The role of extracellular matrix composition in structure and function of bioengineered skeletal muscle

One of the obstacles to the potential clinical utility of bioengineered skeletal muscle is its limited force generation capacity. Since engineered muscle, unlike most native muscle tissue, is composed of relatively short myofibers, we hypothesized that, its force production and transmission would be profoundly influenced by cell-matrix interactions. To test this hypothesis, we systematically varied the matrix protein type (collagen I/fibrin/Matrigel) and concentration in engineered, hydrogel-based neonatal rat skeletal muscle bundles and assessed the resulting tissue structure, generation of contractile force, and intracellular Ca(2+) handling. After two weeks of culture, the muscle bundles consisted of highly aligned and cross-striated myofibers and exhibited standard force-length and force-frequency relationships achieving tetanus at 40?Hz. The use of 2?mg/ml fibrin (control) yielded isometric tetanus amplitude of 1.4?±?0.3?mN as compared to 0.9?±?0.4?mN measured in collagen I-based bundles. Higher fibrin and Matrigel concentrations synergistically yielded further increase in active force generation to 2.8?±?0.5?mN without significantly affecting passive mechanical properties, tetanus-to-twitch ratio, and twitch kinetics. Optimized matrix composition yielded significant cellular hypertrophy (protein/DNA ratio?=?11.4?±?4.1 vs. 6.5?±?1.9?μg/μg in control) and a prolonged Ca(2+) transient half-width (Ca(50)?=?232.8?±?33.3 vs. 101.7?±?19.8?ms). The use of growth-factor-reduced Matrigel, instead of standard Matrigel did not alter the obtained results suggesting enhanced cell-matrix interactions rather than growth factor supplementation as an underlying cause for the measured increase in contractile force. In summary, biomaterial-based manipulation of cell-matrix interactions represents an important target for improving contractile force generation in engineered skeletal muscle.     

Increased left prefrontal activation in patients with unipolar depression: an event-related, parametric, performance-controlled fMRI study

BACKGROUND: Executive deficits associated with frontal lobe dysfunction are prominent in depression. We applied a newly developed WM task to investigate the neural correlates of executive processes with functional magnetic resonance imaging (fMRI) at comparable performance levels analyzing correct trials only. METHODS: We studied 12 partially remitted, medicated inpatients meeting DSM-IV criteria for major depressive disorder and 17 healthy controls. We used a parametric version of a delayed match-to-sample WM task requiring manipulation of verbal material during a delay period in an event-related fMRI design. RESULTS: Depressed patients were generally slower and load-dependently less accurate than healthy controls. Patients showed significantly more activation of left dorsolateral prefrontal cortex with highest cognitive load. Additionally, they showed higher activation in ventromedial prefrontal cortex during the control condition. LIMITATIONS: The fact that patients were taking different antidepressant drugs could limit the explanatory power of the present results. CONCLUSIONS: Increased lateral prefrontal activation despite comparably successful performance - when only correct trials were analyzed - in patients with depression can be interpreted as evidence for compensatory recruitment of prefrontal cortical resources.     

Membrane association of myotubularin-related protein 2 is mediated by a pleckstrin homology-GRAM domain and a coiled-coil dimerization module

Mutations in the myotubularin (MTM)-related protein 2 (MTMR2) gene are responsible for the severe autosomal recessive neuropathy Charcot-Marie-Tooth disease type 4B1. MTMR2 belongs to the MTM family of dual-specific phosphatases that use phosphatidylinositol (PI) 3,5-bisphosphate [PI(3,5)P2] and PI 3-phosphate [PI(3)P] as their substrate. Because these substrates are localized in the membrane bilayer, membrane targeting of Mtmr2 is an important regulatory mechanism. In hypoosmotically stressed COS cells with increased levels of PI(3,5)P2, Mtmr2 is bound to the membrane of vacuoles formed under these conditions. Using several mutant forms of Mtmr2, we identified two domains that are necessary for membrane association: (i) A pleckstrin homology-GRAM domain; and (ii) a coiled-coil module. Protein-lipid overlay assays show that the pleckstrin homology-GRAM domain binds to PI(3,5)P2 and PI(5)P, a substrate and a product of the Mtmr2 enzyme, respectively. We also demonstrate that Mtmr2 forms a dimer and that the C-terminal coiled-coil is responsible for homodimerization, in addition to membrane association. Our data indicate that phosphoinositide-protein interactions, as well as protein-protein interactions, are necessary for the correct regulation of MTMR2.     

Popliteal traumatic arteriovenous fistulas

BACKGROUND: The purpose of this report is to analyze the clinical presentation, diagnosis, and outcome of surgical treatment in patients with popliteal arteriovenous fistulas (AVFs) in order to make trauma surgeons aware of the various issues patients with popliteal AVFs might present. METHODS: From 1991 to 2000, 49 patients were treated for traumatic AVF. Among these patients, seven suffered from popliteal AVF of various durations. The patients were men and ranged in age from 17 to 27 years, with a mean age of 22.4 years. The time from injury to admission to our institutions varied from 5 days to 2 years. A diagnosis of popliteal AVF was made after clinical examinations revealed thrill and bruit over the injury sites. The diagnosis was confirmed in four of the patients after they underwent angiography. Patients with long-standing popliteal AVF underwent cardiology examinations to check for signs of heart failure. All patients with popliteal AVF received surgical treatment. Five patients had major blood vessels reconstructed, one patient had a minor blood vessel ligated, and another patient had a minor blood vessel reconstructed. RESULTS: Five of the seven patients experienced no postoperative difficulties. No serious heart failure occurred; however, there were signs of cardiac overload in three of the five patients. The two remaining patients of the seven underwent leg amputations. However, one of the two patients had a gangrenous foot at admission to our institution, and vascular reconstruction on the other patient was unsuccessful. For all seven patients, the average hospital stay in the vascular surgery department was 16.2 days and the follow-up ranged from 2 to 44 months, with a mean of 21.5 months. CONCLUSION: Trauma of the popliteal space requires special attention, since blood vessel injuries in that zone might result in serious complications. Popliteal traumatic AVFs result in a high rate of leg amputation and long-standing fistulas produce cardiac overload. The presence of thrill and bruit over the injury site should alert the examiner to consider the existence of AVF. Angiography is a reliable diagnostic tool, and should be used in all vitally stable patients. Surgical or nonsurgical closure of AVF will prevent local and systemic complications that might be irreversible in long-standing fistulas.     

Diagnostic potential of neutrophil elastase inhibitor complex in the routine care of critically ill newborn infants

It has been suggested that determination of the neutrophil elastase α1-proteinase inhibitor complex (E-α1PI) improves the diagnosis of bacterial infection in newborns. We evaluated the use of E-α1PI measurements in 143 newborns, consecutively admitted to a tertiary intensive care unit, employing a new random access assay and a sampling procedure that minimises post-collection artefacts. The 95% range for non-infected newborns was 20–110 μg/l up to the 5th day of life and 20–85 μg/l thereafter. The sensitivity as to the diagnosis of culture-proven bloodstream infection was 80% for E-α1PI, 86% for the immature to total neutrophil ratio, 64% for C-reactive protein and 37% for the total white blood cell count. The corresponding specificity amounted to 97%, 85%, 85% and 86%, respectively. E-α1PI increases preceded elevations of C-reactive protein by 18 h. Like C-reactive protein, E-α1PI levels did not distinguish between bloodstream infection and non-bacterial inflammatory responses. Results of E-α1PI became available within 1 h of collection and usually 2–3 h before manual leucocyte counts. Conclusion Determination of neutrophil elastase α1-proteinase inhibitor levels yields diagnostic advantages comparable to those of manual differential counts but provide faster turnaround times. Received: 16 February 2000 / Accepted: 8 March 2000     

Intraoperative common duct pressure and flow measurements

Operative cholangiography is still the most accurate and available method for assessing the presence or absence of stones in the common duct. However, 30 to 40 percent of stones will still be overlooked with cholangiography. To reduce the incidence of overlooked common duct stones we recommend that pressure and flow measurements be obtained before cholangiography. When pressures are high or high normal and flows low, the duct should be explored even in the presence of a normal cholangiogram. Under these circumstances, the incidence of falsepositive pressure flow studies is less than 5 percent even in inexperienced hands.     

Stimulation of hepatic phenylalanine hydroxylase activity but not Pah-mRNA expression upon oral loading of tetrahydrobiopterin in normal mice

Tetrahydrobiopterin (BH4) supplementation in patients with BH4-responsive phenylalanine hydroxylase (PAH) deficiency is an alternative to low-phenylalanine diet. To further investigate hepatic BH4-responsiveness, oral administration of 50 mg BH4/kg/day for 5 weeks was performed in wild-type mice. We observed a 2-fold increase in PAH protein by quantitative Western blot analysis and a 1.7-fold increase in enzyme activity, but no change in Pah-mRNA expression by quantitative real-time PCR analysis in treated mice compared to controls. Our findings support the proposed chemical-chaperone effect of BH4 to protect PAH.     

Bidirectional effects of benzodiazepine binding site ligands in the passive avoidance task: differential antagonism by flumazenil and beta-CCt

Recent research on genetically modified mice has attributed the amnesic effect of benzodiazepines mainly to the alpha1-containing GABA(A) receptor subtypes. The pharmacological approach, using subtype selective ligands, is needed to complement genetic studies. We tested the effects of the non-selective antagonist flumazenil (0-20.0 mg/kg), the preferential alpha1-subunit selective antagonist beta-carboline-3-carboxylate-t-butyl ester (beta-CCt) (0-30.0 mg/kg), the non-selective agonist midazolam (0-2.0 mg/kg), the preferential alpha1-subunit selective agonist zolpidem (0-3.0 mg/kg), and the non-selective inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) (0-2.0 mg/kg) in the one-trial step-through passive avoidance task in rats. The compounds were administered intraperitoneally, before the acquisition test. Flumazenil and beta-CCt did not affect retention performance. Midazolam and zolpidem induced amnesia in a dose-dependent manner. The complete reversal of amnesia was unattainable. The effects of zolpidem were significantly attenuated by the both, flumazenil (10.0 mg/kg) and beta-CCt (30.0 mg/kg); by contrast, only flumazenil was considerably effective when combined with midazolam. DMCM exerted promnesic effects at 0.2mg/kg, in an inverted U-shape manner. Both antagonists tended to abolish this action. The results indicate that some other alpha-subunit(s), in addition to the alpha1-subunit, contribute to the amnesic actions of non-selective benzodiazepine site agonists in the passive avoidance task. On the other hand, a significant part of the DMCM-induced promnesic effect could involve the alpha1-subunit and/or other putative beta-CCt-sensitive binding site(s).