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One of the important mechanisms for the stabilization of soil organic carbon (SOC) is its spatial inaccessibility for microbial biodegradation within soil aggregates. However, little has been documented regarding soil aggregate stability with grassland degradation in the alpine region of the Qinghai‐Tibetan Plateau (QTP). In this study, we used physical and density fractions to elucidate the mechanisms of differences in SOC in non‐degraded and degraded grasslands of two grassland biomes, alpine meadow and alpine steppe in the QTP. There were considerable differences between non‐degraded and degraded grasslands for the soil physical and chemical properties, aggregate distribution and aggregate‐SOC content. The non‐degraded alpine meadow (AMND) had the largest value among all the alpine grasslands for the SOC content of the microaggregate fraction, with values of 31.83 g kg−1. The degraded grasslands showed significantly larger SOC content of macroaggregates than non‐degraded grasslands. The degraded alpine steppe (ASD) had the largest SOC content, with the value of 25.51 g kg−1. The aggregate distribution of the macroaggregate, microaggregate and free silt+clay fractions was consistent with the variation in SOC content of these three aggregate fractions. The fine intra‐aggregate particulate organic matter (fiPOM) content was significantly less in degraded grasslands, indicating that grassland degradation might have disrupted the fiPOM‐C. The increase in CO2 emissions was related to the destruction of soil aggregates in the alpine grasslands of the QTP. Highlights: Effects of alpine grassland degradation on aggregate‐associated SOC were investigated.Used soil aggregate fractions to evaluate the variation of aggregate‐associated SOC.Grassland degradation promoted the destruction of soil aggregates in the alpine grasslands of the QTP.Destruction of soil aggregates promoted CO2 emissions in the degraded alpine grasslands of the QTP. [ABSTRACT FROM AUTHOR]

Abstract Background Orthologs (genes that have diverged after a speciation event) tend to have similar function, and so their prediction has become an important component of comparative genomics and genome annotation. The gold standard phylogenetic analysis approach of comparing available organismal phylogeny to gene phylogeny is not easily automated for genome-wide analysis; therefore, ortholog prediction for large genome-scale datasets is typically performed using a reciprocal-best-BLAST-hits (RBH) approach. One problem with RBH is that it will incorrectly predict a paralog as an ortholog when incomplete genome sequences or gene loss is involved. In addition, there is an increasing interest in identifying orthologs most likely to have retained similar function. Results To address these issues, we present here a high-throughput computational method named Ortholuge that further evaluates previously predicted orthologs (including those predicted using an RBH-based approach) – identifying which orthologs most closely reflect species divergence and may more likely have similar function. Ortholuge analyzes phylogenetic distance ratios involving two comparison species and an outgroup species, noting cases where relative gene divergence is atypical. It also identifies some cases of gene duplication after species divergence. Through simulations of incomplete genome data/gene loss, we show that the vast majority of genes falsely predicted as orthologs by an RBH-based method can be identified. Ortholuge was then used to estimate the number of false-positives (predominantly paralogs) in selected RBH-predicted ortholog datasets, identifying approximately 10% paralogs in a eukaryotic data set (mouse-rat comparison) and 5% in a bacterial data set (Pseudomonas putida – Pseudomonas syringae species comparison). Higher quality (more precise) datasets of orthologs, which we term "ssd-orthologs" (supporting-species-divergence-orthologs), were also constructed. These datasets, as well as Ortholuge software that may be used to characterize other species' datasets, are available at http://www.pathogenomics.ca/ortholuge/ (software under GNU General Public License). Conclusion The Ortholuge method reported here appears to significantly improve the specificity (precision) of high-throughput ortholog prediction for both bacterial and eukaryotic species. This method, and its associated software, will aid those performing various comparative genomics-based analyses, such as the prediction of conserved regulatory elements upstream of orthologous genes.

This paper investigates delivery of encapsulated drug from poly lactic-co-glycolic micro-/nano-particles. Experimental data collected from about 50 papers are analyzed by machine learning algorithms including linear regression, principal component analysis, Gaussian process regression, and artificial neural networks. The focus is to understand the effect of drug solubility, drug molecular weight, particle size, and pH-value of the release matrix/environment on drug release profiles. The results obtained from machine learning is then used as guidelines for designing new in vitro experiments to examine dependence of drug release profiles on those four factors. It is interesting to see that indeed the results of the new in vitro experiments are in basic agreement with the results obtained from machine learning., (© 2025. The Author(s).)

To address the shortcoming of Polyvinyl alcohol (PVA) fibers for food or medical packaging materials including low mechanical strength and poor water resistance, lignin (LN) was used as raw material, acetone/H 2 O as solvent to self-assemble into lignin nanoparticles (LNP) by adverse solvent precipitation approach, and then PVA/LNP composite fibers with different LNP contents were fabricated successfully by wet and dry spinning. Herein, vast hydrophilic hydroxyl groups in PVA decreased owing to the hydrogen bond between LN and PVA, Especially, with only 0.5 wt% loading of LNP into the PVA/LNP fibers, the diameter was 94.4 dtex, tensile strength was 10.1 cN/dtex (1279.8 MPa), initial modulus was 94.7 cN/dtex (12.0 GPa), the crystallinity was 56.7 %, the orientation was 97.1 %, and water contact angle was 103.1°. Compared with pure PVA fibers, the tensile strength of PVA/LNP-0.5 fibers was increased by 44.2 % and the contact angle was increased 37°. This work provides novel insights into obtaining lignin-reinforced PVA composite fibers with strong mechanical properties and excellent water resistance properties, indicating the potential of the PVA/LNP fibers for food or medical packaging application., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Exometabolomics is an approach to assess how microorganisms alter, or react to their environments through the depletion and production of metabolites. It allows the examination of how soil microbes transform the small molecule metabolites within their environment, which can be used to study resource competition and cross-feeding. This approach is most powerful when used with defined media that enable tracking of all metabolites. However, microbial growth media have traditionally been developed for the isolation and growth of microorganisms but not metabolite utilization profiling through Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Here, we describe the construction of a defined medium, the Northen Lab Defined Medium (NLDM), that not only supports the growth of diverse soil bacteria but also is defined and therefore suited for exometabolomic experiments. Metabolites included in NLDM were selected based on their presence in R2A medium and soil, elemental stoichiometry requirements, as well as knowledge of metabolite usage by different bacteria. We found that NLDM supported the growth of 108 of the 110 phylogenetically diverse (spanning 36 different families) soil bacterial isolates tested and all of its metabolites were trackable through LC-MS/MS analysis. These results demonstrate the viability and utility of the constructed NLDM medium for growing and characterizing diverse microbial isolates and communities., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 de Raad, Li, Kuehl, Andeer, Kosina, Hendrickson, Saichek, Golini, Han, Wang, Bowen, Deutschbauer, Arkin, Chakraborty and Northen.)

Stroke is the third leading cause of death in the United States and the leading cause of adult disability. Despite enormous research efforts including many clinical trials, tissue plasminogen activator (tPA) remains the only FDA-approved treatment for acute ischemic stroke. Unfortunately, only 1-3% of stroke patients in the US receive this therapy because of the narrow time window and severe side effects for using tPA. The most deadly and damaging side effect is the risk of intracranial bleeding or hemorrhage. For that reason, the dose of tPA and its overall administration are under tight control, which may compromise the effect of thrombolysis. Studies have been focused on improving the effectiveness of tPA for higher rate of reperfusion, and the safety for less adverse bleeding episode. We studied how metal ions (zinc & iron) affect tPA-induced thrombolysis in vitro and in vivo, and proposed a method to improve the rate of thrombolysis. The amount of hemoglobin in the blood clot lysis was measured by a spectrophotometer. The tPA-induced thrombolysis was measured in vivo in femoral artery. Our results showed that Zn 2+ , Fe 3+ and Fe 2+ inhibited tPA-induced thrombolysis, with Zn 2+ and Fe 2+ being the most effective. Metal ion chelating agent EDTA when it was co-applied with tPA significantly enhanced the tPA-induced thrombolysis. The chelation alone did not have noticeable thrombolytic effect. In in vivo study of tPA-induced thrombosis following femoral artery thrombosis, the co-application of tPA and EDTA achieved significant higher rate of reperfusion than that by tPA treatment alone, suggesting that ion chelation facilitates tPA-induced thrombolysis and potentially improves the safety of tPA application by reducing the necessary dose of tPA application. Our results suggest that the co-application of a chelator and tPA improves the efficacy and, potentially, safety of tPA application, by reducing the necessary dose of tPA for thrombolysis., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

SARS-CoV-2 is a pandemic coronavirus that causes severe respiratory disease (COVID-19) in humans and is responsible for millions of deaths around the world since early 2020. The virus affects the human respiratory cells through its spike (S) proteins located at the outer shell. To monitor the rapid spreading of SARS-CoV-2 and to reduce the deaths from the COVID-19, early detection of SARS-CoV-2 is of utmost necessity. This report describes a flexible colorimetric biosensor capable of detecting the S protein of SARS-CoV-2. The colorimetric biosensor is made of polyurethane (PU)-polydiacetylene (PDA) nanofiber composite that was chemically functionalized to create a binding site for the receptor molecule-nucleocapsid antibody (anti-N) protein of SARS-CoV-2. After the anti-N protein conjugation to the functionalized PDA fibers, the PU-PDA-NHS-anti fiber was able to detect the S protein of SARS-CoV-2 at room temperature via a colorimetric transition from blue to red. The PU-PDA nanofiber-based biosensors are flexible and lightweight and do not require a power supply such as a battery when the colorimetric detection to S protein occurs, suggesting a sensing platform of wearable devices and personal protective equipment such as face masks and medical gowns for real-time monitoring of virus contraction and contamination. The wearable biosensors could significantly power mass surveillance technologies to fight against the COVID-19 pandemic., Supplementary Information: The online version contains supplementary material available at 10.1007/s44164-022-00022-z., Competing Interests: Conflict of interestThe authors declare no competing interests., (© The Author(s), under exclusive licence to Springer Nature Switzerland AG 2022.)

Endophytic nitrogen-fixing (diazotrophic) bacteria are essential members of the microbiome of switchgrass ( Panicum virgatum ), considered to be an important commodity crop in bioenergy production. While endophytic diazotrophs are known to provide fixed atmospheric nitrogen to their host plant, there are many other plant growth-promoting (PGP) capabilities of these organisms to be demonstrated. The diversity of PGP traits across different taxa of switchgrass-colonizing endophytes is understudied, yet critical for understanding endophytic function and improving cultivation methods of important commodity crops. Here, we present the isolation and characterization of three diazotrophic endophytes: Azospirillum agricola R1C, Klebsiella variicola F10Cl, and Raoultella terrigena R1Gly. Strains R1C and F10Cl were isolated from switchgrass and strain R1Gly, while isolated from tobacco, is demonstrated herein to colonize switchgrass. Each strain exhibited highly diverse genomic and phenotypic PGP capabilities. Strain F10Cl and R1Gly demonstrated the highest functional similarity, suggesting that, while endophyte community structure may vary widely based on host species, differences in functional diversity are not a clearly delineated. The results of this study advance our understanding of diazotrophic endophyte diversity, which will allow us to design robust strategies to improve cultivation methods of many economically important commodity crops., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gushgari-Doyle, Schicklberger, Li, Walker and Chakraborty.)

Environmental changes can stress and alter biology at the molecular and cellular level. For example, metal-protein interaction is a classic physic and biological property of nature, which is fundamentally influenced by acidity. Here, we report a unique cellular reprogramming phenomenon in that a brief strong acid treatment induced the expression of pluripotent stem cell (PSC) markers. We used strong acid to briefly challenge mix-cultured gastric cells, and then subcultured survived cells in a normal cell culture medium. We found that survival acid-treated cells expressed PSC markers detected by commonly used pluripotent antibodies such as SSEA-4 and Oct4. In addition, we observed that the survived cells from the acid challenge grew faster during the second and third weeks of subculture and had a relative short doubling time (DT) than the controls. PSC marker-labeled 'older' cells also presented immature cell-like morphology with some having marker Oct4 in the nucleus. Finally, the expression of the markers appeared to be sensitive to metal ion chelation. Removal of the metals during a brief acid treatment reduced pluripotent marker-positive cells, suggesting the dissociation of metals from metal-binding proteins may be a factor involved in the induction of stem cell markers. Our findings reveal that somatic cells appear to possess a plasticity feature to express pluripotent marker proteins or to select cell subpopulations that express pluripotent marker proteins when cells are transiently exposed to strong acid. It opens new directions for understanding conserved regulatory mechanisms involved in cellular survival under stressful stimulation.

Zinc (Zn 2+ ) is stored in the nucleus, endoplasmic reticulum (ER), Golgi apparatus, mitochondria, lysosomes, and zinc-binding proteins. The acidity of the microenvironment affects the binding between zinc and proteins in which zinc become free or loosely bound. In this study, when cells were treated with an acidic medium, we started seeing free zinc 'hot spots' or zincosomes where we found bright zinc fluorescence. The rising free zinc quickly across whole cells with both intensity and distribution were pH-dependent. Interestingly, the nucleus was more sensitive to acidic treatment as the increase of nuclear zinc was faster and higher than the increase of cytosolic zinc. In addition, we re-cultured strong acid-challenged cells in a normal medium. Comparing to the control, these cells exhibited multiple zinc 'hot spots' beside the nucleus, suggesting that free zinc became more extensively distributed. To investigate further the function of zinc in cell shaping and morphological changes, we categorized strong acid-challenged cells into different shapes and found that the proportion of each cell shape had changed after the acid challenge. These acid-induced changes of the cell shape percentage were partially reversed by the reduction of zinc, suggesting that zinc participated in directing the cell shapes and morphologies during cell growth. Our findings reveal that acidic pH affects the dynamics of cellular zinc by making zinc more accessible to cellular compartments and zinc-binding proteins, which provided new insights into understanding the cellular behavior and the function of zinc in it., Competing Interests: None., (IJPPP Copyright © 2021.)

Zinc (Zn 2+ ) is important in cellular processes. In the cell, free zinc is tightly regulated and found in minuscule amounts. However, in an unhealthy cellular environment, such as hypoxia, zinc increases in the cell and zinc overload may occur. Studies have shown that zinc overload causes cellular and mitochondrial stress. Mitochondrial stress affects mitochondrial morphology. In normal cells, mitochondrial morphology resembles a long, tubular shape. In unhealthy cells, mitochondrial morphology resembles fragmented, circular shape. To address whether zinc overload contributes directly to the abnormal changes of mitochondrial morphology, we imaged and analyzed mitochondria that were treated with the application of exogenous zinc. In the first part of the study, exogenous zinc was applied to HeLa cells at 1 µM, 10 µM, 50 µM, 100 µM, or 200 µM zinc chloride along with 10 µM pyrithione. Mitochondrial morphology was analyzed with Mito-Morphology micro in ImageJ. Mitochondrial morphology changed from a healthy tubular shape to an unhealthy circular shape and fragmentation. Mitochondrial morphology changes were observed in a dose-dependent fashion. The second part of the study involved applying the metal ion chelator TPEN after applying 50 µM zinc chloride along with 10 µM pyrithione. TPEN reduced zinc-induced abnormal mitochondrial morphology after zinc treatment. This present study supports that zinc overload may cause morphology changes induced by mitochondrial stress that may lead to cell death., Competing Interests: None., (IJPPP Copyright © 2021.)

Biomaterials to facilitate the restoration of cardiac tissue is of emerging importance. While there are many aspects to consider in the design of biomaterials, mechanical properties can be of particular importance in this dynamically remodeling tissue. This review focuses on one specific processing method, electrospinning, that is employed to generate materials with a fibrous microstructure that can be combined with material properties to achieve the desired mechanical behavior. Current methods used to fabricate mechanically relevant micro-/nanofibrous scaffolds, in vivo studies using these scaffolds as therapeutics, and common techniques to characterize the mechanical properties of the scaffolds are covered. We also discuss the discrepancies in the reported elastic modulus for physiological and pathological myocardium in the literature, as well as the emerging area of in vitro mechanobiology studies to investigate the mechanical regulation in cardiac tissue engineering. Lastly, future perspectives and recommendations are offered in order to enhance the understanding of cardiac mechanobiology and foster therapeutic development in myocardial regenerative medicine.

A subset of presynaptic glutamatergic vesicles in the brain co-releases zinc (Zn 2+ ) with glutamate into the synapse. However, the role of synaptically released Zn 2+ is still under investigation. Here, we studied the effect of Zn 2+ on glutamate homeostasis by measuring the evoked extracellular glutamate level (EGL) and the probability of evoked action potential (P EAP ) at the Zn 2+ -containing or zincergic mossy fiber-CA3 synapses of the rat hippocampus. We found that the application of Zn 2+ (ZnCl 2 ) exerted bidirectional effects on both EGL and P EAP : facilitatory at low concentration (~1 µM) while repressive at high concentration (~50 µM). To determine the action of endogenous Zn 2+ , we also used extracellular Zn 2+ chelator to remove the synaptically released Zn 2+ . Zn 2+ chelation reduced both EGL and P EAP , suggesting that endogenous Zn 2+ has mainly a facilitative role in glutamate secretion on physiological condition. We revealed that calcium/calmodulin-dependent protein kinase II was integral to the mechanism by which Zn 2+ facilitated the release of glutamate. Moreover, a glutamate transporter was the molecular entity for the action of Zn 2+ on glutamate uptake by which Zn 2+ decreases glutamate availability. Taken together, we show a novel action of Zn 2+ , which is to biphasically regulate glutamate homeostasis via Zn 2+ concentration-dependent synaptic facilitation and depression. Thus, co-released Zn 2+ is physiologically important for enhancing weak stimulation, but potentially mitigates excessive stimulation to keep synaptic transmission within optimal physiological range., (© 2020 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

In recent years, Janus materials have become a research hotspot in the field of materials science; however, fabricating inorganic Janus-like nanofibers (NFs) is still a challenge. Herein, we report novel ZnO/NiO Janus-like NFs with efficient photocatalytic performance via an electrospinning method followed by calcination treatment. The morphology, structure, chemical composition and crystallinity of ZnO/NiO Janus-like NFs were studied in detail via SEM, TEM, HRTEM, EDS, FT-IR, XPS and XRD, indicating that the NFs had a perfect Janus-like structure composed of ZnO and NiO. A series of photocatalytic experiments were carried out in aqueous organic dye solutions under 365 nm UV radiation for 1 h, with the degradation rate of malachite green able to reach 96%, proving that the NFs have great potential in the field of organic dye degradation. Furthermore, a reasonable catalytic mechanism for the ZnO/NiO Janus-like NFs was proposed, which was discussed from the view of electron-hole pairs and p-n junctions. In short, the method in our work is expected to become a new way of effectively preparing functional inorganic Janus-like NFs.

Although Janus films of different compositions have been commonly utilized to develop moisture actuators due to the different capabilities of swelling in materials, a sole material with a distinct structural design is also able to provide moisture-actuation. In this study, we simply used graphene oxide (GO) to fabricate a sole GO film with an asymmetric structure which consisted of a wavy layer and a smooth layer. Due to the asymmetric structure and excellent hygroscopicity of the GO material, the asymmetric graphene oxide (AGO) film (2.5 × 0.5 cm2) was responsive to moisture and showed a maximum bending angle change of ≈1800° as the relative humidity (RH) changed. Compared with other reports about moisture actuators, the AGO film exhibited a superior bending capability. Furthermore, we propose a novel mechanism for moisture actuation of the AGO film based on our detailed observations, and a wavy structure has been introduced for showing great potential in bending deformation. Finally, the AGO film was used as a grabber to grab a leaf and it exhibited good capability to twine around a plastic rod. This work provides a novel pathway for the development of moisture-responsive materials for potential applications in robotics, artificial muscles and switches.

Protein kinase C delta (PKCδ) is a Ser/Thr-specific kinase involved in many fundamental cellular processes including growth, differentiation and apoptosis. PKCδ is expressed ubiquitously in all known cell types, and can be activated by diacylglycerol, phorbol esters and other kinases. Multiple lines of evidence have indicated that the mode of activation greatly influences the role PKCδ plays in cellular function. Divalent metal ions, such as zinc are released as a response to cellular stress and injury, often resulting in oxidative damage and cell death. In this study, we evaluate the effect increased concentrations of intracellular zinc has on the phosphorylation state and subcellular localization of PKCδ. More specifically, we demonstrate that intracellular zinc inhibits the phosphorylation of PKCδ at Thr 505 in a concentration-dependent manner and facilitates the translocation of PKCδ from the cytosol to the Golgi complex. Analysis of a PKCδ structural model revealed a potential His-Cys3 zinc-binding domain adjacent to residue Thr 505 and suggests that interaction with a Zn 2+ ion may preclude phosphorylation at this site. This study establishes zinc as a potent modulator of PKCδ function and suggests a novel mechanism by which PKCδ is able to "sense" changes in the concentration of intracellular zinc. These findings illuminate a new paradigm of metal ion-protein interaction that may have significant implications on a broad spectrum of cellular processes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cardiovascular disorder occurs when a local blood clot obstructs an artery or a vein to its surround organs, causing related tissues to lose function and die. It is one of the leading causes of mortality and a major cause of disability. The effect of thrombolysis induced by injecting intravenous thrombolytic agents is critical for reducing tissue damages. Streptokinase (SK) is a widely used thrombolytic agent in the treatment of thromboembolism in the blood vessels. A high unit of streptokinase is used in thrombolytic therapies for thrombotic disorders and could improve tissue reperfusion. It is a potent plasminogen activator. However, safety concerns for the usage of a high unit of streptokinase have been raised for the hemorrhagic transformation. In the present study, we studied how zinc would affect streptokinase-induced thrombolysis in vitro , and proposed a strategy to improve streptokinase's effectiveness in promoting thrombolysis. The mice whole blood was used to form the blood clot in vitro by incubating with calcium at 37°C for 30 minutes. Streptokinase was used for inducing thrombolysis measured with the spectrophotometer. Zinc and its chelator, Ca-EDTA, were applied with streptokinase, respectively. Results showed that the co-application zinc inhibited the thrombolytic effect of streptokinase in a dose-dependent manner. Zinc chelator, Ca-EDTA, significantly increased the effect of streptokinase-induced thrombolysis. Our results suggest that zinc chelation improved the efficiency of streptokinase in thrombolysis. The results may have a significant clinical implication by potentially reducing the adverse effect of streptokinase application., Competing Interests: None.

Polydiacetylenes (PDAs) are conjugative polymers that demonstrate color changes as a response to an external stimulus. In this study, 10,12-pentacosadiynoic acid (PCDA) was mixed with a supporting polymer including poly(ethylene oxide) (PEO) and polyurethane (PU), and the mixture solution was electrospun to construct fiber composites. The electrospun fibers were then photopolymerized using UV irradiation to produce PEO-PDA and PU-PDA nanofiber mats with a fiber diameter ranging from 130 nm to 2.5 μm. The morphologies of both PEO-PDA and PU-PDA nanofibers were dependent on electrospinning parameters such as the ratio of PCDA to PEO or PCDA to PU and the total polymer concentrations. Scanning electron microscopy images showed beaded fibers of PEO-PDA and PU-PDA at 2 and 18 w/v % concentrations, respectively. Smooth fibers were found when the solvent concentration was increased to 3.75 w/v % in PEO-PDA and 25 w/v % in PU-PDA fibers. Both PEO-PDA and PU-PDA nanofiber composites demonstrated excellent colorimetric responses to the presence of Escherichia coli ATCC25922 bacterial cells and the changes in pH as external stimuli. The nanofibers underwent a rapid colorimetric response when exposed directly to E. coli ATCC25922 grown on Luria-Bertani agar. The comparison between the PEO-PDA and PU-PDA suggested that the combination of PEO and PDA is favorable because it provides a sensitive response to the presence of E. coli . The results were compared with samples of a PDA polymer in the absence of a matrix polymer. The colorimetric response was similar when the PDA polymer and the PDA nanofiber composites were exposed to pH changes, and the color change was found to occur at pH 10 and enhanced at pH 11-13. The PDA-containing nanofiber composites showed stronger colorimetric responses than those of the PDA polymer only, suggesting their potential as biosensors and chemosensors., Competing Interests: The authors declare no competing financial interest.

Both zinc (Zn 2+ ) and reactive oxygen species (ROS) have been shown to accumulate during hypoxic-ischemic stress and play important roles in pathological processes. To understand the cross talk between the two of them, here we studied Zn 2+ and ROS accumulation by employing fluorescent probes in HeLa cells to further the understanding of the cause and effect relationship of these two important cellular signaling systems during chemical-ischemia, stimulated by oxygen and glucose deprivation (OGD). We observed two Zn 2+ rises that were divided into four phases in the course of 30 min of OGD. The first Zn 2+ rise was a transient, which was followed by a latent phase during which Zn 2+ levels recovered; however, levels remained above a basal level in most cells. The final phase was the second Zn 2+ rise, which reached a sustained plateau called Zn 2+ overload. Zn 2+ rises were not observed when Zn 2+ was removed by TPEN (a Zn 2+ chelator) or thapsigargin (depleting Zn 2+ from intracellular stores) treatment, indicating that Zn 2+ was from intracellular storage. Damaging mitochondria with FCCP significantly reduced the second Zn 2+ rise, indicating that the mitochondrial Zn 2+ accumulation contributes to Zn 2+ overload. We also detected two OGD-induced ROS rises. Two Zn 2+ rises preceded two ROS rises. Removal of Zn 2+ reduced or delayed OGD- and FCCP-induced ROS generation, indicating that Zn 2+ contributes to mitochondrial ROS generation. There was a Zn 2+ -induced increase in the functional component of NADPH oxidase, p47 phox , thus suggesting that NADPH oxidase may mediate Zn 2+ -induced ROS accumulation. We suggest a new mechanism of cross talk between Zn 2+ and mitochondrial ROS through positive feedback processes that eventually causes excessive free Zn 2+ and ROS accumulations during the course of ischemic stress., (Copyright © 2017 the American Physiological Society.)

Zinc (Zn(2+)) is required for numerous cellular functions. As such, the homeostasis and distribution of intracellular zinc can influence cellular metabolism and signaling. However, the exact distribution of free zinc within live cells remains elusive. Previously we showed the release of zinc from thapsigargin/IP3-sensitive endoplasmic reticulum (ER) storage in cortical neurons. In the present study, we investigated if other cellular organelles also contain free chelatable zinc and function as organelle storage for zinc. To identify free zinc within the organelles, live cells were co-stained with Zinpyr-1, a zinc fluorescent dye, and organelle-specific fluorescent dyes (MitoFluor Red 589: mitochondria; ER Tracker Red: endoplasmic reticulum; BODIPY TR ceramide: Golgi apparatus; Syto Red 64: nucleus). We examined organelles that represent potential storing sites for intracellular zinc. We showed that zinc fluorescence staining was co-localized with MitoFluor Red 589, ER Tracker Red, and BODIPY TR ceramide respectively, suggesting the presence of free zinc in mitochondria, endoplasmic reticulum, and the Golgi apparatus. On the other hand, cytosol and nucleus had nearly no detectable zinc fluorescence. It is known that nucleus contains high amount of zinc binding proteins that have high zinc binding affinity. The absence of zinc fluorescence suggests that there is little free zinc in these two regions. It also indicates that the zinc fluorescence detected in mitochondria, ER and Golgi apparatus represents free chelatable zinc. Taken together, our results support that these organelles are potential zinc storing organelles during cellular zinc homeostasis.

Mitochondrial reactive oxygen species (ROS) are known to accumulate during chemical hypoxia, causing adverse effects on cell function and survival. Recent studies show important role zinc accumulation plays in dysfunction associated with hypoxia. It is well known that ROS accumulation also plays a major role in cellular damage by hypoxia. In this study, fluorescent imaging and pharmacological methods were used in live HeLa cells to determine role of zinc in initial ROS accumulation in mitochondria during chemical hypoxia (oxygen glucose depravation with 4 mM sodium dithionite). Accumulation of both was observed as a very rapid phenomenon with initial rapid zinc increase (zinc wave) within 60 seconds of hypoxia onset and ROS increase within 4.5 minutes. Zinc chelation with TPEN removed the initial zinc wave which in turn abolished ROS accumulation. Influx of exogenous zinc induced rapid ROS accumulation. Inhibition of NADPH oxidase with apocynin, a NADPH oxidase inhibitor, showed significant and prolonged reduction in zinc induced ROS accumulation. We proposed a novel mechanism of intracellular zinc increase that activates NADPH oxidase which in turn triggers mitochondrial ROS production.

Polydiacetylene (PDA) is an attractive conjugated material for use in biosensors due to its unique characteristic of undergoing a blue-to-red color change in response to external stimuli. 10,12-Pentacosadiynoic acid (PCDA) and poly (ethylene oxide) (PEO) were used in this study to develop fiber composites via an electrospinning method at various mass ratios of PEO to PCDA, solution concentrations, and injection speeds. The PEO-PDA fibers in blue phase were obtained via photo-polymerization upon UV-light irritation. High mass ratios of PEO to PCDA, low polymer concentrations of spinning solution, and low injection speeds promoted fine fibers with small diameters and smooth surfaces. The colorimetric transition of the fibers was investigated when the fibers were heated at temperatures ranging from 25 °C to 120 °C. A color switch from blue to red in the fibers was observed when the fibers were heated at temperatures greater than 60 °C. The color transition was more sensitive in the fibers made with a low mass ratio of PEO to PCDA due to high fraction of PDA in the fibers. The large diameter fibers also promoted the color switch due to high reflectance area in the fibers. All of the fibers were analyzed using Fourier transform infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC) and compared before and after the color change occurred. The colorimetric transitional mechanism is proposed to occur due to conformational changes in the PDA macromolecules.

Intracellular zinc release and the generation of reactive oxygen species (ROS) have been reported to be common ingredients in numerous toxic signaling mechanisms in neurons. A key source for intracellular zinc release is its liberation from metallothionein-III (MT-III). MT-III binds and regulates intracellular zinc levels under physiological conditions, but the zinc-binding thiols readily react with certain ROS and reactive nitrogen species (RNS) to result in intracellular zinc liberation. Liberated zinc induces ROS and RNS generation by multiple mechanisms, including the induction of mitochondrial ROS production, and also promotes ROS formation outside the mitochondria by interaction with the enzymes NADPH oxidase and 12-lipoxygenase. Of particular relevance to neuronal injury in the context of ischemia and prolonged seizures, the positive feedback cycle between ROS/RNS generation and increasing zinc liberation will be examined.

Thrombotic cerebral ischemia is one of the leading causes of mortality and chronic disability. Animal models provide an essential tool for understanding the complex cellular and molecular pathophysiology of ischemia and for improving treatment and testing novel neuroprotective drugs in the preclinical setting. In this study, we tested zebrafish as a novel model for thrombotic ischemic brain damage. Zebrafish were intraperitoneally injected with Rose Bengal and light exposure was directed onto the optic tectum region of the brain to induce photothrombosis. After full recovery from anesthesia, zebrafish consistently exhibited abnormal swimming patterns, indicating brain injury from the procedure. The staining of 2,3,5-triphenyltetrazolium chloride (TTC) 24 h after the treatment showed lack of staining of the exposed area of the brain, which further confirmed the ischemic injury. Application of Activase®-tPA improved viability of the brain. The tPA treatment also reduced the occurrence of moving disability as well as the mortality rate, demonstrating that the zebrafish model not only showed focal ischemic injury but also responded well to tPA therapy. Our results suggest that the current photothrombotic method induced focal ischemia in zebrafish and produced consistent brain damage that can be measured by behavioral changes and quantified by histological staining.

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor with strong neuroprotective properties. However, it has limited capacity to cross the blood-brain barrier and thus potentially limiting its protective capacity. Recent studies demonstrated that intranasal drug administration is a promising way in delivering neuroprotective agents to the central nervous system. The current study therefore aimed at determining whether intranasal administration of G-CSF increases its delivery to the brain and its neuroprotective effect against ischemic brain injury. Transient focal cerebral ischemia in rat was induced with middle cerebral artery occlusion. Our resulted showed that intranasal administration is 8-12 times more effective than subcutaneous injection in delivering G-CSF to cerebrospinal fluid and brain parenchyma. Intranasal delivery enhanced the protective effects of G-CSF against ischemic injury in rats, indicated by decreased infarct volume and increased recovery of neurological function. The neuroprotective mechanisms of G-CSF involved enhanced upregulation of HO-1 and reduced calcium overload following ischemia. Intranasal G-CSF application also promoted angiogenesis and neurogenesis following brain ischemia. Taken together, G-CSF is a legitimate neuroprotective agent and intranasal administration of G-CSF is more effective in delivery and neuroprotection and could be a practical approach in clinic.

It is well known that zinc (Zn(2+)) is required for the process of insulin biosynthesis and the maturation of insulin secretory granules in pancreatic beta (β)-cells, and that changes in Zn(2+) levels in the pancreas have been found to be associated with diabetes. Glucose-stimulation causes a rapid co-secretion of Zn(2+) and insulin with similar kinetics. However, we do not know whether Zn(2+) regulates insulin availability and secretion. Here we investigated the effect of Zn(2+) on glucose-stimulated insulin secretion (GSIS) in isolated mouse pancreatic islets. Whereas Zn(2+) alone (control) had no effect on the basal secretion of insulin, it significantly inhibited GSIS. The application of CaEDTA, by removing the secreted Zn(2+) from the extracellular milieu of the islets, resulted in significantly increased GSIS, suggesting an overall inhibitory role of secreted Zn(2+) on GSIS. The inhibitory action of Zn(2+) was mostly mediated through the activities of KATP/Ca(2+) channels. Furthermore, during brief paired-pulse glucose-stimulated Zn(2+) secretion (GSZS), Zn(2+) secretion following the second pulse was significantly attenuated, probably by the secreted endogenous Zn(2+) after the first pulse. Such an inhibition on Zn(2+) secretion following the second pulse was completely reversed by Zn(2+) chelation, suggesting a negative feedback mechanism, in which the initial glucose-stimulated Zn(2+) release inhibits subsequent Zn(2+) secretion, subsequently inhibiting insulin co-secretion as well. Taken together, these data suggest a negative feedback mechanism on GSZS and GSIS by Zn(2+) secreted from β-cells, and the co-secreted Zn(2+) may act as an autocrine inhibitory modulator.

Hydrogen-rich water (HRW) has anti-oxidant activities, and it exerts neuroprotective effects during ischemia-reperfusion brain injury. Parvalbumin and hippocalcin are two calcium buffering proteins, which are involved in neuronal differentiation, maturation and apoptosis. The aim of this study was to investigate whether HRW could moderate parvalbumin and hippocalcin expression during ischemic brain injury and glutamate toxicity-induced neuronal cell death. Focal brain ischemia was induced in male Sprague-Dawley rats by middle cerebral artery occlusion (MCAO). Rats were treated with H2O or HRW (6 ml/kg per rat) before and after MCAO, and cerebral cortical tissues were collected 1, 7 and 14 days after MCAO. Based on our results, HRW treatment was able to reduce brain infarct volume and improve neurological function following ischemic brain injury. In addition, HRW prevented the ischemia-induced reduction of parvalbumin and hippocalcin levels in vivo and also reduced the glutamate toxicity-induced death of neurons, including the dose-dependent reduction of glutamate toxicity-associated proteins in vitro. Moreover, HRW attenuated the glutamate toxicity-induced elevate in intracellular Ca(2+) levels. All these results suggest that HRW could protect against ischemic brain injury and that the maintenance of parvalbumin and hippocalcin levels by HRW during ischemic brain injury might contribute to the neuroprotective effects against neuron damage., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Understanding nanoparticle-surface adhesion is necessary to develop inert tracers for subsurface applications. Here we show that nanoparticles with neutral surface charge may make the best subsurface tracers, and that it may be possible to used SiO2 nanoparticle retention to measure the fraction of solid surface that has positive charge. We show that silica nanoparticles dispersed in NaCl electrolyte solutions are increasingly retained in calcium carbonate (calcite) sand-packed columns as the solution ionic strength increases, but are not retained if they are injected in pure water or Na2SO4 electrolyte solutions. The particles retained in the NaCl experiments are released when the column is flushed with pure water or Na2SO4 solution. AFM measurements on calcite immersed in NaCl solutions show the initial repulsion of a silica colloidal probe as the surface is approached is reduced as the solution ionic strength increases, and that at high ionic strengths it disappears entirely and only attraction remains. These AFM measurements and their interpretation with Derjaguin-Landau-Verwey-Overbeek (DLVO) theory shows the calcite surface charge is always negative for Na2SO4 solutions, but changes from negative to positive in a patchy fashion as the ionic strength of the NaCl solution increases. Since mixed-charge (patchy) surfaces may be common in the subsurface, nanoparticles with near-zero charge may make the best tracers., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Zinc (Zn2+) is an essential element crucial for growth and development, and also plays a role in cell signaling for cellular processes like cell division and apoptosis. In the mammalian pancreas, Zn2+ is essential for the correct processing, storage, secretion, and action of insulin in beta (β)-cells. Insulin is stored inside secretory vesicles or granules, where two Zn2+ ions coordinate six insulin monomers to form the hexameric-structure on which maturated insulin crystals are based. The total Zn2+ content of the mammalian pancreas is among the highest in the body, and Zn2+ concentration reach millimolar levels in the interior of the dense-core granule. Changes in Zn2+ levels in the pancreas have been found to be associated with diabetes. Hence, the relationship between co-stored Zn2+ and insulin undoubtedly is critical to normal β-cell function. The advances in the field of Zn2+ biology over the last decade have facilitated our understanding of Zn2+ trafficking, its intracellular distribution and its storage. When exocytosis of insulin occurs, insulin granules fuse with the β-cell plasma membrane and release their contents, i.e., insulin as well as substantial amount of free Zn2+, into the extracellular space and the local circulation. Studies increasingly indicate that secreted Zn2+ has autocrine or paracrine signaling in β-cells or the neighboring cells. This review discusses the Zn2+ homeostasis in β-cells with emphasis on the potential signaling role of Zn2+ to islet biology.

Diminished or inappropriate secretion of insulin is associated with type II diabetes. The cellular/molecular mechanism coupled with the regulation of insulin secretion is still under intense investigation. Divalent ion zinc (Zn(2+)) is co-packaged and co-secreted with insulin and is intimately involved in the process of insulin biosynthesis and the maturation of insulin secretory granules. The study reported here investigated glucose-stimulated zinc secretion (GSZS) and the effect of zinc on glucose-stimulated insulin secretion (GSIS) in the HIT-T15 pancreatic β-cell line. Zinc secretion was measured using a newly developed fluorescent zinc imaging approach, and the insulin secretion was measured using an enzyme-linked immunosorbent assay. There was apparent granular-like zinc staining in β-cells. The application of glucose induced detectable zinc secretion or GSZS. Like GSIS, GSZS was dependent on the glucose concentration (5-20 mm) and the presence of extracellular calcium. The application of a zinc chelator enhanced GSZS. When brief paired-pulse glucose stimulations, which involve the initial glucose stimulation followed by a second round of glucose stimulation, were applied, zinc secretion or GSZS that followed the first pulse was inhibited. This inhibition was reversed by zinc chelation, suggesting a feedback mechanism on GSZS by zinc secreted from β-cells. Finally, the application of zinc (50 μm) strongly inhibited GSIS as measured by enzyme-linked immunosorbent assay. The present study suggests that insulin secretion is regulated by co-secreted zinc that may act as an autocrine inhibitory modulator.

A study was conducted using zebrafish as a model of hypoxic brain injury to investigate the potential neuroprotective effects of zinc (Zn(2+)) chelation. The accumulation of intracellular Zn(2+) is a significant causal factor of the neuronal injury, and has been implicated in cell death followed by ischemic stroke. In this study, the zebrafish was placed in the hypoxia chamber with an extremely low level of dissolved oxygen (less than 0.8 mg/L), which is similar to the conditions in a complete global ischemic stroke. Approximately 50% of zebrafish died after a short period (≈11 min) of hypoxic treatment, suggesting that this is a responsive model system for use in evaluating treatments for hypoxic brain damage. The application of DEDTC reduced intracellular Zn(2+) accumulation and produced a concentration-dependent effect by increasing the survival rate of zebrafish. Zn(2+) chelation also enhanced zebrafish tolerance for hypoxia. When the brain damages were evaluated with TTC staining, the zebrafish that were treated with DEDTC in hypoxic treatment yielded the improvement of TTC staining that was similar to the healthy zebrafish brain. The results support that rising intracellular Zn(2+) plays a critical role in the neuronal damages, and demonstrate the protective effects of Zn(2+) chelation in hypoxic-ischemic brain injury in zebrafish.

Zinc (Zn(2+)) appears to be intimately involved in insulin metabolism since insulin secretion is correlated with zinc secretion in response to glucose stimulation, but little is known about the regulation of zinc homeostasis in pancreatic beta-cells. This study set out to identify the intracellular zinc transient by imaging free cytosolic zinc in HIT-T15 beta-cells with fluorescent zinc indicators. We observed that membrane depolarization by KCl (30-60 mM) was able to induce a rapid increase in cytosolic concentration of zinc. Multiple zinc transients of similar magnitude were elicited during repeated stimulations. The amplitude of zinc responses was not affected by the removal of extracellular calcium or zinc. However, the half-time of the rising slope was significantly slower after removing extracellular zinc with zinc chelator CaEDTA, suggesting that extracellular zinc affect the initial rising phase of zinc response. Glucose (10 mM) induced substantial and progressive increases in intracellular zinc concentration in a similar way as KCl, with variation in the onset and the duration of zinc mobilization. It is known that the depolarization of beta-cell membrane is coupled with the secretion of insulin. Rising intracellular zinc concentration may act as a critical signaling factor in insulin metabolism of pancreatic beta-cells.

Acute cerebral ischemia is one of the leading causes of mortality and chronic disability. Animal models provide an essential tool for understanding the complex cellular and molecular pathophysiology of hypoxic-ischemia and for testing novel neuroprotective drugs in the pre-clinical setting. In this study we tested zebrafish as a novel model for hypoxic-ischemic brain damage. We built an air-proof chamber where water inside had a low oxygen concentration (0.6-0.8 mg/L) proximate to complete hypoxia. Each zebrafish was placed individually in the hypoxia chamber and was subjected to hypoxia treatment until it became motionless, lying on its side on the bottom of the chamber (time to hypoxia = 679.52 ± 90 seconds, mean ± SD, n =23), followed by transferring into a recovery beaker. Overall, 60.87% of subjects did not recover from hypoxia while 39% survived. The size and distribution of brain injury were determined by triphenyltetrazolium chloride (TTC) staining. Bilateral, moderate to complete TTC decoloration or demarcation of the infarct after 10 minutes of hypoxic treatment was clearly visible in the optic tectum of the optic lobe. The size of the infarct expanded to the deep structure of the optic lobe with longer hypoxic treatments. The zebrafish that survived hypoxia experienced initial twitching followed by unbalanced erratic movements until they regained coordinated, balanced swimming ability. These data indicate that zebrafish are susceptible to hypoxic attack and suggest that the model we present in this study can be used as an alternative model to evaluate hypoxia-induced brain damage.

Zn(2+) ions are a critical component of cellular machinery. The ion is required for the function of many cell components crucial to survival, such as transcription factors, protein synthetic machinery, metabolic enzymes, hormone packaging, among other roles. In stark contrast to the cells' necessity for a sufficient Zn(2+) supply, an excess of free Zn(2+) is a situation that results in acute toxicity. Under normal conditions, free Zn(2+) levels in the cell are extremely low; whereas estimates of free Zn(2+) are in the subpicomolar range. In this way, the detection of elevated intracellular Zn(2+) can be exploited as a highly sensitive and specific signal to indicate neuronal dysfunction. We have shown that the relationship between intracellular Zn(2+) accumulation and the development of cellular injury/death to be ubiquitous among each of five tissue types tested; demonstrating the broad application and utility of the present technique.

Zinc oxide (ZnO) is an active ingredient in sunscreen owing to its properties of broadly filtering the ultraviolet (UV) light spectrum and it is used to protect against the carcinogenic and photodamaging effects of solar radiation on the skin. This study investigated the dissociation of zinc (Zn(2+) ) from ZnO in commercial sunscreens under ultraviolet type B light (UVB) irradiation and assessed the cytotoxicity of Zn(2+) accumulation in human epidermal keratinocytes (HEK). Using Zn(2+) fluorescent microscopy, we observed a significant increase in Zn(2+) when ZnO sunscreens were irradiated by UVB light. The amount of Zn(2+) increase was dependent on both the irradiation intensity as well as on the ZnO concentration. A reduction in cell viability as a function of ZnO concentration was observed with cytotoxic assays. In a real-time cytotoxicity assay using propidium iodide, the treatment of UVB-irradiated ZnO sunscreen caused a late- or delayed-type cytotoxicity in HEK. The addition of a Zn(2+) chelator provided a protective effect against cellular death in all assays. Furthermore, Zn(2+) was found to induce the production of reactive oxygen species (ROS) in HEK. Our data suggest that UVB irradiation produces an increase in Zn(2+) dissociation in ZnO sunscreen and, consequently, the accumulation of free or labile Zn(2+) from sunscreen causes cytotoxicity and oxidative stress., (© 2010 Wiley Periodicals, Inc.)

Ultraviolet (UV) radiation is known to cause oxidative stress, inflammation, DNA damage and apoptotic cell death; however, many details of these malign mechanism have yet to be elucidated. In this study, the exposure of adult human epidermal keratinocytes (HEKa) with UVB (>100 mJ/cm(2)) resulted in the significant increase of intracellular zinc that was released from its storage and was detected by fluorescent zinc indicators. Toxicity testing revealed that UVB-induced zinc release in HEKa is associated with HEKa cell death. Cells that showed elevated intracellular zinc fluorescence upon UVB exposure were also stained by propidium iodide (PI), a traditional viability indicator whose fluorescent signal is as a result of its intercalating with DNA fragments and is unaffected by zinc concentration, showing significant colocalization [Pearson's correlation coefficients r=0.956 (n=6)]. The cytotoxicity of zinc was also determined by an MTT assay after applying the exogenous zinc (ZnCl2) along with its ionophore pyrithione (20 microM) into HEKa culture medium. A significant reduction in cell viability as a function of both zinc concentration and exposure time was observed. The treatments of 1, 10 and 100 microM ZnCl2 with pyrithione demonstrated 2.3, 60 and 84% cell deaths, respectively (control 0.5%) after 30 min. ZnCl2 (100 microM) was also found to induce complete HEKa death after 1 h. Thus, the present study demonstrates that UVB irradiation-induced increased zinc is detrimental to HEKa viability, and zinc may be a necessary step in UVB-induced cell death signaling pathways.

Background: Changes in ionic concentration have a fundamental effect on numerous physiological processes. For example, IP3-gated thapsigargin sensitive intracellular calcium (Ca2+) storage provides a source of the ion for many cellular signaling events. Less is known about the dynamics of other intracellular ions. The present study investigated the intracellular source of zinc (Zn2+) that has been reported to play a role in cell signaling., Results: In primary cultured cortical cells (neurons) labeled with intracellular fluorescent Zn2+ indicators, we showed that intracellular regions of Zn2+ staining co-localized with the endoplasmic reticulum (ER). The latter was identified with ER-tracker Red, a marker for ER. The colocalization was abolished upon exposure to the Zn2+ chelator TPEN, indicating that the local Zn2+ fluorescence represented free Zn2+ localized to the ER in the basal condition. Blockade of the ER Ca2+ pump by thapsigargin produced a steady increase of intracellular Zn2+. Furthermore, we determined that the thapsigargin-induced Zn2+ increase was not dependent on extracellular Ca2+ or extracellular Zn2+, suggesting that it was of intracellular origin. The applications of caged IP3 or IP3-3Kinase inhibitor (to increase available IP3) produced a significant increase in intracellular Zn2+., Conclusions: Taken together, these results suggest that Zn2+ is sequestered into thapsigargin/IP3-sensitive stores and is released upon agonist stimulation.

There is a rising intracellular Zn2+ transient during neuronal ischemic hypoxia (oxygen-glucose deprivation and reoxygenation, OGD/R). The results of our recent works suggest that the OGD/R-induced Zn2+ transient can readily be mistaken for a Ca2+ transient. The aim of this study was to examine the respective functions of Zn2+ and Ca2+ in OGD/R-induced neuronal injury. We showed that [Zn2+]i accumulation was consistently met with the induction of OGD/R-induced cell injury. Ca2+ accumulation induced with high [K+] (to open voltage-gated calcium channels) or ionomycin (a Ca2+ ionophore) caused a moderate neuronal injury that was reduced significantly by the application of the Zn2+ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). In comparison, Zn2+ accumulation, induced with the Zn2+ ionophore pyrithione, resulted in significantly greater injury. The application of nimodipine and MK801 was shown to attenuate neuronal injury only from a mild (10 mins) OGD insult. Neuronal injury from more severe (30 mins) OGD was not mitigated by the ion channel antagonists, whereas treatment with the Zn2+ chelator TPEN did afford significant protection from cell injury. These results indicate Zn2+-mediated damage to be of greater consequence than Ca2+-mediated damage, and collectively support the suggestion that Zn2+ accumulation may be a more significant causal factor of OGD/R-induced neuronal injury.

Vesicular zinc (Zn(2+)) is found in a subset of glutamatergic nerve terminals throughout the mammalian forebrain and is colocalized with glutamate. Despite well-documented neuromodulatory roles, exocytosis of endogenous Zn(2+) from presynaptic terminals has never been directly demonstrated, because existing studies have measured elevated Zn(2+) concentrations by examining the perfusate. Thus, the specific origin of synaptic Zn(2+) remains a controversial subject. Here, we describe synaptic Zn(2+) trafficking between cellular compartments at hippocampal mossy fiber synapses by using the fluorescent indicator Zinpyr-1 to label the hippocampal mossy fiber boutons. We determined endogenous Zn(2+) exocytosis by direct observation of vesicular Zn(2+) as decreasing fluorescence intensity from presynaptic axonal boutons in the stratum lucidum of CA3 during neural activities induced by the stimulation of membrane depolarization. This presynaptic fluorescence gradually returned to a level near baseline after the withdrawal of moderate stimulation, indicating an endogenous mechanism to replenish vesicular Zn(2+). The exocytosis of the synaptic Zn(2+) was also dependent on extracellular Ca(2+) and was sensitive to Zn(2+)-specific chelators. Vesicular Zn(2+) loading was sensitive to the vacuolar-type H(+)-ATPase inhibitor concanamycin A, and our experiments indicated that blockade of vesicular reloading with concanamycin A led to a depletion of that synaptic Zn(2+). Furthermore, synaptic Zn(2+) translocated to the postsynaptic cell body upon release to produce increases in the concentration of weakly bound Zn(2+) within the postsynaptic cytosol, demonstrating a feature unique to ionic substances released during neurotransmission. Our data provide important evidence for Zn(2+) as a substance that undergoes release in a manner similar to common neurotransmitters., ((c) 2007 Wiley-Liss, Inc.)

Although synaptically released, vesicular Zn(2+) has been proposed to play a neuromodulatory or neuronal signaling role at the mossy fiber-CA3 synapse, Zn(2+) release remains controversial, especially when detected using fluorescent imaging. In the present study, we investigated synaptically released Zn(2+) at the mossy fiber (MF) synapse in rat hippocampal slices using three chemically distinct, fluorescent Zn(2+) indicators. The indicators employed for this study were cell membrane impermeable (or extracellular) Newport Green [K(DZn2+) approximatelly 1 microM] , Zinpyr-4 K(DZn2+) approximately 1 nM and FluoZin-3 K(DZn2+) approximately 15 nM, chosen, in part, for their distinct dissociation constants. Among the three indicators, FluoZin-3 was also sensitive to Ca(2+) K(DCa2+) approximately 200-300 microM which was present in the extracellular medium ([Ca(2+)](o)>2mM). Hippocampal slices loaded with either Newport Green or FluoZin-3 showed increases in fluorescence after electrical stimulation of the mossy fiber pathway. These results are consistent with previous studies suggesting the presence of synaptically released Zn(2+) in the extracellular space during neuronal activities; however, the rise in FluoZin-3 fluorescence observed was complicated by the data that the addition of exogenous Zn(2+) onto FluoZin-3 loaded slices gave little change in fluorescence. In the slices loaded with the high-affinity indicator Zinpyr-4, there was little change in fluorescence after mossy fiber activation by electrical stimulation. Further study revealed that the sensitivity of Zinpyr-4 was mitigated by saturation with Zn(2+) contamination from the slice. These data suggest that the sensitivity and selectivity of a probe may affect individual outcomes in a given experimental system.

Much of our current evidence concerning of the role of calcium (Ca2+) as a second messenger comes from its interaction with fluorescent probes; however, many Ca2+ probes also have a higher affinity for another divalent cation: zinc (Zn2+). In this study, using a selective Zn2+ probe (Newport Green), we investigated the accumulation of intracellular Zn2+ transients in acute rat hippocampal slices during ischemia, simulated by oxygen and glucose deprivation (OGD). Subsequent reperfusion with glucose-containing oxygenated medium resulted in an additional increase in intracellular Zn2+. Such observations compelled us to investigate the contribution of Zn2+ to the alleged intracellular Ca2+ overload occurring in ischemia and reperfusion. Using confocal fluorescent microscopy of Calcium Green-1, a widely used Ca2+ indicator, we detected increases in fluorescence intensity during OGD and reperfusion. However, application of a Zn2+ chelator, at the peak of the fluorescence elevation (interpreted as Ca2+ overload), resulted in a significant drop in intensity, suggesting that rising Zn2+ is the primary source of the increasing Calcium Green-1 fluorescence. Finally, staining with the cell viability indicator propidium iodide revealed that Zn2+ is responsible for the ischemic neuronal cell death, because Zn2+ chelation prevented cells from sustaining ischemic damage. Current cellular models of ischemic injury center on Ca2+-mediated excitotoxicity. Our results indicate that Zn2+ elevation contributes to conventionally recognized Ca2+ overload and also suggest that the role of Ca2+ in neurotoxicity described previously using Ca2+ probes may need to be re-examined to determine whether effect previously attributed to Ca2+ could, in part, be attributable to Zn2+.

Investigations into the roles of Ca(2+) and Zn(2+) in cell biology have been facilitated by the development of sensitive fluorometric probes that have enabled the measurement of Ca(2+) or Zn(2+) in both extracellular and intracellular environments. It is critical to be aware of the specificity and relative selectivity of a probe for the targeted ion. Here, we investigated metal-ion responses by screening nominally Zn(2+)- or Ca(2+)-selective fluorophores in solutions containing various concentrations of Ca(2+), as a potential interferent for Zn(2+), or Zn(2+), as a potential interferent for Ca(2+). The results suggested that Zn(2+)-sensitive dyes were more specific for their targeted ion than dyes that targeted Ca(2+). Ca(2+)-sensitive dyes such as Calcium Green-1, Fura-2, and Fluo-3 showed a wide range of interaction with Zn(2+), even responding to Zn(2+) in the presence of high concentrations of Ca(2+). We demonstrate that these Ca(2+) indicators can effectively measure dynamic changes of cytosolic Zn(2+). Our results appeal for a new generation of Ca(2+) fluorophores that are more specific for Ca(2+) over Zn(2+). One implication of these results is that data obtained using Ca(2+)-sensitive dyes may need to be re-examined to determine if results previously attributed to Ca(2+) could, in part, be due to Zn(2+).