Molecular and pharmacological modulation of CALHM1 promote neuroprotection against oxygen and glucose deprivation in a model of hippocampal slices

Calcium homeostasis modulator 1 (CALHM1) is a calcium channel involved in the regulation of cytosolic Ca2+ levels. From a physiological point of view, the open state of CALHM1 depends not only on voltage but also on the extracellular concentration of calcium ([Ca2+]) ions. At low [Ca2+]e or depolarization, the channel is opened, allowing Ca2+ influx; however, high extracellular [Ca2+]e or hyperpolarization promote its resting state. The unique Ca2+ permeation of CALHM1 relates to the molecular events that take place in brain ischemia, such as depolarization and extracellular changes in [Ca2+]e, particularly during the reperfusion phase after the ischemic insult. In this study, we attempted to understand its role in an in vitro model of ischemia, namely oxygen and glucose deprivation, followed by reoxygenation (OGD/Reox). To this end, hippocampal slices from wild-type Calhm1+/+, Calhm1+/-, and Calhm1-/- mice were subjected to OGD/Reox. Our results point out to a neuroprotective effect when CALHM1 is partially or totally absent. Pharmacological manipulation of CALHM1 with CGP37157 reduced cell death in Calhm1+/+ slices but not in that of Calhm1-/- mice after exposure to the OGD/Reox protocol. This ionic protection was also verified by measuring reactive oxygen species production upon OGD/Reox in Calhm1+/+ and Calhm1-/- mice, resulting in a downregulation of ROS production in Calhm1-/- hippocampal slices and increased expression of HIF-1α. Taken together, we can conclude that genetic or pharmacological inhibition of CALHM1 results in a neuroprotective effect against ischemia, due to an attenuation of the neuronal calcium overload and downregulation of oxygen reactive species productionThis research was funded by the Spanish Ministry of Science, Innovation and Universities Ref RTI2018-095793-B-I00 and Comunidad Autónoma de Madrid Ref. B2017/BMD-3827 to MGL. ETN PURINESDX Research and Innovation Agreement Nº 766124. Program under the Marie Sklodowska-Curie and Proyectos Santander-Universidad Autónoma de Madrid 2017, to MFC

Mitochondrial pH Monitored by a New Engineered Green Fluorescent Protein Mutant

We here describe a new molecularly engineered green fluorescent protein chimera that shows a high sensitivity to pH in the alkaline range. This probe was named mtAlpHi, for mitochondrial alkaline pH indicator, and possesses several key properties that render it optimal for studying the dynamics of mitochondrial matrix pH, e.g. it has an apparent pK(a) (pK(a)') around 8.5, it shows reversible and large changes in fluorescence in response to changes in pH (both in vitro and in intact cells), and it is selectively targeted to the mitochondrial matrix. Using mtAlpHi we could monitor pH changes that occur in the mitochondrial matrix in a variety of situations, e.g. treatment with uncouplers or Ca(2+) ionophores, addition of drugs that interfere with ATP synthesis or electron flow in the respiratory chain, weak bases or acids, and receptor activation. We observed heterogeneous pH increases in the mitochondrial matrix during Ca(2+) accumulation by this organelle. Finally, we demonstrate that Ca(2+) mobilization from internal stores induced by ionomycin and A23187 cause a dramatic acidification of the mitochondrial matrix

Effect of the prefermentative addition of copigments on the polyphenolic composition of Tempranillo wines after malolactic fermentation

[EN] The influence of the prefermentative addition of copigments and different winemaking technologies on the polyphenolic composition of Tempranillo red wines after malolactic fermentation was studied. Six experiments dealing with the prefermentative addition of caffeic acid, rutin, (+) catechin, white grape skin tannin, white grape seed tannin and control wines were realised. Three different winemaking technologies (traditional vinification, prefermentative cold maceration at 6-8 A degrees C and cold soak at 0-2 A degrees C with dry ice) were studied. Prefermentative addition of copigments increases anthocyanin copigmentation reactions and produces wines with a greater colour, a higher anthocyanin concentration, a superior contribution of anthocyanins to the colour of the wine, a superior percentage of tannins polymerised with polysaccharides and less astringency. Cold prefermentative maceration increases the extraction of polyphenols, the anthocyanin copigmentation reactions and the polymerisation reactions between tannins and polysaccharides. The effectiveness of the combination of copigments and prefermentative maceration treatments was demonstrated by the increase of the concentration of the polyphenolic compounds.The experimental reporter here is a first part of a project financially supported by the Science and Technology Minister from Spanish Government ( AGL 2006-10723-C02-02) which the authors gratefully acknowledgeAlvarez Cano, MI.; Aleixandre Benavent, JL.; García Esparza, MJ.; Lizama Abad, V.; Aleixandre Tudo, J. (2009). Effect of the prefermentative addition of copigments on the polyphenolic composition of Tempranillo wines after malolactic fermentation. European Food Research and Technology. 228(4):501-510. https://doi.org/10.1007/s00217-008-0957-0S501510228

CALHM1 and its polymorphism P86L differentially control Ca²⁺ homeostasis, mitogen-activated protein kinase signaling, and cell vulnerability upon exposure to amyloid β

The mutated form of the Ca2+ channel CALHM1 (Ca2+ homeostasis modulator 1), P86L-CALHM1, has been correlated with early onset of Alzheimer’s disease (AD). P86L-CALHM1 increases production of amyloid beta (Ab) upon extracellular Ca2+ removal and its subsequent addback. The aim of this study was to investigate the effect of the overexpression of CALHM1 and P86L-CALHM, upon Ab treatment, on the following: (i) the intracellular Ca2+ signal pathway; (ii) cell survival proteins ERK1/2 and Ca2+/cAMP response element binding (CREB); and (iii) cell vulnerability after treatment with Ab. Using aequorins to measure the effect of nuclear Ca2+ concentrations ([Ca2+]n) and cytosolic Ca2+ concentrations ([Ca2+]c) on Ca2+ entry conditions, we observed that baseline [Ca2+]n was higher in CALHM1 and P86L-CALHM1 cells than in control cells. Moreover, exposure to Ab affected [Ca2+]c levels in HeLa cells overexpressing CALHM1 and P86L-CALHM1 compared with control cells. Treatment with Ab elicited a significant decrease in the cell survival proteins p-ERK and p-CREB, an increase in the activity of caspases 3 and 7, and more frequent cell death by inducing early apoptosis in P86L-CALHM1- overexpressing cells than in CALHM1 or control cells. These results suggest that in the presence of Ab, P86L-CALHM1 shifts the balance between neurodegeneration and neuronal survival toward the stimulation of pro-cytotoxic pathways, thus potentially contributing to its deleterious effects in AD.This work was partly supported by the following grants: Ministerio de Economía y Competitividad, FPU Program, Refs. AP2009/0343 (AJMO) and AP2010/1219 (IB). ARN: FIS PI10/01426. MGL: Ministerio de Economía y Competitividad, Ref. SAF2012-23332. MFCA: Consolidación de grupos de investigación UAM-CAM 1004040047. We also thank Fundación Teófilo Hernando, Madrid, Spain, for their continued suppor

Inflammation-related molecules in tears of patients with chronic ocular pain and dry eye disease

Producción CientíficaThe purpose of this study was to analyze inflammation- and pain-related molecules in tears of patients suffering from chronic ocular pain associated with dry eye (DE) and/or a previous corneal refractive surgery (RS). Based on history, symptomatology, and clinical signs, the subjects (n = 180, 51.0 ± 14.7 years, 118 females, 62 males) in this cross-sectional study were assigned to one of five groups: DE and chronic ocular pain after RS (P/DE-RS, n = 52); asymptomatic subjects, i.e., without DE and chronic ocular pain, after RS (A-RS, n = 30); DE and chronic ocular pain without previous RS (P/DE-nonRS, n = 31); DE, no pain, and no previous RS (DE-nonRS, n = 35); and asymptomatic subjects with no previous RS (controls, n = 32). The tear concentrations of 20 cytokines and substance P (SP) were analyzed by immunobead-based assay and enzyme-linked immunosorbent assay, respectively. We found that tear levels of interleukin (IL)-10 and SP were increased in the RS groups. There were significant differences in IL-8/CXCL8 among the five groups. Nerve growth factor (NGF) tear levels were significantly higher in P/DE-RS than in DE-nonRS and controls. IL-9 had the highest percentage of detection in the P/DE-RS and P/DE-nonRS groups, while macrophage inflammatory protein (MIP)-1α, IL-2, and interferon (IFN)-γ were higher in the P/DE-RS, A-RS, and P/DE-nonRS groups. IL-17A was detected only in the A-RS group. Moderate correlations were observed in the A-RS, P/DE-nonRS, DE-nonRS and controls groups. A positive correlation was obtained between growth related oncogene concentration and tear break-up time (rho = 0.550; p = 0.012), while negative correlation was found between monocyte chemoattractant protein-3/CCL7 and conjunctival staining (rho = −0.560; p = 0.001), both in the A-RS group. IL-10 correlated positively with ocular pain intensity (rho = 0.513; p = 0.003) in the P/DE-nonRS group. Regulated on Activation Normal T Cell Expressed and Secreted/CCL5 correlated negatively with conjunctival staining (rho = −0.545; p = 0.001) in the DE-nonRS group. SP correlated negatively with corneal staining (rho = −0.559; p = 0.001) in the controls. In conclusion, chronic ocular pain was associated with higher IL-9 tear levels. IL-10, SP, MIP-1α/CCL3, IL-2, and IFN-γ were associated with previous RS. Higher levels of IL-8/CXCL8, MIP-1α/CCL3, IL-2, and IFN-γ were associated with DE-related inflammation, while NGF levels were related to chronic ocular pain and DE in RS patients. These findings suggest that improved knowledge of tear cytokines and neuromodulators will lead to a more nuanced understanding of how these molecules can serve as biomarkers of chronic ocular pain, leading to better therapeutic and disease management decisions.Ministerio de Ciencia, Innovación y Universidades (grants SAF-2016-77080-P, FPU17/02715 and FPU15/01443

Application of Multivariate Regression Methods to Predict Sensory Quality of Red Wines

Copyright - Czech Academy of Agricultural Sciences The original publication is available on http://agriculturejournals.cz/web/cjfs.htmSeveral multivariate methods including partial least squares (PLS) regression, principal component regression (PCR) or multiple linear regression (MLR) have been applied to predict wine quality, based on the definition of chemical and phenolic parameters of grapes and wines harvested at different ripening levels. Three different models including grape phenolic maturity parameters (grape), wine phenolic parameters (wine) and a combination of grape and wine phenolic parameters (grape + wine) were analysed for each of the wine sensory attributes. The grape parameter model has been presented as the best test to predict wine quality based on sensory scores. On the other hand, wine models showed lower accuracy. The combination of grape and wine parameters presented intermediate results showing sometimes good predictability. Moreover, PLS and PCR appeared as more accurate multivariate methods compared to MLR. Although MLR showed higher correlation coefficients, lower RPD values were observed, displaying thus its lower prediction accuracy. Multivariate calibration statistics appeared as a promising tool to predict wine sensory quality in an easy and inexpensive way.Aleixandre Tudo, J.; Alvarez Cano, MI.; García Esparza, MJ.; Lizama Abad, V.; Aleixandre Benavent, JL. (2015). Application of Multivariate Regression Methods to Predict Sensory Quality of Red Wines. Czech Journal of Food Sciences. 33(3):217-227. https://doi.org/10.17221/370/2014-CJFSS21722733

Volatile compounds and phenolic composition of skins and seeds of 'Cabernet Sauvignon' grapes under different deficit irrigation regimes

[EN] Aroma compounds and skin and seed pohphenols arc determinants of wine composition. The aim of this study was to determine the effect of different post-veraison deficit irrigation strategies on volatile profile and the chemical composition of grape skin and seeds in a 'Cabernet Sauvignon' vineyard in Valencia (Spain). Besides a non-irrigated regime (rainfed), irrigation treatments consisted of replacing 25, 50 and 75 % of the estimated crop evapotranspiration (ETC). When compared to rainfed vines, watering during post-veraison at 75 % of the ETC, decreased concentrations of alcohols but increased those of aldehydes such as hexanal, related to herbaceous (non-desirable) aromas in wines. Irrigating at 25 % or 50 % of ETC resulted in similar concentrations of grape volatile compounds than rainfed vines. There was also a general trend in a reduction in skin to flesh ratio as irrigation regime increased. The concentration of skin anthocyanins and tannins increased with water applications, but seed tannins decreased in the most irrigated regimes. This suggests different effects of water stress on skin and seed polyphenol synthesis and accumulation. For the tannin content, water stress provoked higher tannin mean degree polymerization values, which positively affect must astringency. Under the experimental conditions of the present study, watering at 50 % ETC during post-veraison is the recommended irrigation strategy for optimizing grape composition and improving yield in comparison with rainfed vines.This research was funded by the Spanish Ministry of Economy, Industry and Competitiveness MINECO (FEDER co-financing Projects TRACE 2009_00120 and AGL2014-54201-C4-4-R) and a grant agreement with Celler del Roure Winery.García Esparza, MJ.; Abrisqueta, I.; Escriche Roberto, MI.; Intrigliolo, D.; Alvarez Cano, MI.; Lizama Abad, V. (2018). Volatile compounds and phenolic composition of skins and seeds of 'Cabernet Sauvignon' grapes under different deficit irrigation regimes. VITIS. 57(3):83-91. https://doi.org/10.5073/vitis.2018.57.83-91S839157

The purinergic P2X7 receptor as a potential drug target to combat neuroinflammation in neurodegenerative diseases

Neurodegenerative diseases (NDDs) represent a huge social burden, particularly in Alzheimer's disease (AD) in which all proposed treatments investigated in murine models have failed during clinical trials (CTs). Thus, novel therapeutic strategies remain crucial. Neuroinflammation is a common pathogenic feature of NDDs. As purinergic P2X7 receptors (P2X7Rs) are gatekeepers of inflammation, they could be developed as drug targets for NDDs. Herein, we review this challenging hypothesis and comment on the numerous studies that have investigated P2X7Rs, emphasizing their molecular structure and functions, as well as their role in inflammation. Then, we elaborate on research undertaken in the field of medicinal chemistry to determine potential P2X7R antagonists. Subsequently, we review the state of neuroinflammation and P2X7R expression in the brain, in animal models and patients suffering from AD, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, and retinal degeneration. Next, we summarize the in vivo studies testing the hypothesis that by mitigating neuroinflammation, P2X7R blockers afford neuroprotection, increasing neuroplasticity and neuronal repair in animal models of NDDs. Finally, we reviewed previous and ongoing CTs investigating compounds directed toward targets associated with NDDs; we propose that CTs with P2X7R antagonists should be initiated. Despite the high expectations for putative P2X7Rs antagonists in various central nervous system diseases, the field is moving forward at a relatively slow pace, presumably due to the complexity of P2X7Rs. A better pharmacological approach to combat NDDs would be a dual strategy, combining P2X7R antagonism with drugs targeting a selective pathway in a given NDD.The authors would like to acknowledge the support received from the EU Horizon 2020 Research and Innovation Program under Maria Sklodowska‐Curie (Grant Agreement No. 766124). The authors would also like to thank the support received from the Ministerio de Economía y Competitividad (MINECO, Spain; Grant No. SAF2016‐78892R to Luis Gandía and Antonio G. García) and Fundación Teófilo Hernando

Obesity attenuates the effect of sleep apnea on active TGF-ss 1 levels and tumor aggressiveness in patients with melanoma

Active transforming growth factor-β1 (TGF-β1), a cytokine partially regulated by hypoxia and obesity, has been related with poor prognosis in several tumors. We determine whether obstructive sleep apnea (OSA) increases serum levels of active TGF-β1 in patients with cutaneous melanoma (CM), assess their relationship with melanoma aggressiveness and analyze the factors related to TGF-β1 levels in obese and non-obese OSA patients. In a multicenter observational study, 290 patients with CM were underwent sleep studies. TGF-β1 was increased in moderate-severe OSA patients vs. non-OSA or mild OSA patients with CM. In OSA patients, TGF-β1 levels correlated with mitotic index, Breslow index and melanoma growth rate, and were increased in presence of ulceration or higher Clark levels. In CM patients, OSA was associated with higher TGF-β1 levels and greater melanoma aggressiveness only in non-obese subjects. An in vitro model showed that IH-induced increases of TGF-β1 expression in melanoma cells is attenuated in the presence of high leptin levels. In conclusion, TGF-β1 levels are associated with melanoma aggressiveness in CM patients and increased in moderate-severe OSA. Moreover, in non-obese patients with OSA, TGF-β1 levels correlate with OSA severity and leptin levels, whereas only associate with leptin levels in obese OSA patients