Comprehensive protein interactome analysis of a key RNA helicase: detection of novel stress granule proteins

DDX6 (p54/RCK) is a human RNA helicase with central roles in mRNA decay and translation repression. To help our understanding of how DDX6 performs these multiple functions, we conducted the first unbiased, large-scale study to map the DDX6-centric protein-protein interactome using immunoprecipitation and mass spectrometry. Using DDX6 as bait, we identify a high-confidence and high-quality set of protein interaction partners which are enriched for functions in RNA metabolism and ribosomal proteins. The screen is highly specific, maximizing the number of true positives, as demonstrated by the validation of 81% (47/58) of the RNA-independent interactors through known functions and interactions. Importantly, we minimize the number of indirect interaction partners through use of a nuclease-based digestion to eliminate RNA. We describe eleven new interactors, including proteins involved in splicing which is an as-yet unknown role for DDX6. We validated and characterized in more detail the interaction of DDX6 with Nuclear fragile X mental retardation-interacting protein 2 (NUFIP2) and with two previously uncharacterized proteins, FAM195A and FAM195B (here referred to as granulin-1 and granulin-2, or GRAN1 and GRAN2). We show that NUFIP2, GRAN1, and GRAN2 are not P-body components, but re-localize to stress granules upon exposure to stress, suggesting a function in translation repression in the cellular stress response. Using a complementary analysis that resolved DDX6's multiple complex memberships, we further validated these interaction partners and the presence of splicing factors. As DDX6 also interacts with the E3 SUMO ligase TIF1β, we tested for and observed a significant enrichment of sumoylation amongst DDX6's interaction partners. Our results represent the most comprehensive screen for direct interaction partners of a key regulator of RNA life cycle and localization, highlighting new stress granule components and possible DDX6 functions-many of which are likely conserved across eukaryotes

ADVANCED COMPUTATIONAL ANALYSIS OF DISORDERED MATERIALS AND CLAY MINERALS

Final report describing a three-year research project applying various methods to study the structures of crystalline solids and also apply to polycrystalline disordered materials

Mineralogical In-situ Investigation of Acid-Sulfate Samples from the Rio Tinto River, Spain, with a Portable XRD/XRF Instrument

A field campaign was organized in September 2006 by Centro de Astobiologica (Spain) and Washington University (St Louis, USA) for the geological study of the Rio Tinto river bed sediments using a suite of in-situ instruments comprising an ASD reflectance spectrometer, an emission spectrometer, panoramic and close-up color imaging cameras, a life detection system and NASA's CheMin 4 XRD/XRF prototype. The primary objectives of the field campaign were to study the geology of the site and test the potential of the instrument suite in an astrobiological investigation context for future Mars surface robotic missions. The results of the overall campaign will be presented elsewhere. This paper focuses on the results of the XRD/XRF instrument deployment. The specific objectives of the CheMin 4 prototype in Rio Tinto were to 1) characterize the mineralogy of efflorescent salts in their native environments; 2) analyze the mineralogy of salts and oxides from the modern environment to terraces formed earlier as part of the Rio Tinto evaporative system; and 3) map the transition from hematite-dominated terraces to the mixed goethite/salt-bearing terraces where biosignatures are best preserved

Systemic Myostatin Inhibition via Liver-Targeted Gene Transfer in Normal and Dystrophic Mice

Background: Myostatin inhibition is a promising therapeutic strategy to maintain muscle mass in a variety of disorders, including the muscular dystrophies, cachexia, and sarcopenia. Previously described approaches to blocking myostatin signaling include injection delivery of inhibitory propeptide domain or neutralizing antibodies. Methodology/Principal Findings: Here we describe a unique method of myostatin inhibition utilizing recombinant adenoassociated virus to overexpress a secretable dominant negative myostatin exclusively in the liver of mice. Systemic myostatin inhibition led to increased skeletal muscle mass and strength in control C57 Bl/6 mice and in the dystrophindeficient mdx model of Duchenne muscular dystrophy. The mdx soleus, a mouse muscle more representative of human fiber type composition, demonstrated the most profound improvement in force production and a shift toward faster myosin-heavy chain isoforms. Unexpectedly, the 11-month-old mdx diaphragm was not rescued by long-term myostatin inhibition. Further, mdx mice treated for 11 months exhibited cardiac hypertrophy and impaired function in an inhibitor dose–dependent manner. Conclusions/Significance: Liver-targeted gene transfer of a myostatin inhibitor is a valuable tool for preclinical investigation of myostatin blockade and provides novel insights into the long-term effects and shortcomings of myostatin inhibition o

“Some men deeply hate women, and express that hatred freely”: examining victims’ experiences and perceptions of gendered hate crime

Extensive debate about the place of gender within the hate crime policy domain has been fuelled by national victimisation surveys indicating people’s experiences of ‘gender hate crime’ coupled with Nottinghamshire Police’s decision to begin categorising misogynistic street harassment as a form of hate crime. Drawing on the results of an online survey of 85 respondents, this article explores people’s experiences of gender-related victimisation as ‘hate crimes’. The analysis demonstrates how participants relate their experiences to the hate crime concept, their perceptions on punishment and reporting to the police, and also wider impacts on their recovery processes. This paper provides a timely contribution towards current debates around using the existing hate crime model for addressing crimes motivated by gender hostility

Test and Delivery of the Chemin Mineralogical Instrument for Mars Science Laboratory

The CheMin mineralogical instrument on MSL will return quantitative powder X-ray diffraction data (XRD) and qualitative X-ray fluorescence data (XRF; 14<Z<92) from scooped soil samples and drilled rock powders collected on the Mars surface. The geometry of the source, sample, and detector is shown. A transmission geometry was chosen so that diffracted intensities in the low-20 region (5-15 deg), important for phyllosilicate identification, could be detected

Needs and opportunities in mineral evolution research

Progress in understanding mineral evolution, Earth’s changing near-surface mineralogy through time, depends on the availability of detailed information on mineral localities of known ages and geologic settings. A comprehensive database including this information, employing the mindat.org web site as a platform, is now being implemented. This resource will incorporate software to correlate a range of mineral occurrences and properties vs. time, and it will thus facilitate studies of the chang- ing diversity, distribution, associations, and characteristics of individual minerals as well as mineral groups. The Mineral Evolution Database thus holds the prospect of revealing mineralogical records of important geophysical, geochemical, and biological events in Earth history.Organismic and Evolutionary Biolog

Analysis of Organic Molecules Extracted from Mars Analogues and Influence of Their Mineralogy Using N-Methyl-N-(tert-butyldimethylsilyl)Trifluoroacetamide Derivatization Coupled with Gas Chromatography Mass Spectrometry in Preparation for the Sample Analysis at Mars Derivatization Experiment on the Mars Science Laboratory Mission

The search for complex organic molecules on Mars, including important biomolecules such as amino acids and carboxylic acids will require a chemical extraction and derivatization step to transform these organic compounds into species that are sufficiently volatile to be detected by gas chromatography mass spectrometry (GCMS). We have developed, a one-pot extraction and chemical derivatization protocol using N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) and dimethylformamide (DMF) for the Sample Analysis at Mars (SAM) experiment on the Mars Science Laboratory (MSL). The temperature and duration the derivatization reaction, pre-concentration of chemical derivatives, and gas chromatographic separation parameters have been optimized under SAM instrument design constraints. MTBSTFA/DMF extraction and derivatization at 300 C for several minutes of a variety of terrestrial Mars analogue materials facilitated the detection of amino acids and carboxylic acids in a surface soil sample collected from the Atacama Desert and a carbonate-rich stromatolite sample from Svalbard. However, the rapid reaction of MTBSTFA with water in several analogue materials that contained high abundances of hydrated minerals and the possible deactivation of derivatized compounds by iron oxides, as detected by XRD/XRF using the CheMin field unit Terra, proved to be highly problematic for the direct extraction of organics using MTBSTFA, The combination of pyrolysis and two different chemical derivatization methods employed by SAM should enable a wide range of organic compounds to be detected by GCMS if present on Mars

Dietary elimination of children with food protein induced gastrointestinal allergy – micronutrient adequacy with and without a hypoallergenic formula?

Background: The cornerstone for management of Food protein-induced gastrointestinal allergy (FPGIA) is dietary exclusion; however the micronutrient intake of this population has been poorly studied. We set out to determine the dietary intake of children on an elimination diet for this food allergy and hypothesised that the type of elimination diet and the presence of a hypoallergenic formula (HF) significantly impacts on micronutrient intake. Method: A prospective observational study was conducted on children diagnosed with FPIGA on an exclusion diet who completed a 3 day semi-quantitative food diary 4 weeks after commencing the diet. Nutritional intake where HF was used was compared to those without HF, with or without a vitamin and mineral supplement (VMS). Results: One-hundred-and-five food diaries were included in the data analysis: 70 boys (66.7%) with median age of 21.8 months [IQR: 10 - 67.7]. Fifty-three children (50.5%) consumed a HF and the volume of consumption was correlated to micronutrient intake. Significantly (p <0.05) more children reached their micronutrient requirements if a HF was consumed. In those without a HF, some continued not to achieve requirements in particular for vitamin D and zinc, in spite of VMS. Conclusion: This study points towards the important micronutrient contribution of a HF in children with FPIGA. Children, who are not on a HF and without a VMS, are at increased risk of low intakes in particular vitamin D and zinc. Further studies need to be performed, to assess whether dietary intake translates into actual biological deficiencies