FAD-Linked Autofluorescence and Chemically-Evoked Zinc Changes at Hippocampal Mossy Fiber-CA3 Synapses

Glutamatergic vesicles in hippocampal mossy fiber presynaptic boutons release zinc, which plays a modulatory role in synaptic activity and LTP. In this work, a fluorescence microscopy technique and the fluorescent probe for cytosolic zinc, Newport Green (NG), were applied, in a combined study of autofluorescence and zinc changes at the hippocampal mossy fiber-CA3 synaptic system. In particular, the dynamics of flavoprotein (FAD) autofluorescence signals, was compared to that of postsynaptic zinc signals, elicited both by high K+ (20 mM) and by tetraethylammonium (TEA, 25 mM). The real zinc signals were obtained subtracting autofluorescence values, from corresponding total NG-fluorescence data. Both autofluorescence and zinc-related fluorescence were raised by high K+. In contrast, the same signals were reduced during TEA exposure. It is suggested that the initial outburst of TEA-evoked zinc release might activate ATP-sensitive K+ (KATP) channels, as part of a safeguard mechanism against excessive glutamatergic action. This would cause sustained inhibition of zinc signals and a more reduced mitochondrial state. In favor of the “KATP channel hypothesis”, the KATP channel blocker tolbutamide (250 μM) nearly suppressed the TEA-evoked fluorescence changes. It is concluded that recording autofluorescence from brain slices is essential for the accurate assessment of zinc signals and actions.

Halotolerant Endophytic Bacterium Serratia rubidaea ED1 Enhances Phosphate Solubilization and Promotes Seed Germination

Quinoa is renowned for its nutritional value and ability to withstand harsh environmental conditions such as salinity. In the present work, we isolated 34 phosphate solubilizing endophytic bacteria associated with the roots of quinoa plants. Based on phosphate solubilization efficiency and biochemical characterization, we selected one isolate named ED1. Ribotyping using partial 16S RNA gene analysis revealed that the selected isolate shares 99.7% identity with Serratia rubidaea. Plant growth promoting (PGP) studies showed that the ED1 strain solubilized complexed forms of phosphate (Ca3(PO4)2). Zinc release from ZnO, Zn3(PO4)2, or ZnCO3 revealed the efficient ZnO solubilization by the ED1 strain. Except for proteases, the strain ED1 produced siderophores, cellulase, ammonia and exhibited oligonitrophilic features. Indole acetic acid (IAA) production was detected with and without the L-tryptophan precursor. Next, we demonstrated that the ED1 strain tolerated 1.5 M NaCl final concentration and exhibited intrinsic resistance to seven antibiotics frequently prescribed for medical use. Moreover, we found that ED1 strain withstood 2 mg/L of Cadmium and 1 mg/L of either Nickel or Copper. Furthermore, we observed that S. rubidaea ED1 stimulated quinoa seeds germination and seedlings growth under salt stress conditions. Lastly, we discuss the advantages versus disadvantages of applying the S. rubidaea ED1 strain as a beneficial agent for salty and/or heavy metals contaminated soils.

Geochemical Assessment of Desulphurized Tailings as Cover Material in Cold Climates

It is essential to develop effective mine waste management approaches and mine site reclamation techniques to curtail the adverse effects of mining processes on the natural environment. This study focuses on the use of partially desulphurized tailings as a moisture-retaining layer in an insulation cover with capillary barrier effects (ICCBE). Tailings were obtained from a nickel ultramafic ore processing plant at a mining company located in a continuous permafrost region of northern Québec, Canada. The geochemical response of tailings at two different sulphur contents (0.4 and 0.8 wt%), with and without ICCBEs, was tested by applying eight freeze-thaw and wetting cycles. Desulphurization of the tailings allowed to reduce the content of sulphide minerals by about 90%, from ~22 wt% to around 1.2–2.2 wt%. Column kinetic geochemical tests showed that Ni leaching was significantly reduced to concentrations ranging between 0.01–0.22 mg L−1 compared to 0.63–1.92 mg L−1 from the raw tailings (thanks to the desulphurization process). Zinc release was maintained around 0.04–1.72 mg L−1 compared to 0.4–3.69 mg L−1 from the raw tailing. Although all the columns produced leachates displaying circumneutral to slightly alkaline pH, the columns with ICCBE are expected to prevent acid mine drainage generation longer than the other columns due to reduced sulphide content and a constantly high degree of saturation maintained by capillary barrier effects.

Kupyaphores are counter regulatory zinc homeostatic metallophores required for Mycobacterium tuberculosis colonization

Tuberculosis (TB) patients suffer from progressive and debilitating loss of muscle mass and function, referred to as cachexia. Though a multifactorial condition, cachexia in cancer is promoted by systemic zinc redistribution and accumulation in muscles. Clinical studies with TB patients indeed show zinc dyshomeostasis. We therefore set out to understand mechanisms by which Mycobacterium tuberculosis (Mtb) govern zinc metallostasis at the host-pathogen interface. Here, we report a novel zinc metallophore from Mtb that restores zinc metabolic imbalance. These diisonitrile lipopeptides, named kupyaphores are transiently induced early-on during macrophage infection and also in infected mice lungs. Kupyaphores protects bacteria from host-mediated nutritional deprivation and intoxication. Kupyaphore Mtb mutant strain cannot mobilize zinc and shows reduced fitness in mice. Further, we characterize Mtb encoded isonitrile hydratase that could mediate intracellular zinc release through covalent modification of kupyaphores. Our studies could provide a molecular link between TB-induced altered zinc homeostasis and associated cachexia.

The zinc transporter ZIP9 (Slc39a9) regulates zinc dynamics essential to egg activation in zebrafish

The zinc transporter ZIP9 (SLC39A9) was recently characterized as a membrane androgen receptor in various teleost and mammalian cell models. ZIP9 shows the highest expression in ovaries of teleosts, a tissue in which both androgen signaling and zinc dynamics have significant roles. To examine the role of ZIP9 in ovarian physiology, we generated a ZIP9-mutant zebrafish strain using a CRISPR/Cas9 system. zip9-/- females showed significant reductions in fecundity, embryo viability, and growth of their offspring compared to wildtype (WT) fish. Furthermore, a high proportion of zip9-/- eggs failed to undergo normal chorion elevation during activation. In WT eggs, zinc was detected in cortically-localized vesicles which underwent exocytosis upon activation. zip9-/- eggs showed abnormal cortical vesicle development and had a significantly depressed activation-induced zinc release compared to WT eggs. Moreover, pharmacologically sustained elevation of zinc in WT eggs prior to activation resulted in abnormal chorion elevation similar to that observed in zip9-/- eggs. These results indicate that ZIP9 is essential for proper zinc modulation during zebrafish egg activation and presents the first evidence of zinc modulation during egg activation in a non-mammalian species.

Acidosis, Zinc and HMGB1 in Sepsis: A Common Connection Involving Sialoglycan Recognition

Blood pH is tightly regulated between 7.35-7.45, with values below 7.3 during sepsis being associated with lactic acidosis, low serum zinc, and release of proinflammatory HMGB1 from activated and/or necrotic cells. Using an ex vivo whole blood system to model lactic acidosis, we show that while HMGB1 does not engage leukocyte receptors at physiological pH, lowering pH with lactic acid facilitates binding. At normal pH, micromolar zinc supports plasma sialoglycoprotein binding by HMGB1, which is markedly reduced when pH is adjusted with lactic acid to sepsis levels. Glycan array studies confirmed zinc and pH-dependent HMGB1 binding to sialoglycans typical of plasma glycoproteins. Thus, proinflammatory effects of HMGB1 are suppressed via plasma sialoglycoproteins until drops in pH and zinc release HMGB1 to trigger downstream immune activation.Significance StatementHMGB1 sequestered by plasma sialoglycoproteins at physiological pH is released when pH and zinc concentrations fall in sepsis.

Zinc Determines Dynamical Properties and Aggregation Kinetics of Human Insulin

Protein aggregation is a widespread process leading to deleterious consequences in the organism, with amyloid aggregates being important not only in biology but also for drug design and biomaterial production. Insulin is a protein largely used in diabetes treatment and its amyloid aggregation is at the basis of the so-called insulin-derived amyloidosis. Here we uncover the major role of zinc in both insulin dynamics and aggregation kinetics at low pH, where the formation of different amyloid superstructures (fibrils and spherulites) can be thermally induced. Amyloid aggregation is accompanied by zinc release and the suppression of water-sustained insulin dynamics, as shown by particle-induced X-ray emission and X-ray absorption spectroscopy and by neutron spectroscopy, respectively. Our study shows that zinc binding stabilizes the native form of insulin by facilitating hydration of this hydrophobic protein and suggests that introducing new binding sites for zinc can improve insulin stability and tune its aggregation propensity.Statement of SignificanceLocalized amyloidosis occurs at insulin injection sites for diabetes treatment, leading to deleterious inflammations known as insulin-derived amyloidosis. Amyloid superstructures are also promising candidates in the field of biomaterials. Here we revealed that zinc, coordinated to insulin in the native form, is released upon amyloid aggregation, when insulin forms superstructures known as fibrils and spherulites. Zinc release leads to a full suppression of functionally essential protein dynamics through a modification of the protein’s hydration properties and completely modifies insulin amyloid kinetics. The results suggest that changes in protein hydration upon zinc binding/release modifies both stability and dynamics of insulin and might then be a general strategy to control protein stability and tune protein aggregation into amorphous and ordered superstructures.