Relative mutagenic effectiveness and efficiency of chemical mutagens (Caffeine and EMS) and heavy metals [(Pb(NO3)2 and Cd(NO3)2)] in developing chlorophyll and morphological mutants in lentil.

Mutagenic effectiveness and efficiency are the utmost vital indices to determine the effective and efficient mutagenic concentrations for the generation of high frequency of desirable mutation in mutation breeding. Nevertheless, there are meager study are available for employing effective and efficient concentration of caffeine, EMS, Pb(NO3)2 and Cd(NO3)2 for the crop improvement. Thus, the current study was performed to assess the mutagenic effectiveness and efficiency of caffeine, EMS and heavy metals [Pb(NO3)2 Cd(NO3)2] and to determine the genetic variability of M2 and M3 lentil mutant lines. The frequency of different chlorophyll and chromophyll mutation was found highest at moderate and higher concentrations of chemical mutagens and heavy metals in M1 and M2 generation. The highest effectiveness was in 20 ppm Cd(NO3)2, followed by 20 ppm Pb(NO3)2, 0.10% EMS, and 0.10% caffeine. The present investigation also showed lower doses of caffeine, EMS, Pb(NO3)2, and Cd(NO3)2 were more efficient than higher concentrations, and caffeine was found more efficient followed by EMS, Pb(NO3)2, and Cd(NO3)2. Furthermore, a broad spectrum of viable mutations affecting different morphological characters of the plants viz., leaves, plant height, growth habits, flowers, pods, and seeds in M2 and M3 generation were recorded. Ten morphological mutants showing acceptable agronomic and horticultural features were identified, as genetic resources for further breeding.

Physio-biochemical analysis and molecular characterization of induced lentil mutant lines.

Lens culinaris is a proteinaceous food crop that is consumed worldwide for protein requirements. Mutation breeding has been used to improve protein content, yield, and related traits, as well as to select highly desirable mutants that are economically significant. An investigation of genotypic variation in lentil germplasm was carried out using induced mutagenesis, with caffeine, ethyl methane sulfonate (EMS), lead nitrate, and cadmium nitrate as mutagens that resulted in 18 mutant lines in the M3 generation. For the present study, we analyzed the genetic diversity of lentil mutant lines using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and random amplified polymorphic DNA markers (RAPD). The heterozygosity of RAPD markers per primer ranged from 50.00-90.90% with an average of 71.04%. The genetic divergent analysis was performed using hierarchical clustering (UPGMA), exhibited that these mutant lines were classified mainly into five subpopulation or clusters. A close resemblance with highest genetic coefficient similarity (1.00) were observed between control and mutant H; between mutant M and E; between mutant Q and J2, while more divergent mutants were N2 with mutant B; and mutant R with mutant J1with least genetic coefficient similarity (0.22). Protein and mineral content (Fe, Zn and Cu) were increased significantly in some high yielding mutant lines concerning to the control plant, and showed polymorphic variations in polypeptide chains in terms of banding pattern. Stomatal morphology in high yielding mutants were perceived utilizing scanning electron microscopy (SEM), exhibiting variations in stomatal size, stomatal opening and number of stomata. The present study's promising mutant lines' biological, physiological, and molecular profiles provide a foundation for forthcoming preservation and consumption strategies to broaden the genetic diversity of the breeding population of lentil.

Natural Compounds: Potential Therapeutics for the Inhibition of Cartilage Matrix Degradation in Osteoarthritis.

Osteoarthritis (OA) is the most common degenerative joint disease characterized by enzymatic degradation of the cartilage extracellular matrix (ECM) causing joint pain and disability. There is no disease-modifying drug available for the treatment of OA. An ideal drug is expected to stop cartilage ECM degradation and restore the degenerated ECM. The ECM primarily contains type II collagen and aggrecan but also has minor quantities of other collagen fibers and proteoglycans. In OA joints, the components of the cartilage ECM are degraded by matrix-degrading proteases and hydrolases which are produced by chondrocytes and synoviocytes. Matrix metalloproteinase-13 (MMP-13) and a disintegrin and metalloproteinase with thrombospondin motifs 4 and 5 (ADAMTS5) are the major collagenase and aggrecanase, respectively, which are highly expressed in OA cartilage and promote cartilage ECM degradation. Current studies using various in vitro and in vivo approaches show that natural compounds inhibit the expression and activity of MMP-13, ADAMTS4, and ADAMTS5 and increase the expression of ECM components. In this review, we have summarized recent advancements in OA research with a focus on natural compounds as potential therapeutics for the treatment of OA with emphasis on the prevention of cartilage ECM degradation and improvement of joint health.

Oxidative stress and inflammation in osteoarthritis pathogenesis: Role of polyphenols.

Osteoarthritis (OA) is the most prevalent joint degenerative disease leading to irreversible structural and functional changes in the joint and is a major cause of disability and reduced life expectancy in ageing population. Despite the high prevalence of OA, there is no disease modifying drug available for the management of OA. Oxidative stress, a result of an imbalance between the production of reactive oxygen species (ROS) and their clearance by antioxidant defense system, is high in OA cartilage and is a major cause of chronic inflammation. Inflammatory mediators, such as interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) are highly upregulated in OA joints and induce ROS production and expression of matrix degrading proteases leading to cartilage extracellular matrix degradation and joint dysfunction. ROS and inflammation are interdependent, each being the target of other and represent ideal target/s for the treatment of OA. Plant polyphenols possess potent antioxidant and anti-inflammatory properties and can inhibit ROS production and inflammation in chondrocytes, cartilage explants and in animal models of OA. The aim of this review is to discuss the chondroprotective effects of polyphenols and modulation of different molecular pathways associated with OA pathogenesis and limitations and future prospects of polyphenols in OA treatment.

Proteome Profiling of the Mutagen-Induced Morphological and Yield Macro-Mutant Lines of Nigella sativa L.

In the present investigation, the leaf proteome profile of the macro-mutant lines of Nigella sativa L. was analyzed to identify the key proteins involved in the expression of traits associated with the morphology, seed yield, and content of thymoquinone. In our earlier study, the macro-mutants were generated with contrasting morphological traits and seed yields through induced mutagenesis, using ethyl methyl sulfonate, gamma rays, and combinations of both. Analysis of the leaf proteome of the control and macro-mutant lines of N. sativa showed that twenty-three proteins were differentially expressed. These differentially expressed proteins were sequenced through mass spectrometry and identified using the MASCOT software. On the basis of their function, these proteins were categorized into several groups. Most proteins were found in the categories of signal transduction (18%) and carbon metabolism (18%). A total of 13% of proteins belonged to the categories of energy and metabolism. Proteins in the categories of secondary plant metabolism, stress defense, cytoskeleton, and protein synthesis were also found. The polycomb group protein (FIE1), transcription factor (PRE1), and geranyl diphosphate synthase were notable proteins, in addition to some proteins of signal transduction and carbon metabolism. Expression patterns of the differentially expressed proteins were also studied at the transcript level by using qRT-PCR. Transcriptomics analysis was consistent with the proteomics data. This study shows the changes that take place at the proteomic level through induced mutagenesis, as well as the involvement of some proteins in the expression traits associated with plant height, seed yield, and the thymoquinone content of N. sativa. The identified proteins might help elucidate the metabolic pathways involved in the expression of traits, including seed yield, and the active compounds of medicinal plants.

The large protein 'L' of Peste-des-petits-ruminants virus exhibits RNA triphosphatase activity, the first enzyme in mRNA capping pathway.

Peste-des-petits-ruminants is a highly contagious and fatal disease of goats and sheep caused by non-segmented, negative strand RNA virus belonging to the Morbillivirus genus-Peste-des-petits-ruminants virus (PPRV) which is evolutionarily closely related to Rinderpest virus (RPV). The large protein 'L' of the members of this genus is a multifunctional catalytic protein, which transcribes and replicates the viral genomic RNA as well as possesses mRNA capping, methylation and polyadenylation activities; however, the detailed mechanism of mRNA capping by PPRV L protein has not been studied. We have found earlier that the L protein of RPV has RNA triphosphatase (RTPase), guanylyltransferase (GTase) and methyltransferase activities, and unlike vesicular stomatitis virus (VSV), follows the conventional pathway of mRNA capping. In the present work, using a 5'-end labelled viral RNA as substrate, we demonstrate that PPRV L protein has RTPase activity when present in the ribonucleoprotein complex of purified virus as well as recombinant L-P complex expressed in insect cells. Further, a minimal domain in the C-terminal region (aa1640-1840) of the L protein has been expressed in E. coli and shown to exhibit RTPase activity. The RTPase activity of PPRV L protein is metal-dependent and functions with a divalent cation, either magnesium or manganese. In addition, RTPase associated nucleotide triphosphatase activity (NTPase) of PPRV L protein is also demonstrated. This work provides the first detailed study of RTPase activity and identifies the RTPase domain of PPRV L protein.

Harpagoside suppresses IL-6 expression in primary human osteoarthritis chondrocytes.

There is growing evidence in support of the involvement of inflammatory response in the pathogenesis of osteoarthritis (OA). Harpagoside, one of the bioactive components of Harpagophytum procumbens (Hp), has been shown to possess anti-inflammatory properties. Here we used an in vitro model of inflammation in OA to investigate the potential of harpagoside to suppress the production of inflammatory cytokines/chemokines such as IL-6 and matrix degrading proteases. We further investigated the likely targets of harpagoside in primary human OA chondrocytes. OA chondrocytes were pre-treated with harpagoside before stimulation with IL-1ß. mRNA expression profile of 92 cytokines/chemokines was determined using TaqMan Human Chemokine PCR Array. Expression levels of selected mRNAs were confirmed using TaqMan assays. Protein levels of IL-6 and MMP-13 were assayed by ELISA and immunoblotting. Total protein levels and phosphorylation of signaling proteins were determined by immunoblotting. Cellular localization of IL-6 and c-Fos was performed by immunofluorescence and confocal microscopy. DNA binding activity of c-FOS/AP-1 was determined by ELISA. Harpagoside significantly altered the global chemokine expression profile in IL-1ß-stimulated OA chondrocytes. Expression of IL-6 was highly induced by IL-1ß, which was significantly inhibited by pre-treatment of OA chondrocytes with harpagoside. Harpagoside did not inhibit the IL-1ß-induced activation of NF-κB and C/EBPß transcription factors but suppressed the IL-1ß-triggered induction, phosphorylation, and DNA binding activity of c-FOS, one of the main components of AP-1 transcription factors. Further, harpagoside significantly inhibited the expression of MMP-13 in OA chondrocytes under pathological conditions. siRNA-mediated knockdown of IL-6 resulted in suppressed expression and secretion of MMP-13 directly linking the role of IL-6 with MMP-13 expression. Taken together, the present study suggests that harpagoside exerts a significant anti-inflammatory effect by inhibiting the inflammatory stimuli mediated by suppressing c-FOS/AP-1 activity in OA chondrocytes under pathological conditions. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:311-320, 2017.

The microRNA miR-29a is associated with human immunodeficiency virus latency.

BACKGROUND: Latent reservoirs of HIV-1 provide a major challenge to its cure. There are increasing reports of interplay between HIV-1 replication and host miRNAs. Several host miRNAs, which potentially target the nef-3'LTR region of HIV-1 RNA, including miR-29a, are proposed to promote latency. FINDINGS: We used two established cellular models of HIV-1 latency - the U1 monocytic and J1.1 CD4+ T cell lines to show an inverse relationship between HIV-1 replication and miR-29a levels, which was mediated by the HIV-1 Nef protein. Using a miR-29a responsive luciferase reporter plasmid, an expression plasmid and an anti-miR29a LNA, we further demonstrate increased miR-29a levels during latency and reduced levels following active HIV replication. Finally, we show that miR-29a levels in the PBMCs and plasma of HIV infected persons also correlate inversely with latency and active viral replication. CONCLUSIONS: The levels of miR-29a correlate inversely with active HIV-1 replication in cell culture models and in HIV infected persons. This links miR-29a to viral latency and suggests another approach to activate and destroy latent HIV-1 reservoirs.

Escheriosome-mediated cytosolic delivery of PLK1-specific siRNA: potential in treatment of liver cancer in BALB/c mice.

AIM: In the present study, the anticancer efficacy of a novel escheriosome-based formulation of PLK1-specific siRNA was evaluated against liver cancer in BALB/c mice. MATERIALS & METHODS: The escheriosome-based siRNA nanoparticles were prepared using lipids isolated from Escherichia coli. The escheriosomes were characterized for size, surface charge and stability. The anticancer potential of PLK1-specific siRNA formulation was ascertained on the basis of expression of pro-/anti-apoptotic factors and histopathological studies. RESULTS: The escheriosome-entrapped siRNA was found to be released in surrounding milieu in a sustained manner. The nanoformulation was successful in modulating proapoptotic factors and eventually helped in better survival of the treated animals. CONCLUSION: Our data demonstrate the efficacy of systemically administered siRNA in the treatment of experimental liver cancer. This novel therapeutic strategy may be applicable to a broad range of cancers in patients with the obstinate form of the disease.