Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S,and G2 Phases of the Cell Cycle in Trypanosomatids

Trypanosomatids are the etiologic agents of various infectious diseases in humans. They diverged early during eukaryotic evolution and have attracted attention as peculiar models for evolutionary and comparative studies. Here, we show a meticulous study comparing the incorporation and detection of the thymidine analogs BrdU and EdU in Leishmania amazonensis, Trypanosoma brucei, and Trypanosoma cruzi to monitor their DNA replication. We used BrdU‐ and EdU‐incorporated parasites with the respective standard detection approaches: indirect immunofluorescence to detect BrdU after standard denaturation (2 M HCl) and “click” chemistry to detect EdU. We found a discrepancy between these two thymidine analogs due to the poor detection of BrdU, which is reflected on the estimative of the duration of the cell cycle phases G1, S, and G2. To solve this discrepancy, we increase the exposure of incorporated BrdU using different concentrations of HCl. Using a new value for HCl concentration, we re‐estimated the phases G1, S, G2 + M, and cytokinesis durations, confirming the values found by this approach using EdU. In conclusion, we suggest that the studies using BrdU with standard detection approach, not only in trypanosomatids but also in others cell types, should be reviewed to ensure an accurate estimation of DNA replication monitoring.     

Cytotaxonomy of Eurypyga helias (Gruiformes,Eurypygidae): First Karyotypic Description and Phylogenetic Proximity with Rynochetidae

The sunbittern (Eurypyga helias) is a South American Gruiformes, the only member of Family Eurypigidae. In most phylogenetic proposals, it is placed in a more distant position than other families of the so-called “core Gruiformes”. Different studies based on molecular, morphological and biogeographical data suggest that the Eurypigidae is closely related to the kagu (Rhynochetos jubatus), the only species in Rynochetidae, another family not included in the core Gruiformes. Here, the karyotype of the sunbittern is described for the first time, by classical and molecular cytogenetics, using whole chromosome probes derived from Gallus gallus and Leucopternis albicollis. We found a diploid number of 80, with only one pair of biarmed autosomal macrochromosomes, similar to that observed in the kagu. Chromosome painting revealed that most syntenies found in the avian putative ancestral karyotype (PAK) were conserved in the sunbittern. However, PAK1, PAK2, and PAK5 corresponded to two chromosome pairs each. Probes derived from L. albicollis confirm that fissions in PAK1 and PAK2 were centric, whereas in PAK5 the fission is interstitial. In addition, there is fusion of segments homologous to PAK2q and PAK5. From a phylogenetic point of view, comparisons of our results with two other Gruiformes belonging to family Rallidae suggest that the PAK5q fission might be a synapomorphy for Gruiformes. Fissions in PAK1 and PAK2 are found only in Eurypigidae, and might also occur in Rynochetidae, in view of the similar chromosomal morphology between the sunbittern and the kagu. This suggests a close phylogenetic relationship between Eurypigidae and Rynochetidae, whose common ancestor was separated by the Gondwana vicariancy in South America and New Caledonia, respectively.     

Risk of symptomatic dengue for foreign visitors to the 2014 FIFA World Cup in Brazil

Brazil will host the FIFA World Cup™, the biggest single-event competition in the world, from June 12-July 13 2014 in 12 cities. This event will draw an estimated 600,000 international visitors. Brazil is endemic for dengue. Hence, attendees of the 2014 event are theoretically at risk for dengue. We calculated the risk of dengue acquisition to non-immune international travellers to Brazil, depending on the football match schedules, considering locations and dates of such matches for June and July 2014. We estimated the average per-capita risk and expected number of dengue cases for each host-city and each game schedule chosen based on reported dengue cases to the Brazilian Ministry of Health for the period between 2010-2013. On the average, the expected number of cases among the 600,000 foreigner tourists during the World Cup is 33, varying from 3-59. Such risk estimates will not only benefit individual travellers for adequate pre-travel preparations, but also provide valuable information for public health professionals and policy makers worldwide. Furthermore, estimates of dengue cases in international travellers during the World Cup can help to anticipate the theoretical risk for exportation of dengue into currently non-infected areas.     

Moniliophthora perniciosa Necrosis- and Ethylene-Inducing Protein 2 (MpNep2) as a Metastable Dimer in Solution: Structural and Functional Implications

Understanding how Nep-like proteins (NLPs) behave during the cell cycle and disease progression of plant pathogenic oomycetes, fungi and bacteria is crucial in light of compelling evidence that these proteins play a role in Witches` Broom Disease (WBD) of Theobroma cacao, one of the most important phytopathological problems to afflict the Southern Hemisphere. The crystal structure of MpNep2, a member of the NLP family and the causal agent of WBD, revealed the key elements for its activity. This protein has the ability to refold after heating and was believed to act as a monomer in solution, in contrast to the related homologs MpNep1 and NPP from the oomyceteous fungus Phytophthora parasitica. Here, we identify and characterize a metastable MpNep2 dimer upon over-expression in Escherichia coli using different biochemical and structural approaches. We found using ultra-fast liquid chromatography that the MpNep2 dimer can be dissociated by heating but not by dilution, oxidation or high ionic strength. Small-angle X-ray scattering revealed a possible tail-to-tail interaction between monomers, and nuclear magnetic resonance measurements identified perturbed residues involved in the putative interface of interaction. We also explored the ability of the MpNep2 monomer to refold after heating or chemical denaturation. We observed that MpNep2 has a low stability and cooperative fold that could be an explanation for its structure and activity recovery after stress. These results can provide new insights into the mechanism for MpNep2′s action in dicot plants during the progression of WBD and may open new avenues for the involvement of NLP- oligomeric species in phytopathological disorders.     

Exposure to sub-inhibitory concentrations of cefotaxime enhances the systemic colonization of Salmonella Typhimurium in BALB/c mice

It has been proposed that sub-inhibitory concentrations of antibiotics play a role in virulence modulation. In this study, we evaluated the ability of Salmonella enterica serovar Typhimurium (hereafter S. Typhimurium) to colonize systemically BALB/c mice after exposure to a sub-inhibitory concentration of cefotaxime (CTX). In vivo competition assays showed a fivefold increase in systemic colonization of CTX-exposed bacteria when compared to untreated bacteria. To identify the molecular mechanisms involved in this phenomenon, we carried out a high-throughput genetic screen. A transposon library of S. Typhimurium mutants was subjected to negative selection in the presence of a sub-inhibitory concentration of CTX and genes related to anaerobic metabolism, biosynthesis of purines, pyrimidines, amino acids and other metabolites were identified as needed to survive in this condition. In addition, an impaired ability for oxygen consumption was observed when bacteria were cultured in the presence of a sub-inhibitory concentration of CTX. Altogether, our data indicate that exposure to sub-lethal concentrations of CTX increases the systemic colonization of S. Typhimurium in BALB/c mice in part by the establishment of a fitness alteration conducive to anaerobic metabolism.     

Life-cycle modification in open oceans accounts for genome variability in a cosmopolitan phytoplankton

Emiliania huxleyi is the most abundant calcifying plankton in modern oceans with substantial intraspecific genome variability and a biphasic life cycle involving sexual alternation between calcified 2N and flagellated 1N cells. We show that high genome content variability in Emiliania relates to erosion of 1N-specific genes and loss of the ability to form flagellated cells. Analysis of 185 E. huxleyi strains isolated from world oceans suggests that loss of flagella occurred independently in lineages inhabiting oligotrophic open oceans over short evolutionary timescales. This environmentally linked physiogenomic change suggests life cycling is not advantageous in very large/diluted populations experiencing low biotic pressure and low ecological variability. Gene loss did not appear to reflect pressure for genome streamlining in oligotrophic oceans as previously observed in picoplankton. Life-cycle modifications might be common in plankton and cause major functional variability to be hidden from traditional taxonomic or molecular markers.     

Evaluation of the catalytic specificity,biochemical properties,and milk clotting abilities of an aspartic peptidase from <Emphasis Type="Italic">Rhizomucor miehei</Emphasis>

In this study, we detail the specificity of an aspartic peptidase from Rhizomucor miehei and evaluate the effects of this peptidase on clotting milk using the peptide sequence of k-casein (Abz-LSFMAIQ-EDDnp) and milk powder. Molecular mass of the peptidase was estimated at 37 kDa, and optimum activity was achieved at pH 5.5 and 55 °C. The peptidase was stable at pH values ranging from 3 to 5 and temperatures of up 45 °C for 60 min. Dramatic reductions in proteolytic activity were observed with exposure to sodium dodecyl sulfate, and aluminum and copper (II) chloride. Peptidase was inhibited by pepstatin A, and mass spectrometry analysis identified four peptide fragments (TWSISYGDGSSASGILAK, ASNGGGGEYIFGGYDSTK, GSLTTVPIDNSR, and GWWGITVDRA), similar to rhizopuspepsin. The analysis of catalytic specificity showed that the coagulant activity of the peptidase was higher than the proteolytic activity and that there was a preference for aromatic, basic, and nonpolar amino acids, particularly methionine, with specific cleavage of the peptide bond between phenylalanine and methionine. Thus, this peptidase may function as an important alternative enzyme in milk clotting during the preparation of cheese.     

Dynamics of in vitro acquisition of resistance by Candida parapsilosis to different azoles

Candida parapsilosis is a common isolate from clinical fungal infectious episodes. Resistance of C. parapsilosis to azoles has been increasingly reported. To analyse the development of resistance in C. parapsilosis , four azole-susceptible clinical strains and one American Type Culture Collection type strain were cultured in the presence of fluconazole, voriconazole and posaconazole at different concentrations. The isolates developed variable degrees of azole resistance according to the antifungal used. Fluconazole was the fastest inducer while posaconazole was the slowest. Fluconazole and voriconazole induced resistance to themselves and each other, but not to posaconazole. Posaconazole induced resistance to all azoles. Developed resistance was stable; it could be confirmed after 30 days of subculture in drug-free medium. Azole-resistant isolates revealed a homogeneous population structure; the role of azole transporter efflux pumps was minor after evaluation by microdilution and cytometric assays with efflux pump blockers (verapamil, ibuprofen and carbonyl cyanide 3-chloro-phenylhydrazone). We conclude that the rapid development of azole resistance occurs by a mechanism that might involve mutation of genes responsible for ergosterol biosynthesis pathway, stressed by exposure to antifungals.     

Morphological, physiological and behavioural evaluation of a ‘Mice in Space’ housing system

Environmental conditions likely affect physiology and behaviour of mice used for life sciences research on Earth or in Space. Here, we analysed the effects of cage confinement on the weightbearing musculoskeletal system, behaviour and stress of wild-type mice (C57BL/6JRj, 30 g b.wt., total n = 24) housed for 25 days in a prototypical ground-based and fully automated life support habitat device called “Mice in Space” (MIS). Compared with control housing (individually ventilated cages) the MIS mice revealed no significant changes in soleus muscle size and myofiber distribution (type I vs. II) and quality of bone (3-D microarchitecture and mineralisation of calvaria, spine and femur) determined by confocal and micro-computed tomography. Corticosterone metabolism measured non-invasively (faeces) monitored elevated adrenocortical activity at only start of the MIS cage confinement (day 1). Behavioural tests (i.e., grip strength, rotarod, L/D box, elevated plus-maze, open field, aggressiveness) performed subsequently revealed only minor changes in motor performance (MIS vs. controls). The MIS habitat will not, on its own, produce major effects that could confound interpretation of data induced by microgravity exposure during spaceflight. Our results may be even more helpful in developing multidisciplinary protocols with adequate scenarios addressing molecular to systems levels using mice of various genetic phenotypes in many laboratories. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.