High Ca2 Research Articles

Nernst–Planck–Gaussian finite element modelling of Ca2+ electrodiffusion in amphibian striated muscle transverse tubule–sarcoplasmic reticular triadic junctional domains

IntroductionIntracellular Ca2+ signalling regulates membrane permeabilities, enzyme activity, and gene transcription amongst other functions. Large transmembrane Ca2+ electrochemical gradients and low diffusibility between cell compartments potentially generate short-lived, localised, high-[Ca2+] microdomains. The highest concentration domains likely form between closely apposed membranes, as at amphibian skeletal muscle transverse tubule–sarcoplasmic reticular (T-SR, triad) junctions.Materials and methodsFinite element computational analysis characterised the formation and steady state and kinetic properties of the Ca2+ microdomains using established empirical physiological and anatomical values. It progressively incorporated Fick diffusion and Nernst–Planck electrodiffusion gradients, K+, Cl−, and Donnan protein, and calmodulin (CaM)-mediated Ca2+ buffering. It solved for temporal–spatial patterns of free and buffered Ca2+, Gaussian charge differences, and membrane potential changes, following Ca2+ release into the T-SR junction.ResultsComputational runs using established low and high Ca2+ diffusibility (DCa2+) limits both showed that voltages arising from intracytosolic total [Ca2+] gradients and the counterions little affected microdomain formation, although elevated DCa2+ reduced attained [Ca2+] and facilitated its kinetics. Contrastingly, adopting known cytosolic CaM concentrations and CaM-Ca2+ affinities markedly increased steady-state free ([Ca2+]free) and total ([Ca2+]), albeit slowing microdomain formation, all to extents reduced by high DCa2+. However, both low and high DCa2+ yielded predictions of similar, physiologically effective, [Ca2+-CaM]. This Ca2+ trapping by the relatively immobile CaM particularly increased [Ca2+] at the junction centre. [Ca2+]free, [Ca2+-CaM], [Ca2+], and microdomain kinetics all depended on both CaM-Ca2+ affinity and DCa2+. These changes accompanied only small Gaussian (∼6 mV) and surface charge (∼1 mV) effects on tubular transmembrane potential at either DCa2+.ConclusionThese physical predictions of T-SR Ca2+ microdomain formation and properties are compatible with the microdomain roles in Ca2+ and Ca2+-CaM-mediated signalling but limited the effects on tubular transmembrane potentials. CaM emerges as a potential major regulator of both the kinetics and the extent of microdomain formation. These possible cellular Ca2+ signalling roles are discussed in relation to possible feedback modulation processes sensitive to the μM domain but not nM bulk cytosolic, [Ca2+]free, and [Ca2+-CaM], including ryanodine receptor-mediated SR Ca2+ release; Na+, K+, and Cl− channel-mediated membrane excitation and stabilisation; and Na+/Ca2+ exchange transport.

All-optical mapping of Ca2+ transport and homeostasis in dendrites

Calcium mediates many important signals in dendrites. However, the basic transport properties of calcium in dendrites have been difficult to measure: how far and how fast does a local influx of calcium propagate? We developed an all-optical system for simultaneous targeted Ca2+ import and Ca2+ concentration mapping. We co-expressed a blue light-activated calcium selective channelrhodopsin, CapChR2, with a far-red calcium sensor, FR-GECO1c, in cultured rat hippocampal neurons, and used patterned optogenetic stimulation to introduce calcium into cells with user-defined patterns of space and time. We determined a mean steady-state length constant for Ca2+ transport ϕ ∼ 5.8 μm, a half-life for return to baseline t1/2 ∼ 1.7 s, and an effective diffusion coefficient D ∼ 20 μm2/s, though there were substantial differences in Ca2+ dynamics between proximal and distal dendrites. At high Ca2+ concentration, distal dendrites showed nonlinear activation of Ca2+ efflux, which we pharmacologically ascribed to the NCX1 antiporter. Genetically encoded tools for all-optical mapping of Ca2+ transport and handling provide a powerful capability for studying this important messenger.

Synergistic removal of ultra-fine coal particles and Ca2+/Mg2+ from mine wastewater via dual-stage adsorption prior to membrane filtration

Mining effluent cannot be directly employed for de-dusting purposes in the mines, on account of containing suspended solids (SS), Ca2+, and Mg2+. Although extant methods can eliminate them, yet no facile method has been contrived to simultaneously eradicate ultra-fine coal particles (∼4 μm) and hardness from the mining effluent. As such, a dual-adsorption process prior to membrane filtration was proffered by availing the preliminary adsorption of Ca2+ and Mg2+ to ultra-fine coal particles in the bulk solution and the secondary adsorption of Ca2+ and Mg2+ onto the cake layer. Experimental findings revealed that in contradistinction to the standalone membrane process, the hybrid process could attain a higher SS removal escalating from 99 % to 99.9 % and higher Ca2+ and Mg2+ removal rates (the removal rate for Ca2+ increased from 11.26 % to 22.12 % by 96.45 %, and the removal rate for Mg2+ increased from 11.73 % to 23.02 % by 96.25 %). Compared with other four commercial membranes (PES, CA, PTFE, and PP), the PVDF membrane with an average pore size of 0.22 μm performed the best with respect to the simultaneous removal of SS, Ca2+, and Mg2+. Thereafter, all SS removals exceeded 99.9 % and bore a tenuous connection to operating temperature, flow rate, and transmembrane pressure. Nevertheless, the removal rates of Ca2+ and Mg2+ diminished with the increase of transmembrane pressure, but exhibited a tendency of initially ascending and then descending as accelerating the flow rate. The optimal operating temperature fell within the purview of the acceptable operational temperature (<30 °C) in the coal mine. Hence, the proposed hybrid system provides one novel approach to concomitantly eliminate SS, Ca2+, and Mg2+ by availing not additional chemical reagents but the wastes (ultra-fine coal particles) encompassed in mine wastewater.

Excellent quality acquisition of myofibrillar protein in red shrimp (Solenocera crassicornis) based on regulating the oxidation degree of atmospheric cold plasma treatment.

Myofibrillar protein (MP) is essential for the texture and taste of shrimp surimi products. The reactive oxygen/nitrogen species (ROS/RNS) produced by atmospheric cold plasma (ACP) might cause oxidative modification of MP. In this study, the effect of different ACP treatment times on the properties of red shrimp MP was investigated in detail. The mild oxidation induced by ACP treatment for 1 min (ACP-1 min) promoted the unfolding and refolding of the MP structure, which was manifested as a transition from α-helix to β-sheets. Compared with other groups, more hydrogen bonds and hydrophobic interactions in the ACP-1 min group might enhance the interactions between the myosin heavy chain and actin, which was conducive to the formation of a regular and dense three-dimensional network structure. Person correlation analysis revealed that ROS/RNS mediated the changes in the secondary and tertiary structure of MP. In addition, ACP-1 min has high Ca2+-ATPase activity, which reflected that MP maintained structural integrity. While excessive oxidation in the ACP treatments for 3 min and 5 min reduced MP robustness. The stable internal structure in ACP-1 min group gave the MP excellent texture profile (1.79 mJ of adhesiveness and 0.89 mm of springiness) and rheological characteristics (0.373 MPa of storage modulus). Overall, the excellent quality of MP could be obtained by regulating the oxidation degree of ACP, which would provide valuable information for the in-depth and efficient processing of shrimp products. © 2024 Society of Chemical Industry.

Role of ryanodine receptor cooperativity in Ca2+-wave-mediated triggered activity in cardiomyocytes.

Ca2+ waves are known to trigger delayed after-depolarizations that can cause malignant cardiac arrhythmias. However, modelling Ca2+ waves using physiologically realistic models has remained a major challenge. Existing models with low Ca2+ sensitivity of ryanodine receptors (RyRs) necessitate large release currents, leading to an unrealistically large Ca2+ transient amplitude incompatible with the experimental observations. Consequently, current physiologically detailed models of delayed after-depolarizations resort to unrealistic cell architectures to produce Ca2+ waves with a normal Ca2+ transient amplitude. Here, we address these challenges by incorporating RyR cooperativity into a physiologically detailed model with a realistic cell architecture. We represent RyR cooperativity phenomenologically through a Hill coefficient within the sigmoid function of RyR open probability. Simulations in permeabilized myocytes with high Ca2+ sensitivity reveal that a sufficiently large Hill coefficient is required for Ca2+ wave propagation via the fire-diffuse-fire mechanism. In intact myocytes, propagating Ca2+ waves can occur only within an intermediate Hill coefficient range. Within this range, the spark rate is neither too low, enabling Ca2+ wave propagation, nor too high, allowing for the maintenance of a high sarcoplasmic reticulum load during diastole of the action potential. Moreover, this model successfully replicates other experimentally observed manifestations of Ca2+-wave-mediated triggered activity, including phase 2 and phase 3 early after-depolarizations and high-frequency voltage-Ca2+ oscillations. These oscillations feature an elevated take-off potential with depolarization mediated by the L-type Ca2+ current. The model also sheds light on the roles of luminal gating of RyRs and the mobile buffer ATP in the genesis of these arrhythmogenic phenomena. KEY POINTS: Existing mathematical models of Ca2+ waves use an excessively large Ca2+-release current or unrealistic diffusive coupling between release units. Our physiologically realistic model, using a Hill coefficient in the ryanodine receptor (RyR) gating function to represent RyR cooperativity, addresses these limitations and generates organized Ca2+ waves at Hill coefficients ranging from ∼5 to 10, as opposed to the traditional value of 2. This range of Hill coefficients gives a spark rate neither too low, thereby enabling Ca2+ wave propagation, nor too high, allowing for the maintenance of a high sarcoplasmic reticulum load during the plateau phase of the action potential. Additionally, the model generates Ca2+-wave-mediated phase 2 and phase 3 early after-depolarizations, and coupled membrane voltage with Ca2+ oscillations mediated by the L-type Ca2+ current. This study suggests that pharmacologically targeting RyR cooperativity could be a promising strategy for treating cardiac arrhythmias linked to Ca2+-wave-mediated triggered activity.

Voltage- and Ca2+-inducible PLC activity for analyzing PI(4,5)P2 sensitivity of ion channels in Xenopus oocytes

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key membrane lipid regulating various ion channel activities. Currently, several molecular tools are used to modulate PIP2 levels, each of which has distinct advantages and drawbacks. In this study, we proposed a novel methodology using heterologous Xenopus oocytes to precisely manipulate PIP2 levels using phospholipase C (PLC)-ζ, which hydrolyzes PIP2. Xenopus oocytes injected with PLCζ exhibited notable hyperpolarization-induced Ca2+ influx driven by the increased driving force of Ca2+. High Ca2+ sensitivity of PLCζ facilitated hyperpolarization-induced PLC activity in Xenopus oocytes that was voltage- and Ca2+-dependent. This study demonstrated the regulatory capacity of PLCζ in modulating PIP2-sensitive ion channels, such as the KCNQ2/3 and GIRK channels, in a voltage- and Ca2+-dependent manner. Moreover, activation pathway of PLCζ only requires a two-electrode voltage clamp setup, making it a convenient molecular tool to manipulate PIP2 levels in combination with a voltage-sensing phosphatase (VSP). PLCζ has distinct characteristics and advantages compared to VSP: (1) Hyperpolarization, but not depolarization, reduced the PIP2 levels, (2) PIP2 levels were decreased without any increase in phosphatidylinositol 4-monophosphate (PIP) levels, and (3) PIP2 levels were reduced by Ca2+ administration. Therefore, PLCζ effectively supports understanding how PIP2 regulates ion channels, alongside VSP. Overall, this study highlights the unique characteristics of PLCζ and its distinct advantages in analyzing ion channel regulation by PIP2 and the PLC pathway in Xenopus oocytes.

Carvedilol suppresses coronary artery spasm in A-kinase anchoring protein 150 knockout mouse

Abstract Backgrounds A-kinase anchoring proteins (AKAPs) act as scaffold to regulate activation of protein kinases and subsequent downstream signaling. AKAP 150 interacts with protein kinase A and protein kinase C, and has a critical role in regulating L-type calcium channel activity. We previously reported enhanced phospholipase C activity as an important mechanism of coronary spastic angina (CSA), known as Prinzmetal’s angina. Coronary artery spasm was induced by intravenous ergometrine injection in mouse model of vascular smooth muscle cell (VSMC)-specific enhanced phospholipase C activity concomitantly with enhanced intracellular Ca2+ concentration ([Ca2+]i). Then, we hypothesized that coronary artery spasm would be inhibited by AKAP 150 knockout, however, coronary artery spasm was unexpectedly induced in AKAP 150 knockout (AKAP-KO) mice without affecting [Ca2+]i, suggesting that AKAP-KO mouse is an unique model of CSA independent of [Ca2+]i. Previous report showed that AKAP-KO mice have a high Ca2+/calmodulin-dependent protein kinase (CaMK) II activity, and carvedilol, a non-selective beta-adrenergic receptor antagonist, inhibits CaMK II activity. Aim We tested the hypothesis that carvedilol could prevent coronary artery spasm via inhibiting CaMK II activity, which is a key molecule to regulate VSMC contraction, in AKAP-KO mice. Methods and Results We pretreated 14-18-weeks-old AKAP-KO mice with intraperitoneal injection of carvedilol (2.5mg/kg), propranolol (20mg/kg), or vehicle (control). Coronary artery spasm, evaluated by ST-segment elevation on lead II of body surface electrocardiogram, was induced by intravenous ergometrine injection (30mg/kg) in all controls (5/5, 100%) and all propranolol-treated mice (5/5, 100%), whereas it tended to be inhibited in carvedilol-treated mice (3/6, 50%) (p=0.08 vs control). In isolated VSMCs obtained from the aorta of AKAO-KO mice, the changes in [Ca2+]i from baseline in response to acetylcholine (ACh, 100μM) did not differ among the groups of pretreatment with carvedilol (10μM), propranolol (10μM), or vehicle, indicating that coronary artery spasm in AKAP-KO mice was induced by Ca2+-independent mechanism. In response to ACh, the phosphorylation of CaMK II was tended to be decreased (p=0.07), and the phosphorylation of myosin light chain was decreased (p&amp;lt;0.05) in VSMCs treated with carvedilol compared with control. Conclusions Carvedilol may inhibit coronary artery spasm probably via inhibiting CaMK II activity in AKAP-KO mice. Our results may provide a new paradigm into the pathogenesis of CSA as a model of enhanced Ca2+ sensitivity and important clinical implications for the mechanism of calcium channel antagonist-refractory coronary artery spasm.

Incorporating concrete waste as additive in acidic fermentation–A novel approach for enhanced biohydrogen production and concrete mass reduction

Acidic fermentation (AF) of organic waste/wastewater generates valuable byproducts, including hydrogen, volatile fatty acids, and ethanol; however, its sensitivity to pH drops limits the stability and efficiency of the process. A reversal process, the acidic chemical treatment of concrete waste (CW) to recycle its aggregates, generates calcium hydroxide, which can serve as buffering agent. Meanwhile, integrating both processes can introduce a sustainable win-win approach, which is the aim of this study. To assess this approach, a series of batch AF experiments were conducted at 50 °C and pH 5.5, using glucose as the substrate. Cementitious specimen (1.5 × 1.5 × 0.8 cm) was supplemented to each 200 mL-fermenter. Unamended fermenters, with and without additional NaHCO3-buffering, were used as controls to compare with the amended ones. Introducing cementitious specimens to AF increased H2-production by 2-fold and 1.7-fold compared to controls with and without NaHCO3-addition, respectively, after three consecutive feed-cycles. The surface analysis of incorporated specimen confirmed the Ca, Al, Mg, and Si leaching. The AF efficiency and resulting cementitious mass reduction were further assessed at different organic loads and specimens’ volume, surface area, and porosity (by changing water-to-cement [W/C] ratio). Increasing the organic load from 10 to 20 g-glucose/L resulted in lower H2-production, higher specimen mass reduction (up to ∼32%), and higher Ca2+ release (up to 2 g/L); however, no significant effect was observed when using specimens with higher W/C ratio or surface area. Moreover, the presence of cementitious specimens significantly influenced the microbial composition, leading to notable developments in the abundant genera Thermoanaerobacterium and Bacillus. This study presents a novel approach to sustainably enhancing AF process using CW as both an additive and a treatable substance, with reusable aggregates as a byproduct. It provides valuable insights for optimizing the process and guiding future practical applications. This includes considering various concrete compositions, adjusting organic load conditions, and evaluating long-term stability in larger-scale systems.

Histidine-rich calcium-binding protein: a molecular integrator of cardiac excitation-contraction coupling.

During mammalian cardiomyocyte excitation-contraction coupling, Ca2+ influx through voltage-gated Ca2+ channels triggers Ca2+ release from the sarcoplasmic reticulum (SR) through ryanodine receptor channels. This Ca2+-induced Ca2+ release mechanism controls cardiomyocyte contraction and is exquisitely regulated by SR Ca2+ levels. The histidine-rich calcium-binding protein (HRC) and its aspartic acid-rich paralogue aspolin are high-capacity, low-affinity Ca2+-binding proteins. Aspolin also acts as a trimethylamine N-oxide demethylase. At low intraluminal Ca2+ concentrations, HRC binds to the SR Ca2+-ATPase 2, inhibiting its Ca2+-pumping activity. At high intraluminal Ca2+ levels, HRC interacts with triadin to reduce Ca2+ release through ryanodine receptor channels. This Review analyses the evolution of these Ca2+-regulatory proteins, to gain insights into their roles. It reveals that HRC homologues are present in chordates, annelid worms, molluscs, corals and sea anemones. In contrast, triadin appears to be a chordate innovation. Furthermore, HRC is evolving more rapidly than other cardiac excitation-contraction coupling proteins. This positive selection (or relaxed negative selection) occurs along most of the mammalian HRC protein sequence, with the exception being the C-terminal cysteine-rich region, which is undergoing negative selection. The histidine-rich region of HRC might be involved in pH sensing, as an adaptation to air-breathing, endothermic and terrestrial life. In addition, a cysteine-rich pattern within HRC and aspolin is also found in a wide range of iron-sulfur cluster proteins, suggesting roles in redox reactions and metal binding. The polyaspartic regions of aspolins are likely to underlie their trimethylamine N-oxide demethylase activity, which might be mimicked by the acidic regions of HRCs. These potential roles of HRCs and aspolins await experimental verification.

Cryoprotective effect of magnetic field-assisted freezing in combination with curdlan on myofibrillar protein of Litopenaeus vannamei: Oxidative aggregation, protein structure and thermal stability

To counteract the negative effects of conventional freezing methods on frozen surimi products, the present study investigated the effects of magnetic field (MF) freezing in combination with different amounts of curdlan (CUR) on oxidative denaturation, structural alterations, and thermal stability of myofibrillar protein (MP) in shrimp surimi. The results indicated that the intervention of magnetic fields alone improved the quality of frozen muscle proteins. Under the dual intervention of magnetic field-assisted freezing and CUR, small ice crystals formed with MF6 treatment resulted in significantly lower solubility, turbidity, and mean particle size than the control group (p < 0.05). Moreover, the oxidation of MP was also significantly slowed down by inhibiting the formation of hydrophobic groups and carbonyl groups, maintaining a high content of sulfhydryl groups. MF6 also exhibited a high Ca2+-ATPase activity. The α-helix content, the increase in fluorescence intensity under this condition also tend to make the secondary and tertiary structures of myofibrillar protein more stable. Meanwhile, electrophoretic profiles and CLSM showed that this condition maintains the integrity of the MP. In addition, the MF6 samples also had high protein thermal stability. However, excess CUR reduces the excellence of MP cryoprotection. As a result, MF6 has better protection and improvement effects on protein function, structure and thermal stability, and CUR was also found to have the potential for cryoprotectant development.

SUMOylation-induced membrane localization of TRPV1 suppresses proliferation and migration in gastric cancer cells

Gastric cancer (GC) remains a significant health challenge due to its high mortality rate and the limited efficacy of current targeted therapies. A critical barrier in developing more effective treatments is the lack of understanding of specific mechanisms driving GC progression. This study investigates the role of Transient Receptor Potential Vanilloid 1 (TRPV1), a non-selective cation channel known for its high Ca2+ permeability and tumor-suppressive properties in gastrointestinal cancers. Specifically, we explore the impact of SUMOylation—a dynamic and reversible post-translational modification—on TRPV1’s function in GC. We demonstrate that SUMOylation of TRPV1 inhibits cell proliferation and migration in MGC-803 and AGS GC cells. By mutating amino acids near TRPV1’s existing SUMO motif (slKpE), we created a bidirectional SUMO motif (EψKψE) that enhances TRPV1 SUMOylation, resulting in further suppression of GC cell proliferation and migration. In vivo studies support these findings, showing that TRPV1 SUMOylation prevents spontaneous tumorigenesis in a mouse GC model. Further investigation reveals that TRPV1 SUMOylation increases the protein’s membrane expression by inhibiting its interaction with the adaptor-related protein complex 2 mu 1 subunit (AP2M1). This elevated membrane expression leads to increased intracellular Ca2+ influx, activating the AMP-activated protein kinase (AMPK) pathway, which in turn inhibits the proliferation and migration of GC cells.

Physiological and differential protein expression analyses of the calcium stress response in the Drynaria roosii rhizome

High concentration Ca2+ in karst soil is harmful to agriculture. Some dominant plants can adapt well to karst soil, but how Ca2+ affect plant is unknown. Drynaria roosii is a Ca2+-tolerant fern and its dry rhizome is a common Chinese medicine of Miao nationality in Guizhou, China. This study analyzed the physiological and proteomic characteristics of the rhizome of D. roosii under calcium stress. Physiological results indicated that calcium stress may lead to osmotic stress. Proteomic results showed that 147 differentially expressed proteins (96 increased, 51decreased) were identified under calcium stress, and these proteins mainly involved in signal transduction, protein translation, material transport, antioxidant defense and secondary metabolism. This study will lay a foundation for studying the calcium adaptation mechanism of D. roosii at the molecular level.

Target dispersion of pozzolanic materials nearby hydration-released calcium hydrates to improve the pozzolanic reaction degree

The random distribution of pozzolanic materials (PM) using direct mixing strategy reduces their contact possibility with hydration-released calcium hydroxide (CH), and limits the corresponding reinforcing efficiency to cement-based materials via pozzolanic reaction. In this study, a target dispersion strategy of PM nearby CH was proposed using super absorbent polymers (SAPs) as the carrier. On the one hand, nano silica (NS) and calcined clay (CC), two typical PM, can be encapsulated into SAPs through the hydrogen bonding interaction; on the other hand, the region around SAPs was enriched with more water and higher Ca2+ concentration, which triggered the in situ crystallization of CH. In this case, the contact possibility between the NS or CC (NS/CC) and hydration-released CH was improved by the target dispersion strategy, and resulted in a 60 %–253 % increase in pozzolanic reaction degree (PRD) of NS/CC. Furthermore, the reinforcing effect of NS/CC on compressive strength of cement paste was enhanced by 25 % through this strategy. Therefore, the current study introduces a novel method for increasing the pozzolanic reinforcing efficiency of cement-based materials by turning the random to target dispersion of PM nearby CH.

Probing plant signal processing optogenetically by two channelrhodopsins.

Early plant responses to different stress situations often encompass cytosolic Ca2+ increases, plasma membrane depolarization and the generation of reactive oxygen species1-3. However, the mechanisms by which these signalling elements are translated into defined physiological outcomes are poorly understood. Here, to study the basis for encoding of specificity in plant signal processing, we used light-gated ion channels (channelrhodopsins). We developed a genetically engineered channelrhodopsin variant called XXM 2.0 with high Ca2+ conductance that enabled triggering cytosolic Ca2+ elevations in planta. Plant responses tolight-induced Ca2+ influx through XXM 2.0 were studied side by side with effects caused by an anion efflux through the light-gated anion channelrhodopsin ACR1 2.04. Although both tools triggered membrane depolarizations, their activation led to distinct plant stress responses: XXM 2.0-induced Ca2+ signals stimulated production of reactive oxygen species and defence mechanisms; ACR1 2.0-mediated anion efflux triggered drought stress responses. Our findings imply that discrete Ca2+ signals and anion efflux serve as triggers for specific metabolic and transcriptional reprogramming enabling plants to adapt to particular stress situations. Our optogenetics approach unveiled that within plant leaves, distinct physiological responses are triggered by specific ion fluxes, which are accompanied by similar electrical signals.

Calcium-Mediated Cell Adhesion Enhancement-Based Antimetastasis and Synergistic Antitumor Therapy by Conjugated Polymer-Calcium Composite Nanoparticles.

Strengthening tumor cellular adhesion through regulating the concentration of extracellular Ca2+ is highly challenging and promising for antimetastasis. Herein, a pH-responsive conjugated polymer-calcium composite nanoparticle (PFV/CaCO3/PDA@PEG) is developed for calcium-mediated cell adhesion enhancement-based antimetastasis and reactive oxygen species (ROS)-triggered calcium overload and photodynamic therapy (PDT) synergistic tumor treatment. PFV/CaCO3/PDA@PEG is mainly equipped with conjugated poly(fluorene-co-vinylene) (PFV-COOH)-composited CaCO3 nanoparticles, which can be rapidly decomposed under the tumor acidic microenvironment, effectively releasing Ca2+ and the photosensitizer PFV-COOH. The high extracellular Ca2+ concentration facilitates the generation of dimers between two adjacent cadherin ectodomains, which greatly enhances cell-cell adhesion and suppresses tumor metastasis. The inhibition rates are 97 and 87% for highly metastatic tumor cells 4T1 and MCF-7, respectively. Such a well-designed nanoparticle also contributes to realizing PDT, mitochondrial dysfunction, and ROS-triggered Ca2+ overload synergistic therapy. Furthermore, PFV/CaCO3/PDA@PEG displayed superior in vivo inhibition of 4T1 tumor growth and demonstrated a marked antimetastatic effect by both intravenous and intratumoral injection modes. Thus, this study provides a powerful strategy for calcium-mediated metastasis inhibition for tumor therapy.

Effects of calcium ions and polysaccharides type on transparent exopolymer particle formation and the related fouling mechanisms

Organics and divalent cations are the primary barriers constraining the performance of membrane technology, while the interactions between them and the detailed mechanisms of their impacts are still lacking in-depth analysis. In this study, sodium alginate and xanthan gum were selected as polysaccharides models, and the formation of transparent extracellular polymer particles (TEP) was assessed to examine the effect of Ca2+ and polysaccharides type on membrane fouling from both qualitative and quantitative perspectives. The results revealed that higher Ca2+ concentrations led to a greater abundance of TEP, and the transformation of TEP microstructure is a key factor for the membrane fouling change indicated by specific filtration resistance (SFR). TEP formed by sodium alginate underwent a transformation from amorphous-TEP (a-TEP) form to particle-TEP (p-TEP), corresponding to a unimodal pattern of SFR variation. With increasing Ca2+ concentration, the molecular interactions of xanthan gum became stronger, resulting in larger fibrous a-TEP and a continuous SFR increase. According to the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory, TEP formed by xanthan gum exhibited higher adhesion energy, thus causing more severe membrane fouling. The SFR variation of the TEP system can be satisfactorily explained by the conception of chemical potential change in the filtration process depicted in Flory-Huggins theory. This study is the first work to introduce models regarding chemical potential and TEP microstructure, linking the system chemical potential and TEP microstructure with membrane fouling indicated by SFR. As all, this study provided a new perspective for analyzing the polysaccharide fouling behavior via TEP determination and further enhanced the understanding through thermodynamic analysis.

Mitogen-Activated Protein Kinase Kinase OsMEK2 Positively Regulates Ca2+ Influx and Ferroptotic Cell Death during Rice Immune Responses.

Mitogen-activated protein (MAP) kinase (MAPK) signaling pathway is important in plant immune responses, involved in iron- and reactive oxygen species (ROS)-dependent ferroptotic cell death mediated by Ca2+. High Ca2+ influx triggered iron-dependent ROS accumulation, lipid peroxidation, and subsequent hypersensitive response (HR) cell death in rice (Oryza sativa). Apoplastic Ca2+ chelation by EGTA during avirulent Magnaporthe oryzae infection altered Ca2+, ROS, and Fe2+ accumulation, increasing rice susceptibility to infection. By contrast, acibenzolar-S-methyl (ASM), a plant defense activator, significantly enhanced Ca2+ influx, and H2O2 accumulation, triggering rice ferroptotic cell death during virulent Magnaporthe oryzae infection. Here, we report a novel role of the MAPK signaling pathway in regulating cytoplasmic Ca2+ increase during ferroptotic cell death in rice immunity, using the ΔOsmek2 knockout mutant rice. The knockout of rice OsMEK2 impaired the ROS accumulation, lipid peroxidation, and iron accumulation during avirulent M. oryzae infection. This study has shown that OsMEK2 could positively regulate iron- and ROS-dependent ferroptotic cell death in rice by modulating the expression of OsNADP-ME, OsRBOHB, OsPLC, and OsCNGC. This modulation indicates a possible mechanism for how OsMEK2 participates in Ca2+ regulation in rice ferroptotic cell death, suggesting its broader role in plant immune responses in response to M. oryzae infection.

Additive Manufactured Multiplex Sensing Structures for Attomole-Sensitive Detection of Hypocalcemia

INTRODUCTION In the United States, 30-50% of dairy cows experience metabolic disorders due to nutritional deficiencies, leading to economic losses, especially from reduced milk yield due to hypocalcemia. Calcium is crucial for muscle and nerve function, skeletal strength, and immune function. Laboratory screening costs $60 per sample, and on-site testing costs around $1000. With approximately 33% subclinical and 5% clinical hypocalcemia prevalence in US dairy herds, there's a need for smart, cost-effective sensors. Early diagnosis is vital for disease prevention, relying on rapid, farm-side, accurate assessment of calcium (Ca2+) and phosphate (P) ion levels.Herein, an attomole-sensitive sensor was developed using 3D printed structures to detect ionized phosphate to calcium ratios in milk samples, diagnosing milk fever. Unique 3D printed sensor geometries with wrinkled surfaces provide an unprecedented limit-of-detection of 138 aM. Wrinkled surfaces of poly (3-octyl-thiophene) act as ion-to-electron transducers for Ca2+ and P detection. The P/Ca2+ ratio measurement with potentiometry technique not only indicates early disease but also enables on-site testing without any need for mediator or sample preparation. Our sensor validated using cow’s milk, whole milk, and fetal bovine serum, and results were compared with commercial meters. This low-cost, sensitive, and fast response (~10 s) sensor is a promising tool for in-farm diagnostic facilitating management of dairy cows. SENSOR FABRICATION The manufacturing process begins with 3D CAD design of the sensor base (Figure 3c). The sensor is produced using a high-resolution extrusion-based 3D printer, enables the manufacturing of microstructures. Prints undergo washing process, and resin is cured with UV light (405 nm). Figure 2b shows two distinct microstructure types on the sensor base, amplifying surface area. The lateral pattern enhances surface area, aiding 3D microelectrode formation for analyte diffusion. Besides, random wrinkles form unintentionally due to resin instability during UV exposure, serving as microelectrodes and increasing surface area, leading to enhanced sensitivity. Next, a Kapton shadow mask covers the sensor's surface, and an e-beam evaporator applies a gold thin layer to uncovered regions. Ca2+ and P ion sensing electrodes are modified with POT as the ion-to-electron layer, followed by drop-cast Ca2+ and P ion-selective membrane (ISM) on top of POT layer. The reference electrode is constructed by applying Ag/AgCl paste between two working electrodes, covered with a Nafion layer for stability and Cl¯ leaking prevention. To improve repeatability, each sensor section is treated with a high ion concentration solution of Ca2+ and P. RESULTS AND DISCUSSION Surface characterization of the electrode involved electrochemical and surface analyses. FE-SEM images in Figure 2b, d show the lateral pattern and micro-wrinkles of the sensor base. Cyclic voltammetry investigated electrochemical behavior, revealing distinct peaks during ferro/ferricyanide presence (Figure 2a). Gold catalyzed mediator redox reaction, with the POT layer decreasing peak currents due to increased surface resistance. The ISM, primarily PVC, elevated electrical resistance, and selectively allowing Ca2+ and P to pass through rather than mediators, reducing current. Electrochemical Impedance Spectroscopy confirmed findings, with Nyquist plots showing charge transfer resistance (Rct) in Figure 2c. Open circuit potentiometry (OCP) detected Ca2+ and P ions (Figure 1a, 1c). OCP response increasing with higher Ca2+ concentration and decreasing with elevated P ion concentration. Calibration graphs in Figure 1b, 1d demonstrated a linear relationship between ion concentrations and OCP. OCP measurements across various concentrations, sensor washing, and repeated measurements in Figure 1a, 1c indicated sensors repeatability. Finally, sensor tests with fresh milk samples from 15 lactating Holstein multiparous cows revealed notable variation in P levels compared to Ca2+ concentration. Examining the P/Ca2+ ion ratio in milk samples (Figure 6f) assessed milk fever in dairy cows, indicating health status in terms of hypocalcemia, and hypercalcemia. Note: The high quality images will be provided in the poster. Figure 1

Acidity-Responsive Fe-PDA@CaCO3 Nanoparticles for Photothermal-Enhanced Calcium-Overload- and Reactive-Oxygen-Species-Mediated Tumor Therapy.

Calcium-overload-mediated tumor therapy has received considerable interest in oncology. However, its efficacy has been proven to be inadequate due to insufficient calcium ion concentration at the tumor site coupled with challenges in facilitating efficient calcium uptake by tumors, leading to unsatisfactory therapeutic outcomes. In the present study, calcium carbonate nanoshell mineralized ferric polydopamine nanoparticles (Fe-PDA@CaCO3 NPs) were prepared for achieving Ca2+-overload-mediated tumor therapy. Upon entering the tumor site, the pH-responsive CaCO3 layer, acting as a "Ca2+ storage pool", rapidly degraded and released high quantities of free Ca2+ within the weakly acidic environment. The Fe-PDA core, with its excellent photothermal conversion properties, could significantly increase the temperature upon exposure to near-infrared (NIR) light irradiation, thereby activating the TRPV1 channel and leading to a large influx of released Ca2+ into the cytoplasm. Furthermore, the exposed Fe-PDA core could react with the tumor-overexpressed hydrogen peroxide (H2O2) to efficiently produce hydroxyl radicals (•OH), significantly increasing intracellular reactive oxygen species (ROS) levels and thus inhibiting the activity of the Ca2+ efflux pump, resulting in a high intracellular Ca2+ concentration. Ultimately, the increase in calcium/ROS levels could disrupt mitochondrial homeostasis and activate the apoptosis pathway. The current work presents a promising approach for tumor therapy using photothermal-enhanced calcium-overload-mediated ion interference therapy and chemodynamic therapy.

Naturally derived double-network hydrogels with application as flexible adhesive sensors

Hydrogels have been widely used in the biomedical field, including wearable sensors and biological adhesives. However, achieving a balance between various functionalities, such as wet adhesion, stable conductivity, and biocompatibility, in one customized hydrogel has been a challenging issue. In this study, we developed a multifunctional hydrogel comprising recombinant human collagen (RHC) and aldehyde-modified sodium alginate (Ald-alginate), which was primarily crosslinked through a Schiff-base reaction and metal chelation. Due to the combination of a dynamic covalent crosslinking network (imine linkage between RHC and Ald-alginate) and a dynamic ionic crosslinking network (ionic bonding between Ca2+ and Ald-alginate), the hydrogel exhibited excellent self-healing and injectable behaviors. Benefiting from the high Ca2+ content, the hydrogel also attained antifreezing and conductivity properties. In addition to its excellent conductivity and biocompatibility, the hydrogel exhibited strong wet tissue adhesion ability and could adhere rapidly and strongly to the surfaces of various objects or biological tissues, forming a good sealing environment. Moreover, the hydrogel could be directly adhered to a tissue surface as a flexible sensor to accurately detect physiological signals. The versatility of this multifunctional hydrogel will open new avenues for biomedical applications, such as bioadhesives and biosensing.