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Purpose: The purpose of this study was to evaluate the relation between pelvic fracture patterns and the angiographic findings, and to assess the effectiveness of the embolisation.Methods: This retrospective study, included patients with pelvic fractures and angiographic evaluation. Demographics, Injury Severity Score (ISS), associated injuries, embolisation time, blood units needed, method of treatment and complications were recorded and analysed. Fractures were classified according to the Burgess system.Results: Between 1998 and 2008, 34 patients with pelvic fractures underwent angiographic investigation. Twenty six were males. The mean age was 41 years. Twenty-seven were motor vehicle accidents and seven were falls. There were 11 anterior posterior (APC) fractures, 12 lateral compression (LC), eight vertical shear (VS) patterns and three with combined mechanical injuries. The median ISS was 33.1 (range 5-66). From the 34 who underwent angiography, 29 had positive vascular extravasations. From them, 21 had embolisation alone, two had vascular repair and embolisation, five required vascular repair alone and one patient died while being prepared for embolisation. Five cases were re-embolised. The findings suggested that AP fractures have a higher tendency to bleeding compared with LC fractures. Both had a higher chance of blood loss compared to VS and complex fracture patterns. We reported 57 additional injuries and 65 fractures. The complications were: one non lethal pulmonary embolism, one renal failure, one liver failure, one systemic infection, two deep infections and two psychological disorientations. Seven patients died in hospital.Conclusion: Control of pelvic fracture bleeding is based on the multidisciplinary approach mainly related to hospital facilities and medical personnel's awareness. The morphology of the fracture did not have a predictive value of the vascular lesion and the respective bleeding. [ABSTRACT FROM AUTHOR]

Unlabelled: Cobb described a method of reconstruction in Johnson and Strom Type II tibialis posterior dysfunction (TPD) using a split tibialis anterior musculotendinous graft. We assessed patient function and satisfaction after a modified Cobb reconstruction in a group of patients with a narrow spectrum of dysfunction, examined a modification of the Johnson and Strom classification to emphasize severity of deformity, and assessed the ability of the technique to prevent subsequent fixed deformity. We prospectively followed 32 patients managed by this technique and a translational os calcis osteotomy with early flexible deformity after failed conservative treatment. There were 28 women and four men with unilateral disease. The average followup was 5.1 years. Staging was confirmed clinically and with imaging. The modified surgery involved a bone tunnel in the navicular rather than the medial cuneiform with plaster for 8 weeks followed by orthotics and physiotherapy. All of the osteotomies healed and 29 of the 32 patients could perform a single heel rise test at 12 months. The mean postoperative American Orthopaedic Foot and Ankle Society hindfoot score was 89. One patient had a superficial wound infection and one a temporary dysesthesia of the medial plantar nerve; both resolved. The observations suggest the technique is a comparable method of treating early Johnson and Strom Type II TPD.Level Of Evidence: Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence. [ABSTRACT FROM AUTHOR]

Background. We report a patient with ossification of the Achilles tendon, presenting plain radiographs, ultrasound, power doppler, computed tomography and magnetic resonance findings. The majority of the tendon was ossified, and there was adjacent soft tissue oedema. The appearance of peritendinous oedema associated with ossification has not, to our knowledge, been reported in the literature, and could account for hindfoot pain in cases of tendon ossification. Method. A review of 44 articles on aetiology, histology and imaging was performed. Results. Trauma, either accidental or from surgery, rather than tendinopathy is the likely aetiological factor. Conclusion. The Achilles tendon usually responds to an insult with the development of mature bone, not dystrophic calcification, with no evidence of inflammatory or degenerative changes. [ABSTRACT FROM AUTHOR]

Aim: To compare the radiation dose of cervical spine clearance and body CT in a cohort of unconscious, major trauma patients for three different protocols, comparing spiral to multislice CT. To quantify the radiation exposure effect of the protocols on the lifetime cancer risk. Method: The hospital trauma database was used to find the unconscious (GCS<9), severely injured (Injury Severity Score >15) from 1 January 2001 to 31 December 2003, excluding isolated head injuries. The protocols used for imaging the brain and cervical spine were, including the radiographs performed as a mode: The exposure factors and field of view used were put into the Monte Carlo software, to estimate the CT and radiographic X-ray doses to the body as a whole and the dose to the thyroid associated with each region imaged. The associated nominal additional lifetime cancer risk was assessed. Results: Excluding inter hospital transfers, where data was incomplete, 87 patients survived to be admitted and fulfilled the criteria. In 30 cases, the CT films were missing, the exposure factors were not recorded or no imaging was performed. In a further 21 cases, the X-ray packets were missing. Three patients had brain and cervico-dorsal CT imaging only, leaving 33 cases for evaluation. The effective radiation dose for a spiral CT of the brain using the Toshiba Xpress GX CT scanner was 3.8 mSv. The total effective doses for imaging the brain and cervical spine using the three protocols with the same CT scanner were (S.D. as % of mean): (1) 4.4 mSv (5%), (2) 7.1 mSv (10%) and (3) 8.2 mSv (15%). The corresponding mean thyroid doses were: (1) 8.5 mGy (25%), (2) 48.9 mGy (20%) and (3) 66.5 mSv (20%). The resultant nominal lifetime cancer risks were: (1) 1:4500, (2) 1:2800 and (3) 1:2400. For the Siemens Sensation 16 multislice CT scanner, the total effective doses (S.D. as % of mean) were: (1) 2.3 mSv (10%), (2) 4.3 mSv (25%) and (3) 5.4 mSv (35%). The mean doses to the thyroid were: (1) 5.9 mGy (30%), (2) 36.1 mGy (50%) and (3) 52.4 mGy (40%). The lifetime cancer risks were: (1) 1:8700, (2) 1:4600 and (3) 1:3700. Using the Toshiba spiral CT scanner, the total dose and additional lifetime nominal cancer risk associated with CT of the chest, abdomen and pelvis (CAP) as 16 mSv and 1:1250, respectively. Using the Siemens multislice CT scanner, these were 11.8 mSv and 1:1700. The cancer risk for protocol 1 when combined with a CT scan of the chest, abdomen and pelvis was 1:1000 for the spiral CT scanner and 1:1500 for the multislice CT (MCT) scanner. The cancer risk for protocol 2 with CAP CT using the MCT was 1:1200. The cancer risk for protocol 3 when combined with a CT scan of the chest, abdomen and pelvis was 1:1100 for the multislice CT scanner. Prior to the introduction of the BTS guidelines for cervical clearance, 12% of cases had CT of the body, which increased to 16% post-guidelines. Conclusions: CT of the trunk (chest, abdomen and pelvis) is associated with the greatest risk of inducing a fatal cancer in the severely injured patient with a GCS less than 9. In our institution the multislice CT protocols expose the patient to less radiation than single slice CT, which is contrary to much of the published work to date. CT scanning the thyroid (or whole cervical spine) still has a marked effect on the cancer risk in cervical clearance. Many centres will relax cervical spinal precautions in unconscious trauma patients if the cervical spine CT with reconstructions is normal. CT of the whole cervical spine may be justified in the unconscious, severely injured patient. In conscious trauma patients, the additional lifetime risk may not justify CT of the whole cervical spine as a routine practice. [ABSTRACT FROM AUTHOR]

Chicken meat is contaminated with Salmonella from the gut of infected chickens during slaughter. Eradication of Salmonella from broiler chickens through hygiene measures and/or vaccination is not cost-effective; complementary approaches are required. A mature gut microbiota obstructs Salmonella infection in chickens, and deliberate fortification of colonization resistance through prebiotic feed formulations would benefit public health and poultry production. Prebiotic galactooligosaccharides hastens Salmonella clearance from the gut of infected chickens. To better understand the role of galactooligosaccharides in colonization resistance, broiler chickens were raised on a wheat-soybean meal-based feed, with or without galactooligosaccharides for the first 24 days of life. Chickens were orally challenged with Salmonella enterica serovar Enteritidis at 20 days and the effect of supplementary galactooligosaccharides characterized by profiling Salmonella colonization, gut microbiota, innate immune response, and cecal short-chain fatty acid concentrations. Exposure to dietary galactooligosaccharides shortened the time to clear S . Enteritidis from the ceca. Differential abundance analysis of the cecal microbiota associated Salmonella challenge with a bacterial taxon belonging to the Acidaminococcaceae family ( P < 0.005). Increased cecal concentrations of the short-chain fatty acids propionate and valerate were measured in Salmonella -challenged chickens sustained on either control or galactooligosaccharide-supplemented feed relative to mock-challenged controls; but far greater concentrations were detected in chickens fed a galactooligosaccharide-supplemented diet in early life. The abundance of the Acidaminococcaceae taxon exhibited a positive correlation with the cecal concentrations of propionate (ρ = 0.724, P = 0.008) and valerate (ρ = 0.71, P = 0.013). The absence of cecal pro-inflammatory transcriptional responses suggest that the rapid Salmonella clearance observed for the galactooligosaccharide-supplemented diet was not linked to innate immune function., Importance: Work presented here identifies bacterial taxa responsible for colonization resistance to Salmonella in broiler chickens. Deliberate cultivation of these taxa with prebiotic galactooligosaccharide has potential as a straight-forward, safe, and cost-effective intervention against Salmonella . We hypothesize that catabolism of galactooligosaccharide and its breakdown products by indigenous microorganisms colonizing the chicken gut produce excess levels of propionate. In the absence of gross inflammation, propionate is inimical to Salmonella and hastens intestinal clearance., Competing Interests: N.M.F. was a consultant to Dairy Crest Ltd.

Background/purpose: The purpose of this study was to report a case series of full-thickness macular holes without vitreomacular traction that resolved without surgery., Methods: This study is a retrospective case series of 11 patients who demonstrated closure of full-thickness macular holes without surgical intervention., Results: All full-thickness macular holes closed, with all patients having improvement in visual acuity. All but one of the cases had visual acuity better than 20/40 at last recorded visit. Most cases presented with associated epiretinal membrane (73%), cystoid changes (64%), defects <150 μ m (80%), and resolved within 2 months (91%). Topical anti-inflammatory drops were used in 7 of 11 cases, and dorzolamide was used in one case., Conclusion: Full-thickness macular holes can develop in eyes without the presence of vitreomacular traction. Topical therapy without vitrectomy may be particularly helpful in closure of full-thickness macular holes with associated cystoid macular edema. Holes with a lamellar hole component may spontaneously resolve as part of a retinal remodeling process.

The objective of this work was to assess the consequences of repeated intra-articular injection of monosodium urate (MSU) crystals with inflammasome priming by lipopolysaccharide (LPS) in order to simulate recurrent bouts of gout in rats. Translational imaging was applied to simultaneously detect and quantify injury in different areas of the knee joint. MSU/LPS induced joint swelling, synovial membrane thickening, fibrosis of the infrapatellar fat pad, tidemark breaching, and cartilage invasion by inflammatory cells. A higher sensitivity to mechanical stimulus was detected in paws of limbs receiving MSU/LPS compared to saline-injected limbs. In MSU/LPS-challenged joints, magnetic resonance imaging (MRI) revealed increased synovial fluid volume in the posterior region of the joint, alterations in the infrapatellar fat pad reflecting a progressive decrease of fat volume and fibrosis formation, and a significant increase in the relaxation time T 2 in femoral cartilage, consistent with a reduction of proteoglycan content. MRI also showed cyst formation in the tibia, femur remodeling, and T 2 reductions in extensor muscles consistent with fibrosis development. Repeated intra-articular MSU/LPS injections in the rat knee joint induced pathology in multiple tissues and may be a useful means to investigate the relationship between urate crystal deposition and the development of degenerative joint disease., (© 2022. The Author(s).)

Exercise modulates metabolism and the gut microbiome. Brief exposure to low mT-range pulsing electromagnetic fields (PEMFs) was previously shown to accentuate in vitro myogenesis and mitochondriogenesis by activating a calcium-mitochondrial axis upstream of PGC-1α transcriptional upregulation, recapitulating a genetic response implicated in exercise-induced metabolic adaptations. We compared the effects of analogous PEMF exposure (1.5 mT, 10 min/week), with and without exercise, on systemic metabolism and gut microbiome in four groups of mice: (a) no intervention; (b) PEMF treatment; (c) exercise; (d) exercise and PEMF treatment. The combination of PEMFs and exercise for 6 weeks enhanced running performance and upregulated muscular and adipose Pgc-1α transcript levels, whereas exercise alone was incapable of elevating Pgc-1α levels. The gut microbiome Firmicutes/Bacteroidetes ratio decreased with exercise and PEMF exposure, alone or in combination, which has been associated in published studies with an increase in lean body mass. After 2 months, brief PEMF treatment alone increased Pgc-1α and mitohormetic gene expression and after >4 months PEMF treatment alone enhanced oxidative muscle expression, fatty acid oxidation, and reduced insulin levels. Hence, short-term PEMF treatment was sufficient to instigate PGC-1α-associated transcriptional cascades governing systemic mitohormetic adaptations, whereas longer-term PEMF treatment was capable of inducing related metabolic adaptations independently of exercise., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Improvements in growth performance and health are key goals in broiler chicken production. Inclusion of prebiotic galacto-oligosaccharides (GOS) in broiler feed enhanced the growth rate and feed conversion of chickens relative to those obtained with a calorie-matched control diet. Comparison of the cecal microbiota identified key differences in abundances of Lactobacillus spp. Increased levels of Lactobacillus johnsonii in GOS-fed juvenile birds at the expense of Lactobacillus crispatus were linked to improved performance (growth rate and market weight). Investigation of the innate immune responses highlighted increases of ileal and cecal interleukin-17A (IL-17A) gene expression counterposed to a decrease in IL-10. Quantification of the autochthonous Lactobacillus spp. revealed a correlation between bird performance and L. johnsonii abundance. Shifts in the cecal populations of key Lactobacillus spp. of juvenile birds primed intestinal innate immunity without harmful pathogen challenge. IMPORTANCE Improvements in the growth rate of broiler chickens can be achieved through dietary manipulation of the naturally occurring bacterial populations while mitigating the withdrawal of antibiotic growth promoters. Prebiotic galacto-oligosaccharides (GOS) are manufactured as a by-product of dairy cheese production and can be incorporated into the diets of juvenile chickens to improve their health and performance. This study investigated the key mechanisms behind this progression and pinpointed L. johnsonii as a key species that facilitates the enhancements in growth rate and gut health. The study identified the relationships between the GOS diet, L. johnsonii intestinal populations, and cytokine immune effectors to improve growth., (Copyright © 2020 Richards et al.)

Worldwide Campylobacter jejuni is a leading cause of foodborne disease. Contamination of chicken meat with digesta from C. jejuni -positive birds during slaughter and processing is a key route of transmission to humans through the food chain. Colonization of chickens with C. jejuni elicits host innate immune responses that may be modulated by dietary additives to provide a reduction in the number of campylobacters colonizing the gastrointestinal tract and thereby reduce the likelihood of human exposure to an infectious dose. Here we report the effects of prebiotic galacto-oligosaccharide (GOS) on broiler chickens colonized with C. jejuni when challenged at either an early stage in development at 6 days of age or 20 days old when campylobacters are frequently detected in commercial flocks. GOS-fed birds had increased growth performance, but the levels of C. jejuni colonizing the cecal pouches were unchanged irrespective of the age of challenge. Dietary GOS modulated the immune response to C. jejuni by increasing cytokine IL-17A expression at colonization. Correspondingly, reduced diversity of the cecal microbiota was associated with Campylobacter colonization in GOS-fed birds. In birds challenged at 6 days-old the reduction in microbial diversity was accompanied by an increase in the relative abundance of Escherichia spp. Whilst immuno-modulation of the Th17 pro-inflammatory response did not prevent C. jejuni colonization of the intestinal tract of broiler chickens, the study highlights the potential for combinations of prebiotics, and specific competitors (synbiotics) to engage with the host innate immunity to reduce pathogen burdens., (Copyright © 2020 Flaujac Lafontaine, Richards, Connerton, O’Kane, Ghaffar, Cummings, Fish and Connerton.)

Bacteriophage biocontrol to reduce Campylobacter jejuni levels in chickens can reduce human exposure and disease acquired through the consumption of contaminated poultry products. Investigating changes in the chicken microbiota during phage treatment has not previously been undertaken but is crucial to understanding the system-wide effects of such treatments to establish a sustainable application. A phage cocktail containing two virulent Campylobacter phages was used to treat broiler chickens colonized with C. jejuni HPC5. Campylobacter counts from cecal contents were significantly reduced throughout the experimental period but were most effective 2 days post-treatment showing a reduction of 2.4 log 10 CFU g -1 relative to mock-treated Campylobacter colonized controls. The administered phages replicated in vivo to establish stable populations. Bacteriophage predation of C. jejuni was not found to affect the microbiota structure but selectively reduced the relative abundance of C. jejuni without affecting other bacteria.

Background: Campylobacters are an unwelcome member of the poultry gut microbiota in terms of food safety. The objective of this study was to compare the microbiota, inflammatory responses, and zootechnical parameters of broiler chickens not exposed to Campylobacter jejuni with those exposed either early at 6 days old or at the age commercial broiler chicken flocks are frequently observed to become colonized at 20 days old., Results: Birds infected with Campylobacter at 20 days became cecal colonized within 2 days of exposure, whereas birds infected at 6 days of age did not show complete colonization of the sample cohort until 9 days post-infection. All birds sampled thereafter were colonized until the end of the study at 35 days (mean 6.1 log 10 CFU per g of cecal contents). The cecal microbiota of birds infected with Campylobacter were significantly different to age-matched non-infected controls at 2 days post-infection, but generally, the composition of the cecal microbiota were more affected by bird age as the time post infection increased. The effects of Campylobacter colonization on the cecal microbiota were associated with reductions in the relative abundance of OTUs within the taxonomic family Lactobacillaceae and the Clostridium cluster XIVa. Specific members of the Lachnospiraceae and Ruminococcaceae families exhibit transient shifts in microbial community populations dependent upon the age at which the birds become colonized by C. jejuni. Analysis of ileal and cecal chemokine/cytokine gene expression revealed increases in IL-6, IL-17A, and Il-17F consistent with a Th17 response, but the persistence of the response was dependent on the stage/time of C. jejuni colonization that coincide with significant reductions in the abundance of Clostridium cluster XIVa., Conclusions: This study combines microbiome data, cytokine/chemokine gene expression with intestinal villus, and crypt measurements to compare chickens colonized early or late in the rearing cycle to provide insights into the process and outcomes of Campylobacter colonization. Early colonization results in a transient growth rate reduction and pro-inflammatory response but persistent modification of the cecal microbiota. Late colonization produces pro-inflammatory responses with changes in the cecal microbiota that will endure in market-ready chickens.

The role of mammalian high temperature requirement protease A1 (HTRA1) in somatic stem cell differentiation and mineralized matrix formation remains controversial, having been demonstrated to impart either anti- or pro-osteogenic effects, depending on the in vitro cell model used. The aim of this study was therefore to further evaluate the role of HTRA1 in regulating the differentiation potential and lineage commitment of murine mesenchymal stem cells in vitro, and to assess its influence on bone structure and regeneration in vivo. Our results demonstrated that short hairpin RNA-mediated ablation of Htra1 in the murine mesenchymal cell line C3H10T1/2 increased the expression of several osteogenic gene markers, and significantly enhanced matrix mineralization in response to BMP-2 stimulation. These effects were concomitant with decreases in the expression of chondrogenic gene markers, and increases in adipogenic gene expression and lipid accrual. Despite the profound effects of loss-of-function of HTRA1 on this in vitro osteochondral model, these were not reproduced in vivo, where bone microarchitecture and regeneration in 16-week-old Htra1-knockout mice remained unaltered as compared to wild-type controls. By comparison, analysis of femurs from 52-week-old mice revealed that bone structure was better preserved in Htra1-knockout mice than age-matched wild-type controls. These findings therefore provide additional insights into the role played by HTRA1 in regulating mesenchymal stem cell differentiation, and offer opportunities for improving our understanding of how this multifunctional protease may act to influence bone quality.

The S1 serine protease family is one of the largest and most biologically important protease families. Despite their biomedical significance, generic approaches to generate potent, class-specific, bioactive non-covalent inhibitors for these enzymes are still limited. In this work, we demonstrate that Ahp-cyclodepsipeptides represent a suitable scaffold for generating target-tailored inhibitors of serine proteases. For efficient synthetic access, we developed a practical mixed solid- and solution-phase synthesis that we validated through performing the first chemical synthesis of the two natural products Tasipeptin A and B. The suitability of the Ahp-cyclodepsipeptide scaffold for tailored inhibitor synthesis is showcased by the generation of the most potent human HTRA protease inhibitors to date. We anticipate that our approach may also be applied to other serine proteases, thus opening new avenues for a systematic discovery of serine protease inhibitors., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The osteoinductive properties of prostaglandin E 2 (PGE 2 ) and its signaling pathways have led to suggestions that it may serve as a potential therapeutic strategy for bone loss. However, the prominence of PGE 2 as an inducer of bone formation is attributed primarily to findings from studies using rodent models. In the current study, we investigated the effects of PGE 2 on human bone marrow stromal cell (hBMSC) lineage commitment and determined its mode of action. We demonstrated that PGE 2 treatment of hBMSCs significantly altered the expression profile of several genes associated with osteoblast differentiation (RUNX2 and ALP) and maturation (BGLAP and MGP). This was attributed to the activation of specific PGE 2 receptors, and was associated with increases in cAMP production and sustained AKT phosphorylation. Pharmacological inhibition of exchange protein directly activated by cAMP (Epac), but not protein kinase A (PKA), recovered the mineralization functions of hBMSC-derived osteoblasts treated with PGE 2 and restored AKT phosphorylation, along with the expression levels of RUNX2, ALP, BGLAP and MGP. Our findings therefore provide insights into how PGE 2 influences hBMSC-mediated matrix mineralization, and should be taken into account when evaluating the role of PGE 2 in human bone metabolism.

Although several tendon-selective genes exist, they are also expressed in other musculoskeletal tissues. As cell and tissue engineering is reliant on specific molecular markers to discriminate between cell types, tendon-specific genes need to be identified. In order to accomplish this, we have used RNA sequencing (RNA-seq) to compare gene expression between tendon, bone, cartilage and ligament from horses. We identified several tendon-selective gene markers, and established eyes absent homolog 2 ( EYA2 ) and a G-protein regulated inducer of neurite outgrowth 3 ( GPRIN3 ) as specific tendon markers using RT-qPCR. Equine tendon cells cultured as three-dimensional spheroids expressed significantly greater levels of EYA2 than GPRIN3 , and stained positively for EYA2 using immunohistochemistry. EYA2 was also found in fibroblast-like cells within the tendon tissue matrix and in cells localized to the vascular endothelium. In summary, we have identified EYA2 and GPRIN3 as specific molecular markers of equine tendon as compared to bone, cartilage and ligament, and provide evidence for the use of EYA2 as an additional marker for tendon cells in vitro., Competing Interests: The authors declare no conflict of interest.

Background: Many strategies have been used to improve the visualisation of the ACL including sagittal, coronal oblique sequences, and 3D volume imaging. Nevertheless, the ACL may not always be visualised., Methods: Two hundred and thirty-one consecutive patients (77 females; 154 males; average age 43.5, range 18 to 82 years; 205 with chronic, 20 acute, and 6 acute on chronic symptoms) underwent knee arthroscopy for mechanical symptoms within a week of MRI. After routine orthogonal sequences, if general MRI radiographers, with over four years experience, were not able to identify the presence of the ACL, then two 3D volume sequences and 2D limited sagittal oblique T1 sequences were performed. Patients requiring extra sequences, missed by the radiography technicians, were recalled. The MRI sequences were evaluated in a blinded fashion by three radiologists, and compared to the knee arthroscopy findings, with the normal ACL acting as internal controls. The radiography technicians performed additional ACL sequences in 63 patients (27%); of these, 10 patients had a partial and 12 patients had a complete ACL tear. Only 2 patients (0.6%) were recalled (one with a normal, and one with a full thickness ACL tear)., Results: The filmed ACL evaluation for complete tears and a normal ACL had a sensitivity of 100%, specificity of 97.1% and accuracy of 97.3%, slightly higher than evaluating on the monitor. Volume sequences had specificities and accuracies over 95%, with good intraobserver reliability (Kappa 0.859, 95% CI 0.705-1.0). Experienced radiographers identified most cases requiring supplementary MRI ACL sequences. An additional volume sequence was beneficial when filmed. Use of the monitor can offer some benefits. Limited oblique T1 sequence of the intercondylar notch was unreliable.

Adipogenesis is the process by which mesenchymal stem cells (MSCs) develop into lipid-laden adipocytes. Being the dominant cell type within adipose tissue, adipocytes play a central role in regulating circulating fatty acid levels, which is considered to be of critical importance in maintaining insulin sensitivity. High temperature requirement protease A1 (HTRA1) is a newly recognized regulator of MSC differentiation, although its role as a mediator of adipogenesis has not yet been defined. The aim of this work was therefore to evaluate HTRA1's influence on human MSC (hMSC) adipogenesis and to establish a potential mode of action. We report that the addition of exogenous HTRA1 to hMSCs undergoing adipogenesis suppressed their ability to develop into lipid laden adipocytes. These effects were demonstrated as being reliant on both its protease and PDZ domain, and were mediated through the actions of c-Jun N-terminal kinase and matrix metalloproteinases (MMPs). The relevance of such findings with regards to HTRA1's potential influence on adipocyte function in vivo is made evident by the fact that HTRA1 and MMP-13 were readily identifiable within crown-like structures present in visceral adipose tissue samples from insulin resistant obese human subjects. These data therefore implicate HTRA1 as a negative regulator of MSC adipogenesis and are suggestive of its potential involvement in adipose tissue remodeling under pathological conditions. Stem Cells 2016;34:1601-1614., (© 2016 AlphaMed Press.)

Objectives: To investigate the diagnostic performance of grey scale Ultrasound (US), power Doppler (PD) and US elastography for diagnosing painful patellar tendinopathy, and to establish their relationship with Victorian Institute of Sport Assessment-Patella (VISA-P) scores in a group of volleyball players with and without symptoms of patellar tendinopathy., Design: Cross-sectional study., Methods: Thirty-five volleyball players (70 patellar tendons) were recruited during a national university volleyball competition. Players were imaged with conventional US followed by elastography. The clinical findings of painful patellar tendons were used as the reference standard for diagnosing patellar tendinopathy. In addition, all participants completed the VISA-P questionnaires., Results: Of the 70 patellar tendons, 40 (57.1%) were clinically painful. The diagnostic accuracy of grey scale US, PD and elastography were 60%, 50%, 62.9%, respectively, with sensitivity/specificity of 72.5%/43.3%, 12.5%/100%, and 70%/53.3%, respectively. Combined US elastography and grey scale imaging achieved 82.5% sensitivity, 33.3% specificity and 61.4% accuracy while routine combination technique of PD and grey scale imaging revealed 72.5% sensitivity, 43.3% specificity and 60.0% accuracy. Tendons in players categorized as soft on elastography had statistically significantly greater AP thickness (p<0.001) and lower VISA-P scores (p=0.004) than those categorized as hard. There was no significant association between grey scale US abnormalities (hypoechogenicities and/or fusiform swelling) and VISA-P scores (p=0.098)., Conclusions: Soft tendon properties depicted by US elastography may be more related to patellar tendon symptoms compared to grey scale US abnormalities. The supplementation of US elastography to conventional US may enhance the sensitivity for diagnosing patellar tendinopathy in routine clinical practice., (Copyright © 2015 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.)

All-trans retinoic acid (ATRA) is a potent inducer of osteogenic differentiation in mouse adipose-derived stromal cells (mASCs), although the underlying mechanisms responsible for its mode of action have yet to be completely elucidated. High temperature requirement protease A1 (HtrA1) is a newly recognized modulator of human multipotent stromal cell (MSC) osteogenesis and as such, may play a role in regulating ATRA-dependent osteogenic differentiation of mASCs. In this study, we assessed the influence of small interfering RNA (siRNA)-induced repression of HtrA1 production on mASC osteogenesis and examined its effects on ATRA-mediated mammalian target of rapamycin (mTOR) signaling. Inhibition of HtrA1 production in osteogenic mASCs resulted in a significant reduction of alkaline phosphatase activity and mineralized matrix formation. Western blot analyses revealed the rapid activation of Akt (Ser473) and p70S6K (Thr389) in ATRA-treated mASCs, and that levels of phosphorylated p70S6K were noticeably reduced in HtrA1-deficient mASCs. Further studies using mTOR inhibitor rapamycin and siRNA specific for the p70S6K gene Rps6kb1 confirmed ATRA-mediated mASC osteogenesis as being dependent on p70S6K activation. Finally, transfection of cells with a constitutively active rapamycin-resistant p70S6K mutant could restore the mineralizing capacity of HtrA1-deficient mASCs. These findings therefore lend further support for HtrA1 as a positive mediator of MSC osteogenesis and provide new insights into the molecular mode of action of ATRA in regulating mASC lineage commitment.

While ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1, formerly PARP1) and its enzymatic activity have been shown to be important for reprogramming and differentiation of cells, such as during adipogenesis, their role and mechanism in regulating osteoclastogenesis and bone homeostasis are largely unknown. Here, in cell culture-based RANKL-induced osteoclastogenesis models, we show that silencing of ARTD1 or inhibition of its enzymatic activity enhances osteoclast differentiation and function. As a consequence of ARTD1 silencing or inhibition, the recruitment of p65/RelA to the IL-1β promoter, which is associated with transcriptionally active histone marks, IL-1β expression and inflammasome-dependent secretion of IL-1β are enhanced. This subsequently promotes sustained induction of the transcription factor Nfatc1/A and osteoclastogenesis in an autocrine manner via the IL-1 receptor. In vivo, Artd1-deficient mice display significantly decreased bone mass as a consequence of increased osteoclast differentiation. Accordingly, the expression of osteoclast markers is enhanced in mutant compared to wild-type mice. Together, these results indicate that ARTD1 controls osteoclast development and bone remodelling via its enzymatic activity by modulating the epigenetic marks surrounding the IL-1β promoter and expression of IL-1β and subsequently also Nfatc1/A.

Tenocytes represent a valuable source of cells for the purposes of tendon tissue engineering and regenerative medicine and as such, should possess a high degree of tenogenic differentiation prior to their use in vivo in order to achieve maximal efficacy. In the current report, we identify an efficient means by which to maintain differentiated tenocytes in vitro by employing the hanging drop technique in combination with defined growth media supplements. Equine tenocytes retained a more differentiated state when cultured as scaffold-free microtissue spheroids in low serum-containing medium supplemented with L-ascorbic acid 2-phosphate, insulin and transforming growth factor (TGF)-β1. This was made evident by significant increases in the expression levels of pro-tenogenic markers collagen type I (COL1A2), collagen type III (COL3A1), scleraxis (SCX) and tenomodulin (TNMD), as well as by enhanced levels of collagen type I and tenomodulin protein. Furthermore, tenocytes cultured under these conditions demonstrated a typical spindle-like morphology and when embedded in collagen gels, became highly aligned with respect to the orientation of the collagen structure following their migration out from the microtissue spheroids. Our findings therefore provide evidence to support the use of a biomimetic microtissue approach to culturing tenocytes and that in combination with the defined growth media described, can improve their differentiation status and functional repopulation of collagen matrix., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Piglet neonatal diarrhoea is an important issue in modern pig production and is linked to increased mortality and poor growth rates, affecting long-term pig health, increasing use of medication and cost of production. Saccharomyces cerevisiae var. boulardii (SB) is a probiotic yeast with documented clinical efficacy in the prevention and treatment of diarrhoeal diseases in humans. The objectives of the current study were to evaluate the effect of SB on occurrence and severity of neonatal diarrhoea in piglets, mortality and growth rate. Forty-six litters (606 piglets) were randomly allocated to a control or SB treatment (n=23 per treatment). Within 24 h of farrowing, piglets assigned to the SB treatment received a single oral dose of a paste containing 3.3×10(9) CFU of SB CNCM I-107(9). Piglets from the control litters received a placebo paste. Piglet weight, mortality and diarrhoea were recorded up to day 7 of age. It was shown that numbers of diarrhoea days were significantly correlated with increased mortality rate and reduced weight gain (P<0.05). SB treatment had no effect on growth or mortality in diarrhoeic litters. However, SB-supplemented litters had significantly lower faecal scores, indicating firmer faeces (P<0.01) and fewer numbers of diarrhoeic days (P<0.01) during the 1(st) week of life. Reduction in the number of diarrhoeic litters compared with the control group was observed following the probiotic administration (P<0.05). These results highlight the detrimental effects of neonatal diarrhoea on pre-weaning performance and suggest that SB, by reducing diarrhoea duration and severity, has the potential of improving enteric health in the early stages of life in pigs.

Two main features common to all solid tumors are tissue hypoxia and inflammation, both of which cause tumor progression, metastasis, therapy resistance and increased mortality. Chronic inflammation is associated with increased cancer risk, as demonstrated for inflammatory bowel disease patients developing colon cancer. However, the interplay between hypoxia and inflammation on the molecular level remains to be elucidated. We found that MC-38 mouse colon cancer cells contain functional hypoxic (HIF-1α) and inflammatory (p65/RelA) signaling pathways. In contrast to cells of the myeloid lineage, HIF-1α levels remained unaffected in MC-38 cells treated with LPS, and hypoxia failed to induce NF-κB. A similar regulation of canonical HIF and NF-κB target genes confirmed these results. RNA deep sequencing of HIF-1α and p65/RelA knock-down cells revealed that a surprisingly large fraction of HIF target genes required p65/RelA for hypoxic regulation and a number of p65/RelA target genes required HIF-1α for proinflammatory regulation, respectively. Hypoxia attenuated the inflammatory response to LPS by inhibiting nuclear translocation of p65/RelA independently of HIF-1α, which was associated with enhanced IκBα levels and decreased IKKβ phosphorylation. These data demonstrate that the interaction between hypoxic and inflammatory signaling pathways needs to be considered when designing cancer therapies targeting HIF or NF-κB.

PPARγ-dependent gene expression during adipogenesis is facilitated by ADP-ribosyltransferase D-type 1 (ARTD1; PARP1)-catalyzed poly-ADP-ribose (PAR) formation. Adipogenesis is accompanied by a dynamic modulation of the chromatin landscape at PPARγ target genes by ligand-dependent co-factor exchange. However, how endogenous PPARγ ligands, which have a low affinity for the receptor and are present at low levels in the cell, can induce sufficient co-factor exchange is unknown. Moreover, the significance of PAR formation in PPARγ-regulated adipose tissue function is also unknown. Here, we show that inhibition of PAR formation in mice on a high-fat diet reduces weight gain and cell size of adipocytes, as well as PPARγ target gene expression in white adipose tissue. Mechanistically, topoisomerase II activity induces ARTD1 recruitment to PPARγ target genes, and ARTD1 automodification enhances ligand binding to PPARγ, thus promoting sufficient transcriptional co-factor exchange in adipocytes. Thus, ARTD1-mediated PAR formation during adipogenesis is necessary to adequately convey the low signal of endogenous PPARγ ligand to effective gene expression. These results uncover a new regulatory mechanism of ARTD1-induced ADP-ribosylation and highlight its importance for nuclear factor-regulated gene expression., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

There is much interest in quantifying the nutritional value of UK wheat distillers dried grains with solubles (W-DDGS) for livestock species. A study was designed to evaluate caecal parameters (pH, short chain fatty acids (SCFAs) and bacterial diversity) in layer hens fed on balanced diets containing graded concentrations of W-DDGS. A total of 32 layer hens (Bovans Brown strain at 27 weeks of age) were randomly allocated to one of 4 dietary treatments containing W-DDGS at 0, 60, 120 or 180 g/kg. Each treatment was fed to 8 replicate individually housed layer hens over a 5-d acclimatisation period, followed by a 4-week trial. Individual feed intakes were monitored and all eggs were collected daily for weeks 2, 3 and 4 of the trial, weighed and an assessment of eggshell "dirtiness" made. All hens were culled on d 29 and caecal pH and SCFAs measured. Polymerase chain reaction denaturing gradient gel electrophoresis of the bacterial 16 S rDNA gene was used to assess total bacterial diversity of luminal caecal content from hens fed the 0 and 180 g W-DDGS/kg diets. Unweighted pair group method with arithmetic mean (UPGMA) dendrograms were generated from DGGE banding patterns. Increasing W-DDGS dietary concentrations resulted in a more acidic caecal environment. Caecal SCFAs were unaffected by diet aside from a quadratic effect for molar proportions of isobutyric acid. Diversity profiles of the bacterial 16S rRNA gene from luminal caecal contents were unaffected by W-DDGS inclusion. The results of the current study suggest that W-DDGS can be successfully formulated into nutritionally balanced layer diets (supplemented with xylanase and phytase) at up to 180 g/kg with no detrimental effects to the caecal environment.

Adipose-derived stromal cells (ASCs) are increasingly being used for orthopedic-based tissue engineering approaches due to their ability to readily undergo osteogenic differentiation. In the present study, we used in vitro and in vivo approaches to evaluate the use of ASCs as a treatment strategy for age-related osteoporosis. Molecular, histological and micro-computed tomography (micro-CT) based approaches confirmed that ASCs isolated from 18-week-old osteoporotic senescence-accelerated mice (SAMP6) were capable of undergoing osteogenesis when cultured in either silk fibroin (SF) scaffolds or scaffold-free microtissues (ASC-MT). A single intratibial injection of CM-Dil-labeled isogeneic ASCs or ASC-MT into SAMP6 recipients significantly improved trabecular bone quality after 6 weeks in comparison to untreated contralateral bones, as determined by micro-CT. Injected ASCs could be observed in paraffin wax bone sections at 24 h and 6 weeks post treatment and induced a significant increase in several molecular markers of bone turnover. Furthermore, a significant improvement in the osteogenic potential of osteoporotic patient-derived human bone marrow stromal cells (BMSCs) was observed when differentiated in conditioned culture media harvested from osteoporotic patient-derived human ASCs. These findings therefore support the use of ASCs as an autologous cell-based approach for the treatment of osteoporosis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Summary: In the present study, we evaluated the potential for aminobisphosphonates to enhance the development of bone-forming osteoblasts from progenitor cells isolated from aged female osteoporotic patients. The aminobisphosphonates tested significantly enhanced osteoblast formation and thus lend further insights into their possible mode of action in the treatment of osteoporosis., Introduction: The primary aim of this study was to evaluate the influence of aminobisphosphonates on the osteogenesis of human bone marrow stromal cells (hBMSCs) and mineralization of differentiating bone-forming cells isolated from osteoporotic patients., Methods: The influence of aminobisphosphonate treatment on hBMSC osteogenesis was assessed by the quantitative measurement of alkaline phosphatase (ALP) activity, in addition to quantitative reverse transcription polymerase chain reaction and Western blot analysis of known osteogenic markers. Mineralized matrix formation by hBMSC-derived osteoblasts was visualized and quantified using Alizarin red staining., Results: hBMSC cultures treated with osteogenic medium supplemented with zoledronate demonstrated a significant increase in Alizarin red staining after 3 weeks as compared to cells cultured in osteogenic medium alone. Similarly, cultures of differentiating hBMSCs isolated from patients receiving alendronate treatment also demonstrated an increased propensity for mineralization, even in the absence of further in vitro stimulation by zoledronate. The stimulatory effects of aminobisphosphonate treatment on hBMSC-derived osteoblast-mediated mineralization were independent of any alterations in ALP activity, although significant decreases in the expression levels of osteopontin (SPP1) were evident in hBMSCs following exposure to aminobisphosphonates. Further analysis including Western blotting and loss-of-function studies revealed osteopontin as having a negative influence on the mineralization of differentiating osteoporotic bone-forming cells., Conclusions: The results presented here demonstrate for the first time that aminobisphosphonate treatment of osteoporotic hBMSCs enhances their capacity for osteoblast formation and subsequent mineral deposition, thus supporting the concept of aminobisphosphonates as having an osteoanabolic effect in osteoporosis.