Direct Effects of Toxic Divalent Cations on Contractile Proteins with Implications for the Heart: Unraveling Mechanisms of Dysfunction.

The binding of calcium and magnesium ions to proteins is crucial for regulating heart contraction. However, other divalent cations, including xenobiotics, can accumulate in the myocardium and enter cardiomyocytes, where they can bind to proteins. In this article, we summarized the impact of these cations on myosin ATPase activity and EF-hand proteins, with special attention given to toxic cations. Optimal binding to EF-hand proteins occurs at an ionic radius close to that of Mg2+ and Ca2+. In skeletal Troponin C, Cd2+, Sr2+, Pb2+, Mn2+, Co2+, Ni2+, Ba2+, Mg2+, Zn2+, and trivalent lanthanides can substitute for Ca2+. As myosin ATPase is not a specific MgATPase, Ca2+, Fe2+, Mn2+, Ni2+, and Sr2+ could support myosin ATPase activity. On the other hand, Zn2+ and Cu2 significantly inhibit ATPase activity. The affinity to various divalent cations depends on certain proteins or their isoforms and can alter with amino acid substitution and post-translational modification. Cardiac EF-hand proteins and the myosin ATP-binding pocket are potential molecular targets for toxic cations, which could significantly alter the mechanical characteristics of the heart muscle at the molecular level.

Differential effects of magnesium, calcium, and sodium on Listeria monocytogenes biofilm formation.

Listeria monocytogenes is a gram-positive foodborne pathogen that causes outbreaks of listeriosis associated with a diverse range of foods. L. monocytogenes forms biofilms as a strategy to enhance its survival in the environment. These biofilms then provide a source of contamination in processing plant environments. Cations like magnesium, calcium, and sodium are commonly found in the environment and are important to bacteria to maintain their homeostasis. It is, therefore, valuable to understand the relationship between these cations and biofilm formation. In this study, four isolates of L. monocytogenes from seafood processing environments were used to investigate the influence of magnesium, calcium, and sodium (1, 10, and 50 mM) on biofilms. The isolates selected were defined as being either a low biofilm former, a high biofilm former, an outbreak isolate, and a persistent isolate from the seafood industry. The study showed that the divalent cations magnesium and calcium increased biofilm formation compared with the monovalent cation, sodium. Fifty mM concentrations of the divalent cations significantly enhanced biofilm formation. The cations did not have a significant effect on the initial stages of biofilm formation but appeared to influence the later stages of biofilm development.

Microtiter plate with built-in oxygen sensors: a novel approach to investigate the dynamics of Pseudomonas aeruginosa growth suppression in the presence of divalent cations and antibiotics.

The depletion of dissolved oxygen in a defined synthetic medium can be measured in real time, using a micro-well plate format, associated with a fluorescent plate reader. This technology is appropriate for investigating the effect of antibiotics on cell kinetics because there is a direct correlation between the latter and the amount of dissolved oxygen in the medium of an assay. In this study, the metabolic activity of the opportunistic human pathogen Pseudomonas aeruginosa PA01 was investigated using the OxoPlate OP96U optical sensor technology. The response of P. aeruginosa to aminoglycoside antibiotics when Ca2+and Mg2+ ions are present in the Evans defined synthetic medium was measured. The results revealed that the effect of antibiotics on P. aeruginosa is influenced by the concentration of divalent cations present in the test medium, although the efficiency of Ca2+ in supressing antibiotic activity was found to be greater than that of Mg2+. By comparison to tobramycin, the effect of amikacin is largely inhibited by the Ca2+and Mg2+concentrations. The study results underscore that the reliability of the observation of growth inhibitors is enhanced by the oxygen consumption measurements. Thus, the OxoPlate OP96U system is proven to be an accurate method to test the effectiveness of antibiotic treatments against P. aeruginosa.

ASIC1a senses lactate uptake to regulate metabolism in neurons.

Lactate is a major metabolite largely produced by astrocytes that nourishes neurons. ASIC1a, a Na+ and Ca2+-permeable channel with an extracellular proton sensing domain, is thought to be activated by lactate through chelation of divalent cations, including Ca2+, Mg2+ and Zn2+, that block the channel pore. Here, by monitoring lactate-evoked H+ and Ca2+ transport in cultured mouse cortical and hippocampal neurons, we find that stereo-selective neuronal uptake of L-lactate results in rapid intracellular acidification that triggers H+ extrusion to activate plasma membrane ASIC1a channels, leading to propagating Ca2+ waves into the cytosol and mitochondria. We show that lactate activates ASIC1a at its physiological concentrations, far below that needed to chelate divalent cations. The L-isomer of lactate exerts a much greater effect on ASIC1a-mediated activity than the d-isomer and this stereo-selectivity arises from lactate transporters, which prefer the physiologically common L-lactate. The lactate uptake in turn results in intracellular acidification, which is then followed by a robust acid extrusion. The latter response sufficiently lowers the pH in the vicinity of the extracellular domain of ASIC1a to trigger its activation, resulting in cytosolic and mitochondrial Ca2+ signals that accelerate mitochondrial respiration. Furthermore, blocking ASIC1a led to a robust mitochondrial ROS production induced by L-lactate. Together our results indicate that ASIC1a is a metabolic sensor, which by sensing extracellular pH drop triggered by neuronal lactate uptake with subsequent proton extrusion, transmits a Ca2+ response that is propagated to mitochondria to enhance lactate catabolism and suppress ROS production.

Assembly of Cas7 subunits of Leptospira on the mature crRNA of CRISPR-Cas I-B is modulated by divalent ions.

The spirochete Leptospira interrogans serovar Copenhageni harbors the genetic elements of the CRISPR-Cas type I-B system in its genome. CRISPR-Cas is a CRISPR RNA (crRNA) mediated adaptive immune system in most prokaryotes against mobile genetic elements (MGEs). To eliminate the intruding MGEs, CRISPR-Cas type I systems utilize a Cascade (CRISPR-associated complex for antiviral defense) complex composed of Cas5, Cas6, Cas7, and Cas8 bound with a crRNA. The Cas7 is essentially known to constitute the major component of the Cascade complex. The present study reports the biochemical characterization of the Cas7 (LinCas7) from the CRISPR-Cas type I-B system of L. interrogans serovar Copenhageni. The pure recombinant LinCas7 (rLinCas7) exists as a monomer in the solution by size exclusion chromatography. The rLinCas7 demonstrates an endoDNase activity dependent upon divalent Mg2+ ions, monovalent ions, pH, temperature, and substrate size. Analysis of ribonucleoprotein composite (rLinCas7-crRNA) by electron microscopy and native-PAGE demonstrated that rLinCas7 could oligomerize on the mature CRISPR RNA (crRNA) framework in the presence of Mg2+ ions. The ribonucleoprotein composite attains a helical shape similar to the backbone of the Cascade complex. However, in the absence of Mg2+ ions, rLinCas7 acts as an RNase. The fluorescence spectroscopy disclosed a weak interaction (Kd = 26.81 mM) between rLinCas7 and Mg2+ ions, leading to an overall conformational change in rLinCas7 that modulates the rLinCas7's activity on DNA and RNA substrates. The nuclease activity of LinCas7 characterized in this study aids to the functional divergences among proteins of the Cas7 family from different CRISPR-Cas systems in various organisms.

Revisiting Jatropha curcas Monomeric Esterase: A Dienelactone Hydrolase Compatible with the Electrostatic Catapult Model.

Jatropha curcas contains seeds with a high oil content, suitable for biodiesel production. After oil extraction, the remaining mass can be a rich source of enzymes. However, data from the literature describing physicochemical characteristics for a monomeric esterase from the J. curcas seed did not fit the electrostatic catapult model for esterases/lipases. We decided to reevaluate this J. curcas esterase and extend its characterization to check this apparent discrepancy and gain insights into the enzyme's potential as a biocatalyst. After anion exchange chromatography and two-dimensional gel electrophoresis, we identified the enzyme as belonging to the dienelactone hydrolase family, characterized by a cysteine as the nucleophile in the catalytic triad. The enzyme displayed a basic optimum hydrolysis pH of 9.0 and an acidic pI range, in contrast to literature data, making it well in line with the electrostatic catapult model. Furthermore, the enzyme showed low hydrolysis activity in an organic solvent-containing medium (isopropanol, acetonitrile, and ethanol), which reverted when recovering in an aqueous reaction mixture. This enzyme can be a valuable tool for hydrolysis reactions of short-chain esters, useful for pharmaceutical intermediates synthesis, due to both its high hydrolytic rate in basic pH and its stability in an organic solvent.

Divalent Cation Modulation of Ion Permeation in TMEM16 Proteins.

Intracellular divalent cations control the molecular function of transmembrane protein 16 (TMEM16) family members. Both anion channels (such as TMEM16A) and phospholipid scramblases (such as TMEM16F) in this family are activated by intracellular Ca2+ in the low µM range. In addition, intracellular Ca2+ or Co2+ at mM concentrations have been shown to further potentiate the saturated Ca2+-activated current of TMEM16A. In this study, we found that all alkaline earth divalent cations in mM concentrations can generate similar potentiation effects in TMEM16A when applied intracellularly, and that manipulations thought to deplete membrane phospholipids weaken the effect. In comparison, mM concentrations of divalent cations minimally potentiate the current of TMEM16F but significantly change its cation/anion selectivity. We suggest that divalent cations may increase local concentrations of permeant ions via a change in pore electrostatic potential, possibly acting through phospholipid head groups in or near the pore. Monovalent cations appear to exert a similar effect, although with a much lower affinity. Our findings resolve controversies regarding the ion selectivity of TMEM16 proteins. The physiological role of this mechanism, however, remains elusive because of the nearly constant high cation concentrations in cytosols.

Tumor Microenvironment-Responsive Nanococktails for Synergistic Enhancement of Cancer Treatment via Cascade Reactions.

A combination treatment strategy that relies on the synergetic effects of different therapeutic approaches has been considered to be an effective method for cancer therapy. Herein, a chemotherapeutic drug (doxorubicin, Dox) and a manganese ion (Mn2+) were co-loaded into regenerated silk fibroin-based nanoparticles (NPs), followed by the surface conjugation of phycocyanin (PC) to construct tumor microenvironment-activated nanococktails. The resultant PC-Mn@Dox-NPs showed increased drug release rates by responding to various stimulating factors (acidic pH, hydrogen peroxide (H2O2), and glutathione), revealing that they could efficiently release the payloads (Dox and Mn2+) in tumor cells. The released Dox could not only inhibit the growth of tumor cells but also generated a large amount of H2O2. The elevated H2O2 was decomposed into the highly harmful hydroxyl radicals and oxygen through an Mn2+-mediated Fenton-like reaction. Furthermore, the generated oxygen participated in photodynamic therapy (PDT) and produced abundant singlet oxygen. Our investigations demonstrate that these PC-Mn@Dox-NPs exhibit multiple bioresponsibilities and favorable biosafety. By integrating Dox-induced chemotherapy, Mn2+-mediated chemodynamic therapy, and PC-based PDT via cascade reactions, PC-Mn@Dox-NPs achieved enhanced in vitro and in vivo anticancer efficacies compared to all the mono- or dual-therapeutic approaches. These findings reveal that PC-Mn@Dox-NPs can be exploited as a promising nanococktail for cascade reaction-mediated synergistic cancer treatment.

The Effects of Divalent Cation-Chelated Prion Fibrils on the Immune Response of EOC 13.31 Microglia Cells.

Transmissible spongiform encephalopathies (TSEs) are epidemic neurodegenerative diseases caused by prion proteins; in particular, they are induced by misfolded prion proteins (PrPSc). PrPSc tend to aggregate into insoluble amyloid prion fibrils (fPrPWT), resulting in apoptosis of neuron cells and sequential neurodegeneration. Previous studies indicate that microglia cells play an important role in the innate immune system, and that these cells have good neuroprotection and delay the onset of TSEs. However, microglia can be a double-sided blade. For example, both Cu2+ and Mn2+ can induce microglia activation and secrete many inflammatory cytokines that are fatal to neuron cells. Unfortunately, PrP have cation binding sites at the N-terminus. When PrPSc accumulate during microglial phagocytosis, microglia may change the phenotype to secrete pro-inflammation cytokines, which increases the severity of the disease. Some studies have revealed an increase in the concentration of Mn2+ in the brains of patients. In this study, we treated microglia with fPrPWT and cations and determined IκBα and IL-1ß expression by Western blotting and quantitative polymerase chain reaction. The results showed that Mn-fPrPWT decreased IκBα levels and dramatically increased IL-1ß mRNA expression. In addition, competing binding between Cu2+ and Mn2+ can decrease the effect of Mn-fPrPWT on IκBα and IL-1ß. The effects of divalent cations and fPrPWT in microglia inflammation are also discussed.

Divalent cation-induced conformational changes of influenza virus hemagglutinin.

Divalent cations Cu2+ and Zn2+ can prevent the viral growth in mammalian cells during influenza infection, and viral titers decrease significantly on a copper surface. The underlying mechanisms include DNA damage by radicals, modulation of viral protease, M1 or neuraminidase, and morphological changes in viral particles. However, the molecular mechanisms underlying divalent cation-mediated antiviral activities are unclear. An unexpected observation of this study was that a Zn2+ ion is bound by Glu68 and His137 residues at the head regions of two neighboring trimers in the crystal structure of hemagglutinin (HA) derived from A/Thailand/CU44/2006. The binding of Zn2+ at high concentrations induced multimerization of HA and decreased its acid stability. The acid-induced conformational change of HA occurred even at neutral pH in the presence of Zn2+. The fusion of viral and host endosomal membranes requires substantial conformational changes in HA upon exposure to acidic pH. Therefore, our results suggest that binding of Zn2+ may facilitate the conformational changes of HA, analogous to that induced by acidic pH.

A Single Natural Variation Determines Cytosolic Ca2+-Mediated Hyperthermosensitivity of TRPA1s from Rattlesnakes and Boas.

Transient receptor potential ankyrin 1 from rattlesnakes (rsTRPA1) and boas (bTRPA1) was previously proposed to underlie thermo-sensitive infrared sensing based on transcript enrichment in infrared-sensing neurons and hyper-thermosensitivity expressed in Xenopus oocytes. It is unknown how these TRPA1s show thermosensitivities that overwhelm other thermoreceptors, and why rsTRPA1 is more thermosensitive than bTRPA1. Here, we show that snake TRPA1s differentially require Ca2+ for hyper-thermosensitivity and that predisposition to cytosolic Ca2+ potentiation correlates with superior thermosensitivity. Extracellularly applied Ca2+ upshifted the temperature coefficients (Q10s) of both TRPA1s, for which rsTRPA1, but not bTRPA1, requires cytosolic Ca2+. Intracellular Ca2+ chelation and substitutive mutations of the conserved cytosolic Ca2+-binding domain lowered rsTRPA1 thermosensitivity comparable to that of bTRPA1. Thapsigargin-evoked Ca2+ or calmodulin little affected rsTRPA1 activity or thermosensitivity, implying the importance of precise spatiotemporal action of Ca2+. Remarkably, a single rattlesnake-mimicking substitution in the conserved but presumably dormant cytosolic Ca2+-binding domain of bTRPA1 substantially enhanced thermosensitivity through cytosolic Ca2+ like rsTRPA1, indicating the capability of this single site in the determination of both cytosolic Ca2+ dependence and thermosensitivity. Collectively, these data suggest that Ca2+ is essential for the hyper-thermosensitivity of these TRPA1s, and cytosolic potentiation by permeating Ca2+ may contribute to the natural variation of infrared senses between rattlesnakes and boas.

Characterization of a novel glycoside hydrolase family 46 chitosanase, Csn-BAC, from Bacillus sp. MD-5.

Chitosanases play an important role in chitosan degradation, and the enzymatic degradation products of chitosan show various biological activities. Here, a novel glycoside hydrolase family 46 chitosanase (named Csn-BAC) from Bacillus sp. MD-5 was heterologously expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was purified by Ni-NTA affinity chromatography, and its molecular weight was estimated to be 35 kDa by SDS-PAGE. Csn-BAC showed maximal activity toward colloidal chitosan at pH 7 and 40 °C. The enzymatic activity of Csn-BAC was enhanced by Mn2+, Cu2+ and Co2+ at 1 mM, and by Mn2+ at 5 mM. Thin-layer chromatography and electrospray ionization-mass spectrometry results demonstrated that Csn-BAC exhibited an endo-type cleavage pattern and hydrolyzed chitosan to yield, mainly, (GlcN)2 and (GlcN)3. The enzymatic properties of this chitosanase may make it a good candidate for use in oligosaccharide production-based industries.

Antifungal Activity of Lactic Acid Bacteria Isolates and Their Associations: The Effects of Ca and Mg Divalent Cations.

The study of effects of Ca2+ and Mg2+ on antifungal activity of lactic acid bacteria (LAB) isolates and their associations revealed inducing and inhibiting effects on antifungal activity. The addition of Ca2+ essentially inhibited the antifungal effect of L. rhamnosus MDC9661 but stimulated the activity of RIN-2003-Ls, MDC9632 and MDC9633 strains, as well as their associations. Mg2+ partly increased the inhibitory activity of LAB isolates, while the addition of ions combination did not cause changes of their antifungal activity. The supplementation of Ca2+ stimulated the antifungal effect of most associations against Penicillium sp., Trichoderma viride, Geotrichum candidum, and Aspergillus flavus compared with the native conditions. The addition of Mg2+ induced the antifungal activity of RIN-2003-Ls, MDC9632, MDC9633, and INR-2010-Tsov-G-St combinations. The antifungal effects of most associations were increased in the presence of ions mixture. The natural LAB associations including VKPM B-3386, MDC9632, and MDC9633 could not suppress the growth of any tested mold; however, the supplementation of ions combination revealed their antifungal effect against all kinds of molds. The finding of substantial stimulation of the most LAB associations antifungal effect by metal ions can be basis for creation of new effective antifungal preparations by the supplementation of ions combined mixture.

Effects of Different Energy Substrates and Nickel and Cadmium Ions on the Growth of Acidithiobacillus ferrooxidans and Its Application for Disposal of Ni-Cd Batteries.

In the present work, the effects of different energy substrates and nickel ions (Ni2+) and cadmium ions (Cd2+) on the growth of Acidithiobacillus ferrooxidans (A. ferrooxidans) were investigated. Ferrous sulphate (FeSO4) was the optimum energy substrate for A. ferrooxidans growth, among the selected substrates. When cultured together with FeSO4 and sulphur (S), A. ferrooxidans first oxidised the ferrous ions (Fe2+), and the S was utilised as the concentration of Fe2+ decreased. After adapting to culture with Ni2+ and Cd2+, A. ferrooxidans presented good tolerance to both ions, with the maximum concentration reaching 4.11 g/L Ni2+ and 1.69 g/L Cd2+. A preliminary simulation of industrial application was also performed on used Ni-Cd batteries. With bioleaching, the highest concentrations of Cd2+ and Ni2+ were 3003 mg/L at day 8 and 1863 mg/L at day 14, respectively.

Divalent ion-induced aggregation of gold nanoparticles for voltammetry Immunosensing: comparison of transducer signals in an assay for the squamous cell carcinoma antigen.

A method is described for the electrochemical determination of squamous cell carcinoma (SCC) antigen, and by testing the effect of 30 nm gold nanoparticles (GNPs). Three comparative studies were performed in the presence and absence of GNPs, and with agglomerated GNPs. The divalent ion Ca(II) was used to induce a strong agglomeration of GNPs, as confirmed by colorimetry and voltammetry. Herein, colorimetry was used to test the best amount of salt needed to aggregate the GNPs. Despite, voltammetry was used to determine the status of biomolecules on the sensor. The topography of the surface of ZnO-coated interdigitated electrodes was analyzed by using 3D-nano profilometry, scanning electron microscopy, atomic force microscopy and high-power microscopy. The interaction between SCC antigen and antibody trigger vibrations on the sensor and cause dipole moment, which was measured using a picoammeter with a linear sweep from 0 to 2 V at 0.01 V step voltage. The sensitivity level was 10 fM by 3σ calculation for the dispersed GNP-conjugated antigen. This indicates a 100-fold enhancement compared to the condition without GNP conjugation. However, the sensitivity level for agglomerated GNPs conjugated antibody was not significant with 100 fM sensitivity. Specificity was tested for other proteins in serum, namely blood clotting factor IX, C-reactive protein, and serum albumin. The SCC antigen was quantified in spiked serum and gave recoveries that ranged between 80 and 90%. Graphical abstractSchematic representation of SCC (squamous cell carcinoma) antigen determination using divalent ion induced agglomerated GNPs. Sensitivity increment depends on the occurrence of more SCC antigen and antibody binding event via GNPs integration. Notably, lower detection limit was achieved at femto molar with proper orientation of biological molecules.

The Cap-Snatching SFTSV Endonuclease Domain Is an Antiviral Target.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne virus with 12%-30% case mortality rates and is related to the Heartland virus (HRTV) identified in the United States. Together, SFTSV and HRTV are emerging segmented, negative-sense RNA viral (sNSV) pathogens with potential global health impact. Here, we characterize the amino-terminal cap-snatching endonuclease domain of SFTSV polymerase (L) and solve a 2.4-Å X-ray crystal structure. While the overall structure is similar to those of other cap-snatching sNSV endonucleases, differences near the C terminus of the SFTSV endonuclease suggest divergence in regulation. Influenza virus endonuclease inhibitors, including the US Food and Drug Administration (FDA) approved Baloxavir (BXA), inhibit the endonuclease activity in in vitro enzymatic assays and in cell-based studies. BXA displays potent activity with a half maximal inhibitory concentration (IC50) of ∼100 nM in enzyme inhibition and an EC50 value of ∼250 nM against SFTSV and HRTV in plaque assays. Together, our data support sNSV endonucleases as an antiviral target.

The influence of temperature- and divalent-cation-mediated aggregation of ß-casein on the physical and microstructural properties of ß-casein-stabilised emulsions.

The objective of this study was to assess the influence of self-association of ß-casein (ß-CN) induced by both increasing temperature (5-55 °C) and divalent cation addition (Ca2+ or Mg2+) on the properties of ß-CN-stabilised emulsions. The particle size of 0.5% (w/w) ß-CN in 10 mM imidazole/HCl buffer (pH 6.8) was determined as a function of temperature and addition of divalent cations. Addition of CaCl2 caused a greater increase in protein particle size than MgCl2. Oil-in-water emulsions stabilised with 0.5% (w/w) ß-CN, ß-CN with added CaCl2 or MgCl2 (ß-CN/Ca and ß-CN/Mg, respectively) were also investigated as a function of temperature using light scattering, analytical centrifugation, rheology and confocal laser scanning microscopy (CLSM). Emulsions prepared with ß-CN/Ca flocculated after incubation at 55 °C for 20 min and displayed significantly different physical properties (p < 0.05) compared to emulsions stabilised with ß-CN or ß-CN/Mg in the temperature range 5-55 °C. Based on CLSM analysis and analysis of the interfacial protein load, this flocculation was attributed to the interaction of adsorbed ß-CN between droplets and the interaction of adsorbed and non-adsorbed ß-CN aggregates in the aqueous phase via calcium bridges. Furthermore, the flocculation of ß-CN/Ca emulsions was reversible upon cooling, which is similar to that of ß-CN/Ca in solution. In conclusion, the temperature-dependent behaviour of ß-CN-stabilised emulsions correlated to the temperature-induced aggregation of ß-CN, particularly in the presence of Ca2+. Hence, the stability of ß-CN-stabilised emulsions can be predicted from the extent of ß-CN aggregation in aqueous solution (i.e., aggregate size).

The effects of varying Mg2+ ion concentrations on contractions to the cotransmitters ATP and noradrenaline in the rat vas deferens.

The vas deferens responds to a single electrical pulse with a biphasic contraction caused by cotransmitters ATP and noradrenaline. Removing Mg2+ (normally 1.2 mM) from the physiological salt solution (PSS) enhances the contraction. This study aimed to determine the effect of Mg2+ concentration on nerve cotransmitter-mediated contractions. Rat vasa deferentia were sequentially bathed in increasing (0, 1.2, 3 mM) or decreasing (3, 1.2, 0 mM) Mg2+ concentrations. At each concentration a single field pulse was applied, and the biphasic contraction recorded. Contractions to exogenous noradrenaline 10 µM and ATP 100 µM were also determined. The biphasic nerve-mediated contraction was elicited by ATP and noradrenaline as NF449 (10 µM) and prazosin (100 nM) completely prevented the respective peaks. Taking the contractions in normal PSS (Mg2+ 1.2 mM) as 100%, lowering Mg2+ to 0 mM enhanced the ATP peak to 170 ±â€¯7% and raising Mg2+ to 3 mM decreased it to 39 ±â€¯3%; the noradrenaline peak was not affected by lowering Mg2+ to 0 mM (97 ±â€¯3%) but was decreased to 63 ±â€¯4% in high Mg2+ (3 mM). Contractions to exogenous ATP, but not noradrenaline, were increased in Mg2+ 0 mM and both were inhibited with Mg2+ 3 mM. Changing Mg2+ concentration affects the contractions elicited by the cotransmitters ATP and noradrenaline. The greatest effects were to potentiate the contraction to ATP in Mg2+ 0 mM and to inhibit the contraction to both ATP and noradrenaline in high Mg2+. Future publications should clearly justify any decision to vary the magnesium concentration from normal (1.2 mM) values.

Structure and function of the Toscana virus cap-snatching endonuclease.

Toscana virus (TOSV) is an arthropod-borne human pathogen responsible for seasonal outbreaks of fever and meningoencephalitis in the Mediterranean basin. TOSV is a segmented negative-strand RNA virus (sNSV) that belongs to the genus phlebovirus (family Phenuiviridae, order Bunyavirales), encompassing other important human pathogens such as Rift Valley fever virus (RVFV). Here, we carried out a structural and functional characterization of the TOSV cap-snatching endonuclease, an N terminal domain of the viral polymerase (L protein) that provides capped 3'OH primers for transcription. We report TOSV endonuclease crystal structures in the apo form, in complex with a di-ketoacid inhibitor (DPBA) and in an intermediate state of inhibitor release, showing details on substrate binding and active site dynamics. The structure reveals substantial folding rearrangements absent in previously reported cap-snatching endonucleases. These include the relocation of the N terminus and the appearance of new structural motifs important for transcription and replication. The enzyme shows high activity rates comparable to other His+ cap-snatching endonucleases. Moreover, the activity is dependent on conserved residues involved in metal ion and substrate binding. Altogether, these results bring new light on the structure and function of cap-snatching endonucleases and pave the way for the development of specific and broad-spectrum antivirals.

Influence of divalent cations on the biofouling behaviors of alginate hydrogels.

Alginate is one of the most favorable materials in many biomedical applications. The mechanical properties of alginate hydrogels can be easily tailored by adding different concentrations of divalent cations. In this work, we demonstrate that the method can also notably influence the biofouling behaviors of alginate hydrogels. A series of alginate hydrogels was prepared by tuning the concentrations of two types of divalent cation (Ca2+ or Ba2+). It was found that the biofouling behaviors of the hydrogels exhibited a 'U' curve tendency with the cation concentrations. Interestingly, we found that in optimal conditions ([Ca2+] = 0.9 mM or [Ba2+] = 0.54 mM), the resultant Ca0.9- and Ba0.54-alginate hydrogels were able to achieve negligible adhesion of the proteins and bacteria. Moreover, these two formulations were also able to prevent inflammatory responses at least 4 weeks after subcutaneous implantation in a mouse model. The findings in this work provide more insights into the design and development of appropriate alginate hydrogels for different applications.