The SR-BI Partner PDZK1 Facilitates Hepatitis C Virus Entry

Entry of hepatitis C virus (HCV) into hepatocytes is a multi-step process that involves a number of different host cell factors. Following initial engagement with glycosaminoglycans and the low-density lipoprotein receptor, it is thought that HCV entry proceeds via interactions with the tetraspanin CD81, scavenger receptor class B type I (SR-BI), and the tight-junction proteins claudin-1 (CLDN1) and occludin (OCLN), culminating in clathrin-dependent endocytosis of HCV particles and their pH-dependent fusion with endosomal membranes. Physiologically, SR-BI is the major receptor for high-density lipoproteins (HDL) in the liver, where its expression is primarily controlled at the post-transcriptional level by its interaction with the scaffold protein PDZK1. However, the importance of interaction with PDZK1 to the involvement of SR-BI in HCV entry is unclear. Here we demonstrate that stable shRNA-knockdown of PDZK1 expression in human hepatoma cells significantly reduces their susceptibility to HCV infection, and that this effect can be reversed by overexpression of full length PDZK1 but not the first PDZ domain of PDZK1 alone. Furthermore, we found that overexpression of a green fluorescent protein chimera of the cytoplasmic carboxy-terminus of SR-BI (amino acids 479–509) in Huh-7 cells resulted in its interaction with PDZK1 and a reduced susceptibility to HCV infection. In contrast a similar chimera lacking the final amino acid of SR-BI (amino acids 479–508) failed to interact with PDZK1 and did not inhibit HCV infection. Taken together these results indicate an indirect involvement of PDZK1 in HCV entry via its ability to interact with SR-BI and enhance its activity as an HCV entry factor

Identification of Synaptic Targets of Drosophila Pumilio

Drosophila Pumilio (Pum) protein is a translational regulator involved in embryonic patterning and germline development. Recent findings demonstrate that Pum also plays an important role in the nervous system, both at the neuromuscular junction (NMJ) and in long-term memory formation. In neurons, Pum appears to play a role in homeostatic control of excitability via down regulation of para, a voltage gated sodium channel, and may more generally modulate local protein synthesis in neurons via translational repression of eIF-4E. Aside from these, the biologically relevant targets of Pum in the nervous system remain largely unknown. We hypothesized that Pum might play a role in regulating the local translation underlying synapse-specific modifications during memory formation. To identify relevant translational targets, we used an informatics approach to predict Pum targets among mRNAs whose products have synaptic localization. We then used both in vitro binding and two in vivo assays to functionally confirm the fidelity of this informatics screening method. We find that Pum strongly and specifically binds to RNA sequences in the 3′UTR of four of the predicted target genes, demonstrating the validity of our method. We then demonstrate that one of these predicted target sequences, in the 3′UTR of discs large (dlg1), the Drosophila PSD95 ortholog, can functionally substitute for a canonical NRE (Nanos response element) in vivo in a heterologous functional assay. Finally, we show that the endogenous dlg1 mRNA can be regulated by Pumilio in a neuronal context, the adult mushroom bodies (MB), which is an anatomical site of memory storage

RNA Interference and Single Particle Tracking Analysis of Hepatitis C Virus Endocytosis

Hepatitis C virus (HCV) enters hepatocytes following a complex set of receptor interactions, culminating in internalization via clathrin-mediated endocytosis. However, aside from receptors, little is known about the cellular molecular requirements for infectious HCV entry. Therefore, we analyzed a siRNA library that targets 140 cellular membrane trafficking genes to identify host genes required for infectious HCV production and HCV pseudoparticle entry. This approach identified 16 host cofactors of HCV entry that function primarily in clathrin-mediated endocytosis, including components of the clathrin endocytosis machinery, actin polymerization, receptor internalization and sorting, and endosomal acidification. We next developed single particle tracking analysis of highly infectious fluorescent HCV particles to examine the co-trafficking of HCV virions with cellular cofactors of endocytosis. We observe multiple, sequential interactions of HCV virions with the actin cytoskeleton, including retraction along filopodia, actin nucleation during internalization, and migration of internalized particles along actin stress fibers. HCV co-localizes with clathrin and the ubiquitin ligase c-Cbl prior to internalization. Entering HCV particles are associated with the receptor molecules CD81 and the tight junction protein, claudin-1; however, HCV-claudin-1 interactions were not restricted to Huh-7.5 cell-cell junctions. Surprisingly, HCV internalization generally occurred outside of Huh-7.5 cell-cell junctions, which may reflect the poorly polarized nature of current HCV cell culture models. Following internalization, HCV particles transport with GFP-Rab5a positive endosomes, which is consistent with trafficking to the early endosome. This study presents technical advances for imaging HCV entry, in addition to identifying new host cofactors of HCV infection, some of which may be antiviral targets

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Welfare and health of dairy cattle on out-wintering pads or in cubicle housing with or without cushioned flooring.

End of Project ReportThe first study described in this report involved housing 66 spring calving heifers in one of three systems during the winter, namely, (i) a conventional cubicle house, (ii) a cubicle house with cushioned flooring covering the slats (slat mats) in the passageway and (iii) on a wood-chip out-wintering pad. Behaviour, health and performance indicators were measured on all animals while pregnant from housing in November 2003 until calving in January 2004. Additionally, data were collected on the first 15 animals to calve in each treatment for the first four weeks of lactation in the spring. The slat mats resulted in some improvements to hoof health compared to the conventional cubicle house. Furthermore, it increased feeding times although this had no effect on feed intake or performance. The results also indicated that heifers have a preference for standing on cushioned flooring rather than on concrete during late pregnancy. Both groups indoors differed greatly from the outdoor heifers in several respects. The outdoor animals had healthier feet and were less affected by injuries to the limbs. They also had a more diverse behaviour repertoire and slipped and tripped less. However, their welfare was adversely affected by inclement weather conditions with indications of immunosuppression combined with a reduction in average daily gain being recorded. Furthermore, they were dirtier and spent less time lying down. None of these factors influenced milk yield, quality or composition in early lactation. Welfare problems associated with the pad were weather and management dependent and hence could be addressed by more frequent cleaning of the pad and/or an increase in space allowance combined with the provision of shelter. Hence, the potential for good welfare in dairy heifers was higher on the pad than indoors in a cubicle system even when slat mats were provided. In the second study, 62 autumn calving pluriparous dairy cows were housed in September 2004 in a cubicle system with either solid concrete floors or solid concrete floors covered by a rubber mat and cleaned by an automatic scrapper. Behaviour, locomotion and foot lesion scores were recorded from at least 3 weeks prior to calving until at least 16 weeks post-partum. Furthermore, in-depth measures of oestrous behaviour and reproductive performance were recorded. The cushioned flooring had no effect on sole or white line lesion scores or on dermatitis scores. However, it reduced the rate of wear of the heels in early lactation. Cows on cushioned flooring spent more time standing, but not feeding, at the feed face while cows on concrete stood in the cubicles instead. It appears that where cows have access to spacious, well-designed cubicles they can use them for standing to get relief for their feet from the concrete. Similar to the previous study this also indicates that cows prefer to stand on cushioned flooring than on bare concrete and emphasises the importance of at least providing cows with mats or mattresses in their cubicles. There were no effects of the cushioned flooring on oestrous behaviour or reproductive performance, which was poor in both treatments. It is suggested that the reasons for this were that the cushioned flooring did not provide sufficient traction for the cows and so they were as reluctant as the cows on concrete to perform mounting behaviour.European Union Structural Fun

A comparison of 4 predictive models of calving assistance and difficulty in dairy heifers and cows

The aim of this study was to build and compare predictive models of calving difficulty in dairy heifers and cows for the purpose of decision support and simulation modeling. Models to predict 3 levels of calving difficulty (unassisted, slight assistance, and considerable or veterinary assistance) were created using 4 machine learning techniques: multinomial regression, decision trees, random forests, and neural networks. The data used were sourced from 2,076 calving records in 10 Irish dairy herds. In total, 19.9 and 5.9% of calving events required slight assistance and considerable or veterinary assistance, respectively. Variables related to parity, genetics, BCS, breed, previous calving, and reproductive events and the calf were included in the analysis. Based on a stepwise regression modeling process, the variables included in the models were the dam\u27s direct and maternal calving difficulty predicted transmitting abilities (PTA), BCS at calving, parity; calving assistance or difficulty at the previous calving; proportion of Holstein breed; sire breed; sire direct calving difficulty PTA; twinning; and 2-way interactions between calving BCS and previous calving difficulty and the direct calving difficulty PTA of dam and sire. The models were built using bootstrapping procedures on 70% of the data set. The held-back 30% of the data was used to evaluate the predictive performance of the models in terms of discrimination and calibration. The decision tree and random forest models omitted the effect of twinning and included only subsets of sire breeds. Only multinomial regression and neural networks explicitly included the modeled interactions. Calving BCS, calving difficulty PTA, and previous calving assistance ranked as highly important variables for all 4 models. The area under the receiver operating characteristic curve (ranging from 0.64 to 0.79) indicates that all of the models had good overall discriminatory power. The neural network and multinomial regression models performed best, correctly classifying 75% of calving cases and showing superior calibration, with an average error in predicted probability of 3.7 and 4.5%, respectively. The neural network and multinomial regression models developed are both suitable for use in decision-support and simulation modeling

The effects of altering milking frequency and/or diet in early lactation on the energy balance, production and reproduction of dairy cows.

End of Project ReportIt has been suggested that negative energy balance (NEB) in the immediate post-partum period is potentially an important factor in the association between increasing milk output and declining reproductive performance. The objective of this project was to design an experimental model that could be used to impose different degrees of NEB immediately after calving and to examine the effect of this model on dry matter intake (DMI), milk production, energy balance (EB), metabolic and reproductive hormonal profiles, the onset and pattern of post-partum ovarian cyclicity and reproductive physiology around AI. Two experiments were carried out to evaluate the effects of milking frequency and diet on DMI, production, energy balance and blood metabolites and hormones in the first 4 weeks after calving and subsequent reproduction. Reducing milking frequency from either thrice or twice daily to once daily reduced DMI but also reduced milk production. This resulted in a better EB in once daily milked cows in both experiments, the reduction being significant in the first. Milk production during the 4-week treatment period was reduced by 23 and 20 percent by reducing milking frequency from thrice to once daily in experiments 1 and 2, respectively. There was a reduction of approximately 10 percent in the cumulative yield up to week 20 of lactation in experiment 1 and of approximately 9 percent in total lactation yield in experiment 2. Reducing milking frequency resulted in increased plasma glucose, insulin and IGF-1 concentrations and reduced non-esterified fatty acid (NEFA) and beta hydroxybutyrate (BHB) concentrations. Conception rates to first service or overall pregnancy rates were not different between milking frequency treatments but once daily milking resulted in a shorter interval to first ovulation than thrice daily milking, due to a higher proportion of cows on this treatment ovulating the first post-partum dominant follicle. Increasing the energy density 2 of the diet increased DMI and milk production with no consequent effect on energy balance. Logistic regression on the combined data from the two experiments showed that lower energy intake, greater NEB and lower milk protein content and were significantly associated with poorer conception to first service. Lower plasma IGF-1 concentrations in experiment 2 were also associated with a lower conception rate to first service. A third experiment which investigated protein concentration in the concentrate combined with concentrate feeding level post calving (for two groups of cows in different body condition score at calving) showed no effect of post calving diet on BCS change. Overall the results suggest that reducing milking frequency to once per day during the first 4 weeks of lactation reduces NEB and appears to be a suitable strategy for altering energy balance at this time. However, the short-term reduction in milking frequency immediately post partum reduces total lactation yields. Blood metabolite and hormonal concentrations indicate better energy balance for cows milked once daily. Increasing dietary energy density or reducing the protein content of the diet does not appear to be effective in changing energy balance in early lactation. Decreased NEB in the first 4 weeks post-partum is associated with an improved conception rate to first service

Frameshift mutation in p53 regulator RPL26 is associated with multiple physical abnormalities and a specific pre-ribosomal RNA processing defect in diamond-blackfan anemia

Diamond-Blackfan anemia (DBA) is an inherited form of pure red cell aplasia that usually presents in infancy or early childhood and is associated with congenital malformations in approximately 30-50% of patients. DBA has been associated with mutations in nine ribosomal protein (RP) genes in about 53% of patients. We completed a large-scale screen of 79 RP genes by sequencing 16 RP genes (RPL3, RPL7, RPL8, RPL10, RPL14, RPL17, RPL19, RPL23A, RPL26, RPL27, RPL35, RPL36A, RPL39, RPS4X, RPS4Y1, and RPS21) in 96 DBA probands. We identified a de novo two-nucleotide deletion in RPL26 in one proband associated with multiple severe physical abnormalities. This mutation gives rise to a remarkable ribosome biogenesis defect that affects maturation of both the small and the large subunits. We also found a deletion in RPL19 and missense mutations in RPL3 and RPL23A, which may be variants of unknown significance. Together with RPL5, RPL11, and RPS7, RPL26 is the fourth RP regulating p53 activity that is linked to DBA