Intracellular Free Calcium Levels Research Articles

Vitamin C Drives Reentrant Actin Phase Transition: Biphasic Exocytosis Regulation Revealed by Single-Vesicle Electrochemistry.

Unveiling molecular mechanisms that dominate protein phase dynamics has been a pressing need for deciphering the intricate intracellular modulation machinery. While ions and biomacromolecules have been widely recognized for modulating protein phase separations, effects of small molecules that essentially constitute the cytosolic chemical atmosphere on the protein phase behaviors are rarely understood. Herein, we report that vitamin C (VC), a key small molecule for maintaining a reductive intracellular atmosphere, drives reentrant phase transitions of myosin II/F-actin (actomyosin) cytoskeletons. The actomyosin bundle condensates dissemble in the low-VC regime and assemble in the high-VC regime in vitro or inside neuronal cells, through a concurrent myosin II protein aggregation-dissociation process with monotonic VC concentration increase. Based on this finding, we employ in situ single-cell and single-vesicle electrochemistry to demonstrate the quantitative modulation of catecholamine transmitter vesicle exocytosis by intracellular VC atmosphere, i.e., exocytotic release amount increases in the low-VC regime and decreases in the high-VC regime. Furthermore, we show how VC regulates cytomembrane-vesicle fusion pore dynamics through counteractive or synergistic effects of actomyosin phase transitions and the intracellular free calcium level on membrane tensions. Our work uncovers the small molecule-based reversive protein phase regulatory mechanism, paving a new way to chemical neuromodulation and therapeutic repertoire expansion.

Metabolic profiling of Mytilus coruscus mantle in response of shell repairing under acute acidification.

Mytilus coruscus is an economically important marine bivalve mollusk found in the Yangtze River estuary, which experiences dramatic pH fluctuations due to seasonal freshwater input and suffer from shell fracture or injury in the natural environment. In this study, we used intact-shell and damaged-shell M. coruscus and performed metabolomic analysis, free amino acids analysis, calcium-positive staining, and intracellular calcium level tests in the mantle to investigate whether the mantle-specific metabolites can be induced by acute sea-water acidification and understand how the mantle responds to acute acidification during the shell repair process. We observed that both shell damage and acute acidification induced alterations in phospholipids, amino acids, nucleotides, organic acids, benzenoids, and their analogs and derivatives. Glycylproline, spicamycin, and 2-aminoheptanoic acid (2-AHA) are explicitly induced by shell damage. Betaine, aspartate, and oxidized glutathione are specifically induced by acute acidification. Our results show different metabolic patterns in the mussel mantle in response to different stressors, which can help elucidate the shell repair process under ocean acidification. furthermore, metabolic processes related to energy supply, cell function, signal transduction, and amino acid synthesis are disturbed by shell damage and/or acute acidification, indicating that both shell damage and acute acidification increased energy consumption, and disturb phospholipid synthesis, osmotic regulation, and redox balance. Free amino acid analysis and enzymatic activity assays partially confirmed our findings, highlighting the adaptation of M. coruscus to dramatic pH fluctuations in the Yangtze River estuary.

Protective Effect of Ergothioneine against 7-Ketocholesterol-Induced Mitochondrial Damage in hCMEC/D3 Human Brain Endothelial Cells.

Recent findings have suggested that the natural compound ergothioneine (ET), which is synthesised by certain fungi and bacteria, has considerable cytoprotective potential. We previously demonstrated the anti-inflammatory effects of ET on 7-ketocholesterol (7KC)-induced endothelial injury in human blood-brain barrier endothelial cells (hCMEC/D3). 7KC is an oxidised form of cholesterol present in atheromatous plaques and the sera of patients with hypercholesterolaemia and diabetes mellitus. The aim of this study was to elucidate the protective effect of ET on 7KC-induced mitochondrial damage. Exposure of human brain endothelial cells to 7KC led to a loss of cell viability, together with an increase in intracellular free calcium levels, increased cellular and mitochondrial reactive oxygen species, a decrease in mitochondrial membrane potential, reductions in ATP levels, and increases in mRNA expression of TFAM, Nrf2, IL-1β, IL-6 and IL-8. These effects were significantly decreased by ET. Protective effects of ET were diminished when endothelial cells were coincubated with verapamil hydrochloride (VHCL), a nonspecific inhibitor of the ET transporter OCTN1 (SLC22A4). This outcome demonstrates that ET-mediated protection against 7KC-induced mitochondrial damage occurred intracellularly and not through direct interaction with 7KC. OCTN1 mRNA expression itself was significantly increased in endothelial cells after 7KC treatment, consistent with the notion that stress and injury may increase ET uptake. Our results indicate that ET can protect against 7KC-induced mitochondrial injury in brain endothelial cells.

ORAI Calcium Channels: Regulation, Function, Pharmacology, and Therapeutic Targets.

The changes in intracellular free calcium (Ca2+) levels are one of the most widely regulators of cell function; therefore, calcium as a universal intracellular mediator is involved in very important human diseases and disorders. In many cells, Ca2+ inflow is mediated by store-operated calcium channels, and it is recognized that the store-operated calcium entry (SOCE) is mediated by the two partners: the pore-forming proteins Orai (Orai1-3) and the calcium store sensor, stromal interaction molecule (STIM1-2). Importantly, the Orai/STIM channels are involved in crucial cell signalling processes such as growth factors, neurotransmitters, and cytokines via interaction with protein tyrosine kinase coupled receptors and G protein-coupled receptors. Therefore, in recent years, the issue of Orai/STIM channels as a drug target in human diseases has received considerable attention. This review summarizes and highlights our current knowledge of the Orai/STIM channels in human diseases and disorders, including immunodeficiency, myopathy, tubular aggregate, Stormorken syndrome, York platelet syndrome, cardiovascular and metabolic disorders, and cancers, as well as suggesting these channels as drug targets for pharmacological therapeutic intervention. Moreover, this work will also focus on the pharmacological modulators of Orai/STIM channel complexes. Together, our thoughtful of the biology and physiology of the Orai/STIM channels have grown remarkably during the past three decades, and the next important milestone in the field of store-operated calcium entry will be to identify potent and selective small molecules as a therapeutic agent with the purpose to target human diseases and disorders for patient benefit.

Change of intracellular calcium level causes acute neurotoxicity by antisense oligonucleotides via CSF route

Antisense oligonucleotides (ASOs) are promising therapeutics for intractable central nervous system (CNS) diseases. For this clinical application, neurotoxicity is one of the critical limitations. Therefore, an evaluation of this neurotoxicity from a behavioral perspective is important to reveal symptomatic dysfunction of the CNS and elucidate the underlying molecular mechanism. We here exploited a behavioral analysis method to categorize and quantify the acute neurotoxicity of mice administered with toxic ASOs via intracerebroventricular injection. The toxic ASOs were found to reduce consciousness and locomotor function in mice in a dose-dependent manner. Mechanistically, we analyzed the effects of modulators against receptors or channels, which regulate calcium influx of neurons, on the ASO neurotoxicity. Modulators promoting calcium influx mitigated, whereas those hindering calcium influx increased, invivo neurotoxicity of ASOs in mice. In an invitro assay to evaluate intracellular free calcium levels using rat primary cortical neurons, toxic ASOs reduced the calcium levels. The findings of this study demonstrated the behavioral characteristics of ASO-induced neurotoxicity and revealed that changes in intracellular free calcium levels are a part of the mechanism underlying the neurotoxic effects of ASO.

Nanoelectropulse delivery for cell membrane perturbation and oxidation in human colon adenocarcinoma cells with drug resistance

Ultrashort electric pulses in the nanosecond range (nsPEF) can affect extra- and intracellular lipid structures and can also alternate cell functioning reversibly and irreversibly. Several of the nsPEF effects are due to the abrupt rise in intracellular free calcium levels and calcium ions influx from the outside. Calcium is one of the most important factors in cell proliferation, differentiation, and cell death (apoptosis or necrosis). Manipulating calcium levels using electroporation can have different effects on normal and malignant cells. This study aimed to examine the impact of nsPEFs, combined with 1 mM Ca2+ in human colon adenocarcinoma cell lines: sensitive- LoVo and drug resistant-LoVoDX. In this study 200 pulses of 10 ns and high voltage (12.5–50 kVcm−1) were used. Cell viability was determined by MTT and clonogenic assay. Proteasomal activity, GSH/GSSG assay, ROS production, and PALS-1 protein were evaluated as oxidative stress markers and protein damage. Cell morphology was visualized by AFM, SEM, and confocal microscopy imaging. The results revealed that nsPEF with 1 mM Ca2+ is cytotoxic, particularly for LoVoDX cells, and safe for normal cells. NsPEF provoked ROS release, altered cell polarity, and destabilized cell morphology. These results can be important for future protocols for colon adenocarcinoma using calcium nsPEF.

Construction of a bioluminescence-based assay for bitter taste receptors (TAS2Rs)

In humans, a family of 25 bitter taste receptors (TAS2Rs) mediates bitter taste perception. A common approach to characterize bitter causative agents involves expressing TAS2Rs and the appropriate signal transducers in heterologous cell systems, and monitoring changes in the intracellular free calcium levels upon ligand exposure using a fluorescence-based modality, which typically suffers from a low signal window, and is susceptible to interference by autofluorescence, therefore prohibiting its application to screening of plant or food extracts, which are likely to contain autofluorescent compounds. The aim of this study is to develop and validate a bioluminescence-based intracellular calcium release assay for TAS2Rs that has a better assay performance than a fluorescence-based assay. Furthermore, the bioluminescence-based assay enabled the evaluation of TAS2R agonists within an autofluorescent matrix, highlighting its potential utility in the assessment of the bitterness-inducing properties of plant or food fractions by the food industry. Additionally, improvement to the bioluminescence-based assay for some TAS2Rs was achieved by altering their N-terminal signal sequences, leading to signal window enhancement. Altogether, the bioluminescence-based TAS2R assay can be used to perform functional studies of TAS2Rs, evaluate TAS2R-modulating properties of autofluorescent samples, and facilitate the discovery of compounds that can function as promising bitter taste modulators.

Effect of zinc and copper ions on cadmium-induced toxicity in rat cultured cortical neurons

BackgroundCadmium is a highly toxic heavy metal that is capable of accumulating in the body and causing neurodegeneration. However, the effect of other trace elements on Cd2+ toxicity is currently poorly understood. The aim of this work was to study the effect of Zn2+ and Cu2+ ions on cadmium-induced death of neurons in the cerebral cortex. MethodsThe work was performed on rat cortical primary cultures. The MTT test was used to determine the cytotoxicity effects. Analysis of intracellular Ca2+ concentration was assessed by the Fluo-4 AM calcium indicator that exhibit an increase in fluorescence upon binding Ca2+. MitoSOX Red (mitochondrial superoxide indicator) was used to measuring mitochondrial ROS content in live cells. ResultsIn this article, we show that the administration of CdCl2 (0.005–0.02 mM) for 48 h induced an increase in dose-dependent death rate of cultured cortical neurons. Mature neurons were more sensitive to the damaging effects of Cd2+ than immature ones. ZnCl2 (0.01–0.03 mM) significantly protected neurons from this toxic effect. In contrast to ZnCl2, CuCl2 (0.01 mM) increased cadmium neurotoxicity. Using Fluo-4 AM, measurements of intracellular calcium ions demonstrated that 24 h-exposure to Cd2+ induced intensive increase in Fluo-4 fluorescence in neurons, which was significantly reduced by zinc ions. CuCl2 increased the cadmium-induced Fluo-4 and MitoSOX Red fluorescence in neurons. The chelator of intracellular Ca2+ BAPTA significantly decreased Cd2+-induced intensive increase in Fluo-4 fluorescence in cells. ConclusionThe data obtained by us indicate that Zn2+ and Cu2+ can affect the neurotoxicity of cadmium in different directions: Zn2+ weaken the violation of intracellular calcium homeostasis caused by cadmium, preventing cell death, while Cu2+ potentiate the increase in the level of free intracellular calcium induced by cadmium and the development of mitochondrial dysfunction with an increase in the production of free radicals in differentiated cultured neurons of the cerebral cortex, which ultimately stimulates cytotoxicity.

γδ Tcells license immature B cells to produce a broad range of polyreactive antibodies.

Immature autoreactive B cells are present in all healthy individuals, but it is unclear which signals are required for their maturation into antibody-producing cells. Inducible depletion of γδ Tcells show that direct interaction between γδ Tcells and immature B cells in the spleen support an "innate" transition to mature B cells with a broad range of antigen specificities. IL-4 production of γδ Tcells and cell-to-cell contact via CD30L support B cell maturation and induce genes of the unfolded protein response and mTORC1 signaling. Eight days after invivo depletion of γδ Tcells, increased numbers of B cells are already stuck in the transitional phase and express increased levels of IgD and CD21. Absence of γδ Tcells leads also to reduced levels of serum anti-nuclear autoantibodies, making γδ Tcells an attractive target to treat autoimmunity.

Inhibition of mitochondrial reactive oxygen species improves coronary endothelial function after cardioplegic hypoxia/reoxygenation

ObjectiveCardioplegic ischemia–reperfusion and diabetes mellitus are correlated with coronary endothelial dysfunction and inactivation of small conductance calcium-activated potassium channels. Increased reactive oxidative species, such as mitochondrial reactive oxidative species, may contribute to oxidative injury. Thus, we hypothesized that inhibition of mitochondrial reactive oxidative species may protect coronary small conductance calcium-activated potassium channels and endothelial function against cardioplegic ischemia–reperfusion-induced injury. MethodsSmall coronary arteries and endothelial cells from the hearts of mice with and without diabetes mellitus were isolated and examined by using a cardioplegic hypoxia and reoxygenation model to determine whether the mitochondria-targeted antioxidant Mito-Tempo could protect against coronary endothelial and small conductance calcium-activated potassium channel dysfunction. The microvessels or mouse heart endothelial cells were treated with or without Mito-Tempo (0-10 μM) 5 minutes before and during cardioplegic hypoxia and reoxygenation. Microvascular function was assessed in vitro by vessel myography. K+ currents of mouse heart endothelial cells were measured by whole-cell patch clamp. The levels of intracellular cytosolic free calcium (Ca2+) concentration, mitochondrial reactive oxidative species, and small conductance calcium-activated potassium protein expression of mouse heart endothelial cells were measured by Rhod-2 fluorescence staining, MitoSox, and Western blotting, respectively. ResultsCardioplegic hypoxia and reoxygenation significantly attenuated endothelial small conductance calcium-activated potassium channel activity, caused calcium overload, and increased mitochondrial reactive oxidative species of mouse heart endothelial cells in both the nondiabetic and diabetes mellitus groups. In addition, treating mouse heart endothelial cells with Mito-Tempo (10 μM) reduced cardioplegic hypoxia and reoxygenation-induced Ca2+ and mitochondrial reactive oxidative species overload in both the nondiabetic and diabetes mellitus groups, respectively (P < .05). Treatment with Mito-Tempo (10 μM) significantly enhanced coronary relaxation responses to adenosine 5′-diphosphate and NS309 (P < .05), and endothelial small conductance calcium-activated potassium channel currents in both the nondiabetic and diabetes mellitus groups (P < .05). ConclusionsAdministration of Mito-Tempo improves endothelial function and small conductance calcium-activated potassium channel activity, which may contribute to its enhancement of endothelium-dependent vasorelaxation after cardioplegic hypoxia and reoxygenation.

MO008NEPHROTOXIC MECHANISMS OF GADOLINIUM: IMPLICATIONS FOR THE USE OF GADOLINIUM-BASED CONSTRAST AGENTS

Abstract Background and Aims Gadolinium-based contrast agents are widely used for magnetic resonance imaging, and although they may be considered well tolerated at recommended dosing levels, recent evidences support the deposition of free gadolinium in the tissues, and its slow release into circulation, resulting in long-term toxicity, which is aggravated in renal patients. The kidney, as the major excretion organ of these agents, and particularly the proximal tubule, as a common location of xenobiotics’ bioaccumulation, may be key targets of gadolinium’s deleterious effects. This study aimed at unveiling the nephrotoxic potential and the underlying mechanisms of toxicity of gadolinium, using an in vitro model of normal human proximal tubular cells (HK-2 cell line). Method HK-2 cells were exposed for 24 h to a wide concentration range of gadolinium, in the form of trichloride hexahydrate, and cell viability was assessed to estimate the half maximal effective concentration (EC50) and the subtoxic concentration eliciting 1 % of cell death (EC01). These ECs were further used to determine the effects of gadolinium on the cell’s oxidative status, mitochondrial function, cell death mechanisms and lipid deposition, through variable colorimetric and fluorometric assays. Expression of the pro-inflammatory gene IL6 was also determined through quantitative PCR. Results Gadolinium induced cell death in a concentration-dependent manner, with estimated EC01 and EC50 of 3 and 340 µM, respectively. When compared to control cells, the subtoxic concentration showed no significant effects in terms of reactive oxygen and nitrogen species (ROS and RNS) production, total glutathione levels (tGSH) or total antioxidant status (TAS). On the other hand, the EC50 showed significant disruption of the oxidative status of the cell, with over 60 % depletion of tGSH cell contents (p &amp;lt; 0.01) and a significant decrease of TAS (p &amp;lt; 0.05). However, this effect was not accompanied by an increase, but rather by a significant decline in ROS and RNS production of about 50 % below control (p &amp;lt; 0.0001). At the EC50, but not at EC01, it was also observed the disturbance of the mitochondrial and energetic homeostasis, as showed by the increment of intracellular free calcium levels, hyperpolarization of the mitochondrial membrane, and decay of the ATP levels (p &amp;lt; 0.0001). Cell death induced by gadolinium was characterized by typical morphological changes of late apoptosis and necrosis, with a significant increase in propidium iodide uptake and lactate dehydrogenase leakage (p ≤ 0.0001) at EC50. The presence of neutral lipids-containing vesicles was observed in the cytoplasm of cells exposed to gadolinium using the fluorescent dye BODIPY, already noticeable at the subtoxic concentration. Cells exposed to gadolinium also showed increased expression of IL6, though this effect was only significant at the EC01 (p &amp;lt; 0.05). Conclusion Gadolinium showed marked cytotoxic potential at micromolar levels in HK-2 cells. This cytotoxicity was characterized by increased oxidative stress independent of ROS and RNS production and mitochondrial dysfunction followed by cell death via late apoptosis and necrosis. At a subtoxic concentration, gadolinium was also able to elicit the accumulation of lipidic vesicles within the cells’ cytoplasm, and to trigger a pro-inflammatory response. Although it is still unclear which amount of gadolinium is in fact released from the complexes commonly used as contrast agents, this study shows that gadolinium ion has direct nephrotoxic potential, with noteworthy manifestations at subtoxic concentrations. Acknowledgments This work was supported by Applied Molecular Biosciences Unit (UCIBIO), financed by national funds from FCT/MCTES (UIDB/04378/2020), by North Portugal Regional Coordination and Development Commission (CCDR-N)/NORTE2020/Portugal 2020 (Norte-01-0145-FEDER-000024).

Kisspeptin leads to calcium signaling in cultured rat dorsal root ganglion neurons.

Although kisspeptin and GPR54 have been reported to be expressed in the neurons of the dorsal root ganglion (DRG) of rats, and kisspeptin has been suggested to be involved in pain modulation in rodents, there is no study on the effects and mechanisms of kisspeptin on sensory neurons. Therefore, the aim of this study was to investigate the effects and mechanism of kisspeptin on intracellular free calcium levels in cultured rat DRG neurons. Bath application of kisspeptin-10 increased intracellular free calcium levels ([Ca2+]i). In the absence of extracellular calcium, the kisspeptin induced an attenuated but still significant increase in [Ca2+]i. [Ca2+]i responses persisted in the presence of protein kinase C (PKC) inhibitor. Data from this study revealed that kisspeptin-10 activates [Ca2+]i signaling independent of PKC in cultured rat sensory neurons suggesting that peripheral site is also involved in the pain modulating effect of kisspeptin.

Kisspeptin leads to calcium signaling in cultured rat dorsal root ganglion neurons

Although kisspeptin and GPR54 have been reported to be expressed in the neurons of the dorsal root ganglion (DRG) of rats, and kisspeptin has been suggested to be involved in pain modulation in rodents, there is no study on the effects and mechanisms of kisspeptin on sensory neurons. Therefore, the aim of this study was to investigate the effects and mechanism of kisspeptin on intracellular free calcium levels in cultured rat DRG neurons. Bath application of kisspeptin-10 increased intracellular free calcium levels ([Ca2+]i). In the absence of extracellular calcium, the kisspeptin induced an attenuated but still significant increase in [Ca2+]i. [Ca2+]i responses persisted in the presence of protein kinase C (PKC) inhibitor. Data from this study revealed that kisspeptin-10 activates [Ca2+]i signaling independent of PKC in cultured rat sensory neurons suggesting that peripheral site is also involved in the pain modulating effect of kisspeptin.

Rapid tyrosine phosphorylation and activation of Bruton's tyrosine/Tec kinases in platelets induced by collagen binding or CD32 cross-linking

Stimulation of the platelet nonintegrin collagen receptor, glycoprotein VI, evokes a signaling response similar to that induced by antigen receptor activation in B and T lymphocytes. A key transducer of the lymphocyte signaling pathways is the Bruton's tyrosine kinase (Btk)/Tec kinase family, which connects receptors to the elevation of intracellular-free calcium levels. An important signaling function for Btk in collagen-induced platelet activation in vitro was recently demonstrated by other researchers using Btk-deficient platelets from patients with X-linked agammaglobulinemia (XLA). Since Btk-deficiency does not induce an overt platelet-based bleeding disorder in vivo, collagen receptor responses may include other Btk/Tec kinase family members in normal platelets. Both Btk and Tec had increased tyrosine following stimulation of collagen receptors or CD32 cross-linking. Data from kinetic analyses and inhibitor studies and the use of phosphopeptide-specific antibodies recognizing 2 Btk regulatory phosphorylated tyrosine residues suggest a mechanism for coordinate recruitment of Btk and Tec through the immunoreceptor tyrosine-based activation motif, Src family kinases, and phosphatidylinositol 3-kinase. In XLA platelets, collagen treatment increased tyrosine phosphorylation of Tec and several other signaling proteins, including Lyn, Fyb, Slp-76, and the Wiskott-Aldrich syndrome protein. This indicates that important elements of the collagen signaling pathway proximal and distal to Btk and Tec are preserved despite the lack of functional Btk. The results are consistent with the conclusion that activation of Tec may sustain XLA platelet function in vivo, while some in vitro assays of nonintegrin collagen receptor signaling through the Btk/Tec kinase family reflect the additive dosage of the transducers. (Blood. 2000;95:1663-1670)

Yi-Zhi-Fang-Dai Formula Exerts a Protective Effect on the Injury of Tight Junction Scaffold Proteins in Vitro and in Vivo by Mediating Autophagy through Regulation of the RAGE/CaMKKβ/AMPK/mTOR Pathway.

Alzheimer's disease (AD) is a chronic neurodegeneration disease that is closely related to the abnormal tight junction scaffold proteins (TJ) proteins of the blood-brain barrier (BBB). Recently, Yi-Zhi-Fang-Dai Formula (YZFDF) had exerted a neuronal protective effect against amyloid peptide (Aβ) toxicity. Still, the therapeutic mechanism of YZFDF in restoring Aβ-induced injury of TJ proteins (ZO-1, Occludin, and Claudin-5) remains unclear. This study aimed to explore the underlying mechanism of YZFDF in alleviating the injury of TJ proteins. We examined the impacts of YZFDF on autophagy-related proteins and the histopathology of Aβ in the APP/PS1 double-transgenic male mice. We then performed the free intracellular calcium levels [Ca2+]i analysis and the cognitive behavior test of the AD model. Our results showed that YZFDF ameliorated the injury of TJ proteins by reducing the mRNA transcription and expression of the receptor for advanced glycation end-products (RAGE), the levels of [Ca2+]i, calmodulin-dependent protein kinase β (CaMKKβ), phosphorylated AMP-activated protein kinase (AMPK). Accordingly, YZFDF increased the expression of the phosphorylated mammalian targets of rapamycin (mTOR), leading to inhibition of autophagy (downregulated LC3 and upregulated P62). Moreover, the Aβ1-42 oligomers-induced alterations of autophagy in murine mouse brain capillary (bEnd.3) cells were blocked by RAGE small interfering RNA (siRNA). These results suggest that YZFDF restored TJ proteins' injury by suppressing autophagy via RAGE signaling. Furthermore, YZFDF reduced the pathological precipitation of Aβ in the hippocampus, and improved cognitive behavior impairment of the AD model suggested that YZFDF might be a potential therapeutic candidate for treating AD through RAGE/CaMKKβ/AMPK/mTOR-regulated autophagy pathway.

Ceramic 3D-Printed Titanium Cranioplasty.

Inlay cranioplasties following partial craniectomy in tumor or trauma cases and onlay cranioplasties for reconstructions of residual developmental skull anomalies are frequently performed using CAD-CAM techniques. In this case series, we present a novel cranial implant design, being a combination of 3D-printed titanium grade 23 and calcium phosphate paste (CeTi). The titanium patient-specific implant, manufactured using selective laser melting, has a latticed border with interconnected micropores. The cranioplasty is miniscrew fixed and its border zone subsequently partially filled with calcium phosphate paste to promote osteoinduction and osteoconduction. From April 2017 to April 2019, 8 patients have been treated with such a CeTi implant. The inlay cranioplasties were each time revision surgeries of complicated cases. All implants were successful after a limited follow-up time (range 18-42 months). There were no dehiscences and no infections, and no complaints of thermal conduction. The proposed CeTi cranial implant combines the strength of titanium implants with the biological integration potential of ceramic implants and seems particularly resistant to infection, probably due to the biofunctionalized titanium surface and the antimicrobial activity of elevated intracellular free calcium levels.

Curcumin loaded nanoparticles reversed monocrotophos induced motor impairment and memory deficit: Role of oxidative stress and intracellular calcium level

Monocrotophos is one of the extensively used organophosphorus insecticides in agriculture and animal husbandry in India. It induces neurotoxicity by inhibiting acetyl cholinesterase enzyme and elevating oxidative stress. Curcumin, dietary polyphenol, well known for its antioxidant and anti-inflammatory properties which could elicit protective effect against monocrotophos induced neurobehavioural abnormalities. Therefore this study is aimed to investigate ameliorative effect of curcumin loaded Nanoparticles on monocrotophos induced neurotoxicity.Curcumin loaded PLGA nanoformulation was prepared using nanoprecipitation technique and was characterized using zeta sizer, FTIR and NMR spectroscopy. For in-vivo study, 30 male rats were utilized. Exposure of monocrotophos was done at 2 mg/kg (p.o.) for 28 days. Curcumin bulk and cucurmin loaded Nanoparticles were administered with or without monocrotophos. Effect of monocrotophos exposure on behavioural changes was assessed by analysing spontaneous locomotor activity, motor coordination in rotarod test and contextual fear memory in passive shock avoidance paradigm. Effect on biochemical alteration in blood and brain were estimated.The average particle size of Curcumin loaded Nanoparticles was 114.9 nm and in-vitro release study suggested suitable release of Curcumin in simulated intestinal fluid. Results of in-vivo study indicated, monocrotophos exposure caused a significant increase in reactive oxygen species, thiobarbituric acid reactive substances, and intracellular free calcium levels along with altered behavioural abnormalities in locomotor activity, motor coordination, learning and memory. Decline in antioxidant defence level might suggest reduced host defence response. Treatment with curcumin alone did not significantly reversed monocrotophos induced neurotoxicity in rats. Whilst, treatment with curcumin loaded nanoparticles significantly reduced monocrotophos induced behavioural and biochemical alteration in rat brain.The outcome of the present study suggested improved efficacy of curcumin loaded nanoparticles, possibly via its enhanced bioavailability. Further alleviation of oxidative stress and intracellular free Ca2+ level might be speculated as one of the plausible neuroprotective mechanism of curcumin in this study.

Investigating role of Mycobacterium tuberculosis secretory antigens in altering activation of T cell signaling events in Jurkat T cells.

Mycobacterium tuberculosis is able to survive and persist as an intracellular pathogen by modulating its own metabolism and host immunity. The molecules and mechanisms utilized to accomplish this modulation are not fully understood. The present study elucidates the effects of M. tuberculosis secretory antigens on T-cell-receptor (TCR)/CD28-triggered signaling in Jurkat T-cells. In the present study, intracellular calcium mobilization was investigated in CD3-activated cells in response to M. tuberculosis antigens, Ag85A, early secretory antigenic target-6 (ESAT-6), and H37Rv. The activation of mitogen-activated protein kinases, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p-38 was also analyzed in CD3- and CD28-activated cells by western blotting. Our results showed CD3-triggered modulations in free intracellular calcium levels in Jurkat T-cells in response to M. tuberculosis antigens. In addition, we also noted M. tuberculosis antigens induced downregulation in phosphorylation of ERK1/2 and p-38. Overall, our results proposed that M. tuberculosis secretory antigens, particularly ESAT-6, impede TCR/CD28-induced signaling events which could be responsible for T-cell unresponsiveness, implicated in the progression of disease. The present study demonstrated M. tuberculosis secretory antigens induced alteration of T-cell signaling pathways in unsensitized Jurkat T-cell line which might be implied in T-cell dysfunctioning during the progression of the disease.

Potential anti-neuroblastoma agents from Juniperus oblonga

Neuroblastoma (NB) is a neuroendocrine tumor derived from neural crest cells. Approximately 90% of cases occur in children less than 5 years old. The amplification of MYCN correlates with high-risk neuroblastoma and patients with MYCN amplified showed poorer prognosis than those without MYCN amplification. In this study, three compounds isolated from Juniperus oblonga showed anti-proliferative activity against NB cell lines with and without tetracycline inducible MYCN over-expression which were identified as (−)-deoxypodophyllotoxin (1), (−)-matairesinol (2) and (+)-isocupressic acid (3). The effects of compounds 2 and 3 in NB cells included a decrease in NB cell viability and induction of apoptosis. Compound 1 was more effective in NB cells over-expressing MycN. Compound 1 also showed almost 2-fold induction of intracellular free calcium levels in M2(+) cells, which may indicate a different mechanism of action for this compound. Cytotoxicity studies against the human embryonic kidney cell (HEK-293) showed compounds 1, 2 and 3 were ineffective in the non-cancer cells at concentrations approximating their IC50 against the NB cell lines. These results may lead to safer and more effective treatment options for NB patients especially for those with high-risk NB.

A Mixture of Functional Complex Extracts from Lycium barbarum and Grape Seed Enhances Immunity Synergistically In Vitro and In Vivo

A mixture of multiple ingredients is often more effective than the individual ingredients. The functions of Lycium barbarum polysaccharide (LBP) glycoconjugate and grape seed procyanidins (GSP) are widely known. Here, we investigated the synergistic immune-enhancing activity of LBP and GSP. Atomic force microscopy results suggested that the mixture of LBP and GSP exhibited circular structure unlike LBP alone, and the addition of polyphenols may change the spatial conformation of the sugar chain. The changes in the structure were related to the synergistic effect of the two functional agents on immune recovery. In vitro, the proliferation rate of splenocytes was higher in LBP + GSP group (64.16%), rather than the sum of LBP group (13.01%) and GSP group (43.61%) individually used. This synergistical proliferation of splenocytes may be correlated to the increasing intracellular free calcium levels. Furthermore, the mixture significantly enhanced the immunity in vivo, as evident from the recovery of peripheral white blood cell counts in LBP + GSP group (18.535 × 109 /L) to normal group levels (18.115 × 109 /L) and higher B cell proliferation than normal group (P < 0.05). These results highlight the immune-enhancing activity of the combination of LBP and GSP associated with the structural changes, which may facilitate the development of functional foods with fewer resources but enhanced activities. PRACTICAL APPLICATION: The synergistic effects of LBP and GSP on immunomodulatory were better than the sum of the effects of the individual agents both in vitro and in vivo. Our results may provide a research-based support for the development of related functional products and an insight into the production of food resources with a fewer but more effective functional agents for better results.