Regulation of gene expression by modulating microRNAs through Epigallocatechin-3-gallate in cancer.

Cancer is an intricate ailment that has a higher death rate globally and is characterized by aberrant cell proliferation and metastasis in nature. Since the beginning of healthcare, natural products, especially those derived from plants, have been utilized to support human health. Green tea contains an essential catechin called epigallocatechin gallate, which has anti-proliferative, anti-mutagenic, anti-inflammatory, and antioxidative properties. The anticancer properties of EGCG have been extensively studied using pre-clinical cell culture and animal model systems. Dysregulated miRNA may be a biomarker since it influences the different characteristics of cancer like upholding proliferative signaling, cell death, invasiveness, metastasis, and angiogenesis. EGCG either elevates or lowers the expression of dysregulated miRNAs in cancer. Nonetheless, due to its anticancer properties, greater attention has been paid towards the development of efficient strategies for utilizing EGCG in cancer chemotherapy. This review summarizes the modifying effect of EGCG on miRNAs in cancer after briefly discussing the anticancer mechanisms of EGCG and the function of miRNAs in cancer.

Multifaceted plant growth-promoting traits of indigenous rhizospheric microbes against Phomopsis theae, a causal agent of stem canker in tea plants.

Phomopsis canker is one of the major devastating stem diseases that occur in tea plants caused by the fungal pathogen Phomopsis theae. Rapid development of this disease leads to a capital loss in the tea industry which demands an ecofriendly disease management strategy to control this aggressive pathogen. A total of 245 isolates were recovered from the tea rhizosphere and screened for in vitro plant growth promoting (PGP) traits and antagonism against P. theae. Among them, twelve isolates exhibited multifarious PGP traits including phytohormones, siderophore, hydrogen cyanide, salicylic acid production, phosphate solubilization, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, and antifungal activity. In vitro studies on morphological, biochemical, and phylogenetic analyses classified the selected isolates as Pseudomonas fluorescens (VPF5), Bacillus subtilis (VBS3), Streptomyces griseus (VSG4) and Trichoderma viride (VTV7). Specifically, P. fluorescens VPF5 and B. subtilis VBS3 strains showed the highest level of PGP activities. On the other hand, VBS3 and VTV7 strains showed higher biocontrol efficacy in inhibiting mycelia growth and spore germination of P. theae. A detailed investigation on hydrolytic enzymes produced by antagonistic strains, which degrade the fungus cell wall, revealed that highest amount of chitinase and ß-1,3- glucanase in VTV7 and VBS3 strains. Further, the key antifungal secondary metabolites from these biocontrol agents associated with suppression of P. theae were identified using gas chromatography mass spectrometry. The above study clearly recognized the specific traits in the isolated microbes, which make them good candidates as plant growth-promoting rhizobacteria (PGPR) and biocontrol agents to improve plant growth and health. However, greenhouse trials and field application of these beneficial microbes is required to further confirm their efficacy for the management of stem canker in tea cultivation.

Theaflavin-Containing Black Tea Extract: A Potential DNA Methyltransferase Inhibitor in Human Colon Cancer Cells and Ehrlich Ascites Carcinoma-Induced Solid Tumors in Mice.

Tea is the most popularly consumed beverage in the world. Theaflavin and thearubigins are the key bioactive compounds of black tea that have anticarcinogenic properties as reported in several studies. However, the epigenetic potential of these compounds has not yet been explored. DNA methyltransferase (DNMT) enzymes induce methylation of DNA at cytosine residues and play a significant role in epigenetic regulation and cancer therapy. The present study has explored the role of black tea as a DNMT inhibitor in the prevention of cancer. Herein, the effect of theaflavin has been studied in colon cancer cell line (HCT-116) and EAC-induced solid tumors in mice. It was found that theaflavin prevented cell proliferation and inhibited tumor progression as well. In silico study showed that theaflavin interacted with DNMT1 and DNMT3a enzymes and blocked their activity. Theaflavin also decreased DNMT activity In Vitro and In Vivo as evident from the DNMT activity assay. Results of immunohistochemistry revealed that theaflavin reduced DNMT expression in the tumors of mice. Taken together, our findings showed that theaflavin has a potential role as a DNMT inhibitor in HCT-116 cell line and EAC induced solid tumors in mice.

Synergistic effect of cytokinin and gibberellins stimulates release of dormancy in tea (Camellia sinensis (L.) O. Kuntze) bud.

Reactivation of dormant meristem in banjhi (dormant) shoots is important to enhance the quality and quantity of tea production. The field grown tea bushes were subjected to treatment with dormancy breaking agents such as potassium nitrate (KNO3), thiourea, sodium nitro prusside (SNP), the phytohormones kinetin (Kn) and gibberellins (GA). The efficacy of Kn and GA were comparatively lesser than KNO3 while the combination of Kn and GA (50 and100 ppm respectively) resulted in better dormancy reduction in tea buds. This observation was supported by our results from gene expression study where accumulation patterns of mRNAs corresponding to histones (H2A, H2B, H3 and H4), cyclins (B2, D1 and D3), cyclin-dependent kinase (CDKA), ubiquitination enzymes (FUS, EXT CE2), cyclophilin, E2F, and tubulin were analyzed during growth-dormancy cycles in tea apical buds under the influence of Kn, GA and their combinations. The level of these mRNAs was low in dormant buds, which was significantly increased by foliar application of GA and Kn combination. The present study indicated that the foliar application of GA in combination with Kn will help to improve quality and quantity of tea production by breaking dormancy and stimulating the bud growth.

Pharmacokinetic, toxicokinetic, and bioavailability studies of epigallocatechin-3-gallate loaded solid lipid nanoparticle in rat model.

Epigallocatechin-3-gallate (EGCG), derived from green tea, is an active phytochemical against many types of cancer, cardiovascular, neurological and inflammatory diseases. However, its pharmaceutical activity is limited due to low bioavailability and chemical instability. To overcome these limitations, we fabricated spherical, EGCG loaded solid lipid nanoparticles (SLN-EGCG) as an oral delivery system. The SLN-EGCG showed a hydrodynamic diameter of 300.2 ± 3.8 nm with the drug encapsulation efficiency of 81 ± 1.4%. Additionally, a slow and sustained release of EGCG was noted. Mathematical modeling of release kinetic data suggested that the SLN-EGCG followed the Higuchi model and released EGCG via fickian diffusion method. The data on pharmacokinetic parameters indicated significantly improved bioavailability and protection of EGCG from degradation due to encapsulation into SLN. The SLN-EGCG did not show any acute or sub-chronic toxicity when compared with free EGCG in the rat model. Together these data supported the hypothesis that SLN-EGCG is capable of enhancing the bioavailability and stability of EGCG and can be used as an alternative system for oral administration of EGCG.

Next-Generation Sequencing Reveals the Role of Epigallocatechin-3-Gallate in Regulating Putative Novel and Known microRNAs Which Target the MAPK Pathway in Non-Small-Cell Lung Cancer A549 Cells.

Lung cancer constitutes 85% of non-small cell lung cancer diagnosed cases. MicroRNAs are novel biomarkers that are capable of modulating multiple oncogenic pathways. Epigallocatechin-3-gallate (EGCG) is a potent chemopreventive and chemotherapeutic agent for cancer. We aimed to identify important known and putative novel microRNAs modulated by EGCG in A549 cells using next-generation sequencing and identify their gene targets. Preliminary analysis revealed an IC50 value of 309 µM with G0/G1 phase arrest at 40 µM EGCG treatment. MicroRNA profiling identified 115 known and 4 putative novel microRNAs in 40 µM and 134 known and 3 putative novel microRNAs in 100 µM EGCG-treated A549 cells. The top 10 up-expressed microRNAs were similar between the untreated control and EGCG-treated A549 cells. An up-expression in oncogenic microRNAs, which belong to broadly conserved seed families, were observed in untreated control and EGCG-treated A549 cells. Kyoto Encyclopedia of Genes and Genomes and Protein Analysis Through Evolutionary Relationships pathway analyses of the validated microRNA targeting genes strengthened the hypothesis that EGCG treatment can modulate microRNAs that play a significant role in the MAPK signaling pathway. Expression profile of microRNAs was validation by quantitative real time PCR of randomly selected microRNAs. This study identified signature microRNAs that can be used as novel biomarkers for lung cancer diagnosis.

Silicon alleviates arsenic-induced toxicity in wheat through vacuolar sequestration and ROS scavenging.

Arsenic (As) is a phytotoxic element causing health hazards. This work investigates whether and how silicon (Si) alleviates As toxicity in wheat. The addition of Si under As-stress significantly improved morphophysiological characteristics, total protein, and membrane stability compared to As-stressed plants, suggesting that Si does have critical roles in As detoxification in wheat. Analysis of arsenate reductase activity and phytosiderophore (PS) release reveals their no involvement in the Si-mediated alleviation of As in wheat. Furthermore, Si supplementation in As-stressed plants showed a significant increase of As in roots but not in shoots compared with the plants grown under As stress. Further, gene expression analysis of two chelating molecules, TaPCS1 (phytochelatin synthase) and TaMT1 (metallothionein synthase) showed significant induction due to Si application under As stress compared with As-stressed plants. It is consistent with the physiological observations and suggests that alleviation of As toxicity in rice might be associated with As sequestration in roots leading to reduced As translocation in shoots. Furthermore, increased catalase, peroxidase, and glutathione reductase activities in roots imply the active involvement of reactive oxygen species scavenging for protecting wheat plants from As-induced oxidative injury. The study provides mechanistic evidence on the beneficial effect of Si on As toxicity in wheat plants.

Biochemical and molecular studies on the resistance mechanisms in tea [Camellia sinensis (L.) O. Kuntze] against blister blight disease.

Tea (Camellia sinensis) plantations are exposed to biotic and abiotic stresses. Among the biotic factors, blister blight (BB), caused by Exobasidium vexans, affects the quality and quantity of the product and demands high fungicide application. A long term solution for disease resistance would require the knowledge of the basic molecular and biochemical changes occurring in plant as an attempt to resist the pathogen and limit the spread of the disease which can further help in developing resistant cultivars using biotechnological tools. Thus, gene expression studies using the cDNA based suppressive subtractive hybridization library, characterization of genes for pathogenesis related (PR) proteins [chitinase (CsCHIT), glucanase (CsGLUC), phenylalanine ammonia lyase (CsPAL)] and genes in flavonoid pathway were accessed in the BB resistant and susceptible cultivars, SA6 and TES34, respectively. Further, biochemical analysis of PR and antioxidant enzymes (POX, APX, SOD) involved in BB resistance have been carried out to investigate the potential molecular and biochemical changes. Various stages of pathogen development had varied impact on PR protein, flavonoid pathway and anti-oxidative enzymes and indicates the possible role of reactive oxygen species, lignins, flavonoids, anthocyanins and other synthesized compounds in acting as antimicrobial/antifungal agents in tea cultivars.

Potential neuroprotective properties of epigallocatechin-3-gallate (EGCG).

Neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) enforce an overwhelming social and economic burden on society. They are primarily characterized through the accumulation of modified proteins, which further trigger biological responses such as inflammation, oxidative stress, excitotoxicity and modulation of signalling pathways. In a hope for cure, these diseases have been studied extensively over the last decade to successfully develop symptom-oriented therapies. However, so far no definite cure has been found. Therefore, there is a need to identify a class of drug capable of reversing neural damage and preventing further neural death. This review therefore assesses the reliability of the neuroprotective benefits of epigallocatechin-gallate (EGCG) by shedding light on their biological, pharmacological, antioxidant and metal chelation properties, with emphasis on their ability to invoke a range of cellular mechanisms in the brain. It also discusses the possible use of nanotechnology to enhance the neuroprotective benefits of EGCG.

Comparative genome analysis of Lysinibacillus B1-CDA, a bacterium that accumulates arsenics.

Previously, we reported an arsenic resistant bacterium Lysinibacillus sphaericus B1-CDA, isolated from an arsenic contaminated lands. Here, we have investigated its genetic composition and evolutionary history by using massively parallel sequencing and comparative analysis with other known Lysinibacillus genomes. Assembly of the sequencing reads revealed a genome of ~4.5 Mb in size encompassing ~80% of the chromosomal DNA. We found that the set of ordered contigs contains abundant regions of similarity with other Lysinibacillus genomes and clearly identifiable genome rearrangements. Furthermore, all genes of B1-CDA that were predicted be involved in its resistance to arsenic and/or other heavy metals were annotated. The presence of arsenic responsive genes was verified by PCR in vitro conditions. The findings of this study highlight the significance of this bacterium in removing arsenics and other toxic metals from the contaminated sources. The genetic mechanisms of the isolate could be used to cope with arsenic toxicity.

Elevated concentrations of serum matrix metalloproteinase-2 and -9 and their associations with circulating markers of cardiovascular diseases in chronic arsenic-exposed individuals.

BACKGROUND: Cardiovascular diseases (CVDs) and cancers are the major causes of chronic arsenic exposure-related morbidity and mortality. Matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) are deeply involved in the pathogenesis of CVDs and cancers. This study has been designed to evaluate the interactions of arsenic exposure with serum MMP-2 and MMP-9 concentrations especially in relation to the circulating biomarkers of CVDs. METHODS: A total of 373 human subjects, 265 from arsenic-endemic and 108 from non-endemic areas in Bangladesh were recruited for this study. Arsenic concentrations in the specimens were measured by inductively coupled plasma mass spectroscopy (ICP-MS) and serum MMPs were quantified by immunoassay kits. RESULTS: Serum MMP-2 and MMP-9 concentrations in arsenic-endemic population were significantly (p < 0.001) higher than those in non-endemic population. Both MMPs showed significant positive interactions with drinking water (r s = 0.208, p < 0.001 for MMP-2; r s = 0.163, p < 0.01 for MMP-9), hair (r s = 0.163, p < 0.01 for MMP-2; r s = 0.173, p < 0.01 for MMP-9) and nail (r s = 0.160, p < 0.01 for MMP-2; r s = 0.182, p < 0.001 for MMP-9) arsenic of the study subjects. MMP-2 concentrations were 1.02, 1.03 and 1.05 times, and MMP-9 concentrations were 1.03, 1.06 and 1.07 times greater for 1 unit increase in log-transformed water, hair and nail arsenic concentrations, respectively, after adjusting for covariates (age, sex, BMI, smoking habit and hypertension). Furthermore, both MMPs were increased dose-dependently when the study subjects were split into three (≤10, 10.1-50 and > 50 µg/L) groups based on the regulatory upper limit of water arsenic concentration set by WHO and Bangladesh Government. MMPs were also found to be significantly (p < 0.05) associated with each other. Finally, the concentrations of both MMPs were correlated with several circulating markers related to CVDs. CONCLUSIONS: This study showed the significant positive associations and dose-response relationships of arsenic exposure with serum MMP-2 and MMP-9 concentrations. This study also showed the interactions of MMP-2 and MMP-9 concentrations with the circulating markers of CVDs suggesting the MMP-2 and MMP-9 -mediated mechanism of arsenic-induced CVDs.

Bioremediation of hexavalent chromium (VI) by a soil-borne bacterium, Enterobacter cloacae B2-DHA.

Chromium and chromium containing compounds are discharged into the nature as waste from anthropogenic activities, such as industries, agriculture, forest farming, mining and metallurgy. Continued disposal of these compounds to the environment leads to development of various lethal diseases in both humans and animals. In this paper, we report a soil borne bacterium, B2-DHA that can be used as a vehicle to effectively remove chromium from the contaminated sources. B2-DHA is resistant to chromium with a MIC value of 1000 µg mL(-1) potassium chromate. The bacterium has been identified as a Gram negative, Enterobacter cloacae based on biochemical characteristics and 16S rRNA gene analysis. TOF-SIMS and ICP-MS analyses confirmed intracellular accumulation of chromium and thus its removal from the contaminated liquid medium. Chromium accumulation in cells was 320 µg/g of cells dry biomass after 120-h exposure, and thus it reduced the chromium concentration in the liquid medium by as much as 81%. Environmental scanning electron micrograph revealed the effect of metals on cellular morphology of the isolates. Altogether, our results indicate that B2-DHA has the potential to reduce chromium significantly to safe levels from the contaminated environments and suggest the potential use of this bacterium in reducing human exposure to chromium, hence avoiding poisoning.

Isolation and characterization of a Lysinibacillus strain B1-CDA showing potential for bioremediation of arsenics from contaminated water.

The main objective of this study was to identify and isolate arsenic resistant bacteria that can be used for removing arsenic from the contaminated environment. Here we report a soil borne bacterium, B1-CDA that can serve this purpose. B1-CDA was isolated from the soil of a cultivated land in Chuadanga district located in the southwest region of Bangladesh. The morphological, biochemical and 16S rRNA analysis suggested that the isolate belongs to Lysinibacillus sphaericus. The minimum inhibitory concentration (MIC) value of the isolate is 500 mM (As) as arsenate. TOF-SIMS and ICP-MS analysis confirmed intracellular accumulation and removal of arsenics. Arsenic accumulation in cells amounted to 5.0 mg g(-1) of the cells dry biomass and thus reduced the arsenic concentration in the contaminated liquid medium by as much as 50%. These results indicate that B1-CDA has the potential for remediation of arsenic from the contaminated water. We believe the benefits of implementing this bacterium to efficiently reduce arsenic exposure will not only help to remove one aspect of human arsenic poisoning but will also benefit livestock and native animal species. Therefore, the outcome of this research will be highly significant for people in the affected area and also for human populations in other countries that have credible health concerns as a consequence of arsenic-contaminated water.

Role of epigallocatechin-3- gallate in the regulation of known and novel microRNAs in breast carcinoma cells.

Breast cancer comprises 30% of all cancer cases among the world's women population. MicroRNAs are small, endogenous, non-coding RNAs that regulate cell proliferating and apoptotic pathways by modulating expressions of related genes. Phytochemicals like epigallocatechin-3-gallate (EGCG) are known to have a chemotherapeutic effect on cancer often through the regulation of microRNAs. The aim is to find out the key known and novel miRNAs, which are controlled by EGCG in breast cancer cell line MDA-MB-231. Next-generation sequencing (NGS) revealed 1,258 known and 330 novel miRNAs from untreated and 83 µM EGCG (IC50 value of EGCG) treated cells. EGCG modulated 873 known and 47 novel miRNAs in the control vs. treated sample. The hypothesis of EGCG being a great modulator of miRNAs that significantly control important cancer-causing pathways has been established by analyzing with Kyoto Encyclopedia of Genes and Genomes (KEGG) and Protein Analysis Through Evolutionary Relationships (PANTHER) database. Validation of known and novel miRNA expression differences in untreated vs. treated cells was done using qPCR. From this study, a few notable miRNAs were distinguished that can be used as diagnostics as well as prognostic markers for breast cancer.

Lysinibacillus sphaericus mediates stress responses and attenuates arsenic toxicity in Caenorhabditis elegans.

Exposure to toxic metals alters host response and that leads to disease development. Studies have revealed the effects of metals on microbial physiology, however, the role of metal resistant bacteria on host response to metals is unclear. The hypothesis that xenobiotic interactions between gut microbes and arsenic influence the host physiology and toxicity was assessed in a Caenorhabditis elegans model. The arsenic-resistant Lysinibacillus sphaericus B1CDA was fed to C. elegans to determine the host responses to arsenic in comparison to Escherichia coli OP50 food. L. sphaericus diet extended C. elegans lifespan compared to E. coli diet, with an increased expression of genes involved in lifespan, stress response and immunity (hif-1, hsp-16.2, mtl-2, abf-2, clec-60), as well as reduced fat accumulation. Arsenic-exposed worms fed L. sphaericus also had a longer lifespan than those fed E. coli and had an increased expression of genes involved in cytoprotection, stress resistance (mtl-1, mtl-2) and oxidative stress response (cyp-35A2, isp-1, ctl-2, sod-1), together with a decreased accumulation of reactive oxygen species (ROS). In comparison with E. coli, L. sphaericus B1CDA diet increased C. elegans fitness while detoxifying arsenic induced ROS and extending lifespan.

Dose-response relationship between arsenic exposure and the serum enzymes for liver function tests in the individuals exposed to arsenic: a cross sectional study in Bangladesh.

BACKGROUND: Chronic arsenic exposure has been shown to cause liver damage. However, serum hepatic enzyme activity as recognized on liver function tests (LFTs) showing a dose-response relationship with arsenic exposure has not yet been clearly documented. The aim of our study was to investigate the dose-response relationship between arsenic exposure and major serum enzyme marker activity associated with LFTs in the population living in arsenic-endemic areas in Bangladesh. METHODS: A total of 200 residents living in arsenic-endemic areas in Bangladesh were selected as study subjects. Arsenic concentrations in the drinking water, hair and nails were measured by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). The study subjects were stratified into quartile groups as follows, based on concentrations of arsenic in the drinking water, as well as in subjects' hair and nails: lowest, low, medium and high. The serum hepatic enzyme activities of alkaline phosphatase (ALP), aspartate transaminase (AST) and alanine transaminase (ALT) were then assayed. RESULTS: Arsenic concentrations in the subjects' hair and nails were positively correlated with arsenic levels in the drinking water. As regards the exposure-response relationship with arsenic in the drinking water, the respective activities of ALP, AST and ALT were found to be significantly increased in the high-exposure groups compared to the lowest-exposure groups before and after adjustments were made for different covariates. With internal exposure markers (arsenic in hair and nails), the ALP, AST and ALT activity profiles assumed a similar shape of dose-response relationship, with very few differences seen in the higher groups compared to the lowest group, most likely due to the temporalities of exposure metrics. CONCLUSIONS: The present study demonstrated that arsenic concentrations in the drinking water were strongly correlated with arsenic concentrations in the subjects' hair and nails. Further, this study revealed a novel exposure- and dose- response relationship between arsenic exposure metrics and serum hepatic enzyme activity. Elevated serum hepatic enzyme activities in the higher exposure gradients provided new insights into arsenic-induced liver toxicity that might be helpful for the early prognosis of arsenic-induced liver diseases.

Association between arsenic exposure and plasma cholinesterase activity: a population based study in Bangladesh.

BACKGROUND: Arsenic is a potent pollutant that has caused an environmental catastrophe in certain parts of the world including Bangladesh where millions of people are presently at risk due to drinking water contaminated by arsenic. Chronic arsenic exposure has been scientifically shown as a cause for liver damage, cancers, neurological disorders and several other ailments. The relationship between plasma cholinesterase (PChE) activity and arsenic exposure has not yet been clearly documented. However, decreased PChE activity has been found in patients suffering liver dysfunction, heart attack, cancer metastasis and neurotoxicity. Therefore, in this study, we evaluated the PChE activity in individuals exposed to arsenic via drinking water in Bangladesh. METHODS: A total of 141 Bangladeshi residents living in arsenic endemic areas with the mean arsenic exposure of 14.10 +/- 3.27 years were selected as study subjects and split into tertile groups based on three water arsenic concentrations: low (< 129 microg/L), medium (130-264 microg/L) and high (> 265 microg/L). Study subjects were further sub-divided into two groups ( 50 microg/L) based on the recommended upper limit of water arsenic concentration (50 microg/L) in Bangladesh. Blood samples were collected from the study subjects by venipuncture and arsenic concentrations in drinking water, hair and nail samples were measured by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). PChE activity was assayed by spectrophotometer. RESULTS: Arsenic concentrations in hair and nails were positively correlated with the arsenic levels in drinking water. Significant decreases in PChE activity were observed with increasing concentrations of arsenic in water, hair and nails. The average levels of PChE activity in low, medium and high arsenic exposure groups were also significantly different between each group. Lower levels of PChE activity were also observed in the > 50 microg/L group compared to the

Glutathione Restores Hg-Induced Morpho-Physiological Retardations by Inducing Phytochelatin and Oxidative Defense in Alfalfa.

Mercury (Hg) is toxic to plants, but the effect of glutathione in Hg alleviation was never studied in alfalfa, an important forage crop. In this study, Hg toxicity showed morphological retardation, chlorophyll reduction, and PSII inefficiency, which was restored due to GSH supplementation in alfalfa plants treated with Hg. Results showed a significant increase of Hg, but Fe and S concentrations substantially decreased in root and shoot accompanied by the downregulation of Fe (MsIRT1) and S (MsSultr1;2 and MsSultr1;3) transporters in roots of Hg-toxic alfalfa. However, GSH caused a significant decrease of Hg in the shoot, while the root Hg level substantially increased, accompanied by the restoration of Fe and S status, relative to Hg-stressed alfalfa. The subcellular analysis showed a substantial deposition of Hg in the root cell wall accompanied by the increased GSH and PC and the upregulation of MsPCS1 and MsGSH1 genes in roots. It suggests the involvement of GSH in triggering PC accumulation, causing excess Hg bound to the cell wall of the root, thereby reducing Hg translocation in alfalfa. Bioinformatics analysis showed that the MsPCS1 protein demonstrated one common conserved motif linked to the phytochelatin synthase domain (CL0125) with MtPCS1 and AtMCS1 homologs. These in silico analysis further confirmed the detoxification role of MsPCS1 induced by GSH in Hg-toxic alfalfa. Additionally, GSH induces GSH and GR activity to counteract oxidative injuries provoked by Hg-induced H2O2 and lipid peroxidation. These findings may provide valuable knowledge to popularize GSH-derived fertilizer or to develop Hg-free alfalfa or other forage plants.

Molecular profiling of multidrug-resistant river water isolates: insights into resistance mechanism and potential inhibitors.

Polluted waters are an important reservoir for antibiotic resistance genes and multidrug-resistant bacteria. This report describes the microbial community, antibiotic resistance genes, and the genetic profile of extended spectrum ß-lactamase strains isolated from rivers at, Pune, India. ESBL-producing bacteria isolated from diverse river water catchments running through Pune City were characterized for their antibiotic resistance. The microbial community and types of genes which confer antibiotic resistance were identified followed by the isolation of antibiotic-resistant bacteria on selective media and their genome analysis. Four representative isolates were sequenced using next generation sequencing for genomic analysis. They were identified as Pseudomonas aeruginosa, Escherichia coli, and two isolates were Enterobacter cloacae. The genes associated with the multidrug efflux pumps, such as tolC, macA, macB, adeL, and rosB, were detected in the isolates. As MacAB-TolC is an ABC type efflux pump responsible for conferring resistance in bacteria to several antibiotics, potential efflux pump inhibitors were identified by molecular docking. The homology model of their MacB protein with that from Escherichia coli K12 demonstrated structural changes in different motifs of MacB. Molecular docking of reported efflux pump inhibitors revealed the highest binding affinity of compound MC207-110 against MacB. It also details the potential efflux pump inhibitors that can serve as possible drug targets in drug development and discovery.

AtMBD8 is involved in control of flowering time in the C24 ecotype of Arabidopsis thaliana.

The Arabidopsis thaliana accession C24 is a vernalization-responsive, moderately late flowering ecotype. We report that a mutation in AtMBD8, which encodes a protein with a putative Methyl-CpG-Binding Domain (MBD), in C24 background, results in a delay in flowering time during both long and short days. The atmbd8-1 mutant responded to vernalization as wild type (wt) plants. Consistent with a role in modulation of flowering time, an AtMBD8::GUS-reporter construct was expressed in the shoot meristem region and developing leaves. Full-genome transcriptional profiling revealed very few changes in gene expression between atmbd8-1 and wt plants. The expression level of FLC, the major repressor of transition to flowering, was unchanged in atmbd8-1, and in accordance with that, genes upstream of FLC were unaffected by the mutation. The expression level of CONSTANS, involved in photoperiodic control of flowering, was very similar in atmbd8-1 and wt plants. In contrast, the major promoters of flowering, FT and SOC1, were both downregulated. As FT is a regulator of SOC1, we conclude that AtMBD8 is a novel promoter of flowering that acts upstream of FT in the C24 accession. In contrast to atmbd8-1, the Colombia (Col) SALK T-DNA insertion line, atmbd8-2, did not display a delayed transition to flowering. Transcriptional profiling revealed that a substantial number of genes were differentially expressed between C24 and Col wt seedlings. Several of these genes are also differentially expressed in late flowering mutants. We suggest that these differences contribute to the contrasting effect of a mutation in AtMBD8 in the two ecotypes.