Enzymic, phylogenetic, and structural characterization of the unusual papain-like protease domain of Plasmodium falciparum SERA5.

Serine repeat antigen 5 (SERA5) is an abundant antigen of the human malaria parasite Plasmodium falciparum and is the most strongly expressed member of the nine-gene SERA family. It appears to be essential for the maintenance of the erythrocytic cycle, unlike a number of other members of this family, and has been implicated in parasite egress and/or erythrocyte invasion. All SERA proteins possess a central domain that has homology to papain except in the case of SERA5 (and some other SERAs), where the active site cysteine has been replaced with a serine. To investigate if this domain retains catalytic activity, we expressed, purified, and refolded a recombinant form of the SERA5 enzyme domain. This protein possessed chymotrypsin-like proteolytic activity as it processed substrates downstream of aromatic residues, and its activity was reversed by the serine protease inhibitor 3,4-diisocoumarin. Although all Plasmodium SERA enzyme domain sequences share considerable homology, phylogenetic studies revealed two distinct clusters across the genus, separated according to whether they possess an active site serine or cysteine. All Plasmodia appear to have at least one member of each group. Consistent with separate biological roles for members of these two clusters, molecular modeling studies revealed that SERA5 and SERA6 enzyme domains have dramatically different surface properties, although both have a characteristic papain-like fold, catalytic cleft, and an appropriately positioned catalytic triad. This study provides impetus for the examination of SERA5 as a target for antimalarial drug design.

Sequence and phylogenetic analysis of human T cell lymphotropic virus type 1 from Tumaco, Colombia

Human T cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes leukemia and the neurological disorder HTLV-1 associated myelopathy or tropical spastic paraparesis (HAM/TSP). Infection with this virus - although it is distributed worldwide - is limited to certain endemic areas of the world. Despite its specific distribution and slow mutation rate, molecular epidemiology on this virus has been useful to follow the movements of human populations and routes of virus spread to different continents. In the present study, we analyzed the genetic variability of a region of the env gene of isolates obtained from individuals of African origin that live on the Pacific coast of Colombia. Sequencing and comparison of the fragment with the same fragment from different HTLV-1 isolates showed a variability ranging from 0.8% to 1.2%. Phylogenetic studies permit us to include these isolates in the transcontinental subgroup A in which samples isolated from Brazil and Chile are also found. Further analyses will be necessary to determine if these isolates were recently introduced into the American continent or if they rather correspond to isolates introduced during the Paleolithic period.

Ezrin-Radixin-Moesin-binding Sequence of PSGL-1 Glycoprotein Regulates Leukocyte Rolling on Selectins and Activation of Extracellular Signal-regulated Kinases.

P-selectin glycoprotein ligand-1 (PSGL-1) mediates the capture (tethering) of free-flowing leukocytes and subsequent rolling on selectins. PSGL-1 interactions with endothelial selectins activate Src kinases and spleen tyrosine kinase (Syk), leading to α(L)β(2) integrin-dependent leukocyte slow rolling, which promotes leukocyte recruitment into tissues. In addition, but through a distinct pathway, PSGL-1 engagement activates ERK. Because ezrin, radixin and moesin proteins (ERMs) link PSGL-1 to actin cytoskeleton and because they serve as adaptor molecules between PSGL-1 and Syk, we examined the role of PSGL-1 ERM-binding sequence (EBS) on cell capture, rolling, and signaling through Syk and MAPK pathways. We carried out mutational analysis and observed that deletion of EBS severely reduced 32D leukocyte tethering and rolling on L-, P-, and E-selectin and slightly increased rolling velocity. Alanine substitution of Arg-337 and Lys-338 showed that these residues play a key role in supporting leukocyte tethering and rolling on selectins. Importantly, EBS deletion or Arg-337 and Lys-338 mutations abrogated PSGL-1-induced ERK activation, whereas they did not prevent Syk phosphorylation or E-selectin-induced leukocyte slow rolling. These studies demonstrate that PSGL-1 EBS plays a critical role in recruiting leukocytes on selectins and in activating the MAPK pathway, whereas it is dispensable to phosphorylate Syk and to lead to α(L)β(2)-dependent leukocyte slow rolling.

The role of queen pheromones in social insects: queen control or queen signal?

Queens and workers in social insect colonies can differ in reproductive goals such as colony-level sex allocation and production of males by workers. That the presence of queen(s) often seems to affect worker behaviour in situations of potential conflict has given rise to the idea of queen control over reproduction. In small colonies queen control is possible via direct aggression against workers, but in large colonies queens cannot be effectively aggressive towards all the workers. This, plus evidence that queen-produced chemicals affect worker behaviour, has led to the conclusion that physical intimidation has been replaced by pheromonal queen control, whereby queen(s) chemically manipulate workers into behaving in ways that increase the queen's fitness at the worker's expense. It is argued in this paper, however, that pheromonal queen control has never conclusively been demonstrated and is evolutionarily difficult to justify. Proposed examples of pheromonal control are more likely to be honest signals, with workers' responses increasing their own inclusive fitness. A series of experimental and field studies in which positive results would give prima facie evidence for pheromonal queen control is suggested. Finally, three terms are defined: (1) pheromonal queen control for workers or subordinate queens being chemically manipulated into acting against their own best interests; (2) pheromonal queen signal for situations where workers or subordinate queens react to queen pheromones in ways that increase their, and possibly the queens', inclusive fitness; and (3) pheromonal queen effect where changes in the workers' or subordinate queens' behaviour have an unknown consequence on their inclusive fitness.

Functional complementation of a yeast knockout strain by Schistosoma mansoni Rho1 GTPase in the presence of caffeine, an agent that affects mutants defective in the protein kinase C signal transduction pathway

In a previous study, the Schistosoma mansoni Rho1 protein was able to complement Rho1 null mutant Saccharomyces cerevisiae cells at restrictive temperatures and under osmotic stress (low calcium concentration) better than the human homologue (RhoA). It is known that under osmotic stress, the S. cerevisiae Rho1 triggers two distinct pathways: activation of the membrane 1,3-beta-glucan synthase enzymatic complex and activation of the protein kinase C1 signal transduction pathway, promoting the transcription of response genes. In the present work the SmRho1 protein and its mutants smrho1E97P, smrho1L101T, and smrho1E97P, L101T were used to try to clarify the basis for the differential complementation of Rho1 knockout yeast strain by the human and S. mansoni genes. Experiments of functional complementation in the presence of caffeine and in the presence of the osmotic regulator sorbitol were conducted. SmRho1 and its mutants showed a differential complementation of the yeast cells in the presence of caffeine, since smrho1E97P and smrho1E97P, L101T mutants showed a delay in the growth when compared to the yeast complemented with the wild type SmRho1. However, in the presence of sorbitol and caffeine the wild type SmRho1 and mutants showed a similar complementation phenotype, as they allowed yeast growth in all caffeine concentrations tested.

Challenges of phylogenetic analyses of aDNA sequences

One of the crucial steps of authentication of aDNA sequences is phylogenetic consistency. Amplified sequences should fit into the phylogenetic framework of their supposed origin. An inherent property of aDNA sequences however, is their short sequence length. Additionally, genes for aDNA studies are often chosen by their preservation potential rather than by phylogenetically informative content. This poses potential challenges regarding their analyses, and might result in an inaccurate reflection of the supposed phylogenetic history of the sequence or organism under study. In this paper some fundamental problems of phylogenetic analysis and interpretation of aDNA datasets are discussed. Suggestions for character sampling and treatment of missing data are made. The publication is the result of a talk from the 1st PAMINSA Meeting in Rio de Janeiro, July 2005.

Incompletely resolved phylogenetic trees inflate estimates of phylogenetic conservatism.

The tendency for more closely related species to share similar traits and ecological strategies can be explained by their longer shared evolutionary histories and represents phylogenetic conservatism. How strongly species traits co-vary with phylogeny can significantly impact how we analyze cross-species data and can influence our interpretation of assembly rules in the rapidly expanding field of community phylogenetics. Phylogenetic conservatism is typically quantified by analyzing the distribution of species values on the phylogenetic tree that connects them. Many phylogenetic approaches, however, assume a completely sampled phylogeny: while we have good estimates of deeper phylogenetic relationships for many species-rich groups, such as birds and flowering plants, we often lack information on more recent interspecific relationships (i.e., within a genus). A common solution has been to represent these relationships as polytomies on trees using taxonomy as a guide. Here we show that such trees can dramatically inflate estimates of phylogenetic conservatism quantified using S. P. Blomberg et al.'s K statistic. Using simulations, we show that even randomly generated traits can appear to be phylogenetically conserved on poorly resolved trees. We provide a simple rarefaction-based solution that can reliably retrieve unbiased estimates of K, and we illustrate our method using data on first flowering times from Thoreau's woods (Concord, Massachusetts, USA).

Cutting edge: IL-1α is a crucial danger signal triggering acute myocardial inflammation during myocardial infarction.

Myocardial infarction (MI) induces a sterile inflammatory response that contributes to adverse cardiac remodeling. The initiating mechanisms of this response remain incompletely defined. We found that necrotic cardiomyocytes released a heat-labile proinflammatory signal activating MAPKs and NF-κB in cardiac fibroblasts, with secondary production of cytokines. This response was abolished in Myd88(-/-) fibroblasts but was unaffected in nlrp3-deficient fibroblasts. Despite MyD88 dependency, the response was TLR independent, as explored in TLR reporter cells, pointing to a contribution of the IL-1 pathway. Indeed, necrotic cardiomyocytes released IL-1α, but not IL-1β, and the immune activation of cardiac fibroblasts was abrogated by an IL-1R antagonist and an IL-1α-blocking Ab. Moreover, immune responses triggered by necrotic Il1a(-/-) cardiomyocytes were markedly reduced. In vivo, mice exposed to MI released IL-1α in the plasma, and postischemic inflammation was attenuated in Il1a(-/-) mice. Thus, our findings identify IL-1α as a crucial early danger signal triggering post-MI inflammation.

Inteins in pathogenic fungi: a phylogenetic tool and perspectives for therapeutic applications

Inteins or "internal proteins" are coding sequences that are transcribed and translated with flanking sequences (exteins). After translation, the inteins are excised by an autocatalytic process and the host protein assumes its normal conformation and develops its expected function. These parasitic genetic elements have been found in important, conserved proteins in all three domains of life. Most of the eukaryotic inteins are present in the fungi kingdom and the PRP8 intein is one of the most widespread inteins, occurring in important pathogens such as Cryptococcus neoformans (varieties grubii and neoformans), Cryptococcus gattii, Histoplasma capsulatum and Paracoccidioides brasiliensis. The knowledge of conserved and non-conserved domains in inteins have opened up new opportunities for the study of population variability in pathogenic fungi, including their phylogenetic relationships and recognition or diagnoses of species. Furthermore, inteins in pathogenic fungi should also be considered a promising therapeutic drug target, since once the autocatalytic splicing is inhibited, the host protein, which is typically vital, will not be able to perform its normal function and the fungal cell will not survive or reproduce.

Dengue virus type 3 in Brazil: a phylogenetic perspective

Circulation of a new dengue virus (DENV)-3 genotype was recently described in Brazil and Colombia, but the precise classification of this genotype has been controversial. Here we perform phylogenetic and nucleotide-distance analyses of the envelope gene, which support the subdivision of DENV-3 strains into five distinct genotypes (GI to GV) and confirm the classification of the new South American genotype as GV. The extremely low genetic distances between Brazilian GV strains and the prototype Philippines/L11423 GV strain isolated in 1956 raise important questions regarding the origin of GV in South America.

Phylogenetic reconstruction of transmission events from individuals with acute HIV infection: toward more-rigorous epidemiological definitions.

Phylogenetic reconstructions of transmission events from individuals with acute human immunodeficiency virus (HIV) infection are conducted to illustrate this group's heightened infectivity. Varied definitions of acute infection and assumptions about observed phylogenetic clusters may produce misleading results. We conducted a phylogenetic analysis of HIV pol sequences from 165 European patients with estimated infection dates and calculated the difference between dates within clusters. Nine phylogenetic clusters were observed. Comparison of dates within clusters revealed that only 2 could have been generated during acute infection. Previous analyses may have incorrectly assigned transmission events to the acutely HIV infected when they were more likely to have occurred during chronic infection.

Phylogenetic analysis of Biomphalaria tenagophila (Orbigny, 1835) (Mollusca: Gastropoda)

Mitochondrial DNA of Biomphalaria tenagophila, a mollusc intermediate host of Schistosoma mansoni in Brazil, was sequenced and characterised. The genome size found for B. tenagophila was 13,722 bp and contained 13 messenger RNAs, 22 transfer RNAs (tRNA) and two ribosomal RNAs (rRNA). In addition to sequencing, the mitochondrial DNA (mtDNA) genome organization of B. tenagophila was analysed based on its content and localization of both coding and non-coding regions, regions of gene overlap and tRNA nucleotide sequences. Sequences of protein, rRNA 12S and rRNA 16S nucleotides as well as gene organization were compared between B. tenagophila and Biomphalaria glabrata, as the latter is the most important S. mansoni intermediate host in Brazil. Differences between such species were observed regarding rRNA composition. The complete sequence of the B. tenagophila mitochondrial genome was deposited in GenBank (accession EF433576). Furthermore, phylogenetic relationships were estimated among 28 mollusc species, which had their complete mitochondrial genome deposited in GenBank, using the neighbour-joining method, maximum parsimony and maximum likelihood bootstrap. B. tenagophila was positioned at a branch close to B. glabrata and Pulmonata molluscs, collectively comprising a paraphyletic group, contrary to Opistobranchia, which was positioned at a single branch and constituted a monophyletic group.

Phylogenetic alpha and beta diversities of butterfly communities correlate with climate in the western Swiss Alps

The observation of non-random phylogenetic distribution of traits in communities provides evidence for niche-based community assembly. Environment may influence the phylogenetic structure of communities because traits determining how species respond to prevailing conditions can be phylogenetically conserved. In this study, we investigate the variation of butterfly species richness and of phylogenetic - and -diversities along temperature and plant species richness gradients. Our study indicates that butterfly richness is independently positively correlated to temperature and plant species richness in the study area. However, the variation of phylogenetic - and -diversities is only correlated to temperature. The significant phylogenetic clustering at high elevation suggests that cold temperature filters butterfly lineages, leading to communities mostly composed of closely related species adapted to those climatic conditions. These results suggest that in colder and more severe conditions at high elevations deterministic processes and not purely stochastic events drive the assemblage of butterfly communities.

DNA multigene sequencing of topotypic specimens of the fascioliasis vector Lymnaea diaphana and phylogenetic analysis of the genus Pectinidens (Gastropoda)

Freshwater lymnaeid snails are crucial in defining transmission and epidemiology of fascioliasis. In South America, human endemic areas are related to high altitudes in Andean regions. The species Lymnaea diaphana has, however, been involved in low altitude areas of Chile, Argentina and Peru where human infection also occurs. Complete nuclear ribosomal DNA 18S, internal transcribed spacer (ITS)-2 and ITS-1 and fragments of mitochondrial DNA 16S and cytochrome c oxidase (cox)1 genes of L. diaphana specimens from its type locality offered 1,848, 495, 520, 424 and 672 bp long sequences. Comparisons with New and Old World Galba/Fossaria, Palaearctic stagnicolines, Nearctic stagnicolines, Old World Radix and Pseudosuccinea allowed to conclude that (i) L. diaphana shows sequences very different from all other lymnaeids, (ii) each marker allows its differentiation, except cox1 amino acid sequence, and (iii) L. diaphana is not a fossarine lymnaeid, but rather an archaic relict form derived from the oldest North American stagnicoline ancestors. Phylogeny and large genetic distances support the genus Pectinidens as the first stagnicoline representative in the southern hemisphere, including colonization of extreme world regions, as most southern Patagonia, long time ago. The phylogenetic link of L. diaphana with the stagnicoline group may give light to the aforementioned peculiar low altitude epidemiological scenario of fascioliasis.