Characterization of a glycine-rich protein from Rhipicephalus microplus: tissue expression, gene silencing and immune recognition.

Salivary molecules, as glycine-rich proteins (GRPs), are essential to tick attachment and feeding on the host and are suggested to be involved in the host's immune system evasion, therefore representing natural candidates in the search for protective vaccine antigens. This work shows the molecular characterization of a GRP from Rhipicephalus microplus (RmGRP). The cDNA and putative amino acid sequences were analysed, as well as the transcription level in tick tissues/developmental stages, showing the highest levels of gene expression in 1-day-old larvae and salivary glands of fully engorged females. RmGRP gene silencing resulted in a lower hatching rate of larvae from treated females. In addition, recombinant RmGRP (rRmGRP) was recognized by sera from naturally and experimentally infested bovines, displaying considerable differences among the individuals tested. rRmGRP was recognized by anti-saliva and anti-salivary glands sera, while anti-rRmGRP serum recognized RmGRP in saliva and salivary glands, indicating its secretion into the host. The data collected indicate that RmGRP may present roles other than in the tick-host relationship, especially in embryo development. In addition, the high expression in adult females, antigenicity and presence of shared characteristics with other tick protective GRPs turns RmGRP a potential candidate to compose an anti-tick vaccine cocktail.

The detection of viable vegetative cells of Bacillus sporothermodurans using propidium monoazide with semi-nested PCR.

Bacillus sporothermodurans produces highly heat-resistant spores that can survive ultra-high temperature (UHT) treatment in milk. Therefore, we developed a rapid, specific and sensitive semi-nested touchdown PCR assay combined with propidium monoazide (PMA) treatment for the detection of viable B. sporothermodurans vegetative cells. The semi-nested touchdown PCR alone proved to be specific for B. sporothermodurans, and the achieved detection limit was 4 CFU/mL from bacterial culture and artificially contaminated UHT milk. This method combined with PMA treatment was shown to amplify DNA specifically from viable cells and presented a detection limit of 10(2) CFU/mL in UHT milk. The developed PMA-PCR assay shows applicability for the specific detection of viable cells of B. sporothermodurans from UHT milk. This method is of special significance for applications in the food industry by reducing the time required for the analysis of milk and dairy products for the presence of this microorganism.

Fluoroquinolone and beta-lactam antimicrobials induce different transcriptome profiles in Salmonella enterica persister cells.

Here, we investigate the transcriptome profiles of two S. Enteritidis and one S. Schwarzengrund isolates that present different persister levels when exposed to ciprofloxacin or ceftazidime. It was possible to note a distinct transcript profile among isolates, time of exposure, and treatment. We could not find a commonly expressed transcript profile that plays a role in persister formation after S. enterica exposure to beta-lactam or fluoroquinolone, as only three DEGs presented the same behavior under the conditions and isolates tested. It appears that the formation of persisters in S. enterica after exposure to ciprofloxacin is linked to the overexpression of genes involved in the SOS response (recA), cell division inhibitor (sulA), iron-sulfur metabolism (hscA and iscS), and type I TA system (tisB). On the other hand, most genes differentially expressed in S. enterica after exposure to ceftazidime appeared to be downregulated and were part of the flagellar assembly apparatus, citrate cycle (TCA cycle), glycolysis/gluconeogenesis, carbon metabolism, bacterial secretion system, quorum sensing, pyruvate metabolism pathway, and biosynthesis of secondary metabolites. The different transcriptome profiles found in S. enterica persisters induced by ciprofloxacin and ceftazidime suggest that these cells modulate their response differently according to each stress.

Subcellular localization of X-linked inhibitor of apoptosis protein (XIAP) in cancer: Does that matter?

X-linked inhibitor of apoptosis protein (XIAP) finely tunes the balance between survival and death to control homeostasis. XIAP is found aberrantly expressed in cancer, which has been shown to promote resistance to therapy-induced apoptosis and confer poor outcome. Despite its predominant cytoplasmic localization in human tissues, growing evidence implicates the expression of XIAP in other subcellular compartments in sustaining cancer hallmarks. Herein, we review our current knowledge on the prognostic role of XIAP localization and discuss molecular mechanisms underlying differential biological functions played in each compartment. The comprehension of XIAP subcellular shuttling and functional dynamics might provide the rationale for future anticancer therapeutics.

Differential gene expression patterns in the autogamous plant Hordeum euclaston (Poaceae).

Sib-seedlings of 95 strains of the strictly autogamous grass Hordeum euclaston were analyzed by horizontal polyacrylamide gel electrophoresis for four isoenzyme systems at a specific ontogenetic stage. We found differences in the activity of some genes among individuals of this species. Hence, an ontogenetic analysis was carried out to investigate 12 strains at five ontogenetic stages, to determine the patterns of expression of these genes during development. The differences in the presence versus absence of certain isoenzyme bands may be due to differential regulatory activation in response to environmental differences, as all plants showed the same structural genes, although these genes were active in different tissues and/or times of development. These results indicate the importance of differential gene activation in the metabolic phenotype variability of this strictly autogamous, highly homozygous species. The same structural alleles for isoenzymes showed the active form of the enzymes (phenotypic expression) to be present in different tissues and/or stages of development. Differential isoenzyme gene activation was shown to be directly responsible for the enzymatic variability (metabolic phenotype) presented by the plants, which seem to possess almost no heterozygosis.

The scavenger receptor MARCO is involved in Leishmania major infection by CBA/J macrophages.

CBA/J mice are resistant to Leishmania major infection but are permissive to L. amazonensis infection. In addition, CBA/J macrophages control L. major but not L. amazonensis infection in vitro. Phagocytosis by macrophages is known to determine the outcome of Leishmania infection. Pattern recognition receptors (PRR) adorning antigen presenting cell surfaces are known to coordinate the link between innate and adaptive immunity. The macrophage receptor with collagenous structure (MARCO) is a PRR that is preferably expressed by macrophages and is capable of binding Gram-positive and Gram-negative bacteria. No research on the role of MARCO in Leishmania-macrophage interactions has been reported. Here, we demonstrate, for the first time, that MARCO expression by CBA/J macrophages is increased in response to both in vitro and in vivo L. major infections, but not to L. amazonensis infection. In addition, a specific anti-MARCO monoclonal antibody reduced L. major infection of macrophages by 30%-40% in vitro. The draining lymph nodes of anti-MARCO-treated mice displayed a reduced presence of immunolabelled parasite and parasite antigens, as well as a reduced inflammatory response. These results support the hypothesis that MARCO has a role in macrophage infection by L. major in vitro as well as in vivo.

Pharmacological action of tick saliva upon haemostasis and the neutralization ability of sera from repeatedly infested hosts.

Ticks are blood-feeding arthropods widely distributed in the world and vectors of several diseases. As haematophagy demands evasion strategies and repeatedly infested hosts develop protective immune responses, we investigated the mechanisms of the Rhipicephalus (Boophilus) microplus saliva anti-haemostatic activity and the possible relationship between the acquired natural anti-tick host resistance and anti-haemostatic action. For this purpose, we studied the effects of R. microplus saliva on different pathways of haemostasis and tested whether repeated infested bovine sera (RIBS) are able to abolish salivary anti-haemostatic activities. R. microplus saliva (i) displays inhibitory activity upon collagen-induced platelet aggregation; (ii) inhibits the induction of endothelial pro-coagulant state; and (iii) reduces thrombogenesis in vivo. RIBS were shown to be able to partially block the delay of coagulation and the anti-thrombotic effect of saliva, and to totally abolish the modulation of endothelium activation. Conversely, RIBS has no effect on the inhibition of platelet aggregation. These results show, for the first time, the neutralization ability of sera from acquired resistance hosts against tick anti-haemostatics. Moreover, this is the first report of a haematophagous parasite able to modulate endothelial cell pro-coagulant state, and addresses the presence of anti-platelet and anti-thrombotic activity in R. microplus saliva.

Comparative IgG recognition of tick extracts by sera of experimentally infested bovines.

Enzyme-linked immunosorbent assay (ELISA) and Western blot were used to investigate the pattern of antibody responses of six bovines infested twelve times with Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae) (six heavy infestations followed by six light infestations) against salivary gland, gut and larvae extracts. During heavy infestations, bovine IgG levels were shown to be higher, and a decrease in the number and weight of ticks that completed the parasitic cycle was observed. The pattern changed starting from the seventh infestation, showing a decrease in IgG levels. An initial increase followed by a significant decrease in the proportion of ticks that completed the parasitic cycle was also observed from the seventh infestation. The number of molecules recognized by Western blot was higher from sera collected following heavy infestations than after light infestations, although a great variation in the profiles detected could be seen when the bovines were compared. These results indicate that IgG responses to different tick antigens may not be generally associated with bovine resistance, and that infestation levels modulate the magnitude of humoral responses and possibly the immune mechanisms in the natural acquisition of tick resistance.

High-impact polystyrene/polyaniline membranes for acid solution treatment by electrodialysis: preparation, evaluation, and chemical calculation.

In this study different membranes were produced, aiming to evaluate their use in electrodialysis. These membranes were produced using conventional polymer (high-impact polystyrene) and polyaniline. The membrane characterization was done by FTIR spectroscopy, scanning electron microscopy (SEM), and thermogravimetry (TGA). The studies of the zinc and proton extraction ionic transport through the membranes were evaluated using a three-compartment cell. The results obtained using the produced membranes were compared to the results obtained with the commercial membrane Nafion 450. It was found that a synthesized membrane can be used to recover zinc in acid media. In addition, a preliminary computational essay about the structures of PAni and CSA is presented.

Mapping of quantitative trait locus associated with maize tolerance to high seed drying temperature.

quantitative trait locus (QTL) mapping and identification of traits of agronomic importance is important in the process of molecular marker-assisted selection in breeding programs. The molecular map of maize is well saturated and QTL and simple sequence repeat (SSR) markers have been identified, whereas few markers linked to seed quality traits are included. The present study aimed to identify QTL and the gene action and to quantify the effects of these regions in the phenotypic variation related to maize tolerance to high seed drying temperature. SSR markers and 129 segregating families of F2 plants of the cross of intolerant and tolerant lines were used in regression and composite interval mapping methods. Three maize QTL associated with tolerance to high seed drying temperature were identified and mapped to chromosomes 6 and 8, explaining 39% of the phenotypic variation of the trait with additive, dominance and overdominance gene action. These markers seem to be effectively associated with the evaluated trait, since all were mapped near genes whose expression products were associated with seed desiccation tolerance.

Rickettsia parkeri in free-ranging wild canids from Brazilian Pampa.

Spotted fevers are tick-borne diseases associated with various Rickettsia species. Rickettsia parkeri sensu stricto (s.s.) is the agent of an emerging eschar-associated rickettsiosis in humans from the USA and South American Pampa. Considering that R. parkeri s.s. is restricted to Americas and the potential role of dogs in the epidemiology of the disease, it is thus reasonable to hypothesize that wild canids could be involved in the enzootic cycle of this rickettsiosis. The aim of this work was to investigate the potential role of the wild canids from Pampa, Cerdocyon thous (crab-eating fox) and Lycalopex gymnocercus (Pampas fox), in the ecology of R. parkeri s.s. For that, 32 live-trapped free-ranging wild canids were sampled. Ticks were observed in 30 of the 32 foxes. Of the 292 ticks collected, 22 (7.5%) were positive by PCR for the presence of R. parkeri s.s. DNA. Also, 20 (62%) wild canids showed antibodies against R. parkeri. The results suggest that wild canids are involved in the enzootic cycle of R. parkeri s.s. in the Pampa biome and could be responsible for pathogen (and its vectors) dispersal.

One-step global parameter estimation of kinetic inactivation parameters for Bacillus sporothermodurans spores under static and dynamic thermal processes.

Bacillus sporothermodurans produces highly heat-resistant endospores, that can survive under ultra-high temperature. High heat-resistant sporeforming bacteria are one of the main causes for spoilage and safety of low-acid foods. They can be used as indicators or surrogates to establish the minimum requirements for heat processes, but it is necessary to understand their thermal inactivation kinetics. The aim of the present work was to study the inactivation kinetics under both static and dynamic conditions in a vegetable soup. Ordinary least squares one-step regression and sequential procedures were applied for estimating these parameters. Results showed that multiple dynamic heating profiles, when analyzed simultaneously, can be used to accurately estimate the kinetic parameters while significantly reducing estimation errors and data collection.

Cloning and partial characterization of a Boophilus microplus (Acari: Ixodidae) glutathione S-transferase.

A cDNA of glutathione S-transferase (GST) was isolated from a cDNA library of salivary glands of Boophilus microplus. The recombinant protein was purified by glutathione affinity chromatography and assayed upon the chromogenic substrate CDNB. The 864 bp cloned fragment was sequenced and showed an open reading frame coding for a protein of 220 amino acids. Expression of the GST gene was tested by RT-PCR in tick tissues and larvae mRNA. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to the mammalian class mu GSTs.

Regulation of the glutamate uptake by extracellular calcium.

To determine whether [Ca(2+)](e) modulates glutamate re-uptake, we studied the uptake mechanism into rat cerebrocortical synaptosomes. The removal of extracellular Ca(2+) caused a negative modulation in the uptake mechanism. The calculated K(50) value was 0.185 +/- 0.019 mM (n = 4). The Michaelis-Menten data analysis indicate that absence of Ca(2+) diminished the V(max) kinetic parameter by about 60% without changing significantly the K(m) suggesting a non-competitive mechanism. We also tested the involvement of intracellular Ca(2+) in this phenomenon by trapping BAPTA into the synaptosomal vesicles to control the Ca(2+) concentration. Our results suggest that intracellular Ca(2+) changes have a less predominant role on the glutamate uptake than do extracellular Ca(2+). These findings argue in favor of an important role of extracellular [Ca(2+)] in maintaining the L-glutamate re-uptake mechanism in the mammalian central nervous system.

Stable carbon isotope ratio of tree leaves, boles and fine litter in a tropical forest in Rondônia, Brazil.

Leaves of 208 trees were collected for isotopic analysis together with wood from 36 tree boles and 18 samples of fine litter from a terra-firme forest located at Samuel Ecological Reserve, Rondônia State, in the southwestern Amazon region. The range of δ13C values in leaves was from -28 to -36‰, with an average (±1 SD) of -32.1 ± 1.5‰, which was more negative than the δ13C values of bole samples (-28.4 ± 2.0‰) and fine litter (-28.7 ± 2.0‰). These values are within the range found for tropical and subtropical forests. Pooling the δ13C values for leaf samples from trees of the same height gave averages which were positively correlated with plant height at a highly significant level, with a slope of 0.06 and an intercept of -33.3‰ and a correlation coefficient r 2=0.70 (P<0.001).

Contouring the hip and the abdomen.

This article describes the surgical procedures used for the aesthetic improvements of the flanks and abdomen. A clinical classification for patient selection regarding when to use liposuction, surgery, or both, is included. Isolated flankplasty, isolated abdominoplasty, and combined flanks with abdominoplasty performed in the same stage are reviewed. Finally, secondary problems after traditional abdominoplasty are evaluated. Solutions are described and illustrated.

Detection of genomic variability in different populations of the cattle tick Boophilus microplus in southern Brazil.

DNA from seven isolates of the cattle tick Boophilus microplus was analyzed by restriction fragment length polymorphism (RFLP). Three different cDNA clones, named P-9, P-25 and CP-12, isolated from a B. microplus cDNA library, were used as DNA probes. DNA sequences of P-9 have high similarity to ribosomal genes, whereas P-25 does not show significant homology with known sequences within databases. CP-12 is a cDNA clone encoding a cysteine endopeptidase gene. A limited degree of polymorphism was detected with P-9 and P-25, while CP-12 showed a different pattern of bands for each tick isolate. These findings suggest the existence of a complex genotypic diversity of the tick B. microplus population in endemic regions.

Systematic study of high molecular weight compounds in Amazonian plants by high temperature gas chromatography-mass spectrometry.

The fractions of hexane and dichloromethane extraction from marupá (Simaruba amara) and (Bertholletia excelsa) leaves were analyzed by HT-HRGC (high temperature high resolution gas chromatography) and HT-HRGC coupled to mass spectrometry (HT-HRGC-MS). Several compounds can be characterized including unusual high molecular weight compounds.

[Mesenteric panniculitis]. / Paniculite mesentérica.

A case of mesenteric panniculitis is presented in which the main signal was an abdominal pulsable mass and severe obstruction of the lymphatic vessels of the mesentery. A review of the literature was done.

Serotonergic modulation of face-emotion recognition.

Facial expressions of basic emotions have been widely used to investigate the neural substrates of emotion processing, but little is known about the exact meaning of subjective changes provoked by perceiving facial expressions. Our assumption was that fearful faces would be related to the processing of potential threats, whereas angry faces would be related to the processing of proximal threats. Experimental studies have suggested that serotonin modulates the brain processes underlying defensive responses to environmental threats, facilitating risk assessment behavior elicited by potential threats and inhibiting fight or flight responses to proximal threats. In order to test these predictions about the relationship between fearful and angry faces and defensive behaviors, we carried out a review of the literature about the effects of pharmacological probes that affect 5-HT-mediated neurotransmission on the perception of emotional faces. The hypothesis that angry faces would be processed as a proximal threat and that, as a consequence, their recognition would be impaired by an increase in 5-HT function was not supported by the results reviewed. In contrast, most of the studies that evaluated the behavioral effects of serotonin challenges showed that increased 5-HT neurotransmission facilitates the recognition of fearful faces, whereas its decrease impairs the same performance. These results agree with the hypothesis that fearful faces are processed as potential threats and that 5-HT enhances this brain processing.