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P53 is a transcription factor that controls a variety of genes, primarily involved in cell cycle and other processes related to cell survival and death. We have isolated peptides targeting P53 (protein and domains) using the "phage display" technique. Interestingly, adding ZnCl2 at 5-10 mM in panning solutions helped to recover more plaque-forming units at least at round one of the screening. Subtractive docking analyses were designed by using a pool of common redundant peptides known as parasites. This rationale helped us differentiate between possibly specific and non-specific bindings. We found notable differences in docking characteristics between different sets of peptides either related to different targets or related to zinc-conditions. The set of zinc-related peptides shows advantageous docking profiles: sharper binding for some positions and distinct exclusive bound residues, including the relevant R248 and R273. Zinc would have modulating/helping role in the targeting of protein P53 by phage displayed peptides in addition to an enhancement action on bacterial infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ben Abid et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

This study aims to assess the level of metal contamination and the ecological risk index at the abandoned Zaida Pb/Zn mining site in eastern Morocco and identify native plant species found on the site that can be used in site rehabilitation through phytoremediation strategies. Samples from seven native and abundant plant species at the site, along with their rhizospheric soils, were collected and analyzed using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) to determine the concentrations of various metal(loid)s, including As, Cu, Ni, Cd, Sb, Zn, and Pb. Indicators of soil pollution and ecological risks were also assessed, including the enrichment factor (EF), pollution index (PI), and ecological risk index (ERI). The Biological Accumulation Coefficient (BAC), Translocation Factor (TF), and Biological Concentration Factor (BCF) of plant samples were calculated. The results reveal polymetallic soil contamination, with notably higher concentrations of Pb, Cu and Zn, reaching respectively 5568 mg kg-1 DW, 152 mg kg-1 DW, and 148 mg kg-1 DW, indicating a significant potential ecological risk. The enrichment factor (EF) was also assessed for each metal(loid)s, and the results indicated that the metal contamination was of anthropogenic origin and linked to intensive mining activities in Zaida. These findings are supported by the pollution index (PI) ranging from 1.6 to 10.01, which reveals an extremely high metal(loid)s pollution level. None of the plant species exhibited a hyperaccumulation of metal(loid)s. However, Artemisia herba alba demonstrated a strong capacity to accumulate Pb in its aboveground parts, with a concentration of 468 mg kg-1 DW. Stipa tenacissima, Retama spherocarpa, and Astragalus armatus, showed a significant Pb accumulation in their roots reaching 280, 260, and 256 mg kg-1 DW.respectively. Based on BAC, TF, and BCF, Stipa tenacissima exhibited potential for Ni and Cd phytostabilization, as well as the ability for Zn phytoextraction. Additionally, Artemisia herba alba displayed the capability to phytoextract Cd and had a high propensity to translocate all the studied metal(loid)s. Astragalus armatus has the potential to be used in the phytostabilization of Zn and Ni, as well as for the phytoextraction of As and Sb. These native species from the Zaida site, although not hyperaccumulators, have the potential to contribute significantly to the phytoextraction or phytostabilization of potentially toxic elements (PTEs). Moreover, they can serve as vegetative cover to mitigate the erosion and dispersion of metal(loid)s., Competing Interests: The authors have declared no conflict of interest., (Copyright: © 2024 Diallo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Zinc is an essential metal for cells, but excess amounts are toxic. Other than by regulating the intracellular zinc concentration by zinc uptake or efflux, the mechanisms underlying bacterial resistance to excess zinc are unknown. In the present study, we searched for zinc-resistant mutant strains from the Keio collection, a gene knockout library of Escherichia coli, a model gram-negative bacteria. We found that knockout mutant of RpmJ (L36), a 50S ribosomal protein, exhibited zinc resistance. The rpmJ mutant was sensitive to protein synthesis inhibitors and had altered translation fidelity, indicating ribosomal dysfunction. In the rpmJ mutant, the intracellular zinc concentration was decreased under excess zinc conditions. Knockout of ZntA, a zinc efflux pump, abolished the zinc-resistant phenotype of the rpmJ mutant. RNA sequence analysis revealed that the rpmJ mutant exhibited altered gene expression of diverse functional categories, including translation, energy metabolism, and stress response. These findings suggest that knocking out RpmJ alters gene expression patterns and causes zinc resistance by lowering the intracellular zinc concentration. Knockouts of other ribosomal proteins, including RplA, RpmE, RpmI, and RpsT, also led to a zinc-resistant phenotype, suggesting that deletion of ribosomal proteins is closely related to zinc resistance., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Shirakawa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Zinc oxide nanoparticles (nano-ZnOs) are widely used and possess great potentials in agriculture and biomedicine. It is inevitable for human exposure to these nanoparticles. However, no study had been conducted to investigate the long term effects of nano-ZnOs. This study aimed at investigating effects of nano-ZnOs on development, zinc metabolism and biodistribution of minerals (Zn, Fe, Cu, and Mn) in mice from week 3 to 35. After the characteristics of nano-ZnOs were determined, they were added into the basal diet at 0, 50, 500 and 5000 mg/kg. Results indicated that added 50 and 500 mg/kg nano-ZnOs showed minimal toxicity. However, 5000 mg/kg nano-ZnOs significantly decreased body weight (from week 4 to 16) and increased the relative weights of the pancreas, brain and lung. Added 5000 mg/kg nano-ZnOs significantly increased the serum glutamic-pyruvic transaminase activity and zinc content, and significantly enhanced mRNA expression of zinc metabolism-related genes, including metallothionein 1(32.66 folds), metallothionein 2 (31.42 folds), ZIP8 (2.21folds), ZIP14 (2.45 folds), ZnT1 (4.76 folds), ZnT2 (6.19 folds) and ZnT4 (1.82 folds). The biodistribution determination showed that there was a significant accumulation of zinc in the liver, pancreas, kidney, and bones (tibia and fibula) after receiving 5000 mg/kg nano-ZnO diet, while no significant effects on Cu, Fe, and Mn levels, except for liver Fe content and pancreas Mn level. Our results demonstrated that long term exposure to 50 and 500 mg/kg nano-ZnO diets showed minimal toxicity. However, high dose of nano-ZnOs (5000 mg/kg) caused toxicity on development, and altered the zinc metabolism and biodistribution in mice. [ABSTRACT FROM AUTHOR]

Zinc plays a critical role in many physiological processes, and disruption of zinc homeostasis induces various disorders, such as growth retardation, osteopenia, immune deficiency, and inflammation. However, how the imbalance in zinc homeostasis leads to heart disease is not yet fully understood. Cardiovascular diseases are a major cause of death worldwide, and the development of novel therapeutic targets to treat it is urgently needed. We report that a zinc transporter, ZIP13, regulates cardiovascular homeostasis. We found that the expression level of Zip13 mRNA was diminished in both primary neonatal cardiomyocytes and mouse heart tissues treated with the cardiotoxic agent doxycycline. Primary neonatal cardiomyocytes from Zip13 gene-knockout (KO) mice exhibited abnormal irregular arrhythmic beating. RNA-seq analysis identified 606 differentially expressed genes in Zip13-KO mouse-derived primary neonatal cardiomyocytes and Gene ontology (GO) analysis revealed that both inflammation- and cell adhesion-related genes were significantly enriched. In addition, telemetry echocardiography analysis suggested that arrhythmias were likely to occur in Zip13-KO mice, in which elevated levels of the cardiac fibrosis marker Col1a1, vascular inflammation-related gene eNOS, and Golgi-related molecule GM130 were observed. These results indicate the physiological importance of ZIP13-it maintains cardiovascular homeostasis by resolving inflammation and stress response. Our findings suggest that optimizing ZIP13 expression and/or function may improve cardiovascular disease management., Competing Interests: The authors have declared that no competing interests exist.

Background: Schizophrenia and especially deficit schizophrenia (DSCZ) are characterized by increased activity of neuroimmunotoxic pathways and a generalized cognitive decline (G-CoDe). There is no data on whether the interleukin (IL)-6/IL-23/T helper 17 (IL-6/IL-23/Th17)-axis is more associated with DSCZ than with non-deficit schizophrenia (NDSCZ) and whether changes in this axis are associated with the G-CoDe and the phenome (a factor extracted from all symptom domains) of schizophrenia., Methods: This study included 45 DSCZ and 45 NDSCZ patients and 40 controls and delineated whether the IL-6/IL-23/Th17 axis, trace elements (copper, zinc) and ions (magnesium, calcium) are associated with DSCZ, the G-CoDe and the schizophrenia phenome., Results: Increased plasma IL-23 and IL-6 levels were associated with Th17 upregulation, assessed as a latent vector (LV) extracted from IL-17, IL-21, IL-22, and TNF-α. The IL-6/IL-23/Th17-axis score, as assessed by an LV extracted from IL-23, IL-6, and the Th17 LV, was significantly higher in DSCZ than in NDSCZ and controls. We discovered that 70.7% of the variance in the phenome was explained by the IL-6/IL-23/Th17-axis (positively) and the G-CoDe and IL-10 (both inversely); and that 54.6% of the variance in the G-CoDe was explained by the IL-6/IL-23/Th17 scores (inversely) and magnesium, copper, calcium, and zinc (all positively)., Conclusion: The pathogenic IL-6/IL-23/Th17-axis contributes to the generalized neurocognitive deficit and the phenome of schizophrenia, especially that of DSCZ, due to its key role in peripheral inflammation and neuroinflammation and its consequent immunotoxic effects on neuronal circuits. These clinical impairments are more prominent in subjects with lowered IL-10, magnesium, calcium, and zinc., Competing Interests: he authors have declared that no competing interests exist.

Human alkaline ceramidase 3 (ACER3) is one of three alkaline ceramidases (ACERs) that catalyze the conversion of ceramide to sphingosine. ACERs are members of the CREST superfamily of integral-membrane hydrolases. All CREST members conserve a set of three Histidine, one Aspartate, and one Serine residue. Although the structure of ACER3 was recently reported, catalytic roles for these residues have not been biochemically tested. Here, we use ACER3 as a prototype enzyme to gain insight into this unique class of enzymes. Recombinant ACER3 was expressed in yeast mutant cells that lack endogenous ceramidase activity, and microsomes were used for biochemical characterization. Six-point mutants of the conserved CREST motif were developed that form a Zn-binding active site based on a recent crystal structure of human ACER3. Five point mutants completely lost their activity, with the exception of S77A, which showed a 600-fold decrease compared with the wild-type enzyme. The activity of S77C mutant was pH sensitive, with neutral pH partially recovering ACER3 activity. This suggested a role for S77 in stabilizing the oxyanion of the transition state. Together, these data indicate that ACER3 is a Zn2+-dependent amidase that catalyzes hydrolysis of ceramides via a similar mechanism to other soluble Zn-based amidases. Consistent with this notion, ACER3 was specifically inhibited by trichostatin A, a strong zinc chelator., Competing Interests: The authors have declared that no competing interests exist.

Stress associated proteins (SAPs) in plants have a key role in providing tolerance to multiple abiotic stresses. SAP gene family in Solanum tuberosum has not been fully studied before. This study identified 17 StSAP genes in S. tuberosum which code for A20/AN1 zinc-finger proteins. All the genes were distributed on ten different chromosomes and six segmental duplication events were identified. The SAPs in S. tuberosum and its orthologs in Arabidopsis thaliana were classified into six groups through the phylogenetic analysis. Introns across StSAP genes were identified in four genes. The promotor study of the StSAP genes showed different hormone and stress-related cis-elements that could potentially have a role in environmental stress response. The expression of StSAP genes in response to heat, mannitol, and salt were analyzed through in silico transcriptomic analysis. This study could potentially help in further understanding the functions of SAP genes in S. tuberosum., Competing Interests: The authors have declared that no competing interests exist.

Inflammatory signaling induces barrier dysfunction in retinal-pigmented epithelium (RPE) cells and plays a role in the pathology of age-related macular degeneration (AMD). We studied the role of Zn flux from the endoplasmic reticulum (ER) to the cytoplasm via Zip7 during inflammatory signaling in RPE cells. In ARPE-19 cells, Zip7 inhibition reduced impedance loss, FITC-dextran permeability and cytokine induction caused by challenge with IL-1β/TNF-α. Zip7 inhibition in iPS-derived RPE cells challenged with TNF- α reduced barrier loss in TER assays. In ARPE-19 cells, a Zn ionophore restored cytokine induction and barrier loss in cells challenged with IL-1 β /TNF- α despite Zip7 inhibition. A cell permeable Zn chelator demonstrated that Zn is essential for IL-1 β /TNF- α signaling. ER stress caused by Zip7 inhibition in ARPE-19 cells was found to partially contribute to reducing barrier dysfunction caused by IL-1 β /TNF- α. Overall, it was shown that Zn flux through Zip7 from the ER to the cytoplasm plays a critical role in driving barrier dysfunction caused by inflammatory cytokines in RPE cells., Competing Interests: The authors have declared that no competing interests exist.

Genome-wide association studies (GWAS) are a valuable approach to understanding the genetic basis of complex traits. One of the challenges of GWAS is the translation of genetic association results into biological hypotheses suitable for further investigation in the laboratory. To address this challenge, we introduce Network Interface Miner for Multigenic Interactions (NIMMI), a network-based method that combines GWAS data with human protein-protein interaction data (PPI). NIMMI builds biological networks weighted by connectivity, which is estimated by use of a modification of the Google PageRank algorithm. These weights are then combined with genetic association p-values derived from GWAS, producing what we call 'trait prioritized sub-networks.' As a proof of principle, NIMMI was tested on three GWAS datasets previously analyzed for height, a classical polygenic trait. Despite differences in sample size and ancestry, NIMMI captured 95% of the known height associated genes within the top 20% of ranked sub-networks, far better than what could be achieved by a single-locus approach. The top 2% of NIMMI height-prioritized sub-networks were significantly enriched for genes involved in transcription, signal transduction, transport, and gene expression, as well as nucleic acid, phosphate, protein, and zinc metabolism. All of these sub-networks were ranked near the top across all three height GWAS datasets we tested. We also tested NIMMI on a categorical phenotype, Crohn's disease. NIMMI prioritized sub-networks involved in B- and T-cell receptor, chemokine, interleukin, and other pathways consistent with the known autoimmune nature of Crohn's disease. NIMMI is a simple, user-friendly, open-source software tool that efficiently combines genetic association data with biological networks, translating GWAS findings into biological hypotheses. [ABSTRACT FROM AUTHOR]

Genome maintenance is an essential process in all cells. In prokaryotes, the RadD protein is important for survival under conditions that include DNA-damaging radiation. Precisely how RadD participates in genome maintenance remains unclear. Here we present a high-resolution X-ray crystal structure of ADP-bound Escherichia coli RadD, revealing a zinc-ribbon element that was not modelled in a previous RadD crystal structure. Insights into the mode of nucleotide binding and additional structure refinement afforded by the new RadD model will help to drive investigations into the activity of RadD as a genome stability and repair factor., Competing Interests: The authors have declared that no competing interests exist.

Invasive fungal infections, which kill more than 1.6 million patients each year worldwide, are difficult to treat due to the limited number of antifungal drugs (azoles, echinocandins, and polyenes) and the emergence of antifungal resistance. The transcription factor Crz1, a key regulator of cellular stress responses and virulence, is an attractive therapeutic target because this protein is absent in human cells. Here, we used a CRISPR-Cas9 approach to generate isogenic crz1Δ strains in two clinical isolates of caspofungin-resistant C. glabrata to analyze the role of this transcription factor in susceptibility to echinocandins, stress tolerance, biofilm formation, and pathogenicity in both non-vertebrate (Galleria mellonella) and vertebrate (mice) models of candidiasis. In these clinical isolates, CRZ1 disruption restores the susceptibility to echinocandins in both in vitro and in vivo models, and affects their oxidative stress response, biofilm formation, cell size, and pathogenicity. These results strongly suggest that Crz1 inhibitors may play an important role in the development of novel therapeutic agents against fungal infections considering the emergence of antifungal resistance and the low number of available antifungal drugs., Competing Interests: P.L.P received grants from Astellas, Basilea, MSD and Pfizer and speaker’s fees from Gilead, Basilea, Pfizer and MSD. The other authors declare not have any conflict of interest to disclose.

Emergence of multidrug resistant pathogens is increasing globally at an alarming rate with a need to discover novel and effective methods to cope infections due to these pathogens. Green nanoparticles have gained attention to be used as efficient therapeutic agents because of their safety and reliability. In the present study, we prepared zinc oxide nanoparticles (ZnO NPs) from aqueous leaf extract of Acacia arabica. The nanoparticles produced were characterized through UV-Visible spectroscopy, scanning electron microscopy, and X-ray diffraction. In vitro antibacterial susceptibility testing against foodborne pathogens was done by agar well diffusion, growth kinetics and broth microdilution assays. Effect of ZnO NPs on biofilm formation (both qualitatively and quantitatively) and exopolysaccharide (EPS) production was also determined. Antioxidant potential of green synthesized nanoparticles was detected by DPPH radical scavenging assay. The cytotoxicity studies of nanoparticles were also performed against HeLa cell lines. The results revealed that diameter of zones of inhibition against foodborne pathogens was found to be 16-30 nm, whereas the values of MIC and MBC ranged between 31.25-62.5 μg/ml. Growth kinetics revealed nanoparticles bactericidal potential after 3 hours incubation at 2 × MIC for E. coli while for S. aureus and S. enterica reached after 2 hours of incubation at 2 × MIC, 4 × MIC, and 8 × MIC. 32.5-71.0% inhibition was observed for biofilm formation. Almost 50.6-65.1% (wet weight) and 44.6-57.8% (dry weight) of EPS production was decreased after treatment with sub-inhibitory concentrations of nanoparticles. Radical scavenging potential of nanoparticles increased in a dose dependent manner and value ranged from 19.25 to 73.15%. Whereas cytotoxicity studies revealed non-toxic nature of nanoparticles at the concentrations tested. The present study suggests that green synthesized ZnO NPs can substitute chemical drugs against antibiotic resistant foodborne pathogens., Competing Interests: The authors have declared that no competing interests exist.

This study aimed to evaluate the effect of cultivation conditions in the context of light on the retention of selected vitamins, minerals and polyphenols in the stem and cap of the oyster mushroom (Pleurotus ostreatus L.). Additionally, the effect of the retention of bioactive components on the antioxidant activity of mushroom extracts was evaluated, taking into account the morphological part. Oyster mushrooms grown in the light of 200 lux had higher riboflavin content compared to mushrooms exposed to the light of lower intensity. The thiamine content of the mushrooms dropped with decreasing light intensity during cultivation. The content of biologically active compounds was found to be equal in the stem and the cap. In the case of riboflavin, it was shown that its contents in cap fractions, irrespective of the cultivation method, was statistically significantly higher than in stems. The mineral composition of caps and stems differed from each other. No differences in Zn and Cu content between the morphological parts of the mushroom studied were found. However, it was shown that the stems, regardless of the type of light, contained less iron, magnesium and sodium. Thus, it was observed that limited light exposure caused an increase in the content of total polyphenolic compounds, which did not correlate with antioxidant activity. There was no effect of the light on the antioxidant activity of mushrooms. It was also shown that stem extracts had higher antioxidant activity compared to the extracts obtained from the caps. This findings point to the possibility and potentail of use both fraction of mushrooms in the new food products development., Competing Interests: The authors have declared that no competing interests exist.

Wheat is an important global staple food crop; however, its productivity is severely hampered by changing climate. Erratic rain patterns cause terminal drought stress, which affect reproductive development and crop yield. This study investigates the potential and zinc (Zn) and silicon (Si) to ameliorate terminal drought stress in wheat and associated mechanisms. Two different drought stress levels, i.e., control [80% water holding capacity (WHC) was maintained] and terminal drought stress (40% WHC maintained from BBCH growth stage 49 to 83) combined with five foliar-applied Zn-Si combinations (i.e., control, water spray, 4 mM Zn, 40 mM Si, 4 mM Zn + 40 mM Si applied 7 days after the initiation of drought stress). Results revealed that application of Zn and Si improved chlorophyll and relative water contents under well-watered conditions and terminal drought stress. Foliar application of Si and Zn had significant effect on antioxidant defense mechanism, proline and soluble protein, which showed that application of Si and Zn ameliorated the effects of terminal drought stress mainly by regulating antioxidant defense mechanism, and production of proline and soluble proteins. Combined application of Zn and Si resulted in the highest improvement in growth and antioxidant defense. The application of Zn and Si improved yield and related traits, both under well-watered conditions and terminal drought stress. The highest yield and related traits were recorded for combined application of Zn and Si. For grain and biological yield differences among sole and combined Zn-Si application were statistically non-significant (p>0.05). In conclusion, combined application of Zn-Si ameliorated the adverse effects of terminal drought stress by improving yield through regulating antioxidant mechanism and production of proline and soluble proteins. Results provide valuable insights for further cross talk between Zn-Si regulatory pathways to enhance grain biofortification., Competing Interests: The authors have declared that no competing interests exist.

Malnutrition a health disorders arising due to over or low use of minerals, vitamins and nutritional substances required for proper functioning of body tissues and organs. Zinc (Zn) is the most important mineral required for the normal metabolism of plants and humans. Zinc-deficiency is one of the major cause of malnutrition globally. Maize is highly susceptible to Zn-deficiency and inflicts Zn-deficiency to humans and other animals being nourished on it. This study evaluated the effect of zinc-lysine chelate alone (0.1, 0.5, 1.0 and 1.5%) as seed priming and in combination with Zn-solubilizing bacteria (PMEL-1, PMEL-48, PMEL-57and PMEL-71)) on grain biofortification of autumn maize. The Zn accumulation in different parts (roots, stem, leaves, grains and cob pith) was quantified. Results indicated that Zn contents were 18.5% higher in the seeds primed with 1.5% solution of Zn-lysine chelate and inoculation of ZSB strains compared to control treatments. Seed priming with 1.5% Zn-lysine chelate in combination with ZSB inoculation significantly improved cob diameter and cob length by 16.75% and 42% during 2016 and by 11.36% and 34.35% during 2017. The increase in 100 grains weight over control was 18.4% and 15.27% for 2016 and 2017, respectively. The Zn contents were increased by 15.3%, 15.6%, 49.1%, and 33.0% in grain, cob-pith, stemand roots, respectively compared from control. Thus, the combined application of 1.5% Zn-lysine chelates along with ZSB inoculation could be used for combating malnutrition., Competing Interests: No conflict of interest.

Zinc (Zn) is an important micronutrient for crop plants and essential for human health. The Zn-deficiency is an important malnutrition problem known globally. Biofortified foods could overcome Zn deficiency in humans. Mungbean [Vigna radiata (L.) Wilczek] is an important, pulse crop frequently grown in arid and semi-arid regions of the world. Mungbean could provide essential micronutrients, including Zn to humans. Therefore, it is very important to investigate the impact of Zn fertilization on the yield and grain biofortification of mungbean. Twelve mungbean genotypes (i.e., NM-28, NM-2011, NM-13-1, NM-2006, NM-51, NM-54, NM-19-19, NM-92, NM-121-25, NM-20-21, 7006, 7008) were assessed for their genetic diversity followed by Zn-biofortification, growth and yield under control (0 kg ha-1) and Zn-fertilized (10 kg ha-1) conditions. Data relating to allometric traits, yield components, grain yield and grain Zn contents were recorded. Zinc fertilization improved entire allometric and yield-related traits. Grain yield of different genotypes ranged from 439 to 904 kg ha-1 under control and 536 to 1462 kg ha-1 under Zn-fertilization. Zinc concentration in the grains varied from 15.50 to 45.60 mg kg-1 under control and 18.53 to 64.23 mg kg-1 under Zn-fertilized conditions. The tested genotypes differed in their Zn-biofortification potential. The highest and the lowest grain Zn contents were noted for genotypes NM-28 and NM-121-25, respectively. Significant variation in yield and Zn-biofortification indicated the potential for improvement in mungbean yield and grain Zn-biofortification. The genotypes NM-28 and NM-2006 could be used in breeding programs for improvement in grain Zn concentration due to their high Zn uptake potential. Nonetheless, all available genotypes in the country should be screened for their Zn-biofortification potential., Competing Interests: The authors have declared that no competing interests exist.

N-fixing leguminous species can reach atmospheric dinitrogen gas (N2), having an advantage under N-limited degraded environments. These N-fixers are constantly used as facilitative species. Chlorophyll a fluorescence (ChF) acknowledges how different species take up and use light energy during photosynthesis. These techniques assess stress and performance responses to photosynthesis and are used for the selection of species with potential for reforestation. Six Fabaceae species were selected for this study: three nonfixing species (Cenostigma tocantinum, Senna reticulata and Dipteryx odorata) and three N-fixing species (Clitoria fairchildiana, Inga edulis and Acacia spp.). Variations in chlorophyll fluorescence under high vs. low water and nutrient conditions were studied. Multivariate analysis was performed to detect the effects of seasonality and fertilization on dark-adapted ChF two years after the experiment was established. The correlation among ChF variables and growth, photosynthesis and foliar nutrient concentrations was evaluated. Under high water- and nutrient-availability conditions, plants exhibited an enhanced performance index on absorption basis values correlated with electron transport fluxes. Under drought and nutrient-poor conditions, most species exhibit increased energy dissipation as photoprotection. High interspecific variation was found; therefore, species-specific responses should be considered in future ChF studies. Corroborating the ability to colonize high-light environments, N-fixers showed an increased performance index correlated with electron transport and Zn and N foliar concentrations. Negative correlations were found between photosynthesis and trapped fluxes. Diameter growth was positively correlated with electron transport fluxes. Given the different responses among species, ChF is an effective technique to screen for seasonality, fertilization and species effects and should be considered for use during forest restoration. Finally, the addition of fertilization treatments may facilitate tropical forest restoration due to the importance of nutrients in physiological processes. N-fixers showed high photochemical performance and tolerance to abiotic stress in degraded areas and therefore should be included to support ecosystem biomass restoration., Competing Interests: The authors have declared that no competing interests exist.

Nuclear factor-kappa B (NF-κB) plays a critical role in the host defense against microbial pathogens. Many pathogens modulate NF-κB signaling to establish infection in their host. Salmonella enterica serovar Typhimurium (S. Typhimurium) possesses two type III secretion systems (T3SS-1 and T3SS-2) and directly injects many effector proteins into host cells. It has been reported that some effectors block NF-κB signaling, but the molecular mechanism of the inactivation of NF-κB signaling in S. Typhimurium is poorly understood. Here, we identified seven type III effectors-GogA, GtgA, PipA, SseK1, SseK2, SseK3, and SteE-that inhibited NF-κB activation in HeLa cells stimulated with TNF-α. We also determined that only GogA and GtgA are involved in regulation of the activation of NF-κB in HeLa cells infected with S. Typhimurium. GogA, GtgA, and PipA are highly homologous to one another and have the consensus zinc metalloprotease HEXXH motif. Our experiments demonstrated that GogA, GtgA, and PipA each directly cleaved NF-κB p65, whereas GogA and GtgA, but not PipA, inhibited the NF-κB activation in HeLa cells infected with S. Typhimurium. Further, expressions of the gogA or gtgA gene were induced under the SPI-1-and SPI-2-inducing conditions, but expression of the pipA gene was induced only under the SPI-2-inducing condition. We also showed that PipA was secreted into RAW264.7 cells through T3SS-2. Finally, we indicated that PipA elicits bacterial dissemination in the systemic stage of infection of S. Typhimurium via a T3SS-1-independent mechanism. Collectively, our results suggest that PipA, GogA and GtgA contribute to S. Typhimurium pathogenesis in different ways., Competing Interests: The authors have declared that no competing interests exist.

Copper (Cu), iron (Fe), and zinc (Zn) are essential trace minerals for the reproduction, growth, and immunity of mammalian herbivore populations. We examined the relationships between Cu, Fe, and Zn in soils, common plants, and hepatic stores of two wild herbivores to assess the effects of weather, sex, and population density on the transfer of trace minerals from soils to mammals during the growing season. Soils, grasses, woody browse, hispid cotton rats (Sigmodon hispidus), and white-tailed deer (Odocoileus virginianus) were sampled across 19 sites. Concentrations of Cu, Fe, and Zn in grasses and browse species were not correlated with concentrations of those minerals in soils sampled from the same areas. Leaves of woody browse were higher in Cu, lower in Fe, and similar in Zn when compared with grasses. Available concentrations of soils were positively related to liver Cu and Zn in hispid cotton rats, which was consistent with the short lives and high productivity of these small mammals that rely on grass seed heads. Interactions between soil concentrations and weather also affected liver Cu and Fe in deer, which reflected the greater complexity of trophic transfers in large, long-lived, browsing herbivores. Population density was correlated with liver concentrations of Cu, Fe, and Zn in hispid cotton rats, and concentrations of Cu and Fe in deer. Liver Cu was < 5 mg/kg wet weight in at least 5% of animals at two of eight sites for hispid cotton rats and < 3.8 mg/kg wet weight in at least 5% of animals at three of 12 sites for deer, which could indicate regional limitation of Cu for populations of mammalian herbivores. Our data indicate that supplies of trace minerals may contribute to density dependence of herbivore populations. Local population density may therefore influence the prevalence of deficiency states and disease outbreak that exacerbate population cycles in wild mammals., Competing Interests: The authors have declared that no competing interests exist.

The integral membrane zinc metalloprotease ZMPSTE24 plays a key role in the proteolytic processing of farnesylated prelamin A, the precursor of the nuclear scaffold protein lamin A. Failure of this processing step results in the accumulation of permanently farnesylated forms of prelamin A which cause the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS), as well as related progeroid disorders, and may also play a role in physiological aging. ZMPSTE24 is an intriguing and unusual protease because its active site is located inside of a closed intramembrane chamber formed by seven transmembrane spans with side portals in the chamber permitting substrate entry. The specific features of prelamin A that make it the sole known substrate for ZMPSTE24 in mammalian cells are not well-defined. At the outset of this work it was known that farnesylation is essential for prelamin A cleavage in vivo and that the C-terminal region of prelamin A (41 amino acids) is sufficient for recognition and processing. Here we investigated additional features of prelamin A that are required for cleavage by ZMPSTE24 using a well-established humanized yeast system. We analyzed the 14-residue C-terminal region of prelamin A that lies between the ZMPSTE24 cleavage site and the farnesylated cysteine, as well 23-residue region N-terminal to the cleavage site, by generating a series of alanine substitutions, alanine additions, and deletions in prelamin A. Surprisingly, we found that there is considerable flexibility in specific requirements for the length and composition of these regions. We discuss how this flexibility can be reconciled with ZMPSTE24's selectivity for prelamin A., Competing Interests: The authors have declared that no competing interests exist.

Wheat is one of the best-domesticated cereal crops and one of the vital sources of nutrition for humans. An investigation was undertaken to reveal the potential of novel bio-inoculants enriching micronutrients in shoot and grains of wheat crop to eliminate the hazards of malnutrition. Sole as well as consortia inoculation of bio-inoculants significantly enhanced mineral nutrients including zinc (Zn) and iron (Fe) concentrations in shoot and grains of wheat. Various treatments of bio-inoculants increase Zn and Fe content up to 1-15% and 3-13%, respectively. Sole inoculation of Bacillus aryabhattai (S10) impressively improves the nutritious of wheat. However, the maximum increase in minerals contents of wheat was recorded by consortia inoculation of Paenibacillus polymyxa ZM27, Bacillus subtilis ZM63 and Bacillus aryabhattai S10. This treatment also showed a maximum bacterial population (18 × 104 cfu mL-1) in the rhizosphere. The consortium application of these strains showed up to a 17% increase in yield. It is evident from the results that the consortium application was more effective than sole and co-inoculation. A healthy positive correlation was found between growth, yield, and the accessibility of micronutrients to wheat crops at the harvesting stage. The present investigations revealed the significance of novel bacterial strains in improving the nutritional status of wheat crops. These strains could be used as bio-inoculants for the biofortification of wheat to combat hidden hunger in developing countries., Competing Interests: The authors have declared that no competing interests exist.

Alginetin is the major product formed from pentoses and hexurionic acids. Alginetin is producted by cooking process of food including pection, a naturally-occurring polysacharride found in many plants. However, the biological interaction and toxicity of alginetin are not known at all. The aim of the present study was to investigate the cellular actions of alginetin on rat thymic lymphocytes. The effects of alginetin on the cell were examined using flow cytometry with fluorescent probes. Alginetin increased cellular content of non-protein thiols ([NPT]i) and elevated intracellular Zn2+ levels ([Zn2+]i). Chelation of intracellular Zn2+ reduced the effect of alginetin on [NPT]i, and chelation of external Zn2+ almost completely diminished alginetin-induced elevation of [Zn2+]i, indicating that alginetin treatment increased Zn2+ influx. Increased [NPT]i and [Zn2+]i levels in response to alginetin were positively correlated. Alginetin protected cells against oxidative stress induced by hydrogen peroxide and Ca2+ overload by calcium ionophore. It is considered that the increases in [NPT]i and [Zn2+]i are responsible for the cytoprotective activity of alginetin because NPT attenuates oxidative stress and Zn2+ competes with Ca2+. Alginetin may be produced during manufacturing of jam, which may provide additional health benefits of jam., Competing Interests: The authors have declared that no competing interests exist.

Cellular homeostasis of zinc, an essential element for living organisms, is tightly regulated by a family of zinc transporters. The zinc transporter 7, ZnT7, is highly expressed on the membrane of the Golgi complex of intestinal epithelial cells and goblet cells. It has previously been shown that Znt7 knockout leads to zinc deficiency and decreased weight gain in C57BL/6 mice on a defined diet. However, effects within the colon are unknown. Given the expression profile of Znt7, we set out to analyze the changes in mucin density and gut microbial composition in the mouse large intestine induced by Znt7 knockout. We fed a semi-purified diet containing 30 mg Zn/kg to Znt7-/- mice with their heterozygous and wild type littermates and found a sex specific effect on colonic mucin density, goblet cell number, and microbiome composition. In male mice Znt7 knockout led to increased goblet cell number and mucin density but had little effect on gut microbiome composition. However, in female mice Znt7 knockout was associated with decreased goblet cell number and mucin density, with increased proportions of the microbial taxa, Allobaculum, relative to wild type. The gut microbial composition was correlated with mucin density in both sexes. These findings suggest that a sex-specific relationship exists between zinc homeostasis, mucin production and the microbial community composition within the colon., Competing Interests: The authors have declared that no competing interests exist.

Zinc ions (Zn2+) are important messenger molecules involved in various physiological functions. To maintain the homeostasis of cytosolic Zn2+ concentration ([Zn2+]c), Zrt/Irt-related proteins (ZIPs) and Zn2+ transporters (ZnTs) are the two families of proteins responsible for decreasing and increasing the [Zn2+]c, respectively, by fluxing Zn2+ across the membranes of the cell and intracellular compartments in opposite directions. Most studies focus on the cytotoxicity incurred by a high concentration of [Zn2+]c and less investigate the [Zn2+]c at physiological levels. Zinc oxide-nanoparticle (ZnO-NP) is blood brain barrier-permeable and elevates the [Zn2+]c to different levels according to the concentrations of ZnO-NP applied. In this study, we mildly elevated the [Zn2+]c by ZnO-NP at concentrations below 1 μg/ml, which had little cytotoxicity, in cultured human neuroblastoma SH-SY5Y cells and characterized the importance of Zn2+ transporters in 6-hydroxy dopamine (6-OHDA)-induced cell death. The results show that ZnO-NP at low concentrations elevated the [Zn2+]c transiently in 6 hr, then declined gradually to a basal level in 24 hr. Knocking down the expression levels of ZnT1 (located mostly at the plasma membrane) and ZIP8 (present in endosomes and lysosomes) increased and decreased the ZnO-NP-induced elevation of [Zn2+]c, respectively. ZnO-NP treatment reduced the basal levels of reactive oxygen species and Bax/Bcl-2 mRNA ratios; in addition, ZnO-NP decreased the 6-OHDA-induced ROS production, p53 expression, and cell death. These results show that ZnO-NP-induced mild elevation in [Zn2+]c activates beneficial effects in reducing the 6-OHDA-induced cytotoxic effects. Therefore, brain-delivery of ZnO-NP can be regarded as a potential therapy for neurodegenerative diseases., Competing Interests: The authors have declared that no competing interests exist.

Background: Zinc (Zn) deficiency is one of the most common micronutrient deficiencies worldwide. Accurate estimates of Zn intake would facilitate the design and implementation of effective nutritional interventions., Objective: We sought to improve estimates of dietary Zn intake by evaluating staple crop Zn content and dietary Zn consumption by children under the age of 5 in 9 rural districts of Uganda., Methods: We measured the Zn content of 581 crop samples from household farms and 167 crop samples from nearby markets, and administered food frequency questionnaires to the primary caretakers of 237 children. We estimated Zn consumption using 3 sources of crop Zn content: (i) the HarvestPlus food composition table (FCT) for Uganda, (ii) measurements from household crops, and (iii) measurements from market crops., Results: The Zn content of staple crops varied widely, resulting in significantly different estimates of dietary Zn intake. 41% of children appeared to be at risk when estimates were based on market-sampled crops, 23% appeared at risk when estimates were based on the HarvestPlus FCT, and 16% appeared at risk when estimates were based on samples from household farms., Conclusion: The use of FCTs to calculate Zn intake overestimated the risk of dietary inadequacy for children who primarily consumed staple crops that were produced on household farms, but underestimated the risk for children who primarily consumed staple crops that were purchased at market. More information on the Zn content of staple crops in developing countries could lead to more accurate estimates of dietary intake and associated deficiencies., Competing Interests: The authors have declared that no competing interests exist.

Glyoxalase pathway is the major pathway of methylglyoxal detoxification and is ubiquitously present in all organisms ranging from prokaryotes to eukaryotes. Glyoxalase I (GLYI) and Glyoxalase II (GLYII), the two core enzymes of this pathway work together to neutralize methylglyoxal (MG), a dicarbonyl molecule with detrimental cytotoxicity at higher concentrations. The first step towards the detoxification of MG is catalyzed by GLYI, a metalloenzyme that requires divalent metal ions (either Zn2+ as seen in eukaryotes or Ni2+ as in prokaryotes). However, both Zn2+ and Ni2+ dependent GLYIs have been shown to co-exist in a higher eukaryote i.e. Arabidopsis thaliana. In the present study, we determine the role of both Zn2+ dependent (AtGLYI2) and Ni2+ dependent (AtGLYI3, AtGLYI6) GLYIs from Arabidopsis in salinity stress tolerance. AtGLYI2 overexpressing Arabidopsis plants showed better growth rate while maintaining lower levels of MG under high saline conditions. They were taller with more number of silique formation with respect to their Ni2+ dependent counterparts. Further, lack in germination of Arabidopsis AtGLYI2 mutants in presence of exogenous MG indicates the direct involvement of Zn2+ dependent GLYI in MG detoxification, suggesting Zn2+ dependent GLYI as the main enzyme responsible for MG detoxification and salinity stress tolerance., Competing Interests: The authors have declared that no competing interests exist.

Heavy metal accumulation in edible plants grown in contaminated soils poses a major environmental risk to humans and grazing animals. This study focused on the concentration and speciation of Zn in different edible plants grown in soils contaminated with smelter wastes (Spelter, WV, USA) containing high levels of the metals Zn, Cu, Pb, Cd. Their accumulation was examined in different parts (roots, stem, and leaves) of plants and as a function of growth stage (dry seed, sprouting seed, cotyledon, and leaves) in the root vegetables radish, the leafy vegetable spinach and the legume clover. Although the accumulation of metals varied significantly with plant species, the average metal concentrations were [Zn] > [Pb] > [Cu] > [Cd]. Metal uptake studies were complemented with bulk and micro X-ray absorption spectroscopy (XAS) at Zn K-edge and micro X-ray fluorescence (μXRF) measurements to evaluate the speciation and distribution of Zn in these plant species. Dynamic interplay between the histidine and malate complexation of Zn was observed in all plant species. XRF mapping of spinach leaves at micron spatial resolution demonstrated the accumulation of Zn in vacuoles and leaf tips. Radish root showed accumulation of Zn in root hairs, likely as ZnS nanoparticles. At locations of high Zn concentration in spinach leaves, μXANES suggests Zn complexation with histidine, as opposed to malate in the bulk leaf. These findings shed new light on the dynamic nature of Zn speciation in plants., Competing Interests: The authors have declared that no competing interests exist.

The individual toxicity and bioaccumulation of cadmium, copper and zinc for common carp juveniles was evaluated in a direct comparison in two experimental setups. First, fish were exposed for 10 days to different metal concentrations in order to link metal bioaccumulation to LC50 values (concentration lethal to 50% of the animals) and incipient lethal levels (ILL, concentration where 50% survives indefinitely). Accumulated metals showed a positive dose dependent uptake for cadmium and copper, but not for zinc. Toxicity was in the order cadmium>copper>zinc with 96h LC50 values for cadmium at 0.20±0.16 μM, for copper at 0.77±0.03 μM, and for zinc at 29.89±9.03 μM respectively. For copper, the 96h exposure was sufficient to calculate the incipient lethal level and therefore 96h LC50 and ILL levels were the same, while for cadmium and zinc 5 to 6 days were needed to reach ILL resulting in slightly lower values at 0.16 μM and 28.33 μM respectively. Subsequently, a subacute exposure experiment was conducted, where carp juveniles were exposed to 2 equitoxic concentrations (10% and 50% of LC50 96 h) of the three metals for 1, 3 and 7 days. Again a significant dose-dependent increase in gill cadmium and copper, but not in zinc, was observed during the 7-day exposure. Copper clearly affected sodium levels in gill tissue, while zinc and cadmium did not significantly alter any of the gill electrolytes. The overall histopathological effects (e.g. hyperemia and hypertrophy) of the metal exposures were mild for most of the alterations. Our study showed that copper an cadmium (but not zinc) showed dose dependent metal accumulation, however this bioaccumulation was only correlated with mortality for cadmium. Metal specific alterations were reduced gill sodium levels in copper exposed fish and oedema of the primary epithelium which typically occurred in both levels of zinc exposure., Competing Interests: The authors have declared that no competing interests exist.

Changes in eating behavior of adolescents are associated with high consumption of processed and ultra-processed foods. This study evaluated the association between these foods and the prevalence of inadequate micronutrient intake in adolescents. A cross-sectional study was conducted with 444 adolescents from public schools in the city of Natal, northeastern Brazil. The adolescents' habitual food consumption was evaluated using two 24-hour dietary recalls. Foods were categorized according to the degree of processing (processed and ultra-processed) and distributed into energy quartiles, using the NOVA classification system. Inadequacies in micronutrient intake were assessed using the estimated average requirement (EAR) as the cutoff point. Multivariate logistic regression models were used to estimate the relationship between energy percentage from processed and ultra-processed foods and prevalence of inadequate micronutrient intake. The mean (Standard Deviation (SD)) consumption of total energy from processed foods ranged from 5.8% (1.7%) in Q1 to 20.6% (2.9%) in Q4, while the mean consumption of total energy from ultra-processed foods ranged from 21.4% (4.9%) in Q1 to 61.5% (11.7%) in Q4. The rates of inadequate intake of vitamin D, vitamin E, folate, calcium, and selenium were above 80% for both sexes across all age groups. Energy consumption from processed foods was associated with higher prevalence of inadequate selenium intake (p < 0.01) and lower prevalence of inadequate vitamin B1 intake (p = 0.04). Energy consumption from ultra-processed foods was associated with lower prevalence of inadequate zinc and vitamin B1 intake (p < 0.01 and p = 0.03, respectively). An increase in the proportion of energy obtained from processed and ultra-processed foods may reflect higher prevalence of inadequate selenium intake and lower prevalence of vitamin B1 and zinc inadequacy., Competing Interests: The authors have declared that no competing interests exist.

Corynebacterium diphtheriae is a Gram-positive bacterial pathogen and the causative agent of diphtheria, a severe disease of the upper respiratory tract of humans. Factors required for C. diphtheriae to survive in the human host are not well defined, but likely include the acquisition of essential metals such as zinc. In C. diphtheriae, zinc-responsive global gene regulation is controlled by the Zinc Uptake Regulator (Zur), a member of the Fur-family of transcriptional regulators. In this study, we use transcriptomics to identify zinc-regulated genes in C. diphtheriae by comparing gene expression of a wild-type strain grown without and with zinc supplementation. Zur-regulated genes were identified by comparing wild-type gene expression with that of an isogenic zur mutant. We observed zinc repression of several putative surface proteins, the heme efflux system hrtBA, various ABC transporters, and the non-ribosomal peptide synthetase/polyketide synthase cluster sidAB. Furthermore, increased gene expression in response to zinc was observed for the alcohol dehydrogenase, adhA. Zinc and Zur regulation were confirmed for several genes by complementing the zur deletion and subsequent RT-qPCR analysis. We used MEME to predict Zur binding sites within the promoter regions of zinc- and Zur-regulated genes, and verified Zur binding by electrophoretic mobility shift assays. Additionally, we characterized cztA (dip1101), which encodes a putative cobalt/zinc/cadmium efflux family protein. Deletion of cztA results in increased sensitivity to zinc, but not to cobalt or cadmium. This study advances our knowledge of changes to Zur-dependent global gene expression in response to zinc in C. diphtheriae. The identification of zinc-regulated ABC transporters herein will facilitate future studies to characterize zinc transport in C. diphtheriae., Competing Interests: The authors have declared that no competing interests exist.

Dps proteins (DNA-binding proteins from starved cells) have been found to detoxify H2O2. At their catalytic centers, the ferroxidase center (FOC), Dps proteins utilize Fe2+ to reduce H2O2 and therefore play an essential role in the protection against oxidative stress and maintaining iron homeostasis. Whereas most bacteria accommodate one or two Dps, there are five different Dps proteins in Nostoc punctiforme, a phototrophic and filamentous cyanobacterium. This uncommonly high number of Dps proteins implies a sophisticated machinery for maintaining complex iron homeostasis and for protection against oxidative stress. Functional analyses and structural information on cyanobacterial Dps proteins are rare, but essential for understanding the function of each of the NpDps proteins. In this study, we present the crystal structure of NpDps4 in its metal-free, iron- and zinc-bound forms. The FOC coordinates either two iron atoms or one zinc atom. Spectroscopic analyses revealed that NpDps4 could oxidize Fe2+ utilizing O2, but no evidence for its use of the oxidant H2O2 could be found. We identified Zn2+ to be an effective inhibitor of the O2-mediated Fe2+ oxidation in NpDps4. NpDps4 exhibits a FOC that is very different from canonical Dps, but structurally similar to the atypical one from DpsA of Thermosynechococcus elongatus. Sequence comparisons among Dps protein homologs to NpDps4 within the cyanobacterial phylum led us to classify a novel FOC class: the His-type FOC. The features of this special FOC have not been identified in Dps proteins from other bacterial phyla and it might be unique to cyanobacterial Dps proteins., Competing Interests: The authors have declared that no competing interests exist.

Ternary solution (TSOL) is a novel Zn chelate-based systemic antimicrobial formulation designed for treating citrus bacterial pathogens 'Candidatus Liberibacter asiaticus' and Xanthomonas citri subsp. citri. TSOL is a component of MS3T, a novel multifunctional surface/sub-surface/systemic therapeutic formulation. Antimicrobial activity of TSOL was compared with the antimicrobial compound ZnO against X. citri subsp. citri and 'Ca. L. asiaticus' surrogate Liberibacter crescens in batch cultures. X. citri subsp. citri and L. crescens were also introduced into microfluidic chambers, and the inhibitory action of TSOL against biofilm formation was evaluated. The minimum inhibitory concentration of TSOL for both X. citri subsp. citri and L. crescens was 40ppm. TSOL was bactericidal to X. citri subsp. citri and L. crescens above 150 ppm and 200 ppm, respectively. On the contrary, ZnO was more effective as a bactericidal agent against L. crescens than X. citri subsp. citri. TSOL was more effective in controlling growth and biofilm formation of X. citri subsp. citri in batch cultures compared to ZnO. Time-lapse video imaging microscopy showed that biofilm formation of X. citri subsp. citri was inhibited in microfluidic chambers treated with 60 ppm TSOL. TSOL also inhibited further growth of already formed X. citri subsp. citri and L. crescens biofilms in microfluidic chambers. Leaf spraying of TSOL showed higher plant uptake and systemic movement in citrus (Citrus reshni) plants compared to that of ZnO, suggesting that TSOL is a promising antimicrobial compound to control vascular plant pathogens such as 'Ca. L. asiaticus'., Competing Interests: The authors have declared that no competing interests exist.

The uptake of zinc, which is vital in trace amounts, is tightly controlled in bacteria. For this control, bacteria of the Streptococcaceae group use a Zn(II)-binding repressor named ZitR in lactococci and AdcR in streptococci, while other bacteria use a Zur protein of the Ferric uptake regulator (Fur) superfamily. ZitR and AdcR proteins, characterized by a winged helix-turn-helix DNA-binding domain, belong to the multiple antibiotic resistance (MarR) superfamily, where they form a specific group of metallo-regulators. Here, one such Zn(II)-responsive repressor, ZitR of Lactococcus lactis subspecies cremoris strain MG1363, is characterized. Size Exclusion Chromatography-coupled to Multi Angle Light Scattering, Circular Dichroism and Isothermal Titration Calorimetry show that purified ZitR is a stable dimer complexed to Zn(II), which is able to bind its two palindromic operator sites on DNA fragments. The crystal structure of ZitR holo-form (Zn(II)4-ZitR2), has been determined at 2.8 Å resolution. ZitR is the fourth member of the MarR metallo-regulator subgroup whose structure has been determined. The folding of ZitR/AdcR metallo-proteins is highly conserved between both subspecies (cremoris or lactis) in the Lactococcus lactis species and between species (Lactococcus lactis and Streptococcus pneumoniae or pyogenes) in the Streptococcaceae group. It is also similar to the folding of other MarR members, especially in the DNA-binding domain. Our study contributes to better understand the biochemical and structural properties of metallo-regulators in the MarR superfamily., Competing Interests: The authors have declared that no competing interests exist.

While an ischemic insult poses a lethal danger to myocardial cells, a significant proportion of cardiac myocytes remain viable throughout the ischemic episode and die, paradoxically, only after the blood flow is reinstated. Despite decades of research, the actual chronology of critical events leading to cardiomyocyte death during the reperfusion phase remains poorly understood. Arguably, identification of the pivotal event in this setting is necessary to design effective strategies aimed at salvaging the myocardium after an ischemic attack. Here we used neonatal rat ventricular myocytes (NRVMs) subjected to 20-30 min of simulated ischemia followed by 1 hour of "reperfusion". Using different combinations of spectrally-compatible fluorescent indicators, we analyzed the relative timing of the following events: (1) abnormal increase in cytoplasmic [Ca2+] (TCaCy); (2) abnormal increase in mitochondrial [Ca2+] (TCaMi); (3) loss of mitochondrial inner membrane potential (ΔΨm) indicating mitochondrial permeability transitions (TMPT); (4) sacrolemmal permeabilization (SP) to the normally impermeable small fluorophore TO-PRO3 (TSP). In additional experiments we also analyzed the timing of abnormal uptake of Zn2+ into the cytoplasm (TZnCy) relative to TCaCy and TSP. We focused on those NRVMs which survived anoxia, as evidenced by at least 50% recovery of ΔΨm and the absence of detectable SP. In these cells, we found a consistent sequence of critical events in the order, from first to last, of TCaCy, TCaMi, TMPT, TSP. After detecting TCaCy and TCaMi, abrupt switches between 1.1 mM and nominally zero [Ca2+] in the perfusate quickly propagated to the cytoplasmic and mitochondrial [Ca2+]. Depletion of the sarcoplasmic reticulum with ryanodine (5 μM)/thapsigargin (1 μM) accelerated all events without changing their order. In the presence of ZnCl2 (10-30 μM) in the perfusate we found a consistent timing sequence TCaCy < TZn ≤ TSP. In some cells ZnCl2 interfered with Ca2+ uptake, causing "steps" or "gaps" in the [Ca2+]Cy curve, a phenomenon never observed in the absence of ZnCl2. Together, these findings suggest an evolving permeabilization of NRVM's sarcolemma during reoxygenation, in which the expansion of the pore size determines the timing of critical events, including TMPT., Competing Interests: The authors have declared that no competing interests exist.

Micronutrient deficiency is one of the biggest public health concerns in Bangladesh. As per World Health Organisation (WHO) in the 2016 report, 40% women of reproductive age suffer from anaemia. According to the National Micronutrient Survey 2011-2012, 57% women suffer from zinc deficiency. The objective of the present study was to determine the effectiveness of fortified rice (FFR in addressing anaemia and zinc deficiency among vulnerable women. Baseline and endline surveys were conducted among female Vulnerable Group Development (VDG) beneficiaries in five districts in Bangladesh before and after 12 months of FFR distribution. The intervention group received 30 kg FFR; the control group received 30 kg non-FFR for every month from January 2013 to December 2013. The sample sizes were 870 women (435/group) at baseline and 800 (400/group) at endline. Difference-in-difference (DID) was estimated to measure the effect of FFR on anaemia and serum zinc. In the baseline survey, 39% of the FFR group and 34% of the non-FFR group had anaemia. At endline, 34% of women in the FFR group were anaemic compared to 40.7% in the non-FFR group. At endline, prevalence of anaemia was reduced in the FFR group by 4.8% but increased in the non-FFR group by 6.7%. The DID estimation showed the reduction in anaemia after 12 months of FFR consumption was significant (p = 0.035). The DID in mean haemoglobin level after 12 months of FFR consumption was also statistically significant (p = 0.002). Zinc deficiency decreased by 6% in the FFR group at endline, though the DID was not significant. Most of the respondents of the FFR group reported that they received their entitled rice on a regular basis however only half of the non-FFR respondents received every month in 12 months. Anaemia was significantly associated with not consuming fortified rice, geographical region, older age and heavy menstrual bleeding (P<0.05). FFR reduced anaemia and zinc deficiency prevalence. Replacement of regular rice with FFR in the VGD programme is recommended to reduce anaemia among vulnerable groups., Competing Interests: The authors have declared that no competing interests exist.

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.

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.

Verticillium insectorum J3 was isolated from a local lead-zinc deposit tailing, and its biosorption characteristics and reaction to the toxicities of different Pb(II) and Zn(II) concentrations were investigated. SEM, FTIR, a pH test and a desorption experiment were carried out to identify a possible mechanism. The biosorption of J3 presented an inhibition trend at low concentrations (25-75 mg L-1) and promotion at high concentrations (100-300 mg L-1). J3 absorbed Pb(II) prior to Zn(II) and produced alkaline substances, while mycelial and pellet morphology modifications were important for the removal of Pb(II) and Zn(II) under different stressful conditions (SEM results). Both intracellular accumulation and extracellular absorption may contribute to the removal of Pb(II) at lower concentrations (25-50 mg L-1), although mainly extracellular biosorption occurred at higher concentrations (75-300 mg L-1). However, Zn(II) bioaccumulation occurred at all concentrations assayed. Verticillium insectorum J3 may have evolved active defenses to alleviate the toxicity of heavy metals and proved to be a highly efficient biosorbent, especially for Pb(II) at high concentrations. This study is a useful reference for the development of biotreatment technologies to mitigate heavy metal waste., Competing Interests: Hunan Guozhen Environmental Technology Co., Ltd just provided support in the form of salaries for author [TL], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials.

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.

Plants in arid zones are constantly exposed to drought stress. The ASR protein family (Abscisic, Stress, Ripening) -a subgroup of the late embryogenesis abundant superfamily- is involved in the water stress response and adaptation to dry environments. Tomato ASR1, as well as other members of this family, is an intrinsically disordered protein (IDP) that functions as a transcription factor and a chaperone. Here we employed different biophysical techniques to perform a deep in vitro characterization of ASR1 as an IDP and showed how both environmental factors and in vivo targets modulate its folding. We report that ASR1 adopts different conformations such as α-helix or polyproline type II in response to environmental changes. Low temperatures and low pH promote the polyproline type II conformation (PII). While NaCl increases PII content and slightly destabilizes α-helix conformation, PEG and glycerol have an important stabilizing effect of α-helix conformation. The binding of Zn2+in the low micromolar range promotes α-helix folding, while extra Zn2+ results in homo-dimerization. The ASR1-DNA binding is sequence specific and dependent on Zn2+. ASR1 chaperone activity does not change upon the structure induction triggered by the addition of Zn2+. Furthermore, trehalose, which has no effect on the ASR1 structure by itself, showed a synergistic effect on the ASR1-driven heat shock protection towards the reporter enzyme citrate synthase (CS). These observations prompted the development of a FRET reporter to sense ASR1 folding in vivo. Its performance was confirmed in Escherichia coli under saline and osmotic stress conditions, representing a promising probe to be used in plant cells. Overall, this work supports the notion that ASR1 plasticity is a key feature that facilitates its response to drought stress and its interaction with specific targets., Competing Interests: The authors have declared that no competing interests exist.

The HIV-1 nucleocapsid 7 (NCp7) plays crucial roles in multiple stages of HIV-1 life cycle, and its biological functions rely on the binding of zinc ions. Understanding the molecular mechanism of how the zinc ions modulate the conformational dynamics and functions of the NCp7 is essential for the drug development and HIV-1 treatment. In this work, using a structure-based coarse-grained model, we studied the effects of zinc cofactors on the folding and target RNA(SL3) recognition of the NCp7 by molecular dynamics simulations. After reproducing some key properties of the zinc binding and folding of the NCp7 observed in previous experiments, our simulations revealed several interesting features in the metal ion modulated folding and target recognition. Firstly, we showed that the zinc binding makes the folding transition states of the two zinc fingers less structured, which is in line with the Hammond effect observed typically in mutation, temperature or denaturant induced perturbations to protein structure and stability. Secondly, We showed that there exists mutual interplay between the zinc ion binding and NCp7-target recognition. Binding of zinc ions enhances the affinity between the NCp7 and the target RNA, whereas the formation of the NCp7-RNA complex reshapes the intrinsic energy landscape of the NCp7 and increases the stability and zinc affinity of the two zinc fingers. Thirdly, by characterizing the effects of salt concentrations on the target RNA recognition, we showed that the NCp7 achieves optimal balance between the affinity and binding kinetics near the physiologically relevant salt concentrations. In addition, the effects of zinc binding on the inter-domain conformational flexibility and folding cooperativity of the NCp7 were also discussed.

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein specific for Neisseria and constitutes one of the three main protein antigens of the Bexsero vaccine. Meningococcal and human proteases, cleave NHBA protein upstream or downstream of a conserved Arg-rich region, respectively. The cleavage results in the release of the C-terminal portion of the protein. The C-terminal fragment originating from the processing of meningococcal proteases, referred to as C2 fragment, exerts a toxic effect on endothelial cells altering the endothelial permeability. In this work, we reported that recombinant C2 fragment has no influence on the integrity of human airway epithelial cell monolayers, consistent with previous findings showing that Neisseria meningitidis traverses the epithelial barrier without disrupting the junctional structures. We showed that epithelial cells constantly secrete proteases responsible for a rapid processing of C2 fragment, generating a new fragment that does not contain the Arg-rich region, a putative docking domain reported to be essential for C2-mediated toxic effect. Moreover, we found that the C3-convertase of the alternative complement pathway is one of the proteases responsible for this processing. Overall, our data provide new insights on the cleavage of NHBA protein during meningococcal infection. NHBA cleavage may occur at different stages of the infection, and it likely has a different role depending on the environment the bacterium is interacting with.

Polished rice is poor source of micronutrients, however wide genotypic variability exists for zinc uptake and remobilization and zinc content in brown and polished grains in rice. Two landraces (Chittimutyalu and Kala Jeera Joha) and one popular improved variety (BPT 5204) were grown under zinc sufficient soil and their analyses showed high zinc in straw of improved variety, but high zinc in polished rice in landraces suggesting better translocation ability of zinc into the grain in landraces. Transcriptome analyses of the panicle tissue showed 41182 novel transcripts across three samples. Out of 1011 differentially expressed exclusive transcripts by two landraces, 311 were up regulated and 534 were down regulated. Phosphate transporter-exporter (PHO), proton-coupled peptide transporters (POT) and vacuolar iron transporter (VIT) showed enhanced and significant differential expression in landraces. Out of 24 genes subjected to quantitative real time analyses for confirmation, eight genes showed significant differential expression in landraces. Through mapping, six rice microsatellite markers spanning the genomic regions of six differentially expressed genes were validated for their association with zinc in brown and polished rice using recombinant inbred lines (RIL) of BPT 5204/Chittimutyalu. Thus, this study reports repertoire of genes associated with high zinc in polished rice and a proof concept for deployment of transcriptome information for validation in mapping population and its use in marker assisted selection for biofortification of rice with zinc.

Wheat is an important dietary source of zinc (Zn) and other mineral elements in many countries. Dietary Zn deficiency is widespread, especially in developing countries, and breeding (genetic biofortification) through the HarvestPlus programme has recently started to deliver new wheat varieties to help alleviate this problem in South Asia. To better understand the potential of wheat to alleviate dietary Zn deficiency, this study aimed to characterise the baseline effects of genotype (G), site (E), and genotype by site interactions (GxE) on grain Zn concentration under a wide range of soil conditions in India. Field experiments were conducted on a diverse panel of 36 Indian-adapted wheat genotypes, grown on a range of soil types (pH range 4.5-9.5), in 2013-14 (five sites) and 2014-15 (six sites). Grain samples were analysed using inductively coupled plasma-mass spectrometry (ICP-MS). The mean grain Zn concentration of the genotypes ranged from 24.9-34.8 mg kg-1, averaged across site and year. Genotype and site effects were associated with 10% and 6% of the overall variation in grain Zn concentration, respectively. Whilst G x E interaction effects were evident across the panel, some genotypes had consistent rankings between sites and years. Grain Zn concentration correlated positively with grain concentrations of iron (Fe), sulphur (S), and eight other elements, but did not correlate negatively with grain yield, i.e. no yield dilution was observed. Despite a relatively small contribution of genotype to the overall variation in grain Zn concentration, due to experiments being conducted across many contrasting sites and two years, our data are consistent with reports that biofortifying wheat through breeding is likely to be effective at scale given that some genotypes performed consistently across diverse soil types. Notably, all soils in this study were probably Zn deficient and interactions between wheat genotypes and soil Zn availability/management (e.g. the use of Zn-containing fertilisers) need to be better-understood to improve Zn supply in food systems.

Zinc is required for normal bone development and cartilage formation. The purpose of this study was to assess the effect of with adding organic Zn (alone or phytase inclusion) at the reduced dose to growing male Ross 308 chickens on somatic axis and bone tissue metabolism. 200 one-day old broilers were divided into the negative control group fed diet without Zn or phytase inclusion, positive control group receiving Zn in the 100% of daily recommended dose from ZnO, and two experimental groups fed diet introduced Zn in 25% of daily recommendation as a glycine chelate (Zn-Gly) with or without phytase inclusion (500 FTU·kg-1). Supplemental organic Zn increased bone Zn and Mg content, serum IGF-1, growth hormone and leptin concentration. Additional phytase inclusion increased body weight gain, blood plasma Ca, Fe, Zn and osteocalcin concentration and tibia ash percentage when compared to the Zn-deprived control. Bone geometry, yield and ultimate strengths were enhanced in both organic Zn supplemented groups, and the overall mechanical strength parameters of bone were better in these groups than in the positive control group supplemented with standard dose of inorganic Zn. Also marked improvements in the thickness of articular and the growth plate cartilages as well as real bone volume and thickness of metaphyseal trabeculae were achieved in all broilers fed Zn-supplemented diet irrespective of phytase inclusion, however, the highest cancellous bone mass and the best trabecular structure were noted after ZnO supplementation. In concludion, although dietary organic Zn given to growing broilers in 25% of daily recommended dose improved general bone properties and mechanical strength, the obtained results do not allow to unambiguously state that organic Zn supplementation at this level, even after phytase inclusion, is sufficient for proper bone development.

The development of shark vertebrae and the possible drivers of inter- and intra-specific differences in vertebral structure are poorly understood. Shark vertebrae are used to examine life-history traits related to trophic ecology, movement patterns, and the management of fisheries; a better understanding of their development would be beneficial to many fields of research that rely on these calcified structures. This study used Scanning X-ray Fluorescence Microscopy to observe zinc distribution within vertebrae of ten shark species from five different orders. Zinc was mostly localised within the intermedialis and was generally detected at levels an order of magnitude lower in the corpus calcareum. In most species, zinc concentrations were higher pre-birth mark, indicating a high rate of pre-natal zinc deposition. These results suggest there are inter-specific differences in elemental deposition within vertebrae. Since the deposition of zinc is physiologically-driven, these differences suggest that the processes of growth and deposition are potentially different in the intermedialis and corpus calcareum, and that caution should be taken when extrapolating information such as annual growth bands from one structure to the other. Together these results suggest that the high inter-specific variation in vertebral zinc deposition and associated physiologies may explain the varying effectiveness of ageing methodologies applied to elasmobranch vertebrae.

Lentil is a major cool-season grain legume grown in South Asia, West Asia, and North Africa. Populations in developing countries of these regions have micronutrient deficiencies; therefore, breeding programs should focus more on improving the micronutrient content of food. In the present study, a set of 96 diverse germplasm lines were evaluated at three different locations in India to examine the variation in iron (Fe) and zinc (Zn) concentration and identify simple sequence repeat (SSR) markers that associate with the genetic variation. The genetic variation among genotypes of the association mapping (AM) panel was characterized using a genetic distance-based and a general model-based clustering method. The model-based analysis identified six subpopulations, which satisfactorily explained the genetic structure of the AM panel. AM analysis identified three SSRs (PBALC 13, PBALC 206, and GLLC 563) associated with grain Fe concentration explaining 9% to 11% of phenotypic variation and four SSRs (PBALC 353, SSR 317-1, PLC 62, and PBALC 217) were associated with grain Zn concentration explaining 14%, to 21% of phenotypic variation. These identified SSRs exhibited consistent performance across locations. These candidate SSRs can be used in marker-assisted genetic improvement for developing Fe and Zn fortified lentil varieties. Favorable alleles and promising genotypes identified in this study can be utilized for lentil biofortification.

Mycoremediation is an on-site remediation strategy, which employs fungi to degrade or sequester contaminants from the environment. The present work focused on the bioremediation of soils contaminated with zinc by the use of a native mycorrhizal fungi (AM) called Funneliformis geosporum (Nicol. & Gerd.) Walker & Schüßler. Experiments were performed using Triticum aestivum L. cv. Gemmeza-10 at different concentrations of Zn (50, 100, 200 mg kg-1) and inoculated with or without F. geosporum. The results showed that the dry weight of mycorrhizal wheat increased at Zn stressed plants as compared to the non-Zn-stressed control plants. The concentrations of Zn also had an inhibitory effect on the yield of dry root and shoot of non-mycorrhizal wheat. The photosynthetic pigment fractions were significantly affected by Zn treatments and mycorrhizal inoculation, where in all treatments, the content of the photosynthetic pigment fractions decreased as the Zn concentration increased in the soil. However, the level of minerals of shoots, roots, and grains was greatly influenced by Zn-treatment and by inoculation with F. geosporum. Treatment with Zn in the soil increased Cu and Zn concentrations in the root, shoot and grains, however, other minerals (P, S, K, Ca and Fe) concentration was decreased. Inoculation of wheat with AM fungi significantly reduced the accumulation of Zn and depressed its translocation in shoots and grains of wheat. In conclusion, inoculation with a native F. geosporum-improves yields of wheat under higher levels of Zn and is possible to be applied for the improvement of zinc contaminated soil.

β-cells release hexameric Zn2+-insulin into the extracellular space, but monomeric Zn2+-free insulin appears to be the only biologically active form. The mechanisms implicated in dissociation of the hexamer remain unclear, but they seem to be Zn2+ concentration-dependent. In this study, we investigate the influence of albumin binding to Zn2+ on Zn2+-insulin dissociation into Zn2+-free insulin and its physiological, methodological and therapeutic relevance. Glucose and K+-induced insulin release were analyzed in isolated mouse islets by static incubation and perifusion experiments in the presence and absence of albumin and Zn2+ chelators. Insulin tolerance tests were performed in rats using different insulin solutions with and without Zn2+ and/or albumin. Albumin-free buffer does not alter quantification by RIA of Zn2+-free insulin but strongly affects RIA measurements of Zn2+-insulin. In contrast, accurate determination of Zn2+-insulin was obtained only when bovine serum albumin or Zn2+ chelators were present in the assay buffer solution. Albumin and Zn2+ chelators do not modify insulin release but do affect insulin determination. Preincubation with albumin or Zn2+ chelators promotes the conversion of "slow" Zn2+-insulin into "fast" insulin. Consequently, insulin diffusion from large islets is ameliorated in the presence of Zn2+ chelators. These observations support the notion that the Zn2+-binding properties of albumin improve the dissociation of Zn2+-insulin into subunits after exocytosis, which may be useful in insulin determination, insulin pharmacokinetic assays and islet transplantation.