ORE identifies extreme expression effects enriched for rare variants.

MOTIVATION: Non-coding rare variants (RVs) may contribute to Mendelian disorders but have been challenging to study due to small sample sizes, genetic heterogeneity and uncertainty about relevant non-coding features. Previous studies identified RVs associated with expression outliers, but varying outlier definitions were employed and no comprehensive open-source software was developed. RESULTS: We developed Outlier-RV Enrichment (ORE) to identify biologically-meaningful non-coding RVs. We implemented ORE combining whole-genome sequencing and cardiac RNAseq from congenital heart defect patients from the Pediatric Cardiac Genomics Consortium and deceased adults from Genotype-Tissue Expression. Use of rank-based outliers maximized sensitivity while a most extreme outlier approach maximized specificity. Rarer variants had stronger associations, suggesting they are under negative selective pressure and providing a basis for investigating their contribution to Mendelian disorders. AVAILABILITY AND IMPLEMENTATION: ORE, source code, and documentation are available at https://pypi.python.org/pypi/ore under the MIT license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Reporting of vertebral fragility fractures: can radiologists help reduce the number of hip fractures?

Patients with osteoporotic vertebral fractures are at increased risk of hip fracture. In a cohort of hip fracture patients, many had previous imaging studies showing incidental vertebral fractures. Fifty-four percent of fractures were not reported by the radiologist, highlighting a missed opportunity for diagnosing and treating osteoporosis, thereby preventing further fractures. PURPOSE: Patients with osteoporotic vertebral fragility fractures (VFFs) are at increased risk of future fractures, including hip fractures. Treating osteoporosis in these patients has the potential to reduce the risk of subsequent hip fractures, which are associated with high morbidity, mortality and cost. In this retrospective cohort study, we investigated the reporting and follow-up of VFFs evident on imaging by radiologists at the John Radcliffe Hospital, Oxford. MATERIALS AND METHODS: Data from the local Fracture Liaison Service was used to case-find all incident hip fractures from 2013 presenting to the trust. We then identified patients who had also undergone a radiological procedure that included the thoracic and/or lumbar spine in the previous 6 years. All identified radiological images were re-examined for the presence of VFFs using the Genant semi-quantitative method. RESULTS: Seven hundred and thirty-two patients over the age of 50 with a hip fracture in 2013 were identified. One hundred and fifty-seven patients had previously undergone a radiological procedure involving the spine, and VFFs were identified in 65/157 (41%). Of these, only 30/65 (46%) were reported by a radiologist when the fracture was first visible. 32/35 (91%) of unreported VFFs were from imaging reported by non-musculoskeletal radiologists. Only 16/65 (25%) of patients with a VFF were documented as being on bone-specific therapy at the time of hip fracture. CONCLUSIONS: Our study highlights the under-reporting of osteoporotic vertebral fractures, particularly by non-musculoskeletal radiologists. Better systems for reporting and referring osteoporotic VFFs are necessary to increase the number of patients receiving appropriate osteoporosis treatment.

MRI differential diagnosis of suspected multiple sclerosis.

Diagnosing multiple sclerosis (MS) can be very challenging owing to its variable clinical features and lack of a definitive test. Magnetic resonance imaging (MRI) is a core diagnostic tool in the detection of MS lesions and demonstration of spatial and temporal distribution of disease. Moreover, MRI plays a crucial role in the exclusion of alternative diagnoses of MS. The aim of this review is to describe the typical MRI features of MS and to present a series of common mimics of MS with emphasis on their distinguishing features from MS.

The popliteal fibular ligament in acute knee trauma: patterns of injury on MR imaging.

OBJECTIVE: To describe the patterns of injury associated with injury to the popliteofibular ligament injury. MATERIALS AND METHODS: A retrospective review was performed of 180 MRI scans undertaken for acute knee trauma. Scans were excluded if the time of injury was over 4 weeks from the time of the scan, or if there was a history of septic arthritis, inflammatory arthropathy, previous knee surgery, or significant artefact. An agreed criterion for assessing the structures of the posterolateral ligamentous complex was defined and in each scan, the popliteofibular ligament (PFL) was scored as normal or injured. The menisci, ligaments, and tendons of each knee were also assessed. RESULTS: The mean age was 25.7 years (range, 9-65 years) and 72.2% (n = 130) patients were male. The PFL was injured in 36 cases (20%). There is a significant association between PFL injury and ACL rupture (p = 0.0001), ITB injury (p = 0.0001), PCL injury (p = 0.0373), in addition to associations with injury to other posterolateral corner structures including the lateral collateral ligament (p = 0.0001), biceps femoris tendon (p = 0.0014), and popliteus tendon (p = 0.0014). Of our series of PFL injuries, nine cases (25%) were associated with further injuries of posterolateral corner structures and in 27 cases (75%) the PFL was the only posterolateral corner structure torn. CONCLUSIONS: PFL injury is not uncommon in acute knee trauma and is associated with significant internal derangement of the knee, especially anterior cruciate ligament rupture, ITB sprain, and injury to other structures within the posterolateral corner.

Posterior circulation infarction in patients with traumatic cervical spinal cord injury and its relationship to vertebral artery injury.

STUDY DESIGN: Prospective study. OBJECTIVE: To ascertain the prevalence of posterior circulation stroke in traumatic chronic spinal cord injured (SCI) patients and associated traumatic vertebral artery injuries (VAI). METHODS: All adult patients with cervical SCI and American Spinal Injury Association Impairment Scale (AIS) grade A or B referred for follow-up magnetic resonance imaging of their spinal cord were invited to take part in the study between January 2010 and December 2012 at the National Spinal Injury Centre. Two additional sequences were added to the existing imaging protocol to evaluate the brain and vertebral arteries. RESULTS: Ninety-eight patients were recruited. All imaging were analysed independently by three consultant radiologists. Posterior circulation infarcts were noted in seven (7%) patients. Significant VAI was noted in 13 patients (13%) with 10 occlusions and 3 with high-grade stenosis. However, only one patient had co-existent posterior circulation infarct and significant VAI. CONCLUSION: There is an increased prevalence of posterior circulation infarction in SCI patients. The relationship with associated traumatic VAI requires further investigation.

Validating a threshold of ocular gaze deviation for the prediction of acute ischaemic stroke.

AIM: To determine a threshold at which the degree of ocular gaze deviation (OGD) on axial imaging is highly specific for the prediction of acute ischaemic stroke. MATERIALS AND METHODS: A retrospective analysis of 517 patients who had received MRI with diffusion-weighted imaging (DWI) for suspected acute stroke was performed. The degree of OGD was measured in all patients and the presence and location of infarction determined. The difference in OGD between groups was compared using the independent t-test for normally distributed data and the Mann-Whitney test for non-normal data. The sensitivity and specificity for degrees of OGD in the prediction of acute infarction was calculated using a receiver operating curve (ROC) analysis. RESULTS: The imaging of 448 patients meeting the inclusion criteria was reviewed. Acute infarct was demonstrated in 34.8% (n=156). There was a significant difference in the degree of OGD between patients with an acute infarct and those without evidence of acute ischaemia (p<0.001). ROC curve analysis for OGD demonstrated area under the curve (AUC) = 0.619 with increasing degrees of OGD more specific for acute infarct. OGD >11.95° had a sensitivity of 17% and specificity of 95.9% in predicting acute infarction. CONCLUSION: Significant OGD>11.95° has a high specificity for acute infarct. This threshold may provide a helpful additional sign in the detection of subtle acute infarct, particularly on axial CT brain imaging.

CAML regulates Bim-dependent thymocyte death.

Appropriate control of apoptosis during T lymphocyte differentiation is critical for destruction of T cells bearing potentially autoreactive or useless immuno-receptors and for survival of those T cells bearing antigen receptors that may recognize foreign proteins. Despite the well-established importance of thymocyte survival, the exact signals regulating thymocyte apoptosis have not been fully elucidated. Here, we show that thymocytes lacking the endoplasmic reticulum protein calcium-modulating cyclophilin ligand (CAML) failed to undergo normal T-cell development and exhibited dramatically increased rates of apoptosis. In vitro, CAML-deficient thymocytes accumulated high levels of reactive oxygen species (ROS) and underwent abnormally accelerated death in response to several cytotoxic stimuli, including treatment with etoposide, cytokine deprivation, or Fas ligation. Although neither p53 deletion nor loss of Fas rescued the survival and continued development of CAML-deficient thymocytes, removal of the pro-apoptotic BH3-only Bcl-2 family member Bim significantly restored their survival. This work reveals CAML to be a critically important regulator of ROS- and Bim-dependent thymocyte death.

A reproducible method for the enumeration of functional (cytokine producing) versus non-functional peptide-specific cytotoxic T lymphocytes in human peripheral blood.

One of the most difficult laboratory challenges in the field of therapeutic cancer vaccines has been the development of uncomplicated/reproducible methods for the quantification of vaccine immunization efficacy in peripheral blood of cancer patients. Existing methods are limited by lack of functional information (tetramers), difficulties with standardization/reproducibility [enzyme-linked immunosorbent spot (ELISPOT)] and reliance on endogenous (sample-specific) antigen presentation (cytokine flow cytometry). Herein we present a reproducible method utilizing an artificial antigen-presenting cell platform for flow cytometry-based quantification of the frequency and activation status of peptide-specific cytotoxic T lymphocytes. The methodology [currently presented for cytomegalovirus human leucocyte antigen (HLA)-A2 cognant peptide antigens] allows simultaneous ex vivo quantification of activated (cytokine-producing) and inactive tetramer-positive T cells following HLA class I/peptide/CD28 stimulation independent of endogenous antigen presentation. The simplicity and reliability of the assay provide for high-throughput applications and automation. The utility and application of this method are discussed.

Infrared intensities of some SiH-containing compounds in the gas-phase.

Infrared intensities measured in the gas-phase are reported for CH3SiH3, CH3SiD3, (CH3)2SiH2, (CH3)2SiD2, (SiH3)2CH2, (SiD3)2CH2, Si2H6, SiH2Cl2 and (SiH3)2O. These are compared with theoretical estimates from HF, MP2 and B3LYP calculations with the 6-311G** basis set. Literature values of nuCH intensities per bond from 18 compounds correlate linearly with the values calculated at MP2 and B3LYP levels: the corresponding HF plot is slightly curved. The new HC(Si) data fit these correlations adequately. In similar plots for SiH stretching intensity, the point for SiH2Cl2 is displaced, especially at the HF level. The lack of relation of nuCH or nuSiH intensity to Mulliken atomic charge points to the effect of varying atomic charge flux in the parameter thetamu/thetar. Anomalies associated with nuSiH intensities influenced by chlorine or OR substitution and previously explained by d(pi)-p(pi) bonding are attributed instead to charge flux variation. For silyl groups, deformation band intensities are roughly additive according to the number of such groups. However, this is not the case for the methyl symmetric deformation bands in methyl and dimethyl silanes.

The analysis of costimulatory receptor signaling cascades in normal T lymphocytes using in vitro gene transfer and reporter gene analysis.

Ligation of the antigen receptor and costimulatory receptors on the surface of T lymphocytes initiates intracellular signals that regulate cell-cycle progression and cell differentiation. To effectively manipulate the activation of T cells for immunotherapeutic applications, it will be important to understand how these signaling pathways are integrated to control specific gene transcription events. Here we describe a novel transient transfection procedure that efficiently introduces DNA into non-dividing normal human and murine T lymphocytes while maintaining high cell viability. Using this technique, reporter genes can be introduced to characterize intracellular signaling pathways that regulate specific gene transcription events in normal T-lymphocyte populations. We show that the CD28 receptor can be differentially coupled to downstream signaling pathways in different T-lymphocyte populations. In addition, we demonstrate that a gene encoding a tagged constitutively active mitogen-activated kinase kinase-1 protein can be transfected and rapidly expressed to regulate the expression of Bcl-2 in normal thymocytes.

Infrared and quantum-chemical studies of the structure and vibrations of trisilylamine.

New infrared spectra are reported for variously labelled trisilylamines. Quantum-chemical (QC) calculations of structure and force field have been made at HF, MP2 and B3LYP levels, each with the 6-31G* and 6-311G** basis sets. At each level, a minimum in the potential surface occurs at the C3h configuration. No evidence was found for a significant variation in SiH bond length with orientation. The appearance of two bands in the infrared spectrum of N(SiH3)3 in the 2nuSiH region is explained by local mode theory in terms of transitions to (200) and (110) levels. In the gas phase, signal averaging appears to occur in the nuSiH region in the infrared spectrum, but not in the Raman. In solid films, both IR and Raman spectra indicate the presence of a range of SiH bond strengths, corresponding to an absence of any site symmetry. Each complete QC calculated force field was fitted to the frequencies observed for N(SiH3)3 and N(SiD3)3, using nine independent scale factors. An interaction force constant between nu(as)NSi3 and delta(s)SiH3 motions required further adjustment. Unobserved frequencies in the d0 and d9 species are predicted. The out-of-plane skeletal bending mode is expected to lie between 170 and 200 cm(-1). Unscaled SiH3 torsional frequencies vary from 64 cm(-1) upwards. The effect of the presence of three internal rotors on the spectra throughout calls for theoretical study.

Promoter clearance by RNA polymerase II is an extended, multistep process strongly affected by sequence.

We have characterized RNA polymerase II complexes halted from +16 to +49 on two templates which differ in the initial 20 nucleotides (nt) of the transcribed region. On a template with a purine-rich initial transcript, most complexes halted between +20 and +32 become arrested and cannot resume RNA synthesis without the SII elongation factor. These arrested complexes all translocate upstream to the same location, such that about 12 to 13 bases of RNA remain in each of the complexes after SII-mediated transcript cleavage. Much less arrest is observed over this same region with a second template in which the initially transcribed region is pyrimidine rich, but those complexes which do arrest on the second template also translocate upstream to the same location observed with the first template. Complexes stalled at +16 to +18 on either template do not become arrested. Complexes stalled at several locations downstream of +35 become partially arrested, but these more promoter-distal arrested complexes translocate upstream by less than 10 nt; that is, they do not translocate to a common, far-upstream location. Kinetic studies with nonlimiting levels of nucleoside triphosphates reveal strong pausing between +20 and +30 on both templates. These results indicate that promoter clearance by RNA polymerase II is at least a two-step process: a preclearance escape phase extending up to about +18 followed by an unstable clearance phase which extends over the formation of 9 to 17 more bonds. Polymerases halted during the clearance phase translocate upstream to the preclearance location and arrest in at least one sequence context.

Dendritic cell modulation by 1alpha,25 dihydroxyvitamin D3 and its analogs: a vitamin D receptor-dependent pathway that promotes a persistent state of immaturity in vitro and in vivo.

Dendritic cells (DCs) play a central role in regulating immune activation and responses to self. DC maturation is central to the outcome of antigen presentation to T cells. Maturation of DCs is inhibited by physiological levels of 1alpha,25 dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] and a related analog, 1alpha,25(OH)(2)-16-ene-23-yne-26,27-hexafluoro-19-nor-vitamin D(3) (D(3) analog). Conditioning of bone marrow cultures with 10(-10) M D(3) analog resulted in accumulation of immature DCs with reduced IL-12 secretion and without induction of transforming growth factor beta1. These DCs retained an immature phenotype after withdrawal of D(3) analog and exhibited blunted responses to maturing stimuli (CD40 ligation, macrophage products, or lipopolysaccharide). Resistance to maturation depended on the presence of the 1alpha,25(OH)(2)D(3) receptor (VDR). In an in vivo model of DC-mediated antigen-specific sensitization, D(3) analog-conditioned DCs failed to sensitize and, instead, promoted prolonged survival of subsequent skin grafts expressing the same antigen. To investigate the physiologic significance of 1alpha,25(OH)(2)D(3)/VDR-mediated modulation of DC maturity we analyzed DC populations from mice lacking VDR. Compared with wild-type animals, VDR-deficient mice had hypertrophy of subcutaneous lymph nodes and an increase in mature DCs in lymph nodes but not spleen. We conclude that 1alpha,25(OH)(2)D(3)/VDR mediates physiologically relevant inhibition of DC maturity that is resistant to maturational stimuli and modulates antigen-specific immune responses in vivo.

Maturation versus death of developing double-positive thymocytes reflects competing effects on Bcl-2 expression and can be regulated by the intensity of CD28 costimulation.

Immature double-positive (DP) thymocytes mature into CD4(+)CD8(-) cells in response to coengagement of TCR with any of a variety of cell surface "coinducer" receptors, including CD2. In contrast, DP thymocytes are signaled to undergo apoptosis by coengagement of TCR with CD28 costimulatory receptors, but the molecular basis for DP thymocyte apoptosis by TCR plus CD28 coengagement is not known. In the present study, we report that TCR plus CD28 coengagement does not invariably induce DP thymocyte apoptosis but, depending on the intensity of CD28 costimulation, can induce DP thymocyte maturation. We demonstrate that distinct but interacting signal transduction pathways mediate DP thymocyte maturation signals and DP thymocyte apoptotic signals. Specifically, DP maturation signals are transduced by the extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and up-regulate expression of the antiapoptotic protein Bcl-2. In contrast, the apoptotic response stimulated by CD28 costimulatory signals is mediated by ERK/MAPK-independent pathways. Importantly, when TCR-activated thymocytes are simultaneously coengaged by both CD28 and CD2 receptors, CD28 signals can inhibit ERK/MAPK-dependent Bcl-2 protein up-regulation. Thus, there is cross-talk between the signal transduction pathways that transduce apoptotic and maturation responses, enabling CD28-initiated signal transduction pathways to both stimulate DP thymocyte apoptosis and also negatively regulate maturation responses initiated by TCR plus CD2 coengagement.

Potent inhibition of dendritic cell differentiation and maturation by vitamin D analogs.

We show that the immunosuppressive effects of 1alpha, 25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) are due, in part, to inhibition of the T cell stimulatory functions of dendritic cells (DCs). Addition of 10(-12) and 10(-8) M 1alpha,25(OH)(2)D(3) to murine DC cultures resulted in a concentration-dependent reduction in levels of class II MHC and the co-stimulatory ligands B7-1, B7-2, and CD40 without affecting the number of DCs generated. Higher concentrations of 1alpha,25(OH)(2)D(3) reduced DC yield. The capacity of DCs to induce proliferation of purified allogeneic T cells was reduced by 1alpha,25(OH)(2)D(3). The vitamin D(3) analog, 1alpha,25(OH)(2)-16-ene-23-yne-26,27-hexafluoro-19-nor -D(3), exerted identical effects at 100-fold lower concentrations. Inhibition of DC maturation and stimulatory function was absent in cultures from mice genetically lacking vitamin D receptors (VDR). Vitamin D analogs effectively reduce DC function via VDR-dependent pathways.

Inhibition of interleukin-1-stimulated NF-kappaB RelA/p65 phosphorylation by mesalamine is accompanied by decreased transcriptional activity.

Nuclear factor kappaB (NF-kappaB) is an inducible transcription factor that regulates genes important in immunity and inflammation. The activity of NF-kappaB is highly regulated: transcriptionally active NF-kappaB proteins are sequestered in the cytoplasm by inhibitory proteins, IkappaB. A variety of extracellular signals, including interleukin-1 (IL-1), activate NF-kappaB by inducing phosphorylation and degradation of IkappaB, allowing nuclear translocation and DNA binding of NF-kappaB. Many of the stimuli that activate NF-kappaB by inducing IkappaB degradation also cause phosphorylation of the NF-kappaB RelA (p65) polypeptide. The transactivating capacity of RelA is positively regulated by phosphorylation, suggesting that in addition to cytosolic sequestration by IkappaB, phosphorylation represents another mechanism for control of NF-kappaB activity. In this report, we demonstrate that mesalamine, an anti-inflammatory aminosalicylate, dose-dependently inhibits IL-1-stimulated NF-kappaB-dependent transcription without preventing IkappaB degradation or nuclear translocation and DNA binding of the transcriptionally active NF-kappaB proteins, RelA, c-Rel, or RelB. Mesalamine was found to inhibit IL-1-stimulated RelA phosphorylation. These data suggest that pharmacologic modulation of the phosphorylation status of RelA regulates the transcriptional activity of NF-kappaB, independent of nuclear translocation and DNA binding. These findings highlight the importance of inducible phosphorylation of RelA in the control of NF-kappaB activity.