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1.  The HIV-Brazil Cohort Study: Design, Methods and Participant Characteristics 
PLoS ONE  2014;9(5):e95673.
Background
The HIV-Brazil Cohort Study was established to analyze the effectiveness of combination antiretroviral therapy (cART) and the impact of this treatment on morbidity, quality of life (QOL) and mortality. The study design, patients’ profiles and characteristics of cART initiation between 2003 and 2010 were described.
Methodology/Principal Findings
Since 2003, the HIV-Brazil Cohort has been following HIV-infected adults receiving cART at 26 public health care facilities, using routine clinical care data and self-reported QOL questionnaires. When not otherwise available, data are obtained from national information systems. The main outcomes of interest are diseases related or unrelated to HIV; suppression of viral replication; adverse events; virological, clinical and immunological failures; changes in the cART; and mortality. For the 5,061 patients who started cART between 2003 and 2010, the median follow-up time was 4.1 years (IQR 2.2–5.9 years) with an 83.4% retention rate. Patient profiles were characterized by a predominance of men (male/female ratio 1.7∶1), with a mean age of 36.9 years (SD 9.9 years); 55.2% had been infected with HIV via heterosexual contact. The majority of patients (53.4%) initiated cART with a CD4+ T-cell count ≤200 cells/mm3. The medications most often used in the various treatment regimens were efavirenz (59.7%) and lopinavir/ritonavir (18.2%). The proportion of individuals achieving viral suppression within the first 12 months of cART use was 77.4% (95% CI 76.1–78.6). Nearly half (45.4%) of the patients presented HIV-related clinical manifestations after starting cART, and the AIDS mortality rate was 13.9 per 1,000 person-years.
Conclusions/Significance
Results from cART use in the daily practice of health services remain relatively unknown in low- and middle-income countries, and studies with the characteristics of the HIV-Brazil Cohort contribute to minimizing these shortcomings, given its scope and patient profile, which is similar to that of the AIDS epidemic in the country.
doi:10.1371/journal.pone.0095673
PMCID: PMC4006775  PMID: 24789106
2.  Monitoring persistence of the entomopathogenic fungus Metarhizium anisopliae under simulated field conditions with the aim of controlling adult Aedes aegypti (Diptera: Culicidae) 
Parasites & Vectors  2014;7:198.
Background
Entomopathogenic fungi are potential candidates for use in integrated vector management, with recent emphasis aimed at developing adult mosquito control methods. Here we investigated the persistence of the fungus Metarhizium anisopliae when tested against female A. aegypti under field conditions.
Methods
Black cotton cloths impregnated with M. anisopliae conidia, formulated in vegetable oil + isoparaffin, were maintained on a covered veranda for up to 30 days. At specific times, pieces of the cloths were removed, placed in Tween 80 and the resuspended conidia were sprayed directly onto mosquitoes. The persistence of conidia impregnated on black cloths using three different carriers was evaluated in test rooms. Fifty mosquitoes were released into each room and after a 5 day period, the surviving insects were captured. Another 50 insects were then released into each room. The capacity of the fungus at reducing mosquito survival was evaluated over a total of 35 days.
Results
Conidia extracted from cloths maintained on the veranda for 2 to 18 days remained virulent, with 28 to 60% mosquito survival observed. Mosquito survival following exposure to fungus impregnated cloths showed that fungus + Tween caused similar reductions to that of fungus + vegetable oil. Mosquitoes exposed to the formulation fungus + vegetable oil had survival rates of 36% over the first 5 days of the experiment. Following the release of the second cohort of mosquitoes (6-11days), survival increased to 50%. The survival of the 12–17 day cohort (78%) was statistically equal to that of the controls (84%). Formulation of the fungus in vegetable oil + isoparaffin increased the persistence of the fungus, with the 18–23 day cohort (64% survival) still showing statistical differences to that of the controls (87% survival).
Conclusions
The potential of entomopathogenic fungi for the control of adult A. aegypti was confirmed under field conditions. Vegetable oil + isoparaffin formulations of M. anisopliae significantly increased the effectiveness of the fungus, thus reducing the need for frequent changes of black cloths in residences. Our future aim is to obtain effective control of mosquito populations, with cloths only needing to being replaced once a month.
doi:10.1186/1756-3305-7-198
PMCID: PMC4021620  PMID: 24766705
Persistence; Viability; Vector; Dengue; Oil formulation
3.  Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis 
Background
Molecular biological methods have become increasingly relevant to the diagnosis and control of infectious diseases, such as leishmaniasis. Since various factors may affect the sensitivity of PCR assays, including DNA yield and purity, an optimal extraction method is pivotal. Losses of a parasite’s DNA during extraction may significantly impair its detection by PCR and lead to false-negative results. This study proposes a triplex PCR assay targeting the parasite’s DNA, an external control (pUC18) and an internal control (G3PD) for accurate diagnosis of leishmaniasis.
Results
Two primer pairs were designed to detect the plasmid pUC18 and a triplex PCR assay targeting the Leishmania braziliensis kinetoplast DNA, the external control and the internal control was standardized. The triplex PCR assay was assessed for its ability to detect the three target DNA fragments simultaneously.
PCR products from pUC18 DNA resulted in bands of 368 (P1) and 316 (P2) base pairs (bp). The triplex PCR optimized with the chosen external control system (P1) allowed the simultaneous detection of the internal control (G3PD – 567 bp) as well as of small quantities (10 pg) of the target parasite’s DNA, detected by amplification of a 138 bp product.
Conclusions
The new tool standardized herein enables a more reliable interpretation of PCR results, mainly by contributing to quality assurance of leishmaniasis diagnosis. Furthermore, after simple standardization steps, this protocol could be applied to the diagnosis of other infectious diseases in reference laboratories. This triplex PCR enables the assessment of small losses during the DNA extraction process, problems concerning DNA degradation (sample quality) and the detection of L. braziliensis kDNA.
doi:10.1186/1678-9199-20-16
PMCID: PMC4012836  PMID: 24808911
Extraction control; Multiplex PCR; pUC18; Leishmaniasis; Diagnosis; False-negative result
4.  Trypanosoma cruzi Cell Death Induced by the Morita-Baylis-Hillman Adduct 3-Hydroxy-2-Methylene-3-(4-Nitrophenylpropanenitrile) 
PLoS ONE  2014;9(4):e93936.
Chagas disease, caused by the protozoan Trypanosoma cruzi, remains a serious health concern due to the lack of effective vaccines or satisfactory treatment. In the search for new compounds against this neglected disease, we have previously demonstrated that the compound 3-Hydroxy-2-methylene-3-(4-nitrophenylpropanenitrile) (MBHA3), derived from the Morita-Baylis-Hillman reaction, effectively caused a loss of viability in both the epimastigote and trypomastigote forms. However, the mechanisms of parasite death elicited by MBHA3 remain unknown. The aim of this study was to better understand the morphophysiological changes and the mechanism of cell death induced by MBHA3 treatment on T. cruzi. To perform this analysis, we used confocal microscopy and flow cytometry to monitor the fluorescent probes such as annexin-V/propidium iodide (AV/PI), calcein-AM/ethidium homodimer (CA/EH), acridine orange (AO) and rhodamine 123 (Rho 123). Lower concentrations of MBHA3 led to alterations in the mitochondrial membrane potential and AO labeling, but did not decrease the viability of the epimastiogote forms, as determined by the CA/EH and AV/PI assays. Conversely, treatment with higher concentrations of MBHA3 led to extensive plasma membrane damage, loss of mitochondrion membrane potential, DNA fragmentation and acidification of the cytoplasm. Our findings suggest that at higher concentrations, MBHA3 induces T. cruzi epimastigote death by necrosis in a mitochondrion-dependent manner.
doi:10.1371/journal.pone.0093936
PMCID: PMC3979736  PMID: 24714638
5.  C-Reactive Protein: Clinical and Epidemiological Perspectives 
An important etiopathogenic component of cardiovascular disease is atherosclerosis, with inflammation being an essential event in the pathophysiology of all clinical pictures it comprises. In recent years, several molecules implicated in this process have been studied in order to assess cardiovascular risk in both primary and secondary prevention. C-reactive protein is a plasmatic protein of the pentraxin family and an acute phase reactant, very useful as a general inflammation marker. Currently, it is one of the most profoundly researched molecules in the cardiovascular field, yet its clinical applicability regarding cardiovascular risk remains an object of discussion, considered by some as a simple marker and by others as a true risk factor. In this sense, numerous studies propose its utilization as a predictor of cardiovascular risk through the use of high-sensitivity quantification methods for the detection of values <1 mg/L, following strict international guidelines. Increasing interest in these clinical findings has led to the creation of modified score systems including C-reactive protein concentrations, in order to enhance risk scores commonly used in clinical practice and offer improved care to patients with cardiovascular disease, which remains the first cause of mortality at the worldwide, national, and regional scenarios.
doi:10.1155/2014/605810
PMCID: PMC3932642  PMID: 24653858
6.  Mini-Arc for the Treatment of Female Stress Urinary Incontinence: Long-Term Prospective Evaluation by Patient Reported Outcomes 
ISRN Urology  2014;2014:659383.
Single-incision slings were introduced in the surgical treatment of female stress urinary incontinence (SUI) to lessen the morbidity associated with traditional midurethral slings. However, long-term reports on patient satisfaction are still scarce. This study describes the outcome of women treated with Mini-Arc at a mean follow-up of 45 months. In a previous report on 105 women with 15-month mean follow-up, 84 (80%) were found cured and 12 (11%) improved. Now, with a mean follow-up of 45 months, cured/improved patients were reassessed by telephone and completed Patient Global Impression of Improvement (PGI-I), Patient Global Impression of Severity (PGI-S), rated their improvement in a 0–100 scale, and answered if they would recommend the procedure. At 45-month follow-up, 73 women cured/improved were available for evaluation. Over 80% of the cured patients rated the improvement of SUI by the PGI-I as “very much better” or “much better,” reported their urinary tract condition to be “normal” on PGI-S, and described their improvement >70%. Ninety percent would recommend this procedure to a friend. The improved-patient population is very small (n = 7). This study shows that the majority of patients cured/improved after Mini-Arc placement maintain a high degree of satisfaction at a long-term evaluation.
doi:10.1155/2014/659383
PMCID: PMC3918723  PMID: 24579053
7.  Convexity meningiomas enhanced by sodium fluorescein 
Background:
The resection of the meningiomas surrounding the dura is an important goal during the removal of a convexity meningioma. The authors present the first application of sodium fluorescein (SF) as a tool for tumor and dural tail identification in convexity meningiomas.
Methods:
Five frontal convexity meningiomas operated on between December 2012 and April 2013 were included. After initial dissection a dose of 1 g of the SF, 20% was injected into a peripheral vein. Tumor and dural tail were removed using the correlation between magnetic resonance imaging (MRI) findings and transoperative SF enhancement.
Results:
Simpson Grade 1 removal was obtained in three cases, grade 2 in one atypical meningioma and grade zero in one case. SF dural tail enhancement was positive in all cases and histologic analysis evidenced involvement of the dura by tumors.
Conclusion:
SF enhancement was evident in meningiomas and dura surrounding the lesions. Histologic analysis confirmed dural involvement. SF could represent an universally available fluorescent tool for meningioma surgery.
doi:10.4103/2152-7806.124978
PMCID: PMC3927087  PMID: 24575318
Fluorescent marker; grade zero removal; meningioma; surgical resection
8.  Correction: Depth Cues and Perceived Audiovisual Synchrony of Biological Motion 
PLoS ONE  2014;9(1):10.1371/annotation/d0e27a68-ad6d-452f-bfca-337487fc933c.
doi:10.1371/annotation/d0e27a68-ad6d-452f-bfca-337487fc933c
PMCID: PMC3882173
9.  Interaction of Mycobacterium leprae with Human Airway Epithelial Cells: Adherence, Entry, Survival, and Identification of Potential Adhesins by Surface Proteome Analysis 
Infection and Immunity  2013;81(7):2645-2659.
This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.
doi:10.1128/IAI.00147-13
PMCID: PMC3697615  PMID: 23670556
10.  Depth Cues and Perceived Audiovisual Synchrony of Biological Motion 
PLoS ONE  2013;8(11):e80096.
Background
Due to their different propagation times, visual and auditory signals from external events arrive at the human sensory receptors with a disparate delay. This delay consistently varies with distance, but, despite such variability, most events are perceived as synchronic. There is, however, contradictory data and claims regarding the existence of compensatory mechanisms for distance in simultaneity judgments.
Principal Findings
In this paper we have used familiar audiovisual events – a visual walker and footstep sounds – and manipulated the number of depth cues. In a simultaneity judgment task we presented a large range of stimulus onset asynchronies corresponding to distances of up to 35 meters. We found an effect of distance over the simultaneity estimates, with greater distances requiring larger stimulus onset asynchronies, and vision always leading. This effect was stronger when both visual and auditory cues were present but was interestingly not found when depth cues were impoverished.
Significance
These findings reveal that there should be an internal mechanism to compensate for audiovisual delays, which critically depends on the depth information available.
doi:10.1371/journal.pone.0080096
PMCID: PMC3828238  PMID: 24244617
11.  Transcriptional Activity, Chromosomal Distribution and Expression Effects of Transposable Elements in Coffea Genomes 
PLoS ONE  2013;8(11):e78931.
Plant genomes are massively invaded by transposable elements (TEs), many of which are located near host genes and can thus impact gene expression. In flowering plants, TE expression can be activated (de-repressed) under certain stressful conditions, both biotic and abiotic, as well as by genome stress caused by hybridization. In this study, we examined the effects of these stress agents on TE expression in two diploid species of coffee, Coffea canephora and C. eugenioides, and their allotetraploid hybrid C. arabica. We also explored the relationship of TE repression mechanisms to host gene regulation via the effects of exonized TE sequences. Similar to what has been seen for other plants, overall TE expression levels are low in Coffea plant cultivars, consistent with the existence of effective TE repression mechanisms. TE expression patterns are highly dynamic across the species and conditions assayed here are unrelated to their classification at the level of TE class or family. In contrast to previous results, cell culture conditions per se do not lead to the de-repression of TE expression in C. arabica. Results obtained here indicate that differing plant drought stress levels relate strongly to TE repression mechanisms. TEs tend to be expressed at significantly higher levels in non-irrigated samples for the drought tolerant cultivars but in drought sensitive cultivars the opposite pattern was shown with irrigated samples showing significantly higher TE expression. Thus, TE genome repression mechanisms may be finely tuned to the ideal growth and/or regulatory conditions of the specific plant cultivars in which they are active. Analysis of TE expression levels in cell culture conditions underscored the importance of nonsense-mediated mRNA decay (NMD) pathways in the repression of Coffea TEs. These same NMD mechanisms can also regulate plant host gene expression via the repression of genes that bear exonized TE sequences.
doi:10.1371/journal.pone.0078931
PMCID: PMC3823963  PMID: 24244387
12.  Exergaming as a Strategic Tool in the Fight against Childhood Obesity: A Systematic Review 
Journal of Obesity  2013;2013:438364.
Improper use of electronic media is considered a major contributing factor to childhood obesity. However, exergames, a new generation of active games, have made it possible to combine electronic entertainment with physical exercise. The purpose of this systematic review was to analyze the use of exergaming as a strategic tool in the fight against childhood obesity. Information was retrieved from the databases SciELO, LILACS, Pubmed, Ebsco, and Science Direct, using the search words “egames,” “exergames,” “exergaming,” “new generation of video games,” “active video games,” “energy expenditure,” “body composition,” and “physical activity” in English and Portuguese, covering the period January 2008 to April 2012. Nine articles met the inclusion criteria. Exergaming was found to increase physical activity levels, energy expenditure, maximal oxygen uptake, heart rate, and percentage of physical activity engaged in and to reduce waist circumference and sedentary screen time. Thus, exergaming may be considered a highly relevant strategic tool for the adoption of an active and healthy lifestyle and may be useful in the fight against childhood obesity.
doi:10.1155/2013/438364
PMCID: PMC3844237  PMID: 24319594
15.  Eosinophilic Esophagitis in Brazilian Pediatric Patients 
We examined 11 pediatric patients with eosinophilic esophagitis with a tardy diagnosis. The symptoms were initially thought to be related to other diseases, leading to the use of inadequate therapeutic approaches. The patients were between 3 and 17 years old (mean 7.8 ± 3.8 years), and 8 of the patients were male. Common symptoms included abdominal pain, regurgitation, difficulty in gaining weight, vomiting, dysphagia, and coughing. The mean age for the onset of symptoms was 4.3 ± 2.9 years. Endoscopic findings included normal mucosa in five (45%) patients, thickening of the mucosa with longitudinal grooves in three (27%), erosive esophagitis in two (18%), and a whitish stippling in one (9%) patient. Treatment included the use of a topical corticosteroid for 10 patients. In eight (73%) cases, the treatment made the symptoms disappear. Ten patients underwent histopathological management after treatment, with a decrease in the number of eosinophils.
doi:10.4137/CMPed.S12733
PMCID: PMC3785386  PMID: 24106430
esophagitis; eosinophilia; diagnostic
16.  Testing fungus impregnated cloths for the control of adult Aedes aegypti under natural conditions 
Parasites & Vectors  2013;6:256.
Background
Entomopathogenic fungi could be useful tools for reducing populations of the dengue mosquito Aedes aegypti. Here the efficiency of fungus (Metarhizium anisopliae) impregnated cloths (with and without imidacloprid [IMI]) was evaluated against adult A. aegypti in simulated human dwellings. Behaviour of mosquitoes in the presence of black cloths was also investigated.
Findings
When mosquitoes were released into the test rooms, the lowest survival rates (38%) were seen when five black cloths impregnated with conidia of ESALQ 818 + 10 ppm IMI were fixed under tables and chairs. This result was significantly lower than the survival rate recorded when cloths were impregnated with ESALQ 818 alone (44%) or ESALQ 818 + 0.1 ppm IMI (43%). Blood fed A. aegypti had lower landing frequencies on black cloths than sucrose fed insects during the first 24 h following feeding, which may have been due to reduced flight activity. Few mosquitoes (4-5%) were observed to land on the cloths during the hours of darkness. The landing pattern of sucrose-fed mosquitoes on non-treated and fungus-treated cloths was similar.
Conclusion
The synergism between M. anisopliae and IMI significantly reduced Aedes survival in simulated field conditions. The use of fungus impregnated cloths is a promising point source application method for the control of adult A. aegypti.
doi:10.1186/1756-3305-6-256
PMCID: PMC3848359  PMID: 24010874
Virulence; Field testing; Fungus; Insecticide; Vector; Dengue; Blood feeding; Behaviour
17.  Potential biomarkers for the clinical prognosis of severe dengue 
Memórias do Instituto Oswaldo Cruz  2013;108(6):755-762.
Currently, several assays can confirm acute dengue infection at the point-of-care. However, none of these assays can predict the severity of the disease symptoms. A prognosis test that predicts the likelihood of a dengue patient to develop a severe form of the disease could permit more efficient patient triage and treatment. We hypothesise that mRNA expression of apoptosis and innate immune response-related genes will be differentially regulated during the early stages of dengue and might predict the clinical outcome. Aiming to identify biomarkers for dengue prognosis, we extracted mRNA from the peripheral blood mononuclear cells of mild and severe dengue patients during the febrile stage of the disease to measure the expression levels of selected genes by quantitative polymerase chain reaction. The selected candidate biomarkers were previously identified by our group as differentially expressed in microarray studies. We verified that the mRNA coding for CFD, MAGED1, PSMB9, PRDX4 and FCGR3B were differentially expressed between patients who developed clinical symptoms associated with the mild type of dengue and patients who showed clinical symptoms associated with severe dengue. We suggest that this gene expression panel could putatively serve as biomarkers for the clinical prognosis of dengue haemorrhagic fever.
doi:10.1590/0074-0276108062013012
PMCID: PMC3970693  PMID: 24037198
dengue; biomarkers; apoptosis; innate immunity; quantitative real-time PCR
18.  Primary proximal urethral adenocarcinoma: Case report and brief review 
Primary urethral cancer in females is rare. It has a poor prognosis. The published data on this topic are limited, composed mostly of small case series. This paper presents a case of an advanced adenocarcinoma of the urethra, intestinal type, treated with anterior exenteration.
doi:10.5489/cuaj.399
PMCID: PMC3713150  PMID: 23914268
19.  Correction: Dengue Virus Type 3 Adaptive Changes during Epidemics in São Jose de Rio Preto, Brazil, 2006–2007 
PLoS ONE  2013;8(6):10.1371/annotation/5b2477b7-f68d-4800-a17e-fd85f5ad8311.
doi:10.1371/annotation/5b2477b7-f68d-4800-a17e-fd85f5ad8311
PMCID: PMC3741030
20.  The Genome of Anopheles darlingi, the main neotropical malaria vector 
Marinotti, Osvaldo | Cerqueira, Gustavo C. | de Almeida, Luiz Gonzaga Paula | Ferro, Maria Inês Tiraboschi | Loreto, Elgion Lucio da Silva | Zaha, Arnaldo | Teixeira, Santuza M. R. | Wespiser, Adam R. | Almeida e Silva, Alexandre | Schlindwein, Aline Daiane | Pacheco, Ana Carolina Landim | da Silva, Artur Luiz da Costa | Graveley, Brenton R. | Walenz, Brian P. | Lima, Bruna de Araujo | Ribeiro, Carlos Alexandre Gomes | Nunes-Silva, Carlos Gustavo | de Carvalho, Carlos Roberto | Soares, Célia Maria de Almeida | de Menezes, Claudia Beatriz Afonso | Matiolli, Cleverson | Caffrey, Daniel | Araújo, Demetrius Antonio M. | de Oliveira, Diana Magalhães | Golenbock, Douglas | Grisard, Edmundo Carlos | Fantinatti-Garboggini, Fabiana | de Carvalho, Fabíola Marques | Barcellos, Fernando Gomes | Prosdocimi, Francisco | May, Gemma | de Azevedo Junior, Gilson Martins | Guimarães, Giselle Moura | Goldman, Gustavo Henrique | Padilha, Itácio Q. M. | Batista, Jacqueline da Silva | Ferro, Jesus Aparecido | Ribeiro, José M. C. | Fietto, Juliana Lopes Rangel | Dabbas, Karina Maia | Cerdeira, Louise | Agnez-Lima, Lucymara Fassarella | Brocchi, Marcelo | de Carvalho, Marcos Oliveira | Teixeira, Marcus de Melo | Diniz Maia, Maria de Mascena | Goldman, Maria Helena S. | Cruz Schneider, Maria Paula | Felipe, Maria Sueli Soares | Hungria, Mariangela | Nicolás, Marisa Fabiana | Pereira, Maristela | Montes, Martín Alejandro | Cantão, Maurício E. | Vincentz, Michel | Rafael, Miriam Silva | Silverman, Neal | Stoco, Patrícia Hermes | Souza, Rangel Celso | Vicentini, Renato | Gazzinelli, Ricardo Tostes | Neves, Rogério de Oliveira | Silva, Rosane | Astolfi-Filho, Spartaco | Maciel, Talles Eduardo Ferreira | Ürményi, Turán P. | Tadei, Wanderli Pedro | Camargo, Erney Plessmann | de Vasconcelos, Ana Tereza Ribeiro
Nucleic Acids Research  2013;41(15):7387-7400.
Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vector–human and vector–parasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles-darlingi.
doi:10.1093/nar/gkt484
PMCID: PMC3753621  PMID: 23761445
21.  Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus  
Memórias do Instituto Oswaldo Cruz  2013;108(4):494-500.
In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL) are transferred to the haemolymphatic lipophorin (Lp) and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80%) and NL (20%). The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs.
doi:10.1590/0074-0276108042013016
PMCID: PMC3970613  PMID: 23827998
lipid metabolism; lipoprotein; lipid transport; phospholipids; midgut
22.  Dengue Virus Type 3 Adaptive Changes during Epidemics in São Jose de Rio Preto, Brazil, 2006–2007 
PLoS ONE  2013;8(5):e63496.
Global dengue virus spread in tropical and sub-tropical regions has become a major international public health concern. It is evident that DENV genetic diversity plays a significant role in the immunopathology of the disease and that the identification of polymorphisms associated with adaptive responses is important for vaccine development. The investigation of naturally occurring genomic variants may play an important role in the comprehension of different adaptive strategies used by these mutants to evade the human immune system. In order to elucidate this role we sequenced the complete polyprotein-coding region of thirty-three DENV-3 isolates to characterize variants circulating under high endemicity in the city of São José de Rio Preto, Brazil, during the onset of the 2006-07 epidemic. By inferring the evolutionary history on a local-scale and estimating rates of synonymous (dS) and nonsynonimous (dN) substitutions, we have documented at least two different introductions of DENV-3 into the city and detected 10 polymorphic codon sites under significant positive selection (dN/dS > 1) and 8 under significant purifying selection (dN/dS < 1). We found several polymorphic amino acid coding sites in the envelope (15), NS1 (17), NS2A (11), and NS5 (24) genes, which suggests that these genes may be experiencing relatively recent adaptive changes. Furthermore, some polymorphisms correlated with changes in the immunogenicity of several epitopes. Our study highlights the existence of significant and informative DENV variability at the spatio-temporal scale of an urban outbreak.
doi:10.1371/journal.pone.0063496
PMCID: PMC3646734  PMID: 23667626
23.  Cadmium Tolerance and Removal from Cunninghamella elegans Related to the Polyphosphate Metabolism 
The aim of the present work was to study the cadmium effects on growth, ultrastructure and polyphosphate metabolism, as well as to evaluate the metal removal and accumulation by Cunninghamella elegans (IFM 46109) growing in culture medium. The presence of cadmium reduced growth, and a longer lag phase was observed. However, the phosphate uptake from the culture medium increased 15% when compared to the control. Moreover, C. elegans removed 70%–81% of the cadmium added to the culture medium during its growth. The C. elegans mycelia showed a removal efficiency of 280 mg/g at a cadmium concentration of 22.10 mg/L, and the removal velocity of cadmium was 0.107 mg/h. Additionally, it was observed that cadmium induced vacuolization, the presence of electron dense deposits in vacuoles, cytoplasm and cell membranes, as well as the distinct behavior of polyphosphate fractions. The results obtained with C. elegans suggest that precipitation, vacuolization and polyphosphate fractions were associated to cadmium tolerance, and this species demonstrated a higher potential for bioremediation of heavy metals.
doi:10.3390/ijms14047180
PMCID: PMC3645682  PMID: 23538844
heavy metal; cadmium; polyphosphate; tolerance; Cunninghamella elegans
24.  Extreme environments as potential drivers of convergent evolution by exaptation: the Atacama Desert Coastal Range case 
We have recently discovered a variety of unrelated phototrophic microorganisms (two microalgae and one cyanobacteria) in specialized terrestrial habitats at The Coastal Range of the Atacama Desert. Interestingly, morphological and molecular evidence suggest that these three species are all recent colonists that came from aquatic habitats. The first case is Cyanidiales inhabiting coastal caves. Cyanidiales are microalgae that are commonly found in warm acid springs, but have also been recently discovered as cave flora in Italy. The case is Dunaliella biofilms colonizing spider webs in coastal caves; Dunaliella are microalgae typically found in hypersaline habitats. The third case is Chroococcidiopsis, a genus of Cyanobacteria commonly found in deserts around the world that has also been described in warm springs. Thus, we show that the traits found in the closest ancestors of the aforementioned species (which inhabited other unrelated extreme environments) seem to be now useful for the described species in their current subaerial habitats and may likely correspond to cases of exaptations. Altogether, the Coastal Range of the Atacama Desert may be considered as a place where key steps on the colonization of land by phototrophic organisms seem to be being repeated by convergent evolution of extant microalgae and Cyanobacteria.
doi:10.3389/fmicb.2012.00426
PMCID: PMC3526103  PMID: 23267354
Atacama Desert; convergent evolution; photrophic eukaryotes
25.  Relationship Between Group Cohesion and Anxiety in Soccer 
Journal of Human Kinetics  2012;34:119-127.
Group cohesion in sport is a widely spread theme today. Research has found cohesion to be influenced by several individual and group components. Among the cognitive variables that relate to cohesion we found competitive anxiety. The purpose of this study was to examine the relation between task cohesion (ATG-T, and GI-T) and competitive state anxiety (A-state), and also if there would be a relation between cohesion and self-confidence. Participants were 366 football players of both genders male and female, aged between 15 to 23 years old, from Portugal’s championships. Cohesion was measured using the Portuguese version of the Group Environment Questionnaire, and to assess competitive anxiety, we used the Portuguese version of the Competition State Anxiety Inventory 2. Our results show that female athletes report experiencing more cognitive anxiety and less self-confidence than male athletes. Only cognitive anxiety relates in a significantly negative way with the perception of cohesion (GI-T e ATG-T) in the total number of participants and in male athletes. Relatively to the somatic anxiety, it only relates negatively with the perception of the integration of the group in the total number of participants and in the male gender.
doi:10.2478/v10078-012-0071-z
PMCID: PMC3590828  PMID: 23487008
Group Dynamics; cohesion; anxiety; soccer

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