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The serious side effects caused by chemotherapeutics and the development of cancer chemoresistance represent the most significant limitations in the treatment of cancer. Some alternative approaches have been developed in recent years, which are based on natural compounds, and have allowed important advances in cancer therapeutics. During the last 50 years, sponges have been considered a promising source of natural products from the marine environment, representing ~30% of all marine natural products. Among sponges, the Mediterranean species

A disproportional large number of neurodevelopmental disorders (NDDs) is caused by variants in genes encoding transcription factors and chromatin modifiers. However, the functional interactions between the corresponding proteins are only partly known. Here, we show that KDM5C, encoding a H3K4 demethylase, is at the intersection of transcriptional axes under the control of three regulatory proteins ARX, ZNF711 and PHF8. Interestingly, mutations in all four genes (KDM5C, ARX, ZNF711 and PHF8) are associated with X-linked NDDs comprising intellectual disability as a core feature. in vitro analysis of the KDM5C promoter revealed that ARX and ZNF711 function as antagonist transcription factors that activate KDM5C expression and compete for the recruitment of PHF8. Functional analysis of mutations in these genes showed a correlation between phenotype severity and the reduction in KDM5C transcriptional activity. The KDM5C decrease was associated with a lack of repression of downstream target genes Scn2a, Syn1 and Bdnf in the embryonic brain of Arx-null mice. Aiming to correct the faulty expression of KDM5C, we studied the effect of the FDA-approved histone deacetylase inhibitor suberanilohydroxamic acid (SAHA). In Arx-KO murine ES-derived neurons, SAHA was able to rescue KDM5C depletion, recover H3K4me3 signalling and improve neuronal differentiation. Indeed, in ARX/alr-1-deficient Caenorhabditis elegans animals, SAHA was shown to counteract the defective KDM5C/rbr-2-H3K4me3 signalling, recover abnormal behavioural phenotype and ameliorate neuronal maturation. Overall, our studies indicate that KDM5C is a conserved and druggable effector molecule across a number of NDDs for whom the use of SAHA may be considered a potential therapeutic strategy.

The interest in dietary polyphenols in recent years has greatly increased due to their antioxidant bioactivity with preventive properties against chronic diseases. Polyphenols, by modulating different cellular functions, play an important role in neuroprotection and are able to neutralize the effects of oxidative stress, inflammation, and apoptosis. Interestingly, all these mechanisms are involved in neurodegeneration. Although polyphenols display differences in their effectiveness due to interindividual variability, recent studies indicated that bioactive polyphenols in food and beverages promote health and prevent age-related cognitive decline. Polyphenols have a poor bioavailability and their digestion by gut microbiota produces active metabolites. In fact, dietary bioactive polyphenols need to be modified by microbiota present in the intestine before being absorbed, and to exert health preventive effects by interacting with cellular signalling pathways. This literature review includes an evaluation of the literature in English up to December 2019 in PubMed and Web of Science databases. A total of 307 studies, consisting of research reports, review articles and articles were examined and 146 were included. The review highlights the role of bioactive polyphenols in neurodegeneration, with a particular emphasis on the cellular and molecular mechanisms that are modulated by polyphenols involved in protection from oxidative stress and apoptosis prevention.

Background The gut-brain axis is considered a neuroendocrine system, which connects the brain and gastrointestinal tract and plays an important role in stress response. The homeostasis of gut-brain axis is important for health conditions and its alterations are associated to neurological disorders and neurodegenerative diseases. Method We selected recent papers analysing the association among alterations in the homeostasis of the gut-brain axis and neurological disorders. In addition, we described how bioactive natural molecules - such as polyphenols - by influencing gut microbiota composition may help rescue neural signalling pathways impaired in neurodegenerative diseases. Results Recent studies show that gut microbiota is a dynamic ecosystem that can be altered by external factors such as diet composition, antibiotics or xenobiotics. Gut bacterial community plays a key role in maintaining normal brain functions. Metagenomic analyses have elucidated that the relationship between gut and brain, either in normal or in pathological conditions, reflects the existence of a "microbiota-gut-brain" axis. Gut microbiota composition can be influenced by dietary ingestion of probiotics or natural bioactive molecules such as prebiotics and polyphenols. Their derivatives coming from microbiota metabolism can affect both the gut bacterial composition and brain biochemistry. Conclusion This review highlights the role of gut microbiota in regulating regulates brain biochemistry and the role of microbiota metabolites on neuropathologies. Dietary ingestion of probiotics, prebiotics and polyphenols affect gut microbiota composition underlining the key role played by specific metabolites not only in the gut microbiota composition but also in the brain health maintenance.

Ultraviolet (UV) radiations are responsible for skin photoaging inducing alteration of the molecular and cellular pathways resulting in dryness and reduction of skin elasticity. In this study, we investigated, in vitro, the antiaging and antioxidant effects of hyaluronan formulations based hydrogel. Skinkò E, an intradermic formulation composed of hyaluronic acid (HA), minerals, amino acids, and vitamins, was compared with the sole HA of the same size. For this purpose, HaCaT cells were subjected to UV-A radiations and H2O2 exposure and then treated with growth medium (CTR) combined with M-HA or Skinkò E to evaluate their protective ability against stressful conditions. Cells reparation was evaluated using a scratch in vitro model and Time-Lapse Video Microscopy. A significant protective effect for Skinkò E was shown with respect to M-HA. In addition, Skinkò E increased cell reparation. Therefore, NF-kB, SOD-2, and HO-1 were significantly reduced at the transcriptional and protein level. Interestingly, γ-H2AX and protein damage assay confirmed the protection by hyaluronans tested against oxidative stress. G6pdΔ ES cell line, highly susceptible to oxidative stress, was used as a further cellular model to assess the antioxidant effect of Skinkò E. Western blotting analyses showed that the treatment with this new formulation exerts marked antioxidant action in cells exposed to UV-A and H2O2. Thus, the protective and reparative properties of Skinkò E make it an interesting tool to treat skin aging.

Neurological disorders can be associated with protein glycosylation abnormalities. Rett syndrome is a devastating genetic brain disorder, mainly caused by de novo loss-of-function mutations in the methylCpG binding protein 2 (MECP2) gene. Although its pathogenesis appears to be closely associated with a redox imbalance, no information on glycosylation is available. Glycoprotein detection strategies (i.e., lectin-blotting) were applied to identify target glycosylation changes in the whole brain of Mecp2 mutant murine models of the disease. Remarkable glycosylation pattern changes for a peculiar 50 kDa protein, i.e., the N-linked brain nucleotide pyrophosphatase-5 were evidenced, with decreased N-glycosylation in the presymptomatic and symptomatic mutant mice. Glycosylation changes were rescued by selected brain Mecp2 reactivation. Our findings indicate that there is a causal link between the amount of Mecp2 and the N-glycosylation of NPP-5. (C) 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Huntington’s disease (HD) has traditionally been described as a disorder purely of the brain, however evidence indicates that peripheral abnormalities are also commonly seen. Among others, severe unintended body weight loss represents a prevalent and often debilitating feature of HD pathology, with no therapies available. It correlates with disease progression and significantly affects the quality of life of HD patients. Curcumin, a naturally occurring polyphenol with multiple therapeutic properties, has been validated to exert important beneficial effects under health conditions as well as in different pathological settings, including neurodegenerative and gastrointestinal (GI) disorders. Here, we investigated the potential therapeutic action that curcumin-supplemented diet may exert on central and peripheral dysfunctions in R6/2 mice, a well-characterized HD animal model which recapitulates some features of human pathology. Maintenance of normal motor function, protection from neuropathology and from GI dysfunction, preservation of GI emptying, and conserved intestinal contractility, proved the beneficial role of life-long dietary curcumin in HD and corroborated the potential of the compound to be exploited to alleviate very debilitating symptoms associated with the disease.

Background Huntington Disease (HD) has traditionally been described as a disorder purely of the brain, however emerging evidence indicates that peripheral abnormalities are also commonly seen. Among others, unintended body weight loss represents a hallmark of peripheral HD pathology. It correlates with disease progression and significantly affects the quality of life of HD patients. Although the underlying molecular mechanism is still unknown, evidence suggests that body weight loss seems not to be secondary to inadequate nutrition or to chorea, but rather attributable to changes in the metabolism and defective nutrients absorption along the intestinal tract. Curcumin, a naturally occurring polyphenol, has been validated to exert important beneficial effects in a multitude of gastrointestinal dysfunction and neurodegenerative processes similar to those occurring in HD. Although its therapeutic effect in HD is still questionable, recent evidence indicates that curcumin significantly improves neuropathology as well neurochemical and neurobehavioral defects in a mouse model of the disease, however whether it may be suited to be developed to treat gastrointestinal dysfunction in the disease is not clear yet. Aim In this study, we aimed to investigate the potential beneficial effects that the chronic administration of curcumin may have on gastrointestinal dysfunction commonly occurring in R6/2 mice as the disease progresses. Method Curcumin along with bioperine (bioavailability enhancing-agent) was orally administered to females 10 days before pregnancy and during the entire gestation period up to the end of lactation. Starting from the third week of age, pups were daily treated with 40 mg/kg curcumin for 7 weeks. KCl-induced contraction was evaluated in isolated intestinal tract from both curcumin and vehicle-treated mice. Results Our preliminary data demonstrate that chronic administration of curcumin is safe and well tolerated in R6/2 mice. It prevents the gradual weight loss despite no changes in food intake were observed. From the functional point of view, curcumin considerably ameliorates the intestinal phenotype and restores the normal intestinal reactivity compared to untreated R6/2 mice.

Embryonic stem (ES) cells, combining self-renewal ability with wide range tissue-specific cell differentiation, represent one of the most powerful model systems in basic research, drug discovery and biomedical applications. In the field of drug development, ES cells are instrumental in high-throughput/content screening (HTS/HCS) for the evaluation of large compound libraries to test biological activity and toxic properties. Since it is a high priority to test new compounds in vitro, before starting animal and human treatments, there is an increasing demand for new in vitro models that can be used in HTS/HCS to facilitate drug development. In order to achieve this objective, several methods for ES cell self-renewal or differentiation have been evaluated to assess their compatibility with HTS/HCS. This review describes protocols used to screen molecules able to maintain self-renewal or to induce differentiation in ectodermal, mesodermal, endodermal, and their derivative cell lines. © 2014 Springer Science+Business Media New York.

Blood-brain barrier (BBB) breakdown, due to the concomitant disruption of the tight junctions (TJs), normally required for the maintenance of BBB function, and to the altered transport of molecules between blood and brain and vice-versa, has been suggested to significantly contribute to the development and progression of different brain disorders including Huntington’s disease (HD). Although the detrimental consequence the BBB breakdown may have in the clinical settings, the timing of its alteration remains elusive for many neurodegenerative diseases. In this study we demonstrate for the first time that BBB disruption in HD is not confined to established symptoms, but occurs early in the disease progression. Despite the obvious signs of impaired BBB permeability were only detectable in concomitance with the onset of the disease, signs of deranged TJs integrity occur precociously in the disease and precede the onset of overt symptoms. To our perspective this finding may add a new dimension to the horizons of pathological mechanisms underlying this devastating disease, however much remains to be elucidated for understanding how specific BBB drug targets can be approached in the future.

The use of Embryonic Stem Cells (ESCs) holds considerable promise both for drug discovery programs and the treatment of degenerative disorders in regenerative medicine approaches. Nevertheless, the successful use of ESCs is still limited by the lack of efficient control of ESC self-renewal and differentiation capabilities. In this context, the possibility to modulate ESC biological properties and to obtain homogenous populations of correctly specified cells will help developing physiologically relevant screens, designed for the identification of stem cell modulators. Here, we developed a high throughput screening-suitable ESC neural differentiation assay by exploiting the Cell (maker) robotic platform and demonstrated that neural progenies can be generated from ESCs in complete automation, with high standards of accuracy and reliability. Moreover, we performed a pilot screening providing proof of concept that this assay allows the identification of regulators of ESC neural differentiation in full automation.

Objective Rett syndrome (RTT) is a neurological disorder and a leading cause of mental retardation in females. It is caused by mutations in methyl-CpG-binding protein 2 (MeCP2) gene and more rarely in cyclin-dependent kinase-like 5 (CDKL5) and forkhead box protein G1 (FOXG1) genes. Increased oxidative stress (OS) has been documented in MeCP2-RTT patients. Here, we evaluated the levels of 4-hydroxynonenal plasma protein adducts (4HNE-PAs) in MeCP2-, CDKL5-, and FOXG1-RTT and in their clinical variants. Design and methods 4HNE-PAs were determined by Western blot in plasma from healthy subjects and RTT patients. Results 4HNE-PAs levels were increased in MeCP2- and CDKL5-related RTT but not in FOXG1-related RTT. Conclusion These results showed that OS is present in RTT clinical variants and could play a key role in RTT pathogenesis. Under the OS point of view FOXG1-related RTT appears to be distinct from the MeCP2/CDKL5, suggesting a distinct mechanism involved in its pathogenesis.

Rett syndrome (RTT, MIM 312750) is a rare and orphan progressive neurodevelopmental disorder affecting girls almost exclusively, with a frequency of 1/15,000 live births of girls. The disease is characterized by a period of 6 to 18 months of apparently normal neurodevelopment, followed by early neurological regression, with a progressive loss of acquired cognitive, social, and motor skills. RTT is known to be caused in 95% of the cases by sporadic de novo loss-of-function mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene encoding methyl-CpG binding protein 2 (MeCP2), a nuclear protein able to regulate gene expression. Despite almost two decades of research into the functions and role of MeCP2, little is known about the mechanisms leading from MECP2 mutation to the disease. Oxidative stress (OS) is involved in the pathogenic mechanisms of several neurodevelopmental and neurodegenerative disorders, although in many cases it is not clear whether OS is a cause or a consequence of the pathology. Fairly recently, the presence of a systemic OS has been demonstrated in RTT patients with a strong correlation with the patients' clinical status. The link between MECP2 mutation and the redox imbalance found in RTT is not clear. Animal studies have suggested a possible direct correlation between Mecp2 mutation and increased OS levels. In addition, the restoration of Mecp2 function in astrocytes significantly improves the developmental outcome of Mecp2-null mice and reexpression of Mecp2 gene in the brain of null mice restored oxidative damage, suggesting that Mecp2 loss of function can be involved in oxidative brain damage. Starting from the evidence that oxidative damage in the brain of Mecp2-null mice precedes the onset of symptoms, we evaluated whether, based on the current literature, the dysfunctions described in RTT could be a consequence or, in contrast, could be caused by OS. We also analyzed whether therapies that at least partially treated some RTT symptoms can play a role in defense against OS. At this stage we can propose that OS could be one of the main causes of the dysfunctions observed in RTT. In addition, the major part of the therapies recommended to alleviate RTT symptoms have been shown to interfere with oxidative homeostasis, suggesting that MeCP2 could somehow be involved in the protection of the brain from OS.

Embryonic stem (ES) cells can differentiate in vitro into a variety of cell types. Efforts to produce endodermal cell derivatives, including lung, liver and pancreas, have been met with modest success. Understanding how the endoderm originates from ES cells is the first step to generate specific cell types for therapeutic purposes. Recently, it has been demonstrated that inhibition of Myc or mTOR induces endodermal differentiation. Both Myc and mTOR are known to be activators of the Pentose Phosphate Pathway (PPP). We found that, differentely from wild type (wt), ES cells unable to produce pentose sugars through PPP differentiate into endodermal precursors in cell culture conditions generally non-permissive to generate them. The same effect was observed when wt ES cells were differentiated in presence of chemical inhibitors of the PPP. These data highlight a new role for metabolism. Indeed, to our knowledge, it is the first time that modulation of a metabolic pathway is described to be crucial in determining ES cell fate.

Glucose-6-phosphate dehydrogenase plays a crucial role in protection from redox-stress-induced apoptosis

The onset of resistance to drug-induced apoptosis of tumour cells is a major problem in cancer therapy. We studied a drug-selected clone of promyelocytic HL-60 cells, called HCW-2, which display a complex resistance to a wide variety of apoptosis-inducing agents and we found that these cells show a dramatic increase in the expression of heat shock proteins (Hsps) 70 and 27, while the parental cell line does not. It is known that stress proteins such as Hsps can confer resistance to a variety of damaging agents other than heat shock, such as TNF-alpha, monocyte-induced cytotoxicity, and also play a role in resistance to chemotherapy. This elevated expression of Hsps is paralleled by an increased activity of mitochondrial metabolism and pentose phosphate pathway, this latter leading to high levels of glucose-6-phosphate dehydrogenase and, consequently, of glutathione. Thus, the apoptotic-deficient phenotype is likely because of the presence of high levels of stress response proteins and GSH, which may confer resistance to apoptotic agents, including chemotherapy drugs. Moreover, the fact that in HCW-2 cells Hsp70 are mainly localised in mitochondria may account for the increased performances of mitochondrial metabolism. These observations could have some implications for the therapy of cancer, and for the design of combined strategies that act on antioxidant defences of the neoplastic cell.

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway that is responsible for the generation of NADPH, which is required in many detoxifying reactions. We have recently demonstrated that G6PD expression is induced by a variety of chemical agents acting at different steps in the biochemical pathway controlling the intracellular redox status. Although we obtained evidence that the oxidative stress-mediated enhancement of G6PD expression is a general phenomenon, the functional significance of such G6PD induction after oxidant insult is still poorly understood. In this report, we used a GSH-depleting drug that determines a marked decrease in the intracellular pool of reduced glutathione and a gradual but notable increase in G6PD expression. Both effects are seen soon after drug addition. Once G6PD activity has reached the maximum, the GSH pool is restored. We suggest and also provide the first direct evidence that G6PD induction serves to maintain and regenerate the intracellular GSH pool. We used HeLa cell clones stably transfected with the human G6PD gene that display higher G6PD activity than the parent HeLa cells. Although the activities of glutathione peroxidase, glutathione reductase, and catalase were comparable in all strains, the concentrations of GSH were significantly higher in G6PD-overexpressing clones. A direct consequence of GSH increase in these cells is a decreased reactive oxygen species production, which makes these cells less sensitive to the oxidative burst produced by external stimuli. Indeed, all clones that constitutively overexpress G6PD exhibited strong protection against oxidants-mediated cell killing. We also observe that NF-kappa B activation, in response to tumor necrosis factor-alpha treatment, is strongly reduced in human HeLa cells overexpressing G6PD.

Purpose Electrically active supports provide new horizons for bio-sensing and artificial organ design. Cell-based electrochemical biosensors can be used as bio-microactuators, applied to the biorobotics. Microchip-based bioassay systems can provide real-time cell analysis for preclinical drug design or for intelligent drug delivery devices. In regenerative medicine, electrically active supports can be used as bio-reactors to monitor cell activity, optimize the stem cell differentiation and control cell and tissue morphology. Biocompatibility and direct interaction of the electrically active surface with the cell surface is a critical aspect of this technology. Methods In this work embryonic stem cells (AK7 ES) have been cultivated on the surface of thin films achieved through the evaporation of two aromatic compounds (T6 and PDI-8CN2) of particular interest for the fabrication of organic field-effect transistors (OFET). One of the potential advantages offered by the application of OFETs as bio-electronic supports is that they represent a powerful tool for the detection of bio-signals because their electrically active surface is an organic film. Results The cell morphology on T6 and PDI-8CN 2 surface shows to be similar to the usual cell appearance, as obtained when standard culture support (petri dish) are employed. Moreover, our experimental results demonstrate that stem cells can be lead to differentiation up to “beating” cardiomyocytes even on these electrically-active organic films. Conclusions This investigation encourages the perspective to develop OFET-based biosensors in order to accurately characterize stem cells during the cardiac differentiation process and eventually increase their differentiation efficiency.

Background Curcumin, the active ingredient in turmeric, has been validated to harbour important beneficial effects for a multitude of inflammatory-based diseases and also for neurodegenerative processes similar to those occurring in HD. To this regard, it has been recently described that curcumin significantly improves neuropathology and positively affects some of the neurochemical and neurobehavioral disease-related defects in a mouse model of HD. Although the mechanism of its neuroprotective action is still not completely understood, it has been hypothesised to act majorly through anti-inflammatory and anti-oxidative activities. Aim Here, our purpose is to investigate whether such specific properties of curcumin may potentially play a role in the control of the gradual body weight loss and in the accumulation of redox metals commonly observed in HD. We also want to examine whether the described beneficial effect of curcumin in HD is a shared mechanism across multiple HD models and not confined only to a single or few systems. Methods Curcumin along with bioperine (bioavailability enhancing-agent) was orally administered to females 10 days before pregnancy and during the entire gestation period up to the end of lactation. Starting from the third week of age, pups were daily treated with 40 mg/kg curcumin for 7 weeks. Results In line with other studies, so far, our preliminary data confirmed that curcumin has an outstanding safety profile with no adverse effect also in R6/2 mice. Evaluation of any potential beneficial effects of the treatment is in progress. Conclusion Curcumin is well tolerated also in R6/2 HD mice and, based on its therapeutic properties, it could conceivably represent an attractive possibility to develop a valuable dietary supplement in neurodegenerative diseases such as HD.

We review here some recent data about Glucose-6-phosphate dehydrogenase (G6PD), the housekeeping X-linked gene encoding the first enzyme of the pentose phosphate pathway (PPP), a NADPH-producing dehydrogenase. This enzyme has been popular among clinicians, biochemists, geneticists and molecular biologists because it is the most common form of red blood cell enzymopathy. G6PD deficient erythrocytes do not generate NADPH in any other way than through the PPP and for this reason they are more susceptible than any other cells to oxidative damage. Moreover, this enzyme has also been of crucial importance in many significant discoveries; indeed, G6PD polymorphisms have been instrumental in studying X-inactivation in the human species, as well as in establishing the clonal nature of certain tumors. G6PD deficiency, generally considered as a mild and benign condition, is significantly disadvantageous in certain environmental conditions like in presence of certain drugs. Nevertheless, G6PD deficiency has been positively selected by malaria, and recent knowledge seems to show that it also confers an advantage against the development of cancer, reduces the risk of coronary diseases and has a beneficial effect in terms of longevity.

International audience; Intellectual disability (ID) and epilepsy often occur together and have a dramatic impact on the development and quality of life of the affected children. Polyalanine (polyA)-expansion-encoding mutations of aristaless-related homeobox (ARX) cause a spectrum of X-linked ID (XLID) diseases and chronic epilepsy, including infantile spasms. We show that lysine-specific demethylase 5C (KDM5C), a gene known to be mutated in XLID-affected children and involved in chromatin remodeling, is directly regulated by ARX through the binding in a conserved noncoding element. We have studied altered ARX carrying various polyA elongations in individuals with XLID and/or epilepsy. The changes in polyA repeats cause hypomorphic ARX alterations, which exhibit a decreased trans-activity and reduced, but not abolished, binding to the KDM5C regulatory region. The altered functioning of the mutants tested is likely to correlate with the severity of XLID and/or epilepsy. By quantitative RT-PCR, we observed a dramatic Kdm5c mRNA downregulation in murine Arx-knockout embryonic and neural stem cells. Such Kdm5c mRNA diminution led to a severe decrease in the KDM5C content during in vitro neuronal differentiation, which inversely correlated with an increase in H3K4me3 signal. We established that ARX polyA alterations damage the regulation of KDM5C expression, and we propose a potential ARX-dependent path acting via chromatin remodeling.

Intellectual Disability (ID) and Epilepsy often occur together with a dramatic impact on the development and quality of life of the affected children. PolyAlanine expansion mutations of Aristaless-related homeobox gene (ARX) cause a spectrum of X-chromosome phenotypes with ID (XLID) and various forms of malignant paediatric epilepsy. We here present that lysine (K)-specific demethylase 5C (KDM5C/JARID1C/SMCX), a known XLID gene involved in chromatin remodeling, is an ARX-dependent target, demonstrating a functional link between these two XLID/Epilepsy genes. Combining luciferase reporter and DNA binding assays, we reveal that ARX directly binds a CNE in the 5' region of KDM5C. Our investigation into the functional properties of ARX with PolyAlanine expansions have demonstrated that changes in the repeat units cause a decreased trans-activating activity and a reduced, but not abolished, binding to the KDM5C regulatory region. In the Arx KO ES disease model, a decreased level of Kdm5C mRNA and protein has been shown to be associated with an increased level of H3K4me3, a defect that could compromise cyclical rounds of methylation-demethylation and consequently the genes in DNA "off" and "on". These data suggest that ARX and KDM5C may be functionally connected in specific neuronal paths, which once identified, could facilitate the dissection of the peculiar neurophenotypes observed in XLID and Epilepsy patients. Overall, a role for chromatin packaging in ARX diseases is an attractive hypothesis as it expands our horizons of the modes of PolyAlanine pathogenesis, however further study it required.

Several lines of evidence have provided clues for the pivotal role played by the KRAB- containing zinc finger protein ZFP57 in the establishment and maintenance of the asymmetric epigenetic state, both DNA methylation and histone tails specific modifications, of the several tens of imprinted loci in mouse. ZFP57 associates with KAP1, the general corepressor of the abundant KRAB-ZFPs family, a scaffold for the recruitment of chromatin modifiers that include the histone H3 methyltransferase SETDB1 and heterochromatin protein-1 (HP1) isoforms, thus promoting H3 trimethylation at lysine 9 and chromoproteins assembly at targeted sites. Further, DNA methyltransferases are also present in KAP1 complexes, a finding that correlates with the requirement for ZFP57 to mantain DNA methylation at targeted sites. We have established in murine ES cells the epigenomic profile of the endogenous Zfp57 factor along with those of the associated KAP1, SETDB1, HP1g and the complex-dependent H3K9 trimethylation. We will report on the DNA binding motifs inference, features of bound genomic targets along with the gene association analysis that support potential pivotal roles of the locus-specific targeting ZFP57 factor in shaping the epigenetic state of multiple genes in addition to imprinted loci, through histone modification, Hp1-mediated heterochromatinization and DNA methylation.

Evidence of enhanced oxidative stress (O.S.) and lipid peroxidation has been reported in patients with Rett syndrome (RTT), a relatively rare neurodevelopmental disorder progressing in 4-stages, and mainly caused by loss-of-function mutations in the methyl-CpG-binding protein 2. No effective therapy for preventing or arresting the neurologic regression in the disease in its various clinical presentations is available. Based on our prior evidence of enhanced O.S. and lipid peroxidation in RTT patients, herein we tested the possible therapeutic effects of omega-3 polyunsaturated fatty acids (omega-3 PUFAs), known antioxidants with multiple effects, on the clinical symptoms and O.S. biomarkers in the earliest stage of RTT. A total of 20 patients in stage I were randomized (n = 10 subjects per arm) to either oral supplementation with omega-3 PUFAs-containing fish oil (DHA: 72.9 +/- A 8.1 mg/kg b.w./day; EPA: 117.1 +/- A 13.1 mg/kg b.w./day; total omega-3 PUFAs: 246.0 +/- A 27.5 mg/kg b.w./day) for 6 months or no treatment. Primary outcomes were potential changes in clinical symptoms, with secondary outcomes including variations for five O.S. markers in plasma and/or erythrocytes (nonprotein bound iron, F-2-dihomo-isoprostanes, F-3-isoprostanes, F-4-neuroprostanes, and F-2-isoprostanes). A significant reduction in the clinical severity (in particular, motor-related signs, nonverbal communication deficits, and breathing abnormalities) together with a significant decrease in all the examined O.S. markers was observed in the omega-3 PUFAs supplemented patients, whereas no significant changes were evidenced in the untreated group. For the first time, these findings strongly suggest that a dietary intervention in this genetic disease at an early stage of its natural history can lead to a partial clinical and biochemical rescue.

Aims Rett syndrome (RTT) is a rare neurodevelopmental disorder frequently linked to methyl-CpG-binding protein 2 ( MeCP2 ) gene mutations. RTT is associated with a 300-fold increased risk of sudden cardiac death. Rhythm abnormalities and cardiac dysautonomia do not to fully account for cardiac mortality. Conversely, heart function in RTT has not been explored to date. Recent data indicate a previously unrecognized role of MeCP2 in cardiomyocytes development. Besides, increased oxidative stress markers (OS) have been found in RTT. We hypothesized that (i) RTT patients present a subclinical biventricular dysfunction and (ii) the myocardial dysfunction correlate with OS. Methods and results We evaluated typical ( n = 72) and atypical ( n = 20) RTT female and healthy controls ( n = 92). Main outcome measurements were (i) echocardiographic biventricular systo-diastolic parameters; (ii) correlation between echocardiographic measures and OS levels, i.e. plasma and intra-erythrocyte non-protein-bound iron (NPBI) and plasma F2-Isoprostanes (F2-IsoPs). A significant reduction in longitudinal biventricular function (tricuspid annular plane systolic excursion, mitral annular plane systolic excursion, S′ of lateral and septal mitral annulus, S′ of tricuspidal annulus) was evidenced in RTT patients vs. controls. No significant changes in the LV ejection fraction were found. Peak-early filling parameters (E, E′ of lateral mitral annulus, E′ of tricuspidal annulus) and right ventricular systolic pressure were reduced. A-wave, E/A, and E/E′ were normal. OS markers were increased, but only F2-IsoPs correlated to LV systolic dysfunction. Conclusion These data indicate a previously unrecognized subclinical systo-diastolic biventricular myocardial dysfunction in typical and atypical RTT patients. A reduced preload is evidenced. The biventricular dysfunction is partially related to OS damage.

In an effort to investigate the subtelomeric region of the X chromosome among Orientals, five DNA sites in the F8C and G6PD genes were analyzed in a sample of 46 chromosomes belonging to the Chinese Li population, an ethnic group characterized by a high prevalence of G6PD deficiency. The DNA sites analyzed, which are highly polymorphic in other populations, have a low degree of heterozygosity in the Li sample and, furthermore, the distribution of the corresponding haplotypes is very different from that previously observed in Italian populations. Interestingly, three unrelated Li G6PD-deficient variants analyzed at the DNA level have the 1376G-->T mutation characteristic of G6PDCanton and they share the same haplotype, including the sites mentioned above, as well as eight DNA polymorphisms in the red/green color vision pigment genes located proximal to G6PD on chromosome X.

A stem cell is a specific kind of cell that has the unique capacity to renew itself and to give rise to specialized cell type. In terms of potentially, stem cells can be classified in three types: • Totipotent: is the ability to form all cell types, including the extra-embryonic tissues. In mammals, the fertilized egg, zygote and the first 2, 4, 8, 16 blastomeres from the early, are examples of totipotent cells. • Pluripotency: is the ability to differentiate into several cell types derived from any of the three germ layers (ectoderm, mesoderm, endoderm), but they are unable to produce extra-embryonic tissues. Cells from the inner cell mass of blastocyst are pluripotent. • Multipotent: cells can form a small number of tissues that are restricted to a particular germ layer origin: e.g. blood cells or bone cells. In according to their source, stem cells are categorized in embryonic or adult (Fig. 1): • Embryonic stem cells (ES cells) are derived from the inner cell mass of the blastocyst (an early stage embryo) and have a high proliferative capabilities and differ from other stem cells because they have the ability to generate derivatives of all three germ layers. Embryonic stem cells have been shown to contribute to all cell lineages, including the germ line, following microinjection studies in murine embryos which give rise to chimeras (Bradley et al., 1984; Nagy et al., 1990). In vitro, murine ES cells can be propagated indefinitely in an undifferentiated state, under specific culture conditions they can differentiated into specific cell types. • Adult stem cells are undifferentiated cells found among differentiated cells of a specific tissue, including bone marrow (de Haan, 2002), skin (Watt, 2001), intestinal epithelium (Potten, 1998), liver (Theise et al., 1999), retina (Tropepe et al., 2000), central nervous system (Okano, 2002), pancreas (Ramiya et al., 2000) and skeletal muscle (Seale et al., 2001). They typically can differentiate into a relatively limited number of cell types. There is no doubt that stem cells have the potential to treat many human afflictions, including cancer, diabetes, neurodegeneration, as well as for studying basic developmental biology, and intensive screening of drug and toxic (Watt and Driskell, 2010).

Rett syndrome (RTT) is a pervasive development disorder, mainly caused by mutations in the methyl-CpG binding protein 2 (MeCP2) gene. No reliable biochemical markers of the disease are available. Here we assess F₄-neuroprostanes (F₄-NeuroPs), lipid peroxidation products of the docosahexaenoic acid, as a novel disease marker in RTT and correlate it with clinical presentation, MeCP2 mutation type, and disease progression. In addition, we investigate on the impact of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) supplementation on F₄-NeuroPs levels.A case-control study design was used. A cohort of RTT patients (n=144) exhibiting different clinical presentations, disease stages, and MeCP2 gene mutations were evaluated. F₄-NeuroPs were measured in free form using a GC/NICI-MS/MS technique. Plasma F₄-NeuroPs levels in patients were compared to healthy controls and related to RTT forms, disease progression, and response to ω-3 PUFAs supplementation.Plasma F₄-NeuroPs levels were i) higher in RTT than in controls; ii) increased with the severity of neurological symptoms; iii) significantly elevated during the typical disease progression; iv) higher in MeCP2-nonsense as compared to missense mutation carriers; v) higher in typical RTT as compared to RTT variants; and vi) decreased in response to 12 months ω-3 PUFAs oral supplementation.Quantification of plasma F₄-NeuroPs provides a novel RTT marker, related to neurological symptoms severity, mutation type and clinical presentation.

The molecular mechanisms controlling mouse embryonic stem cell (ESC) metastability, i.e. their capacity to fluctuate between different states of pluripotency, are not fully resolved. We developed and used a novel automation platform, the Cell(maker), to screen a library of metabolites on two ESC-based phenotypic assays (i.e. proliferation and colony phenotype) and identified two metabolically related amino acids, namely L-proline (L-Pro) and L-ornithine (L-Orn), as key regulators of ESC metastability. Both compounds, but mainly L-Pro, force ESCs toward a novel epiblast stem cell (EpiSC)-like state, in a dose-and time-dependent manner. Unlike EpiSCs, L-Pro-induced cells (PiCs) contribute to chimeric embryos and rely on leukemia inhibitor factor (LIF) to self-renew. Furthermore, PiCs revert to ESCs or differentiate randomly upon removal of either L-Pro or LIF, respectively. Remarkably, PiC generation depends on both L-Pro metabolism (uptake and oxidation) and Fgf5 induction, and is strongly counteracted by antioxidants, mainly L-ascorbic acid (vitamin C, Vc). ESCs PiCs phenotypic transition thus represents a previously undefined dynamic equilibrium between pluripotent states, which can be unbalanced either toward an EpiSC-like or an ESC phenotype by L-Pro/L-Orn or Vc treatments, respectively. All together, our data provide evidence that ESC metastability can be regulated at a metabolic level.

Background Rett syndrome (RTT) is a pervasive development disorder, mainly caused by mutations in the methyl-CpG binding protein 2 (MeCP2) gene. No reliable biochemical markers of the disease are available. Here we assess F4-neuroprostanes (F4-NeuroPs), lipid peroxidation products of the docosahexaenoic acid, as a novel disease marker in RTT and correlate it with clinical presentation, MeCP2 mutation type, and disease progression. In addition, we investigate on the impact of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) supplementation on F4-NeuroPs levels. Methods A case–control study design was used. A cohort of RTT patients (n = 144) exhibiting different clinical presentations, disease stages, and MeCP2 gene mutations were evaluated. F4-NeuroPs were measured in free form using a GC/NICI-MS/MS technique. Plasma F4-NeuroPs levels in patients were compared to healthy controls and related to RTT forms, disease progression, and response to ω-3 PUFAs supplementation. Results Plasma F4-NeuroPs levels were i) higher in RTT than in controls; ii) increased with the severity of neurological symptoms; iii) significantly elevated during the typical disease progression; iv) higher in MeCP2-nonsense as compared to missense mutation carriers; v) higher in typical RTT as compared to RTT variants; and vi) decreased in response to 12 months ω-3 PUFAs oral supplementation. Conclusions Quantification of plasma F4-NeuroPs provides a novel RTT marker, related to neurological symptoms severity, mutation type and clinical presentation.

Biotechnologies such as high-throughput screening (HTS) enable evaluation of large compound libraries for their biological activity and toxic properties. In the field of drug development, embryonic stem (ES) cells have been instrumental in HTS for testing the effect of new compounds. We report an innovative method in one step to differentiate ES cells in neurons and glial cells. The four different neuronal subtypes, gamma-aminobutyric acid (GABA)-ergic, dopaminergic, serotonergic, and motor neurons, are formed in culture. This protocol is adaptable to small wells and is highly reproducible, as indicated by the Z-factor value. Moreover, by using either leukemia inhibitory factor (LIF) or recombinant Cripto protein in our culture conditions, we provide evidence that this protocol is suitable for testing the effect of different molecules on neuronal differentiation of ES cells. Finally, thanks to the simplicity in carrying out the experiment, this method provides the possibility of following the morphological evolution of the in vitro differentiating neuronal cells by timelapse videomicroscopy. Our experimental system provides a powerful tool for testing the effect of different substances on survival and/or differentiation of neuronal and glial cells in an HTS-based approach. Furthermore, using genetically modified ES cells, it would be possible to screen for drugs that have a therapeutic effect on specific neuronal pathologies or to follow, by time-lapse videomicroscopy, their ability to in vitro differentiate.