Zinc Transporter Research Articles

AtIAR1 is a Zn transporter that regulates auxin metabolism in Arabidopsis thaliana.

Root growth in Arabidopsis is inhibited by exogenous auxin-amino acid conjugates, and mutants resistant to one such conjugate [indole-3-acetic acid (IAA)-Ala] map to a gene (AtIAR1) that is a member of a metal transporter family. Here, we test the hypothesis that AtIAR1 controls the hydrolysis of stored conjugated auxin to free auxin through zinc transport. AtIAR1 complements a yeast mutant sensitive to zinc, but not manganese- or iron-sensitive mutants, and the transporter is predicted to be localized to the endoplasmic reticulum/Golgi in plants. A previously identified Atiar1 mutant and a non-expressed T-DNA mutant both exhibit altered auxin metabolism, including decreased IAA-glucose conjugate levels in zinc-deficient conditions and insensitivity to the growth effect of exogenous IAA-Ala conjugates. At a high concentration of zinc, wild-type plants show a novel enhanced response to root growth inhibition by exogenous IAA-Ala which is disrupted in both Atiar1 mutants. Furthermore, both Atiar1 mutants show changes in auxin-related phenotypes, including lateral root density and hypocotyl length. The findings therefore suggest a role for AtIAR1 in controlling zinc release from the secretory system, where zinc homeostasis plays a key role in regulation of auxin metabolism and plant growth regulation.

Mechanisms of Cadmium Neurotoxicity

Cadmium is a heavy metal that increasingly contaminates food and drink products. Once ingested, cadmium exerts toxic effects that pose a significant threat to human health. The nervous system is particularly vulnerable to prolonged, low-dose cadmium exposure. This review article provides an overview of cadmium’s primary mechanisms of neurotoxicity. Cadmium gains entry into the nervous system via zinc and calcium transporters, altering the homeostasis for these metal ions. Once within the nervous system, cadmium disrupts mitochondrial respiration by decreasing ATP synthesis and increasing the production of reactive oxygen species. Cadmium also impairs normal neurotransmission by increasing neurotransmitter release asynchronicity and disrupting neurotransmitter signaling proteins. Cadmium furthermore impairs the blood–brain barrier and alters the regulation of glycogen metabolism. Together, these mechanisms represent multiple sites of biochemical perturbation that result in cumulative nervous system damage which can increase the risk for neurological and neurodegenerative disorders. Understanding the way by which cadmium exerts its effects is critical for developing effective treatment and prevention strategies against cadmium-induced neurotoxic insult.

Chemistry meets biology in the coordination dynamics of metalloproteins

Metal sites in proteins are often presented in an idealized way that does not capture the intrinsic dynamic behavior of the protein or the extrinsic factors that affect changes in the coordination of the metal ion in biological space and time. The bioinorganic chemistry possible in healthy and diseased living organisms is limited by prevailing pH values, redox potentials, and availability and concentrations of metal ions and ligands. Changes in any of these parameters and protein-protein or protein-ligand interactions can result in differences in the type of metal ion bound, metal occupancy, and coordination number or geometry. This article addresses the plasticity and complexity of metal coordination in proteins when these parameters are considered. It uses three examples of zinc sites with sulfur donor atoms from cysteines in mammalian proteins: alcohol dehydrogenases, metallothioneins, and zinc transporters of the ZnT (SLC30A) family. Coordination dynamics of the metal sites in these proteins has different purposes; in alcohol dehydrogenases for the metal ion to perform its different roles in the catalytic cycle, in metallothioneins for serving as a metal buffer, and in ZnT zinc transporters for sensing metal ions and moving them through the protein and thus biological membranes. Defining the biological and chemical parameters that determine and affect coordination dynamics of metal ions in proteins will inform future investigations of metalloproteins.

Investigation of the activity of a novel tropolone in osteosarcoma.

Osteosarcoma (OS) is a primary malignant bone tumor characterized by frequent metastasis, rapid disease progression, and a high rate of mortality. Treatment options for OS have remained largely unchanged for decades, consisting primarily of cytotoxic chemotherapy and surgery, thus necessitating the urgent need for novel therapies. Tropolones are naturally occurring seven-membered non-benzenoid aromatic compounds that possess antiproliferative effects in a wide array of cancer cell types. MO-OH-Nap is an α-substituted tropolone that has activity as an iron chelator. Here, we demonstrate that MO-OH-Nap activates all three arms of the unfolded protein response (UPR) pathway and induces apoptosis in a panel of human OS cell lines. Co-incubation with ferric chloride or ammonium ferrous sulfate completely prevents the induction of apoptotic and UPR markers in MO-OH-Nap-treated OS cells. MO-OH-Nap upregulates transferrin receptor 1 (TFR1) protein levels, as well as TFR1, divalent metal transporter 1 (DMT1), iron-regulatory proteins (IRP1, IRP2), ferroportin (FPN), and zinc transporter 14 (ZIP14) transcript levels, demonstrating the impact of MO-OH-Nap on iron-homeostasis pathways in OS cells. Furthermore, MO-OH-Nap treatment restricts the migration and invasion of OS cells in vitro. Lastly, metabolomic profiling of MO-OH-Nap-treated OS cells revealed distinct changes in purine and pyrimidine metabolism. Collectively, we demonstrate that MO-OH-Nap-induced cytotoxic effects in OS cells are dependent on the tropolone's ability to alter cellular iron availability and that this agent exploits key metabolic pathways. These studies support further evaluation of MO-OH-Nap as a novel treatment for OS.

Abstract 14145: The Zinc Transporter ZIP7 Plays a Critical Role in Ferrotposis Induced by Ischemia/Reperfusion in Mouse Hearts

Background: Although ferroptosis has been proposed to play a role in myocardial ischemia/reperfusion (I/R) injury, the exact molecular mechanism by which ferroptosis is induced by I/R remains unclear. Hypothesis: We tested the hypothesis that upregulation of the zinc transporter ZIP7 at reperfusion triggers ferroptosis by downregulating the mitochondrial GPX4 (mtGPX4) via the guanine-rich sequence-binding factor 1 (GRSF1) and increasing mitochondrial iron accumulation through inhibition of the mitochondrial iron importer degradation. Aims: To determine if ZIP7 plays a role in I/R-induced ferroptosis and to investigate the underlying molecular mechanism. Methods: Mouse hearts were subjected to I/R in vivo . The cardiac-specific Slc39a7 conditional knockout mice (ZIP7 cKO) were generated by adopting the CRISPR/Cas9 system. Adult mouse cardiomyocytes were isolated enzymatically. Ferroptosis was evaluated by detecting PTGS2, MDA, and BODIPY C11. Mitochondrial Fe 2+ was determined with Mito-FerroGreen fluorescent probe. Results: I/R upregulated ZIP7 expression and induced ferroptosis as indicated by the increases in PTGS2 mRNA expression (290.90 ± 52.78 % of control), MDA (152.10 ± 15.07 % of control) and BODIPY C11 (130.68 ± 5.52 % of control) levels, which was prevented by ZIP7 cKO. ZIP7 cKO also mitigated doxorubicin-induced ferroptosis. ZIP7 cKO upregulated mtGPX4 expression (123.40±3.26 % of I/R) and increased mitochondrial GRSF1 expression (150.90±8.51 % of I/R) at reperfusion. In addition, knockout of GRSF1 inhibited GPX4 expression (54.73±5.44 % of control). Moreover, ZIP7 cKO prevented the accumulation of Fe 2+ within mitochondria (44.15±2.46 % of I/R) and promoted the degradation of SLC25A37 (62.57±8.03% of I/R) and SLC25A28 (69.82±3.10 % of I/R) at reperfusion. Conclusions: Upregulation of ZIP7 contributes to the induction of ferroptosis at reperfusion presumably by reducing mtGPX4 expression via GRSF1and increasing Fe 2+ accumulation within mitochondria through inhibition of SLC25A37 and SLC25A28 degradation.

Toxicity effects of zinc supply on growth revealed by physiological and transcriptomic evidences in sweet potato (Ipomoea batatas (L.) Lam)

Zinc toxicity affects crop productivity and threatens food security and human health worldwide. Unfortunately, the accumulation patterns of zinc and the harmful effects of excessive zinc on sweet potato have not been well explored. In the present research, two genotypes of sweet potato varieties with different accumulation patterns of zinc were selected to analyze the effects of excessive zinc on sweet potato via hydroponic and field cultivation experiments. The results indicated that the transfer coefficient was closely related to the zinc concentration in the storage roots of sweet potato. Excessive zinc inhibited the growth of sweet potato plants by causing imbalanced mineral concentrations, destroying the cellular structure and reducing photosynthesis. Furthermore, a total of 17,945 differentially expressed genes were identified in the two genotypes under zinc stress by transcriptomic analysis. Differentially expressed genes involved in the absorption and transport of zinc, defense networks and transcription factors played important roles in the response to zinc stress. In conclusion, this study provides a reference for the selection of sweet potato varieties in zinc contaminated soil and lays a foundation for investigating the tolerance of sweet potato to excessive zinc, which is meaningful for environmental safety and human health.

373 Effects of Different Trace Mineral Supplementation Strategies on Liver Mineral Status and Gene Expression of At-Risk Mineral Deficient Feedlot Cattle

Abstract Grazing lands in the western US are deficient in several trace minerals known to be critical for livestock health and performance. Mineral supplementation is often not possible since animals graze rough terrain, making mineral delivery logistically difficult. Therefore, trace minerals are often supplemented during the growing and feedlot period to maximize cattle health and production. However, morbidity and mortality rates are often greater in these mineral deficient cattle, at least in part due to their mineral deficiencies, and producers welcome input on best practices for mineral supplementation. This study aimed to evaluate how different mineral supplementation strategies affect liver mineral concentrations and abundance of mRNA involved in mineral transport in mineral deficient cattle receiving different mineral supplementation strategies. Sixty mineral deficient feedlot steers were obtained and initially stratified by weight into one of four treatment groups (n = 15): no mineral supplementation (CON); dietary mineral supplementations at industry recommended levels (DM); multimin injection at labeled dose (MM); or a combination of the MM and DM treatments (MM+DM). Treatments were given for the first 40 days of the trial and steers were fed ad libitum in pens equipped with GrowSafe bunks. Liver biopsies were conducted on days 0, 5, 10, 20, 30 and 40 to assess liver mineral concentrations of copper (Cu), manganese (Mn), selenium (Se), and zinc (Zn). Total mRNA was isolated from liver biopsies on days 0, 20, and 40 and mRNA abundance of genes involved in trace mineral transport was measured. The liver mineral data was analyzed using the PROC MIXED procedure of SAS with day as a repeated measure to evaluate the fixed effect of treatment. A series of contrasts were also conducted to determine whether mRNA abundance differed between the mineral treatments and the CON. There was a treatmentxday interaction (P < 0.05) on the Cu concentration where the MM treatment peaked at day 20 and then started to decrease towards day 40. All mineral treatments increased (P < 0.05) the liver Se concentration and mRNA abundance of zinc transporter (ZNT) 7, ZNT9, and Mn superoxide dismutase (MnSOD) compared with the CON. Steers receiving the DM treatment increased (P < 0.05) the mRNA abundance of Zrt- and Irt-like proteins (ZIP) 1, ZNT1, ZNT3, and ZNT4 compared with the CON. When compared with the CON treatment, the DM and MM+DM had a tendency (P = 0.07) to increase the abundance of divalent metal transporter 1 (DMT1) and ZNT1, as well as the liver Mn concentration. Overall, both the MM+DM and DM treatments improved mineral status by increasing liver mineral concentrations. However, the DM treatment was more effective at increasing the mRNA abundance of mineral transporters in the liver. Further research is needed to determine which pathways were influenced by an improved trace mineral status.

The NLRX1-SLC39A7 complex orchestrates mitochondrial dynamics and mitophagy to rejuvenate intervertebral disc by modulating mitochondrial Zn2+ trafficking

ABSTRACT Intervertebral disc degeneration (IDD) is the most critical pathological factor in the development of low back pain. The maintenance of nucleus pulposus (NP) cell and intervertebral disc integrity benefits largely from well-controlled mitochondrial quality, surveilled by mitochondrial dynamics (fission and fusion) and mitophagy, but the outcome is cellular context-dependent that remain to be clarified. Our studies revealed that the loss of NLRX1 is correlated with NP cell senescence and IDD progression, which involve disordered mitochondrial quality. Further using animal and in vitro tissue and cell models, we demonstrated that NLRX1 could facilitate mitochondrial quality by coupling mitochondrial dynamic factors (p-DNM1L, L-OPA1:S-OPA1, OMA1) and mitophagy activity. Conversely, mitochondrial collapse occurred in NLRX1-defective NP cells and switched on the compensatory PINK1-PRKN pathway that led to excessive mitophagy and aggressive NP cell senescence. Mechanistically, NLRX1 was originally shown to interact with zinc transporter SLC39A7 and modulate mitochondrial Zn2+ trafficking via the formation of an NLRX1-SLC39A7 complex on the mitochondrial membrane of NP cells, subsequently orchestrating mitochondrial dynamics and mitophagy. The restoration of NLRX1 function by gene overexpression or pharmacological agonist (NX-13) treatment showed great potential for regulating mitochondrial fission with synchronous fusion and mitophagy, thus sustaining mitochondrial homeostasis, ameliorating NP cell senescence and rejuvenating intervertebral discs. Collectively, our findings highlight a working model whereby the NLRX1-SLC39A7 complex coupled mitochondrial dynamics and mitophagy activity to surveil and target damaged mitochondria for degradation, which determines the beneficial function of the mitochondrial surveillance system and ultimately rejuvenates intervertebral discs. Abbreviations: 3-MA: 3-methyladenine; Baf-A1: bafilomycin A1; CDKN1A/p21: cyclin dependent kinase inhibitor 1A; CDKN2A/p16: cyclin dependent kinase inhibitor 2A; DNM1L/DRP1: dynamin 1 like; EdU: 5-Ethynyl-2’-deoxyuridine; HE: hematoxylin-eosin; IDD: intervertebral disc degeneration; IL1B/IL-1β: interleukin 1 beta; IL6: interleukin 6; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MKI67/Ki67: marker of proliferation Ki-67; LBP: low back pain; MMP: mitochondrial membrane potential; MFN1: mitofusin 1; MFN2: mitofusin 2; MFF: mitochondrial fission factor; NP: nucleus pulposus; NLRX1: NLR family member X1; OMA1: OMA1 zinc metallopeptidase; OPA1: OPA1 mitochondrial dynamin like GTPase; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxidative species; SASP: senescence-associated secretory phenotype; SA-GLB1/β-gal: senescence-associated galactosidase beta 1; SO: safranin o; TBHP: tert-butyl hydroperoxide; TP53/p53: tumor protein p53; SLC39A7/ZIP7: solute carrier family 39 member 7; TOMM20: translocase of outer mitochondrial membrane 20; TIMM23: translocase of inner mitochondrial membrane 23.

Role of transcription factor complex OsbHLH156-OsIRO2 in regulating manganese, copper, and zinc transporters in rice.

Iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn) are essential micronutrients that are necessary for plant growth and development, but can be toxic at supra-optimal levels. Plants have evolved a complex homeostasis network that includes uptake, transport, and storage of these metals. It was shown that the transcription factor (TF) complex OsbHLH156-OsIRO2 is activated under Fe deficient conditions and acts as a central regulator on Strategy II Fe acquisition. In this study, the role of the TF complex on Mn, Cu, and Zn uptake was evaluated. While Fe deficiency led to significant increases in shoot Mn, Cu, and Zn concentrations, the increases of these divalent metal concentrations were significantly suppressed in osbhlh156 and osiro2 mutants, suggesting that the TF complex plays roles on Mn, Cu, and Zn uptake and transport. An RNA-sequencing assay showed that the genes associated with Mn, Cu, and Zn uptake and transport were significantly suppressed in the osbhlh156 and osiro2 mutants. Transcriptional activation assays demonstrated that the TF complex could directly bind to the promoters of OsIRT1, OsYSL15, OsNRAMP6, OsHMA2, OsCOPT1/7, and OsZIP5/9/10, and activate their expression. In addition, the TF complex is required to activate the expression of nicotianamine (NA) and 2'-deoxymugineic acid (DMA) synthesis genes, which in turn facilitate the uptake and transport of Mn, Cu, and Zn. Furthermore, OsbHLH156 and OsIRO2 promote Cu accumulation to partially restore the Fe-deficiency symptoms. Taken together, OsbHLH156 and OsIRO2 TF function as core regulators not only in Fe homeostasis, but also in Mn, Cu, and Zn accumulation.

Mycoplasma synoviae lipid-associated membrane proteins identification and expression changes when exposed to chicken cells.

Mycoplasma synoviae is a significant cause of respiratory disease and synovitis among chickens, and has an adverse economic impact on broiler breeding efforts. The present study was designed to develop a systematic understanding of the role that M. synoviae lipid-associated membrane proteins (LAMPs) may play in the virulence of this pathogen. Bioinformatics tools were used to identify 146 predicted membrane proteins and lipoproteins in the M. synoviae proteome. Then, Triton X-114 was used to extract LAMPs that were subsequently identified via LC-MS/MS. This approach enabled the detection of potential LAMPs, and the top 200 most abundant proteins detected using this strategy were subject to further analysis. M. synoviae cells (100 MOI) were exposed to chicken fibroblasts (DF-1) and macrophages (HD-11) in a 1:1 mixed culture. Analysis of LAMP transcripts identified 72 up-regulated LAMP genes which were analyzed in depth by bioinformatics. GO analysis revealed these genes to be enriched in the nucleotide binding, sulfur amino acid transmembrane transporter activity, tRNA binding, rRNA modification, and transition metal ion transport pathways. Moreover, KEGG enrichment analysis suggested that these genes were enriched in the biosynthesis of secondary metabolites, carbon metabolism, glycolysis/gluconeogenesis, and nitrogen metabolism pathways.

A machine learning approach to identifying Salmonella stress response genes in isolates from poultry processing

We explored the potential of machine learning to identify significant genes associated with Salmonella stress response during poultry processing using whole genome sequencing (WGS) data. The Salmonella isolates (n = 177) used in this study were obtained from various chicken sources (skin before chiller, chicken carcass before chiller, frozen chicken, and post-chill chicken carcass). Six machine learning algorithms (random forest, neural network, cost-sensitive learning, logit boost, and support vector machine linear and radial kernels) were trained on Salmonella WGS data, and model fit was assessed using standard evaluation metrics such as the area under the receiver operating characteristic (AUROC) curve and confusion matrix statistics. All models achieved high performances based on the AUROC metric, with logit boost showing the best performance with an AUROC score of 0.904, sensitivity of 0.889, and specificity of 0.920. The significant genes identified included ybtX, which encodes a Yersiniabactin-associated zinc transporter, and the transferase-encoding genes yccK and thiS. Additionally, genes coding for cold (cspA, cspD, and cspE) and heat shock (rpoH and rpoE) responses were identified. Other significant genes included those involved in lipopolysaccharide biosynthesis (irp1, waaD, rfc, and rfbX), DNA repair and replication (traI), biofilm formation (ccdA and fyuA), and cellular metabolism (irtA).

226 Modulation of Zinc, Copper and Iron Transporters in Weaned Pigs Fed Different Dietary Zinc/Copper Ratios

Abstract This study evaluated the impact of different dietary zinc/copper ratios on the gene expression of zinc (Zn), copper (Cu), and iron (Fe) transporters in jejunum and liver of weaned pigs. Piglets (n = 160) were weaned at 21 days of age (d 21; 7.81 ± 0.25 kg) and fed a basal post-weaning diet (without antibiotics) supplemented (n = 40/treatment) with 100 (LZn) or 3,000 (HZn) mg/kg of Zn as ZnO in combination with 6 (LCu) or 130 (HCu) mg/kg of Cu as CuSO4 until d 42. Piglets were sacrificed at d 21, d 28, d 35, and d 42 (n = 10 animals∙treatment -1∙day-1) for harvesting jejunum and liver samples. The mRNA expression of Zn, Cu, and Fe transporters was evaluated by quantitative real-time PCR. Data were analyzed using a 2x2x3 factorial design (two Zn levels, two Cu levels, and three slaughter days) and results are presented as fold-change relative to pre-treatment values (d 21). For Zn-related genes, jejunum Zip4 expression was least for HZn from d 28 (Zn effect; P ≤ 0.01; Table 1) and Zip5 was greatest for HZn at d 28 (Zn effect; P = 0.04). On d 42, the greatest values for jejunal Zip4 were observed with LZnHCu (interaction Zn x Cu x time; P = 0.05). For Znt1 in jejunum and liver, mRNA expression was greatest for HZn from d 28 (Zn effect; P ≤ 0.01) whereas Zip5 was greatest in liver for HZn at d 28 and d 35 (Zn effect; P ≤ 0.04). On d 35, the least values for hepatic Zip5 were observed with LZnHCu (interaction Zn x Cu x time; P = 0.04). For Cu-related genes, no treatment effect (P ≥ 0.12) was detected for Crt1 and Atox1 in jejunum and liver. In both tissues, Mt3 expression was greatest for HZn from d 28 (Zn effect; P ≤ 0.01). No treatment effect was detected (P = 0.53) for Atp7a expression in jejunum. However, for Atp7b in liver, values were greatest for HZn from d 35 (Zn effect; P ≤ 0.01). For Fe-related genes, no treatment effect (P ≥ 0.18) was detected for Heph, Tfrc and Fpn1 in jejunum and for Dmt1, Tfrc and Hamp in liver. In jejunum, Dmt1 was greatest for LZn at d 28 and d 42 (Zn effect; P ≤ 0.01) whereas Fth1 was greatest in HZn groups from d28 (Zn effect; P ≤ 0.01) and for HCu at d42 (Cu effect; P = 0.04). In liver, Fth1 and Fpn1 were greatest for HZn from d28 (Zn effect; P ≤ 0.02). In conclusion, among the present different dietary zinc/copper ratios, most treatment effects on intestinal and hepatic Zn, Cu and Fe transporters originated from dietary ZnO levels suggesting a major contribution of Zn in modulating the metabolism of these trace minerals in post-weaning piglets.

The efficiency and safety evaluation of hemoglobin hydrolysate as a non-heme iron fortifier

Hemoglobin hydrolysate is derived from the enzymatic degradation of hemoglobin. This work aimed to evaluate whether hemoglobin hydrolysate promotes the absorption of non-heme iron and the safety of absorbed iron in mice by analyzing the iron binding content, iron circulation, and liver homeostasis. We found that hemoglobin hydrolysate promoted the absorption of non-heme iron with high efficiency in duodenum by spontaneously binding non-heme iron during digestion, and increased hepatic iron content by up-regulating divalent metal transporter 1, zinc transporter 14, but hepatic iron content only increased at 3 weeks. Duodenal iron entered the blood through ferroportin without restriction at 3 weeks, and excessive iron entered the liver and then affected the hepatocyte membranes permeability and lipid synthesis through oxidative stress. With the prolongation of dietary intervention, the up-regulated hepcidin acted on the ferroportin to restrict excess iron from entering the blood, and then the hepatic homeostasis recovered. In addition, hemoglobin hydrolysate enhanced the hepatic antioxidant capacity. Taken together, hemoglobin hydrolysate has a strong ability to promote the absorption of non-heme iron in vivo, and the absorbed iron is relatively safe due to the regulation of hepcidin.

The directed evolution of NDM-1.

β-Lactam antibiotics are among the most frequently prescribed therapeutic agents. A common mechanism of resistance toward β-lactam antibiotics is the production of β-lactamases. These enzymes are capable of hydrolyzing the β-lactam bond, rendering the drug inactive. Among the four described classes, the metallo- β-lactamases (MBLs, class B) employ one or two zinc ions in the active site for catalysis. One of the three most clinically relevant MBLs is New Delhi Metallo- β-Lactamase (NDM-1). The current study sought to investigate the in vitro protein evolution of NDM-1 β-lactamase using error-prone polymerase chain reaction. Evaluation revealed that variants were not found to confer higher levels of resistance toward meropenem based on amino acid substitutions. Thus, we postulate that increases in transcription or changes in zinc transport may be clinically more relevant to meropenem resistance than amino acid substitutions.

Genome- and transcriptome-wide characterization of ZIP gene family reveals their potential role in radish (Raphanus sativus) response to heavy metal stresses

Radish is an important root vegetable crop susceptible to heavy metal (HM) stresses. Zinc- and iron-regulated transporter-like proteins play vital roles in transporting metal ion to enhance HM tolerance or decrease detoxification in plants. In this study, 27 out of 28 RsZIP genes were unevenly located on eight chromosomes with exception of chromosome 3 (Chr.3). The evolutionary pattern divided the whole RsZIP proteins into 18 subgroups. Furthermore, four tandem and six WGD/segmental duplication events were identified, while 24 pairs containing 16 RsZIP and 13 AtZIP genes were orthologous. Moreover, the expression levels of 17 RsZIP genes including RsZIP10b,c and RsIRT1a,b were significantly decreased under 200 mg/L CdCl2 and 1000 mg/L Pb(NO3)2 treatment. RT-qPCR analysis indicated that four RsZIP genes (RsIRT1d, RsIRT1e, RsIAR1 and RsZIP10d) were rapidly induced at 2 h under Pb and Cd treatment, while both RsZIP1 and RsZIP10b were downregulated. The results of yeast functional complementation indicated that RsIRT1d and RsIRT1e confer yeast Pb but no Cd tolerance, suggesting that they might play an important role in the transport of metal ions. These results could provide valuable insights into the characteristics of ZIP family genes and facilitate further exploring the molecular mechanism underlying HM stress responses in radish and other root vegetable crops.

Insight in lag phase of Listeria monocytogenes during enrichment through proteomic and transcriptomic responses

The dynamics of the enrichment-based detection procedure of the foodborne pathogen Listeria monocytogenes from food still remains poorly understood. This enrichment is crucial in the reliable detection of this pathogen and more insight into the recovery mechanism during this step is important to advance our understanding of lag phase behaviour during enrichment. In this study we combined transcriptomic and proteomic analyses to better understand the physiological processes within the lag phase of L. monocytogenes during enrichment. Upon transfer of BHI-cultured stationary phase L. monocytogenes cells to half-Fraser enrichment broth (HFB), motility-associated genes and proteins were downregulated, while expression of metal uptake transporters, resuscitation-promoting factors that stimulate growth from dormancy, antibiotic efflux pumps and oxidative stress proteins were upregulated. Next to this, when cells with a heat stress history were cultured in enrichment broth, proteins necessary for recovery were upregulated with functions in DNA-damage repair, protein refolding, cell-wall repair, and zinc transport. Proteomic results pointed to possible factors that support shortening the lag duration, including the addition of 10 µM zinc and the addition of spent HFB containing presumed concentrations of resuscitation-promoting factors. However, these interventions did not lead to biologically relevant reduction of lag phase. Also, when cells were enriched in spent HFB, final cell concentrations were similar to enrichments in fresh HFB, indicating that the enrichment broth seems not to lack critical substrates. Concludingly, this study gives insight into the proteomic changes in the lag phase during enrichment and shows that supplementation of HFB is not the best strategy to optimize the current enrichment method.

Large-scale exome array summary statistics resources for glycemic traits to aid effector gene prioritization.

Genome-wide association studies for glycemic traits have identified hundreds of loci associated with these biomarkers of glucose homeostasis. Despite this success, the challenge remains to link variant associations to genes, and underlying biological pathways. To identify coding variant associations which may pinpoint effector genes at both novel and previously established genome-wide association loci, we performed meta-analyses of exome-array studies for four glycemic traits: glycated hemoglobin (HbA1c, up to 144,060 participants), fasting glucose (FG, up to 129,665 participants), fasting insulin (FI, up to 104,140) and 2hr glucose post-oral glucose challenge (2hGlu, up to 57,878). In addition, we performed network and pathway analyses. Single-variant and gene-based association analyses identified coding variant associations at more than 60 genes, which when combined with other datasets may be useful to nominate effector genes. Network and pathway analyses identified pathways related to insulin secretion, zinc transport and fatty acid metabolism. HbA1c associations were strongly enriched in pathways related to blood cell biology. Our results provided novel glycemic trait associations and highlighted pathways implicated in glycemic regulation. Exome-array summary statistic results are being made available to the scientific community to enable further discoveries.

Antibiotic- and metal-resistant endophytes inhabit Armeria maritima hyperaccumulator

Abstract Background and aims Recent research has recognized the presence of metal-resistant bacteria in plants and their role in phytoremediation intensification. However, information on the antibiotic resistance profile of those bacteria remains scarce. This study, describes the first isolation of endophytic bacteria from green parts of Armeria maritima growing on mine-tailing soil in southern Poland, and presents the resistance profile of these microorganisms. Methods Bacteria were isolated from internal tissues of Armeria maritima and characterized. Minimal Inhibitory Concentration (MIC) of metals was determined by the plate dilution method using (CH3COO)2Pb and ZnSO4 supplemented medium; antibiotic susceptibility was determined by disk diffusion method according to EUCAST version 11.0; the whole genome sequencing was performed using the MiSeq platform (Illumina). The physicochemical properties of soil were evaluated according to European Standards. Results Toxic metal-resistant bacteria were isolated from the green parts of Armeria maritima. The endophytes were identified as Pseudomonas spp. The annotated bacterial genomes carried genes encoding numerous metal ion transporters, metal reducing enzymes and efflux pump components. The bacteria were resistant to streptomycin, fosfomycin and ß-lactams. Moreover, genome analysis revealed the presence of MacAB-TolC efflux pump genes conferring resistance to macrolides, the multidrug efflux pumps AcrAB-TolC and MexAB-OprM. Conclusion Armeria maritima is inhabited by endophytic bacteria identified as Pseudomonas species that are resistant to metals and to antibiotics. Under the One Health concept the contamination of soil and plants with ARB and ARGs should be monitored and limited and a regulatory framework for safety use of bacterial bioinoculants should be established.

Leukemia cells accumulate zinc for oncofusion protein stabilization

Acute promyelocytic leukemia (APL) and chronic myeloid leukemia (CML) are both hematological malignancies characterized by genetic alterations leading to the formation of oncofusion proteins. The classical chromosomal aberrations in APL and CML result in the PML-RARα and BCR-ABL1 oncofusion proteins, respectively. Interestingly, our flow cytometric analyses revealed elevated free intracellular zinc levels in various leukemia cells, which may play a role in stabilizing oncofusion proteins in leukemia and thus support cell proliferation and malignancy. Long-term zinc deficiency resulted in the degradation of PML-RARα in NB4 cells (APL cell line) and of BCR-ABL1 in K562 cells (CML cell line). This degradation may be explained by increased caspase 3 activity observed in zinc deficient cells, whereas zinc reconstitution normalized the caspase 3 activity and abolished zinc deficiency-induced oncofusion protein degradation. In NB4 cells, fluorescence microscopic images further indicated enlarged and enriched lysosomes during zinc deficiency, suggesting increased rates of autophagy. Moreover, NB4 cells exhibited increased expression of the zinc transporters ZIP2, ZIP10 and ZnT3 during zinc deficiency and revealed excessive accumulation of zinc in contrast to healthy peripheral blood mononuclear cells (PBMCs), when zinc was abundantly available extracellularly. Our results highlight the importance of altered zinc homeostasis for some characteristics in leukemia cells, uncover potential pathways underlying the effects of zinc deficiency in leukemia cells, and provide potential alternative strategies by which oncofusion proteins can be degraded.

Cloning and biological function analysis of Nramps in blueberry

The natural resistance-associated macrophage protein (Nramp) family comprises crucial membrane transport proteins that play vital roles in the transport and reuse of heavy metal ions in plants. Limited information is currently available on the Nramp family in blueberry (Vaccinium spp.; Vc). Elucidation of the mechanism underlying mineral-element deficiency and heavy metal toxicity in blueberry is essential for the sustainable development of the blueberry industry. In this study, three Nramp genes (VcNramp4, VcNramp6, and VcNramp8.2) were cloned and identified from blueberry, and their functions were analyzed in transgenic Arabidopsis thaliana and yeast. Sequence analysis showed that the VcNramp genes encoded 321–455 amino acids and contained 6–8 transmembrane structural domains. Expression analysis showed that VcNramp genes were significantly up-regulated under iron (Fe) deficiency. The expression of VcNramp genes was most significantly up-regulated in the roots, by 2.05-fold relative to the control group. VcNramp4 and VcNramp6 expression in yeast and A. thaliana alleviated Fe deficiency stress. Interestingly, yeast expressing VcNramp6 showed a 1.13- to 1.38-fold increase in Fe accumulation at different medium pH levels, which was significantly higher than that of yeast expressing the other VcNramp genes. All VcNramp genes respond to changes in Fe ion concentration. Among them, VcNramp6 can effectively ameliorate Fe deficiency symptoms in plants. The expression of VcNramp genes exhibited distinct tissue specificity. Notably, the expression levels of VcNramp6 and VcNramp8.2 in the stems was only 2 %–10 % of that in the roots. The results illustrated the Fe nutrient inefficiency of blueberry. Furthermore, all identified VcNramp genes responded to mild zinc or cadmium stress, but their expression levels decreased under severe zinc or cadmium stress.