Supplementation with Moringa oleifera Lam leaf and seed flour during the pregnancy and lactation period of Wistar rats: Maternal evaluation of initial and adult neurobehavioral development of the rat progeny.

ETHNOPHARMACOLOGICAL RELEVANCE: Moringa oleifera Lam. (M. oleifera) is a tree species of Indian origin popularly known as the "tree of life". In various cultures, it is used by pregnant women to increase milk production, yet studies on its effects during pregnancy and lactation are lacking. AIM OF THE STUDY: To evaluate the nutraceutical aspects of flours produced from the leaves and seeds of M. oleifera, and to evaluate the effect of supplementation of pregnant Wistar rats during the gestation and lactation period, with the aim of studying the weight gain and neonatal parameters of the pregnant rats, as well as effects on the neurobehavioral development and memory in their offspring. MATERIALS AND METHODS: The flour supplementation was conducted at a concentration of 100 mg per kg of animal body weight. For the memory tests, the Open Field Habituation test was performed and repeated after seven days. The Object Recognition test was conducted with the animal exposed to the open field in short and long familiarization sessions. The data obtained were analyzed using Kruskal-Wallis tests for non-parametric data and one-way and two-way ANOVA for parametric data. RESULTS: Flour produced from both the leaf and seed of M. oleifera was found to contain significant amounts of nutrients (protein, fibre, carbohydrates, etc.), making them suitable for supplementation. The exposure of pregnant rats to M. oleifera leaf and seed flours did not affect weight gain, did not have harmful effects on the birth of offspring, and did not result in abortions or mutations in the offspring. Regarding the supplemented group's offspring, early maturation of the senses in the offspring compared to the control group was observed in all tests were conducted; indicating that supplementation positively impacted cognitive development. Further, the offspring of the supplemented rats presented reduced locomotion and greater exploration of new objects compared to the control group offspring, indicating positive effects on learning. CONCLUSION: This study describes for the first time the beneficial effects on pregnant Wistar rats and their offspring of maternal supplementation with flour products from the leaves and seeds of M. oleifera.

Nutritional, Physicochemical and Structural Parameters of Mauritia flexuosa Fruits and By-Products for Biotechnological Exploration of Sustainable Goods.

Research background: Commercialization of Mauritia flexuosa (buriti) fruits in Brazil is at an early stage. Herein, we evaluate the nutritional value of pulp, peel and endocarp samples from buriti fruits, perform macroscopic and microscopic evaluations and analyze their physicochemical properties. Experimental approach: Size and mass, pH, sugar and protein contents, soluble/insoluble fiber, total titratable acidity and energy value of the samples were analyzed. Mineral profiling was performed by energy dispersive X-ray fluorescence spectrometry, and fatty acids and phytosterols were determined by gas chromatography-mass spectrometry. Samples were also submitted to differential scanning calorimetry coupled to a thermal analyzer, and microstructure, morphology, surface and viscosity were evaluated by scanning electron microscopy (SEM) and X-ray diffraction (XRD) with copper radiation. Rheological behavior was also studied. Results and conclusions: Lyophilized pulp had higher nutritional content of minerals, proteins, carbohydrates and energy than in natura pulp. Lyophilized pulp and its by-products showed suitable yields (>17.31%) and low a w, and potassium, manganese and monounsaturated fatty acid contents. Peels showed elevated amounts of saturated and polyunsaturated fatty acids and phytosterols (ß-sitosterol and stigmasterol), and endothermic behavior. The reductions of calcium, magnesium and manganese ranging from 18.5 to 22.7% were observed following the lyophilization. Drying processes generated semi-crystalline powders. Both peels and endocarp contained higher amounts of insoluble fiber and lower contents of sugars. Similar results were obtained by microscopic morphological analysis, differential scanning calorimetry and XRD analysis. Pulp and endocarp exhibited pseudoplastic non-Newtonian behavior, and flow behavior index values were lower than 1, while peels presented dilatant behaviour. Thus, physicochemical and nutritional characterization of pulp and by-products, such as peels and endocarp, are essential to support scientific research and exploration of new sustainable products. Novelty and scientific contribution: Processing and conservation techniques, like lyophilization, maintain the good quality of nutritional contents and bioactive compounds of buriti whole fruits, and can be used to extend their shelf life, preserve alimentary characteristics and provide wider purposes and availability. Such parameters may generate income and food security for local and regional communities.

A screen of kinase inhibitors reveals a potential role of Chk1 in regulating Hydra head regeneration and maintenance.

The cnidarian Hydra possesses remarkable regenerative capabilities which allow it to regrow lost or damaged body parts in a matter of days. Given that many key regulators of regeneration and development are evolutionarily conserved, Hydra is a valuable model system for studying the fundamental molecular mechanisms underlying these processes. In the past, kinase inhibitors have been useful tools for determining the role of conserved signaling pathways in Hydra regeneration and patterning. Here, we present a systematic screen of a commercially available panel of kinase inhibitors for their effects on Hydra regeneration. Isolated Hydra gastric segments were exposed to 5 µM of each kinase inhibitor and regeneration of the head and foot regions were scored over a period of 96 hours. Of the 80 kinase inhibitors tested, 28 compounds resulted in abnormal regeneration. We directed our focus to the checkpoint kinase 1 (Chk1) inhibitor, SB 218078, considering the role of Chk1 in G2 checkpoint regulation and the importance of G2-paused cells in Hydra regeneration. We found that Hydra exposed to SB 218078 were unable to regenerate the head and maintain head-specific structures. Furthermore, SB 218078-treated Hydra displayed a reduction in the relative proportion of epithelial cells; however, no differences were seen for interstitial stem cells or their derivatives. Lastly, exposure to SB 218078 appeared to have no impact on the level of mitosis or apoptosis. Overall, our study demonstrates the feasibility of kinase inhibitor screens for studying Hydra regeneration processes and highlights the possible role for Hydra Chk1 in head regeneration and maintenance.

Insulin signalling in mushroom body neurons regulates feeding behaviour in Drosophila larvae.

Whereas the pivotal role of insulin signalling in cell division, growth and differentiation is well documented, its role in the regulation of neuronal function and behaviour has recently become the focus of intense investigation. The simple organization of the Drosophila larval brain and the availability of genetic tools to impair the function of insulin receptor signalling in a spatially specific manner makes Drosophila an attractive model to investigate the role of the insulin pathway in specific behaviours. Here, we show that impairment of insulin signalling in the mushroom body neurons, a structure involved in associative learning, impairs feeding behaviour in the Drosophila larva.

Menin links the stress response to genome stability in Drosophila melanogaster.

BACKGROUND: The multiple endocrine neoplasia type I gene functions as a tumor suppressor gene in humans and mouse models. In Drosophila melanogaster, mutants of the menin gene (Mnn1) are hypersensitive to mutagens or gamma irradiation and have profound defects in the response to several stresses including heat shock, hypoxia, hyperosmolarity and oxidative stress. However, it is not known if the function of menin in the stress response contributes to genome stability. The objective of this study was to examine the role of menin in the control of the stress response and genome stability. METHODOLOGY/PRINCIPAL FINDINGS: Using a test of loss-of-heterozygosity, we show that Drosophila strains lacking a functional Mnn1 gene or expressing a Mnn1 dsRNA display increased genome instability in response to non-lethal heat shock or hypoxia treatments. This is also true for strains lacking all Hsp70 genes, implying that a precise control of the stress response is required for genome stability. While menin is required for Hsp70 expression, the results of epistatic studies indicate that the increase in genome instability observed in Mnn1 lack-of-function mutants cannot be accounted for by mis-expression of Hsp70. Therefore, menin may promote genome stability by controlling the expression of other stress-responsive genes. In agreement with this notion, gene profiling reveals that Mnn1 is required for sustained expression of all heat shock protein genes but is dispensable for early induction of the heat shock response. CONCLUSIONS/SIGNIFICANCE: Mutants of the Mnn1 gene are hypersensitive to several stresses and display increased genome instability when subjected to conditions, such as heat shock, generally regarded as non-genotoxic. In this report, we describe a role for menin as a global regulator of heat shock gene expression and critical factor in the maintenance of genome integrity. Therefore, menin links the stress response to the control of genome stability in Drosophila melanogaster.

The Drosophila gene RanBPM functions in the mushroom body to regulate larval behavior.

BACKGROUND: In vertebrates, Ran-Binding Protein in the Microtubule Organizing Center (RanBPM) appears to function as a scaffolding protein in a variety of signal transduction pathways. In Drosophila, RanBPM is implicated in the regulation of germ line stem cell (GSC) niche organization in the ovary. Here, we addressed the role of RanBPM in nervous system function in the context of Drosophila larval behavior. METHODOLOGY/PRINCIPAL FINDINGS: We report that in Drosophila, RanBPM is required for larval feeding, light-induced changes in locomotion, and viability. RanBPM is highly expressed in the Kenyon cells of the larval mushroom body (MB), a structure well studied for its role in associative learning in Drosophila and other insects. RanBPM mutants do not display major disruption in nervous system morphology besides reduced proliferation. Expression of the RanBPM gene in the Kenyon cells is sufficient to rescue all behavioral phenotypes. Through genetic epistasis experiments, we demonstrate that RanBPM participates with the Drosophila orthologue of the Fragile X Mental Retardation Protein (FMRP) in the development of neuromuscular junction (NMJ). CONCLUSIONS/SIGNIFICANCE: We demonstrate that the RanBPM gene functions in the MB neurons for larval behavior. Our results suggest a role for this gene in an FMRP-dependent process. Taken together our findings point to a novel role for the MB in larval behavior.

Mutual regulation of the Drosophila disconnected (disco) and Distal-less (Dll) genes contributes to proximal-distal patterning of antenna and leg.

The Drosophila disconnected (disco) gene encodes a C(2)H(2)-type zinc finger transcription factor required for the development of the central and peripheral nervous systems. We report that disco participates in a positive feedback loop with the Dll gene, a master regulator of ventral appendage development. Dll function is not only required for proper disco expression in antenna and leg discs, but is also sufficient for ectopic expression of disco in the developing retina and wing imaginal discs. Conversely, disco gene function is required for the maintenance of Dll expression. We show that Dll phenotypes are partially rescued by the up-regulation of disco expression in the Dll domain. Reduction in disco gene function disrupts antenna and leg development, and the phenotypes closely resemble that produced by Dll alleles. These observations demonstrate that disco plays a fundamental role in the Dll-dependent patterning of antenna and leg, perhaps as a regulator of Dll gene expression.

Role of serotonergic neurons in the Drosophila larval response to light.

BACKGROUND: Drosophila larval locomotion consists of forward peristalsis interrupted by episodes of pausing, turning and exploratory behavior (head swinging). This behavior can be regulated by visual input as seen by light-induced increase in pausing, head swinging and direction change as well as reduction of linear speed that characterizes the larval photophobic response. During 3rd instar stage, Drosophila larvae gradually cease to be repelled by light and are photoneutral by the time they wander in search for a place to undergo metamorphosis. Thus, Drosophila larval photobehavior can be used to study control of locomotion. RESULTS: We used targeted neuronal silencing to assess the role of candidate neurons in the regulation of larval photobehavior. Inactivation of DOPA decarboxylase (Ddc) neurons increases the response to light throughout larval development, including during the later stages of the 3rd instar characterized by photoneutral response. Increased response to light is characterized by increase in light-induced direction change and associated pause, and reduction of linear movement. Amongst Ddc neurons, suppression of the activity of corazonergic and serotonergic but not dopaminergic neurons increases the photophobic response observed during 3rd instar stage. Silencing of serotonergic neurons does not disrupt larval locomotion or the response to mechanical stimuli. Reduced serotonin (5-hydroxytryptamine, 5-HT) signaling within serotonergic neurons recapitulates the results obtained with targeted neuronal silencing. Ablation of serotonergic cells in the ventral nerve cord (VNC) does not affect the larval response to light. Similarly, disruption of serotonergic projections that contact the photoreceptor termini in the brain hemispheres does not impact the larval response to light. Finally, pan-neural over-expression of 5-HT1A Dro receptors, but not of any other 5-HT receptor subtype, causes a significant decrease in the response to light of 3rd instar larvae. CONCLUSION: Our data demonstrate that activity of serotonergic and corazonergic neurons contribute to the control of larval locomotion by light. We conclude that this control is carried out by 5-HT neurons located in the brain hemispheres, but does not appear to occur at the photoreceptor level and may be mediated by 5-HT1A Dro receptors. These findings provide new insights into the function of 5-HT neurons in Drosophila larval behavior as well as into the mechanisms underlying regulation of larval response to light.

The flamenco locus controls the gypsy and ZAM retroviruses and is required for Drosophila oogenesis.

In Drosophila, the as yet uncloned heterochromatic locus flamenco (flam) controls mobilization of the endogenous retrovirus gypsy through the repeat-associated small interfering (rasi) RNA silencing pathway. Restrictive alleles (flamR) downregulate accumulation of gypsy transcripts in the somatic follicular epithelium of the ovary. In contrast, permissive alleles (flamP) are unable to repress gypsy. DIP1, the closest transcription unit to a flam-insertional mutation, was considered as a good candidate to be a gypsy regulator, since it encodes a dsRNA-binding protein. To further characterize the locus we analyzed P-induced flam mutants and generated new mutations by transposon mobilization. We show that flam is required somatically for morphogenesis of the follicular epithelium, the tissue where gypsy is repressed. This developmental activity is necessary to control gypsy and another retroelement, ZAM. We also show that flam is not DIP1, as none of the new permissive mutants affect the DIP1 coding sequence. In addition, two deletions removing DIP1 coding sequences do not affect any of the flamenco functions. Our results suggest that flamenco extends proximally to DIP1, spanning >130 kb of transposon-rich heterochromatin. We propose a model explaining the multiple functions of this large heterochromatic locus.

Kinematic analysis of Drosophila larval locomotion in response to intermittent light pulses.

We report a quantitative analysis of the modulation of locomotion by light in the Drosophila larva. The photobehavior of wild type larvae and of larvae carrying mutations altering various aspects of locomotion was evaluated in an assay that exposes individual animals to intermittent pulses of dark and light (ON/OFF assay). The application of the Dynamic Image Analysis System for the analysis of larval movement in the ON/OFF assay allowed a detailed description of the behavioral repertoire underlying the modulation of larval motion by light. We established that the larval response to light is characterized by decreased frequency of peristaltic contractions in addition to increased direction change and pausing. Moreover, using various mutant strains we show that this approach allows the detection of a response to light in mutant larvae whose locomotion is severely reduced and uncoordinated, mutants that would otherwise have been considered non-responders in this assay.

Isotermas de dessorção da casca do maracujá (Passiflora edulis Sims): determinação experimental e avaliação de modelos matemáticos / Desorption isotherms of passion fruit peel (Passiflora edulis Sims): experimental determination and mathematical model evaluation

A produção de proteínas microbianas sobre a casca de maracujá visando à produção de suplemento protéico para animais é uma alternativa encontrada para o grande desperdício desse resíduo agroindustrial. Através das isotermas de dessorção da casca do maracujá, foi verificado que as condições de umidade inicial para o crescimento de microrganismos neste resíduo por meio da fermentação semi-sólida deverá ser acima de 55 por cento (base úmida) e a faixa de umidade ideal para o armazenamento do resíduo em torno de 5,3 por cento (base úmida).

The microbial protein production on passion fruit peel shell as an animal protein supplement production is an alternative found out to avoid the large wastage of agri-industrial residues. Through the desorption isotherms of passion fruit peel, it is verified that for the microorganism growth by solid-state fermentation on this residue, the initial moisture content condition should be above 55 percent (wet basis). The ideal moisture content range for residue storage is around 5.3 percent (wet basis).

Menin is a regulator of the stress response in Drosophila melanogaster.

Menin, the product of the multiple endocrine neoplasia type I gene, has been implicated in several biological processes, including the control of gene expression and apoptosis, the modulation of mitogen-activated protein kinase pathways, and DNA damage sensing or repair. In this study, we have investigated the function of menin in the model organism Drosophila melanogaster. We show that Drosophila lines overexpressing menin or an RNA interference for this gene develop normally but are impaired in their response to several stresses, including heat shock, hypoxia, hyperosmolarity and oxidative stress. In the embryo subjected to heat shock, this impairment was characterized by a high degree of developmental arrest and lethality. The overexpression of menin enhanced the expression of HSP70 in embryos and interfered with its down-regulation during recovery at the normal temperature. In contrast, the inhibition of menin with RNA interference reduced the induction of HSP70 and blocked the activation of HSP23 upon heat shock, Menin was recruited to the Hsp70 promoter upon heat shock and menin overexpression stimulated the activity of this promoter in embryos. A 70-kDa inducible form of menin was expressed in response to heat shock, indicating that menin is also regulated in conditions of stress. The induction of HSP70 and HSP23 was markedly reduced or absent in mutant embryos harboring a deletion of the menin gene. These embryos, which did not express the heat shock-inducible form of menin, were also hypersensitive to various conditions of stress. These results suggest a novel role for menin in the control of the stress response and in processes associated with the maintenance of protein integrity.

Genetic dissection of trophic interactions in the larval optic neuropil of Drosophila melanogaster.

The larval visual system of Drosophila melanogaster consists of two bilateral clusters of 12 photoreceptors, which express Rhodopsin 5 and 6 (Rh5 and Rh6) in a non-overlapping manner. These neurons send their axons in a fascicle, the larval optic nerve (LON), which terminates in the larval optic neuropil. The LON is required for the development of a serotonergic arborization originating in the central brain and for the development of the dendritic tree of the circadian pacemakers, the small ventral lateral neurons (LNv) [Malpel, S., Klarsfeld, A., Rouyer, F., 2002. Larval optic nerve and adult extra-retinal photoreceptors sequentially associate with clock neurons during Drosophila brain development. Development 129, 1443-1453; Mukhopadhyay, M., Campos, A.R., 1995. The larval optic nerve is required for the development of an identified serotonergic arborization in Drosophila melanogaster. Dev. Biol., 169, 629-643]. Here, we show that both Rh5- and Rh6-expressing fibers overlap equally with the 5-HT arborization and that it, in turn, also contacts the dendritic tree of the LNv. The experiments described here aimed at determining whether Rh5- or Rh6-expressing fibers, as well as the LNv, influence the development of this serotonergic arborization. We conclude that Rh6-expressing fibers play a unique role in providing a signal required for the outgrowth and branching of the serotonergic arborization. Moreover, the innervation of the larval optic neuropil by the 5-HT arborization depends on intact Rac function. A possible role for these serotonergic processes in modulating the larval circadian rhythmicity and photoreceptor function is discussed.

Photic input pathways that mediate the Drosophila larval response to light and circadian rhythmicity are developmentally related but functionally distinct.

The Drosophila melanogaster larval photosensory organ that mediates the response to light consists of bilaterally symmetrical clusters of 12 photoreceptors. These are distinguished on the basis of expression of the rhodopsins Rh5 and Rh6. The Rh6-expressing cells correspond to the Hofbauer-Buchner (H-B) eyelet found later in the posterior margin of the adult compound eye and recently shown to function as an input pathway in the entrainment of circadian rhythmicity in adult Drosophila. In addition, the axons of the larval photoreceptors are found in intimate association with a subset of the main circadian pacemaker neurons located in the developing accessory medulla, the small ventral lateral neurons (LNv). The observed spatial overlap between components of the circadian circuitry, input pathway, and pacemaker neurons-and the larval visual organ-suggest a functional relationship between these two photosensory input pathways. In this study we determined the requirement of specific rhodopsin-expressing photoreceptors including the presumptive H-B eyelet and pacemaker neurons in the larval locomotory response to visual stimuli. Our results demonstrate that two of the most important components of the neuronal circuitry underlying circadian rhythmicity in Drosophila, namely, the extraretinal H-B cluster and the circadian pacemakers, while in intimate association with the larval visual system are not required for the larval motor response to light.

A novel double-stranded RNA-binding protein, disco interacting protein 1 (DIP1), contributes to cell fate decisions during Drosophila development.

We report the identification of the Disco Interacting Protein 1 (DIP1) gene isolated in a yeast interaction trap screen using the zinc finger protein disconnected (disco) as a bait. DIP1 encodes a protein containing two double-stranded RNA binding domains (dsRBD). Consistent with the presence of dsRBD, DIP1 binds dsRNA or structured RNAs in Northwestern assays. DIP1 is found in nuclear subdomains resembling speckles known to accumulate transcription and splicing factors. In early embryos, nuclear localization of DIP1 protein coincides with the onset of zygotic gene expression. Later in development DIP1 expression is decreased in dividing cells in different tissues. Overexpression of DIP1 in the eye-antennal imaginal disc, early in embryonic and larval development, causes the formation of supernumerary structures in the head capsule. A role for DIP1 in epigenetic mechanisms that lead to the establishment and/or maintenance of cell fate specification is discussed.

The accessory subunit of DNA polymerase gamma is essential for mitochondrial DNA maintenance and development in Drosophila melanogaster.

DNA polymerase gamma, Pol gamma, is the key replicative enzyme in animal mitochondria. The Drosophila enzyme is a heterodimer comprising catalytic and accessory subunits of 125 kDa and 35 kDa, respectively. Both subunits have been cloned and characterized in a variety of model systems, and genetic mutants of the catalytic subunit were first identified in Drosophila, as chemically induced mutations that disrupt larval behavior (tamas). Mutations in the gene encoding the accessory subunit have not yet been described in any organism. Here, we report the consequences of null mutations upon mitochondrial DNA (mtDNA) replication and morphology, cell proliferation, and organismal viability. Mutations in the accessory subunit cause lethality during early pupation, concomitant with loss of mtDNA and mitochondrial mass, and reduced cell proliferation in the central nervous system. Surprisingly, the function of the central nervous system and muscle, as assessed in a locomotion assay, are only marginally affected. This finding is in contrast to our previous findings that disruption in the function of the catalytic subunit causes severe reduction in larval locomotion. We discuss our results in the context of current hypotheses for the function of the accessory subunit in mtDNA replication.