Zinc Transporter Research Articles

E‐Field Effects on Day‐To‐Day Variations of Geomagnetic Mid‐Latitude Sporadic E Layers

AbstractThis study investigates the effects of the electric fields (E‐fields) on day‐to‐day variations in geomagnetic mid‐latitude sporadic E layers (EsLs) using a numerical ionospheric model. It is widely accepted that geomagnetic mid‐latitude EsL dynamics depend largely on wind shear variations. In contrast, E‐field effects on geomagnetic mid‐latitude EsLs have not been investigated extensively and are still little known. Previous observations have reported that E‐fields change EsL heights by only several kilometers. However, no studies have investigated E‐field effects on day‐to‐day EsL variations. Recently, our numerical ionospheric model succeeded generally in reproducing day‐to‐day EsL variations driven by winds at geomagnetic mid‐latitudes. In this study, using the ionospheric model, we conducted EsL simulations including both E‐fields and winds, to elucidate how E‐fields affect day‐to‐day EsL variations at geomagnetic mid‐latitudes. It was found that zonal and meridional/vertical E‐fields change the vertical EsL dynamics above ∼110 km. Below ∼130 km, where most Es layers appear, vertical/meridional E‐fields are more effective for EsL dynamics than zonal E‐fields. During the day, E‐fields pump metal ions from the lower ionosphere to more than 110 km. Pumping facilitates the formation of daytime EsLs above ∼110 km. In the evening, E‐fields drive downward ion motion and increase the descending EsL density and occurrence. E‐fields affect day‐to‐day EsL variations at geomagnetic mid‐latitudes by altering background metal ion distribution and transport.

PTFS12-06-23 Investigation of Cellular Zinc Influx in In Vitro Senescent Enterocyte Models

PTFS12-06-23 Investigation of Cellular Zinc Influx in In Vitro Senescent Enterocyte Models

Nicotianamine: A Key Player in Metal Homeostasis and Hyperaccumulation in Plants

Nicotianamine (NA) is a low-molecular-weight N-containing metal-binding ligand, whose accumulation in plant organs changes under metal deficiency or excess. Although NA biosynthesis can be induced in vivo by various metals, this non-proteinogenic amino acid is mainly involved in the detoxification and transport of iron, zinc, nickel, copper and manganese. This review summarizes the current knowledge on NA biosynthesis and its regulation, considers the mechanisms of NA secretion by plant roots, as well as the mechanisms of intracellular transport of NA and its complexes with metals, and its role in radial and long-distance metal transport. Its role in metal tolerance is also discussed. The NA contents in excluders, storing metals primarily in roots, and in hyperaccumulators, accumulating metals mainly in shoots, are compared. The available data suggest that NA plays an important role in maintaining metal homeostasis and hyperaccumulation mechanisms. The study of metal-binding compounds is of interdisciplinary significance, not only regarding their effects on metal toxicity in plants, but also in connection with the development of biofortification approaches to increase the metal contents, primarily of iron and zinc, in agricultural plants, since the deficiency of these elements in food crops seriously affects human health.

Effects of multi-heavy metal composite pollution on microorganisms around a lead-zinc mine in typical karst areas, southwest China

Heavy metal pollution poses a serious hazard to the soil bacterial community. The purpose of this study is to understand the characteristics of soil heavy metal pollution in lead-zinc mines in karst areas and the response of Pb, Zn, Cd, and As-induced composite pollution to soil microorganisms. This paper selected soil samples from the lead-zinc mining area of Xiangrong Mining Co., Ltd., Puding County, Guizhou Province, China. The soil in the mining area is contaminated by multiple heavy metals such as Pb, Zn, Cd and As. The average levels of Pb, Zn, Cd and As in the Pb-Zn mining soil were 14.5, 7.8, 5.5 and 4.4 times higher than the soil background in this area, respectively. Bacterial community structures and functions were analyzed using 16 S rRNA high-throughput sequencing technology and the PICRUSt method. A total of 19 bacterial phyla, 34 classes and 76 orders were detected in the tested soil. At the phylum level, the Proteobacteria are the dominant flora of the soil in the tailings reservoir area of the lead-zinc mine, respectively GWK1 (49.64%), GWK2 (81.89%), GWK3 (95.16%); and for the surrounding farmland soil, the Proteobacteria, Actinobacteriota, Acidobacteriota, Chloroflexi and Firmicutes are the most abundant in five bacterial groups. RDA analyses revealed that the heavy metal pollution of the lead-zinc mining area has a significant impact on the diversity of soil microorganisms. As the distance from the mining area increased, the heavy metal comprehensive pollution and potential risk value decreased, and the bacterial diversity increased. Additionally, various types of heavy metals have different effects on bacterial communities, and soil heavy metal content will also change the bacterial community structure. Proteobacteria positively related to Pb, Cd, and Zn, therefore, Proteobacteria were highly resistant to heavy metals. PICRUSt analysis suggested that heavy metals significantly affect the metabolic function of microorganisms. Microorganisms might generate resistance and enable themselves to survive by increasing the transport of metal ions and excreting metal ions. These results can be used as a basis for the microbial remediation of heavy metal-contaminated farmland in mining areas.

Differentiation and decreased genetic diversity in field contaminated oysters Crassostrea hongkongensis: Identification of selection signatures

The extent to which chemical contamination affects the population structure and genetic diversity of natural populations remains elusive. Here, we used the whole-genome resequencing and transcriptome to diagnose the effects of long-term exposure to multiple elevated chemical pollutants on the population differentiation and genetic diversity in oysters Crassostrea hongkongensis in a typically polluted Pearl River Estuary (PRE) of Southern China. Population structure revealed an obvious differentiation between the PRE oysters and those collected from a nearby clean Beihai (BH) individuals, while no significant differentiation was observed among individuals collected from the three pollution sites within PRE due to the high gene flow. The decreased genetic diversity in the PRE oysters reflected the long-term effects of chemical pollutants. Selective sweeps between BH and PRE oysters revealed that chemical defensome genes, including glutathione S-transferase, zinc transporter, were responsible for their differentiation, sharing common metabolic process of other pollutants. Combined with the genome-wide association analysis, 25 regions containing 77 genes were identified to be responsible for the direct selection regions of metals. Linkage disequilibrium blocks and haplotypes within these regions provided the biomarkers of permanent effects. Our results provide important insights to the genetic mechanisms underlying the rapid evolution under chemical contamination in marine bivalves.

Pan-transcriptomic Profiling Demarcates Serendipita Indica-Phosphorus Mediated Tolerance Mechanisms in Rice Exposed to Arsenic Toxicity

Inadvertent accumulation of arsenic (As) in rice (Oryza sativa L.) is a concern for people depending on it for their subsistence, as it verily causes epigenetic alterations across the genome as well as in specific cells. To ensure food safety, certain attempts have been made to nullify this highest health hazard encompassing physiological, chemical and biological methods. Albeit, the use of mycorrhizal association along with nutrient reinforcement strategy has not been explored yet. Mechanisms of response and resistance of two rice genotypes to As with or without phosphorus (P) nutrition and Serendipita indica (S. indica; S.i) colonization were explored by root transcriptome profiling in the present study. Results revealed that the resistant genotype had higher auxin content and root plasticity, which helped in keeping the As accumulation and P starvation response to a minimum under alone As stress. However, sufficient P supply and symbiotic relationship switched the energy resources towards plant’s developmental aspects rather than excessive root proliferation. Higher As accumulating genotype (GD-6) displayed upregulation of ethylene signaling/biosynthesis, root stunting and senescence related genes under As toxicity. Antioxidant defense system and cytokinin biosynthesis/signaling of both genotypes were strengthened under As + S.i + P, while the upregulation of potassium (K) and zinc (Zn) transporters depicted underlying cross-talk with iron (Fe) and P. Differential expression of phosphate transporters, peroxidases and GSTs, metal detoxification/transport proteins, as well as phytohormonal metabolism were responsible for As detoxification. Taken together, S. indica symbiosis fortified with adequate P-fertilizer can prove to be effective in minimizing As acquisition and accumulation in rice plants.

Steric Hindrance-Induced Dehydration Promotes Cation Selectivity in Trans-Subnanochannel Transport.

Dehydration is a basic phenomenon in ion transport through confined nanochannels, but how it affects ion trans-membrane selectivity has not been understood due to a lack of characterization techniques and suitable pore structures. Herein, hydration number distributions of typical alkali metal ions were characterized by combining uniform subnanochannels of ZIF-8-based membranes with the in situ liquid time-of-flight secondary ion mass spectrometry (ToF-SIMS) technique, revealing that steric hindrance induced ion dehydration through neutral confined ZIF-8 windows. The reduction in size due to partial dehydration increased the intrapore velocity for monovalent cations. The highest entropy value with maximum size changes resulting from dehydration drove fast and efficient selective transport of Li+ over other alkaline metal ions, leading to a Li+/Rb+ selectivity of 5.2. The dehydration at the entrance of membrane pores was shown to account for the majority of overall barriers, being a dominant element for ion transport. High hydration energy (>1500 kJ/mol) hindered the dehydration and transport of typical alkaline earth metal ions, achieving ultrahigh monovalent/bivalent cation selectivity (∼104). These findings uncover the crucial role of dehydration energy barriers and size-based entropy barriers in ion selectivity of trans-subnanochannel transport, providing guidelines for designing selective membranes with specific pore sizes to promote the dehydration of desired solutes.

Endophytic Bacillus sp. AP10 harboured in Arabis paniculata mediates plant growth promotion and manganese detoxification

Phytoremediation of heavy metal-polluted soils assisted by plant-associated endophytes, is a suitable method for plant growth and manganese (Mn) removal in contaminated soils. This investigation was conducted to evaluate the Mn-resistant endophytic resources of the Mn hyperaccumulator Arabis paniculata and their functions in the phytoremediation of Mn2+ toxicity. This study isolated an endophytic bacterium with high Mn resistance and indole-3-acetic acid (IAA) production form A. paniculata and identified it as Bacillus sp. AP10 using 16 S rRNA gene sequencing analysis. The effects of Bacillus sp. AP10 on the alleviation of Mn2+ toxicity in Arabidopsis thaliana seedlings and the molecular mechanisms were further investigated using biochemical tests and RNA-seq analysis. Under Mn2+ stress, Bacillus sp. AP10 increased the biomass, chlorophyll content and the translocation factor (TF) values of Mn in the aerial parts, while decreased the malondialdehyde (MDA) content of A. thaliana seedlings compared with that of control plants. The differentially expressed genes (DEGs) and enrichment analysis showed that Bacillus sp. AP10 could significantly increase the expression of key genes involved in cell-wall loosening, which may improve plant growth under Mn stress. Superoxide dismutase (SOD)-encoding genes were detected as DEGs after AP10 treatment. Moreover, AP10 regulated the expression of genes responsible for phenylpropanoid pathway, which may promote antioxidant flavonoids accumulation for reactive oxygen species (ROS) scavenging to improve Mn tolerance. The activation of ATP-binding cassette (ABC) transporter gene expression especially ABCB1 after AP10 stimulation, explained the elevation of metal ion binding or transport related to enhanced Mn accumulation in plants. Futhermore, AP10 might alleviate Mn toxicity through enhancing abscisic acid (ABA) responsive gene expression and ABA biosynthesis. These findings provide new insights into the functions and regulatory mechanism of Bacillus sp. AP10 in promoting plant growth, and tolerance, improving Mn accumulation and alleviating Mn2+ toxicity in plants. The application of Bacillus sp. AP10 as potential phytoremediators may be a promising strategy in Mn2+ contaminated fields. Availability of data and materialsThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Integration of Physiological, Transcriptomic and Metabolomic Reveals Molecular Mechanism of Paraisaria dubia Response to Zn2+ Stress.

Utilizing mycoremediation is an important direction for managing heavy metal pollution. Zn2+ pollution has gradually become apparent, but there are few reports about its pollution remediation. Here, the Zn2+ remediation potential of Paraisaria dubia, an anamorph of the entomopathogenic fungus Ophiocordyceps gracilis, was explored. There was 60% Zn2+ removed by Paraisaria dubia mycelia from a Zn2+-contaminated medium. To reveal the Zn2+ tolerance mechanism of Paraisaria dubia, transcriptomic and metabolomic were executed. Results showed that Zn2+ caused a series of stress responses, such as energy metabolism inhibition, oxidative stress, antioxidant defense system disruption, autophagy obstruction, and DNA damage. Moreover, metabolomic analyses showed that the biosynthesis of some metabolites was affected against Zn2+ stress. In order to improve the tolerance to Zn2+ stress, the metabolic mechanism of metal ion transport, extracellular polysaccharides (EPS) synthesis, and microcycle conidiation were activated in P. dubia. Remarkably, the formation of microcycle conidiation may be triggered by reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) signaling pathways. This study supplemented the gap of the Zn2+ resistance mechanism of Paraisaria dubia and provided a reference for the application of Paraisaria dubia in the bioremediation of heavy metals pollution.

1481-P: Islet Cell Autoantibody Formation and Its Effect on Insulin Requirement after Total Pancreatectomy with Islet Autotransplantation (TPIAT)

Introduction: Long term insulin requirements following TPIAT range from insulin independence to full (high) dose daily insulin (>0.5 units/kg/day). Markers of islet cell autoimmunity are used in children to predict type 1 diabetes risk. However, no studies have explored whether an association exists between islet cell autoimmunity and insulin requirement following TPIAT in children. We hypothesized that children with islet cell autoantibody formation were more likely to have a persistent insulin requirement following TPIAT. Methods: All children who underwent TPIAT between 2015 and 2021 at our institution were eligible for inclusion. Islet cell autoimmunity was defined as positive islet cell autoantibody screen for one or more of the following types of autoantibodies: Glutamic Acid Decarboxylase (GAD), Islet Antigen-2 (IA-2), Insulin (IAA), or Zinc Transporter 8 (ZnT8). Patients were screened prior to TPIAT and every six months following TPIAT for up to three years. In all, 73 patients were included in the analysis. Total daily insulin dose was determined for each patient at each time interval. Chi-square or Fisher’s exact tests were used to compare proportions. Results: Of patients with 12-month data (n=38), 47% of islet cell autoantibody negative patients were off insulin vs 26% of antibody positive patients (p=0.19). This trend was significant at 18, 24, and 36 months after TPIAT. At 18 months, 90% (9/10) of antibody negative patients were off insulin vs 29% (4/14) of antibody positive patients (p=0.005). At 24 months, 73% (8/11) of antibody negative patients were off insulin vs 23% (3/13) of antibody positive patients (p=0.04). At 36 months, 100% (8/8) of antibody negative patients were off insulin compared to 0% (0/9) of antibody positive patients (p<0.0001). Conclusions: The presence of pancreatic islet cell antibodies was associated with persistent insulin requirement after TPIAT. Disclosure A.R.Lavik: None. C.M.O.Lowe: None. S.E.Tellez: None. L.Hornung: None. C.Heinzman: None. J.D.Nathan: None. M.Abu-el-haija: None. D.A.Elder: None.

Zinc supplementation and ractopamine hydrochloride impact gene expression of zinc transporters in finishing beef steers

Zinc is a trace mineral of interest for optimizing growth in feedlot cattle due to its roles in many physiological functions, including growth. Twenty-four Angus-cross steers (467 ± 13 kg) were used to assess the effects of supplemental Zn and ractopamine hydrochloride (RAC) on trace mineral concentrations and muscle gene expression. Four GrowSafe-equipped pens were randomly assigned to treatments (1 pen of six steers/treatment): 0 (CON), 60 (LOW), 120 (MED) or 180 (HI) mg supplemental Zn/kg DM (Availa-Zn, Zinpro). Dietary Zn treatments were initiated on d 0 and RAC supplementation (300 mg·steer·-1·d-1; Actogain45, Zoetis) began on d 53. Blood, liver and muscle (longissimus thoracis) samples were collected from all steers on d -4, 48, and 67. The LOW treatment was removed from gene expression analyses due to < 3 steers being represented for 14 of 22 genes. Data were analyzed using ProcMixed of SAS with the fixed effect of treatment and steer as the experimental unit; orthogonal linear and quadratic contrast statements were used to compare treatments. On d 48 and 67, there were linear and quadratic trends for plasma Zn to be greater in Zn-supplemented steers than CON (P ≤ 0.10). On d 48, there was a tendency for a quadratic decrease on the expression of SLC30A4 (P ≤ 0.07) but no other differences due to treatment. On d 67, several genes involved in Zn transport and storage (MTA1, SLC39A7, SLC39A8, SLC39A9, SLC39A10, SLC39A13) were decreased (P ≤ 0.08), suggesting increased growth influences intracellular Zn trafficking and demands.

Impact of an SLC30A8 loss-of-function variant on the pancreatic distribution of zinc and manganese: laser ablation-ICP-MS and positron emission tomography studies in mice.

Common variants in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8 (expressed largely in pancreatic islet alpha and beta cells), are associated with altered risk of type 2 diabetes. Unexpectedly, rare loss-of-function (LoF) variants in the gene, described in heterozygous individuals only, are protective against the disease, even though knockout of the homologous SLC30A8 gene in mice leads to unchanged or impaired glucose tolerance. Here, we aimed to determine how one or two copies of the mutant R138X allele in the mouse SLC30A8 gene impacts the homeostasis of zinc at a whole-body (using non-invasive 62Zn PET imaging to assess the acute dynamics of zinc handling) and tissue/cell level [using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to map the long-term distribution of zinc and manganese in the pancreas]. Following intravenous administration of [62Zn]Zn-citrate (~7 MBq, 150 μl) in wild-type (WT), heterozygous (R138X+/-), and homozygous (R138X+/+) mutant mice (14-15 weeks old, n = 4 per genotype), zinc dynamics were measured over 60min using PET. Histological, islet hormone immunohistochemistry, and elemental analysis with LA-ICP-MS (Zn, Mn, P) were performed on sequential pancreas sections. Bulk Zn and Mn concentration in the pancreas was determined by solution ICP-MS. Our findings reveal that whereas uptake into organs, assessed using PET imaging of 62Zn, is largely unaffected by the R138X variant, mice homozygous of the mutant allele show a substantial lowering (to 40% of WT) of total islet zinc, as anticipated. In contrast, mice heterozygous for this allele, thus mimicking human carriers of LoF alleles, show markedly increased endocrine and exocrine zinc content (1.6-fold increase for both compared to WT), as measured by LA-ICP-MS. Both endocrine and exocrine manganese contents were also sharply increased in R138X+/- mice, with smaller increases observed in R138X+/+ mice. These data challenge the view that zinc depletion from the beta cell is the likely underlying driver for protection from type 2 diabetes development in carriers of LoF alleles. Instead, they suggest that heterozygous LoF may paradoxically increase pancreatic β-cell zinc and manganese content and impact the levels of these metals in the exocrine pancreas to improve insulin secretion.

Unbalancing Zur (FurB)-mediated homeostasis in Anabaena sp. PCC7120: Consequences on metal trafficking, heterocyst development and biofilm formation.

Zinc is required for the activity of many enzymes and plays an essential role in gene regulation and redox homeostasis. In Anabaena (Nostoc) sp. PCC7120, the genes involved in zinc uptake and transport are controlled by the metalloregulator Zur (FurB). Comparative transcriptomics of a zur mutant (Δzur) with the parent strain unveiled unexpected links between zinc homeostasis and other metabolic pathways. A notable increase in the transcription of numerous desiccation tolerance-related genes, including genes involved in the synthesis of trehalose and the transference of saccharide moieties, among many others, was detected. Biofilm formation analysis under static conditions revealed a reduced capacity of Δzur filaments to form biofilms compared to the parent strain, and such capacity was enhanced when Zur was overexpressed. Furthermore, microscopy analysis revealed that zur expression is required for the correct formation of the envelope polysaccharide layer in the heterocyst, as Δzur cells showed reduced staining with alcian blue compared to Anabaena sp. PCC7120. We suggest that Zur is an important regulator of the enzymes involved in the synthesis and transport of the envelope polysaccharide layer, influencing heterocyst development and biofilm formation, both relevant processes for cell division and interaction with substrates in its ecological niche.

Metabolic Plasticity Endows Mixotrophic Organisms with High Tolerance to Cadmium and Special Potential for Recovering Cadmium-Contaminated Aquatic Environment.

Heavy metal pollution in waters causes serious stress to aquatic ecosystems. Several autotrophs with strong tolerance are extensively used to adsorb heavy metals, but their use may be limited by the specific conditions of polluted waters due to their single nutrition mode. By contrast, mixotrophs possess strong environmental adaptability due to their plastic metabolic modes. However, studies focusing on mixotroph's resistance and its underlying mechanism in response to heavy metals and their bioremediation potentials are currently lacking. In this study, we investigated the population, phytophysiological, and transcriptomic (RNA-Seq) responses of a typical and common mixotrophic organism, Ochromonas, to cadmium exposure and then evaluated their capacity to remove cadmium under mixotrophic condition. Compared with autotrophy, mixotrophic Ochromonas enhanced photosynthetic performances under short-time cadmium exposure and subsequently shifted to stronger resistance with increasing exposure time. Transcriptomic analyses suggested that the genes related to photosynthesis, ATP production, ECM components, and scavenging of reactive oxygen species and damaged organelles were upregulated to boost mixotrophic Ochromonas resistance to cadmium. Consequently, the harm of metal exposure was eventually reduced and cellular stability was maintained. Approximately, 70% of cadmium at 2.4 mg L-1 cadmium was removed by mixotrophic Ochromonas in the end, benefiting from upregulated genes associated with the transport of metal ions. Hence, mixotrophic Ochromonas tolerance to cadmium can be attributed to multiple pathways of energy metabolism and effective transport of metal ions. Collectively, this study advanced a better understanding of the unique mechanism of heavy metal resistance in mixotrophs and their potential use in recovering cadmium-contaminated aquatic ecosystems. IMPORTANCE Mixotrophs widely living in aquatic ecosystems possess unique ecological roles and strong environmental adaptability due to their plastic metabolic modes; however, little is known about their underlying resistance mechanism and bioremediation potential in response to environmental stresses. For the first time, this work investigated how mixotrophs respond to metal pollutants through physiological, population dynamics, and transcriptional regulation, and highlighted the unique underlying mechanism of mixotrophs to resist and remove heavy metal, thereby advancing our understanding of the potentials of mixotrophs in recovering metal-contaminated aquatic environments. These unique properties in mixotrophs are essential for the long-term functional stability of aquatic ecosystems.

Anti-Islet Autoantibodies in Type 1 Diabetes.

Anti-islet autoantibodies serve as key markers in immune-mediated type 1 diabetes (T1D) and slowly progressive T1D (SPIDDM), also known as latent autoimmune diabetes in adults (LADA). Autoantibodies to insulin (IAA), glutamic acid decarboxylase (GADA), tyrosine phosphatase-like protein IA-2 (IA-2A), and zinc transporter 8 (ZnT8A) are currently employed in the diagnosis, pathological analysis, and prediction of T1D. GADA can also be detected in non-diabetic patients with autoimmune diseases other than T1D and may not necessarily reflect insulitis. Conversely, IA-2A and ZnT8A serve as surrogate markers of pancreatic β-cell destruction. A combinatorial analysis of these four anti-islet autoantibodies demonstrated that 93-96% of acute-onset T1D and SPIDDM cases were diagnosed as immune-mediated T1D, while the majority of fulminant T1D cases were autoantibody-negative. Evaluating the epitopes and immunoglobulin subclasses of anti-islet autoantibodies help distinguish between diabetes-associated and non-diabetes-associated autoantibodies and is valuable for predicting future insulin deficiency in SPIDDM (LADA) patients. Additionally, GADA in T1D patients with autoimmune thyroid disease reveals the polyclonal expansion of autoantibody epitopes and immunoglobulin subclasses. Recent advancements in anti-islet autoantibody assays include nonradioactive fluid-phase assays and the simultaneous determination of multiple biochemically defined autoantibodies. Developing a high-throughput assay for detecting epitope-specific or immunoglobulin isotype-specific autoantibodies will facilitate a more accurate diagnosis and prediction of autoimmune disorders. The aim of this review is to summarize what is known about the clinical significance of anti-islet autoantibodies in the pathogenesis and diagnosis of T1D.

A CRE/DRE dual recombinase transgenic mouse reveals synaptic zinc-mediated thalamocortical neuromodulation.

Synaptic zinc is a neuromodulator that shapes synaptic transmission and sensory processing. The maintenance of synaptic zinc is dependent on the vesicular zinc transporter, ZnT3. Hence, the ZnT3 knockout mouse has been a key tool for studying the mechanisms and functions of synaptic zinc. However, the use of this constitutive knockout mouse has notable limitations, including developmental, compensatory, and brain and cell type specificity issues. To overcome these limitations, we developed and characterized a dual recombinase transgenic mouse, which combines the Cre and Dre recombinase systems. This mouse allows for tamoxifen-inducible Cre-dependent expression of exogenous genes or knockout of floxed genes in ZnT3-expressing neurons and DreO-dependent region and cell type-specific conditional ZnT3 knockout in adult mice. Using this system, we reveal a neuromodulatory mechanism whereby zinc release from thalamic neurons modulates N-methyl-d-aspartate receptor activity in layer 5 pyramidal tract neurons, unmasking previously unknown features of cortical neuromodulation.

Loss of zinc transporters ZIP1 and ZIP3 augments platelet reactivity in response to thrombin and accelerates thrombus formation in vivo.

Zinc (Zn2+) is considered as important mediator of immune cell function, thrombosis and haemostasis. However, our understanding of the transport mechanisms that regulate Zn2+ homeostasis in platelets is limited. Zn2+ transporters, ZIPs and ZnTs, are widely expressed in eukaryotic cells. Using mice globally lacking ZIP1 and ZIP3 (ZIP1/3 DKO), our aim was to explore the potential role of these Zn2+ transporters in maintaining platelet Zn2+ homeostasis and in the regulation of platelet function. While ICP-MS measurements indicated unaltered overall Zn2+ concentrations in platelets of ZIP1/3 DKO mice, we observed a significantly increased content of FluoZin3-stainable free Zn2+, which, however, appears to be released less efficiently upon thrombin-stimulated platelet activation. On the functional level, ZIP1/3 DKO platelets exhibited a hyperactive response towards threshold concentrations of G protein-coupled receptor (GPCR) agonists, while immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptor agonist signalling was unaffected. This resulted in enhanced platelet aggregation towards thrombin, bigger thrombus volume under flow ex vivo and faster in vivo thrombus formation in ZIP1/3 DKO mice. Molecularly, augmented GPCR responses were accompanied by enhanced Ca2+ and PKC, CamKII and ERK1/2 signalling. The current study thereby identifies ZIP1 and ZIP3 as important regulators for the maintenance of platelet Zn2+ homeostasis and function.

Monodisperse covalent organic nanosheets by in-situ oxidation method for efficient ion/molecule separation

Two-dimensional (2D) covalent organic nanosheets (CONs) are layered crystalline porous polymers comprising periodically extended graphene-like structures, and have great application potential in membrane separation field due to the advantages of abundant optional monomers, tunable pore size and structure, facilely-tailored functionality and pre-designed properties. However, unlike graphene oxide (GO) membranes, it is hard to directly prepare 2D CON membranes by vacuum filtration because of the difficulties in preparation of ultra-thin, large-sized and well-dispersed 2D CONs. Herein, an improved buffering interlayer interface method was proposed to realize a facile one-step synthesis of GO-like CON oxides by using H2O2 as an in-situ oxidant in the buffer layer. The stereoscopic fulcrum-oxygen sites introduced into the structure of CONs lead to weaken interactions between CON layers and increased hydrophilicity, which contributes to subsequent preparation of monodisperse nanosheets. The membranes fabricated with the as-prepared monodisperse nanosheets can realize high-efficiency transport of metal ions with a precise charge-discriminated group separation ability and efficient ion rejection. The strategy of in-situ oxidation to prepare monodisperse CONs proposed in this study provides a valuable approach for the preparation of novel functionalized CONs and the achievement of fast membrane separation performance.

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay.

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

Abstract B075: Anti-human LIV-1 antibody drug conjugate for treatment of metastatic prostate cancer

Abstract In the worldwide male population, prostate cancer has the most frequent malignancy; out of all the cancer-related causes of deaths, it is also one of the most common. With the advance of new therapies of androgen receptor pathway inhibitors, patient survival rates have improved significantly. There are still tremendous unmet needs, however, for the patients with treat-refractory metastatic castration-resistant prostate cancer (mCRPC). LIV-1, also known as SLC39A6 or ZIP6, is a member of the zinc transporter family and was first identified as an estrogen-inducible gene in breast cancer. Previous studies with immunohistochemical (IHC) analysis showed that LIV-1 is expressed by estrogen receptor-positive (ER+), hormone-treated tumors (both primary and metastatic sites) and ER-/PR-/Her2- (triple-negative) breast cancers. These studies also showed that in healthy human tissues, LIV-1 expression is limited to hormonally-regulated organs (prostate, uterus, and breast). The broad expression of LIV-1 in prostate and breast cancer tumors in combination with the limited expression in vital organs makes LIV-1 an excellent target for an antibody-drug conjugate (ADC). We generated an ADC, BRY812, consisting of a humanized anti-LIV-1 mAb conjugated to monomethyl auristatin E (MMAE), via a novel conjugation method that prevents MMAE from coming off of the antibody during the circulation. The PK studies in rat and monkey showed that the free MMAE released from ADC is 1 to 2 log lower compared with approved ADCs prepared by the standard conjugation method. Low free MMAE concentration significantly lowers the risk of off target toxicity by the ADC. In vitro and in vivo studies demonstrated the antitumor activity of BRY812 for the treatment of prostate and ER+ and triple-negative breast cancers with a better safety profile and a larger therapeutic window. The humanized LIV-1 ADC, BRY812 is currently in preclinical toxicity studies and will advance to First-in-Human trials in April, 2023. Citation Format: Xiaobei Zhao, Gang Chen. Anti-human LIV-1 antibody drug conjugate for treatment of metastatic prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B075.