μM MnCl2 Research Articles

Hyperaccumulation of metal in the apoplast contributes to the tolerance of the phytoremediator Pistia stratiotes L. to manganese-contaminated water

Phytoremediation of manganese (Mn)-contaminated water requires the selection of Mn-tolerant species. This study reports on physiological changes and Mn bioaccumulation in the aquatic macrophyte Pistia stratiotes cultivated under various MnCl2 concentrations: control, 80, 340, 600, 1000, 2000, and 4000 µM. Few visual symptoms of Mn toxicity, such as chlorosis, were observed after 10 days, especially in plants treated with 2000 and 4000 µM MnCl2. High Mn accumulation was recorded, with maximum values of 23,700 and 24,600 µg g−1 DW in the shoots and roots, respectively, at 4000 µM Mn, contrasting with 825.01 and 1587.53 µg g−1 DW in control plants. Cellular fractioning showed that Mn in shoots and roots was mainly associated with the cell wall, with approximately 90% of the Mn in roots detected in the apoplast. There were no significant changes in net CO2 assimilation or respiratory rates after 5 and 10 days of Mn exposure. These results demonstrate that P. stratiotes is a Mn hyperaccumulator species with excellent phytoremediation potential, as shown by its high bioaccumulation capacity and its ability to maintain photosynthetic efficiency under Mn stress.

Effect of Melissa officinalis L. leaf extract on manganese-induced cyto-genotoxicity on Allium cepa L.

Although the antioxidant properties of Melissa officinalis extract (Mox) are widely known, little work has focused on its protective capacity against heavy metal stress. The primary objective of this study was to determine the potential of Mox to mitigate manganese (II) chloride (MnCI2)-induced cyto-genotoxicity using the Allium and comet assays. Physiological, genotoxic, biochemical and anatomical parameters as well as the phenolic composition of Mox were examined in Allium cepa (L.). Application of 1000 µM MnCl2 reduced the rooting percentage, root elongation, weight gain, mitotic index and levels of chlorophyll a and chlorophyll b pigments compared to the control group. However, it increased micronuclei formation, chromosomal abnormality frequencies, tail DNA percentage, proline amount, lipid peroxidation level and meristematic damage severity. The activities of superoxide dismutase and catalase also increased. Chromosomal aberrations induced by MnCl2 were fragment, sticky chromosome, vagrant chromosome, unequal distribution of chromatin and bridge. Application of 250 mg/L Mox and 500 mg/L Mox along with MnCl2 significantly alleviated adverse effects dose dependently. The antioxidant activity bestowed by the phenolic compounds in Mox assisted the organism to combat MnCl2 toxicity. Consequently, Mox exerted remarkable protection against MnCl2 toxicity and it needs to be investigated further as a potential therapeutic option.

A citrate efflux transporter important for manganese distribution and phosphorus uptake in rice.

The plant citrate transporters, functional in mineral nutrient uptake and homeostasis, usually belong to the multidrug and toxic compound extrusion transporter family. We identified and functionally characterized a rice (Oryza sativa) citrate transporter, OsCT1, which differs from known plant citrate transporters and is structurally close to rice silicon transporters. Domain analysis depicted that OsCT1 carries a bacterial citrate-metal transporter domain, CitMHS. OsCT1 showed citrate efflux activity when expressed in Xenopus laevis oocytes and is localized to the cell plasma membrane. It is highly expressed in the shoot and reproductive tissues of rice, and its promoter activity was visible in cells surrounding the vasculature. The OsCT1 knockout (KO) lines showed a reduced citrate content in the shoots and the root exudates, whereas overexpression (OE) line showed higher citrate exudation from their roots. Further, the KO and OE lines showed variations in the manganese (Mn) distribution leading to changes in their agronomical traits. Under deficient conditions (Mn-sufficient conditions followed by 8 days of 0 μm MnCl2 · 4H2 O treatment), the supply of manganese towards the newer leaf was found to be obstructed in the KO line. There were no significant differences in phosphorus (P) distribution; however, P uptake was reduced in the KO and increased in OE lines at the vegetative stage. Further, experiments in Xenopus oocytes revealed that OsCT1 could efflux citrate with Mn. In this way, we provide insights into a mechanism of citrate-metal transport in plants and its role in mineral homeostasis, which remains conserved with their bacterial counterparts.

Quantitative Proteomics Reveals Manganese Alleviates Heat Stress of Broiler Myocardial Cells via Regulating Nucleic Acid Metabolism.

Heat stress threatens severely cardiac function by caused myocardial injury in poultry. Our previous study has showed that manganese (Mn) has a beneficial effect on heat-stress resistance of broiler. Therefore, we tried to confirm the alleviation mechanism through proteomic analysis after heat stress exposure to primary broiler myocardial cells pretreated with Mn. The experiment was divided into four groups: CON group (37 °C, cells without any treatment), HS group (43 °C, cells treatment with heat stress for 4 h), HS+MnCl2 group (cells treated with 20 μM MnCl2 before heat stress), and HS+Mn-AA group (cells treated with 20 μM Mn compound amino acid complex before heat stress). Proteome analysis using DIA identified 300 differentially expressed proteins (DEPs) between CON group and HS group; 93 and 121 DEPs were identified in inorganic manganese treatment group and organic manganese treatment group, respectively; in addition, there were 53 DEPs identified between inorganic and organic manganese group. Gene Ontology (GO) analysis showed that DEPs were mainly involved in binding, catalytic activity, response to stimulus, and metabolic process. DEPs of manganese pretreatment involved in a variety of biological regulatory pathways, and significantly influenced protein processing and repair in endoplasmic reticulum, apoptosis, and DNA replication and repair. These all seem to imply that manganese may help to resist cell damage induced by heat stress by regulating key node proteins. These findings contribute to a better understanding of the effects of manganese on overall protein changes during heat-stress and the possible mechanisms, as well as how to better use manganese to protect heart function in high temperature.

Assessment of Cardiac Toxicity of Manganese Chloride for Cardiovascular Magnetic Resonance

MRI is widely used in cardiology to characterize the structure and function of the heart. Currently, gadolinium-based contrast agents are widely used to improve sensitivity and specificity of diagnostic images. Recently, Manganese, a calcium analogue, has emerged as a complementary contrast agent with the potential to reveal remaining viable cells within altered tissue. Imaging applications may be limited by substantial toxicity of manganese. Indeed, cardiac safety of manganese is not yet comprehensively assessed. In this study we investigated the effect of MnCl2 (1–100 µM) on cardiac function. Hemodynamic function was determined ex vivo using an isolated working rat heart preparation. HL-1 cardiac myocytes were used to investigate cell viability (calcein AM) and calcium cycling (Cal-520 a.m.). Rat ventricular cardiomyocytes were dissociated by enzymatic digestion. Action potentials and calcium currents were recorded using the patch clamp technique. MRI experiments were performed at 1.5T on formalin-fixed rat hearts, previously perfused with MnCl2. MnCl2 perfusion from 1 up to 100 µM in isolated working hearts did not alter left ventricular hemodynamic parameters. Contractility and relaxation index were not altered up to 50 µM MnCl2. In HL-1 cardiac myocytes, incubation with increasing concentrations of MnCl2 did not impact cell viability. The amplitude of the calcium transients were significantly reduced at 50 and 100 µM MnCl2. In freshly isolated ventricular myocytes, action potential duration at 20, 50 and 90% of repolarization were not modified up to 10 µM of MnCl2. L-type calcium current amplitude was significantly decreased by 50 and 100 µM of MnCl2. MRI on heart perfused with 25 and 100 µM of MnCl2 showed a dose dependent decrease in the T1 relaxation time. In conclusion, our results show that low concentrations of MnCl2 (up to 25 µM) can be used as a contrast agent in MRI, without significant impact on cardiac hemodynamic or electrophysiology parameters.

Assessment of cardiac toxicity of manganese chloride for cardiovascular magnetic resonance

MRI is widely used in cardiology to visualize the structure and function of the heart. Currently gadolinium is the contrast agents use to improve sensitivity and specificity of diagnostic images, enabling the assessment of scar and fibrosis. Manganese-based contrast molecules are being developed with the hope to improve the diagnosis of cardiac pathologies. Manganese, a calcium analogue, allows detection of viable cells within altered tissue. However, the cardiac safety of manganese is not fully assessed. To characterize the effect of MnCl2 on cardiac function and electrical parameters. Hemodynamic function was determined ex vivo using the isolated working rat heart preparation. HL-1 cell line was used to investigate cell viability (loading with calcein AM) and calcium cycling (loading with CAL-520 AM). Rat ventricular cardiomyocytes were dissociated by enzymatic digestion. Action potentials and calcium currents were recorded using the patch clamp technique. The effect of ascending concentrations of MnCl2 (1, 10, 25, 50 and 100 μM) were evaluated. MRI experiments (1.5 T and 9.4 T) were performed on formalin-fixed rat hearts, previously perfused with 25 or 100 μM of MnCl2. Left ventricular hemodynamic parameters from isolated working hearts show that heart rate is not modified by μM [MnCl2]. Similar observation was made for contractility index and aortic flow up to 50 μM; 100 μM MnCl2 induced a significant decrease of the two parameters. At the cellular level, μM [MnCl2] did not impact HL-1 cell viability. The amplitude of the calcium transients was significantly reduced only at 100 μM of MnCl2 ( P < 0.05). In isolated ventricular myocytes, action potential duration at 20%, 50% and 90% of repolarization were not modified up to 10 μM of MnCl2. A small but significant decrease of action potential duration was observed at 25, 50 and 100 μM (APD90 decrease by 23 ± 6% vs control). Peak L-type calcium current amplitude was significantly decreased by 50 and 100 μM of MnCl2. MRI on heart perfused with 25 μM of MnCl2 (no impact hemodynamic function) and 100 μM of MnCl2 showed a dose dependent decrease in the T1 relaxation time, a parameter for altered and disease state. Our results show that μM [MnCl2] (up to 25 μM) can be used as a contrast agent in MRI, without effects on cardiac hemodynamic parameters. MRI with manganese should therefore enable visualization of viable and stunned myocardium.

Protective role of m6A binding protein YTHDC2 on CCNB2 in manganese-induced spermatogenesis dysfunction

Human infertility has become the third largest serious disease in the world, seriously affecting the quality of human fertility. Studies have shown that manganese (Mn) can accumulate in the testis through the blood-testicular barrier and damage the male reproductive system. However, the mechanism has not been explored clearly. Recent studies have reported that YTH domain-containing 2 (YTHDC2) can regulate reproductive function. However, none has explored the role of YTHDC2 in Mn-induced reproductive toxicity. The present study investigated whether YTHDC2/CyclinB2 (CCNB2) pathway participates in Mn-induced reproductive toxicity using Kunming mice, spermatogonia, and the seminal plasma of male workers. The mice were received intraperitoneal (i.p.) injections of 0, 12.5, 25, and 50 mg/kg MnCl2 once daily for 2 weeks. The cells were treated with 0, 100, 200 and 400 μM MnCl2 for 24 h. Here, we found that occupational Mn exposure significantly increased Mn levels in the seminal plasma of male workers, while decreased sperm density, semen quality, and the levels of YTHDC2, CCNB1, and CCNB2. We found that Mn can inhibit the YTHDC2/CCNB2 signaling pathway and block the G2/M phase of the cell cycle. Moreover, the morphology of cells and the histomorphology of mice testis were injured. Notably, over-expression (OE) of YTHDC2 increased CCNB2 levels, reduced cell cycle arrest, and improved reproductive toxicity after Mn exposure. These findings suggest that the YTHDC2/CCNB2 signaling pathway participates in Mn-induced reproductive toxicity, and OE of YTHDC2 can mitigate the toxicity of Mn.

Neuroprotective effects of disubstituted dithiolethione ACDT against manganese-induced toxicity in SH-SY5Y cells

Dithiolethiones are lipophilic, organosulfur compounds that activate the Nrf2 transcription factor causing an upregulation of various phase II antioxidant enzymes. A disubstituted dithiolethione 5-amino-3-thioxo-3H-(1,2) dithiole-4-carboxylic acid ethyl ester (ACDT) retains the functional pharmacophore while also containing modifiable functional groups. Neuroprotection against autoimmune encephalomyelitis in vivo and 6-hydroxy dopamine (a model for Parkinson's disease) in vitro have been previously reported with ACDT. Manganese (Mn) is a metal essential for metabolic processes at low concentrations. Overexposure and accumulation of Mn leads to a neurological condition called manganism which shares pathophysiological sequelae with parkinsonism. Here we hypothesized ACDT to be protective against manganese-induced cytotoxicity. SH-SY5Y human neuroblastoma cells exposed to 300 μM MnCl2 displayed approximately 50% cell death, and a 24-h pretreatment with 75 μM ACDT significantly reversed this cytotoxicity. ACDT pretreatment was also found to increase total GSH levels (2.18-fold) and the protein levels of NADPH:quinone oxidoreductase-1 (NQO1) enzyme (6.33-fold), indicating an overall increase in the cells' antioxidant defense stores. A corresponding 2.32-fold reduction in the level of Mn-induced reactive oxygen species was also observed in cells pretreated with ACDT. While no changes were observed in the protein levels of apoptotic markers Bax and Bcl-2, pretreatment with 75 μM ACDT led to a 2.09-fold downregulation of ZIP14 import transporter, indicating a potential reduction in the cellular uptake of Mn as an additional neuroprotective mechanism. These effects did not extend to other transporters like the divalent metal transporter 1 (DMT1) or ferroportin. Collectively, ACDT showed substantial neuroprotection against Mn-induced cytotoxicity, opening a path for dithiolethiones as a potential novel therapeutic option against heavy metal neurotoxicity.

Effects of Excess Manganese on the Xylem Sap Protein Profile of Tomato (Solanum lycopersicum) as Revealed by Shotgun Proteomic Analysis.

Metal toxicity is a common problem in crop species worldwide. Some metals are naturally toxic, whereas others such as manganese (Mn) are essential micro-nutrients for plant growth but can become toxic when in excess. Changes in the composition of the xylem sap, which is the main pathway for ion transport within the plant, is therefore vital to understanding the plant’s response(s) to metal toxicity. In this study we have assessed the effects of exposure of tomato roots to excess Mn on the protein profile of the xylem sap, using a shotgun proteomics approach. Plants were grown in nutrient solution using 4.6 and 300 µM MnCl2 as control and excess Mn treatments, respectively. This approach yielded 668 proteins reliably identified and quantified. Excess Mn caused statistically significant (at p ≤ 0.05) and biologically relevant changes in relative abundance (≥2-fold increases or ≥50% decreases) in 322 proteins, with 82% of them predicted to be secretory using three different prediction tools, with more decreasing than increasing (181 and 82, respectively), suggesting that this metal stress causes an overall deactivation of metabolic pathways. Processes most affected by excess Mn were in the oxido-reductase, polysaccharide and protein metabolism classes. Excess Mn induced changes in hydrolases and peroxidases involved in cell wall degradation and lignin formation, respectively, consistent with the existence of alterations in the cell wall. Protein turnover was also affected, as indicated by the decrease in proteolytic enzymes and protein synthesis-related proteins. Excess Mn modified the redox environment of the xylem sap, with changes in the abundance of oxido-reductase and defense protein classes indicating a stress scenario. Finally, results indicate that excess Mn decreased the amounts of proteins associated with several signaling pathways, including fasciclin-like arabinogalactan-proteins and lipids, as well as proteases, which may be involved in the release of signaling peptides and protein maturation. The comparison of the proteins changing in abundance in xylem sap and roots indicate the existence of tissue-specific and systemic responses to excess Mn. Data are available via ProteomeXchange with identifier PXD021973.

Effect of Manganese Chloride and of Cotreatment with Cadmium Chloride on the In Vitro Proliferative, Motile and Invasive Behavior of MDA-MB231 Breast Cancer Cells.

We examined the dose–response effect of MnCl2 on the proliferative behavior of triple-negative breast cancer MDA-M231 cells vs. immortalized HB2 cells from breast epithelium taken as nontumoral counterparts. We also tested the effect of MnCl2 on tumor cell invasiveness in vitro by evaluating the relative invasion indexes through Boyden chamber assays. Moreover, we checked whether cotreatment with both MnCl2 and CdCl2 could modify the observed biological response by MDA-MB231 cells. Our results show a promotional impact of MnCl2 on cell proliferation, with 5 µM concentration inducing the more pronounced increase after 96-h exposure, which is not shared by HB2 cells. Exposure to 5 µM MnCl2 induced also an elevation of the relative invasion index of cancer cells. The Mn-mediated stimulatory effects were counteracted by cotreatment with CdCl2. These data support the concept that human exposure to high environmental concentrations of Mn may increase the risk of carcinogenesis and metastasis by prompting the expansion and dissemination of triple-negative breast cancer cells. On the other hand, the Mn-counteracting anticancer property of Cd looks promising and deserves a more detailed characterization of the involved intracellular targets aimed to the molecular modeling of specific antineoplastic agents against malignant breast cancer spreading.

Size-selective filtration of the atrial wall estimated from the accumulation of tracers in the kidney of the mussel Mytilus galloprovincialis.

In order to determine the molecular weight cut-off (MWCO) for the atrial wall filtration into kidneys of the Mytilus galloprovincialis, we employed five magnetic resonance (MR) tracers: manganese chloride (Mn2+), gadolinium chloride (Gd3+), manganese-ethylenediaminetetraacetic acid (MnEDTA), gadolinium-diethylenetriamine pentaacetic acid (GdDTPA) and oligomer-based contrast agent (CH3-DTPA-Gd). After injection of the MR tracers (1 or 2 mmol l-1×0.1ml) into the visceral mass, T1-weighted MR imaging (T1w-MRI) and the longitudinal relaxation rates (1/T1=R1) were measured at 20°C. The MR tracers were distributed uniformly in the visceral mass within 1 h after injection. The T1w-MRI intensity and R1 of the kidney (R1K) were increased by Mn2+ and MnEDTA, with urine concentrations estimated at 210 and 65 µmoll-1, respectively. The rest of the tracers showed only minimal or no increase. When the mussels were additionally incubated in seawater with 10 µmoll-1 MnCl2, R1K was increased in the GdDTPA group, but not in the GdCl3 group. Therefore, Gd3+ might have inhibited renal accumulation of Mn2+ and Gd3+ Incubation in seawater with 10 µmoll-1 MnEDTA showed no increase in the R1K, but additional incubation with 10 µmoll-1 MnCl2 caused an increase in R1K It is suggested that injected MnEDTA was filtrated as MnEDTA per se, and not likely separated into free Mn2+ Thus, we concluded that the MWCO of the atrial wall of the M. galloprovincialis is around 0.5 kDa, which is almost 1/100 of that for vertebrate animals, and suggests a reduction in efforts to reabsorb metabolites and osmolytes from the urine.

Manganese is a Deinococcus radiodurans growth limiting factor in rich culture medium.

To understand the effects triggered by Mn2+ on Deinococcus radiodurans, the proteome patterns associated with different growth phases were investigated. In particular, under physiological conditions we tested the growth rate and the biomass yield of D. radiodurans cultured in rich medium supplemented or not with MnCl2. The addition of 2.5-5.0 µM MnCl2 to the medium neither altered the growth rate nor the lag phase, but significantly increased the biomass yield. When higher MnCl2 concentrations were used (10-250 µM), biomass was again found to be positively affected, although we did observe a concentration-dependent lag phase increase. The in vivo concentration of Mn2+ was determined in cells grown in rich medium supplemented or not with 5 µM MnCl2. By atomic absorption spectroscopy, we estimated 0.2 and 0.75 mM Mn2+ concentrations in cells grown in control and enriched medium, respectively. We qualitatively confirmed this observation using a fluorescent turn-on sensor designed to selectively detect Mn2+in vivo. Finally, we investigated the proteome composition of cells grown for 15 or 19 h in medium to which 5 µM MnCl2 was added, and we compared these proteomes with those of cells grown in the control medium. The presence of 5 µM MnCl2 in the culture medium was found to alter the pI of some proteins, suggesting that manganese affects post-translational modifications. Further, we observed that Mn2+ represses enzymes linked to nucleotide recycling, and triggers overexpression of proteases and enzymes linked to the metabolism of amino acids.

Manganese transport and toxicity in polarized WIF-B hepatocytes.

Manganese (Mn) toxicity arises from nutritional problems, community and occupational exposures, and genetic risks. Mn blood levels are controlled by hepatobiliary clearance. The goals of this study were to determine the cellular distribution of Mn transporters in polarized hepatocytes, to establish an in vitro assay for hepatocyte Mn efflux, and to examine possible roles the Mn transporters would play in metal import and export. For these experiments, hepatocytoma WIF-B cells were grown for 12-14 days to achieve maximal polarity. Immunoblots showed that Mn transporters ZIP8, ZnT10, ferroportin (Fpn), and ZIP14 were present. Indirect immunofluorescence microscopy localized Fpn and ZIP14 to WIF-B cell basolateral domains whereas ZnT10 and ZIP8 associated with intracellular vesicular compartments. ZIP8-positive structures were distributed uniformly throughout the cytoplasm, but ZnT10-positive vesicles were adjacent to apical bile compartments. WIF-B cells were sensitive to Mn toxicity, showing decreased viability after 16 h exposure to >250 μM MnCl2. However, the hepatocytes were resistant to 4-h exposures of up to 500 μM MnCl2 despite 50-fold increased Mn content. Washout experiments showed time-dependent efflux with 80% Mn released after a 4 h chase period. Hepcidin reduced levels of Fpn in WIF-B cells, clearing Fpn from the cell surface, but Mn efflux was unaffected. The secretory inhibitor, brefeldin A, did block release of Mn from WIF-B cells, suggesting vesicle fusion may be involved in export. These results point to a possible role of ZnT10 to import Mn into vesicles that subsequently fuse with the apical membrane and empty their contents into bile. NEW & NOTEWORTHY Polarized WIF-B hepatocytes express manganese (Mn) transporters ZIP8, ZnT10, ferroportin (Fpn), and ZIP14. Fpn and ZIP14 localize to basolateral domains. ZnT10-positive vesicles were adjacent to apical bile compartments, and ZIP8-positive vesicles were distributed uniformly throughout the cytoplasm. WIF-B hepatocyte Mn export was resistant to hepcidin but inhibited by brefeldin A, pointing to an efflux mechanism involving ZnT10-mediated uptake of Mn into vesicles that subsequently fuse with and empty their contents across the apical bile canalicular membrane.

Involvement of gap junctions in astrocyte impairment induced by manganese exposure

Glutamate excitotoxicity, characterized as excessive glutamate stress, is considered to be involved in cerebral ischaemia, brain trauma, and neurodegenerative diseases such as Parkinson’s disease and Alzheimer's disease. Glutamate homeostasis disruption was highlighted in Mn neurotoxicity caused by high levels of Mn. Astrocytes, accounting for approximately 50% of the neuronal cells in the central nervous system and maintain glutamate homeostasis, are sensitive to neurotoxicity induced by Mn exposure. Astrocytes are tightly coupled with gap junctions (GJ), which are comprised of connexins, mainly connexin43 (Cx43). The gap junctional intercellular communication (GJIC) pathway allows small signal molecules, such as glutamate, ATP (adenosine triphosphate, ATP) and tropic factors, etc., to transfer between adjacent cells. Evidence has shown that astrocytes execute the bystander effect during cell death through the GJIC pathway. However, the pathogenic mechanism of the gap junction underlying glutamate neurotoxicity induced by manganese exposure has not been elucidated yet. In the present study, primary astrocytes were cultured and then exposed to different levels of Mn (ranging from 0 to 1000 μM) for 4/16 h to investigate the function of the GJIC in apoptosis induced by Mn. The cellular toxicity was confirmed by cell viability and apoptotic percentage through MTT assay and flow cytometry (FC). The levels of intracellular/extracellular glutamate were measured by high-performance liquid chromatography (HPLC). The fluorescent dye, Lucifer Yellow (LY), was used to assess the status of gap junctions among astrocytes after Mn exposure. The protein/gene expression of major gap junctional forming protein, Cx43, was also investigated. Cell viability was distinctly reduced when exposed to 500 and 1000 μM MnCl2 compared with control cells at both time points. The percentage of apoptosis was significantly increased among all detected Mn levels (125, 500 and 1000 μM MnCl2) of exposure (p < 0.05) with a concentration-dependent manner at either time point. Mn administration for 4/16 h also caused a remarkable intracellular/extracellular glutamate increase in a concentration-dependent manner for extracellular glutamate levels (p < 0.01). Gap junctions were prominently inhibited by Mn with Cx43 protein shown as shortening of the LY dye transfer distance at both time points. In-cell western blot indicated that Mn caused a decrease in Cx43 protein/gene expression in a dose-dependent manner. These results suggested that the gap junction intercellular communication and its forming protein, Cx43, are likely involved in glutamate excitotoxicity induced by Mn exposure.

Bacterial cyclic β-(1,2)-glucans sequester iron to protect against iron-induced toxicity.

Cellular iron homeostasis is critical for survival and growth. Bacteria employ a variety of strategies to sequester iron from the environment and to store intracellular iron surplus that can be utilized in iron-restricted conditions while also limiting the potential for the production of iron-induced reactive oxygen species (ROS). Here, we report that membrane-derived oligosaccharide (mdo) glucan, an intrinsic component of Gram-negative bacteria, sequesters the ferrous form of iron. Iron-binding, uptake, and localization experiments indicated that both secreted and periplasmic β-(1,2)-glucans bind iron specifically and promote growth under iron-restricted conditions. Xanthomonas campestris and Escherichia coli mutants blocked in the production of β-(1,2)-glucan accumulate low amounts of intracellular iron under iron-restricted conditions, whereas they exhibit elevated ROS production and sensitivity under iron-replete conditions. Our results reveal a critical role of glucan in intracellular iron homeostasis conserved in Gram-negative bacteria.

Manganese-calcium intermixing facilitates heteroepitaxial growth at the [formula omitted] calcite-water interface

Abstract In situ atomic force microscopy (AFM) measurements were performed to probe surface precipitates that formed on the 10 1 ¯ 4 surface of calcite (CaCO3) single crystals following reaction with Mn2+-bearing aqueous solutions. Three-dimensional epitaxial islands were observed to precipitate and grow on the surfaces. In situ time-sequenced measurements demonstrated that the growth rates were commensurate with those obtained for epitaxial islands formed on calcite crystals reacted with Cd2+-bearing aqueous solutions of the same range in supersaturation with respect to the pure metal carbonate phase. This finding was unexpected as rhodochrosite (MnCO3) and calcite display a 10% lattice mismatch, based on the area of their 10 1 ¯ 4 surface unit cells, whereas the lattice mismatch is only 4% for otavite (CdCO3) and calcite. Coatings of varying thicknesses were therefore synthesized by reacting calcite single crystals in calcite-equilibrated aqueous solutions with up to 250 μM MnCl2. Ex situ X-ray photoelectron spectroscopy (XPS), scanning transmission electron microscopy (STEM), energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), X-ray reflectivity (XRR), and AFM measurements of the reacted crystals demonstrated the formation of an epitaxial (Mn,Ca)CO3 solid solution. The epitaxial solid solution had a spatially complex composition, whereby the first few nanometers were rich in Ca and the Mn content increased with distance from the original calcite surface, culminating in a topmost region of almost pure MnCO3 for the thickest coatings. The effective lattice mismatch was therefore much smaller than the nominal mismatch thus explaining the measured growth rates. These findings highlight the strong influence played by the substrate on the composition of surface precipitates in aqueous conditions.

An improved glycerol biosensor with an Au-FeS-NAD-glycerol-dehydrogenase anode

An improved glycerol biosensor was developed via direct attachment of NAD+-glycerol dehydrogenase coenzyme-apoenzyme complex onto supporting gold electrodes, using novel inorganic iron (II) sulfide (FeS)-based single molecular wires. Sensing performance factors, i.e., sensitivity, a detection limit and response time of the FeS and conventional pyrroloquinoline quinone (PQQ)-based biosensor were evaluated by dynamic constant potential amperometry at 1.3V under non-buffered conditions. For glycerol concentrations ranging from 1 to 25mM, a 77% increase in sensitivity and a 53% decrease in detection limit were observed for the FeS-based biosensor when compared to the conventional PQQ-based counterpart. The electrochemical behavior of the FeS-based glycerol biosensor was analyzed at different concentrations of glycerol, accompanied by an investigation into the effects of applied potential and scan rate on the current response. Effects of enzyme stimulants ((NH4)2SO4 and MnCl2·4H2O) concentrations and buffers/pH (potassium phosphate buffer pH 6–8, Tris buffer pH 8–10) on the current responses generated by the FeS-based glycerol biosensor were also studied. The optimal detection conditions were 0.03M (NH4)2SO4 and 0.3µm MnCl2·4H2O in non-buffered aqueous electrolyte under stirring whereas under non-stirring, Tris buffer at pH 10 with 0.03M (NH4)2SO4 and 30µm MnCl2·4H2O were found to be optimal detection conditions. Interference by glucose, fructose, ethanol, and acetic acid in glycerol detection was studied. The observations indicated a promising enhancement in glycerol detection using the novel FeS-based glycerol sensing electrode compared to the conventional PQQ-based one. These findings support the premise that FeS-based bioanodes are capable of biosensing glycerol successfully and may be applicable for other enzymatic biosensors.

Evaluation of the Amount of the Manganese Entry in the Neurons for Activity‐Induced Manganese‐Enhanced MRI

SUMMARYThe activity‐induced manganese‐enhanced MRI (AIM‐MRI) is one of the candidate methods for measuring the neuronal activity noninvasively in vivo. This method has the potential to measure the history of the neuronal activity, since the Mn2+ can enter the active neuron through voltage‐dependent Ca2+ channels. However, the relation between the neuronal activity and the accumulation of Mn2+ in the cell has not been clear. At first, we confirmed that the longitudinal relaxation time of H+ (T1) measured by means of MRI apparatus was related to the [Mn2+] using the pseudo intracellular phantoms with various concentrations of Mn2+. The inverse of T1 (1/T1) was proportional to [Mn2+], thus [Mn2+] can be measured by MRI quantitatively. Next, we investigated the relation between the neuronal activity and the accumulation of Mn2+ in the striatal GABAergic neurons using the method of the Mn2+ quenching of Fura‐2 LR, a family of Fura fluorescent Ca2+ indicators. The amount of the Ca2+ influx was correlated to the neuronal activity evoked by tetanic stimulations with various frequencies. In the same slice preparation, the amounts of the fluorescence quench induced by the tetanic stimulations under the condition of 50 μM MnCl2 administration were recorded. As a result, the amount of the quench was proportional to the amount of the Ca2+ influx. These results suggested that a cell, which has higher activity, accumulated larger amount of Mn2+ in the neuron. Thus, our results supported that AIM‐MRI can measure the history of the neuronal activities in the whole brain noninvasively in vivo.

ARGINASE-BASED AMPEROMETRIC BIOSENSOR FOR MANGANESE IONS ANALYSIS

The development of simple cost-effective sensitive enzymatic methods for analysis of toxic metallic ions is an actual problem. Promising tools for elaboration of such methods are Mn2+-dependent enzymes. A novel manganese(II)-sensitive amperometric bi-enzyme biosensor based on of recombinant human arginase I (arginase) isolated from the gene-engineered strain of methylotrophic yeast Hansenula polymorpha and commercial urease is described. The biosensing layer with urease and apo-enzyme of arginase was placed onto a polyaniline-Nafion composite platinum electrode. The developed sensor revealed a high sensitivity to Mn2+-ions – 9200±20 A/(M∙m2)with the apparent Michaelis-Menten constant derived from Mn2+-ions calibration curve of 11.5±1.0 µM. A linear concentration range was observed from 1 µM to 6,5 µM MnCl2, a limit of detection being of 0.15 µM and a response time – 2.5 min. The proposed biosensor may be useful to monitor manganese compounds in laboratories of medicine, food industry and environmental control service.

The Bacterial Tyrosine Kinase Activator TkmA Contributes to Biofilm Formation Largely Independently of the Cognate Kinase PtkA in Bacillus subtilis.

In Bacillus subtilis, biosynthesis of exopolysaccharide (EPS), a key biofilm matrix component, is regulated at the posttranslational level by the bacterial tyrosine kinase (BY-kinase) EpsB. EpsB, in turn, relies on the cognate kinase activator EpsA for activation. A concerted role of a second BY-kinase-kinase activator pair, PtkA and TkmA, respectively in biofilm formation was also indicated in previous studies. However, the exact functions of PtkA and TkmA in biofilm formation remain unclear. In this work, we show that the kinase activator TkmA contributes to biofilm formation largely independently of the cognate kinase, PtkA. We further show that the biofilm defect caused by a ΔtkmA mutation can be rescued by complementation by epsA, suggesting a functional overlap between TkmA and EpsA and providing a possible explanation for the role of TkmA in biofilm formation. We also show that the importance of TkmA in biofilm formation depends largely on medium conditions; the biofilm defect of ΔtkmA is very severe in the biofilm medium LBGM (lysogenic broth [LB] supplemented with 1% [vol/vol] glycerol and 100 μM MnSO4) but marginal in another commonly used biofilm medium, MSgg (5 mM potassium phosphate [pH 7.0], MOPS [100 mM morpholinepropanesulfonic acid] [pH 7.0], 2 mM MgCl2, 700 μM CaCl2, 50 μM MnCl2, 50 μM FeCl3, 1 μM ZnCl2, 2 μM thiamine, 0.5% glycerol, 0.5% glutamic acid, 50 μg/ml tryptophan, 50 μg/ml threonine, and 50 μg/ml phenylalanine). The molecular basis for the medium dependence is likely due to differential expression of tkmA and epsA in the two different media and complex regulation of these genes by both Spo0A and DegU. Our studies provide genetic evidence for possible cross talk between a BY-kinase activator (TkmA) and a noncognate kinase (EpsB) and an example of how environmental conditions may influence such cross talk in regulating biofilm formation in B. subtilis. In bacteria, biosynthesis of secreted polysaccharides is often regulated by bacterial tyrosine kinases (BY-kinases). BY-kinases, in turn, rely on cognate kinase activators for activation. In this study, we investigated the role of a BY-kinase activator in biofilm formation in Bacillus subtilis. We present evidence that different BY-kinase activators may functionally overlap each other, as well as an example of how activities of the BY-kinase activators may be highly dependent on environmental conditions. Our study broadens the understanding of the complexity of regulation of the BY-kinases/kinase activators and the influence on bacterial cell physiology.