The antibody loci of the domestic goat (Capra hircus)

The domestic goat (Capra hircus) is an important ruminant species both as a source of antibody-based reagents for research and biomedical applications and as an economically important animal for agriculture, particularly for developing nations that maintain most of the global goat population. Characterization of the loci encoding the goat immune repertoire would be highly beneficial for both vaccine and immune reagent development. However, in goat and other species whose reference genomes were generated using short-read sequencing technologies, the immune loci are poorly assembled as a result of their repetitive nature. Our recent construction of a long-read goat genome assembly (ARS1) has facilitated characterization of all three antibody loci with high confidence and comparative analysis to cattle. We observed broad similarity of goat and cattle antibody-encoding loci but with notable differences that likely influence formation of the functional antibody repertoire. The goat heavy-chain locus is restricted to only four functional and nearly identical IGHV genes, in contrast to the ten observed in cattle. Repertoire analysis indicates that light-chain usage is more balanced in goats, with greater representation of kappa light chains (~ 20-30%) compared to that in cattle (~ 5%). The present study represents the first characterization of the goat antibody loci and will help inform future investigations of their antibody responses to disease and vaccination

The evolution of the natural killer complex; a comparison between mammals using new high-quality genome assemblies and targeted annotation.

Natural killer (NK) cells are a diverse population of lymphocytes with a range of biological roles including essential immune functions. NK cell diversity is in part created by the differential expression of cell surface receptors which modulate activation and function, including multiple subfamilies of C-type lectin receptors encoded within the NK complex (NKC). Little is known about the gene content of the NKC beyond rodent and primate lineages, other than it appears to be extremely variable between mammalian groups. We compared the NKC structure between mammalian species using new high-quality draft genome assemblies for cattle and goat; re-annotated sheep, pig, and horse genome assemblies; and the published human, rat, and mouse lemur NKC. The major NKC genes are largely in the equivalent positions in all eight species, with significant independent expansions and deletions between species, allowing us to propose a model for NKC evolution during mammalian radiation. The ruminant species, cattle and goats, have independently evolved a second KLRC locus flanked by KLRA and KLRJ, and a novel KLRH-like gene has acquired an activating tail. This novel gene has duplicated several times within cattle, while other activating receptor genes have been selectively disrupted. Targeted genome enrichment in cattle identified varying levels of allelic polymorphism between the NKC genes concentrated in the predicted extracellular ligand-binding domains. This novel recombination and allelic polymorphism is consistent with NKC evolution under balancing selection, suggesting that this diversity influences individual immune responses and may impact on differential outcomes of pathogen infection and vaccination

Trends in clinical development timeframes for anti-viral drugs launched in the UK, 1981-2014: a retrospective observational study

OBJECTIVES: Recent decades have witnessed the development of highly innovative new antiviral drug therapies. However, there are concerns that rising costs and lengthening development times could have implications for future patient access to innovative new drugs. We sought to establish whether the time taken for the clinical development of new antiviral drugs launched in the UK had increased since the 1980s. DESIGN AND SETTING: Retrospective observational study of all new antiviral drugs licensed for use in the UK. PRIMARY AND SECONDARY OUTCOME MEASURES: Duration of clinical development (from initiation of studies in humans to receipt of Marketing Authorisation), subdivided into clinical trial and regulatory approval periods by the date of Marketing Authorisation Application. RESULTS: 48 new antiviral drugs were licensed for use in the UK between 1981 and 2014 (inclusive), over half (54%) initially for HIV infection. The overall mean duration of clinical development was 77.2 months, of which 64.6 months was spent in clinical trials before regulatory submission. The total time in clinical development increased from 41.7 months for drugs licensed 1981–1992 to 91.7 months for drugs licensed 2004–2014. This increase was accounted for by an increase in the clinical trials period and not the regulatory approval period, for which there was no observable trend. Drugs initially licensed to treat hepatitis C had a longer duration of clinical development than those indicated for other viral infections. However, the, initially shorter clinical development durations of drugs indicated for HIV infection increased more rapidly across the study period than those indicated for other viral infections. CONCLUSIONS: The time spent by antiviral drugs in clinical development has increased markedly in recent decades despite many initiatives to speed access to innovative new drugs. However, this represents only one part of the translational research pathway, and a complete picture of development timeframes is lacking

Acute phosphate depletion and in vitro rat proximal tubule injury: Protection by glycine and acidosis

Acute phosphate depletion and in vitro rat proximal tubule injury: Protection by glycine and acidosis.The effects of phosphate (PO4) removal from Krebs Henseleit buffer on freshly isolated rat proximal tubules (rPT) were assessed by measuring Ca2+ uptake (nmol/mg protein), cellular adenosine triphosphate (ATP) (nmol/mg), tissue K+ content (nmol/mg) and lactate dehydrogenase (LDH) as an index of cell integrity. Ca2+ uptake increased by 50% in rPT incubated in zero PO4 medium as compared to control (2.6 ±0.1 vs. 3.9 ±0.19, P < 0.001) and LDH release increased 2.5-fold from 14.2 ±0.6 to 31.6 ±1.6%, P < 0.001. Neither verapamil (200 µM) nor mepacrine (50 µM) reduced Ca2+ uptake or decreased LDH release suggesting that the increased Ca2+ uptake was not occurring through potential operated channels and that phospholipase-induced cell injury was not the cause of increased LDH release. Either glycine (2 mM) or extracellular fluid acidosis (pH 7.06), however, significantly diminished rPT injury and Ca2+ uptake. Specifically, as compared to the increased LDH released in untreated, PO4-depleted rPT, LDH release was diminished significantly by glycine treatment (31.0 ±0.9 vs. 15.5 ±1.6%, P < 0.001) or acidosis (30.3 ±0.04 vs. 19.2 ±0.9%, P < 0.01). Ca2+ uptake did not increase in glycine treated tubules (2.6 ±0.1 vs. 2.8 ±0.2 nmol/mg, NS) or in the presence of acidosis (2.6 ±0.1 vs. 2.97 ±0.17 nmol/mg, NS). ATP concentrations were markedly reduced by PO4 depletion (2.8±0.2 vs. 4.8±0.3 nmol/mg, P < 0.001) and remained at low levels during either acidosis or glycine-induced protection. ATP depletion was accompanied by loss of K+ from rPT and this was only modestly attenuated by either glycine or acidosis. Total cell PO4 was not significantly altered, however, perchloric acid (PCA) extractable free PO4 was reduced significantly (33.3 ±4.5 to 15.9 ±3.5 nmol/mg, P < 0.01). The rPT injury, associated with acute PO4 depletion, may be related to Ca2+ uptake since Ca2+ uptake and LDH release were both attenuated by glycine administration or acidosis

A problem-solving approach to value-adding decision making in construction design

Purpose – To illustrate the use of a Value Adding Toolbox by construction industry designers when addressing customer value expectations using problem solving. Design/methodology/approach – Focused literature review establishes the need for construction industry design solutions to deliver customer value and a Value Adding Toolbox is proposed in response. Case studies validate Toolbox use and one illustrative example is provided. Interviews with prospective Toolbox users identify barriers to adoption and inform a recommended approach to organisational adoption. Findings – The Toolbox is found to be effective at helping construction designers to solve technical design problems with regard to customer expectations of value. However, designers are found to be initially reluctant to adopt the new tool. Organisation learning is therefore required to establish the importance of customer value satisfaction as a prerequisite to Toolbox adoption by designers. Originality/value – This paper provides a useful insight into the practical application of problemsolving tools by construction designers to better understand customer needs

Surgery for Hilar cholangiocarcinoma: the Newcastle-upon-Tyne Liver Unit experience

Background: Hilar cholangiocarcinoma (HCCA) arises from the confluence of the common hepatic duct and has a poor prognosis. If resectable, an extended left (eLH) or right hemihepatectomy (eRH) is usually required to provide oncological clearance. We reviewed outcomes for patients with HCCA managed at our centre. Methods: Electronic records of patients referred to our centre for HCCA were retrospectively reviewed. The Kaplan–Meier method was used to estimate overall survival (OS) with the log rank test used for significance (p &lt; 0.05). A Cox regression was performed to ascertain factors that may influence survival. Results: 156 HCCA patients were identified (44 resected versus 112 non-resected). Resected patients had longer OS compared to non-resected patients (50.3 versus 9.8 months, p &lt; 0.001). Patients who underwent an eLH (n = 15) had significantly longer OS at 3 years compared to eRH patients (67.7 vs. 42.1%, respectively; p = 0.007). An eLH was an independent predictor of survival (HR 0.43, p = 0.04). Lymph node positivity (n = 23, hazard ratio 1.72, p = 0.027) and the presence of microvascular invasion (n = 28, hazard ratio 1.78, p = 0.047) were independent predictors of mortality. The frequency of lymph node positivity and microvascular invasion did not differ between eLH and eRH patients (p &gt; 0.05). Conclusion: Patients undergoing an eLH for HCCA have significantly better long-term outcomes compared to those undergoing eRH, independent of other pathological variables. The functional liver remnant (FLR) is usually smaller following eRH, resulting in a higher risk of post-operative liver failure. Combining CT volumetry with PVE may result in better prediction and optimisation of the FLR in the context of eRH for HCCA. Novel findings: An extended left hemihepatectomy is an independent predictor of survival; investigation into the precise interaction between left- and right-sided resections and pre- and post-embolization liver volume is warranted

Earthquake relocations and InSAR analysis following the June 12th 2011 eruption of Nabro volcano, Afar

Nabro volcano sits on the southern part of Danakil block to the east of the Afar depression, on the Arabian plate. On the 12th June 2011, Nabro volcano suddenly erupted after being inactive for 10,000 years. The eruption caused a 17-km-long lava flow, a 15-km-high ash cloud, and ranks as one of the largest emissions of SO2 since the Mt. Pinatubo (1991) event. This eruption creates an important opportunity to use seismicity and surface deformation measurements to understand the subsurface magmatic system and deformation of a hazardous, off axis caldera during continental rupture. We installed a network of 8 seismometers around Nabro caldera which began recording on the 31st August and tasked SAR acquisitions from TerraSAR-X (TSX) and Cosmo-SkyMed (CSK) satellites. The SAR images used for this study post date the eruption. We used TSX stripmap mode images from ascending and descending orbits. Using a small baseline approach, we used 25 images acquired between the 1st July 2011 to the 5th October 2012 on descending orbit 046, to create 34 interferograms. We complemented these with 19 images from ascending orbit 130 spanning the 6th July 2011 to the 10th October 2012 from ascending orbit 130, which we used to create 21 interferograms. We produced a velocity ratemap and timeseries using π-RATE showing subsidence of up to 25cm/yr centred on Nabro. We used a Monte-Carlo hybrid downhill simplex technique to invert the dataset and found the best fitting solution as a mogi source at 6.9 ±1.1 km depth, and located at a 13.35 (lat) and 41.69 (long). The time dependence observed is consistent with a viscoelastic relaxation around the magma chamber, following depletion. Concurrent with the TSX acquisitions, CSK imaged the volcano on a descending track between 26th June 2011 and 18th July 2012 within the ASI project SAR4Volcanoes, and 64 images were used to produce 171 interferograms which were inverted to form a timeseries using a SBAS approach. This dataset has an overall subsidence signal, but the time series shows a shorter wavelength fluctuation of ground deformation, which is not apparent in the TSX data. We processed the seismic signals detected by the temporary local network and by a seismic station within a permanent regional array, to provide hypocentre locations for the period September-October, 2011. We used Hypoinverse-2000 to provide preliminary locations for events, which were then relocated using HypoDD. Absolute error after Hypoinverse-2000 processing was approximately ±2 and ±4 km in the horizontal and the vertical directions, respectively. Using HypoDD, relative errors were reduced to ±20 and ±30 m in the horizontal and vertical directions, respectively. The hypocentres show clusters of activity as well as aseismic regions. The majority of the earthquakes are located at the active vent, with fewer events located on the flanks. There is a smaller cluster of events to the south-west of Nabro beneath neighbouring Mallahle volcanic caldera, despite no eruption occurring here nor any post-eruptive deformation. This may imply some stress triggering mechanism or some pressure connection between the magma system of the two calderas. We present both the seismic and InSAR datasets as a joint approach to understand this eruption, as well as further implications for other ‘quiet calderas’

Loss of equilibrative nucleoside transporter 1 in mice leads to progressive ectopic mineralization of spinal tissues resembling diffuse idiopathic skeletal hyperostosis in humans

Diffuse idiopathic skeletal hyperostosis (DISH) is a noninflammatory spondyloarthropathy, characterized by ectopic calcification of spinal tissues. Symptoms include spine pain and stiffness, and in severe cases dysphagia and spinal cord compression. The etiology of DISH is unknown and there are no specific treatments. Recent studies have suggested a role for purine metabolism in the regulation of biomineralization. Equilibrative nucleoside transporter 1 (ENT1) transfers hydrophilic nucleosides, such as adenosine, across the plasma membrane. In mice lacking ENT1, we observed the development of calcified lesions resembling DISH. By 12 months of age, ENT1-/- mice exhibited signs of spine stiffness, hind limb dysfunction, and paralysis. Micro-computed tomography (μCT) revealed ectopic mineralization of paraspinal tissues in the cervical-thoracic region at 2 months of age, which extended to the lumbar and caudal regions with advancing age. Energy-dispersive X-ray microanalysis of lesions revealed a high content of calcium and phosphorus with a ratio similar to that of cortical bone. At 12 months of age, histological examination of ENT1-/- mice revealed large, irregular accumulations of eosinophilic material in paraspinal ligaments and entheses, intervertebral discs, and sternocostal articulations. There was no evidence of mineralization in appendicular joints or blood vessels, indicating specificity for the axial skeleton. Plasma adenosine levels were significantly greater in ENT1 -/- mice than in wild-type, consistent with loss of ENT1 - a primary adenosine uptake pathway. There was a significant reduction in the expression of Enpp1, Ank, and Alpl in intervertebral discs from ENT1-/- mice compared to wild-type mice. Elevated plasma levels of inorganic pyrophosphate in ENT1-/- mice indicated generalized disruption of pyrophosphate homeostasis. This is the first report of a role for ENT1 in regulating the calcification of soft tissues. Moreover, ENT1-/- mice may be a useful model for investigating pathogenesis and evaluating therapeutics for the prevention of mineralization in DISH and related disorders. © 2013 American Society for Bone and Mineral Research. Copyright © 2013 American Society for Bone and Mineral Research

DiffSplice: The Genome-Wide Detection of Differential Splicing Events with RNA-Seq

The RNA transcriptome varies in response to cellular differentiation as well as environmental factors, and can be characterized by the diversity and abundance of transcript isoforms. Differential transcription analysis, the detection of differences between the transcriptomes of different cells, may improve understanding of cell differentiation and development and enable the identification of biomarkers that classify disease types. The availability of high-throughput short-read RNA sequencing technologies provides in-depth sampling of the transcriptome, making it possible to accurately detect the differences between transcriptomes. In this article, we present a new method for the detection and visualization of differential transcription. Our approach does not depend on transcript or gene annotations. It also circumvents the need for full transcript inference and quantification, which is a challenging problem because of short read lengths, as well as various sampling biases. Instead, our method takes a divide-and-conquer approach to localize the difference between transcriptomes in the form of alternative splicing modules (ASMs), where transcript isoforms diverge. Our approach starts with the identification of ASMs from the splice graph, constructed directly from the exons and introns predicted from RNA-seq read alignments. The abundance of alternative splicing isoforms residing in each ASM is estimated for each sample and is compared across sample groups. A non-parametric statistical test is applied to each ASM to detect significant differential transcription with a controlled false discovery rate. The sensitivity and specificity of the method have been assessed using simulated data sets and compared with other state-of-the-art approaches. Experimental validation using qRT-PCR confirmed a selected set of genes that are differentially expressed in a lung differentiation study and a breast cancer data set, demonstrating the utility of the approach applied on experimental biological data sets. The software of DiffSplice is available at http://www.netlab.uky.edu/p/bioinfo/DiffSplice