The extreme N-terminal domain of a hordeivirus TGB1 movement protein mediates its localization to the nucleolus and interaction with fibrillarin

The hordeiviral movement protein encoded by the first gene of the triple gene block (TGBp1) of Poa semilatent virus (PSLV), interacts with viral genomic RNAs to form RNP particles which are considered to be a form of viral genome capable of cell-to-cell and long-distance transport in infected plants. The PSLV TGBp1 contains a C-terminal NTPase/helicase domain (HELD) and an N-terminal extension region consisting of two structurally and functionally distinct domains: an extreme N-terminal domain (NTD) and an internal domain (ID). This study demonstrates that transient expression of TGBp1 fused to GFP in Nicotiana benthamiana leaves results in faint but obvious fluorescence in the nucleolus in addition to cytosolic distribution. Mutagenesis of the basic amino acids inside the NTD clusters A ¹¹⁶KSKRKKKNKK¹²⁵ and B ¹⁷⁵KKATKKESKKQTK¹⁸⁷ reveals that these clusters are indispensable for nuclear and nucleolar targeting of PSLV TGBp1 and may contain nuclear and nucleolar localization signals or their elements. The PSLV TGBp1 is able to bind to fibrillarin, the major nucleolar protein (AtFib2 from Arabidopsis thaliana) in vitro. This protein–protein interaction occurs between the glycine-arginine-rich (GAR) domain of fibrillarin and the first 82 amino acid residues of TGBp1. The interaction of TGBp1 with fibrillarin is also visualized in vivo by bimolecular fluorescence complementation (BiFC) during co-expression of TGBp1 or its deletion mutants, and fibrillarin as fusions to different halves of YFP in N. benthamiana plants. The sites responsible for nuclear/nucleolar localization and fibrillarin binding, have been located within the intrinsically disordered TGBp1 NTD. These data could suggest that specific functions of hordeivirus TGBp1 may depend on its interaction with nucleolar components.     

Interpreting evidence: why values can matter as much as science

Despite increasing awareness of the ways in which non-epistemic values play roles in science, many scientists remain reluctant to acknowledge values at stake in their own work. Even when research clearly relates to risk assessment and establishing public policy, contexts in which the presence of values is less likely to be contentious, scientists tend to present such research as merely involving empirical questions about what the evidence is. As a result, debates over policy-related science tend to be framed as purely epistemic debates over the state of the evidence. We argue that this neglects the important ways that ethical and social values play legitimate roles in judgments about what we take to be evidence for a particular policy. Using the case of recent disputes about the relative safety of home birth, we argue that although the debate has been framed as a purely scientific one about the empirical evidence for home birth, it actually involves disagreements about underlying value assumptions. If our claims are correct, then in order to move the debate forward, scientists will need to engage in a critical discussion about the values at stake.     

Saliva samples are a viable alternative to blood samples as a source of DNA for high throughput genotyping

ABSTRACT: BACKGROUND: The increasing trend for incorporation of biological sample collection within clinical trials requires sample collection procedures which are convenient and acceptable for both patients and clinicians. This study investigated the feasibility of using saliva-extracted DNA in comparison to blood-derived DNA, across two genotyping platforms: Applied Biosystems Taqman TM and Illumina Beadchip TM genome-wide arrays. METHOD: Patients were recruited from the Pharmacogenetics of Breast Cancer Chemotherapy (PGSNPS) study. Paired blood and saliva samples were collected from 79 study participants. The Oragene DNA Self-Collection kit (DNAgenotek(R)) was used to collect and extract DNA from saliva. DNA from EDTA blood samples (median volume 8 ml) was extracted by GenProbe, Livingstone, UK. DNA yields, standard measures of DNA quality, genotype call rates and genotype concordance between paired, duplicated samples were assessed. RESULTS: Total DNA yields were lower from saliva (mean 24 ug, range 0.2-52 ug) than from blood (mean 210 ug, range 58-577 ug) and a 2-fold difference remained after adjusting for the volume of biological material collected. Protein contamination and DNA fragmentation measures were greater in saliva DNA. 78/79 saliva samples yielded sufficient DNA for use on Illumina Beadchip arrays and using Taqman assays. Four samples were randomly selected for genotyping in duplicate on the Illumina Beadchip arrays. All samples were genotyped using Taqman assays. DNA quality, as assessed by genotype call rates and genotype concordance between matched pairs of DNA was high (>97%) for each measure in both blood and saliva-derived DNA. CONCLUSION: We conclude that DNA from saliva and blood samples is comparable when genotyping using either Taqman assays or genome-wide chip arrays. Saliva sampling has the potential to increase participant recruitment within clinical trials, as well as reducing the resources and organisation required for multicentre sample collection.     

Exploring the pan-surfome of Streptococcus suis: Looking for common protein antigens

Streptococcus suis is a swine and human pathogen for which no commercial vaccine is still available. Conserved and broadly distributed surface proteins have become the chosen targets for the development of efficacious vaccines that could overcome the problems of non-heterologous protection of bacterins or capsule polysaccharide-based vaccines. In this work, we have analyzed by proteomics a collection of 39 strains obtained from infected pigs. The isolates belonged to 19 of the most prevalent serotypes during the last years. We have applied the "shaving" approach to define the "pan-surfome" or the set of both common and unique surface proteins identified in such strains. This set was constituted by 113 proteins. We have categorized them for their potential for further vaccination studies, based on their distribution among strains and their a priori accessibility to antibodies. According to these criteria, the cell-wall protein SsnA appears to be the best candidate from this list, as it was that with the widest distribution among the analyzed pathogen types, it showed to be highly immunogenic and highly accessible to antibodies, as demonstrated by flow cytometry.     

The retinoic acid receptor-related orphan nuclear receptor γ1 (RORγ1): a novel player determinant of insulin sensitivity in morbid obesity

The orphan nuclear receptors (ONRs), retinoic acid receptor-related orphan receptor γ-1 (RORγ1) and peroxisome proliferator-activated receptor γ-2 (PPARγ2), are central mediators controlling adipocyte (AD) differentiation. Through their distinct tissue distribution and specific target gene activation, ONRs control diverse aspects of fatty acid metabolism and insulin sensitivity. Adding further complexity, obesity begets resistance to insulin signals and can ultimately result in diabetes. In this study, we investigate whether there are differences in the RORγ1 and PPARγ2 expression in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) from morbid obesity (MO) individuals either insulin resistant (high-IR MO) or insulin sensitivity (low-IR MO). Our results indicate for the first time in human the RORγ1 mRNA and protein expression levels and activation with coactivator, such as peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) were higher in the VAT from high-IR MO. In contrast, PPARγ2 expression and activation were higher in the VAT from low-IR MO. In this way, we have also found a positive association between RORγ1 mRNA and protein expression with many components of metabolic syndrome, with a strong dependence of insulin and HOMA(IR) index in VAT, but not in SAT. Our data suggest that RORγ1 may be added to the growing list of nuclear receptors in adipose tissue use to modulate the insulin resistance associated to the obesity. Measurement of RORγ1 and PPARγ2 in adipose tissue might be useful for evaluating the outcomes of various clinical interventions for obesity-related diabetes type II.     

Genetic analysis of the individual contribution to virulence of the type III effector inventory of Pseudomonas syringae pv. phaseolicola

Several reports have recently contributed to determine the effector inventory of the sequenced strain Pseudomonas syringae pv. phaseolicola (Pph) 1448a. However, the contribution to virulence of most of these effectors remains to be established. Genetic analysis of the contribution to virulence of individual P. syringae effectors has been traditionally hindered by the lack of phenotypes of the corresponding knockout mutants, largely attributed to a high degree of functional redundancy within their effector inventories. In support of this notion, effectors from Pseudomonas syringae pv. tomato (Pto) DC3000 have been classified into redundant effector groups (REGs), analysing virulence of polymutants in the model plant Nicotiana benthamiana. However, using competitive index (CI) as a virulence assay, we were able to establish the individual contribution of AvrPto1(Pto) (DC3000) to Pto DC3000 virulence in tomato, its natural host, even though typically, contribution to virulence of AvrPto1 is only shown in strains also lacking AvrPtoB (also called HopAB2), a member of its REG. This report raised the possibility that even effectors targeting the same defence signalling pathway may have an individual contribution to virulence, and pointed out to CI assays as the means to establish such a contribution for individual effectors. In this work, we have analysed the individual contribution to virulence of the majority of previously uncharacterised Pph 1448a effectors, by monitoring the development of disease symptoms and determining the CI of single knockout mutants at different stages of growth within bean, its natural host. Despite their potential functional redundancy, we have found individual contributions to virulence for six out of the fifteen effectors analysed. In addition, we have analysed the functional relationships between effectors displaying individual contribution to virulence, highlighting the diversity that these relationships may present, and the interest of analysing their functions within the context of the infection.     

Ih current is necessary to maintain normal dopamine fluctuations and sleep consolidation in Drosophila

HCN channels are becoming pharmacological targets mainly in cardiac diseases. But apart from their well-known role in heart pacemaking, these channels are widely expressed in the nervous system where they contribute to the neuron firing pattern. Consequently, abolishing Ih current might have detrimental consequences in a big repertoire of behavioral traits. Several studies in mammals have identified the Ih current as an important determinant of the firing activity of dopaminergic neurons, and recent evidences link alterations in this current to various dopamine-related disorders. We used the model organism Drosophila melanogaster to investigate how lack of Ih current affects dopamine levels and the behavioral consequences in the sleep:activity pattern. Unlike mammals, in Drosophila there is only one gene encoding HCN channels. We generated a deficiency of the DmIh core gene region and measured, by HPLC, levels of dopamine. Our data demonstrate daily variations of dopamine in wild-type fly heads. Lack of Ih current dramatically alters dopamine pattern, but different mechanisms seem to operate during light and dark conditions. Behaviorally, DmIh mutant flies display alterations in the rest:activity pattern, and altered circadian rhythms. Our data strongly suggest that Ih current is necessary to prevent dopamine overproduction at dark, while light input allows cycling of dopamine in an Ih current dependent manner. Moreover, lack of Ih current results in behavioral defects that are consistent with altered dopamine levels.     

RNA binding is more critical to the suppression of silencing function of Cucumber mosaic virus 2b protein than nuclear localization

Previously, we found that silencing suppression by the 2b protein and six mutants correlated both with their ability to bind to double-stranded (ds) small RNAs (sRNAs) in vitro and with their nuclear/nucleolar localization. To further discern the contribution to suppression activity of sRNA binding and of nuclear localization, we have characterized the kinetics of in vitro binding to a ds sRNA, a single-stranded (ss) sRNA, and a micro RNA (miRNA) of the native 2b protein and eight mutant variants. We have also added a nuclear export signal (NES) to the 2b protein and assessed how it affected subcellular distribution and suppressor activity. We found that in solution native protein bound ds siRNA, miRNA, and ss sRNA with high affinity, at protein:RNA molar ratios ~2:1. Of the four mutants that retained suppressor activity, three showed sRNA binding profiles similar to those of the native protein, whereas the remaining one bound ss sRNA at a 2:1 molar ratio, but both ds sRNAs with 1.5-2 times slightly lower affinity. Three of the four mutants lacking suppressor activity failed to bind to any sRNA, whereas the remaining one bound them at far higher ratios. NES-tagged 2b protein became cytoplasmic, but suppression activity in patch assays remained unaffected. These results support binding to sRNAs at molar ratios at or near 2:1 as critical to the suppressor activity of the 2b protein. They also show that cytoplasmically localized 2b protein retained suppressor activity, and that a sustained nuclear localization was not required for this function.     

Monitoring the occurrence of indoor fungi in a hospital

BackgroundThere is a lack of standardized protocols for assessing the presence of indoor fungi. It is thus difficult to compare results from different studies or to measure the effect of indoor fungal presence on occupants.AimsThe aim of the present work was to evaluate the presence of airborne fungal propagules within a hospital taking into account the influence of environmental factors.MethodsThe study was conducted in a hospital over a period of two years. Two portable aerobiological samplers were used: one capturing propagules onto a sticky surface, and the other onto a culture medium consisting of Sabouraud dextrose agar in Petri dishes, supplemented with chloramphenicol. Sampling was performed indoors at four sites (two on the ground floor and two on the third floor, each consisting of an open ward and a closed room). Samples were also taken outdoors. The following factors were considered for fungus occurrence: season, weather conditions, number of people present in the wards, the insulation of the indoor sites and the existence of construction works on the two floors. We carried out 60 ten-minute samples, weekly during the spring (24 samples), and fortnightly for the rest of the year (36 samples).ResultsA total of 2456 colony forming units (CFU) were obtained, with mean propagule concentrations of 107 CFU/m³ outdoors and 24 CFU/m³ indoors. 35330 counts were recorded for propagules. The mean concentrations were 2473 propagules/m³ outdoors and 790 indoors. A statistically significant positive correlation was found between the number of people in one of the wards and fungus occurrence, and the occurrence in both ground floor and third floor rooms was positively correlated with outdoor levels. These showed a seasonal pattern with peaks in summer. Indoors, however, the peaks appeared in spring and autumn. Outdoor construction activities affected the propagule loads but not the number of CFU.ConclusionsThe indoor fungus occurrence in the hospital was independent of meteorological conditions and of insulation from outside of the indoor sites selected, but was correlated with the season and number of people in the third floor ward. Outdoor construction activities affected values of indoor propagules, although seasonality could mask their effect.