Calcium Activity Research Articles

Hepatocyte CHRNA4 mediates the MASH-promotive effects of immune cell-produced acetylcholine and smoking exposure in mice and humans

Metabolic dysfunction-associated steatohepatitis (MASH) is a leading risk factor for liver cirrhosis and hepatocellular carcinoma. Here, we report that CHRNA4, a subunit of nicotinic acetylcholine receptors (nAChRs), is an accelerator of MASH progression. CHRNA4 also mediates the MASH-promotive effects induced by smoking. Chrna4 was expressed specifically in hepatocytes and exhibited increased levels in mice and patients with MASH. Elevated CHRNA4 levels were positively correlated with MASH severity. We further revealed that during MASH development, acetylcholine released from immune cells or nicotine derived from smoking functioned as an agonist to activate hepatocyte-intrinsic CHRNA4, inducing calcium influx and activation of inflammatory signaling. The communication between immune cells and hepatocytes via the acetylcholine-CHRNA4 axis led to the production of a variety of cytokines, eliciting inflammation in liver and promoting the pathogenesis of MASH. Genetic and pharmacological inhibition of CHRNA4 protected mice from diet-induced MASH. Targeting CHRNA4 might be a promising strategy for MASH therapeutics.

Single-cell RNA sequencing uncovers the cell type-dependent transcriptomic changes in the retrosplenial cortex after peripheral nerve injury

The retrosplenial cortex (RSC) is a vital area for storing remote memory and has recently been found to undergo broad changes after peripheral nerve injury. However, little is known about the role of RSC in pain regulation. Here, we examine the involvement of RSC in the pain of mice with nerve injury. Notably, reducing the activities of calcium-/calmodulin-dependent protein kinase type II-positive splenial neurons chemogenetically increases paw withdrawal threshold and extends thermal withdrawal latency in mice with nerve injury. The single-cell or single-nucleus RNA-sequencing results predict enhanced excitatory synaptic transmissions in RSC induced by nerve injury. Local infusion of 1-naphthyl acetyl spermine into RSC to decrease the excitatory synaptic transmissions relieves pain and induces conditioned place preference. Our data indicate that RSC is critical for regulating physiological and neuropathic pain. The cell type-dependent transcriptomic information would help understand the molecular basis of neuropathic pain.

In vivo neuronal deficits and microglia structural dynamics in the course of tau pathology and during antibody treatment

AbstractBackgroundWe crossed mouse models Thy‐1GCaMP6, Cx3cr1CreER:tdTomatoflox and Cx3cr1GFP with PS19 P301S tauopathy mice to visualize neuronal calcium and microglia structural dynamics in vivo during the progression of tau pathology and after tau immunotherapy.MethodBy using two‐photon live imaging in head‐restrained mice attached to a custom‐made, free‐floating treadmill, we examined calcium activity in L2/3 pyramidal neurons and microglia structural dynamics in the motor cortex during tau pathology progression and after two intravenous antibody injections targeting the truncated Asp421 epitope of tau.ResultL2/3 pyramidal neurons from young Thy‐1GCaMP6:wild‐type mice (1‐6 months old) increased their somatic activity during running periods as expected, but their littermate Thy‐1GCaMP6:PS19+/− mice did not. Importantly, acute tau immunotherapy restored neuronal function after two intravenous antibody injections (100 µg each) in Thy‐1GCaMP6:PS19+/− mice (5‐6 months old), whereas control antibody had no effect. Although tau pathology is evident in the cortex and hippocampus in 2‐3 months old Cx3cr1GFP/+:PS19+/− mice and Cx3cr1CreER/+:tdTomatoflox:PS19+/− mice, their microglia morphology did not differ from control littermate Cx3cr1GFP/+:wild‐type mice.ConclusionOur current findings indicate that functional deficits in cortical neurons associated with tau pathology start early in PS19 mice and can be alleviated by two acute tau antibody injections. Also, those neuronal functional impairments can be detected without accompanying structural changes in microglia, which suggests that neuronal deficits linked to tau accumulation precede microglial activation. Studies on the potential influence of tau antibody treatment on microglia morphology in vivo are underway.

Phase formation and hydration of alumina cements on the basis of cobalt‐molybdenum‐containing waste

AbstractThe subject of the research is features of synthesis of alumina cements on the basis of compositions of system CaO – CoO – Al2O3 – MoO3 with use of cobalt‐molybdenum‐containing waste for reception of refractory materials with a complex of the set properties. The processes of phase formation in raw mixtures have been investigated. X‐ray phase studies of clinkers fired at different temperatures and holding times proved that as a result of the interaction of the raw materials of the mixture in the material synthesizes a mixture of hydraulically active calcium mono‐ and di‐aluminate and refractory cobalt spinel, which provides the resulting complex high strength, accelerated curing time, fire resistance. The absence of phases corresponding to molybdenum compounds is explained by the fact that they are part of hydraulically active aluminates as limited solid solutions, deforming the crystal lattice and increasing hydraulic activity. Thus, it was found that the high strength of cement is due to the presence of calcium hydroaluminates such as C2AH8, aluminum hydroxide, as well as unhydrated grains of calcium aluminates, which will contribute to further long‐term strength. It is this coexistence of phases in both the crystalline and colloidal states provide high strength cement stone.

Enhancing soil resilience and crop physiology with biochar application for mitigating drought stress in durum wheat (Triticumdurum)

The use of biochar has recently garnered significant attention as an agricultural management technique highly endorsed by the scientific community. Biochar, owing to its high carbon content, contributes to increased organic matter storage in the soil, consequently enhancing crop growth. This study aimed to elucidate changes in physicochemical soil fertility and durum wheat (Triticum durum) var. Vitron production under the influence of three biochar doses (0 g/kg, 5 g/kg, and 15 g/kg of soil) in combination with varying levels of drought stress (100 %, 80 %, 40 %, and 20 % of field capacity 'FC'). Notably, we observed a substantial increase in all physicochemical soil parameters, except for active calcium carbonate equivalent (ACCE), which displayed lower values (8.78 ± 1.43 %) in soils treated with biochar compared to control soil (15.69 ± 4.03 %). The biochar dose of 5 g/kg yielded the highest moisture content (8.81 %) and pH value (7.83). However, the highest organic matter content (4.89 ± 0.17 %) and total calcium carbonate equivalent ‘TCCE’ (3.67 ± 0.48 %) were observed with the dose 15 g/kg. Nevertheless, regarding plant growth, no improvements were observed in terms of height and above-ground biomass (AGB). Conversely, leaf surface area exhibited significant changes with biochar application, along with an increase in chlorophyll pigment content. On the other hand, drought stress significantly hindered plant height, AGB, and leaf water reserves, resulting in values of 13.48 ± 1.60 cm, 1.57 ± 0.31g/plant, and 41.79 ± 1.67 %, respectively. The interaction between biochar and water stress appeared to mitigate and limit the impact of stress. Notably, an enhancement in organic matter storage and soil water reserves was observed. For example, the moisture content in the control soil was 6.95 %, while it increased to 12.76 % for 15g biochar/kg and 80 % FC. A similar trend was observed for organic matter, TCCE, and electrical conductivity. This effect positively influenced chlorophyll a and b content, as well as leaf water content. However, when stress was combined with biochar amendment, plant height and AGB decreased. The addition of biochar improved soil fertility and physiological parameters of wheat plants. Nevertheless, when combined with water stress, especially in cases of reduced water reserves, productivity did not witness any significant improvements.

Bone Fracture in Rett Syndrome: Mechanisms and Prevention Strategies.

The present study aimed to evaluate the burden and management of fragility fractures in subjects with Rett syndrome. We searched all relevant medical literature from 1 January 1986 to 30 June 2023 for studies under the search term "Rett syndrome and fracture". The fracture frequency ranges from a minimum of 13.9% to a maximum of 36.1%. The majority of such fractures occur in lower limb bones and are associated with low bone mineral density. Anticonvulsant use, joint contractures, immobilization, low physical activity, poor nutrition, the genotype, and lower calcium and vitamin D intakes all significantly impair skeletal maturation and bone mass accrual in Rett syndrome patients, making them more susceptible to fragility fractures. This review summarizes the knowledge on risk factors for fragility fracture in patients with Rett syndrome and suggests a possible diagnostic and therapeutic care pathway for improving low bone mineral density and reducing the risk of fragility fractures. The optimization of physical activity, along with adequate nutrition and the intake of calcium and vitamin D supplements, should be recommended. In addition, subjects with Rett syndrome and a history of fracture should consider using bisphosphonates.

Production and characterization of fruit jam with activated pectin using wild hawthorn puree (Crataegus monogyna Jacq.)

In this study, we developed a technology for the production of jelly confiture based on natural hawthorn puree. The use of activated low-esterified hawthorn pectin in the recipe was shown to increase the complexing ability of the product in relation to zinc and lead ions, making it suitable for functional preventive nutrition. The final product was characterised by a bright orange-yellow colour, a distinct hawthorn taste and odour, and a gelatinous consistency. Evaluation of the product based on organoleptic, physicochemical, and microbiological indicators showed that it had high nutritional and physiological value due to its retention of native micronutrients, high content of potassium, magnesium, and calcium, and antioxidant activity. The use of this product in a daily diet can satisfy the daily need for physiologically active substances and food ingredients. Overall, the use of natural puree from wild hawthorn fruit in the technology of jelly confiture allows for the expansion of raw material sources in the production of confectionery products with high nutritional and physiological value.

Parathyroid carcinoma case-report in a patient with multinodular goiter and chronic kidney disease: difficulties in diagnosis and treatment (observation from practice)

We would like to present a clinical case of severe primary hyperparathyroidism due to a parathyroid carcinoma of atypical location in a patient with chronic kidney disease of complex etiology and multinodular goiter. Patient S., 59 years old, was followed-up for a long time in tertiary referral hospitals for “chronic tubulointerstitial nephritis with nephrosclerosis”, secondary hyperparathyroidism due to chronic kidney disease (CKD) G3–4, osteoporosis, and a multinodular euthyroid colloid goiter. In July 2021 she was referred to the Endocrinology Research Centre in order to clarify the diagnosis because of the persistence of an extremely high level of parathyroid hormone (PTH) despite cinacalcet treatment. During examination, primary hyperparathyroidism, a left parathyroid gland lesion, multinodular goiter with subclinical thyrotoxicosis, and vitamin D deficiency were diagnosed. After the removal of the left parathyroid gland lesion (histologically confirmed parathyroid carcinoma) and a left-sided hemithyroidectomy, hypocalcemia («hungry bone syndrome») developed, but the level of parathyroid hormone remained elevated. After 3–18 months after surgery, no data for relapse of primary hyperparathyroidism was obtained. The persistent moderate increase in PTH was regarded as secondary hyperparathyroidism in CKD and hypocalcemia. Complex therapy of osteoporosis with the antiresorptive drug denosumab, vitamin D and its active metabolite, calcium preparations, and parathyroidectomy led to a significant increase in bone mineral density (BMD) and no repeated fractures 18 months after surgery.Conclusion. In patients with pre-dialysis CKD and high PTH levels, it is necessary to make a differential diagnosis between primary (PHPT) and secondary hyperparathyroidism (SHPT). Severe manifestations of primary hyperparathyroidism can be suspicious for parathyroid carcinoma.

Calcined waste shells as a promising, eco‐friendly adsorbent, antimicrobial, food preservative, and food packaging material: A mini review

AbstractConventionally, waste shells from aquaculture are dumped as a low‐value byproduct, which deteriorates ecosystems and threatens public health. Therefore, the reuse of waste shells is a pivotal issue. Calcination (≥700°C) can thermally convert calcium carbonate (CaCO3; the main component in waste shells) into active calcium oxide (CaO). This calcined shell powder has adsorption properties, antimicrobial properties, and biocompatibility, which can be used as a functional biofiller in food packaging. Calcined shell powder is also ecofriendly and nontoxic at the concentrations required for food processing and packaging, and has been approved as a food additive in Japan. Thus, this review comprehensively discusses the calcination process, compositions, adsorption and antimicrobial properties (and the underlying mechanisms), and applications of calcined shell powder in food processing and packaging.Practical applicationsThe active ingredient of calcined shell powder is active calcium oxide (CaO), which has functional abilities such as adsorption and antimicrobial properties, and is also ecofriendly and nontoxic to humans at the concentrations required for food processing and packaging. Its application as functional filler for food preservation and packaging films, the benefits, and the related mechanisms behind was the finding of this study.

Efficacy of calcium hydroxide-loaded poly(lactic-co-glycolic acid) biodegradable nanoparticles as an intracanal medicament against endodontopathogenic microorganisms in a multi-species biofilm model.

This study aimed to evaluate the antimicrobial activity of calcium hydroxide-loaded poly(lactic-co-glycolic acid) nanoparticles (CH-loaded PLGA NPs) on multi-species biofilms. Human root blocks were prepared (n = 40), and multi-species suspensions of Candida albicans, Enterococcus faecalis and Streptococcus gordonii were incubated within the root canals for 21 days. Canals (n = 10/group) were then medicated with saline solution (negative control), chlorhexidine (positive control), calcium hydroxide and CH-loaded PLGA NPs for 7 days. Samples taken from the 0.1 mm root canal dentin were collected, and cell growth was detected by culture on BHI agar. The viable cell count of the Ca(OH)2, chlorhexidine gel and CH-loaded PLGA NPs group was significantly lower than the normal saline group (p < 0.001). CH-loaded PLGA NPs demonstrated a significant lower viable cell than Ca(OH)2 (p < 0.001); it has potential as a medicament for endodontic therapy.

CNSC-38. CHARACTERIZATION OF THE FUNCTIONAL RELEVANCE OF INTRA- AND INTERTUMORAL NETWORKS IN PRIMARY BREAST CANCER

Abstract Despite intensive efforts to improve current therapeutical strategies, breast cancer continues to be a life-shortening diagnosis for a significant number of affected women. Considering that the innervation density of breast cancers negatively correlates with the prognosis, the emerging field of Cancer Neuroscience, which deals with the reciprocal interactions between the nervous system and cancer cells, represents an attractive novel research direction. In fact, studies on breast cancer brain metastases have proven that the metastatic cells receive growth stimuli by hijacking healthy glutamatergic synapses. Beyond being capable of forming direct glutamatergic cancer-neuron synapses, primary brain tumor cells also connect to a multicellular intercommunicating network that contributes to therapy resistance. Whether similar mechanisms also apply to tumors outside the brain – accountable for the majority of cancer cases – remains unknown. Here, we aim at unraveling the functional role of intratumoral networks and the influence of (potential) direct primary breast cancer cell-neuron interactions. We hypothesize that purinergic signaling as a universal synaptic signaling mechanism enhances tumor growth. Through the application of ex vivo whole-organ tissue-clearing and nerve-labeling approaches, we noticed prevalent sensory and sympathetic innervation in the healthy and diseased murine mammary gland. Furthermore, we measured tumor cell proliferation rates in an in vitro model of primary murine sensory DRG-neurons co-cultured with breast cancer stem cells and compared these to monoculture controls. By implanting a mammary imaging window over the growing primary tumor, we longitudinally track the calcium activity of breast cancer cells in their natural microenvironment in the living mouse. We thereby are able to record co-activity of neighboring cells indicative of intratumoral network communication in primary breast cancers. Overall, our data support the existence of functional intra- and intertumoral breast cancer cell networks as an alternative therapeutical target. Future work needs to concentrate on unequivocally detecting direct neuron-cancer cell contacts.

Abstract 15822: Cardiac Effects of EDG-7500, a Novel Cardiac Sarcomere Regulator: In vitro and in vivo Evidence for Slowing Isovolumic Contraction and Improved Ventricular Compliance

Background: Hypertrophic Cardiomyopathy (HCM) is a disease where excess acto-myosin crossbridge formation results in hyperdynamic contraction, LV hypertrophy, and diastolic dysfunction due to increased myocardial stiffness. Conventional negative inotropes alleviate, to some degree, the systolic alteration in HCM, but do not improve LV filling. EDG-7500 is a small molecule that preferentially decreases diastolic tension and slows the velocity of myocardial force generation by regulating, but not inhibiting, cardiac myosin. This study assessed pharmacodynamic responses to EDG-7500. Methods: Myofilament biomechanics and sarcomere structure were evaluated on LV permeabilized pig fibers. In vivo, dogs were instrumented for arterial pressure and LV pressure-volume recordings. Data were obtained with vehicle, EDG-7500 (n = 5; 0.3 mg/kg IV), or metoprolol (2-4 mg/kg IV, n = 3); systemic and LV hemodynamics and load-independent function were examined. *:P&lt;0.05 for EDG-7500 vs. vehicle Results: X-ray diffraction studies showed EDG-7500 preserved the myosin head population competent to form crossbridges as well as the recruitment of reserve myosin-heads during calcium activation. EDG-7500 (1μM) slowed crossbridge formation, with a limited reduction in maximal force (-6±2%) and decreased diastolic tension. At 0.3 mg/kg, EDG-7500 slowed early contraction (PEP: +10±1%*) and reduced the peak rate of LV pressure development (+dP/dtmax: -8 ± 1%*) with minimal reductions in peak end systolic elastance (Emax: -4±1%*). Systemic mean arterial pressure and cardiac output were unchanged. EDG-7500 increased LV end-diastolic volume (+5 ± 1%*) and preserved LV end-diastolic pressure (EDP: -8 ± 3%, 9 ± 1 to 8 ± 1 mmHg). A downward and rightward shift of the pressure-volume relationship indicated improved LV compliance. β-AR blockade did not elicit this pro-compliant effect (EDP: +26 ± 12%). Conclusions: Cardiac sarcomere regulation with EDG-7500 has a unique cardiovascular profile characterized by the overall preservation of the myosin motor-head population and a slowing of early cardiac contraction and improved LV distensibility. This cardiovascular profile could have salutary effects in patients with HCM or patients with impaired diastolic function.

Remineralizing gel with calcium lactate for the prevention of caries in children

The article presents the results of a single-center open clinical trial of the use of the remineralizing gel «PRESIDENT Minerals gel 0+». The effectiveness of the drug in children in the period of mixed dentition with a compensated form of caries was studied.Materials and methods. The study involved 19 children, mean age 8,3 years. The duration of the study is 1 month.Clinical indices and laboratory parameters were determined: DMF+df, OHI-S, PMA, the degree of enamel electrometry, calcium, glucose, total protein and antioxidant activity of saliva (AOA). Organoleptic properties were determined on a 5-point scale.Results. After one month of using the remineralizing gel, a decrease in the hygiene index by 23,27%, a decrease in the electrometric evaluation of enamel by 15,58% were revealed. In saliva, a decrease in the level of protein by 33,13% and an increase in the content of calcium by 53,33% were revealed. The organoleptic properties of the gel were evaluated on a five-point scale at 4,31±0,22 points.Conclusion. «PRESIDENT 0+ Minerals gel» with calcium lactate and hydroxyapatite has a remineralizing effect, good organoleptic properties and can be recommended for caries prevention in children.

Targeting Mitochondrial Calcium Uptake to Eradicate Venetoclax-Resistant Acute Myeloid Leukemia Stem Cells

Introduction Previously, we demonstrated that human leukemia stem cells (LSCs) are preferentially reliant on oxidative phosphorylation (OXPHOS) for survival. Furthermore, inhibition of BCL2 with venetoclax (ven) acts to suppress OXPHOS, leading to the eradication of LSCs. While the activity of ven in targeting LSCs is well-established, nearly all patients eventually develop disease that is resistant to treatment. Thus, in the present study, we sought to better understand the biological properties of ven-resistant LSCs. We reasoned that the expression of known BCL2 interacting proteins in ven-sensitive (ven-S) vs. ven-resistant (ven-R) would provide insights on resistance mechanisms. Initial findings showed that one of the most prevalent molecular features observable in ven resistant LSCs is up-regulation of calcium signaling. As calcium is a critical modulator of multiple enzymes involved in OXPHOS, we hypothesized that the maintenance of high mitochondrial calcium could contribute to circumventing the effects of ven treatment. Thus, the present study was designed to test the functional role of calcium in ven-R LSCs and to determine whether targeting mitochondrial calcium transport represents a therapeutic vulnerability. Methods CITE-Seq analysis was performed on 25 AML patient specimens and analyzed using TotalVI. All studies were conducted in primary human AML specimens with LSCs defined by a ROS-low phenotype. Mitochondrial calcium measurements were done using Rhod2-AM. OXPHOS activity was measured through seahorse and enzyme activity assays. Genetic perturbations were done using siRNA via electroporation and confirmed with western blot. LSC function was assessed by colony formation assays and PDX experiments. PDX experiments were also used to determine efficacy of tumor reduction in ven-R AML samples. Results To identify unique features of ven resistant LSCs, we performed single cell RNA-seq on known ven-S vs. ven-R specimens. As shown in Fig 1, resistant LSCs had higher expression of genes involved in calcium transport into the mitochondria, which was reflected by increased steady-state mitochondrial calcium levels compared to ven-S LSCs. Thus, we investigated targeting of the calcium uniporter, MCU, as a means to reduce mitochondrial calcium levels. Treatment of primary LSCs with pharmacological MCU inhibitors (Ru265, MCUi4), as well as genetic inhibition of MCU with siRNAs, led to decreased mitochondrial calcium levels, suppression of calcium dependent TCA cycle dehydrogenase activity, reduced OXPHOS activity, and impaired LSC function as measured by colony assays (CFU) and engraftment in immune deficient mice. Importantly, MCU inhibition did not significantly impair normal stem/progenitor cells. To develop a clinically relevant system, we investigated bio-available agents known to inhibit MCU. Intriguingly, the only known FDA-approved direct MCU inhibitor is the chemotherapy agent, mitoxantrone (mitox). We performed a series of studies to assess the impact of mitox on ven-R LSCs. Our findings demonstrate that venetoclax resistant LSCs are exquisitely sensitive to mitox. In CFU assays, doses as low 1.0nM show strong inhibitory activity. Further, treatment of immune deficient mice bearing human xenografts (Fig 2), showed significant reduction of bulk disease in primary mice, and strong suppression of LSCs in secondary transplants. Notably, DNA damage was not present in LSCs treated with mitox as measured by gamma H2AX staining. Additionally, parallel studies with the related agents, doxorubicin and etoposide, showed no inhibition of CFU, mitochondrial calcium, or OXPHOS activity. Lastly, no discernable effect on normal hematopoietic stem cell activity was evident. Conclusions Our findings suggest that ven-R LSCs have adapted to their unique cellular demands by increasing basal mitochondrial calcium levels to drive OXPHOS. Based on this finding, we propose that inhibition of mitochondrial calcium uptake impairs multiple enzymes required for OXPHOS, which in turns leads to eradication of LSCs. Importantly, we also show that the well-established chemotherapy agent mitoxantrone can effectively inhibit MCU at nanomolar concentrations and is a highly potent against ven-R LSCs. We are currently working to establish a clinical trial in which low-dose mitox will be deployed to augment ven-based therapy.

Frass from Black Soldier Fly Larvae, Hermetia illucens, as a Possible Functional Dietary Ingredient in Channel Catfish Feed

Frass–the by-product of the larva meal industry–was recently shown to be a sustainable ingredient for use in diets of several fish species. Additionally, because of its possible immunomodulatory activity and anti-microbial properties, frass may have great potential as an organic method of pathogen control in aquaculture. Five diets containing 0, 5, 10, 20 and 30% frass from black soldier fly larvae, Hermetia illucens, were fed to channel catfish (5.24 ± 0.04 g) in quadruplicate aquaria to apparent satiation twice daily. At the end of the 10-week feeding trial, blood samples were collected from all groups to measure hematological and immune parameters, and to determine the effects of dietary frass on resistance to Flavobacterium covae infection. Hematological parameters (red blood cell count, hemoglobin, and hematocrit)—but not white blood cell count—were improved with the inclusion of frass. Serum glucose levels were significantly lower in fish on the diet with frass than fish on the diet without frass. Fish fed the highest dietary levels of frass (30%) had a significantly higher serum cholesterol level than fish on the control diet. Serum complement activity was significantly higher in fish on diets containing frass at levels of 10% and 20%. No significant differences were observed in other measure serum components including albumin, alkaline phosphatase, alanine aminotransferase, amylase, calcium, phosphorus, potassium, total protein, globulin, thyroxine and lysozyme activity. Even though overall mortality was low (0–17%), fish on the diets containing frass at levels 20% or more showed significantly higher survival than that of control fish or fish on lower levels of dietary frass. The use of frass in the catfish diet may prove beneficial by improving hematological parameters, and select serum immune effectors, and the overall resistance of juvenile channel catfish against F. covae infection.

Platelet Mitochondria Calcium Uniporter Regulates ITAM-Dependent Platelet Activation and Signaling

Introduction: The ability of platelets to multitask is critical to arrest bleeding after injury and for the development of arterial thrombosis, which underlies myocardial infarction and stroke. Platelet activation relies heavily on changes in cytoplasmic calcium flux. However, little is known on the role mitochondrial calcium flux directly plays in platelet activation. In other cells, release of calcium from intracellular stores results in calcium entry into channels located in the outer mitochondrial membrane. Once calcium enters the intermembrane space, entry into the mitochondrial matrix is regulated by a multimeric complex, composed of channel-forming subunits and regulatory elements called the mitochondrial calcium uniporter (MCU). MCU has been extensively studied in cardiac tissue where calcium flux through MCU is critical for regulating bioenergetics, ROS formation, and cytoplasmic calcium levels. As these processes are important for platelet activation, these studies would indicate mitochondrial calcium flux may regulate platelet activation. However, the role of MCU in platelet function and activation is poorly understood. Aim: The goals of the current study are 1) to examine the role of MCU and mitochondrial calcium flux in platelet function and 2) to establish if mitochondrial calcium regulates thrombosis. Methods: We generated a platelet-specific MCU-deficient mouse (MCU fl/fl-PF4-cre, KO) and compared them to littermate wild-type controls (MCU fl/fl, WT). Platelet function including activation, aggregation, and mitochondrial calcium flux in response to PAR4 activating peptide (PAR) and ADP (P2Y12), both GPCRs or to collagen (GPVI) and rhodocytin (CLEC-2), both ITAMs, were examined. In addition, we examined ex vivo platelet adhesion under arterial and venous shear to collagen and in vivo thrombosis using a ferric chloride carotid artery model. Results: Mice deficient inplatelet MCU were viable and fertile. In addition, platelet-specific MCU deletion did not alter platelet counts, mean platelet volume, or platelet half-life. MCU KO platelets had significantly reduced (p&amp;lt;0.05) GPVI and CLEC-2-dependent aIIbb3 activation and P-selectin expression while platelet activation in response to P2Y12 and PAR4 stimulation was unchanged. Consistent with our activation results, platelet aggregation was significantly reduced in response to collagen and rhodocytin (p&amp;lt;0.05) in MCU KO platelets, but not thrombin or ADP. MCU KO platelets adhered significantly less (p=0.0012) to collagen compared to MCU WT platelets under arterial and venous shear conditions. In vivo, MCU KO mice had longer occlusion time compared to the WT (p=0.0044). Mechanistically, mitochondrial calcium flux was significantly reduced (p&amp;lt;0.05) in MCU KO platelets compared to WT platelets after GPVI stimulation, but not PAR4 activation. Furthermore, mitochondrial reactive oxygen species (ROS) generation was significantly reduced in MCU KO platelets compared to WT platelets (p=0.0097) after GPVI-dependent activation while PAR4 activation induced no change in mitochondrial ROS. Mitochondrial ROS is known to regulate signaling in other cells. Consistent with this hypothesis, we observed a significant reduction in the ITAM signaling molecules pSyK (p=0.0084) and pPLCγ2 (p=0.012) in MCU KO platelets after GPVI stimulation. Furthermore, inhibiting mitochondrial ROS using MitoTempo, a specific mitochondrial ROS inhibitor, decreased aggregation (p=0.0004) as well as downstream signaling, including pSyk (p=0.0046) and pPLCγ2 (p=0.0493) in WT platelets when treated with a GPVI agonist. In parallel, treating MCU KO platelets with H 2O 2 to induce ROS production increased platelet aggregation (p=0.0036) after GPVI activation. Conclusion(s): Platelet MCU mediates platelet activation and thrombosis in an ITAM-dependent manner by regulating mitochondrial calcium flux and ROS generation as well as downstream ITAM signaling through GPVI and CLEC-2. Our data support a novel role for mitochondria and mitochondria calcium flux in regulating ITAM-dependent platelet activation and demonstrate platelet MCU as a novel anti-platelet target to reduce thrombosis

Neural signal propagation atlas of Caenorhabditis elegans

Establishing how neural function emerges from network properties is a fundamental problem in neuroscience1. Here, to better understand the relationship between the structure and the function of a nervous system, we systematically measure signal propagation in 23,433 pairs of neurons across the head of the nematode Caenorhabditis elegans by direct optogenetic activation and simultaneous whole-brain calcium imaging. We measure the sign (excitatory or inhibitory), strength, temporal properties and causal direction of signal propagation between these neurons to create a functional atlas. We find that signal propagation differs from model predictions that are based on anatomy. Using mutants, we show that extrasynaptic signalling not visible from anatomy contributes to this difference. We identify many instances of dense-core-vesicle-dependent signalling, including on timescales of less than a second, that evoke acute calcium transients—often where no direct wired connection exists but where relevant neuropeptides and receptors are expressed. We propose that, in such cases, extrasynaptically released neuropeptides serve a similar function to that of classical neurotransmitters. Finally, our measured signal propagation atlas better predicts the neural dynamics of spontaneous activity than do models based on anatomy. We conclude that both synaptic and extrasynaptic signalling drive neural dynamics on short timescales, and that measurements of evoked signal propagation are crucial for interpreting neural function.

Meta-Analysis of the Efficacy of Levosimendan in the Treatment of Severe Sepsis Complicated with Septic Cardiomyopathy.

Sepsis is a medical condition characterized by acute organ dysfunction and uncontrolled inflammation. Organ dysfunction in sepsis is the primary cause of mortality in patients with myocardial dysfunction. Levosimendan is a vasodilating and inotropic agent used in patients with acute heart failure and has resulted in decreased morbidity and mortality in these patients. Our main objective is to examine levosimendan's efficacy in treating severe sepsis complicated with septic cardiomyopathy. We systematically searched five databases, PubMed, Web of Science, Embase, Cochrane Library and BioMed Central, for articles and publications from their inception to 2023. Our study adopted the PICOS approach in identifying suitable publications during the systematic search. Inclusion criteria included randomized, controlled trials utilizing levosimendan in adult patients diagnosed with septic shock or severe sepsis. We excluded non-English publications and non-randomized controlled trials. The Newcastle-Ottawa scale (NOS) scale was used to assess the methodological quality, while the risk of bias was assessed through the Cochrane Risk of bias tool. All statistical analyses were performed using RevMan version 5.4. Eight studies met the eligibility criteria and were included in the analysis. There was a statistically significant positive effect on cardiac input in patients treated with levosimendan compared to those treated with dobutamine (p < 0.001). Similarly, there were positive effects on left ventricular ejection fraction (LVEF) (p < 0.001) and left ventricular stroke work index (LVSWI) (p < 0.001). We observed a significant reduction in mortality (p < 0.01) and serum levels of lactic acid (p < 0.01). Levosimendan is a calcium sensitizer associated with an influx of calcium ions and activation of ATP-dependent potassium channels that increases myocardial contractility contractions, enhances vasodilation and improves oxygen supply to the cells and tissues. Levosimendan is highly efficacious and safe in the management of sepsis and sepsis-induced cardiomyopathy.

Aberrant DJ-1 expression underlies L-type calcium channel hypoactivity in dendrites in tuberous sclerosis complex and Alzheimer's disease.

L-type voltage-gated calcium (Ca2+) channels (L-VGCC) dysfunction is implicated in several neurological and psychiatric diseases. While a popular therapeutic target, it is unknown whether molecular mechanisms leading to disrupted L-VGCC across neurodegenerative disorders are conserved. Importantly, L-VGCC integrate synaptic signals to facilitate a plethora of cellular mechanisms; however, mechanisms that regulate L-VGCC channel density and subcellular compartmentalization are understudied. Herein, we report that in disease models with overactive mammalian target of rapamycin complex 1 (mTORC1) signaling (or mTORopathies), deficits in dendritic L-VGCC activity are associated with increased expression of the RNA-binding protein (RBP) Parkinsonism-associated deglycase (DJ-1). DJ-1 binds the mRNA coding for the alpha and auxiliary Ca2+ channel subunits CaV1.2 and α2δ2, and represses their mRNA translation, only in the disease states, specifically preclinical models of tuberous sclerosis complex (TSC) and Alzheimer's disease (AD). In agreement, DJ-1-mediated repression of CaV1.2/α2δ2 protein synthesis in dendrites is exaggerated in mouse models of AD and TSC, resulting in deficits in dendritic L-VGCC calcium activity. Finding of DJ-1-regulated L-VGCC activity in dendrites in TSC and AD provides a unique signaling pathway that can be targeted in clinical mTORopathies.

Effects of vitrification solution supplemented with platelet-rich plasma in rat ovarian tissue cryopreservation.

The subject of this study was to investigate the utility of platelet-rich plasma (PRP) in the cryopreservation process to reduce cryodamage and increase tissue viability. Twenty-one female Wistar rats were randomly allocated to three groups. In Group 1 (G1), rats were not subjected to vitrification (n = 7). Group 2 (G2) was the vitrification group in which PRP was added to the basic vitrification solution (n = 7). Group 3 (G3) was the vitrification group in which fetal bovine serum was added to the basic vitrification solution (n = 7). Warmed tissues were evaluated with histochemical (HC) and immunohistochemical (IHC) staining, the TUNEL method, immunofluorescence (IF) staining, and biochemical analyses. The percentages of IHC staining, TUNEL method positivity, and IF staining were significantly higher in G2 compared to both G1 and G3 (P < 0.05). G2 ovaries exhibited a significant increase in both malondialdehyde and catalase values in comparison to G1 (P < 0.05). In HC staining, degenerations in primary and secondary follicles and in ovarian tissue were more common in the PRP-supplemented group. The calcium used in PRP activation was suspected to have increased the degeneration and prevented the possible positive effects of PRP. To the best of our knowledge, PRP-supplemented vitrification solution was used for the first time in the literature in this study in whole rat ovarian tissue vitrification. If PRP is to be used as a component in vitrification solution for rat ovarian tissue, the use of lower amounts of calcium or different methods in PRP activation, or the use of nonactivated PRP, should be considered from the beginning.