High-content screen in human pluripotent cells identifies miRNA-regulated pathways controlling pluripotency and differentiation

Background: By post-transcriptionally regulating multiple target transcripts, microRNAs (miRNAs or miR) play important biological functions. H1 embryonic stem cells (hESCs) and NTera-2 embryonal carcinoma cells (ECCs) are two of the most widely used human pluripotent model cell lines, sharing several characteristics, including the expression of miRNAs associated to the pluripotent state or with differentiation. However, how each of these miRNAs functionally impacts the biological properties of these cells has not been systematically evaluated. Methods: We investigated the effects of 31 miRNAs on NTera-2 and H1 hESCs, by transfecting miRNA mimics. Following 3-4 days of culture, cells were stained for the pluripotency marker OCT4 and the G2 cell-cycle marker Cyclin B1, and nuclei and cytoplasm were co-stained with Hoechst and Cell Mask Blue, respectively. By using automated quantitative fluorescence microscopy (i.e., high-content screening (HCS)), we obtained several morphological and marker intensity measurements, in both cell compartments, allowing the generation of a multiparametric miR-induced phenotypic profile describing changes related to proliferation, cell cycle, pluripotency, and differentiation. Results: Despite the overall similarities between both cell types, some miRNAs elicited cell-specific effects, while some related miRNAs induced contrasting effects in the same cell. By identifying transcripts predicted to be commonly targeted by miRNAs inducing similar effects (profiles grouped by hierarchical clustering), we were able to uncover potentially modulated signaling pathways and biological processes, likely mediating the effects of the microRNAs on the distinct groups identified. Specifically, we show that miR-363 contributes to pluripotency maintenance, at least in part, by targeting NOTCH1 and PSEN1 and inhibiting Notch-induced differentiation, a mechanism that could be implicated in na\uefve and primed pluripotent states. Conclusions: We present the first multiparametric high-content microRNA functional screening in human pluripotent cells. Integration of this type of data with similar data obtained from siRNA screenings (using the same HCS assay) could provide a large-scale functional approach to identify and validate microRNA-mediated regulatory mechanisms controlling pluripotency and differentiation

Plant litter dynamics in the forest-stream interface: Precipitation is a major control across tropical biomes

Riparian plant litter is a major energy source for forested streams across the world and its decomposition has repercussions on nutrient cycling, food webs and ecosystem functioning. However, we know little about plant litter dynamics in tropical streams, even though the tropics occupy 40% of the Earth's land surface. Here we investigated spatial and temporal (along a year cycle) patterns of litter inputs and storage in multiple streams of three tropical biomes in Brazil (Atlantic forest, Amazon forest and Cerrado savanna), predicting major differences among biomes in relation to temperature and precipitation regimes. Precipitation explained most of litter inputs and storage, which were generally higher in more humid biomes (litterfall: 384, 422 and 308 g m-2 y-1, storage: 55, 113 and 38 g m-2, on average in Atlantic forest, Amazon and Cerrado, respectively). Temporal dynamics varied across biomes in relation to precipitation and temperature, with uniform litter inputs but seasonal storage in Atlantic forest streams, seasonal inputs in Amazon and Cerrado streams, and aseasonal storage in Amazon streams. Our findings suggest that litter dynamics vary greatly within the tropics, but point to the major role of precipitation, which contrasts with the main influence of temperature in temperate areas. © 2017 The Author(s)

Stigma diversity in tropical legumes with considerations on stigma classification

The great stigma diversity in angiosperms implies a choice of criteria for stigma classification, which nowadays is characterized as dry (= little or no secretory surface and exudate retained by the cuticle and/or protein pellicle), wet (= conspicuous secretory surface, abundant in fluid exudate) and semidry (exudate retained by cuticle and/or protein pellicle). Despite being a very species-rich family, whose representatives exhibit a wide floral variation, no comparative studies of stigma diversity have been done for the whole Leguminosae. In order to assess the stigma morphological diversity in legumes and to evaluate the criteria used in the main classifications of the stigma, we compared the stigma morphology in 15 distinct legume lineages. In addition, we evaluated the stigma classification in other 152 legume species whose morphology was already described in the literature. Stigmas were removed from floral buds and flowers and processed for analyses under scanning electron and light microscopes. The stigma of the study legumes exhibits quite variable morphology, mainly concerning the diameter, the occurrence of an orifice or a furrow, the coating, the cellular composition, and the occurrence, chemical nature and release mechanism of the exudate. This diversity appears to be related mainly to the evolutionary history of the group and also to the selective pressures exerted by different types of pollen and pollinator. More conflicting criteria for stigma classification lie in defining the semidry type, found mostly in papilionoids. For better stigma classification we suggest that stigma morphology be evaluated at the time prior to anthesis, when the cuticle is not yet broken and the exudate is not exposed or drained. In addition, several techniques should be employed for a better classification801129CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP142876/2008-9; 301960/2009-7; 152759/2010-7sem informação2002/11834-5; 2008/57487-0; 2009/01057-

Características biométricas de mudas de Tabebula chrysotricha(standl.) formadas em diferentes substratos e soluções de fertirrigação, quando plantadas em campo Biometric characterizations of Tabebula chrysotricha (standl.) seedlings formed in different substrates and fertirrigation solutions, when planted in fields

Este trabalho objetivou estudar as características biométricas de plantas de ipê-amarelo (Tabebuia chrysotricha Standl.) formadas, na fase de viveiro, em função de quatro substratos, variando as soluções de fertirrigação. Para compor os substratos, foram utilizadas fibras de coco fibrosa e granulada, formando os tratamentos 100% fibrosa, 60% fibrosa e 40% granulada, 40% fibrosa e 60% granulada e 100% granulada. A adubação de base foi igual em todos os tratamentos, e as soluções de adubação variaram, a fim de se obterem soluções completas com condutividades elétricas de 1,06 dS m-1; 2,12 dS m-1; 3,2 dS m-1; e 4,24 dS m¹. As sementes foram colocadas diretamente nos tubetes (120 mL) contendo os respectivos substratos e receberam as fertirrigações por subsuperfície uma vez por semana, respeitando-se os tratamentos de adubação. Quando as mudas atingiram aproximadamente 20 cm de altura, elas foram realocadas sob tela de 9% de sombreamento, onde permaneceram até o plantio, em Taubaté, SP. Mudas produzidas em fibra de coco 100% granulada alcançaram, em campo, maiores alturas, diâmetros de coleto e número de folhas. Apesar das diferentes soluções de fertirrigação aplicadas, as alturas de parte aérea se igualaram a partir dos 167 dias após o plantio em campo. Recomendase a produção de mudas de ipê-amarelo (T. chrysotricha) em substratos contendo fibra de coco granulada e soluções de fertirrigação com condutividade elétrica de 1,06 dS m-1.<br>The work aimed to study the formation of Tabebuia chrysotricha Standl. seedlings in function of four substrates, varying the fertirrigation solutions. To compose the substrate, fibrous and granulated coconut fiber was used, obtaining the following treatments: 100% fibrous, 60% fibrous and 40% granulated, 40% fibrous and 60% granulated and 100% granulated. The base fertilization was the same for all treatments and the solutions of fertilization varied in order to obtain complete solutions with electric conductivities of 1.06 dS m-1, 2.12 dS m-1, 3.2 dS m-1 and 4.25 dS m-1. The propagative material was sowed directly into plastic containers (120mL) with the respective substrates. The fertilization was received through sub irrigation once a week, respecting the treatments of fertilizations. When the seedlings reached 20cm of height, approximately, they were replaced under black screens (9% of shading), where they remained until planting in Taubaté-SP. Seedlings produced in coconut fiber 100% granulate reached, in the field, higher heights, stem diameters and number of leaves. In function of applied solutions, heights of the aerial part were homogeneous starting from 167 days after planting. The production of T. chrysotricha seedlings is recommended in granulated coconut fiber substrate and fertilizer solutions with electric conductivity of 1.06 dS m-1