Generation of two induced pluripotent stem cell (iPSC) lines from p.F508del Cystic Fibrosis patients.

Cystic Fibrosis (CF) is a monogenic, lethal disease caused by mutations in the cystic fibrosis transmembrane conductance (CFTR) gene. Here we report the production of CF-iPS cell lines from two different p.F508del homozygous female patients (Table 1). Two different primary cell types, skin fibroblasts and keratinocytes, were transfected with retroviral cocktails containing four: c-MYC, KLF4, OCT4 and SOX2 (MKOS) or three: KLF4, OCT4 and SOX2 (KOS) reprogramming factors. Two fibroblast-derived MKOS lines are described in the main text. The lines carry the p.F508del mutation, have a normal karyotype, express pluripotency markers and are able to differentiate into the three germ layers.

Effects of Resveratrol and Other Polyphenols on the Most Common Brain Age-Related Diseases.

BACKGROUND: With global increase in elderly population, modern societies must find strategies to reduce the consequences of aging process; thereby decreasing the incidence of age-related neurodegenerative diseases. Oxidative stress and recently inflammation, have been pointed out as the leading causes of brain aging. Thereby, the consumption or administration of antioxidant and anti-inflammatory molecules, such as polyphenols, is a beneficial strategy recommended for preventing brain aging and several brain age-related diseases. METHODS AND RESULTS: Several studies suggest that long term consumption of dietary polyphenols offers protection against development of neurodegenerative diseases. These beneficial effects are in part due to their antioxidant and anti-inflammatory properties, together with their positive role in the modulation of processes involved in the physiopathology of several neurodegenerative diseases (e.g., epigenetic factors, amyloid deposition, cholinesterase inhibition, autophagy, and neurotrophic factors, among others). Altogether, these molecules open the door to the research of new neuroprotective strategies. This review summarizes the latest discoveries in how polyphenols can exert positive effects on brain health in aging, emphasizing those effects on the diseases that most commonly affect the brain during aging: Parkinson's Disease (PD), Alzheimer's disease (AD), dementia and depression. Moreover, within are addressed the epigenetic effects of polyphenols as possible mediators in their positive effects on brain health, and the future challenges of research in this topic Conclusion: In brief, this review presents a report of state-of the art knowledge regarding the positive influences of polyphenols on the most common brain age-related diseases as well as in healthy brain aging.

Generation of replication-proficient influenza virus NS1 point mutants with interferon-hyperinducer phenotype.

The NS1 protein of influenza A viruses is the dedicated viral interferon (IFN)-antagonist. Viruses lacking NS1 protein expression cannot multiply in normal cells but are viable in cells deficient in their ability to produce or respond to IFN. Here we report an unbiased mutagenesis approach to identify positions in the influenza A NS1 protein that modulate the IFN response upon infection. A random library of virus ribonucleoproteins containing circa 40 000 point mutants in NS1 were transferred to infectious virus and amplified in MDCK cells unable to respond to interferon. Viruses that activated the interferon (IFN) response were subsequently selected by their ability to induce expression of green-fluorescent protein (GFP) following infection of A549 cells bearing an IFN promoter-dependent GFP gene. Using this approach we isolated individual mutant viruses that replicate to high titers in IFN-compromised cells but, compared to wild type viruses, induced higher levels of IFN in IFN-competent cells and had a reduced capacity to counteract exogenous IFN. Most of these viruses contained not previously reported NS1 mutations within either the RNA-binding domain, the effector domain or the linker region between them. These results indicate that subtle alterations in NS1 can reduce its effectiveness as an IFN antagonist without affecting the intrinsic capacity of the virus to multiply. The general approach reported here may facilitate the generation of replication-proficient, IFN-inducing virus mutants, that potentially could be developed as attenuated vaccines against a variety of viruses.

An unbiased genetic screen reveals the polygenic nature of the influenza virus anti-interferon response.

UNLABELLED: Influenza A viruses counteract the cellular innate immune response at several steps, including blocking RIG I-dependent activation of interferon (IFN) transcription, interferon (IFN)-dependent upregulation of IFN-stimulated genes (ISGs), and the activity of various ISG products; the multifunctional NS1 protein is responsible for most of these activities. To determine the importance of other viral genes in the interplay between the virus and the host IFN response, we characterized populations and selected mutants of wild-type viruses selected by passage through non-IFN-responsive cells. We reasoned that, by allowing replication to occur in the absence of the selection pressure exerted by IFN, the virus could mutate at positions that would normally be restricted and could thus find new optimal sequence solutions. Deep sequencing of selected virus populations and individual virus mutants indicated that nonsynonymous mutations occurred at many phylogenetically conserved positions in nearly all virus genes. Most individual mutants selected for further characterization induced IFN and ISGs and were unable to counteract the effects of exogenous IFN, yet only one contained a mutation in NS1. The relevance of these mutations for the virus phenotype was verified by reverse genetics. Of note, several virus mutants expressing intact NS1 proteins exhibited alterations in the M1/M2 proteins and accumulated large amounts of deleted genomic RNAs but nonetheless replicated to high titers. This suggests that the overproduction of IFN inducers by these viruses can override NS1-mediated IFN modulation. Altogether, the results suggest that influenza viruses replicating in IFN-competent cells have tuned their complete genomes to evade the cellular innate immune system and that serial replication in non-IFN-responsive cells allows the virus to relax from these constraints and find a new genome consensus within its sequence space. IMPORTANCE: In natural virus infections, the production of interferons leads to an antiviral state in cells that effectively limits virus replication. The interferon response places considerable selection pressure on viruses, and they have evolved a variety of ways to evade it. Although the influenza virus NS1 protein is a powerful interferon antagonist, the contributions of other viral genes to interferon evasion have not been well characterized. Here, we examined the effects of alleviating the selection pressure exerted by interferon by serially passaging influenza viruses in cells unable to respond to interferon. Viruses that grew to high titers had mutations at many normally conserved positions in nearly all genes and were not restricted to the NS1 gene. Our results demonstrate that influenza viruses have fine-tuned their entire genomes to evade the interferon response, and by removing interferon-mediated constraints, viruses can mutate at genome positions normally restricted by the interferon response.

Complementary roles of tumor necrosis factor alpha and interferon gamma in inducible microglial nitric oxide generation.

Proinflammatory cytokines and pathogen components activate microglia to release several substances such as nitric oxide (NO) produced after the induction of type II nitric oxide synthase (iNOS). The present study was designed to elucidate the interaction between the proinflammatory cytokines interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) on iNOS expression and NO production in microglial cells. In primary mouse microglial cells exposure to IFN-gamma (5 and 10 ng/ml; 48 h) or TNF-alpha (20 ng/ml; 48 h) alone were unable to induce iNOS expression; however, when cells were exposed to both cytokines together, the expression of this enzyme and the NO production in culture media were found significantly increased. In the BV-2 microglial cell line, IFN-gamma and TNF-alpha were shown to cooperate in nuclear factor kappa B (NF-kappa B) activation, an essential transcription factor for iNOS gene transcription. Importantly, IFN-gamma induced NF-kappa B binding to DNA was totally dependent on the endogenous TNF-alpha released via MEK/ERK signalling pathway. Thus, exposure of BV-2 cells to IFN-gamma in the presence of the selective MEK inhibitor U0126 or a neutralizing anti-TNF-alpha antibody significantly reduced IFN-gamma dependent NF-kappa B activation and iNOs expression. In addition, by activating the Jak/STAT pathway IFN-gamma potentiated TNF-alpha induced NF-kappa B binding to DNA and activated additional transcription factors (i.e. IRF-1) known to be essential for iNOs gene expression. The present findings demonstrate that the proinflammatory cytokines IFN-gamma and TNF-alpha have complementary roles on iNOS expression in microglial cells and this might be relevant to understand the molecular mechanisms of microglial activation associated with the pathogenesis of several neuroinflammatory disorders in which increased levels of IFN-gamma and TNF-alpha have been reported.

Vascular endothelial growth factor protects spinal cord motoneurons against glutamate-induced excitotoxicity via phosphatidylinositol 3-kinase.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective death of motoneurons. Recently, vascular endothelial growth factor (VEGF) has been identified as a neurotrophic factor and has been implicated in the mechanisms of pathogenesis of ALS and other neurological diseases. The potential neuroprotective effects of VEGF in a rat spinal cord organotypic culture were studied in a model of chronic glutamate excitotoxicity in which glutamate transporters are inhibited by threohydroxyaspartate (THA). Particularly, we focused on the effects of VEGF in the survival and vulnerability to excitotoxicity of spinal cord motoneurons. VEGF receptor-2 was present on spinal cord neurons, including motoneurons. Chronic (3 weeks) treatment with THA induced a significant loss of motoneurons that was inhibited by co-exposure to VEGF (50 ng/mL). VEGF activated the phosphatidylinositol 3-kinase/Akt (PI3-K/Akt) signal transduction pathway in the spinal cord cultures, and the effect on motoneuron survival was fully reversed by the specific PI3-K inhibitor, LY294002. VEGF also prevented the down-regulation of Bcl-2 and survivin, two proteins implicated in anti-apoptotic and/or anti-excitotoxic effects, after THA exposure. Together, these findings indicate that VEGF has neuroprotective effects in rat spinal cord against chronic glutamate excitotoxicity by activating the PI3-K/Akt signal transduction pathway and also reinforce the hypothesis of the potential therapeutic effects of VEGF in the prevention of motoneuron degeneration in human ALS.

Stimulation of mitogen-activated protein kinase kinases (MEK1/2) by mu-, delta- and kappa-opioid receptor agonists in the rat brain: regulation by chronic morphine and opioid withdrawal.

Opioid addiction modulates the extracellular signal-regulated kinase (ERK) leading to synaptic plasticity in the brain. ERK1/2 are stimulated by mitogen-activated protein kinase kinases (MEK1/2), but little is known about the regulation of MEK activity by opioid drugs. This study was designed to assess the acute effects of selective mu-, delta-, and kappa-opioid receptor agonists, as well as those induced by chronic morphine and opioid withdrawal, on the content of phosphorylated MEK1/2 in the rat brain. Sufentanil (1-30 microg/kg, 30-120 min) induced dose- and time-dependent increases in MEK1/2 phosphorylation in the cerebral cortex and corpus striatum (30-177%) through a naloxone-sensitive mechanism. Morphine (100 mg/kg, 2 h) also augmented MEK1/2 phosphorylation in the both brain regions (50-70%). Similarly, the selective delta-opioid receptor agonist SNC-80 (10 mg/kg, 30 min) increased MEK1/2 activity in the cortex (60%) that was antagonized by naltrindole. In contrast, the selective kappa-opioid receptor agonist (-)-U50488H (10 mg/kg, 30-120 min) did not modify significantly MEK1/2 phosphorylation in the cortex. Chronic morphine (10-100 mg/kg, 5 days) was not associated with alterations in the content of phosphorylated MEK1/2 in the brain (induction of tachyphylaxis to the acute effects). In morphine-dependent rats, however, naloxone (2 mg/kg)-precipitated withdrawal (2-6 h) induced robust increases in MEK1/2 phosphorylation in cortex (27-49%) and striatum (83-123%). Spontaneous opioid withdrawal (24 h) in morphine-dependent rats did not alter MEK1/2 activity in the brain. The findings may be relevant in the context of the pivotal role played by the MEK/ERK pathway in various long-lasting forms of synaptic plasticity associated with opioid addiction.