The Role of RCK/p54 Enzymatic Activity during Hepatitis C Infection

Hepatitis C virus (HCV) infected 30,500 people in the United States alone in 2014. As a positive-sense single-stranded RNA virus, the HCV genome facilitates translation and replication of the virus within the host cell. During infection, HCV subverts numerous host proteins to aid in viral production. One such protein is DDX6, a DEAD-box helicase that is associated with the decapping complex and microRNA Induced Silencing Complex (miRISC). Previous studies in our lab have shown that DDX6 is required for HCV expression. Specifically, DDX6 modulates the interaction of miR-122 with the HCV 5’ UTR and promotes stability and replication of the viral genome. To elucidate which enzymatic activities of DDX6 are required to promote HCV gene expression, we used site-directed mutagenesis to disrupt the enzymatic activities of DDX6. Specifically, we mutated the ATPase, RNA binding and helicase domains within 3x-Flag-DDX6Δsi plasmid DNA. We next used lentivirus transduction to stably express the 3xFlag-tagged DDX6 mutants in Huh7 cells, in which expression of endogenous DDX6 was deleted by gene editing using CRISPR-Cas9. Analysis by immunofluorescence staining and confocal microscopy of uninfected cells, shows wild-type 3xFlag-DDX6 localizing in processing bodies, while the enzymatic mutants show diffuse cytoplasmic staining. DDX6 stable cell lines were infected with the HCV infectious clone JFH-1, and at three-days post-infection cells were harvested and viral and cellular protein and RNA abundance analyzed by western and northern blotting, respectively. Surprisingly, only DDX6 knock-out cells expressing the wild-type 3xFlag-DDX6 were infected with HCV. These data show that the enzymatic activities of DDX6 are critical for HCV infection. Since RNA helicases unwind RNA, it is possible that the helicase and RNA binding activities of RCK/p54 are necessary to remodel the complex secondary structures found on the 5’ and 3’ untranslated regions of the HCV genome, and may play an important role in the transition between translation and replication

Clinical pain assessment in the horse

Accurate pain assessment is fundamental to optimal pain management, representing a major welfare concern. Pain assessment has received considerable attention in farm, laboratory and companion animals, however, there is little objective equine pain research. This study aimed to objectively identify behavioural indicators of pain, examining both acute post-operative (castration) and chronic (laminitis) pain.Male thoroughbred horses (n=T0/group) underwent castration or sham castration (control) performed under either standing surgical sedation (SS) or general anaesthesia (GA). Horses were monitored for 24 hours pre-operatively and 48 hours post¬ operatively. Additionally, seven acute laminitic horses and paired age, sex and breedmatched controls were monitored for up to five days. Assessments were made using time-lapse video recording and direct observation of undisturbed spontaneous behaviour and evoked human interaction behaviours. Data were acquired using The Observer™ and analysed using generalised mixed effects (GME) and discriminant analysis (DA).GA and SS castrates spent more time with their ears back and displayed a higher frequency of stepping away than controls in interactive tests (P<0.017, GME). Head level with withers increased post-operatively in SS castrate, but not control horses (P<0.001, GME). Additionally, sham GA resulted in increased inattentive behaviour and hindlimb resting and reduced 'head up' and recumbency (P<0.039, GME). Laminitic horses showed reduced hindlimb resting and walking with increased lying, 'head level' and forelimb lifting compared to controls (P<0.046, GME). Accuracy of discrimination (DA) between 'painful' and 'pain-free' horses was >78.6% in acute and chronic pain.We identified behavioural parameters indicative of pain and discomfort in acute and chronic pain states. Acute pain may be most accurately identified through the examination of evoked behaviour, whilst changes in spontaneous behaviour appear more altered in chronic pain

Impact Evaluation of the SADA Millennium Villages Project in Northern Ghana: Endline Summary Report

The Millennium Villages Project (MVP) aims to demonstrate how the Millennium Development Goals (MDGs) could be achieved locally through an integrated approach to development. While the MDGs have now been superseded by the Sustainable Development Goals (SDGs, 2016–30), there remains a consistent thread to the MDGs around issues such as eradicating poverty, preventing avoidable deaths and improving education. Furthermore, the interconnected nature of the SDGs means the MVP model also has relevance for those seeking to address extreme poverty by taking an integrated approach to sustainable development. This report summarises the findings from what we believe to be the first independent impact evaluation of the MVP approach. It is hoped that the evidence and analysis will be of relevance to a wide range of actors in international development.Department for International Development (DFID

Depletion of stromal cells expressing fibroblast activation protein-α from skeletal muscle and bone marrow results in cachexia and anemia.

Fibroblast activation protein-α (FAP) identifies stromal cells of mesenchymal origin in human cancers and chronic inflammatory lesions. In mouse models of cancer, they have been shown to be immune suppressive, but studies of their occurrence and function in normal tissues have been limited. With a transgenic mouse line permitting the bioluminescent imaging of FAP(+) cells, we find that they reside in most tissues of the adult mouse. FAP(+) cells from three sites, skeletal muscle, adipose tissue, and pancreas, have highly similar transcriptomes, suggesting a shared lineage. FAP(+) cells of skeletal muscle are the major local source of follistatin, and in bone marrow they express Cxcl12 and KitL. Experimental ablation of these cells causes loss of muscle mass and a reduction of B-lymphopoiesis and erythropoiesis, revealing their essential functions in maintaining normal muscle mass and hematopoiesis, respectively. Remarkably, these cells are altered at these sites in transplantable and spontaneous mouse models of cancer-induced cachexia and anemia. Thus, the FAP(+) stromal cell may have roles in two adverse consequences of cancer: their acquisition by tumors may cause failure of immunosurveillance, and their alteration in normal tissues contributes to the paraneoplastic syndromes of cachexia and anemia