Application of multivariate data analysis for the classification of two dimensional gel images in Neuroproteomics

Two-dimensional gel electrophoresis (2DE) still plays a key role in proteomics for exploring the protein content of complex biological mixtures. However, the development of fully automatic strategies in extracting interpretable information from gel images is still a challenging task. In this work, we present a computational strategy aiming at an automatic classification of the discriminant patterns emerging from separation images intended as fingerprints of the correspondent biological conditions. The method was applied to gel images acquired in a study on motor neuron diseases: 33 2DE maps generated from samples of cerebrospinal fluid were processed (26 pathologic and 7 control subjects). Quantitative image descriptors were extracted and fitted to a partial least squares-discriminant analysis (PLSDA) assessing the chance to classify the samples. Moreover, the model was able to identify gel areas that most differ through the clinical categories. Combining multivariate statistical techniques with 2DEs may represent a valid tool to extract informative protein patterns. This kind of approach can contribute to the development of a system of screening to discriminate different clinical conditions on the basis of the overall patterns emerging from the maps, representing a useful complementary analysis in the routine of a proteomic laboratory. © 2011 Mazzara S, et al

Improved Label-Free LC-MS Analysis by Wavelet-Based Noise Rejection

Label-free LC-MS analysis allows determining the differential expression level of proteins in multiple samples, without the use of stable isotopes. This technique is based on the direct comparison of multiple runs, obtained by continuous detection in MS mode. Only differentially expressed peptides are selected for further fragmentation, thus avoiding the bias toward abundant peptides typical of data-dependent tandem MS. The computational framework includes detection, alignment, normalization and matching of peaks across multiple sets, and several software packages are available to address these processing steps. Yet, more care should be taken to improve the quality of the LC-MS maps entering the pipeline, as this parameter severely affects the results of all downstream analyses. In this paper we show how the inclusion of a preprocessing step of background subtraction in a common laboratory pipeline can lead to an enhanced inclusion list of peptides selected for fragmentation and consequently to better protein identification

HIF factors cooperate with PML-RARα to promote acute promyelocytic leukemia progression and relapse

Acute promyelocytic leukemia (APL) is epitomized by the chromosomal translocation t(15;17) and the resulting oncogenic fusion protein PML-RARα. Although acting primarily as a transcriptional repressor, PML-RARα can also exert functions of transcriptional co-activation. Here, we find that PML-RARα stimulates transcription driven by HIF factors, which are critical regulators of adaptive responses to hypoxia and stem cell maintenance. Consistently, HIF-related gene signatures are upregulated in leukemic promyelocytes from APL patients compared to normal promyelocytes. Through in vitro and in vivo studies, we find that PML-RARα exploits a number of HIF-1α-regulated pro-leukemogenic functions that include cell migration, bone marrow (BM) neo-angiogenesis and self-renewal of APL blasts. Furthermore, HIF-1α levels increase upon treatment of APL cells with all-trans retinoic acid (ATRA). As a consequence, inhibiting HIF-1α in APL mouse models delays leukemia progression and exquisitely synergizes with ATRA to eliminate leukemia-initiating cells (LICs)

Hypoxia inducible factor-1β regulates a pro-invasive phenotype in acute monocytic leukemia

Hypoxia inducible transcription factors (HIFs) are the main regulators of adaptive responses to hypoxia and are often activated in solid tumors, but their role in leukemia is less clear. In acute myeloid leukemia (AML), in particular, controversial new findings indicate that HIF-1β can act either as an oncogene or a tumor suppressor gene, and this may depend on the stage of leukemia development and/or the AML sub-type. In this study, we find that HIF-1β promotes leukemia progression in the acute monocytic leukemia sub-type of AML through activation of an invasive phenotype. By applying a list of validated HIF-1β-target genes to different AML sub-types, we identified a HIF-1β signature that typifies acute monocytic leukemia when compared with all other AML sub-types. We validated expression of this signature in cell lines and primary cells from AML patients. Interestingly, this signature is enriched for genes that control cell motility at different levels. As a consequence, inhibiting HIF- 1β impaired leukemia cell migration, chemotaxis, invasion and transendothelial migration in vitro, and this resulted in impaired bone marrow homing and leukemia progression in vivo. Our data suggest that in acute monocytic leukemia an active HIF-1β-dependent pro-invasive pathway mediates the ability of leukemic cells to migrate and invade extramedullary sites and may be targeted to reduce leukemia dissemination

A complex selection signature at the human AVPR1B gene

<p>Abstract</p> <p>Background</p> <p>The vasopressin receptor type 1b (<it>AVPR1B</it>) is mainly expressed by pituitary corticotropes and it mediates the stimulatory effects of AVP on ACTH release; common <it>AVPR1B </it>haplotypes have been involved in mood and anxiety disorders in humans, while rodents lacking a functional receptor gene display behavioral defects and altered stress responses.</p> <p>Results</p> <p>Here we have analyzed the two exons of the gene and the data we present suggest that <it>AVPR1B </it>has been subjected to natural selection in humans. In particular, analysis of exon 2 strongly suggests the action of balancing selection in African populations and Europeans: the region displays high nucleotide diversity, an excess of intermediate-frequency alleles, a higher level of within-species diversity compared to interspecific divergence and a genealogy with common haplotypes separated by deep branches. This relatively unambiguous situation coexists with unusual features across exon 1, raising the possibility that a nonsynonymous variant (Gly191Arg) in this region has been subjected to directional selection.</p> <p>Conclusion</p> <p>Although the underlying selective pressure(s) remains to be identified, we consider this to be among the first documented examples of a gene involved in mood disorders and subjected to natural selection in humans; this observation might add support to the long-debated idea that depression/low mood might have played an adaptive role during human evolution.</p

Protein fingerprints of cultured CA3-CA1 hippocampal neurons: comparative analysis of the distribution of synaptosomal and cytosolic proteins

<p>Abstract</p> <p>Background</p> <p>All studies aimed at understanding complex molecular changes occurring at synapses face the problem of how a complete view of the synaptic proteome and of its changes can be efficiently met. This is highly desirable when synaptic plasticity processes are analyzed since the structure and the biochemistry of neurons and synapses get completely reshaped. Because most molecular studies of synapses are nowadays mainly or at least in part based on protein extracts from neuronal cultures, this is not a feasible option: these simplified versions of the brain tissue on one hand provide an homogeneous pure population of neurons but on the other yield only tiny amounts of proteins, many orders of magnitude smaller than conventional brain tissue. As a way to overcome this limitation and to find a simple way to screen for protein changes at cultured synapses, we have produced and characterized two dimensional electrophoresis (2DE) maps of the synaptic proteome of CA3-CA1 hippocampal neurons in culture.</p> <p>Results</p> <p>To obtain 2D maps, hippocampal cultures were mass produced and after synaptic maturation, proteins were extracted following subfractionation procedures and separated by 2D gel electrophoresis. Similar maps were obtained for the crude cytosol of cultured neurons and for synaptosomes purified from CA3-CA1 hippocampal tissue. To efficiently compare these different maps some clearly identifiable reference points were molecularly identified by mass spectrometry and immunolabeling methods. This information was used to run a differential analysis and establish homologies and dissimilarities in these 2D protein profiles.</p> <p>Conclusion</p> <p>Because reproducible fingerprints of cultured synapses were clearly obtained, we believe that our mapping effort could represent a simple tool to screen for protein expression and/or protein localization changes in CA3-CA1 hippocampal neurons following plasticity.</p

Epileptiform Activity and Cognitive Deficits in SNAP-25+/− Mice are Normalized by Antiepileptic Drugs

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a protein that participates in the regulation of synaptic vesicle exocytosis through the formation of the soluble NSF attachment protein receptor complex and modulates voltage-gated calcium channels activity. The Snap25 gene has been associated with schizophrenia, attention deficit hyperactivity disorder, and bipolar disorder, and lower levels of SNAP-25 have been described in patients with schizophrenia. We used SNAP-25 heterozygous (SNAP-25+/−) mice to investigate at which extent the reduction of the protein levels affects neuronal network function and mouse behavior. As interactions of genotype with the specific laboratory conditions may impact behavioral results, the study was performed through a multilaboratory study in which behavioral tests were replicated in at least 2 of 3 distinct European laboratories. Reductions of SNAP-25 levels were associated with a moderate hyperactivity, which disappeared in the adult animals, and with impaired associative learning and memory. Electroencephalographic recordings revealed the occurrence of frequent spikes, suggesting a diffuse network hyperexcitability. Consistently, SNAP-25+/− mice displayed higher susceptibility to kainate-induced seizures, paralleled by degeneration of hilar neurons. Notably, both EEG profile and cognitive defects were improved by antiepileptic drugs. These results indicate that reduction of SNAP-25 expression is associated to generation of epileptiform discharges and cognitive dysfunctions, which can be effectively treated by antiepileptic drug

A systems biology approach to investigate the mechanism of action of trabectedin in a model of myelomonocytic leukemia

This study was designed to investigate the mode of action of trabectedin in myelomonocytic leukemia cells by applying systems biology approaches to mine gene expression profiling data and pharmacological assessment of the cellular effects. Significant enrichment was found in regulons of target genes inferred for specific transcription factors, among which MAFB was the most upregulated after treatment and was central in the transcriptional network likely to be relevant for the specific therapeutic effects of trabectedin against myelomonocytic cells. Using the Connectivity Map, similarity among transcriptional signatures elicited by treatment with different compounds was investigated, showing a high degree of similarity between transcriptional signatures of trabectedin and those of the topoisomerase I inhibitor, irinotecan, and an anti-dopaminergic antagonist, thioridazine. The study highlights the potential importance of systems biology approaches to generate new hypotheses that are experimentally testable to define the specificity of the mechanism of action of drugs.The Pharmacogenomics Journal advance online publication, 13 December 2016; doi:10.1038/tpj.2016.76

Association between SNAP-25 gene polymorphisms and cognition in autism: functional consequences and potential therapeutic strategies.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is involved in different neuropsychiatric disorders, including schizophrenia and attention-deficit/hyperactivity disorder. Consistently, SNAP-25 polymorphisms in humans are associated with hyperactivity and/or with low cognitive scores. We analysed five SNAP-25 gene polymorphisms (rs363050, rs363039, rs363043, rs3746544 and rs1051312) in 46 autistic children trying to correlate them with Childhood Autism Rating Scale and electroencephalogram (EEG) abnormalities. The functional effects of rs363050 single-nucleotide polymorphism (SNP) on the gene transcriptional activity, by means of the luciferase reporter gene, were evaluated. To investigate the functional consequences that SNAP-25 reduction may have in children, the behaviour and EEG of SNAP-25(+/-) adolescent mice (SNAP-25(+/+)) were studied. Significant association of SNAP-25 polymorphism with decreasing cognitive scores was observed. Analysis of transcriptional activity revealed that SNP rs363050 encompasses a regulatory element, leading to protein expression decrease. Reduction of SNAP-25 levels in adolescent mice was associated with hyperactivity, cognitive and social impairment and an abnormal EEG, characterized by the occurrence of frequent spikes. Both EEG abnormalities and behavioural deficits were rescued by repeated exposure for 21 days to sodium salt valproate (VLP). A partial recovery of SNAP-25 expression content in SNAP-25(+/-) hippocampi was also observed by means of western blotting. A reduced expression of SNAP-25 is responsible for the cognitive deficits in children affected by autism spectrum disorders, as presumably occurring in the presence of rs363050(G) allele, and for behavioural and EEG alterations in adolescent mice. VLP treatment could result in novel therapeutic strategies

Nivel de la ejecución presupuestal y su incidencia en el logro del programa de incentivos en la Municipalidad Distrital de Baños del Inca durante el periodo 2016

RESUMEN: El presente trabajo tiene como objetivo evaluar el comportamiento de la ejecución presupuestal en el cumplimiento de las metas institucionales dentro del ejercicio presupuestal referidas al Presupuesto Institucional de Apertura, Presupuesto Institucional Modificado, presupuesto ejecutada por fuentes de financiamiento, categoría presupuestal y proyectos de inversión social. Es por ello la importancia del presente trabajo de investigación para mostrar las dificultades con respecto a la ejecución del presupuesto del programa de Incentivos de la Municipalidad Distrital de Baños del Inca, que conllevan a la no satisfacción de acuerdo a las necesidades de la población y por ende no logra alcanzar las metas y objetivos de dicha municipalidad. Para la investigación se ha utilizado los métodos deductivo, descriptivo, analítico y comparativo para evaluar la problemática de la ejecución presupuestal. El objetivo general de la investigación es: Evaluar el nivel de cumplimiento de los planes de ejecución presupuestal y su incidencia en el logro del programa de incentivos de la Municipalidad Distrital de Baños del Inca en el periodo 2016. PALABRAS CLAVE: ejecución presupuestal, logro del Programa de Incentivos; Municipalidad Distrital de Baños del Inca