Comprehensive Proteomics Analysis of Polyhydroxyalkanoate (PHA) Biology in Pseudomonas putida KT2440: The Outer Membrane Lipoprotein OprL is a Newly Identified Phasin.

Pseudomonas putida KT2440 is an important bioplastic-producing industrial microorganism capable of synthesizing the polymeric carbon-rich storage material, polyhydroxyalkanoate (PHA). PHA is sequestered in discrete PHA granules, or carbonosomes, and accumulates under conditions of stress, for example, low levels of available nitrogen. The pha locus responsible for PHA metabolism encodes both anabolic and catabolic enzymes, a transcription factor, and carbonosome-localized proteins termed phasins. The functions of phasins are incompletely understood but genetic disruption of their function causes PHA-related phenotypes. To improve our understanding of these proteins, we investigated the PHA pathways of P.putida KT2440 using three types of experiments. First, we profiled cells grown in nitrogen-limited and nitrogen-excess media using global expression proteomics, identifying sets of proteins found to coordinately increase or decrease within clustered pathways. Next, we analyzed the protein composition of isolated carbonosomes, identifying two new putative components. We carried out physical interaction screens focused on PHA-related proteins, generating a protein-protein network comprising 434 connected proteins. Finally, we confirmed that the outer membrane protein OprL (the Pal component of the Pal-Tol system) localizes to the carbonosome and shows a PHA-related phenotype and therefore is a novel phasin. The combined datasets represent a valuable overview of the protein components of the PHA system in P.putida highlighting the complex nature of regulatory interactions responsive to nutrient stress.

An aggregation inhibitor specific to oligomeric intermediates of Aß42 derived from phage display libraries of stable, small proteins.

The self-assembly of amyloid ß peptide (Aß) to fibrillar and oligomeric aggregates is linked to Alzheimer's disease. Aß binders may serve as inhibitors of aggregation to prevent the generation of neurotoxic species and for the detection of Aß species. A particular challenge involves finding binders to on-pathway oligomers given their transient nature. Here we construct two phage­display libraries built on the highly inert and stable protein scaffold S100G, one containing a six-residue variable surface patch and one harboring a seven-residue variable loop insertion. Monomers and fibrils of Aß40 and Aß42 were separately coupled to silica nanoparticles, using a coupling strategy leading to the presence of oligomers on the monomer beads, and they were used in three rounds of affinity selection. Next-generation sequencing revealed sequence clusters and candidate binding proteins (SXkmers). Two SXkmers were expressed as soluble proteins and tested in terms of aggregation inhibition via thioflavin T fluorescence. We identified an SXkmer with loop­insertion YLTIRLM as an inhibitor of the secondary nucleation of Aß42 and binding analyses using surface plasmon resonance technology, Förster resonance energy transfer, and microfluidics diffusional sizing imply an interaction with intermediate oligomeric species. A linear peptide with the YLTIRLM sequence was found inhibitory but at a lower potency than the more constrained SXkmer loop. We identified an SXkmer with side-patch VI-WI-DD as an inhibitor of Aß40 aggregation. Remarkably, our data imply that SXkmer-YLTIRLM blocks secondary nucleation through an interaction with oligomeric intermediates in solution or at the fibril surface, which is a unique inhibitory mechanism for a library-derived inhibitor.

Macrophage Galactose Lectin Contributes to the Regulation of FVIII (Factor VIII) Clearance in Mice-Brief Report.

BACKGROUND: Although most plasma FVIII (Factor VIII) circulates in complex with VWF (von Willebrand factor), a minority (3%-5%) circulates as free-FVIII, which is rapidly cleared. Consequently, 20% of total FVIII may be cleared as free-FVIII. Critically, the mechanisms of free-FVIII clearance remain poorly understood. However, recent studies have implicated the MGL (macrophage galactose lectin) in modulating VWF clearance. METHODS: Since VWF and FVIII share similar glycosylation, we investigated the role of MGL in FVIII clearance. FVIII binding to MGL was assessed in immunosorbent and cell-based assays. In vivo, FVIII clearance was assessed in MGL1-/- and VWF-/-/FVIII-/- mice. RESULTS: In vitro-binding studies identified MGL as a novel macrophage receptor that binds free-FVIII in a glycan-dependent manner. MGL1-/- and MGL1-/- mice who received an anti-MGL1/2 blocking antibody both showed significantly increased endogenous FVIII activity compared with wild-type mice (P=0.036 and P<0.0001, respectively). MGL inhibition also prolonged the half-life of infused FVIII in FVIII-/- mice. To assess whether MGL plays a role in the clearance of free FVIII in a VWF-independent manner, in vivo clearance experiments were repeated in dual VWF-/-/FVIII-/- mice. Importantly, the rapid clearance of free FVIII in VWF-/-/FVIII-/- mice was significantly (P=0.012) prolonged in the presence of anti-MGL1/2 antibodies. Finally, endogenous plasma FVIII levels in VWF-/- mice were significantly increased following MGL inhibition (P=0.016). CONCLUSIONS: Cumulatively, these findings demonstrate that MGL plays an important role in regulating macrophage-mediated clearance of both VWF-bound FVIII and free-FVIII in vivo. We propose that this novel FVIII clearance pathway may be of particular clinical importance in patients with type 2N or type 3 Von Willebrand disease.

A chromatin-independent role of Polycomb-like 1 to stabilize p53 and promote cellular quiescence.

Polycomb-like proteins 1-3 (PCL1-3) are substoichiometric components of the Polycomb-repressive complex 2 (PRC2) that are essential for association of the complex with chromatin. However, it remains unclear why three proteins with such apparent functional redundancy exist in mammals. Here we characterize their divergent roles in both positively and negatively regulating cellular proliferation. We show that while PCL2 and PCL3 are E2F-regulated genes expressed in proliferating cells, PCL1 is a p53 target gene predominantly expressed in quiescent cells. Ectopic expression of any PCL protein recruits PRC2 to repress the INK4A gene; however, only PCL2 and PCL3 confer an INK4A-dependent proliferative advantage. Remarkably, PCL1 has evolved a PRC2- and chromatin-independent function to negatively regulate proliferation. We show that PCL1 binds to and stabilizes p53 to induce cellular quiescence. Moreover, depletion of PCL1 phenocopies the defects in maintaining cellular quiescence associated with p53 loss. This newly evolved function is achieved by the binding of the PCL1 N-terminal PHD domain to the C-terminal domain of p53 through two unique serine residues, which were acquired during recent vertebrate evolution. This study illustrates the functional bifurcation of PCL proteins, which act in both a chromatin-dependent and a chromatin-independent manner to regulate the INK4A and p53 pathways.

Collagen X Marker Levels are Decreased in Individuals with Achondroplasia.

Collagen X marker (CXM) is a degradation fragment of collagen type X. It is a real-time biomarker of height velocity with established norms. Plasma C-type natriuretic peptide (CNP) and NTproCNP levels have also been found to correlate with growth velocity in the general population and are elevated in individuals with achondroplasia compared with age- and sex-matched controls. Collagen X marker levels in people with fibroblast growth factor receptor 3 (FGFR3)-opathies have never been systematically measured. The objective of this study was to measure CXM in a population of dwarfism caused by FGFR3-opathies. Using the same cohort in which CNP and NTproCNP levels were previously measured, archived serum aliquots from 63 children with achondroplasia, six with hypochondroplasia, and two with thanatophoric dysplasia had CXM concentrations measured. Results were plotted against age- and sex-specific norms, and standard deviation scores were plotted for comparison between clinical diagnoses. CXM levels were significantly decreased (p < 0.0001) in children with achondroplasia compared with age- and sex-matched controls. Temporal patterns of change in CXM levels were sex-dependent. As the FGFR3 pathway was more constitutively active, CXM levels decreased. New tools are emerging to study impact of skeletal dysplasia on growth plate regulation and function.

A Hybrid Architecture (CO-CONNECT) to Facilitate Rapid Discovery and Access to Data Across the United Kingdom in Response to the COVID-19 Pandemic: Development Study.

BACKGROUND: COVID-19 data have been generated across the United Kingdom as a by-product of clinical care and public health provision, as well as numerous bespoke and repurposed research endeavors. Analysis of these data has underpinned the United Kingdom's response to the pandemic, and informed public health policies and clinical guidelines. However, these data are held by different organizations, and this fragmented landscape has presented challenges for public health agencies and researchers as they struggle to find relevant data to access and interrogate the data they need to inform the pandemic response at pace. OBJECTIVE: We aimed to transform UK COVID-19 diagnostic data sets to be findable, accessible, interoperable, and reusable (FAIR). METHODS: A federated infrastructure model (COVID - Curated and Open Analysis and Research Platform [CO-CONNECT]) was rapidly built to enable the automated and reproducible mapping of health data partners' pseudonymized data to the Observational Medical Outcomes Partnership Common Data Model without the need for any data to leave the data controllers' secure environments, and to support federated cohort discovery queries and meta-analysis. RESULTS: A total of 56 data sets from 19 organizations are being connected to the federated network. The data include research cohorts and COVID-19 data collected through routine health care provision linked to longitudinal health care records and demographics. The infrastructure is live, supporting aggregate-level querying of data across the United Kingdom. CONCLUSIONS: CO-CONNECT was developed by a multidisciplinary team. It enables rapid COVID-19 data discovery and instantaneous meta-analysis across data sources, and it is researching streamlined data extraction for use in a Trusted Research Environment for research and public health analysis. CO-CONNECT has the potential to make UK health data more interconnected and better able to answer national-level research questions while maintaining patient confidentiality and local governance procedures.

Flow Cytometry and Fecal Indicator Bacteria Analyses for Fingerprinting Microbial Pollution in Karst Aquifer Systems.

Microbial pollution of aquifers is a persistent water quality problem globally which poses significant risks to public health. Karst aquifer systems are exceptionally vulnerable to pollution from fecal contamination sources as a result of rapid recharge of water from the surface via discrete pathways linked to highly conductive, solutionally enlarged conduits alongside strong aquifer heterogeneity. Consequently, rapid changes in microbial water quality, which are difficult to monitor with expensive and time-consuming conventional microbiological methods, are a major concern in karst environments. This study examined flow cytometric (FCM) fingerprinting of bacterial cells in groundwater together with fecal indicator bacteria (FIB) at nine separate karst springs of varying catchment size over a 14 month period in order to assess whether such a technique can provide faster and more descriptive information about microbial pollution through such karst aquifer systems. Moreover, the data have also been evaluated with respect to the potential of using turbidity as an easy-to-measure proxy indicator of microbial pollution in a novel way. We argue that FCM provides additional data from which enhanced insights into fecal pollution sources and its fate and transport in such karst catchments can be gained. We also present valuable new information on the potential and limitations of turbidity as an indicator of fecal groundwater contamination in karst. FCM has the potential to become a more widely used tool in the field of contaminant hydrogeology.

Examination of the effect of niosome preparation methods in encapsulating model antigens on the vesicle characteristics and their ability to induce immune responses.

Niosome nanoparticles can be prepared using different methods, each of which can affect the size and homogeneity of the prepared particles. The aim of this study was to establish if the method of preparation impacted on the prepared vesicles when loaded with a model protein and the type of immune responses induced to the vaccine antigen. Niosomes were prepared using both the traditional thin film hydration (TFH) technique and the microfluidic mixing (MM) technique. Influenza antigen was then entrapped in the niosomes and formulations tested for their ability to induce in vivo immune responses in immunised BALB/c mice. Niosomes prepared by MM had a mean size of 157 ± 1.8 nm and were significantly more uniform compared with the niosomes prepared using TFH (mean size 388 ± 10 nm). Niosomes play a key role as an adjuvant to help raise high antibody immune responses. This was confirmed in this study since animals treated with both types of niosomes and antigen were more responsive than unentrapped (free) antigen. Cytokine analysis showed that the TFH niosomes induced a Th1 immune response by raising IgG2a and high levels of IFN-É£, while the MM niosomes induced a Th2 immune response by inducing IgG1 (p < .05). These results confirmed that the method of preparation of the niosomes nanoparticles induced different immune responses and the average particle size of the niosomes differed depending on the method of manufacture. This indicates that particle size and uniformity are of importance and should be taken into consideration when designing an oral based delivery system for vaccine delivery.

Effect of Intra-articular Platelet-Rich Plasma vs Placebo Injection on Pain and Medial Tibial Cartilage Volume in Patients With Knee Osteoarthritis: The RESTORE Randomized Clinical Trial.

Importance: Most clinical guidelines do not recommend platelet-rich plasma (PRP) for knee osteoarthritis (OA) because of lack of high-quality evidence on efficacy for symptoms and joint structure, but the guidelines emphasize the need for rigorous studies. Despite this, use of PRP in knee OA is increasing. Objective: To evaluate the effects of intra-articular PRP injections on symptoms and joint structure in patients with symptomatic mild to moderate radiographic medial knee OA. Design, Setting, and Participants: This randomized, 2-group, placebo-controlled, participant-, injector-, and assessor-blinded clinical trial enrolled community-based participants (n = 288) aged 50 years or older with symptomatic medial knee OA (Kellgren and Lawrence grade 2 or 3) in Sydney and Melbourne, Australia, from August 24, 2017, to July 5, 2019. The 12-month follow-up was completed on July 22, 2020. Interventions: Interventions involved 3 intra-articular injections at weekly intervals of either leukocyte-poor PRP using a commercially available product (n = 144 participants) or saline placebo (n = 144 participants). Main Outcomes and Measures: The 2 primary outcomes were 12-month change in overall average knee pain scores (11-point scale; range, 0-10, with higher scores indicating worse pain; minimum clinically important difference of 1.8) and percentage change in medial tibial cartilage volume as assessed by magnetic resonance imaging (MRI). Thirty-one secondary outcomes (25 symptom related and 6 MRI assessed; minimum clinically important difference not known) evaluated pain, function, quality of life, global change, and joint structures at 2-month and/or 12-month follow-up. Results: Among 288 patients who were randomized (mean age, 61.9 [SD, 6.5] years; 169 [59%] women), 269 (93%) completed the trial. In both groups, 140 participants (97%) received all 3 injections. After 12 months, treatment with PRP vs placebo injection resulted in a mean change in knee pain scores of -2.1 vs -1.8 points, respectively (difference, -0.4 [95% CI, -0.9 to 0.2] points; P = .17). The mean change in medial tibial cartilage volume was -1.4% vs -1.2%, respectively (difference, -0.2% [95% CI, -1.9% to 1.5%]; P = .81). Of 31 prespecified secondary outcomes, 29 showed no significant between-group differences. Conclusions and Relevance: Among patients with symptomatic mild to moderate radiographic knee OA, intra-articular injection of PRP, compared with injection of saline placebo, did not result in a significant difference in symptoms or joint structure at 12 months. These findings do not support use of PRP for the management of knee OA. Trial Registration: Australian New Zealand Clinical Trials Registry Identifier: ACTRN12617000853347.

Resolving the Interactome of the Human Macrophage Immunometabolism Regulator (MACIR) with Enhanced Membrane Protein Preparation and Affinity Proteomics.

Expression of the macrophage immunometabolism regulator gene (MACIR) is associated with severity of autoimmune disease pathology and with the regulation of macrophage biology through unknown mechanisms. The encoded 206 amino acid protein lacks homology to any characterized protein sequence and is a disordered protein according to structure prediction algorithms. To identify interactions of MACIR with proteins from all subcellular compartments, a membrane solubilization buffer is employed, that together with a high affinity EF hand based pull down method, increases the resolution of quantitative mass spectrometry analysis with significant enrichment of interactions from membrane bound nuclear and mitochondrial compartments compared to samples prepared with radioimmunoprecipitation assay buffer. A total of 63 significant interacting proteins are identified and interaction with the nuclear transport receptor TNPO1 and the trafficking proteins UNC119 homolog A and B are validated by immunoprecipitation. Mutational analysis in two candidate nuclear localization signal motifs in the MACIR amino acid sequence shows the interaction with TNPO1 is likely via a non-classical proline/tyrosine-nuclear localization signal motif (aa98-117). It is shown that employing a highly specific and high affinity pull down method that performs efficiently in this glycerol and detergent rich buffer is a powerful approach for the analysis of uncharacterized protein interactomes.

COTI-2 reactivates mutant p53 and inhibits growth of triple-negative breast cancer cells.

PURPOSE: Triple-negative breast cancer (TNBC) currently lacks an approved targeted therapy. The tumour suppressor TP53 gene is mutated in approximately 80% of TNBC cases. COTI-2 is a third-generation thiosemicarbazone engineered for high efficacy and low toxicity which acts by reactivating mutant p53 to a WT form. The aim of this study was to investigate COTI-2 as a targeted therapy for TNBC patients. METHODS: Using a panel of 18 breast cell lines, we carried out MTT assay. p53 protein folding was determined by immunofluorescent staining with the p53 mutant-specific antibody PAb240 and the p53 WT-specific PAb1620. Surface plasmon resonance was used to determine binding affinity of COTI-2 to full length (FL) p53, and the DNA-binding domain (DBD). Flow cytometry was used to measure apoptosis. RESULTS: TNBC cell lines were significantly more responsive to COTI-2 than non-TNBC cell lines (p = 0.04). Furthermore, lower IC50 values were found in p53 mutant compared to p53 WT cells (p = 0.001). COTI-2 was shown to bind to FL and DBD of mutant p53. Treatment resulted in an increase in staining with PAb1620 which coincided with a decrease in staining with PAb240, suggesting refolding of the mutant protein. In addition, COTI-2 was found to induce apoptosis in TNBC cell lines. CONCLUSION: We conclude that targeting mutant p53 with COTI-2 is a potential approach for treating p53-mutated TNBC.

Exercise for the intervertebral disc: a 6-month randomised controlled trial in chronic low back pain.

BACKGROUND CONTEXT: Muscle, bone and tendon respond anabolically to mechanical forces. Whether the intervertebral disc (IVD) can benefit from exercise is unclear. PURPOSE: To examine whether exercise can beneficially affect IVD characteristics. STUDY DESIGN/SETTING: This is a single-blinded 6-month randomised controlled trial (ACTRN12615001270505) in an exercise and physiotherapy clinic. PATIENT SAMPLE: Forty patients with chronic non-specific low back pain (NSCLBP) are included in this study. OUTCOME MEASURES: The primary outcome was lumbar IVD T2 time (MRI). Secondary outcomes included IVD diffusion coefficient and IVD expansion with short-duration lying. METHODS: Twenty patients progressively loaded their lumbar IVDs (exercise) via an exercise programme involving progressive upright aerobic and resistance exercises targeting the trunk and major muscle groups and were compared to twenty patients who performed motor control training and manual therapy (control). Testing occurred at baseline, 3 months and 6 months. RESULTS: Seventeen exercise and fifteen control patients completed the interventions. There were no group-by-time differences in T2 time of the entire IVD (exercise 94.1 ± 10.0 ms vs. control 96.5 ± 9.3 ms, p = 0.549). Exercise patients had shorter T2 time in the posterior annulus at 6 months (82.7 ± 6.8 ms vs. 85.1 ± 8.0 ms, p = 0.028). Exercise patients showed higher L5/S1 apparent diffusion coefficients and decreased IVD height at 3 months (both p ≤ 0.050). After adjustments for multiple comparisons, differences lost statistical significance. Per-protocol and intent-to-treat analyses yielded similar findings. CONCLUSIONS: This trial found that 6 months of exercise did not benefit the IVD of people with NSCLBP. Based on this index study, future studies could investigate the effect of exercise on IVD in different populations, with different types, durations and/or intensities of exercise, and using different IVD markers. These slides can be retrieved under Electronic Supplementary Material.

Innate activation of human primary epithelial cells broadens the host response to Mycobacterium tuberculosis in the airways.

Early events in the human airways determining whether exposure to Mycobacterium tuberculosis (Mtb) results in acquisition of infection are poorly understood. Epithelial cells are the dominant cell type in the lungs, but little is known about their role in tuberculosis. We hypothesised that human primary airway epithelial cells are part of the first line of defense against Mtb-infection and contribute to the protective host response in the human respiratory tract. We modelled these early airway-interactions with human primary bronchial epithelial cells (PBECs) and alveolar macrophages. By combining in vitro infection and transwell co-culture models with a global transcriptomic approach, we identified PBECs to be inert to direct Mtb-infection, yet to be potent responders within an Mtb-activated immune network, mediated by IL1ß and type I interferon (IFN). Activation of PBECs by Mtb-infected alveolar macrophages and monocytes increased expression of known and novel antimycobacterial peptides, defensins and S100-family members and epithelial-myeloid interactions further shaped the immunological environment during Mtb-infection by promoting neutrophil influx. This is the first in depth analysis of the primary epithelial response to infection and offers new insights into their emerging role in tuberculosis through complementing and amplifying responses to Mtb.

Contact-Related Strain of Quadratus Femoris, Obturator Externus, and Inferior Gemellus in an Australian Football Player: A Case Report.

CONTEXT: Muscle injuries of the hip stabilizers are considered rare in sport. OBJECTIVE: This report presents a previously unreported case of a contact injury resulting in acute strain of quadratus femoris, obturator externus, and inferior gemellus in an amateur Australian rules football player. DESIGN: Level 4-case report. CASE PRESENTATION: A player was tackled ipsilateral to the injured leg, while in hip flexion in a lunged position. The case describes the diagnostic process, initial management, and return to play for this athlete. RESULTS: Following rehabilitation, the player was able to return to sport at 8 weeks without ongoing issues. CONCLUSIONS: A literature search for sports-related contact injuries to either muscle returned only one result. All other documented cases of injury to these muscle groups are confined to noncontact mechanisms or delayed presentations. Despite conventional teaching, the action of the deep external rotators of the hip appears to be positionally dependent. Knowledge of this type of injury and mechanism may be useful for future clinical reasoning and differential diagnosis in patients with this type of presentation.

Identification of α-helix 4 (α4) of Rab11a as a novel Rab11-binding domain (RBD): Interaction of Rab11a with the Prostacyclin Receptor.

The cellular trafficking of numerous G protein-coupled receptors (GPCRs) is known to be regulated by Rab proteins that involves a direct protein:protein interaction between the receptor and the GTPase. In the case of the human prostacyclin receptor (hIP), it undergoes agonist-induced internalization and subsequent Rab11a-dependent recyclization involving an interaction between a Rab11-binding domain (RBD) localized within its carboxyl-tail domain with Rab11a. However, the GPCR-interacting domain on Rab11a itself is unknown. Hence, we sought to identify the region within Rab11a that mediates its interaction with the RBD of the hIP. The α4 helix region of Rab11 was identified as a novel binding domain for the hIP, a site entirely distinct from the Switch I/Switch II -regions that act as specific binding domain for most other Rab and Ras-like GTPase interactants. Specifically, Glu138 within α4 helix of Rab11a appears to contact with key residues (e.g. Lys304) within the RBD of the hIP, where such contacts differ depending on the agonist-activated versus -inactive status of the hIP. Through mutational studies, supported by in silico homology modelling of the inactive and active hIP:Rab11a complexes, a mechanism is proposed to explain both the constitutive and agonist-induced binding of Rab11a to regulate intracellular trafficking of the hIP. Collectively, these studies are not only the first to identify α4 helix of Rab11a as a protein binding domain on the GTPase but also reveal novel mechanistic insights into the intracellular trafficking of the hIP, and potentially of other members of the GPCR superfamily, involving Rab11-dependent mechanisms.

Simplifying G Protein-Coupled Receptor Isolation with a Calcium-Dependent Fragment Complementation Affinity System.

The process of isolating recombinant G protein-coupled receptors from membrane preparations is challenging because the process requires solubilization in detergent micelles and multistep affinity chromatography protocols. Solubilization buffers contain high concentrations of salts, detergents, and glycerol that create stringent conditions necessary to stabilize the receptor but in which affinity chromatography resins perform poorly, and these resins also require the addition of eluting agents that complicate downstream assays. To simplify this process we have developed a high affinity fragment complementation molecular switch as a highly specific system for receptor capture in solubilization buffer with a calcium chelation-based elution step releasing functional protein in a simple buffer. Here we describe in detail the design, methodology, interpretation, and limitations of this novel affinity chromatography system in the isolation and purification of the cannabinoid G protein-coupled receptor CB2, in comparison with commercially available systems. This powerful tool may be applied to any recombinant membrane bound protein and can be further optimized to enhance the yield and purity of the most challenging protein targets for study.

Intervertebral disc status is associated with vertebral marrow adipose tissue and muscular endurance.

PURPOSE: Low back pain is a major public health issue. Identifying factors associated with better intervertebral disc (IVD) characteristics gives insight into IVD metabolism and highlights intervention targets for improvement of IVD health. This cross-sectional study investigates whether IVD T2-relaxation time on magnetic resonance imaging (MRI) is associated with vertebral fat fraction (VFF; to quantify marrow adipose tissue), trunk muscle cross-sectional area (CSA), muscle function and physical activity (PA). METHODS: Seventy-nine healthy subjects (35 males, 44 females) without history of spinal disease were included. Lumbar IVDs T2-relaxation time, lumbar VFF and CSA of multifidus, erector spinae, quadratus lumborum, and psoas muscles were quantified via MRI. Isometric trunk flexion and extension endurance times as well as habitual PA levels and exposure to occupational spine risk factors were documented. Pearson-partial correlations adjusted for anthropometric differences by controlling for vertebral body height. RESULTS: Higher IVD T2-time correlated with: (a) lower VFF (r = - 0.27, p < 0.05), (b) greater trunk extensor muscle endurance (r = 0.37, p < 0.01), and (c) greater trunk flexor muscle endurance (r = 0.30, p < 0.01) but not with muscle CSA. Lower VFF also correlated with greater extensor muscle endurance (r = - 0.26, p < 0.05) and habitual PA (MET-mins per week) (r = - 0.24, p < 0.05). CONCLUSION: This is the first study to show that better IVD hydration is associated with lower VFF and that greater physical activity is associated with favourable levels of vertebral marrow adipose tissue in young healthy individuals. Reduced vertebral marrow adipose tissue may specifically improve IVD hydration via improved nutrient supply. These slides can be retrieved under Electronic Supplementary Material.

Efficacy of intra-articular injections of platelet-rich plasma as a symptom- and disease-modifying treatment for knee osteoarthritis - the RESTORE trial protocol.

BACKGROUND: Knee osteoarthritis (OA) causes substantial pain, physical dysfunction and impaired quality of life. There is no cure for knee OA, and for some people, the disease may involve progressive symptomatic and structural deterioration over time. Platelet-rich plasma (PRP) is a therapeutic agent that aims to address underlying biological processes responsible for OA pathogenesis. As such, it has the potential to improve both symptoms and joint structure. The aim of this clinical trial is to determine whether a series of injections of PRP into the knee joint will lead to a significantly greater reduction in knee pain, and less loss of medial tibial cartilage volume over 12 months when compared to a series of placebo saline injections in people with knee OA. METHODS: This will be a two-group, superiority, randomised, participant-, interventionist- and assessor-blinded, placebo-controlled trial. Two hundred and eighty-eight participants aged over 50 years with painful knee OA and mild to moderate structural change on x-ray (Kellgren and Lawrence grade 2 and 3) will be randomly allocated to receive either three PRP injections or three normal saline injections into the knee joint at weekly intervals. The primary outcomes will be 12-month change in average overall knee pain severity (numeric rating scale) and medial tibial cartilage volume (magnetic resonance imaging (MRI)). Secondary outcomes include additional measures of knee pain and other symptoms, function in daily living and sport and recreation, quality of life, participant-perceived global ratings of change, and other MRI structural outcomes including meniscal and cartilage morphology, synovitis, effusion, bone marrow lesions and cartilage defects. A range of additional measures will be recorded, and a separate health economic evaluation will be performed. DISCUSSION: The findings from this study will help determine whether PRP improves both clinical and structural knee OA outcomes over 12 months when compared to a series of placebo saline injections. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry reference: ACTRN12617000853347 . Prospectively registered 9th of June 2017.

Ligand-directed targeting of lymphatic vessels uncovers mechanistic insights in melanoma metastasis.

Metastasis is the most lethal step of cancer progression in patients with invasive melanoma. In most human cancers, including melanoma, tumor dissemination through the lymphatic vasculature provides a major route for tumor metastasis. Unfortunately, molecular mechanisms that facilitate interactions between melanoma cells and lymphatic vessels are unknown. Here, we developed an unbiased approach based on molecular mimicry to identify specific receptors that mediate lymphatic endothelial-melanoma cell interactions and metastasis. By screening combinatorial peptide libraries directly on afferent lymphatic vessels resected from melanoma patients during sentinel lymphatic mapping and lymph node biopsies, we identified a significant cohort of melanoma and lymphatic surface binding peptide sequences. The screening approach was designed so that lymphatic endothelium binding peptides mimic cell surface proteins on tumor cells. Therefore, relevant metastasis and lymphatic markers were biochemically identified, and a comprehensive molecular profile of the lymphatic endothelium during melanoma metastasis was generated. Our results identified expression of the phosphatase 2 regulatory subunit A, α-isoform (PPP2R1A) on the cell surfaces of both melanoma cells and lymphatic endothelial cells. Validation experiments showed that PPP2R1A is expressed on the cell surfaces of both melanoma and lymphatic endothelial cells in vitro as well as independent melanoma patient samples. More importantly, PPP2R1A-PPP2R1A homodimers occur at the cellular level to mediate cell-cell interactions at the lymphatic-tumor interface. Our results revealed that PPP2R1A is a new biomarker for melanoma metastasis and show, for the first time to our knowledge, an active interaction between the lymphatic vasculature and melanoma cells during tumor progression.

Connective tissue injury in calf muscle tears and return to play: MRI correlation.

OBJECTIVE: The aim of our study was to assess a group of patients with calf muscle tears and evaluate the integrity of the connective tissue boundaries and interfaces. Further, we propose a novel MRI grading system based on integrity of the connective tissue and assess any correlation between the grading score and time to return to play. We have also reviewed the anatomy of the calf muscles. MATERIALS AND METHODS: We retrospectively evaluated 100 consecutive patients with clinical suspicion and MRI confirmation of calf muscle injury. We evaluated each calf muscle tear with MRI for the particular muscle injured, location of injury within the muscle and integrity of the connective tissue structure at the interface. The muscle tears were graded 0-3 depending on the degree of muscle and connective tissue injury. The time to return to play for each patient and each injury was found from the injury records and respective sports doctors. RESULTS: In 100 patients, 114 injuries were detected. Connective tissue involvement was observed in 63 out of 100 patients and failure (grade 3 injury) in 18. Mean time to return to play with grade 0 injuries was 8 days, grade 1 tears was 17 days, grade 2 tears was 25 days and grade 3 tears was 48 days (p<0.001). CONCLUSION: The integrity of the connective tissue can be used to estimate and guide the time to return to play in calf muscle tears.