Human brain harbors single nucleotide somatic variations in functionally relevant genes possibly mediated by oxidative stress

Somatic variation in DNA can cause cells to deviate from the preordained genomic path in both disease and healthy conditions. Here, using exome sequencing of paired tissue samples, we show that the normal human brain harbors somatic single base variations measuring up to 0.48% of the total variations. Interestingly, about 64% of these somatic variations in the brain are expected to lead to non-synonymous changes, and as much as 87% of these represent G:C>T:A transversion events. Further, the transversion events in the brain were mostly found in the frontal cortex, whereas the corpus callosum from the same individuals harbors the reference genotype. We found a significantly higher amount of 8-OHdG (oxidative stress marker) in the frontal cortex compared to the corpus callosum of the same subjects (p<0.01), correlating with the higher G:C>T:A transversions in the cortex. We found significant enrichment for axon guidance and related pathways for genes harbouring somatic variations. This could represent either a directed selection of genetic variations in these pathways or increased susceptibility of some loci towards oxidative stress. This study highlights that oxidative stress possibly influence single nucleotide somatic variations in normal human brain

Co-expression network of neural-differentiation genes shows specific pattern in schizophrenia

Background: Schizophrenia is a neurodevelopmental disorder with genetic and environmental factors contributing to its pathogenesis, although the mechanism is unknown due to the difficulties in accessing diseased tissue during human neurodevelopment. The aim of this study was to find neuronal differentiation genes disrupted in schizophrenia and to evaluate those genes in post-mortem brain tissues from schizophrenia cases and controls. Methods: We analyzed differentially expressed genes (DEG), copy number variation (CNV) and differential methylation in human induced pluripotent stem cells (hiPSC) derived from fibroblasts from one control and one schizophrenia patient and further differentiated into neuron (NPC). Expression of the DEG were analyzed with microarrays of post-mortem brain tissue (frontal cortex) cohort of 29 schizophrenia cases and 30 controls. A Weighted Gene Co-expression Network Analysis (WGCNA) using the DEG was used to detect clusters of co-expressed genes that werenon-conserved between adult cases and controls brain samples. Results: We identified methylation alterations potentially involved with neuronal differentiation in schizophrenia, which displayed an over-representation of genes related to chromatin remodeling complex (adjP = 0.04). We found 228 DEG associated with neuronal differentiation. These genes were involved with metabolic processes, signal transduction, nervous system development, regulation of neurogenesis and neuronal differentiation. Between adult brain samples from cases and controls there were 233 DEG, with only four genes overlapping with the 228 DEG, probably because we compared single cell to tissue bulks and more importantly, the cells were at different stages of development. The comparison of the co-expressed network of the 228 genes in adult brain samples between cases and controls revealed a less conserved module enriched for genes associated with oxidative stress and negative regulation of cell differentiation. Conclusion: This study supports the relevance of using cellular approaches to dissect molecular aspects of neurogenesis with impact in the schizophrenic brain. We showed that, although generated by different approaches, both sets of DEG associated to schizophrenia were involved with neocortical development. The results add to the hypothesis that critical metabolic changes may be occurring during early neurodevelopment influencing faulty development of the brain and potentially contributing to further vulnerability to the illness.We thank the patients, doctors and nurses involved with sample collection and the Stanley Medical Research Institute. This research was supported by either Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq #17/2008) and Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ). MM (CNPq 304429/2014-7), ACT (FAPESP 2014/00041-1), LL (CAPES 10682/13-9) HV (CAPES) and BP (PPSUS 137270) were supported by their fellowshipsinfo:eu-repo/semantics/publishedVersio

Human cerebral organoids and fetal brain tissue share proteomic similarities

The limited access to functional human brain tissue has led to the development of stem cell-based alternative models. The differentiation of human pluripotent stem cells into cerebral organoids with self-organized architecture has created novel opportunities to study the early stages of the human cerebral formation. Here we applied state-of-the-art label-free shotgun proteomics to compare the proteome of stem cell-derived cerebral organoids to the human fetal brain. We identified 3,073 proteins associated with different developmental stages, from neural progenitors to neurons, astrocytes, or oligodendrocytes. The major protein groups are associated with neurogenesis, axon guidance, synaptogenesis, and cortical brain development. Glial cell proteins related to cell growth and maintenance, energy metabolism, cell communication, and signaling were also described. Our data support the variety of cells and neural network functional pathways observed within cell-derived cerebral organoids, confirming their usefulness as an alternative model. The characterization of brain organoid proteome is key to explore, in a dish, atypical and disrupted processes during brain development or neurodevelopmental, neurodegenerative, and neuropsychiatric diseases7CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFINANCIADORA DE ESTUDOS E PROJETOS - FINEPFUNDAÇÃO CARLOS CHAGAS FILHO DE AMPARO À PESQUISA DO ESTADO DO RIO DE JANEIRO - FAPERJFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPNão temNão temNão temNão tem14/21035-0; 16/07332-7; 13/08711-3; 14/10068-4JN, VS-C, and DM-D-S are supported by the São Paulo Research Foundation (FAPESP) grants 14/21035-0, 16/07332-7, 13/08711-3, and 14/10068-4. CS was recipient of a CAPES-FAPERJ Postdoc fellowship. Other funds are provided by the National Council for Scientific and Technological Development (CNPq), the Instituto Nacional de Ciência e Tecnologia de Neurociência Translacional (INCT-INNT), Foundation for Research Support in the State of Rio de Janeiro (FAPERJ), Coordination for the Improvement of Higher Education Personnel (CAPES), Brazilian Funding Authority for Studies and Projects (FINEP), and Brazilian Development Bank (BNDES

Análise bibliométrica da produção científica brasileira sobre Tecnologia de Construção e Edificações na base de dados Web of Science

Resumo Este estudo trata da análise da produção científica brasileira sobre tecnologias de construção e edificações por meio de indicadores bibliométricos. A área foi escolhida em razão de sua relevância para o desenvolvimento econômico do país. A bibliometria é um método de análise quantitativa para a pesquisa científica. Os dados elaborados por meio dos estudos bibliométricos mensuram a contribuição do conhecimento científico derivado das publicações em determinadas áreas. Os indicadores de produção são úteis para o planejamento e a execução de políticas públicas, e para o conhecimento da comunidade científica sobre o sistema em que está inserida. O objetivo deste trabalho é estudar a produção científica brasileira na área da construção e tecnologia da construção civil a partir da análise de artigos científicos publicados em periódicos e anais de congressos indexados na base de dados Web of Science (WoS). Os dados foram coletados utilizando como critério de seleção a categoria "Construction & Building Technology", sendo analisados dados de 910 artigos de autores brasileiros no período de 1982 a 2014. A interpretação dos dados permite confirmar o expressivo crescimento da produção científica brasileira e da grande contribuição da região Sudeste para essa evolução

Aneuploidy and Confined Chromosomal Mosaicism in the Developing Human Brain

BACKGROUND: Understanding the mechanisms underlying generation of neuronal variability and complexity remains the central challenge for neuroscience. Structural variation in the neuronal genome is likely to be one important mechanism for neuronal diversity and brain diseases. Large-scale genomic variations due to loss or gain of whole chromosomes (aneuploidy) have been described in cells of the normal and diseased human brain, which are generated from neural stem cells during intrauterine period of life. However, the incidence of aneuploidy in the developing human brain and its impact on the brain development and function are obscure. METHODOLOGY/PRINCIPAL FINDINGS: To address genomic variation during development we surveyed aneuploidy/polyploidy in the human fetal tissues by advanced molecular-cytogenetic techniques at the single-cell level. Here we show that the human developing brain has mosaic nature, being composed of euploid and aneuploid neural cells. Studying over 600,000 neural cells, we have determined the average aneuploidy frequency as 1.25-1.45% per chromosome, with the overall percentage of aneuploidy tending to approach 30-35%. Furthermore, we found that mosaic aneuploidy can be exclusively confined to the brain. CONCLUSIONS/SIGNIFICANCE: Our data indicates aneuploidization to be an additional pathological mechanism for neuronal genome diversification. These findings highlight the involvement of aneuploidy in the human brain development and suggest an unexpected link between developmental chromosomal instability, intercellural/intertissular genome diversity and human brain diseases

Protocol for the establishment of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells.

Serine integrases (Ints) are a family of site-specific recombinases (SSRs) encoded by some bacteriophages to integrate their genetic material into the genome of a host. Their ability to rearrange DNA sequences in different ways including inversion, excision, or insertion with no help from endogenous molecular machinery, confers important biotechnological value as genetic editing tools with high host plasticity. Despite advances in their use in prokaryotic cells, only a few Ints are currently used as gene editors in eukaryotes, partly due to the functional loss and cytotoxicity presented by some candidates in more complex organisms. To help expand the number of Ints available for the assembly of more complex multifunctional circuits in eukaryotic cells, this protocol describes a platform for the assembly and functional screening of serine-integrase-based genetic switches designed to control gene expression by directional inversions of DNA sequence orientation. The system consists of two sets of plasmids, an effector module and a reporter module, both sets assembled with regulatory components (as promoter and terminator regions) appropriate for expression in mammals, including humans, and plants. The complete method involves plasmid design, DNA delivery, testing and both molecular and phenotypical assessment of results. This platform presents a suitable workflow for the identification and functional validation of new tools for the genetic regulation and reprogramming of organisms with importance in different fields, from medical applications to crop enhancement, as shown by the initial results obtained. This protocol can be completed in 4 weeks for mammalian cells or up to 8 weeks for plant cells, considering cell culture or plant growth time.Na publicação: Eduardo O. Melo; Daniela M. Bittencourt; Elibio Rech

Normal Human Pluripotent Stem Cell Lines Exhibit Pervasive Mosaic Aneuploidy

Human pluripotent stem cell (hPSC) lines have been considered to be homogeneously euploid. Here we report that normal hPSC – including induced pluripotent - lines are karyotypic mosaics of euploid cells intermixed with many cells showing non-clonal aneuploidies as identified by chromosome counting, spectral karyotyping (SKY) and fluorescent in situ hybridization (FISH) of interphase/non-mitotic cells. This mosaic aneuploidy resembles that observed in progenitor cells of the developing brain and preimplantation embryos, suggesting that it is a normal, rather than pathological, feature of stem cell lines. The karyotypic heterogeneity generated by mosaic aneuploidy may contribute to the reported functional and phenotypic heterogeneity of hPSCs lines, as well as their therapeutic efficacy and safety following transplantation

The cyanobacterial saxitoxin exacerbates neural cell death and brain malformations induced by zika virus

The northeast (NE) region of Brazil commonly goes through drought periods, which favor cyanobacterial blooms, capable of producing neurotoxins with implications for human and animal health. The most severe dry spell in the history of Brazil occurred between 2012 and 2016. Coincidently, the highest incidence of microcephaly associated with the Zika virus (ZIKV) outbreak took place in the NE region of Brazil during the same years. In this work, we tested the hypothesis that saxitoxin (STX), a neurotoxin produced in South America by the freshwater cyanobacteria Raphidiopsis raciborskii, could have contributed to the most severe Congenital Zika Syndrome (CZS) profile described worldwide. Quality surveillance showed higher cyanobacteria amounts and STX occurrence in human drinking water sup-plies of NE compared to other regions of Brazil. Experimentally, we described that STX dou-bled the quantity of ZIKV-induced neural cell death in progenitor areas of human brain organoids, while the chronic ingestion of water contaminated with STX before and during gestation caused brain abnormalities in offspring of ZIKV-infected immunocompetent C57BL/6J mice. Our data indicate that saxitoxin-producing cyanobacteria is overspread in water reservoirs of the NE and might have acted as a co-insult to ZIKV infection in Brazil. These results raise a public health concern regarding the consequences of arbovirus outbreaks happening in areas with droughts and/or frequent freshwater cyanobacterial blooms.Fil: Pedrosa, Carolina da S. G.. D’Or Institute for Research and Education; BrasilFil: Souza, Leticia R. Q.. D’Or Institute for Research and Education; BrasilFil: Gomes, Tiago A.. Universidade Federal do Rio de Janeiro; Brasil. Instituto Oswaldo Cruz; BrasilFil: de Lima, Caroline V. F.. D’Or Institute for Research and Education; BrasilFil: Ledur, Pitia F.. D’Or Institute for Research and Education; BrasilFil: Karmirian, Karina. D’Or Institute for Research and Education; Brasil. Universidade Federal do Rio de Janeiro; BrasilFil: Barbeito Andrés, Jimena. Universidade Federal do Rio de Janeiro; Brasil. Universidad Nacional Arturo Jauretche. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos; ArgentinaFil: Costa, Marcelo do N.. Universidade Federal do Rio de Janeiro; BrasilFil: Higa, Luiza M.. Universidade Federal do Rio de Janeiro; BrasilFil: Rossi, Átila D.. Universidade Federal do Rio de Janeiro; BrasilFil: Bellio, Maria. Universidade Federal do Rio de Janeiro; BrasilFil: Tanuri, Amilcar. Universidade Federal do Rio de Janeiro; BrasilFil: Prata Barbosa, Arnaldo. D’Or Institute for Research and Education; BrasilFil: Tovar Moll, Fernanda. D’Or Institute for Research and Education; Brasil. Universidade Federal do Rio de Janeiro; BrasilFil: Garcez, Patricia P.. Universidade Federal do Rio de Janeiro; BrasilFil: Lara, Flavio A.. Instituto Oswaldo Cruz; BrasilFil: Molica, Renato J. R.. Universidad Federal Rural Pernambuco; BrasilFil: Rehen, Stevens K.. D’Or Institute for Research and Education; Brasil. Universidade Federal do Rio de Janeiro; Brasi

Genome-wide expression assay comparison across frozen and fixed postmortem brain tissue samples

<p>Abstract</p> <p>Background</p> <p>Gene expression assays have been shown to yield high quality genome-wide data from partially degraded RNA samples. However, these methods have not yet been applied to postmortem human brain tissue, despite their potential to overcome poor RNA quality and other technical limitations inherent in many assays. We compared cDNA-mediated annealing, selection, and ligation (DASL)- and <it>in vitro </it>transcription (IVT)-based genome-wide expression profiling assays on RNA samples from artificially degraded reference pools, frozen brain tissue, and formalin-fixed brain tissue.</p> <p>Results</p> <p>The DASL-based platform produced expression results of greater reliability than the IVT-based platform in artificially degraded reference brain RNA and RNA from frozen tissue-based samples. Although data associated with a small sample of formalin-fixed RNA samples were poor when obtained from both assays, the DASL-based platform exhibited greater reliability in a subset of probes and samples.</p> <p>Conclusions</p> <p>Our results suggest that the DASL-based gene expression-profiling platform may confer some advantages on mRNA assays of the brain over traditional IVT-based methods. We ultimately consider the implications of these results on investigations of neuropsychiatric disorders.</p