Genetics of ankylosing spondylitis - Insights into pathogenesis

Ankylosing spondylitis (AS), an immune-mediated arthritis, is the prototypic member of a group of conditions known as spondyloarthropathies that also includes reactive arthritis, psoriatic arthritis and enteropathic arthritis. Patients with these conditions share a clinical predisposition for spinal and pelvic joint dysfunction, as well as genetic associations, notably with HLA-B*27. Spondyloarthropathies are characterized by histopathological inflammation in entheses (regions of high mechanical stress where tendons and ligaments insert into bone) and in the subchondral bone marrow, and by abnormal osteoproliferation at involved sites. The association of AS with HLA-B*27, first described >40 years ago, led to hope that the cause of the disease would be rapidly established. However, even though many theories have been advanced to explain how HLA-B*27 is involved in AS, no consensus about the answers to this question has been reached, and no successful treatments have yet been developed that target HLA-B27 or its functional pathways. Over the past decade, rapid progress has been made in discovering further genetic associations with AS that have shed new light on the aetiopathogenesis of the disease. Some of these discoveries have driven translational ideas, such as the repurposing of therapeutics targeting the cytokines IL-12 and IL-23 and other factors downstream of this pathway. AS provides an excellent example of how hypothesis-free research can lead to major advances in understanding pathogenesis and to the development of innovative therapeutic strategies.

Identification of IDUA and WNT16 phosphorylation-related non-synonymous polymorphisms for bone mineral density in meta-analyses of genome-wide association studies

Protein phosphorylation regulates a wide variety of cellular processes. Thus, we hypothesize that single-nucleotide polymorphisms (SNPs) that may modulate protein phosphorylation could affect osteoporosis risk. Based on a previous conventional genome-wide association (GWA) study, we conducted a three-stage meta-analysis targeting phosphorylation-related SNPs (phosSNPs) for femoral neck (FN)-bone mineral density (BMD), total hip (HIP)-BMD, and lumbar spine (LS)-BMD phenotypes. In stage 1, 9593 phosSNPs were meta-analyzed in 11,140 individuals of various ancestries. Genome-wide significance (GWS) and suggestive significance were defined by α = 5.21 × 10–6 (0.05/9593) and 1.00 × 10–4, respectively. In stage 2, nine stage 1–discovered phosSNPs (based on α = 1.00 × 10–4) were in silico meta-analyzed in Dutch, Korean, and Australian cohorts. In stage 3, four phosSNPs that replicated in stage 2 (based on α = 5.56 × 10–3, 0.05/9) were de novo genotyped in two independent cohorts. IDUA rs3755955 and rs6831280, and WNT16 rs2707466 were associated with BMD phenotypes in each respective stage, and in three stages combined, achieving GWS for both FN-BMD (p = 8.36 × 10–10, p = 5.26 × 10–10, and p = 3.01 × 10–10, respectively) and HIP-BMD (p = 3.26 × 10–6, p = 1.97 × 10–6, and p = 1.63 × 10–12, respectively). Although in vitro studies demonstrated no differences in expressions of wild-type and mutant forms of IDUA and WNT16B proteins, in silico analyses predicts that WNT16 rs2707466 directly abolishes a phosphorylation site, which could cause a deleterious effect on WNT16 protein, and that IDUA phosSNPs rs3755955 and rs6831280 could exert indirect effects on nearby phosphorylation sites. Further studies will be required to determine the detailed and specific molecular effects of these BMD-associated non-synonymous variants. © 2015 American Society for Bone and Mineral Research.

ERAP2 functional knockout in humans does not alter surface heavy chains or HLA-B27, inflammatory cytokines or endoplasmic reticulum stress markers

Introduction Single nucleotide polymorphisms in ERAP2 are strongly associated with ankylosing spondylitis (AS). One AS-associated single nucleotide polymorphism, rs2248374, causes a truncated ERAP2 protein that is degraded by nonsense-mediated decay. Approximately 25% of the populations of European ancestry are therefore natural ERAP2 knockouts. We investigated the effect of this associated variant on HLA class I allele presentation, surface heavy chains, endoplasmic reticulum (ER) stress markers and cytokine gene transcription in AS. Methods Patients with AS and healthy controls with either AA or GG homozygous status for rs2248374 were studied. Antibodies to CD14, CD19-ECD, HLA-A-B-C, Valpha7.2, CD161, anti-HC10 and anti-HLA-B27 were used to analyse peripheral blood mononuclear cells. Expression levels of ER stress markers (GRP78 and CHOP) and proinflammatory genes (tumour necrosis factor (TNF), IL6, IL17 and IL22) were assessed by qPCR. Results There was no significant difference in HLAclass I allele presentation or major histocompatibility class I heavy chains or ER stress markers GRP78 and CHOP or proinflammatory gene expression between genotypes for rs2248374 either between cases, between cases and controls, and between controls. Discussion Large differences were not seen in HLAB27 expression or cytokine levels between subjects with and without ERAP2 in AS cases and controls. This suggests that ERAP2 is more likely to influence AS risk through other mechanisms.

Mutations in known monogenic high bone mass loci only explain a small proportion of high bone mass cases

High bone mass (HBM) can be an incidental clinical finding; however, monogenic HBM disorders (eg, LRP5 or SOST mutations) are rare. We aimed to determine to what extent HBM is explained by mutations in known HBM genes. A total of 258 unrelated HBM cases were identified from a review of 335,115 DXA scans from 13 UK centers. Cases were assessed clinically and underwent sequencing of known anabolic HBM loci: LRP5 (exons 2, 3, 4), LRP4 (exons 25, 26), SOST (exons 1, 2, and the van Buchem's disease [VBD] 52-kb intronic deletion 3'). Family members were assessed for HBM segregation with identified variants. Three-dimensional protein models were constructed for identified variants. Two novel missense LRP5 HBM mutations ([c.518C>T; p.Thr173Met], [c.796C>T; p.Arg266Cys]) were identified, plus three previously reported missense LRP5 mutations ([c.593A>G; p.Asn198Ser], [c.724G>A; p.Ala242Thr], [c.266A>G; p.Gln89Arg]), associated with HBM in 11 adults from seven families. Individuals with LRP5 HBM ( approximately prevalence 5/100,000) displayed a variable phenotype of skeletal dysplasia with increased trabecular BMD and cortical thickness on HRpQCT, and gynoid fat mass accumulation on DXA, compared with both non-LRP5 HBM and controls. One mostly asymptomatic woman carried a novel heterozygous nonsense SOST mutation (c.530C>A; p.Ser177X) predicted to prematurely truncate sclerostin. Protein modeling suggests the severity of the LRP5-HBM phenotype corresponds to the degree of protein disruption and the consequent effect on SOST-LRP5 binding. We predict p.Asn198Ser and p.Ala242Thr directly disrupt SOST binding; both correspond to severe HBM phenotypes (BMD Z-scores +3.1 to +12.2, inability to float). Less disruptive structural alterations predicted from p.Arg266Cys, p.Thr173Met, and p.Gln89Arg were associated with less severe phenotypes (Z-scores +2.4 to +6.2, ability to float). In conclusion, although mutations in known HBM loci may be asymptomatic, they only account for a very small proportion ( approximately 3%) of HBM individuals, suggesting the great majority are explained by either unknown monogenic causes or polygenic inheritance.

Reproducibility of scratch assays is affected by the initial degree of confluence: Experiments, modelling and model selection

Scratch assays are difficult to reproduce. Here we identify a previously overlooked source of variability which could partially explain this difficulty. We analyse a suite of scratch assays in which we vary the initial degree of confluence (initial cell density). Our results indicate that the rate of re-colonisation is very sensitive to the initial density. To quantify the relative roles of cell migration and proliferation, we calibrate the solution of the Fisher–Kolmogorov model to cell density profiles to provide estimates of the cell diffusivity, D, and the cell proliferation rate, λ. This procedure indicates that the estimates of D and λ are very sensitive to the initial density. This dependence suggests that the Fisher–Kolmogorov model does not accurately represent the details of the collective cell spreading process, since this model assumes that D and λ are constants that ought to be independent of the initial density. Since higher initial cell density leads to enhanced spreading, we also calibrate the solution of the Porous–Fisher model to the data as this model assumes that the cell flux is an increasing function of the cell density. Estimates of D and λ associated with the Porous–Fisher model are less sensitive to the initial density, suggesting that the Porous–Fisher model provides a better description of the experiments.

Prolonged consumption of sucrose in a binge-like manner, alters the morphology of medium spiny neurons in the nucleus accumbens shell

The modern diet has become highly sweetened, resulting in unprecedented levels of sugar consumption, particularly among adolescents. While chronic long-term sugar intake is known to contribute to the development of metabolic disorders including obesity and type II diabetes, little is known regarding the direct consequences of long-term, binge-like sugar consumption on the brain. Because sugar can cause the release of dopamine in the nucleus accumbens (NAc) similarly to drugs of abuse, we investigated changes in the morphology of neurons in this brain region following short- (4 weeks) and long-term (12 weeks) binge-like sucrose consumption using an intermittent two-bottle choice paradigm. We used Golgi-Cox staining to impregnate medium spiny neurons (MSNs) from the NAc core and shell of short- and long-term sucrose consuming rats and compared these to age-matched water controls. We show that prolonged binge-like sucrose consumption significantly decreased the total dendritic length of NAc shell MSNs compared to age-matched control rats. We also found that the restructuring of these neurons resulted primarily from reduced distal dendritic complexity. Conversely, we observed increased spine densities at the distal branch orders of NAc shell MSNs from long-term sucrose consuming rats. Combined, these results highlight the neuronal effects of prolonged binge-like intake of sucrose on NAc shell MSN morphology.

Impact of resistance exercise on ribosome biogenesis is acutely regulated by post-exercise recovery strategies

Muscle hypertrophy occurs following increased protein synthesis, which requires activation of the ribosomal complex. Additionally, increased translational capacity via elevated ribosomal RNA (rRNA) synthesis has also been implicated in resistance training-induced skeletal muscle hypertrophy. The time course of ribosome biogenesis following resistance exercise (RE) and the impact exerted by differing recovery strategies remains unknown. In the present study, the activation of transcriptional regulators, the expression levels of pre-rRNA, and mature rRNA components were measured through 48 h after a single-bout RE. In addition, the effects of either low-intensity cycling (active recovery, ACT) or a cold-water immersion (CWI) recovery strategy were compared. Nine male subjects performed two bouts of high-load RE randomized to be followed by 10 min of either ACT or CWI. Muscle biopsies were collected before RE and at 2, 24, and 48 h after RE. RE increased the phosphorylation of the p38-MNK1-eIF4E axis, an effect only evident with ACT recovery. Downstream, cyclin D1 protein, total eIF4E, upstream binding factor 1 (UBF1), and c-Myc proteins were all increased only after RE with ACT. This corresponded with elevated abundance of the pre-rRNAs (45S, ITS-28S, ITS-5.8S, and ETS-18S) from 24 h after RE with ACT. In conclusion, coordinated upstream signaling and activation of transcriptional factors stimulated pre-rRNA expression after RE. CWI, as a recovery strategy, markedly blunted these events, suggesting that suppressed ribosome biogenesis may be one factor contributing to the impaired hypertrophic response observed when CWI is used regularly after exercise.

Impact of exercise selection on hamstring muscle activation

Objective: To determine the extent to which different strength training exercises selectively activate the commonly injured biceps femoris long head (BFLH) muscle. Methods: This two-part observational study recruited 24 recreationally active males. Part 1 explored the amplitudes and the ratios of lateral to medial hamstring (BF/MH) normalised electromyography (nEMG) during the concentric and eccentric phases of 10 common strength training exercises. Part 2 used functional magnetic resonance imaging (fMRI) to determine the spatial patterns of hamstring activation during two exercises which i) most selectively, and ii) least selectively activated the BF in part 1. Results: Eccentrically, the largest BF/MH nEMG ratio was observed in the 45° hip extension exercise and the lowest was observed in the Nordic hamstring (NHE) and bent-knee bridge exercises. Concentrically, the highest BF/MH nEMG ratio was observed during the lunge and 45° hip extension and the lowest was observed for the leg curl and bent-knee bridge. fMRI revealed a greater BFLH to semitendinosus activation ratio in the 45° hip extension than the NHE (p<0.001). The T2 increase after hip extension for BFLH, semitendinosus and semimembranosus muscles were greater than that for BFSH (p<0.001). During the NHE, the T2 increase was greater for the semitendinosus than for the other hamstrings (p≤0.002). Conclusion: This investigation highlights the non-uniformity of hamstring activation patterns in different tasks and suggests that hip extension exercise more selectively activates the BFLH while the NHE preferentially recruits the semitendinosus. These findings have implications for strength training interventions aimed at preventing hamstring injury.

Craving mediates stress in predicting lapse during alcohol dependence treatment

Background Stress, craving, and depressed mood have all been implicated in alcohol use treatment lapses. Few studies have examined all 3 factors. Progress has been limited because of difficulties with craving assessment. The Alcohol Craving Experience Questionnaire (ACE) is a new measure of alcohol craving. It is both psychometrically sound and conceptually rigorous. This prospective study examines a stress–treatment response model that incorporates mediation by craving and moderation by depressed mood and pharmacotherapy. Methods Five hundred and thirty-nine consecutively treated alcohol-dependent patients voluntarily participated in an abstinence-based 12-week cognitive-behavioral therapy (CBT) program at a hospital alcohol and drug outpatient clinic. Measures of stress, craving, depressed mood, and alcohol dependence severity were administered prior to treatment. Treatment lapse and treatment dropout were assessed over the 12-week program duration. Results Patients reporting greater stress experienced stronger and more frequent cravings. Stronger alcohol craving predicted lapse, after controlling for dependence severity, stress, depression, and pharmacotherapy. Alcohol craving mediated stress to predict lapse. Depressed mood and anticraving medication were not significant moderators. Conclusions Among treatment seeking, alcohol-dependent patients, craving mediated the relationship between stress and lapse. The effect was not moderated by depressed mood or anticraving medication.

The complete mitochondrial genome of the eastern grey kangaroo (Macropus giganteus)

We present the complete mitochondrial genome (accession number: LK995454) of an iconic Australian species, the eastern grey kangaroo (Macropus giganteus). The mitogenomic organization is consistent with other marsupials, encoding 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes, an origin of light strand replication and a control region or Dloop. No repetitive sequences were detected in the control region. The M. giganteus mitogenome exemplifies a combination of tRNA gene order and structural peculiarities that appear to be unique to marsupials. We present a maximum likelihood phylogeny based on complete mitochondrial protein and RNA coding sequences that confirms the phylogenetic position of the grey kangaroo among macropodids.

Geomolecular dating and the origin of placental mammals

In modern evolutionary divergence analysis the role of geological information extends beyond providing a timescale, to informing molecular rate variation across the tree. Here I consider the implications of this development. I use fossil calibrations to test the accuracy of models of molecular rate evolution for placental mammals, and reveal substantial misspecification associated with life history rate correlates. Adding further calibrations to reduce dating errors at specific nodes unfortunately tends to transfer underlying rate errors to adjacent branches. Thus, tight calibration across the tree is vital to buffer against rate model errors. I argue that this must include allowing maximum bounds to be tight when good fossil records permit, otherwise divergences deep in the tree will tend to be inflated by the interaction of rate errors and asymmetric confidence in minimum and maximum bounds. In the case of placental mammals I sought to reduce the potential for transferring calibration and rate model errors across the tree by focusing on well-supported calibrations with appropriately conservative maximum bounds. The resulting divergence estimates are younger than others published recently, and provide the long-anticipated molecular signature for the placental mammal radiation observed in the fossil record near the 66 Ma Cretaceous–Paleogene extinction event.

The emerging world of small ORFs

Small open reading frames (sORFs) are an often overlooked feature of plant genomes. Initially found in plant viral RNAs and considered an interesting curiosity, an increasing number of these sORFs have been shown to encode functional peptides or play a regulatory role. The recent discovery that many of these sORFs initiate with start codons other than AUG, together with the identification of functional small peptides encoded in supposedly noncoding primary miRNA transcripts (pri-miRs), has drastically increased the number of potentially functional sORFs within the genome. Here we review how advances in technology, notably ribosome profiling (RP) assays, are complementing bioinformatics and proteogenomic methods to provide powerful ways to identify these elusive features of plant genomes, and highlight the regulatory roles sORFs can play.

Guided respiration mindfulness therapy: Development and evaluation of a brief therapist training program

The present paper describes the development and evaluation of a standardized multi-component therapist training program in guided respiration mindfulness therapy (GRMT). GMRT is a manual-based, experimental clinical intervention involving concentrated focus on sustained self-regulation of breathing, application of mindfulness to emergent somatic experience and relaxation. Therapists (n = 61) new to the approach attended a 2-day experiential workshop and were evaluated pre-post workshop for change in intervention knowledge, as well as change in mindfulness. These trainees also participated in post-workshop focus group sessions to explore perception of the intervention. A subset of 40 therapists participated in a second training component, and 14 of these were rated for competent delivery of the intervention during participation in a clinical trial. During training, therapists personally received the treatment giving the opportunity to assess treatment session (n = 283) impact on sense of wellbeing. Results indicated a brief focused training program can equip therapists with basic knowledge and skills required to deliver the standardized manual-based treatment. Qualitative analysis of focus group sessions showed that therapists endorsed the intervention for clinical use and found it personally beneficial. This research provides a foundation for further evaluation of clinical effectiveness of the intervention.