Internal Calcium Research Articles

Mathematical model for IP$_{3}$ dependent calcium oscillations and mitochondrial associate membranes in non-excitable cells

Theoretical studies on calcium oscillations within the cytosolic [Ca$^{2+}$], and mitochondria [Ca$^{2+}$]$_{mit}$ have been conducted using a mathematical model-based approach. The model incorporates the mechanism of calcium-induced calcium release (CICR) through the activation of inositol-trisphosphate receptors (IPR), with a focus on the endoplasmic reticulum (ER) as an internal calcium store. The production of 1,4,5 inositol-trisphosphate (IP$_{3}$) through the phospholipase \(C\) isoforms and its degradation via Ca$^{2+}$ are considered, with IP$_{3}$ playing a crucial role in modulating calcium release from the ER. The model includes a simple kinetic mechanism for mitochondrial calcium uptake, release and physical connections between the ER and mitochondria, known as mitochondrial associate membrane complexes (MAMs), which influence cellular calcium homeostasis. Bifurcation analysis is used to explore the different dynamic properties of the model, identifying various regimes of oscillatory behavior and how these regimes change in response to different levels of stimulation, highlighting the complex regulatory mechanisms governing intracellular calcium signaling.

Evaluation of the efficacy of different irrigation activation techniques in removing of calcium hydroxide on teeth with the simulated internal root resorption cavity: a confocal laser scanning microscope analysis.

The aim of this study is to evaluate the effect of different irrigation activation methods on root canal sealer penetration in teeth with simulated internal root resorption (IRR) and calcium hydroxide (CH) applied using a confocal laser scanning microscope (CLSM). 60 incisors with a single root and a single canal were included in the study. IRR cavities were created in the middle third of the root canal, and CH was placed. The samples were randomly divided into 4 groups (n = 15) according to the irrigation activation method to be tested: standard needle irrigation (SNI), sonic activation (EDDY), photon-induced photoacoustic flow (PIPS), and shock wave enhanced emission photoacoustic flow (SWEEPS). After irrigation activation applications, the root canals were obturated. Sections of 1.0 ± 0.1mm were taken from the apical, middle, and coronal regions of each sample. The penetration area (µm2) and maximum penetration depth (µm) of the root canal sealer were examined by CLSM and analyzed using ImageJ software. Statistical analysis was performed with a one-way ANOVA and post-hoc Tukey test at the P < 0.05 significance level. Among all irrigation activation methods tested, both the penetration area and maximum penetration depth of the root canal sealer were greater in the coronal region than in the apical region (p < 0.05). In the IRR region, there was no difference in terms of maximum penetration depth between PIPS and SWEEPS (p > 0.05), it was highest in SWEEPS (p < 0.05). PIPS and SWEEPS were better than other irrigation activation methods in the penetration of root canal sealer in the resorption areas of teeth with IRR.

Unveiling the impact of carbonation treatment on BOF slag hydration activity: Formation and development of the barrier layer

Carbonation treatment is one of the most promising methods to improve the volume stability of basic oxygen furnace (BOF) slag, but the reasons for the reduced activity of heavily carbonated BOF slag have been rarely investigated. This study prepared carbonated BOF slag under the condition of CO2 concentration at 99 % and pressure at 3.0 MPa, and evaluated its activity through various methods. The mechanism behind activity inhibition was revealed. Results showed that the maximum CO2 sequestration of BOF slag was 13.4 % within 3 h. Highly carbonated BOF slag exhibits lower hydration activity than uncarbonated slag, with a 15.9 % decrease in strength activity index and a 61.0 % reduction in cumulative hydration heat release. After carbonation treatment, a 1–5μm thick CaCO3 barrier layer accumulates on the surface of BOF slag, affecting slag hydration even with accelerated hydration. This barrier layer inhibits the efflux of internal calcium ions and can persist long-term during the hydration without being affected. Furthermore, carbonation treatment increased the leaching risk of heavy metal ions, particularly Mn, by 175-fold.

Effect of asiaticoside on systolic blood pressure and relaxation of isolated thoracic aorta of rats

To investigate the effect of asiaticoside on blood pressure and relaxation of thoracic aorta in rats and explore the underlying mechanism. SD rats treated with 50 and 100 mg/kg asiaticoside by daily gavage for 2 weeks were monitored for systolic blood pressure changes, and histological changes of the thoracic aorta were evaluated using HE staining. In isolated rat endothelium-intact and endothelium-denuded thoracic aorta rings, the effects of asiaticoside on relaxation of the aortic rings were tested at baseline and following norepinephrine (NE)- and KCl-induced constriction. The vascular relaxation effect of asiaticoside was further observed in NE-stimulated endothelium-intact rat aortic rings pretreated with L-nitroarginine methyl ester, indomethacin, zinc protoporphyrin Ⅸ, tetraethyl ammonium chloride, glibenclamide, barium chloride, Iberiotoxin, 4-aminopyridine, or TASK-1-IN-1. The aortic rings were treated with KCl and NE followed by increasing concentrations of CaCl2 to investigate the effect of asiaticoside on vasoconstriction induced by external calcium influx and internal calcium release. Asiaticoside at 50 and 100 mg/kg significantly lowered systolic blood pressure in rats without affecting the thoracic aorta histomorphology. While not obviously affecting resting aortic rings with intact endothelium, asiaticoside at 100 mg/kg induced significant relaxation of the rings constricted by KCl and NE, but its effects differed between endothelium-intact and endothelium-denuded rings. In endothelium-intact aortic rings pretreated with indomethacin, ZnPP Ⅸ, barium chloride, glyburide, TASK-1-IN-1 and 4-aminopyridine, asiaticoside did not produce significant effect on NE-induced vasoconstriction, and tetraethylammonium, Iberiotoxin and L-nitroarginine methyl ester all inhibited the relaxation effect of asiaticoside. In KCland NE-treated rings, asiaticoside obviously inhibited CaCl2-induced vascular contraction. Asiaticoside induces thoracic aorta relaxation by mediating high-conductance calcium-activated potassium channel opening, promoting nitric oxide release from endothelial cells and regulating Ca2+ influx and outflow, thereby reducing systolic blood pressure in rats.

Glioblastoma and Internal Carotid Artery Calcium Score: A Possible Novel Prognostic Partnership?

Purpose: Clinical evidence suggests an association between comorbidities and outcome in patients with glioblastoma (GBM). We hypothesised that the internal carotid artery (ICA) calcium score could represent a promising prognostic biomarker in a competing risk analysis in patients diagnosed with GBM. Methods: We validated the use of the ICA calcium score as a surrogate marker of the coronary calcium score in 32 patients with lung cancer. Subsequently, we assessed the impact of the ICA calcium score on overall survival in GBM patients treated with radio-chemotherapy. Results: We analysed 50 GBM patients. At the univariate analysis, methyl-guanine-methyltransferase gene (MGMT) promoter methylation (p = 0.048), gross total tumour resection (p = 0.017), and calcium score (p = 0.011) were significant prognostic predictors in patients with GBM. These three variables also maintained statistical significance in the multivariate analysis. Conclusions: the ICA calcium score could be a promising prognostic biomarker in GBM patients.

A biophysically comprehensive model of urothelial afferent neurons: implications for sensory signalling in urinary bladder.

The urothelium is the innermost layer of the bladder wall; it plays a pivotal role in bladder sensory transduction by responding to chemical and mechanical stimuli. The urothelium also acts as a physical barrier between urine and the outer layers of the bladder wall. There is intricate sensory communication between the layers of the bladder wall and the neurons that supply the bladder, which eventually translates into the regulation of mechanical activity. In response to natural stimuli, urothelial cells release substances such as ATP, nitric oxide (NO), substance P, acetylcholine (ACh), and adenosine. These act on adjacent urothelial cells, myofibroblasts, and urothelial afferent neurons (UAN), controlling the contractile activity of the bladder. There is rising evidence on the importance of urothelial sensory signalling, yet a comprehensive understanding of the functioning of the urothelium-afferent neurons and the factors that govern it remains elusive to date. Until now, the biophysical studies done on UAN have been unable to provide adequate information on the ion channel composition of the neuron, which is paramount to understanding the electrical functioning of the UAN and, by extension, afferent signalling. To this end, we have attempted to model UAN to decipher the ionic mechanisms underlying the excitability of the UAN. In contrast to previous models, our model was built and validated using morphological and biophysical properties consistent with experimental findings for the UAN. The model included all the channels thus far known to be expressed in UAN, including; voltage-gated sodium and potassium channels, N, L, T, P/Q, R-type calcium channels, large-conductance calcium-dependent potassium (BK) channels, small conductance calcium-dependent (SK) channels, Hyperpolarisation activated cation (HCN) channels, transient receptor potential melastatin (TRPM8), transient receptor potential vanilloid (TRPV1) channel, calcium-activated chloride(CaCC) channels, and internal calcium dynamics. Our UAN model a) was constrained as far as possible by experimental data from the literature for the channels and the spiking activity, b) was validated by reproducing the experimental responses to current-clamp and voltage-clamp protocols c) was used as a base for modelling the non-urothelial afferent neurons (NUAN). Using our models, we also gained insights into the variations in ion channels between UAN and NUAN neurons.

Theoretical calculation of NMDA receptor desensitization and intracellular calcium determination through simulation

One of the crucial processes in preventing cellular damage due to excitotoxicity involves the calcium-mediated desensitization of the N-methyl-D-aspartate (NMDA) receptor. The opening of NMDA channels results in the influx of Na+ and Ca2+ ions and efflux of K+ ions. This condition leads to an increase in intracellular calcium ([Ca2+]i), thereby diminishing the NMDA current and preventing damage induced by constant stimulation of the receptor by glutamate. In this study, a simulator based on a phenomenological mathematical model was developed, employing the Visual Basic 6.0 language for the Windows® environment. Input variables encompass intracellular calcium, peak currents (Ip), and steady-state currents (Iss). Input currents were derived from previously published electrophysiological recordings. The simulator adeptly reproduces the NMDA receptor desensitization phenomenon under varying experimental conditions and accurately calculates internal calcium with a 10% margin of error. It proves valuable in scenarios where simultaneous recordings of NMDA current and [Ca2+]i via fluorescence labeling techniques are impractical. In the realm of education, it enables students to conduct virtual experiments, providing an immersive introduction to the topic.

Hijacking of internal calcium dynamics by intracellularly residing viral rhodopsins.

Rhodopsins are ubiquitous light-driven membrane proteins with diverse functions, including ion transport. Widely distributed, they are also coded in the genomes of giant viruses infecting phytoplankton where their function is not settled. Here, we examine the properties of OLPVR1 (Organic Lake Phycodnavirus Rhodopsin) and two other type 1 viral channelrhodopsins (VCR1s), and demonstrate that VCR1s accumulate exclusively intracellularly, and, upon illumination, induce calcium release from intracellular IP3-dependent stores. In vivo, this light-induced calcium release is sufficient to remote control muscle contraction in VCR1-expressing tadpoles. VCR1s natively confer light-induced Ca2+ release, suggesting a distinct mechanism for reshaping the response to light of virus-infected algae. The ability of VCR1s to photorelease calcium without altering plasma membrane electrical properties marks them as potential precursors for optogenetics tools, with potential applications in basic research and medicine.

Nano-plastics and gastric health: Decoding the cytotoxic mechanisms of polystyrene nano-plastics size

Gastrointestinal diseases exert a profound impact on global health, leading to millions of healthcare interventions and a significant number of fatalities annually. This, coupled with escalating healthcare expenditures, underscores the need for identifying and addressing potential exacerbating factors. One emerging concern is the pervasive presence of microplastics and nano-plastics in the environment, largely attributed to the indiscriminate usage of disposable plastic items. These nano-plastics, having infiltrated our food chain, pose a potential threat to gastrointestinal health. To understand this better, we co-cultured human gastric fibroblasts (HGF) with polystyrene nano-plastics (PS-NPs) of diverse sizes (80, 500, 650 nm) and meticulously investigated their cellular responses over a 24-hour period. Our findings revealed PS particles were ingested by the cells, with a notable increase in ingestion as the particle size decreased. The cellular death induced by these PS particles, encompassing both apoptosis and necrosis, showcased a clear dependence on both the particle size and its concentration. Notably, the larger PS particles manifested more potent cytotoxic effects. Further analysis indicated a concerning reduction in cellular membrane potential, alongside a marked increase in ROS levels upon PS particles exposure. This suggests a significant disruption of mitochondrial function and heightened oxidative stress. The larger PS particles were especially detrimental in causing mitochondrial dysfunction. In-depth exploration into the PS particles impact on genes linked with the permeability transition pore (PTP) elucidated that these PS particles instigated an internal calcium rush. This surge led to a compromise in the mitochondrial membrane potential, which in tandem with raised ROS levels, further catalyzed DNA damage and initiated cell death pathways. In essence, this study unveils the intricate mechanisms underpinning cell death caused by PS particles in gastric epithelial cells and highlighting the implications of PS particles on gastrointestinal health. The revelations from this research bear significant potential to shape future healthcare strategies and inform pertinent environmental policies.

Heat Transfer and Melting Characteristics of Calcium‐Cored Wire in Molten Steel

To accurately describe the heat transfer and melting characteristics of a calcium‐cored wire in molten steel, the finite difference method is used in theoretical calculations to solve the relationship between the melting temperature, radius, and time. Numerical simulations are performed using the Eulerian–Eulerian method to characterize the temperature distribution at different periods. The results show that the radius of the calcium wire and steel shell both decrease and the melting rate gradually increases. The calcium‐cored wire has a fish‐scale temperature distribution, with the temperature decreasing from a maximum at the front down to the tail end and increasing outward from the center axis. The temperature of the wire core is 1120 K at 0.272 s, indicating the complete melting of the pure internal calcium. The temperature of the calcium wire–steel shell interface reaches 1793 K at 0.979 s and remains from 1645 to 1757 K at the front of calcium wire. The steel shell melts through, and calcium flows into the molten steel in liquid form. The numerical simulation results generally agree with the theoretical calculations, and the reliability of the model is verified by comparison with published studies.

Localization of PDE4D, HCN1 channels, and mGluR3 in rhesus macaque entorhinal cortex may confer vulnerability in Alzheimer's disease.

Alzheimer's disease cortical tau pathology initiates in the layer II cell clusters of entorhinal cortex, but it is not known why these specific neurons are so vulnerable. Aging macaques exhibit the same qualitative pattern of tau pathology as humans, including initial pathology in layer II entorhinal cortex clusters, and thus can inform etiological factors driving selective vulnerability. Macaque data have already shown that susceptible neurons in dorsolateral prefrontal cortex express a "signature of flexibility" near glutamate synapses on spines, where cAMP-PKA magnification of calcium signaling opens nearby potassium and hyperpolarization-activated cyclic nucleotide-gated channels to dynamically alter synapse strength. This process is regulated by PDE4A/D, mGluR3, and calbindin, to prevent toxic calcium actions; regulatory actions that are lost with age/inflammation, leading to tau phosphorylation. The current study examined whether a similar "signature of flexibility" expresses in layer II entorhinal cortex, investigating the localization of PDE4D, mGluR3, and HCN1 channels. Results showed a similar pattern to dorsolateral prefrontal cortex, with PDE4D and mGluR3 positioned to regulate internal calcium release near glutamate synapses, and HCN1 channels concentrated on spines. As layer II entorhinal cortex stellate cells do not express calbindin, even when young, they may be particularly vulnerable to magnified calcium actions and ensuing tau pathology.

Analysis of the amount of lost fluids, some blood components and mineral salts in volleyball under hot weather conditions

The research objective was to study the amount of lost fluids, some blood components and mineral salts in volleyball players under hot weather conditions. The sample of the present study was composed of 12 volleyball players of Al-Sinaa Club (Baghdad, Iraq) in the 2022/2023 season. The variables analyzed in this study were: Heart rate before and after exercise, internal and external body temperature before and after exertion, potassium ion, sodium ion, calcium ion, and the amount of fluid lost (the player's weight) before and after the exercise. The tests were conducted at a temperature between 42-47 degrees Celsius. The maximum anaerobic exercise was performed with volleyball. The results showed that to play volleyball under hot weather conditions (42-47 degrees Celsius) significantly increased the following variables in volleyball players: heart rate, external body temperature, internal body temperature, sodium ion and calcium ion. However, potassium ion significantly decreased. Therefore, training in hot weather has positive physical repercussions. The authors recommend to train in different weather conditions.

Abstract 5801: Inhibition of muscarinic acetylcholine receptors in glioma stem cells blocks tumor progression

Abstract Primary gliomas arising within the brain are the deadliest form of brain cancer and account for 78% of all malignant brain tumors. Glioma patients have a 5 percent five-year survival rate and drug-resistant tumors often recur following surgical resection and treatment with radiation and/or chemotherapy. The cancer stem cell hypothesis suggests the presence of a subset of undifferentiated cells, namely glioma stem cells (GSCs), in the heterogenous tumor mass responsible for disease progression. Understanding mechanisms responsible for maintaining GSCs and developing strategies to deplete the GSC population could improve glioma treatment and prognosis. Some GSCs are similar to oligodendrocyte precursor cells (OPCs), which are observed in neural development and the adult brain. OPCs are prone to malignant transformation and are believed to be a cell of origin for glioma. Recent findings indicate that the well-characterized neurotransmitter acetylcholine (ACh) maintains the primitive state of normal OPCs via muscarinic ACh receptors (mAChRs) preventing maturation and cell cycle exit. We hypothesized that cholinergic signaling may also maintain the primitive state of OPC-like GSCs. We analyzed publicly available single nuclei RNASeq data of patient glioblastoma samples and observed high expression of CHRM3, encoding the M3 mAChR, in OPC-like cells of the proneural subtype of glioma. Studies in mouse OPC-like GSCs confirmed high levels of CHRM3 expression and demonstrated that ACh generated voltage changes and rapid (&amp;lt; 1 second) increases in cytosolic calcium from internal calcium stores. Exposure to a brain permeant FDA-approved anti-muscarinic drug targeting M3mAChR suppressed proliferation, evoked calcium release, and the activation of intracellular second messengers PKC and ERK. Pharmacologic inhibition of mAChRs in established patient derived glioma grafts prevented re-initiation of tumors in subsequent host animals compared with vehicle treated tumors. These studies suggest that the cholinergic microenvironment maintains GSCs in a manner similar to normal OPCs and provides a platform for repositioning available small molecule mAChR antagonists for treatment of glioma. Citation Format: Sumyuktha V. Anand, Min K. Lee, Alexander G. Skorput, Isabella B. Fox, Alison L. Young, Brock C. Christensen, Allan Gulledge, Matthew C. Havrda. Inhibition of muscarinic acetylcholine receptors in glioma stem cells blocks tumor progression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5801.

Reactive oxygen species in status epilepticus.

It has long been recognized that status epilepticus can cause considerable neuronal damage, and this has become one of its defining features. The mechanisms underlying this damage are less clear. Excessive activation of NMDA receptors results in large rises in internal calcium, which eventually lead to neuronal death. Between NMDA receptor activation and neuronal death are a number of intermediary steps, key among which is the generation of free radicals and reactive oxygen and nitrogen species. Although it has long been thought that mitochondria are the primary source for reactive oxygen species, more recent evidence has pointed to a prominent role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, an enzyme localized in cell membranes. There is burgeoning in vivo and in vitro evidence that therapies that target the production or removal of reactive oxygen species are not only effective neuroprotectants following status epilepticus, but also potently antiepileptogenic. Moreover, combining therapies targeted at inhibiting NADPH oxidase and at increasing endogenous antioxidants seems to offer the greatest benefits.

Saccharin and aspartame excite rat retinal neurons.

Retinal sensitivity to a variety of artificial sweeteners was tested by monitoring changes in internal free calcium in isolated retinal neurons using Fluo3. Several ligands, including aspartame and saccharin elevated internal free calcium. The effects of these ligands were mediated by both ligand-gated membrane channels and G-protein coupled receptors. We explored the receptors responsible for this phenomenon. Surprisingly, mRNA for subunits of the sweet taste receptor dimer (T1R2 and T1R3) were found in retina. Interestingly, knockdown of T1R2 reduced the response to saccharin but not aspartame. But TRPV1 channel antagonists suppressed the responses to aspartame. The results indicate that artificial sweeteners can increase internal free calcium in the retinal neurons through multiple pathways. Furthermore, aspartame reduced the b-wave, but not the a-wave, of the electroretinogram, indicating disruption of communication between photoreceptors and second order neurons.

Roles of Cholesterol and PtdIns(4,5)P2 in the Regulation of STIM1-Orai1 Channel Function.

Calcium is one of the most prominent second messengers. It is involved in a wide range of functions at the single-cell level but also in modulating regulatory mechanisms in the entire organism. One process mediating calcium signaling involves hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by the phospholipase-C (PLC). Thus, calcium and PtdIns(4,5)P2 are intimately intertwined two second-messenger cascades that often depend on each other. Another relevant lipid associated with calcium signaling is cholesterol. Both PtdIns(4,5)P2 and cholesterol play key roles in the formation and maintenance of specialized signaling nanodomains known as lipid rafts. Lipid rafts are particularly important in calcium signaling by concentrating and localizing calcium channels such as the Orai1 channel. Depletion of internal calcium stores is initiated by the production of inositol-1,4,5-trisphosphate (IP3). Calcium depletion from the ER induces the oligomerization of STIM1, which binds Orai1 and initiates calcium influx into the cell. In the present review, we analyzed the complex interactions between cholesterol, PtdIns(4,5)P2, and the complex formed by the Orai1 channel and the signaling molecule STIM1. We explore some of the complex mechanisms governing calcium homeostasis and phospholipid metabolism, as well as the interaction between these two apparently independent signaling cascades.

Dual-frequency ultrasound enhances functional neuron differentiation from neural stem cells by modulating Ca2+ dynamics and the ERK1/2 signaling pathway.

Our previous study demonstrated that ultrasound is able to promote differentiation on neural stem cells (NSCs), and dual-frequency ultrasound promotes this effect due to enhanced acoustic cavitation compared with single-frequency ultrasound. However, the underlying biological reasons have not been well disclosed. The purpose of this study was to investigate the underlying bioeffects, mechanisms and signaling pathways of dual-frequency ultrasound on NSC differentiation.The morphology, neurite outgrowth, and differentiation percentages were investigated under various dual-frequency simulation parameters with exposure periods varying from 5 to 15 min. Morphological observations identified that dual-frequency ultrasound stimulation promoted ultrasound dose-dependent neurite outgrowth. In particular, cells exposed for 10 min/2 days showed optimal neurite outgrowth and neuron differentiation percentages. In addition, live cell calcium images showed that dual-frequency ultrasound enhanced the internal calcium content of the cells, and calcium ions entering cells from the extracellular environment could be observed. Dual frequency ultrasound exposure enhanced extracellular calcium influx and upregulated extracellular signal-regulated kinases 1/2 (ERK1/2) expression. Observations from immunostaining and protein expression examinations also identified that dual-frequency ultrasound promoted brain-derived neurotrophic factor (BDNF) secretion from astrocytes derived from NSCs. In summary, evidence supports that dual-frequency ultrasound effectively enhances functional neuron differentiation via calcium channel regulation via the downstream ERK1/2 pathway and promotes BDNF secretion to serve as feedback to cascade neuron differentiation. The results may provide an alternative for cell-based therapy in brain injury.

Long-term depression–inductive stimulation causes long-term potentiation in mouse Purkinje cells with a mutant thyroid hormone receptor

Thyroid hormones (THs) regulate gene expression by binding to nuclear TH receptors (TRs) in the cell. THs are indispensable for brain development. However, we have little knowledge about how congenital hypothyroidism in neurons affects functions of the central nervous system in adulthood. Here, we report specific TH effects on functional development of the cerebellum by using transgenic mice overexpressing a dominant-negative TR (Mf-1) specifically in cerebellar Purkinje cells (PCs). Adult Mf-1 mice displayed impairments in motor coordination and motor learning. Surprisingly, long-term depression (LTD)-inductive stimulation caused long-term potentiation (LTP) at parallel fiber (PF)-PC synapses in adult Mf-1 mice, although there was no abnormality in morphology or basal properties of PF-PC synapses. The LTP phenotype was turned to LTD in Mf-1 mice when the inductive stimulation was applied in an extracellular high-Ca2+ condition. Confocal calcium imaging revealed that dendritic Ca2+ elevation evoked by LTD-inductive stimulation is significantly reduced in Mf-1 PCs but not by PC depolarization only. Single PC messenger RNA quantitative analysis showed reduced expression of SERCA2 and IP3 receptor type 1 in Mf-1 PCs, which are essential for mGluR1-mediated internal calcium release from endoplasmic reticulum in cerebellar PCs. These abnormal changes were not observed in adult-onset PC-specific TH deficiency mice created by adeno-associated virus vectors. Thus, we propose the importance of TH action during neural development in establishing proper cerebellar function in adulthood, independent of its morphology. The present study gives insight into the cellular and molecular mechanisms underlying congenital hypothyroidism-induced dysfunctions of central nervous system and cerebellum.

Depleted Calcium Stores and Increased Calcium Entry in Rod Photoreceptors of the Cacna2d4 Mouse Model of Cone-Rod Dystrophy RCD4.

Unidentified pathogenetic mechanisms and genetic and clinical heterogeneity represent critical factors hindering the development of treatments for inherited retinal dystrophies. Frameshift mutations in Cacna2d4, which codes for an accessory subunit of voltage-gated calcium channels (VGCC), cause cone-rod dystrophy RCD4 in patients, but the underlying mechanisms remain unknown. To define its pathogenetic mechanisms, we investigated the impact of a Cacna2d4 frameshift mutation on the electrophysiological profile and calcium handling of mouse rod photoreceptors by patch-clamp recordings and calcium imaging, respectively. In mutant (MUT) rods, the dysregulation of calcium handling extends beyond the reduction in calcium entry through VGCC and surprisingly involves internal calcium stores' depletion and upregulation of calcium entry via non-selective cationic channels (CSC). The similar dependence of CSC on basal calcium levels in WT and MUT rods suggests that the primary defect in MUT rods lies in defective calcium stores. Calcium stores' depletion, leading to upregulated calcium and sodium influx via CSC, represents a novel and, so far, unsuspected consequence of the Cacna2d4 mutation. Blocking CSC may provide a novel strategy to counteract the well-known pathogenetic mechanisms involved in rod demise, such as the reticulum stress response and calcium and sodium overload due to store depletion.

Nanosecond PEF Induces Oxidative Stress and Apoptosis via Proteasomal Activity Inhibition in Gastric Adenocarcinoma Cells with Drug Resistance.

Nanosecond (ns) pulsed electric field (PEF) is a technology in which the application of ultra-short electrical pulses can be used to disrupt the barrier function of cell plasma and internal membranes. Disruptions of the membrane integrity cause a substantial imbalance in cell homeostasis in which oxidative stress is a principal component. In the present study, nsPEF-induced oxidative stress was investigated in two gastric adenocarcinoma cell lines (EPG85-257P and EPG85-257RDB) which differ by their sensitivity to daunorubicin. Cells were exposed to 200 pulses of 10 ns duration, with the amplitude and pulse repetition frequency at 1 kHz, with electric field intensity varying from 12.5 to 50 kV/cm. The electroporation buffer contained either 1 mM or 2 mM calcium chloride. CellMask DeepRed visualized cell plasma permeabilization, Fluo-4 was used to visualize internal calcium ions content, and F-actin was labeled with AlexaFluor®488 for the cytoskeleton. The cellular viability was determined by MTT assay. An alkaline and neutral comet assay was employed to detect apoptotic and necrotic cell death. The luminescent method estimated the modifications in GSSG/GSH redox potential and the imbalance of proteasomal activity (chymotrypsin-, trypsin- and caspase-like). The reactive oxygen species (ROS) level was measured by flow cytometry using dihydroethidium (DHE) dye. Morphological visualization indicated cell shrinkage, affected cell membranes (characteristic bubbles and changed cell shape), and the reorganization of actin fibers with sites of its dense concentration; the effect was more intense with the increasing electric field strength. The most significant decrease in cell viability and GSSG/GSH redox potential was noted at the highest amplitude of 50 kV/cm, and calcium ions amplified this effect. nsPEF, particularly with calcium ions, inhibited proteasomal activities, resulting in increased protein degradation. nsPEF increased the percentage of apoptotic cells and ROS levels. The EPG85-257 RDB cell line, which is resistant to standard chemotherapy, was more sensitive to applied nsPEF protocols. The applied nsPEF method disrupted the metabolism of cancer cells and induced apoptotic cell death. The nsPEF ability to cause apoptosis, oxidative stress, and protein degradation make the nsPEF methodology a suitable alternative to current anticancer pharmacological methods.