Effect of methylene blue on the genomic response to reperfusion injury induced by cardiac arrest and cardiopulmonary resuscitation in porcine brain

<p>Abstract</p> <p>Background</p> <p>Cerebral ischemia/reperfusion injury is a common secondary effect of cardiac arrest which is largely responsible for postresuscitative mortality. Therefore development of therapies which restore and protect the brain function after cardiac arrest is essential. Methylene blue (MB) has been experimentally proven neuroprotective in a porcine model of global ischemia-reperfusion in experimental cardiac arrest. However, no comprehensive analyses have been conducted at gene expression level.</p> <p>Methods</p> <p>Pigs underwent either untreated cardiac arrest (CA) or CA with subsequent cardiopulmonary resuscitation (CPR) accompanied with an infusion of saline or an infusion of saline with MB. Genome-wide transcriptional profiling using the Affymetrix porcine microarray was performed to 1) gain understanding of delayed neuronal death initiation in porcine brain during ischemia and after 30, 60 and 180 min following reperfusion, and 2) identify the mechanisms behind the neuroprotective effect of MB after ischemic injury (at 30, 60 and 180 min).</p> <p>Results</p> <p>Our results show that restoration of spontaneous circulation (ROSC) induces major transcriptional changes related to stress response, inflammation, apoptosis and even cytoprotection. In contrast, the untreated ischemic and anoxic insult affected only few genes mainly involved in intra-/extracellular ionic balance. Furthermore, our data show that the neuroprotective role of MB is diverse and fulfilled by regulation of the expression of soluble guanylate cyclase and biological processes accountable for inhibition of apoptosis, modulation of stress response, neurogenesis and neuroprotection.</p> <p>Conclusions</p> <p>Our results support that MB could be a valuable intervention and should be investigated as a therapeutic agent against neural damage associated with I/R injury induced by cardiac arrest.</p

Cancer Biomarker Discovery: The Entropic Hallmark

Background: It is a commonly accepted belief that cancer cells modify their transcriptional state during the progression of the disease. We propose that the progression of cancer cells towards malignant phenotypes can be efficiently tracked using high-throughput technologies that follow the gradual changes observed in the gene expression profiles by employing Shannon's mathematical theory of communication. Methods based on Information Theory can then quantify the divergence of cancer cells' transcriptional profiles from those of normally appearing cells of the originating tissues. The relevance of the proposed methods can be evaluated using microarray datasets available in the public domain but the method is in principle applicable to other high-throughput methods. Methodology/Principal Findings: Using melanoma and prostate cancer datasets we illustrate how it is possible to employ Shannon Entropy and the Jensen-Shannon divergence to trace the transcriptional changes progression of the disease. We establish how the variations of these two measures correlate with established biomarkers of cancer progression. The Information Theory measures allow us to identify novel biomarkers for both progressive and relatively more sudden transcriptional changes leading to malignant phenotypes. At the same time, the methodology was able to validate a large number of genes and processes that seem to be implicated in the progression of melanoma and prostate cancer. Conclusions/Significance: We thus present a quantitative guiding rule, a new unifying hallmark of cancer: the cancer cell's transcriptome changes lead to measurable observed transitions of Normalized Shannon Entropy values (as measured by high-throughput technologies). At the same time, tumor cells increment their divergence from the normal tissue profile increasing their disorder via creation of states that we might not directly measure. This unifying hallmark allows, via the the Jensen-Shannon divergence, to identify the arrow of time of the processes from the gene expression profiles, and helps to map the phenotypical and molecular hallmarks of specific cancer subtypes. The deep mathematical basis of the approach allows us to suggest that this principle is, hopefully, of general applicability for other diseases

Inhibition of human immunodeficiency virus type 1 replication by nuclear chimeric anti-HIV ribozymes in a human T lymphoblastoid cell line

Human immunodeficiency virus (HIV) infection represents one of the most challenging systems for gene therapy. Thanks to the extended knowledge of the molecular biology of the HIV life cycle, many different strategies have been developed including transdominant modifications of HIV proteins, RNA decoys, antisense RNA, ribozymes, and intracellular antibody fragments. In this paper, we have tested in a human T lymphoblastoid cell line the antiviral activity of ribozymes specifically designed to co-localize inside the nucleus with the Rev pre-mRNA before it is spliced and transported to the cytoplasm. This result was obtained by inserting the ribozyme in the spliceosomal U1 small nuclear RNA (snRNA) and in a derivative that has perfect complementarity with the 5' splice site of the Rev pre-mRNA. These ribozymes were tested in human T cell clones and were shown to be very efficient in inhibiting viral replication. Not only were the p24 levels in the culture medium drastically reduced but so were the intracellular HIV transcripts. Control disabled ribozymes enabled us to show the specificity of the ribozyme activity. Therefore, these constructs have potential utility for gene therapy of HIV-1 infection

Il fascicolo elettronico

Il volume raccoglie i contributi dei relatori che hanno partecipato al seminario di studi sul \u201cfascicolo elettronico\u201d, svoltosi a Roma il 9 luglio 2010 presso l\u2019Accademia dei Lincei, i quali hanno trattato i temi dell\u2019organizzazione dei contenuti digitali e della formazione della memoria digitale sotto tutti gli aspetti: sotto il profilo archivistico, precisando la natura, le caratteristiche e la funzione del fascicolo ai fini di una corretta gestione documentale; in rapporto ai vincoli imposti dalla normativa vigente; con riferimento alle raccomandazioni internazionali sulla gestione informatica dei documenti; sotto il profilo dell\u2019innovazione connessa alla particolare natura del fascicolo elettronico; sotto l\u2019aspetto applicativo, illustrando le funzioni di un ERMS (Electronic Record Management System)

Gene correction of integrin b4-dependent PA-JEB keratinocytes establishes a role for b4 tyrosines 1422 and 1440 in hemidesmosome assembly.

The cytoplasmic domain of beta4 integrin contains two pairs of fibronectin-like repeats separated by a connecting segment. The connecting segment harbors a putative tyrosine activation motif in which tyrosines 1422 and 1440 are phosphorylated in response to alpha6beta4 binding to laminin-5. Primary beta4-null keratinocytes, obtained from a newborn suffering from lethal junctional epidermolysis bullosa, were stably transduced with retroviruses carrying a full-length beta4 cDNA or a beta4 cDNA with phenylalanine substitutions at Tyr-1422 and Tyr-1440. Hemidesmosome assembly was evaluated on organotypic skin cultures. beta4-corrected keratinocytes were indistinguishable from normal cells in terms of alpha6beta4 expression, the localization of hemidesmosome components, and hemidesmosome structure and density, suggesting full genetic and functional correction of beta4-null keratinocytes. In cultures generated from beta4(Y1422F/Y1440F) keratinocytes, beta4 mutants as well as alpha6 integrin, HD1/plectin, and BP180 were not concentrated at the dermal-epidermal junction. Furthermore, the number of hemidesmosomes was strikingly reduced as compared with beta4-corrected keratinocytes. The rare hemidesmosomes detected in beta4(Y1422F/Y1440F) cells were devoid of sub-basal dense plates and of inner cytoplasmic plaques with keratin filament insertion. Collectively, our data demonstrate that the beta4 tyrosine activation motif is not required for the localization of alpha6beta4 at the keratinocyte plasma membrane but is essential for optimal assembly of bona fide hemidesmosomes