Intracellular Calcium Concentration Research Articles

Functional characterization of OR51B5 and OR1G1 in human lung epithelial cells as potential drug targets for non-type 2 lung diseases

Abstract Background Hypersensitivity to odorants like perfumes can induce or promote asthma with non-type 2 inflammation for which therapeutic options are limited. Cell death of primary bronchial epithelial cells (PBECs) and the release of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8 are key in the pathogenesis. Extra-nasal olfactory receptors (ORs) can influence cellular processes involved in asthma. This study investigated the utility of ORs in epithelial cells as potential drug targets in this context. Methods We used the A549 cell line and primary bronchial epithelial cells using air–liquid interface culture system (ALI-PBECs). OR expression was investigated by RT-PCR, Western blot, and Immunofluorescence. Effects of OR activation by specific ligands on intracellular calcium concentration, cAMP, Phospholipase C (PLC), cell viability, and IL-6 and IL-8 secretion were analyzed by calcium imaging, enzyme immunoassays, Annexin V/ propidium iodide -based fluorescence-activated cell staining or by ELISA, respectively. Results By screening A549 cells, the OR51B5 agonists Farnesol and Isononyl Alcohol and the OR1G1 agonist Nonanal increased intracellular Ca2 + . OR51B5 and OR1G1 mRNAs and proteins were detected. Both receptors showed a preferential intracellular localization. OR51B5- but not OR1G1-induced Ca2 + dependent on both cAMP and PLC signaling. Farnesol, Isononyl Alcohol, and Nonanal, all reduced cell viability and induced IL-8 and IL-6 release. The data were verified in ALI-PBECs. Conclusion ORs in the lung epithelium might be involved in airway-sensitivity to odorants. Their antagonism could represent a promising strategy in treatment of odorant-induced asthma with non-type 2 inflammation. Graphical Abstract

CNSC-13. CROSSTALK BETWEEN SENSORY NEURONAL ACTIVITY AND TUMOR CELLS PROMOTES MALIGNANCY AND CANCER PAIN

Abstract BACKGROUND Nerve infiltration of solid tumors is associated with poor prognosis in multiple cancer types. Understanding the role of neuronal activity in cancer progression offers innovative approaches for treating malignancies and cancer-associated neurological issues such as pain. Pain perception is mediated by the activity of peripheral sensory neurons. Many cancer types induce pain, and sensory innervation could support tumor growth, raising the interesting hypothesis that cancer pain-associated neuronal activity promotes malignancies. Our study aims to test the hypothesis using malignant peripheral nerve sheath tumor (MPNST) as the experimental platform. METHODS in vivo allograft models were established by implanting mouse MPNST cells into the sciatic nerves of male and female mice, followed by performing pain behavior assays (von Frey, Hargreaves, and conditioned place preference tests) and tumor growth measurements. Primary mouse dorsal root ganglion (DRG) sensory neuron cultures were prepared for calcium imaging and neuron-tumor co-culture experiments. RESULTS MPNST tumor-bearing mice exhibited hypersensitivity to noxious and non-noxious stimuli (evoked pain) and ongoing pain, compared to the sham controls. MPNST-conditioned media increased intracellular calcium concentration and ATF3 (indicator for nerve injury/stress) expression in primary sensory neuron culture, suggesting that MPNST tumor cell secretory factors may induce pain by triggering sensory neuron injury/stress responses that increase sensory neuron activity/sensitivity. Reciprocally, we found that stimulation of sensory neuron activity accelerates MPNST tumor growth. The conditioned media from stimulated sensory neurons increased tumor proliferation (EdU assay) in vitro, suggesting that sensory neuron activity-induced paracrine factors increase MPNST cell growth. CONCLUSIONS MPNST is a devastating cancer that grows within peripheral nerves which induce intractable pain. Our findings demonstrate a feedforward cycle where the cancer cells induce sensory neuron hypersensitivity/hyperactivity (pain) which in turn further accelerates tumor growth. Future studies will identify the molecular mechanisms underlying this crosstalk between MPNST and sensory neuron activity.

Quinoline- and pyrimidine-based allosteric modulators of the sarco/endoplasmic reticulum calcium ATPase.

Small-molecule allosteric activators of the enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA) hold promise as novel experimental tools to manipulate intracellular calcium concentrations and as therapeutic agents to treat medical conditions associated with elevated cytosolic calcium levels. Here, we synthesized and characterized 20 analogs of the known allosteric SERCA activator CDN1163 and tested their ability to stimulate SERCA activity. The structures of the compounds varied in the alkyl group of the parent scaffold's ether moiety as well as in the composition of the nitrogenous aromatic ring system. The most active compounds exhibited potencies in the sub-micromolar range while increasing enzyme activity by more than 25%. The observed structure-activity relationships indicated that bulky alkyl groups in the ether moiety along with a quinoline ring methyl substituent were beneficial for activity. Replacement of the quinoline by a pyrimidine ring system reduced activity. To conceive a potential mechanism of action, we generated a molecular model of the transition state of SERCA when undergoing the rate-limiting step of its catalytic cycle. Subsequent blind docking with CDN1163 identified a high-affinity binding site close to the enzyme's ATP binding pocket, suggesting that the activators may accelerate SERCA's catalytic cycle by aiding in ATP binding and positioning.

Protein disulfide isomerase 1 (PDIA1) regulates platelet-derived extracellular vesicle release

BackgroundProtein disulfide isomerase 1 (PDIA1) and 3 (PDIA3) regulate platelet activation and thrombus formation. However, their role in the formation of platelet-derived extracellular vesicles (pEVs) remains unknown. AimTo characterise the effects of PDIA1 and PDIA3 inhibition on pEV formation in washed murine platelets in response to platelet glycoprotein VI (GPVI) receptor or intracellular calcium signal activation. MethodsWashed platelets were isolated from C57BL/6 mice and activated using convulxin or the calcium ionophore A23187. Then, the resulting pEVs were analysed using nano flow cytometry (FC), platelet aggregation was measured by FC and a 96-well plate-based assay, and intracellular free calcium concentration ([Ca2+]i) was measured by indicator fluorescence. Platelet PDIs were blocked by a classic selective PDIA1 inhibitor (bepristat 2a) and sulphonamides of aziridine-2-carboxylic acid derivatives, novel PDI inhibitors relatively selective for PDIA1 or PDIA3 (C-3389 and C-3399, respectively). Clinically relevant antiplatelet drugs were used for comparison. ResultsConvulxin and A23187 concentration-dependently induced pEV formation. However, unlike convulxin, platelet activation by A23187 did not stimulate their aggregation. Bepristat 2a, C-3389 and C-3399 inhibited convulxin-induced pEV release accompanied by the reduction of [Ca2+]i. In contrast, only bepristat 2a inhibited A23187-induced pEV release, but without effect on [Ca2+]i. Cangrelor and tirofiban, but not acetylsalicylic acid (ASA), inhibited convulxin-induced pEV release, but neither of them inhibited A23187-induced pEV release. ConclusionThe inhibition of PDIA1 represents a novel approach to inhibit pEV formation by a mechanism independent of platelet aggregation and calcium signaling.

Distribution and calcium signaling function of somatostatin receptor subtypes in rat pituitary

The somatostatin (SST) receptor family controls pituitary hormone secretion, but the distribution and specific roles of these receptors on the excitability and voltage-gated calcium signaling of hormone producing pituitary cells have not been fully characterized. Here we show that the rat pituitary gland expressed Sstr1, Sstr2, Sstr3, and Sstr5 receptor genes in a cell type-specific manner: Sstr1 and Sstr2 in thyrotrophs, Sstr3 in gonadotrophs and lactotrophs, Sstr2, Sstr3, and Sstr5 in somatotrophs, and none in corticotrophs and melanotrophs. Most gonadotrophs and thyrotrophs spontaneously fired high-amplitude single action potentials, which were silenced by SST without affecting intracellular calcium concentrations. In contrast, lactotrophs and somatotrophs spontaneously fired low-amplitude plateau-bursting action potentials in conjunction with calcium transients, both of which were silenced by SST. Moreover, SST inhibited GPCR-induced voltage-gated calcium signaling and hormone secretion in all cell types expressing SST receptors, but the inhibition was more pronounced in somatotrophs. The pattern of inhibition of electrical activity and calcium signaling was consistent with both direct and indirect inhibition of voltage-gated calcium channels, the latter being driven by cell type-specific hyperpolarization. These results indicate that the action of SST in somatotrophs is enhanced by the expression of several types of SST receptors and their slow desensitization, that SST may play a role in the electrical resynchronization of gonadotrophs, thyrotrophs, and lactotrophs, and that the lack of SST receptors in corticotrophs and melanotrophs keeps them excitable and ready to responses to stress.

The negative electrostatic potential of the coordination between calcium and carboxyl/oxygen group in calcium-peptide complexes contributes to calcium utilization

The coordination electrostatic potential has significantly influenced the binding energy of calcium-peptide coordination complexes. However, its impact on calcium bioavailability is unclear. A calcium-binding peptide (CBP) DEDEQIPSHPPR, purified from soybean yogurt was selected. The study selected tetrapeptide residues of acidic amino acids that play an important role in CBP and designed sixteen tetrapeptides rich in carboxyl oxygen atoms to investigate the influence of electrostatic potential on calcium-peptide binding. The results indicated that DEDE exhibited the strongest binding energy (−375.27 kcal/mol) and binding capacity (464.11 ± 2.14 mg/g) with calcium ions compared to other peptides. Moreover, DEDE also increased intracellular calcium concentration the most, being 2.04 times higher than CaCl2. Calcium ions coordinated with carboxyl oxygen atoms possessing negative electrostatic potential, which correlated positively with the bioavailability of calcium ions. This electrostatic potential analysis provides a theoretical basis for developing and screening bioactive peptides with high calcium bioavailability.

Nanoparticles encapsulating phosphatidylinositol derivatives promote neuroprotection and functional improvement in preclinical models of ALS via a long-lasting activation of TRPML1 lysosomal channel

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease currently incurable, in which motor neuron degeneration leads to voluntary skeletal muscle atrophy. Molecularly, ALS is characterized by protein aggregation, synaptic and organellar dysfunction, and Ca2+ dyshomeostasis. Of interest, autophagy dysfunction is emerging as one of the main putative targets of ALS therapy. A tune regulation of this cleansing process is affordable by a proper stimulation of TRPML1, one of the main lysosomal channels. However, TRPML1 activation by PI(3,5)P2 has low open probability to remain in an active conformation. To overcome this drawback we developed a lipid-based formulation of PI(3,5)P2 whose putative therapeutic potential has been tested in in vitro and in vivo ALS models.Pharmacodynamic properties of PI(3,5)P2 lipid-based formulations (F1 and F2) on TRPML1 activity have been characterized by means of patch-clamp electrophysiology and Fura-2AM video-imaging in motor neuronal cells. Once selected for the ability to stabilize TRPML1 activity, the most effective preparation F1 was studied in vivo to measure neuromuscular function and survival of SOD1G93A ALS mice, thereby establishing its therapeutic profile.F1, but not PI(3,5)P2 alone, stabilized the open state of the lysosomal channel TRPML1 and increased the persistence of intracellular calcium concentration ([Ca2+]i). Then, F1 was effective in delaying motor neuron loss, improving innervated endplants and muscle performance in SOD1G93A mice, extending overall lifespan by an average of 10 days. Of note F1 prevented gliosis and autophagy dysfunction in ALS mice by restoring PI(3,5)P2 level.Our novel self-assembling lipidic formulation for PI(3,5)P2 delivery exerts a neuroprotective effect in preclinical models of ALS mainly regulating dysfunctional autophagy through TRPML1 activity stabilization.

Quantification of Sarcoplasmic Reticulum Ca2+ Release in Primary Ventricular Cardiomyocytes.

In the heart, ion channels generate electrical currents that signal muscle contraction through changes in intracellular calcium concentration, i.e., [Ca2+]. The cardiac ryanodine receptor type 2 (RyR2) is the predominant ion channel responsible for increasing intracellular [Ca2+] by releasing Ca2+ from the sarcoplasmic reticulum (SR). Timely Ca2+ release is necessary for appropriate cardiac function, and dysfunction can cause or contribute to life-threatening diseases such as arrhythmia. Quantification of SR-Ca2+ release in the form of sparks and waves can provide valuable insight into RyR2 opening, and factors that influence or regulate channel function. Here, we provide a series of protocols that outline processes for (1) obtaining high-quality isolated cardiomyocytes, (2) preparing samples for experimentally investigating factors that influence RyR2 function, and (3) data acquisition and analysis. Notably, our protocols leverage the potency of the recently developed myosin ATPase inhibitor, Mavacamten. This affords the opportunity to characterize the effects of small molecules or reconstituted proteins/enzymes on RyR2-Ca2+ release events across a range of [Ca2+]. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Cardiomyocyte isolation from mouse Basic Protocol 2: Preparation of cardiomyocytes for Ca2+ imaging Basic Protocol 3: Confocal microscopy and quantitative Ca2+ analysis using SparkMaster 2.

Food grade titanium dioxide induced endoplasmic reticulum stress in colon cells: Comparison between normal and colorectal carcinoma cells

BackgroundFood-grade titanium dioxide (E171) has been under scrutiny in the last decade since its possible adverse effects; however, the cellular mechanisms underlying E171 toxicity have not been thoroughly described. AimWe aimed to compare the effects of E171 on endoplasmic reticulum (ER) homeostasis in normal and cancer colon cells. Experimental designWe exposed normal, carcinoma, and adenocarcinoma cells to 0.1, 1, 10, 50 and 100 μg/cm2 of E171 for 24, 48 and 72 h, and we evaluated ER stress, cell viability, titanium uptake, intracellular calcium concentration, and gene expression related to unfolded protein response (UPR) and chaperone pathways. ResultsCell viability decreased only after 72 h of exposure to 100 μg/cm2 of E171. Adenocarcinoma cells internalized higher titanium amounts than normal and carcinoma cells, but the effects in ER distribution, intracellular calcium concentration, and gene expression were similar among the three cell lines. The expression of UPR and chaperone pathways were downregulated at the lowest concentrations but upregulated at the highest concentrations in the three cell lines. ConclusionE171 induces ER stress through alterations in ER distribution, intracellular calcium, and UPR and chaperone protein pathways.

Loss of Stim2 in zebrafish induces glaucoma-like phenotype.

Calcium is involved in vision processes in the retina and implicated in various pathologies, including glaucoma. Rod cells rely on store-operated calcium entry (SOCE) to safeguard against the prolonged lowering of intracellular calcium ion concentrations. Zebrafish that lacked the endoplasmic reticulum Ca2+ sensor Stim2 (stim2 knockout [KO]) exhibited impaired vision and lower light perception-related gene expression. We sought to understand mechanisms that are responsible for vision impairment in stim2 KO zebrafish. The single-cell RNA (scRNA) sequencing of neuronal cells from brains of 5days postfertilization larvae distinguished 27 cell clusters, 10 of which exhibited distinct gene expression patterns, including amacrine and γ-aminobutyric acid (GABA)ergic retinal interneurons and GABAergic optic tectum cells. Five clusters exhibited significant changes in cell proportions between stim2 KO and controls, including GABAergic diencephalon and optic tectum cells. Transmission electron microscopy of stim2 KO zebrafish revealed decreases in width of the inner plexiform layer, ganglion cells, and their dendrites numbers (a hallmark of glaucoma). GABAergic neuron densities in the inner nuclear layer, including amacrine cells, as well as photoreceptors significantly decreased in stim2 KO zebrafish. Our study suggests a novel role for Stim2 in the regulation of neuronal insulin expression and GABAergic-dependent vision causing glaucoma-like retinal pathology.

Nociceptive TRP channels function as molecular target for several antifungal drugs.

Topically applied antifungal agents can induce adverse effects, such as pain and irritation. The transient receptor potential (TRP) channels-TRPA1 and TRPV1-mainly expressed in sensory neurons, act as sensors for detecting irritants. This study aims to evaluate the involvement of nociceptive channels in topical antifungal-induced pain and irritation. We tested nine topical antifungals belonging five classes: isoconazole, econazole, miconazole, clotrimazole, and ketoconazole as imidazoles; liranaftate as a thiocarbamate; terbinafine as an allylamine; amorolfine as a morpholine; and butenafine as a benzylamine. Intracellular calcium concentrations ([Ca2+]i) and membrane currents in response to antifungals were measured to estimate channel activity using heterologously expressing cells and isolated mouse sensory neurons. In mouse TRPA1-expressing cells, all the tested drugs induced an increase in [Ca2+]i, which was abrogated or reduced by a TRPA1 blocker. Although many drugs evoked the TRPA1-nonspecific [Ca2+]i response at high concentrations, responses to clotrimazole, ketoconazole, and liranaftate were TRPA1 specific and elicited current responses in TRPA1-expressing cells. In mouse TRPV1-expressing cells, clotrimazole and ketoconazole elicited [Ca2+]i and current responses. In mouse sensory neurons, liranaftate-induced increase in [Ca2+]i was abrogated by a TRPA1 blocker and Trpa1 deletion. Responses to ketoconazole were inhibited by TRPA1 and TRPV1 blockers and by the genetic deletion of either channel. These results suggest that topical antifungal-induced pain and irritation are attributable to the activation of nociceptive TRPA1 and/or TRPV1 channel/s. Consequently, caution should be exercised in the use of topical antifungals with symptoms of pain.

Gintonin Stimulates Glucose Uptake in Myocytes: Involvement of Calcium and Extracellular Signal-Regulated Kinase Signaling.

Ginseng has anti-hyperglycemic effects. Gintonin, a glycolipoprotein derived from ginseng, also stimulates insulin release from pancreatic beta cells. However, the role of gintonin in glucose metabolism within skeletal muscle is unknown. Here, we showed the effect of gintonin on glucose uptake, glycogen content, glucose transporter (GLUT) 4 expression, and adenosine triphosphate (ATP) content in C2C12 myotubes. Gintonin (3-30 μg/mL) dose-dependently stimulated glucose uptake in myotubes. The expression of GLUT4 on the cell membrane was increased by gintonin treatment. Treatment with 1-3 μg/mL of gintonin increased glycogen content in myotubes, but the content was decreased at 30 μg/mL of gintonin. The ATP content in myotubes increased following treatment with 10-100 μg/mL gintonin. Gintonin transiently elevated intracellular calcium concentrations and increased the phosphorylation of extracellular signal-regulated kinase (ERK). Gintonin-induced transient calcium increases were inhibited by treatment with the lysophosphatidic acid receptor inhibitor Ki16425, the phospholipase C inhibitor U73122, and the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate. Gintonin-stimulated glucose uptake was decreased by treatment with U73122, the intracellular calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester, and the ERK inhibitor PD98059. These results show that gintonin plays a role in glucose metabolism by increasing glucose uptake through transient calcium increases and ERK signaling pathways. Thus, gintonin may be beneficial for glucose metabolism control.

Measuring GPCR-Induced Intracellular Calcium Signaling Using a Quantitative High-Throughput Assay.

Alterations in intracellular calcium are integral to signal transduction pathways for many G-protein-coupled receptors, but this signaling is not well studied. This is mostly due to a lack of reliable, robust, high-throughput, quantitative methods to monitor intracellular calcium concentrations in live cells. Recently, we developed a reliable, robust, quantitative method to measure intracellular calcium levels in which HEK293 cell suspensions loaded with Fura-2/AM are placed in 96-well plates. Minimum and maximum intracellular calcium levels, which are required for converting fluorescence into calcium concentrations, are calibrated using EGTA to chelate calcium and ionomycin to load calcium into cells, respectively. Fluorescence is monitored with a PHERAstar FS plate reader. We provide a detailed method for this high-throughput assay that can be used to quantitate intracellular calcium in endogenous and exogenously (stable or transient) expressed GPCRs in HEK293 cells.

Regulation of vascular smooth muscle cell contraction during early postnatal ontogenesis

Growth of the body in early postnatal ontogenesis is associated with changes in the functioning of many organ systems, including the cardiovascular system. The circulatory system of newborns is characterized by numerous structural and functional features, which at the systemic level is manifested in a significantly lower level of blood pressure. This review describes the differences in the mechanisms of regulation of vascular smooth muscle cell contraction in early postnatal ontogenesis and in adulthood, including age-related changes in the functioning of ion channels, which activity affects membrane potential level and intracellular concentration of calcium ions, as well as changes in calcium sensitivity of the contractile apparatus. The final section of the review discusses the connection between the mechanisms regulating contraction and differentiation of vascular smooth muscle cells during maturation.

Effect of Magnetic Field and Magnetic Nanoparticles on Choice of Endothelial Cell Phenotype

Background. Endothelial cells as participants in angiogenesis choose their phenotype as tip cells (leading, migratory) or stalk cells (following). It has been experimentally found and theoretically modeled that rapid oscillations in intracellular calcium concentration play a key role in controlling phenotype selection and possible vessel architecture. In addition, the intracellular calcium concentration in endothelial cells is known to be regulated by mechanical wall shear stress induced by blood flow, which controls mechanosensitive calcium ion channel gating. Experimental methods of controlling mechanosensitive ion channel gating in external magnetic fields with application of magnetic nanoparticles are developed that affect magnetic nanoparticles artificially attached to cell membranes. Objective. A key question is raised about the possibility of controlled selection of endothelial cell phenotype in external magnetic fields due to the presence of artificial or biogenic magnetic nanoparticles embedded in the cell membrane. Methods. The magnetic wall shear stress is calculated due to the influence of the external magnetic field on the magnetic nanoparticles embedded in the cell membrane, which controls the mechanosensitive calcium ion pathways. Numerical modeling of oscillations in intracellular calcium concentration in endothelial cells and determination of their final phenotype was carried out taking into account intercellular communication. The python programming language and scipy, py-pde, matplotlib packages of the python programming language were used for numerical modeling. Results. The magnetic field flux density and frequency ranges of a uniform rotating magnetic field, as well as the magnitude of the gradient and the frequency of a non-uniform oscillating magnetic field were calculated for controlling the amplitude and frequency of intracellular calcium concentration oscillations in endothelial cells, as well as the selection of their phenotype. It opens the perspective of controlling angiogenesis and vessel architecture. Conclusions. Phenotype selection by endothelial cells can be controlled in a uniform rotating external magnetic field, as well as in a non-homogeneous oscillating magnetic field.

Protein kinase C (PKC) inhibitor Calphostin C activates PKC in a light-dependent manner at high concentrations via the production of singlet oxygen

Calphostin C (Cal-C) is a protein kinase C (PKC) inhibitor that binds to its C1 domain. The aim of the present study was to elucidate the action of Cal-C in addition to PKC inhibition. First, we confirmed that Cal-C at low concentrations (<200 nM) inhibit phorbol ester-induced PKC translocation and G-protein-coupled receptor (GPCR)-mediated PKC activation. Cal-C at higher concentrations (>2 μM) increased intracellular calcium ion concentrations ([Ca2+]i) in a concentration-dependent manner. The origin of this increase is the mobilization of the endoplasmic reticulum (ER), which does not involve GPCR or ryanodine receptors. Cal-C at high concentrations also cause structural changes in the ER, such as the formation of vacuoles and aggregates, and calcium leakage from the ER. At 2 μM, Cal-C translocated a calcium-sensitive PKCα. Studies using a C-kinase activity reporter and a myristoylated alanine-rich protein kinase C substrate fused with green fluorescent protein (GFP) have also revealed that Cal-C at high concentrations activate PKC in living cells. Additionally, the PKC-activating effects of Cal-C were light-dependent. Finally, studies using Si-DMA, an indicator of singlet oxygen, showed that Cal-C at high concentrations generated singlet oxygen, causing structural changes in the ER and leakage of calcium into the cytosol, which triggered PKC activation. This study confirms the novel action of Cal-C, solely considered a PKC inhibitor. Cal-C acted as a PKC inhibitor at low concentrations and a PKC activator at high concentrations by generating singlet oxygen in a light-dependent manner, suggesting that Cal-C can be used in photodynamic therapy.

Synergistic effect of cerium chloride and calcium chloride alters calcium signaling in keratinocytes to promote epidermal differentiation.

Epidermal keratinocytes undergo morphological and functional changes during differentiation, eventually being enucleated to become corneocytes. Calcium has been shown to be involved in various cellular functions of epidermal cells, including proliferation, differentiation, and apoptosis. Cerium is a lanthanide-series element and rare earth metal. For skin, cerium oxide has been investigated for use in absorbing UV and promoting wound healing. However, the functions and physiological effects of inorganic cerium on the skin have rarely been investigated. Here, we focused on cerium's function in epidermal keratinocytes and its interaction with calcium by investigating their effects on cell differentiation and intracellular calcium concentration. This study showed that applying cerium chloride to epidermal keratinocytes altered calcium signaling. It also suggested that cerium and calcium induced an increase in intracellular calcium concentration and promoted keratinocyte differentiation.

Evaluating the Reparative Potential of Secretome from Patient-Derived Induced Pluripotent Stem Cells during Ischemia–Reperfusion Injury in Human Cardiomyocytes

During a heart attack, ischemia causes losses of billions of cells; this is especially concerning given the minimal regenerative capability of cardiomyocytes (CMs). Heart remuscularization utilizing stem cells has improved cardiac outcomes despite little cell engraftment, thereby shifting focus to cell-free therapies. Consequently, we chose induced pluripotent stem cells (iPSCs) given their pluripotent nature, efficacy in previous studies, and easy obtainability from minimally invasive techniques. Nonetheless, using iPSC secretome-based therapies for treating injured CMs in a clinical setting is ill-understood. We hypothesized that the iPSC secretome, regardless of donor health, would improve cardiovascular outcomes in the CM model of ischemia–reperfusion (IR) injury. Episomal-generated iPSCs from healthy and dilated cardiomyopathy (DCM) donors, passaged 6–10 times, underwent 24 h incubation in serum-free media. Protein content of the secretome was analyzed by mass spectroscopy and used to treat AC16 immortalized CMs during 5 h reperfusion following 24 h of hypoxia. IPSC-derived secretome content, independent of donor health status, had elevated expression of proteins involved in cell survival pathways. In IR conditions, iPSC-derived secretome increased cell survival as measured by metabolic activity (p &lt; 0.05), cell viability (p &lt; 0.001), and maladaptive cellular remodelling (p = 0.052). Healthy donor-derived secretome contained increased expression of proteins related to calcium contractility compared to DCM donors. Congruently, only healthy donor-derived secretomes improved CM intracellular calcium concentrations (p &lt; 0.01). Heretofore, secretome studies mainly investigated differences relating to cell type rather than donor health. Our work suggests that healthy donors provide more efficacious iPSC-derived secretome compared to DCM donors in the context of IR injury in human CMs. These findings illustrate that the regenerative potential of the iPSC secretome varies due to donor-specific differences.

Inhibiting Ca2+ channels in Alzheimer's disease model mice relaxes pericytes, improves cerebral blood flow and reduces immune cell stalling and hypoxia.

Early in Alzheimer's disease (AD), pericytes constrict capillaries, increasing their hydraulic resistance and trapping of immune cells and, thus, decreasing cerebral blood flow (CBF). Therapeutic approaches to attenuate pericyte-mediated constriction in AD are lacking. Here, using in vivo two-photon imaging with laser Doppler and speckle flowmetry and magnetic resonance imaging, we show that Ca2+ entry via L-type voltage-gated calcium channels (CaVs) controls the contractile tone of pericytes. In AD model mice, we identifed pericytes throughout the capillary bed as key drivers of an immune reactive oxygen species (ROS)-evoked and pericyte intracellular calcium concentration ([Ca2+]i)-mediated decrease in microvascular flow. Blocking CaVs with nimodipine early in disease progression improved CBF, reduced leukocyte stalling at pericyte somata and attenuated brain hypoxia. Amyloid β (Aβ)-evoked pericyte contraction in human cortical tissue was also greatly reduced by CaV block. Lowering pericyte [Ca2+]i early in AD may, thus, offer a therapeutic strategy to enhance brain energy supply and possibly cognitive function in AD.

Studying Drosophila Larval Behavior in Agarose Channels.

Larvae of the fruit fly Drosophila melanogaster are a popular and tractable model system for studying the development and function of sensorimotor circuits, thanks to the relative numerical simplicity of their nervous system and the wealth of available genetic tools to manipulate the anatomy, activity, and function of specific cell types. Researchers studying the role of a particular gene or cell type in sensorimotor circuit activity or function may wish to observe the effects of an experimental manipulation during behavior in the intact animal. Observing these effects, which may include changes in the intracellular calcium concentration or movement of small numbers of neurons, muscles, etc., typically requires high-spatial-resolution imaging, which poses several difficulties in the freely crawling larva. Freely crawling larvae can move quickly and with changeable heading, making manual or automatic tracking challenging; additionally, they may make three-dimensional movements, such as rearing, that can degrade imaging focus. These challenges are potentially solvable using advanced imaging and algorithmic tracking setups, but cost, space, or development time may be prohibitive. This protocol describes a simple and cost-effective method for placing larvae inside agarose channels, thereby restricting larval crawling to a single dimension and enabling higher-magnification time-series imaging of fluorescently labeled structures during many cycles of locomotion. By using larvae that express fluorescent calcium indicators in cells of interest, researchers can apply this method to study the effects of experimental manipulations on neural or muscular activity during behavior in the intact animal.