Intracellular Zinc Research Articles

Sensitive visual detection of intracellular zinc ions based on signal-on polydopamine carbon dots

The concentration of intracellular zinc ions is a significant clinical parameter for diagnosis. However, it is still a challenge for direct visual detection of zinc ions in cells at single-cell level. To address this issue, herein, water-soluble amino-rich polydopamine carbon quantum dots (PDA-CQDs) were successfully synthesized, with strong blue-green fluorescence as the probes for zinc ions detection in cells. The structure and properties of PDA-CQDs were confirmed by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transformed infrared (FT-IR), UV–visible spectrophotometry (UV–vis), and fluorescence spectroscopy. Importantly, by successfully linking salicylaldehyde (SA) to PDA-CQDs via nucleophilic reaction, the FL quenching and Zn ions induced FL-recovering system was built up, thus offering a signal-on platform for the detection of zinc ions. This PDA-CQDs-SA nanoprobe can be applied for the detection of Zn2+ with a detection limit of 0.09 μM, with good biocompatibility confirmed using cytotoxicity assay. Of significance, the results of fluorescence bioimaging showed that PDA-CQDs-SA is able to detect Zn2+ in single-cell visually, with the detection limit of Zn ions in cells as low as 0.11 μM per cell, which was confirmed using flow cytometry. Therefore, this work offers a potential probe for Zn2+ detection in cells at single-cell level, towards the precise diagnosis of zinc ions related diseases.

Zinc Accumulation Aggravates Cerebral Ischemia/Reperfusion Injury Through Inducing Endoplasmic Reticulum Stress.

Zinc is highly enriched in the central nervous system. Numerous evidences suggest that high concentration of zinc acts as a critical mediator of neuronal death in the ischemic brain, however, the possible mechanisms of neurotoxicity of zinc during cerebral ischemia/reperfusion (I/R) remain elusive. Endoplasmic reticulum (ER) is a storage location of intracellular zinc. ER stress related genes were up-regulated during zinc-induced neuronal death in vascular-type senile dementia. In the present study, we investigated whether intracellular accumulated zinc aggravates I/R injury through ER stress and ER stress-associated apoptosis. Male Sprague-Dawley rats were subjected to 90min middle cerebral artery occlusion (MCAO) and received either vehicle or zinc chelator TPEN 15mg/kg. The expression of ER stress related factors glucose-regulated protein 78 (GRP78) and phosphorylated eukaryotic initiation factor 2α (p-eIF2α), ER stress related apoptotic proteins CCAAT-enhancer-binding protein homologous protein (CHOP) and caspase-12, as well as anti-apoptotic factor B-cell lymphoma-2 (Bcl-2) were assessed 24h after reperfusion. Our results showed that the levels of GRP78 and p-eIF2α, as well as CHOP and caspase-12, were increased in ischemic brain, indicating that cerebral I/R triggers ER stress. Furthermore, GRP78, CHOP and caspase-12 were all colocalized with the zinc-specific dyes NG, suggesting that there is certain relationship between cytosolic labile zinc and ER stress following cerebral ischemia. Chelating zinc with TPEN reversed the expression of GRP78, p-eIF2α in ischemic rats. Moreover, CHOP and NeuN double staining positive cells, as well as caspase-12 and TUNEL double staining positive cells were also decreased after TPEN treatment, indicating that chelating zinc might inhibit ER stress and decreased ER stress associated neuronal apoptosis. In addition, TPEN treatment reversed the downregulated level of Bcl-2, which localized in the ER membrane and involved in the dysfunction of ER, confirming that the anti-apoptosis effects of chelating zinc following I/R are exerted via inhibition of the ER stress. Taken together, this study demonstrated that excessive zinc activates ER stress and zinc induced neuronal cell death is at least partially due to ER stress specific neuronal apoptosis in ischemic penumbra, which may provide an important mechanism of cerebral I/R injury.

Zinc transporter ZIP12 maintains zinc homeostasis and protects spermatogonia from oxidative stress during spermatogenesis

BackgroundOverwhelming evidences suggest oxidative stress is a major cause of sperm dysfunction and male infertility. Zinc is an important non-enzymatic antioxidant with a wide range of biological functions and plays a significant role in preserving male fertility. Notably, zinc trafficking through the cellular and intracellular membrane is mediated by specific families of zinc transporters, i.e., SLC39s/ZIPs and SLC30s/ZnTs. However, their expression and function were rarely evaluated in the male germ cells. The aim of this study is to determine and characterize the crucial zinc transporter responsible for the maintenance of spermatogenesis.MethodsThe expression patterns of all 14 ZIP members were characterized in the mouse testis. qRT-PCR, immunoblot and immunohistochemistry analyses evaluated the ZIP12 gene and protein expression levels. The role of ZIP12 expression was evaluated in suppressing the sperm quality induced by exposure to an oxidative stress in a spermatogonia C18–4 cell line. Zip12 RNAi transfection was performed to determine if its downregulation altered cell viability and apoptosis in this cell line. An obese mouse model fed a high-fat-diet was employed to determine if there is a correlation between changes in the ZIP12 expression level and sperm quality.ResultsThe ZIP12 mRNA and protein expression levels were higher than those of other ZIP family members in both the mouse testis and other tissues. Importantly, the ZIP12 expression levels were very significantly higher in both mice and human spermatogonia and spermatozoa. Moreover, the testicular ZIP12 expression levels significantly decreased in obese mice, which was associated with reduced sperm zinc content, excessive sperm ROS generation, poor sperm quality and male subfertility. Similarly, exposure to an oxidative stress induced significant declines in the ZIP12 expression level in C18–4 cells. Knockdown of ZIP12 expression mediated by transfection of a ZIP12 siRNA reduced both the zinc content and viability whereas apoptotic activity increased in the C18–4 cell line.ConclusionsThe testicular zinc transporter ZIP12 expression levels especially in spermatogonia and spermatozoa are higher than in other tissues. ZIP12 may play a key role in maintaining intracellular zinc content at levels that reduce the inhibitory effects of rises in oxidative stress on spermatogonia and spermatozoa viability during spermatogenesis which help counteract declines in male fertility.

Loss of MBD2 ameliorates LPS-induced alveolar epithelial cell apoptosis and ALI in mice via modulating intracellular zinc homeostasis.

Apoptosis of alveolar epithelial cells is a critical initial link in the pathogenesis of acute lung injury (ALI), recent studies have revealed that Methyl-CpG binding domain protein 2 (MBD2) was involved in the execution of apoptosis, yet its role in ALI remained unclear. In the present study, we aim to explore the role and mechanism of MBD2 in the pathogenesis of ALI. We have found that MBD2 expression, in parallel to apoptosis, increased in alveolar epithelial cells of mice treated with LPS, knockout of MBD2 reduced apoptosis and protected mice from LPS-induced ALI. In MLE-12 cells, a cell line of murine alveolar epithelial cells, LPS induced MBD2 expression and apoptosis in a dose- and time-dependent manner. Knockdown of MBD2 with shRNA alleviated, while overexpression of MBD2 increased LPS-induced apoptosis. Mechanistically, intracellular zinc level decreased when MLE-12 cells were treated with LPS. MBD2knockdown restored intracellular zinc level after LPS treatment, and MBD2 overexpression further aggravated LPS-induced intracellular zinc loss. Metal transcription factor 1 (MTF1) is a critical transcription factor in charge of intracellular zinc efflux. LPS treatment induced MTF1 expression both in vivo and in vitro. Inhibition of MTF1 reduced LPS-induced apoptosis in MLE-12 cells. MBD2 could bind to the promoter region of MTF1 and promote MTF1 expression. Collectively, these data indicated that loss of MBD2-ameliorated LPS-induced alveolar epithelial cell apoptosis and ALI in mice via modulating intracellular zinc homeostasis by upregulating MTF1.

Vitamin D3 and zinc synergistically induce regulatory T cells and suppress interferon-γ production in mixed lymphocyte culture

Although vitamin D3 (cholecalciferol) and zinc are known to individually shift the immune response towards tolerance, little is known about the effect of their combined administration. This work contributes to understanding the combined action of zinc and vitamin D3 in different in vitro models for immune reactions. Zinc and vitamin D3 in combination boosted the differentiation into Foxp3+CD4+ T cells (Treg). Vitamin D3 alone reduced the percentage of CD4+T-bet+ T cells (TH1). In mixed lymphocyte culture (MLC), therapeutic concentrations of vitamin D3 and zinc in combination suppressed interferon-γ (IFN-γ) secretion more strongly than the single agent treatment. This effect was also detected for a combination of subtherapeutic concentrations of both vitamin D3 and zinc. Vitamin D3, even at nanomolar concentrations, increased intracellular zinc levels. PBMC (peripheral blood mononuclear cells) of individuals at risk of zinc deficiency responded to vitamin D3 treatment with a higher mRNA expression of Zip13. In PBMC, both agents reduced activation-induced IL-17 secretion. In summary, this study shows, for the first time, a vitamin D3-induced upregulation of CD4+Foxp3+ T cells in MLC. The data propose a model where zinc augments the effect of vitamin D3 in certain therapeutic and subtherapeutic concentrations. Lower concentrations of both vitamin D3 and zinc could be used for effective treatment, thus reducing possible side effects from vitamin D3 and zinc. Vitamin D3 and zinc in combination may be a promising and cheap option to treat dysregulated immune response in various conditions.

Expression and Distribution of Free Zinc in Penile Erectile Tissue.

Several studies have shown that zinc has a significant influence on erectile function. However, no studies evaluating the cellular distribution of free zinc in penile erectile tissue have been performed. Therefore, this study aimed to test whether free zinc is present in penile tissue and whether it may be involved in the electrical stimulation (ES)-induced penile erection. The subjects for this study were 26 young (8-week-old) male C57BL/6J mice. After the cavernous nerve was exposed through a midline stomach incision, 14 mice received ES of the cavernous nerve (ES group), whereas 12 mice did not (control group). Intracavernous pressure (ICP) (consisting of 10 V at a duration of 1 min, frequency of 12 Hz and a pulse width of 1 m/s) was recorded during ES. Immediately after ICP was recorded, penile tissues were harvested for histological and biochemical analysis, including analysis of zinc transporter 3 (ZnT3) and intracellular free zinc levels. The expression of neuronal nitric oxide synthase (nNOS) and endothelial NOS (eNOS) in penile tissue was significantly greater in the ES group than in the control group (p=0.036 and 0.016, respectively). And then, ZnT3 and intracellular free zinc were present in the penile tissue of both groups. However, ZnT3 immunofluorescence in the ES group was more intense in the dorsal nerve bundle (22% increase, p=0.032). The ES group also showed higher intensity N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ) fluorescence signals indicative of intracellular free zinc level in the penile tissue compared to the control group (49% increase in dorsal nerve bundle, p=0.001; 50% increase in corpus cavernosum, p=0.001). The results of the study supported the expression and distribution of free zinc in penile tissue and increased levels after penile erection. Therefore, this study provides anatomical evidence for the potential role of free zinc in penile erection.

Dynamic changes of intracellular zinc ion level during maturation, fertilization, activation, and development in mouse oocytes.

Although it is well known that calcium oscillations are required for fertilization in all mammalian species studied to date, recent studies also showed the ejection of zinc into the extracellular milieu in a series of coordinated events, called "zinc spark," during mammalian fertilization. These results led us to the hypothesis that a zinc ion-dependent signal is important for oocyte maturation, fertilization (activation), and further embryonic development. In this study, we evaluated the amounts and localization of intracellular zinc ions during maturation, fertilization, activation, and embryonic development in mouse oocytes. Our results show that abundant zinc ions are present in both immature and mature oocytes. After in vitro fertilization, the amounts of zinc ions were dramatically decreased at the pronuclear (PN) stage. Artificial activation by cycloheximide, SrCl2 , and TPEN also reduced the amounts of zinc ions in the PN stage. On the other hand, PN embryos derived from sperm injection still showed high level of zinc ions. However, the amounts of zinc ions rapidly increased at the blastocysts regardless of activation method. We showed here that the amounts of zinc ions dramatically changed during maturation, fertilization, activation, and development in mouse oocytes.

Intracellular Zinc Signaling Influences NMDA Receptor Function by Enhancing the Interaction of ZnT1 with GluN2A

Intracellular Zinc Signaling Influences NMDA Receptor Function by Enhancing the Interaction of ZnT1 with GluN2A

Effects of changes in ambient oxygen levels and hypoxia-reoxygenation on intracellular zinc levels in human coronary artery endothelial cells

Effects of changes in ambient oxygen levels and hypoxia-reoxygenation on intracellular zinc levels in human coronary artery endothelial cells

Human Epidermal Zinc Concentrations after Topical Application of ZnO Nanoparticles in Sunscreens.

Zinc oxide nanoparticle (ZnO NP)-based sunscreens are generally considered safe because the ZnO NPs do not penetrate through the outermost layer of the skin, the stratum corneum (SC). However, cytotoxicity of zinc ions in the viable epidermis (VE) after dissolution from ZnO NP and penetration into the VE is ill-defined. We therefore quantified the relative concentrations of endogenous and exogenous Zn using a rare stable zinc-67 isotope (67Zn) ZnO NP sunscreen applied to excised human skin and the cytotoxicity of human keratinocytes (HaCaT) using multiphoton microscopy, zinc-selective fluorescent sensing, and a laser-ablation inductively coupled plasma–mass spectrometry (LA-ICP-MS) methodology. Multiphoton microscopy with second harmonic generation imaging showed that 67ZnO NPs were retained on the surface or within the superficial layers of the SC. Zn fluorescence sensing revealed higher levels of labile and intracellular zinc in both the SC and VE relative to untreated skin, confirming that dissolved zinc species permeated across the SC into the VE as ionic Zn and significantly not as ZnO NPs. Importantly, the LA-ICP-MS estimated exogenous 67Zn concentrations in the VE of 1.0 ± 0.3 μg/mL are much lower than that estimated for endogenous VE zinc of 4.3 ± 0.7 μg/mL. Furthermore, their combined total zinc concentrations in the VE are much lower than the exogenous zinc concentration of 21 to 31 μg/mL causing VE cytotoxicity, as defined by the half-maximal inhibitory concentration of exogenous 67Zn found in human keratinocytes (HaCaT). This speaks strongly for the safety of ZnO NP sunscreens applied to intact human skin and the associated recent US FDA guidance.

The cytotoxicity of zinc oxide nanoparticles to 3D brain organoids results from excessive intracellular zinc ions and defective autophagy.

Although the neurotoxicity of ZnO nanoparticles (NPs) has been evaluated in animal and nerve cell culture models, these models cannot accurately mimic human brains. Three-dimensional (3D) brain organoids based on human-induced pluripotent stem cells have been developed to study the human brains, but this model has rarely been used to evaluate NP neurotoxicity. We used 3D brain organoids that express cortical layer proteins to investigate the mechanisms of ZnO NP-induced neurotoxicity. Cytotoxicity caused by high levels of ZnO NPs (64μg/mL) correlated with high intracellular Zn ion levels but not superoxide levels. Exposure to a non-cytotoxic concentration of ZnO NPs (16μg/mL) increased the autophagy-marker proteins LC3B-II/I but decreased p62 accumulation, whereas a cytotoxic concentration of ZnO NPs (64μg/mL) decreased LC3B-II/I proteins but did not affect p62 accumulation. Fluorescence micro-optical sectioning tomography revealed that 64μg/mL ZnO NPs led to decreases in LC3B proteins that were more obvious at the outer layers of the organoids, which were directly exposed to the ZnO NPs. In addition to reducing LC3B proteins in the outer layers, ZnO NPs increased the number of micronuclei in the outer layers but not the inner layers (where LC3B proteins were still expressed). Adding the autophagy flux inhibitor bafilomycin A1 to ZnO NPs increased cytotoxicity and intracellular Zn ion levels, but adding the autophagy inducer rapamycin only slightly decreased cellular Zn ion levels. We conclude that high concentrations of ZnO NPs are cytotoxic to 3D brain organoids via defective autophagy and intracellular accumulation of Zn ions.

Effect of Different Zinc Sources and Concentrations on the Biomass Yield of Saccharomyces cerevisiae Yeast.

Zinc incorporation and the growth of Saccharomyces cerevisiae were investigated in a culture supplemented with three inorganic zinc salts, i.e. zinc sulphate, zinc chloride and zinc nitrate. The cultivation was performed on a yeast extract peptone dextrose (YEPD) broth medium. The growth of yeast was carried out at different concentrations of zinc, i.e. 0, 30, 60, 90 and 120mg 100ml-1. It was found that the addition of different zinc sources at 30mg 100ml-1 concentration produced higher biomass yield ranging 1.00-1.03g from 100ml-1 of cultivation medium, while higher zinc concentration in the medium caused significantly lower yields of yeast biomass. The amount of zinc in yeast cells was determined by an atomic absorption spectrometer (AAS). The highest amount of zinc in yeast cells was achieved when added in the form of zinc sulphate at a concentration of 120mg 100ml-1. The increment of intracellular zinc was up to 9889.67mgkg-1 of biomass.

Zinc antagonizes iron-regulation of tyrosine hydroxylase activity and dopamine production in Drosophila melanogaster

BackgroundDopamine (DA) is a neurotransmitter that plays roles in movement, cognition, attention, and reward responses, and deficient DA signaling is associated with the progression of a number of neurological diseases, such as Parkinson’s disease. Due to its critical functions, DA expression levels in the brain are tightly controlled, with one important and rate-limiting step in its biosynthetic pathway being catalyzed by tyrosine hydroxylase (TH), an enzyme that uses iron ion (Fe2+) as a cofactor. A role for metal ions has additionally been associated with the etiology of Parkinson’s disease. However, the way dopamine synthesis is regulated in vivo or whether regulation of metal ion levels is a component of DA synthesis is not fully understood. Here, we analyze the role of Catsup, the Drosophila ortholog of the mammalian zinc transporter SLC39A7 (ZIP7), in regulating dopamine levels.ResultsWe found that Catsup is a functional zinc transporter that regulates intracellular zinc distribution between the ER/Golgi and the cytosol. Loss-of-function of Catsup leads to increased DA levels, and we showed that the increased dopamine production is due to a reduction in zinc levels in the cytosol. Zinc ion (Zn2+) negatively regulates dopamine synthesis through direct inhibition of TH activity, by antagonizing Fe2+ binding to TH, thus rendering the enzyme ineffective or non-functional.ConclusionsOur findings uncovered a previously unknown mechanism underlying the control of cellular dopamine expression, with normal levels of dopamine synthesis being maintained through a balance between Fe2+ and Zn2+ ions. The findings also provide support for metal modulation as a possible therapeutic strategy in the treatment of Parkinson’s disease and other dopamine-related diseases.

Slc39a2-Mediated Zinc Homeostasis Modulates Innate Immune Signaling in Phenylephrine-Induced Cardiomyocyte Hypertrophy

Zinc dyshomeostasis has been involved in the pathogenesis of cardiac hypertrophy; however, the dynamic regulation of intracellular zinc and its downstream signaling in cardiac hypertrophy remain largely unknown. Using Zincpyr1 staining, we found a significant decrease of intracellular Zinc concentration in phenylephrine (PE)-induced hypertrophy of neonatal rat ventricular myocytes (NRVMs). We then screened SLC39 family members responsible for zinc uptake and identified Slc39a2 as the only one altered by PE treatment. Slc39a2 knockdown in NRVMs reduced the intracellular Zinc level, and exacerbated the hypertrophic responses to PE treatment. In contrast, adenovirus-mediated Slc39a2 overexpression enhanced zinc uptake and suppressed PE-induced Nppb expression. RNA sequencing analysis showed a pro-hypertrophic transcriptome reprogramming after Slc39a2 knockdown. Interestingly, the innate immune signaling pathways, including NOD signaling, TOLL-like receptor, NFκB, and IRFs, were remarkably enriched in the Slc39a2-regulated genes. Slc39a2 deficiency enhanced the phosphorylation of P65 NFκB and STAT3, and reduced the expression of IκBα. Finally, the expression of IRF7 was significantly increased by Slc39a2 knockdown, which was in turn suppressed by IRF7 knockdown. Our data demonstrate that zinc homeostasis mediated by a Slc39a2/IRF7 regulatory circuit contributes to the alteration of innate immune signaling in cardiomyocyte hypertrophy.

Dengue virus replication enhances labile zinc pools by modulation of ZIP8

Zinc-dependent viral proteins rely on intracellular zinc homeostasis for successful completion of infectious life-cycle. Here, we report that the intracellular labile zinc levels were elevated at early stages of dengue virus (DENV) infection in hepatic cells and this increase in free zinc was abolished in cells infected with UV-inactivated virus or with a DENV replication inhibitor implicating a role for zinc homeostasis in viral RNA replication. This change in free zinc was mediated by zinc transporter, ZIP8, as siRNA-mediated knockdown of ZIP8 resulted in abrogation of increase in free zinc levels leading to significant reduction in DENV titers suggesting a crucial role for ZIP8 in early stages of DENV replication. Furthermore, elevated free zinc levels correlated with high copy numbers of dengue genome in peripheral blood leukocytes obtained from dengue patients compared to healthy controls suggesting a critical role for zinc homeostasis in dengue infection.Take AwaysDengue virus utilises cellular zinc homeostasis during replication of its RNA.ZIP8 upregulates free zinc levels during dengue virus replication.Enhanced viremia associates with elevated intracellular free zinc in dengue.

The role of zinc transporter proteins as predictive and prognostic biomarkers of hepatocellular cancer.

Identification of the key processes involved in the tumor progression, malignancy and the molecular factors which are responsible for the transition of the cirrhotic cells to the tumor cells, contribute to the detection of biomarkers for diagnosis of hepatocellular carcinoma (HCC) at an early stage. According to clinical data, HCC is mostly characterized by a significant decrease in zinc levels. It is strongly implied that zinc deficiency is the major event required in the early stages of tumor formation and development of malignancy. Due to this reason, the definition of the molecular players which have a role in zinc homeostasis and cellular zinc level could give us a clue about the transition state of the cirrhosis to hepatic tumor formation. Despite the well-known implications of zinc in the development of HCCthe correlation of the expression of zinc transporter proteins with tumor progression and malignancy remain largely unknown. In the present study, we evaluated in detail the relationship of zinc deficiency on the prognosis of early HCC patients. In this study, we aimed to test the potential zinc transporters which contribute tothe transformation of cirrhosis to HCCand the progression of HCC. Among the 24 zinc transporter proteins, the proteins to be examined were chosen by using Gene Expression Profiling Interactive Analysis (GEPIA) webpage and RNA-seq analysis using TCGA database. ZIP14 and ZIP5 transporters were found as common differentially expressed genes from both bioinformatic analyses. ZnT1, ZnT7 and ZIP7 transporters have been associated with tumor progression. Relative abundance of ZnT1, ZIP5 and ZIP14 protein level was determined by immunohistochemistry (IHC) in surgically resected liver specimens from 16 HCC patients at different stages. IHC staining intensity was analyzed by using ImageJ software and scored with the histological scoring (H-score) method. The staining of ZnT1 was significantly higher in Grade III comparing to Grade II and Grade I. On the contrary, ZIP14 staining decreased almost 10-foldcomparing to Grade Iand Grade II. ZIP5 staining was detected almost 2-fold higher in cirrhosis than HCC. But ZnT1 staining was observed almost 3-fold lower in cirrhosis comparing to HCC. Intracellular free zinc level was measured by flow cytometry in Hep40 and Snu398 cells using FluoZin-3 dye. The intracellular free zinc level was almost 9-fold decreased in poor differentiated Snu398 HCC cells comparing to well differentiated Hep40 HCC cells.This report establishes for the first time the correlation between the expression pattern of ZIP14, ZnT1 and ZIP5 and significant zinc deficiency which occurs concurrently with the advancing of malignancy. Our results provide new molecular insight into ZnT1, ZIP14 and ZIP5 mediated regulation of cellular zinc homeostasis and indicate that zinc transporters might be important factors and events in HCC malignancy, which can lead to the development of early biomarkers.

Zinc-mediated activation of CREB pathway in proliferation of pulmonary artery smooth muscle cells in pulmonary hypertension

BackgroundTranscription factor CREB is involved in the development of pulmonary hypertension (PH). However, little is known about the role and regulatory signaling of CREB in PH.MethodsA series of techniques, including bioinformatics methods, western blot, cell proliferation and luciferase reporter assay were used to perform a comprehensive analysis of the role and regulation of CREB in proliferation of pulmonary artery smooth muscle cells (PASMCs) in PH.ResultsUsing bioinformatic analysis of the differentially expressed genes (DEGs) identified in the development of monocrotaline (MCT)- and hypoxia-induced PH, we found the overrepresentation of CRE-containing DEGs. Western blot analysis revealed a sustained increase in total- and phosphorylated-CREB in PASMCs isolated from rats treated with MCT. Similarly, an enhanced and prolonged serum-induced CREB phosphorylation was observed in hypoxia-pretreated PASMCs. The sustained CREB phosphorylation in PASMCs may be associated with multiple protein kinases phosphorylated CREB. Additionally, hierarchical clustering analysis showed reduced expression of the majority of CREB phosphatases in PH, including regulatory subunits of PP2A, Ppp2r2c and Ppp2r3a. Cell proliferation analysis showed increased PASMCs proliferation in MCT-induced PH, an effect relied on CREB-mediated transcriptional activity. Further analysis revealed the raised intracellular labile zinc possibly from ZIP12 was associated with reduced phosphatases, increased CREB-mediated transcriptional activity and PASMCs proliferation.ConclusionsCREB pathway was overactivated in the development of PH and contributed to PASMCs proliferation, which was associated with multiple protein kinases and/or reduced CREB phosphatases and raised intracellular zinc. Thus, this study may provide a novel insight into the CREB pathway in the pathogenesis of PH.B6vttFt3ef-LGEoxvha7NyVideo abstract

476 Effect of Holding Period on Viability, Acrosome Status and Intracellular Zinc of Boar Sperm During Liquid Storage

Abstract Sperm capacitation results in acrosomal remodeling, increased membrane fluidity, plasma membrane fusability and hyperactivated sperm. Distinct sperm zinc signatures reflect intracellular zinc content, capacitation status, and viability. Therefore, flow cytometric assessment could be a useful tool to evaluate sperm quality and longevity during liquid storage of boar semen. Cooled semen (17°C) from commercial boars (n = 12) were assessed at Day-1, -4 and -7 after collection for motility (IVOS II, Hamilton Thorne, Beverly, MA), intracellular zinc and viability. Samples were stained with 8 µg/mL Hoecsht-33342 (Hoe), 1 µM FluoZinTM-3-AM (Zinc), 10 µM propidium iodide (PI) and 5.6 µg/mL PNA-Alexa647TM (Molecular Probes, Eugene, OR), incubated for 30 min, washed with PBS, and aditionally incubated for 30 min. Acquisition and analysis of 20000 events were performed using Bio-Rad ZE5 Cell Analyzer (Hercules, CA) and FLowJoTM (Ashland, OR). Hoe+ events were gated into Zinc/PI plot to determine viable sperm with high Zinc (Zinc+/PI-). Likewise, Hoe+ events were gated into PI/PNA plot and then into Zinc histogram to detect viable-intact-acrosome with high Zinc (Zn+/PI-/PNA-). Data were analyzed by mixed model for repeated measures, Tukey-adjusted pairwise comparisons and Pearson’s correlation. Viable sperm at Day-1 (86.0±3.1; emmean±SM) and Day-4 (84.7±2.8) showed similar percentage of Zinc+ sperm, and both significantly higher compared to Day-7 (52.2±2.8). Also, viable-intact-acrosome sperm displayed similar percentage of Zinc+ sperm at Day-1 (93.8±3.4) and Day-4 (91.6±3.4) and both higher than sperm at Day-7 (64.5±3.1; P < 0.0001). Percentages of Zinc+ sperm were higher (P < 0.001) when viable sperm had intact acrosome within Day. Percentages of Zinc+ sperm strongly correlated to viable-intact-acrosome sperm (r=0.76, P < 0.0001) and total motility (r=0.65, P < 0.0001). We conclude that sperm having intact plasmatic membrane and acrosome display higher intracellular zinc content which is related to capacitated status, remaining non-capacitated until at least to Day-4 after collection in the tested extender.

ZnS@BSA Nanoclusters Potentiate Efficacy of Cancer Immunotherapy.

Although immunotherapy such as immune checkpoint inhibitors has shown promising efficacy in cancer treatment, the responsiveness among patients is relatively limited. Activation of the cyclic guanosine monophosphate-adenosine monophosphate synthase/interferon gene stimulator (cGAS/STING) signaling pathway to upregulate innate immunity has become an emerging strategy for enhancing tumor immunotherapy. Herein, ZnS@BSA (bovine serum albumin) nanoclusters synthesized via a self-assembly approach are reported, where the released zinc ions under acidic tumor microenvironment significantly enhance cGAS/STING signals. Meanwhile, intracellular zinc ions can produce reactive oxygen species, which is further facilitated by the generated H2 S gas from ZnS@BSA via specifically inhibiting catalase in hepatocellular carcinoma cells. It is found that the nanoclusters activate the cGAS/STING signals in mice, which promotes the infiltration of CD8+ T cells at the tumor site and cross-presentation of dendritic cells, leading to an improved immunotherapy efficacy against hepatocellular carcinoma.

Zinc-chelating postsynaptic density-95 N-terminus impairs its palmitoyl modification.

Chemical synaptic transmission represents the most sophisticated dynamic process and is highly regulated with optimized neurotransmitter balance. Imbalanced transmitters can lead to transmission impairments, for example, intracellular zinc accumulation is a hallmark of degenerating neurons. However, the underlying mechanisms remain elusive. Postsynaptic density protein-95 (PSD-95) is a primary postsynaptic membrane-associated protein and the major scaffolding component in the excitatory postsynaptic densities, which performs substantial functions in synaptic development and maturation. Its membrane association induced by palmitoylation contributes largely to its regulatory functions at postsynaptic sites. Unlike other structural domains in PSD-95, the N-terminal region (PSD-95NT) is flexible and interacts with various targets, which modulates its palmitoylation of two cysteines (C3/C5) and glutamate receptor distributions in postsynaptic densities. PSD-95NT contains a putative zinc-binding motif (C2H2) with undiscovered functions. This study is the first effort to investigate the interaction between Zn2+ and PSD-95NT. The NMR titration of 15 N-labeled PSD-95NT by ZnCl2 was performed and demonstrated Zn2+ binds to PSD-95NT with a binding affinity (Kd ) in the micromolar range. The zinc binding was confirmed by fluorescence and mutagenesis assays, indicating two cysteines and two histidines (H24, H28) are critical residues for the binding. These results suggested the concentration-dependent zinc binding is likely to influence PSD-95 palmitoylation since the binding site overlaps the palmitoylation sites, which was verified by the mimic PSD-95 palmitoyl modification and intact cell palmitoylation assays. This study reveals zinc as a novel modulator for PSD-95 postsynaptic membrane association by chelating its N-terminal region, indicative of its importance in postsynaptic signaling.