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Immobilization-induced skeletal unloading results in muscle atrophy and rapid bone loss, thereby increasing the risk of falling and the need for implant therapy in patients with extended bed rest or neuromuscular injuries. Skeletal unloading causes bone loss by altering bone growth and resorption, suggesting that implant performance might be affected. To test this, we focused on early events in implant osseointegration. We used the rat sciatic neurectomy-induced disuse model under two different settings. In Study 1, 16 Sprague Dawley rats (SD) were separated into control, sham operated+cast immobilization, and sciatic neurectomy+casting groups; titanium implants with multiscale microtextured topography and hydrophilic chemistry (modSLA) were inserted in the distal femoral metaphysis. Neurectomy surgeries and casting were performed at the same surgical setting as implant placement; rats were euthanized 4 weeks post-implantation. In Study 2, we established the unloaded condition before implantation. A total of 12 SD rats were divided into control and sciatic+femoral neurectomy groups. A total of 24 days after sciatic and femoral neurectomy surgery, rats received implants. Study 2 rats were euthanized at 4 weeks post-implantation. MicroCT and histomorphometry showed that trabecular bone and osseointegration were reduced when disuse was established before implantation. Osteoblasts isolated from Study 1 sciatic neurectomy tibial bones exhibited impaired differentiation on modSLA culture disks, revealing a possible mechanism responsible for the decreased osseointegration observed in the Study 2 rats. This study addressed the importance of considering the mechanical unloading and muscle function history before implant insertion and suggests that implant performance was reduced due to poor cellular ability to regenerate., (© 2024 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Objective : Volumetric muscle loss results in intramuscular axotomy, denervating muscle distal to the injury and leading to paralysis, denervation, and loss of muscle function. Once the nerve is damaged, paralyzed skeletal muscle will atrophy and accumulate noncontractile connective tissue. The objective of this study was to determine differences in connective tissue, atrophy, and inflammatory signaling between two paralysis models, botulinum toxin (Botox), which blocks acetylcholine transmission while keeping nerves intact, and neurectomy, which eliminates all nerve-to-muscle signaling. Approach : Twenty male Sprague Dawley rats were randomized and received a sciatic-femoral neurectomy (SFN), Botox-induced muscle paralysis of the proximal femur muscles, quadriceps femoris, hamstrings, and calf muscles (BTX), or sham. Muscle force was measured 52 days postsurgery, and samples were collected for histology, protein, and mRNA assays. Results: SFN and BTX decreased twitch and tetanic force, decreased fiber size by twofold, and increased myogenic expression compared with controls. SFN increased the levels of all major extracellular matrix proteins correlating with fibrosis [ e.g., laminin, fibronectin, and collagen type(s) I, III, VI]. SFN also increased profibrotic and proinflammatory mRNA compared with BTX and controls. Innovation : SFN and BTX were similar in gross morphology and functional deficiencies. However, SFN exhibited a higher amount of fibrosis in histological sections and immunoblotting. The present study shows evidence that nerve signaling changes NF-κB and TGF-β signaling, warranting future studies to determine the mechanisms involved. Conclusion: These data indicate that nerve signaling may influence fibrogenesis following denervation, but the mechanisms involved may differ as a function of the method of paralysis.

Advanced glycation end-products (AGEs) stochastically accrue in skeletal muscle and on collagen over an individual's lifespan, stiffening the muscle and modifying the stem cell (MuSC) microenvironment while promoting proinflammatory, antiregenerative signaling via the receptor for advanced glycation end-products (RAGEs). In the present study, a novel in vitro model was developed of this phenomenon by cross linking a 3-D collagen scaffold with AGEs and investigating how myoblasts responded to such an environment. Briefly, collagen scaffolds were incubated with d-ribose (0, 25, 40, 100, or 250 mM) for 5 days at 37°C. C2C12 immortalized mouse myoblasts were grown on the scaffolds for 6 days in growth conditions for proliferation, and 12 days for differentiation and fusion. Human primary myoblasts were also used to confirm the C2C12 data. AGEs aberrantly extended the DNA production stage of C2C12s (but not in human primary myoblasts) which is known to delay differentiation in myogenesis, and this effect was prevented by RAGE inhibition. Furthermore, the differentiation and fusion of myoblasts were disrupted by AGEs, which were associated with reductions in integrins and suppression of RAGE. The addition of S100b (RAGE agonist) recovered the differentiation and fusion of myoblasts, and the addition of RAGE inhibitors (FPS-ZM1 and Azeliragon) inhibited the differentiation and fusion of myoblasts. Our results provide novel insights into the role of the AGE-RAGE axis in skeletal muscle aging, and future work is warranted on the potential application of S100b as a proregenerative factor in aged skeletal muscle. NEW & NOTEWORTHY Collagen cross-linked by advanced glycation end-products (AGEs) induced myoblast proliferation but prevented differentiation, myotube formation, and RAGE upregulation. RAGE inhibition occluded AGE-induced myoblast proliferation, while the delivery of S100b, a RAGE ligand, recovered fusion deficits.

Reduced skeletal loading associated with many conditions, such as neuromuscular injuries, can lead to bone fragility and may threaten the success of implant therapy. Our group has developed a botulinum toxin A (botox) injection model to imitate disease-reduced skeletal loading and reported that botox dramatically impaired the bone formation and osseointegration of titanium implants. Semaphorin 3A (sema3A) is an osteoprotective factor that increases bone formation and inhibits bone resorption, indicating its potential therapeutic role in improving osseointegration in vivo. We first evaluated the sema3A effect on whole bone morphology following botox injections by delivering sema3A via injection. We then evaluated the sema3A effect on the osseointegration of titanium implants with two different surface topographies by delivering sema3A to cortical bone defect sites prepared for implant insertion and above the implants after insertion using a copper-free click hydrogel that polymerizes rapidly in situ. Implants had hydrophobic smooth surfaces (PT) or multiscale biomimetic micro/nano topography (SLAnano). Sema3A rescued the botox-impaired bone formation. Furthermore, biomimetic Ti implants improved the bone-to-implant contact (BIC) and mechanical properties of the integrated bone in the botox-treated rats, which sema3A enhanced. This study demonstrated the value of biomimetic approaches combining multiscale topography and biologics in improving the clinical outcomes of implant therapy.

Transfection of chondrocytes with microRNA-451(miR-451), present in growth zone cartilage of the growth plate, upregulates production of enzymes association with extracellular matrix degradation. miR-451 is also present in articular cartilage and exacerbates IL-1β effects in articular chondrocytes. Moreover, when osteoarthritis (OA) was induced in Sprague Dawley rats via bilateral anterior cruciate ligament transection (ACLT), miR-451 expression was increased in OA cartilage compared to control, suggesting its inhibition might be used to prevent or treat OA. To examine the prophylactic and therapeutic potential of inhibiting miR-451, we evaluated treatment with miR-451 power inhibitor (451-PI) at the onset of joint trauma and treatment after OA had developed. The prophylactic animal cohort received twice-weekly intra-articular injections of either 451-PI or a negative control (NC-PI) beginning on post-surgical day 3. OA was allowed to develop for 24 days in the therapeutic cohort before beginning injections. All rats were killed on day 45. Micro-CT, histomorphometrics, OARSI scoring, and muscle force testing were performed on samples. 451-PI mitigated OA progression compared to NC-PI limbs in the prophylactic cohort based on histomorphometric analysis and OARSI scoring, but no differences were detected by micro-CT. 451-PI treatment beginning 24 days post-surgery was not able to reduce OA severity. Prophylactic administration of 451-PI mitigates OA progression in a post-trauma ACLT rat model supporting its potential to prevent OA development following an ACLT injury clinically., (© 2022. The Author(s).)

Volumetric muscle loss (VML) is the acute loss of muscle mass due to trauma. Such injuries occur primarily in the extremities and are debilitating, as there is no clinical treatment to restore muscle function. Pro-inflammatory advanced glycation end-products (AGEs) and the soluble receptor for advanced glycation end-products (RAGE) are known to increase in acute trauma patient's serum and are correlated with increased injury severity. However, it is unclear whether AGEs and RAGE increase in muscle post-trauma. To test this, we used decellularized muscle matrix (DMM), a pro-myogenic, non-immunogenic extracellular matrix biomaterial derived from skeletal muscle. We delivered adipose-derived stromal cells (ASCs) and primary myoblasts to support myogenesis and immunomodulation (N = 8 rats/group). DMM non-seeded and seeded grafts were compared to empty defect and sham controls. Then, 56 days after surgery muscle force was assessed, histology characterized, and protein levels for AGEs, RAGE, p38 MAPK, and myosin heavy chains were measured. Overall, our data showed improved muscle regeneration in ASC-treated injury sites and a regulation of RAGE and p38 MAPK signaling, while myoblast-treated injuries resulted in minor improvements. Taken together, these results suggested that ASCs combined with DMM provides a pro-myogenic microenvironment with immunomodulatory capabilities and indicates further exploration of RAGE signaling in VML.

Metabolic bone is highly innervated by both sensory and sympathetic nerves. In addition to skeletal development, neural regulation participates in local bone remodeling, which is important for successful osseointegration of titanium implants. Neurectomy is a model used to investigate the lack of neural function on bone homeostasis, but the relative impacts of direct denervation to bone or denervation-induced muscle paralysis are less well defined. To investigate this difference, we used two nerve intervention models, sciatic and femoral neurectomy (SFN) v. botox-induced muscle paralysis (BTX) and assessed the resulting femoral bone phenotype and Ti implant osseointegration. Male Sprague Dawley rats (19) were randomly divided into three groups: implant control (n = 5), SFN (n = 7), and BTX (n = 7). Ti implants (microrough/hydrophilic [modSLA], Institut Straumann AG) were placed in the distal metaphysis of each femur on day 24 post-SFN or BTX. Bone and muscle were examined on day 28 after implant insertion. Both nerve intervention models impaired osseointegration. MicroCT and histology indicated that both models had reduced trabecular bone formation. Only BTX reduced cortical bone formation and increased cortical bone porosity. BTX resulted in more bone loss characterized by the least trabecular and cortical bone, as well as osseointegration. Osteoblasts isolated from the tibia exhibited a model-specific phenotype when they were grown on Ti substrates in vitro. Neurectomy caused more severe muscle atrophy than botox injection. These results indicate that neural regulation directly modulates bone formation and osseointegration. Muscle paralysis modulated the effects of loss of neural inputs into bone, supporting the hypothesis that mechanical loading of bone is a factor in achieving successful osseointegration. The different effects of botox and neurectomy on bone phenotype indicated that the sensory and sympathetic nerves had a role in the osseointegration process., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Advanced age causes skeletal muscle to undergo deleterious changes including muscle atrophy, fast-to-slow muscle fiber transition, and an increase in collagenous material that culminates in the age-dependent muscle wasting disease known as sarcopenia. Advanced glycation end-products (AGEs) non-enzymatically accumulate on the muscular collagens in old age via the Maillard reaction, potentiating the accumulation of intramuscular collagen and stiffening the microenvironment through collagen cross-linking. This review contextualizes known aspects of skeletal muscle extracellular matrix (ECM) aging, especially the role of collagens and AGE cross-linking, and underpins the motor nerve's role in this aging process. Specific directions for future research are also discussed, with the understudied role of AGEs in skeletal muscle aging highlighted. Despite more than a half century of research, the role that intramuscular collagen aggregation and cross-linking plays in sarcopenia is well accepted yet not well integrated with current knowledge of AGE's effects on muscle physiology. Furthermore, the possible impact that motor nerve aging has on intramuscular cross-linking and muscular AGE levels is posited.

Matrix vesicles (MVs) are extracellular organelles produced by growth plate cartilage cells in a zone-specific manner. MVs are similar in size to exosomes, but they are tethered to the extracellular matrix (ECM) via integrins. Originally associated with matrix calcification, studies now show that they contain matrix processing enzymes and microRNA that are specific to their zone of maturation. MVs produced by costochondral cartilage resting zone (RC) chondrocytes are enriched in microRNA 503 whereas those produced by growth zone (GC) chondrocytes are enriched in microRNA 122. MVs are packaged by chondrocytes under hormonal and factor regulation and release of their contents into the ECM is also under hormonal control, suggesting that their microRNA might have a regulatory role in growth plate proliferation and maturation. To test this, we selected a subset of these enriched microRNAs and transfected synthetic mimics back into RC and GC cells. Transfecting growth plate chondrocytes with select microRNA produced a broad range of phenotypic responses indicating that MV-based microRNAs are involved in the regulation of these cells. Specifically, microRNA 122 drives both RC and GC cells toward a proliferative phenotype, stabilizes the matrix and inhibits differentiation whereas microRNA 22 exerts control over regulatory factor production. This study demonstrates the strong regulatory capability possessed by unique MV enriched microRNAs on growth plate chondrocytes and their potential for use as therapeutic agents., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: To examine whether genetic variability plays a role in skeletal muscle response to disuse., Methods: We examined skeletal muscle response to disuse in five different strains of mice: CAST/EiJ, NOD/ShiLtJ, NZO/HILtJ, 129S1/SvImJ and A/J. Mice had one limb immobilized by a cast for three weeks., Results: Response to immobilization was dependent on the strain of mice. Skeletal muscle mass/body weight was decreased by immobilization in all strains except 1291/SvImJ. Immobilization decreased absolute skeletal muscle mass in quadriceps and gastrocnemius in NOD/ShiltJ and NZO/HILtJ mice. Three weeks of immobilization resulted in an increase in quadriceps levels of atrogenes in CAST/EiJ. Immobilization resulted in an increase in quadriceps and gastrocnemius levels of Myh4 in CAST/EiJ. A similar trend was observed for Myh7 in gastrocnemius muscle. Immobilization resulted in a decrease of the p-p70S6K1/total p706SK1 ratio in quadriceps of NOD/ShiLtJ mice and the gastrocnemius of A/J mice. Immobilization did not affect the p-4EBP1/total 4EBP1 ratio in quadriceps of any of the strains examined. However, the p-4EBP1/total 4EBP1 ratio in gastrocnemius was greater in immobilized, relative to control, limbs in CAST/EiJ mice., Conclusion: Genetic variability affects the response of skeletal muscle to disuse., Competing Interests: The authors have no conflict of interest.

Decellularized tissues are biocompatible materials that engraft well, but the age of their source has not been explored for clinical translation. Advanced glycation end products (AGEs) are chemical cross-links that accrue on skeletal muscle collagen in old age, stiffening the matrix and increasing inflammation. Whether decellularized biomaterials derived from aged muscle would suffer from increased AGE collagen cross-links is unknown. We characterized gastrocnemii of 1-, 2-, and 20-month-old C57BL/6J mice before and after decellularization to determine age-dependent changes to collagen stiffness and AGE cross-linking. Total and soluble collagen was measured to assess if age-dependent increases in collagen and cross-linking persisted in decellularized muscle matrix (DMM). Stiffness of aged DMM was determined using atomic force microscopy. AGE levels and the effect of an AGE cross-link breaker, ALT-711, were tested in DMM samples. Our results show that age-dependent increases in collagen amount, cross-linking, and general stiffness were observed in DMM. Notably, we measured increased AGE-specific cross-links within old muscle, and observed that old DMM retained AGE cross-links using ALT-711 to reduce AGE levels. In conclusion, deleterious age-dependent modifications to collagen are present in DMM from old muscle, implying that age matters when sourcing skeletal muscle extracellular matrix as a biomaterial.

Background: Platelet-rich-plasma (PRP) is used to treat knee osteoarthritis; however, mechanistic evidence of PRP effectiveness for pain relief is limited., Objective: To assess molecular biomarkers and mesenchymal stem cells (MSCs) in synovial fluid during PRP treatment of the osteoarthritic knee joint., Design: Single blinded, randomized, placebo controlled pilot study., Setting: Veterans Affairs Medical Center., Participants: Seventeen participants with mild to moderate knee osteoarthritis were randomized in a 2:1 placebo-controlled ratio, receiving PRP or saline (placebo) intra-articular injection into the knee joint., Methods: Knee synovial fluid was analyzed before the respective injections and again 10 days following injection. Participants were followed up to 12 months completing visual analog scale (VAS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) questionnaires at intervals over that period., Main Outcome Measures: The effects of PRP on synovial protein and MSC gene expression levels were measured by multiplex enzyme-linked immunosorbent assay and quantitative polymerase chain reaction., Results: Novel biomarkers including levels of interleukin (IL)-5, IL-6, IL-10, and tumor necrosis factor-α were measured in synovial fluid 10 days after PRP treatment. Altered gene expression profiles in MSCs from patients treated with PRP were observed for matrix metalloproteinases and inflammatory markers (IL-6, IL-8, CCL2, TNF-α). A2M protease was significantly increased following PRP treatment (P = .005). WOMAC scores declined for up to 3 months from baseline levels and remained low at 6 and 12 months in the PRP group. In contrast, WOMAC scores for patients receiving the saline injection were relatively unchanged for up to 12 months., Conclusions: We report significant changes for the biomarker A2M (P = .005) as well as differences in expression of cellular markers and postulate that PRP modulates the local knee synovial environment by altering the inflammatory milieu, matrix degradation, and angiogenic growth factors. The PRP treatment group had less pain and stiffness and improved function scores., (© 2021 American Academy of Physical Medicine and Rehabilitation.)

Products developed for skeletal muscle regeneration frequently incorporate allogeneic and xenogeneic materials to elicit a regenerative response to heal skeletal muscle wounds. To avoid graft rejection in preclinical studies, immunodeficient rodents are used. Whether the immunodeficiency alters the host response to the material in skeletal muscle has not been studied. In this study, we hypothesized that an allogeneic acellular skeletal muscle grafts implanted in an immunodeficient rat (RNU, Foxn1 -deficient) would exhibit better new muscle fiber formation compared to grafts implanted in immunocompetent Sprague Dawley (SD) rats. Decellularized SD skeletal muscle matrix (DMM) was implanted in the gastrocnemius (N = 8 rats/group). 56 days after surgery, animal gait was examined and animals were euthanized. Muscle force was assessed and fiber number as well as immune cell infiltrate was measured by histomorphometry and immunohistochemistry. Animal gait and percent recovery of muscle force were unchanged in both groups, but newly regenerated muscle fibers increased in RNU rats. Macrophage staining for CD68 was higher in RNU rats than in SD rats. These data show differences in muscle regeneration between animal models using the same biomaterial treatment, but these differences could not be ascribed to the immune response. Overall, our data provide awareness that more studies are needed to understand how host responses to biomaterials differ based on the animal model used.

Viewing other people with distinctive accuracy-the degree to which personality impressions correspond with targets' unique characteristics-often predicts positive interpersonal experiences, including liking and relationship satisfaction. Does this hold in the context of first dates, or might distinctive accuracy have negative links with romantic interest in such evaluative settings? We examined this question using two speed-dating samples (Sample 1: N = 172, N = 2,407 dyads; Sample 2: N = 397, N = 1,849 dyads). Not surprisingly, positive impressions of potential dating partners were strongly associated with greater romantic interest. In contrast, distinctively accurate impressions were associated with significantly less romantic interest. This association was even stronger for potential partners whose personalities were less romantically appealing, specifically, those lower in extraversion. In sum, on a first date, distinctive accuracy tends to be paired with lower romantic interest. The potential implications of distinctive accuracy for romantic interest and of romantic interest for distinctive accuracy are discussed.

Regenerative medicine treatments for severe skeletal muscle injuries are limited, resulting in persistent functional deficits. Clinical options include neglecting the wound with the expectation that fibrosis will develop or using an autologous muscle graft with minimal functional improvement. A regenerative matrix can be used, but muscle fiber development on these matrices remains a challenge in vivo. Here, we explored the fundamental mechanisms that mediate cell-substrate signaling and its effect on cell-cell communication during myoblast fusion and tube formation to improve outcomes following implantation of matrices used to stimulate muscle regeneration. We previously reported that integrin-α7 was increased on anisotropic biomaterials, suggesting a role for α7β1 signaling in myoblast communication via connexin 43 and M-cadherin. Our results demonstrated that α7 silencing blocked expression of myogenic differentiation factor 1 (Myod), myogenin (Myog), myogenic factor 6 (Myf6), myosin heavy chain type 1 (Myh1), and transmembrane protein 8c (Tmem8c), indicating that myoblast fusion was inhibited. Expression of α5 and M-cadherin decreased but β1 and connexin 43 increased. We examined protein production and observed reduced extracellular-signal regulated kinase 1/2 (ERK) in α7-silenced cells that correlated with upregulation of connexin 43 and M-cadherin, suggesting a compensatory pathway. These results indicate that α7 signaling plays a critical role in ex vivo fusion and implicates a relationship with connexin 43 and M-cadherin.

Implanted polymer scaffolds can induce inflammation leading to the foreign body response (FBR), fibrosis, and implant failure. Thus, it is important to understand how immune cells interact with scaffolds to mitigate inflammation and promote a regenerative response. We previously demonstrated that macrophage phenotype is modulated by fiber and pore diameters of an electrospun scaffold. However, it is unclear if this effect is consistent among other innate immune cells. Mast cells are inflammatory sentinels that play a vital role in the FBR of implanted biomaterials, as well as angiogenesis. We determined if altering electrospun scaffold architecture modulates mast cell responses, with the goal of promoting regenerative cell-scaffold interactions. Polydioxanone (PDO) scaffolds were made from 60 mg/mL or 140 mg/mL PDO solutions, yielding structures with divergent fiber and pore diameters. Mouse mast cells plated on these scaffolds were activated with IL-33 or lipopolysaccharide (LPS). Relative to the 60 mg/mL scaffold, 140 mg/mL scaffolds yielded less IL-6 and TNF, and greater VEGF secretion. Pores >4-6 μm elicited less IL-6 and TNF secretion. IL-33-induced VEGF regulation was more complex, showing effects of both pore size and fiber diameter. These data indicate parameters that can predict mast cell responses to scaffolds, informing biomaterial design to increase wound healing and diminish implant rejection. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 884-892, 2019., (© 2019 Wiley Periodicals, Inc.)

Endochondral bone formation is a precise and highly ordered process whose exact regulatory framework is still being elucidated. Multiple regulatory pathways are known to be involved. In some cases, regulation impacts gene expression, resulting in changes in chondrocyte phenotypic expression and extracellular matrix synthesis. Rapid regulatory mechanisms are also involved, resulting in release of enzymes, factors and micro RNAs stored in extracellular matrisomes called matrix vesicles. Vitamin D metabolites modulate endochondral development via both genomic and rapid membrane-associated signaling pathways. 1α,25-dihydroxyvitamin D3 [1α,25(OH) 2 D 3 ] acts through the vitamin D receptor (VDR) and a membrane associated receptor, protein disulfide isomerase A3 (PDIA3). 24R,25-dihydroxyvitamin D3 [24R,25(OH) 2 D 3 ] affects primarily chondrocytes in the resting zone (RC) of the growth plate, whereas 1α,25(OH) 2 D 3 affects cells in the prehypertrophic and upper hypertrophic cell zones (GC). This includes genomically directing the cells to produce matrix vesicles with zone specific characteristics. In addition, vitamin D metabolites produced by the cells interact directly with the matrix vesicle membrane via rapid signal transduction pathways, modulating their activity in the matrix. The matrix vesicle payload is able to rapidly impact the extracellular matrix via matrix processing enzymes as well as providing a feedback mechanism to the cells themselves via the contained micro RNAs., (Copyright © 2017. Published by Elsevier Inc.)

Current strategies to treat volumetric muscle loss use primarily pedicle or free muscle transfers, but these grafts fail to adequately regenerate functional tissue. Decellularized soft tissue grafts possess physical and chemical cues to promote muscle regeneration, suggesting their potential for use in large muscle defects. In this study, we developed a decellularized muscle matrix (DMM) graft using rat gastrocnemius. Anisotropy and chemical components of the extracellular matrix were retained, including laminin, fibronectin, and collagen. We compared the ability of DMM, autologous muscle grafts (clinical standard), and type I collagen plugs (negative control) to support muscle regeneration. DMM supported regeneration over a 56-day period in 1 × 1 cm and 1.5 × 1 cm gastrocnemius defects in rats. Muscle function tests demonstrated improved muscle recovery in rats with DMM grafts when compared to collagen. Histological sections were assessed using morphometrics and immunostaining. DMM supported muscle regeneration with less fibrosis and more de novo neuromuscular receptors than either autograft or collagen. Overall, our results indicate that DMM may be used as a muscle replacement graft based on its ability to improve muscle function recovery, promote muscle regeneration, and support new neuromuscular junctions.

Volumetric muscle loss is debilitating and involves extensive rehabilitation. One approach to accelerate healing, rehabilitation, and muscle function is to repair damaged skeletal muscle using regenerative medicine strategies. In sports medicine and orthopedics, a common clinical approach is to treat minor to severe musculoskeletal injuries with platelet-rich plasma (PRP) injections. While these types of treatments have become commonplace, there are limited data demonstrating their effectiveness. The goal of this study was to determine the effect of PRP on myoblast gene expression and protein production when incorporated into a polymer fiber. To test this, we generated extracellular matrix mimicking scaffolds using aligned polydioxanone (PDO) fibers containing lyophilized PRP (SmartPReP ® 2, Harvest Technologies Corporation, Plymouth, MA). Scaffolds with PRP caused a dose-dependent increase in myogenin and myosin heavy chain but did not affect myogenic differentiation factor-1 (MyoD). Integrin α7β1D decreased and α5β1A did not change in response to PRP scaffolds. ERK inhibition decreased myogenin and increased Myod on the PDO-PRP scaffolds. Taken together, these data suggest that alignment and PRP produce a substrate-dependent, ERK-dependent, and dose-dependent effect on myogenic differentiation.

Chondrocytes at different maturation states in the growth plate produce matrix vesicles (MVs), membrane organelles found in the extracellular matrix, with a wide range of contents, such as matrix processing enzymes and receptors for hormones. We have shown that MVs harvested from growth zone (GC) chondrocyte cultures contain abundant small RNAs, including miRNAs. Here, we determined whether RNA also exists in MVs produced by less mature resting zone (RC) chondrocytes and, if so, whether it differs from the RNA in MVs produced by GC cells. Our results showed that RNA, small RNA specifically, was present in RC-MVs, and it was well-protected from RNase by the phospholipid membrane. A group of miRNAs was enriched in RC-MVs compared RC-cells, suggesting that miRNAs are selectively packaged into MVs. High throughput array and RNA sequencing showed that ~39% miRNAs were differentially expressed between RC-MVs and GC-MVs. Individual RT-qPCR also confirmed that miR-122-5p and miR-150-5p were expressed at significantly higher levels in RC-MVs compared to GC-MVs. This study showed that growth plate chondrocytes at different differentiation stages produce different MVs with different miRNA contents, further supporting extracellular vesicle miRNAs play a role as "matrisomes" that mediate the cell-cell communication in cartilage and bone development.

People's reports of their thoughts, feelings, and behaviors are used in many fields of biomedical and social science. When these states have been studied over time, researchers have often observed an unpredicted and puzzling decrease with repeated assessment. When noted, this pattern has been called an "attenuation effect," suggesting that the effect is due to bias in later reports. However, the pattern could also be consistent with an initial elevation bias. We present systematic, experimental investigations of this effect in four field studies (study 1: n = 870; study 2: n = 246; study 3: n = 870; study 4: n = 141). Findings show clear support for an initial elevation bias rather than a later decline. This bias is larger for reports of internal states than for behaviors and for negative mental states and physical symptoms than for positive states. We encourage increased awareness and investigation of this initial elevation bias in all research using subjective reports., Competing Interests: The authors declare no conflict of interest.

Regulating soft tissue repair to prevent fibrosis and promote regeneration is central to creating a microenvironment conducive to soft tissue development. Macrophages play an important role in this process. The macrophage response can be modulated using biomaterials, altering cytokine and growth factor secretion to promote regeneration. Electrospun polydioxanone (PDO) fiber scaffolds promoted an M2 phenotype when macrophages were cultured on large diameter, highly porous scaffolds, but an M1 phenotype on smaller diameter fibers. In this study, we investigated whether incorporation of galectin-1, an immunosuppressive protein that enhances muscle regeneration, could promote the M2 response. Galectin-1 was incorporated into large and small fiber PDO scaffolds during electrospinning. Galectin-1 incorporation increased arginase-1 and reduced iNOS and IL-6 production in mouse bone-marrow derived macrophages compared with PDO alone for both scaffold types. Inhibition of ERK mitogen-activated protein kinase did not alter galectin-1 effects on arginase-1 and iNOS expression, but reversed IL-6 suppression, indicating that IL-6 is mediated by a different mechanism. Our results suggest that galectin-1 can be used to modulate macrophage commitment to a pro-regenerative M2 phenotype, which may positively impact tissue regeneration when using small diameter PDO scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2562-2571, 2017., (© 2017 Wiley Periodicals, Inc.)

Unlabelled: The aligned structural environment in skeletal muscle is believed to be a crucial component in functional muscle regeneration. Myotube formation is increased on aligned biomaterials, but we do not fully understand the mechanisms that direct this enhanced fusion. Previous studies indicate that the α7 integrin subunit is upregulated during myoblast differentiation, suggesting that signaling via α7β1 mediates the effect of alignment. To test this hypothesis, we took advantage of an in vitro model using random and aligned polydioxanone (PDO) matrices and C2C12 myoblasts. We measured expression and production of myoblast markers: paired box-7 (Pax7), myogenic differentiation factor-1 (MyoD), myogenin (MyoG), myogenic factor-6 (Myf6), and myosin heavy chain (MyHC). To examine the role of α7β1 signaling, we measured expression and production of α7, α5, and β1 and myoblast markers in wild type cells and in cells silenced for α7 and assessed effects of silencing on myogenic differentiation. Downstream signaling via ERK1/2 mitogen activated protein kinase (MAPK) was examined using a specific MEK1/2 inhibitor. Alignment increased mRNAs and protein for early (MyoD) and late (MyoG, MyHC) myoblast markers in comparison to non-aligned matrices, and these levels corresponded with increased α7 protein. α7-silencing reduced MyoG and MyHC protein in cells cultured on tissue culture polystyrene and aligned PDO matrices compared to wild type cells. Inhibition of ERK1/2 blocked effects of alignment. These data suggest that alignment regulates myogenic differentiation via α7β1 integrin signaling and ERK1/2 mediated gene expression., Statement of Significance: Muscle regeneration in severe muscle injuries is complex, requiring a sequence of events to promote healing and not fibrosis. Aligned biomaterials that recapitulate muscle environments hold potential to facilitate regeneration, but it is important to understand cell-substrate signaling to form functional muscle. A critical component of muscle signaling is integrin α7β1, where mice lacking α7 exhibit a dystrophic phenotype and impaired regeneration. Here, we report the role of α7β1 signaling in myoblast differentiation on aligned biomaterials. α7-silenced myoblasts were found to regulate myogenic differentiation and demonstrate defective fusion. Our data shows reduced levels of myogenin and myosin heavy chain protein, while MyoD remains unchanged. These results support the hypothesis that α7β1 signaling plays a role in substrate-dependent tissue engineering strategies., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The ability to expand and direct both precursor and stem cells towards a differential fate is considered extremely advantageous in tissue engineering. Platelet-rich plasma (PRP) possesses a milieu of growth factors and cytokines, which have the potential to have either a differentiative or proliferative influence on the cell type tested. Here, we investigated the effect of PRP on C2C12 myoblasts. A range of PRP concentrations in differentiation media was used to determine whether a concentration dependence existed, while PRP embedded in fibres of aligned electrospun polydioxanone and polycaprolactone was used to determine whether this presence of fibres would cause any differences in response. In both cases, it was found that late myogenic markers were suppressed after 7 days in culture. However, an early differentiation marker, MyoD, was upregulated during this same time period. The results from this study represent the ability of PRP to have an influence over both myogenic proliferation and differentiation, a factor which could prove useful in future studies involved with skeletal muscle tissue engineering., (Copyright © 2013 John Wiley & Sons, Ltd.)

The benefits of close relationships for mental and physical health are well documented. One of the mechanisms presumed to underlie these effects is social support, whereby close others provide practical and emotional assistance in times of need. Although there is no doubt that generalized perceptions of support availability are beneficial, research examining actual instances of support receipt has found unexpectedly mixed results. Receiving support sometimes has positive effects, but null or even negative effects are common. In this article, we review our multimethod program of research that seeks to understand and explain the costs of receiving social support. We focus on reductions in the recipient's sense of relationship equity and self-efficacy as mechanisms of this effect and examine a number of other moderating factors. Although we have found that receiving support incurs costs on average, there is considerable variability yet to be explained. Using diary data from 312 persons preparing to take a challenging exam, we examined the potential of individual differences in neuroticism, agreeableness, and attachment insecurity to explain variability in experienced support costs. We close with new questions about why received support may be beneficial or benign in some situations while being especially toxic in others., (© 2013 Wiley Periodicals, Inc.)

Although research has shown that attachment anxiety is detrimental to ongoing relationships, less is known about whether and how it affects the earliest stages of relationship initiation. How does attachment anxiety affect an initial interaction with a potential relationship partner? The present investigation explored the interpersonal outcomes associated with attachment anxiety in the context of various relational opportunities, testing a mediational model whereby interpersonal displays characteristic of state social anxiety-social disengagement and manifest anxiety-were proposed as mechanisms of interpersonal failure. In Study 1, participants engaged in speed-dating. In Study 2, participants were videotaped introducing themselves to an attractive, single, preferred-sex confederate who would ostensibly be deciding whether to meet them. In Study 3, participants were videotaped having a semistructured 40-min interaction with an attractive, friendly, single, preferred-sex confederate. Across all 3 contexts, attachment anxiety was associated with negative interpersonal outcomes, mediated by displays of social disengagement (Study 2) and manifest anxiety (Studies 1 and 3). The negative displays and outcomes associated with attachment anxiety were expressed behaviorally as verbal disfluencies and interpersonal awkwardness (Study 3). Overall, attachment anxiety was a robust predictor of interpersonal failure when presented with a relational opportunity. Such failures will reinforce the negative expectations underpinning state social anxiety, making it harder for more anxiously attached individuals to initiate and develop the satisfying relationships that might over time help them overcome their relational insecurity.

For blood vessel tissue engineering, an ideal vascular graft should possess excellent biocompatibility and mechanical properties. For this study, a elastic material of poly (L-lactic acid-co-ε-caprolactone) (P(LLA-CL)), collagen and chitosan blended scaffold at different ratios were fabricated by electrospinning. Upon fabrication, the scaffolds were evaluated to determine the tensile strength, burst pressure, and dynamic compliance. In addition, the contact angle and endothelial cell proliferation on the scaffolds were evaluated to demonstrate the structures potential to serve as a vascular prosthetic capable of in situ regeneration. The collagen/chitosan/P(LLA-CL) scaffold with the ratio of 20:5:75 reached the highest tensile strength with the value of 16.9 MPa, and it was elastic with strain at break values of ~112%, elastic modulus of 10.3 MPa. The burst pressure strength of the scaffold was greater than 3365 mmHg and compliance value was 0.7%/100 mmHg. Endothelial cells proliferation was significantly increased on the blended scaffolds versus the P(LLA-CL). Meanwhile, the endothelial cells were more adherent based on the increase in the degree of cell spreading on the surface of collagen/chitosan/P(LLA-CL) scaffolds. Such blended scaffold especially with the ratio of 20:5:75 thus has the potential for vascular graft applications., (Copyright © 2012 Wiley Periodicals, Inc.)

Objective: Humans face an enduring conflict between desires to affiliate with others but to protect the self; effective social functioning often requires reconciling the resulting ambivalence between these motives. Attachment anxiety is characterized by chronically heightened concerns about affiliation and self-protection; we investigated how anxious individuals' chronic relational ambivalence affects interpersonal behavior., Method: We used the Prisoner's Dilemma and the Assurance Game to examine how the ambivalence associated with attachment anxiety affects pro-social behavior, comparing chronic attachment anxiety with both chronic (Study 1; N = 94) and contextually activated (Study 2; N = 56) security., Results: Chronic attachment anxiety was associated with ambivalent behavior in the social dilemma games. Specifically, the chronically anxious were mistrustfully inconsistent in their strategic choices and took more time to make these choices. However, priming the chronically anxious with attachment security decreased ambivalence by promoting more fluent cooperative behavior., Conclusions: To our knowledge, these are the first studies to examine the effect of the anxiously attached's chronic relational ambivalence on pro-social behavior. These findings illustrate that the simultaneous activation of affiliation and self-protection can have interpersonal consequences, increasing mistrust and hesitance. Importantly, however, we were able to attenuate these effects by priming felt security., (© 2012, Wiley Periodicals, Inc.)

The purpose of this study was to create seamless, acellular, small diameter bioresorbable arterial grafts that attempt to mimic the extracellular matrix and mechanical properties of native artery using synthetic and natural polymers. Silk fibroin, collagen, elastin, and polycaprolactone (PCL) were electrospun to create a tri-layered structure for evaluation. Dynamic compliance testing of the electrospun grafts ranged from 0.4-2.5%/100 mmHg, where saphenous vein (1.5%/100 mmHg) falls within this range. Increasing PCL content caused a gradual decrease in medial layer compliance, while changes in PCL, elastin, and silk content in the adventitial layer had varying affects. Mathematical modeling was used to further characterize these results. Burst strength results ranged from 1614-3500 mmHg, where some exceeded the capacity of the pressure regulator. Four week degradation studies demonstrated no significant changes in compliance or burst strength, indicating that these grafts could withstand the initial physiological conditions without risk of degradation. Overall, we were able to manufacture a multi-layered graft that architecturally mimics the native vascular wall and mechanically matches the gold standard of vessel replacement, saphenous vein., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Electrospun non-woven structures have the potential to form bioresorbable vascular grafts that promote tissue regeneration in situ as they degrade and are replaced by autologous tissue. Current bioresorbable grafts lack appropriate regeneration potential since they do not have optimal architecture, and their fabrication must be altered by the manipulation of process parameters, especially enhancing porosity. We describe here an air-impedance process where the solid mandrel is replaced with a porous mandrel that has pressurized air exiting the pores to impede fiber deposition. The mandrel design, in terms of air-flow rate, pore size, and pore distribution, allows for control over fiber deposition and scaffold porosity, giving greater cell penetration without a detrimental loss of mechanical properties or structural integrity., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery under pulsatile deformations. The goal of this study was to mimic the structure of native artery by fabricating a multi-layered electrospun conduit composed of poly(caprolactone) (PCL) with the addition of elastin and collagen with blends of 45-45-10, 55-35-10, and 65-25-10 PCL-ELAS-COL to demonstrate mechanical properties indicative of native arterial tissue, while remaining conducive to tissue regeneration. Whole grafts and individual layers were analyzed using uniaxial tensile testing, dynamic compliance, suture retention, and burst strength. Compliance results revealed that changes to the middle/medial layer changed overall graft behavior with whole graft compliance values ranging from 0.8 - 2.8 % / 100 mmHg, while uniaxial results demonstrated an average modulus range of 2.0 - 11.8 MPa. Both modulus and compliance data displayed values within the range of native artery. Mathematical modeling was implemented to show how changes in layer stiffness affect the overall circumferential wall stress, and as a design aid to achieve the best mechanical combination of materials. Overall, the results indicated that a graft can be designed to mimic a tri-layered structure by altering layer properties.

The process of electrospinning has proven to be highly beneficial for use in a number of tissue-engineering applications due to its ease of use, flexibility and tailorable properties. There have been many publications on the creation of aligned fibrous structures created through various forms of electrospinning, most involving the use of a metal target rotating at high speeds. This work focuses on the use of a variation known as airgap electrospinning, which does not use a metal collecting target but rather a pair of grounded electrodes equidistant from the charged polymer solution to create highly aligned 3D structures. This study involved a preliminary investigation and comparison of traditionally and airgap electrospun silk-fibroin-based ligament constructs. Structures were characterized with SEM and alignment FFT, and underwent porosity, permeability, and mechanical anisotropy evaluation. Preliminary cell culture with human dermal fibroblasts was performed to determine the degree of cellular orientation and penetration. Results showed airgap electrospun structures to be anisotropic with significantly increased porosity and cellular penetration compared to their traditionally electrospun counterparts.

Initiating a romantic relationship invokes an approach-avoidance conflict between the desire for affiliation and the fear of rejection; optimally, people should selectively approach potential partners who reciprocate their interest. This may be difficult for anxiously attached people: They may be unpopular, and their ambivalence could lead to either a fearfully selective approach at the cost of missed opportunities or an unselective, indiscriminate approach at the cost of increasing rejection. Using a speed-dating paradigm, data were collected from 116 participants, and a signal detection framework was applied to examine the outcomes. For anxious participants, speed-dating attendance was motivated by loneliness. At speed dating, they were unpopular and unselective; they missed fewer opportunities but made more failed attempts. Anxious men made fewer matches than nonanxious men, whereas anxious women were buffered by having a response bias toward saying "yes" to potential partners. Attachment anxiety predicted outcomes above and beyond the powerful impact of attractiveness.

Throughout native artery, collagen, and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery under pulsatile deformations. The goal of this study was to mimic the structure of native artery by fabricating a multi-layered electrospun conduit composed of poly(caprolactone) (PCL) with the addition of elastin and collagen with blends of 45-45-10, 55-35-10, and 65-25-10 PCL-ELAS-COL to demonstrate mechanical properties indicative of native arterial tissue, while remaining conducive to tissue regeneration. Whole grafts and individual layers were analyzed using uniaxial tensile testing, dynamic compliance, suture retention, and burst strength. Compliance results revealed that changes to the middle/medial layer changed overall graft behavior with whole graft compliance values ranging from 0.8 to 2.8%/100 mm Hg, while uniaxial results demonstrated an average modulus range of 2.0-11.8 MPa. Both modulus and compliance data displayed values within the range of native artery. Mathematical modeling was implemented to show how changes in layer stiffness affect the overall circumferential wall stress, and as a design aid to achieve the best mechanical combination of materials. Overall, the results indicated that a graft can be designed to mimic a tri-layered structure by altering layer properties., (Copyright 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The process of electrospinning has seen a resurgence of interest in the last few decades which has led to a rapid increase in the amount of research devoted to its use in tissue engineering applications. Of this research, the area of cardiovascular tissue engineering makes up a large percentage, with substantial resources going towards the creation of bioresorbable vascular grafts composed of electrospun nanofibers of collagen and other biopolymers. These bioresorbable grafts have compositions that allow for the in situ remodeling of the structure, with the eventual replacement of the graft with completely autologous tissue. This review will highlight some of the work done in the field of electrospinning for cardiovascular applications, with an emphasis on the use of biopolymers such as collagens, elastin, gelatin, fibrinogen, and silk fibroin, as well as biopolymers used in combination with resorbable synthetic polymers.

Extracellular matrices are arranged with a specific geometry based on tissue type and mechanical stimulus. For blood vessels in the body, preferential alignment of fibers is in the direction of repetitive force. Electrospinning is a controllable process which can result in fiber alignment and randomization depending on the parameters utilized. In this study, arterial grafts composed of polycaprolactone (PCL), polydioxanone (PDO) and silk fibroin in blends of 100:0 and 50:50 for both PCL:silk and PDO:silk were investigated to determine if fibers could be controllably aligned using a mandrel rotational speed ranging from 500 to 8000 revolutions per minute (RPM). Results revealed that large- and small-diameter mandrels produced different degrees of fiber alignment based on a fast Fourier transform of scanning electron microscope images. Uniaxial tensile testing further demonstrated scaffold anisotropy through changes in peak stress, modulus and strain at break at mandrel rotational speeds of 500 and 8000 RPM, causing peak stress and modulus for PCL to increase 5- and 4.5-fold, respectively, as rotational speed increased. Additional mechanical testing was performed on grafts using dynamic compliance, burst strength and longitudinal strength displaying that grafts electrospun at higher rotational rates produced stiffer conduits which had lower compliance and higher burst strength compared to the lower mandrel rotational rate. Scaffold properties were found to depend on several parameters in the electrospinning process: mandrel rotational rate, polymer type, and mandrel size. Vascular scaffold design under anisotropic conditions provided interesting insights and warrants further investigation.

The purpose of this study was to enhance the mechanical properties and slow the degradation of an electrospun fibrinogen scaffold, while maintaining the scaffold's high level of bioactivity. Three different cross-linkers were used to achieve this goal: glutaraldehyde vapour, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in ethanol and genipin in ethanol. Scaffolds with a fibrinogen concentration of 120 mg ml(-1) were electrospun and cross-linked with one of the aforementioned cross-linkers. Mechanical properties were determined through uniaxial tensile testing performed on scaffolds incubated under standard culture conditions for 1 day, 7 days and 14 days. Cross-linked scaffolds were seeded with human foreskin fibroblasts (BJ-GFP-hTERT) and cultured for 7, 14 and 21 days, with histology and scanning electron microscopy performed upon completion of the time course. Mechanical testing revealed significantly increased peak stress and modulus values for the EDC and genipin cross-linked scaffolds, with significantly slowed degradation. However, cross-linking with EDC and genipin was shown to have some negative effect on the bioactivity of the scaffolds as cell migration throughout the thickness of the scaffold was slowed.

This study aims at evaluating the importance of the two-component regulatory system VicRK to virulence of the horse pathogen Streptococcus equi subspecies equi and the potential of a vicK mutant as a live vaccine candidate using mouse infection models. The vicK gene was deleted by gene replacement. The DeltavicK mutant is attenuated in virulence in both subcutaneous and intranasal infections in mice. DeltavicK grows less slowly than the parent strain but retains the ability of S. equi to resist to phagocytosis by polymorphoneuclear leukocytes, suggesting that the vicK deletion causes growth defect. DeltavicK infection protects mice against reinfection with a wild-type S. equi strain. Intranasal DeltavicK infection induces production of anti-SeM mucosal IgA and systemic IgG. These results indicate that VicRK is important to S. equi growth and virulence and suggest that DeltavicK has the potential to be developed as a live S. equi vaccine.

This study characterizes the cross-linking of electrospun elastin and the mechanical properties of suture-reinforced 1.5mm internal diameter electrospun tubes composed of blended polydioxanone (PDO) and soluble elastin. Several tube configurations were tested to assess the effects of reinforcement on tube mechanical properties. Between the electrospun layers of each double-layered prosthetic, zero, one or two 6-0 sutures were wound, maintaining 1mm spacing with a pitch of 9 degrees . Single-layered tubes without suture were also examined. Samples were cross-linked and tested for compliance and burst strength. Compliance decreased significantly (p <0.05) and burst strength significantly increased (p <0.01) with reinforcement. Uncross-linked tubes were also tested to determine the effects of cross-linking. Results demonstrated that cross-linking significantly decreases burst strength (p <0.01), while decreases in compliance for cross-linked tubes were not significant. Cross-linked suture-reinforced PDO-elastin tubes had burst pressures more than 10 times greater than normal systolic pressures and exhibited a range of compliance values, including those matching native artery. These tubes display many characteristics of the "ideal" small-diameter graft, having mechanical properties that can be tailored to match those desired in vascular replacement applications.

Aims: To evaluate prospectively the impact of an appendix ultrasound (US) service on the clinical management of patients presenting with suspected acute appendicitis., Materials and Methods: The referring clinician completed a proforma for patients presenting with suspected acute appendicitis. Two visual analogue scales assessed clinical suspicion before and after knowledge of laboratory results. The clinician also indicated if they intended to operate had US been unavailable. During a 3-year period, 327 patients were examined by graded-compression US and diagnosed "positive" or "negative" for acute appendicitis. Findings were correlated with histopathology results. The referring clinician completed a retrospective audit questionnaire to assess user satisfaction., Results: Clinical suspicion was altered by knowledge of laboratory results. The decision to operate if US had been unavailable, was "yes" in 70 cases (group A), "no" in 231 (group B), and incomplete in 26 (group C). In group A, 31 patients (44.3%) had a negative US and 25 avoided surgery. US identified 39 cases of appendicitis and 37 appendicectomies confirmed appendicitis in 34 cases. In group B, 72 (31.2%) patients had a positive US and 66 appendicectomies confirmed 51 cases of appendicitis. The sensitivity of US was 94.7% in group A, 93.3% in group B and 93.8% overall. Specificity was 90.6% in group A, 91.2% in group B and 91.3% overall. US findings were contrary to intended surgical management in 103 cases. Management was altered in 97 cases (32.2%), with a positive outcome in 85 (28.2%). The referrers found US of appendix very useful in planning appropriate management., Conclusion: US of the appendix increases diagnostic accuracy, alters management and is more sensitive and specific than clinical impression, either alone, or in conjunction with laboratory results.

Background: Heme is a preferred iron source of bacterial pathogens. Streptococcus equi subspecies equi is a bacterial pathogen that causes strangles in horses. Whether S. equi has a heme acquisition transporter is unknown., Results: An S. equi genome database was blasted with the heme binding proteins Shp and HtsA of Streptococcus pyogenes, and found that S. equi has the homologue of Shp (designated SeShp) and HtsA (designated SeHtsA). Tag-free recombinant SeShp and SeHtsA and 6xHis-tagged SeHtsA (SeHtsAHis) were prepared and characterized. Purified holoSeShp and holoSeHtsA bind Fe(II)-protoporphyrin IX (heme) and Fe(III)-protoporphyrin IX (hemin) in a 1:1 stoichiometry, respectively, and are designated hemoSeShp and hemiSeHtsA. HemiSeShp and hemiSeHtsAHis can be reconstituted from apoSeShp and apoSeHtsAHis and hemin. HemoSeShp is stable in air and can be oxidized to hemiSeShp by ferricyanide. HemiSeHtsA can be reduced into hemoSeHtsA, which autoxidizes readily. HemoSeShp rapidly transfers its heme to apoSeHtsAHis. In addition, hemoSeShp can also transfer its heme to apoHtsA, and hemoShp is able to donate heme to apoSeHtsAHis., Conclusion: The primary structures, optical properties, oxidative stability, and in vitro heme transfer reaction of SeShp and SeHtsA are very similar to those of S. pyogenes Shp and HtsA. The data suggest that the putative cell surface protein SeShp and lipoprotein SeHtsA are part of the machinery to acquire heme in S. equi. The results also imply that the structure, function, and functional mechanism of the heme acquisition machinery are conserved in S. equi and S. pyogenes.

The genome of the human pathogen group A Streptococcus (GAS) encodes the transporters MtsABC, FtsABCD, and HtsABC to take up ferric and manganese ions, ferric ferrichrome, and heme, respectively. The GAS genome also encodes two metalloregulators PerR and MtsR. To understand the regulation of the expression of these transporters, the mtsR and perR deletion mutants of a GAS serotype M1 strain were generated, and the effects of the deletions and Fe(3+), Mn(2+), and Zn(2+) on the expression of mtsA, htsA, and ftsB were examined. Mn(2+) dramatically depresses mtsA transcription and levels of the MtsA protein but does not downregulate the expression of htsA and ftsB. Fe(3+) decreases the expression of mtsA and htsA but has no effect on ftsB expression. Zn(2+) has no effect on the expression of all three genes. The deletion of mtsR abolishes the Mn(2+)- and Fe(3+)-induced depression of mtsA expression and the Fe(3+)-dependent decrease in htsA expression. The deletion of mtsR does not significantly alter GAS virulence in a mouse model of subcutaneous infection. The deletion of perR does not affect the expression of the genes in response to the metal ions. MtsR binds to the mts promoter region in the presence of Mn(2+) or Fe(2+). The results indicate that MtsR differentially regulates the expression of mtsABC and htsABC.

An electrospun cardiovascular graft composed of polydioxanone (PDO) and elastin has been designed and fabricated with mechanical properties to more closely match those of native arterial tissue, while remaining conducive to tissue regeneration. PDO was chosen to provide mechanical integrity to the prosthetic, while elastin provides elasticity and bioactivity (to promote regeneration in vitro/in situ). It is the elastic nature of elastin that dominates the low-strain mechanical response of the vessel to blood flow and prevents pulsatile energy from being dissipated as heat. Uniaxial tensile and suture retention tests were performed on the electrospun grafts to demonstrate the similarities of the mechanical properties between the grafts and native vessel. Dynamic compliance measurements produced values that ranged from 1.2 to 5.6%/100 mmHg for a set of three different mean arterial pressures. Results showed the 50:50 ratio to closely mimic the compliance of native femoral artery, while grafts that contained less elastin exceeded the suture retention strength of native vessel. Preliminary cell culture studies showed the elastin-containing grafts to be bioactive as cells migrated through their full thickness within 7 days, but failed to migrate into pure PDO scaffolds. Electrospinning of the PDO and elastin-blended composite into a conduit for use as a small diameter vascular graft has extreme potential and warrants further investigation as it thus far compares favorably to native vessel.

The regulator VicR of the two-component regulatory system VicRK is essential in several Gram-positive bacteria. However, the authors were able to generate an unconditional vicR insertional mutant of group A Streptococcus. This mutant grew well in rich media but not in non-immune human blood and serum, had attenuated virulence, and was unstable in mice. Complementation of the mutant with vicR expressed in trans restored its phenotype to wild-type. A vicK deletion mutant had a phenotype similar to that of the vicR mutant. Phagocytosis and killing of the vicR mutant were normal, suggesting that VicRK does not regulate processes involved in evasion of host defence. Microarray analysis showed that vicR inactivation down-regulated the transcription of 13 genes, including putative cell wall hydrolase gene pcsB and spy1058-1060, which encode a putative phosphotransferase system enzyme II for carbohydrate transport, and upregulated the expression of five genes, including spy0183 and spy0184, which encode an osmoprotectant transporter OpuA. Consistent with microarray analysis, the vicR mutant took up more of the osmoprotectants betaine and proline and was sensitive to osmotic stress, indicating that vicR inactivation induced osmotic stress and increased susceptibility to osmotic pressure. Additionally, a spy1060 deletion mutant also displayed attenuated virulence. These results suggest that VicRK regulates processes involved in cell wall metabolism, nutrient uptake, and osmotic protection.

Background: The Streptococcus pyogenes or Group A Streptococcus (GAS) genome encodes three ABC transporters, namely, FtsABCD, MtsABC, and HtsABC, which share homology with iron transporters. MtsABC and HtsABC are believed to take up ferric (Fe3+) and manganese ions and heme, respectively, while the specificity of FtsABCD is unknown., Results: Recombinant FtsB, the lipoprotein component of FtsABCD, was found to bind Fe3+ ferrichrome in a 1:1 stoichiometry. To investigate whether FtsABCD transports Fe3+ ferrichrome, GAS isogenic strains defective in lipoprotein gene ftsB and permease gene ftsC were generated, and the effects of the mutations on uptake of Fe3+ ferrichrome were examined using radioactive 55Fe3+ ferrichrome. FtsB was produced in the wild-type strain but not in the ftsB mutant, confirming the ftsB inactivation. While wild-type GAS took up 3.6 x 10(4) Fe3+ ferrichrome molecules per bacterium per min at room temperature, the ftsB and ftsC mutants did not have a detectable rate of Fe3+ ferrichrome uptake. The inactivation of ftsB or ftsC also decreased 55Fe3+ ferrichrome uptake by >90% under growth conditions in the case of limited uptake time. Complementation of the ftsB mutant with a plasmid carrying the ftsB gene restored FtsB production and 55Fe3+ ferrichrome association at higher levels compared with the parent strain. The inactivation of mtsA and htsA and Fe-restricted conditions enhanced the production of FtsB and Fe3+ ferrichrome uptake., Conclusion: The FtsB protein bound Fe3+ ferrichrome, and inactivation of ftsB or ftsC, but not htsA or mtsA, diminished Fe3+ ferrichrome uptake, indicating that FtsABCD, but not HtsABC and MtsABC, is the transporter that takes up Fe3+ ferrichrome in GAS. Fe acquisition systems are virulence factors in many bacterial pathogens and are attractive vaccine candidates. The elucidation of the FtsABCD specificity advances the understanding of Fe acquisition processes in GAS and may help evaluating the GAS Fe acquisition systems as vaccine candidates.

Objective: This study describes the sonographic features of sacral perineural cysts that initially presented as adnexal complex cystic masses on transvaginal sonography., Conclusion: Perineural cysts may have a complex cystic appearance, including septation and internal debris, on transvaginal sonography. The extraovarian, extraperitoneal, and posterior location on real-time sonography are suggestive features.

Epiploic appendagitis and segmental omental infarction are more frequently encountered with the increased use of abdominal ultrasound and Computed tomography (CT) in the radiological assessment of the patient who presents clinically with acute abdominal pain. Recognition of specific imaging abnormalities enables the radiologist to make the correct diagnosis. This is important, as the appropriate management of both conditions is often conservative. Follow-up imaging features correlate with clinical improvement.