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Sclerostin (SOST) is a Wnt signaling inhibitor detrimental to osteogenic differentiation and bone mineral acquisition. While control of SOST action delays the pathogenesis of skeletal disorders, the effects of SOST vaccination on the estrogen deficiency-induced bone deterioration remain elusive. In this study, we generated a SOST-Fc fusion protein which was composed of a SOST peptide Pro-Asn-Ala-Ile-Gly along with an IgG Fc fragment. SOST-Fc vaccination increased serum anti-SOST antibody levels and reduced serum SOST concentrations in mice. In vitro, anti-SOST serum attenuated the SOST-induced inhibition of osteogenic gene expression in osteoblast cultures. Administration with SOST-Fc increased serum levels of bone formation marker osteocalcin and alleviated the ovariectomy escalation of serum resorption markers CTX-1 and TRAP5b concentrations. It remarkably lessened the estrogen deficiency-mediated deterioration of bone mineral density, morphometric characteristics of trabecular bone, and mechanical strength of femurs and lumbar spines. The SOST-Fc-treated skeletal tissue exhibited moderate responses to the adverse actions of ovariectomy to bone mineral accretion, osteoclast surface, trabecular separation, and fatty marrow histopathology. SOST-Fc treatment increased serum osteoclast-inhibitory factor osteoprotegrin levels in conjunction with strong Wnt3a, β-catenin, and TCF4 immunostaining in osteoblasts, whereas it weakened the estrogen deficiency enhancement of osteoclast-promoting factor receptor activator of nuclear factor-κB ligand. Taken together, blockade of SOST action by SOST-Fc vaccination sustains Wnt signaling, which harmonizes bone mineral accretion and resorption reactions and thereby ameliorates ovariectomy-induced bone loss. This study highlights SOST-Fc fusion protein as a new molecular therapeutic potential for preventing from osteoporotic disorders. [ABSTRACT FROM AUTHOR]

Highlights: [•] A cell recycling two-tank system was used for continuous production of bioethanol. [•] Each tank fermentor equipped with a settler for recycling of flocculent yeast. [•] The system was effective for ethanol production at high dilution rates. [•] The productivity was tremendously enhanced compared with batch fermentation. [ABSTRACT FROM AUTHOR]

Abstract: Excessive glucocorticoid administration accelerates osteoblast apoptosis and skeletal deterioration. Heat shock proteins (HSPs) regulate metabolic activities in osteoblastic cells. This study characterized the biological significance of HSP60 in glucocorticoid-induced bone loss. Rats were treated with glucocorticoid, HSP60 antisense oligonucleotides, or adenovirus-mediated HSP60 gene transfer. Bone mineral density, metaphyseal trabecular micro-architecture, and fragility were analyzed by dual X-ray absorptiometry, micro-computed tomography, and material testing, respectively. Differential proteomic profiles of bone tissue extracts were detected by bi-dimensional electrophoresis and mass spectrometry. Survival and proapoptotic signal transduction were quantified by immunoblotting. Glucocorticoid-treated rats had low bone mineral density and metaphyseal trabecular microstructure in association with downregulation of collagen 1α1 and HSP60 expressions in bone tissue. Gain of HSP60 function by adenovirus-mediated HSP60 gene transfer abrogated the deleterious effects of glucocorticoid treatment on bone mass, trabecular microstructure, and mechanical strength. Enhancement of HSP60 signaling attenuated the glucocorticoid-induced loss of trabecular bone volume, mineral acquisition reactions and osteoblast surface. HSP60 gene transfer activated ERK and Akt and reduced Bax and cytochrome c release, as well as caspase-3 cleavage, which attenuated the inhibitory effects of glucocorticoid treatment on osteoblast survival. Loss of HSP60 function by HSP60 antisense oligonucleotides accelerated mitochondrial apoptotic programs and osteoblast apoptosis. Knockdown of HSP60 induced loss of bone mass, micro-architecture integrity, and mechanical property. Taken together, loss of HSP60 signaling contributes to the glucocorticoid-induced enhancement of pro-apoptotic reactions, thereby accelerating osteoblast apoptosis and bone mass loss. Enhancement of HSP60 function is beneficial for protecting bone tissue against the glucocorticoid-induced inhibition of bone cell viability and bone formation. [Copyright &y& Elsevier]

Abstract: Computer-aided process/solvent design is introduced to find a feasible biocompatible solvent for an extractive fermentation and separation process. The designed biocompatible solvent serves as both the extractant for extractive fermentation and the entrainer for extractive distillation, to yield water-free ethanol. Several goals, such as maximizing production rate and extraction efficiency, and limiting solvent utilization, are simultaneously considered in the optimal solvent design problem. Thus, the design was formulated as a mixed-integer nonlinear optimization problem. A two-phase computational scheme was introduced to solve the problem. The mixed-integer hybrid differential evolution (MIHDE) algorithm was first applied to solve the problem in order to obtain a feasible solution. The feasible solution was then served as an initial starting point for the mixed-integer sequential quadratic programming (MISQP) solver to numerically confirm that the optimal design was achieved. We have compared the crisp and fuzzy approaches to the design problem. The fuzzy goal attainment approach is able to address goal trade-offs and yield an overall satisfactory grade for the problem. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we considered a multi-stage integrated extractive fermentation with cell recycling for ethanol production using the genetically engineered Sacchromyces yeast 1400 (pLNH33), which can utilize glucose and xylose as carbon sources to produce ethanol. Each stage consists of a stirred-tank bioreactor, a cell settler and an extractor. A generalized mathematical model was formulated to express the multi-stage integrated process. The aim of the optimization problem was to obtain the maximum overall productivity and conversions subject to the interval inequality constraints for the residual glucose and xylose concentrations and the total sugar supply. A fuzzy goal attainment method was applied to the multiobjective problem in order to achieve the maximum satisfaction for all design requirements. From the computational results, the integrated extractive fermentation with cell recycling (involving the extraction of ethanol from the extractor in situ to alleviate product inhibition) led to an optimal overall productivity that was 8.0% higher than that obtained by the method of continuous fermentation with cell recycling, and about 13-fold higher than that obtained by the method of continuous fermentation without cell recycling. [Copyright &y& Elsevier]

Abstract: Intensive bone cell apoptosis contributes to osteonecrosis of femoral head (ONFH). Dickkopf-1 (DKK1) reportedly mediates various types of skeletal disorders. This study investigated whether DKK1 was linked to the occurrence of ONFH. Thirty-nine patients with various stages of ONFH were recruited. Bone specimens were harvested from 34 ONFH patients underwent hip arthroplasty, and from 10 femoral neck fracture patients. Bad, Bcl2 TNFα, DKK1, Wnt3a, LRP5, and Axin1 expressions were analyzed by quantitative RT-PCR and ELISA. Apoptotic cells were assayed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labelling (TUNEL). Primary bone-marrow mesenchymal cells were treated with DKK1 RNA interference and recombinant DKK1 protein. ONFH patients with the histories of being administrated corticosteroids and excessive alcohol consumption had significantly higher Bad and DKK1 mRNA expressions in bone tissue and DKK1 abundances in serum than femoral neck fracture patients. Bone cells adjacent to osteonecrotic bone displayed strong DKK1 immunoreactivity and TUNEL staining. Increased DKK1 expression in bone tissue and serum correlated with Bad expression and TUNEL staining. Serum DKK1 abundance correlated with the severity of ONFH. The DKK1 RNA interference and recombinant DKK1 protein regulated Bad expression and apoptosis of primary bone-marrow mesenchymal cells. Knock down of DKK1 reduced dexamethasone-induced apoptosis of mesenchymal cells. Taken together, promoted DKK1 expression was associated with bone cell apoptosis in the occurrence of ONFH patients with the histories of corticosteroid and alcohol intake and progression of ONFH. DKK1 expression in injured tissue provides new insight into ONFH pathogenesis. [Copyright &y& Elsevier]

Abstract: Problems of parameter estimation of nonlinear bioreaction systems are in general formulated as function optimization problems and are known to be frequently ill-conditioned and multimodal. While the optimization problems are defined on a large parameter search space, only few evolutionary algorithms are able to find a global solution to the large parameter search problem. In this study, a geometric mean mutation was embedded to hybrid differential evolution to replace a gene of the selected individual outside the assigned region. The replaced individuals were then applied to a differential mutation strategy to yield a perturbed individual. The benefit of using a large parameter search space to an inverse problem is to reduce the kinetic model complexity to yield a more compact formulation. Two inverse problems and twelve static benchmark problems with the large parameter search space are employed to illustrate the effectiveness of the proposed method. [Copyright &y& Elsevier]

Abstract: Aims: Long-term glucocorticoid administration is known to induce bone deterioration. Glycogen synthase kinase-3β (GSK-3β) signaling reportedly participates in bone remodeling. This study investigated whether GSK-3β inhibitor could regulate glucocorticoid-induced inhibition of osteoblast differentiation in vitro or bone mass in vivo. Main methods: MC3T3-E1 osteoblasts were treated with kinase-inactive GSK-3β mutant and 6-bromoindirubin-3′-oxim (BIO) and then exposed to 1µM dexamethasone. Survival and osteoblast differentiation of cell cultures were assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling, quantitative RT-PCR, and von Kossa staining. Mineral density, biomechanical properties and microenvironments of BIO- and glucocorticoid-treated rat bone tissues were analyzed using dual-energy X-ray absorptiometry, material testing, and histomorphometry, respectively. Key findings: Glucocorticoid decreased levels of phosphorylated Ser9-GSK-3β and β-catenin in osteoblast cultures. Kinase-inactive GSK-3β mutant and BIO treatments attenuated dexamethasone-induced inhibition of β-catenin, Runx2 abundance, and osteoblast differentiation but suppressed glucocorticoid-induced apoptosis of cell cultures. Exogenous BIO treatment alleviated methylprednisolone-induced impairment of mineral density, biomechanical strength, trabecular bone volume, osteoblast surface, and bone formation rate of rat bone tissue. BIO treatment also attenuated glucocorticoid-induced promotion of osteoclast surface and marrow adipocyte volume in bone tissue. Bone cells adjacent to glucocorticoid-stressed bone tissue displayed strong phosphorylated Ser9-GSK-3β and β-catenin immunostaining following BIO treatment. Significance: Inhibition of GSK-3β abrogated glucocorticoid-induced bone loss by increasing β-catenin- and Runx2-mediated osteoblast differentiation. Controlling GSK-3β signaling in bone cells may be a strategy for preventing glucocorticoid-induced osteopenia. [Copyright &y& Elsevier]

Purpose: To evaluate the effect and its mechanism of aminoguanidine (AG) on small-bowel protection after whole-abdominal irradiation (WAI) in rats. Methods and Materials: Male Sprague-Dawley rats (300–400 g) subjected to 12 Gy WAI were used for the study. Aminoguanidine at a dose of 50–800 mg/kg was administered by the gavage route 2 h before WAI. Mucosal damage of small bowel was evaluated by the grade of diarrhea and crypt survival; oxidative stress was determined by the level of 8-hydroxy 2′-deoxyguanosine (8-OHdG) with immunohistochemistry (IHC). Nitrosative stress was evaluated by the expression of inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine (3-NT) with IHC, and systemic and portal vein NOx (nitrite + nitrate) levels were measured and compared with and without AG treatment after WAI. Results: Aminoguanidine showed a dose-dependent effect against WAI-induced diarrhea. Aminoguanidine at a dose of 400 mg/kg had the best protective effect, from 92% to 17% (p = 0.002). Aminoguanidine increased crypt survival from 23% to 46% (p = 0.003). It also significantly attenuated 8-OHdG expression but not 3-NT and iNOS expression at both 4 and 8 h after 12-Gy WAI. Aminoguanidine did not alter the portal vein NOx levels 4 and 8 h after 12-Gy WAI. Conclusion: Aminoguanidine has a radioprotective effect against radiation-induced small-bowel damage due to its antioxidant effect but not inhibition of nitric oxide production. Dietary AG may have a potentially protective effect on the small intestine of patients subjected to pelvic and abdominal radiotherapies. [Copyright &y& Elsevier]

Abstract: In this study, the computer-aided process/solvent design is introduced to find an optimal biocompatible solvent and to maximize the ethanol production rate simultaneously for the single- or double-stage extractive fermentation process with cell recycling. Such a process/solvent design problem is formulated as a mixed-integer nonlinear programming problem that is solved by mixed-integer hybrid differential evolution in order to obtain a global design. The double-stage process can use a smaller amount of fresh solvent to increase ethanol productivity compared with that of the single-stage process, but it will also decrease overall conversion. Comparing the case studies, the simultaneous process/solvent design could yield higher overall ethanol productivity than that of the process design. The maximum ethanol production rate for the double-stage extractive fermentation with cell recycling was about 10-fold higher than that of continuous fermentation and about twofold higher than that of continuous fermentation with cell recycling. [Copyright &y& Elsevier]

Abstract: Osteonecrosis of the femoral head (ONFH) is a skeletal disorder characterized by ischemic deterioration, bone marrow edema and eventually femoral head collapse. The systemic regulation of ONFH in adult patients has not been examined. Serum proteomic is an innovative tool that potentially detects simultaneous expressions of serum proteins in pathological contexts. We compared the serum proteome profiles of 11 adult patients with ONFH (3 females and 8 males) and 11 healthy volunteers (3 females and 8 males). The proteins in the aliquots of sera were subjected to isoelectric focusing, two-dimensional gel electrophoresis and silver staining. The protein spots were matched and quantified using an imaging analysis system. The differentially expressed protein spots were subjected to in-gel trypsin digestion. The peptide mass fingerprints were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF) and a bioinformation search. We found that ONFH patients showed significantly higher abundances of kininogen 1 variant, complement factor C3 precursor, and complement factor H and lower levels of antithrombin III chain B, apolipoprotein A--IV precursor, and gelsolin isoform α precursor. These proteins of interest were reported to modulate thrombotic/fibrinolytic reactions, oxidative stress, vessel injury, tissue necrosis or cell apoptosis in several tissue types under pathological contexts. Taken together, the occurrence of ONFH was associated with various serum protein expressions. Our high--throughput serum proteomic findings indicated that multiple pathological reactions presumably occurred in ONFH. [Copyright &y& Elsevier]

In this study, crisp and fuzzy multiple-goal optimization approaches are respectively introduced to design an optimal biocompatible solvent to a two-stage extractive fermentation with cell recycling for ethanol production. When designing a biocompatible solvent for the extractive fermentation process, many issues, such as extractive efficiency, conversion, amount of solvent utilized and so on, have to be considered. An interactive multiple-goal design procedure is introduced to determine a trade-off result in order to satisfy such contradicted goals. Both approaches could be iterated to solve the interactive multiple-goal design problem in order to yield a trade-off result. However, the crisp optimization design is a tedious task that requires the designer to provide various pairs of the upper bounds for the design problem to obtain the corresponding solution. The fuzzy optimization approach is able to be trade-off several goals simultaneously and to yield the overall satisfactory grade for the product/process design problem. [Copyright &y& Elsevier]

In this study, crisp and flexible optimization approaches are, respectively, introduced to design an optimal biocompatible solvent for an extractive fermentation process. The optimal design problem is formulated as a mixed-integer nonlinear programming model in which performance requirements of the compounds are reflected in the objective and the constraints. In general, the requirements for the objective and constraints are not rigid; consequently, the flexible or fuzzy optimization approach is applied to soften the rigid requirement for maximization of the extraction efficiency and to consider the mass flow rate and biocompatibility of solvent as the softened inequality constraints to the solvent design problem. Having elicited the membership function for the objective function and the constraint, the optimal solvent design problem can be formulated as a flexible goal attainment problem. Mixed-integer hybrid differential evolution is applied to solve the problem in order to find a satisfactory design. [Copyright &y& Elsevier]

Abstract: We analyze the growth dynamics and production of an industrially interesting amino acid in a recombinant Escherichia coli strain, using in parallel two alternative modeling frameworks, namely S-systems and lin-log models. These models have identical steady-state solutions, but differ in their representation of transients. The models were parameterized with data from two bioprocesses that differed in their initial culture concentrations and then used to predict responses under yet a different initial culture regimen. Among the S-system models, several alternatives representing slightly different pathway structures, were tested. All were found to be capable of capturing the dynamics of all variables in the test systems and also of predicting the dynamic responses under new conditions. The lin-log models also captured the dynamics, but not as well as the S-system models. A probable reason for the inferior performance of lin-log models is their intrinsic property of not representing situations well where variable concentrations are moderately small. [Copyright &y& Elsevier]

Abstract: Dickkopf-1 (DKK1) has been found to act as a potent Wnt signaling-inhibitory factor for regulating skeletal disorders. We investigated whether modulation of DKK1 expression by end-capped phosphorothioate DKK1 antisense oligonucleotide could alter estrogen loss-induced bone loss. Ovariectomized or sham-operated rats were given 20 μg/kg/day DKK1 sense or antisense oligonucleotide or vehicle for 28 days. Femurs and tibiae were dissected to assess bone mass, biomechanical strength, immunohistochemistry and ex vivo osteoclast formation. We found that DKK1 antisense oligonucleotide significantly abrogated the suppressing effect of ovariectomy on weight, mineral content, mineral density and peak load of femurs. DKK1 antisense oligonucleotide treatment reduced ovariectomy promotion of ex vivo osteoclast differentiation of primary M-CSF-dependent bone marrow macrophages. Histomorphometric observation demonstrated that DKK1 antisense oligonucleotide treatment increased osteoblast number and impaired ovariectomy-promoted trabecular bone loss and osteoclast number in bone tissue. Osteoblastic cells adjacent to endosteum of trabecular bone and chondrocytes at calcified cartilage expressed intensive DKK1 and RANKL and weak OPG immunostaining in ovariectomized rat bone microenvironments. Osteogenic cells and chondral cells displayed weak DKK1, RANKL and OPG expression of bone tissue after DKK1 antisense oligonucleotide treatment. Taken together, attenuation of DKK1 expression in ovariectomized rat bone tissue alleviated loss of bone mass and biomechanical property. The regulatory action of DKK1 antisense oligonucleotide treatment on bone tissue appeared to suppress the promoting effect of estrogen deficiency on osteoclastogenesis-stimulatory factor RANKL expression and osteoclast differentiation. Control of DKK1 signaling can be used in the future as an alternative strategy for protecting estrogen deficiency induction of bone loss. [Copyright &y& Elsevier]

Abstract: A two-phase design approach is introduced to determine the optimal feed rate, fed glucose concentration and fermentation time to maximize protein productivity using recombinant Escherichia coli BL21 (pBAW2) strain. The first phase is applied to determine a primary S-system kinetic model using batch time-series data. Two runs were carried out in the second phase to achieve the maximum protein productivity for the fed-batch fermentation process. The computational results using the S-system kinetic model obtained from the second run are in better agreement with the experiments than those using the kinetic model obtained from batch time-series data. For cross-validation, two extra fed-batch experiments with different feed strategies were carried out for comparison with the optimal fed-batch result. From the experimental results, this approach could improve productivity by at least 3%. [Copyright &y& Elsevier]

Abstract: In this study, we have established a suitable model to describe the dynamic characteristic of an aspartase-overproducing Escherichia coli strain. A traditional Monod model and power-law models are respectively applied to describe the dynamic behaviors. When the model structure was selected, the parameter values of the model should be determined by the global/local optimization method. There are two major challenges, numerical integration for differential equations and finding global parameter values. In this study, we introduce hybrid differential evolution, which is a variant of genetic algorithms, to avoid obtaining a premature solution. In addition, we apply a modified collocation method to avoid numerical integration. The Monod''s model could only predict the growth characteristic of the recombinant E. coli, qualitatively. The S-system representation could suit for constructing the model structure of the microbial growth. [Copyright &y& Elsevier]

Vascular endothelial growth factor (VEGF) is an important regulator for angiogenesis and endochondral bone formation. Although low-intensity pulsed ultrasound (US) has been recently used for accelerating fracture healing, the effect of US stimulation on angiogenic factor production by osteoblasts remains undetermined. Here, we found that US elevation of VEGF-A expression in human osteoblasts to be mediated by nitric oxide (NO) and hypoxia-inducible factor-1α (HIF-1α). Human osteoblasts were treated with or without US stimulation (200 μs pulse, 1 kHz at 30 mW/cm2) for 20 min. Cells were subjected to assessment of VEGF-A expression, NO production, nitric oxide synthase (NOS) catalytic activities, and HIF-1α transactivation. Results showed that US significantly increased VEGF-A mRNA and protein levels in 6 h. US augmentation of VEGF level was transcriptionally mediated. Early inhibition of NO production, but not calcium or prostaglandin E2, significantly reduced US-enhanced VEGF-A levels. Osteoblasts responded to US treatment by increasing NO production, NOS catalytic activities, iNOS immunoexpression, nuclear HIF-1α activation, and binding to the VEGF-A promoter. Inhibition of NOS activity by N-nitro-l-arginine methyl ester (l-NAME) or blockade of guanylate cyclase activity by ODQ reduced US-augmented HIF-1α transactivation and VEGF-A levels. Conditioned medium harvested from US-treated osteoblasts promoted tube formation of human umbilical vein endothelial cells (HUVEC). Monoclonal VEGF-A antibody neutralization or l-NAME pretreatment reduced the promoting effect of conditioned medium on angiogenesis of HUVEC. Together, these findings show that NO plays an important role in mediating extracellular stimuli released by US and triggering intracellular response of osteoblasts to produce angiogenic factor after US treatment. [Copyright &y& Elsevier]

Study is aimed to elucidate the effect of porcine leukocyte antimicrobial protein on the proliferation of Staphylococcus aureus and Escherichia coli inoculated in ground meats. The antimicrobial proteins were isolated from porcine blood using 0.2 M sodium acetate extraction and cation-exchange column chromatography. Antimicrobial protein preparation consisted predominantly of 7.5 and 6 kDa molecules. Immunoblotting demonstrated that the 7.5 kDa molecule displayed defensin immuno-reactivity, a unique blood granulocyte antimicrobial protein. Both 7.5 and 6 kDa molecules could decrease apparent proliferation of test cultures. Sterilization (121 °C, 30 min) and proteolytic enzymes (pepsin, trypsin, and chymotrypsin) could significantly decrease bacteriocidal activities of antimicrobial protein preparation. Furthermore, antimicrobial protein preparation enhanced exposure of intercellular nucleotide of test cultures. Antimicrobial protein preparation inhibition of S. aureus and E. coli growth was concentration and time dependence. Adding 160 μg/g antimicrobial protein preparation to ground ham meat and sausage mince could significantly (P<0.05) hurdle viable colony formation of test cultures at 6 and 12 h, respectively. Taken together, porcine leukocyte antimicrobial protein preparation has potential to inhibit proliferation of S. aureus and E. coli inoculated in ground meat via induction of bacterolysis, suggesting this antimicrobial protein preparation be an alternative natural bio-preservation source for ground meat products. [Copyright &y& Elsevier]

In this study, we consider a two-stage fermenter with cell recycling including an extractor to produce lactic acid. When the extractor was omitted, the proposed process was simplified to two special processes. Under the same operating conditions, we compared the overall lactic acid productivity and glucose conversion for the three processes. The proposed fermentation process was more efficient than the other processes. To simultaneously obtain the maximum productivity and conversion, the problem is formulated as a fuzzy multiobjective optimization problem. The fuzzy decision is introduced to convert the multiobjective fuzzy optimization problem into the fuzzy goal attainment problem. Hybrid differential evolution is applied to solve the fuzzy goal attainment problem to obtain a global Pareto solution. [Copyright &y& Elsevier]

In this study, we have introduced a fuzzy decision-making approach to design a multi-objective optimal design problem of a multiproduct batch chemical plant. In the crisp decision-making approach, the designer is first to solve the multi-objective optimization problem, and then to check whether each optimal objective function value satisfies the rigid preference goal. In real world application, the preference goal for each objective function is an interval bound not a rigid value so that the problem becomes a fuzzy goal optimization problem. A monotonic increasing or decreasing membership function is used to define the degree of satisfaction for each objective function so that the fuzzy goal optimization problem is then represented as an augmented minimax problem formulated as mixed-integer nonlinear programming (MINLP) models. To obtain a unique solution, we have introduced the mixed-integer hybrid differential evolution (MIHDE) to solve the MINLP problems. The MIHDE can straightforwardly solve the problem without any mathematical model transformation. Two examples were used to illustrate the applicability of the proposed interactive algorithm. [Copyright &y& Elsevier]

• Miro1 over-expression of WJMSCs enhanced intercellular communications and promoted cell proliferation in mitochondria-defective fibroblasts. • Miro1 over-expressed WJMSCs improved mitochondrial membrane potential and cell proliferation rates in mitochondria-defective fibroblasts. • Miro1 over-expressed WJMSCs decreased rotenone-induced apoptosis in mitochondria-defective fibroblasts. • Miro1 over-expressed WJMSCs reduced ROS production and helped restore bioenergetics in mitochondria-defective fibroblasts. • The potential effectiveness of a Miro1-targeted WJMSC-based therapy for restoring mitochondrial function is promising, thereby offering a novel therapeutic approach for MELAS and other mitochondria-related diseases. Mitochondria are important for maintaining cellular energy metabolism and regulating cellular senescence. Mitochondrial DNA (mtDNA) encodes subunits of the OXPHOS complexes which are essential for cellular respiration and energy production. Meanwhile, mtDNA variants have been associated with the pathogenesis of neurodegenerative diseases, including MELAS, for which no effective treatment has been developed. To alleviate the pathological conditions involved in mitochondrial disorders, mitochondria transfer therapy has shown promise. Wharton's jelly mesenchymal stem cells (WJMSCs) have been identified as suitable mitochondria donors for mitochondria-defective cells, wherein mitochondrial functions can be rescued. Miro1 participates in mitochondria trafficking by anchoring mitochondria to microtubules. In this study, we identified Miro1 over-expression as a factor that could help to enhance the efficiency of mitochondrial delivery. More specifically, we reveal that Miro1 over-expressed WJMSCs significantly improved intercellular communications, cell proliferation rates, and mitochondrial membrane potential, while restoring mitochondrial bioenergetics in mitochondria-defective fibroblasts. Furthermore, Miro1 over-expressed WJMSCs decreased rates of induced apoptosis and ROS production in MELAS fibroblasts; although, Miro1 over-expression did not rescue mtDNA mutation ratios nor mitochondrial biogenesis. This study presents a potentially novel therapeutic strategy for treating mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), and other diseases associated with dysfunctional mitochondria, while the pathophysiological relevance of our results should be further verified by animal models and clinical studies. [ABSTRACT FROM AUTHOR]

Abstract: The synthesis of N-butylphthalimide (PTR) from the reaction of 1-bromobutane and potassium salt of phthalimide (PTK) was carried out successfully in a solid–liquid phase-transfer catalytic condition (SL-PTC) instead of reaction in a liquid–liquid two-phase medium. No water is required in the reaction system. The advantage of using SL-PTC to synthesize organic chemicals is to avoid a serious hydration of potassium salt of phthalimide from the reaction of 1-bromobutane and potassium salt of phthalimide in a liquid–liquid two-phase solution. The reaction is greatly enhanced in the solid–liquid solution catalyzed by quaternary ammonium salts. The reaction mechanism is proposed and verified by examining the experimental evidence. A kinetic model is proposed in which a pseudo first-order rate law is sufficient to describe the results. Kinetics of the reaction, including the effects of agitation speed, amount of acetonitrile, organic solvents, amount of tetrabutylammonium bromide, quaternary ammonium salts, amount of water and temperature on the conversion of 1-bromobutane and the apparent rate constant (k app) were investigated in detail. Rational explanations to account for the phenomena on the results were made. [Copyright &y& Elsevier]

Abstract: This work describes the etherification of 4,4′-bis(chloromethyl)-1,1′-biphenyl and 1-butanol catalyzed by a new multi-site phase-transfer catalyst as carried out in an alkaline solution/organic solvent two-phase medium. The new multi-site phase-transfer catalysts were obtained from the reaction of 4,4′-bis(chloromethyl)-1,1′-biphenyl and tertiary amines. During or after completing the reaction, the mono-substituted product (4,4′-(butoxymethyl)-1,1′-biphenyl) and di-substituted product (4,4′-bis(butoxymethyl)-1,1′-biphenyl) are both produced. A rational mechanism of the etherification was proposed based on the experimental observation and a kinetic model was developed. The two apparent rate constants of the organic-phase reactions were obtained via experimental data. The reaction is greatly enhanced by adding a small quantity of the new multi-site phase-transfer catalysts. This new novel phase-transfer catalyst exhibits higher reactivity than the “single-site” quaternary ammonium salts. Preliminary studies on the kinetics and the effect of various parameters such as agitation speed, amount of organic solvent, multi-site catalysts, potassium hydroxide, water, organic solvents, inorganic salt and temperature were investigated in detail. Rational explanations of the results are made. [Copyright &y& Elsevier]

In this paper, mixed-integer hybrid differential evolution (MIHDE) is developed to deal with the mixed-integer optimization problems. This hybrid algorithm contains the migration operation to avoid candidate individuals clustering together. We introduce the population diversity measure to inspect when the migration operation should be performed so that the user can use a smaller population size to obtain a global solution. A mixed coding representation and a rounding operation are introduced in MIHDE so that the hybrid algorithm is not only used to solve the mixed-integer nonlinear optimization problems, but also used to solve the real and integer nonlinear optimization problems. Some numerical examples are tested to illustrate the performance of the proposed algorithm. Numerical examples show that the proposed algorithm converges to better solutions than the conventional genetic algorithms. [Copyright &y& Elsevier]

Shock wave treatment is believed to improve bone healing after fracture. The purpose of this study was to evaluate the effect of shock wave treatment on bone mass and bone strength after fracture of the femur in a rabbit model. A standardized closed fracture of the right femur was created with a three-point bending method in 24 New Zealand white rabbits. Animals were randomly divided into three groups: (1) control (no shock wave treatment), (2) low-energy (shock wave treatment at 0.18 mJ/mm2 energy flux density with 2000 impulses), and (3) high-energy (shock wave treatment at 0.47 mJ/mm2 energy flux density with 4000 impulses). Bone mass (bone mineral density (BMD), callus formation, ash and calcium contents) and bone strength (peak load, peak stress and modulus of elasticity) were assessed at 12 and 24 weeks after shock wave treatment. While the BMD values of the high-energy group were significantly higher than the control group (P = 0.021), the BMD values between the low-energy and control groups were not statistically significant (P = 0.358). The high-energy group showed significantly more callus formation (P < 0.001), higher ash content (P < 0.001) and calcium content (P = 0.003) than the control and low-energy groups. With regard to bone strength, the high-energy group showed significantly higher peak load (P = 0.012), peak stress (P = 0.015) and modulus of elasticity (P = 0.011) than the low-energy and control groups. Overall, the effect of shock wave treatment on bone mass and bone strength appears to be dose dependent in acute fracture healing in rabbits. [Copyright &y& Elsevier]

Abstract Ethnopharmacological relevance Sijunzi decoction is a well-known traditional Chinese medicine (TCM) commonly used for invigorating vital energy and for the enhancement of immunity. Modified Sijunzi decoctions have been extensively used to treat cachexia and improve the quality of life of cancer patients undergoing chemotherapy. Aim of the study This study was aimed to provide comprehensive evidence for the anti-cachectic effect of a modified Sijunzi decoction (Zhen-Qi; ZQ-SJZ) and characterize its anti-cachectic mechanism, especially in cisplatin-induced muscle atrophy. Materials and methods We employed a Lewis lung carcinoma (LLC)-induced cancer cachectic mouse model to demonstrate the anti-cachectic effect of ZQ-SJZ. Moreover, we provided an in vitro C2C12 myotube formation model to investigate the effect of ZQ-SJZ in hampering cisplatin-induced muscle atrophy. Results The administration of ZQ-SJZ can recover tumor- and/or cisplatin-induced body weight loss, intestinal mucosal damage, as well as forelimb grip strength and myofiber size. The administration of ZQ-SJZ also significantly prolonged the survival of LLC-induced cachectic mice under cisplatin treatment. Mechanistically, ZQ-SJZ increased the levels of myogenic proteins, such as myosin heavy chain (MyHC) and myogenin, and decreased the atrophy-related protein, atrogin-1, in cisplatin-treated C2C12 myotubes in vitro. In addition, cisplatin-induced mitochondria dysfunction could be hampered by the co-administration of ZQ-SJZ, by which it recovered the cisplatin-mediated decrease in PGC-1α and PKM1 levels. Conclusions The administration of ZQ-SJZ can recover tumor- and/or cisplatin-induced cachectic conditions and significantly prolong the survival of LLC-induced cachectic mice under cisplatin treatment. The profound effect of ZQ-SJZ in hampering tumor- and/or cisplatin-induced cachexia may be due to its modulation of the mitochondrial function and subsequent myogenesis. Taken together, these results demonstrated the anti-cachectic mechanism of ZQ-SJZ and its potential use as a palliative strategy to improve the efficacy of chemotherapy. Graphical abstract fx1 [ABSTRACT FROM AUTHOR]

Histone hypermethylation represses gene transcription, which affects cartilage homeostasis or joint remodeling. Trimethylation of lysine 27 of histone 3 (H3K27me3) changes epigenome signatures, regulating tissue metabolism. This study aimed to investigate whether loss of H3K27me3 demethylase Kdm6a function affected osteoarthritis development. We revealed that chondrocyte-specific Kdm6a knockout mice developed relatively long femurs and tibiae as compared to wild-type mice. Kdm6a deletion mitigated osteoarthritis symptoms, including articular cartilage loss, osteophyte formation, subchondral trabecular bone loss, and irregular walking patterns of destabilized medial meniscus-injured knees. In vitro , loss of Kdm6a function compromised the loss in expression of key chondrocyte markers Sox9, collagen II, and aggrecan and improved glycosaminoglycan production in inflamed chondrocytes. RNA sequencing showed that Kdm6a loss changed transcriptomic profiles, which contributed to histone signaling, NADPH oxidase, Wnt signaling, extracellular matrix, and cartilage development in articular cartilage. Chromatin immunoprecipitation sequencing uncovered that Kdm6a knockout affected H3K27me3 binding epigenome, repressing Wnt10a and Fzd10 transcription. Wnt10a was, among others, functional molecules regulated by Kdm6a. Forced Wnt10a expression attenuated Kdm6a deletion-induced glycosaminoglycan overproduction. Intra-articular administration with Kdm6a inhibitor GSK-J4 attenuated articular cartilage erosion, synovitis, and osteophyte formation, improving gait profiles of injured joints. In conclusion, Kdm6a loss promoted transcriptomic landscapes contributing to extracellular matrix synthesis and compromised epigenetic H3K27me3-mediated promotion of Wnt10a signaling, preserving chondrocytic activity to attenuate osteoarthritic degeneration. We highlighted the chondroprotective effects of Kdm6a inhibitor for mitigating the development of osteoarthritic disorders. [ABSTRACT FROM AUTHOR]

Glucocorticoid treatment reportedly increases the morbidity of osteoporotic or osteonecrotic disorders. Exacerbated bone acquisition and escalated marrow adipogenesis are prominent pathological features of glucocorticoid-mediated skeletal disorders. MicroRNAs reportedly modulate tissue metabolism and remodeling. This study was undertaken to investigate the biological roles of microRNA-29a (miR-29a) in skeletal and fat metabolism in the pathogenesis of glucocorticoid-induced osteoporosis. Transgenic mice overexpressing miR-29a precursor or wild-type mice were given methylprednisolone. Bone mass, microarchitecture and histology were assessed by dual energy X-ray absorptiometry, μCT and histomorphometry. Differential gene expression and signaling components were delineated by quantitative RT-PCR and immunoblotting. Glucocorticoid treatment accelerated bone loss and marrow fat accumulation in association with decreased miR-29a expression. The miR-29a transgenic mice had high bone mineral density, trabecular microarchitecture and cortical thickness. miR-29a overexpression mitigated the glucocorticoid-induced impediment of bone mass, skeletal microstructure integrity and mineralization reaction and attenuated fatty marrow histopathology. Ex vivo, miR-29a increased osteogenic differentiation capacity and alleviated the glucocorticoid-induced promotion of adipocyte formation in primary bone-marrow mesenchymal progenitor cell cultures. Through inhibition of histone deacetylase 4 (HDAC4) expression, miR-29a restored acetylated Runx2 and β-catenin abundances and reduced RANKL, leptin and glucocorticoid receptor expression in glucocorticoid-mediated osteoporosis bone tissues. Taken together, glucocorticoid suppression of miR-29a signaling disturbed the balances between osteogenic and adipogenic activities, and thereby interrupted bone formation and skeletal homeostasis. miR-29a inhibition of HDAC4 stabilized the acetylation state of Runx2 and β-catenin that ameliorated the detrimental effects of glucocorticoid on mineralization and lipogenesis reactions in bone tissue microenvironments. This study highlighted emerging skeletal-anabolic actions of miR-29a signaling in the progression of glucocorticoid-induced bone tissue destruction. Sustaining miR-29a actions is beneficial in protecting against glucocorticoid-mediated osteoporosis. [ABSTRACT FROM AUTHOR]

Abstract: Excess glucocorticoid treatment induces loss of osteoblast differentiation. Post-translational modification of β-catenin reportedly regulates osteogenic activities in bone cells. This study was undertaken to test whether miR-29a signaling regulates the acetylation status of β-catenin in the glucocorticoid-mediated osteoblast dysfunction. Murine osteoblast cultures were incubated under osteogenic conditions with or without supraphysiological glucocorticoid, miR-29a precursor, antisense oligonucleotides or histone deacetylase 4 (HDAC4) RNA interferences. Osteoblast differentiation was determined by alkaline phosphatase activity, calcium deposition, and von Kossa stain. β-Catenin acetylation and miR-29a transcription were detected by immunoblotting, chromatin immunoprecipitation and quantitative PCR. Protein interaction was detected by fluorescence protein ligation assay. Supraphysiological glucocorticoid treatment repressed osteoblast differentiation and induced loss of miR-29a expression and acetylated β-catenin levels in osteoblast cultures. Gain of miR-29a function attenuated the deleterious effects of glucocorticoid on osteogenic gene expression and mineralized nodule formation, whereas knockdown of miR-29a signaling accelerated loss of osteoblast differentiation capacity. miR-29a reduced HDAC4 signaling and attenuated the glucocorticoid-mediated β-catenin deacetylation and ubiquitination and restored nuclear β-catenin levels. Glucocorticoid-induced loss of miR-29a signaling occurred through transcriptional and translational regulation. Interruption of HDAC4 signaling attenuated the glucocorticoid-induced hypoacetylation of histone H3 at lysine 9 (H3K9Ac) and restored the enrichment of H3K9Ac in miR-29a proximal promoter region and miR-29a transcription in cell cultures. Taken together, excess glucocorticoid-induced loss of miR-29a signaling accelerates β-catenin deacetylation and ubiquitination that impairs osteogenic activities of osteoblast cultures. miR-29a and HDAC4 reciprocal regulation of H3K9 acetylation contributes to the acetylation status of β-catenin and miR-29a expression. Enhancement of miR-29a signaling is an alternative strategy for protecting against the adverse actions of excess glucocorticoid on differentiation capacity of osteogenic cells. [Copyright &y& Elsevier]

Abstract: Background: Angiotensin II (AngII) reportedly enhances stem cell proliferation, and type 1 angiotensin II receptor (AT1R) antagonists reduce angiogenesis in a rodent hindlimb ischemic model. Whether AT1R antagonists can alter the angiogenic activity of bone-marrow mesenchymal stem cells (BMSCs) is unknown. The purpose of this study is to investigate whether AT1R antagonists can alter the angiogenic activity of BMSCs and explore the potential mechanism for such an action. Methods: Mouse BMSCs were isolated and treated with AngII, an AT1R antagonist, and a type 2 angiotensin II receptor (AT2R) antagonist. Angiogenic activity of BMSCs was detected by vascular endothelial growth factor (VEGF) secretion and tube formation of human umbilical vein endothelial cells (HUVECs). BMSCs isolated from enhanced green fluorescent protein (eGFP)-transgenic mice were allografted into ischemic hindlimbs in mice. Results: The BMSCs constitutively expressed AT1Rs and AT2Rs. AngII treatment significantly increased VEGF secretion by BMSCs. Valsartan (AT1R antagonist) but not PD123319 (AT2R antagonist) treatment attenuated the AngII-induced promotion of VEGF synthesis by BMSCs. The AngII and AngII receptor antagonist control of angiogenic activity of BMSCs were confirmed by tube formation of HUVECs. AngII treatment promoted phosphorylated Ser473 Akt abundance in BMSCs. RNA interference of an isoform of AT1R, valsartan, and wortmannin treatments attenuated AngII-induced Akt phosphorylation. Allograft of BMSCs significantly increased blood flow and VEGF expression in the gastrocnemius muscles of ischemic hindlimbs, which was attenuated after valsartan treatment. Conclusions: AT1R antagonists, via AT-1R/PI3K/Akt pathways, impair the AngII-induced promotion of angiogenic activity of mouse BMSCs. [Copyright &y& Elsevier]

• Systemic gardiquimod and CpG ODN did not change baseline ABR thresholds. • CpG ODN increased kanamycin-induced ABR threshold shifts and OHC loss. • The combination of CpG ODN and kanamycin elevated gene expression of proinflammatory cytokines. • CpG ODN increased Iba1+ macrophage infiltration in the cochlea. The Toll-like receptor (TLR) signaling pathway is the key regulator of the innate immune system in response to systemic infection. Several studies have reported that the systemic TLR4 agonist lipopolysaccharide exacerbates aminoglycoside ototoxicity, but the influence of virus-associated TLR7 and TLR9 signaling cascades on the cochlea is unclear. The present study aimed to investigate the auditory effects of systemic TLR7 and TLR9 agonists during chronic kanamycin treatment. CBA/CaJ mice received the TLR7 agonist gardiquimod or TLR9 agonist CpG oligodeoxynucleotides (ODN) one day before kanamycin injection and on the 5th and 10th days during a 14-day course of kanamycin treatment. We observed that systemic gardiquimod or CpG ODN alone did not affect the baseline auditory brainstem response (ABR) threshold. Three weeks after kanamycin treatment, gardiquimod did not significantly change ABR threshold shifts, whereas CpG ODN significantly increased kanamycin-induced ABR threshold shifts. Furthermore, outer hair cell (OHC) evaluation revealed that CpG ODN reduced distortion product otoacoustic emission amplitudes and increased kanamycin-induced OHC loss. CpG ODN significantly elevated cochlear Irf-7, Tnf-α, Il-1 , and Il-6 transcript levels. In addition, an increased number of Iba-1+ cells, which represented activated macrophages, was observed in the cochlea treated with CpG ODN. Our results indicated that systemic CpG ODN exacerbated kanamycin-induced ototoxicity and increased cochlear inflammation. This study implies that patients with underlying virus infection may experience more severe aminoglycoside-induced hearing loss if it occurs. [ABSTRACT FROM AUTHOR]

Abstract: Supraphysiological glucocorticoid administration accelerates loss of survival and differentiation in osteoblastic cells, thereby increasing the risks of osteopenic or osteonecrotic disorders. Neuroendocrine component type 1 cannabinoid receptor (CB1) is found to regulate bone mass. This study characterized the biological role of CB1 in glucocorticoid-induced suppression of osteoblast differentiation. Murine MC3T3-E1 osteoblasts were incubated under osteogenic conditions in the presence or absence of 1μM glucocorticoid, RNA interference, CB1 antagonist AM251, and agonist WIN55212-2. Cell survival was detected by formazan synthesis and TUNEL staining. Osteoblast differentiation was quantified by mineralized matrix accumulation and expression of the osteogenic factors Runx2 and osteocalcin. Expression of signaling molecules was assessed by immunoblotting. Glucocorticoid increased CB1 expression in association with decreased osteocalcin expression and mineralized nodule deposition. CB1 RNA interference and AM251 attenuated the deleterious actions of glucocorticoid treatment on survival and osteogenic activities, whereas activating CB1 by WIN55212-2 impaired osteoblast differentiation. CB1 signaling regulated JNK, ERK, GSK-3β, and Akt activation as well as Runx2 and IGF-I expression. Inhibition of GSK-3β by the kinase-inactive GSK-3β mutant or activation of ERK by the active MEK-1 mutant abrogated glucocorticoid-induced inhibition of osteoblast differentiation. Glucocorticoid-induced CB1 expression occurred via glucocorticoid receptor-dependent transcriptional and translational regulation. Gain of Runx2 function and loss of MKP-1 action attenuated glucocorticoid-induced enhancement of CB1 expression. Taken together, CB1 regulation of ERK and GSK-3β-dependent pathways participates in glucocorticoid inhibition of Runx2 signaling and osteoblast differentiation. Runx2 reciprocally regulates glucocorticoid-induced promotion of CB1 signaling. Our findings provide new insights into the role of the neuroendocrine component CB1 in glucocorticoid-induced osteoblast dysfunction. [Copyright &y& Elsevier]

Abstract: Background: Contracture of the deltoid muscle is an uncommon disorder. The symptoms usually are nonspecific and the diagnosis may be missed, especially when combined with other shoulder disorders, such as rotator cuff lesions. Few reports have described the surgical treatment of combined deltoid contracture and a torn rotator cuff. The purpose of this study was to share our experiences in the diagnosis and treatment of patients, who sustained deltoid contracture combined with rotator cuff tearing. Materials and methods: Between April 2001 and December 2006, 18 consecutive patients underwent concomitant treatment for distal release of deltoid contracture and repair of a torn rotator cuff. The mean age at operation was 55.1 years. There were eight female and ten male patients. The acromial type, winging angle of the scapula and thickest diameter of the deltoid fibrotic band were measured using preoperative magnetic resonance imaging studies. The abduction-contracture angle, extension-contracture angle, horizontal-adduction angle and Constant and Murley scores were measured preoperatively and at the latest follow-up. Results: There were nine complete rotator cuff tears and nine partial tears. At an average of 5 years and 3 months’ follow-up, the mean abduction-contracture angle significantly improved from 27° to 0° (p <0.001), the mean extension-contracture angle improved from 13° to 0° (p <0.001), and, the mean horizontal-adduction angle improved from 8° to 44° (p <0.001). The mean Constant score also improved from 69 points to 95 points (p <0.001). Conclusions: If a symptomatic torn rotator cuff and deltoid contracture co-exist, simultaneous operative treatment of both conditions is highly recommended. [Copyright &y& Elsevier]

Abstract: The Internet enables learners to be brought together where they can cooperate in learning in groups without space and time limitations. It is, however, quite a challenge to form ideal groups in a short time and ensure satisfactory interaction for learners in cyberspace. In this study, we propose a useful grouping method to help teachers improve group-learning in e-learning by first establishing effective groups with rules based on data mining, and then facilitating student interaction using a system that monitors members’ communication status. Field observations and quantitative evidence show the validity and practicability of the proposed method. [Copyright &y& Elsevier]

Extracorporeal shock waves (ESW) have recently been used in bone repair. Extracellular signal-regulated kinase (ERK) and p38 kinase are found to act as important mediators for osteogenic factor and mechanical-stimulated proliferation and differentiation of bone-forming cells. A previous study reported that ESW promoted healing of segmental defects in rats by inducing bone morophogenetic proteins (Bone 32 (2003) 387–396) and stimulating osteogenic differentiation of mesenchymal stem cells. In this study, we found that ERK and p38 activation was involved in ESW-augmented bone regeneration of segmental defects. ESW treatment (0.16 mJ/mm2, 1 Hz, 500 impulses) rapidly promoted [3H]-thymidine uptake in 1 day and progressively increased alkaline phosphatase activity, collagen I, II, and osteocalcin synthesis in callus organ culture within 14 days after treatment. Results of [γ-32P]-phosphotransferase activity assay showed that ERK and p38 in calluses were rapidly activated 1 day and 7 days after ESW treatment, respectively. Histological observation showed that segmental defects subjected to ESW treatment underwent typical bone formation (mesenchymal cell aggregation, hypertrophic cartilage, and endochondral/intramembrane ossification). Intensive bone formation coincided with evident expression of phosphorylated ERK and p38. Moreover, expression of phosphorylated ERK persisted in mesenchymal, chondral, and osteoblastic cells at newly developed bone and cartilage, and the expression of activated p38 was evident on chondral cells located at hypertrophic cartilage. Our findings suggest that mitogen-activated protein kinases (MAPK) regulate the stimulation of biophysical ESW, triggering mitogenic and osteogenic responses in the defects. ERK phosphorylation is active throughout the period of ESW-induced bone regeneration. p38 activation most likely plays an important role in signaling cartilage formation in callus. [Copyright &y& Elsevier]

Purpose: To study the pathomechanism of shock wave injuries to the femoral artery, vein and nerve in dogs.Materials and methods: High-energy shock wave (0.47 mJ/mm2 energy flux density) were applied to the right femoral artery, vein and nerve in nine mongrels, and the left side was used as control. Macroscopic examinations including structure integrity, edema, discolouration and extravasation; and microscopic examinations including detachment of the intima layer, disruption of the medium layer, capillary congestion, neutrophil margination and extravasation of the outer layer and the surrounding tissues were performed in 2 h, and in 4 days, respectively, after shock wave application.Results: The most serious injuries included disruption of the medium layer with separation from the adventitia layer of the femoral artery. The injuries to the femoral vein and nerve predominantly involved the outer adventitia layer and the surrounding tissues. Mild nerve bundle swelling was noted in most cases. There were capillary congestion, neutrophil margination and extravasation indicative of inflammatory tissue reaction after shock wave application.Conclusion: High-energy shock waves caused serious injuries to the femoral artery, vein and nerve, especially the femoral artery and inflammatory reaction to the surrounding tissues. [Copyright &y& Elsevier]