Phytochemical profiling and nephroprotective potential of ethanolic leaf extract of Polyalthia longifolia against cisplatin-induced oxidative stress in rat model.

ETHNOPHARMACOLOGICAL RELEVANCE: Kidney problems are becoming more common globally and are considered a major health issue in the modern world with high mortality rate. Polyalthia longifolia (Sonn.) Thwaites is a tropical ethnomedicinal plant used to treat various diseases like diabetes, hypertension and urinary disorders and possess antioxidant and anti-inflammatory properties. AIM OF THE STUDY: This study aimed to investigate the phytochemical composition of 70% ethanolic leaf extract of Polyalthia longifolia (Sonn.) Thwaites (PL) and evaluates its nephroprotective effects against cisplatin-induced nephrotoxicity in Wistar rats. MATERIALS AND METHODS: The leaves of PL were extracted with 70% ethanol and performed the phytochemical profiling using Liquid Chromatography-Mass Spectrometry (LC-MS). The nephroprotective effect of PL leaf extract was evaluated at three doses (150, 300 and 600 mg/kg, p.o.) for 14 days against cisplatin toxicity (16 mg/kg, i.p., once) in male Wistar rats. Body and kidney weight indices, kidney function markers and lipid profile markers in serum, and oxidative stress markers in kidney tissue were performed along with the histopathological analysis of kidney. RESULTS: The LC-MS chromatograph confirmed the presence of various phytocompounds include N-Methylhernagine (aporphine alkaloid), 4-Acetamidobutanoic acid (gamma amino acid) and choline, etc. in the PL leaf extract. Exposure of cisplatin (16 mg/kg, i.p., once only) to the animals significantly elevated the levels of kidney functional markers (i.e. serum urea, uric acid, creatinine) and the lipid markers (triglyceride and total cholesterol) in blood circulation with depletion of serum albumin which were reversed by the therapy of PL leaf extract (150, 300 and 600 mg/kg) in dose-dependent manner. The altered level of body and kidney weight in cisplatin treated group was also restored by the therapy. PL leaf extract effectively improved the antioxidant defense system of kidney at all doses by restoring the levels of tissue glutathione, superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase with the dose-dependent reduction of lipid peroxidation against cisplatin-induced renal oxidative stress. The histopathological observations also showed the significant recovery in cellular morphology after PL treatment when compared to the cisplatin toxicity group. The highest dose 600 mg/kg of PL leaf extract showed more pronounced renal recovery (p < 0.001) followed by other two doses, which was similar to the silymarin treatment group (a reference drug) against nephrotoxicity. CONCLUSION: The results of this study revealed the nephroprotective effects of PL leaves against cisplatin-induced nephrotoxicity by reversing the level of biochemical markers and mitigating oxidative stress as well as improving the architecture of renal tissues. This renal protection by PL might be due to the synergistic effect of its phytoconstituents and antioxidant efficacy.

Cereblon-based Bifunctional Degrader of SOS1, BTX-6654, Targets Multiple KRAS Mutations and Inhibits Tumor Growth.

Mutations within the oncogene KRAS drive an estimated 25% of all cancers. Only allele-specific KRAS G12C inhibitors are currently available and are associated with the emergence of acquired resistance, partly due to upstream pathway reactivation. Given its upstream role in the activation of KRAS, son of sevenless homolog 1 (SOS1), has emerged as an attractive therapeutic target. Agents that target SOS1 for degradation could represent a potential pan-KRAS modality that may be capable of circumventing certain acquired resistance mechanisms. Here, we report the development of two SOS1 cereblon-based bifunctional degraders, BTX-6654 and BTX-7312, cereblon-based bifunctional SOS1 degraders. Both compounds exhibited potent target-dependent and -specific SOS1 degradation. BTX-6654 and BTX-7312 reduced downstream signaling markers, pERK and pS6, and displayed antiproliferative activity in cells harboring various KRAS mutations. In two KRAS G12C xenograft models, BTX-6654 degraded SOS1 in a dose-dependent manner correlating with tumor growth inhibition, additionally exhibiting synergy with KRAS and MEK inhibitors. Altogether, BTX-6654 provided preclinical proof of concept for single-agent and combination use of bifunctional SOS1 degraders in KRAS-driven cancers.

Ameliorative impact of herbal formulation -Majoon-Dabeed-ul-ward and Sharbat-e-Deenar against CCl4 induced liver toxicity via regulation of antioxidant enzymes and oxidative stress.

Polyherbal Unani formulations have been used in the treatment of liver diseases for a long time. (Ibrahim M, Khaja MN, Aara A, Khan AA, Habeeb MA, Devi YP, Narasu ML, Habibullah CM. Hepatoprotective activity of Sapindus mukorossi and Rheum emodi extracts: in vitro and in vivo studies. World J Gastroenterol. 2008:14:2566-2571.) The aim of the present study was to investigate comparative hepatoprotective potential of Majoon-e-Dabeed-ul-ward (MD) and Sharbat-e-Deenar (SD) against CCl4 induced subchronic hepatic toxicity. In vivo study, albino rats were divided into 5 groups. Group I was control; Group II was experimental control treated with CCl4 (0.15 mL/kg, i.p. for 21 days); Groups III-IV treated with SD (2 mL/kg, p.o.) and MD (1,000 mg/kg, p.o.) for 5 days following CCl4 intoxication as in group 2 respectively; and Group V was positive control treated with silymarin (50 mg/kg, p.o.). In vitro hepatoprotective activity of SD and MD (25, 50, and 100 µg/mL) was assessed by SRB assay and flow cytometry analysis. CCl4 exposure significantly elevated the release of hepatic enzymes i.e. AST, ALT, LDH, and SALP in serum and lipid peroxidation in liver tissue which all these parameters were reversed after SD and MD administration. Therapy for 5 days also normalized the levels of antioxidant enzymes i.e. catalase, SOD, GPx, GR, tissue GSH, and aniline hydroxylase in CCl4 treated group. DNA damage and histological alterations caused by CCl4 were restored towards normal group. In vitro study showed protective effect of SD and MD against CCl4 treated HepG2 cell lines and rat hepatocytes. The results suggested that MD has a significant hepatoprotective potential and regulatory effect on oxidative stress than SD against CCl4 induced hepatotoxicity, and that this effect may be related to its antioxidant activity.

Small molecule allosteric inhibitors of RORγt block Th17-dependent inflammation and associated gene expression in vivo.

Retinoic acid receptor-related orphan nuclear receptor (ROR) γt is a member of the RORC nuclear hormone receptor family of transcription factors. RORγt functions as a critical regulator of thymopoiesis and immune responses. RORγt is expressed in multiple immune cell populations including Th17 cells, where its primary function is regulation of immune responses to bacteria and fungi through IL-17A production. However, excessive IL-17A production has been linked to numerous autoimmune diseases. Moreover, Th17 cells have been shown to elicit both pro- and anti-tumor effects. Thus, modulation of the RORγt/IL-17A axis may represent an attractive therapeutic target for the treatment of autoimmune disorders and some cancers. Herein we report the design, synthesis and characterization of three selective allosteric RORγt inhibitors in preclinical models of inflammation and tumor growth. We demonstrate that these compounds can inhibit Th17 differentiation and maintenance in vitro and Th17-dependent inflammation and associated gene expression in vivo, in a dose-dependent manner. Finally, RORγt inhibitors were assessed for efficacy against tumor formation. While, RORγt inhibitors were shown to inhibit tumor formation in pancreatic ductal adenocarcinoma (PDAC) organoids in vitro and modulate RORγt target genes in vivo, this activity was not sufficient to delay tumor volume in a KP/C human tumor mouse model of pancreatic cancer.

Targeting transcriptional coregulator OCA-B/Pou2af1 blocks activated autoreactive T cells in the pancreas and type 1 diabetes.

The transcriptional coregulator OCA-B promotes expression of T cell target genes in cases of repeated antigen exposure, a necessary feature of autoimmunity. We hypothesized that T cell-specific OCA-B deletion and pharmacologic OCA-B inhibition would protect mice from autoimmune diabetes. We developed an Ocab conditional allele and backcrossed it onto a diabetes-prone NOD/ShiLtJ strain background. T cell-specific OCA-B loss protected mice from spontaneous disease. Protection was associated with large reductions in islet CD8+ T cell receptor specificities associated with diabetes pathogenesis. CD4+ clones associated with diabetes were present but associated with anergic phenotypes. The protective effect of OCA-B loss was recapitulated using autoantigen-specific NY8.3 mice but diminished in monoclonal models specific to artificial or neoantigens. Rationally designed membrane-penetrating OCA-B peptide inhibitors normalized glucose levels and reduced T cell infiltration and proinflammatory cytokine expression in newly diabetic NOD mice. Together, the results indicate that OCA-B is a potent autoimmune regulator and a promising target for pharmacologic inhibition.

Biological attenuation of arsenic and nitrate in a suspended growth denitrifying-sulphidogenic bioreactor and stability check of arsenic-laden biosolids.

Co-occurrence of arsenic and nitrate in groundwater sources at a wide range of concentrations is reported. In this work, performance of suspended growth semi-batch reactor was assessed for co-removal of arsenic and nitrate from simulated groundwater to meet the drinking water standards in the absence of iron. The bioreactor was inoculated with mixed bacterial culture and operated in the absence of oxygen for more than 450 days under varying influent arsenate (200-800 µg/L), nitrate concentrations (50-250 mg/L), and hydraulic retention time of 3-6 days. Complete nitrate removal was observed at all tested concentrations. Arsenic removal was found to meet drinking water standards from initial concentrations and up to 600 µg/L. The extended toxicity characteristic leaching procedure leaching experiments indicated that arsenic-laden biosolids would not constitute a hazardous waste. The arsenic leaching was found to increase with an increase in dissolved oxygen and the final leachate concentrations of arsenic were below 150 µg/L. The leaching experiments suggested maintaining non-alkaline conditions for minimum arsenic release from arsenic biosolids formed under sulphidogenic conditions. This study is the first to report that nitrate and arsenic can be simultaneously removed to meet drinking standards in a suspended growth bioreactor.

Bacterially-assisted recovery of cadmium and nickel as their metal sulfide nanoparticles from spent Ni-Cd battery via hydrometallurgical route.

Soaring demand for technology metals (e.g., Cd, Ni) and its ever-depleting primary resources ask for alternative recovery from secondary sources. Ni-Cd battery is one such source that can abridge the gap between demand and supply of such metals. Biogenic recovery, being environmentally benign, is explored for Cd and Ni recovery to manage the menace of spent Ni-Cd battery. Studies with 20, 40 and 60 mg/L Cd2+ initial concentrations in batch mode (in triplicates) at pH 7.0 ± 0.2, 30 ± 0.5 °C and 120 rpm were conducted using sulfate-reducing bacteria for 10 days. Analysis of extracellular polymeric substance revealed that protein secretion was enhanced, thereby forming Cd-EPS binding. Biosolids were collected and freeze-dried for morphological analysis viz. FESEM/EDX, PXRD and TEM, which revealed the formation of CdS nanoparticles (JCPDS card #00-042-1411) in range of 2-6 nm. Similarly, combined effect with 5, 10 and 20 mg/L Ni2+ at 20 mg/L Cd2+ were also investigated. Furthermore, to test the efficacy for real field application, spent Ni-Cd battery was dismantled and its powder was characterized, digested with concentrated HCl at 70 °C and was fed in batch mode after cooling, wherein nanoparticles of Ni and Cd sulfides were formed that has potential as semi-conducting material.

A practical approach on reuse of drinking water treatment plant residuals for fluoride removal.

The sustainable management of the voluminous waste from drinking water treatment plants has motivated environmental researchers towards several reuse options. Water treatment residues (WTR) are proven adsorbent for remediation of many water- and soil-borne anions (perchlorate, selenium and arsenic), and may be able to remove fluoride from contaminated water. In this study, the sustainable reuse of the freely available waste of the drinking water treatment plants, namely WTR, was explored for their fluoride removal potential to meet drinking water standards. WTR was characterized by specific surface area, Fourier transform infrared (FT-IR), scanning electron microscopy and X-ray powder diffraction (XRD). Batch adsorption experiments were conducted as a function of WTR dose, contact time, agitation speed, initial fluoride concentration, initial temperature and water pH to get best adsorption capacity. About 90% fluoride removal (from initial 5.0 mg/L) was observed within 2 h contact time at WTR dose of 28 g/L. Also, WTR effectively removed fluoride in the pH range of 5-8, whereas removal efficiency decreased at pH 9 or higher. The adsorption equilibrium was established within 120-150 min. Adsorption isotherm data were best fit to Langmuir (R 2 = 0.984) and Freundlich models (R 2 = 0.983), while adsorption kinetic study exhibited that second-order kinetic model was followed with rate constant of 0.038 g/mg min. The FT-IR and XRD analyses affirmed that the metal hydroxyl and metal oxide groups contributed to the fluoride removal. The experimental results show the promising potential of WTR as an adsorbent in fluoride removal from real contaminated groundwater.

Development of a sulfidogenic bioreactor system for removal of co-existent selenium, iron and nitrate from drinking water sources.

The present study showed for the first time that selenium, iron, and nitrate could be simultaneously removed in a sulfidogenic bioreactor to meet drinking water standards. A bioreactor inoculated with mixed bacterial consortium was operated for around 330 days in anoxic environment at 30 °C under varying combination of influent selenate (200-1000 µg/L as selenium), and iron (3-10 mg/L) in presence of 50 mg/L of nitrate. Required amount of acetic acid (as carbon source) and sulfate were supplied and the reactor was operated at different empty bed contact time (EBCT) of 45-120 min. Along with complete removal of nitrate, the reactor removed both selenium and iron to meet the drinking water standards. Field emission transmission electron microscopy (FETEM) and X-ray diffraction (XRD) analyses confirmed the formation of selenium sulfide (SeS), achavalite (FeSe) and pyrite (FeS2), which were the possible removal mechanisms of selenium and iron. Thus, this study exhibited that selenium, iron, and nitrate can be simultaneously removed to meet the drinking water standards in a sulfidogenic bioreactor.

Transcription factor Oct1 protects against hematopoietic stress and promotes acute myeloid leukemia.

A better understanding of the development and progression of acute myelogenous leukemia (AML) is necessary to improve patient outcome. Here we define roles for the transcription factor Oct1/Pou2f1 in AML and normal hematopoiesis. Inappropriate reactivation of the CDX2 gene is widely observed in leukemia patients and in leukemia mouse models. We show that Oct1 associates with the CDX2 promoter in both normal and AML primary patient samples, but recruits the histone demethylase Jmjd1a/Kdm3a to remove the repressive H3K9me2 mark only in malignant specimens. The CpG DNA immediately adjacent to the Oct1 binding site within the CDX2 promoter exhibits variable DNA methylation in healthy control blood and bone marrow samples, but complete demethylation in AML samples. In MLL-AF9-driven mouse models, partial loss of Oct1 protects from myeloid leukemia. Complete Oct1 loss completely suppresses leukemia but results in lethality from bone marrow failure. Loss of Oct1 in normal hematopoietic transplants results in superficially normal long-term reconstitution; however, animals become acutely sensitive to 5-fluorouracil, indicating that Oct1 is dispensable for normal hematopoiesis but protects blood progenitor cells against external chemotoxic stress. These findings elucidate a novel and important role for Oct1 in AML.

Bio-attenuation of arsenic and iron coupled with nitrate remediation in multi-oxyanionic system: Batch and column studies.

Co-occurrence of arsenic and iron along with nitrate in groundwater makes the trio an onerous combination both in terms of potability and treatment. To meet drinking water guidelines, batch and column laboratory trials were conducted on simulated and bore-well water for attenuation of arsenic (1000 µg/L), iron (5 mg/L) and nitrate (150 mg/L). Increment in sulphate showed a direct individual impact on iron removal, meeting WHO guidelines. The bio-kinetic parameters were in the range of: µmax = 0.079-0.551/d, Ks = 116.18-645.19 mg/L, Kd = 0.0009-0.0077/d and Y = 0.034-0.094 mg MLVSS/mg COD. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) confirmed orpiment precipitation and/or co-precipitation with mackinawite are the key mechanisms for arsenic and iron attenuation. Column experiments were conducted by charging simulated groundwater containing arsenic (500 µg/L), nitrate (50 mg/L), sulphate (25 mg/L) and iron (3 mg/L) in an acetate (105 mg/L as COD) fed flow-through bioreactor at constant empty bed contact time of 60 min. Profile sampling illustrated segregation of different terminal electron accepting zones following thermodynamic yield for sequential removal of different oxyanions. This study showed the importance of considering microbially mediated terminal electron-accepting processes (TEAP) for multi-oxyanion removal in engineered systems.

Effects of backwashing strategy and dissolved oxygen on arsenic removal to meet drinking water standards in a sulfidogenic attached growth reactor.

Efficiency and feasibility of two backwashing methods (water-nitrogen and water-air assisted) on arsenic and its co-pollutants removal were assessed through running a sulfidogenic attached growth reactor (AGR) treating arsenic spiked simulated groundwater for about 600 days. Replacing water with nitrogen assisted backwashing (WNAB) by water with air assisted backwashing (WAAB) introduced dissolved oxygen (DO) as an additional electron acceptor, which required an increased empty bed contact time (EBCT) to retain the entire terminal electron accepting zones (DO, nitrate, arsenate and sulfate) within the reactor. Removal of arsenic to below 10 µg/L required a longer EBCT at higher influent DO in backwash water. Notably, MiSeq sequencing analysis confirmed the presence of diverse bacterial community on biofilm which can utilize multiple terminal electron acceptors present in the bioreactor.

Concurrent removal of nitrate, arsenic and iron from simulated and real-life groundwater to meet drinking water standards: Effects of operational and environmental parameters.

The aim of this work was to study concurrent removal of nitrate, arsenic and iron in an attached growth reactor (AGR) based on bio-sulphidogenesis treating simulated and real-life ground water. A lab-scale bioreactor system was monitored for a period of 511 days under conditions identical to those prevailing at full-scale to assess the relative influence of empty bed contact time (EBCT) (20-90 min), backwash strategies (water-nitrogen and water-air), temperature (20-50 °C), pH (6.6-8.4) and shut down on reactor performance and recovery. Complete removal of nitrate (50 mg/L) and over 95% removal of iron (3 mg/L) occurred. Arsenic removal efficiency was around 99% (500 µg/L) and treated water arsenic concentration was in compliance with the World Health Organization and Indian Standard of 10 µg/L. Port sampling along the depth of bioreactor shows shifting of terminal electron accepting process zones at lower EBCT of 20 min and after air assisted backwashing. The temperature range of 20-50 °C and pH range of 6.6-8.4 were applicable for arsenic removal in natural conditions. Precipitated biosolids were analysed using electron microscopy. Biogenic sulphides resulted in the precipitation of arsenosulphides and iron sulphides, which concurrently removed arsenic and iron. This study suggests that a sulphidogenic bioreactor may help to set the basis for concurrent removal of nitrate, arsenic and iron from real-life groundwater using mixed biofilm bacterial community.

Investigation on stability and leaching characteristics of mixtures of biogenic arsenosulphides and iron sulphides formed under reduced conditions.

Arsenic is removed from aqueous phase through precipitation as arsenosulphides and/or co-precipitation and adsorption on iron sulphides. Studies were carried out to ascertain the stability of reduced biogenic arsenic and iron sulphide precipitates formed in an attached growth reactor (AGR) through a series of experiments based on Toxicity Characteristic Leaching Procedure (TCLP), aging and long term leaching tests. About half of the AGR was initially added with waste activated carbon (WAC) to support the growth of mixed microbial consortia and used for treatment of arsenic and iron contaminated simulated groundwater. The X-ray diffraction (XRD), X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) spectroscopy results indicated that the biosolids were mainly composed of arsenosulphides and iron sulphides. While TCLP and aging tests were conducted in anoxic as well as oxic conditions with the aim to evaluate stability of biomass containing biogenic sulphides, long term leaching test was conducted through supply of aerated distilled water to evaluate the stability of spent WAC as well. Results generated from the research indicate that the concentration of leached arsenic never exceeded 123 µg/L under all conditions tested, thus biosolids not imposing an environmental hazard.

Enforcement of developmental lineage specificity by transcription factor Oct1.

Embryonic stem cells co-express Oct4 and Oct1, a related protein with similar DNA-binding specificity. To study the role of Oct1 in ESC pluripotency and transcriptional control, we constructed germline and inducible-conditional Oct1-deficient ESC lines. ESCs lacking Oct1 show normal appearance, self-renewal and growth but manifest defects upon differentiation. They fail to form beating cardiomyocytes, generate neurons poorly, form small, poorly differentiated teratomas, and cannot generate chimeric mice. Upon RA-mediated differentiation, Oct1-deficient cells induce lineage-appropriate developmentally poised genes poorly while lineage-inappropriate genes, including extra-embryonic genes, are aberrantly expressed. In ESCs, Oct1 co-occupies a specific set of targets with Oct4, but does not occupy differentially expressed developmental targets. Instead, Oct1 occupies these targets as cells differentiate and Oct4 declines. These results identify a dynamic interplay between Oct1 and Oct4, in particular during the critical window immediately after loss of pluripotency when cells make the earliest developmental fate decisions.

Pask integrates hormonal signaling with histone modification via Wdr5 phosphorylation to drive myogenesis.

PAS domain containing protein kinase (Pask) is an evolutionarily conserved protein kinase implicated in energy homeostasis and metabolic regulation across eukaryotic species. We now describe an unexpected role of Pask in promoting the differentiation of myogenic progenitor cells, embryonic stem cells and adipogenic progenitor cells. This function of Pask is dependent upon its ability to phosphorylate Wdr5, a member of several protein complexes including those that catalyze histone H3 Lysine 4 trimethylation (H3K4me3) during transcriptional activation. Our findings suggest that, during myoblast differentiation, Pask stimulates the conversion of repressive H3K4me1 to activating H3K4me3 marks on the promoter of the differentiation gene myogenin (Myog) via Wdr5 phosphorylation. This enhances accessibility of the MyoD transcription factor and enables transcriptional activation of the Myog promoter to initiate muscle differentiation. Thus, as an upstream kinase of Wdr5, Pask integrates signaling cues with the transcriptional network to regulate the differentiation of progenitor cells.

Oct1 and OCA-B are selectively required for CD4 memory T cell function.

Epigenetic changes are crucial for the generation of immunological memory. Failure to generate or maintain these changes will result in poor memory responses. Similarly, augmenting or stabilizing the correct epigenetic states offers a potential method of enhancing memory. Yet the transcription factors that regulate these processes are poorly defined. We find that the transcription factor Oct1 and its cofactor OCA-B are selectively required for the in vivo generation of CD4(+) memory T cells. More importantly, the memory cells that are formed do not respond properly to antigen reencounter. In vitro, both proteins are required to maintain a poised state at the Il2 target locus in resting but previously stimulated CD4(+) T cells. OCA-B is also required for the robust reexpression of multiple other genes including Ifng. ChIPseq identifies ∼50 differentially expressed direct Oct1 and OCA-B targets. We identify an underlying mechanism involving OCA-B recruitment of the histone lysine demethylase Jmjd1a to targets such as Il2, Ifng, and Zbtb32. The findings pinpoint Oct1 and OCA-B as central mediators of CD4(+) T cell memory.

Pluripotency transcription factor Oct4 mediates stepwise nucleosome demethylation and depletion.

The mechanisms whereby the crucial pluripotency transcription factor Oct4 regulates target gene expression are incompletely understood. Using an assay system based on partially differentiated embryonic stem cells, we show that Oct4 opposes the accumulation of local H3K9me2 and subsequent Dnmt3a-mediated DNA methylation. Upon binding DNA, Oct4 recruits the histone lysine demethylase Jmjd1c. Chromatin immunoprecipitation (ChIP) time course experiments identify a stepwise Oct4 mechanism involving Jmjd1c recruitment and H3K9me2 demethylation, transient FACT (facilitates chromatin transactions) complex recruitment, and nucleosome depletion. Genome-wide and targeted ChIP confirms binding of newly synthesized Oct4, together with Jmjd1c and FACT, to the Pou5f1 enhancer and a small number of other Oct4 targets, including the Nanog promoter. Histone demethylation is required for both FACT recruitment and H3 depletion. Jmjd1c is required to induce endogenous Oct4 expression and fully reprogram fibroblasts to pluripotency, indicating that the assay system identifies functional Oct4 cofactors. These findings indicate that Oct4 sequentially recruits activities that catalyze histone demethylation and depletion.

Reversal of methylmercury-induced oxidative stress, lipid peroxidation, and DNA damage by the treatment of N-acetyl cysteine: a protective approach.

This study was designed to evaluate the protective effect of N-acetyl cysteine in reducing methylmercury (MeHg)-induced oxidative stress, lipid peroxidation, DNA damage in liver, kidney, and brain, and their ability to restore altered hepatic, renal, and other biochemical variables. Male Sprague-Dawley rats (150±10 g) were randomly divided into three groups. Group 1 served as the control. Groups 2 and 3 were administered methylmercury (1 mg kg⁻¹ orally, 5 days/week) for 12 weeks, and group 2 served as the experimental control. Group 3 received N-acetyl cysteine (0.6 mg kg⁻¹ intraperitoneally, two days/week) for 12 weeks after methylmercury exposure. Methylmercury exposure caused a significant rise in bilirubin, gamma-glutamyl transpeptidase, protein, triglycerides, cholesterol, urea, creatinine, uric acid, and blood urea nitrogen, with a concomitant decrease in albumin content, reduced glutathione level and acetyl cholinesterase activity, antioxidant enzymes such as glutathione reductase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, and adenosine triphosphatase. However, lipid peroxidation level, metallothionein expression, and DNA damage with increment of tail length were observed after methylmercury intoxication. N-acetyl cysteine, a widely available, nontoxic amino acid derivative, is a promising antioxidant with a wide spectrum of biological functions. The ability of N-acetyl cysteine to enhance mercury excretion and its wide availability in clinical use indicate that it may be an ideal therapeutic agent against methylmercury poisoning.

Efficient chromatin immunoprecipitation using limiting amounts of biomass.

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results. Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions(1,2) , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully(3,4) , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol. This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available(5-8) in that we extensively cover the later, analysis phase.