Trypanosoma cruzi (Kinetoplastida Trypanosomatidae): ecology of the transmission cycle in the wild environment of the Andean valley of Cochabamba, Bolivia

An active Trypanosoma cruzi transmission cycle maintained by wild rodents in the Andean valleys of Cochabamba Bolivia is described. Wild and domestic Triatoma infestans with 60% infection with T. cruzi were found and was evidenced in 47.5% (rodents) and 26.7% (marsupial) by parasitological and/or serologycal methods. Phyllotis ocilae and the marsupial species Thylamys elegans, are the most important reservoirs followed by Bolomys lactens and Akodon boliviensis. In spite of both genotypes (TCI and TCII) being prevalent in Bolivia, in our study area only T. cruzi I is being transmitted. Our data suggest that wild T. infestans and wild small mammals play an important role in the maintenance of the transmission cycle of T. cruzi. Furthermore, the finding of high prevalence of T. cruzi infection in wild T. infestans point to the risk of the dispersion of Chagas' disease.     

Regulation of Vacuolar Proton-translocating ATPase Activity and Assembly by Extracellular pH

Vacuolar proton-translocating ATPases (V-ATPases) are responsible for organelle acidification in all eukaryotic cells. The yeast V-ATPase, known to be regulated by reversible disassembly in response to glucose deprivation, was recently reported to be regulated by extracellular pH as well (Padilla-López, S., and Pearce, D. A. (2006) J. Biol. Chem. 281, 10273–10280). Consistent with those results, we find 57% higher V-ATPase activity in vacuoles isolated after cell growth at extracellular pH of 7 than after growth at pH 5 in minimal medium. Remarkably, under these conditions, the V-ATPase also becomes largely insensitive to reversible disassembly, maintaining a low vacuolar pH and high levels of V₁ subunit assembly, ATPase activity, and proton pumping during glucose deprivation. Cytosolic pH is constant under these conditions, indicating that the lack of reversible disassembly is not a response to altered cytosolic pH. We propose that when alternative mechanisms of vacuolar acidification are not available, maintaining V-ATPase activity becomes a priority, and the pump is not down-regulated in response to energy limitation. These results also suggest that integrated pH and metabolic inputs determine the final assembly state and activity of the V-ATPase.     

Exploring Nitrilase Sequence Space for Enantioselective Catalysis

Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 10⁶ to 10¹⁰ members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.     

The viral mimic, polyinosinic:polycytidylic acid, induces fever in rats via an interleukin-1-dependent mechanism

Polyinosinic:polycytidylic acid (poly I:C) is a synthetic double-stranded RNA that is used experimentally to model viral infections in vivo. Previous studies investigating the inflammatory properties of this agent in rodents demonstrated that it is a potent pyrogen. However, the mechanisms underlying this response have not been fully elucidated. In the current study, we examined the effects of peripheral administration of poly I:C on body temperature and cytokine production. Male rats were implanted with biotelemetry devices and randomly assigned to one of the following three groups: poly I:C + saline, poly I:C + interleukin-1 receptor antagonist (IL-1ra), or saline + saline. Maximal fever of 1.6 degrees C above baseline was observed 3 h after an intraperitoneal injection of poly I:C (750 microg/kg). Pretreatment with IL-1ra diminished this response by >50% (maximum body temperature = 0.6 degrees C above baseline). Plasma IL-6 concentration increased fivefold 2 h post-poly I:C compared with saline-injected rats; levels returned to baseline 4 h postinjection. Pretreatment with IL-1ra prevented this rise in IL-6. Plasma tumor necrosis factor (TNF)-alpha was also increased more than fourfold 2 h postinjection but remained unaffected by IL-1ra treatment. IL-1beta and cyclooxygenase-2 mRNA were significantly upregulated in the hypothalamus of poly I:C-treated animals. Finally, poly I:C decreased food intake by 30%, but this response was not altered by pretreatment with IL-1ra. These results suggest that poly I:C induces fever, but not anorexia, through an IL-1 and prostaglandin-dependent mechanism.     

Association between DNA Damage Response and Repair Genes and Risk of Invasive Serous Ovarian Cancer

Background

We analyzed the association between 53 genes related to DNA repair and p53-mediated damage response and serous ovarian cancer risk using case-control data from the North Carolina Ovarian Cancer Study (NCOCS), a population-based, case-control study.

Methods/Principal Findings

The analysis was restricted to 364 invasive serous ovarian cancer cases and 761 controls of white, non-Hispanic race. Statistical analysis was two staged: a screen using marginal Bayes factors (BFs) for 484 SNPs and a modeling stage in which we calculated multivariate adjusted posterior probabilities of association for 77 SNPs that passed the screen. These probabilities were conditional on subject age at diagnosis/interview, batch, a DNA quality metric and genotypes of other SNPs and allowed for uncertainty in the genetic parameterizations of the SNPs and number of associated SNPs. Six SNPs had Bayes factors greater than 10 in favor of an association with invasive serous ovarian cancer. These included rs5762746 (median OR(odds ratio)_{per allele} = 0.66; 95% credible interval (CI) = 0.44–1.00) and rs6005835 (median OR_{per allele}  = 0.69; 95% CI  = 0.53–0.91) in CHEK2, rs2078486 (median OR_{per allele}  = 1.65; 95% CI = 1.21–2.25) and rs12951053 (median OR_{per allele}  = 1.65; 95% CI = 1.20–2.26) in TP53, rs411697 (median OR _{rare homozygote}  = 0.53; 95% CI  = 0.35 – 0.79) in BACH1 and rs10131 (median OR _{rare homozygote}  =  not estimable) in LIG4. The six most highly associated SNPs are either predicted to be functionally significant or are in LD with such a variant. The variants in TP53 were confirmed to be associated in a large follow-up study.

Conclusions/Significance

Based on our findings, further follow-up of the DNA repair and response pathways in a larger dataset is warranted to confirm these results.     

Influence of membrane-cortex linkers on the extrusion of membrane tubes

The cell membrane is an inhomogeneous system composed of phospholipids, sterols, carbohydrates, and proteins that can be directly attached to underlying cytoskeleton. The protein linkers between the membrane and the cytoskeleton are believed to have a profound effect on the mechanical properties of the cell membrane and its ability to reshape. Here, we investigate the role of membrane-cortex linkers on the extrusion of membrane tubes using computer simulations and experiments. In simulations, we find that the force for tube extrusion has a nonlinear dependence on the density of membrane-cortex attachments: at a range of low and intermediate linker densities, the force is not significantly influenced by the presence of the membrane-cortex attachments and resembles that of the bare membrane. For large concentrations of linkers, however, the force substantially increases compared with the bare membrane. In both cases, the linkers provided membrane tubes with increased stability against coalescence. We then pulled tubes from HEK cells using optical tweezers for varying expression levels of the membrane-cortex attachment protein Ezrin. In line with simulations, we observed that overexpression of Ezrin led to an increased extrusion force, while Ezrin depletion had a negligible effect on the force. Our results shed light on the importance of local protein rearrangements for membrane reshaping at nanoscopic scales.     

Leishmania UDP-sugar Pyrophosphorylase: THE MISSING LINK IN GALACTOSE SALVAGE?

The Leishmania parasite glycocalyx is rich in galactose-containing glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.     

Insertion of Epicatechin Gallate into the Cytoplasmic Membrane of Methicillin-resistant Staphylococcus aureus Disrupts Penicillin-binding Protein (PBP) 2a-mediated ��-Lactam Resistance by Delocalizing PBP2

Epicatechin gallate (ECg) sensitizes methicillin-resistant Staphylococcus aureus (MRSA) to oxacillin and other β-lactam agents; it also reduces the secretion of virulence-associated proteins, prevents biofilm formation, and induces gross morphological changes in MRSA cells without compromising the growth rate. MRSA is resistant to oxacillin because of the presence of penicillin-binding protein 2a (PBP2a), which allows peptidoglycan synthesis to continue after oxacillin-mediated acylation of native PBPs. We show that ECg binds predominantly to the cytoplasmic membrane (CM), initially decreasing the fluidity of the bilayer, and induces changes in gene expression indicative of an attempt to preserve and repair a compromised cell wall. On further incubation, the CM is reorganized; the amount of lysylphosphatidylglycerol is markedly reduced, with a concomitant increase in phosphatidylglycerol, and the proportion of branched chain fatty acids increases, resulting in a more fluid structure. We found no evidence that ECg modulates the enzymatic activity of PBP2a through direct binding to the protein but determined that PBP2 is delocalized from the FtsZ-anchored cell wall biosynthetic machinery at the septal division site following intercalation into the CM. We argue that many features of the ECg-induced phenotype can be explained by changes in the fluid dynamics of the CM.     

Population structure and phylogeography reveal pathways of colonization by a migratory marine reptile (Chelonia mydas) in the central and eastern Pacific

Climate, behavior, ecology, and oceanography shape patterns of biodiversity in marine faunas in the absence of obvious geographic barriers. Marine turtles are an example of highly migratory creatures with deep evolutionary lineages and complex life histories that span both terrestrial and marine environments. Previous studies have focused on the deep isolation of evolutionary lineages (>3 mya) through vicariance; however, little attention has been given to the pathways of colonization of the eastern Pacific and the processes that have shaped diversity within the most recent evolutionary time. We sequenced 770 bp of the mtDNA control region to examine the stock structure and phylogeography of 545 green turtles from eight different rookeries in the central and eastern Pacific. We found significant differentiation between the geographically separated nesting populations and identified five distinct stocks (F_ST = 0.08–0.44, P < 0.005). Central and eastern Pacific Chelonia mydas form a monophyletic group containing 3 subclades, with Hawaii more closely related to the eastern Pacific than western Pacific populations. The split between sampled central/eastern and western Pacific haplotypes was estimated at around 0.34 mya, suggesting that the Pacific region west of Hawaii has been a more formidable barrier to gene flow in C. mydas than the East Pacific Barrier. Our results suggest that the eastern Pacific was colonized from the western Pacific via the Central North Pacific and that the Revillagigedos Islands provided a stepping‐stone for radiation of green turtles from the Hawaiian Archipelago to the eastern Pacific. Our results fit with a broader paradigm that has been described for marine biodiversity, where oceanic islands, such as Hawaii and Revillagigedo, rather than being peripheral evolutionary “graveyards”, serve as sources and recipients of diversity and provide a mechanism for further radiation.     

Soil seed bank in a patchy vegetation of coastal sandy plains in southeastern Brazil

Large seed banks have been found in tropical dry forests and also in habitats with high seasonality in rainfall. However, patchily structured vegetation could induce great spatial variation in the seed bank. We characterized the seed bank in a patchy vegetation of restinga, a common type of coastal vegetation found in the Atlantic forest biome. We also evaluated whether there is any spatial variation between the litter and soil layer, bare sand, and the edge and center of vegetation patches with distinct species dominance. We found 104 seeds/m² in the seed bank using a 5‐cm‐depth sampling. Seven out of 16 species found in the restinga seed bank germinated; two of these were found in the early stages of vegetation patches. We found a higher number of seeds at the edge than in the center of vegetation patches. However, there were no significant differences in the number of seeds in the seed bank between the litter and soil layer, and between vegetation patches with distinct species dominance. Bare sandy soils had lower seed bank densities than vegetation patches. A small seed bank size might be explained by the low proportion of seeds from herbaceous and woody species, which are pioneers in the Atlantic forest. However, seed bank might play an important role in the early stages of the successional process, due to the occurrence of the few species that are able to colonize new young vegetation patches.