Numb/Numbl-Opo antagonism controls retinal epithelium morphogenesis by regulating integrin endocytosis

Polarized trafficking of adhesion receptors plays a pivotal role in controlling cellular behavior during morphogenesis. Particularly, clathrin-dependent endocytosis of integrins has long been acknowledged as essential for cell migration. However, little is known about the contribution of integrin trafficking to epithelial tissue morphogenesis. Here we show how the transmembrane protein Opo, previously described for its essential role during optic cup folding, plays a fundamental role in this process. Through interaction with the PTB domain of the clathrin adaptors Numb and Numbl via an integrin-like NPxF motif, Opo antagonizes Numb/Numbl function and acts as a negative regulator of integrin endocytosis in vivo. Accordingly, numb/numbl gain-of-function experiments in teleost embryos mimic the retinal malformations observed in opo mutants. We propose that developmental regulator Opo enables polarized integrin localization by modulating Numb/Numbl, thus directing the basal constriction that shapes the vertebrate retina epithelium.

Analysis of opo cis-regulatory landscape uncovers Vsx2 requirement in early eye morphogenesis

The self-organized morphogenesis of the vertebrate optic cup entails coupling the activation of the retinal gene regulatory network to the constriction-driven infolding of the retinal epithelium. Yet the genetic mechanisms underlying this coordination remain largely unexplored. Through phylogenetic footprinting and transgenesis in zebrafish, here we examine the cis-regulatory landscape of opo, an endocytosis regulator essential for eye morphogenesis. Among the different conserved enhancers identified, we isolate a single retina-specific element (H6_10137) and show that its activity depends on binding sites for the retinal determinant Vsx2. Gain- and loss-of-function experiments and ChIP analyses reveal that Vsx2 regulates opo expression through direct binding to this retinal enhancer. Furthermore, we show that vsx2 knockdown impairs the primary optic cup folding. These data support a model by which vsx2, operating through the effector gene opo, acts as a central transcriptional node that coordinates neural retina patterning and optic cup invagination in zebrafish.

Isolation and characterization of plastid terminal oxidase gene from carrot and its relation to carotenoid accumulation

Carrot (Daucus carota L.) is a biennial plant that accumulates considerable amounts of carotenoid pigments in the storage root. To better understand the molecular mechanisms for carotenoid accumulation in developing storage roots, plastid terminal oxidase (PTOX) cDNA was isolated and selected for reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Present in photosynthetic species, PTOX is a plastid-located, nucleus encoded plastoquinone (PQ)-O2 oxidoreductase (plastioquinol oxidase). The enzyme is known to play a role as a cofactor for phytoene desaturase, and consequently plays a key role in the carotenoid biosynthesis pathway. A single PTOX gene was identified (DcPTOX) in carrot. DcPTOX encodes a putative protein with 366 amino acids that contains the typical structural features of PTOXs from higher plants. The expression of DcPTOX was analysed during the development of white, yellow, orange, red, and purple carrot roots, along with five genes known to be involved in the carotenoid biosynthesis pathway, PSY2, PDS, ZDS1, LCYB1, and LCYE. Expression analysis revealed the presence of DcPTOX transcripts in all cultivars, and an increase of transcripts during the time course of the experiment, with differential expression among cultivars in early stages of root growth. Our results demonstrated that DcPTOX showed a similar profile to that of other carotenoid biosynthetic genes with high correlation to all of them. The preponderant role of PSY in the biosynthesis of carotenoid pigments was also confirmed.

Timing expression profile of carotenoids pathway genes in maize grains for characterization of landrace germoplasm from south Brazil.

2011

Caracteres morfo-agronômicos e bioquímicos de clones elite de mandioca de mesa com raízes de polpas amarelada e rosada.

A mandioca (Manihot esculenta Crantz) é considerada uma espécie relevante como fonte alimentícia para a população mundial, principalmente para os países subdesenvolvidos e emergentes. A mandioca é fornecedora de energia a partir do amido acumulado em suas raízes de reserva, mas é também importante destacar a presença dos carotenóides com atividade antioxidante. Nesse contexto, o presente trabalho teve como objetivo caracterizar, por meio de descritores morfológicos, agronômicos e bioquímicos, clones elite de mandioca de mesa de polpa aparelhada e rosada do programa de melhoramento genético de mandioca da Embrapa Cerrados. Foram caracterizados durante duas safras, 13 clones de mandioca de mesa com polpa amarelada e 8 clones com polpa rosada, em comparação com a variedade testemunha IAC 576-70 (BGMC 753). Para avaliar as características morfológicas foram obtidos 40 descritores qualitativos para cada clone. Tanto nos clones de polpas amarelada quanto naqueles de raízes de polpas rosada, houve diferenças morfológicas, demostrando que nenhum clone apresentou 100% de similaridade. O fator ano/safra não influenciou a expressão fenotípica dos caracteres aferidos. Com base no coeficiente cofenético, verificou-se elevado ajuste entre a representação gráfica via dendrograma de r = 0,80 nas raízes de polpa amarelada e r = 0,92 na rosada e a matriz de dissimilaridade genética. Entre os caracteres aferidos, os que apresentaram maior entropia nas raízes amarelada foram, a coloração da epiderme externa, forma do lóbulo central da folha e cor do córtex da raiz, ao passo que na rosada foi à cor do disco, forma do lóbulo central e cor do pecíolo. Foi realizada também a caracterização com base na altura da planta, altura da primeira ramificação, peso da parte aérea sem a cepa, produtividade em raízes, índices de amido nas raízes determinados por meio do método da balança hidrostática, tempo para a cocção e teor de ácido cianídrico nas raízes. Com base nos caracteres avaliados, os clones que se destacaram com polpa amarelada e rosada respectivamente, no caractere altura da primeira ramificação (273/08 e 259/08) e (390/08, 345/08 e a testemunha IAC 576-70), altura da planta (90/08, 272/08, 273/08, 497/08, 259/08 e 450/08) e (390/08, 345/08 e 378/08), peso da parte aérea sem a cepa (94/08 e 272/08) e (390/08, 406/08, 390/08, 378/08 e 341/08), porcentagem de amido nas raízes (26/08, 272/08, 259/08 e 450/08) e (378/08, 413/08, 390/08 e a testemunha IAC 576-70), produtividade de raízes (215/08) e (testemunha IAC 576-70, 341/08, 406/08, 390/08 e 387/08). Com relação ao tempo de cocção na safra 2011/2012, todos os clones necessitaram de tempo inferior a 30 minutos. Em relação ao teor de carotenóides totais nas raízes os clones de amarelada que se destacaram foram 91/08, 94/08, 215/08, 246/08, 272/08 e 497/08, e, naqueles de raízes rosada, os clones 406/08 e 341/08. Em relação ao teor de proteínas nas raízes amarelada, os clones 26/08, 90/08 e 91/08, foram os melhores enquanto nas raízes rosada se destacaram os clones 406/08 e a testemunha IAC 576-70. Os teores de HCN nas raízes de reserva de mandioca foram inferiores a 100 mg kg-1 em todos os clones avaliados. Diferenças significativas entre clones de mandioca de polpas amarelada e rosada foram verificadas para todas as características agronômicas, morfológicas e bioquímicas avaliadas. Os clones tiveram bom desempenho nas avaliações para o cultivo comercial na região do Cerrado e, alguns destes, têm potencial para utilização no melhoramento visando o incremento de carotenóides. ABSTRACT: Cassava (Manihot esculenta Crantz) is considered a relevant species as a food source for the world's population, particularly for developing and emerging countries. The cassava is a provider of energy from starch accumulated in their reserve roots, but it is also important to highlight the presence of carotenoids with antioxidant activity. In this context, this study aimed to characterize, using morphological, agronomic and biochemical, descriptors elite clones from sweet cassava of yellowish and pinkish pulps from the cassava breeding program at Embrapa Cerrados. They were characterized for two crops, 13 edible cassava clones with yellowish pulp and 8 clones with pinkish pulp, compared with the control variety IAC 576-70 (BGMC 753). To evaluate the morphological characteristics were obtained 40 qualitative descriptors for each clone. Both clones the yellowish pulp as those the roots the pinkish pulp, there was morphological differences among clones, showing that no clone showed 100% similarity. The year / crop factor did not influence the phenotypic expression of measured characters. Based on cofenetic coefficient, was found high fit between the graphical representation via dendrogram of r = 0.80 in the roots of yellowish pulp and r = 0.92 in the pinkish of genetic dissimilarity matrix. Among the measured characters, those with the highest entropy in the yellowish roots were, the color of the outer epidermis, the central lobe shape of the leaf and root cortex color, whereas the pinkish was the color to disc, central lobe shape and petiole color. We also performed the characterization based on plant height, the first branch point, and shoot weight without strain, productivity in roots, and index of starch in the roots determines by the method of hydrostatic balance, time for cooking and acid cyanide content in the roots. Based on the evaluated characters, clones stood out with pulps yellowish and pinkish respectively, characters height of the first branch (273/08 and 259/08) and (390/08, 345/08 and the witness IAC 576-70), plant height (90 / 08, 272/08, 273/08, 497/08, 259/08 and 450/08) and (390/08, 345/08 and 378/08), shoot weight without strain (94/08 and 272/08) and (390/08, 406/08, 390/08, 378/08 and 341/08), percentage of starch in the roots (26/08, 272/08, 259/08 and 450/08) and (378/08, 413/08, 390/08 and the witness IAC 576-70), roots of productivity (215/08) and (witnesses IAC 576-70, 341/08, 406/08, 390/08 and 387/08). Regarding the cooking time in the 2011/2012 harvest, all clones showed time less than 30 minutes. Regarding the total carotenoid content in the pulps clones of yellowish roots that stood out were 91/08, 94/08, 215/08, 246/08, 272/08 and 497/08, and, those the clones with pulp pinkish 406/08 and 341/08. Regarding the protein content in yellowish roots the clones 26/08, 90/08 and 91/08, was the best while the pinkish roots highlight clones 406/08 and witness IAC 576-70. The levels of HCN in reserve roots of cassava were less than 100 mg kg-1em all evaluated clones. Significant differences between yellowish and pinkish of pulps cassava clones were checked for all agronomic, morphological and biochemical characteristics evaluated. The clones had well in the ratings for commercial cultivation in the Cerrado region and some of these, clones has potential for use in breeding aimed at increase of carotenoids.

Natural variation in expression of genes associated with carotenoid biosynthesis and accumlation in cassava (Manihot esculenta Crantz) storage root.

2016

Antioxidant Activity Of Hydrophilic And Lipophilic Extracts Of Brazilian Blueberries.

Hydrophilic and lipophilic extracts of ten cultivars of Highbush and Rabbiteye Brazilian blueberries (Vaccinium corymbosum L. and Vacciniumashei Reade, respectively) that are used for commercial production were analysed for antioxidant activity by the FRAP, ORAC, ABTS and β-carotene-linoleate methods. Results were correlated to the amounts of carotenoids, total phenolics and anthocyanins. Brazilian blueberries had relatively high concentration of total phenolics (1,622-3,457 mg gallic acid equivalents per 100 g DW) and total anthocyanins (140-318 mg cyanidin-3-glucoside equivalents per 100 g DW), as well as being a good source of carotenoids. There was a higher positive correlation between the amounts of these compounds and the antioxidant activity of hydrophilic compared to lipophilic extracts. There were also significant differences in the level of bioactive compounds and antioxidant activities between different cultivars, production location and year of cultivation.

Molecular Diversity Of Fungal And Bacterial Communities In The Marine Sponge Dragmacidon Reticulatum.

The present work aimed to investigate the diversity of bacteria and filamentous fungi of southern Atlantic Ocean marine sponge Dragmacidon reticulatum using cultivation-independent approaches. Fungal ITS rDNA and 18S gene analyses (DGGE and direct sequencing approaches) showed the presence of representatives of three order (Polyporales, Malasseziales, and Agaricales) from the phylum Basidiomycota and seven orders belonging to the phylum Ascomycota (Arthoniales, Capnodiales, Dothideales, Eurotiales, Hypocreales, Pleosporales, and Saccharomycetales). On the other hand, bacterial 16S rDNA gene analyses by direct sequencing approach revealed the presence of representatives of seven bacterial phyla (Cyanobacteria, Proteobacteria, Actinobacteria, Bacteroidetes, Lentisphaerae, Chloroflexi, and Planctomycetes). Results from statistical analyses (rarefaction curves) suggested that the sampled clones covered the fungal diversity in the sponge samples studied, while for the bacterial community additional sampling would be necessary for saturation. This is the first report related to the molecular analyses of fungal and bacterial communities by cultivation-independent approaches in the marine sponges D. reticulatum. Additionally, the present work broadening the knowledge of microbial diversity associated to marine sponges and reports innovative data on the presence of some fungal genera in marine samples.

Carotenóides: propriedades, aplicações e biotransformação para formação de compostos de aroma

Carotenoids are widely distributed in nature, providing yellow, orange or red color in a great number of vegetables, microorganisms and in some animals. Carotenoids act as biological antioxidants and seem to play an important role in human health by protecting cells and tissues from the damaging effects of free radicals and singlet oxygen. Several authors describe the oxidative cleavage of carotenoids in flavor compounds as occuring through chemical or photochemical degradations or through biotechnological processes. Biotransformation of carotenoids seems to be a reasonable alternative to produce flavor compounds since these compounds are considered 'natural' ingredients. In this work we describe the properties of some carotenoids, as well as biotechnological approaches to obtain its oxyfunctionalized derivatives.

Caracterização físico-química de filmes comestíveis de amido adicionado de acerola (Malphigia emarginata D.C.)

Edibles films are an alternative to synthetic materials used for packing food products. Barbados cherry is rich in vitamin C and carotenoids. The aim of this study was to characterize and develop films by casting from cassava starch, lyophilized Barbados cherry pulp and glycerol. The films were characterized with respect to thickness, water vapor permeability (WVP), water solubility, vitamin C, carotene and mechanical properties. The interaction of pulp and glycerol reduced film thickness. An increase in pulp concentration up to 60% increased WVP but beyond this concentration reduced both WVP and solubility leading to an increased level of vitamin C and β carotene in the films.

Desenvolvimento e validação de método espectrofotométrico para determinação de corante à base de luteína adicionado em iogurte desnatado

A simple analytical method for extraction and quantification of lutein colorant added to yogurt was developed and validated. The method allowed complete extraction of carotenoids using tetrahydrofuran in vortex, followed by centrifugation, partition to diethyl ether/petroleum ether, and drying. The carotenoids dissolved in ethanol were quantified by UV-Vis spectrophotometry. This method showed linearity in the range tested (1.41-13.42 µg g-1), limits of detection and quantification of 0.42 and 1.28 µg g-1, respectively, low relative standard deviation (3.4%) and recovery ranging from 95 to 103%. The method proved reliable for quantification of lutein added to yogurt.

Physiological limitations in two sugarcane varieties under water suppression and after recovering

Increasing water scarcity and depleted water productivity in irrigated soils are inducing farmers to adopt improved varieties, such as those with high-capacity tolerance. The use of tolerant varieties of sugarcane might substantially avoid the decline of productivity under water deficit. This research aimed to evaluate the harmful effects of drought on the physiology of two sugarcane varieties (RB867515 and RB962962) during the initial development. Young plants were subjected to irrigation suspension until total stomata closure, and then rewatered. Significant reduction on stomatal conductance, transpiration, and net photosynthesis were observed. RB867515 showed a faster stomatal closure while RB962962 slowed the effects of drought on the gas exchanges parameters with a faster recovering after rewatering. Accumulation of carbohydrates, amino acids, proline, and protein in the leaves and roots of the stressed plants occurred in both varieties, substantially linked to reduction of the leaf water potential. Due to the severity of stress, this accumulation was not enough to maintain the cell turgor pressure, so relative water content was diminished. Water stress affected the contents of chlorophyll (a, b, and total) in both varieties, but not the levels of carotenoids. There was a significant reduction in dry matter under stress. In conclusion, RB962962 variety endured stressed conditions more than RB867515, since it slowed down the damaging effects of drought on the gas exchanges. In addition, RB962962 presented a faster recovery than RB867515, a feature that qualifies it as a variety capable of enduring short periods of drought without major losses in the initial stage of its development.

The Sperm Of Hexagenia (pseudeatonica) Albivitta Walker (ephemeroptera: Fossoriae: Ephemeridae)

This study describes the sperm morphology of the mayfly Hexagenia (Pseudeatonica) albivitta (Ephemeroptera). Its spermatozoon measures approximately 30 μm of which 9 μm corresponds to the head. The head is composed of an approximately round acrosomal vesicle and a cylindrical nucleus. The nucleus has two concavities, one in the anterior tip, where the acrosomal vesicle is inserted and a deeper one at its base, where the flagellum components are inserted. The flagellum is composed of an axoneme, a mitochondrion and a dense rod adjacent to the mitochondrion. A centriolar adjunct is also observed surrounding the axoneme in the initial portion of the flagellum and extends along the flagellum for at least 2 μm, surrounding the axoneme in a half-moon shape. The axoneme is the longest component of the flagellum, and it follows the 9+9+0 pattern, with no central pair of microtubules. At the posterior region of the flagellum, the mitochondrion has a dumb-bell shape in cross sections that, together with the rectangular mitochondrial-associated rod, is responsible for the flattened shape of the flagellum. An internal membrane is observed surrounding both mitochondrion and its associated structure.

HOXB5 expression in oral squamous cell carcinoma

Human HOX genes encode transcription factors that act as master regulators of embryonic development. They are important in several processes such as cellular morphogenesis and differentiation. The HOXB5 gene in particular has been reported in some types of neoplasm, but not in oral cancer. OBJECTIVE: The present study investigated the expression of HOXB5 in oral squamous cell carcinoma (SCC) and in non-tumoral adjacent tissues, focusing on verifying its possible role as a broad tumor-associated gene and its association with histopathological and clinical (TNM) characteristics. MATERIAL AND METHODS: RT-PCR was performed to amplify HOXB5 mRNA in 15 OSCCs and adjacent non-tumoral epithelium. A possible association with TNM and histopathologic data was verifed by the chi-square and post-hoc t-test. RESULTS: HOXB5 was amplifed in 60% non-tumoral epithelium and in 93.3% carcinomas. No statistically signifcant differences were found regarding the HOXB5 mRNA expression and TNM or histological grade. CONCLUSION: HOXB5 is expressed in OSCCs and its role in cancer progression should be further investigated.