Divalent Cation Chelator Research Articles

A moldable hydrogel based on sericin and Zn2+/F- dual-doped hydroxyapatite promotes skull defect repair through the synergistic effects of immunoregulation, enhanced angiogenesis and osteogenesis

Skull defect repair typically involves multiple stages, including immunomodulation, angiogenesis, osteogenic differentiation, and biomineralization, etc. Most existing therapeutic biomaterials fail to show functional effects across all these stages, leading to unsatisfactory repair effects. To address this challenge, an organic–inorganic hybrid HT(SA)HAp moldable hydrogel with the aforementioned functional properties was developed. The moldable hydrogel consisted of phenylboronic acid-grafted hyaluronic acid, tannic acid, alendronate sodium-grafted sericin (Ser-AL), and hydroxyapatite co-doped with 5 % Zn and 10 % F (Zn5%/F10%-HAp). These materials were cross-linked due to phenylborate ester bonds, metal-phenol interactions, and the AL-mediated chelation of divalent metal cations. Sericin-mediated immunomodulation serves as a precursor to micro-environmental regulation, reducing inflammation at the site of injury and creating an environment conducive to angiogenesis and osteogenic differentiation. The degradation products of the hydrogel’s organic skeleton promoted the proliferation of mesenchymal stem cells and vascular endothelial cells. The enhanced paracrine effect of mesenchymal stem cells and the release of Zn2+ from modified Zn5%/F10%-HAp promoted angiogenesis, while the degradation products of these materials ensured continued promotion of osteogenic differentiation and biomineralization. This organic–inorganic hybrid hydrogel had the synergistic effects of immunoregulation, enhanced angiogenesis, osteogenic differentiation, and biomineralization, which could significantly accelerate the repair of skull defects, and demonstrate completely repaired skull defects at 8 weeks. Thus, this multifunctional moldable hydrogel provided an effective and stable treatment strategy for the rapid repair of skull defects.

Extractable latex yield from Taraxacum kok-saghyz roots is enhanced by increasing rubber particle buoyancy

Taraxacum kok-saghyz (TK) is a leading alternative rubber-producing plant that produces natural rubber (NR) latex in laticifers within its roots. Unlike the tropical rubber tree, Hevea brasiliensis, TK can be grown in temperate regions, such as the northern United States, as the basis of a domestic rubber production industry. To support such an industry, efficient, scalable rubber extraction techniques are needed. In this study, aqueous extraction was used to recover TK natural rubber latex (TNRL) from four harvests of greenhouse-produced TK roots with a new technique that improves rubber particle buoyancy in root homogenate by chelation. The new method includes harvest, cleaning, and homogenization of freshly harvested roots, followed by centrifugation, vacuum collection of latex, and purification. Treatment of homogenate with ethylenediaminetetraacetic acid (EDTA), a divalent cation chelator, allowed more than twice as much latex to be extracted from fresh homogenate. EDTA-treated rubber particles contained lower concentrations of divalent cations than untreated ones in fresh homogenate and homogenate stored for two weeks. Long term (three months) storage of root homogenate similarly yielded more latex than fresh homogenate and had low divalent cation rubber particle concentrations, indicating that heavy cations had slowly leached from the particles into the aqueous medium during storage. Rubber particle size was larger in this treatment suggestive of rubber particle agglomeration but this was prevented and reversed by EDTA. Removal of divalent cations via chelation increased particle packing density of particles in concentrated latex and appeared to increase the gel content of dried latex samples. Molecular characterization of dried samples of purified TNRL established that the rubber was of the high molecular weight needed for product manufacturing.

Mechanism of Lactiplantibacillus plantarum regulating Ca2+ affecting the replication of PEDV in small intestinal epithelial cells.

Porcine epidemic diarrhea virus (PEDV) mainly invades the small intestine and promotes an inflammatory response, eventually leading to severe diarrhea, vomiting, dehydration, and even death of piglets, which seriously threatens the economic development of pig farming. In recent years, researchers have found that probiotics can improve the intestinal microenvironment and reduce diarrhea. At the same time, certain probiotics have been shown to have antiviral effects; however, their mechanisms are different. Herein, we aimed to investigate the inhibitory effect of Lactiplantibacillus plantarum supernatant (LP-1S) on PEDV and its mechanism. We used IPEC-J2 cells as a model to assess the inhibitory effect of LP-1S on PEDV and to further investigate the relationship between LP-1S, Ca2+, and PEDV. The results showed that a divalent cation chelating agent (EGTA) and calcium channel inhibitors (Bepridil hydrochloride and BAPTA-acetoxymethylate) could inhibit PEDV proliferation while effectively reducing the intracellular Ca2+ concentration. Furthermore, LP-1S could reduce PEDV-induced loss of calcium channel proteins (TRPV6 and PMCA1b), alleviate intracellular Ca2+ accumulation caused by PEDV infection, and promote the balance of intra- and extracellular Ca2+ concentrations, thereby inhibiting PEDV proliferation. In summary, we found that LP-1S has potential therapeutic value against PEDV, which is realized by modulating Ca2+. This provides a potential new drug to treat PEDV infection.

Epithelium dynamics differ in time and space when exposed to the permeation enhancers penetramax and EGTA. A head-to-head mechanistic comparison

Absorption of therapeutic peptides like glucagon-like peptide or insulin for diabetes therapy upon oral administration is highly restricted by the tight junction (TJ) proteins interconnecting the cells comprising the intestinal epithelium. An approach to improve transepithelial permeation of such biopharmaceuticals via the paracellular pathway is to use functional excipients, which transiently modulate the TJs. Here, we investigated the membrane-interacting peptide, penetramax, and the divalent cation chelator, ethylene glycol tetraacetic acid (EGTA) at different concentrations, to reveal and compare their cellular modes of action when increasing the transepithelial permeation of drug macromolecules. The epithelial integrity was studied in real time along with dextran permeation across differentiated epithelial Caco-2 cell monolayers. TJ protein expression and cytoskeleton organization were investigated during and after exposure to penetramax or EGTA. Based on orthogonal methods, we show that penetramax acts by a mechanism that immediately and transiently widens the paracellular space, resulting in size selective permeant passage and with subsequent reconstitution of the epithelium. At the same time, the expression and organization of different TJ proteins are modulated reversibly. In contrast, the effect of EGTA on modulating the paracellular space is slower and TJ protein unspecific, and without clear permeant size selectivity. Overall, these data provide in-depth insights for understanding intestinal barrier dynamics of importance when evaluating new or existing excipients for oral delivery of biopharmaceuticals, such as peptide therapeutics.

Enhancing volatile fatty acids accumulation through anaerobic co-fermentation of excess sludge and sodium citrate: Divalent cation chelation and carbon source supplement

This work developed an innovative anaerobic co-fermentation strategy of excess sludge to enhance volatile fatty acids (VFAs) production and carbon recovery relying on the sodium citrate (SC) functions of divalent cation bridging impairing and biodegradable substrate supplement. It was demonstrated that SC greatly improved rate constant and equilibrium performances of both sludge hydrolysis and VFAs accumulation, suggesting that the 0.4 g/g SS was the optimum SC dosage. The SC was partly absorbed in sludge solid and chelated structural divalent cations, which greatly facilitated sludge hydrolysis with the SCOD/TCOD of 28.20%. Meanwhile, the residual SC in liquid phase contributed to VFAs bio-generation as carbon substrates. Thereby, the VFAs accumulation was improved to 387.72 mg COD/g VSS in a short-term anaerobic co-fermentation (2 d), approximately 7.64 times higher than that without SC addition. The VFAs were mainly composed by acetate (58.86–81.76%) and propionate (7.97–16.28%). In the whole anaerobic co-fermentation process, remarkable carbon source of 1785.74 mg/L was released. Acetate was the dominant composition in total soluble carbon sources with the percentage of 44.45–83.98%, manifesting the bio-availability of fermentative liquid. In this case, the bio-available carbon recovery rate of 38.04% was achieved. Moreover, mechanism of the enhanced anaerobic co-fermentation was illustrated and the economic benefits were clarified.

Greater than pH 8: The pH dependence of EDTA as a preservative of high molecular weight DNA in biological samples.

Ethylenediaminetetraacetic acid (EDTA) is a divalent cation chelator and chemical preservative that has been shown to be the active ingredient of the popular DNA preservative DESS. EDTA may act to reduce DNA degradation during tissue storage by sequestering divalent cations that are required by nucleases naturally occurring in animal tissues. Although EDTA is typically used between pH 7.5 and 8 in preservative preparations, the capacity of EDTA to chelate divalent cations is known to increase with increasing pH. Therefore, increasing the pH of EDTA-containing preservative solutions may improve their effectiveness as DNA preservatives. To test this hypothesis, we stored tissues from five aquatic species in 0.25 M EDTA adjusted to pH 8, 9, and 10 for 12 months at room temperature before DNA isolation. For comparison, tissues from the same specimens were also stored in 95% ethanol. DNA extractions performed on tissues preserved in EDTA pH 9 or 10 resulted in as great or greater percent recovery of high molecular weight DNA than did extractions from tissues stored at pH 8. In all cases examined, percent recovery of high molecular weight DNA from tissues preserved in EDTA pH 10 was significantly better than that observed from tissues preserved in 95% ethanol. Our results support the conclusion that EDTA contributes to DNA preservation in tissues by chelating divalent cations and suggest that preservative performance can be improved by increasing the pH of EDTA-containing DNA preservative solutions.

The antifungal potential of the chelating agent EDTA against postharvest plant pathogen Botrytis cinerea

Botrytis cinerea is a phytopathogenic fungus that causes gray mold, a major postharvest disease of fruits and vegetables. Chemical fungicides remain the main solution to control Botrytis disease, but concerns have raised about their safety to environment and human health, and there is an increasing need for development of more effective and less toxic treatments. In this study the divalent cation chelating agent ethylenediaminetetraacetic acid (EDTA) exhibited marked antifungal activity against B. cinerea, including inhibition of spore germination, mycelial growth, infection cushion formation, stimulation of cell death, and impairment of fungal virulence. These adverse effects of EDTA could be reversed by the addition of calcium ion, implying that metal ion chelation is involved in the fungicidal mechanism. Bean leaf and tomato fruit protection assay indicated that EDTA treatment led to a significant reduction of infection by B. cinerea. Furthermore, the antifungal activity of EDTA was significantly enhanced when used in combination with fenhexamid. These findings suggest that EDTA could be a promising tool to control B. cinerea, and application of EDTA may reduce the use of conventional chemical fungicides.

Divalent cation chelation enhancing carbon migration and recovery from anaerobic fermentation of waste activated sludge

Recently, the short-chain fatty acids (SCFAs) production with carbon recovery from waste activated sludge has received increasing attention. Despite that numerous approaches have been investigated, the divalent cation chelation towards enhancing anaerobic fermentation and the carbon migration fates has been rarely reported. This work attempted to chelate structural divalent cations from sludge flocs by sodium pyrophosphate (SP) at the dosages of 0–0.6 g/g SS, facilitating sludge disintegration and hydrolysis with particulate organic matters solubilization. At the optimal SP dosage of 0.4 g/g SS, the chemical oxygen demand (COD) and carbon source content in sludge solid were reduced by 45.24 % and 35.50 % within 1-day treatment, leading to considerable sludge hydrolysis and carbon release performances with soluble COD/total COD of 23.21 % and carbon release rate of 26.94 % in 2-day anaerobic fermentation, respectively. Numerous SCFAs of 237.43 mg COD/g VSS were produced by 4-day anaerobic fermentation, with the overall carbon recovery rate of 21.19 %, which were dominantly composed by acetate and propionate. Meanwhile, the SP-inhibited biogas generation also contributed to SCFAs accumulation. The divalent cation chelation obviously improved rate constant and equilibrium performances of anaerobic fermentation process, which were 1.81–5.21 and 1.36–2.82 times higher than those without SP, respectively. The carbon migration pattern was proposed by interphase carbon source balance and the mechanism was also illustrated. The crucial novelty was to propose an innovative and economic sludge hydrolysis pattern by divalent cation chelation towards improving carbon recovery.

Inhibition and Activation of Humoral Phenoloxidase and Their Effect on Divalent Cations in the Hemolymph of Freshwater Prawn Macrobrachium rosenbergii (De Man, 1879)

The aim of the present study is to examine phenoloxidase (PO), a copper-containing enzyme that plays a crucial role in the immune responses of arthropods, especially crustaceans and insects. However, PO activity is present in the plasma of the hemolymph without pathogenic infections. Here we report the PO activity in the plasma of M. rosenbergii was spectrophotometrically studied. The phenylthiourea (PTU) inhibition of PO activity was found to be the highest at a 10 mM concentration. The determination of PO activity was also highest activated by trypsin (2 mg.ml-1), detergents like sodium dodecyl sulphate (SDS; 8 mg.ml-1) and laminarin (4 mg.ml-1) enzyme expression. The effect of divalent cations on PO activity like sodium chloride (100 mM), calcium chloride, magnesium chloride, manganese chloride, and potassium chloride (10 mM) affected the activation of PO activity in trypsin, SDS, and laminarin. However, these divalent cations only slightly affected PTU inhibition of PO activity. In addition to calcium chloride, the PO activity with trypsin and laminarin is more or less the same as with control, but a great enhancement was found in SDS, which indicates that the combination of SDS with calcium chloride is required for increasing the PO activity in M. rosenbergii. The concentration of ethylenediaminetetraacetic acid (EDTA) of 1–10 mM has the maximum inhibition in PTU and activation of trypsin, SDS, and laminarin, which indicates that EDTA is a divalent cation chelator that affects the PO activity in M. rosenbergii. Hence, an attempt has been made to optimize the conditions for inhibitor, activators, divalent cations, EDTA sensitivity, and phenoloxidase activity in the plasma of the freshwater prawn M. rosenbergii.

Effects of Axonal Demyelination, Inflammatory Cytokines and Divalent Cation Chelators on Thalamic HCN Channels and Oscillatory Bursting.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that is characterized by the progressive loss of oligodendrocytes and myelin and is associated with thalamic dysfunction. Cuprizone (CPZ)-induced general demyelination in rodents is a valuable model for studying different aspects of MS pathology. CPZ feeding is associated with the altered distribution and expression of different ion channels along neuronal somata and axons. However, it is largely unknown whether the copper chelator CPZ directly influences ion channels. Therefore, we assessed the effects of different divalent cations (copper; zinc) and trace metal chelators (EDTA; Tricine; the water-soluble derivative of CPZ, BiMPi) on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that are major mediators of thalamic function and pathology. In addition, alterations of HCN channels induced by CPZ treatment and MS-related proinflammatory cytokines (IL-1β; IL-6; INF-α; INF-β) were characterized in C57Bl/6J mice. Thus, the hyperpolarization-activated inward current (Ih) was recorded in thalamocortical (TC) neurons and heterologous expression systems (mHCN2 expressing HEK cells; hHCN4 expressing oocytes). A number of electrophysiological characteristics of Ih (potential of half-maximal activation (V0.5); current density; activation kinetics) were unchanged following the extracellular application of trace metals and divalent cation chelators to native neurons, cell cultures or oocytes. Mice were fed a diet containing 0.2% CPZ for 35 days, resulting in general demyelination in the brain. Withdrawal of CPZ from the diet resulted in rapid remyelination, the effects of which were assessed at three time points after stopping CPZ feeding (Day1, Day7, Day25). In TC neurons, Ih was decreased on Day1 and Day25 and revealed a transient increased availability on Day7. In addition, we challenged naive TC neurons with INF-α and IL-1β. It was found that Ih parameters were differentially altered by the application of the two cytokines to thalamic cells, while IL-1β increased the availability of HCN channels (depolarized V0.5; increased current density) and the excitability of TC neurons (depolarized resting membrane potential (RMP); increased the number of action potentials (APs); produced a larger voltage sag; promoted higher input resistance; increased the number of burst spikes; hyperpolarized the AP threshold), INF-α mediated contrary effects. The effect of cytokine modulation on thalamic bursting was further assessed in horizontal slices and a computational model of slow thalamic oscillations. Here, IL-1β and INF-α increased and reduced oscillatory bursting, respectively. We conclude that HCN channels are not directly modulated by trace metals and divalent cation chelators but are subject to modulation by different MS-related cytokines.

ASIC1a senses lactate uptake to regulate metabolism in neurons

Lactate is a major metabolite largely produced by astrocytes that nourishes neurons. ASIC1a, a Na+ and Ca2+-permeable channel with an extracellular proton sensing domain, is thought to be activated by lactate through chelation of divalent cations, including Ca2+, Mg2+ and Zn2+, that block the channel pore. Here, by monitoring lactate-evoked H+ and Ca2+ transport in cultured mouse cortical and hippocampal neurons, we find that stereo-selective neuronal uptake of L-lactate results in rapid intracellular acidification that triggers H+ extrusion to activate plasma membrane ASIC1a channels, leading to propagating Ca2+ waves into the cytosol and mitochondria. We show that lactate activates ASIC1a at its physiological concentrations, far below that needed to chelate divalent cations. The L-isomer of lactate exerts a much greater effect on ASIC1a-mediated activity than the d-isomer and this stereo-selectivity arises from lactate transporters, which prefer the physiologically common L-lactate. The lactate uptake in turn results in intracellular acidification, which is then followed by a robust acid extrusion. The latter response sufficiently lowers the pH in the vicinity of the extracellular domain of ASIC1a to trigger its activation, resulting in cytosolic and mitochondrial Ca2+ signals that accelerate mitochondrial respiration. Furthermore, blocking ASIC1a led to a robust mitochondrial ROS production induced by L-lactate. Together our results indicate that ASIC1a is a metabolic sensor, which by sensing extracellular pH drop triggered by neuronal lactate uptake with subsequent proton extrusion, transmits a Ca2+ response that is propagated to mitochondria to enhance lactate catabolism and suppress ROS production.

Abstract P334: Biophysical Properties Of A Novel Mitochondrial Large Ubiquitous Non-selective Amiloride Sensitive (luna) Current

Inner mitochondrial membrane (IMM) ion channels and transporters account for communication of the matrix with the intermembrane space (IMS) and the cytosol. Transport of solutes and ions is keep under strict regulation mainly because small changes in solute concentrations could generate changes in mitochondrial volume or membrane potential (ΔΨ m ), interrupting ATP synthesis and leading to mitochondrial damage. The list of recently discovered mitochondrial ion channels has been growing in the past decades. In this work, using the patch-clamp technique we observed the activity of a novel mitochondrial current, named here LUNA current, in mitoplasts (IMM striped of outer membrane) from mouse liver, spleen, brain and heart, as well as established cell lines. LUNA is a novel non-selective cation current (K + >Na + >NMDG + >H + ) active at depolarized membrane potentials. The basal activity of whole-mitoplast LUNA currents from wild type mice hearts changed from 445±106 pA/pF to 1232±287 pA/pF upon chelation of external divalent cations (Ca 2+ and Mg 2+ ). Moreover, the activity of LUNA is independent of the mitochondrial Ca 2+ uniporter and of the non-selective reactive oxygen species modulator channel (ROMO1). In the heart, the activity of LUNA was enhanced in both the Tfam -KO mice, which have impaired electron transport chain (ETC) activity and are a model for mitochondrial cardiomyopathies, and mice with cardiac pressure overload due to transverse aortic constriction (TAC) compared to sham-operated hearts (729±197; n=7 vs 283±137 pA/pF). LUNA current is reversibly inhibited by amiloride with no sensitivity to the vast majority of common K + , Na + and Ca 2+ channels and ETC inhibitors. The molecular identity of mitochondrial LUNA current remains to be determined.

Novel Protocols for Scalable Production of High Quality Purified Small Extracellular Vesicles from Bovine Milk

Extracellular Vesicles (EVs) are cell-secreted nanovesicles that have unique potential for encapsulating and targeting “difficult-to-drug” therapeutic cargos. Milk provides an enriched source of EVs, and of particular interest to the drug delivery field, small EVs. Small EVs are distinguished from large EVs by membrane components, biogenesis mechanism and downstream functionality - in particular, small EVs are primarily composed of exosomes, which show high stability in vivo and naturally function in the targeted delivery of biological materials to cells. Moreover, bovine milk is abundantly produced by the dairy industry, widely consumed, and generally well tolerated by humans. Importantly, there is evidence that milk exosomes and small EVs are efficiently taken up into the circulation from the gut, providing the opportunity for their use in administration of therapeutics such as microRNAs or peptides not typically available via an oral route. Unfortunately, present methods for isolation do not efficiently separate EVs from milk proteins, resulting in contamination that is not desirable in a clinical-grade therapeutic. Herein, we present novel EV purification methods focused on optimized timing and levels of temperature and divalent cation chelation. Incorporation of these solubilization steps into centrifugation- and tangential flow filtration-based methods provide large amounts of purified small EVs at ultra-dense concentrations, which are substantially free from contaminating milk proteins. Remarkably, these ultra-dense isolates equal 10 to 15% of the starting volume of milk indicating a prodigious rate of small EV production by mammary glands. Our approach enables gentle, scalable production of ultrastructurally and functionally intact small EVs from milk, providing a path to their industrial scale purification for oral delivery of therapeutic biologics and small drugs.

Abstract 2626: Exosomes derived from the BT-20 human breast cancer cell line express E-selectin ligands

Abstract Exosomes are small extracellular vesicles (40-150 nm in diameter) secreted by cells and initially proposed as a mechanism to eliminate cellular waste. More recently, exosomes have been reported to participate in cancer metastasis because of their ability to mediate local and distant cell-to-cell communication. Furthermore, exosomes create cancer-favored microenvironments, potentially enhancing cancer metastasis. However, it is unknown whether exosomes interact with E-selectin, an adhesion molecule expressed by vascular endothelium and shown to be involved in metastasis. Our lab has previously shown that BT-20 human breast cancer cells adhesively interact with endothelial E-selectin via several E-selectin ligands such as sialofucosylated CD44 variants, Mac-2BP, and gangliosides. Therefore, we hypothesize that exosomes secreted by the BT-20 human breast cancer cell line interact with E-selectin. To investigate this hypothesis, exosomes were generated by culturing cells in serum-depleted media and isolated by a series of ultracentrifugation steps. Molecular size analysis using dynamic light scattering revealed that the size range of the isolated particles was similar to the expected size of exosomes. To detect CD9 (an exosome marker) and E-selectin ligands, both breast cancer whole cell lysate and exosome lysate were tested by Western blot. The higher expression of CD9 in isolated particles than the whole cell lysates confirmed that the isolated particles were exosomes. When Western blots were labeled with recombinant E-selectin/Fc chimera with Ca2+, bands were detected at approximately 130 kDa, indicating the presence of E-selectin ligands. As a negative control, the divalent cation chelator EDTA was added to the primary labeling incubation. Bands were not detected in these negative control experiments, indicating no E-selectin interactions in the absence of Ca2+. Human IgG (hIgG) was also used as a negative control in Western blots and showed no signal with exosome lysates, as expected. In comparison, experiments were also performed with Hs578T breast cancer cells, a cell line that our lab has shown does not express E-selectin ligands. Exosomes isolated from Hs578T cells did not express E-selectin ligands, consistent with whole cells. Altogether, these results suggest that BT-20 human breast cancer exosomes express E-selectin ligands. Future work will focus on identifying and characterizing the exosome E-selectin ligands, as well as investigating other cancer cells that express E-selectin ligands. Ultimately, understanding the interaction between exosomes and E-selectin can lead to new diagnostic and therapeutic strategies for cancer metastasis. Citation Format: Yinan Huang, Christian A. Showalter, Monica M. Burdick. Exosomes derived from the BT-20 human breast cancer cell line express E-selectin ligands [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2626.

Fluoxetine Induces Apoptotic and Oxidative Neuronal Death Associated with The Influx of Copper Ions in Cultured Neuronal Cells.

We examined the effect of fluoxetine, a selective serotonin reuptake inhibitor antidepressant, on neuronal viability in mouse cortical near-pure neuronal cultures. Addition of fluoxetine to the media for 24 hours induced neuronal death in a concentration-dependent manner. To delineate the mechanisms of fluoxetine-induced neuronal death, we investigated the effects of trolox, cycloheximide (CHX), BDNF, z-VAD-FMK, and various metal-chelators on fluoxetine-induced neuronal death. Neuronal death was assessed by MTT assay. The addition of 20 µM fluoxetine to the media for 24 hours induced 60–70% neuronal death, which was associated with the hallmarks of apoptosis, chromatin condensation and DNA laddering. Fluoxetine-induced death was significantly attenuated by CHX, BDNF, or z-VAD-FMK. Treatment with antioxidants, trolox and ascorbate, also markedly attenuated fluoxetine-induced death. Interestingly, some divalent cation chelators (EGTA, Ca-EDTA, and Zn-EDTA) also markedly attenuated the neurotoxicity. Fluoxetine-induced reactive oxygen species (ROS) generation was measured using the fluorescent dye 2′,7′-dichlorofluorescin diacetate. Trolox and bathocuproine disulfonic acid (BCPS), a cell membrane impermeable copper ion chelator, markedly attenuated the ROS production and neuronal death. However, deferoxamine, an iron chelator, did not affect ROS generation or neurotoxicity. We examined the changes in intracellular copper concentration using a copper-selective fluorescent dye, Phen Green FL, which is quenched by free copper ions. Fluoxetine quenched the fluorescence in neuronal cells, and the quenching effect of fluoxetine was reversed by co-treatment with BCPS, however, not by deferoxamine. These findings demonstrate that fluoxetine could induce apoptotic and oxidative neuronal death associated with an influx of copper ions.

Bacteria poration on modified boron-doped diamond electrode surfaces induced by divalent cation chelation

This work showed that divalent ions from the outer membrane of bacteria can be chelated byN-propyl-2-hydroxyacetamide functional groups that were immobilized on boron-doped diamond electrodes.

Controlled deprotection and release of a small molecule from a compartmented synthetic tissue module

Synthetic tissues built from communicating aqueous droplets offer potential applications in biotechnology, however, controlled release of their contents has not been achieved. Here we construct two-droplet synthetic tissue modules that function in an aqueous environment. One droplet contains a cell-free protein synthesis system and a prodrug-activating enzyme and the other a small-molecule prodrug analog. When a Zn2+-sensitive protein pore is made in the first droplet, it allows the prodrug to migrate from the second droplet and become activated by the enzyme. With Zn2+ in the external medium, the activated molecule is retained in the module until it is released on-demand by a divalent cation chelator. The module is constructed in such a manner that one or more, potentially with different properties, might be incorporated into extended synthetic tissues, including patterned materials generated by 3D-printing. Such modules will thereby increase the sophistication of synthetic tissues for applications including controlled multidrug delivery.

Ex vivo observation of insect hemocyte behavior against beads and nematodes in the presence of insect plasma

Recognition of foreign targets by insect hemocytes is a crucial first step for insect immunity against invading multicellular organisms in the hemocoel. To understand the mechanism of recognition, we observed the hemocyte behavior of Galleria mellonella Linnaeus (Lepidoptera: Pyralidae) larvae against beads and the nonparasitic nematode Caenorhabditis elegans (Maupas) (Rhabditida: Rhabditidae) in the presence of plasma ex vivo using time-lapse microscopy. Both granular cells and plasmatocytes adhered to and spread on the surface of beads and nematodes. In addition, the spread plasmatocytes actively moved over the beads and nematodes. These results suggest that not only granular cells but also plasmatocytes can recognize foreign targets in the presence of insect plasma and that spread plasmatocytes can actively search for foreign targets. Hemocyte adhesion to beads and nematodes ex vivo was similar to that of the in vivo 1 h after injection. A divalent cation chelator inhibited the spreading and adhesion of plasmatocytes ex vivo, but it did not affect the adhesion of granular cells. The present method enables the analysis of acute hemocyte response against foreign targets in the presence of plasma.

Adeno-Associated Virus VP1u Exhibits Protease Activity.

Adeno-associated viruses (AAVs) are being developed for gene delivery applications, with more than 100 ongoing clinical trials aimed at the treatment of monogenic diseases. In this study, the unique N-terminus of AAV capsid viral protein 1 (VP1u), containing a canonical group XIII PLA2 enzyme domain, was observed to also exhibit proteolytic activity. This protease activity can target casein and gelatin, two standard substrates used for testing protease function but does not self-cleave in the context of the capsid or target globular proteins, for example, bovine serum albumin (BSA). However, heated BSA is susceptible to VP1u-mediated cleavage, suggesting that disordered proteins are substrates for this protease function. The protease activity is partially inhibited by divalent cation chelators ethylenediaminetetraacetic acid (EDTA) and ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA), and human alpha-2-macroglobulin (A2M), a non-specific protease inhibitor. Interestingly, both the bovine pancreatic (group VIIA) and bee venom (group III) PLA2 enzymes also exhibit protease function against casein. This indicates that PLA2 groups, including VP1u, have a protease function. Amino acid substitution of the PLA2 catalytic motif (76HD/AN) in the AAV2 VP1u resulted in attenuation of protease activity, suggesting that the protease and PLA2 active sites are related. However, the amino acid substitution of histidine H38, which is not involved in PLA2 function, to alanine, also affects protease activity, suggesting that the active site/mechanism of the PLA2 and protease function are not identical.

Atomic structure of the translation regulatory protein NS1 of bluetongue virus.

Bluetongue virus (BTV) non-structural protein 1 (NS1) regulates viral protein synthesis and exists as tubular and non-tubular forms in infected cells, but how tubules assemble and how protein synthesis is regulated are unknown. Here, we report near-atomic resolution structures of two NS1 tubular forms determined by cryo-electron microscopy. The two tubular forms are different helical assemblies of the same NS1 monomer, consisting of an amino-terminal foot, a head and body domains connected to an extended carboxy-terminal arm, which wraps atop the head domain of another NS1 subunit through hydrophobic interactions. Deletion of the C terminus prevents tubule formation but not viral replication, suggesting an active non-tubular form. Two zinc-finger-like motifs are present in each NS1 monomer, and tubules are disrupted by divalent cation chelation and restored by cation addition, including Zn2+, suggesting a regulatory role of divalent cations in tubule formation. In vitro luciferase assays show that the NS1 non-tubular form upregulates BTV mRNA translation, whereas zinc-finger disruption decreases viral mRNA translation, tubule formation and virus replication, confirming a functional role for the zinc-fingers. Thus, the non-tubular form of NS1 is sufficient for viral protein synthesis and infectious virus replication, and the regulatory mechanism involved operates through divalent cation-dependent conversion between the non-tubular and tubular forms.