Psoralen and ultraviolet A light treatment directly affects phosphatidylinositol 3-kinase signal transduction by altering plasma membrane packing

Psoralen and ultraviolet A light (PUNTA) are used to kill pathogens in blood products and as a treatment of aberrant cell proliferation in dermatitis, cutaneous T-cell lymphoma, and graft versus-host disease. DNA damage is well described, but the direct effects of PUVA on cell signal transduction are poorly understood. Because platelets are anucleate and contain archetypal signal transduction machinery, they are ideally suited to address this. Lipidomics on platelet membrane extracts showed that psoralen forms adducts with unsaturated carbon bonds of fatty acyls in all major phospholipid classes after PUVA. Such adducts increased lipid packing as measured by a blue shift of an environment-sensitive fluorescent probe in model liposomes. Furthermore, the interaction of these liposomes with lipid order-sensitive proteins like amphipathic lipid-packing sensor and a-synuclein was inhibited by PUVA. In platelets, PUVA caused poor membrane binding of Akt and Bruton's tyrosine kinase effectors following activation of the collagen glycoprotein VI and thrombin protease-activated receptor (PAR) 1. This resulted in defective Akt phosphorylation despite unaltered phosphatidylinositol 3,4,5-trisphosphate levels. Downstream integrin activation was furthermore affected similarly by PUVA following PAR1 (effective half-maximal concentration (EC), 8.4 +/- 1.1 versus 4.3 +/- 1.1 mu M) and glycoprotein VI (EC50, 1.61 +/- 0.85 versus 0.26 +/- 0.21 mu g/ml) but not PAR4 (EC50, 50 +/- 1 versus 58 +/- 1 mu m) signal transduction. Our findings were confirmed in T-cells ftom graft-versus-host disease patients treated with extracorporeal photopheresis, a form of systemic PUVA. In conclusion, PUVA increases the order of lipid phases by covalent modification of phospholipids, thereby inhibiting membrane recruitment of effector kinases

Evaluation of automated sample preparation, retention time locked GC-MS and automated data analysis for the metabolomic study of Arabidopsis species

One of the most critical parameters in GC-MS application to metabolimic studies is the sample preparation. Often a combination of oximation and silylation is used and this method is applicable to acids (e.g. fatty acids), sterols, amines, amino acids, sugars, etc. Automation of the derivatisation process enhances repeatability and the time between sample preparation and analysis can be kept short and constant. For analysis, retention time locked (RTL) gas chromatography/mass spectrometry was used. For data analysis, AMDIS deconvolution followed by 2 multivariate statistical approaches were used and compared. Arabidopsis thaliana, a popular model organism in plant biology and genetics, was used for testing. The data analysis software allowed the detection of several features that were up- or downregulated in the different species

nsP4 is a major determinant of Alphavirus replicase activity and template selectivity

Alphaviruses have positive-strand RNA genomes containing two open reading frames (ORFs). The first ORF encodes the nonstructural (ns) polyproteins P123 and P1234 that act as precursors for the subunits of the viral RNA replicase (nsP1 to nsP4). Processing of P1234 leads to the formation of a negative-strand replicase consisting of nsP4 (RNA polymerase) and P123 components. Subsequent processing of P123 results in a positive-strand replicase. The second ORF encoding the structural proteins is expressed via the synthesis of a subgenomic RNA. Alphavirus replicase is capable of using template RNAs that contain essential cis-active sequences. Here, we demonstrate that the replicases of nine alphaviruses, expressed in the form of separate P123 and nsP4 components, are active. Their activity depends on the abundance of nsP4. The match of nsP4 to its template strongly influences efficient subgenomic RNA synthesis. nsP4 of Barmah Forest virus (BFV) formed a functional replicase only with matching P123, while nsP4s of other alphaviruses were compatible also with several heterologous P123s. The P123 components of Venezuelan equine encephalitis virus and Sindbis virus (SINV) required matching nsP4s, while P123 of other viruses could form active replicases with different nsP4s. Chimeras of Semliki Forest virus, harboring the nsP4 of chikungunya virus, Ross River virus, BFV, or SINV were viable. In contrast, chimeras of SINV, harboring an nsP4 from different alphaviruses, exhibited a temperature-sensitive phenotype. These findings highlight the possibility for formation of new alphaviruses via recombination events and provide a novel approach for the development of attenuated chimeric viruses for vaccination strategies. </p

A Radial Velocity Study of Composite-Spectra Hot Subdwarf Stars with the Hobby-Eberly Telescope

Many hot subdwarf stars show composite spectral energy distributions indicative of cool main sequence companions. Binary population synthesis (BPS) models demonstrate such systems can be formed via Roche lobe overflow or common envelope evolution but disagree on whether the resulting orbital periods will be long (years) or short (days). Few studies have been carried out to assess the orbital parameters of these spectroscopic composite binaries; current observations suggest the periods are long. To help address this problem, we selected fifteen moderately-bright (V~13) hot subdwarfs with F-K dwarf companions and monitored their radial velocities (RVs) from January 2005 to July 2008 using the bench-mounted Medium Resolution Spectrograph on the Hobby-Eberly Telescope (HET). Here we describe the details of our observing, reduction, and analysis techniques and present preliminary results for all targets. By combining the HET data with recent observations from the Mercator telescope, we are able to calculate precise orbital solutions for three systems using more than 6 years of observations. We also present an up-to-date period histogram for all known hot subdwarf binaries, which suggests those with F-K main sequence companions tend to have orbital periods on the order of several years. Such long periods challenge the predictions of conventional BPS models, although a larger sample is needed for a thorough assessment of the models' predictive success. Lastly, one of our targets has an eccentric orbit, implying some composite-spectrum systems might have formerly been hierarchical triple systems, in which the inner binary merged to create the hot subdwarf.Comment: Published in The Astrophysical Journal, Volume 758, Issue 1, article id. 58 (2012). References updated and Equation (5) corrected. 12 pages, 5 figures, 5 table

Cross-utilisation of template RNAs by alphavirus replicases

Most alphaviruses (family Togaviridae) including Sindbis virus (SINV) and other human pathogens, are transmitted by arthropods. The first open reading frame in their positive strand RNA genome encodes for the non-structural polyprotein, a precursor to four separate subunits of the replicase. The replicase interacts with cis-acting elements located near the intergenic region and at the ends of the viral RNA genome. A trans-replication assay was developed and used to analyse the template requirements for nine alphavirus replicases. Replicases of alphaviruses of the Semliki Forest virus complex were able to cross-utilize each other's templates as well as those of outgroup alphaviruses. Templates of outgroup alphaviruses, including SINV and the mosquito-specific Eilat virus, were promiscuous; in contrast, their replicases displayed a limited capacity to use heterologous templates, especially in mosquito cells. The determinants important for efficient replication of template RNA were mapped to the 5' region of the genome. For SINV these include the extreme 5'- end of the genome and sequences corresponding to the first stem-loop structure in the 5' untranslated region. Mutations introduced in these elements drastically reduced infectivity of recombinant SINV genomes. The trans-replicase tools and approaches developed here can be instrumental in studying alphavirus recombination and evolution, but can also be applied to study other viruses such as picornaviruses, flaviviruses and coronaviruses.</p

The acquisitive–conservative axis of leaf trait variation emerges even in homogeneous environments

The acquisitive-conservative axis of plant ecological strategies results in a pattern of leaf trait covariation that captures the balance between leaf construction costs and plant growth potential. Studies evaluating trait covariation within species are scarcer, and have mostly dealt with variation in response to environmental gradients. Little work has been published on intraspecific patterns of leaf trait covariation in the absence of strong environmental variation.Methods: We analysed covariation of four leaf functional traits (SLA: specific leaf area, LDMC: leaf dry matter content, Ft: force to tear, and Nm: leaf nitrogen content) in six Poaceae and four Fabaceae species common in the dry Chaco forest of Central Argentina, growing in the field and in a common garden. We compared intraspecific covariation patterns (slopes, correlation and effect size) of leaf functional traits with global interspecific covariation patterns. Additionally, we checked for possible climatic and edaphic factors that could affect the intraspecific covariation pattern.Key Results: We found negative correlations for the LDMC-SLA, Ft-SLA, LDMC-Nm , and Ft-Nm trait pairs. This intraspecific covariation pattern found both in the field and in the common garden and not be explained by climatic or edaphic variation in the field follows the expected acquisitive-conservative axis. At the same time, we found quantitative differences in slopes among different species, and between these intraspecific patterns and the interspecific ones. Many of these differences seem to be idiosyncratic, but some appear consistent among species (e.g.all the intraspecific LDMC-SLA and LDMC-Nm slopes tend to be shallower than the global).Conclusions: Our study indicates that the acquisitive-conservative leaf functional trait covariation pattern occurs at the intraspecific level even in the absence of relevant environmental variation in the field. This suggests a high degree of variation-covariation in leaf functional traits not driven by environmental variables.Fil: Gorne, Lucas Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales; ArgentinaFil: Díaz, Sandra Myrna. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales; ArgentinaFil: Minden, Vanessa. University of Oldenburg; Alemania. Vrije Universiteit Brussel; BélgicaFil: Onoda, Yusuke. Kyoto University. School of Agriculture; JapónFil: Kramer, Koen. Wageningen University; Países BajosFil: Muir, Christopher. University Of Hawaii; Estados UnidosFil: Michaletz, Sean T. University of British Columbia; CanadáFil: Lavorel, Sandra. Centre National de la Recherche Scientifique; FranciaFil: Sharpe, Joanne. Sharplex Services, Edgecomb; Estados UnidosFil: Jansen, Steven. Universitat Ulm; AlemaniaFil: Slot, Martijn. Smithsonian Tropical Research Institute; PanamáFil: Chacon, Maximiliano Eduardo. Universidad de Costa Rica; Costa RicaFil: Boenisch, Gerhard. Max Planck Institute For Biogeochemistry; Alemani

Reflections as a settler person doing research in collaboration with Indigenous peoples.

Objectives To reflect on my position as a settler person in Australia and ensure Indigenous voices are prioritised throughout my research, as part of a co-designed, Aboriginal-led study which aims to understand trends in the removal of Indigenous children born in Western Australia using data linkage and qualitative research. Approach As a non-Indigenous person, it is important to reflect on my cultural background and acknowledge my limited understanding of the cultural context of the Indigenous communities represented in the data. Listening to Indigenous voices and collaborating with Indigenous peoples at all stages of my research – from my PhD supervisor to investigators on the broader study, to members of the community and policy reference groups – will be key to improve my understanding of the data from a system and context I am unfamiliar with. Results Collaboration has been cyclical, with results from the qualitative research and discussion with the reference groups informing the initial quantitative research direction. Findings from this research were presented back to the groups, resulting in further questions and directions to explore. The journey so far has been one of learning and understanding the skills I have and the role they can play whilst acknowledging the limits of my own knowledge and the need for Indigenous voices to guide the research in order to be doing research with Indigenous peoples, rather than on them. Conclusion Co-design with Indigenous peoples is critical for doing research which affects them or uses data from their communities. Understanding my own cultural background and acknowledging the limitations of my experience continues to be important for honest and meaningful collaboration

Cumulative incidence of child protection system contacts among a cohort of Western Australian Aboriginal children born 2000 to 2013

Background: Reducing the over-representation of Aboriginal children in the child protection system is a key target for the Australian government. Objective: We aimed to provide more recent evidence on the population-level cumulative incidence of contacts for Aboriginal children with child protective services (CPS) in Western Australia (WA). Participants and Setting: Linked administrative data was provided for WA CPS between 2000 and 2015 for 33,709 Aboriginal children born in WA between 2000 and 2013. Methods: Descriptive summaries and cumulative incidence estimates were used to examine changes in CPS contact trends over time and within sibling groups. Results: There was an increase in early-childhood contacts for children born more recently, with 7.6 % and 2.3 % of children born in 2000–2001 having a notification and placement in out-of-home care by age one, respectively, compared to 15.1 % and 4.3 % of children born in 2012–2013. Among sibling groups where at least one sibling had a CPS contact, approximately half of children had their first contacts on the same date as another sibling. For children born after one of their siblings had been placed in out-of-home care, 31.9 % had themselves been placed in out-of-home care by age one. Conclusions: Multiple children tend to be placed into out-of-home care when at least one sibling is, which is likely to have a significant impact on families affected. The additional risk of placement also carries over to children born after the first removal in a sibling group, highlighting the need for further support to prevent future removals