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Ca 2+ homeostasis is crucial for the entire life of eukaryotic cells from the beginning to the end. Mishandling in Ca 2+ homeostasis is indeed linked with a large number of pathological conditions. Thus, the possibility to specifically monitor cellular calcium fluxes in different subcellular compartments represents a key tool to deeply understand the mechanisms involved in cellular dysfunctions. To cope with this need, several Ca 2+ indicators have been developed allowing to accurately measure both basal Ca 2+ concentration and agonist-induced Ca 2+ signals in a wide spectrum of organelles. Among these, the genetically encoded GFP-based indicators are routinely used to measure Ca 2+ transients thanks to their ability to change their spectral properties in response to Ca 2+ binding. In this chapter, we will describe a protocol that utilizes the GCaMP6f probe targeted to mitochondria (4mtGCaMP) to measure mitochondrial calcium levels in resting conditions in HeLa cells. This method allows to easily and quickly register alterations of mitochondrial Ca 2+ homeostasis in different cell populations and experimental settings, representing a precious tool to unravel the pathological pathways leading to pathogenic conditions.

Basal-like breast cancer (BLBC) is the most aggressive subtype with a poor clinical outcome; however, the molecular mechanisms underlying aggressiveness in BLBC remain poorly understood. Methods: The effects of gamma-aminobutyrate aminotransferase (ABAT) on GABA receptors, Ca 2+ -NFAT1 axis, and cancer cell behavior were assessed by Ca 2+ imaging, Western blotting, immunostaining, colony formation, and migration and invasion assays. We elucidated the relationship between ABAT and Snail by luciferase reporter and ChIP assays. The effect of ABAT expression on BLBC cells was determined by in vitro and in vivo tumorigenesis and a lung metastasis mouse model. Results: We showed that, compared to other subtypes, ABAT was considerably decreased in BLBC. Mechanistically, ABAT expression was downregulated due to Snail-mediated repression leading to increased GABA production. GABA then elevated intracellular Ca 2+ concentration by activating GABA-A receptor (GABA A ), which contributed to the efficient activation of NFAT1 in BLBC cells. ABAT expression resulted in inhibition of tumorigenicity, both in vitro and in vivo , and metastasis of BLBC cells. Thus, loss of ABAT contributed to BLBC aggressiveness by activating the Ca 2+ -NFAT1 axis. In breast cancer patients, loss of ABAT expression was strongly correlated with large tumor size, high grade and metastatic tendency, poor survival, and chemotherapy resistance. Conclusions: Our findings have provided underlying molecular details for the aggressive behavior of BLBC. The Snail-mediated downregulation of ABAT expression in BLBC provides tumorigenic and metastatic advantages by activating GABA-mediated Ca 2+ -NFAT1 axis. Thus, our results have identified potential prognostic indicators and therapeutic targets for this challenging disease., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Divalent cations can change the actual electrical potential at the outer surface of the plasma membrane. They do so by the so-called Gouy-Chapman-Stern effect which is due to the electrical "masking" that certain ions, especially divalents, can exert onto the electrically negative charged polar heads of the membrane phospholipids.Chondroitin sulfates can chelate free calcium ions to a different extent based on the spatial arrangement of their sulfate groups and can thus alter the actual availability of screening divalent ions at the outer membrane surface.Voltage-dependent ion channels sense the actual potential difference between the two sides of the plasma membrane and are thus exquisite and extremely sensitive "devices" able to react to changes in the electrical potential across the membrane.Hence, by recording the shift in the activation curve of well-known voltage-dependent ionic channels it will be possible to study the physical effect of ECM chondroitin sulfates on membrane conductances.

A chemotrophic, aerobic bacterial strain, Bacillus subtilis B2, was used to produce amylase by submerged fermentation under different light sources. SDS-PAGE indicated that the 55 kDa enzyme belonged to the α-amylase group. B2 was incubated in basal media with 1% soluble starch (pH 7.0) under blue, green, red, and white light-emitting diodes (LEDs), and white fluorescent light. Fermentation under blue LEDs maximized amylase production (180.59 ± 1.6 U/mL at 24 h). Production at 48 h increased to 310.56 ± 1.6 U/mL with 5% glucose as a simple carbon source and to 300.51 ± 1.7 U/mL with 5% groundnut oil cake as an agricultural waste substrate. Activity and stability of the amylase were greatest at pH 7.0 and 45-55 °C. Na + , Ca 2+ , Mg 2+ , Co 2+ , Ba 2+ , and K + increased activity, while Ni 2+ , Hg 2+ , Mn 2+ , Cu 2+ , Fe 3+ , and Zn 2+ inhibited activity. EDTA, PMSF and DTNB reduced activity by 50% or more, while tetrafluoroethylene and 1,10-phenanthroline reduced activity by 30%. The amylase was highly tolerant of the surfactants, compatible with organic solvents, oxidizing agents and the reducing agents reduced activity. These properties suggest utility of amylase produced by B. subtilis B2 under blue LED-mediated fermentation for industrial applications.

Fourier-transform infrared spectroscopy (FTIR) is a powerful tool for examining the metal coordination of the side chain COO - groups of Glu and Asp on Ca 2+ -binding proteins in solution. The behavior of COO - symmetric stretch can be investigated by using protein samples in H 2 O solution. However, it is difficult to obtain information about the behavior of the COO - antisymmetric stretch in H 2 O solution, because the COO - antisymmetric stretching band overlaps with the amide II band. Therefore, to obtain reliable infrared spectra in the region of COO - antisymmetric stretch, exchangeable protons in the protein should be completely deuterated by incubating the apoprotein dissolved in D 2 O under mild heating conditions.

Calcium ion (Ca 2+ ) is a ubiquitous intracellular messenger able to generate versatile intracellular signals that modulate a large variety of functions in virtually every cell type. Chemical and genetic biosensors, targeted to different subcellular compartments, have been developed and continuously improved to monitor Ca 2+ dynamics in living cells. Here we describe the usage of Förster resonance energy transfer (FRET)-based Cameleon probes to investigate Ca 2+ influx across the plasma membrane (PM) or Ca 2+ release from the main intracellular Ca 2+ store, the endoplasmic reticulum (ER).

The store-operated calcium (Ca 2+ ) entry (SOCE) pathway is an essential Ca 2+ signaling pathway in non-excitable cells that serve many physiological functions. SOCE is mediated through the plasma membrane (PM) protein, Orai1, and the endoplasmic reticulum protein, stromal interaction molecule 1 (STIM1). One of the most well-established methods to study SOCE is using the Ca 2+ -sensing dye, fura-2. Here we describe a detailed protocol on how to use fura-2 to study Ca 2+ signaling from SOCE in human embryonic kidney (HEK) cells.

In brain networks, neurons are constantly involved in a dynamic interaction with the other cell populations and, particularly, with astrocytes, the most abundant glial cells in the brain. Astrocytes respond to neurotransmitters with Ca 2+ elevations which represent a key event in the modulation of local brain circuits played by these glial cells. Due to technical limitations, the study of Ca 2+ signal dynamics in astrocytes has focused for decades almost exclusively on somatic and perisomatic regions. Accordingly, Ca 2+ signal in astrocytic fine protrusions, which are in close contact with the synapse, has been poorly investigated. Over the last years, the diffusion of novel tools such as the viral vector gene delivery of genetically encoded Ca 2+ indicators (GECI), the optogenetics, and multiphoton laser scanning microscopy has boosted significantly our capability to study astrocytic Ca 2+ signals in the different subcellular compartments. Here we report a protocol that combines these techniques to study astrocyte Ca 2+ signaling in response to somatostatin (SST)-expressing interneurons, one of the main classes of GABAergic inhibitory interneurons.

Functional contribution of S(E)R-mitochondria coupling to normal cellular processes is crucial and any alteration in S(E)R-mitochondria axis may be responsible for the onset of diseases. Mitochondrial free Zn 2+ level in cardiomyocytes ([Zn 2+ ] Mit ) is lower comparison to either its cytosolic or S(E)R level under physiological condition. However, there is little information about distribution of Zn 2+ -transporters on mitochondria and role of Zn 2+ -dependent mitochondrial-function associated with [Zn 2+ ] Mit . Since we recently have shown how hyperglycemia (HG)-induced changes in ZIP7 and ZnT7 contribute to Zn 2+ -transport across S(E)R and contribute to S(E)R-stress in the heart, herein, we hypothesized that these transporters can also be localized to mitochondria and affect the S(E)R-mitochondria coupling, and thereby contribute to cellular Zn 2+ -muffling between S(E)R-mitochondria in HG-cells. Mitochondrial localizations of ZIP7 and ZnT7 were demonstrated using fluorescence technique while they were confirmed in isolated mitochondrial fractions using biochemical analysis. Markedly decreased ZIP7 and increased ZnT7 levels were measured in isolated mitochondrial fractions from either HG- or doxorubicin, DOX (as positive control)-treated cardiomyocytes. Significantly increases in [Zn 2+ ] Mit and ROS production levels and depolarized mitochondrial membrane potential were also measured in HG cells. The expression levels of some key proteins, responsible for proper S(E)R-mitochondria coupling such as Mfn-1, Fis-1, OPA1, BAP31, STIM1 and PML in either HG- or DOX-cells were supported our above hypothesis, strongly. Overall, this study provides an important description about the role of ZIP7 and ZnT7, localized to both mitochondria and S(E)R and contribute to cellular Zn 2+ -muffling between cellular-compartments in HG or hypertrophic cardiomyocytes via affecting S(E)R-mitochondria coupling. Any alteration in this axis and/or cellular [Zn 2+ ] may provide new insight for prevention/therapy of HF in diabetes and/or hypertrophy., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

Lysosomes are emerging as calcium store organelles that can modulate various intracellular processes such as the regulation of nutrient signaling through the activation of TFEB, a master gene for lysosomal function, or very specialized functions like lysosomal exocytosis. Here, we describe two different techniques that can be used to study these processes. In the case report, we described two studies where these methodologies allowed us to unravel the role of calcineurin in the dephosphorylation of TFEB as well as the involvement of TFEB in lysosomal exocytosis, respectively.

Ions are important regulators for the cellular function of many proteins. This holds particularly true for S100 proteins whose function is not only calcium-dependent but also appears to be modulated by other divalent cations such as zinc, manganese, or copper. One way ions are thought to influence the function of S100 proteins (and any protein in general) is by changing their three-dimensional organization, through modifications in either their monomeric shape, their oligomeric state, or both. X-ray crystallography is a very powerful technique to study the effect of ions on the 3D architecture of macromolecules since it gives a direct visualization of where ions bind and how the protein structure is affected upon ion binding. Taking the example of human S100A8, I describe here the complete procedure to obtain a highly pure and homogenous S100 protein sample, crystallize it in the presence of divalent cations, and derive a 3D structural model from diffraction images. I further detail computational methods used to determine precisely the nature and position of the divalent cations within S100A8 structure. This methodology can easily be applied to any ion-binding protein, provided that the ion anomalous scattering properties allow to identify it unambiguously.

Cellular signaling represents an evolution of biological systems to sense external stimuli and communicate extracellular microenvironment to the intracellular compartments. The processes underlying molecular signaling have been widely studied due to their important cellular functions. There are numerous techniques available to quantitate the different molecules involved in cellular processes. Among them, calcium is a ubiquitous signaling molecule involved in many biological pathways. Over time the methods to measure intracellular calcium have advanced to better understand its role as a second messenger. In this chapter, we introduce a method to study a single cilium, a mechanosensor that elicits a calcium signaling cascade. To successfully observe the calcium changes in this thin cylindrical-like projection from the cell surface, we utilize a genetically encoded sensor with a high spatial and temporal resolution. In addition, the probe must be localized to the ciliary compartment in order to observe the intraciliary calcium signaling dynamics. To this end, a cilium targeting genetically encoded indicator is used to observe calcium fluxes in both cytoplasm and cilioplasm., (© 2019 Elsevier Inc. All rights reserved.)

Ca 2+ ion is universally considered the most versatile second messenger responsible for decoding and regulating the majority of the signaling pathways within the cell. The study of intracellular Ca 2+ concentration ([Ca 2+ ] i ) dynamics is consequently of primary importance for the interpretation of cellular biology. This chapter will present a relatively simple, largely diffused, and nevertheless robust method to measure variations of [Ca 2+ ] i by the use of the Ca 2+ -sensitive chemical dye Fura-2. A general protocol for the assessment of [Ca 2+ ] i in adherent cells, applicable to a variety of cell systems, will be first presented. Then, the implementation of Fura-2 to detect [Ca 2+ ] i in two specific cell types, namely, human adrenocortical cells and primary skin fibroblasts, will be discussed in more particulars. Finally, the procedure to monitor Ca 2+ influx through the plasma membrane using Fura-2 will be described.

Studying Ca 2+ dynamics in protozoan parasites is not an easy task. Loading of parasites with commonly used Ca 2+ fluorescent dyes (such as Fuo4-AM) remains as the major protocol to measure the Ca 2+ oscillations inside the cell. In this chapter, we describe an alternative method to study Ca 2+ signaling in Plasmodium falciparum parasite. This method employs the construction of transgenic parasites (through standard molecular biology techniques), selection of the transfected population, and use of those parasites in spectrofluorometric Ca 2+ assays.

Mitochondria accumulate significant amounts of calcium when cytosolic calcium is elevated above the resting levels of 50-100 nM during signaling events. This calcium uptake is primarily mediated by a macromolecular protein assembly called mitochondrial calcium uniporter (MCU) that resides in the mitochondrial inner membrane. In 2004, we applied patch-clamp technique for the first time to record calcium currents from the mitochondrial inner membrane and proved unequivocally that MCU is a highly selective calcium channel. This chapter describes how patch-clamp technique can be applied to record the Ca 2+ uniporter currents from the mitochondrial inner membrane, isolation of mitochondria from the heart tissue, and preparation of mitoplast using French Press. We also discuss advantages of patch-clamp technique as compared to other methods of determining mitochondrial uniporter activity and important considerations in applying patch-clamp technique to such a small subcellular organelle. With small variations in the bath and pipette solution composition, the same methodology can be applied to study any other currents (e.g., H + or Cl - ) from the mitochondrial inner membrane.

One of the pathological site effects in excitotoxic activation is Zn2+ overload to postsynaptic neurons. Such an effect is considered to be equivalent to the glutamate component of excitotoxicity. Excessive uptake of Zn2+ by active voltage-dependent transport systems in these neurons may lead to significant neurotoxicity. The aim of this study was to investigate whether and which antagonists of the voltage gated calcium channels (VGCC) might modify this Zn2+-induced neurotoxicity in neuronal cells. Our data demonstrates that depolarized SN56 neuronal cells may take up large amounts of Zn2+ and store these in cytoplasmic and mitochondrial sub-fractions. The mitochondrial Zn2+ excess suppressed pyruvate uptake and oxidation. Such suppression was caused by inhibition of pyruvate dehydrogenase complex, aconitase and NADP-isocitrate dehydrogenase activities, resulting in the yielding of acetyl-CoA and ATP shortages. Moreover, incoming Zn2+ increased both oxidized glutathione and malondialdehyde levels, known parameters of oxidative stress. In depolarized SN56 cells, nifedipine treatment (L-type VGCC antagonist) reduced Zn2+ uptake and oxidative stress. The treatment applied prevented the activities of PDHC, aconitase and NADP-IDH enzymes, and also yielded the maintenance of acetyl-CoA and ATP levels. Apart from suppression of oxidative stress, N- and P/Q-type VGCCs presented a similar, but weaker protective influence. In conclusion, our data shows that in the course of excitotoxity, impairment to calcium homeostasis is tightly linked with an excessive neuronal Zn2+ uptake. Hence, the VGCCs types L, N and P/Q share responsibility for neuronal Zn2+ overload followed by significant energy-dependent neurotoxicity. Moreover, Zn2+ affects the target tricarboxylic acid cycle enzymes, yields acetyl-CoA and energy deficits as well., Competing Interests: The authors have declared that no competing interests exist.

In gastrointestinal smooth muscle, acetylcholine induced muscle contraction is biphasic, initial peak followed by sustained contraction. Contraction is regulated by phosphorylation of 20 kDa myosin light chain (MLC) at Ser19, interaction of actin and myosin, and actin polymerization. The present study characterized the signaling mechanisms involved in actin polymerization during initial and sustained muscle contraction in response to muscarinic M3 receptor activation in gastric smooth muscle cells by targeting the effectors of initial (phospholipase C (PLC)-β/Ca2+ pathway) and sustained (RhoA/focal adhesion kinase (FAK)/Rho kinase pathway) contraction. The initial Ca2+ dependent contraction and actin polymerization is mediated by sequential activation of PLC-β1 via Gαq, IP3 formation, Ca2+ release and Ca2+ dependent phosphorylation of proline-rich-tyrosine kinase 2 (Pyk2) at Tyr402. The sustained Ca2+ independent contraction and actin polymerization is mediated by activation of RhoA, and phosphorylation of FAK at Tyr397. Both phosphorylation of Pyk2 and FAK leads to phosphorylation of paxillin at Tyr118 and association of phosphorylated paxillin with the GEF proteins p21-activated kinase (PAK) interacting exchange factor α, β (α and β PIX) and DOCK 180. These GEF proteins stimulate Cdc42 leading to the activation of nucleation promoting factor N-WASP (neuronal Wiskott-Aldrich syndrome protein), which interacts with actin related protein complex 2/3 (Arp2/3) to induce actin polymerization and muscle contraction. Acetylcholine induced muscle contraction is inhibited by actin polymerization inhibitors. Thus, our results suggest that a novel mechanism for the regulation of smooth muscle contraction is mediated by actin polymerization in gastrointestinal smooth muscle which is independent of MLC20 phosphorylation., Competing Interests: The authors have declared that no competing interests exist.

The dental caries pathogen Streptococcus mutans can ferment a variety of sugars to produce organic acids. Exposure of S. mutans to certain nonmetabolizable carbohydrates, such as xylitol, impairs growth and can cause cell death. Recently, the presence of a sugar-phosphate stress in S. mutans was demonstrated using a mutant lacking 1-phosphofructokinase (FruK) that accumulates fructose-1-phosphate (F-1-P). Here, we studied an operon in S. mutans , sppRA , which was highly expressed in the fruK mutant. Biochemical characterization of a recombinant SppA protein indicated that it possessed hexose-phosphate phosphohydrolase activity, with preferences for F-1-P and, to a lesser degree, fructose-6-phosphate (F-6-P). SppA activity was stimulated by Mg 2+ and Mn 2+ but inhibited by NaF. SppR, a DeoR family regulator, repressed the expression of the sppRA operon to minimum levels in the absence of the fructose-derived metabolite F-1-P and likely also F-6-P. The accumulation of F-1-P, as a result of growth on fructose, not only induced sppA expression, but it significantly altered biofilm maturation through increased cell lysis and enhanced extracellular DNA release. Constitutive expression of sppA , via a plasmid or by deleting sppR , greatly alleviated fructose-induced stress in a fruK mutant, enhanced resistance to xylitol, and reversed the effects of fructose on biofilm formation. Finally, by identifying three additional putative phosphatases that are capable of promoting sugar-phosphate tolerance, we show that S. mutans is capable of mounting a sugar-phosphate stress response by modulating the levels of certain glycolytic intermediates, functions that are interconnected with the ability of the organism to manifest key virulence behaviors. IMPORTANCE Streptococcus mutans is a major etiologic agent for dental caries, primarily due to its ability to form biofilms on the tooth surface and to convert carbohydrates into organic acids. We have discovered a two-gene operon in S. mutans that regulates fructose metabolism by controlling the levels of fructose-1-phosphate, a potential signaling compound that affects bacterial behaviors. With fructose becoming increasingly common and abundant in the human diet, we reveal the ways that fructose may alter bacterial development, stress tolerance, and microbial ecology in the oral cavity to promote oral diseases., (Copyright © 2018 American Society for Microbiology.)

Histone deacetylases (HDACs) catalyze the hydrolysis of Ɛ-acetyl-lysine residues of histones. Removal of acetyl groups results in condensation of chromatin structure and repression of gene expression. Human class I, II, and IV HDACs are said to be zinc-dependent in that they require divalent zinc ions to catalyze the deacetylase reaction. HDACs are considered potential targets for the treatment of cancer due to their role in regulating transcription. They are also thought to play important roles in the development of organisms such as honey bees. The fatty acid, 10-hydroxy-2E-decenoic acid (10-HDA), which can account for up to 5% of royal jelly composition has been reported as an HDAC inhibitor. The crystal structure of the HDAC3:SMRT complex possesses two monovalent cations (MVCs) labeled as potassium with one MVC binding site near the active site Zn(II) and the second MVC binding site ≥20 Å from the active site Zn(II). We report here the inhibitory effects of excess Zn(II) on the catalytic activity of histone deacetylase 3 (HDAC3) bound to the deacetylase activating domain of nuclear receptor corepressor 2 (NCOR2). We also report the effects of varying concentrations of potassium ions where [K+] up to 10 mM increase HDAC3 activity with a maximum kcat/KM of approximately 80,000 M-1s-1 while [K+] above 10 mM inhibit HDAC3 activity. The inhibition constant (Ki) of 10-HDA was determined to be 5.32 mM. The regulatory effects of zinc, potassium, and 10-HDA concentration on HDAC3 activity suggest a strong correlation between these chemical species and epigenetic control over Apis mellifera caste differentiation among other control mechanisms., Competing Interests: The authors have declared that no competing interests exist.

Background: The plasma membrane Na + /Ca 2+ -exchanger (NCX) has recently been shown to regulate Ca 2+ -dependent N-methyl-D-aspartate receptor (NMDAR) desensitization, suggesting a tight interaction of NCXs and NMDARs in lipid nanoclasters or "rafts". To evaluate possible role of this interaction we studied effects of Li + on NMDA-elicited whole-cell currents and Ca 2+ responses of rat cortical neurons in vitro before and after cholesterol extraction by methyl-β-cyclodextrin (MβCD)., Results: Substitution Li + for Na + in the external solution caused a concentration-dependent decrease of steady-state NMDAR currents from 440 ± 71 pA to 111 ± 29 pA in 140 mM Na + and 140 mM Li + , respectively. The Li + inhibition of NMDAR currents disappeared in the absence of Ca 2+ in the external solution (Ca 2+ -free), suggesting that Li + enhanced Ca 2+ -dependent NMDAR desensitization. Whereas the cholesterol extraction with MβCD induced a decrease of NMDAR currents to 136 ± 32 pA in 140 mM Na + and 46 ± 15 pA in 140 mM Li + , the IC 50 values for the Li + inhibition were similar (about 44 mM Li + ) before and after this procedure. In the Ca 2+ -free Na + solution the steady-state NMDAR currents after the cholesterol extraction were 47 ± 6% of control values. Apparently this amplitude decrease was not Ca 2+ -dependent. In the Na + solution containing 1 mM Ca 2+ the Ca 2+ -dependent NMDAR desensitization was greater when cholesterol was extracted. Obviously, this procedure promoted its development. In agreement, Li + and KB-R7943, an inhibitor of NCX, both considerably reduced NMDA-activated Ca 2+ responses. The cholesterol extraction itself caused a decrease of NMDA-activated Ca 2+ responses and, in addition, abolished the effects of Li + and KB-R7943. The cholesterol loading into the plasma membrane caused a recovery of the KB-R7943 effects., Conclusions: Taken together our data suggest that NCXs downregulate the Ca 2+ -dependent NMDAR desensitization. Most likely, this is determined by a tight functional interaction of NCX and NMDAR molecules because of their co-localization in membrane lipid rafts. The destruction of these rafts is accompanied by an enhancement of NMDAR desensitization and a loss of NCX-selective agent effects on NMDARs.

Aims: Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) predisposes to ventricular tachyarrhythmias (VTs) during high heart rates due to physical or psychological stress. The essential role of catecholaminergic effects on ventricular cardiomyocytes in this situation is well documented, but the importance of heart rate per se for arrhythmia initiation in CPVT1 is largely unexplored., Methods and Results: Sixteen CPVT1 patients performed a bicycle stress-test. Occurrence of VT triggers, i.e. premature ventricular complexes (PVC), depended on high heart rate, with individual thresholds. Atrial pacing above the individual PVC threshold in three patients did not induce PVCs. The underlying mechanism for the clinical observation was explored using cardiomyocytes from mice with the RyR2-R2474S (RyR2-RS) mutation, which exhibit exercise-induced VTs. While rapid pacing increased the number of Ca2+ waves in both RyR2-RS and wild-type (p<0.05), β-adrenoceptor (βAR) stimulation induced more Ca2+ waves in RyR2-RS (p<0.05). Notably, Ca2+ waves occurred despite decreased sarcoplasmic reticulum (SR) Ca2+ content in RyR2-RS (p<0.05), suggesting increased cytosolic RyR2 Ca2+ sensitivity. A computational model of mouse ventricular cardiomyocyte electrophysiology reproduced the cellular CPVT1 phenotype when RyR2 Ca2+ sensitivity was increased. Importantly, diastolic fluctuations in phosphorylation of RyR2 and SR Ca2+ content determined Ca2+ wave initiation. These factors were modulated towards increased propensity for arrhythmia initiation by increased pacing rates, but even more by βAR stimulation., Conclusion: In CPVT1, VT propensity depends on individual heart rate thresholds for PVCs. Through converging data from clinical exercise stress-testing, cellular studies and computational modelling, we confirm the heart rate-independent pro-arrhythmic effects of βAR stimulation in CPVT1, but also identify an independent and synergistic contribution from effects of high heart rate., Competing Interests: The authors have declared that no competing interests exist.

Mycobacterium leprae uptakes various bivalent metal ions via different transporters in host species. Uptake of Cu 2+ and Zn 2+ are essential for generation of superoxide dismutases and catalases, which provide defense against reactive oxygen species mediated death of this pathogen in macrophages. Furthermore, it has also been noticed that levels of different bivalent metal ions (Ca 2+ , Mg 2+ , Cu 2+ and Zn 2+ ) in blood serum are altered in leprotic patients. Mycobacterium leprae HSP18 is an immunodominant antigen which helps in growth and survival of Mycobacterium leprae in host species. A possible link can exist between HSP18 and aberration of bivalent metal ion homeostasis. Therefore, we investigated the interaction of these four bivalent metal ions with HSP18 and found that the protein only interacts with Zn 2+ and Cu 2+ . Such association process is reversible and moderately high affinity in nature with unit binding stoichiometry. Theoretical studies revealed that the most probable site for Zn 2+ -binding lies in the N-terminal domain; While, the same for Cu 2+ -binding lies in the "α-crystallin domain" of HSP18. Binding of Zn 2+ /Cu 2+ to HSP18 brings about subtle changes in the secondary and tertiary structure of HSP18 but are distinctly opposite in nature. While Zn 2+ causes oligomeric association, Cu 2+ leads to oligomeric dissociation of HSP18. Structural stability, surface hydrophobicity and chaperone activity of HSP18 are enhanced on Zn 2+ binding, while all of them are reduced upon Cu 2+ binding. Altogether, metal ions binding to HSP18 regulate its function which may have far reaching effect on the survival and pathogenicity of Mycobacterium leprae in host species., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Xenograft, namely bone-derived biological apatite (BAp), is widely recognized as a favorable biomaterial in bone tissue engineering owing to its biodegradability, biocompatibility, and osteoconductive properties. Substitutions of endogenous trace ions are thought to improve the osteogenic capacity of xenograft compared with synthetic hydroxyapatite (HAp). In order to modify the physicochemical and biological properties of apatite, different approaches to induce trace ion incorporation have been widely considered. In this study, we demonstrated that the incorporation of fluoride ions into porcine bone-derived biological apatite (pBAp) contributes to altered crystal morphology of the apatite, the sustained release of fluoride, and the in situ release of endogenous trace ions (e.g., magnesium and calcium) into the peripheral tissue microenvironment. This ionic balanced perimaterial microenvironment not only led to superior proliferation and osteogenic differentiation of rat bone mesenchymal stem cells (rBMSCs), but also accelerated new bone formation of the calvarial defect on a rat model via the activation of Wnt/β-catenin signaling. These promising observations may be attributed to the controlled release of endogenous trace ions from the xenograft to the peripheral tissue microenvironment driven by fluoride ion incorporation. Lastly, this study may provide a new insight to strengthen the osteogenicity of xenografts for clinical applications in the future.

Modulation of RNA structure is essential in the life cycle of RNA viruses. Immediate replication upon infection requires RNA unwinding to ensure that RNA templates are not in intra- or intermolecular duplex forms. The calicivirus NS3, one of the highly conserved nonstructural (NS) proteins, has conserved motifs common to helicase superfamily 3 among six genogroups. However, its biological functions are not fully understood. In this study we report the oligomeric state and the nucleotide triphosphatase (NTPase) and RNA chaperone activities of the recombinant full-length NS3 derived from murine norovirus (MNV). The MNV NS3 has an Mg 2+ -dependent NTPase activity, and site-directed mutagenesis of the conserved NTPase motifs blocked enzyme activity and viral replication in cells. Further, the NS3 was found via fluorescence resonance energy transfer (FRET)-based assays to destabilize double-stranded RNA in the presence of Mg 2+ or Mn 2+ in an NTP-independent manner. However, the RNA destabilization activity was not affected by mutagenesis of the conserved motifs of NTPase. These results reveal that the MNV NS3 has an NTPase-independent RNA chaperone-like activity, and that a FRET-based RNA destabilization assay has the potential to identify new antiviral drugs targeting NS3.

Reduced cardiac contractility during heart failure (HF) is linked to impaired Ca 2+ release from Ryanodine Receptors (RyRs). We investigated whether this deficit can be traced to nanoscale RyR reorganization. Using super-resolution imaging, we observed dispersion of RyR clusters in cardiomyocytes from post-infarction HF rats, resulting in more numerous, smaller clusters. Functional groupings of RyR clusters which produce Ca 2+ sparks (Ca 2+ release units, CRUs) also became less solid. An increased fraction of small CRUs in HF was linked to augmented 'silent' Ca 2+ leak, not visible as sparks. Larger multi-cluster CRUs common in HF also exhibited low fidelity spark generation. When successfully triggered, sparks in failing cells displayed slow kinetics as Ca 2+ spread across dispersed CRUs. During the action potential, these slow sparks protracted and desynchronized the overall Ca 2+ transient. Thus, nanoscale RyR reorganization during HF augments Ca 2+ leak and slows Ca 2+ release kinetics, leading to weakened contraction in this disease., Competing Interests: TK, Jv, NM, PL, MF, JA, EN, AC, IS, OS, AE, GL, WL No competing interests declared, (© 2018, Kolstad et al.)

Spinal muscular atrophy (SMA) is a severe genetic disorder that manifests in progressive neuromuscular degeneration. SMA originates from loss-of-function mutations of the SMN1 (Survival of Motor Neuron 1) gene. Recent evidence has implicated peripheral deficits, especially in skeletal muscle, as key contributors to disease progression in SMA. In this study we generated myogenic cells from two SMA-affected human embryonic stem cell (hESC) lines with deletion of SMN1 bearing two copies of the SMN2 gene and recapitulating the molecular phenotype of Type 1 SMA. We characterized myoblasts and myotubes by comparing them to two unaffected, control hESC lines and demonstrate that SMA myoblasts and myotubes showed altered expression of various myogenic markers, which translated into an impaired in vitro myogenic maturation and development process. Additionally, we provide evidence that these SMN1 deficient cells display functional deficits in cholinergic calcium signaling response, glycolysis and oxidative phosphorylation. Our data describe a novel human myogenic SMA model that might be used for interrogating the effect of SMN depletion during skeletal muscle development, and as model to investigate biological mechanisms targeting myogenic differentiation, mitochondrial respiration and calcium signaling processes in SMA muscle cells., Competing Interests: NH, DH, SS, CT, KM, ME, FM were employed by F. Hoffmann-La Roche while conducting the work on this project and declare no conflict of interest. SP, AS, SL, CP, MHB, US were employed by Genea Biocells while conducting the work on this project and declare no conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The structure of the actin filament is known at a resolution that has allowed the architecture of protein components to be unambiguously assigned. However, fully understanding the chemistry of the system requires higher resolution to identify the ions and water molecules involved in polymerization and ATP hydrolysis. Here, we find experimental evidence for the association of cations with the surfaces of G-actin in a 2.0-Å resolution X-ray structure of actin bound to a Cordon-Bleu WH2 motif and in previously determined high-resolution X-ray structures. Three of four reoccurring divalent cation sites were stable during molecular dynamics (MD) simulations of the filament, suggesting that these sites may play a functional role in stabilizing the filament. We modeled the water coordination at the ATP-bound Mg 2+ , which also proved to be stable during the MD simulations. Using this model of the filament with a hydrated ATP-bound Mg 2+ , we compared the cumulative probability of an activated hydrolytic water molecule approaching the γ-phosphorous of ATP, in comparison with G-actin, in the MD simulations. The cumulative probability increased in F-actin in line with the activation of actin's ATPase activity on polymerization. However, inclusion of the cations in the filament lowered cumulative probability, suggesting the rate of hydrolysis may be linked to filament flexibility. Together, these data extend the possible roles of Mg 2+ in polymerization and the mechanism of polymerization-induced activation of actin's ATPase activity., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

The gliotransmitter glutamate in different brain regions modulates neuronal excitability and synaptic transmission through a variety of mechanisms. Among the hallmarks of astrocytic glutamate release are the slow depolarizing inward currents (SICs) in neurons mediated by N-methyl-d-aspartate receptor activation. Different stimuli that evoke Ca 2+ elevations in astrocytes induce neuronal SICs suggesting a Ca 2+ -dependent exocytotic glutamate release mechanism of SIC generation. To gain new insights into this mechanism, we investigated the relationship between astrocytic Ca 2+ elevations and neuronal SICs in mouse hippocampal slice preparations. Here we provide evidence that SICs, occurring either spontaneously or following a hypotonic challenge, are unchanged in the virtual absence of Ca 2+ signal changes at astrocytic soma and processes, including spatially restricted Ca 2+ microdomains. SICs are also unchanged in the presence of Bafilomycin A1 that after prolonged slice incubation depletes glutamate from astrocytic vesicles. We also found that hemichannels and TREK family channels-that recent studies proposed to mediate astrocytic glutamate release - are not involved in SIC generation. SICs are reduced by the volume-sensitive anion channel antagonists diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), quinine and fluoxetine, suggesting a possible contribution of these channels in SIC generation. Direct measurements of astrocytic glutamate release further confirm that hypotonicity-evoked gliotransmission is impaired following DIDS, quinine and fluoxetine while the exocytotic release of glutamate-that is proposed to mediate synaptic transmission modulation by astrocytes-remains unaffected. In conclusion, our data provide evidence that the release of glutamate generating SICs occurs independently on exocytotic Ca 2+ -dependent glutamate release mechanism., (© 2018 Wiley Periodicals, Inc.)

Iron transport through the duodenum is regulated by carrier proteins, one of which is the ubiquitously distributed divalent metal transporter (DMT1) which is responsible for the uptake of iron across the apical surface of the duodenal enterocyte. The crystallographic structure of Staphylococcus capitis divalent metal ion transporter (ScaDMT1) was obtained and it was used as a template for the construction of a homology model of human divalent metal transporter (hDMT1). The binding site for hDMT1 was determined by using SiteMap as well as molecular docking studies on ScaDMT1. The differences in binding modes between ScaDMT1 and hDMT1 were noted for a set of 7 iron containing compounds, including ferrous sulphate. Diffusion of ferrous ion was observed during the course of molecular dynamic simulation which corresponded to the postulated mechanism of iron transport. Further, the dock scores correlated well with relative bioavailabilities of the iron compounds. The study confirmed the efficacy of the in silico model which could be used for future studies on the absorption of micronutrients., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Heavy metal-associated (HMA) domains bind metal ions at its Cys-x-x-Cys (CxxC) motif and constitute an intracellular network for trafficking of metal ions for utilization and detoxification. We thus expect that novel metalloproteins can be identified by screening proteins interacting with a HMA domain. In this study, we performed yeast two-hybrid screening of the human proteome and found an uncharacterized protein encoded as open reading frame 123 in chromosome 1 (C1orf123) that can interact specifically with the HMA domain of a copper chaperone for superoxide dismutase (CCSdI). Our X-ray structural analysis of C1orf123 further revealed that it binds a Zn2+ ion in a tetrahedral coordination with four thiolate groups from two conserved CxxC motifs. For the interaction between C1orf123 and CCSdI, the CxxC motifs in both C1orf123 and CCSdI were required, implying metal-mediated interaction through the CxxC motifs. Notably, C1orf123 did not interact with several other HMA domains containing CxxC motifs, supporting high specificity in the interaction between C1orf123 and CCSdI. Based upon these results, we further discuss functional and structural significance of the interaction between C1orf123 and CCS., Competing Interests: The authors have declared that no competing interests exist.

The calcium binding protein ALG-2 is upregulated in several types of cancerous tissues and cancer cell death may be a consequence of ALG-2 downregulation. Novel research suggests that ALG-2 is involved in membrane repair mechanisms, in line with several published studies linking ALG-2 to processes of membrane remodeling and transport, which may contribute to the fitness of cells or protect them from damage. To investigate the involvement of ALG-2 in cell recovery after membrane damage we disrupted the PDCD6 gene encoding the ALG-2 protein in DT-40 cells and exposed them to electroporation. ALG-2 knock-out cells were more sensitive to electroporation as compared to wild type cells. This phenotype could be reversed by reestablishing ALG-2 expression confirming that ALG-2 plays an important role in cell recovery after plasma membrane damage. We found that overexpression of wild type ALG-2 but not a mutated form unable to bind Ca2+ partially protected HeLa cells from digitonin-induced cell death. Further, we were able to inhibit the cell protective function of ALG-2 after digitonin treatment by adding a peptide with the ALG-2 binding sequence of ALIX, which has been proposed to serve as the ALG-2 downstream target in a number of processes including cell membrane repair. Our results suggest that ALG-2 may serve as a novel therapeutic target in combination with membrane damaging interventions., Competing Interests: The authors have declared that no competing interests exist.

The prion protein (PrP) is a cell surface protein that in disease misfolds and becomes infectious causing Creutzfeldt-Jakob disease in humans, scrapie in sheep, and chronic wasting disease in deer and elk. Little is known regarding the dimerization of PrP and its role in disease. We developed a bioluminescent prion assay (BPA) to quantify PrP dimerization by bimolecular complementation of split Gaussia luciferase (GLuc) halves that are each fused to PrP. Fusion constructs between PrP and N- and C-terminal GLuc halves were expressed on the surface of RK13 cells (RK13-DC cells) and dimerized to yield a bioluminescent signal that was decreased in the presence of eight different antibodies to PrP. Dimerization of PrP was independent of divalent cations and was induced under stress. Challenge of RK13-DC cells with seven different prion strains did not lead to detectable infection but was measurable by bioluminescence. Finally, we used BPA to screen a compound library for compounds inhibiting PrP dimerization. One of the most potent compounds to inhibit PrP dimerization was JTC-801, which also inhibited prion replication in RML-infected ScN2a and SMB cells with an EC 50 of 370 nM and 220 nM, respectively. We show here that BPA is a versatile tool to study prion biology and to identify anti-prion compounds.

Glucose isomerase (GI) catalyzes the reversible enzymatic isomerization of d-glucose and d-xylose to d-fructose and d-xylulose, respectively. This is one of the most important enzymes in the production of high-fructose corn syrup (HFCS) and biofuel. We recently determined the crystal structure of GI from S. rubiginosus (SruGI) complexed with a xylitol inhibitor in one metal binding mode. Although we assessed inhibitor binding at the M1 site, the metal binding at the M2 site and the substrate recognition mechanism for SruGI remains the unclear. Here, we report the crystal structure of the two metal binding modes of SruGI and its complex with glucose. This study provides a snapshot of metal binding at the SruGI M2 site in the presence of Mn 2+ , but not in the presence of Mg 2+ . Metal binding at the M2 site elicits a configuration change at the M1 site. Glucose molecule can only bind to the M1 site in presence of Mn 2+ at the M2 site. Glucose and Mn 2+ at the M2 site were bridged by water molecules using a hydrogen bonding network. The metal binding geometry of the M2 site indicates a distorted octahedral coordination with an angle of 55-110°, whereas the M1 site has a relatively stable octahedral coordination with an angle of 85-95°. We suggest a two-step sequential process for SruGI substrate recognition, in Mn 2+ binding mode, at the M2 site. Our results provide a better understanding of the molecular role of the M2 site in GI substrate recognition., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Alphavirus non-structural protein, nsP1 has a distinct molecular mechanism of capping the viral RNAs than the conventional capping mechanism of host. Thus, alphavirus capping enzyme nsP1 is a potential drug target. nsP1 catalyzes the methylation of guanosine triphosphate (GTP) by transferring the methyl group from S-adenosylmethionine (SAM) to a GTP molecule at its N7 position with the help of nsP1 methyltransferase (MTase) followed by guanylylation (GT) reaction which involves the formation of m 7 GMP-nsP1 covalent complex by nsP1 guanylyltransferase (GTase). In subsequent reactions, m 7 GMP moiety is added to the 5' end of the viral ppRNA by nsP1 GTase resulting in the formation of cap0 structure. In the present study, chikungunya virus (CHIKV) nsP1 MTase and GT reactions were confirmed by an indirect non-radioactive colorimetric assay and western blot assay using an antibody specific for the m 7 G cap, respectively. The purified recombinant CHIKV nsP1 has been used for the development of a rapid and sensitive non-radioactive enzyme linked immunosorbent assay (ELISA) to identify the inhibitors of CHIKV nsP1. The MTase reaction is followed by GT reaction and resulted in m 7 GMP-nsP1 covalent complex formation. The developed ELISA nsP1 assay measures this m 7 GMP-nsP1 complex by utilizing anti-m 7 G cap monoclonal antibody. The mutation of a conserved residue Asp63 to Ala revealed its role in nsP1 enzyme reaction. Inductively coupled plasma mass spectroscopy (ICP-MS) was used to determine the presence of magnesium ions (Mg 2+ ) in the purified nsP1 protein. The divalent metal ion selectivity and investigation show preference for Mg 2+ ion by CHIKV nsP1. Additionally, using the developed ELISA nsP1 assay, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA) and ribavirin were determined and the IC 50 values were estimated to be 2.69 μM, 5.72 μM and 1.18 mM, respectively., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Malaria parasites export many proteins into their host erythrocytes and increase membrane permeability to diverse solutes. Although most solutes use a broad-selectivity channel known as the plasmodial surface anion channel, increased Ca ++ uptake is mediated by a distinct, poorly characterised mechanism that appears to be essential for the intracellular parasite. Here, we examined infected cell Ca ++ uptake with a kinetic fluorescence assay and the virulent human pathogen, Plasmodium falciparum. Cell surface labelling with N-hydroxysulfosuccinimide esters revealed differing effects on transport into infected and uninfected cells, indicating that Ca ++ uptake at the infected cell surface is mediated by new or altered proteins at the host membrane. Conditional knockdown of PTEX, a translocon for export of parasite proteins into the host cell, significantly reduced infected cell Ca ++ permeability, suggesting involvement of parasite-encoded proteins trafficked to the host membrane. A high-throughput chemical screen identified the first Ca ++ transport inhibitors active against Plasmodium-infected cells. These novel chemical scaffolds inhibit both uptake and parasite growth; improved in vitro potency at reduced free [Ca ++ ] is consistent with parasite killing specifically via action on one or more Ca ++ transporters. These inhibitors should provide mechanistic insights into malaria parasite Ca ++ transport and may be starting points for new antimalarial drugs., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA.)

The aim of this study was the evaluation of activities of versatile peroxidase (VP) in cultures of Clonostachys sp. (Gliocladium sp.) strains during biotransformation of 0.2% alkali lignin (AL). The principal component analysis (PCA) method was applied to determine the main factors and correlation between biotransformation of 0.2% AL, activity of VPs, pH value, and Clonostachys sp. strains. The biotransformation of 0.2% AL in cultures of microscopic fungi included decolorization (medium lightening) and colorization (darkening of the medium). The versatile peroxidase synthesized by the microscopic fungi tested showed activity for oxidation of Mn(II) and guaiacol, but the activity for oxidation of guaiacol in the presence of Mn(II) was significantly higher. The PCA analysis indicated a strong correlation between biotransformation of 0.2% AL and pH and between oxidation of Mn(II), guaiacol without and in the presence of Mn(II) ions, strains, and pH.

How the presence of Ca 2+ ions at the aqueous TiO 2 interface influences the binding modes of two experimentally identified titania-binding peptides, Ti-1 and Ti-2, is investigated using replica exchange with solute tempering molecular dynamics simulations. The findings are compared with available experimental data, and the results are contrasted with those obtained under NaCl solution conditions. For Ti-1, Ca 2+ ions enhance the adsorption of the negatively charged Asp8 residue in this sequence to the negatively charged surface, via Asp–Ca 2+ –TiO 2 bridging. This appears to generate a nonlocal impact on the adsorption of Lys12 in Ti-1, which then pins the peptide to the surface via direct surface contact. For Ti-2, fewer residues were predicted to adsorb directly to the surface in CaCl 2 , compared with predictions made for NaCl solution, possibly due to competition between the other peptide residues and Ca 2+ ions to adsorb to the surface. This reduction in direct surface contact gives rise to a more extensive solvent-mediated contact for Ti-2. In general, the presence of Ca 2+ ions resulted in a loss of conformational diversity of the surface-adsorbed conformational ensembles of these peptides, compared to counterpart data predicted for NaCl solution. The findings provide initial insights into how peptide–TiO 2 interactions might be tuned at the molecular level via modification of the salt composition of the liquid medium.

Bordetella pertussis, the causative agent of whooping cough, has the capability to survive inside the host cells. This process requires efficient adaptation of the pathogen to the intracellular environment and the associated stress. Among the proteins produced by the intracellular B. pertussis we identified a protein (BP0414) that shares homology with MgtC, a protein which was previously shown to be involved in the intracellular survival of other pathogens. To explore if BP0414 plays a role in B. pertussis intracellular survival a mutant strain defective in the production of this protein was constructed. Using standard in vitro growth conditions we found that BP0414 is required for B. pertussis growth under low magnesium availability or low pH, two environmental conditions that this pathogen might face within the host cell. Intracellular survival studies showed that MgtC is indeed involved in B. pertussis viability inside the macrophages. The use of bafilomycin A1, which inhibits phagosome acidification, abolished the survival defect of the mgtC deficient mutant strain suggesting that in intracellular B. pertussis the role of MgtC protein is mainly related to the bacterial adaptation to the acidic conditions found inside the of phagosomes. Overall, this work provides an insight into the importance of MgtC in B. pertussis pathogenesis and its contribution to bacterial survival within immune cells., Competing Interests: The authors have declared that no competing interests exist.

The large yybP-ykoY family of bacterial riboswitches is broadly distributed phylogenetically. Previously, these gene-regulatory RNAs were proposed to respond to Mn 2+ . X-ray crystallography revealed a binuclear cation-binding pocket. This comprises one hexacoordinate site, with six oxygen ligands, which preorganizes the second, with five oxygen and one nitrogen ligands. The relatively soft nitrogen ligand was proposed to confer affinity for Mn 2+ , but how this excludes other soft cations remained enigmatic. By subjecting representative yybP-ykoY riboswitches to diverse cations in vitro, we now find that these RNAs exhibit limited transition metal ion selectivity. Among the cations tested, Cd 2+ and Mn 2+ bind most tightly, and comparison of three new Cd 2+ -bound crystal structures suggests that these riboswitches achieve selectivity by enforcing heptacoordination (favored by high-spin Cd 2+ and Mn 2+ , but otherwise uncommon) in the softer site. Remarkably, the Cd 2+ - and Mn 2+ -selective bacterial transcription factor MntR also uses heptacoordination within a binuclear site to achieve selectivity., (Published by Elsevier Ltd.)

It is well accepted that the introduction of negative supercoils locally unwinds the DNA double helix, influencing thus the activity of proteins. Despite the use of recent methods of molecular dynamics simulations to model the DNA supercoiling-induced DNA deformation, the precise extent and location of unpaired bases induced by the negative supercoiling have never been investigated at the nucleotide level. Our goals in this study were to use radiolabeled double-stranded DNA mini-circles (dsMCs) to locate the unpaired bases on dsMCs whose topology ranged from relaxed to hyper-negatively supercoiled states, and to characterize the binding of proteins involved in the DNA metabolism. Our results show that the Nuclease SI is nearly ten times more active on hyper-negatively supercoiled than relaxed DNA. The structural changes responsible for this stimulation of activity were mapped for the first time with a base pair resolution and shown to be subtle and distributed along the entire sequence. As divalent cations modify the DNA topology, our binding studies were conducted with or without magnesium. Without magnesium, the dsMCs topoisomers mostly differ by their twist. Under these conditions, the Escherichia coli topoisomerase I weakly binds relaxed dsMCs and exhibits a stronger binding on negatively and hyper-negatively supercoiled dsMCs than relaxed dsMCs, with no significant difference in the binding activity among the supercoiled topoisomers. For the human replication protein A (hRPA), the more negatively supercoiled is the DNA, the better the binding, illustrating the twist-dependent binding activity for this protein. The presence of magnesium permits the dsMCs to writhe upon introduction of negative supercoiling and greatly modifies the binding properties of the hRPA and Escherichia coli SSB on dsMCs, indicating a magnesium-dependent DNA binding behavior. Finally, our experiments that probe the topology of the DNA in the hRPA-dsMC complexes show that naked and hRPA-bound dsMCs have the same topology., Competing Interests: The authors have declared that no competing interests exist.

We used the nanometer-wide tubules of the transverse tubular (t)-system of human skeletal muscle fibers as sensitive sensors for the quantitative monitoring of the Ca 2+ -handling properties in the narrow junctional cytoplasmic space sandwiched between the tubular membrane and the sarcoplasmic reticulum cisternae in single muscle fibers. The t-system sealed with a Ca 2+ -sensitive dye trapped in it is sensitive to changes in ryanodine receptor (RyR) Ca 2+ leak, the store operated calcium entry flux, plasma membrane Ca pump, and sodium-calcium exchanger activities, thus making the sealed t-system a nanodomain Ca 2+ sensor of Ca 2+ dynamics in the junctional space. The sensor was used to assess the basal Ca 2+ -handling properties of human muscle fibers obtained by needle biopsy from control subjects and from people with a malignant hyperthermia (MH) causative RyR variant. Using this approach we show that the muscle fibers from MH-susceptible individuals display leakier RyRs and a greater capacity to extrude Ca 2+ across the t-system membrane compared with fibers from controls. This study provides a quantitative way to assess the effect of RyR variants on junctional membrane Ca 2+ handling under defined ionic conditions., Competing Interests: The authors declare no conflict of interest.

Regulated exocytosis, which underlies many intercellular signaling events, is a tightly controlled process often triggered by calcium ion(s) (Ca 2+ ). Despite considerable insight into the central components involved, namely, the core fusion machinery [soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE)] and the principal Ca 2+ sensor [C2-domain proteins like synaptotagmin (Syt)], the molecular mechanism of Ca 2+ -dependent release has been unclear. Here, we report that the Ca 2+ -sensitive oligomers of Syt1, a conserved structural feature among several C2-domain proteins, play a critical role in orchestrating Ca 2+ -coupled vesicular release. This follows from pHluorin-based imaging of single-vesicle exocytosis in pheochromocytoma (PC12) cells showing that selective disruption of Syt1 oligomerization using a structure-directed mutation (F349A) dramatically increases the normally low levels of constitutive exocytosis to effectively occlude Ca 2+ -stimulated release. We propose a parsimonious model whereby Ca 2+ -sensitive oligomers of Syt (or a similar C2-domain protein) assembled at the site of docking physically block spontaneous fusion until disrupted by Ca 2+ Our data further suggest Ca 2+ -coupled vesicular release is triggered by removal of the inhibition, rather than by direct activation of the fusion machinery., Competing Interests: The authors declare no conflict of interest.

Reinvestigation of the metabolite profile in a disruptant of the quinoprotein dehydrogenase (orf23) gene revealed that the Orf23 protein catalyzes dehydrogenation of the C23-C25 lactate moiety to pyruvate during lankacidin biosynthesis in Streptomyces rochei 7434AN4. The dehydrogenase activity was expressed and detected in a soluble fraction of the Streptomyces lividans recombinant harboring orf23. The Orf23 protein preferentially converts lankacidinol to lankacidin C in the presence of pyrroloquinoline quinone (PQQ). Other lankacidinol derivatives, lankacidinol A and iso-lankacidinol, were also converted to the corresponding C-24 keto compounds, lankacidin A (=sedecamycin) and iso-lankacidin C. Addition of various divalent metal cations, especially Ca 2+ , enhanced the dehydrogenase activity, whereas EDTA completely inhibited. These findings confirmed that the quinoprotein dehydrogenase Orf23 functions at the final oxidation step of lankacidin biosynthesis., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

A synthetic derivative, GnRH [6-D-Phe], stable against enzymatic degradation, self-assembles and forms nanostructures and fibrils upon a pH shift in the presence of different concentrations of Zn 2+ in vitro. Attenuated Total Reflection Fourier Transform Infrared spectroscopy (ATR-FTIR) revealed the existence of higher order assembly of Zn 2+ : GnRH [6-D-Phe]. Nuclear Magnetic Resonance spectroscopy (NMR) indicated a weak interaction between Zn 2+ and GnRH [6-D-Phe]. Atomic Force Microscopy (AFM) showed the existence of GnRH [6-D-Phe] oligomers and fibrils. Molecular Dynamic (MD) simulation of the 10:1 Zn 2+ : GnRH [6-D-Phe] explored the interaction and dimerization processes. In contrast to already existing short peptide fibrils, GnRH [6-D-Phe] nanostructures and fibrils form in a Tris-buffered pH environment in a controlled manner through a temperature reduction and a pH shift. The lyophilized Zn 2+ : GnRH [6-D-Phe] assembly was tested as a platform for the sustained delivery of GnRH [6-D-Phe] and incorporated into two different oil vehicle matrices. The in vitro release was slow and continuous over 14 days and not influenced by the oil matrix.

Human KLK8/neuropsin, a kallikrein-related serine peptidase, is mostly expressed in skin and the hippocampus regions of the brain, where it regulates memory formation by synaptic remodeling. Substrate profiles of recombinant KLK8 were analyzed with positional scanning using fluorogenic tetrapeptides and the proteomic PICS approach, which revealed the prime side specificity. Enzyme kinetics with optimized substrates showed stimulation by Ca 2+ and inhibition by Zn 2+ , which are physiological regulators. Crystal structures of KLK8 with a ligand-free active site and with the inhibitor leupeptin explain the subsite specificity and display Ca 2+ bound to the 75-loop. The variants D70K and H99A confirmed the antagonistic role of the cation binding sites. Molecular docking and dynamics calculations provided insights in substrate binding and the dual regulation of activity by Ca 2+ and Zn 2+ , which are important in neuron and skin physiology. Both cations participate in the allosteric surface loop network present in related serine proteases. A comparison of the positional scanning data with substrates from brain suggests an adaptive recognition by KLK8, based on the tertiary structures of its targets. These combined findings provide a comprehensive picture of the molecular mechanisms underlying the enzyme activity of KLK8.

Repeated activation of N-Methyl-d-aspartate receptors (NMDARs) causes a Ca 2+ -dependent reduction in NMDAR-mediated current in dopamine (DA) neurons of the substantia nigra pars compacta (SNc) in one week old rats; however, a Ca 2+ -dependent regulatory protein has not been identified. The role of the Ca 2+ -dependent cysteine protease, calpain, in mediating NMDAR current rundown was investigated. In brain slices from rats aged postnatal day 7-9 ('P7'), bath application of either of the membrane permeable calpain inhibitors, N-Acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN, 20 μM) or MDL-28170 (30 μM) significantly reduced whole-cell NMDAR current rundown. To investigate the role of the calpain-2 isoform, the membrane permeable calpain-2 inhibitor, Z-Leu-Abu-CONH-CH2-C6H3 (3, 5-(OMe)2 (C2I, 200 nM), was applied; C2I application significantly reduced whole cell NMDAR current rundown. Interestingly, ALLN but not C2I significantly reduced rundown of NMDA-EPSCs. These results suggest the calpain-2 isoform mediates Ca 2+ -dependent regulation of extrasynaptic NMDAR current in the first postnatal week, while calpain-1 might mediate rundown of synaptic NMDAR currents. One week later in postnatal development, at P12-P16 ('P14'), there was significantly less rundown in SNc-DA neurons, and no significant effect on rundown of either Ca 2+ chelation or treatment with the calpain inhibitor, ALLN, suggesting that the rundown observed in SNc-DA neurons from two week-old rats might be Ca 2+ -independent. In conclusion, Ca 2+ -dependent rundown of extrasynaptic NMDAR currents in SNc DA neurons involves calpain-2 activation, but Ca 2+ - and calpain-2-dependent NMDAR current rundown is developmentally regulated., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Memantine and ketamine are NMDA receptor (NMDAR) open channel blockers that are thought to act via similar mechanisms at NMDARs, but exhibit divergent clinical effects. Both drugs act by entering open NMDARs and binding at a site deep within the ion channel (the deep site) at which the endogenous NMDAR channel blocker Mg 2+ also binds. Under physiological conditions, Mg 2+ increases the IC 50 s of memantine and ketamine through competition for binding at the deep site. Memantine also can inhibit NMDARs after associating with a second site accessible in the absence of agonist, a process termed second site inhibition (SSI) that is not observed with ketamine. Here we investigated the effects of 1 mM Mg 2+ on recovery from inhibition by memantine and ketamine, and on memantine SSI, of the four main diheteromeric NMDAR subtypes. We found that: recovery from memantine inhibition depended strongly on the concentration of memantine used to inhibit the NMDAR response; Mg 2+ accelerated recovery from memantine and ketamine inhibition through distinct mechanisms and in an NMDAR subtype-dependent manner; and Mg 2+ occupation of the deep site disrupted memantine SSI in a subtype-dependent manner. Our results support the hypothesis that memantine associates with, but does not inhibit at the second site. After associating with the second site, memantine can either slowly dissociate directly to the extracellular solution, or transit to the deep site, resulting in typical channel block. Memantine's relatively slow dissociation from the second site underlies the dependence of NMDAR recovery from inhibition on both memantine concentration and on Mg 2+ ., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Despite dissemination of information regarding the harm on fetal development of smoking while pregnant, the number of pregnancies associated with nicotine exposure appears to have stagnated. Presence of nicotine during neural formulation is associated with a higher susceptibility of drug dependence, suggesting an altered development of neurons in circuits involved in saliency and motivation. The laterodorsal tegmental nucleus (LDT) plays a role in coding stimuli valence via afferents to mesolimbic nuclei. Accordingly, alterations in development of neural mechanisms in the LDT could be involved in vulnerability to drug dependency. Therefore, we examined the effect of prenatal nicotine exposure (PNE) on glutamatergic functioning of LDT neurons in mouse brain slices using whole-cell, patch clamp concurrent with fluorescence-based calcium imaging. PNE was associated with larger amplitudes of AMPA-induced currents, and greater AMPA-mediated rises in intracellular calcium. AMPA/NMDA ratios and the AMPA-current rectification index were lower and higher, respectively, consistent with changes in the functionality of AMPA receptors in the PNE, which was substantiated by a greater inhibition of evoked and spontaneous glutamatergic synaptic events by a selective inhibitor of GluA2-lacking AMPA receptors. Paired pulse ratios showed a decreased probability of glutamate release from presynaptic inputs, and fluorescent imaging indicated a decreased action potential-dependent calcium increase associated with PNE. When taken together, our data suggest that PNE alters LDT glutamatergic functioning, which could alter output to mesolimbic targets. Such an alteration could play a role in altered coding of relevancy of drug stimuli that could enhance risk for development of drug dependency., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Introduction: Ca2+ release from sarcoplasmic reticulum (SR) is known to contribute to automaticity via the cytoplasmic Na+-Ca2+ exchanger (NCX). Mitochondria participate in Ca2+ cycling. We studied the role of mitochondrial Ca2+ flux in ventricular spontaneous electrical activity., Methods: Spontaneously contracting mouse embryonic stem cells (ESC)-derived ventricular cardiomyocytes (CMs) were differentiated from wild type and ryanodine receptor type 2 (RYR2) knockout mouse ESCs and differentiated for 19-21 days. Automaticity was also observed in human induced pluripotent stem cell (hiPSC)-derived ventricular CMs differentiated for 30 days, and acute isolated adult mouse ventricular cells in ischemic simulated buffer. Action potentials (APs) were recorded by perforated whole cell current-clamp. Cytoplasmic and mitochondrial Ca2+ transients were determined by fluorescent imaging., Results: In mouse ESC-derived ventricular CMs, spontaneous beating was dependent on the L-type Ca2+ channel, cytoplasmic NCX and mitochondrial NCX. Spontaneous beating was modulated by SR Ca2+ release from RYR2 or inositol trisphosphate receptors (IP3R), the pacemaker current (If) and mitochondrial Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU). In RYR2 knockout mouse ESC-derived ventricular CMs, mitochondrial Ca2+ flux influenced spontaneous beating independently of the SR Ca2+ release from RYR2, and the mitochondrial effect was dependent on IP3R SR Ca2+ release. Depolarization of mitochondria and preservation of ATP could terminate spontaneous beating. A contribution of mitochondrial Ca2+ flux to automaticity was confirmed in hiPSC-derived ventricular CMs and ischemic adult mouse ventricular CMs, confirming the findings across species and cell maturity levels., Conclusions: Mitochondrial and sarcolemma NCX fluxes are required for ventricular automaticity. Mitochondrial Ca2+ uptake plays a modulatory role. Mitochondrial Ca2+ uptake through MCU is influenced by IP3R-dependent SR Ca2+ release., Competing Interests: The authors have declared that no competing interests exist.

The removal of harmful metals from the intestinal environment can be inhibited by various ions which can interfere with the adsorption of target metal ions. Therefore, it is important to understand the ion selectivity and adsorption mechanism of the adsorbent. In this study, we estimated the adsorption properties of Lactobacillus casei JCM1134 by analyzing the correlation between its maximum adsorption level (q max ) for seven metals and their ion characteristics. Some metal ions showed altered adsorption levels by L. casei JCM1134 as culture growth time increased. Although it was impossible to identify specific adsorption components, adsorption of Sr and Ba may depend on capsular polysaccharide levels. The maximum adsorption of L. casei JCM1134 (9 h of growth in culture) for divalent metal ions was in the following order: Cu 2+  > Ba 2+  > Sr 2+  > Cd 2+  > Co 2+  > Mg 2+  > Ni 2+ . The q max showed a high positive correlation with the ionic radius. Because this tendency is similar to adsorption occurring through an ion exchange mechanism, it was inferred that an ion exchange mechanism contributed greatly to adsorption by L. casei JCM1134. Because the decrease in the amount of adsorption due to prolonged culture time was remarkable for metals with a large ion radius, it is likely that the adsorption components involved in the ion exchange mechanism decomposed over time. These results and analytical concept may be helpful for designing means to remove harmful metals from the intestinal tract.