Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression.

The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP); and 29 deletion alleles (DEL), usually a deletion between the translational start and the 3' UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ) and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels

The effect of phase chemistry on the extent of strengthening mechanisms in model Ni-Cr-Al-Ti-Mo based superalloys

The exceptional mechanical properties of polycrystalline nickel-based superalloys arise through various concurrent strengthening mechanisms. Whilst these mechanisms are generally understood, consensus has yet to be established on the precise contribution of each to the overall alloy strength. Furthermore, changes in alloy chemistry influence several different mechanisms, making the assessment of individual alloying elements complex. In this study, a series of model quinary Ni-based superalloys has been investigated to systematically study the effect of varying Mo content on the contributing strengthening mechanisms. Using microstructural data, the yield strength was modelled by summing the individual effects of solid solution in both the γ and γ ' phases, coherency, grain boundary and precipitation strengthening. The total predicted yield stress increased with Mo content despite the diminishing contribution of precipitation strengthening. It is shown that solid solution strengthening of the ordered γ' precipitate phase is a key contributor to the overall strength, and that variations in composition between the tertiary and secondary γ ' lead to significant changes in mechanical properties that should be accounted for in models of alloy strength.Funding was provided by the EPSRC/Rolls-Royce Strategic Partnership under EP/M005607/1 and EP/H022309/1. The Oxford Atom Probe facility was funded by the EPSRC under EP/M022803/1. E. I. Galindo-Nava would like to acknowledge the Royal Academy of Engineering for his fellowship funding. Neutron diffraction beam time was supported through the Canadian Neutron Beam Centre under Experiment number 1258

Influence of powder-bed temperature on the microstructure and mechanical properties of Ti-6Al-4V produced by selective laser melting

Advanced characterisation techniques were used on LPBF Ti-6Al-4V samples produced on a heated base plate. When the substrate temperature is 100{\deg}C the elongation is 6\%, which increases and peaks at 10\% at 570{\deg}C, then sharply decreases to zero ductility at 770{\deg}C. At 100{\deg}C, a heavily strained and twinned microstructure, primarily composed of {\alpha}+{\alpha}', was observed and it was comparable to asbuilt microstructures obtained by conventional LPBF methods. At higher temperatures, twins are no longer present and instead nano-scale {\beta} precipitates are observed within {\alpha}' and {\alpha}, as well as dislocation networks (570{\deg}C) and tangles (770{\deg}C). Solute segregation at crystal defects was observed in all pre-heating conditions. Al and V segregation at microtwins was observed in the 100{\deg}C sample, reporting for the first time `selective' and mutually exclusive Al- and V-rich regions forming in adjacent twins. V segregation at dislocations was observed in the 570{\deg}C and 770{\deg}C samples, consistent with the higher preheating temperatures. High O contents were measured in all samples but with apparent opposing effects. At 100{\deg}C and 570{\deg}C was estimated to be below the critical threshold for O embrittlement and locally aids in maintaining a strength high by solid solution strengthening, whereas at 770{\deg}C it was above the threshold, therefore failing in a brittle fashion. Based on these observations, the initial increase in ductility from 100{\deg}C to 570{\deg}C is attributed to a reduction in microtwins and the dislocation networks acting as `soft barriers' for slip within a coarser microstructure. The lack of ductility at 770{\deg}C was attributed to local solute redistribution causing dislocation pinning and an increase of O content in this sample

Effect of Substrate Bed Temperature on Solute Segregation and Mechanical Properties in Ti–6Al–4V Produced by Laser Powder Bed Fusion

Titanium alloys are particularly sensitive to temperature during additive manufacturing processes, due to their dual phase microstructure and sensitivity to oxygen uptake. In this paper, laser powder bed fusion (LPBF) was used in conjunction with a heated substrate bed at 100 °C, 570 °C and 770 °C to produce specimens of Ti–6Al–4V, to investigate the change in mechanical properties and segregation of alloying elements. An initial increase in ductility was observed when increasing the temperature from 100 °C to 570 °C, followed by a significant loss in ductility when samples were produced at 770 °C. A suite of multi-scale characterisation techniques revealed that the as-printed microstructure was drastically different across the range of temperatures. At 100 °C, α + α′ phases were identified. Deformation twinning was extensively observed in the a phase, with Al and V segregating at the twin interfaces. At 570 °C (the most ductile sample), α′, α and nano-particles of β were observed, with networks of entangled dislocations showing V segregation. At 770 °C, no martensitic α′ was identified. The microstructure was an α + β microstructure and an increased volume fraction of tangled dislocations with localised V segregation. Thermodynamic modelling based on the Gibbs-free energy of formation showed that the increased V concentration at dislocations was insufficient to locally nucleate β phase. However, b-phase nucleation at grain boundaries (not dislocations) caused pinning of grain boundaries, impeding slip and leading to a reduction in ductility. It is likely that the increased O-content within specimens printed at increased temperatures also played a key role in high-temperature embrittlement. Building operations are therefore best performed below sub-transus temperatures, to encourage the growth of strengthening phases via solute segregation, and the build atmosphere must be tightly controlled to reduce oxygen uptake within the samples

Nightly treatment of primary insomnia with prolonged release melatonin for 6 months: a randomized placebo controlled trial on age and endogenous melatonin as predictors of efficacy and safety

<p>Background: Melatonin is extensively used in the USA in a non-regulated manner for sleep disorders. Prolonged release melatonin (PRM) is licensed in Europe and other countries for the short term treatment of primary insomnia in patients aged 55 years and over. However, a clear definition of the target patient population and well-controlled studies of long-term efficacy and safety are lacking. It is known that melatonin production declines with age. Some young insomnia patients also may have low melatonin levels. The study investigated whether older age or low melatonin excretion is a better predictor of response to PRM, whether the efficacy observed in short-term studies is sustained during continued treatment and the long term safety of such treatment.</p> <p>Methods: Adult outpatients (791, aged 18-80 years) with primary insomnia, were treated with placebo (2 weeks) and then randomized, double-blind to 3 weeks with PRM or placebo nightly. PRM patients continued whereas placebo completers were re-randomized 1:1 to PRM or placebo for 26 weeks with 2 weeks of single-blind placebo run-out. Main outcome measures were sleep latency derived from a sleep diary, Pittsburgh Sleep Quality Index (PSQI), Quality of Life (World Health Organzaton-5) Clinical Global Impression of Improvement (CGI-I) and adverse effects and vital signs recorded at each visit.</p> <p>Results: On the primary efficacy variable, sleep latency, the effects of PRM (3 weeks) in patients with low endogenous melatonin (6-sulphatoxymelatonin [6-SMT] ≤8 μg/night) regardless of age did not differ from the placebo, whereas PRM significantly reduced sleep latency compared to the placebo in elderly patients regardless of melatonin levels (-19.1 versus -1.7 min; P = 0.002). The effects on sleep latency and additional sleep and daytime parameters that improved with PRM were maintained or enhanced over the 6-month period with no signs of tolerance. Most adverse events were mild in severity with no clinically relevant differences between PRM and placebo for any safety outcome.</p> <p>Conclusions: The results demonstrate short- and long-term efficacy and safety of PRM in elderly insomnia patients. Low melatonin production regardless of age is not useful in predicting responses to melatonin therapy in insomnia. The age cut-off for response warrants further investigation.</p&gt

Cord blood calcium, phosphate, magnesium, and alkaline phosphatase gestational age-specific reference intervals for preterm infants

<p>Abstract</p> <p>Background</p> <p>The objective was to determine the influence of gestational age, maternal, and neonatal variables on reference intervals for cord blood bone minerals (calcium, phosphate, magnesium) and related laboratory tests (alkaline phosphatase, and albumin-adjusted calcium), and to develop gestational age specific reference intervals based on infants without influential pathological conditions.</p> <p>Methods</p> <p>Cross-sectional study. 702 babies were identified as candidates for this study in a regional referral neonatal unit. After exclusions (for anomalies, asphyxia, maternal magnesium sulfate administration, and death), relationships were examined between cord blood serum laboratory analytes (calcium, phosphate, magnesium, alkaline phosphatase, and albumin-adjusted calcium) with gestation age and also with maternal and neonatal variables using multiple linear regression. Infants with influential pathological conditions were omitted from the development of gestational age specific reference intervals for the following categories: 23-27, 28-31, 32-34, 35-36 and > 36 weeks.</p> <p>Results</p> <p>Among the 506 preterm and 54 terms infants included in the sample. Phosphate, magnesium, and alkaline phosphatase in cord blood serum decreased with gestational age, calcium increased with gestational age. Those who were triplets, small for gestational age, and those whose mother had pregnancy-induced hypertension were influential for most of the analytes. The reference ranges for the preterm infants ≥ 36 weeks were: phosphate 1.5 to 2.6 mmol/L (4.5 to 8.0 mg/dL), calcium: 2.1 to 3.1 mmol/L (8.3 to 12.4 mg/dL); albumin-adjusted calcium: 2.3 to 3.2 mmol/L (9.1 to 12.9 mg/dL); magnesium 0.6 to 1.0 mmol/L (1.4 to 2.3 mg/dL), and alkaline phosphatase 60 to 301 units/L.</p> <p>Conclusions</p> <p>These data suggest that gestational age, as well as potentially pathogenic maternal and neonatal variables should be considered in the development of reference intervals for preterm infants.</p

Varespladib and cardiovascular events in patients with an acute coronary syndrome: the VISTA-16 randomized clinical trial

IMPORTANCE: Secretory phospholipase A2(sPLA2) generates bioactive phospholipid products implicated in atherosclerosis. The sPLA2inhibitor varespladib has favorable effects on lipid and inflammatory markers; however, its effect on cardiovascular outcomes is unknown. OBJECTIVE: To determine the effects of sPLA2inhibition with varespladib on cardiovascular outcomes. DESIGN, SETTING, AND PARTICIPANTS: A double-blind, randomized, multicenter trial at 362 academic and community hospitals in Europe, Australia, New Zealand, India, and North America of 5145 patients randomized within 96 hours of presentation of an acute coronary syndrome (ACS) to either varespladib (n = 2572) or placebo (n = 2573) with enrollment between June 1, 2010, and March 7, 2012 (study termination on March 9, 2012). INTERVENTIONS: Participants were randomized to receive varespladib (500 mg) or placebo daily for 16 weeks, in addition to atorvastatin and other established therapies. MAIN OUTCOMES AND MEASURES: The primary efficacy measurewas a composite of cardiovascular mortality, nonfatal myocardial infarction (MI), nonfatal stroke, or unstable angina with evidence of ischemia requiring hospitalization at 16 weeks. Six-month survival status was also evaluated. RESULTS: At a prespecified interim analysis, including 212 primary end point events, the independent data and safety monitoring board recommended termination of the trial for futility and possible harm. The primary end point occurred in 136 patients (6.1%) treated with varespladib compared with 109 patients (5.1%) treated with placebo (hazard ratio [HR], 1.25; 95%CI, 0.97-1.61; log-rank P = .08). Varespladib was associated with a greater risk of MI (78 [3.4%] vs 47 [2.2%]; HR, 1.66; 95%CI, 1.16-2.39; log-rank P = .005). The composite secondary end point of cardiovascular mortality, MI, and stroke was observed in 107 patients (4.6%) in the varespladib group and 79 patients (3.8%) in the placebo group (HR, 1.36; 95% CI, 1.02-1.82; P = .04). CONCLUSIONS AND RELEVANCE: In patients with recent ACS, varespladib did not reduce the risk of recurrent cardiovascular events and significantly increased the risk of MI. The sPLA2inhibition with varespladib may be harmful and is not a useful strategy to reduce adverse cardiovascular outcomes after ACS. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01130246. Copyright 2014 American Medical Association. All rights reserved

Effect of Audiovisual Training on Monaural Spatial Hearing in Horizontal Plane

The article aims to test the hypothesis that audiovisual integration can improve spatial hearing in monaural conditions when interaural difference cues are not available. We trained one group of subjects with an audiovisual task, where a flash was presented in parallel with the sound and another group in an auditory task, where only sound from different spatial locations was presented. To check whether the observed audiovisual effect was similar to feedback, the third group was trained using the visual feedback paradigm. Training sessions were administered once per day, for 5 days. The performance level in each group was compared for auditory only stimulation on the first and the last day of practice. Improvement after audiovisual training was several times higher than after auditory practice. The group trained with visual feedback demonstrated a different effect of training with the improvement smaller than the group with audiovisual training. We conclude that cross-modal facilitation is highly important to improve spatial hearing in monaural conditions and may be applied to the rehabilitation of patients with unilateral deafness and after unilateral cochlear implantation