Inflammation-driven bone formation in a mouse model of ankylosing spondylitis: Sequential not parallel processes

Background Ankylosing spondylitis (AS) is an immune-mediated arthritis particularly targeting the spine and pelvis and is characterised by inflammation, osteoproliferation and frequently ankylosis. Current treatments that predominately target inflammatory pathways have disappointing efficacy in slowing disease progression. Thus, a better understanding of the causal association and pathological progression from inflammation to bone formation, particularly whether inflammation directly initiates osteoproliferation, is required. Methods The proteoglycan-induced spondylitis (PGISp) mouse model of AS was used to histopathologically map the progressive axial disease events, assess molecular changes during disease progression and define disease progression using unbiased clustering of semi-quantitative histology. PGISp mice were followed over a 24-week time course. Spinal disease was assessed using a novel semi-quantitative histological scoring system that independently evaluated the breadth of pathological features associated with PGISp axial disease, including inflammation, joint destruction and excessive tissue formation (osteoproliferation). Matrix components were identified using immunohistochemistry. Results Disease initiated with inflammation at the periphery of the intervertebral disc (IVD) adjacent to the longitudinal ligament, reminiscent of enthesitis, and was associated with upregulated tumor necrosis factor and metalloproteinases. After a lag phase, established inflammation was temporospatially associated with destruction of IVDs, cartilage and bone. At later time points, advanced disease was characterised by substantially reduced inflammation, excessive tissue formation and ectopic chondrocyte expansion. These distinct features differentiated affected mice into early, intermediate and advanced disease stages. Excessive tissue formation was observed in vertebral joints only if the IVD was destroyed as a consequence of the early inflammation. Ectopic excessive tissue was predominantly chondroidal with chondrocyte-like cells embedded within collagen type II- and X-rich matrix. This corresponded with upregulation of mRNA for cartilage markers Col2a1, sox9 and Comp. Osteophytes, though infrequent, were more prevalent in later disease. Conclusions The inflammation-driven IVD destruction was shown to be a prerequisite for axial disease progression to osteoproliferation in the PGISp mouse. Osteoproliferation led to vertebral body deformity and fusion but was never seen concurrent with persistent inflammation, suggesting a sequential process. The findings support that early intervention with anti-inflammatory therapies will be needed to limit destructive processes and consequently prevent progression of AS.

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.

Identification of a novel FGFRL1 MicroRNA target site polymorphism for bone mineral density in meta-analyses of genome-wide association studies

MicroRNAs (miRNAs) are critical post-transcriptional regulators. Based on a previous genome-wide association (GWA) scan, we conducted a polymorphism in microRNAs' Target Sites (poly-miRTS)-centric multistage meta-analysis for lumbar spine (LS)-, total hip (HIP)-, and femoral neck (FN)-bone mineral density (BMD). In stage I, 41,102 poly-miRTSs were meta-analyzed in 7 cohorts with a genome-wide significance (GWS) α=0.05/41,102=1.22×10-6. By applying α=5×10-5 (suggestive significance), 11 poly-miRTSs were selected, with FGFRL1 rs4647940 and PRR5 rs3213550 as top signals for FN-BMD (P-value=7.67×10-6 and 1.58×10-5) in gender-combined sample. In stage II in silico replication (two cohorts), FGFRL1 rs4647940 was the only signal marginally replicated for FN-BMD (P-value=5.08×10-3) at α=0.10/11=9.09×10-3. PRR5 rs3213550 was also selected based on biological significance. In stage III de novo genotyping replication (two cohorts), FGFRL1 rs4647940 was the only signal significantly replicated for FN-BMD (P-value=7.55×10-6) at α=0.05/2=0.025 in gender-combined sample. Aggregating three stages, FGFRL1 rs4647940 was the single stage I-discovered and stages II- and III-replicated signal attaining GWS for FN-BMD (P-value=8.87×10-12). Dual-luciferase reporter assays demonstrated that FGFRL1 3' untranslated region harboring rs4647940 appears to be hsa-miR-140-5p's target site. In a zebrafish microinjection experiment, dre-miR-140-5p is shown to exert a dramatic impact on craniofacial skeleton formation. Taken together, we provided functional evidence for a novel FGFRL1 poly-miRTS rs4647940 in a previously known 4p16.3 locus, and experimental and clinical genetics studies have shown both FGFRL1 and hsa-miR-140-5p are important for bone formation. © The Author 2015. Published by Oxford University Press. All rights reserved.

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.

Whole-exome re-sequencing in a family quartet identifies POP1 mutations as the cause of a novel skeletal dysplasia

Recent advances in DNA sequencing have enabled mapping of genes for monogenic traits in families with small pedigrees and even in unrelated cases. We report the identification of disease-causing mutations in a rare, severe, skeletal dysplasia, studying a family of two healthy unrelated parents and two affected children using whole-exome sequencing. The two affected daughters have clinical and radiographic features suggestive of anauxetic dysplasia (OMIM 607095), a rare form of dwarfism caused by mutations of RMRP. However, mutations of RMRP were excluded in this family by direct sequencing. Our studies identified two novel compound heterozygous loss-of-function mutations in POP1, which encodes a core component of the RNase mitochondrial RNA processing (RNase MRP) complex that directly interacts with the RMRP RNA domains that are affected in anauxetic dysplasia. We demonstrate that these mutations impair the integrity and activity of this complex and that they impair cell proliferation, providing likely molecular and cellular mechanisms by which POP1 mutations cause this severe skeletal dysplasia. © 2011 Glazov et al.

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.

Partial foot amputations may not always be worth the risk of complications

To the Editor: Dillon and colleagues present a challenging perspective on the evidence comparing partial foot amputation (PFA) and below-knee amputation (BKA) outcomes.1,2 Australia's diabetes-related major amputation rates have only recently reduced to international levels3 and we fear that any oversimplistic perspectives may be detrimental to these improved rates and, importantly, to our patients. Thus, we believe these articles1,2 should be read cognisant of some important points...

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.

Modeling transport through an environment crowded by a mixture of obstacles of different shapes and sizes

Many biological environments are crowded by macromolecules, organelles and cells which can impede the transport of other cells and molecules. Previous studies have sought to describe these effects using either random walk models or fractional order diffusion equations. Here we examine the transport of both a single agent and a population of agents through an environment containing obstacles of varying size and shape, whose relative densities are drawn from a specified distribution. Our simulation results for a single agent indicate that smaller obstacles are more effective at retarding transport than larger obstacles; these findings are consistent with our simulations of the collective motion of populations of agents. In an attempt to explore whether these kinds of stochastic random walk simulations can be described using a fractional order diffusion equation framework, we calibrate the solution of such a differential equation to our averaged agent density information. Our approach suggests that these kinds of commonly used differential equation models ought to be used with care since we are unable to match the solution of a fractional order diffusion equation to our data in a consistent fashion over a finite time period.

Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells

Although the importance of clathrin- and caveolin-independent endocytic pathways has recently emerged, key aspects of these routes remain unknown. Using quantitative ultrastructural approaches, we show that clathrin-independent carriers (CLICs) account for approximately three times the volume internalized by the clathrin-mediated endocytic pathway, forming the major pathway involved in uptake of fluid and bulk membrane in fibroblasts. Electron tomographic analysis of the 3D morphology of the earliest carriers shows that they are multidomain organelles that form a complex sorting station as they mature. Proteomic analysis provides direct links between CLICs, cellular adhesion turnover, and migration. Consistent with this, CLIC-mediated endocytosis of key cargo proteins, CD44 and Thy-1, is polarized at the leading edge of migrating fibroblasts, while transient ablation of CLICs impairs their ability to migrate. These studies provide the first quantitative ultrastructural analysis and molecular characterization of the major endocytic pathway in fibroblasts, a pathway that provides rapid membrane turnover at the leading edge of migrating cells.

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.

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.