MAPKKK-independent regulation of the Hog1 stress-activated protein kinase in Candida albicans

The Hog1 stress-activated protein kinase regulates both stress responses and morphogenesis in Candida albicans and is essential for the virulence of this major human pathogen. Stress-induced Hog1 phosphorylation is regulated by the upstream MAPKK, Pbs2, which in turn is regulated by the MAPKKK, Ssk2. Here, we have investigated the role of phosphorylation of Hog1 and Pbs2 in Hog1-mediated processes in C. albicans. Mutation of the consensus regulatory phosphorylation sites of Hog1 (Thr-174/Tyr-176) and Pbs2 (Ser-355/Thr-359), to nonphosphorylatable residues, resulted in strains that phenocopied hog1Δ and pbs2Δ cells. Consistent with this, stress-induced phosphorylation of Hog1 was abolished in cells expressing nonphosphorylatable Pbs2 (Pbs2(AA)). However, mutation of the consensus sites of Pbs2 to phosphomimetic residues (Pbs2(DD)) failed to constitutively activate Hog1. Furthermore, Ssk2-independent stress-induced Hog1 activation was observed in Pbs2(DD) cells. Collectively, these data reveal a previously uncharacterized MAPKKK-independent mechanism of Hog1 activation in response to stress. Although Pbs2(DD) cells did not exhibit high basal levels of Hog1 phosphorylation, overexpression of an N-terminal truncated form of Ssk2 did result in constitutive Hog1 activation, which was further increased upon stress. Significantly, both Pbs2(AA) and Pbs2(DD) cells displayed impaired stress resistance and attenuated virulence in a mouse model of disease, whereas only Pbs2(AA) cells exhibited the morphological defects associated with loss of Hog1 function. This indicates that Hog1 mediates C. albicans virulence by conferring stress resistance rather than regulating morphogenesis.     

Population genetics and microevolution of clinical Candida glabrata reveals recombinant sequence types and hyper-variation within mitochondrial genomes,virulence genes,and drug targets

Candida glabrata is the second most common etiological cause of worldwide systemic candidiasis in adult patients. Genome analysis of 68 isolates from 8 hospitals across Scotland, together with 83 global isolates, revealed insights into the population genetics and evolution of C. glabrata. Clinical isolates of C. glabrata from across Scotland are highly genetically diverse, including at least 19 separate sequence types that have been recovered previously in globally diverse locations, and 1 newly discovered sequence type. Several sequence types had evidence for ancestral recombination, suggesting transmission between distinct geographical regions has coincided with genetic exchange arising in new clades. Three isolates were missing MATα1, potentially representing a second mating type. Signatures of positive selection were identified in every sequence type including enrichment for epithelial adhesins thought to facilitate fungal adhesin to human epithelial cells. In patent microevolution was identified from 7 sets of recurrent cases of candidiasis, revealing an enrichment for nonsynonymous and frameshift indels in cell surface proteins. Microevolution within patients also affected epithelial adhesins genes, and several genes involved in drug resistance including the ergosterol synthesis gene ERG4 and the echinocandin target FKS1/2, the latter coinciding with a marked drop in fluconazole minimum inhibitory concentration. In addition to nuclear genome diversity, the C. glabrata mitochondrial genome was particularly diverse, with reduced conserved sequence and conserved protein-encoding genes in all nonreference ST15 isolates. Together, this study highlights the genetic diversity within the C. glabrata population that may impact virulence and drug resistance, and 2 major mechanisms generating this diversity: microevolution and genetic exchange/recombination.     

High-resolution structural and thermodynamic analysis of extreme stabilization of human procarboxypeptidase by computational protein design

Recent efforts to design de novo or redesign the sequence and structure of proteins using computational techniques have met with significant success. Most, if not all, of these computational methodologies attempt to model atomic-level interactions, and hence high-resolution structural characterization of the designed proteins is critical for evaluating the atomic-level accuracy of the underlying design force-fields. We previously used our computational protein design protocol RosettaDesign to completely redesign the sequence of the activation domain of human procarboxypeptidase A2. With 68% of the wild-type sequence changed, the designed protein, AYEdesign, is over 10 kcal/mol more stable than the wild-type protein. Here, we describe the high-resolution crystal structure and solution NMR structure of AYEdesign, which show that the experimentally determined backbone and side-chains conformations are effectively superimposable with the computational model at atomic resolution. To isolate the origins of the remarkable stabilization, we have designed and characterized a new series of procarboxypeptidase mutants that gain significant thermodynamic stability with a minimal number of mutations; one mutant gains more than 5 kcal/mol of stability over the wild-type protein with only four amino acid changes. We explore the relationship between force-field smoothing and conformational sampling by comparing the experimentally determined free energies of the overall design and these focused subsets of mutations to those predicted using modified force-fields, and both fixed and flexible backbone sampling protocols.     

Nuclear estrogen and progesterone receptors in the oviduct of heifers under natural and superovulated estrous cycles

Oviducts from 22 crossbred heifers were examined for the presence of nuclear estrogen (ERalpha) and progesterone (PR) receptors at different phases (estrus, metaestrus and diestrus) of naturally occurring estrous cycles and estrous cycles during which superovulation was induced. Receptors were detected by immunohistochemistry in the epithelial cells, connective tissue and muscular layer of oviductal infundibulum, ampulla, ampullary/isthmic transition and isthmus. Epithelial ERalpha was found along the entire oviduct regardless of the cycle phase and of the circulating concentrations of 17beta-estradiol (E(2)) and progesterone (P(4)). Epithelial PR was found mainly at the ampullary-isthmic transition and isthmus and more intensely at the estrus phase but their amount was not correlated with P(4) concentrations. ERalpha in the connective tissue was more abundant at the infundibulum and ampulla, regardless of the phase of the estrous cycle and of E(2) and P(4) circulating concentrations. PR in the connective tissue was found mostly at the ampulla, regardless of the estrous cycle phase but no correlations were found between amount and P(4) concentrations. ERalpha staining intensity in the muscular layer was similar at all phases of the estrous cycle and at all anatomical segments of the oviducts. However, PR staining was more intense at the isthmus during the metaestrus phase and it was negatively correlated with P(4) concentrations. In general, data from the present research suggest that P(4) exerts an inhibitory role upon ERalpha and PR. Also, no differences were found between animals subjected or not to superovulation.     

Stages of development of the floral secretory disk in Tapirira guianensis Aubl. (Anacardiaceae), a dioecious species

The goal of this study was to analyse possible structural and ultrastructural differences between the secretory disk of male and functionally female flowers of Tapirira guianensis (Anacardiaceae) at different developmental stages. Studies were carried out using light, scanning and transmission electron microscopy. Biochemical tests were employed to determine the proportion of sugars in the nectar of the floral morphotypes: they were found to be similar, both predominantly composed of sucrose. In addition to sugars, lipids and phenolic substances were identified in anthetic flowers; thus, the secretory disk is a mixed secretion gland, also called a sensu lato nectary. During anthesis, granulocrine and eccrine secretory mechanisms occur in both floral morphotypes. After anthesis and fertilization of the functionally female flower, only the lipophilic and phenolic secretion continues until the early stages of fruit development. An intrastaminal secretory disk that produces both nectar and lipids is reported for the first time in Anacardiaceae. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 533–544.     

c.G2114A MYH9 mutation (DFNA17) causes non-syndromic autosomal dominant hearing loss in a Brazilian family

We studied a family presenting 10 individuals affected by autosomal dominant deafness in all frequencies and three individuals affected by high frequency hearing loss. Genomic scanning using the 50k Affymetrix microarray technology yielded a Lod Score of 2.1 in chromosome 14 and a Lod Score of 1.9 in chromosome 22. Mapping refinement using microsatellites placed the chromosome 14 candidate region between markers D14S288 and D14S276 (8.85 cM) and the chromosome 22 near marker D22S283. Exome sequencing identified two candidate variants to explain hearing loss in chromosome 14 [PTGDR – c.G894A:p.R298R and PTGER2 – c.T247G:p.C83G], and one in chromosome 22 [MYH9, c.G2114A:p.R705H]. Pedigree segregation analysis allowed exclusion of the PTGDR and PTGER2 variants as the cause of deafness. However, the MYH9 variant segregated with the phenotype in all affected members, except the three individuals with different phenotype. This gene has been previously described as mutated in autosomal dominant hereditary hearing loss and corresponds to DFNA17. The mutation identified in our study is the same described in the prior report. Thus, although linkage studies suggested a candidate gene in chromosome 14, we concluded that the mutation in chromosome 22 better explains the hearing loss phenotype in the Brazilian family.     

The interferon type I signature towards prediction of non-response to rituximab in rheumatoid arthritis patients

Introduction

B cell depletion therapy is efficacious in rheumatoid arthritis (RA) patients failing on tumor necrosis factor (TNF) blocking agents. However, approximately 40% to 50% of rituximab (RTX) treated RA patients have a poor response. We investigated whether baseline gene expression levels can discriminate between clinical non-responders and responders to RTX.

Methods

In 14 consecutive RA patients starting on RTX (test cohort), gene expression profiling on whole peripheral blood RNA was performed by Illumina^® HumanHT beadchip microarrays. Supervised cluster analysis was used to identify genes expressed differentially at baseline between responders and non-responders based on both a difference in 28 joints disease activity score (ΔDAS28 < 1.2) and European League against Rheumatism (EULAR) response criteria after six months RTX. Genes of interest were measured by quantitative real-time PCR and tested for their predictive value using receiver operating characteristics (ROC) curves in an independent validation cohort (n = 26).

Results

Genome-wide microarray analysis revealed a marked variation in the peripheral blood cells between RA patients before the start of RTX treatment. Here, we demonstrated that only a cluster consisting of interferon (IFN) type I network genes, represented by a set of IFN type I response genes (IRGs), that is, LY6E, HERC5, IFI44L, ISG15, MxA, MxB, EPSTI1 and RSAD2, was associated with ΔDAS28 and EULAR response outcome (P = 0.0074 and P = 0.0599, respectively). Based on the eight IRGs an IFN-score was calculated that reached an area under the curve (AUC) of 0.82 to separate non-responders from responders in an independent validation cohort of 26 patients using Receiver Operator Characteristics (ROC) curves analysis according to ΔDAS28 < 1.2 criteria. Advanced classifier analysis yielded a three IRG-set that reached an AUC of 87%. Comparable findings applied to EULAR non-response criteria.

Conclusions

This study demonstrates clinical utility for the use of baseline IRG expression levels as a predictive biomarker for non-response to RTX in RA.     

Inhibiting citrullination in rheumatoid arthritis: taking fuel from the fire

Citrullination is a post-translational modification catalysed by peptidylarginine deiminase and is a common feature of inflammation. The presence of anti-citrullinated protein/peptide antibodies (ACPA), however, is unique to rheumatoid arthritis. Several lines of evidence suggest that ACPA are important in the pathogenesis of rheumatoid arthritis. A popular hypothesis for this pathogenesis is a two-hit model. The first hit gives rise to ACPA, and the second hit, an unrelated episode of synovial inflammation accompanied by citrullination, is perpetuated by the pre-existing antibodies. This model suggests that reducing citrullination might ameliorate disease. Recent findings indicate that citrullination closely correlates with inflammation, and that glucocorticoids decrease peptidylarginine deiminase expression independent of their other anti-inflammatory effects.     

Genetic variability in mitochondrial and nuclear genes of Larus dominicanus (Charadriiformes,Laridae) from the Brazilian coast

Several phylogeographic studies of seabirds have documented low genetic diversity that has been attributed to bottleneck events or individual capacity for dispersal. Few studies have been done in seabirds on the Brazilian coast and all have shown low genetic differentiation on a wide geographic scale. The Kelp Gull is a common species with a wide distribution in the Southern Hemisphere. In this study, we used mitochondrial and nuclear markers to examine the genetic variability of Kelp Gull populations on the Brazilian coast and compared this variability with that of sub-Antarctic island populations of this species. Kelp Gulls showed extremely low genetic variability for mitochondrial markers (cytb and ATPase) and high diversity for a nuclear locus (intron 7 of the β-fibrinogen). The intraspecific evolutionary history of Kelp Gulls showed that the variability found in intron 7 of the β-fibrinogen gene was compatible with the variability expected under neutral evolution but suggested an increase in population size during the last 10,000 years. However, none of the markers revealed evidence of a bottleneck population. These findings indicate that the recent origin of Kelp Gulls is the main explanation for their nuclear diversity, although selective pressure on the mtDNA of this species cannot be discarded.     

Low Primate Diversity and Abundance in Northern Amazonia and its Implications for Conservation

Large areas of the Rio Negro basin in Amazonia are covered by continuous tracts of tropical forest, but have few primate species. This is anomalous considering the general relationship between area and number of species. One possibility is that much of the forest is unsuitable habitat for most primates and the area of suitable habitat is much less than the forested area. This has important consequences for the design of reserves and predictions of the consequences of climate change, which tend to be based on broad categories based on satellite images, and not on information of species distributions within those broad categories. The study was conducted through diurnal and nocturnal line‐transect surveys in the Biodiversity Research Program 25‐km² permanent grid in Viruá National Park, which has vegetation associations typical of much of northern Amazonia. The highest primate diversity and abundances occurred in tall terra firme forests (58%), whereas inundated forests and scrublands, which cover 42 percent of the survey grid and 90.8 percent of the Viruá National Park, have virtually no primates. This suggests that parks and reserves in northwestern Amazonia will have to be very large to maintain viable populations of most primates and their ecological interactions, and that very broad habitat categories are not sufficient to make predictions about actual and future suitability of areas for primate conservation.