triptolide, Oleacein, Curcumin, Resveratrol, Solarmargine, Allicin, aqueous extract or pulp of Açai, Quercetin, and Genistein. We also draw attention to some other compounds including Zanthoxylum bungeanum, Canna genus rhizome, Fuzi-ganjiang herb pair, Aronia melanocarpa, Peppermint, and Gingerol that are effective against oxidative stress and inflammation-related disorders, however, have no known effect on ncRNAs. Lastly, we touched on the many ncRNAs that were found to play a role in oxidative stress and inflammation-related disorders but have not yet been investigated as targets of a natural product. Shedding more light into these two last points of shadow will be of great interest in the valorization of natural compounds in the control and therapy of oxidative stress- and inflammation-associated disorders

Upregulated bovine tuberculosis microRNAs Trigger oncogenic pathways: An In silico perception

Background/Objective: Although microRNA (miRNA)-directed regulation of bovine tuberculosis (bTB) has already been reported, very little is known about the incited pathways and genes. We profiled bTB-upregulated miRNAs through an in silico methodology. Methods: The data of upregulated miRNAs in bTB versus healthy controls were collected and clustered into three groups by their tissue specificity as follows: G1 (mammary gland-specific): bta-miR-146a; G2 (peripheral blood mononuclear cell-specific): bta-miR-155; and G3 (alveolar macrophage-specific): bta-miR-146a, bta-miR-155, bta-miR-142-5p, bta-miR-423-3p, bta-miR-21-5p, bta-miR-27a-3p, bta-miR-99b, bta-miR-147, bta-miR-223, and bta-let-7i. The miRNA–mRNA interaction network was defined by TargetScan. The gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways of these transcripts were examined. Results: The results illustrate the induction of pathways in cancer, highly enriched, and unanimous to all three gene sets (G1, G2, and G3). Mitogen-activated protein kinase and PI3K-Akt signaling were specific to G2 and G3 with fibroblast growth factors formed the key factors. Conclusion: The inferred cancer cascades denote a probable modulation of innate immune response in an infectious state. These baseline pictures could lay the ground for further substantive studies

Triptolide induces growth inhibition and apoptosis of human laryngocarcinoma cells by enhancing p53 activities and suppressing E6-mediated p53 degradation.

Triptolide, an active compound extracted from Chinese herb Leigongteng (Tripterygium wilfordii Hook F.), shows a broad-spectrum of anticancer activity through its cytotoxicity. However, the efficacy of triptolide on laryngocarcinoma rarely been evaluated, and the mechanism by which triptolide-induced cellular apoptosis is still not well understood. In this study, we found that triptolide significantly inhibited the laryngocarcinoma HEp-2 cells proliferation, migration and survivability. Triptolide induces HEp-2 cell cycle arrest at the G1 phase and apoptosis through intrinsic and extrinsic pathways since both caspase-8 and -9 are activated. Moreover, triptolide enhances p53 expression by increasing its stability via down-regulation of E6 and E6AP. Increased p53 transactivates down-stream target genes to initiate apoptosis. In addition, we found that short time treatment with triptolide induced DNA damage, which was consistent with the increase in p53. Furthermore, the cytotoxicity of triptolide is decreased by p53 knockdown or use of caspases inhibitor. In conclusion, our results demonstrated that triptolide inhibits cell proliferation and induces apoptosis in laryngocarcinoma cells by enhancing p53 expression and activating p53 functions through induction of DNA damage and suppression of E6 mediated p53 degradation. These studies indicate that triptolide is a potential anti-laryngocarcinoma drug

Triptolide enhanced the anti-tumor effect of radiation on laryngocarcinoma cells.

<p>(A) The combination use of triptolide with radiation showed more inhibitory effect on the HEp-2 cells viability. Cells were seeded into 96 well plates with a density of 5000 cells per well. After pro-treatment with 10nM triptolide for 5h, cells were treated with various doses of X-ray radiation. Cell viability was detected with CCK8 assay. (B) The combination use of triptolide with radiation showed more inhibitory effect on the HEp-2 cells survivability. After pro-treatment with 10nM triptolide for 5h and radiated with 4 Gy, cells were then trypsonsized and plated in 60 mm plates with a density of 1000 cells per plate. 2 to 3 weeks later, cells were fixed and stained, and the numbers of colonies were counted and the survival fractions were calculated. </p

Triptolide induced apoptosis in laryngocarcinoma cell <i>in</i><i>vitro</i>.

<p>(A) Cell cycle analysis of HEp-2 cells by Flow cytometry. After treated with various doses of triptolide, HEp-2 cells were fixed and stained with propidium iodide (PI). DNA content was detected by Flow cytometry. (B) Apoptotic analysis of HEp-2 cells by Flow cytometry. HEp-2 cells were treated with various doses of triptolide and incubated with AV-FITC (green) and PI (red). Stained cells were analyzed by Flow cytometry. Percentage of intact cells (AV-/PI−) and different stages apoptotic cells (AV+/PI−, AV+/PI+ and AV-/PI+) are presented. (C) Western blot analysis of caspase-8/9/3 proteins in triptolide-treated laryngocarcinoma cell. HEp-2 cells were treated with indicated doses of triptolide for 24 h, the procaspase-8/9/3, PARP and their cleaved products were indicated. β-actin was used as a loading control. </p

Schematic diagram of hypothetic mechanisms underlying triptolide induction of apoptosis in laryngocarcinoma cells.

<p>Triptolide causes DNA damage to induce p53 transcription. Meanwhile triptolide enhances p53 protein stability by suppression of the E6-mediated p53 ubiquitinylation-mediated degradation. Elevated p53 initiates cell apoptosis through death receptors pathways and mitochondrial pathway via activating transcription and non-transcriptional functions (see Discussion for detail). </p

Triptolide up-regulated p53 mRNA level and enhanced p53 protein stabilization in laryngocarcinoma cells.

<p>(A) Triptolide enhanced p53 mRNA level in HEp-2 cells. Cells were treated with indicated doses of triptolide for 24h, p53 mRNA levels were determined by qRT-PCR using the specific primers. (B) Triptolide showed weak influence on the p53 mRNA stability. Cells were treated with 25μg/ml actinomycin D (AD) with or without 50 nM triptolide for indicated times, p53 and β-actin mRNA levels were determined by qRT-PCR and relative p53 mRNA level were presented. (C) Triptolide stabilized p53 protein level in HEp-2 cells. Cells were treated with 50μg/ml cycloheximide (CHX) with or without 50 nM triptolide for indicated times, p53 and β-actin protein levels were determined by western blot. (D) Effect of triptolide on p53 ubiquitination. HEp-2 cells were treated with 50 nM triptolide for indicated times, cell lysates were immunoprecipitated with p53 antibody and immunoblotted with Ub antibody. p53 and β-actin protein level were also presented. (E) Triptolide reduced E6 and E6AP expression in laryngocarcinoma cell. (F) Effect of triptolide on the interaction of p53 with E6 and E6AP. Cells were treated with indicated doses of triptolide and immunoprecipitated with p53 antibody, E6 and E6AP were immune-blotted. (G) and (H) Triptolide reduced E6 and E6AP expression in Hela and TC-1 cells. </p