Septin 2 interacts with dengue virus replication complex proteins and participates in virus replication in mosquito cells.

Septins are a family of GTP-binding proteins identified in insects and mammals. Septins are components of the cytoskeleton and participate in cytokinesis, chromosomal segregation, intracellular vesicular traffic, and response to pathogens. Human septin 6 was identified as necessary for hepatitis C virus replication. Information about host factors necessary for flavivirus replication in mosquitoes is scarce. Thus, the role of septins in the replicative cycle of dengue virus in Aedes spp. derived cells was investigated. Through bioinformatic analysis, sequences of septin-like proteins were identified. Infected mosquito cells showed increased expression of Sep2. Colocalization analysis, proximity ligation and immunoprecipitation assays indicated that Sep2 interacts with proteins E, NS3 and NS5, but not NS1. Immunoelectron microscopy evidenced the presence of AalSep2 in replicative complexes. Finally, silencing of Sep2 expression resulted in a significant decrease in virus progeny, indicating that Sep2 is a host factor participating in dengue virus replication in mosquito cells.

Development of the indirect flight muscles of Aedes aegypti, a main arbovirus vector.

BACKGROUND: Flying is an essential function for mosquitoes, required for mating and, in the case of females, to get a blood meal and consequently function as a vector. Flight depends on the action of the indirect flight muscles (IFMs), which power the wings beat. No description of the development of IFMs in mosquitoes, including Aedes aegypti, is available. METHODS: A. aegypti thoraces of larvae 3 and larvae 4 (L3 and L4) instars were analyzed using histochemistry and bright field microscopy. IFM primordia from L3 and L4 and IFMs from pupal and adult stages were dissected and processed to detect F-actin labelling with phalloidin-rhodamine or TRITC, or to immunodetection of myosin and tubulin using specific antibodies, these samples were analyzed by confocal microscopy. Other samples were studied using transmission electron microscopy. RESULTS: At L3-L4, IFM primordia for dorsal-longitudinal muscles (DLM) and dorsal-ventral muscles (DVM) were identified in the expected locations in the thoracic region: three primordia per hemithorax corresponding to DLM with anterior to posterior orientation were present. Other three primordia per hemithorax, corresponding to DVM, had lateral position and dorsal to ventral orientation. During L3 to L4 myoblast fusion led to syncytial myotubes formation, followed by myotendon junctions (MTJ) creation, myofibrils assembly and sarcomere maturation. The formation of Z-discs and M-line during sarcomere maturation was observed in pupal stage and, the structure reached in teneral insects a classical myosin thick, and actin thin filaments arranged in a hexagonal lattice structure. CONCLUSIONS: A general description of A. aegypti IFM development is presented, from the myoblast fusion at L3 to form myotubes, to sarcomere maturation at adult stage. Several differences during IFM development were observed between A. aegypti (Nematoceran) and Drosophila melanogaster (Brachyceran) and, similitudes with Chironomus sp. were observed as this insect is a Nematoceran, which is taxonomically closer to A. aegypti and share the same number of larval stages.

Intestinal response to dietary manganese depletion in Drosophila.

Manganese is considered essential for animal growth. Manganese ions serve as cofactors to three mitochondrial enzymes: superoxide dismutase (Sod2), arginase and glutamine synthase, and to glycosyltransferases residing in the Golgi. In Drosophila melanogaster, manganese has also been implicated in the formation of ceramide phosphoethanolamine, the insect's sphingomyelin analogue, a structural component of cellular membranes. Manganese overload leads to neurodegeneration and toxicity in both humans and Drosophila. Here, we report specific absorption and accumulation of manganese during the first week of adulthood in flies, which correlates with an increase in Sod2 activity during the same period. To test the requirement of dietary manganese for this accumulation, we generated a Drosophila model of manganese deficiency. Due to the lack of manganese-specific chelators, we used chemically defined media to grow the flies and deplete them of the metal. Dietary manganese depletion reduced Sod2 activity. We then examined gene and protein expression changes in the intestines of manganese depleted flies. We found adaptive responses to the presumed loss of known manganese-dependent enzymatic activities: less glutamine synthase activity (amination of glutamate to glutamine) was compensated by 50% reduction in glutaminase (deamination of glutamine to glutamate); less glycosyltransferase activity, predicted to reduce protein glycosylation, was compensated by 30% reduction in lysosomal mannosidases (protein deglycosylating enzymes); less ceramide phosphoethanolamine synthase activity was compensated by 30% reduction in the Drosophila sphingomyeline phospodiesterase, which could catabolize ceramide phosphoethanolamine in flies. Reduced Sod2 activity, predicted to cause superoxide-dependent iron-sulphur cluster damage, resulted in cellular iron misregulation.

Lys48 ubiquitination during the intraerythrocytic cycle of the rodent malaria parasite, Plasmodium chabaudi.

Ubiquitination tags proteins for different functions within the cell. One of the most abundant and studied ubiquitin modification is the Lys48 polyubiquitin chain that modifies proteins for their destruction by proteasome. In Plasmodium is proposed that post-translational regulation is fundamental for parasite development during its complex life-cycle; thus, the objective of this work was to analyze the ubiquitination during Plasmodium chabaudi intraerythrocytic stages. Ubiquitinated proteins were detected during intraerythrocytic stages of Plasmodium chabaudi by immunofluorescent microscopy, bidimensional electrophoresis (2-DE) combined with immunoblotting and mass spectrometry. All the studied stages presented protein ubiquitination and Lys48 polyubiquitination with more abundance during the schizont stage. Three ubiquitinated proteins were identified for rings, five for trophozoites and twenty for schizonts. Only proteins detected with a specific anti- Lys48 polyubiquitin antibody were selected for Mass Spectrometry analysis and two of these identified proteins were selected in order to detect the specific amino acid residues where ubiquitin is placed. Ubiquitinated proteins during the ring and trophozoite stages were related with the invasion process and in schizont proteins were related with nucleic acid metabolism, glycolysis and protein biosynthesis. Most of the ubiquitin detection was during the schizont stage and the Lys48 polyubiquitination during this stage was related to proteins that are expected to be abundant during the trophozoite stage. The evidence that these Lys48 polyubiquitinated proteins are tagged for destruction by the proteasome complex suggests that this type of post-translational modification is important in the regulation of protein abundance during the life-cycle and may also contribute to the parasite cell-cycle progression.

IDENTIFICATION AND EXPRESSION ANALYSIS OF TWO 14-3-3 PROTEINS IN THE MOSQUITO Aedes aegypti, AN IMPORTANT ARBOVIRUSES VECTOR.

The 14-3-3 proteins are evolutionarily conserved acidic proteins that form a family with several isoforms in many cell types of plants and animals. In invertebrates, including dipteran and lepidopteran insects, only two isoforms have been reported. 14-3-3 proteins are scaffold molecules that form homo- or heterodimeric complexes, acting as molecular adaptors mediating phosphorylation-dependent interactions with signaling molecules involved in immunity, cell differentiation, cell cycle, proliferation, apoptosis, and cancer. Here, we describe the presence of two isoforms of 14-3-3 in the mosquito Aedes aegypti, the main vector of dengue, yellow fever, chikungunya, and zika viruses. Both isoforms have the conserved characteristics of the family: two protein signatures (PS1 and PS2), an annexin domain, three serine residues, targets for phosphorylation (positions 58, 184, and 233), necessary for their function, and nine alpha helix-forming segments. By sequence alignment and phylogenetic analysis, we found that the molecules correspond to Ɛ and ζ isoforms (Aeae14-3-3ε and Aeae14-3-3ζ). The messengers and protein products were present in all stages of the mosquito life cycle and all the tissues analyzed, with a small predominance of Aeae14-3-3ζ except in the midgut and ovaries of adult females. The 14-3-3 proteins in female midgut epithelial cells were located in the cytoplasm. Our results may provide insights to further investigate the functions of these proteins in mosquitoes.

Protein phosphorylation during Plasmodium berghei gametogenesis.

Plasmodium gametogenesis within the mosquito midgut is a complex differentiation process involving signaling mediated by phosphorylation, which modulate metabolic routes and protein synthesis required to complete this development. However, the mechanisms leading to gametogenesis activation are poorly understood. We analyzed protein phosphorylation during Plasmodium berghei gametogenesis in vitro in serum-free medium using bidimensional electrophoresis (2-DE) combined with immunoblotting (IB) and antibodies specific to phosphorylated serine, threonine and tyrosine. Approximately 75 protein exhibited phosphorylation changes, of which 23 were identified by mass spectrometry. These included components of the cytoskeleton, heat shock proteins, and proteins involved in DNA synthesis and signaling pathways among others. Novel phosphorylation events support a role for these proteins during gametogenesis. The phosphorylation sites of six of the identified proteins, HSP70, WD40 repeat protein msi1, enolase, actin-1 and two isoforms of large subunit of ribonucleoside reductase were investigated using TiO2 phosphopeptides enrichment and tandem mass spectrometry. In addition, transient exposure to hydroxyurea, an inhibitor of ribonucleoside reductase, impaired male gametocytes exflagellation in a dose-dependent manner, and provides a resource for functional studies.

AealRACK1 expression and localization in response to stress in C6/36 HT mosquito cells.

The Receptor for Activated C Kinase 1 (RACK1), a scaffold protein member of the tryptophan-aspartate (WD) repeat family, folds in a seven-bladed ß-propeller structure that permits the association of proteins to form active complexes. Mosquitoes of the genus Aedes sp., are vectors of virus producing important diseases such as: dengue, chikungunya and yellow fever. Based on the highly conserved gene sequence of AeaeRACK1 of the mosquito Aedes aegypti we characterized the mRNA and protein of the homologous AealRACK1 from the Ae. albopictus-derived cell line C6/36 HT. Two protein species differing in MW/pI values were observed at 35kDa/8.0 and 36kDa/6.5. The behavior of AealRACK1 was studied inducing stress with serum deprivation and the glucocorticoid dexamethasone. Both stressors induced increase of the expression of AealRACK1 mRNA and proteins. In serum-deprived cells AealRACK1 protein was located cortically near the plasma membrane in contrast to dexamethasone-treated cells where the protein formed a dotted pattern in the cytoplasm. In addition, 33 protein partners were identified by immunoprecipitation and mass spectrometry. Most of the identified proteins were ribosomal, involved in signaling pathways and stress responses. Our results suggest that AealRACK1 in C6/36 HT cells respond to stress increasing its synthesis and producing phosphorylated activated form. BIOLOGICAL SIGNIFICANCE: Insect cells adapt to numerous environmental stressors, including chemicals and invasion of pathogenic microorganisms among others, coordinating cellular and organismal responses. Individual cells sense the environment using receptors that trigger signaling pathways that regulate expression of specific effector proteins and/or cellular responses as movement or secretion. In the coordination of responses to stress, scaffold proteins are pivotal molecules that recruit other proteins forming active complexes. The Receptor for Activated C Kinase 1 (RACK1) is the best studied member of the conserved tryptophan-aspartate (WD) repeat family. RACK1 folds in a seven-bladed ß-propeller structure and it could be activated during stress, participating in different signaling pathways. The presence and activities of RACK1 in mosquitoes had not been documented before, in this work the molecule is demonstrated in an Aedes albopictus-derived cell line and its reaction to stress is observed under the effect of serum deprivation and the presence of glucocorticoid analog dexamethasone, a chemical used to cause stress in vitro.

Differential effects of rapid eye movement sleep deprivation and immobilization stress on blood lymphocyte subsets in rats.

OBJECTIVES: There is growing evidence of the relationship between sleep and the immune response. Studies aimed at elucidating the function of rapid eye movement (REM) sleep have found it difficult to separate the effects due to REM sleep deprivation and the effects due to the stress produced by the deprivation procedure. It has been claimed that immobilization is the main stressor that the animals have to face during the deprivation process. In this study, we analyzed the effects of short-term (24 h) and long-term (240 h) REM sleep deprivation on the distribution of lymphocyte subsets in the peripheral blood of rats. In addition, these effects were compared with those obtained after both short- and long-term stress by immobilization. METHODS: Lymphocyte population bearing surface markers such as CD5 (T cells), CD45RA (B cells), CD4 (T helper/inducer cells), CD8 (T suppressor/cytotoxic cells) and CD161 (NK cells) were analyzed using monoclonal antibodies. Lymphocyte subsets were assessed by flow cytometry. RESULTS: Both short- and long-term REM sleep deprivation decreased the percentage of T lymphocytes and induced a significant increase in NK cells. Short-term immobilization induced only a significant increase in the percentage of B lymphocytes and a decrease in the percentage of T lymphocytes, while long-term immobilization did not elicit any change. CONCLUSION: The present results support the notion that REM sleep deprivation and immobilization stress each exert particular effects on the immune system. These data suggest that the characteristics of the immune response will depend on the nature of the behavioral manipulation.