Effects of muscle fibre shortening on the characteristics of surface motor unit potentials.

Traditionally, studies dealing with muscle shortening have concentrated on assessing its impact on conduction velocity, and to this end, electrodes have been located between the end-plate and tendon regions. Possible morphologic changes in surface motor unit potentials (MUPs) as a result of muscle shortening have not, as yet, been evaluated or characterized. Using a convolutional MUP model, we investigated the effects of muscle shortening on the shape, amplitude, and duration characteristics of MUPs for different electrode positions relative to the fibre-tendon junction and for different depths of the MU in the muscle (MU-to-electrode distance). It was found that the effects of muscle shortening on MUP morphology depended not only on whether the electrodes were between the end-plate and the tendon junction or beyond the tendon junction, but also on the specific distance to this junction. When the electrodes lie between the end-plate and tendon junction, it was found that (1) the muscle shortening effect is not important for superficial MUs, (2) the sensitivity of MUP amplitude to muscle shortening increases with MU-to-electrode distance, and (3) the amplitude of the MUP negative phase is not affected by muscle shortening. This study provides a basis for the interpretation of the changes in MUP characteristics in experiments where both physiological and geometrical aspects of the muscle are varied.

Micropropagação do híbrido PExSC-52 e da cultivar Smooth Cayenne de abacaxizeiro

A produção de mudas de abacaxizeiro de qualidade, em curto período, exige a otimização de protocolos de multiplicação in vitro. O objetivo deste trabalho foi testar cinco combinações de fitorreguladores, baseadas em resultados de pesquisa publicados com micropropagação de abacaxizeiro, na multiplicação in vitro do híbrido PExSC-52 e da cultivar Smooth Cayenne. Gemas axilares foram cultivadas em meio MS contendo benzilaminopurina (BAP) 2 mg/L; BAP 2 mg/L + ácido naftaleno acético (ANA) 2 mg/L; cinetina (CIN) 2 mg/L; CIN 2 mg/L + ANA 2 mg/L e CIN 5 mg/L + ácido indolacético (AIA) 5 mg/L. No híbrido, BAP 2 mg/L proporcionou maior proliferação de brotos por explante por subcultura e taxa de multiplicação crescente até a quarta subcultura. BAP 2 mg/L + ANA 2 mg/L multiplicou melhor a cv. Smooth Cayenne. Quando se utilizou CIN praticamente não ocorreu proliferação de brotos. Brotações do híbrido provenientes da quarta subcultura em presença de BAP 2 mg/L e BAP 2 mg/L + ANA 2 mg/L foram enraizados em meio MS contendo: ANA 1 mg/L; ANA 1 mg/L + CIN 2 mg/L; ANA 0,5 mg/L + CIN 2 mg/L; CIN 2 mg/L e sem fitorregulador. Brotos multiplicados em BAP enraizaram melhor que aqueles multiplicados em BAP + ANA.

Combined intermittent hypoxia and surface muscle electrostimulation as a method to increase peripheral blood progenitor cell concentration.

Background: Our goal was to determine whether short-term intermittent hypoxia exposure, at a level well tolerated by healthy humans and previously shown by our group to increase EPO and erythropoiesis, could mobilizehematopoietic stem cells (HSC) and increase their presence in peripheral circulation. Methods: Four healthy male subjects were subjected to three different protocols: one with only a hypoxic stimulus (OH), another with a hypoxic stimulus plus muscle electrostimulation (HME) and the third with only muscle electrostimulation (OME). Intermittent hypobaric hypoxia exposureconsisted of only three sessions of three hours at barometric pressure 540 hPa (equivalent to an altitude of 5000 m) for three consecutive days, whereas muscular electrostimulation was performed in two separate periods of 25 min in each session. Blood samples were obtained from an antecubital vein on three consecutive days immediately before the experiment and 24 h, 48 h, 4 days and 7 days after the last day of hypoxic exposure. Results: There was a clear increase in the number of circulating CD34+ cells after combined hypobaric hypoxia and muscular electrostimulation. This response was not observed after the isolated application of the same stimuli. Conclusion: Our results open a new application field for hypobaric systems as a way to increase efficiency in peripheral HSC collection.

Combined intermittent hypoxia and surface muscle electrostimulation as a method to increase peripheral blood progenitor cell concentration.

Background: Our goal was to determine whether short-term intermittent hypoxia exposure, at a level well tolerated by healthy humans and previously shown by our group to increase EPO and erythropoiesis, could mobilizehematopoietic stem cells (HSC) and increase their presence in peripheral circulation. Methods: Four healthy male subjects were subjected to three different protocols: one with only a hypoxic stimulus (OH), another with a hypoxic stimulus plus muscle electrostimulation (HME) and the third with only muscle electrostimulation (OME). Intermittent hypobaric hypoxia exposureconsisted of only three sessions of three hours at barometric pressure 540 hPa (equivalent to an altitude of 5000 m) for three consecutive days, whereas muscular electrostimulation was performed in two separate periods of 25 min in each session. Blood samples were obtained from an antecubital vein on three consecutive days immediately before the experiment and 24 h, 48 h, 4 days and 7 days after the last day of hypoxic exposure. Results: There was a clear increase in the number of circulating CD34+ cells after combined hypobaric hypoxia and muscular electrostimulation. This response was not observed after the isolated application of the same stimuli. Conclusion: Our results open a new application field for hypobaric systems as a way to increase efficiency in peripheral HSC collection.

High-Resolution Mass Spectrometry applied to the identification of new metabolites and transformation products of quinolones in chicken muscle tissues

The aim of this work was the identification of new metabolites and transformation products (TPs) in chicken muscle from Enrofloxacin (ENR), Ciprofloxacin (CIP), Difloxacin (DIF) and Sarafloxacin (SAR), which are antibiotics that belong to the fluoroquinolones family. The stability of ENR, CIP, DIF and SAR standard solutions versus pH degradation process (from pH 1.5 to 8.0, simulating the pH since the drug is administered until its excretion) and freeze-thawing (F/T) cycles was tested. In addition, chicken muscle samples from medicated animals with ENR were analyzed in order to identify new metabolites and TPs. The identification of the different metabolites and TPs was accomplished by comparison of mass spectral data from samples and blanks, using liquid chromatography coupled to quadrupole time-of-flight (LC-QqToF) and Multiple Mass Defect Filter (MMDF) technique as a pre-filter to remove most of the background noise and endogenous components. Confirmation and structure elucidation was performed by liquid chromatography coupled to linear ion trap quadrupole Orbitrap (LC-LTQ-Orbitrap), due to its mass accuracy and MS/MS capacity for elemental composition determination. As a result, 21 TPs from ENR, 6 TPs from CIP, 14 TPs from DIF and 12 TPs from SAR were identified due to the pH shock and F/T cycles. On the other hand, 14 metabolites were identified from the medicated chicken muscle samples. Formation of CIP and SAR, from ENR and DIF, respectively, and the formation of desethylene-quinolone were the most remarkable identified compounds.

Muscle moment-arms: a key element in muscle-force estimation.

A clear and rigorous definition of muscle moment-arms in the context of musculoskeletal systems modelling is presented, using classical mechanics and screw theory. The definition provides an alternative to the tendon excursion method, which can lead to incorrect moment-arms if used inappropriately due to its dependency on the choice of joint coordinates. The definition of moment-arms, and the presented construction method, apply to musculoskeletal models in which the bones are modelled as rigid bodies, the joints are modelled as ideal mechanical joints and the muscles are modelled as massless, frictionless cables wrapping over the bony protrusions, approximated using geometric surfaces. In this context, the definition is independent of any coordinate choice. It is then used to solve a muscle-force estimation problem for a simple 2D conceptual model and compared with an incorrect application of the tendon excursion method. The relative errors between the two solutions vary between 0% and 100%.

In-vivo performance of high-density collagen gel tubes for urethral regeneration in a rabbit model.

Congenital malformations or injuries of the urethra can be treated using existing autologous tissue, but these procedures are sometimes associated with severe complications. Therefore, tissue engineering may be advantageous for generating urethral grafts. We evaluated engineered high-density collagen gel tubes as urethral grafts in 16 male New Zealand white rabbits. The constructs were either acellular or seeded with autologous smooth muscle cells, isolated from an open bladder biopsy. After the formation of a urethral defect by excision, the tissue-engineered grafts were interposed between the remaining urethral ends. No catheter was placed postoperatively. The animals were evaluated at 1 or 3 months by contrast urethrography and histological examination. Comparing the graft caliber to the control urethra at 3 months, a larger caliber was found in the cell-seeded grafts (96.6% of the normal caliber) than in the acellular grafts (42.3%). Histology of acellular and cell-seeded grafts did not show any sign of inflammation, and spontaneous regrowth of urothelium could be demonstrated in all grafts. Urethral fistulae, sometimes associated with stenosis, were observed, which might be prevented by urethral catheter application. High-density collagen gel tubes may be clinically useful as an effective treatment of congenital and acquired urethral pathologies.

Peroxisome proliferator-activated receptor β/δ: a master regulator of metabolic pathways in skeletal muscle

Skeletal muscle is considered to be a major site of energy expenditure and thus is important in regulating events affecting metabolic disorders. Over the years, both in vitro and in vivo approaches have established the role of peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) in fatty acid metabolism and energy expenditure in skeletal muscles. Pharmacological activation of PPARβ/δ by specific ligands regulates the expression of genes involved in lipid use, triglyceride hydrolysis, fatty acid oxidation, energy expenditure, and lipid efflux in muscles, in turn resulting in decreased body fat mass and enhanced insulin sensitivity. Both the lipid-lowering and the anti-diabetic effects exerted by the induction of PPARβ/δ result in the amelioration of symptoms of metabolic disorders. This review summarizes the action of PPARβ/δ activation in energy metabolism in skeletal muscles and also highlights the unexplored pathways in which it might have potential effects in the context of muscular disorders. Numerous preclinical studies have identified PPARβ/δ as a probable potential target for therapeutic interventions. Although PPARβ/δ agonists have not yet reached the market, several are presently being investigated in clinical trials.

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration.

Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-kappaB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.

Tracheo-carinal reconstructions using extrathoracic muscle flaps.

OBJECTIVES: Prospective evaluation of tracheo-carinal airway reconstructions using pedicled extrathoracic muscle flaps for closing airway defects after non-circumferential resections and after carinal resections as part of the reconstruction for alleviation of anastomotic tension. METHODS: From January 1996 to June 2006, 41 patients underwent tracheo-carinal airway reconstructions using 45 extrathoracic muscle flaps (latissimus dorsi, n=25; serratus anterior, n=18; pectoralis major, n=2) for closing airway defects resulting from (a) bronchopleural fistulas (BPF) with short desmoplastic bronchial stumps after right upper lobectomy (n=1) and right-sided (pleuro) pneumonectomy (n=13); (b) right (n=9) and left (n=3) associated with partial carinal resections for pre-treated centrally localised tumours; (c) partial non-circumferential tracheal resections for pre-treated tracheal tumours, tracheo-oesophageal fistulas (TEF) and chronic tracheal injury with tracheomalacia (n=11); (d) carinal resections with the integration of a muscle patch in specific parts of the anastomotic reconstruction for alleviation of anastomotic tension (n=4). The airway defects ranged from 2 x 1 cm to 8 x 4 cm and involved up to 50% of the airway circumference. The patients were followed by clinical examination, repeated bronchoscopy, pulmonary function testing and CT scans. The minimum follow-up time was 6 months. RESULTS: Ninety-day mortality was 7.3% (3/41 patients). Four patients (9.7%) sustained muscle flap necrosis requiring re-operation and flap replacement without subsequent mortality, airway dehiscence or stenosis. Airway dehiscence was observed in 1/41 patients (2.4%) and airway stenosis in 1/38 surviving patients (2.6%) responding well to topical mitomycin application. Follow-up on clinical grounds, by CT scans and repeated bronchoscopy, revealed airtight, stable and epithelialised airways and no recurrence of BPF or TEF in all surviving patients. CONCLUSIONS: Tracheo-carinal airway defects can be closed by use of pedicled extrathoracic muscle flaps after non-circumferential resections and after carinal resections with the muscle patch as part of the reconstruction for alleviation of anastomotic tension.

High pressure induced changes in beef muscle proteome: correlation with quality parameters

The relationship between pressure induced changes on individual proteins and selected quality parameters in bovine longissimus thoracis et lumborum (LTL) muscle was studied. Pressures ranging from 200 to 600 MPa at 20 °C were used. High pressure processing (HPP) at pressures above 200 MPa induced strong modifications of protein solubility, meat colour and water holding capacity (WHC). The protein profiles of non-treated and pressure treated meat were observed using two dimensional electrophoresis. Proteins showing significant differences in abundance among treatments were identified by mass spectrometry. Pressure levels above 200 MPa strongly modified bovine LTL proteome with main effects being insolubilisation of sarcoplasmic proteins and solubilisation of myofibrillar proteins. Sarcoplasmic proteins were more susceptible to HPP effects than myofibrillar. Individual protein changes were significantly correlated with protein solubility, L*, b* and WHC, providing further insights into the mechanistic processes underlying HPP influence on quality and providing the basis for the future development of protein markers to assess the quality of processed meats.

Immunohistochemical analysis of bone morphological protein signaling pathway in human myometrium.

We assessed by immunohistochemistry the expression of the phosphorylated (activated) form of Smad1 and 5 (P-SMAD1/5), of Noggin and of two smooth muscle cell markers (α-SMA and SM22) in a series of human myometrium samples and in a smooth muscle cell line derived from human myometrium (HUt-SMC, PromoCell, USA). Myometrium samples were removed from two cadavers (a fetus at 26weeks of gestation and a neonate) and from ten non-menopausal women who underwent hysterectomy for adenomyosis and leiomyoma. P-SMAD1/5 expression was never detected in myometrium (both normal and pathological specimens), but only as a nuclear positive staining in glandular and luminal epithelial cells in sections in which also the endometrial mucosa was present. Noggin was strongly expressed especially in myometrium and adenomyosis samples from non-menopausal patients in comparison to the neonatal and fetal myometrium specimens in which muscle cells were less positive. In more than 95% of HUt-SMCs, α-SMA and Desmin were co-expressed, indicating a pure smooth muscle phenotype. When progesterone was added to the culture medium, no P-SMAD1/5 expression was detected, whereas the expression Noggin and SM22, a marker of differentiated smooth muscle cells, increased by 3 fold (p=0.002) and 4.3 fold (p=0.001), respectively (p=0.002). Our results suggest that, in non-menopausal normal human myometrium, the BMP pathway might be inhibited and that this inhibition might be enhanced by progesterone, which increases the differentiation of smooth muscle cells (SM22 levels). These findings could help in the identification of new mechanisms that regulate uterine motility.

Extracellular deposition of matrilin-2 controls the timing of the myogenic program during muscle regeneration.

Here, we identify a role for the matrilin-2 (Matn2) extracellular matrix protein in controlling the early stages of myogenic differentiation. We observed Matn2 deposition around proliferating, differentiating and fusing myoblasts in culture and during muscle regeneration in vivo. Silencing of Matn2 delayed the expression of the Cdk inhibitor p21 and of the myogenic genes Nfix, MyoD and Myog, explaining the retarded cell cycle exit and myoblast differentiation. Rescue of Matn2 expression restored differentiation and the expression of p21 and of the myogenic genes. TGF-β1 inhibited myogenic differentiation at least in part by repressing Matn2 expression, which inhibited the onset of a positive-feedback loop whereby Matn2 and Nfix activate the expression of one another and activate myoblast differentiation. In vivo, myoblast cell cycle arrest and muscle regeneration was delayed in Matn2(-/-) relative to wild-type mice. The expression levels of Trf3 and myogenic genes were robustly reduced in Matn2(-/-) fetal limbs and in differentiating primary myoblast cultures, establishing Matn2 as a key modulator of the regulatory cascade that initiates terminal myogenic differentiation. Our data thus identify Matn2 as a crucial component of a genetic switch that modulates the onset of tissue repair.