Traumatic brain injury and bone healing: radiographic and biomechanical analyses of bone formation and stability in a combined murine trauma model

Introduction:The combination of traumatic brain injury (TBI) and long-bone fractures has previously been reported to lead to exuberant callus formation. The aim of this experimental study was to radiographically and biomechanically study the effect of TBI on bone healing in a mouse model.Materials and methods:138 female C57/Black6N mice were assigned to four groups (fracture (Fx) / TBI / combined trauma (Fx/TBI) / controls). Femoral osteotomy and TBI served as variables: osteotomies were stabilized with external fixators, TBI was induced with controlled cortical impact injury. During an observation period of four weeks, in vivo micro-CT scans of femora were performed on a weekly basis. Biomechanical testing of femora was performed ex vivo.Results:The combined-trauma group showed increased bone volume, higher mineral density, and a higher rate of gap bridging compared to the fracture group. The combined-trauma group showed increased torsional strength at four weeks.Discussion:TBI results in an increased formation of callus and mineral density compared to normal bone healing in mice. This fact combined with a tendency towards accelerated gap bridging leads to increased torsional strength. The present study underscores the empirical clinical evidence that TBI stimulates bone healing. Identification of underlying pathways could lead to new strategies for bone-stimulating approaches in fracture care.     

A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex

A key obstacle to understanding neural circuits in the?cerebral cortex is that of unraveling the diversity of GABAergic interneurons. This diversity poses general questions for neural circuit analysis: how are these interneuron cell types generated and assembled into stereotyped local circuits and how do they differentially contribute to circuit operations that underlie cortical functions ranging from perception to cognition? Using genetic engineering in mice, we have generated and characterized approximately 20 Cre and inducible CreER knockin driver lines that reliably target major classes and lineages of GABAergic neurons. More select populations are captured by intersection of Cre and Flp drivers. Genetic targeting allows reliable identification, monitoring, and manipulation of cortical GABAergic neurons, thereby enabling a systematic and comprehensive analysis from cell fate specification, migration, and connectivity, to their functions in network dynamics and behavior. As such, this approach will accelerate the study of GABAergic circuits throughout the mammalian brain.     

Neural circuitry that governs Drosophila male courtship behavior

Male-specific fruitless (fru) products (Fru(M)) are both necessary and sufficient to "hardwire" the potential for male courtship behavior into the Drosophila nervous system. Fru(M) is expressed in approximately 2% of neurons in the male nervous system, but not in the female. We have targeted the insertion of GAL4 into the fru locus, allowing us to visualize and manipulate the Fru(M)-expressing neurons in the male as well as their counterparts in the female. We present evidence that these neurons are directly and specifically involved in male courtship behavior and that at least some of them are interconnected in a circuit. This circuit includes olfactory neurons required for the behavioral response to sex pheromones. Anatomical differences in this circuit that might account for the dramatic differences in male and female sexual behavior are not apparent.     

E2 conjugating enzymes must disengage from their E1 enzymes before E3-dependent ubiquitin and ubiquitin-like transfer

During ubiquitin ligation, an E2 conjugating enzyme receives ubiquitin from an E1 enzyme and then interacts with an E3 ligase to modify substrates. Competitive binding experiments with three human E2-E3 protein pairs show that the binding of E1s and of E3s to E2s are mutually exclusive. These results imply that polyubiquitination requires recycling of E2 for addition of successive ubiquitins to substrate.     

On the influence of mechanical conditions in osteochondral defect healing

Despite the introduction of new surgical techniques, the treatment of cartilage defects remains challenging. Delay or complete failure of cartilage healing is associated with problems in biological regeneration. The influence of mechanical conditions on this process, however, remains unevaluated. Osteochondral defects were generated on the left femoral condyle in 18 Yucatan minipigs. After 4, 6 and 12 weeks the defect filling, trabecular orientation and bone density were compared to the intact contralateral side. The mechanical straining during this period was then analyzed using an adaptive finite element technique. Histologically, the osteochondral defects showed bone resorption at the base and bone formation from the circumference. At 12 weeks, the macroscopically healed specimens showed fibrous cartilage formation, a minimally organized trabecular structure and increased trabecular volume fraction compared to the controls (p < 0.002). The amount of cancellous, cartilagineous, and fibrous tissue and the defect size as measured in histomorphometric analysis for the three time points (4, 6 and 12 weeks) was comparable in magnitude to that predicted by finite element analysis. The simulated osteochondral healing process was not fully capable of re-establishing a hyaline-like cartilage layer. The correlation between simulation and histology allows identification of mechanical factors that appear to have a larger impact on the healing of osteochondral defects than previously considered.     

Structural, biochemical, and functional characterization of the calcium sensor neurocalcin delta in the inner retinal neurons and its linkage with the rod outer segment membrane guanylate cyclase transduction system

This study documents the detailed biochemical, structural, and functional identity of a novel Ca(2+)-modulated membrane guanylate cyclase transduction system in the inner retinal neurons. The guanylate cyclase is the previously characterized ROS-GC1 from the photoreceptor outer segments (PROS), and its new modulator is neurocalcin delta. At the membrane, the myristoylated form of neurocalcin delta senses submicromolar increments in free Ca(2+), binds to its specific ROS-GC1 domain, and stimulates the cyclase. Neurocalcin delta is not present in PROS, indicating the absence of the pathway in the outer segments and the dissociation of its linkage with phototransduction. Thus, the pathway is linked specifically with the visual transduction machinery in the secondary neurons of the retina. With the inclusion of this pathway, the findings broaden the understanding of the existing mechanisms showing how ROS-GC1 is able to receive and transduce diverse Ca(2+) signals into the cell-specific generation of second-messenger cyclic GMP in the retinal neurons.     

Prototype foamy virus envelope glycoprotein leader peptide processing is mediated by a furin-like cellular protease, but cleavage is not essential for viral infectivity

Analogous to cellular glycoproteins, viral envelope proteins contain N-terminal signal sequences responsible for targeting them to the secretory pathway. The prototype foamy virus (PFV) envelope (Env) shows a highly unusual biosynthesis. Its precursor protein has a type III membrane topology with both the N and C terminus located in the cytoplasm. Coexpression of FV glycoprotein and interaction of its leader peptide (LP) with the viral capsid is essential for viral particle budding and egress. Processing of PFV Env into the particle-associated LP, surface (SU), and transmembrane (TM) subunits occur posttranslationally during transport to the cell surface by yet-unidentified cellular proteases. Here we provide strong evidence that furin itself or a furin-like protease and not the signal peptidase complex is responsible for both processing events. N-terminal protein sequencing of the SU and TM subunits of purified PFV Env-immunoglobulin G immunoadhesin identified furin consensus sequences upstream of both cleavage sites. Mutagenesis analysis of two overlapping furin consensus sequences at the PFV LP/SU cleavage site in the wild-type protein confirmed the sequencing data and demonstrated utilization of only the first site. Fully processed SU was almost completely absent in viral particles of mutants having conserved arginine residues replaced by alanines in the first furin consensus sequence, but normal processing was observed upon mutation of the second motif. Although these mutants displayed a significant loss in infectivity as a result of reduced particle release, no correlation to processing inhibition was observed, since another mutant having normal LP/SU processing had a similar defect.     

A survey of formal methods for determining the centre of rotation of ball joints

The determination of an accurate centre of rotation (CoR) from segment marker positions is of interest across a wide range of applications, but particularly for clinical gait analysis and for estimating the hip joint centre during surgical intervention of the knee, for limb alignment purposes. For the first time in this survey of formal methods, we classify, analyse and compare different methods (geometric, algebraic, bias compensated algebraic, and Pratt sphere fit methods, as well as the centre transformation technique, the Holzreiter approach, the helical pivot technique, the Schwartz transformation techniques, the minimal amplitude point method and the Stoddart approach) for the determination of spherical joint centres from marker position data. In addition, we propose a new method, the symmetrical CoR estimation or SCoRE, in which the coordinates of the joint centre must only remain constant relative to each segment, thus not requiring the assumption that one segment should remain at rest. For each method, 1000 CoR estimations were analysed with the application of isotropic, independent and identically distributed Gaussian noise (standard deviation 0.1cm) to each of the marker positions, to all markers on the segment simultaneously and the two in combination. For the test conditions used here, most techniques were capable of determining the CoR to within 0.3 cm, as long as the spherical range of motion (RoM) of the joint was 45 degrees or more. Under the most stringent conditions tested, however, the SCoRE was capable of best determining the CoR, to within approximately 1.2mm with a RoM of 20 degrees . The correct selection and application of these methodologies should help improve the accuracy of surgical navigation and clinical kinematic measurement.     

Novel frequenin-modulated Ca2+-signaling membrane guanylate cyclase (ROS-GC) transduction pathway in bovine hippocampus

Frequenin is a member of the neuronal Ca²⁺ sensor protein family, implicated in being the modulator of the neurotransmitter release, potassium channels, phosphatidylinositol signaling pathway and the Ca²⁺-dependent exocytosis of dense-core granules in the PC12 cells. Frequenin exhibits these biological activities through its Ca²⁺ myristoyl switch, yet the switch is functionally inactive. These structural and functional traits of frequenin have been derived through the use of recombinant frequenin. In the present study, frequenin (BovFrq) native to the bovine hippocampus has been purified, sequenced for its 9 internal fragments, cloned, and studied. The findings show that structure of the BovFrq is identical to its form present in chicken, rat, mouse and human, indicating its evolutionary conservation. Its Ca²⁺ myristoyl switch is active in the hippocampus. And, BovFrq physically interacts and turns on yet undisclosed ONE-GC-like ROS-GC membrane guanylate cyclase transduction machinery in the hippocampal neurons. This makes BovFrq a new Ca²⁺-sensor modulator of a novel ROS-GC transduction machinery. The study demonstrates the presence and mechanistic features of this cyclic GMP signaling pathway in the hippocampal neurons, and also provides one more support for the evolving concept where the Ca²⁺-modulated membrane guanylate cyclase transduction machinery in its variant forms is a central operational component of all neurons.