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Find video protocols related to scientific articles indexed in Pubmed.
An insight into the transcriptome of the digestive tract of the bloodsucking bug, Rhodnius prolixus.
PLoS Negl Trop Dis
PUBLISHED: 01-01-2014
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The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet.
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Silencing of maternal heme-binding protein causes embryonic mitochondrial dysfunction and impairs embryogenesis in the blood sucking insect Rhodnius prolixus.
J. Biol. Chem.
PUBLISHED: 08-28-2013
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The heme molecule is the prosthetic group of many hemeproteins involved in essential physiological processes, such as electron transfer, transport of gases, signal transduction, and gene expression modulation. However, heme is a pro-oxidant molecule capable of propagating reactions leading to the generation of reactive oxygen species. The blood-feeding insect Rhodnius prolixus releases enormous amounts of heme during host blood digestion in the midgut lumen when it is exposed to a physiological oxidative challenge. Additionally, this organism produces a hemolymphatic heme-binding protein (RHBP) that transports heme to pericardial cells for detoxification and to growing oocytes for yolk granules and as a source of heme for embryo development. Here, we show that silencing of RHBP expression in female fat bodies reduced total RHBP circulating in the hemolymph, promoting oxidative damage to hemolymphatic proteins. Moreover, RHBP knockdown did not cause reduction in oviposition but led to the production of heme-depleted eggs (white eggs). A lack of RHBP did not alter oocyte fecundation. However, produced white eggs were nonviable. Embryo development cellularization and vitellin yolk protein degradation, processes that normally occur in early stages of embryogenesis, were compromised in white eggs. Total cytochrome c content, cytochrome c oxidase activity, citrate synthase activity, and oxygen consumption, parameters that indicate mitochondrial function, were significantly reduced in white eggs compared with normal dark red eggs. Our results showed that reduction of heme transport from females to growing oocytes by RHBP leads to embryonic mitochondrial dysfunction and impaired embryogenesis.
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Transcriptome and gene expression profile of ovarian follicle tissue of the triatomine bug Rhodnius prolixus.
Insect Biochem. Mol. Biol.
PUBLISHED: 03-17-2011
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Insect oocytes grow in close association with the ovarian follicular epithelium (OFE), which escorts the oocyte during oogenesis and is responsible for synthesis and secretion of the eggshell. We describe a transcriptome of OFE of the triatomine bug Rhodnius prolixus, a vector of Chagas disease, to increase our knowledge of the role of FE in egg development. Random clones were sequenced from a cDNA library of different stages of follicle development. The transcriptome showed high commitment to transcription, protein synthesis, and secretion. The most abundant cDNA was a secreted (S) small, proline-rich protein with maximal expression in the vitellogenic follicle, suggesting a role in oocyte maturation. We also found Rp45, a chorion protein already described, and a putative chitin-associated cuticle protein that was an eggshell component candidate. Six transcripts coding for proteins related to the unfolded-protein response (UPR) by were chosen and their expression analyzed. Surprisingly, transcripts related to UPR showed higher expression during early stages of development and downregulation during late stages, when transcripts coding for S proteins participating in chorion formation were highly expressed. Several transcripts with potential roles in oogenesis and embryo development are also discussed. We propose that intense protein synthesis at the FE results in reticulum stress (RS) and that lowering expression of a set of genes related to cell survival should lead to degeneration of follicular cells at oocyte maturation. This paradoxical suppression of UPR suggests that ovarian follicles may represent an interesting model for studying control of RS and cell survival in professional S cell types.
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Effect of GSK-3 activity, enzymatic inhibition and gene silencing by RNAi on tick oviposition and egg hatching.
Parasitology
PUBLISHED: 05-26-2010
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Glycogen synthase kinase-3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism in mammals. It has been shown to be highly conserved among several organisms, mainly in the catalytic domain region. This enzyme has already been described in Rhipicephalus (Boophilus) microplus and the ovaries of females appeared to be the major site of GSK-3 transcription. The treatment with GSK-3 specific inhibitor (alsterpaullone, bromo-indirubin-oxime 6 and indirubin-3-oxime) caused a reduction in oviposition and egg hatching in completely engorged female ticks. The effect was more pronounced in partially engorged females when alsterpaullone was administrated by artificial capillary feeding. Moreover, GSK-3 gene silencing by RNAi in partially engorged females reduced significantly both oviposition and hatching. The study of tick embryogenesis and proteins that participate in this process has been suggested as an important means for the development of novel strategies for parasite control. GSK-3 is an essential protein involved in embryonic processes and for this reason it has already been suggested as a possible antigen candidate for tick control.
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In vivo uptake of a haem analogue Zn protoporphyrin IX by the human malaria parasite P. falciparum-infected red blood cells.
Cell Biol. Int.
PUBLISHED: 05-25-2010
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The cellular traffic of haem during the development of the human malaria parasite Plasmodium falciparum, through the stages R (ring), T (trophozoite) and S (schizonts), was investigated within RBC (red blood cells). When Plasmodium cultures were incubated with a fluorescent haem analogue, ZnPPIX (Zn protoporphyrin IX) the probe was seen at the cytoplasm (R stage), and the vesicle-like structure distribution pattern was more evident at T and S stages. The temporal sequence of ZnPPIX uptake by P. falciparum-infected erythrocytes shows that at R and S stages, a time-increase acquisition of the porphyrin reaches the maximum fluorescence distribution after 60 min; in contrast, at the T stage, the maximum occurs after 120 min of ZnPPIX uptake. The difference in time-increase acquisition of the porphyrin is in agreement with a maximum activity of haem uptake at the T stage. To gain insights into haem metabolism, recombinant PfHO (P. falciparum haem oxygenase) was expressed, and the conversion of haem into BV (biliverdin) was detected. These findings point out that, in addition to haemozoin formation, the malaria parasite P. falciparum has evolved two distinct mechanisms for dealing with haem toxicity, namely, the uptake of haem into a cellular compartment where haemozoin is formed and HO activity. However, the low Plasmodium HO activity detected reveals that the enzyme appears to be a very inefficient way to scavenge the haem compared with the Plasmodium ability to uptake the haem analogue ZnPPIX and delivering it to the food vacuole.
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Serotonin regulates an acyl-CoA-binding protein (ACBP) gene expression in the midgut of Rhodnius prolixus.
Insect Biochem. Mol. Biol.
PUBLISHED: 01-06-2010
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Acyl-CoA esters have many intracellular functions, acting as energy source, substrate for metabolic processes and taking part in cell signaling. The acyl-CoA-binding protein (ACBP), a highly conserved 10 kDa intracellular protein, binds long- and medium-chain acyl-CoA esters with very high affinity, directing them to specific metabolic routes and protecting them from hydrolysis. An ACBP gene sequence was identified in the genome of Rhodnius prolixus. This ACBP gene (RpACBP-1) was expressed in all analyzed tissues and quantitative PCR showed that expression was highest in posterior midgut. In this tissue, ACBP gene expression increased in the first day after blood meal ( approximately 10-fold) and then decreased to unfed levels in the seventh day after meal. Injection of serotonin (5-hydroxytryptamine; 5-HT), a neuroamine released in the hemolymph after the start of feeding, increased the expression of this gene in the midgut of unfed females, reaching levels similar to those observed in fed insects. This effect of injected 5-HT was inhibited by spiperone, an antagonist of 5-HT mammalian receptors, that was also able to block the physiological increase in RpACBP-1 expression observed after feeding. Injection of cholera toxin or dibutyryl-cAMP also resulted in the stimulation of this gene expression. These data reveal a transcriptional regulatory mechanism in R. prolixus, that is triggered by 5-HT. In this way, a novel role for 5-HT is proposed, as a regulator of ACBP gene expression and, consequently, taking part in the control of lipid metabolism.
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Blood-feeding induces reversible functional changes in flight muscle mitochondria of Aedes aegypti mosquito.
PLoS ONE
PUBLISHED: 04-27-2009
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Hematophagy poses a challenge to blood-feeding organisms since products of blood digestion can exert cellular deleterious effects. Mitochondria perform multiple roles in cell biology acting as the site of aerobic energy-transducing pathways, and also an important source of reactive oxygen species (ROS), modulating redox metabolism. Therefore, regulation of mitochondrial function should be relevant for hematophagous arthropods. Here, we investigated the effects of blood-feeding on flight muscle (FM) mitochondria from the mosquito Aedes aegypti, a vector of dengue and yellow fever.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.