A two-step species-specific 16S rRNA PCR assay for the detection of Taylorella equigenitalis in horses

<p/> <p>A two-step PCR assay was developed for the molecular detection of Taylorella equigenitalis, a Gram-negative genital bacterial pathogen in horses. Two specific oligonucleotide primers (TE16SrRNABCHf [25mer] and TE16SrRNABCHr [29mer]) were designed from multiple alignments of the 16S rRNA gene loci of several closely related taxa, including T. asinigenitalis. Subsequent enhanced surveillance of 250 Thoroughbred animals failed to detect the presence of this organism directly from clinical swabs taken from the genital tract of mares and stallions. Such a molecular approach offers a sensitive and specific alternative to conventional culture techniques, and has the potential to lead to improved diagnosis and subsequent management of horses involved in breeding programmes.</p

PKA Phosphorylation of NDE1 Is DISC1/PDE4 Dependent and Modulates Its Interaction with LIS1 and NDEL1

Nuclear distribution factor E-homolog 1 (NDE1), Lissencephaly 1 (LIS1), and NDE-like 1 (NDEL1) together participate in essential neurodevelopmental processes, including neuronal precursor proliferation and differentiation, neuronal migration, and neurite out-growth. NDE1/LIS1/NDEL1 interacts with Disrupted in Schizophrenia 1 (DISC1) and the cAMP-hydrolyzing enzyme phosphodiesterase 4 (PDE4). DISC1, PDE4, NDE1, and NDEL1 have each been implicated as genetic risk factors for major mental illness. Here, we demonstrate that DISC1 and PDE4 modulate NDE1 phosphorylation by cAMP-dependent protein kinase A (PKA) and identify a novel PKA substrate site on NDE1 at threonine-131 (T131). Homology modeling predicts that phosphorylation at T131 modulates NDE1–LIS1 and NDE1–NDEL1 interactions, which we confirm experimentally. DISC1–PDE4 interaction thus modulates organization of the NDE1/NDEL1/LIS1 complex. T131-phosphorylated NDE1 is present at the postsynaptic density, in proximal axons, within the nucleus, and at the centrosome where it becomes substantially enriched during mitosis. Mutation of the NDE1 T131 site to mimic PKA phosphorylation inhibits neurite outgrowth. Thus PKA-dependent phosphorylation of the NDE1/LIS1/NDEL1 complex is DISC1–PDE4 modulated and likely to regulate its neural functions

Proteomic Analysis Reveals Age-related Changes in Tendon Matrix Composition, with Age- and Injury-specific Matrix Fragmentation

Energy storing tendons, such as the human Achilles and equine superficial digital flexor tendon (SDFT), are highly prone to injury, the incidence of which increases with aging. The cellular and molecular mechanisms that result in increased injury in aged tendons are not well established but are thought to result in altered matrix turnover. However, little attempt has been made to fully characterize the tendon proteome nor determine how the abundance of specific tendon proteins changes with aging and/or injury. The aim of this study was, therefore, to assess the protein profile of normal SDFTs from young and old horses using label-free relative quantification to identify differentially abundant proteins and peptide fragments between age groups. The protein profile of injured SDFTs from young and old horses was also assessed. The results demonstrate distinct proteomic profiles in young and old tendon, with alterations in the levels of proteins involved in matrix organization and regulation of cell tension. Furthermore, we identified several new peptide fragments (neopeptides) present in aged tendons, suggesting that there are age-specific cleavage patterns within the SDFT. Proteomic profile also differed between young and old injured tendon, with a greater number of neopeptides identified in young injured tendon. This study has increased the knowledge of molecular events associated with tendon aging and injury, suggesting that maintenance and repair of tendon tissue may be reduced in aged individuals and may help to explain why the risk of injury increases with aging

DISC1 regulates N-methyl-D-aspartate receptor dynamics:abnormalities induced by a Disc1 mutation modelling a translocation linked to major mental illness

Abstract The neuromodulatory gene DISC1 is disrupted by a t(1;11) translocation that is highly penetrant for schizophrenia and affective disorders, but how this translocation affects DISC1 function is incompletely understood. N-methyl-D-aspartate receptors (NMDAR) play a central role in synaptic plasticity and cognition, and are implicated in the pathophysiology of schizophrenia through genetic and functional studies. We show that the NMDAR subunit GluN2B complexes with DISC1-associated trafficking factor TRAK1, while DISC1 interacts with the GluN1 subunit and regulates dendritic NMDAR motility in cultured mouse neurons. Moreover, in the first mutant mouse that models DISC1 disruption by the translocation, the pool of NMDAR transport vesicles and surface/synaptic NMDAR expression are increased. Since NMDAR cell surface/synaptic expression is tightly regulated to ensure correct function, these changes in the mutant mouse are likely to affect NMDAR signalling and synaptic plasticity. Consistent with these observations, RNASeq analysis of the translocation carrier-derived human neurons indicates abnormalities of excitatory synapses and vesicle dynamics. RNASeq analysis of the human neurons also identifies many differentially expressed genes previously highlighted as putative schizophrenia and/or depression risk factors through large-scale genome-wide association and copy number variant studies, indicating that the translocation triggers common disease pathways that are shared with unrelated psychiatric patients. Altogether, our findings suggest that translocation-induced disease mechanisms are likely to be relevant to mental illness in general, and that such disease mechanisms include altered NMDAR dynamics and excitatory synapse function. This could contribute to the cognitive disorders displayed by translocation carriers

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Autosomal dominant STAT6 Gain of function causes severe atopy associated with lymphoma

The transcription factor STAT6 (Signal Transducer and Activator of Transcription 6) is a key regulator of Th2 (T-helper 2) mediated allergic inflammation via the IL-4 (interleukin-4) JAK (Janus kinase)/STAT signalling pathway. We identified a novel heterozygous germline mutation STAT6 c.1255G > C, p.D419H leading to overactivity of IL-4 JAK/STAT signalling pathway, in a kindred affected by early-onset atopic dermatitis, food allergy, eosinophilic asthma, anaphylaxis and follicular lymphoma. STAT6 D419H expression and functional activity were compared with wild type STAT6 in transduced HEK293T cells and to healthy control primary skin fibroblasts and peripheral blood mononuclear cells (PBMC). We observed consistently higher STAT6 levels at baseline and higher STAT6 and phosphorylated STAT6 following IL-4 stimulation in D419H cell lines and primary cells compared to wild type controls. The pSTAT6/STAT6 ratios were unchanged between D419H and control cells suggesting that elevated pSTAT6 levels resulted from higher total basal STAT6 expression. The selective JAK1/JAK2 inhibitor ruxolitinib reduced pSTAT6 levels in D419H HEK293T cells and patient PBMC. Nuclear staining demonstrated increased STAT6 in patient fibroblasts at baseline and both STAT6 and pSTAT6 after IL-4 stimulation. We also observed higher transcriptional upregulation of downstream genes (XBP1 and EPAS1) in patient PBMC. Our study confirms STAT6 gain of function (GOF) as a novel monogenetic cause of early onset atopic disease. The clinical association of lymphoma in our kindred, along with previous data linking somatic STAT6 D419H mutations to follicular lymphoma suggest that patients with STAT6 GOF disease may be at higher risk of lymphomagenesis

Understanding the Role of DISC1 in Psychiatric Disease and during Normal Development

The biology of schizophrenia is complex with multiple hypotheses (dopamine, glutamate, neurodevelopmental) well supported to underlie the disease.Pathways centered on the risk factor “disrupted in schizophrenia1”(DISC1)maybe able to explain and unite these disparate hypotheses and will be the topic of this mini-symposium preview. Nearly a decade after its original identification at the center of a translocation breakpoint in a large Scottish family that was associated with major psychiatric disease, we are starting to obtain credible insights into its function and role in disease etiology. This preview will highlight a number of exciting areas of current DISC1 research that are revealing roles for DISC1 during normal brain development and also in the disease state. Together these different threads will provide a timely and exciting overview of the DISC1 field and its potential in furthering our understanding of psychiatric diseases and in developing new therapies

Comparative evaluation of aqueous humor viscosity

OBJECTIVE: To evaluate aqueous humor viscosity in the raptor, dog, cat, and horse, with a primary focus on the barred owl (Strix varia). ANIMALS STUDIED: Twenty-six raptors, ten dogs, three cats, and one horse. PROCEDURE: Animals were euthanized for reasons unrelated to this study. Immediately, after horizontal and vertical corneal dimensions were measured, and anterior chamber paracentesis was performed to quantify anterior chamber volume and obtain aqueous humor samples for viscosity analysis. Dynamic aqueous humor viscosity was measured using a dynamic shear rheometer (AR 1000 TA Instruments, New Castle, DE, USA) at 20 °C. Statistical analysis included descriptive statistics, unpaired t-tests, and Tukey\u27s test to evaluate the mean ± standard deviation for corneal diameter, anterior chamber volume, and aqueous humor viscosity amongst groups and calculation of Spearman\u27s coefficient for correlation analyses. RESULTS: The mean aqueous humor viscosity in the barred owl was 14.1 centipoise (cP) ± 9, cat 4.4 cP ± 0.2, and dog 2.9 cP ± 1.3. The aqueous humor viscosity for the horse was 1 cP. CONCLUSIONS: Of the animals evaluated in this study, the raptor aqueous humor was the most viscous. The aqueous humor of the barred owl is significantly more viscous than the dog (P \u3c 0.0001). The aqueous humor viscosity of the raptor, dog, cat, and horse can be successfully determined using a dynamic shear rheometer

Semantic Alignment between ICD-11 and SNOMED CT

Due to fundamental differences in design and editorial policies, semantic interoperability between two de facto standard terminologies in the healthcare domain-the International Classification of Diseases (ICD) and SNOMED CT (SCT), requires combining two different approaches: (i) axiom-based, which states logically what is universally true, using an ontology language such as OWL; (ii) rule-based, expressed as queries on the axiom-based knowledge. We present the ICD-SCT harmonization process including: a) a new architecture for ICD-11, b) a protocol for the semantic alignment of ICD and SCT, and c) preliminary results of the alignment applied to more than half the domain currently covered by the draft ICD-1