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The removal of algal organic matter (AOM) through water treatment processes is a major approach of reducing the formation of disinfection by-products (DBP). Here, the formation of DBP from AOM in karst water under different combination of potassium permanganate (KMnO4) and polyaluminium chloride (PACl) was investigated. The effect of divalent ions (Ca2+ and Mg2+) on DBP formation was traced by AOM chemistry variations. For DBP formation after KMnO4 preoxidation, total carbonaceous DBPs (C-DBPs) decreased by 12.9% but nitrogen-containing DBPs (N-DBPs) increased by 18.8%. Conversely, the C-DBPs further increased by 3.3% but N-DBPs reduced by 10.7% after the addition of PACl besides KMnO4 preoxidation. The variations of aromatic protein-like, soluble microbial products-like compounds and ultraviolet absorbance at 254 nm (UV254) were highly correlated with the formation of DBPs, which suggest aromatic substances strongly affect DBP behaviors at different treatment conditions. In the presence of divalent ions (Ca2+ = 135.86 mg/L, Mg2+ = 18.51 mg/L), the combination of KMnO4 and PACl was more effective in controlling DBP formation compared to the situation without Ca2+ and Mg2+. Specifically, trichloromethane formation was largely inhibited compared to the other tested DBPs, which may refer to complexation of electron-donating groups via divalent ions. While Ca2+ and Mg2+ may not affect the nature of α-carbon and amine groups, so the variation of haloacetonitriles (HANs) was not obvious. The study enhances the understanding of the DBP formation patterns, transformation of carbon and nitrogen by preoxidation-coagulation (KMnO4-PACl) treatment in algae-laden karst water.

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Sensitive biomaterials subjected to surface modification require delicate methods to preserve their structures and key properties. These include collagen-sealed polyester vascular prostheses. For their functionalization, coating with polycatecholamines (PCAs) can be used. PCAs change some important biological properties of biomaterials, e.g., hydrophilicity, bioactivity, antibacterial activity, and drug binding. The coating process can be stimulated by oxidants, organic solvents, or process conditions. However, these factors may change the properties of the PCA layer and the matrix itself. In this work, collagen-sealed vascular grafts were functionalized with a poly(l-DOPA) (PLD) layer using novel seawater-inspired ion combination as an accelerator, compared to the sodium periodate, Cu2+/H2O2 mixture, and accelerator-free reference methods. Then, poly(l-DOPA) was used as the interface for antibiotic binding. The coated prostheses were characterized (SEM, FIB-SEM, FTIR, UV/vis), and their important functional parameters (mechanical, antioxidant, hemolytic, and prothrombotic properties, bioactivity, stability in human blood and simulated body fluid (SBF), antibiotic binding, release, and antibacterial activity) were compared. It was found that although sodium periodate increased the strength and drug-binding capacity of the prosthesis, it also increased the blood hemolysis risk. Cu2+/H2O2 destabilized the mechanical properties of the coating and the graft. The seawater-inspired ion-accelerated method was efficient, stable, and matrix- and human blood-friendly, and it stimulated hydroxyapatite formation on the prosthesis surface. The results lead to the conclusion that selection of the PCA formation accelerator should be based on a careful analysis of the biological properties of medical devices. They also suggest that the ion-accelerated method of PLD coating on medical devices may be highly effective and safer than the oxidant-accelerated coating method.

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This study examined how the concentration of asphaltene and divalent ions in various salinities affects the interfacial tension (IFT) between a model oil/brine using the pendant drop method. The oleic phase consisted of a mixture of toluene and n-heptane (heptol), to which asphaltene was added to investigate how asphaltene molecules affect the surface properties. The base fluid was prepared with a salinity of 40,000 ppm, and two additional solutions with concentrations of 4000 ppm (low salinity) and 80,000 ppm (high salinity) were created. The results revealed that increasing the concentration of asphaltene within certain salinity ranges led to a decrease in IFT. The lowest IFT was observed at the 40,000 ppm salinity level, indicating that at this optimal salinity, the maximum asphaltene concentration migrated to the heptol/brine interface, reducing the IFT from 23 mN/m to 16 mN/m. Additionally, a 0.5 % wt of asphaltene demonstrated a significant concentration of micellization of natural surfactants, suggesting that the interface was nearly saturated with asphaltene. Consequently, concentrations higher than this value did not significantly alter the IFT. In the final part of the study, the impact of divalent ions was investigated, revealing that as the concentration of Ca2+ ions increased up to fourfold, the IFT decreased to 15 mN/m, about 10 % less than the base case. This value represented the lowest IFT compared to Mg2+ and SO42-. Moreover, modeling the results indicated that the relaxation time decreased with increasing salinity, suggesting that higher salinity accelerated the process of asphaltene absorption at the interface.

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In this study, we examined the modulation of algae removal and algal organic matter (AOM) chemistry by potassium permanganate and poly-aluminum chloride (KMnO4-PAC) in simulated karst water. Specifically, we verified the compositional changes of AOM sourcing from Chlorella sp. and Pseudanabaena sp. in response to the presence of divalent ions (Ca2+ and Mg2+). Aromatic protein and soluble microbial products were identified as the primary AOM components. Divalent ions accelerated dissolved organic carbon (DOC) and UV254 removal, particularly with Pseudanabaena sp. greater than Chlorella sp. (P < 0.05). Surface morphology analysis manifested that the removal of filamentous Pseudanabaena sp. was more feasible in comparison to globular Chlorella sp.. Our results highlight the significance of divalent ions in governing chemical behaviors and subsequent removal of both algae and AOM. This study upscales the understanding of the interactions among divalent ions, algae and AOM during preoxidation and coagulation process in algae-laden karst water.

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Divalent ions have a negative effect on the obtained power and efficiency of the reverse electrodialysis (RED) process when using natural waters. These effects can largely be attributed to the interaction between the various ions and the membranes, resulting in a decreased membrane voltage, an increased membrane resistance, and uphill transport of divalent ions. The aim of this study was to investigate the causes of these differences and, if possible, to find underlying causes. The approach mainly followed that in literature articles that specifically focused on the effect of divalent ions on RED. It transpired that seven publications were useful because the methodology was well described and sufficient data was published. I found two widely shared misconceptions. The first concerns the role of the stack voltage in uphill transport of divalent ions; itis often thought that the open circuit voltage (OCV) must be taken into account, but it is plausible that the voltage under working conditions is the critical factor. The second debatable point concerns the methodology used to make a series of solutions to study the effect of divalent ions. Typically, solutions with a constant number of moles of salt are used; however, it is better to make a series with a constant ratio of equivalents of those salts. Moreover, it is plausible that the decreased voltage can be explained by the inherently lower Donnan potential of multi-charged ions and that increased resistance is caused by the fact that divalent ions-with a lower mobility there than the monovalent ions-occupy relatively much of the available space in the gel phase of the membrane. While both resistance and voltage play a decisive role in RED and probably also in other membrane processes like electrodialysis (ED), it is remarkable that there are so few publications that focus on measurements on individual membranes. The implications of these results is that research on the effect of divalent ions in RED, ED and similar processes needs to be more structured in the future. Relatively simple procedures can be developed for the determination of membrane resistance in solutions of mixtures of mono- and divalent salts. The same applies to determining the membrane potential. The challenge is to arrive at a standard method for equipment, methodology, and the composition of the test solutions.

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Citrobacter werkmanii, an aerobe and mesophilic Proteobacterium, is universal in industrial putrefaction, coastal water, and human blood. Our previous studies have discovered that outer membrane protein X (OmpX) of C. werkmanii is involved in calcium response, but the underlying mechanisms and its molecular characteristics remain elusive. To that end, the ompX gene was deleted from the genome of C. werkmanii and its phenotypic variations were thoroughly investigated in conjunction with the wild type (WT) and complementary strains using biochemical and molecular techniques such as RNA-Seq, respectively. The results demonstrated that deleting ompX reduces biofilm formation on polystyrene and glass surfaces. Meanwhile, ΔompX's swimming ability but not for its twitching or swarming abilities, was also reduced on semi-solid plates compared with WT, which was caused by inhibition of flagellar assembly genes, such as flgC, flhB, and fliE, etc. Furthermore, ompX inactivation altered susceptibility to various bactericide classes, as well as responses to Ca2+ and Mg2+ stress. In addition, when compared to WT, ΔompX captures a total of 1,357 deferentially expressed genes (DEGs), of which 465 were up-regulated and 892 were down-regulated, which can be enriched into various GO ontology and KEGG pathway terms. Furthermore, ompX, as well as ompD and ompW, can be modulated at the transcriptional levels by rbsR and tdcA. Overall, the ompX gene contributed to a variety of biological functions in C. werkmanii and could be served as a targeted site for controlling biofilm formation and developing new bactericides.

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Bisphosphonates are drugs that are used to treat osteoporosis that causes the low mineral density of the bones. These drugs can be delivered in several ways, but each method has disadvantages. Materials with high potential as carriers of these drugs are zeolites with divalent ions. The aim of this study was to investigate the effect of divalent cations (calcium, magnesium, zinc) and drug type (risedronate, zoledronate) on sorption and release of the drug for osteoporosis. It was proved that drug sorption occurs on all zeolites presented in this work. Risedronate sorption was highest in zinc zeolite and lowest in calcium zeolite. In the case of zoledronate, sorption was most effective in magnesium zeolite and the least effective in zinc zeolite. Very large differences in drug release profiles were also observed. Risedronate was released several times longer than zoledronate. The diversity of the results indicates that the examined materials can be used in different types of drug delivery systems. They can be used, for example, intravenously or in the form of implants due to the different release profiles. Furthermore, the proposed carriers also release magnesium and calcium ions which are used in the prevention of osteoporosis, and zinc ions which have antibacterial properties.

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This study investigates the influence of co-existing ions on the salinity gradient power generation performance of the reverse electrodialysis (RED) using three different commercial ion exchange membrane pairs. The feed solutions, including the mixture of two different salts, were prepared with 90 wt.% of NaCl and 10 wt.% of LiCl, KCl, CaCl2, MgCl2 or Na2SO4 by keeping the salt ratio between high concentrate solution and low concentrate solution constant as 1:30 (g/g) at various flow velocities (50, 125 and 200 mL/min). It was observed that the divalent ions exhibited a negative impact on the performance of the RED system due to their high valence and low ionic mobility depending on their high hydrated radius and low diffusion coefficients compared to those of the monovalent ions. On the other hand, the effect of the monovalent ions differed according to the properties of ion exchange membranes used in the RED stack. When the power generation performances of ion exchange membrane pairs employed in the RED stack were compared, it was considered that Neosepta AMX and CMX membranes provided the highest power density due to their low membrane thicknesses, low electrical resistances, and relatively high ion exchange capacities compared to other two commercial ion exchange membrane pairs.

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The genus Citrobacter is commonly found in environmental and industrial settings, some members of which have been used for bioremediation of heavy metals owing to the absorption ability of their biofilms. Although our previous studies have found that the outer membrane protein A (OmpA) contributes to the process of Citrobacter werkmanii biofilm formation, the underlying mechanisms remain elusive. Therefore, we deleted ompA from the genome of C. werkmanii and investigated its phenotypes in comparison to the wild type strain (WT) and the complementary strain using biochemical and molecular techniques including RNA-Seq. Our results demonstrated that the deletion of ompA led to an increase in biofilm formation on both polystyrene and glass surfaces due to upregulation of some biofilm formation related genes. Meanwhile, swimming ability, which is mediated by activation of flagellar assembly genes, was increased on semi-solid plates in the ∆ompA strain when compared with WT. Additionally, inactivation of ompA also caused increased 1,2-benzisothiazolin-3-one (BIT) resistance, differential responses to Ca2+ stress, curli protein expression and cellulose production. Finally, ∆ompA caused differential expression of a total of 1470 genes when compared with WT, of which 146 were upregulated and 1324 were downregulated. These genes were classified into different Gene Ontology (GO) and KEGG pathways. In summary, ompA in C. werkmanii contributes to a variety of biological functions and may act as a target site to modulate biofilm formation. KEY POINTS: • ompA is a negative regulator for biofilm formation by C. werkmanii. • ompA inhibits swimming motility of C. werkmanii. • ompA deletion causes different expression profiles in C. werkmanii.

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Both the small and large subunits of the ribosome, the molecular machine that synthesizes proteins, are complexes of ribosomal RNAs (rRNAs) and a number of proteins. In bacteria, the small subunit has a single 16S rRNA whose folding is the first step in its assembly. The central domain of the 16S rRNA folds independently, driven either by Mg2+ ions or by interaction with ribosomal proteins. To provide a quantitative description of ion-induced folding of the ∼350-nucleotide rRNA, we carried out extensive coarse-grained molecular simulations spanning Mg2+ concentration between 0 and 30 mM. The Mg2+ dependence of the radius of gyration shows that globally the rRNA folds cooperatively. Surprisingly, various structural elements order at different Mg2+ concentrations, indicative of the heterogeneous assembly even within a single domain of the rRNA. Binding of Mg2+ ions is highly specific, with successive ion condensation resulting in nucleation of tertiary structures. We also predict the Mg2+-dependent protection factors, measurable in hydroxyl radical footprinting experiments, which corroborate the specificity of Mg2+-induced folding. The simulations, which agree quantitatively with several experiments on the folding of a three-way junction, show that its folding is preceded by formation of other tertiary contacts in the central junction. Our work provides a starting point in simulating the early events in the assembly of the small subunit of the ribosome.

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A novel deodorizer that is capable of selectively eliminating the odorous chemicals, such as ammonia, trimethylamine, hydrogen sulfide and methyl mercaptan, is described. The deodorizer is a nanostructured aerogel by nature, consisting of 2,2-6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidized cellulose nanofibrils (CNF), transition metal divalent cations (M2+), and multi-walled carbon nanotubes (CNT) as the constitutive elements. CNF are firstly mixed with M2+ (M2+, in this paper, typifies Ni2+, Co2+ and Cu2+) to form CNF-M2+ complexes, monodispersed CNT is then mixed to prepare CNT/CNF-M2+ waterborne slurries; CNT/CNF-M2+ hybridized aerogels are finally obtained via freezing-drying of the CNT/CNF-M2+ waterborne slurries. The CNT/CNF-M2+ aerogels are a foam-like structure consisting of CNF and CNT as backbones and M2+ as linkers. The aerogels show higher capabilities (in comparison with activated carbon) for selectively adsorbing ammonia, trimethylamine, hydrogen sulfide and methyl mercaptan. Computing simulations suggest a theoretical conclusion that the odorous chemicals are absorbed in a preferring manner of bimolecular absorptions via the M2+ moieties. The CNT/CNF-M2+ hybridized aerogels are lightweight, eco-friendly, and easy to produce in industrial scales. Our new finding, as is described in this paper, demonstrates potential applications of the TEMPO-oxidized CNF to the field of deodorizations.

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BACKGROUND: Divalent cations are able to interact with exopolysaccharides (EPS) and thus are capable to modify the structure and composition of dental biofilm. At the moment, little is known about the adsorption of metals by cariogenic EPS; thus, the aim of the present study was to evaluate the effect of divalent ions (calcium, magnesium, and zinc) on the growth and biofilm formation of mutans streptococci and on the dissolution of hydroxyapatite as well as to investigate their binding to the bacterial EPS. RESULTS: S. mutans strains used in this study show the highest tolerance towards calcium of the ions tested. Growth parameters showed no differences to control condition for both strains up to 100 mM; revealing natural tolerance to higher concentration of calcium in the surroundings. Although excessive levels of calcium did not impair the growth parameters, it also did not have a positive effect on biofilm formation or its binding affinity to EPS. Magnesium-saturated environment proved to be counterproductive as strains were able to dissolve more Ca2+ from the tooth surface in the presence of magnesium, therefore releasing excessive amounts of Ca2+ in the environment and leading to the progression of the disease. Thus, this supports the idea of self-regulation, when more Ca2+ is released, more calcium is bound to the biofilm strengthening its structure and however, also less is left for remineralization. Zinc inhibited bacterial adhesion already at low concentrations and had a strong antibacterial effect on the strains as well as on calcium dissolution; leading to less biofilm and less EPS. Additionally, Zn2+ had almost always the lowest affinity to all EPS; thus, the unbound zinc could also still remain in the surrounding environment and keep its antimicrobial properties. CONCLUSION: It is important to maintain a stable relationship between calcium, magnesium and zinc as excessive concentrations of one can easily destroy the balance between the three in cariogenic environment and lead to progression of the disease.

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The objective of this study was to assess the influence of self-association of ß-casein (ß-CN) induced by both increasing temperature (5-55 °C) and divalent cation addition (Ca2+ or Mg2+) on the properties of ß-CN-stabilised emulsions. The particle size of 0.5% (w/w) ß-CN in 10 mM imidazole/HCl buffer (pH 6.8) was determined as a function of temperature and addition of divalent cations. Addition of CaCl2 caused a greater increase in protein particle size than MgCl2. Oil-in-water emulsions stabilised with 0.5% (w/w) ß-CN, ß-CN with added CaCl2 or MgCl2 (ß-CN/Ca and ß-CN/Mg, respectively) were also investigated as a function of temperature using light scattering, analytical centrifugation, rheology and confocal laser scanning microscopy (CLSM). Emulsions prepared with ß-CN/Ca flocculated after incubation at 55 °C for 20 min and displayed significantly different physical properties (p < 0.05) compared to emulsions stabilised with ß-CN or ß-CN/Mg in the temperature range 5-55 °C. Based on CLSM analysis and analysis of the interfacial protein load, this flocculation was attributed to the interaction of adsorbed ß-CN between droplets and the interaction of adsorbed and non-adsorbed ß-CN aggregates in the aqueous phase via calcium bridges. Furthermore, the flocculation of ß-CN/Ca emulsions was reversible upon cooling, which is similar to that of ß-CN/Ca in solution. In conclusion, the temperature-dependent behaviour of ß-CN-stabilised emulsions correlated to the temperature-induced aggregation of ß-CN, particularly in the presence of Ca2+. Hence, the stability of ß-CN-stabilised emulsions can be predicted from the extent of ß-CN aggregation in aqueous solution (i.e., aggregate size).

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Hydroxyectoine, an ectoine derivative, is the most common compatible solute in halophilic microorganisms for resisting harsh environments. Compatible solutes can be utilized in fields such as cosmetics, medicine, and biochemistry. Moderately halophilic microorganisms produce much less hydroxyectoine as compared with ectoine. In this study, we first evaluate the effect of medium formulation (i.e., yeast extract (YE) medium and high yeast extract (HYE) medium) on hydroxyectoine production. In addition, an investigation of hydroxyectoine production by Halomonas salina under optimal conditions for vital factors (i.e., iron and α-ketoglutarate) and hydroxylase activity was also carried out. As a result, hydroxyectoine production was obviously elevated (0.9 g/L to 1.8 g/L) when the HYE medium was utilized. Furthermore, hydroxyectoine production further increased to 2.4 g/L when both the α-ketoglutarate and iron factors were added to the HYE medium in the early stationary phase. In addition, we found that ectoine hydroxylase activity increased more when a combination of iron and α-ketoglutarate was used than when either was used alone. The results showed that the alteration of iron and α-ketoglutarate clearly stimulated the expression of ectoine hydroxylase, which in turn affected hydroxyectoine synthesis. This study also showed that hydroxyectoine production was further raised from 2.4 g/L to 2.9 g/L when 50 mM of α-ketoglutarate and 1 mM of iron were added to the HYE medium. Ultimately, the experimental results showed using the optimal conditions further elevated the hydroxyectoine production yield to 2.90 g/L, which was over 3-fold higher than the best results obtained from the original medium.

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Hydrogels are widely used as debriding agents on account of providing a moist environment for wound healing, however the lack of mechanical strength, angiogenesis and antibacterial property limits their applications. In this study, we synthesized novel divalent ion cross-liking hydrogels (copper, zinc, strontium and calcium) and compared the mechanical performance, swelling ratio, antibacterial properties and biocompatibility in vitro and vivo. Thereinto, among the four divalent ions cross-linked hydrogels, copper ion crosslinking exhibited the maximum breaking strength, while strontium and zinc ion cross-linked hydrogels exhibited an excellent mechanical strength. In addition, the swelling ratio and pore size of no-ion cross-linked hydrogels was larger than ion cross-kinked hydrogels. In vitro, the improvements on wound healing after hydrogel application were evaluated by histological and molecular assays by detecting VEGF and TGF-ß expression. In vitro and in vivo study results showed that zinc cross-kinked hydrogel had a spectrum of antibacterial activities, cell viability, mechanical strength and the ability of wound closure by promoting fibroblasts migration, vascularization, collagen deposition and the formation of granulation tissue.

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Octopus bimaculoides is an important commercially fished species in the California Peninsula with aquaculture potential; however, to date limited information is available regarding its digestive physiology. The objective of this study was focused on biochemically characterizing the main enzymes involved in the digestion of O. bimaculoides. Optimum pH, temperature and thermostability were determined for amylases, lipases, trypsin and chymotrypsin; optimum pH and protease inhibitor effect were assessed for acidic and alkaline proteases, and the effect of divalent ions on trypsin and chymotrypsin activity was evaluated in enzymatic extracts from the digestive (DG) and salivary glands (SG) and crop gastric juices (GJ). High amylase activity was detected in GD and GJ whereas this activity is very low in other cephalopods. Salivary glands had the greatest activity in most of the enzyme groups, showing the importance of this organ in digestion. Optimum pH was different depending on the organ and enzyme analyzed. The optimum pH in DG was 3 showing the predominance of acidic proteases in the digestion process. All enzymes were resistant and stable at high temperatures in contrast with other marine species. Trypsin and chymotrypsin activity were highly incremented with the presence of Mg2+, Co2+, Cu2+ and Zn2+ in some tissues. The inhibitor assay showed the importance of serine proteases, metalloproteases and aspartic proteases in the digestive process of this species. This study is the first in assessing the main digestive enzymes of O. bimaculoides and in remarking the importance of other digestive enzyme groups besides proteases in octopuses.

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Optical emission behaviours of lysozyme and bovine serum albumin, from bulk and thin film geometry, were studied in the presence of three different divalent ions (Mg2+ , Ca2+ or Ba2+ ) using different spectroscopic [steady-state fluorescence, UV-Vis and Fourier transform infra-red (FTIR)] techniques. Additionally, protein thin films on silicon surfaces were prepared and morphological studies were carried out using atomic force microscopy. Dynamic quenching was mainly identified for both proteins in the presence of Mg2+ , Ca2+ and Ba2+ ions. The molecular conformation of the proteins was modified in thin films compared with that in solution, consequently quenching efficiencies also varied. ATR-FTIR studies confirmed the conformational changes of proteins in the presence of all divalent ions. All metal ions used were divalent in nature and belonged to the same group of the periodic table but, depending on their individual characteristics such as electron affinity, ionic radius, etc., the magnitude of the protein and hydrated ion interaction varied and accordingly the quenching efficiency was modified. Quenching was maximum for Ca2+ ions, followed by the other two ions. Our study clearly illustrates the geometry-dependent physical and biological functions of proteins.

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The Langmuir monolayers of L-α-phosphatidylcholine from egg yolk were studied by Wilhelmy method. The surface pressure versus molecular area isotherm of lipid on pure water and different subphase (with a presence of divalent ions: Sr2+, Cd2+, Ba2+, Pb2+) was obtained. The limiting area of the isotherms depends on the presence of subphase ions. The addition of divalent ions to the subphase stabilized the monolayers and increased the limiting areas of the monolayer. During the compression in monolayer complexes of 1:1 and 2:1 stoichiometry between L-α-phosphatidylcholine from egg yolk and divalent ions are formed. We used the equilibrium theory to describe the behavior of monolayer components at the air/water interface. An equilibrium theory to describe the behavior of monolayer components at the air/water interface was developed in order to obtain the stability constants and area occupied by one molecule of LMe2+ or L2Me2+ complexes, and complex formation energy (Gibbs free energy) values. These mathematically derived and experimentally confirmed values are of great importance for the interpretation of phenomena occurring in lipid monolayers and bilayers.

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This work aimed to study the influence of pH (3.5 and 7.0) and CaCl2 and MgCl2 addition on heat-set gelation of a quinoa protein isolate at 10% and 15% (w/w). The protein isolate obtained was composed mainly of 11S globulin as was observed by electrophoresis and mass spectrometry analysis. Heat-set gelation occurred at both pH values studied. Nevertheless, the gels formed at pH 3.5 were more viscoelastic and denser than those formed at pH 7.0, that was coarser and presented syneresis. The CaCl2 and MgCl2 addition increased the gel strength during rheological analysis at pH 3.5, possibly due to the formation of fiber-like connections in the gel network. At pH 7.0, the divalent salts resulted in weaker gels formed by agglomerates, suggesting a neutralization of the protein surface charges. The differences in quinoa protein gelation were attributed to solubility, and the flexibility of proteins secondary structure at the pH studied.

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BACKGROUND: One of the main challenges in snakebite envenomation treatment is the development of stable, versatile and efficient anti-venom therapies. Local myotoxicity in accidents involving snakes from the Bothrops genus is still a consequence of serum therapy inefficient neutralization that may lead to permanent sequelae in their victims. One of the classes of toxins that participate in muscle necrosis is the PLA2-like proteins. The aim of this work was to investigate the role of zinc ions in the inhibition of PLA2-like proteins and to advance the current knowledge of their action mechanism. METHODS: Myographic and electrophysiological techniques were used to evaluate the inhibitory effect of zinc ions, isothermal titration calorimetry assays were used to measure the affinity between zinc ions and the toxin and X-ray crystallography was used to reveal details of this interaction. RESULTS: We demonstrated that zinc ions can effectively inhibit the toxin by the interaction with two different sites, which are related to two different mechanism of inhibition: preventing membrane disruption and impairing the toxin state transition. Furthermore, structural study presented here included an additional step in the current myotoxic mechanism improving the comprehension of the allosteric transition that PLA2-like proteins undergo to exert their function. CONCLUSIONS: Our findings show that zinc ions are inhibitors of PLA2-like proteins and suggest two different mechanisms of inhibition for these ions. GENERAL SIGNIFICANCE: Zinc is a new candidate that can assist in anti-venom treatments and can promote the design of new and even more accurate structure-based inhibitors for PLA2-like proteins.

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