Analysis of Oct4-dependent transcriptional networks regulating self-renewal and pluripotency in human embryonic stem cells

The POU domain transcription factor OCT4 is a key regulator of pluripotency in the early mammalian embryo and is highly expressed in the inner cell mass of the blastocyst. Consistent with its essential role in maintaining pluripotency, Oct4 expression is rapidly downregulated during formation of the trophoblast lineage. To enhance our understanding of the molecular basis of this differentiation event in humans, we used a functional genomics approach involving RNA interference-mediated suppression of OCT4 function in a human ESC line and analysis of the resulting transcriptional profiles to identify OCT4-dependent genes in human cells. We detected altered expression of >1,000 genes, including targets regulated directly by OCT4 either positively (NANOG, SOX2, REX1, LEFTB, LEFTA/EBAF DPPA4, THY1, and TDGF1) or negatively (CDX2, EOMES, BMP4, TBX18, Brachyury [T], DKK1, HLX1, GATA6, ID2, and DLX5), as well as targets for the OCT4-associated stem cell regulators SOX2 and NANOG. Our data set includes regulators of ACTIVIN, BMP, fibroblast growth factor, and WNT signaling. These pathways are implicated in regulating human ESC differentiation and therefore further validate the results of our analysis. In addition, we identified a number of differentially expressed genes that are involved in epigenetics, chromatin remodeling, apoptosis, and metabolism that may point to underlying molecular mechanisms that regulate pluripotency and trophoblast differentiation in humans. Significant concordance between this data set and previous comparisons between inner cell mass and trophectoderm in human embryos indicates that the study of human ESC differentiation in vitro represents a useful model of early embryonic differentiation in humans

Nijmegen Breakage Syndrome fibroblasts and iPSCs: cellular models for uncovering disease-associated signaling pathways and establishing a screening platform for anti-oxidants.

Nijmegen Breakage Syndrome (NBS) is associated with cancer predisposition, premature aging, immune deficiency, microcephaly and is caused by mutations in the gene coding for NIBRIN (NBN) which is involved in DNA damage repair. Dermal-derived fibroblasts from NBS patients were reprogrammed into induced pluripotent stem cells (iPSCs) in order to bypass premature senescence. The influence of antioxidants on intracellular levels of ROS and DNA damage were screened and it was found that EDHB-an activator of the hypoxia pathway, decreased DNA damage in the presence of high oxidative stress. Furthermore, NBS fibroblasts but not NBS-iPSCs were found to be more susceptible to the induction of DNA damage than their healthy counterparts. Global transcriptome analysis comparing NBS to healthy fibroblasts and NBS-iPSCs to embryonic stem cells revealed regulation of P53 in NBS fibroblasts and NBS-iPSCs. Cell cycle related genes were down-regulated in NBS fibroblasts. Furthermore, oxidative phosphorylation was down-regulated and glycolysis up-regulated specifically in NBS-iPSCs compared to embryonic stem cells. Our study demonstrates the utility of NBS-iPSCs as a screening platform for anti-oxidants capable of suppressing DNA damage and a cellular model for studying NBN de-regulation in cancer and microcephaly

JNK Signalling Regulates Self-Renewal of Proliferative Urine-Derived Renal Progenitor Cells via Inhibition of Ferroptosis

With a global increase in chronic kidney disease patients, alternatives to dialysis and organ transplantation are needed. Stem cell-based therapies could be one possibility to treat chronic kidney disease. Here, we used multipotent urine-derived renal progenitor cells (UdRPCs) to study nephrogenesis. UdRPCs treated with the JNK inhibitor—AEG3482 displayed decreased proliferation and downregulated transcription of cell cycle-associated genes as well as the kidney progenitor markers—SIX2, SALL1 and VCAM1. In addition, levels of activated SMAD2/3, which is associated with the maintenance of self-renewal in UdRPCs, were decreased. JNK inhibition resulted in less efficient oxidative phosphorylation and more lipid peroxidation via ferroptosis, an iron-dependent non-apoptotic cell death pathway linked to various forms of kidney disease. Our study is the first to describe the importance of JNK signalling as a link between maintenance of self-renewal and protection against ferroptosis in SIX2-positive renal progenitor cells

Human Amniocytes Are Receptive to Chemically Induced Reprogramming to Pluripotency

Restoring pluripotency using chemical compounds alone would be a major step forward in developing clinical-grade pluripotent stem cells, but this has not yet been reported in human cells. We previously demonstrated that VPA_ AFS cells, human amniocytes cultivated with valproic acid (VPA) acquired functional pluripotency while remaining distinct from human embryonic stem cells (hESCs), questioning the relationship between the modulation of cell fate and molecular regulation of the pluripotency network. Here, we used single-cell analysis and functional assays to reveal that VPA treatment resulted in a homogeneous population of self-renewing non-transformed cells that fulfill the hallmarks of pluripotency, i.e., a short G1 phase, a dependence on glycolytic metabolism, expression of epigenetic modifications on histones 3 and 4, and reactivation of endogenous OCT4 and downstream targets at a lower level than that observed in hESCs. Mechanistic insights into the process of VPA-induced reprogramming revealed that it was dependent on OCT4 promoter activation, which was achieved independently of the PI3K (phosphatidylinositol 3-kinase)/ AKT/ mTOR (mammalian target of rapamycin) pathway or GSK3 beta inhibition but was concomitant with the presence of acetylated histones H3K9 and H3K56, which promote pluripotency. Our data identify, for the first time, the pluripotent transcriptional and molecular signature and metabolic status of human chemically induced pluripotent stem cells

Cross-species hybridisation of human and bovine orthologous genes on high density cDNA microarrays

BACKGROUND: Cross-species gene-expression comparison is a powerful tool for the discovery of evolutionarily conserved mechanisms and pathways of expression control. The usefulness of cDNA microarrays in this context is that broad areas of homology are compared and hybridization probes are sufficiently large that small inter-species differences in nucleotide sequence would not affect the analytical results. This comparative genomics approach would allow a common set of genes within a specific developmental, metabolic, or disease-related gene pathway to be evaluated in experimental models of human diseases. The objective of this study was to investigate the feasibility and reproducibility of cross-species analysis employing a human cDNA microarray as probe. RESULTS: As a proof of principle, total RNA derived from human and bovine fetal brains was used as a source of labelled targets for hybridisation onto a human cDNA microarray composed of 349 characterised genes. Each gene was spotted 20 times representing 6,980 data points thus enabling highly reproducible spot quantification. Employing high stringency hybridisation and washing conditions, followed by data analysis, revealed slight differences in the expression levels and reproducibility of the signals between the two species. We also assigned each of the genes into three expression level categories- i.e. high, medium and low. The correlation co-efficient of cross hybridisation between the orthologous genes was 0.94. Verification of the array data by semi-quantitative RT-PCR using common primer sequences enabled co-amplification of both human and bovine transcripts. Finally, we were able to assign gene names to previously uncharacterised bovine ESTs. CONCLUSIONS: Results of our study demonstrate the harnessing and utilisation power of comparative genomics and prove the feasibility of using human microarrays to facilitate the identification of co-expressed orthologous genes in common tissues derived from different species

Níveis de fósforo e potássio em relação a diferentes coberturas de solo e deferentes estratificações de profundidades de coletas de solo.

O objetivo deste trabalho foi avaliar a interferência de diferentes coberturas de solo nos níveis de Fósforo e de Potássio em diferentes estratificações de coleta de solo, e mensurar qual a diferença entre as diferentes coberturas e se as profundidades de coleta são influenciadas pelas coberturas.FertBio 2010

Laser-stimulated Synthesis of Large Nanostructured Fractal Silver Aggregates

A Laser-stimulated synthesis of large silver nanoaggregates consisting of hundreds to thousands of nanoparticles is investigated. It is shown that the morphology of the synthesized nanostructures can be controlled with light and monitored via the evolution of the colloid absorption spectra. A solution method is demonstrated that enables production of a bulk amount of metal nanoaggregates, which are of paramount importance for subwavelength concentration and dramatic enhancement of the electromagnetically-induced processes at the nanoscale.Comment: 10 pages, 8 figures, 13 reference

Patogenicidade de isolados de Fusarium spp. EM SOJA/ Pathogenicity of Fusarium spp. isolates at soybean.

Diferentes espécies de fungos habitantes do solo, incluindo Fusarium, Rhizoctonia, Sclerotium, Macrophomina e Pythium infectam culturas de importância econômica e causam consideráveis perdas. Na área experimental da Embrapa Agrossilvipastoril, foram retiradas amostras durante a estação chuvosa do ano, na safra 2015/16, para caracterização da população de fungos fitoparasitas habitantes do solo, nos diferentes sistemas de integração lavoura pecuária floresta (iLPF). Com o intuito de verificar a patogenicidade de isolados de Fusarium spp obtidos, conduziu-se o teste de patogenicidade em soja. Vasos contendo uma mistura autoclavada de solo e areia (1:3), foram semeados com a variedade TMG 132. Após 10 dias da semeadura, efetuou-se o desbaste, deixando-se duas plantas por vaso para a inoculação. Seis isolados de Fusarium spp (40P1; 02B, 20P1, 08P5, 35B e 10B) foram selecionados aleatoriamente para o teste. A inoculação do fungo foi realizada pela técnica do palito de dente. Os palitos foram esterilizados e colocados sobre placas de Petri contendo B.D.A, onde os isolados foram semeados. Aos 7 dias após a incubação, o micélio colonizou os palitos. 15 dias após a semeadura procedeu-se a inoculação, introduzindo o palito colonizado no coleto da planta. A testemunha recebeu palitos de dente não inoculados. Após inoculação as plantas foram deixadas em câmara úmida por 48 hs. Em seguida foram mantidas em sala climatizada com temperatura de 25oC e com irrigação manual. Após 21 dias da inoculação procedeu-se a avaliação, através de observação dos sintomas. A avaliação foi qualitativa, constatando-se ou não a ocorrência de murcha. Dos seis isolados testados (40P1; 02B, 20P1, 08P5, 35B e 10B), três (02B, 20P1 e 08P5), foram patogênicos às plantas de soja. A próxima etapa da pesquisa é a identificação, através de técnicas moleculares, das espécies dos isolados de Fusarium spp. utilizados nesse trabalho

Carbono da biomassa microbiana e respiração basal em solo sob integração lavoura pecuária floresta.

Este trabalho objetivou avaliar a atividade da comunidade microbiana total de solos sob o sistema de integração lavoura pecuária floresta, por meio dos teores de Carbono da Biomassa Microbiana e Respiração Basal.FertBio 2010

Produtividade da soja cultivada sobre diferentes coberturas de solo, e diferentes adubações.

O objetivo deste trabalho foi avaliar a interferência de diferentes coberturas de solo nas características agronômicas e na produtividade da cultura da soja, e mensurar a necessidade da adubação química da cultura da soja quando esta for cultivada sob diferentes coberturas.FertBio 2010