The Past and Present of an Estuarine-Resident Fish,the “Four-Eyed Fish” Anableps anableps (Cyprinodontiformes,Anablepidae), Revealed by mtDNA Sequences

Historical events, such as changes in sea level during the Pleistocene glacial cycles, had a strong impact on coastal habitats, limiting connectivity and promoting the genetic divergence of various species. In this study, we evaluated the influence of climate oscillations and the possibility of estuary function as a barrier to gene flow among populations of the four-eyed fish, Anableps anableps. This species is fully estuarine-resident, has internal fertilization, is viviparous and does not migrate across long distances. These features make the four-eyed fish an excellent model for the study of evolutionary processes related to genetic differentiation of species and populations in estuaries. The evolutionary history of A. anableps was inferred from phylogeographic and population analyses using sequences of the mitochondrial DNA Control Region of 13 populations distributed in the Amazon and Northeast Coast of Brazil from Calcoene (Amapa) to Parnaiba (Piaui). The 83 retrieved haplotypes show a pattern of four distinct mitochondrial lineages, with up to 3.4% nucleotide divergence among them. The evolutionary reconstruction suggests that these lineages diverged recently in the late Pleistocene/early Holocene after the Atlantic Ocean reaching current levels. Analysis of variability, neutrality and the genetic expansion pattern revealed that the lineages have distinct characteristics, which were shaped by the different geomorphological features of coastal regions combined with sea level oscillations over a very long period of time. Only few neighboring populations show a discreet gene flow. This study may also be helpful for designing new experiments to better understand the geomorphological evolutionary history of the estuaries of the Amazon and the Northeast Coast of Brazil using estuarine-resident species as a model.     

17-AAG Kills Intracellular Leishmania amazonensis while Reducing Inflammatory Responses in Infected Macrophages

Background

Leishmaniasis is a neglected endemic disease with a broad spectrum of clinical manifestations. Pentavalent antimonials have been the treatment of choice for the past 70 years and, due to the emergence of resistant cases, the efficacy of these drugs has come under scrutiny. Second-line drugs are less efficacious, cause a range of side effects and can be costly. The formulation of new generations of drugs, especially in developing countries, has become mandatory.

Methodology/Principal Findings

We investigated the anti-leishmanial effect of 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), an HSP90 inhibitor, in vitro. This inhibitor is currently in clinical trials for cancer treatment; however, its effects against intracellular Leishmania remain untested. Macrophages infected with L. amazonensis were treated with 17-AAG (25–500 nM) and parasite load was quantified using optical microscopy. Parasite load declined in 17-AAG-treated macrophages in a dose- and time-dependent manner. Intracellular parasite death became irreversible after 4 h of treatment with 17-AAG, and occurred independent of nitric oxide (NO) and superoxide (O₂ ⁻) production. Additionally, intracellular parasite viability was severely reduced after 48 h of treatment. Interestingly, treatment with 17-AAG reduced pro-inflammatory mediator production, including TNF-α, IL-6 and MCP-1, yet IL-12 remained unaffected. Electron microscopy revealed morphological alterations, such as double-membrane vacuoles and myelin figures at 24 and 48 h after 17-AAG treatment.

Conclusions/Significance

The HSP90 inhibitor, 17-AAG, possesses high potency under low dosage and reduces both pro-inflammatory and oxidative molecule production. Therefore, further studies are warranted to investigate this inhibitor’s potential in the development of new generations of anti-leishmanials.     

Selection and optimization of PCR-based methods for the detection of Histoplasma capsulatum var. capsulatum

BackgroundCurrent methods for the laboratory diagnosis of histoplasmosis are problematic in terms of their sensitivity, specificity and runtime.ObjectivesThus, in this study, we sought to select and optimize methods for the detection of Histoplasma capsulatum var. capsulatum by polymerase chain reaction (PCR).MethodsThree DNA extraction methods and three PCR methods were evaluated. We optimised the concentration of the components of this PCR reaction and determined its sensitivity and specificity using blood samples to which H. capsulatum had been added.ResultsThe DNA extraction method that yielded the highest-quality DNA used silica membranes (DNeasy Blood &; Tissue Kit, Qiagen, Hilden, Germany), and the amplification method with the best detection capacity used a target gene encoding a 100-kDa protein. Our optimisation of the PCR conditions indicated that the reaction works over a significant range of component concentrations; in addition, it was able to detect H. capsulatum better than traditional culture techniques, with a detection limit of only 10 pg of DNA.ConclusionsIn our experimental conditions, the PCR method selected in this work (instead of nested-PCR) is a tool sensitive enough for the diagnosis of histoplasmosis.     

Birth of normal infants after transfer of embryos that were twice vitrified/warmed at cleavage stages: Report of two cases

The role of cryopreservation in assisted reproductive technology programs has increased within the last years allowing the transfer of a limited number of embryos and the storage of the remaining for future use. The reduction in the number of transferred embryos decreases the frequency of multiple pregnancy rates and of ovarian hyperstimulation syndrome while the cumulative pregnancy rate can be maximized. Moreover, as not all embryos will survive the warming process more cleavage stage embryos are warmed to improve selection for transfer. Therefore, surplus good quality cleavage stage embryos and/or blastocysts must be re-vitrified for further transfer to achieve pregnancy. To our knowledge, there have been no reports demonstrating that human embryos can be successfully vitrified/warmed twice at the cleavage stage. Thus we report two successful pregnancies and deliveries of healthy babies after transfer of embryos that were twice vitrified/warmed at 2–4 cells stage.     

Coxiella burnetii, the agent of Q fever in Brazil: its hidden role in seronegative arthritis and the importance of molecular diagnosis based on the repetitive element IS1111 associated with the transposase gene

Coxiella burnetii is the agent of Q fever , an emergent worldwide zoonosis of wide clinical spectrum. Although C. burnetii infection is typically associated with acute infection, atypical pneumonia and flu-like symptoms, endocarditis, osteoarticular manifestations and severe disease are possible, especially when the patient has a suppressed immune system; however, these severe complications are typically neglected. This study reports the sequencing of the repetitive element IS1111 of the transposase gene of C. burnetii from blood and bronchoalveolar lavage (BAL) samples from a patient with severe pneumonia following methotrexate therapy, resulting in the molecular diagnosis of Q fever in a patient who had been diagnosed with active seronegative polyarthritis two years earlier. To the best of our knowledge, this represents the first documented case of the isolation of C. burnetii DNA from a BAL sample.     

The distribution pattern of Lutzomyia longipalpis (Diptera: Psychodidae) in the peridomiciles of a sector with canine and human visceral leishmaniasis transmission in the municipality of Dracena, São Paulo, Brazil

The specimen distribution pattern of a species can be used to characterise a population of interest and also provides area-specific guidance for pest management and control. In the municipality of Dracena, in the state of S?o Paulo, we analysed 5,889 Lutzomyia longipalpis specimens collected from the peridomiciles of 14 houses in a sector where American visceral leishmaniasis (AVL) is transmitted to humans and dogs. The goal was to analyse the dispersion and a theoretical fitting of the species occurrence probability. From January-December 2005, samples were collected once per week using CDC light traps that operated for 12-h periods. Each collection was considered a sub-sample and was evaluated monthly. The standardised Morisita index was used as a measure of dispersion. Adherence tests were performed for the log-series distribution. The number of traps was used to adjust the octave plots. The quantity of Lu. longipalpis in the sector was highly aggregated for each month of the year, adhering to a log-series distribution for 11 of the 12 months analysed. A sex-stratified analysis demonstrated a pattern of aggregated dispersion adjusted for each month of the year. The classes and frequencies of the traps in octaves can be employed as indicators for entomological surveillance and AVL control.     

Detection of the first incidence of Akodon paranaensis naturally infected with the Jabora virus strain (Hantavirus) in Brazil

We characterised hantaviruses circulating in different Akodon rodent species collected in midwestern Santa Catarina (SC), southern Brazil, where the Jabora hantavirus (JABV) strain was first identified in Akodon montensis. Genetic and phylogenetic analyses based on a partial S segment indicated that, in SC, Akodon paranaensis and A. montensis carried the same type of hantavirus. Additionally, we conducted the first genomic characterisation of the complete S segment from the Brazilian JABV strain. This is the first report of A. paranaensis infected with the JABV.     

Purification and Biological Activities of Abelmoschus esculentus Seed Lectin

The Abelmoschus esculentus (Malvaceae) plant originated in Africa and has spread across a number of tropic countries, including northeastern Brazil. The plant has been used to treat various disorders, such as cancer, microbial infections, hypoglycemia, constipation, urine retention and inflammation. The lectin of A. esculentus (AEL) was isolated by precipitation with ammonium sulfate at a saturation level of 30/60 and purified by ion exchange chromatography (Sephacel-DEAE). The electrophoresis (SDS-PAGE) profile of the AEL showed two protein bands of apparent molecular mass of approximately 15.0 and 21.0?kDa. The homogenity of the protein was confirmed by electrospray mass spectrometry (ESI-MS), which revealed the presence of a 10.29-kDa monomer and a 20.58-kDa dimer. The AEL exhibits agglutinating activity against rabbit (74.41 UH/mP) and human type ABO erythrocytes (21.00 UH/mP). This activity does not require the presence of divalent cations and is specifically inhibited by lactose, fructose and mannose. The intravenous treatment with 0.01, 0.1 and 1?mg/kg of AEL inhibited the paw edema elicited by carrageenan by approximately 15, 22 and 44?%, respectively, but not that induced by dextran. In addition, treatment with 0.1, 1 and 10?mg/kg of AEL also inhibited the abdominal writhing induced by acetic acid by approximately 52, 57 and 69?%, respectively. In conclusion, AEL is a new lectin with a molecular mass of 20.0?kDa, which is -composed of a 10.291-Da monomer and a 20.582-kDa dimer, that exhibits anti-inflammatory, antinociceptive and hemagglutinating activities. In addition, the lectin hemagglutinating property is both metallo-independent and associated with the lectin domain.     

Contemporary practices of strength and conditioning coaches in professional soccer

This study describes the contemporary practices of strength and conditioning coaches in professional soccer. Fifty-two strength and conditioning coaches from professional leagues across 18 countries completed an online survey, consisting of 45 questions, with eight sections: (a) background information, (b) muscular strength and power development, (c) speed development, (d) plyometrics, (e) flexibility development, (f) physical testing, (g) technology use, and (h) programing. A frequency analysis was used to assess and report responses to fixed response questions, and thematic-analysis used for open-ended questions to create clear, identifiable and distinct themes. All strength and conditioning coaches were educated to degree level or higher, 65% held strength and conditioning certifications and 54% held soccer coaching certifications. Concentric (100%) and eccentric (98%) modes of resistance were the most commonly prescribed, whereas the squat (including variations) (52%) was deemed the most important exercise for soccer players. Hang clean (33%) and multiple hops/lunges (89%) were the most programed Olympic weightlifting and plyometric exercises. Global Positioning Systems (94%) were the most utilized technology-based equipment. Time, scheduling and fixtures were the biggest issues faced, which made it difficult to periodize training programs and apply appropriate training loads. Furthermore, strength and conditioning coaches would like to further integrate technology to comprehensively monitor and test players, while also believing that technology will continue to be developed and integrated in the future. Strength and conditioning coaches from professional soccer can use the information from this study to review current practices and also provide ideas for diversifying or modifying future practices.     

The Leishmania infantum cytosolic SIR2-related protein 1 (LiSIR2RP1) is an NAD+ -dependent deacetylase and ADP-ribosyltransferase

Proteins of the SIR2 (Silent Information Regulator 2) family are characterized by a conserved catalytic domain that exerts unique NAD(+)-dependent deacetylase activity on histones and various other cellular substrates. Previous reports from us have identified a Leishmania infantum gene encoding a cytosolic protein termed LiSIR2RP1 (Leishmania infantum SIR2-related protein 1) that belongs to the SIR2 family. Targeted disruption of one LiSIR2RP1 gene allele led to decreased amastigote virulence, in vitro as well as in vivo. In the present study, attempts were made for the first time to explore and characterize the enzymatic functions of LiSIR2RP1. The LiSIR2RP1 exhibited robust NAD(+)-dependent deacetylase and ADP-ribosyltransferase activities. Moreover, LiSIR2RP1 is capable of deacetylating tubulin, either in dimers or, when present, in taxol-stabilized microtubules or in promastigote and amastigote extracts. Furthermore, the immunostaining of parasites revealed a partial co-localization of alpha-tubulin and LiSIR2RP1 with punctate labelling, seen on the periphery of both promastigote and amastigote stages. Isolated parasite cytoskeleton reacted with antibodies showed that part of LiSIR2RP1 is associated to the cytoskeleton network of both promastigote and amastigote forms. Moreover, the Western blot analysis of the soluble and insoluble fractions of the detergent of promastigote and amastigote forms revealed the presence of alpha-tubulin in the insoluble fraction, and the LiSIR2RP1 distributed in both soluble and insoluble fractions of promastigotes as well as amastigotes. Collectively, the results of the present study demonstrate that LiSIR2RP1 is an NAD(+)-dependent deacetylase that also exerts an ADP-ribosyltransferase activity. The fact that tubulin could be among the targets of LiSIR2RP1 may have significant implications during the remodelling of the morphology of the parasite and its interaction with the host cell.