The synthetic TRPML1 agonist ML-SA1 rescues Alzheimer-related alterations of the endosomal-autophagic-lysosomal system.

Abnormalities in the endosomal-autophagic-lysosomal (EAL) system are an early event in Alzheimer's disease (AD) pathogenesis. However, the mechanisms underlying these abnormalities are unclear. The transient receptor potential channel mucolipin 1(TRPML1, also known as MCOLN1), a vital endosomal-lysosomal Ca2+ channel whose loss of function leads to neurodegeneration, has not been investigated with respect to EAL pathogenesis in late-onset AD (LOAD). Here, we identify pathological hallmarks of TRPML1 dysregulation in LOAD neurons, including increased perinuclear clustering and vacuolation of endolysosomes. We reveal that induced pluripotent stem cell (iPSC)-derived human cortical neurons expressing APOE ε4, the strongest genetic risk factor for LOAD, have significantly diminished TRPML1-induced endolysosomal Ca2+ release. Furthermore, we found that blocking TRPML1 function in primary neurons by depleting the TRPML1 agonist PI(3,5)P2 via PIKfyve inhibition, recreated multiple features of EAL neuropathology evident in LOAD. This included increased endolysosomal Ca2+ content, enlargement and perinuclear clustering of endolysosomes, autophagic vesicle accumulation and early endosomal enlargement. Strikingly, these AD-like neuronal EAL defects were rescued by TRPML1 reactivation using its synthetic agonist ML-SA1. These findings implicate defects in TRPML1 in LOAD EAL pathogenesis and present TRPML1 as a potential therapeutic target.

Implication of Oxysterols in Infectious and Non-Communicable Inflammatory Diseases.

Oxysterols, derived from cholesterol oxidation, are formed either by autoxidation, via enzymes, or by both processes [...].

Impact of cancer cachexia on respiratory muscle function and the therapeutic potential of exercise.

Cancer cachexia is defined as a multi-factorial syndrome characterised by an ongoing loss of skeletal muscle mass and progressive functional impairment, estimated to affect 50-80% of patients and responsible for 20% of cancer deaths. Elevations in the morbidity and mortality rates of cachectic cancer patients has been linked to respiratory failure due to atrophy and dysfunction of the ventilatory muscles. Despite this, there is a distinct scarcity of research investigating the structural and functional condition of the respiratory musculature in cancer, with the majority of studies exclusively focusing on limb muscle. Treatment strategies are largely ineffective in mitigating the cachectic state. It is now widely accepted that an efficacious intervention will likely combine elements of pharmacology, nutrition and exercise. However, of these approaches, exercise has received comparatively little attention. Therefore, it is unlikely to be implemented optimally, whether in isolation or combination. In consideration of these limitations, the current review describes the mechanistic basis of cancer cachexia and subsequently explores the available respiratory- and exercise-focused literature within this context. The molecular basis of cachexia is thoroughly reviewed. The pivotal role of inflammatory mediators is described. Unravelling the mechanisms of exercise-induced support of muscle via antioxidant and anti-inflammatory effects in addition to promoting efficient energy metabolism via increased mitochondrial biogenesis, mitochondrial function and muscle glucose uptake provide avenues for interventional studies. Currently available pre-clinical mouse models including novel transgenic animals provide a platform for the development of multi-modal therapeutic strategies to protect respiratory muscles in people with cancer.

Compliance of the fish outflow tract is altered by thermal acclimation through connective tissue remodelling.

To protect the gill capillaries from high systolic pulse pressure, the fish heart contains a compliant non-contractile chamber called the bulbus arteriosus which is part of the outflow tract (OFT) which extends from the ventricle to the ventral aorta. Thermal acclimation alters the form and function of the fish atria and ventricle to ensure appropriate cardiac output at different temperatures, but its impact on the OFT is unknown. Here we used ex vivo pressure-volume curves to demonstrate remodelling of passive stiffness in the rainbow trout (Oncorhynchus mykiss) bulbus arteriosus following more than eight weeks of thermal acclimation to 5, 10 and 18°C. We then combined novel, non-biased Fourier transform infrared spectroscopy with classic histological staining to show that changes in compliance were achieved by changes in tissue collagen-to-elastin ratio. In situ gelatin zymography and SDS-PAGE zymography revealed that collagen remodelling was underpinned, at least in part, by changes in activity and abundance of collagen degrading matrix metalloproteinases. Collectively, we provide the first indication of bulbus arteriosus thermal remodelling in a fish and suggest this remodelling ensures optimal blood flow and blood pressure in the OFT during temperature change.

Polyphenols of the Mediterranean Diet and Their Metabolites in the Prevention of Colorectal Cancer.

The Mediterranean diet is a central element of a healthy lifestyle, where polyphenols play a key role due to their anti-oxidant properties, and for some of them, as nutripharmacological compounds capable of preventing a number of diseases, including cancer. Due to the high prevalence of intestinal cancer (ranking second in causing morbidity and mortality), this review is focused on the beneficial effects of selected dietary phytophenols, largely present in Mediterranean cooking: apigenin, curcumin, epigallocatechin gallate, quercetin-rutine, and resveratrol. The role of the Mediterranean diet in the prevention of colorectal cancer and future perspectives are discussed in terms of food polyphenol content, the effectiveness, the plasma level, and the importance of other factors, such as the polyphenol metabolites and the influence of the microbiome. Perspectives are discussed in terms of microbiome-dependency of the brain-second brain axis. The emergence of polyphenol formulations may strengthen the efficiency of the Mediterranean diet in the prevention of cancer.

7-Ketocholesterol: Effects on viral infections and hypothetical contribution in COVID-19.

7-Ketocholesterol, which is one of the earliest cholesterol oxidization products identified, is essentially formed by the auto-oxidation of cholesterol. In the body, 7-ketocholesterol is both provided by food and produced endogenously. This pro-oxidant and pro-inflammatory molecule, which can activate apoptosis and autophagy at high concentrations, is an abundant component of oxidized Low Density Lipoproteins. 7-Ketocholesterol appears to significantly contribute to the development of age-related diseases (cardiovascular diseases, age-related macular degeneration, and Alzheimer's disease), chronic inflammatory bowel diseases and to certain cancers. Recent studies have also shown that 7-ketocholesterol has anti-viral activities, including on SARS-CoV-2, which are, however, lower than those of oxysterols resulting from the oxidation of cholesterol on the side chain. Furthermore, 7-ketocholesterol is increased in the serum of moderately and severely affected COVID-19 patients. In the case of COVID-19, it can be assumed that the antiviral activity of 7-ketocholesterol could be counterbalanced by its toxic effects, including pro-oxidant, pro-inflammatory and pro-coagulant activities that might promote the induction of cell death in alveolar cells. It is therefore suggested that this oxysterol might be involved in the pathophysiology of COVID-19 by contributing to the acute respiratory distress syndrome and promoting a deleterious, even fatal outcome. Thus, 7-ketocholesterol could possibly constitute a lipid biomarker of COVID-19 outcome and counteracting its toxic effects with adjuvant therapies might have beneficial effects in COVID-19 patients.

Alterations in the Ca2+ toolkit in oesophageal adenocarcinoma.

Aim: To investigate alterations in transcription of genes, encoding Ca2+ toolkit proteins, in oesophageal adenocarcinoma (OAC) and to assess associations between gene expression, tumor grade, nodal-metastatic stage, and patient survival. Methods: The expression of 275 transcripts, encoding components of the Ca2+ toolkit, was analyzed in two OAC datasets: the Cancer Genome Atlas [via the University of Alabama Cancer (UALCAN) portal] and the oesophageal-cancer, clinical, and molecular stratification [Oesophageal Cancer Clinical and Molecular Stratification (OCCAMS)] dataset. Effects of differential expression of these genes on patient survival were determined using Kaplan-Meier log-rank tests. OAC grade- and metastatic-stage status was investigated for a subset of genes. Adjustment for the multiplicity of testing was made throughout. Results: Of the 275 Ca2+-toolkit genes analyzed, 75 displayed consistent changes in expression between OAC and normal tissue in both datasets. The channel-encoding genes, N-methyl-D-aspartate receptor 2D (GRIN2D), transient receptor potential (TRP) ion channel classical or canonical 4 (TRPC4), and TRP ion channel melastatin 2 (TRPM2) demonstrated the greatest increase in expression in OAC in both datasets. Nine genes were consistently upregulated in both datasets and were also associated with improved survival outcomes. The 6 top-ranking genes for the weighted significance of altered expression and survival outcomes were selected for further analysis: voltage-gated Ca2+ channel subunit α 1D (CACNA1D), voltage-gated Ca2+ channel auxiliary subunit α2 δ4 (CACNA2D4), junctophilin 1 (JPH1), acid-sensing ion channel 4 (ACCN4), TRPM5, and secretory pathway Ca2+ ATPase 2 (ATP2C2). CACNA1D, JPH1, and ATP2C2 were also upregulated in advanced OAC tumor grades and nodal-metastatic stages in both datasets. Conclusions: This study has unveiled alterations of the Ca2+ toolkit in OAC, compared to normal tissue. Such Ca2+ signalling findings are consistent with those from studies on other cancers. Genes that were consistently upregulated in both datasets might represent useful markers for patient diagnosis. Genes that were consistently upregulated, and which were associated with improved survival, might be useful markers for patient outcome. These survival-associated genes may also represent targets for the development of novel chemotherapeutic agents.

Oxiapoptophagy: A type of cell death induced by some oxysterols.

Oxysterols are oxidized forms of cholesterol generated from cholesterol by auto-oxidation, enzymatic processes, or both. Some of them (7-ketocholesterol, 7ß-hydroxycholesterol and 24(S)-hydroxycholesterol), when used at cytotoxic concentrations on different cell types from different species (mesenchymal bone marrow cells, monocytic cells and nerve cells), induce a type of cell death associated with OXIdative stress and several characteristics of APOPTOsis and autoPHAGY, defined as oxiapoptophagy. Oxidative stress is associated with overproduction of ROS, increased antioxidant enzyme activities, lipid peroxidation and protein carbonylation. Apoptosis is associated with activation of the mitochondrial pathway, opening of the mitochondrial permeability pore, loss of mitochondrial membrane potential, caspase-3 activation, PARP degradation, nuclear condensation and/or fragmentation. Autophagy is characterized by autophagic vacuoles revealed by monodansylcadaverine staining and transmission electron microscopy, plus increased ratio of LC-3II/LC-3I. In addition, morphological, topographical and functional changes of the peroxisome are observed. LINKED ARTICLES: This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.

Prevention by Dietary Polyphenols (Resveratrol, Quercetin, Apigenin) Against 7-Ketocholesterol-Induced Oxiapoptophagy in Neuronal N2a Cells: Potential Interest for the Treatment of Neurodegenerative and Age-Related Diseases.

The Mediterranean diet is associated with health benefits due to bioactive compounds such as polyphenols. The biological activities of three polyphenols (quercetin (QCT), resveratrol (RSV), apigenin (API)) were evaluated in mouse neuronal N2a cells in the presence of 7-ketocholesterol (7KC), a major cholesterol oxidation product increased in patients with age-related diseases, including neurodegenerative disorders. In N2a cells, 7KC (50 µM; 48 h) induces cytotoxic effects characterized by an induction of cell death. When associated with RSV, QCT and API (3.125; 6.25 µM), 7KC-induced toxicity was reduced. The ability of QCT, RSV and API to prevent 7KC-induced oxidative stress was characterized by a decrease in reactive oxygen species (ROS) production in whole cells and at the mitochondrial level; by an attenuation of the increase in the level and activity of catalase; by attenuating the decrease in the expression, level and activity of glutathione peroxidase 1 (GPx1); by normalizing the expression, level and activity of superoxide dismutases 1 and 2 (SOD1, SOD2); and by reducing the decrease in the expression of nuclear erythroid 2-like factor 2 (Nrf2) which regulates antioxidant genes. QCT, RSV and API also prevented mitochondrial dysfunction in 7KC-treated cells by counteracting the loss of mitochondrial membrane potential (ΨΔm) and attenuating the decreased gene expression and/or protein level of AMP-activated protein kinase α (AMPKα), sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) implicated in mitochondrial biogenesis. At the peroxisomal level, QCT, RSV and API prevented the impact of 7KC by counteracting the decrease in ATP binding cassette subfamily D member (ABCD)3 (a peroxisomal mass marker) at the protein and mRNA levels, as well as the decreased expresssion of genes associated with peroxisomal biogenesis (Pex13, Pex14) and peroxisomal ß-oxidation (Abcd1, Acox1, Mfp2, Thiolase A). The 7KC-induced decrease in ABCD1 and multifunctional enzyme type 2 (MFP2), two proteins involved in peroxisomal ß-oxidation, was also attenuated by RSV, QCT and API. 7KC-induced cell death, which has characteristics of apoptosis (cells with fragmented and/or condensed nuclei; cleaved caspase-3; Poly(ADP-ribose) polymerase (PARP) fragmentation) and autophagy (cells with monodansyl cadaverine positive vacuoles; activation of microtubule associated protein 1 light chain 3-I (LC3-I) to LC3-II, was also strongly attenuated by RSV, QCT and API. Thus, in N2a cells, 7KC induces a mode of cell death by oxiapoptophagy, including criteria of OXIdative stress, APOPTOsis and autoPHAGY, associated with mitochondrial and peroxisomal dysfunction, which is counteracted by RSV, QCT, and API reinforcing the interest for these polyphenols in prevention of diseases associated with increased 7KC levels.

Inhibitory Properties of Aldehydes and Related Compounds against Phytophthora infestans-Identification of a New Lead.

The pathogen Phytophthora infestans is responsible for catastrophic crop damage on a global scale which totals billions of euros annually. The discovery of new inhibitors of this organism is of paramount agricultural importance and of critical relevance to food security. Current strategies for crop treatment are inadequate with the emergence of resistant strains and problematic toxicity. Natural products such as cinnamaldehyde have been reported to have fungicidal properties and are the seed for many new discovery research programmes. We report a probe of the cinnamaldehyde framework to investigate the aldehyde subunit and its role in a subset of aromatic aldehydes in order to identify new lead compounds to act against P. infestans. An ellipticine derivative which incorporates an aldehyde (9-formyl-6-methyl ellipticine, 34) has been identified with exceptional activity versus P. infestans with limited toxicity and potential for use as a fungicide.

Synthesis and Evaluation of Novel Ellipticines and Derivatives as Inhibitors of Phytophthora infestans.

The pathogen Phytophthora infestans is responsible for worldwide catastrophic crop damage and discovery of new inhibitors of this organism is of paramount agricultural and industrial importance. Current strategies for crop treatment are inadequate with limitations of efficacy and market alternatives. Ellipticines have recently been reported to have fungicidal properties and have been assessed against P. infestans growth with promising results. We hereby report a probe of the ellipticine framework to investigate the alkyl subunit and screen a set ellipticines and derivatives to identify new lead compounds to act against P. infestans. A series of ellipticinium salt derivatives have been identified with exceptional growth inhibitory activity and apparent lack of toxicity towards a human cell-line surpassing the effect of known and marketed fungicides. This report identifies the potential of this natural product derivative as a novel fungicide.

Polycystic Kidney Disease Ryanodine Receptor Domain (PKDRR) Proteins in Oomycetes.

In eukaryotes, two sources of Ca2+ are accessed to allow rapid changes in the cytosolic levels of this second messenger: the extracellular medium and intracellular Ca2+ stores, such as the endoplasmic reticulum. One class of channel that permits Ca2+ entry is the transient receptor potential (TRP) superfamily, including the polycystic kidney disease (PKD) proteins, or polycystins. Channels that release Ca2+ from intracellular stores include the inositol 1,4,5-trisphosphate/ryanodine receptor (ITPR/RyR) superfamily. Here, we characterise a family of proteins that are only encoded by oomycete genomes, that we have named PKDRR, since they share domains with both PKD and RyR channels. We provide evidence that these proteins belong to the TRP superfamily and are distinct from the ITPR/RyR superfamily in terms of their evolutionary relationships, protein domain architectures and predicted ion channel structures. We also demonstrate that a hypothetical PKDRR protein from Phytophthora infestans is produced by this organism, is located in the cell-surface membrane and forms multimeric protein complexes. Efforts to functionally characterise this protein in a heterologous expression system were unsuccessful but support a cell-surface localisation. These PKDRR proteins represent potential targets for the development of new "fungicides", since they are of a distinctive structure that is only found in oomycetes and not in any other cellular organisms.

7-Ketocholesterol and 7ß-hydroxycholesterol: In vitro and animal models used to characterize their activities and to identify molecules preventing their toxicity.

Oxysterols are molecules derived by the oxidation of cholesterol and can be formed either by auto-oxidation, enzymatically or by both processes. Among the oxysterols formed by auto-oxidation, 7-ketocholesterol and 7ß-hydroxycholesterol are the main forms generated. These oxysterols, formed endogenously and brought in large quantities by certain foods, have major cytotoxic properties. They are powerful inducers of oxidative stress, inducing dysfunction of organelles (mitochondria, lysosomes and peroxisomes) that can cause cell death. These molecules are often identified in increased amounts in common pathological states such as cardiovascular diseases, certain eye conditions, neurodegenerative disorders and inflammatory bowel diseases. To oppose the cytotoxic effects of these molecules, it is important to know their biological activities and the signaling pathways they affect. Numerous cell models of the vascular wall, eye, brain, and digestive tract have been used. Currently, to counter the cytotoxic effects of 7-ketocholesterol and 7ß-hydroxycholesterol, natural molecules and oils, often associated with the Mediterranean diet, as well as synthetic molecules, have proved effective in vitro. Bioremediation approaches and the use of functionalized nanoparticles are also promising. At the moment, invertebrate and vertebrate models are mainly used to evaluate the metabolism and the toxicity of 7-ketocholesterol and 7ß-hydroxycholesterol. The most frequently used models are mice, rats and rabbits. In order to cope with the difficulty of transferring the results obtained in animals to humans, the development of in vitro alternative methods such as organ/body-on-a-chip based on microfluidic technology are hopeful integrative approaches.

Processive Recoding and Metazoan Evolution of Selenoprotein P: Up to 132 UGAs in Molluscs.

Selenoproteins typically contain a single selenocysteine, the 21st amino acid, encoded by a context-redefined UGA. However, human selenoprotein P (SelenoP) has a redox-functioning selenocysteine in its N-terminal domain and nine selenium transporter-functioning selenocysteines in its C-terminal domain. Here we show that diverse SelenoP genes are present across metazoa with highly variable numbers of Sec-UGAs, ranging from a single UGA in certain insects, to 9 in common spider, and up to 132 in bivalve molluscs. SelenoP genes were shaped by a dynamic evolutionary process linked to selenium usage. Gene evolution featured modular expansions of an ancestral multi-Sec domain, which led to particularly Sec-rich SelenoP proteins in many aquatic organisms. We focused on molluscs, and chose Pacific oyster Magallana gigas as experimental model. We show that oyster SelenoP mRNA with 46 UGAs is translated full-length in vivo. Ribosome profiling indicates that selenocysteine specification occurs with ∼5% efficiency at UGA1 and approaches 100% efficiency at distal 3' UGAs. We report genetic elements relevant to its expression, including a leader open reading frame and an RNA structure overlapping the initiation codon that modulates ribosome progression in a selenium-dependent manner. Unlike their mammalian counterparts, the two SECIS elements in oyster SelenoP (3'UTR recoding elements) do not show functional differentiation in vitro. Oysters can increase their tissue selenium level up to 50-fold upon supplementation, which also results in extensive changes in selenoprotein expression.

Profile of Fatty Acids, Tocopherols, Phytosterols and Polyphenols in Mediterranean Oils (Argan Oils, Olive Oils, Milk Thistle Seed Oils and Nigella Seed Oil) and Evaluation of their Antioxidant and Cytoprotective Activities.

BACKGROUND: The effects of vegetable oils on human health depend on their components. Therefore, their profiles of lipid nutrients and polyphenols were determined. OBJECTIVE: To establish and compare the fatty acid, tocopherol, phytosterol and polyphenol profiles of Mediterranean oils: cosmetic and dietary argan oils (AO; Morocco: Agadir, Berkane); olive oils (OO; Morocco, Spain, Tunisia); milk thistle seed oils (MTSO; Tunisia: Bizerte, Sousse, Zaghouane); nigella seed oil (NSO). METHODS: The biochemical profiles were determined by gas chromatography-flame ionization, high performance liquid chromatography and gas chromatography, coupled with mass spectrometry as required. The antioxidant and cytoprotective activities were evaluated with the KRL (Kit Radicaux Libres) and the fluorescein diacetate tests on nerve cells treated with 7-ketocholesterol (7KC). RESULTS: The fatty acid profile revealed high linoleic acid (C18:2 n-6) content in AO, OO, MTSO and NSO. The highest levels of oleic acid (C18:1 n-9) were found in AO and OO. The tocopherol profile showed that Agadir AO contained the highest amount of α-tocopherol, also present at high level in MTSO and Tunisian OO; Berkane AO was rich in γ-tocopherol. The phytosterol profile indicated that ß-sitosterol was predominant in the oils, except AO; spinasterol was only present in AO. Polyphenol profiles underlined that OO was the richest in polyphenols; hydroxytyrosol was only found in OO; few polyphenols were detected in AO. The oils studied have antioxidant activities, and all of them, except NSO, prevented 7KC-induced cell death. The antioxidant characteristics of AO were positively correlated with procatechic acid and compestanol levels. CONCLUSION: Based on their biochemical profiles, antioxidant and cytoprotective characteristics, AO, OO, and MTSO are potentially beneficial to human health.

Dimethyl fumarate and monomethyl fumarate attenuate oxidative stress and mitochondrial alterations leading to oxiapoptophagy in 158N murine oligodendrocytes treated with 7ß-hydroxycholesterol.

Oxidative stress and mitochondrial dysfunction contribute to the pathogenesis of neurodegenerative diseases and favor lipid peroxidation, leading to increased levels of 7ß-hydroxycholesterol (7ß-OHC) which induces oxiapoptophagy (OXIdative stress, APOPTOsis, autoPHAGY). The cytoprotective effects of dimethylfumarate (DMF), used in the treatment of relapsing remitting multiple sclerosis and of monomethylfumarate (MMF), its main metabolite, were evaluated on murine oligodendrocytes 158 N exposed to 7ß-OHC (50 µM, 24 h) with or without DMF or MMF (25 µM). The activity of 7ß-OHC in the presence or absence DMF or MMF was evaluated on several parameters: cell adhesion; plasma membrane integrity measured with propidium iodide (PI), trypan blue and fluoresceine diacetate (FDA) assays; LDH activity; antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)); generation of lipid peroxidation products (malondialdehyde (MDA), conjugated dienes (CDs)) and protein oxidation products (carbonylated proteins (CPs)); reactive oxygen species (ROS) overproduction conducted with DHE and DHR123. The effect on mitochondria was determined with complementary criteria: measurement of succinate dehydrogenase activity, evaluation of mitochondrial potential (ΔΨm) and mitochondrial superoxide anions (O2●-) production using DiOC6(3) and MitoSOX, respectively; quantification of mitochondrial mass with Mitotracker Red, and of cardiolipins and organic acids. The effects on mitochondrial and peroxisomal ultrastructure were determined by transmission electron microscopy. Intracellular sterol and fatty acid profiles were determined. Apoptosis and autophagy were characterized by staining with Hoechst 33,342, Giemsa and acridine orange, and with antibodies raised against caspase-3 and LC3. DMF and MMF attenuate 7ß-OHC-induced cytotoxicity: cell growth inhibition; decreased cell viability; mitochondrial dysfunction (decrease of succinate dehydrogenase activity, loss of ΔΨm, increase of mitochondrial O2●- production, alteration of the tricarboxilic acid (TCA) cycle, and cardiolipins content); oxidative stress induction (ROS overproduction, alteration of GPx, CAT, and SOD activities, increased levels of MDA, CDs, and CPs); changes in fatty acid and cholesterol metabolism; and cell death induction (caspase-3 cleavage, activation of LC3-I in LC3-II). Ultrastructural alterations of mitochondria and peroxisomes were prevented. These results demonstrate that DMF and MMF prevent major dysfunctions associated with neurodegenerative diseases: oxidative stress, mitochondrial dysfunction, apoptosis and autophagy.

Biotin attenuation of oxidative stress, mitochondrial dysfunction, lipid metabolism alteration and 7ß-hydroxycholesterol-induced cell death in 158N murine oligodendrocytes.

Mitochondrial dysfunction and oxidative stress are involved in neurodegenerative diseases associated with an enhancement of lipid peroxidation products such as 7ß-hydroxycholesterol (7ß-OHC). It is, therefore, important to study the ability of 7ß-OHC to trigger mitochondrial defects, oxidative stress, metabolic dysfunctions and cell death, which are hallmarks of neurodegeneration, and to identify cytoprotective molecules. The effects of biotin were evaluated on 158N murine oligodendrocytes, which are myelin synthesizing cells, exposed to 7ß-OHC (50 µM) with or without biotin (10 and 100 nM) or α-tocopherol (positive control of cytoprotection). The effects of biotin on 7ß-OHC activities were determined using different criteria: cell adhesion; plasma membrane integrity; redox status. The impact on mitochondria was characterized by the measurement of transmembrane mitochondrial potential (ΔΨm), reactive oxygen species (ROS) overproduction, mitochondrial mass, quantification of cardiolipins and organic acids. Sterols and fatty acids were also quantified. Cell death (apoptosis, autophagy) was characterized by the enumeration of apoptotic cells, caspase-3 activation, identification of autophagic vesicles, and activation of LC3-I into LC3-II. Biotin attenuates 7ß-OHC-induced cytotoxicity: loss of cell adhesion was reduced; antioxidant activities were normalized. ROS overproduction, protein and lipid oxidation products were decreased. Biotin partially restores mitochondrial functions: attenuation of the loss of ΔΨm; reduced levels of mitochondrial O2•- overproduction; normalization of cardiolipins and organic acid levels. Biotin also normalizes cholesterol and fatty acid synthesis, and prevents apoptosis and autophagy (oxiapoptophagy). Our data support that biotin, which prevents oligodendrocytes damages, could be useful in the treatment of neurodegeneration and demyelination.

Calcium Signaling in Oomycetes: An Evolutionary Perspective.

Oomycetes are a family of eukaryotic microbes that superficially resemble fungi, but which are phylogenetically distinct from them. These organisms cause major global economic losses to agriculture and fisheries, with representative pathogens being Phytophthora infestans, the cause of late potato blight and Saprolegnia diclina, the instigator of "cotton molds" in fish. As in all eukaryotes, cytoplasmic Ca(2+) is a key second messenger in oomycetes, regulating life-cycle transitions, controlling motility and chemotaxis and, in excess, leading to cell-death. Despite this, little is known about the molecular mechanisms regulating cytoplasmic Ca(2+) concentrations in these organisms. Consequently, this review analyzed the presence of candidate calcium channels encoded within the nine oomycete genomes that are currently available. This revealed key differences between oomycetes and other eukaryotes, in particular the expansion and loss of different channel families, and the presence of a phylum-specific group of proteins, termed the polycystic kidney disease tandem ryanodine receptor domain (PKDRR) channels.

A Novel Selectable Islet 1 Positive Progenitor Cell Reprogrammed to Expandable and Functional Smooth Muscle Cells.

Disorders affecting smooth muscle structure/function may require technologies that can generate large scale, differentiated and contractile smooth muscle cells (SMC) suitable for cell therapy. To date no clonal precursor population that provides large numbers of differentiated SMC in culture has been identified in a rodent. Identification of such cells may also enhance insight into progenitor cell fate decisions and the relationship between smooth muscle precursors and disease states that implicate differentiated SMC. In this study, we used classic clonal expansion techniques to identify novel self-renewing Islet 1 (Isl-1) positive primitive progenitor cells (PPC) within rat bone marrow that exhibited canonical stem cell markers and preferential differentiation towards a smooth muscle-like fate. We subsequently used molecular tagging to select Isl-1 positive clonal populations from expanded and de novo marrow cell populations. We refer to these previously undescribed cells as the PPC given its stem cell marker profile, and robust self-renewal capacity. PPC could be directly converted into induced smooth muscle cells (iSMC) using single transcription factor (Kruppel-like factor 4) knockdown or transactivator (myocardin) overexpression in contrast to three control cells (HEK 293, endothelial cells and mesenchymal stem cells) where such induction was not possible. iSMC exhibited immuno- and cytoskeletal-phenotype, calcium signaling profile and contractile responses similar to bona fide SMC. Passaged iSMC could be expanded to a scale sufficient for large scale tissue replacement. PPC and reprogramed iSMC so derived may offer future opportunities to investigate molecular, structure/function and cell-based replacement therapy approaches to diverse cardiovascular, respiratory, gastrointestinal, and genitourinary diseases that have as their basis smooth muscle cell functional aberrancy or numerical loss. Stem Cells 2016;34:1354-1368.

Effects of cholesterol oxides on cell death induction and calcium increase in human neuronal cells (SK-N-BE) and evaluation of the protective effects of docosahexaenoic acid (DHA; C22:6 n-3).

Some oxysterols are associated with neurodegenerative diseases. Their lipotoxicity is characterized by an oxidative stress and induction of apoptosis. To evaluate the capacity of these molecules to trigger cellular modifications involved in neurodegeneration, human neuronal cells SK-N-BE were treated with 7-ketocholesterol, 7α- and 7ß-hydroxycholesterol, 6α- and 6ß-hydroxycholesterol, 4α- and 4ß-hydroxycholesterol, 24(S)-hydroxycholesterol and 27-hydroxycholesterol (50-100µM, 24h) without or with docosahexaenoic acid (50µM). The effects of these compounds on mitochondrial activity, cell growth, production of reactive oxygen species (ROS) and superoxide anions (O2(-)), catalase and superoxide dismutase activities were determined. The ability of the oxysterols to induce increases in Ca(2+) was measured after 10min and 24h of treatment using fura-2 videomicroscopy and Von Kossa staining, respectively. Cholesterol, 7-ketocholesterol, 7ß-hydroxycholesterol, and 24(S)-hydroxycholesterol (100µM) induced mitochondrial dysfunction, cell growth inhibition, ROS overproduction and cell death. A slight increase in the percentage of cells with condensed and/or fragmented nuclei, characteristic of apoptotic cells, was detected. With 27-hydroxycholesterol, a marked increase of O2(-) was observed. Increases in intracellular Ca(2+) were only found with 7-ketocholesterol, 7ß-hydroxycholesterol, 24(S)-hydroxycholesterol and 27-hydroxycholesterol. Pre-treatment with docosahexaenoic acid showed some protective effects depending on the oxysterol considered. According to the present data, 7-ketocholesterol, 7ß-hydroxycholesterol, 24(S)-hydroxycholesterol and 27-hydroxycholesterol could favor neurodegeneration by their abilities to induce mitochondrial dysfunctions, oxidative stress and/or cell death associated or not with increases in cytosolic calcium levels.