Toxic Accumulation Research Articles

MANF, a structurally unique redox-sensitive chaperone, restores ER-protein folding in the ischemic heart

Rationale: In cardiomyocytes, most secreted and membrane proteins are synthesized and folded in the sarcoplasmic/endoplasmic reticulum (SR/ER). We previously showed that during ischemia, decreased oxygen creates a reducing environment in the SR/ER, preventing protein disulfide isomerases (PDIs) from forming disulfide bonds in nascent proteins, causing ER stress– the toxic accumulation of unfolded proteins which contributes to cardiomyocyte death. Upon ER stress, the transcription factor, ATF6 induces chaperones that restore SR/ER protein folding. We found that ATF6 induces mesencephalic astrocyte-derived neurotrophic factor (MANF), a recently identified protein of unknown function. MANF is structurally unique, so its function cannot be inferred from other proteins. Since MANF ATF6-inducible, ER-localized, and contains a conserved redox-sensitive motif found in PDIs, we hypothesized that MANF is a redox-sensitive chaperone that optimizes cardiomyocyte viability during ischemia. Methods: The redox status of MANF during reductive ER stress, and ability to bind misfolded proteins during ischemia were assessed in neonatal rat ventricular myocytes (NRVM). The ability of recombinant MANF to suppress misfolded protein aggregation was examined in an in vitro chaperone assay. Finally, the effects of MANF loss-of-function in the ischemic heart, in vivo, were determined by generating a transgenic mouse that expresses a cardiomyocyte-specific MANF-targeted microRNA. Results: In NRVM subjected to ER stress MANF was as sensitive to changes in ER redox status as the sentinel PDI, PDIA1 and formed disulfide-linked complexes with misfolded proteins during ischemia-mediated ER stress. Under reducing conditions, recombinant MANF suppressed aggregation of model misfolded proteins, in vitro. MANF knockdown in the heart, in vivo, increased damage from myocardial infarction, and an AAV9-based gene therapy approach rescued the effects of MANF deficiency, in vivo. Conclusions: MANF is a redox-sensitive SR/ER-resident chaperone that is a critical contributor to SR/ER protein folding during the adaptive ER stress response and decreases tissue damage in the ischemic heart.

Enhancing monellin production by Pichia pastoris at low cell induction concentration via effectively regulating methanol metabolism patterns and energy utilization efficiency.

In heterologous protein productions by P. pastoris, methanol induction is generally initiated when cell concentration reaches very high density. The alternative strategy by initiating methanol induction at lower cells concentration was also reported to be effective in easing DO control, reducing toxic by-metabolites accumulation and increasing targeted proteins titers. However, the methanol/energy regulation mechanisms are seldom reported. We theoretically analyzed the methanol/energy metabolisms in protein expression process with the strategies of initiating induction at higher or lower cells concentrations, using monellin production as a prototype. When initiating induction at lower cells concentration and controlling induction temperature at 30°C, monellin concentration reached the highest levels of 2.62~2.71 g/L, which was 2.5~4.9 fold of those obtained with the strategy of initiating induction at higher cells concentration. With the desired induction strategy, 1) carbon metabolism ratio directing into the precursors synthesis route for monellin production reached the highest level of 65%, carbon metabolism ratios towards to precursors synthesis and ATP regeneration routes were regulated at relatively balanced levels; 2) monellin synthesis was completely cell growth associated, with the largest associated coefficient and higher specific growth rate; 3) theoretical NADH (energy) utilization efficiency η was the highest, and η stayed high levels (≥0.8) during most period (89%) within induction phase to supply sufficient energy in supporting monellin synthesis.

In silico prediction of multiple-category classification model for cytochrome P450 inhibitors and non-inhibitors using machine-learning method

The cytochrome P450 (CYP) enzyme superfamily is involved in phase I metabolism which chemically modifies a variety of substrates via oxidative reactions to make them more water-soluble and easier to eliminate. Inhibition of these enzymes leads to undesirable effects, including toxic drug accumulations and adverse drug–drug interactions. Hence, it is necessary to develop in silico models that can predict the inhibition potential of compounds for different CYP isoforms. This study focused on five major CYP isoforms, including CYP1A2, 2C9, 2C19, 2D6 and 3A4, that are responsible for more than 90% of the metabolism of clinical drugs. The main aim of this study is to develop a multiple-category classification model (MCM) for the major CYP isoforms using a Laplacian-modified naïve Bayesian method. The dataset composed of more than 4500 compounds was collected from the PubChem Bioassay database. VolSurf+ descriptors and FCFP_8 fingerprint were used as input features to build classification models. The results demonstrated that the developed MCM using Laplacian-modified naïve Bayesian method was successful in classifying inhibitors and non-inhibitors for each CYP isoform. Moreover, the accuracy, sensitivity and specificity values for both training and test sets were above 80% and also yielded satisfactory area under the receiver operating characteristic curve and Matthews correlation coefficient values.

Rhodol-derived Colorimetric and Fluorescent Probe with the Receptor of Carbonothioate for the Specific Detection of Mercury Ions.

Developing some methods that can simply and effectively detect mercury ions (Hg2+) in the environment and biological systems are very important due to the problems of high toxicity and biological accumulation. Herein, we report a simple rhodol-derived colorimetric and fluorescent probe rhodol-Hg with a recognition receptor of carbonothioate for the specific determination of Hg2+. The color of probe rhodol-Hg solution changed remarkably from colorless to pink in the presence of Hg2+, thus rhodol-Hg could act as a "naked-eye" probe for Hg2+. Additionally, this probe exhibited high selectivity and ultrasensitivity in aqueous solution with the limit of detection (LOD) of 1.4 nM toward Hg2+, and the linear range was 0 - 0.8 μM determined by turn-on fluorescence spectrometry. Importantly, this probe has been successfully used for the detection of Hg2+ in environmental waters and living cells.

SBMA (Kennedy’s Disease) and ALS: Development of disease-modifying therapy

Spinal and bulbar muscular atrophy (SBMA) is an adult-onset neuromuscular disease caused by the expansion of a trinucleotide CAG repeat in the androgen receptor (AR) gene. The ligand-dependent nuclear accumulation of pathogenic AR protein is central to the pathogenesis. Leuprorelin, a luteinizing hormone-releasing hormone (LHRH) analogue that suppresses testosterone production from testis, inhibits toxic accumulation of pathogenic AR, thereby mitigating histopathological and behavioral impairments in a mouse model of SBMA. A randomized placebo-controlled multi-centric phase 3 clinical trial showed an effect of the drug on motor functions, particularly there was a significant improvement of swallowing function. In addition, a long-term follow-up study compared with natural history group of patients revealed that the progression of motor dysfunction and occurrence of pneumoniae due to swallowing dysfunction were very much diminished with leuprorelin treatment.

The Removal of Lead from Aqueous Solution Using Almond Green Hull (Prunus amygdalus-Fascionello) Waste Material Magnetized with Fe3O4

Background: Nowadays, with extensive industrialization, pollution of heavy metals in the environment has increased significantly, which is considered a serious environmental risk for toxicity, carcinogenicity, and biological accumulation. Meanwhile, lead is one of the most important systemic toxic heavy metals, which attacks most organs of the body after adsorption. The aim of this research was to evaluate the efficiency of green almond hull (Prunus amygdalus- Fascionello) magnetized with Fe3O4 to remove lead from aqueous solution. Methods: In this study, the structural characteristic of green almond hull magnetized with Fe3O4 was investigated using a scanning electron microscope (SEM) and vibrating sample magnetometer (VSM) techniques. Also, the effect of different parameters, including pH (2 to 10), mixing speed (0 to 250 rpm), adsorbent dose (1 to 7 g/L), initial concentration (10 to 100 mg/L), and contact time (5 to 200 minutes) was examined to study lead adsorption process by the desired adsorbent. Finally, the experimental data obtained from the equilibrium tests were analyzed using Freundlich and Langmuir isotherms models. Results: The results of this study showed that the maximum removal efficiency was obtained at pH 9 and adsorbent dose of 5 g/L. The removal efficiency was reduced by increasing the initial concentration of lead, and increasing contact time and mixing speed. The maximum lead adsorption at optimal conditions was 91.34%. The results also showed that the resulting data often followed the Langmuir isotherm. Conclusions: According to the results, it could be concluded that green almond hull (Prunus amygdalus-Fascionello) magnetized with Fe3O4 could be used as an affordable and proper adsorbent in removal of lead from aqueous solutions.

Toxic elements and energy accumulation in topsoil and plants of spruce ecosystems

The objective of this research was to evaluate trends and relationships of energy and toxic elements accumulation in A-horizon (the depth of 0–5 cm) of soils and in selected plants of the hemioligotrophic (Dystric Cambisols) and oligotrophic (Skeletic-Rustic Podzols) spruce ecosystems situated along transect (750–1110 m a.s.l.) in the NP Slovenský raj (Eastern Slovakia). The results showed that EU limit values of risk elements in agricultural soils were exceeded for Cu and Cd at the altitude of 750–760 m a.s.l., and in case of Cd also above 1000 m a.s.l. Relationship between energy and toxic elements in soils revealed that with an increasing amount of energy, contents of Zn and Cu significantly declined with altitude (r > –0.5 or r > –0.9). The background value of Cu was exceeded in all plants, that of Zn for Dryopteris filix-mas and Rubus idaeus. Furthermore, excessive accumulation of Cd was revealed by all plants. Cu contents in soils were dominant in determining Cu uptake for Vaccinium myrtillus (r > 0.5); Zn and Cd for V. myrtillus (r > 0.6), D. filix-mas (r > 0.5 or r > 0.8) and Fagus sylvatica (r > –0.8 or r > –0.5); Zn also for R. idaeus species (r > 0.4). The soil-plant transfer coefficients higher than 1 hinted R. idaeus on the plots at the altitude of 960 m a.s.l. (Cd 1.1, Cu 1.2, Zn 3.1), which appears as an excellent native indicator of forest ecosystem contamination.

Mir-193 targets ALDH2 and contributes to toxic aldehyde accumulation and tyrosine hydroxylase dysfunction in cerebral ischemia/reperfusion injury.

MicroRNAs (miRNAs, miR) play a fundamental role in cerebral ischemia/reperfusion (I/R) injury. However, the role of miRNAs in toxic aldehyde and tyrosine accumulation is not fully elucidated. We constructed a cerebral I/R rat model and found that overexpression of miR-193 was associated with the accumulation of 4-Hydroxynonenal (4-HNE), Malondialdehyde (MDA), and tyrosine, and with the decrease of aldehyde dehydrogenase (ALDH2), tyrosine hydroxylase (TH), and dopamine. To unveil the molecular mechanism of the miR-193-mediated phenotype in I/R injury as described above, we performed bioinformatic analysis and found that ALDH2 was a potential target of miR-193. Through in vitro experiments (such as miR-193 mimic/inhibitor transfection, luciferase reporter gene plasmid transfection, and 4-HNE exposure) and in vivo infusion of miR-193 agomir, we demonstrated that miR-193 directly suppressed the expression of ALDH2 and led to toxic aldehyde accumulation, resulting in dysfunction of tyrosine hydroxylase. The present study suggests that the overexpression of miR-193 in a rat model exacerbated brain injury due to the following sequential process: targeted suppression of ALDH2, aldehyde accumulation, and tyrosine hydroxylase dysfunction, leading to tyrosine accumulation and insufficiency of dopamine synthesis.

P-body proteins regulate transcriptional rewiring to promote DNA replication stress resistance

mRNA-processing (P-) bodies are cytoplasmic granules that form in eukaryotic cells in response to numerous stresses to serve as sites of degradation and storage of mRNAs. Functional P-bodies are critical for the DNA replication stress response in yeast, yet the repertoire of P-body targets and the mechanisms by which P-bodies promote replication stress resistance are unknown. In this study we identify the complete complement of mRNA targets of P-bodies during replication stress induced by hydroxyurea treatment. The key P-body protein Lsm1 controls the abundance of HHT1, ACF4, ARL3, TMA16, RRS1 and YOX1 mRNAs to prevent their toxic accumulation during replication stress. Accumulation of YOX1 mRNA causes aberrant downregulation of a network of genes critical for DNA replication stress resistance and leads to toxic acetaldehyde accumulation. Our data reveal the scope and the targets of regulation by P-body proteins during the DNA replication stress response.

Quercetin Attenuates Manganese-Induced Neuroinflammation by Alleviating Oxidative Stress through Regulation of Apoptosis, iNOS/NF-κB and HO-1/Nrf2 Pathways.

Manganese (Mn) is an essential trace element required for the development of human body and acts as an enzyme co-factor or activator for various reactions of metabolism. While essential in trace amounts, excessive Mn exposure can result in toxic accumulations in human brain tissue and resulting extrapyramidal symptoms called manganism similar to idiopathic Parkinson’s disease (PD). Quercetin (QCT) has been demonstrated to play an important role in altering the progression of neurodegenerative diseases by protecting against oxidative stress. This study aimed to investigate the protective effect of QCT on Mn-induced neurotoxicity and the underlying mechanism in SK-N-MC human neuroblastoma cell line and Sprague-Dawley (SD) male rat brain. The results showed that Mn treatment significantly decreased the cell viability of SK-N-MC cell and increased the release of lactate dehydrogenase (LDH), which was attenuated by QCT pretreatment at 10 and 20 µg/mL. Compared to the Mn alone group, QCT pretreatment significantly attenuated Mn-induced oxidative stress, mitochondrial dysfunction and apoptosis. Meanwhile, QCT pretreatment markedly downregulated the NF-κB but upregulated the heme oxygenase-1 (HO-1) and Nrf2 proteins, compared to the Mn alone group. Our result showed the beneficial effect of QCT on hematological parameters against Mn in rat brain. QCT decrease reactive oxygen species (ROS) and protein carbonyl levels and increased Cu/Zn-superoxide dismutase (SOD) activity induced in Mn-treated rats. QCT administration caused a significant reduction in the Mn-induced neuroinflammation by inhibiting the expression of inflammatory markers such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). QCT lowered the Mn elevated levels of various downstream apoptotic markers, including Bax, cytochrome c, cleaved caspase-3 and polymerase-1 (PARP-1), while QCT treatment upregulated anti-apoptotic Bcl-2 proteins and prevented Mn-induced neurodegeneration. Furthermore, administration of QCT (25 and 50 mg/kg) to Mn-exposed rats showed improvement of histopathological alteration in comparison to Mn-treated rats. Moreover, administration of QCT to Mn-exposed rats showed significant reduction of 8-hydroxy-2′-deoxyguanosine (8-OHdG), Bax, activated caspase-3 and PARP-1 immunoreactivity. These results indicate that QCT could effectively inhibit Mn induced apoptosis and inflammatory response in SK-N-MC cells and SD rats, which may involve the activation of HO-1/Nrf2 and inhibition of NF-κB pathway.

A transmission electron microscopy (TEM) study of silver nanoparticles associated with mine waste from New Caledonian nickel deposits: potential origins of silver toxicity in a World Heritage Site

The nature of silver nanoparticles associated with nickel-laterite ore deposits in New Caledonia (SW Pacific Ocean) is explored using transmission electron microscopy. The silver nanoparticles, dispersed in a matrix of amorphous silica, display complex textures such as linear aggregation and bimodal diameter distributions of 3–12 and 20–60 nm for one sample and 2–30 and 150–650 nm for the other, indicative of Ostwald ripening. These features as well as chemical heterogeneity (31–82% Ag—normalized X-ray energy dispersive spectrum), destructive assemblage interfaces, and amorphous and nanoparticle silica phases are consistent with those observed in epithermal gold deposits, potentially indicating a similar origin. Silver is highly toxic to a wide variety of organisms, and therefore, its significant presence in material directly related to mine runoff is a major threat to the New Caledonian environment, particularly the lagoons, which are listed as a World Heritage Site. Potential exists that the silver nanoparticles observed here provide the key to the origin of the toxic silver accumulation observed in New Caledonian reef organisms.

Phytochelatin Synthase has Contrasting Effects on Cadmium and Arsenic Accumulation in Rice Grains

Phytochelatin (PC) synthesis has been well demonstrated as a major metal tolerance mechanism in Arabidopsis thaliana, whereas its contribution to long-distance element transport especially in monocots remains elusive. Using rice as a cereal model, we examined physiological roles of Oryza sativa phytochelatin synthase 1 (OsPCS1) in the distribution and detoxification of arsenic (As) and cadmium (Cd), two toxic elements associated with major food safety concerns. First, we isolated four different transcript variants of OsPCS1 as well as one from OsPCS2. Quantitative real-time reverse transcription–PCR (RT-PCR) of each OsPCS transcript in rice seedlings suggested that expression of OsPCS1full, the longest OsPCS1 variant, was most abundant, followed by OsPCS2. Heterologous expression of OsPCS variants in PCS-deficient mutants of Schizosaccharomyces pombe and A. thaliana suggested that OsPCS1full possessed PCS activity in response to As(III) and Cd while the activity of other PCS variants was very low. To address physiological functions in toxic element tolerance and accumulation, two independent OsPCS1 mutant rice lines (a T-DNA and a Tos17 insertion line) were identified. The OsPCS1 mutants exhibited increased sensitivity to As(III) and Cd in hydroponic experiments, showing the importance of OsPCS1-dependent PC synthesis for rice As(III) and Cd tolerance. Elemental analyses of rice plants grown in soil with environmentally relevant As and Cd concentrations showed increased As accumulation and decreased Cd accumulation in grains of the T-DNA line. The Tos17 mutant also exhibited the reduced Cd accumulation phenotype. These contrasting effects on As and Cd distribution to grains suggest the existence of at least partially distinct PC-dependent pathways for As and Cd.

Repeated AAV-mediated gene transfer by serotype switching enables long-lasting therapeutic levels of hUgt1a1 enzyme in a mouse model of Crigler-Najjar Syndrome Type I.

Adeno-associated virus (AAV) -mediated gene therapy is a promising strategy to treat liver-based monogenic diseases. However, two major obstacles limit its success: first, vector dilution in actively dividing cells, such as hepatocytes in neonates/children, due to the non-integrating nature of the vector; second, development of an immune response against the transgene and/or viral vector. Crigler-Najjar Syndrome Type I is a rare monogenic disease with neonatal onset, caused by mutations in the liver-specific UGT1 gene, with toxic accumulation of unconjugated bilirubin in plasma, tissues and brain. To establish an effective and long lasting cure, we applied AAV-mediated liver gene therapy to a relevant mouse model of the disease. Repeated gene transfer to adults by AAV-serotype switching, upon neonatal administration, resulted in lifelong correction of total bilirubin (TB) levels in both genders. In contrast, vector loss over time was observed after a single neonatal administration. Adult administration resulted in lifelong TB levels correction in male, but not female Ugt1-/- mice. Our findings demonstrate that neonatal AAV-mediated gene transfer to the liver supports a second transfer of the therapeutic vector, by preventing the induction of an immune response and supporting the possibility to improve AAV-therapeutic efficacy by repeated administration.

Early and consistent overexpression of ADRM1 in ovarian high-grade serous carcinoma

BackgroundOvarian carcinoma is highly dependent on the ubiquitin proteasome system (UPS), but its clinical response to treatment with the proteasome inhibitor bortezomib has been disappointing. This has driven exploration of alternate approaches to target the UPS in ovarian cancer. Recently, proteasome inhibitors targeting the 19S regulatory particle-associated RPN13 protein have been described, such as RA190. RPN13, which is encoded by ADRM1, facilitates the recognition by the proteasome of its polyubiquinated substrates. Inhibition of RPN13 produces a rapid, toxic accumulation of polyubiquitinated proteins in ovarian and other cancer cells, triggering apoptosis.Here, we sought to determine if RPN13 is available as a target in precursors of ovarian/fallopian tube cancer as well as all advanced cases, and the impact of increased ADRM1 gene copy number on sensitivity of ovarian cancer to RA190.MethodsADRM1 mRNA was quantified by RNAscope in situ hybridization and RPN13 protein detected by immunohistochemistry in high grade serous carcinoma (HGSC) of the ovary and serous tubal intraepithelial carcinoma (STIC). Amplification of ADRM1 and sensitivity to RA190 were determined in ovarian cancer cell lines.ResultsHere, we demonstrate that expression of ADRM1mRNA is significantly elevated in STIC and HGSC as compared to normal fallopian tube epithelium. ADRM1 mRNA and RPN13 were ubiquitously and robustly expressed in ovarian carcinoma tissue and cell lines. No correlation was found between ADRM1 amplification and sensitivity of ovarian cancer cell lines to RA190, but all were susceptible.ConclusionsRPN13 can potentially be targeted by RA190 in both in situ and metastatic ovarian carcinoma. Ovarian cancer cell lines are sensitive to RA190 regardless of whether the ADRM1 gene is amplified.

ATP-binding cassette transporter G1 and apolipoprotein M are ‘Novel players’ in cholesterol transport at the blood-brain barrier

Aim: Impaired cholesterol/lipoprotein metabolism is linked to neurodegenerative diseases. Cerebral cholesterol homeostasis is maintained by the highly efficient blood-brain barrier (BBB) and flux of the oxysterols 24(S)- and 27-hydroxycholesterol, potent liver-X-receptor (LXR) activators. HDL/apolipoproteins support cerebral lipid transfer and loss of ATP-binding cassette transporters (ABC)G1 and G4 results in toxic accumulation of oxysterols in the brain. The HDL-associated apolipoprotein (apo)M is positively correlated with pre-ß HDL formation in plasma; its presence and function in the brain was thus far unknown. Using an in vitro model of the BBB we examined expression, regulation and functions of ABCG1, ABCG4, and apoM in primary cerebrovascular endothelial cells.

Private: Toxic heavy metal accumulation in dietary vegetables and associated health risks due to wastewater irrigation in Peshawar, Pakistan

Private: Toxic heavy metal accumulation in dietary vegetables and associated health risks due to wastewater irrigation in Peshawar, Pakistan

LpxK Is Essential for Growth of Acinetobacter baumannii ATCC 19606: Relationship to Toxic Accumulation of Lipid A Pathway Intermediates.

Acinetobacter baumannii ATCC 19606 can grow without lipid A, the major component of lipooligosaccharide. However, we previously reported that depletion of LpxH (the fourth enzyme in the lipid A biosynthetic pathway) prevented growth of this strain due to toxic accumulation of lipid A pathway intermediates. Here, we explored whether a similar phenomenon occurred with depletion of LpxK, a kinase that phosphorylates disaccharide 1-monophosphate (DSMP) at the 4' position to yield lipid IVA. An A.baumannii ATCC 19606 derivative with LpxK expression under the control of an isopropyl β-d-1-thiogalactopyranoside (IPTG)-regulated expression system failed to grow without induction, indicating that LpxK is essential for growth. Light and electron microscopy of LpxK-depleted cells revealed morphological changes relating to the cell envelope, consistent with toxic accumulation of lipid A pathway intermediates disrupting cell membranes. Using liquid chromatography-mass spectrometry (LCMS), cellular accumulation of the detergent-like pathway intermediates DSMP and lipid X was shown. Toxic accumulation was further supported by restoration of growth upon chemical inhibition of LpxC (upstream of LpxK and the first committed step of lipid A biosynthesis) using CHIR-090. Inhibitors of fatty acid synthesis also abrogated the requirement for LpxK expression. Growth rescue with these inhibitors was possible on Mueller-Hinton agar but not on MacConkey agar. The latter contains outer membrane-impermeable bile salts, suggesting that despite growth restoration, the cell membrane permeability barrier was not restored. Therefore, LpxK is essential for growth of A.baumannii, since loss of LpxK causes accumulation of detergent-like pathway intermediates that inhibit cell growth. IMPORTANCEAcinetobacter baumannii is a Gram-negative pathogen for which new therapies are needed. The lipid A biosynthetic pathway has several potential enzyme targets for the development of anti-Gram-negative agents (e.g., LpxC). However, A.baumannii ATCC 19606 can grow in the absence of LpxC and, correspondingly, of lipid A. In contrast, we show that cellular depletion of LpxK, a kinase occurring later in the pathway, inhibits growth. Growth inhibition results from toxic accumulation of lipid A pathway intermediates, since chemical inhibition of LpxC or fatty acid biosynthesis rescues cell growth upon loss of LpxK. Overall, this suggests that targets such as LpxK can be essential for growth even in those Gram-negative bacteria that do not require lipid A biosynthesis per se. This strain provides an elegant tool to derive a better understanding of the steps in a pathway that is the focus of intense interest for the development of novel antibacterials.

Abstract 2: MANF, A Structurally Unique Redox-Sensitive Chaperone, Restores ER Protein Folding in the Ischemic Heart.

Rationale: In cardiomyocytes, most secreted and membrane proteins are synthesized and folded in the sarcoplasmic/endoplasmic reticulum (SR/ER). We previously showed that during myocardial ischemia, decreased oxygen creates a reducing environment in the SR/ER, preventing protein disulfide isomerases (PDIs) from forming disulfide bonds in nascent proteins, causing ER stress, i.e. the toxic accumulation of unfolded proteins which contributes to cardiomyocyte death. In response to ER stress, the transcription factor, ATF6 induces chaperones that restore SR/ER protein folding. We found that ATF6 also induces mesencephalic astrocyte-derived neurotrophic factor (MANF), a recently identified protein of unknown function. MANF is structurally unique, so its function cannot be inferred from other proteins. Since MANF is induced by ATF6, is ER-localized, and contains a conserved redox-sensitive motif found in PDIs, we hypothesized that MANF is a redox-sensitive chaperone that optimizes cardiomyocyte viability during ischemia. Methods: The redox status of MANF during reductive ER stress and the ability of MANF to bind misfolded proteins during ischemia were assessed in neonatal rat ventricular myocytes (NRVM). The ability of recombinant MANF to suppress aggregation of misfolded proteins was examined in an in vitro chaperone assay. Finally, the effects of MANF loss-of-function in the ischemic heart, in vivo , were determined by generating a transgenic mouse model that expresses a cardiomyocyte-specific MANF-targeted microRNA. Results: In NRVM subjected to ER stress MANF was as sensitive to changes in ER redox status as the sentinel PDI, PDIA1. Moreover, MANF formed disulfide-linked complexes with misfolded proteins during ischemia-mediated ER stress. Under reducing conditions, recombinant MANF suppressed aggregation of model misfolded proteins, in vitro . MANF knockdown in the heart, in vivo , increased damage from myocardial infarction, and an AAV9-based gene therapy approach rescued the effects of MANF deficiency, in vivo. Conclusions: MANF is a redox-sensitive SR/ER-resident chaperone that is a critical contributor to SR/ER protein folding during the adaptive ER stress response and decreases tissue damage in the ischemic heart.

Accumulation of Microcystin (LR, RR and YR) in Three Freshwater Bivalves in Microcystis aeruginosa Bloom Using Dual Isotope Tracer.

Stable isotope tracers were first applied to evaluate the Microcystis cell assimilation efficiency of Sinanodonta bivalves, since the past identification method has been limited to tracking the changes of each chl-a, clearity, and nutrient. The toxicity profile and accumulation of MC-LR, -RR and -YR in different organs (foot and digestive organs) from the three filter-feeders (Sinanodonta woodiana, Sinanodonta arcaeformis, and Unio douglasiae) were assessed under the condition of toxigenic cyanobacteria (Microcystis aeruginosa) blooms through an in situ pond experiment using 13C and 15N dual isotope tracers. Chl-a concentration in the manipulated pond was dramatically decreased after the beginning of the second day, ranging from 217.5 to 15.6 μg·L−1. The highest amount of MCs was incorporated into muscle and gland tissues in U. douglasiae during the study period, at nearly 2 or 3 times higher than in S. woodiana and S. arcaeformis. In addition, the incorporated 13C and 15N atom % in the U. douglasiae bivalve showed lower values than in other bivalves. The results demonstrate that U. douglasiae has less capacity to assimilate toxic cyanobacteria derived from diet. However, the incorporated 13C and 15N atom % of S. arcaeformis showed a larger feeding capacity than U. douglasiae and S. woodiana. Our results therefore also indicate that S. arcaeformis can eliminate the toxin more rapidly than U. douglasiae, having a larger detoxification capacity.

Effect of alkaline material on phytotoxicity and bioavailability of Cu, Cd, Pb and Zn in stabilized sewage sludge

ABSTRACTThe availability and phytotoxicity of heavy metals in sewage sludge is the key restrictive factor that limits sludge application. In this study, municipal sewage sludge was stabilized by alkaline slag or coal fly ash, then the leaching characteristic and fraction distributions of Cu, Cd, Pb and Zn were studied, and their effects on seed germination, root length, and plant accumulating were compared. The results showed that mixed sewage sludge with alkaline slag and coal fly ash decreased the percentage of available heavy metals in sewage sludge. The percentage of exchangeable Cu, Pb, Zn and Cd in stabilized sewage sludge (S1–S4) was 1.50–8.67 times lower than that in SS-only treatment. Leachate Cd, Cu, Pb and Zn from stabilized SS was much lower than the limit threshold. The addition of alkaline materials to SS reduced the inhibitory effect on seed germination and root growth. Pearson’s correlation analysis indicated that there was a significant negative correlation between pH and available heavy metals, while a significant positive correlation between the percentage of exchangeable fraction of heavy metals, the leaching potential, tissue concentration and accumulation of heavy metal and toxicity for seed germination was observed.