Systematics of the Oswaldoi Complex (Anopheles, Nyssorhynchus) in South America

Abstract Background Effective malaria control relies on accurate identification of those Anopheles mosquitoes responsible for the transmission of Plasmodium parasites. Anopheles oswaldoi s.l. has been incriminated as a malaria vector in Colombia and some localities in Brazil, but not ubiquitously throughout its Neotropical range. This evidence together with variable morphological characters and genetic differences supports that An. oswaldoi s.l. compromises a species complex. The recent fully integrated redescription of An. oswaldoi s.s. provides a solid taxonomic foundation from which to molecularly determine other members of the complex. Methods DNA sequences of the Second Internal Transcribed Spacer (ITS2 - rDNA) (n = 192) and the barcoding region of the Cytochrome Oxidase I gene (COI - mtDNA) (n = 110) were generated from 255 specimens of An. oswaldoi s.l. from 33 localities: Brazil (8 localities, including the lectotype series of An. oswaldoi), Ecuador (4), Colombia (17), Trinidad and Tobago (1), and Peru (3). COI sequences were analyzed employing the Kimura-two-parameter model (K2P), Bayesian analysis (MrBayes), Mixed Yule-Coalescent model (MYC, for delimitation of clusters) and TCS genealogies. Results Separate and combined analysis of the COI and ITS2 data sets unequivocally supported four separate species: two previously determined (An. oswaldoi s.s. and An. oswaldoi B) and two newly designated species in the Oswaldoi Complex (An. oswaldoi A and An. sp. nr. konderi). The COI intra- and inter-specific genetic distances for the four taxa were non-overlapping, averaging 0.012 (0.007 to 0.020) and 0.052 (0.038 to 0.064), respectively. The concurring four clusters delineated by MrBayes and MYC, and four independent TCS networks, strongly confirmed their separate species status. In addition, An. konderi of Sallum should be regarded as unique with respect to the above. Despite initially being included as an outgroup taxon, this species falls well within the examined taxa, suggesting a combined analysis of these taxa would be most appropriate. Conclusions: Through novel data and retrospective comparison of available COI and ITS2 DNA sequences, evidence is shown to support the separate species status of An. oswaldoi s.s., An. oswaldoi A and An. oswaldoi B, and at least two species in the closely related An. konderi complex (An. sp. nr. konderi, An. konderi of Sallum). Although An. oswaldoi s.s. has never been implicated in malaria transmission, An. oswaldoi B is a confirmed vector and the new species An. oswaldoi A and An. sp. nr. konderi are circumstantially implicated, most likely acting as secondary vectors.

Genetic variability and demographic history of Heliothis virescens (Lepidoptera: Noctuidae) populations from Brazil inferred by mtDNA sequences

Intra-and inter-population genetic variability and the demographic history of Heliothis virescens (F.) populations were evaluated by using mtDNA markers (coxI, coxII and nad6) with samples from the major cotton-and soybean-producing regions in Brazil in the growing seasons 2007/08, 2008/09 and 2009/10. AMOVA indicated low and non-significant genetic structure, regardless of geographical scale, growing season or crop, with most of genetic variation occurring within populations. Clustering analyzes also indicated low genetic differentiation. The haplotype network obtained with combined datasets resulted in 35 haplotypes, with 28 exclusive occurrences, four of them sampled only from soybean fields. The minimum spanning network showed star-shaped structures typical of populations that underwent a recent demographic expansion. The recent expansion was supported by other demographic analyzes, such as the Bayesian skyline plot, the unimodal distribution of paired differences among mitochondrial sequences, and negative and significant values of neutrality tests for the Tajima's D and Fu's F-S parameters. In addition, high values of haplotype diversity ((H) over cap) and low values of nucleotide diversity (pi), combined with a high number of low frequency haplotypes and values of theta(pi)<theta(W), suggested a recent demographic expansion of H. virescens populations in Brazil. This demographic event could be responsible for the low genetic structure currently found; however, haplotypes present uniquely at the same geographic regions and from one specific host plant suggest an initial differentiation among H. virescens populations within Brazil.

Polymorphic toxin systems: Comprehensive characterization of trafficking modes, processing, mechanisms of action, immunity and ecology using comparative genomics

Background: Proteinaceous toxins are observed across all levels of inter-organismal and intra-genomic conflicts. These include recently discovered prokaryotic polymorphic toxin systems implicated in intra-specific conflicts. They are characterized by a remarkable diversity of C-terminal toxin domains generated by recombination with standalone toxin-coding cassettes. Prior analysis revealed a striking diversity of nuclease and deaminase domains among the toxin modules. We systematically investigated polymorphic toxin systems using comparative genomics, sequence and structure analysis. Results: Polymorphic toxin systems are distributed across all major bacterial lineages and are delivered by at least eight distinct secretory systems. In addition to type-II, these include type-V, VI, VII (ESX), and the poorly characterized "Photorhabdus virulence cassettes (PVC)", PrsW-dependent and MuF phage-capsid-like systems. We present evidence that trafficking of these toxins is often accompanied by autoproteolytic processing catalyzed by HINT, ZU5, PrsW, caspase-like, papain-like, and a novel metallopeptidase associated with the PVC system. We identified over 150 distinct toxin domains in these systems. These span an extraordinary catalytic spectrum to include 23 distinct clades of peptidases, numerous previously unrecognized versions of nucleases and deaminases, ADP-ribosyltransferases, ADP ribosyl cyclases, RelA/SpoT-like nucleotidyltransferases, glycosyltranferases and other enzymes predicted to modify lipids and carbohydrates, and a pore-forming toxin domain. Several of these toxin domains are shared with host-directed effectors of pathogenic bacteria. Over 90 families of immunity proteins might neutralize anywhere between a single to at least 27 distinct types of toxin domains. In some organisms multiple tandem immunity genes or immunity protein domains are organized into polyimmunity loci or polyimmunity proteins. Gene-neighborhood-analysis of polymorphic toxin systems predicts the presence of novel trafficking-related components, and also the organizational logic that allows toxin diversification through recombination. Domain architecture and protein-length analysis revealed that these toxins might be deployed as secreted factors, through directed injection, or via inter-cellular contact facilitated by filamentous structures formed by RHS/YD, filamentous hemagglutinin and other repeats. Phyletic pattern and life-style analysis indicate that polymorphic toxins and polyimmunity loci participate in cooperative behavior and facultative 'cheating' in several ecosystems such as the human oral cavity and soil. Multiple domains from these systems have also been repeatedly transferred to eukaryotes and their viruses, such as the nucleo-cytoplasmic large DNA viruses. Conclusions: Along with a comprehensive inventory of toxins and immunity proteins, we present several testable predictions regarding active sites and catalytic mechanisms of toxins, their processing and trafficking and their role in intra-specific and inter-specific interactions between bacteria. These systems provide insights regarding the emergence of key systems at different points in eukaryotic evolution, such as ADP ribosylation, interaction of myosin VI with cargo proteins, mediation of apoptosis, hyphal heteroincompatibility, hedgehog signaling, arthropod toxins, cell-cell interaction molecules like teneurins and different signaling messengers.

Trypanosoma cruzi mitochondrial maxicircles display species- and strain-specific variation and a conserved element in the non-coding region

Abstract Background The mitochondrial DNA of kinetoplastid flagellates is distinctive in the eukaryotic world due to its massive size, complex form and large sequence content. Comprised of catenated maxicircles that contain rRNA and protein-coding genes and thousands of heterogeneous minicircles encoding small guide RNAs, the kinetoplast network has evolved along with an extreme form of mRNA processing in the form of uridine insertion and deletion RNA editing. Many maxicircle-encoded mRNAs cannot be translated without this post-transcriptional sequence modification. Results We present the complete sequence and annotation of the Trypanosoma cruzi maxicircles for the CL Brener and Esmeraldo strains. Gene order is syntenic with Trypanosoma brucei and Leishmania tarentolae maxicircles. The non-coding components have strain-specific repetitive regions and a variable region that is unique for each strain with the exception of a conserved sequence element that may serve as an origin of replication, but shows no sequence identity with L. tarentolae or T. brucei. Alternative assemblies of the variable region demonstrate intra-strain heterogeneity of the maxicircle population. The extent of mRNA editing required for particular genes approximates that seen in T. brucei. Extensively edited genes were more divergent among the genera than non-edited and rRNA genes. Esmeraldo contains a unique 236-bp deletion that removes the 5'-ends of ND4 and CR4 and the intergenic region. Esmeraldo shows additional insertions and deletions outside of areas edited in other species in ND5, MURF1, and MURF2, while CL Brener has a distinct insertion in MURF2. Conclusion The CL Brener and Esmeraldo maxicircles represent two of three previously defined maxicircle clades and promise utility as taxonomic markers. Restoration of the disrupted reading frames might be accomplished by strain-specific RNA editing. Elements in the non-coding region may be important for replication, transcription, and anchoring of the maxicircle within the kinetoplast network.

Effect of pre-flaring and file size on the accuracy of two electronic apex locators

Objective: This ex vivo study evaluated the effect of pre-flaring and file size on the accuracy of the Root ZX and Novapex electronic apex locators (EALs). Material and methods: The actual working length (WL) was set 1 mm short of the apical foramen in the palatal root canals of 24 extracted maxillary molars. The teeth were embedded in an alginate mold, and two examiners performed the electronic measurements using #10, #15, and #20 K-files. The files were inserted into the root canals until the "0.0" or "APEX" signals were observed on the LED or display screens for the Novapex and Root ZX, respectively, retracting to the 1.0 mark. The measurements were repeated after the pre-flaring using the S1 and SX Pro-Taper instruments. Two measurements were performed for each condition and the means were used. Intra-class correlation coefficients (ICCs) were calculated to verify the intra-and inter-examiner agreement. The mean differences between the WL and electronic length values were analyzed by the three-way ANOVA test (p<0.05). Results: ICCs were high (>0.8) and the results demonstrated a similar accuracy for both EALs (p>0.05). Statistically significant accurate measurements were verified in the pre-flared canals, except for the Novapex using a #20 K-file. Conclusions: The tested EALs showed acceptable accuracy, whereas the pre-flaring procedure revealed a more significant effect than the used file size.

Pre, intra and post-ischemic hypothermic neuroprotection in temporary focal cerebral ischemia in rats: morphometric analysis

Objective: To evaluate the neuroprotection of mild hypothermia, applied in different moments, in temporary focal cerebral ischemia in rats. Methods: Rats was divided into Control (C), Sham (S), Ischemic-control(IC), Pre-ischemic Hypothermia (IH1), Intra-ischemic Hypothermia (IH2), and Post-ischemic Hypothermia (IH3) groups. Morphometry was performed using the KS400 software (Carl Zeiss (R)) in coronal sections stained by Luxol Fast Blue. Ischemic areas and volumes were obtained. Results: Statistically, blue areas showed difference for C vs. IC, IC vs. IH1 and IC vs. IH2 (p=0.0001; p=0.01; p=0.03), and no difference between C vs. S, IC vs. IH3 and IH vs. IH2 (p=0.39; p=0.85; p=0.63). Red areas showed difference between C vs. IC, IC vs. IH1 and IC vs. IH2 (p=0.0001; p=0.009; p=0.03), and no difference between C vs. S, IC vs. IH3 and IH1 vs. IH2 (p=0.48; p=0.27; p=0.68). Average ischemic areas and ischemic volumes showed difference between IC vs. IH1 and IC vs. IH2 (p=0.0001 and p=0.0011), and no difference between IC vs. IH3 and IH1 vs. IH2 (p=0.57; p=0.79). Conclusion: Pre-ischemic and intra-ischemic hypothermia were shown to be similarly neuroprotective, but this was not true for post-ischemic hypothermia.

Two structurally discrete GH7-cellobiohydrolases compete for the same cellulosic substrate fiber

Background: Cellulose consisting of arrays of linear beta-1,4 linked glucans, is the most abundant carbon-containing polymer present in biomass. Recalcitrance of crystalline cellulose towards enzymatic degradation is widely reported and is the result of intra-and inter-molecular hydrogen bonds within and among the linear glucans. Cellobiohydrolases are enzymes that attack crystalline cellulose. Here we report on two forms of glycosyl hydrolase family 7 cellobiohydrolases common to all Aspergillii that attack Avicel, cotton cellulose and other forms of crystalline cellulose. Results: Cellobiohydrolases Cbh1 and CelD have similar catalytic domains but only Cbh1 contains a carbohydrate-binding domain (CBD) that binds to cellulose. Structural superpositioning of Cbh1 and CelD on the Talaromyces emersonii Cel7A 3-dimensional structure, identifies the typical tunnel-like catalytic active site while Cbh1 shows an additional loop that partially obstructs the substrate-fitting channel. CelD does not have a CBD and shows a four amino acid residue deletion on the tunnel-obstructing loop providing a continuous opening in the absence of a CBD. Cbh1 and CelD are catalytically functional and while specific activity against Avicel is 7.7 and 0.5 U. mg prot-1, respectively specific activity on pNPC is virtually identical. Cbh1 is slightly more stable to thermal inactivation compared to CelD and is much less sensitive to glucose inhibition suggesting that an open tunnel configuration, or absence of a CBD, alters the way the catalytic domain interacts with the substrate. Cbh1 and CelD enzyme mixtures on crystalline cellulosic substrates show a strong combinatorial effort response for mixtures where Cbh1 is present in 2: 1 or 4: 1 molar excess. When CelD was overrepresented the combinatorial effort could only be partially overcome. CelD appears to bind and hydrolyze only loose cellulosic chains while Cbh1 is capable of opening new cellulosic substrate molecules away from the cellulosic fiber. Conclusion: Cellobiohydrolases both with and without a CBD occur in most fungal genomes where both enzymes are secreted, and likely participate in cellulose degradation. The fact that only Cbh1 binds to the substrate and in combination with CelD exhibits strong synergy only when Cbh1 is present in excess, suggests that Cbh1 unties enough chains from cellulose fibers, thus enabling processive access of CelD.

Work and Inter-subjectivity: a theoretical reflection on its dialectics in the field of health and nursing

This theoretical reflection intends to show the inter-subjective relationship that takes place in health and nursing practices under the following theoretical perspectives: Institutional Analysis, Psychodynamics of Labor and the Theory of Communicative Action, with an emphasis on the latter. Linking these concepts to the Marxist approach to work in the field of health emerges from recognizing the need for its continuous reconstruction-in this case, with a view to understand the interaction and communication intrinsic to work in action. The theory of Communicative Action seeks to consider these two inextricable dimensions: work as productive action and as interaction. The first corresponds to instrumental action based on technical rules with a production-guided rationale. The second refers to the interaction that takes place as communicative action and seeks understanding among subjects. We assume that adopting this theoretical perspective in the analysis of health and nursing practices opens new possibilities for clarifying its social and historical process and inter-subjective connections.

Simultaneous Quantification of Lycopene, beta-Carotene, Retinol and alpha-Tocopherol in Plasma after a Simple Extraction Procedure: Stability Study and Application to Human Volunteers

A method for the simultaneous quantification of lycopene, beta-carotene, retinol and alpha-tocopherol by high-performance liquid chromatography (HPLC) with Vis/fluorescence detection with isocratic elution was optimized and validated. The method consists of a rapid and simple liquid-liquid extraction procedure and a posterior quantification of extracted supernatants by HPLC. Aliquots of plasma were stored at -20 degrees C for three months for stability study. The methodology was applied to samples from painters and individuals not exposed to paints (n = 75). The assay was linear for all vitamins (r > 0.99). Intra-and inter-run precisions were obtained with coefficient of variation smaller than 5%. The accuracies ranged from 0.29 to -5.80% and recoveries between 92.73 and 101.97%. Plasma samples and extracted supernatants were stable for 60 days at -20 degrees C. A significant decrease of lycopene, beta-carotene and retinol concentrations in plasma from exposed individuals compared to non-exposed individuals (p < 0.05) was observed. The method is simple, reproducible, precise, accurate and sensitive, and can be routinely utilized in clinical laboratories.

Three-dimensional Doppler evaluation of single spherical samples from the placenta: intra- and interobserver reliability

Objective To evaluate the intra- and interobserver reliability of assessment of three-dimensional power Doppler (3D-PD) indices from single spherical samples of the placenta. Methods Women with singleton pregnancies at 2440 weeks' gestation were included. Three scans were independently performed by two observers; Observer 1 performed the first and third scan, intercalated by the scan of Observer 2. The observers independently analyzed the 3D-PD datasets that they had previously acquired using four different methods, each using a spherical sample: random sample extending from basal to chorionic plate; random sample with 2 cm3 of volume; directed sample to the region subjectively determined as containing more color Doppler signals extending from basal to chorionic plate; or directed sample with 2 cm3 of volume. The vascularization index (VI), flow index (FI) and vascularization flow index (VFI) were evaluated in each case. The observers were blinded to their own and each other's results. Additional evaluation was performed according to placental location: anterior, posterior and fundal or lateral. Intra- and interobserver reliability was assessed by intraclass correlation coefficients (ICC). Results Ninety-five pregnancies were included in the analysis. All three placental 3D-PD indices showed only weak to moderate reliability (ICC < 0.66 and ICC < 0.48, intra- and interobserver, respectively). The highest values of ICC were observed when using directed spherical samples from basal to chorionic plate. When analyzed by placental location, we found lower ICCs for lateral and fundal placentae compared to anterior and posterior ones. Conclusion Intra- and interobserver reliability of assessment of placental 3D-PD indices from single spherical samples in pregnant women greater than 24 weeks' gestation is poor to moderate, and clinical usefulness of these indices is likely to be limited. Copyright (c) 2012 ISUOG. Published by John Wiley & Sons, Ltd.

Human Endogenous Retrovirus K(HML-2) Gag and Env specific T-cell responses are not detected in HTLV-I-infected subjects using standard peptide screening methods

Abstract Background An estimated 10–20 million individuals are infected with the retrovirus human T-cell leukemia virus type 1 (HTLV-1). While the majority of these individuals remain asymptomatic, 0.3-4% develop a neurodegenerative inflammatory disease, termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HAM/TSP results in the progressive demyelination of the central nervous system and is a differential diagnosis of multiple sclerosis (MS). The etiology of HAM/TSP is unclear, but evidence points to a role for CNS-inflitrating T-cells in pathogenesis. Recently, the HTLV-1-Tax protein has been shown to induce transcription of the human endogenous retrovirus (HERV) families W, H and K. Intriguingly, numerous studies have implicated these same HERV families in MS, though this association remains controversial. Results Here, we explore the hypothesis that HTLV-1-infection results in the induction of HERV antigen expression and the elicitation of HERV-specific T-cells responses which, in turn, may be reactive against neurons and other tissues. PBMC from 15 HTLV-1-infected subjects, 5 of whom presented with HAM/TSP, were comprehensively screened for T-cell responses to overlapping peptides spanning HERV-K(HML-2) Gag and Env. In addition, we screened for responses to peptides derived from diverse HERV families, selected based on predicted binding to predicted optimal epitopes. We observed a lack of responses to each of these peptide sets. Conclusions Thus, although the limited scope of our screening prevents us from conclusively disproving our hypothesis, the current study does not provide data supporting a role for HERV-specific T-cell responses in HTLV-1 associated immunopathology.

Simultaneous quantification of lycopene, β-carotene, retinol and α -tocopherol in plasma after a simple extraction procedure: stability study and application to human volunteers

A method for the simultaneous quantification of lycopene, β-carotene, retinol and α-tocopherol by high-performance liquid chromatography (HPLC) with Vis/fluorescence detection with isocratic elution was optimized and validated. The method consists of a rapid and simple liquid-liquid extraction procedure and a posterior quantification of extracted supernatants by HPLC. Aliquots of plasma were stored at -20°C for three months for stability study. The methodology was applied to samples from painters and individuals not exposed to paints (n = 75). The assay was linear for all vitamins (r > 0.99). Intra- and inter-run precisions were obtained with coefficient of variation smaller than 5%. The accuracies ranged from 0.29 to -5.80% and recoveries between 92.73 and 101.97%. Plasma samples and extracted supernatants were stable for 60 days at -20°C. A significant decrease of lycopene, β-carotene and retinol concentrations in plasma from exposed individuals compared to non-exposed individuals (p < 0.05) was observed. The method is simple, reproducible, precise, accurate and sensitive, and can be routinely utilized in clinical laboratories.

Overtraining is associated with DNA damage in blood and skeletal muscle cells of Swiss mice

Abstract: Background: The alkaline version of the single-cell gel (comet) assay is a useful method for quantifying DNA damage. Although some studies on chronic and acute effects of exercise on DNA damage measured by the comet assay have been performed, it is unknown if an aerobic training protocol with intensity, volume, and load clearly defined will improve performance without leading to peripheral blood cell DNA damage. In addition, the effects of overtraining on DNA damage are unknown. Therefore, this study aimed to examine the effects of aerobic training and overtraining on DNA damage in peripheral blood and skeletal muscle cells in Swiss mice. To examine possible changes in these parameters with oxidative stress, we measured reduced glutathione (GSH) levels in total blood, and GSH levels and lipid peroxidation in muscle samples. Results: Performance evaluations (i.e., incremental load and exhaustive tests) showed significant intra and inter-group differences. The overtrained (OTR) group showed a significant increase in the percentage of DNA in the tail compared with the control (C) and trained (TR) groups. GSH levels were significantly lower in the OTR group than in the C and TR groups. The OTR group had significantly higher lipid peroxidation levels compared with the C and TR groups. Conclusions Aerobic and anaerobic performance parameters can be improved in training at maximal lactate steady state during 8 weeks without leading to DNA damage in peripheral blood and skeletal muscle cells or to oxidative stress in skeletal muscle cells. However, overtraining induced by downhill running training sessions is associated with DNA damage in peripheral blood and skeletal muscle cells, and with oxidative stress in skeletal muscle cells and total blood.

A quantitative TaqMan PCR assay for the detection of Ureaplasma diversum

Ureaplasma diversum in veterinary studies is an undesirable microbe, which may cause infection in bulls and may result in seminal vesiculitis, balanopostitis, and alterations in spermatozoids, whereas in cows, it may cause placentitis, fetal alveolitis, abortion, and birth of weak calves. U. diversum is released through organic secretions, especially semen, preputial and vaginal mucus, conjunctival secretion, and milk. The aim of the present study was to develop a TaqMan probe, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of U. diversum from genital swabs of bovines. Primers and probes specific to U. diversum 16S rRNA gene were designed. The specificity, detection limit, intra- and inter-assay variability of qPCR to detect this ureaplasma was compared with the results of the conventional PCR assay (cPCR). Swabs of vaginal mucus from 169 cows were tested. The qPCR assay detected as few as 10 copies of U. diversum and was 100-fold more sensitive than the cPCR. No cross-reactivity with other Mollicutes or eubacteria was observed. U. diversum was detected in 79 swabs (46.42%) by qPCR, while using cPCR it was detected in 42 (25%) samples. The difference in cPCR and qPCR ureaplasma detection between healthy and sick animals was not statistically significant. But the U. diversum load in samples from animals with genital disorders was higher than in healthy animals. The qPCR assay developed herein is highly sensitive and specific for the detection and quantification of U. diversum in vaginal bovine samples.

A new overtraining protocol for mice based on downhill running sessions

The purpose of the present study was to verify whether a downhill running protocol was able to induce non-functional overreaching in > 75% of mice. Mice were divided into control (C), trained (TR) and overtrained (OTR) groups. Bodyweight and food intake were recorded weekly. The incremental load test (ILT) and the exhaustive test (ET) were used to measure performance before and after aerobic training and overtraining protocols. Although the bodyweight of the OTR group was lower than that of the C group at the end of Week 7, the food intake of the OTR group was higher than that of the C and TR groups at the end of Week 8. Evaluation of results from the ILT and ET revealed significant intra- and inter-group differences: whereas the parameters measured by both tests increased significantly in the TR group, they were significantly decreased in the OTR group. In conclusion, this new overtraining protocol based on downhill running sessions induced non-functional overreaching in 100% of mice.