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Incidence of mental health disorders are rising in modernity, with psychological stress linked to a propensity for developing various chronic diseases due to a relative inability of the body to counter the allostatic load on cellular level. Despite these high rates of comorbidities associated with posttraumatic stress disorder (PTSD), there is still a lack of understanding in terms of the peripheral effects of PTSD on tissue level. Therefore, the purpose of this study was to profile basal dermal fibroblast functional status in PTSD using a wide range of markers involved in the cell-to-cell communication facilitated by fibroblasts. Primary dermal fibroblasts derived from patients diagnosed with PTSD (n = 11) and matched trauma exposed controls (i.e. who did not develop PTSD, n = 10) were cultured using standard techniques. The patients and controls were matched based on age, sex, body-mass index (BMI) and lifestyle. The growth rate, population doubling time, cell surface marker expression (CD31, FNDC5) (flow cytometry), secretome (TIMP-2, MMP-9) (ELISAs), intracellular signalling capacity (Fluo-4 Ca2+ flux) and gene expression (IL-6, IL-10, PTX-3, iNOS, Arg1) were compared between groups. The data illustrated significant PTSD-associated fibroblast conditioning resulting in a blunted signalling capacity. This observation highlights the importance of including tissue-specific investigations in future studies focused on elucidating the association between PTSD and subsequent risk for somatic disease. • PTSD conditions fibroblasts in the periphery. • Fibroblast signalling capacity is generally blunted in PTSD. • Data suggest PTSD-associated fibroblast desensitization to stimuli. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal stem cells (MSCs) or skeletal stem cells (SSCs) play a major role in tissue repair due to their robust ability to differentiate into osteoblasts, chondrocytes, and adipocytes. Complex cell signaling cascades tightly regulate this differentiation. In osteogenic differentiation, Runt‐related transcription factor 2 (RUNX2) and ALP activity are essential. Furthermore, during the latter stages of osteogenic differentiation, mineral formation mediated by the osteoblast occurs with the secretion of a collagenous extracellular matrix and calcium deposition. Activation of nuclear factor erythroid 2‐related factor 2 (NRF2), an important transcription factor against oxidative stress, inhibits osteogenic differentiation and mineralization via modulation of RUNX2 function; however, the exact role of NRF2 in osteoblastogenesis remains unclear. Here, we demonstrate that NRF2 activation in human bone marrow‐derived stromal cells (HBMSCs) suppressed osteogenic differentiation. NRF2 activation increased the expression of STRO‐1 and KITLG (stem cell markers), indicating NRF2 protects HBMSCs stemness against osteogenic differentiation. In contrast, NRF2 activation enhanced mineralization, which is typically linked to osteogenic differentiation. We determined that these divergent results were due in part to the modulation of cellular calcium flux genes by NRF2 activation. The current findings demonstrate a dual role for NRF2 as a HBMSC maintenance factor as well as a central factor in mineralization, with implications therein for elucidation of bone formation and cellular Ca2+ kinetics, dystrophic calcification and, potentially, application in the modulation of bone formation. [ABSTRACT FROM AUTHOR]

The mantle muscle of common cuttlefish, Sepia officinalis, is responsible both for high‐magnitude and rapid movements for locomotion, as well as sustained ventilation, which require specific metabolic, electrophysiological, and structural organization. Young cuttlefish have a highly oxidative phenotype and a rapid growth rate. Here, we show high rates of oxygen consumption and protein synthesis in juveniles, and these rates decay exponentially over the first few weeks of growth. This is associated with considerable citrate synthase activity (relative to larger cuttlefish) but a lack of glucose metabolism based on zero uptake of glucose by isolated muscle sheets and minimal activity of hexokinase (similar to larger animals). In contrast to glucose metabolism in the heart, glucose metabolism in these muscle sheets was not stimulated by extracellular taurine. Previous research revealed an unusual ion channel complement in mantle myocytes, the most notable feature of which is the lack of a Na+ current during depolarization. Because this adaptation is not consistent across the coleoid clade, we investigated excitation–contraction coupling. Here, mantle energetics and contractility, including the individual components of the total Ca2+ flux driving contraction, were studied. Results indicate that the majority of Ca2+ current underlying contractile stress development capacity in cuttlefish juveniles is not mediated by dihydropyridine‐sensitive L‐type channels, in contrast to their adult counterparts, and the sarcoplasmic reticulum contributes little to routine contractility. We had previously noted an influence of physiological levels of taurine in limiting cardiac contractility but found no analogous sensitivity in mantle muscle. Finally, transmission electron microscopy of subcellular architecture revealed the presence of sarcoplasmic tubular aggregates, suggesting that oxidative inhibition of sarcoplasmic reticulum function limits its role in this life stage. [ABSTRACT FROM AUTHOR]

ABSTRACT Apicomplexan parasites mobilize ionic calcium (Ca2+) from intracellular stores to promote microneme secretion and facilitate motile processes including gliding motility, invasion, and egress. Recently, a multipass transmembrane protein, ICM1, was found to be important for calcium mobilization in Plasmodium falciparum and P. berghei. Comparative genomics and phylogenetics have revealed putative ICM orthologs in Toxoplasma gondii and other apicomplexans. T. gondii possesses two ICM-like proteins, which we have named TgICM1-L (TGGT1_305470) and TgICM2-L (TGGT1_309910). TgICM1-L and TgICM2-L localized to undefined puncta within the parasite cytosol. TgICM1-L and TgICM2-L are individually dispensable in tachyzoites, suggesting a potential compensatory relationship between the two proteins may exist. Surprisingly, mutants lacking both TgICM1-L and TgICM2-L are fully viable, exhibiting no obvious defects in growth, microneme secretion, invasion, or egress. Furthermore, loss of TgICM1-L, TgICM2-L, or both does not impair the parasite’s ability to mobilize Ca2+. These findings suggest that additional proteins may participate in Ca2+ mobilization or import in Apicomplexa, reducing the dependence on ICM-like proteins in T. gondii. Collectively, these results highlight similar yet distinct mechanisms of Ca2+ mobilization between T. gondii and Plasmodium.IMPORTANCECa2+ signaling plays a crucial role in governing apicomplexan motility; yet, the mechanisms underlying Ca2+ mobilization from intracellular stores in these parasites remain unclear. In Plasmodium, the necessity of ICM1 for Ca2+ mobilization raises the question of whether this mechanism is conserved in other apicomplexans. Investigation into the orthologs of Plasmodium ICM1 in T. gondii revealed a differing requirement for ICM proteins between the two parasites. This study suggests that T. gondii employs ICM-independent mechanisms to regulate Ca2+ homeostasis and mobilization. Proteins involved in Ca2+ signaling in apicomplexans represent promising targets for therapeutic development.

Abstract Background Calcium (Ca2+) signaling regulates various vital cellular functions, including integrin activation and cell migration. Store-operated calcium entry (SOCE) via calcium release-activated calcium (CRAC) channels represents a major pathway for Ca2+ influx from the extracellular space in multiple cell types. The impact of JAK2-V617F and CALR mutations which are disease initiating in myeloproliferative neoplasms (MPN) on SOCE, calcium flux from the endoplasmic reticulum (ER) to the cytosol, and related key signaling pathways in the presence or absence of erythropoietin (EPO) or thrombopoietin (TPO) is poorly understood. Thus, this study aimed to elucidate the effects of these mutations on the aforementioned calcium dynamics, in cellular models of MPN. Methods Intracellular Ca2+ levels were measured over a time frame of 0–1080 s in Fura-2 AM labeled myeloid progenitor 32D cells expressing various mutations (JAK2-WT/EpoR, JAK2-V617F/EpoR; CALR-WT/MPL, CALR-ins5/MPL, and del52/MPL). Basal Ca2+ concentrations were assessed from 0–108 s. Subsequently, cells were stimulated with EPO/TPO in Ca2+-free Ringer solution, measuring Ca2+ levels from 109–594 s (store depletion). Then, 2 mM of Ca2+ buffer resembling physiological concentrations was added to induce SOCE, and Ca2+ levels were measured from 595–1080 s. Fura-2 AM emission ratios (F340/380) were used to quantify the integrated Ca2+ signal. Statistical significance was assessed by unpaired Student's t-test or Mann–Whitney-U-test, one-way or two-way ANOVA followed by Tukey's multiple comparison test. Results Following EPO stimulation, the area under the curve (AUC) representing SOCE significantly increased in 32D-JAK2-V617F cells compared to JAK2-WT cells. In TPO-stimulated CALR cells, we observed elevated Ca2+ levels during store depletion and SOCE in CALR-WT cells compared to CALR-ins5 and del52 cells. Notably, upon stimulation, key components of the Ca2+ signaling pathways, including PLCγ-1 and IP3R, were differentially affected in these cell lines. Hyper-activated PLCγ-1 and IP3R were observed in JAK2-V617F but not in CALR mutated cells. Inhibition of calcium regulatory mechanisms suppressed cellular growth and induced apoptosis in JAK2-V617F cells. Conclusions This report highlights the impact of JAK2 and CALR mutations on Ca2+ flux (store depletion and SOCE) in response to stimulation with EPO and TPO. The study shows that the JAK2-V617F mutation strongly alters the regulatory mechanism of EpoR/JAK2-dependent intracellular calcium balance, affecting baseline calcium levels, EPO-induced calcium entry, and PLCγ-1 signaling pathways. Our results reveal an important role of calcium flux in the homeostasis of JAK2-V617F positive cells.

Calcium flux has been successfully verified to play an important role in the malignant proliferation and progression of brain tumors, which can serve as an important diagnosis guide. However, clinical diagnosis based on calcium information remains challenging because of the highly complex and heterogeneous features in calcium signals. Here we propose a calcium feature-based tumor diagnosis and treatment guidance platform (CA-TDT-GP) using random forest analysis framework for the efficient prediction of complex tumor behaviors for clinical therapy guidance. Multiple important features associated with brain tumor biological malignancy were screened out through comprehensive feature importance analysis. It provided useful guidance for understanding the biological process and the selection of drugs of brain tumors. Further clinical validation confirmed the accurate prediction of tumor biological characteristics by the model, with a coefficient of determination of over 0.86 in the same cohort of patients and over 0.77 for the new cohort of patients. We further verified the clinical malignant assessment by this model, which performed a 100% prediction match with diagnosed WHO grades, indicating great potential of the platform for clinical guidance. This promising model provides a new diagnostic and therapeutic tool for brain tumor research and preclinical treatment. [ABSTRACT FROM AUTHOR]

Background: Calcium (Ca2+) signaling regulates various vital cellular functions, including integrin activation and cell migration. Store-operated calcium entry (SOCE) via calcium release-activated calcium (CRAC) channels represents a major pathway for Ca2+ influx from the extracellular space in multiple cell types. The impact of JAK2-V617F and CALR mutations which are disease initiating in myeloproliferative neoplasms (MPN) on SOCE, calcium flux from the endoplasmic reticulum (ER) to the cytosol, and related key signaling pathways in the presence or absence of erythropoietin (EPO) or thrombopoietin (TPO) is poorly understood. Thus, this study aimed to elucidate the effects of these mutations on the aforementioned calcium dynamics, in cellular models of MPN. Methods: Intracellular Ca2+ levels were measured over a time frame of 0–1080 s in Fura-2 AM labeled myeloid progenitor 32D cells expressing various mutations (JAK2-WT/EpoR, JAK2-V617F/EpoR; CALR-WT/MPL, CALR-ins5/MPL, and del52/MPL). Basal Ca2+ concentrations were assessed from 0–108 s. Subsequently, cells were stimulated with EPO/TPO in Ca2+-free Ringer solution, measuring Ca2+ levels from 109–594 s (store depletion). Then, 2 mM of Ca2+ buffer resembling physiological concentrations was added to induce SOCE, and Ca2+ levels were measured from 595–1080 s. Fura-2 AM emission ratios (F340/380) were used to quantify the integrated Ca2+ signal. Statistical significance was assessed by unpaired Student's t-test or Mann–Whitney-U-test, one-way or two-way ANOVA followed by Tukey's multiple comparison test. Results: Following EPO stimulation, the area under the curve (AUC) representing SOCE significantly increased in 32D-JAK2-V617F cells compared to JAK2-WT cells. In TPO-stimulated CALR cells, we observed elevated Ca2+ levels during store depletion and SOCE in CALR-WT cells compared to CALR-ins5 and del52 cells. Notably, upon stimulation, key components of the Ca2+ signaling pathways, including PLCγ-1 and IP3R, were differentially affected in these cell lines. Hyper-activated PLCγ-1 and IP3R were observed in JAK2-V617F but not in CALR mutated cells. Inhibition of calcium regulatory mechanisms suppressed cellular growth and induced apoptosis in JAK2-V617F cells. Conclusions: This report highlights the impact of JAK2 and CALR mutations on Ca2+ flux (store depletion and SOCE) in response to stimulation with EPO and TPO. The study shows that the JAK2-V617F mutation strongly alters the regulatory mechanism of EpoR/JAK2-dependent intracellular calcium balance, affecting baseline calcium levels, EPO-induced calcium entry, and PLCγ-1 signaling pathways. Our results reveal an important role of calcium flux in the homeostasis of JAK2-V617F positive cells. [ABSTRACT FROM AUTHOR]

Given bone cells' sensitive response to mechanical loading (ML), exercise and vibration therapies are used for improving bone health. The osteoblasts are functional bone cells that control the bone formation. Although osteocyte is the most sensitive bone cell to ML and regulates the function of both osteoblast and osteoclast, it is meaningful to reveal the direct effect of ML on osteoblast. The osteoblast culture system plays an important role in the study of the influence mechanism of ML on osteoblasts, due to the better control of ML's nature and bio-environment of osteoblasts. Our review describes the classic models of osteoblast culture corresponding to the ML derived from daily activities as well as from the vibration therapy. We also discussed the molecular structure, location, and bio-activity of the mechanoreceptor of osteoblast. ML can directly stimulate the mechanoreceptor by changing the structure of membrane proteins and cytomembrane potential. Calcium flux, Akt, ERK and Rho/ROCK signals occur following the activation of mechanoreceptor and regulate osteogenic transcription and cytoskeleton reorganization to affect mechanosensitivity, differentiation, and proliferation of osteoblast. These facts support that the regulating function of ML on osteoblasts is independent of other bio-active factors and help us gain further insights into ML in the studies of culture system and molecular mechanism, which is of great significance to treat ML as an adjuvant therapy for bone health. [ABSTRACT FROM AUTHOR]

Echinacea purpurea (L.) Moench is a medicinal plant commonly used for the treatment of upper respiratory tract infections, the common cold, sore throat, migraine, colic, stomach cramps, and toothaches and the promotion of wound healing. Based on the known pharmacological properties of essential oils (EOs), we hypothesized that E. purpurea EOs may contribute to these medicinal properties. In this work, EOs from the flowers of E. purpurea were steam-distilled and analyzed by gas chromatography–mass spectrometry (GC–MS), GC with flame-ionization detection (GC–FID), and chiral GC–MS. The EOs were also evaluated for in vitro antimicrobial and innate immunomodulatory activity. About 87 compounds were identified in five samples of the steam-distilled E. purpurea EO. The major components of the E. purpurea EO were germacrene D (42.0 ± 4.61%), α-phellandrene (10.09 ± 1.59%), β-caryophyllene (5.75 ± 1.72%), γ-curcumene (5.03 ± 1.96%), α-pinene (4.44 ± 1.78%), δ-cadinene (3.31 ± 0.61%), and β-pinene (2.43 ± 0.98%). Eleven chiral compounds were identified in the E. purpurea EO, including α-pinene, sabinene, β-pinene, α-phellandrene, limonene, β-phellandrene, α-copaene, β-elemene, β-caryophyllene, germacrene D, and δ-cadinene. Analysis of E. purpurea EO antimicrobial activity showed that they inhibited the growth of several bacterial species, although the EO did not seem to be effective for Staphylococcus aureus. The E. purpurea EO and its major components induced intracellular calcium mobilization in human neutrophils. Additionally, pretreatment of human neutrophils with the E. purpurea EO or (+)-δ-cadinene suppressed agonist-induced neutrophil calcium mobilization and chemotaxis. Moreover, pharmacophore mapping studies predicted two potential MAPK targets for (+)-δ-cadinene. Our results are consistent with previous reports on the innate immunomodulatory activities of β-caryophyllene, α-phellandrene, and germacrene D. Thus, this study identified δ-cadinene as a novel neutrophil agonist and suggests that δ-cadinene may contribute to the reported immunomodulatory activity of E. purpurea. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb) infection is initiated by inhalation of bacteria into lung alveoli, where they are phagocytosed by resident macrophages. Intracellular Mtb replication induces the death of the infected macrophages and the release of bacterial aggregates. Here, we show that these aggregates can evade phagocytosis by killing macrophages in a contact‐dependent but uptake‐independent manner. We use time‐lapse fluorescence microscopy to show that contact with extracellular Mtb aggregates triggers macrophage plasma membrane perturbation, cytosolic calcium accumulation, and pyroptotic cell death. These effects depend on the Mtb ESX‐1 secretion system, however, this system alone cannot induce calcium accumulation and macrophage death in the absence of the Mtb surface‐exposed lipid phthiocerol dimycocerosate. Unexpectedly, we found that blocking ESX‐1‐mediated secretion of the EsxA/EsxB virulence factors does not eliminate the uptake‐independent killing of macrophages and that the 50‐kDa isoform of the ESX‐1‐secreted protein EspB can mediate killing in the absence of EsxA/EsxB secretion. Treatment with an ESX‐1 inhibitor reduces uptake‐independent killing of macrophages by Mtb aggregates, suggesting that novel therapies targeting this anti‐phagocytic mechanism could prevent the propagation of extracellular bacteria within the lung. Synopsis: The uptake of Mycobacterium tuberculosis (Mtb) by lung macrophages is followed by intracellular replication, cell death, and release of Mtb aggregates. This study shows that extracellular Mtb aggregates can also induce macrophage death in a contact‐dependent but uptake‐independent manner. Contact with extracellular Mtb aggregates induces plasma membrane perturbation, calcium accumulation, and pyroptotic cell death in macrophages.These events are driven by the Mtb type VII secretion system ESX‐1 and by the surface‐exposed lipid PDIM.In the absence of ESX‐1 effector EsxA/EsxB secretion, the uptake‐independent killing is mediated by the ESX‐1 secreted protein EspB.Treatment with a small molecule that inhibits ESX‐1‐mediated secretion reduces uptake‐independent killing of macrophages by Mtb aggregates. [ABSTRACT FROM AUTHOR]

In this paper, we present a calcium dynamics model that firmly orchestrates exchanges of calcium flux through intracellular/extracellular sources of calcium to investigate cellular activities and calcium homeostasis. The generalized two-dimensional space-time reaction-diffusion model is formulated and analyzed for a wide range of calcium flux. The analytical technique is applied to solve the model by use of the fractional integral transform and some special functions. The existence and uniqueness of the proposed model are discussed. Finally, the obtained results are presented graphically to show the significance of calcium flux by amalgamating the memory of cells and prove the efficiency of the method. • We developed a calcium dynamics model to investigate the interplay of calcium flux based on a reaction-diffusion equation. • We present the computational technique to derive the model's solution using integral transform and special functions. • The existence and uniqueness of the calcium dynamics model are discussed. • We estimated the concentration profile in the presence/absence of VGCC, ER leak, SERCA pump, NCX, and mitochondrial flux. • The modulation of calcium flux provides a noteworthy effect due to the intermediate memory of cells. [ABSTRACT FROM AUTHOR]

Electrical stimulation is effective for bone healing. Several studies have shown that electrical stimulation affects cell migration and proliferation. This study examined whether electrical stimulation enhances the osteogenic differentiation of mouse clonal mesenchymal stem cells (cMSCs). Mouse cMSCs were exposed to electrical stimulation at three different voltages (1 V/cm, 0.5 V/cm, and 0.05 V/cm) for one hour per day. The applied electrical stimulation did not affect the cell viability. The alkaline phosphatase (ALP) activity was increased significantly at day 14 after electrical stimulation with 0.5 V/cm for one hour per day. The genes related to osteogenic differentiation were examined at days three, seven, and 14 after exposure to the three electrical stimuli. Osteocalcin (OC) expression was increased dramatically under all three-voltage conditions. These increases were closely related to the electrically stimulated increases in the intracellular calcium concentrations, assessed by Fluo-4 AM on day 14 after exposure to 0.05 V/cm for one hour per day. Furthermore, the expression of calcineurin/NFAT pathway-related genes was upregulated significantly after electrical stimulation. Thus, electrical stimulation enhances the osteogenic differentiation of mouse cMSCs by increasing the intracellular calcium concentration. [ABSTRACT FROM AUTHOR]

Introduction: Calcium flux is the master second messenger that influences the proliferation–apoptosis balance. The ability of calcium flux alterations to reduce cell growth makes ion channels interesting targets for therapy. Among all, we focused on transient receptor potential vanilloid 1, a ligand-gated cation channel with selectivity for calcium. Its involvement in hematological malignancies is poorly investigated, especially in the field of chronic myeloid leukemia, a malignancy characterized by the accumulation of immature cells.Methods: FACS analysis, Western blot analysis, gene silencing, and cell viability assay were performed to investigate the activation of transient receptor potential vanilloid 1, by N-oleoyl-dopamine, in chronic myeloid leukemia cell lines.Results: We demonstrated that the triggering of transient receptor potential vanilloid 1 inhibits cell growth and promotes apoptosis of chronic myeloid leukemia cells. Its activation induced calcium influx, oxidative stress, ER stress, mitochondria dysfunction, and caspase activation. Interestingly, a synergistic effect exerted by N-oleoyl-dopamine and the standard drug imatinib was found.Conclusion: Overall, our results support that transient receptor potential vanilloid 1 activation could be a promising strategy to enhance conventional therapy and improve the management of chronic myeloid leukemia.

Dental pain, which is the main reason for patients consulting dentists, is classified as a public health concern. The study of cellular and molecular mechanisms contributing to pain is a fundamental element for developing new analgesics. By using a selective antagonist in an in vitro model, this study aimed to establish the role of TRPV-1 in human odontoblast-like cells (OLCs) as a therapeutic target for dental pain mediated by noxious thermal and osmotic stimuli. OLCs were differentiated from dental pulp mesenchymal cells and TRPV1 expression was evaluated. Activation of TRPV-1 was determined by evaluating changes in calcium concentration after stimulation with mannitol and xylitol hyperosmotic solutions or DMEM heated at 45 °C, using the fluorescent calcium probe Fluo-4 AM. In addition, changes in fluorescence (F/F0) due to calcium flux were evaluated using fluorometry and flow cytometry. Simultaneously, the cells were co-stimulated with the selective antagonist capsazepine (CZP). OLCs expressed DSPP and DMP-1, confirming their cellular phenotype. TRPV1 was expressed, and its activation by different stimuli produced an increase in cytosolic Ca2+ which was reduced by the antagonist. Both methods used to evaluate TRPV1 activation through the measurement of calcium probe fluorescence showed similar patterns. These results suggest that TRPV-1 modulation using an antagonist can be implemented as a pharmacological strategy for managing dental pain mediated by hyperosmotic and thermal stimuli. [Display omitted] • Odontoblast-like cells (OLCs) culture is a reliable model that can be used in functional studies. • TRPV1 is expressed in OLCs, and hyperosmotic and thermal stimuli produced their activationwhich was reduced by capsazepine. • Fluorometry and Flow Cytometry are useful methods to evaluate TRPV1 activation and measurement of Ca2+ fluxes. [ABSTRACT FROM AUTHOR]

Objective: To explore whether hydrogen sulphide (H2S) could protect human periodontal ligament stem cells (PDLSCs)from senescence and the possible underlying mechanisms. Methods: Cell cycle assay and Ki-67 assay were used to measure proliferation of PDLSCs. Real-time polymerase chain reaction (PCR) was used to measure cellular senescence-related pl6 and p21. Calcium influx was detected by measurement ofCa2+ imaging. In addition, we analysed the possible mechanisms underlying H2S acting on PDLSCs by microarray. Results: The cell proliferation rate of aging PDLSCs decreased significantly. The expression of cellular senescence-related pi 6 and p21 significantly increased in aging PDLSCs. H2S donor (GYY4137) treatment increased the proliferation rate of senescence PDLSCs. Furthermore, the donor of H2S treatment effectively prevented cell cycle arrest of PDLSCs during the aging process and inhibited the expression of cellular senescence-related markers. Mechanically, H2S donor treatment could activate the calcium influx in PDLSCs. Moreover, pretreatment with TRPV4 inhibitors significantly attenuated the calcium influx induced by H2S donor treatment in PDLSCs. It also alleviated the protective effect of H2S on the senescence of PDLSCs. Conclusion: H2S alleviated the senescence of human PDLSCs by TRPV4 channel mediated calcium flux. These results provide a potential strategy to deal with cell aging and may facilitate cell therapy for oral diseases. [ABSTRACT FROM AUTHOR]

Cancer utilization of large glutamine equivalents contributes to diverging glucose-6-P flux toward the pentose phosphate shunt (PPP) to feed the building blocks and the antioxidant responses of rapidly proliferating cells. In addition to the well-acknowledged cytosolic pathway, cancer cells also run a largely independent PPP, triggered by hexose-6P-dehydrogenase within the endoplasmic reticulum (ER), whose activity is mandatory for the integrity of ER–mitochondria networking. To verify whether this reticular metabolism is dependent on glutamine levels, we complemented the metabolomic characterization of intermediates of the glucose metabolism and tricarboxylic acid cycle with the estimation of proliferating activity, energy metabolism, redox damage, and mitochondrial function in two breast cancer cell lines. ER-PPP activity and its determinants were estimated by the ER accumulation of glucose analogs. Glutamine shortage decreased the proliferation rate despite increased ATP and NADH levels. It depleted NADPH reductive power and increased malondialdehyde content despite a marked increase in glucose-6P-dehydrogenase. This paradox was explained by the deceleration of ER-PPP favored by the decrease in hexose-6P-dehydrogenase expression coupled with the opposite response of its competitor enzyme glucose-6P-phosphatase. The decreased ER-PPP activity eventually hampered mitochondrial function and calcium exchanges. These data configure the ER-PPP as a powerful, unrecognized regulator of cancer cell metabolism and proliferation. [ABSTRACT FROM AUTHOR]

Propolis is a resinous mixture of substances collected and processed from various botanical sources by honeybees. Black poplar (Populus balsamifera L.) buds are one of the primary sources of propolis. Despite their reported therapeutic properties, little is known about the innate immunomodulatory activity of essential oils from P. balsamifera and propolis. In the present studies, essential oils were isolated from the buds of P. balsamifera and propolis collected in Montana. The main components of the essential oil from P. balsamifera were E-nerolidol (64.0%), 1,8-cineole (10.8%), benzyl benzoate (3.7%), α-terpinyl acetate (2.7%), α-pinene (1.8%), o-methyl anisol (1.8%), salicylaldehyde (1.8%), and benzyl salicylate (1.6%). Likewise, the essential oil from propolis was enriched with E-nerolidol (14.4%), cabreuva oxide-VI (7.9%), α-bisabolol (7.1%), benzyl benzoate (6.1%), β-eudesmol (3.6%), T-cadinol (3.1%), 2-methyl-3-buten-2-ol (3.1%), α-eudesmol (3.0%), fokienol (2.2%), nerolidol oxide derivative (1.9%), decanal (1.8%), 3-butenyl benzene (1.5%), 1,4-dihydronaphthalene (1.5%), selina-4,11-diene (1.5%), α-cadinol (1.5%), linalool (1.4%), γ-cadinene (1.4%), 2-phenylethyl-2-methyl butyrate (1.4%), 2-methyl-2-butenol (1.3%), octanal (1.1%), benzylacetone (1.1%), and eremoligenol (1.1%). A comparison between P. balsamifera and propolis essential oils demonstrated that 22 compounds were found in both essential oil samples. Both were enriched in E-nerolidol and its derivatives, including cabreuva oxide VI and nerolidol oxides. P. balsamifera and propolis essential oils and pure nerolidol activated Ca2+ influx in human neutrophils. Since these treatments activated neutrophils, the essential oil samples were also evaluated for their ability to down-regulate the neutrophil responses to subsequent agonist activation. Indeed, treatment with P. balsamifera and propolis essential oils inhibited subsequent activation of these cells by the N-formyl peptide receptor 1 (FPR1) agonist fMLF and the FPR2 agonist WKYMVM. Likewise, nerolidol inhibited human neutrophil activation induced by fMLF (IC50 = 4.0 μM) and WKYMVM (IC50 = 3.7 μM). Pretreatment with the essential oils and nerolidol also inhibited human neutrophil chemotaxis induced by fMLF, again suggesting that these treatments down-regulated human neutrophil responses to inflammatory chemoattractants. Finally, reverse pharmacophore mapping predicted several potential kinase targets for nerolidol. Thus, our studies have identified nerolidol as a potential anti-inflammatory modulator of human neutrophils. [ABSTRACT FROM AUTHOR]

Understanding the mechanisms controlling platelet function is crucial for exploring potential therapeutic targets related to atherothrombotic pathologies and primary hemostasis disorders. Our research, which focuses on the role of platelet mitochondria and Ca2+ fluxes in platelet activation, the formation of the procoagulant phenotype, and thrombosis, has significant implications for the development of new therapeutic strategies. Traditionally, Ca2+-dependent cellular signaling has been recognized as a determinant process throughout the platelet activation, controlled primarily by store-operated Ca2+ entry and the PLC-PKC signaling pathway. However, despite the accumulated knowledge of these regulatory mechanisms, the effectiveness of therapy based on various commonly used antiplatelet drugs (such as acetylsalicylic acid and clopidogrel, among others) has faced challenges due to bleeding risks and reduced efficacy associated with the phenomenon of high platelet reactivity. Recent evidence suggests that platelet mitochondria could play a fundamental role in these aspects through Ca2+-dependent mechanisms linked to apoptosis and forming a procoagulant phenotype. In this context, the present review describes the latest advances regarding the role of platelet mitochondria and Ca2+ fluxes in platelet activation, the formation of the procoagulant phenotype, and thrombosis. [Display omitted] • Frailty syndrome is a geriatric clinical condition affected by alterations in platelet functionality. • iCa2+-dependent signaling is essential for platelet activation and thrombosis. • Ca2+-dependent platelet mitochondrial metabolism is associated with procoagulant formation phenotype and risk of bleeding. • Alterations in mitochondrial function and Ca2+fluxes are associated with a high platelet reactivity and a low response to ASA. [ABSTRACT FROM AUTHOR]

Echinacea purpurea (L.) Moench is a medicinal plant commonly used for the treatment of upper respiratory tract infections, the common cold, sore throat, migraine, colic, stomach cramps, and toothaches and the promotion of wound healing. Based on the known pharmacological properties of essential oils (EOs), we hypothesized that E. purpurea EOs may contribute to these medicinal properties. In this work, EOs from the flowers of E. purpurea were steam-distilled and analyzed by gas chromatography–mass spectrometry (GC–MS), GC with flame-ionization detection (GC–FID), and chiral GC–MS. The EOs were also evaluated for in vitro antimicrobial and innate immunomodulatory activity. About 87 compounds were identified in five samples of the steam-distilled E. purpurea EO. The major components of the E. purpurea EO were germacrene D (42.0 ± 4.61%), α-phellandrene (10.09 ± 1.59%), β-caryophyllene (5.75 ± 1.72%), γ-curcumene (5.03 ± 1.96%), α-pinene (4.44 ± 1.78%), δ-cadinene (3.31 ± 0.61%), and β-pinene (2.43 ± 0.98%). Eleven chiral compounds were identified in the E. purpurea EO, including α-pinene, sabinene, β-pinene, α-phellandrene, limonene, β-phellandrene, α-copaene, β-elemene, β-caryophyllene, germacrene D, and δ-cadinene. Analysis of E. purpurea EO antimicrobial activity showed that they inhibited the growth of several bacterial species, although the EO did not seem to be effective for Staphylococcus aureus. The E. purpurea EO and its major components induced intracellular calcium mobilization in human neutrophils. Additionally, pretreatment of human neutrophils with the E. purpurea EO or (+)-δ-cadinene suppressed agonist-induced neutrophil calcium mobilization and chemotaxis. Moreover, pharmacophore mapping studies predicted two potential MAPK targets for (+)-δ-cadinene. Our results are consistent with previous reports on the innate immunomodulatory activities of β-caryophyllene, α-phellandrene, and germacrene D. Thus, this study identified δ-cadinene as a novel neutrophil agonist and suggests that δ-cadinene may contribute to the reported immunomodulatory activity of E. purpurea.

Left atrial appendage (LAA) thrombus detachment resulting in intracranial embolism is a major complication of atrial fibrillation (AF). Endocardial endothelial cell (EEC) injury leads to thrombosis, whereas autophagy protects against EEC dysfunction. However, the role and underlying mechanisms of autophagy in EECs during AF have not been elucidated. In this study, we isolated EECs from AF model mice and observed reduced autophagic flux and intracellular calcium concentrations in EECs from AF mice. In addition, we detected an increased expression of the mechanosensitive protein PLXND1 in the cytomembranes of EECs. PLXND1 served as a scaffold protein to bind with ORAI1 and further decreased ORAI1-mediated calcium influx. The decrease in the calcium influx-mediated phosphorylation of CAMK2 is associated with the inhibition of autophagy, which results in EEC dysfunction in AF. Our study demonstrated that the change in PLXND1 expression contributes to intracellular calcium dyshomeostasis, which inhibits autophagy flux and results in EEC dysfunction in AF. This study provides a potential intervention target for EEC dysfunction to prevent and treat intracardiac thrombosis in AF and its complications. [ABSTRACT FROM AUTHOR]

Apicomplexan parasites mobilize ionic calcium (Ca 2+ ) from intracellular stores to promote microneme secretion and facilitate motile processes including gliding motility, invasion, and egress. Recently, a multipass transmembrane protein, ICM1, was found to be i mportant for c alcium m obilization in Plasmodium falciparum and P. berghei . Comparative genomics and phylogenetics have revealed putative ICM orthologs in Toxoplasma gondii and other apicomplexans. T. gondii possesses two ICM-like proteins, which we have named TgICM1-L (TGGT1_305470) and TgICM2-L (TGGT1_309910). TgICM1-L and TgICM2-L localized to undefined puncta within the parasite cytosol. TgICM1-L and TgICM2-L are individually dispensable in tachyzoites, suggesting a potential compensatory relationship between the two proteins may exist. Surprisingly, mutants lacking both TgICM1-L and TgICM2-L are fully viable, exhibiting no obvious defects in growth, microneme secretion, invasion, or egress. Furthermore, loss of TgICM1-L, TgICM2-L, or both does not impair the parasite's ability to mobilize Ca 2+ . These findings suggest that additional proteins may participate in Ca 2+ mobilization or import in Apicomplexa, reducing the dependence on ICM-like proteins in T. gondii . Collectively, these results highlight similar yet distinct mechanisms of Ca 2+ mobilization between T. gondii and Plasmodium .IMPORTANCECa 2+ signaling plays a crucial role in governing apicomplexan motility; yet, the mechanisms underlying Ca 2+ mobilization from intracellular stores in these parasites remain unclear. In Plasmodium , the necessity of ICM1 for Ca 2+ mobilization raises the question of whether this mechanism is conserved in other apicomplexans. Investigation into the orthologs of Plasmodium ICM1 in T. gondii revealed a differing requirement for ICM proteins between the two parasites. This study suggests that T. gondii employs ICM-independent mechanisms to regulate Ca 2+ homeostasis and mobilization. Proteins involved in Ca 2+ signaling in apicomplexans represent promising targets for therapeutic development., Competing Interests: The authors declare no conflict of interest.

Backgrounds: Lumbar laminectomy is usually utilized for lumbar disc herniation (LDH), but also causes epidural fibrosis (EF) process associated with abnormal proliferation of fibroblasts. FAM172A is associated with ER stress and cell proliferation, but its mechanism was unclear, especially in the process of EF. Methods: Therefore, the regulation of FAM172A on the calcium flux and autophagy in fibroblasts were investigated by inducing ER stress with tunicamycin and upexpression or downexpression of FAM172A. The calcium flux was determined using Fluo‐3, and autophagy was examined with immunofluorescence or western blot for LC3, Beclin‐1, ATG‐5, and p62. Moreover, the apoptotic protein of Bax and Bcl‐2 was detected, too. Furthermore, the laminectomy model was constructed and then dealt with overexpression of FAM172A. Results: Tunicamycin‐induced endoplasmic reticulum (ER) stress and autophagy process in fibroblasts were associated with the calcium flux regulated by FAM172A, especially in EF cells. Besides, tunicamycin induced autophagy and suppressed cell apoptosis of fibroblasts. Furthermore, FAM72A repressed the proliferation of fibroblasts and the process of EF in the laminectomy model through the mediation of the autophagy process. Conclusions: Tunicamycin‐induced endoplasmic reticulum (ER) stress in fibroblasts was associated with calcium flux mediated by FAM172A. FAM72A participated in the autophagy regulation of fibroblasts and maybe the key interaction regulator of apoptosis and autophagy in fibroblasts, especially for epidural scar cells. [ABSTRACT FROM AUTHOR]

Despite their reported therapeutic properties, not much is known about the immunomodulatory activity of essential oils present in Artemisia species. We isolated essential oils from the flowers and leaves of five Artemisia species: A. tridentata, A. ludoviciana, A. dracunculus, A. frigida, and A. cana. The chemical composition of the Artemisia essential oil samples had similarities and differences as compared to those previously reported in the literature. The main components of essential oils obtained from A. tridentata, A. ludoviciana, A. frigida, and A. cana were camphor (23.0–51.3%), 1,8-cineole (5.7–30.0%), camphene (1.6–7.7%), borneol (2.3–14.6%), artemisiole (1.2–7.5%), terpinen-4-ol (2.0–6.9%), α-pinene (0.8–3.9%), and santolinatriene (0.7–3.5%). Essential oils from A. dracunculus were enriched in methyl chavicol (38.8–42.9%), methyl eugenol (26.1–26.4%), terpinolene (5.5–8.8%), (E/Z)-β-ocimene (7.3–16.0%), β-phellandrene (1.3–2.2%), p-cymen-8-ol (0.9–2.3%), and xanthoxylin (1.2–2.2%). A comparison across species also demonstrated that some compounds were present in only one Artemisia species. Although Artemisia essential oils were weak activators of human neutrophils, they were relatively more potent in inhibiting subsequent neutrophil Ca2+ mobilization with N-formyl peptide receptor 1 (FPR1) agonist fMLF- and FPR2 agonist WKYMVM, with the most potent being essential oils from A. dracunculus. Further analysis of unique compounds found in A. dracunculus showed that farnesene, a compound with a similar hydrocarbon structure as lipoxin A4, inhibited Ca2+ influx induced in human neutrophils by fMLF (IC50 = 1.2 μM), WKYMVM (IC50 = 1.4 μM), or interleukin 8 (IC50 = 2.6 μM). Pretreatment with A. dracunculus essential oils and farnesene also inhibited human neutrophil chemotaxis induced by fMLF, suggesting these treatments down-regulated human neutrophil responses to inflammatory chemoattractants. Thus, our studies have identified farnesene as a potential anti-inflammatory modulator of human neutrophils. [ABSTRACT FROM AUTHOR]

Abstract Backgrounds Lumbar laminectomy is usually utilized for lumbar disc herniation (LDH), but also causes epidural fibrosis (EF) process associated with abnormal proliferation of fibroblasts. FAM172A is associated with ER stress and cell proliferation, but its mechanism was unclear, especially in the process of EF. Methods Therefore, the regulation of FAM172A on the calcium flux and autophagy in fibroblasts were investigated by inducing ER stress with tunicamycin and upexpression or downexpression of FAM172A. The calcium flux was determined using Fluo‐3, and autophagy was examined with immunofluorescence or western blot for LC3, Beclin‐1, ATG‐5, and p62. Moreover, the apoptotic protein of Bax and Bcl‐2 was detected, too. Furthermore, the laminectomy model was constructed and then dealt with overexpression of FAM172A. Results Tunicamycin‐induced endoplasmic reticulum (ER) stress and autophagy process in fibroblasts were associated with the calcium flux regulated by FAM172A, especially in EF cells. Besides, tunicamycin induced autophagy and suppressed cell apoptosis of fibroblasts. Furthermore, FAM72A repressed the proliferation of fibroblasts and the process of EF in the laminectomy model through the mediation of the autophagy process. Conclusions Tunicamycin‐induced endoplasmic reticulum (ER) stress in fibroblasts was associated with calcium flux mediated by FAM172A. FAM72A participated in the autophagy regulation of fibroblasts and maybe the key interaction regulator of apoptosis and autophagy in fibroblasts, especially for epidural scar cells.

Thrombin-activated receptor-1 induces angiogenesis, cell proliferation, and invasion in tumors. The protein-activated receptor-1 (PAR1) is a G-protein coupled receptor that can be catalyzed by blood-derived serine proteases. Though Protease-activated receptor 1 plays an imperative role in coagulation and hemostasis, recent studies have found that PAR1 activation also affects the central nervous system (CNS) in addition to the vasculature. The present study focused on examining the prevalence of PAR1 in two human gliomas cell lines, D54 and U87, as well as the downstream signaling pathway. PAR1 was significantly more expressed in D54 cells than in U87 cells. An increase in PAR1 activity caused an increase in ERK1/2 activity, along with increased migration and invasion. SCH79797 (antagonist) significantly reduced the migration and invasion of the glioma cell lines. These cell lines' migration and invasion upon activator and inhibitor stimulation were related to PAR1 expression. In cells, Ca + 2 concentration ([Ca + 2]i) is altered by PAR1. Thrombin and TFLLR both induced an increase in [Ca + 2]i that was inhibited by SCH79797 (PAR1 inhibitor). The study concludes that PAR1 signaling contributes to the progression of gliomas. PAR1 and its downstream signaling may act as potential therapeutic targets for the prevention of glioma formation.

Nanoparticles (NPs) generate localized magnetic forces during magnetic stimulation, which can, in turn, modulate neuronal excitability and regulate downstream signaling in neurons. In agreement with this idea, under static magnetic field stimulation (SMS), gold-coated superparamagnetic iron oxide (Au-SPIO) core–shell nanoparticles (NPs) can promote and guide the direction of neurite outgrowth. Inspired by these promising results, this study investigates how SMS on Au-SPIO (SMS-Au-SPIO) affects the physiology of midbrain neurons. Transmission electron microscopy (TEM) images showed quasispherical shapes and a diameter of 20 ± 4 nm of Au-SPIO NPs synthesized by forming an Au layer on SPIO using a hydroxylamine hydrochloride-assisted seed growth method. We found that SMS enhanced intracellular uptake of Au-SPIO and that SMS-Au-SPIO resulted in a delayed blockade of an L-type voltage-gated Ca2+ channel (VGCC) in midbrain neurons. Specifically, the frequency of spontaneous L-type VGCC-induced Ca2+ fluxes was significantly reduced in midbrain neurons exposed to either SMS or Au-SPIO or SMS-Au-SPIO. A power spectrum density analysis of Ca2+ fluxes showed that SMS decreased Ca2+ fluxes amplitudes (<0.1 Hz) before and after L-type VGCC blockade. By contrast, SMS-Au-SPIO decreased Ca2+ flux amplitudes only after L-type VGCC blockade, suggesting a modulation of L-type VGCC by SMS-Au-SPIO. Finally, while SMS alone induced apoptosis of dopaminergic (DA) neurons, SMS-Au-SPIO did not. Thus, SMS and SMS-Au-SPIO differentially modulate L-type VGCC-mediated Ca2+ fluxes, and downstream apoptotic signaling in midbrain neurons, implying the possible application of SMS-Au-SPIO as a drug delivery strategy to treat Parkinson's disease. [ABSTRACT FROM AUTHOR]

Wounding induces a calcium wave and disrupts the calcium gradient across the epidermis but mechanisms mediating calcium and downstream signalling, and longer‐term wound healing responses are incompletely understood. As expected, live‐cell confocal imaging of Fluo‐4‐loaded normal human keratinocytes showed an immediate increase in [Ca2+]i at the wound edge that spread as a calcium wave (8.3 µm/s) away from the wound edge with gradually diminishing rate of rise and amplitude. The amplitude and area under the curve of [Ca2+]i flux was increased in high (1.2 mM) [Ca2+]o media. 18α‐glycyrrhetinic acid (18αGA), a gap‐junction inhibitor or hexokinase, an ATP scavenger, blocked the wound‐induced calcium wave, dependent in part on [Ca2+]o. Wounding in a high [Ca2+]o increased nuclear factor of activated T‐cells (NFAT) but not NFkB activation, assessed by dual‐luciferase receptor assays compared to unwounded cells. Treatment with 18αGA or the store‐operated channel blocker GSK‐7975A inhibited wound‐induced NFAT activation, whereas treatment with hexokinase did not. Real‐time cell migration analysis, measuring wound closure rates over 24 h, revealed that 18αGA essentially blocked wound closure whereas hexokinase and GSK‐7975A showed relatively minimal effects. Together these data indicate that while both gap‐junction communication and ATP release from damaged cells are important in regulating the wound‐induced calcium wave, long‐term transcriptional and functional responses are dominantly regulated by gap‐junction communication. [ABSTRACT FROM AUTHOR]

N-methyl-D-aspartate (NMDA) receptors are critical for higher-order nervous system function, but in previously published protocols to convert human induced pluripotent stem cells (iPSCs) to mature neurons, functional NMDA receptors (NMDARs) are often either not reported or take an extended time to develop. Here, we describe a protocol to convert human iPSC-derived neural progenitor cells (NPCs) to mature neurons in only 37 days. We demonstrate that the mature neurons express functional NMDARs exhibiting ligand-activated calcium flux, and we document the presence of NMDAR-mediated electrically evoked postsynaptic current. In addition to being more rapid than previous procedures, our protocol is straightforward, does not produce organoids which are difficult to image, and does not involve co-culture with rodent astrocytes. This could enhance our ability to study primate/human-specific aspects of NMDAR function and signaling in health and disease. [ABSTRACT FROM AUTHOR]

N-methyl-D-aspartate (NMDA) receptors are critical for higher-order nervous system function, but in previously published protocols to convert human induced pluripotent stem cells (iPSCs) to mature neurons, functional NMDA receptors (NMDARs) are often either not reported or take an extended time to develop. Here, we describe a protocol to convert human iPSC-derived neural progenitor cells (NPCs) to mature neurons in only 37 days. We demonstrate that the mature neurons express functional NMDARs exhibiting ligand-activated calcium flux, and we document the presence of NMDAR-mediated electrically evoked postsynaptic current. In addition to being more rapid than previous procedures, our protocol is straightforward, does not produce organoids which are difficult to image, and does not involve co-culture with rodent astrocytes. This could enhance our ability to study primate/human-specific aspects of NMDAR function and signaling in health and disease.

Propolis is a resinous mixture of substances collected and processed from various botanical sources by honeybees. Black poplar (Populus balsamifera L.) buds are one of the primary sources of propolis. Despite their reported therapeutic properties, little is known about the innate immunomodulatory activity of essential oils from P. balsamifera and propolis. In the present studies, essential oils were isolated from the buds of P. balsamifera and propolis collected in Montana. The main components of the essential oil from P. balsamifera were E-nerolidol (64.0%), 1,8-cineole (10.8%), benzyl benzoate (3.7%), α-terpinyl acetate (2.7%), α-pinene (1.8%), o-methyl anisol (1.8%), salicylaldehyde (1.8%), and benzyl salicylate (1.6%). Likewise, the essential oil from propolis was enriched with E-nerolidol (14.4%), cabreuva oxide-VI (7.9%), α-bisabolol (7.1%), benzyl benzoate (6.1%), β-eudesmol (3.6%), T-cadinol (3.1%), 2-methyl-3-buten-2-ol (3.1%), α-eudesmol (3.0%), fokienol (2.2%), nerolidol oxide derivative (1.9%), decanal (1.8%), 3-butenyl benzene (1.5%), 1,4-dihydronaphthalene (1.5%), selina-4,11-diene (1.5%), α-cadinol (1.5%), linalool (1.4%), γ-cadinene (1.4%), 2-phenylethyl-2-methyl butyrate (1.4%), 2-methyl-2-butenol (1.3%), octanal (1.1%), benzylacetone (1.1%), and eremoligenol (1.1%). A comparison between P. balsamifera and propolis essential oils demonstrated that 22 compounds were found in both essential oil samples. Both were enriched in E-nerolidol and its derivatives, including cabreuva oxide VI and nerolidol oxides. P. balsamifera and propolis essential oils and pure nerolidol activated Ca2+ influx in human neutrophils. Since these treatments activated neutrophils, the essential oil samples were also evaluated for their ability to down-regulate the neutrophil responses to subsequent agonist activation. Indeed, treatment with P. balsamifera and propolis essential oils inhibited subsequent activation of these cells by the N-formyl peptide receptor 1 (FPR1) agonist fMLF and the FPR2 agonist WKYMVM. Likewise, nerolidol inhibited human neutrophil activation induced by fMLF (IC50 = 4.0 μM) and WKYMVM (IC50 = 3.7 μM). Pretreatment with the essential oils and nerolidol also inhibited human neutrophil chemotaxis induced by fMLF, again suggesting that these treatments down-regulated human neutrophil responses to inflammatory chemoattractants. Finally, reverse pharmacophore mapping predicted several potential kinase targets for nerolidol. Thus, our studies have identified nerolidol as a potential anti-inflammatory modulator of human neutrophils.

Keywords: calcium flux; cross-linking; Ly49 receptors; MHC class I; NK cell EN calcium flux cross-linking Ly49 receptors MHC class I NK cell 2531 2534 4 10/07/21 20211001 NES 211001 Simultaneous triggering of NK1.1 and MHC class I on NK cells gives a higher Ca SP 2+ sp flux response compared to triggering the NK1.1 receptor alone. MHC class I molecules are not only ligands for inhibitory NK cell receptors, but may also inhibit NK cell function themselves [3-5]. GLO:BVO/01oct21:eji5164-fig-0001.jpg PHOTO (COLOR): 1 MHC class I cross-linking triggers Ca2+ flux in mouse NK cells and provides an additive stimulatory signal in combination with NK1.1. MHC class I molecules co-stimulate NK1.1 signaling and enhance Ca2+ flux in murine NK cells. [Extracted from the article]

Piezo proteins (Piezo1 and Piezo2) are recently identified mechanically activated cation channels in eukaryotic cells and associated with physiological responses to touch, pressure, and stretch. In particular, human RBCs express Piezo1 on their membranes, and mutations of Piezo1 have been linked to hereditary xerocytosis. To date, however, physiological functions of Piezo1 on normal RBCs remain poorly understood. Here, we show that Piezo1 regulates mechanotransductive release of ATP from human RBCs by controlling the shear-induced calcium (Ca(2+)) influx. We find that, in human RBCs treated with Piezo1 inhibitors or having mutant Piezo1 channels, the amounts of shear-induced ATP release and Ca(2+) influx decrease significantly. Remarkably, a critical extracellular Ca(2+) concentration is required to trigger significant ATP release, but membrane-associated ATP pools in RBCs also contribute to the release of ATP. Our results show how Piezo1 channels are likely to function in normal RBCs and suggest a previously unidentified mechanotransductive pathway in ATP release. Thus, we anticipate that the study will impact broadly on the research of red cells, cellular mechanosensing, and clinical studies related to red cell disorders and vascular disease.

Despite their reported therapeutic properties, not much is known about the immunomodulatory activity of essential oils present in Artemisia species. We isolated essential oils from the flowers and leaves of five Artemisia species: A. tridentata, A. ludoviciana, A. dracunculus, A. frigida, and A. cana. The chemical composition of the Artemisia essential oil samples had similarities and differences as compared to those previously reported in the literature. The main components of essential oils obtained from A. tridentata, A. ludoviciana, A. frigida, and A. cana were camphor (23.0–51.3%), 1,8-cineole (5.7–30.0%), camphene (1.6–7.7%), borneol (2.3–14.6%), artemisiole (1.2–7.5%), terpinen-4-ol (2.0–6.9%), α-pinene (0.8–3.9%), and santolinatriene (0.7–3.5%). Essential oils from A. dracunculus were enriched in methyl chavicol (38.8–42.9%), methyl eugenol (26.1–26.4%), terpinolene (5.5–8.8%), (E/Z)-β-ocimene (7.3–16.0%), β-phellandrene (1.3–2.2%), p-cymen-8-ol (0.9–2.3%), and xanthoxylin (1.2–2.2%). A comparison across species also demonstrated that some compounds were present in only one Artemisia species. Although Artemisia essential oils were weak activators of human neutrophils, they were relatively more potent in inhibiting subsequent neutrophil Ca2+ mobilization with N-formyl peptide receptor 1 (FPR1) agonist fMLF- and FPR2 agonist WKYMVM, with the most potent being essential oils from A. dracunculus. Further analysis of unique compounds found in A. dracunculus showed that farnesene, a compound with a similar hydrocarbon structure as lipoxin A4, inhibited Ca2+ influx induced in human neutrophils by fMLF (IC50 = 1.2 μM), WKYMVM (IC50 = 1.4 μM), or interleukin 8 (IC50 = 2.6 μM). Pretreatment with A. dracunculus essential oils and farnesene also inhibited human neutrophil chemotaxis induced by fMLF, suggesting these treatments down-regulated human neutrophil responses to inflammatory chemoattractants. Thus, our studies have identified farnesene as a potential anti-inflammatory modulator of human neutrophils.

Understanding the molecular functions of less-studied proteins is an important task of life science research. Despite reports of basic leucine zipper and W2 domain-containing protein 2 (BZW2) promoting cancer progression first emerging in 2017, little is known about its molecular function. Using a quantitative proteomic approach to identify its interacting proteins, we found that BZW2 interacts with both endoplasmic reticulum (ER) and mitochondrial proteins. We thus hypothesized that BZW2 localizes to and promotes the formation of ER-mitochondria contact sites and that such localization would promote calcium transport from ER to the mitochondria and promote ATP production. Indeed, we found that BZW2 localized to ER-mitochondria contact sites and that BZW2 knockdown decreased ER-mitochondria contact, mitochondrial calcium levels, and ATP production. These findings provide key insights into molecular functions of BZW2, the potential role of BZW2 in cancer progression, and highlight the utility of interactome data in understanding the function of less-studied proteins., Competing Interests: Conflict of interest H. L. is a founder and consultant for Sedec Therapeutics., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Recruitment of leukocytes to sites of acute inflammation is guided by spatial and temporal cues that ensure appropriate cell numbers infiltrate the tissue at precise locations to protect it from infection and initiate repair. On inflamed endothelium, neutrophil rolling via selectins elicits cytosolic calcium release from endoplasmic reticulum (ER)‐stores that are synergistic with chemokine signaling to activate formation of high affinity (HA) LFA‐1 bonds to ICAM‐1, which is necessary to anchor cells against the drag force of blood flow. Bond tension on LFA‐1 within the area of adhesive contact with endothelium elicits calcium entry through calcium release‐activated calcium channel protein 1 (Orai‐1) membrane channels that in turn activate neutrophil shape change and migration. We hypothesized that mechanotransduction via LFA‐1 is mediated by assembly of a cytosolic molecular complex consisting of Kindlin‐3, receptor for activated C kinase 1 (RACK1), and Orai1. Initiation of Ca2+ flux at sites of adhesive contact required a threshold level of shear stress and increased with the magnitude of bond tension transduced across as few as 200 HA LFA‐1. A sequential mechanism triggered by force acting on LFA‐1/Kindlin‐3 precipitated dissociation of RACK1, which formed a concentration gradient above LFA‐1 bond clusters. This directed translocation of ER proximal to Orai1, where binding of inositol 1,4,5‐triphosphate receptor type 1 and activation via stromal interaction molecule 1 elicited Ca flux and subsequent neutrophil shape change and motility. We conclude that neutrophils sense adhesive traction on LFA‐1 bonds on a submicron scale to direct calcium influx, thereby ensuring sufficient shear stress of blood flow is present to trigger cell arrest and initiate transmigration at precise regions of vascular inflammation. [ABSTRACT FROM AUTHOR]

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. In the past years, new therapeutic approaches (e.g., ibrutinib or venetoclax) have been established and greatly improved treatment of CLL. However, complete control or cure of the disease have not been reached so far. Thus, reliable prognostic markers are an imperative for treatment decisions. Recent studies have revealed an essential role for B cell receptor (BCR) signaling in the pathogenesis, prognosis, and therapy of CLL. A heterogeneous response to receptor stimulation with anti‐IgM treatment culminating in different calcium flux capabilities has been demonstrated by several authors. However, the methods employed have not reached clinical application. Here, we report on a flow cytometry‐based assay to evaluate calcium flux capabilities in CLL and demonstrate that compromised BCR signaling with diminished calcium flux is associated with a significantly better clinical outcome and progression free survival. In summary, our data strongly support the role of compromised BCR signaling as an important prognostic marker in CLL and establish a novel diagnostic tool for its assessment in clinical settings. [ABSTRACT FROM AUTHOR]

Thyroid hormone exerts a large number of influences on the cardiovascular system. Increased thyroid hormone action increases the force and speed of systolic contraction and the speed of diastolic relaxation and these are largely beneficial effects. Furthermore, thyroid hormone has marked electrophysiological effects increasing heart rate and the propensity for atrial fibrillation and these effects are largely mal-adaptive. In addition, thyroid hormone markedly increases cardiac angiogenesis and decreases vascular tone. These multiple thyroid hormone effects are largely mediated by the action of nuclear based thyroid hormone receptors (TR) the thyroid hormone receptor alpha and beta. TRα is the predominant isoform in the heart. Rapid nongenomic thyroid hormone effects also occur, which can be clearly demonstrated in ex-vivo experiments. Some of the most marked thyroid hormone effects in cardiac myocytes involve influences on calcium flux, with thyroid hormone promoting expression of the gene encoding the calcium pump of the sarcoplasmic reticulum (SERCa2). In contrast, in hypothyroid animals phospholamban levels, which inhibit the SERCa2 pump, are increased. In addition, marked effects are exerted on the calcium channel of the sarcoplasmic reticulum the ryanodine channel. Related to myofibrillar proteins, myosin heavy chain alpha is increased by T3 and MHC beta is decreased. Complex and interesting interactions occur between cardiac hypertrophy induced by excess thyroid hormone action and cardiac hypertrophy occurring with heart failure. The thyroid hormone mediated cardiac hypertrophy in its initial phases presents a physiological hypertrophy with increases in SERCa2 levels and decreased expression of MHC beta. In contrast, pressure overload induced heart failure leads to a “pathological” cardiac hypertrophy which is largely mediated by activation of the calcineurin system and the MAPkinases signaling system. Recent evidence indicates that heart failure can lead to a downregulation of the thyroid hormone signaling system in the heart. In the failing heart, decreases of thyroid hormone receptor levels occur. In addition, serum levels of T4 and T3 are decreased with heart failure in the frame of the non-thyroidal illness syndrome. The decrease in T3 serves as an indicator for a bad prognosis in the heart failure patient being linked to increased mortality. In animal models, it can be shown that in pressure overload-induced cardiac hypertrophy a decrease of thyroid hormone receptor levels occurs. Cardiac function can be improved by increasing expression of thyroid hormone receptors mediated by adeno-associated virus based gene transfer. The failing heart may develop a “hypothyroid” status contributing to diminished cardiac contractile function.

Little is known about the immunomodulatory activity of essential oils isolated from Juniperus species. Thus, we isolated essential oils from the cones and leaves of eight juniper species found in Montana and in Kazakhstan, including J. horizontalis, J. scopolorum, J. communis, J. seravschanica, J. sabina, J. pseudosabina, J. pseudosabina subsp. turkestanica, and J. sibirica. We report here the chemical composition and innate immunomodulatory activity of these essential oils. Compositional analysis of the 16 samples of Juniper essential oils revealed similarities and differences between our analyses and those previously reported for essential oils from this species. Our studies represent the first analysis of essential oils isolated from the cones of four of these Juniper species. Several essential oil samples contained high levels of cedrol, which was fairly unique to three Juniper species from Kazakhstan. We found that these essential oils and pure (+)-cedrol induced intracellular Ca2+ mobilization in human neutrophils. Furthermore, pretreatment of human neutrophils and N-formyl peptide receptor 1 and 2 (FPR1 and FPR2) transfected HL60 cells with these essential oils or (+)-cedrol inhibited agonist-induced Ca2+ mobilization, suggesting these responses were desensitized by this pretreatment. In support of this conclusion, pretreatment with essential oils from J. seravschanica cones (containing 16.8% cedrol) or pure (+)-cedrol inhibited human neutrophil chemotaxis to N-formyl peptide. Finally, reverse pharmacophore mapping predicted several potential kinase targets for cedrol. Thus, our studies have identified cedrol as a novel neutrophil agonist that can desensitize cells to subsequent stimulation by N-formyl peptide.

NK cell‐mediated Ab‐dependent cellular cytotoxicity (ADCC) is increasingly recognized to play an important role in cancer immunotherapy, transplant rejection, and autoimmunity. However, several aspects of the molecular interactions of IgG subclasses with the Fc‐gamma receptor IIIA (FcγRIIIA)/CD16a expressed on NK cells remain unknown. The aim of the current study was to further analyze the role of IgG subclasses and FCGR3A V158F single nucleotide polymorphism (SNP) on Ca2+ signaling and NK cell‐mediated ADCC against Daudi target cells in vitro. NK cells were isolated from donors with different FCGR3A SNP. The affinity of rituximab IgG subclasses to CD20 expressed on Daudi cells showed similar dissociation constant as tested by flow cytometry. Induction of Ca2+ signaling, degranulation, intracellular cytokine production, and ADCC was demonstrated for IgG1 and IgG3, to a lesser degree also for IgG4, but not for IgG2. Compared to NK cells carrying the low‐affinity (FF) variant for the FCGR3A V158F SNP, binding of IgG1 and IgG3 to NK cells carrying the high‐affinity (VV) and VF SNP variants was two‐ to threefold higher. Variations of FCGR3A SNP among the eight tested donors (1 VV, 3FF, and 4VF) revealed no significant differences of Ca2+ signaling and degranulation; however, ADCC was somewhat weaker in donors with the low‐affinity FF variation. In conclusion, this is the first study correlating Ca2+ signaling and NK cell‐mediated ADCC triggered by the four IgG subclasses with the FCGR3A V158F SNP. Our findings indicate important differences in the interactions of IgG subclasses with FcγRIIIA/CD16a but no major impact of FCGR3A SNP and may therefore help to better correlate the functional properties of particular engineered therapeutic antibodies in vitro with individual differences of their clinical efficacy. [ABSTRACT FROM AUTHOR]

Temperature is an essential physical factor that affects the plant life cycle. Almost all plant species have evolved a robust signal transduction system that enables them to sense changes in the surrounding temperature, relay this message and accordingly adjust their metabolism and cellular functions to avoid heat stress-related damage. Wheat (Triticum aestivum), being a cool-season crop, is very sensitive to heat stress. Any increase in the ambient temperature, especially at the reproductive and grain-filling stages, can cause a drastic loss in wheat yield. Heat stress causes lipid peroxidation due to oxidative stress, resulting in the damage of thylakoid membranes and the disruption of their function, which ultimately decreases photosynthesis and crop yield. The cell membrane/plasma membrane plays prominent roles as an interface system that perceives and translates the changes in environmental signals into intracellular responses. Thus, membrane lipid composition is a critical factor in heat stress tolerance or susceptibility in wheat. In this review, we elucidate the possible involvement of calcium influx as an early heat stress-responsive mechanism in wheat plants. In addition, the physiological implications underlying the changes in lipid metabolism under high-temperature stress in wheat and other plant species will be discussed. In-depth knowledge about wheat lipid reprograming can help develop heat-tolerant wheat varieties and provide approaches to solve the impact of global climate change. [ABSTRACT FROM AUTHOR]

Constipation is a common bowel disease in adults with the symptoms of dry stool or difficulty passing stool. Compared with medication therapy, patients show more compliance with the diet therapy, and thus the diet therapy normally exhibits better therapeutic effect. Allium mongolicum Regel s a perennial herb of Liliaceae native to Mongolia, Kazakhstan, and China, which is traditionally used for constipation. In this paper, we partly clarify the effectiveness of A. mongolicum on constipation from two aspects, including maintaining colon water content and increasing intestinal transit. In loperamide-induced constipation mice model, nine days oral administration of A. mongolicum 50% ethanolic extract increased luminal side water content and regulated intestinal movement rhythm to normalize stools. The activity at least partly related to down-regulation of colon aquaporins 3 (AQP3) expression, and up-regulation and activation of G protein alpha (Gα) and phosphoinositide 3-kinases (PI3K). Further, activities on intestine movements were tested using compounds isolated from A. mongolicum. Three kinds of major flavonoids significantly increased cellular calcium flux in HCT116 cells and promoted mice intestine smooth muscle contraction. The activity may be related to M choline receptor, μ opioid receptor, 5-HT3 receptor, and inositol 1,4,5-trisphosphate (IP3) receptor. [ABSTRACT FROM AUTHOR]

Cytosolic calcium (c Ca2+) entry into mitochondria is facilitated by the mitochondrial membrane potential (ΔΨm), an electrochemical gradient generated by the electron transport chain (ETC). Is has been assumed that as long as mutations that affect the ETC do not affect the ΔΨm, the mitochondrial Ca2+ (m Ca2+) homeostasis remains normal. We show that knockdown of NDUFAF3 and SDHB reduce ETC activity altering m Ca2+ efflux and influx rates while ΔΨm remains intact. Shifting the equilibrium toward lower [Ca2+] m accumulation renders cells resistant to death. Our findings reveal an unexpected relationship between complex I and II with the m Ca2+ homeostasis independent of ΔΨm. • NDUFAF3 and SDHA are essential for complex I and II assembly respectively and its absence decrease OXPHOS. • Knockdown of NDUFAF3 and SDHB altered mitochondrial Ca2+ efflux and influx rates in absence of ΔΨm loss. • Shifting the equilibrium toward lower concentration of mitochondrial Ca2+ accumulation renders cells resistant to death. [ABSTRACT FROM AUTHOR]

Rhododendron (Ericaceae) extracts contain flavonoids, chromones, terpenoids, steroids, and essential oils and are used in traditional ethnobotanical medicine. However, little is known about the immunomodulatory activity of essential oils isolated from these plants. Thus, we isolated essential oils from the flowers and leaves of R. albiflorum (cascade azalea) and analyzed their chemical composition and innate immunomodulatory activity. Compositional analysis of flower (REOFl) versus leaf (REOLv) essential oils revealed significant differences. REOFl was comprised mainly of monoterpenes (92%), whereas sesquiterpenes were found in relatively low amounts. In contrast, REOLv was primarily composed of sesquiterpenes (90.9%), with a small number of monoterpenes. REOLv and its primary sesquiterpenes (viridiflorol, spathulenol, curzerene, and germacrone) induced intracellular Ca2+ mobilization in human neutrophils, C20 microglial cells, and HL60 cells transfected with N-formyl peptide receptor 1 (FPR1) or FPR2. On the other hand, pretreatment with these essential oils or component compounds inhibited agonist-induced Ca2+ mobilization and chemotaxis in human neutrophils and agonist-induced Ca2+ mobilization in microglial cells and FPR-transfected HL60 cells, indicating that the direct effect of these compounds on [Ca2+]i desensitized the cells to subsequent agonist activation. Reverse pharmacophore mapping suggested several potential kinase targets for these compounds; however, these targets were not supported by kinase binding assays. Our results provide a cellular and molecular basis to explain at least part of the beneficial immunotherapeutic properties of the R. albiflorum essential oils and suggest that essential oils from leaves of this plant may be effective in modulating some innate immune responses, possibly by inhibition of neutrophil migration.

Natural killer (NK) cell function is regulated by a balance between activating and inhibitory receptors, but the details of this receptor interplay are not extensively understood. We developed a flow cytometry-based assay system in which Ca2+ flux downstream of antibody-mediated activating receptor triggering was studied in the presence or absence of inhibitory receptor co-crosslinking. We show that the inhibitory influence on activating receptor-induced Ca2+ flux is quantitatively regulated, both on murine and human NK cells. Furthermore, both activating and inhibitory receptors operate in an additive way, suggesting that a fine-tuned balance between activating and inhibitory receptors regulate proximal NK cell signaling. We also demonstrate that murine NK cell expression of H2Dd lowered the capacity of Ly49A to deliver inhibitory signals after antibody crosslinking, suggesting that the cis interaction between H2Dd and Ly49A reduce the signaling capacity of Ly49A in this setting. Finally, we show that priming of NK cells by IL-15 rapidly augments Ca2+ flux after activating receptor signaling without attenuating the potential of inhibitory receptors to reduce Ca2+ flux. Our data shed new light on NK cell inhibition and raises new questions for further studies.

Initiation of T cell antigen receptor (TCR) signaling is dependent on Lck, a Src family kinase. The Src-like adaptor protein (SLAP) contains Src homology (SH)3 and SH2 domains, which are highly homologous to those of Lck and other Src family members. Because of the structural similarity between Lck and SLAP, we studied its potential role in TCR signaling. Here, we show that SLAP is expressed in T cells, and that when expressed in Jurkat T cells it can specifically inhibit TCR signaling leading to nuclear factor of activated T cells (NFAT)-, activator protein 1 (AP-1)-, and interleukin 2-dependent transcription. The SH3 and SH2 domains of SLAP are required for maximal attenuation of TCR signaling. This inhibitory activity can be bypassed by the combination of phorbol myristate acetate (PMA) and ionomycin, suggesting that SLAP acts proximally in the TCR signaling pathway. SLAP colocalizes with endosomes in Jurkat and in HeLa cells, and is insoluble in mild detergents. In stimulated Jurkat cells, SLAP associates with a molecular signaling complex containing CD3zeta, ZAP-70, SH2 domain-containing leukocyte protein of 76 kD (SLP-76), Vav, and possibly linker for activation of T cells (LAT). These results suggest that SLAP is a negative regulator of TCR signaling.

In addition to a role in translation, AIMP1 is secreted to affect various immune cells, such as macrophages, dendritic cells, B cells, and natural killer cells. However, the direct effects of AIMP1 on T cells have not yet been reported. In this study, we investigated whether AIMP1 could modulate T cell responses directly. Results revealed that AIMP1 significantly inhibited T cell receptor (TCR)-dependent activation and proliferation of CD4 T cells, as well as decreased TCR stimuli-induced Ca2+ influx in CD4 T cells. In addition, microscopic analysis revealed that lipid raft association in response to TCR engagement was significantly reduced in the presence of AIMP1, and the phosphorylation of PLCγ and PI3K was also down-regulated in CD4 T cells by AIMP1. Furthermore, AIMP1 specifically enhanced the differentiation of regulatory T (Treg) cells, while it had no effect on T helper type 1 (Th1), type 2 (Th2), and type 17 (Th17) cell differentiation. Collectively, these results indicate that AIMP1 affects T cells directly by down-regulating TCR signaling complex formation and inducing Treg cell differentiation in CD4 T cells. • Aminoacyl tRNA synthetase complex interacting multifunctional protein 1 (AIMP1) inhibits TCR mediated activation and proliferation of CD4 T cells. • AIMP1 regulates Ca2+ influx and lipid raft aggregation in TCR-stimulated CD4 T cells. • The inhibitory function of AIMP1 on TCR signaling specifically enhances Treg cell differentiation. [ABSTRACT FROM AUTHOR]