Influx Channels Research Articles

Positive Interaction between H2O2 and Ca2+ Mediates Melatonin-Induced CBF Pathway and Cold Tolerance in Watermelon (Citrullus lanatus L.).

Cold stress is a major environmental factor that detrimentally affects plant growth and development. Melatonin has been shown to confer plant tolerance to cold stress through activating the C-REPEAT BINDING FACTOR (CBF) pathway; however, the underlying modes that enable this function remain obscure. In this study, we investigated the role of H2O2 and Ca2+ signaling in the melatonin-induced CBF pathway and cold tolerance in watermelon (Citrullus lanatus L.) through pharmacological, physiological, and genetic approaches. According to the results, melatonin induced H2O2 accumulation, which was associated with the upregulation of respiratory burst oxidase homolog D (ClRBOHD) during the early response to cold stress in watermelon. Besides, melatonin and H2O2 induced the accumulation of cytoplasmic free Ca2+ ([Ca2+]cyt) in response to cold. This was associated with the upregulation of cyclic nucleotide-gated ion channel 2 (ClCNGC2) in watermelon. However, blocking of Ca2+ influx channels abolished melatonin- or H2O2-induced CBF pathway and cold tolerance. Ca2+ also induced ClRBOHD expression and H2O2 accumulation in early response to cold stress in watermelon. Inhibition of H2O2 production in watermelon by RBOH inhibitor or in Arabidopsis by AtRBOHD knockout compromised melatonin-induced [Ca2+]cyt accumulation and melatonin- or Ca2+-induced CBF pathway and cold tolerance. Overall, these findings indicate that melatonin induces RBOHD-dependent H2O2 generation in early response to cold stress. Increased H2O2 promotes [Ca2+]cyt accumulation, which in turn induces H2O2 accumulation via RBOHD, forming a reciprocal positive-regulatory loop that mediates melatonin-induced CBF pathway and subsequent cold tolerance.

Channeling of Carbon Flux Towards Carotenogenesis in Botryococcus braunii: A Media Engineering Perspective

Microalgae, due to their unique properties, gained attention for producing promising feedstocks having high contents of proteins, antioxidants, carotenoids, and terpenoids for applications in nutraceutical and pharmaceutical industries. Optimizing production of the high-value renewables (HVRs) in microalgae requires an in-depth understanding of their functional relationship of the genes involved in these metabolic pathways. In the present study, bioinformatic tools were employed for characterization of the protein-encoding genes of methyl erythritol phosphate (MEP) pathway involved in carotenoid and squalene biosynthesis based upon their conserved motif/domain organization. Our analysis demonstrates nearly 200 putative genes showing a conservation pattern within divergent microalgal lineages. Furthermore, phylogenomic studies confirm the close evolutionary proximity among these microalgal strains in the carotenoid and squalene biosynthetic pathways. Further analysis employing STRING predicts interactions among two rate-limiting genes, i.e., phytoene synthase (PSY) and farnesyl diphosphate farnesyl synthase (FPPS), which are specifically involved in the synthesis of carotenoids and squalene. Experimentally, to understand the carbon flux of these rate-limiting genes involved in carotenogenesis, an industrial potential strain, namely, Botryococcus braunii, was selected in this study for improved biomass productivity (i.e., 100 mg L–1 D–1) along with enhanced carotenoid content [0.18% dry cell weight (DCW)] when subjected to carbon supplementation. In conclusion, our approach of media engineering demonstrates that the channeling of carbon flux favors carotenogenesis rather than squalene synthesis. Henceforth, employing omics perspectives will further provide us with new insights for engineering regulatory networks for enhanced production of high-value carbon biorenewables without compromising growth.

Reactive Oxygen Species Downregulate Transient Receptor Potential Melastatin 6 Expression Mediated by the Elevation of miR-24-3p in Renal Tubular Epithelial Cells.

Background: A low level of serum magnesium ion (Mg2+) is associated with type 2 diabetes mellitus (T2D). However, the molecular mechanism of Mg2+ deficiency has not been fully clarified. The current study sought to assesses the effect of reactive oxygen species on the expression of Mg2+ channels and miRNA. Methods: The expression of Mg2+ channels and miRNA were examined by real-time polymerase chain reaction. Intracellular Mg2+ concentration was measured by Magnesium Green fluorescence measurement. Results: The mRNA level of transient receptor potential melastatin 6 (TRPM6), which functions as Mg2+ influx channel in the distal convoluted tubule (DCT) of the kidney, was decreased by glycated albumin (GA), but not by insulin in rat renal tubule-derived NRK-52E cells. The mRNA levels of TRPM7, a homologue of TRPM6, and CNNM2, a Mg2+ efflux transporter located at the basolateral membrane of DCT, were changed by neither GA nor insulin. The generation of reactive oxygen species (ROS) was increased by GA. Hydrogen peroxide (H2O2) dose-dependently decreased TRPM6 mRNA, but it inversely increased the reporter activity of TRPM6. H2O2 accelerated the degradation of TRPM6 mRNA in actinomycin D assay without affecting TRPM7 and CNNM2 mRNA expressions. Nine miRNAs were considered as candidates for the regulator of stability of TRPM6 mRNA. Among them, miR-24-3p expression was increased by H2O2. The H2O2-induced reduction of TRPM6 mRNA was rescued by miR-24-3p siRNA. Magnesium Green fluorescence measurement showed that Mg2+ influx is suppressed by H2O2, which was rescued by an antioxidant and miR-24-3p siRNA. Conclusions: We suggest that GA decreases TRPM6 expression mediated by the elevation of ROS and miR-24-3p in renal tubular epithelial cells of T2D.

Collagen fiber membrane-derived chemically and mechanically durable superhydrophobic membrane for high-performance emulsion separation

Developing high-performance separation membrane with good durability is a highly desired while challenging issue. Herein, we reported the successful fabrication of chemically and mechanically durable superhydrophobic membrane that was prepared by embedding UiO-66 as size-sieving sites within the supramolecular fiber structure of collagen fiber membrane (CFM), followed by the polydimethylsiloxane (PDMS) coating. The as-prepared CFM/UiO-66(12)/PDMS membrane featured capillary effect-enhanced separation flux and homogeneous porous channels guaranteed high separation efficiency. When utilized as double-layer separation membranes, this new type of composite membranes separated various surfactant stabilized water-in-oil microemulsions and nanoemulsions, with the separation efficiency high up to 99.993 % and the flux as high as 973.3 L m− 2 h− 1. Compared with commercial polytetrafluoro ethylene (PTFE) membrane, the advantage of the double-layer CFM/UiO-66(12)/PDMS membranes in separation flux was evident, which exhibited one order of magnitude higher than that of commercial PTFE membrane. The CFM/UiO-66(12)/PDMS membrane was acid-alkali tolerant, UV-aging resistant and reusable for emulsion separation. Notably, the CFM/UiO-66(12)/PDMS membrane was mechanically durable against strong mechanical abrasion, which was still capable of separating diverse water-in-oil emulsions after the abrasion with sandpaper and assembled as double-layer separation membranes. We anticipate that the combination of CFM and metal organic frameworks (MOFs) is an effective strategy for fabricating high-performance separation membrane with high mechanical and chemical durability.Graphical

Parameter dependencies of the experimental nitrogen concentration required for detachment on ASDEX Upgrade and JET

• An experimental scaling law to predict the threshold nitrogen concentration required for detachment is presented based on spectroscopic measurements of N II emission. • A least squares regression show that the threshold nitrogen concentration required for detachment depends strongly on the midplane separatrix density, with an exponent of −2.7 on AUG and −2.4 on JET, and approximately linearly with the power crossing the separatrix. • These two parameter dependencies are consistent with measurements on both ASDEX Upgrade and JET. • Equivalent predictions based on the well-known Lengyel model over-predict the absolute measured nitrogen concentration and show a weaker dependence on the separatrix density. While current tokamak experiments are beginning to use real-time feedback control systems to manage the plasma exhaust, future tokamaks still require validation of theoretical models used to predict the threshold impurity concentration required to sufficiently reduce the power and particle fluxes to the divertor. This work exploits new spectroscopic measurements of the divertor nitrogen concentration, c N , in partially detached N 2 -seeded H-mode plasmas on ASDEX Upgrade (AUG) and JET with the ITER-Like Wall (JET-ILW) to test the parameter dependencies of the power flowing to the outer divertor, P div , outer , and the separatrix electron density, n e , sep . A least-squares regression of the AUG measurements demonstrates that the threshold c N required for detachment scales as c N ∝ P div , outer 1.19 ± 0.32 n e , sep - 2.77 ± 0.36 . This scaling of n e , sep is also consistent with the measurements from JET which, at constant P div , outer , show c N ∝ n e , sep - 2.43 ± 0.27 . The dependencies of P div , outer and n e , sep is demonstrated over at least a factor of two change in both parameters and indicates a stronger dependence on n e , sep in comparison to the Lengyel model, which could be due to the assumption in this model that the heat flux channel width is independent of density. This first assessment of detachment scaling with impurity seeding highlights the need for further analysis of the systematic uncertainties of the measurement and more consistent scenarios from more tokamaks to investigate the machine size scaling.

Leaf Age-Dependent Effects of Boron Toxicity in Two Cucumis melo Varieties

Boron (B) is an essential nutrient for plant growth and development, exhibiting extremely narrow margins between deficiency and toxicity. B toxicity is devastating for productivity and apparent for a continuously increasing part of agricultural land, under the influence of on-going climate change. In this study, the effects of increased B supply (by using H3BO3) were addressed by examining critical physiological responses of young and mature leaves, which were devoid of toxicity symptoms, in two melon varieties (Armenian cucumbers, cantaloupes). B was primarily translocated through the transpiration stream, and secondarily via the active cell membrane transport system. The B distribution pattern was independent of leaf age, and remained rather unchanged under increased B supply. Armenian cucumbers, exhibiting higher leaf B levels, underwent an enhanced adverse impact on (root and shoot) growth, photosynthetic pigment content, cellular membrane integrity, and also exhibited attenuated antioxidant defense stimulation. Notably, and unlike other abiotic stressors, no evidence of B toxicity-induced systemic reaction was apparent. B toxicity greatly enhanced the transcription of the genes coding for borate influx and efflux channels, an effect that was mostly evident in mature leaves. In conclusion, shoot physiological responses to B toxicity are highly localized. Moreover, the obstruction of the diffusion and the B translocation to the aerial organs under increased B supply is genotype-dependent, governing plant physiological responses.

O-GlcNAcylation homeostasis controlled by calcium influx channels regulates multiple myeloma dissemination

BackgroundMultiple myeloma (MM) cell motility is a critical step during MM dissemination throughout the body, but how it is regulated remains largely unknown. As hypercalcemia is an important clinical feature of MM, high calcium (Ca2+) and altered Ca2+ signaling could be a key contributing factor to the pathological process.MethodsBioinformatics analyses were employed to assess the clinical significance of Ca2+ influx channels in clinical specimens of smoldering and symptomatic MM. Functional and regulatory roles of influx channels and downstream signaling in MM cell migration and invasion were conducted and experimental MM dissemination was examined in a xenograft mouse model using in vivo live imaging and engraftment analysis.ResultsInhibition of TRPM7, ORAI1, and STIM1 influx channels, which are highly expressed in MM patients, and subsequent blockage of Ca2+ influx by CRISPR/Cas9 and small molecule inhibitors, effectively inhibit MM cell migration and invasion, and attenuate the experimental MM dissemination. Mechanistic studies reveal a nutrient sensor O-GlcNAcylation as a downstream regulator of Ca2+ influx that specifically targets cell adhesion molecules. Hyper-O-GlcNAcylation following the inhibition of Ca2+ influx channels induces integrin α4 and integrin β7 downregulation via ubiquitin-proteasomal degradation and represses the aggressive MM phenotype.ConclusionsOur findings unveil a novel regulatory mechanism of MM cell motility via Ca2+ influx/O-GlcNAcylation axis that directly targets integrin α4 and integrin β7, providing mechanistic insights into the pathogenesis and progression of MM and demonstrating potential predictive biomarkers and therapeutic targets for advanced MM.

Cross diagnostics measurements of heat load profiles on the lower tungsten divertor of WEST in L-mode experiments

WEST is a full metallic tokamak with an extensive set of diagnostics for heat load measurements. In this paper, heat loads on the lower divertor of WEST are investigated using two independent methods. A first method relies on the thermal inversion of temperature measurements from arrays of thermal sensors embedded a few millimeters below the surface, while the second consists in the inversion of black body surface temperatures measured by infra-red (IR) thermography. The challenge of IR based temperature measurements in the full metal environment of WEST is addressed through a simplified model, allowing to correct for global reflections and low surface emissivities of tungsten surfaces. A large database (>100 L-mode discharges) is investigated. It is found that the energy absorbed by an outer divertor tile during a plasma discharge is closely estimated by the two diagnostics, over a large set of experimental conditions. A similar match is also found for the peak heat flux value on the outer target. The toroidal modulation of target heat loads by magnetic ripple is found to be consistent with the geometrical projection of a parallel heat flux component. Additionally, the heat flux channel width at the target is found to scale linearly with the magnetic flux expansion as expected. These observations give confidence in the robustness of the data from both diagnostics, and confirm the simple geometrical rules at use in the description of heat flux deposition on divertor targets. However, it is shown that the heat flux channel width estimated from infra-red thermography is about three times lower than the width estimated from embedded measurements, which is still under investigation.

Adaptive laboratory evolution of the fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for improved solvent tolerance

Cyanobacteria hold promise as cell factories for the photoautotrophic conversion of carbon dioxide to useful chemicals. For the eventual commercial viability of such processes, cyanobacteria need to be engineered for (i) efficient channeling of carbon flux toward the product of interest and (ii) improved product tolerance, the latter being the focus of this study. We chose the recently reported, fast-growing, high light and CO2 tolerant cyanobacterium Synechococcus elongatus PCC 11801 for adaptive laboratory evolution. In two parallel experiments that lasted over 8400h of culturing and 100 serial passages, S.elongatus PCC 11801 was evolved to tolerate 5g/L n-butanol or 30g/L 2,3-butanediol representing a 100% improvement in concentrations tolerated. The evolved strains retained alcohol tolerance even after being passaged several times without the alcohol stress suggesting that the changes were permanent. Whole genome sequencing of the n-butanol evolved strains revealed mutations in a number of stress responsive genes encoding translation initiation factors, RpoB and an ABC transporter. In 2,3-butanediol evolved strains, genes for ClpC, a different ABC transporter, glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase were found to be mutated. Furthermore, the evolved strains showed significant improvement in tolerance toward several other alcohols. Notably, the n-butanol evolved strain could tolerate up to 32g/L ethanol, thereby making it a promising host for photosynthetic production of biofuels via metabolic engineering.

Comparative assessment of the effects of 3D printed feed spacers on process performance in MD systems

In this study, process enhancements achieved by the use of 3D printed feed spacers based on triply periodic minimal surfaces (TPMS) were comparatively assessed using the two most common membrane distillation (MD) configurations: air gap (AGMD) and direct contact (DCMD). The MD performance was assessed based on the impact of spacer design (TPMS vs. commercial spacer) on flux and feed channel pressure drop, and their consequent impact on the levelized cost of water (COW). The studied spacer architectures led to a minimal improvement (≤17%) in AGMD flux, much lower than that achieved in DCMD (≤57%). Consequently, for a waste heat-operated MD process, the spacer-induced channel pressure drop became the most influential cost bottleneck on COW. Generally, the contribution of the pumping cost to the total operating cost was found to be greater for DCMD than AGMD. Thus, the COW of a waste heat operated DCMD is more sensitive to a decrease in spacer-induced channel pressure drop than in an AGMD. For an MD process operated with additional heat cost, the flux improvement achieved using TPMS spacers reduces the thermal energy component of the operating cost. Ultimately, this predominantly contributes to a lower COW.

Annexin 1 Is a Component of eATP-Induced Cytosolic Calcium Elevation in Arabidopsis thaliana Roots.

Extracellular ATP (eATP) has long been established in animals as an important signalling molecule but this is less understood in plants. The identification of Arabidopsis thaliana DORN1 (Does Not Respond to Nucleotides) as the first plant eATP receptor has shown that it is fundamental to the elevation of cytosolic free Ca2+ ([Ca2+]cyt) as a possible second messenger. eATP causes other downstream responses such as increase in reactive oxygen species (ROS) and nitric oxide, plus changes in gene expression. The plasma membrane Ca2+ influx channels involved in eATP-induced [Ca2+]cyt increase remain unknown at the genetic level. Arabidopsis thaliana Annexin 1 has been found to mediate ROS-activated Ca2+ influx in root epidermis, consistent with its operating as a transport pathway. In this study, the loss of function Annexin 1 mutant was found to have impaired [Ca2+]cyt elevation in roots in response to eATP or eADP. Additionally, this annexin was implicated in modulating eATP-induced intracellular ROS accumulation in roots as well as expression of eATP-responsive genes.

SOLPS-ITER drift modelling of ITER burning plasmas with narrow near-SOL heat flux channels

Abstract SOLPS-ITER code simulations with fluid drifts activated are used to examine the consequences for divertor performance, under burning plasma conditions, of a reduction in scrape-off layer (SOL) cross-field transport such that the outboard midplane SOL parallel heat flux width, λq is reduced to the very low values predicted by the experimental scaling in [T. Eich et al., Nucl. Fusion 53 (2013) 093031] for high plasma current operation on ITER. The decrease of λq from the standard value (~3.5 mm) used in the ITER SOLPS modeling database, to λq ~ 1–2 mm expected from the scaling, leads to a strong narrowing of the operational window in terms of divertor heat loading limits. To maintain similar levels of sub-divertor neutral pressure, pn (one of the key divertor operational parameters) as those obtained with standard transport, higher levels of neon impurity seeding are required when transport is reduced, yielding higher Zeff at the upstream separatrix and requiring further integrated modelling to assess the impact on confinement. The simulations also demonstrate that the strong increase in in-out target peak power loading asymmetry at low pn seen for standard transport when drifts are switched on in the code is preserved, and in fact worsens, for low transport. This significantly reduces margins for power handling control at lower pn if heat flux channels will be narrow on ITER.

Silicon flow from root to shoot in pepper: a comprehensive in silico analysis reveals a potential linkage between gene expression and hormone signaling that stimulates plant growth and metabolism.

BackgroundSilicon (Si) is categorized as a quasi-essential element for plants thanks to the benefits on growth, development and metabolism in a hormetic manner. Si uptake is cooperatively mediated by Lsi1 and Lsi2. Nevertheless, Lsi channels have not yet been identified and characterized in pepper (Capsicum annuum), while genes involved in major physiological processes in pepper are Si-regulated. Furthermore, Si and phytohormones may act together in regulating plant growth, metabolism and tolerance against stress. Our aim was to identify potential synergies between Si and phytohormones stimulating growth and metabolism in pepper, based on in silico data.MethodsWe established a hydroponic system to test the effect of Si (0, 60, 125 and 250 mg L−1 Si) on the concentrations of this element in different pepper plant tissues. We also performed an in silico analysis of putative Lsi genes from pepper and other species, including tomato (Solanum lycopersicum), potato (Solanum tuberosum) and Arabidopsis thaliana, to look for cis-acting elements responsive to phytohormones in their promoter regions. With the Lsi1 and Lsi2 protein sequences from various plant species, we performed a phylogenetic analysis. Taking into consideration the Lsi genes retrieved from tomato, potato and Arabidopsis, an expression profiling analysis in different plant tissues was carried out. Expression of Si-regulated genes was also analyzed in response to phytohormones and different plant tissues and developmental stages in Arabidopsis.ResultsSi concentrations in plant tissues exhibited the following gradient: roots > stems > leaves. We were able to identify 16 Lsi1 and three Lsi2 genes in silico in the pepper genome, while putative Lsi homologs were also found in other plant species. They were mainly expressed in root tissues in the genomes analyzed. Both Lsi and Si-regulated genes displayed cis-acting elements responsive to diverse phytohormones. In Arabidopsis, Si-regulated genes were transcriptionally active in most tissues analyzed, though at different expressed levels. From the set of Si-responsive genes, the NOCS2 gene was highly expressed in germinated seeds, whereas RABH1B, and RBCS-1A, were moderately expressed in developed flowers. All genes analyzed showed responsiveness to phytohormones and phytohormone precursors.ConclusionPepper root cells are capable of absorbing Si, but small amounts of this element are transported to the upper parts of the plant. We could identify putative Si influx (Lsi1) and efflux (Lsi2) channels that potentially participate in the absorption and transport of Si, since they are mainly expressed in roots. Both Lsi and Si-regulated genes exhibit cis-regulatory elements in their promoter regions, which are involved in phytohormone responses, pointing to a potential connection among Si, phytohormones, plant growth, and other vital physiological processes triggered by Si in pepper.

The analysis of RBMK neutron flux profile distortions using operational data history

In this research the operational data history was analysed. The data set given represents RBMK-1000 parameters for one month of the reactor operation. Axial flux sensors data and channel power data taken from database were considered. The transients of axial and radial-azimuthal neutron flux distributions were visualized. For the flux distribution data from 72 core channels with axial flux sensors the axial offset transients were obtained. In the operational data considered xenon-induced power oscillations were revealed. Phase and amplitude transients of the oscillations were calculated. The dynamics of phase and amplitude were visualized over the reactor core cross-sections. The values of quantities were displayed with different color shades. The iodine-135 and xenon-135 concentrations recovery problem solution was considered. Formulas for concentrations recovery were derived. The recovery procedure obtained is based on Cauchy problem solution calculation process. The dependence of accuracy of the recovered transients on the amount of the available operational data was studied. The dependency graph of calculation error on the amount of operational data was plot. The recovery of xenon-135 concentration was carried out based on the operational data history of the power unit where the phenomenon of xenon-induced power oscillations was observed.

Arsenic-induced differential expression of oxidative stress and secondary metabolite content in two genotypes of Andrographis paniculata

The present study explores the differential responses of two genotypes (APwC: wild collection and APMS: mass selection line) of A. paniculata against the three application rates of arsenic (42, 126, and 200 mg kg−1). The oxidative enzymes, As accumulation in different tissues, plant growth, and content of pharmacologically important ent-labdane-related diterpenes (ent-LRDs) of the two genotypes were evaluated in the study. Results demonstrated that As uptake significantly reduced plant biomass in APwC and APMS by 5–41.5% and 9–33% in a dose-response manner, respectively. The APMS exhibited lower bioconcentration and translocation factors, higher As tolerance index, and higher content of ent-LRDs as compared to APWC. As treatment induced a decrease in the sum of four metabolite content of APMS (1.43 times) and an increase in that of APWC (1.12 times) as compared to control. Likewise, variance in the production of 5,7,2′,3′-tetramethoxyflavanone, and stress enzymes was also observed between APwC and APMS. The increase in the expression of ApCPS2 suggested its involvement in channeling of metabolic flux towards the biosynthesis of ent-LRDs under As stress.

Peripheral Pain Modulation of Chrysaora pacifica Jellyfish Venom Requires Both Ca2+ Influx and TRPA1 Channel Activation in Rats.

The venom of jellyfish triggers severe dermal pain along with inflammation and tissue necrosis, and occasionally, induces internal organ dysfunction. However, the basic mechanisms underlying its cytotoxic effects are still unknown. Here, we report one of the mechanisms involved in peripheral pain modulation associated with inflammatory and neurotoxic oxidative signaling in rats using the venom of jellyfish, Chrysaora pacifica (CpV). This jellyfish is identified by brown tentacles carrying nematocysts filled with cytotoxic venom that induces severe pain, pruritus, tentacle marks, and blisters. The subcutaneous injection of CpV into rat forepaws in behavioral tests triggered nociceptive response with a decreased threshold for mechanical pain perception. These responses lasted up to 48h and were completely blocked by verapamil and TTA-P2, T-type Ca2+ channel blockers, or HC030031, a transient receptor potential cation ankyrin 1 (TRPA1) channel blocker, while another Ca2+ channel blocker, nimodipine, was ineffective. Also, treatment with Ca2+ chelators (EGTA and BaptaAM) significantly alleviated the CpV-induced pain response. These results indicate that CpV-induced pain modulation may require both Ca2+ influx through the T-type Ca2+ channels and activation of TRPA1 channels. Furthermore, CpV induced Ca2+-mediated oxidative neurotoxicity in the dorsal root ganglion (DRG) and cortical neurons dissociated from rats, resulting in decreased neuronal viability and increased intracellular levels of ROS. Taken together, CpV may activate Ca2+-mediated oxidative signaling to produce excessive ROS acting as an endogenous agonist of TRPA1 channels in the peripheral terminals of the primary afferent neurons, resulting in persistent inflammatory pain. These findings provide strong evidence supporting the therapeutic effectiveness of blocking oxidative signaling against pain and cytotoxicity induced by jellyfish venom.

Valproic Acid Synergizes With Cisplatin and Cetuximab in vitro and in vivo in Head and Neck Cancer by Targeting the Mechanisms of Resistance.

Recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) is a devastating malignancy with a poor prognosis. The combination of cisplatin (CDDP) plus cetuximab (CX) is one of the standard first-line treatments in this disease. However, this therapeutic regimen is often associated with high toxicity and resistance, suggesting that new combinatorial strategies are needed to improve its therapeutic index. In our study, we evaluated the antitumor effects of valproic acid (VPA), a well-known antiepileptic agent with histone deacetylase inhibitory activity, in combination with CDDP/CX doublet in head and neck squamous cell carcinoma (HNSCC) models. We demonstrated, in HNSCC cell lines, but not in normal human fibroblasts, that simultaneous exposure to equitoxic doses of VPA plus CDDP/CX resulted in a clear synergistic antiproliferative and pro-apoptotic effects. The synergistic antitumor effect was confirmed in four different 3D-self-assembled spheroid models, suggesting the ability of the combined approach to affect also the cancer stem cells compartment. Mechanistically, VPA enhanced DNA damage in combination treatment by reducing the mRNA expression of ERCC Excision Repair 1, a critical player in DNA repair, and by increasing CDDP intracellular concentration via upregulation at transcriptional level of CDDP influx channel copper transporter 1 and downregulation of the ATPAse ATP7B involved in CDDP-export. Valproic acid also induced a dose-dependent downregulation of epidermal growth factor receptor (EGFR) expression and of MAPK and AKT downstream signaling pathways and prevent CDDP- and/or CX-induced EGFR nuclear translocation, a well-known mechanism of resistance to chemotherapy. Indeed, VPA impaired the transcription of genes induced by non-canonical activity of nuclear EGFR, such as cyclin D1 and thymidylate synthase. Finally, we confirmed the synergistic antitumor effect also in vivo in both heterotopic and orthotopic models, demonstrating that the combined treatment completely blocked HNSCC xenograft tumors growth in nude mice. Overall, the introduction of a safe and generic drug such as VPA into the conventional treatment for R/M HNSCC represents an innovative and feasible antitumor strategy that warrants further clinical evaluation. A phase II clinical trial exploring the combination of VPA and CDDP/CX in R/M HNSCC patients is currently ongoing in our institute.

Abstract 5223: Valproic acid, by preventing cisplatin/cetuximab-induced EGFR nuclear translocation and increasing cisplatin uptake, potentiates the antitumor effect of the combination treatment in head and neck squamous cell carcinomas

Abstract INTRODUCTION: Recurrent metastatic (R/M) squamous cell carcinoma of the head and neck (HNSCC) is a devastating malignancy with a poor prognosis and the combination of cisplatin (CDDP) plus cetuximab (CX) is one of the gold standard for first-line treatment. However, this therapy is often associated with toxicity and resistance, suggesting that new combinatorial strategies are needed to improve the therapeutic index of this regimen. In our study, we evaluated the synergistic antitumor effect of valproic acid (VPA), an anticonvulsant compound with histone deacetylase inhibitor (HDACi) activity, in combination with CDDP/CX in HNSCC models in vitro and in vivo. METHODS: Synergistic anti-proliferative effects were assessed on HNSCC Cal27, FaDu and Cal33 cell lines and BJ-hTERT normal fibroblast, by calculating combination index (CI) accordingly to Chou and Talalay method. Apoptosis was measured by flow cytometry analysis and caspase assay. Tumor spheroids were obtained by low attach systems and scored with luminescence 3D-cell viability assay. In vivo experiment was performed on xenograft models in athymic mice. RESULTS: We demonstrated, in HNSCC cells, but not in normal human fibroblasts, that simultaneous exposure to equitoxic doses of VPA plus CDDP/CX results in a clear synergistic antiproliferative and proapoptotic effect. Interestingly, in order to better recapitulate tumor growth complexity compared to 2D monolayers conditions, the synergistic antitumor effect was confirmed in four different 3D-self-assembled spheroid models, as well as in in vivo Cal27 xenograft model. Mechanistically, VPA induced a dose-dependent down-regulation of EGFR expression/activation affecting its downstream canonical pathway (pAKT and pMAPK), which plays a driver role in HNSCC. Moreover, we demonstrated that VPA was able to prevent the CDDP and/or CX induced EGFR nuclear translocation, a well-known mechanism of resistance to chemotherapy. Indeed, VPA impaired the transcription of cyclin D1 and DNA repair genes, regulated by non-canonical activity of nuclear EGFR, thus increasing DNA damage induced by CDDP/CX combination. Moreover, VPA was able to enhance the sensitivity to CDDP, by upregulating, at transcriptional level, the CDDP influx channel copper transporter 1 (CTR1). CONCLUSIONS: The introduction of a safe and generic drug such as VPA into the conventional treatment for R/M HNSCC, represents an innovative and feasible antitumor strategy that warrants further clinical evaluation. Indeed, we are currently enrolling patients in a phase-2 clinical trial in order to explore whether the addition of VPA to the standard combination CDDP/CX can increase the response rate in patients with R/M HNSCC. Citation Format: Federica Iannelli, Andrea Ilaria Zotti, Maria Serena Roca, Laura Grumetti, Tania Moccia, Carlo Vitagliano, Chiara Ciardiello, Francesca Bruzzese, Alessandra Leone, Francesco Caponigro, Franco Ionna, Francesco Longo, Elena Di Gennaro, Alfredo Budillon. Valproic acid, by preventing cisplatin/cetuximab-induced EGFR nuclear translocation and increasing cisplatin uptake, potentiates the antitumor effect of the combination treatment in head and neck squamous cell carcinomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5223.

Mitochondrial Ca2+ regulation in the etiology of heart failure: physiological and pathophysiological implications.

Heart failure (HF) represents one of the leading causes of cardiovascular diseases with high rates of hospitalization, morbidity and mortality worldwide. Ample evidence has consolidated a crucial role for mitochondrial injury in the progression of HF. It is well established that mitochondrial Ca2+ participates in the regulation of a wide variety of biological processes, including oxidative phosphorylation, ATP synthesis, reactive oxygen species (ROS) generation, mitochondrial dynamics and mitophagy. Nonetheless, mitochondrial Ca2+ overload stimulates mitochondrial permeability transition pore (mPTP) opening and mitochondrial swelling, resulting in mitochondrial injury, apoptosis, cardiac remodeling, and ultimately development of HF. Moreover, mitochondria possess a series of Ca2+ transport influx and efflux channels, to buffer Ca2+ in the cytoplasm. Interaction at mitochondria-associated endoplasmic reticulum membranes (MAMs) may also participate in the regulation of mitochondrial Ca2+ homeostasis and plays an essential role in the progression of HF. Here, we provide an overview of regulation of mitochondrial Ca2+ homeostasis in maintenance of cardiac function, in an effort to identify novel therapeutic strategies for the management of HF.

A novel TRPM7/O-GlcNAc axis mediates tumour cell motility and metastasis by stabilising c-Myc and caveolin-1 in lung carcinoma

BackgroundCalcium is an essential signal transduction element that has been associated with aggressive behaviours in several cancers. Cell motility is a prerequisite for metastasis, the major cause of lung cancer death, yet its association with calcium signalling and underlying regulatory axis remains an unexplored area.MethodsBioinformatics database analyses were employed to assess correlations between calcium influx channels and clinical outcomes in non-small cell lung cancer (NSCLC). Functional and regulatory roles of influx channels in cell migration and invasion were conducted and experimental lung metastasis was examined using in vivo live imaging.ResultsHigh expression of TRPM7 channel correlates well with the low survival rate of patients and high metastatic potential. Inhibition of TRPM7 suppresses cell motility in various NSCLC cell lines and patient-derived primary cells and attenuates experimental lung metastases. Mechanistically, TRPM7 acts upstream of O-GlcNAcylation, a post-translational modification and a crucial sensor for metabolic changes. We reveal for the first time that caveolin-1 and c-Myc are favourable molecular targets of TRPM7/O-GlcNAc that regulates NSCLC motility. O-GlcNAcylation of caveolin-1 and c-Myc promotes protein stability by interfering with their ubiquitination and proteasomal degradation.ConclusionsTRPM7/O-GlcNAc axis represents a potential novel target for lung cancer therapy that may overcome metastasis.