Intracellular Calcium Accumulation Research Articles

Marine-derived antimicrobial peptide piscidin-1 triggers extrinsic and intrinsic apoptosis in oral squamous cell carcinoma through reactive oxygen species production and inhibits angiogenesis

Cancer of the head and neck encompasses a wide range of cancers, including oral and oropharyngeal cancers. Oral cancer is often diagnosed at advanced stages and has a dismal prognosis. Piscidin-1, a marine antimicrobial peptide (AMP) containing approximately 22 amino acids, also exhibits significant anticancer properties. We investigated the possible anti-oral cancer effects of piscidin-1 and clarified the mechanisms underlying these effects. We treated the oral squamous cell carcinoma cell lines OC2 and SCC4 with piscidin-1. Cell viability and the expression of different hallmark apoptotic molecules, including reactive oxygen species (ROS), were tested using the appropriate MTT assay, flow cytometry and western blotting assays, and human umbilical vein endothelial cell (HUVEC) wound healing, migration, and tube formation (angiogenesis) assays. Piscidin-1 increases cleaved caspase 3 levels to induce apoptosis. Piscidin-1 also increases ROS levels and intensifies oxidative stress in the endoplasmic reticulum and mitochondria, causing mitochondrial dysfunction. Additionally, it decreases the oxygen consumption rates and activity of mitochondrial complexes I–V. As expected, the antioxidants MitoTEMPOL and N-acetylcysteine reduce piscidin-1–induced ROS generation and intracellular calcium accumulation. Piscidin-1 also inhibits matrix metalloproteinase (MMP)-2/-9 expression in HUVECs, affecting migration and tube formation angiogenesis. We demonstrated that piscidin-1 can promote apoptosis via both intrinsic and extrinsic apoptotic pathways and findings indicate that piscidin-1 has anti-proliferative and anti-angiogenic properties in oral cancer treatment. Our study on piscidin-1 thus provides a basis for future translational anti-oral cancer drug research and a new theoretical approach for anti-oral cancer clinical research.

Omega-conotoxin MVIIA reduces neuropathic pain after spinal cord injury by inhibiting N-type voltage-dependent calcium channels on spinal dorsal horn.

Spinal cord injury (SCI) leads to the development of neuropathic pain. Although a multitude of pathological processes contribute to SCI-induced pain, excessive intracellular calcium accumulation and voltage-gated calcium-channel upregulation play critical roles in SCI-induced pain. However, the role of calcium-channel blockers in SCI-induced pain is unknown. Omega-conotoxin MVIIA (MVIIA) is a calcium-channel blocker that selectively inhibits N-type voltage-dependent calcium channels and demonstrates neuroprotective effects. Therefore, we investigated spinal analgesic actions and cellular mechanisms underlying the analgesic effects of MVIIA in SCI. We used SCI-induced pain model rats and conducted behavioral tests, immunohistochemical analyses, and electrophysiological experiments (in vitro whole-cell patch-clamp recording and in vivo extracellular recording). A behavior study suggested intrathecal MVIIA administration in the acute phase after SCI induced analgesia for mechanical allodynia. Immunohistochemical experiments and in vivo extracellular recordings suggested that MVIIA induces analgesia in SCI-induced pain by directly inhibiting neuronal activity in the superficial spinal dorsal horn. In vitro whole-cell patch-clamp recording showed that MVIIA inhibits presynaptic N-type voltage-dependent calcium channels expressed on primary afferent Aδ-and C-fiber terminals and suppresses the presynaptic glutamate release from substantia gelatinosa in the spinal dorsal horn. In conclusion, MVIIA administration in the acute phase after SCI may induce analgesia in SCI-induced pain by inhibiting N-type voltage-dependent calcium channels on Aδ-and C-fiber terminals in the spinal dorsal horn, resulting in decreased neuronal excitability enhanced by SCI-induced pain.

Effects of Extracellular Calcium Concentration on Hepatic Ischemia-Reperfusion Injury in a Rat Model.

Hypocalcemia is frequently identified during liver transplant. However, supplementation of extracellular calcium could induce increased intracellular calcium concentration, as a potential factor for injury to the liver graft. We evaluated the effects of regulating extracellular calcium concentrations on hepatic ischemia-reperfusion injury. We randomly divided 24 Sprague-Dawley rats into 3 groups: group C received normal saline (n = 8), group L received citrate to induce hypocalcemia (n = 8), and group L-Co received citrate followed by calcium gluconate to ameliorate hypocalcemia (n = 8). Liver enzyme levels and extracellular calcium were measured before surgery, 1 hour after ischemia, and 2 hours after reperfusion. The primary outcome was liver enzyme levels measured 2 hours after reperfusion. In addition, we evaluated intracellular calcium levels, lactate dehydrogenase activity, and histopathological results in liver tissue. Three groups demonstrated significant differences in extracellular calcium concentrations, but intracellular calcium concentrations in liver tissue were not significantly different. Group L showed significantly lower mean arterial pressure than other groups at 1 hour after ischemia (93.6 ± 20.8 vs 69.4 ± 14.2 vs 86.6 ± 10.4 mmHg; P = .02, for group C vs L vs L-Co, respectively). At 2 hours after reperfusion, group L showed significantly higher liver enzymes than other groups (aspartate aminotransferase 443.0 ± 353.2 vs 952.3 ± 94.8 vs 502.4 ± 327.3 U/L, P = .01; and alanine aminotransferase 407.9 ± 406.5 vs 860.6 ± 210.9 vs 333.9 ± 304.2 U/L, P = .02; for group C vs L vs L-Co, respectively). However, no significant difference was shown in lactate dehydrogenase and histological liver injury grade. Administering calcium to rats with hypocalcemia did not increase intracellular calcium accumulation but instead resulted in less hepatic injury compared with rats with low extracellular calcium concentrations in this rat model study.

Effects of Daidzein, Tempeh, and a Probiotic Digested in an Artificial Gastrointestinal Tract on Calcium Deposition in Human Osteoblast-like Saos-2 Cells.

Adequate calcium intake is crucial for the prevention and treatment of bone-related issues. Developing a nutritional source of readily bioavailable calcium is particularly significant for individuals deficient in this essential element and at risk of developing osteoporosis. This research aimed to evaluate the impact of tempeh (T), daidzein (D), and Lactobacillus acidophilus (LA) within a simulated intestinal environment consisting of Caco-2 epithelial and Saos-2 cells, focusing on their implications for bone mineralization mechanisms. In the initial phase, calcium bioaccessibility from calcium citrate (CaCt), LA, D, the daidzein combination D-CaCt-LA (D1:1:1), and the tempeh combination T-CaCt-LA (T1:1:1) was assessed through digestion simulation. The calcium content of both untreated and digested samples was determined using atomic absorption spectrometry (AAS). In the subsequent stage, the digested samples were used to induce intestinal absorption in differentiated enterocyte-like Caco-2 cells. The permeable fractions were then evaluated in a culture of osteoblast-like Saos-2 cells. Preliminary cellular experiments employed the MTT assay to assess cytotoxicity. The results indicated that the analyzed products did not influence the deposition of extracellular calcium in Saos-2 cells cultured without mineralization stimulators. The combined formulations of permeable fractions of digested CaCt, LA, D, and T demonstrated the capacity to enhance the proliferation of Saos-2 cells. In Saos-2 cells, D, D1:1:1, and LA showed no discernible impact on intracellular calcium accumulation, whereas T and T1:1:1 reduced the calcium deposits. Additionally, mRNA transcripts and alkaline phosphatase (ALP) activity levels in Saos-2 cells cultured without mineralization induction were unaffected by the analyzed products. An examination of the products revealed no discernible effect on ALP activity or mRNA expression during Saos-2 cell differentiation. Our findings suggest that tempeh, daidzein, and L. acidophilus did not positively impact cellular calcium deposition in Saos-2 cells. However, tempeh, daidzein and its combination, and L. acidophilus might enhance the process of osteogenic differentiation in Saos-2 cells. Nevertheless, this study did not identify any synergistic impact on calcium deposition and the process of osteogenic differentiation in Saos-2 cells of isoflavones and probiotics.

A newly synthesized flavone avoids COMT-catalyzed methylation and mitigates myocardial ischemia/reperfusion injury in H9C2 cells via JNK and P38 pathways.

Luteolin is a flavone that provides defense against myocardial ischemia/reperfusion (I/R) injury. However, this compound is subjected to methylation mediated by catechol-O-methyltransferase (COMT), thus influencing its pharmacological effect. To synthesize a new flavone from luteolin that avoids COMT-catalyzed methylation and find out the protective mechanism of LUA in myocardial I/R injury. Luteolin and 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) were used to synthesize the new flavone known as LUAAPH-1 (LUA). Then, the myocardial ischemia/reperfusion injury cell model was established using H9c2 cells to detect the effect in myocardial ischemia/reperfusion regulation and to identify the underlying mechanism. Pretreatment with LUA (20 μmol/l) substantially increased cell viability while reducing cell apoptosis rate and caspase-3 expression induced by I/R, and the protective effect of LUA on cell viability was stronger than diosmetin, which is the major methylated metabolite of luteolin. In addition, intracellular reactive oxygen species (ROS) production and calcium accumulation were both inhibited by LUA. Furthermore, we identified that LUA markedly relieved the promotive effects of I/R stimulation upon JNK and p38 phosphorylation. LUT pretreatment conveys significant cardioprotective effects after myocardial I/R injury, and JNK and p38 MAPK signaling pathway may be involved.

Serine racemase expression in the pyramidal layer of the hippocampus of rat in the hypertensive encephalopathy modeling

AbstractBackgroundExcessive influence of glutamate on NMDA receptors mediates a cascade of biochemical changes leading to the development of intracellular acidosis, accumulation of free intracellular calcium and activation of a number of enzymes with the formation of free radicals. D‐serine acts as a co‐agonist of NMDA receptors and is involved in learning and memory under normal conditions. However, it has been suggested to play a role in glutamate damage in a number of CNS diseases. The formation of D‐serine in neurons and glial cells occurs with the participation of the enzyme serine racemase (SR) from L‐serine. An increase in the content of SR and the product of its enzymatic reaction, D‐serine, is considered as a process preceding excitotoxicity.MethodHypertensive encephalopathy was modeled on 12‐month‐old rats by the method of exposure to negative longitudinal g‐forces in the caudocranial vector with a force of 9 G for 5 minutes with an interval of 12 hours for 28 days. The following indicators were assessed: behavioral responses, cognitive and mnestic functions, the relative number of neurons with signs of damage, and the level of expression of serine racemase.ResultA morphometric study of the pyramidal layer of the hippocampus revealed a significant increase in the relative area of the immunopositive material in CA1 by 5.5% compared with the control (p<0.05). A feature of the expression of serine racemase in the experimental group was the translocation of immunopositive material, which was determined not only in the cytoplasm of perikaryons, but also in the dendrites of pyramidal neurons. In addition, the nature of expression changed in the experimental group to a moderately pronounced one, and in some pyramidal neurons to a pronounced one. Violation of the cytoarchitectonic of the CA1 zone was manifested in a less compact arrangement of pyramidal neurons, a pattern of spongiosis due to pericellular and perivascular edema, and the acquisition of increased tortuosity by neuronal dendrites.ConclusionHypertensive encephalopathy modeling demonstrates a sharp increase in the relative number of neurons with signs of damage, a decrease in the specific

Assessment of Biochemical and Neuroactivities of Cultural Filtrate from Trichoderma harzianum in Adjusting Electrolytes and Neurotransmitters in Hippocampus of Epileptic Rats.

Epilepsy is a serious chronic neurological disorder, which is accompanied by recurrent seizures. Repeated seizures cause physical injuries and neuronal dysfunction and may be a risk of cancer and vascular diseases. However, many antiepileptic drugs (AEDs) have side effects of mood alteration or neurocognitive function, a reduction in neuron excitation, and the inhibition of normal activity. Therefore, the present study aimed to evaluate the effect of secondary metabolites of Trichoderma harzianum cultural filtrate (ThCF) when adjusting different electrolytes and neurotransmitters in the hippocampus of epileptic rats. Cytotoxicity of ThCF against LS-174T cancer cells was assessed using a sulforhodamine B (SRB) assay. Quantitative estimation for some neurotransmitters, electrolytes in sera or homogenate of hippocampi tissues, and mRNA gene expression for ion or voltage gates was assessed by quantitative Real-Time PCR. Treatment with ThCF reduces the proliferative percentage of LS-174T cells in a concentration-dependent manner. ThCF administration improves hyponatremia, hyperkalemia, and hypocalcemia in the sera of the epilepticus model. ThCF rebalances the elevated levels of many neurotransmitters and reduces the release of GABA and acetylcholine-esterase. Also, treatments with ThCF ameliorate the downregulation of mRNA gene expression for some gate receptors in hippocampal homogenate tissues and recorded a highly significant elevation in the expression of SCN1A, CACNA1S, and NMDA. Secondary metabolites of Trichoderma (ThCF) have cytotoxic activity against LS-174T (colorectal cancer cell line) and anxiolytic-like activity through a GABAergic mechanism of action and an increase in GABA as inhibitory amino acid in the selected brain regions and reduced levels of NMDA and DOPA. The present data suggested that ThCF may inhibit intracellular calcium accumulation by triggering the NAADP-mediated Ca2+ signaling pathway. Therefore, the present results suggested further studies on the molecular pathway for each metabolite of ThCF, e.g., 6-pentyl-α-pyrone (6-PP), harzianic acid (HA), and hydrophobin, as an alternative drug to mitigate the side effects of AEDs.

Modulation of SK Channels via Calcium Buffering Tunes Intrinsic Excitability of Parvalbumin Interneurons in Neuropathic Pain: A Computational and Experimental Investigation.

Parvalbumin-expressing interneurons (PVINs) play a crucial role within the dorsal horn of the spinal cord by preventing touch inputs from activating pain circuits. In both male and female mice, nerve injury decreases PVINs' output via mechanisms that are not fully understood. In this study, we show that PVINs from nerve-injured male mice change their firing pattern from tonic to adaptive. To examine the ionic mechanisms responsible for this decreased output, we used a reparametrized Hodgkin-Huxley type model of PVINs, which predicted (1) the firing pattern transition is because of an increased contribution of small conductance calcium-activated potassium (SK) channels, enabled by (2) impairment in intracellular calcium buffering systems. Analyzing the dynamics of the Hodgkin-Huxley type model further demonstrated that a generalized Hopf bifurcation differentiates the two types of state transitions observed in the transient firing of PVINs. Importantly, this predicted mechanism holds true when we embed the PVIN model within the neuronal circuit model of the spinal dorsal horn. To experimentally validate this hypothesized mechanism, we used pharmacological modulators of SK channels and demonstrated that (1) tonic firing PVINs from naive male mice become adaptive when exposed to an SK channel activator, and (2) adapting PVINs from nerve-injured male mice return to tonic firing on SK channel blockade. Our work provides important insights into the cellular mechanism underlying the decreased output of PVINs in the spinal dorsal horn after nerve injury and highlights potential pharmacological targets for new and effective treatment approaches to neuropathic pain.SIGNIFICANCE STATEMENT Parvalbumin-expressing interneurons (PVINs) exert crucial inhibitory control over Aβ fiber-mediated nociceptive pathways at the spinal dorsal horn. The loss of their inhibitory tone leads to neuropathic symptoms, such as mechanical allodynia, via mechanisms that are not fully understood. This study identifies the reduced intrinsic excitability of PVINs as a potential cause for their decreased inhibitory output in nerve-injured condition. Combining computational and experimental approaches, we predict a calcium-dependent mechanism that modulates PVINs' electrical activity following nerve injury: a depletion of cytosolic calcium buffer allows for the rapid accumulation of intracellular calcium through the active membranes, which in turn potentiates SK channels and impedes spike generation. Our results therefore pinpoint SK channels as potential therapeutic targets for treating neuropathic symptoms.

Subchronic pulmonary toxicity of ambient particles containing cement production–related elements

Chronic respiratory disease is among the most common non-communicable diseases, and particulate materials (PM) are a major risk factor. Meanwhile, evidence of the relationship between the physicochemical characteristics of PM and pulmonary toxicity mechanism is still limited. Here, we collected particles (CPM) from the air of a port city adjacent to a cement factory, and we found that the CPM contained various elements, including heavy metals (such as arsenic, thallium, barium, and zirconium) which are predicted to have originated from a cement plant adjacent to the sampling site. We also delivered the CPM intratracheally to mice for 13 weeks to investigate the pulmonary toxicity of inhaled CPM. CPM-induced chronic inflammatory lesions with an increased total number of cells in the lung of mice. Meanwhile, among inflammatory mediators measured in this study, levels of IL-1β, TNF-α, CXCL-1, and IFN-γ were elevated in the treated group compared with the controls. Considering that the alveolar macrophage (known as dust cell) is a professional phagocyte that is responsible for the clearance of PM from the respiratory surfaces, we also investigated cellular responses following exposure to CPM in MH-S cells, a mouse alveolar macrophage cell line. CPM inhibited cell proliferation and formed autophagosome-like vacuoles. Intracellular calcium accumulation and oxidative stress, and altered expression of pyrimidine metabolism- and olfactory transduction-related genes were observed in CPM-treated cells. More interestingly, type I-LC3B and full-length PARP proteins were not replenished in CPM-treated cells, and cell cycle changes, apoptotic and necrotic cell death, and caspase-3 cleavage were not significantly detected in cells exposed to CPM. Taken together, we conclude that dysfunction of alveolar macrophages may contribute to CPM-induced pulmonary inflammation. In addition, given the possible transformation of heart tissue observed in CPM-treated mice, we suggest that further study is needed to clarify the systemic pathological changes and the molecular mechanisms following chronic exposure to CPM.

Pathogenetic mechanisms of cognitive impairment in cerebrovascular pathology and prospects for their correction using nootropic and neuroprotective agents

The article presents modern ideas about the pathogenesis of cognitive disorders in cerebrovascular pathology at the cellular level. Dysfunction of neurovascular units is associated with impaired microcirculation, hypoxia, deficiency of energy resources, development of neuroinflammation, increased nitric oxide synthesis and oxidative stress, glutamate excitotoxicity, intracellular calcium accumulation, endothelial dysfunction, impaired circulation of cerebrovascular fluid, venous outflow from the cranial cavity and utilization of brain metabolic products, including misshaped proteins. The above determines the possibility of the combined development of cerebrovascular and neurodegenerative diseases, primarily Alzheimer’s disease. Currently, mixed (vascularneurodegenerative) brain damage is considered as the main cause of cognitive disorders, which is confirmed by the data of post-mortem studies. Pathogenetic therapy of dementia with cholinesterase inhibitors and memantine does not eliminate the cognitive defect, but only slows down its progression. The impossibility of restoring the premorbid level of daily activity of the patient in the treatment of cognitive impairment at the stage of dementia dictates the need for the use of adjuvant nootropic and neuroprotective agents until the breakdown of the functional reserve, that is, at the stage of moderate cognitive impairment. Nicotinoyl gamma-aminobutyric acid has nootropic, tranquilizing, psychostimulant and antioxidant properties. Studies of the last decade have proven the ability of nicotinoyl gamma-aminobutyric acid to suppress neuroinflammation and apoptosis of cells of the central nervous system, increase the expression of angiogenic and cytoskeletal proteins, normalize the permeability of the blood-brain barrier, which can be used to improve the function of neurovascular units and correct vascular-neurodegenerative cognitive impairment. A small number of drug interactions with nicotinoyl gamma-aminobutyric acid allows it to be included in the complex therapy of comorbid patients.

Vx-809, a CFTR Corrector, Acts through a General Mechanism of Protein Folding and on the Inflammatory Process

Correct protein folding is the basis of cellular well-being; thus, accumulation of misfolded proteins within the endoplasmic reticulum (ER) leads to an imbalance of homeostasis that causes stress to the ER. Various studies have shown that protein misfolding is a significant factor in the etiology of many human diseases, including cancer, diabetes, and cystic fibrosis. Misfolded protein accumulation in the ER triggers a sophisticated signal transduction pathway, the unfolded protein response (UPR), which is controlled by three proteins, resident in ER: IRE1α, PERK, and ATF6. Briefly, when ER stress is irreversible, IRE1α induces the activation of pro-inflammatory proteins; PERK phosphorylates eIF2α which induces ATF4 transcription, while ATF6 activates genes encoding ER chaperones. Reticular stress causes an alteration of the calcium homeostasis, which is released from the ER and taken up by the mitochondria, leading to an increase in the oxygen radical species production, and consequently, to oxidative stress. Accumulation of intracellular calcium, in combination with lethal ROS levels, has been associated with an increase of pro-inflammatory protein expression and the initiation of the inflammatory process. Lumacaftor (Vx-809) is a common corrector used in cystic fibrosis treatment which enhances the folding of mutated F508del-CFTR, one of the most prevalent impaired proteins underlying the disease, promoting a higher localization of the mutant protein on the cell membrane. Here, we demonstrate that this drug reduces the ER stress and, consequently, the inflammation that is caused by such events. Thus, this molecule is a promising drug to treat several pathologies that present an etiopathogenesis due to the accumulation of protein aggregates that lead to chronic reticular stress.

The Neuroprotective and Antinociceptive effect of Antidiabetic 3-(2-chlorophenyl)-4-imino-5-phenyl-2-(2-methoxyphenyl)-2H,3H,5H-[1,2,5]thiadiazolidin-1-oxide (CIPMTO) in Streptozotocin-induced diabetic neuropathic pain in rats: primary proof of concept

In this study the neuroprotective and antinociceptive effect of a newly synthesized chemical moiety, a thiazolidin derivative, 3-(2-chlorophenyl)-4-imino-5-phenyl-2-(2-methoxyphenyl)-2H,3H,5H-[1,2,5]thiadiazolidin-1-oxide (CIPMTO) in streptozotocin (STZ) induced diabetic neuropathic pain is examined in rat therapeutic model. Multiple behavior and biochemical parameters were assessed on the therapeutic model to understand the role of CIPMTO. In this study, male Wistar rats (220-250 g) were used (7 groups, one group was not given any kind of chemical treatment (normal control). All other 6 groups were given a dose of STZ. Carboxymethylcellulose (CMC) was used as a vehicle for CIPMTO in this study. Out of those 6 groups, 1 group was given no other treatment (negative control), 1 group was given CMC only (Vehicle control), 3 groups were treated with different doses of CIPMTO, and 1 group was treated with pregabalin (positive control). Thereafter multiple behavioral studies and biochemical testing were carried out. CIPMTO is found to attenuate the STZ induced behavioral and biochemical changes in a dose-dependent manner (5, 10, and 15 mg/kg) as compared to that of the pregabalin-treated group. CIPMTO also reduced STZ-induced diabetic NP, which is attributable to its neuroprotective potential via pharmacological activities such as free radical scavenging, anti-lipid peroxidation, and inhibition of intracellular calcium accumulation. The current study shows that CIPMTO has neuroprotective and antinociceptive properties, as well as the ability to reduce neuropathic pain. Though further research and testing could be conducted for establishing the fact.

Uptake and toxicity of cerium dioxide nanoparticles with different aspect ratio

Cerium dioxide nanoparticles (CeONPs) have been extensively applied in research for future energy development due to two common oxidation states on their surface. Considering that shape (aspect ratio) is a key determinant of NPs-induced toxicity, we compared the toxicity of hexagonal (H)- and rod-shaped (R)-CeONPs in mice. At 24 h after pharyngeal aspiration, both types of CeONPs recruited surrounding immune cells (monocytes and neutrophils) into the lung, and R-CeONPs induced a more severe pulmonary inflammatory response compared with H-CeONPs. To identify an indicator to predict pulmonary inflammatory responses at the cellular level, we also investigated their responses in alveolar macrophage cells. At 24 h after treatment, both types of CeONPs were mainly located within the vacuoles (partially, in the lysosome) in the cytoplasm. Mitochondrial damage, intracellular calcium accumulation, and increased NO production were observed in cells exposed to both types of CeONPs, ultimately resulting in a decrease in cell viability. More interestingly, both types of CeONPs formed multinucleated giant cells. Meanwhile, contrary to when suspended in deionized water, R-CeONPs were strongly aggregated with a negative charge in cell culture media, whereas H-CeONPs were relatively well-dispersed with a positive charge. R-CeONPs-induced lysosomal extension was also recovered by premix with negatively charged DNA, and even NPs suspended in cell culture media without cells were detected under the FACS system, suggesting interference by protein corona. Therefore, we suggest that shape (aspect ratio) is an important factor determining inhaled NPs-induced pathology and that the effect of the surface charge and protein corona should be carefully considered in interpreting results derived from in vitro tests. Furthermore, we propose that the relationship between the formation of multinucleated giant cells and the inflammatory response of inhaled CeONPs should be further studied.

Comparison of toxicity and cellular responses following pulmonary exposure to different types of nanofibers

Pulmonary effects of inhaled microfibers are an emerging public health concern. In this study, we investigated toxicity following pulmonary exposure to synthetic polyethylene oxide fibroin (PEONF) and silk fibroin (SFNF) nanofibers and the cellular responses. When instilled intratracheally weekly for four weeks, body weight gain was significantly reduced in female mice exposed to the higher dose of SFNF when compared with the control group. The total number of cells in the lungs was more significant in all treated groups than in the control, whereas the relative portion of neutrophils and eosinophils increased significantly only in female mice exposed to SFNF. Both types of nanofibers induced notable pathological changes and increased pulmonary expression of MCP-1α, CXCL1, and TGF-β. More importantly, blood calcium, creatinine kinase, sodium, and chloride concentration were affected significantly, showing sex- and material-dependent differences. The relative portion of eosinophils increased only in SFNF-treated mice. In addition, both types of nanofibers induced necrotic and late apoptotic cell death in alveolar macrophages after 24 h of exposure, with accompanying oxidative stress, increased NO production, cell membrane rupture, intracellular organelle damage, and intracellular calcium accumulation. Additionally, multinucleated giant cells were formed in cells exposed to PEONF or SFNF. Taken together, the findings indicate that inhaled PEONF and SFNF may cause systemic adverse health effects with lung tissue damage, showing differences by sex- and material. Furthermore, PEONF- and SFNF-induced inflammatory response may be partly due to the low clearance of dead (or damaged) pulmonary cells and the excellent durability of PEONF and SFNF.

Malignant Hyperthermia: Clinical Management

Malignant hyperthermia is a subclinical myopathy in which general anesthesia triggers an uncontrollable contraction of skeletal muscle that leads to a life-threatening hypercatabolic state and an increase in body temperature. The disease is primarily autosomal dominant; mutations in receptors (especially ryanodine receptor type 1) predispose to volatile anesthetic agents or succinylcholine causing an accumulation of intracellular calcium in skeletal muscle that leads to its overactivation and hypermetabolism. In the acute setting, diagnosis is based mainly on clinical presentation and end-tidal capnography, which reveals an increase in end-tidal CO2. Initial treatment measures include discontinuation of the agent causing the reaction and administration of dantrolene. In non-acute settings, there are specific diagnostic tools such as the caffeine-halothane contracture test to confirm suspected cases. MH is a lethal disease and has a high mortality rate if not treated early.

Inhibition of Plasma Membrane Calcium Pump Influences Intracellular Calcium Signaling Pathways in Breast Cancer.

The plasma membrane calcium pump (PMCA) is an important transporter that maintains intracellular calcium concentration ([Ca2+]i). It allows the calcium (Ca2+) from inside the cell to go out of the cell through the plasma membrane. For this, it cooperates with the proteins in the cell. The aim of this study is to demonstrate the effect of PMCA on intracellular calcium signaling in breast cancer cells. In this study, PMCA was inhibited by orthovanadate (OV), and changes in Calmodulin (CaM), Calcineurin (CaN) and cMyc proteins were demonstrated. Intracellular calcium accumulation was measured when PMCA was inhibited in MDA-MB-231 cells. At the same time, it was observed that the cell movement decreased with time. Over time, CaN and CaM were slightly suppressed, and cMyc protein was not expressed. As a result, when PMCA protein is targeted correctly in breast cancer cells, it has an indirect effect on cancer-promoting proteins.

Mitochondrial regulation of acute extrafollicular B‐cell responses to COVID‐19 severity

BackgroundPatients with COVID‐19 display a broad spectrum of manifestations from asymptomatic to life‐threatening disease with dysregulated immune responses. Mechanisms underlying the detrimental immune responses and disease severity remain elusive.MethodsWe investigated a total of 137 APs infected with SARS‐CoV‐2. Patients were divided into mild and severe patient groups based on their requirement of oxygen supplementation. All blood samples from APs were collected within three weeks after symptom onset. Freshly isolated PBMCs were investigated for B cell subsets, their homing potential, activation state, mitochondrial functionality and proliferative response. Plasma samples were tested for cytokine concentration, and titer of Nabs, RBD‐, S1‐, SSA/Ro‐ and dsDNA‐specific IgG.ResultsWhile critically ill patients displayed predominantly extrafollicular B cell activation with elevated inflammation, mild patients counteracted the disease through the timely induction of mitochondrial dysfunction in B cells within the first week post symptom onset. Rapidly increased mitochondrial dysfunction, which was caused by infection‐induced excessive intracellular calcium accumulation, suppressed excessive extrafollicular responses, leading to increased neutralizing potency index and decreased inflammatory cytokine production. Patients who received prior COVID‐19 vaccines before infection displayed significantly decreased extrafollicular B cell responses and mild disease.ConclusionOur results reveal an immune mechanism that controls SARS‐CoV‐2‐induced detrimental B cell responses and COVID‐19 severity, which may have implications for viral pathogenesis, therapeutic interventions and vaccine development.

Urocortin-1 Is Chondroprotective in Response to Acute Cartilage Injury via Modulation of Piezo1.

Post-traumatic OA (PTOA) is often triggered by injurious, high-impact loading events which result in rapid, excessive chondrocyte cell death and a phenotypic shift in residual cells toward a more catabolic state. As such, the identification of a disease-modifying OA drug (DMOAD) that can protect chondrocytes from death following impact injury, and thereby prevent cartilage degradation and progression to PTOA, would offer a novel intervention. We have previously shown that urocortin-1 (Ucn) is an essential endogenous pro-survival factor that protects chondrocytes from OA-associated pro-apoptotic stimuli. Here, using a drop tower PTOA-induction model, we demonstrate the extent of Ucn’s chondroprotective role in cartilage explants exposed to excessive impact load. Using pathway-specific agonists and antagonists, we show that Ucn acts to block load-induced intracellular calcium accumulation through blockade of the non-selective cation channel Piezo1 rather than TRPV4. This protective effect is mediated primarily through the Ucn receptor CRF-R1 rather than CRF-R2. Crucially, we demonstrate that the chondroprotective effect of Ucn is maintained whether it is applied pre-impact or post-impact, highlighting the potential of Ucn as a novel DMOAD for the prevention of injurious impact overload-induced PTOA.

The strength of antigenic and IL-2 signals epigenetically programs functional memory CD8+ T cells

Abstract Priming of naïve CD8+ T cells requires three signals: 1) cognate antigen, 2) co-stimulation, and 3) cytokines. These signals orchestrate CD8+ T cell differentiation. Particularly, strong T cell receptor (TCR) signals induce a more robust effector response, while weak TCR signals preferentially favor memory CD8+ T cells. However, memory cells induced upon varied TCR signaling strengths have been reported to be functionally similar. In this work, we show that, in contrast to this assumption, the strength of T cell receptor (TCR) signaling determines the requirement for interleukin-2 (IL-2) signals to form a pool of memory CD8+ T cells that are functionally different. We found that upon secondary antigen encounter, memory CD8+ T cells induced upon priming with lower TCR and IL-2 signals exhibited delayed cell cycle entry, impaired expansion and intracellular calcium accumulation compared to those primed with strong TCR and IL-2 signals. Furthermore, we also establish that the combination of both robust TCR and IL-2 signals, but not either one alone, are necessary to induce genome-wide changes in chromatin accessibility in regions targeting a wide range of biological processes consistent with their greater functional fitness. These notably included greater chromatin accessibility in promoters of genes encoding for stem cell, cell cycle and calcium-related proteins. Taken together, these findings demonstrate that modulating TCR strength and IL-2 signaling at the time of CD8+ T cell priming, lead to the differentiation of functionally distinct pools of memory CD8+ T cells. Supported by T32GM7288

T cell receptor and IL-2 signaling strength control memory CD8+ T cell functional fitness via chromatin remodeling

Cognate antigen signal controls CD8+ T cell priming, expansion size and effector versus memory cell fates, but it is not known if and how it modulates the functional features of memory CD8+ T cells. Here we show that the strength of T cell receptor (TCR) signaling controls the requirement for interleukin-2 (IL-2) signals to form a pool of memory CD8+ T cells that competitively re-expand upon secondary antigen encounter. Combining strong TCR and intact IL-2 signaling during priming synergistically induces genome-wide chromatin accessibility in regions targeting a wide breadth of biological processes, consistent with greater T cell functional fitness. Chromatin accessibility in promoters of genes encoding for stem cell, cell cycle and calcium-related proteins correlates with faster intracellular calcium accumulation, initiation of cell cycle and more robust expansion. High-dimensional flow-cytometry analysis of these T cells also highlights higher diversity of T cell subsets and phenotypes with T cells primed with stronger TCR and IL-2 stimulation than those primed with weaker strengths of TCR and/or IL-2 signals. These results formally show that epitope selection in vaccine design impacts memory CD8+ T cell epigenetic programming and function.