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

A Cost Analysis of the 1-2-3 Pap Intervention

Background: Cervical cancer places a substantial economic burden on our healthcare system. The three-dose human papillomavirus (HPV) vaccine series is a cost-effective intervention to prevent HPV infection and resultant cervical cancer. Despite its efficacy, completion rates are low in young women aged 18 through 26 years. 1-2-3 Pap is a video intervention tested and proven to increase HPV vaccination completion rates. Purpose: To provide the full scope of available evidence for 1-2-3 Pap, this study adds economic evidence to the intervention’s efficacy. This study tested the economies of scale hypothesis that the cost of 1-2-3 Pap intervention per number of completed HPV vaccine series would decrease when offered to more women in the target population. Methods: Using cost and efficacy data from the Rural Cancer Prevention Center, a cost analysis was done through a hypothetical adaptation scenario in rural Kentucky. Results: Assuming the same success rate as in the efficacy study, the 1-2-3 Pap adaptation scenario would cover 1000 additional women aged 18 through 26 years (344 in efficacy study; 1346 in adaptation scenario), and almost three times as many completed series (130 in efficacy study; 412 in adaptation scenario) as in the original 1-2-3 Pap efficacy study. Implications: Determination of the costs of implementing 1-2-3 Pap is vital for program expansion. This study provides practitioners and decision makers with objective measures for scalability

Adeno-Assocated Virus (AAV) Serotype 9 Provides Global Cardiac Gene Transfer Superior to AAV1, AAV6, AAV7, and AAV8 in the Mouse and Rat

Heart disease is the leading cause of morbidity and mortality. Cardiac gene transfer may serve as a novel therapeutic approach. This investigation was undertaken to compare cardiac tropisms of adeno-associated virus (AAV) serotypes 1, 6, 7, 8, and 9. Neonatal mice were injected with 2.5 × 1011 genome copies (GC) of AAV serotype 1, 6, 7, 8, or 9 expressing LacZ under the control of the constitutive chicken β-actin promoter with cytomegalovirus enhancer promoter via intrapericardial injection and monitored for up to 1 year. Adult rats were injected with 5 × 1011 GC of the AAV vectors via direct cardiac injection and monitored for 1 month. Cardiac distribution of LacZ expression was assessed by X-Gal histochemistry, and β-galactosidase activity was quantified in a chemiluminescence assay. Cardiac functional data and biodistribution data were also collected in the rat. AAV9 provided global cardiac gene transfer stable for up to 1 year that was superior to other serotypes. LacZ expression was relatively cardiac specific, and cardiac function was unaffected by gene transfer. AAV9 provides high-level, stable expression in the mouse and rat heart and may provide a simple alternative to the creation of cardiac-specific transgenic mice. AAV9 should be used in rodent cardiac studies and may be the vector of choice for clinical trials of cardiac gene transfer

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

Tissue response to dental implants with platform switching

La evolución en los diseños de los sistemas de implantes y en la configuración de los pilares protési- cos ha desarrollado el concepto de plataforma reducida que comprende la colocación de un pilar más estrecho que la plataforma del implante para aumentar su distancia de la interfase hueso-implante. La plataforma reducida es considerada un factor importante para preservar la estabilidad del hueso crestal y de los tejidos blandos y asegurar el éxito de los implantes dentales a largo plazo. La plataforma redu- cida reduce las fuerzas oclusales y la contaminación bacteriana en la interfase entre el hueso crestal y el implante. Los estudios experimentales en animales y clínicos en pacientes muestran su eficacia para prevenir la pérdida ósea y de los tejidos blandos periimplantarios Conclusiones. La introducción de la plataforma reducida constituye un importante campo en la investi- gación experimental en implantología oral y en el tratamiento clínico con implantes para mantener los tejidos periimplantarios.The evolution of designs of implant systems and abutments configurations has developed the concept of platform-switching that involves the connection of a narrower abutment to the platform implant to allow horizontal distance of the interface bone-implant. Platform-switching is considered an important factor to preserve the stability of crestal bone and soft tissue ensuring the success of dental implants in the long-term follow-up. Platform-switching reduces the forces of occlusal loading and bacterial conta- mination in the interface between the crestal bone and the implant. Experimental studies in animals and clinical studies in patients showed that implants with platform-switching have demonstrated the effecti- veness to prevent peri-implant bone loss and subsequent soft tissue loss. Conclusions. The platform-switching constitute an important field for research of experimental implant dentistry and clinical implant treatment in the maintenance of peri-implant tissue

Transcendocardinal Delivery of AAV6 Results in Highly Efficient and Global Cardiac Gene Transfer in Rhesus Macaques

Heart disease is the leading cause of morbidity and mortality, and cardiac gene transfer has potential as a novel therapeutic approach. We previously demonstrated safe and efficient gene transfer to the canine heart using a percutaneous transendocardial injection procedure to deliver self-complementary (sc) adeno-associated virus 6 (AAV6) vector. In the present study, we proceed with our vertical translation study to evaluate cardiac gene transfer in nonhuman primates (NHPs). We screened approximately 30 adult male rhesus macaques for the presence of neutralizing antibodies against AAV6, AAV8, and AAV9, and then selected seven monkeys whose antibody titers against these three serotypes were lower than 1/5. The animals were then randomized to receive either scAAV6 (n =3), scAAV8 (n =1), or scAAV9 (n =3) vector expressing the enhanced green fluorescent protein (EGFP) reporter gene at a dose of 5.4×1012 genome copies/kg, which was administered according to a modified version of our previously developed transendocardial injection procedure. One animal treated with scAAV6 died secondary to esophageal intubation. The remaining animals were euthanized 7 days after gene transfer, at which time tissue was collected for analysis of EGFP expression, histopathology, and biodistribution of the vector genome. We found that (i) transendocardial delivery of AAV is safe in the NHP, (ii) AAV6 and AAV8 provide efficient cardiac gene transfer at similar levels and are superior to AAV9, and (iii) AAV6 is more cardiac-specific than AAV8 and AAV9. The results of this NHP study may help guide the development AAV vectors for the treatment of cardiovascular disease in humans

“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