Toxicity effect of the acaricide fipronil in semi-engorged females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae): preliminary determination of the minimum lethal concentration and LC(50)

Chemical acaricides, especially fipronil (active ingredient of Frontline®), are still the most effective method to control tick populations. In this study, the effectiveness of fipronil was assessed in semi-engorged females of the tick Rhipicephalus sanguineus. A protocol for an in vitro bioassay (AIT) was developed, and the LC₅₀ (lethal concentration 50%) and 95% confidence interval were determined. Ticks were immersed in Petri dishes with different concentrations of fipronil or distilled water for 2 min, dried, and placed in an incubator for 7 days. Dead R. sanguineus females treated with the 14 concentrations of fipronil were counted daily. Mortality results were compared with the Probit analysis, and the LC₅₀ and 95% confidence interval were calculated, g (95): LC₅₀ = 9.647 (4.711 to 13.470). This study was aimed at developing a more appropriate and updated protocol for an in vitro bioassay (AIT – adult immersion test), and providing information on the toxic potential of fipronil (elimination of ectoparasites with lower concentrations) and sensitivity of ticks, especially R. sanguineus, a pest of great interest, due to its occurrence in urban environments.     

NMII Forms a Contractile Transcellular Sarcomeric Network to Regulate Apical Cell Junctions and Tissue Geometry

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Highlights? NMII within epithelial junctions assembles into a chain of muscle-like sarcomeres. ? Sarcomeres are paired across the junction to form transcellular contractile units. ? NMII isoforms show differential expression across heterotypic cell junctions. ? Coordinated sarcomere contractility modulates apical cell shape and tissue geometry     

Effects of ricinoleic acid esters from castor oil of Ricinus communis on the vitellogenesis of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) ticks

This study examines the effects of ricinoleic acid esters from Ricinus communis castor oil on the vitellogenesis of Rhipicephalus sanguineus ticks attached to hosts that were fed with commercial rabbit food containing these esters. The oocytes of ticks from the treatment group (TG) showed cytoplasmic changes that inhibited the development of oocytes I and II to the advanced stages (IV and V) in addition to preventing the maturation of oocytes V, resulting in small ones. In addition, sperm was not observed in ampoules. Our findings confirm the acaricide potential of ricinoleic acid esters.     

Antigen-presenting cells in draining lymph nodes of goats repeatedly infested by the Cayenne tick <Emphasis Type="Italic">Amblyomma cajennense</Emphasis> nymphs

Resistance to tick feeding has been previously shown to be an acquired, immunologically mediated phenomenon in goats, associated with cutaneous basophilia to nymphs of Amblyomma cajennense, the Cayenne tick, after repeated infestations. On the other hand, it is well known that antigen-presenting cells (APCs) play an important role in the host immune reaction to tick infestations. The most able APCs for Th cells are the well defined dendritic cells, mononuclear phagocytes and B-lymphocytes. Immunohistochemical analysis of draining lymph nodes of goats repeatedly infested with nymphs of the ixodid tick A. cajennense to search for APCs was done. Pre-scapular lymph nodes draining the tick attachment sites were collected 15 days after both the first and third infestations. Tick infestations resulted in increased number of CD21⁺ B lymphocytes in lymph nodes after the tertiary infestation. However, the number of CD11b⁺ and CD11c⁺ cells were not altered after the successive infestations. Lower numbers of CD11c⁺ cells had infiltrated lymph nodes responsible for draining the tick infested skin. These findings suggest that acquired immunity of goats against nymphs of A. cajennense is possibly established by B lymphocytes during the first infestation and that APCs may play a key role in this mechanism.     

Secretory process of salivary glands of female Amblyomma cajennense (Acari: Ixodidae) ticks fed on resistant rabbits

Ticks have great economic and health importance since infested animals have reduced milk and meat production, and, besides that, they are expensive ectoparasites to control. While feeding, ticks can transmit to their hosts a large amount of pathogens, including Rickettsia rickettsii responsible for the ??spotted fever?? or ??fever of the mountains.?? It is known that animals infested with ticks or artificially immunized with their salivary gland extracts develop resistance, which is related to a decrease in engorged female weight, in egg-laying by adults, in egg viability and, in some cases, in the capacity of pathogens transmission. The present study aimed to examine morpho-histochemically the female salivary glands of semi and engorged Amblyomma cajennense fed on resistant rabbits. The results revealed that acinus I had no changes when compared to that of females fed on naive rabbits. The c cells of acinus II showed signs of early degeneration, which may result in feeding efficiency decrease. In acinus III d cells, activity time was longer. Such occurrence was associated with the time of female fixation, which increased in females fed on resistant hosts.     

Myoglobin-H2O2 catalyzes the oxidation of β-ketoacids to α-dicarbonyls: mechanism and implications in ketosis

Acetoacetate (AA) and 2-methylacetoacetate (MAA) are accumulated in metabolic disorders such as diabetes and isoleucinemia. Here we examine the mechanism of AA and MAA aerobic oxidation initiated by myoglobin (Mb)/H₂O₂. We propose a chemiluminescent route involving a dioxetanone intermediate whose thermolysis yields triplet α-dicarbonyl species (methylglyoxal and diacetyl). The observed ultraweak chemiluminescence increased linearly on raising the concentration of either Mb (10-500 μM) or AA (10-100 mM). Oxygen uptake studies revealed that MAA is almost a 100-fold more reactive than AA. EPR spin-trapping studies with MNP/MAA revealed the intermediacy of an α-carbon-centered radical and acetyl radical. The latter radical, probably derived from triplet diacetyl, is totally suppressed by sorbate, a well-known quencher of triplet carbonyls. Furthermore, an EPR signal assignable to MNP-AA^• adduct was observed and confirmed by isotope effects. Oxygen consumption and α-dicarbonyl yield were shown to be dependent on AA or MAA concentrations (1-50 mM) and on H₂O₂ or tert-butOOH added to the Mb-containing reaction mixtures. That ferrylMb is involved in a peroxidase cycle acting on the substrates is suggested by the reaction pH profiles and immunospin-trapping experiments. The generation of radicals and triplet dicarbonyl products by Mb/H₂O₂/β-ketoacids may contribute to the adverse health effects of ketogenic unbalance.     

The FMRP/GRK4 mRNA interaction uncovers a new mode of binding of the Fragile X mental retardation protein in cerebellum

Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is caused by the silencing of the FMR1 gene encoding an RNA-binding protein (FMRP) mainly involved in translational control. We characterized the interaction between FMRP and the mRNA of GRK4, a member of the guanine nucleotide-binding protein (G protein)-coupled receptor kinase super-family, both in vitro and in vivo. While the mRNA level of GRK4 is unchanged in the absence or in the presence of FMRP in different regions of the brain, GRK4 protein level is increased in Fmr1-null cerebellum, suggesting that FMRP negatively modulates the expression of GRK4 at the translational level in this brain region. The C-terminal region of FMRP interacts with a domain of GRK4 mRNA, that we called G4RIF, that is folded in four stem loops. The SL1 stem loop of G4RIF is protected by FMRP and is part of the S1/S2 sub-domain that directs translation repression of a reporter mRNA by FMRP. These data confirm the role of the G4RIF/FMRP complex in translational regulation. Considering the role of GRK4 in GABAB receptors desensitization, our results suggest that an increased GRK4 levels in FXS might contribute to cerebellum-dependent phenotypes through a deregulated desensitization of GABAB receptors.     

Pivotal role of Toll-like receptors 2 and 4, its adaptor molecule MyD88, and inflammasome complex in experimental tubule-interstitial nephritis

Tubule-interstitial nephritis (TIN) results in decreased renal function and interstitial inflammation, which ultimately leads to fibrosis. Excessive adenine intake can cause TIN because xanthine dehydrogenase (XDH) can convert this purine into an insoluble compound, which precipitates in the tubuli. Innate immune sensors, such as Toll-like receptors (TLR) and inflammasome complex, play a crucial role in the initiation of inflammation. The aim of this study was to evaluate the roles of TLR-2 and -4, Myd88 and inflammasome complex in an experimental model of TIN. Here, we show that wild-type (WT) mice fed adenine-enriched food exhibited significant renal dysfunction and enhanced cellular infiltration accompanied by collagen deposition. They also presented higher gene and protein expression of pro-inflammatory cytokines. In contrast, TLR-2, -4, MyD88, ASC and Caspase-1 KO mice showed renoprotection associated with expression of inflammatory molecules at levels comparable to controls. Furthermore, treatment of WT animals with allopurinol, an XDH inhibitor, led to reduced levels of uric acid, oxidative stress, collagen deposition and a downregulation of the NF-kB signaling pathway. We concluded that MyD88 signaling and inflammasome participate in the development of TIN. Furthermore, inhibition of XDH seems to be a promising way to therapeutically target the developing inflammatory process.     

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.     

Polymeric Nanoparticles as Oral Delivery Systems for Encapsulation and Release of Polyphenolic Compounds: Impact on Quercetin Antioxidant Activity & Bioaccessibility

A delivery system containing polymeric (Eudragit) nanoparticles has been developed for encapsulation and controlled release of bioactive flavonoids (quercetin). Nanoparticles were fabricated using a solvent displacement method. Particle size, morphology, and charge were measured by light scattering, electron microscopy and ??-potential. Encapsulation efficiency (EE) and release profiles were determined using electrochemical methods. Molecular interactions within the particle matrix were characterized by X-ray diffraction, differential scanning calorimetry, and infrared spectroscopy. Antioxidant properties of free and encapsulated quercetin were analyzed by TBARS and fluorescence spectroscopy. Bioaccessibility of quercetin was evaluated using an in vitro digestion model. Relatively small (d????370?nm) anionic polymeric nanoparticles were formed containing quercetin in a non-crystalline form (EE????67?%). The main interaction between quercetin and Eudragit was hydrogen bonding. Encapsulated quercetin remained stable during 6?months storage and maintained its antioxidant activity. Quercetin bioaccessibility within simulated small intestinal conditions was improved by encapsulation. The knowledge obtained from this study will facilitate the rational design and fabrication of polymeric nanoparticles as oral delivery systems for encapsulation, protection, and release of bioactive compounds.