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A subset of transcription factors like Gli2 and Oct1 are bipotential--they can activate or repress the same target, in response to changing signals from upstream genes. Some previous studies implied that the sex-determination protein TRA-1 might also be bipotential; here we confirm this hypothesis by identifying a co-factor, and use it to explore how the structure of a bipotential switch changes during evolution. First, null mutants reveal that C. briggsae TRR-1 is required for spermatogenesis, RNA interference implies that it works as part of the Tip60 Histone Acetyl Transferase complex, and RT-PCR data show that it promotes the expression of Cbr-fog-3, a gene needed for spermatogenesis. Second, epistasis tests reveal that TRR-1 works through TRA-1, both to activate Cbr-fog-3 and to control the sperm/oocyte decision. Since previous studies showed that TRA-1 can repress fog-3 as well, these observations demonstrate that it is bipotential. Third, TRR-1 also regulates the development of the male tail. Since Cbr-tra-2 Cbr-trr-1 double mutants resemble Cbr-tra-1 null mutants, these two regulatory branches control all tra-1 activity. Fourth, striking differences in the relationship between these two branches of the switch have arisen during recent evolution. C. briggsae trr-1 null mutants prevent hermaphrodite spermatogenesis, but not Cbr-fem null mutants, which disrupt the other half of the switch. On the other hand, C. elegans fem null mutants prevent spermatogenesis, but not Cel-trr-1 mutants. However, synthetic interactions confirm that both halves of the switch exist in each species. Thus, the relationship between the two halves of a bipotential switch can shift rapidly during evolution, so that the same phenotype is produce by alternative, complementary mechanisms., Competing Interests: The authors have declared that no competing interests exist.

One marker for early-onset hip arthritis is femoral acetabular impingement. The current standard way of quantifying impingement is manual calculation of anatomical measures on plain radiographs, including the α-angle. Such measurements are user-dependent and prone to error. We provided a robust computational alternative and proposed using numerical fitting of geometrical shapes. We applied least-squares fitting of an ellipse to the femoral head contour and used the difference between the ellipse axes as a quantification method. The results showed a good correlation between the new measure and previous definitions of the α-angle.

Objective: Older persons have levels of frailty and disability that place them at far greater risk of harm in disasters than younger adults, making it crucial for all agency planners, public health workers, and health care providers to be aware of age-appropriate considerations of preparedness and response. This research evaluated the knowledge and utilization intentions of participants of a geriatric emergency preparedness and response (GEPR) continuing education program that was designed to provide this training., Methods: A qualitative training evaluation was conducted using course evaluation questionnaires from GEPR workshops offered in 2009 in 7 nationwide cities through a partnership of the University of Texas Center for Biosecurity and Public Health Preparedness with the Texas Consortium Geriatric Education Center at Baylor College of Medicine, Houston., Results: Evaluations were completed by 292 participants. Respondents were able to name specific information learned, give examples of applications of the material, and devise plans for further action. Participants believed that their ability to do their jobs had increased, and they provided insightful suggestions instructive to both trainers and to leaders of health care organizations., Conclusions: The results of the evaluation suggest that GEPR workshops are effective in increasing the knowledge of participants and their intentions to use it. There is both a need and a demand for this training. Developers and trainers should provide participants with sufficient time for absorbing the information, consider tailoring the training to the local context, and provide opportunity for practical application, particularly planning exercises.

Purpose: Asymmetric stress imposed on the shoulder can lead to anterior shoulder instability in young athletes who perform repetitive overhead motions. A common treatment, surgical anterior capsule tightening, assumes that the instability is caused by abnormal anterior laxity. This study investigated the possibility that one element of overall imbalance, posterior capsular tightness, could be an underlying reason for shoulder instability. Surgical navigation technology, which is more accurate than whole-body motion-capture systems, was used to study anterior translational motions., Method: The study was used four cadaver shoulders, with the scapula and rotator cuff muscles intact. Opto-electronic surgical navigation localization devices were mounted on the scapula and humerus to accurately capture positions and orientations. The shoulders were passively moved through 7 motions, 5 of simple angulation and 2 combinations of clinical interest. Each motion was repeated in 4 different soft-tissue states: rotator cuff intact, capsule intact, and surgically induced capsular tightnesses of 5 and 10mm., Results: The shoulders had significantly greater anterior translation when the posterior capsule was artificially tightened (p < 0.05); this was particularly in movements that combined abduction with internal or external rotation, which are typical overhead sports motions. Overall translation was indifferent to whether the shoulders were intact or dissected down to the capsule, as was translation during flexion was indifferent to dissection state (p > 0.95)., Conclusion: Surgical navigation technology can easily be used to analyze cadaveric shoulder motion, with opportunities for adaptation to anesthetized patients. Results suggest that the inverse of artificial tightening, such as surgical release of the posterior capsule, may be an effective minimally invasive treatment of chronic shoulder dislocation subsequent to sports motions.

Only 10% of metastatic melanoma patients survive 5 years, even though many can achieve substantial tumor reduction by surgical resection and/or radiation therapy and/or systemic therapy. An effective, nontoxic, consolidation immunotherapy could benefit such patients. We initiated a randomized trial to compare 2 promising patient-specific immunotherapy cell products. Patients had to have a diagnosis of metastatic melanoma and availability of an autologous melanoma cell line. Patients were stratified by whether their most advanced stage had been regional or distant metastases, and by whether they had measurable disease at the time of treatment, then they were randomized to receive irradiated autologous proliferating tumor cells or autologous dendritic cells (DC) loaded with antigens from such cells. Both products were injected subcutaneously in 500 µg of granulocyte-macrophage colony stimulating factor, weekly for 3 weeks and then monthly for 5 months. Patients in the 2 arms did not differ in baseline characteristics. All patients received prescribed therapy. Treatment was well tolerated. At the time of initial analysis, with no patients lost to follow-up, 50% of patients deceased, and all surviving patients followed for at least 6 months after randomization, survival is superior in the DC arm (hazard ratio, 0.27; 95% confidence interval, 0.098-0.729) with median survival not reached versus 15.9 months, and 2-year survival rates of 72% versus 31% (P=0.007). This trial provides evidence that a DC vaccine is associated with longer survival compared with a tumor cell vaccine, and is consistent with previous data suggesting a survival benefit from this patient-specific immunotherapy.

The recently identified human metapneumovirus (HMPV) is a worldwide respiratory virus affecting all age groups and causing pneumonia and bronchiolitis in severe cases. Despite its clinical significance, no specific antiviral agents have been approved for treatment of HMPV infection. Unlike the case for most paramyxoviruses, the fusion proteins (F) of a number of strains, including the clinical isolate CAN97-83, can be triggered by low pH. We recently reported that residue H435 in the HRB linker domain acts as a pH sensor for HMPV CAN97-83 F, likely through electrostatic repulsion forces between a protonated H435 and its surrounding basic residues, K295, R396, and K438, at low pH. Through site-directed mutagenesis, we demonstrated that a positive charge at position 435 is required but not sufficient for F-mediated membrane fusion. Arginine or lysine substitution at position 435 resulted in a hyperfusogenic F protein, while replacement with aspartate or glutamate abolished fusion activity. Studies with recombinant viruses carrying mutations in this region confirmed its importance. Furthermore, a second region within the F(2) domain identified as being rich in charged residues was found to modulate fusion activity of HMPV F. Loss of charge at residues E51, D54, and E56 altered local folding and overall stability of the F protein, with dramatic consequences for fusion activity. As a whole, these studies implicate charged residues and potential electrostatic interactions in function, pH sensing, and overall stability of HMPV F.

Viral fusion proteins are intriguing molecular machines that undergo drastic conformational changes to facilitate virus-cell membrane fusion. During fusion a hydrophobic region of the protein, termed the fusion peptide (FP), is inserted into the target host cell membrane, with subsequent conformational changes culminating in membrane merger. Class I fusion proteins contain FPs between 20 and 30 amino acids in length that are highly conserved within viral families but not between. To examine the sequence dependence of the Hendra virus (HeV) fusion (F) protein FP, the first eight amino acids were mutated first as double, then single, alanine mutants. Mutation of highly conserved glycine residues resulted in inefficient F protein expression and processing, whereas substitution of valine residues resulted in hypofusogenic F proteins despite wild-type surface expression levels. Synthetic peptides corresponding to a portion of the HeV F FP were shown to adopt an α-helical secondary structure in dodecylphosphocholine micelles and small unilamellar vesicles using circular dichroism spectroscopy. Interestingly, peptides containing point mutations that promote lower levels of cell-cell fusion within the context of the whole F protein were less α-helical and induced less membrane disorder in model membranes. These data represent the first extensive structure-function relationship of any paramyxovirus FP and demonstrate that the HeV F FP and potentially other paramyxovirus FPs likely require an α-helical structure for efficient membrane disordering and fusion.

Purpose: Percutaneous scaphoid fixation (PSF) is growing in popularity as a treatment option for non-displaced fractures. Success of this procedure demands high-precision screw placement, which can be difficult to achieve with standard 2D imaging. This study aimed to develop and test a system for computer-assisted navigation using volume slicing of 3D cone-beam computed tomography (CBCT)., Methods: The navigated technique involved a distinctive workflow in which a 3D CBCT imager was calibrated preoperatively, circumventing the need for intraoperative patient-based registration. Intraoperatively, a 3D CBCT image was acquired for both preoperative planning and direct navigation using volume-rendered slices. An in vitro study was conducted to compare the navigated approach to two conventional fluoroscopic methods for volar PSF. The surgical goal was to insert a guide wire to maximize both length and central placement., Results: There was no significant difference in the mean central placement of guide wire, although the variance in central placement was significantly lower using VS navigation (P < 0.01). The lengths of the drill paths were significantly longer for the VS-navigated group compared with one 2D group (P < 0.1). Each navigated trial required only one drilling attempt and resulted in less radiation exposure than conventional C-arm (P < 0.01)., Conclusions: Volume-sliced navigation achieved a more repeatable and reliable central pin placement, with fewer drilling attempts than conventional 2D techniques. Volume-sliced navigation had a higher number of drill paths within the optimal zone maximizing both length of the path and depth from the surface.

Human metapneumovirus (HMPV) is a major worldwide respiratory pathogen that causes acute upper and lower respiratory tract disease. The mechanism by which this virus recognizes and gains access to its target cell is still largely unknown. In this study, we addressed the initial steps in virus binding and infection and found that the first binding partner for HMPV is heparan sulfate (HS). While wild-type CHO-K1 cells are permissive to HMPV infection, mutant cell lines lacking the ability to synthesize glycosaminoglycans (GAGs), specifically, heparan sulfate proteoglycans (HSPGs), were resistant to binding and infection by HMPV. The permissiveness to HMPV infection was also abolished when CHO-K1 cells were treated with heparinases. Importantly, using recombinant HMPV lacking both the G and small hydrophobic (SH) proteins, we report that this first virus-cell binding interaction is driven primarily by the fusion protein (HMPV F) and that this interaction is needed to establish a productive infection. Finally, HMPV binding to cells did not require β1 integrin expression, and RGD-mediated interactions were not essential in promoting HMPV F-mediated cell-to-cell membrane fusion. Cells lacking β1 integrin, however, were less permissive to HMPV infection, indicating that while β1 integrins play an important role in promoting HMPV infection, the interaction between integrins and HMPV occurs after the initial binding of HMPV F to heparan sulfate proteoglycans.

While work with viral fusion proteins has demonstrated that the transmembrane domain (TMD) can affect protein folding, stability, and membrane fusion promotion, the mechanism(s) remains poorly understood. TMDs could play a role in fusion promotion through direct TMD-TMD interactions, and we have recently shown that isolated TMDs from three paramyxovirus fusion (F) proteins interact as trimers using sedimentation equilibrium (SE) analysis (E. C. Smith, et al., submitted for publication). Immediately N-terminal to the TMD is heptad repeat B (HRB), which plays critical roles in fusion. Interestingly, addition of HRB decreased the stability of the trimeric TMD-TMD interactions. This result, combined with previous findings that HRB forms a trimeric coiled coil in the prefusion form of the whole protein though HRB peptides fail to stably associate in isolation, suggests that the trimeric TMD-TMD interactions work in concert with elements in the F ectodomain head to stabilize a weak HRB interaction. Thus, changes in TMD-TMD interactions could be important in regulating F triggering and refolding. Alanine insertions between the TMD and HRB demonstrated that spacing between these two regions is important for protein stability while not affecting TMD-TMD interactions. Additional mutagenesis of the C-terminal end of the TMD suggests that β-branched residues within the TMD play a role in membrane fusion, potentially through modulation of TMD-TMD interactions. Our results support a model whereby the C-terminal end of the Hendra virus F TMD is an important regulator of TMD-TMD interactions and show that these interactions help hold HRB in place prior to the triggering of membrane fusion.

Purpose: Periacetabular osteotomies are a family of surgical procedures used to treat hip dysplasia. In a periacetabular osteotomy, the operating surgeon aims to increase acetabular coverage of the femoral head. The surgical correction has mechanical goals of increasing the stability of the joint and to improving the pressure distribution across the acetabulum. Although it is known that bone will remodel under changing load at the microstructural level, it is unclear whether there is any gross remodeling of the acetabulum or the femoral head in response to the change in loading following a periacetabular osteotomy. This observational study aims to quantify the shape of operative and contralateral hip joint surfaces pre and postoperatively to determine whether there are gross morphological changes in the shape of any of the bony articular surfaces of the joint., Methods: Preoperative and postoperative computed tomography (CT) scans were segmented as triangulated meshes. The bony articular surfaces of these meshes were then isolated. The vertices of these surfaces were fit to spheres and to general ellipsoids and, in the case of the acetabulum, examined in anatomical coordinate frames to look for changes between pre and postoperative segmentations., Results: Spherical fit results were consistent preoperatively and postoperatively, with small changes in the radii of the spheres of best fit for both operative and nonoperative hips. Ellipsoid fitting showed variations between preoperative and postoperative scans in both eccentricity and orientation., Conclusions: Because there is no clear evidence of gross articular surface remodeling, periacetabular osteotomy for an adult should be planned with the expectation that the patient's existing articular structure will be preserved.

Hendra virus is a highly pathogenic paramyxovirus classified as a biosafety level four agent. The fusion (F) protein of Hendra virus is critical for promoting viral entry and cell-to-cell fusion. To be fusogenically active, Hendra virus F must undergo endocytic recycling and cleavage by the endosomal/lysosomal protease cathepsin L, but the route of Hendra virus F following internalization and the recycling signals involved are poorly understood. We examined the intracellular distribution of Hendra virus F following endocytosis and showed that it is primarily present in Rab5- and Rab4-positive endosomal compartments, suggesting that cathepsin L cleavage occurs in early endosomes. Hendra virus F transmembrane domain (TMD) residues S490 and Y498 were found to be important for correct Hendra virus F recycling, with the hydroxyl group of S490 and the aromatic ring of Y498 important for this process. In addition, changes in association of isolated Hendra virus F TMDs correlated with alterations to Hendra virus F recycling, suggesting that appropriate TMD interactions play an important role in endocytic trafficking.

Self-fertile hermaphrodites have evolved independently several times in the genus Caenorhabditis [1, 2]. These XX hermaphrodites make smaller sperm than males [3, 4], which they use to fertilize their own oocytes. Because larger sperm outcompete smaller sperm in nematodes [3-5], it had been assumed that this dimorphism evolved in response to sperm competition. However, we show that it was instead caused by a developmental bias. When we transformed females of the species Caenorhabditis remanei into hermaphrodites [6], their sperm were significantly smaller than those of males. Because this species never makes hermaphrodites in the wild, this dimorphism cannot be due to selection. Instead, analyses of the related nematode Caenorhabditis elegans suggest that this dimorphism might reflect the development of sperm within the distinct physiological environment of the hermaphrodite gonad. These results reveal a new mechanism for some types of developmental bias-the effects of a novel physical location alter the development of ectopic cells in predictable ways., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

This article presents a novel approach that incorporates an ultrasound slice-thickness profile into a filtered, weighted-least-square framework to improve the reconstruction accuracy of a real-time freehand calibration system. An important part of the system is a slice-thickness calibration device that aids in the extraction of the slice thickness across a wide range of imaging depths. Extensive experiments were conducted on a 10,000-image dataset to evaluate the effects of the framework on the calibration accuracy. The results showed that three-dimensional (3-D) reconstruction errors were significantly reduced in every experiment (p < 0.001). Real-time testing showed that the proposed method worked effectively with a small number of input images, suggesting great potential for intraoperative use where only a limited number of data may be available. This new framework can enable efficient quality control of calibration accuracy in real-time operating-room use., (Copyright © 2011 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

The paramyxovirus family includes important human pathogens such as measles, mumps, respiratory syncytial virus, and the recently emerged, highly pathogenic Hendra and Nipah viruses. The viral fusion (F) protein plays critical roles in infection, promoting both the virus-cell membrane fusion events needed for viral entry as well as cell-cell fusion events leading to syncytia formation. We describe the surprising finding that addition of the short epitope HA tag to the cytoplasmic tail (CT) of the Hendra virus F protein leads to a significant increase in the extent of cell-cell membrane fusion. This increase was not due to alterations in surface expression, cleavage state, or association with lipid microdomains. Addition of a Myc tag of similar length did not alter Hendra F protein fusion activity, indicating that the observed stimulation was not solely a result of lengthening the CT. Three tyrosine residues within the HA tag were critical for the increase in the extent of fusion, suggesting C-terminal tyrosines may modulate Hendra fusion activity. The effects of addition of the HA tag varied with other fusion proteins, as parainfluenza virus 5 F-HA showed a decreased level of surface expression and no stimulation of fusion. These results indicate that additions to the C-terminal end of the F protein CT can modulate protein function in a sequence specific manner, reinforcing the need for careful analysis of epitope-tagged glycoproteins. In addition, our results implicate C-terminal tyrosine residues in the modulation of the membrane fusion reaction promoted by these viral glycoproteins.

We tested the registration stability of individualized templates in a consecutive study with 80 patients undergoing hip-resurfacing surgery. These templates physically encode registration and navigation parameters but do not require a computer during the actual surgery. The surgical target was the placement of the femoral guidance pin during hip resurfacing, which is a difficult and highly variable task using conventional instruments. The drill trajectory for the guidance pin of the femoral component was planned on a 3D computer model of the femur derived from a preoperative computed tomography (CT) scan. A surface-matched drilling template was designed to perform mechanical registration on the bone surface and had a hole for the drill guide; the template was created using a rapid prototyping machine. Intraoperatively, the individualized template was positioned on the patient anatomy and the pin was drilled into the femoral neck. The final achieved pin orientation and position were measured using an optoelectronic CT-based navigation system. The measured mean deviation between planned and actual central pin alignment of 0.05° in valgus and 2.8° in anteversion shows that the proposed individualized templates for hip resurfacing have reliable registration.

There is a growing body of evidence to suggest the arthritic hip is an irregularly-shaped, aspherical joint, especially in severely pathological cases. Current methods used to study the shape and motion of the hip in-vivo, are invasive and impractical. This study aimed to assess whether a plastic model of the hip joint can be accurately made from a pelvic CT scan. A cadaver hemi-pelvis was CT imaged and segmented from which a 3D plastic model of the proximal femur and hemi-pelvis were fabricated using rapid-prototyping. Both the plastic model and the cadaver were then imaged using a high-resolution laser scanner. A three-way shape analysis was performed to compare the goodness-of-fit between the cadaver, image segmentation, and the plastic model. Overall, we obtained sub-millimeter fit accuracy between all three hip representations. Shape fit was least favorable in areas where the boundary between cartilage and bone is difficult to distinguish. We submit that rapid-prototyping is an accurate and efficient mechanism for obtaining 3D specimens as a means to further study the irregular geometry of the hip.

Background: One of the challenges in natural orifice transluminal endoscopic surgery (NOTES) is spatial orientation. The Queen's NOTES group has devised a novel method of orientation by using a magnetic device that passes within an endoscope channel allowing for 3-dimensional imaging of the shape and orientation of the endoscope., Objective: To assess the feasibility and utility of a novel orientation device., Design: Randomized, controlled trial., Setting: Animal research laboratory study on four 25-kg pigs., Intervention: The device was tested by 6 endoscopists and 6 laparoscopic surgeons. Starting at the gastrotomy, the time to identify 4 targets was recorded. Participants were required to identify and touch the gallbladder, the fallopian tube, a clip on the abdominal wall, and the liver edge. Use of the orientation device was randomized for each session., Main Outcome Measurements: Time to identify targets with and without the device. Secondary analysis assessed differences between medical specialties and level of training., Results: The mean time to identify all 4 targets with the device was 75.08 ± 42.68 seconds versus 100.20 ± 60.70 seconds without the device (P <.001). The mean time to identify all 4 targets on the first attempt was 102.29 ± 61.36 seconds versus 72.99 ± 40.19 seconds on the second attempt (P <.001). No differences based on specialty or level of training were identified., Limitations: Small sample size and simplicity of tasks., Conclusion: Regardless of randomization order, both groups were faster with the device. These encouraging results warrant further study using more complex scenarios., (Copyright © 2011 American Society for Gastrointestinal Endoscopy. Published by Mosby, Inc. All rights reserved.)

Although dysesthesia is a common surgical complication, there is no accepted method for quantitatively tracking its progression. To address this, two types of computer vision technologies were tested in a total of four configurations. Surface regions on plastic models of limbs were delineated with colored tape, imaged, and compared with computed tomography scans. The most accurate system used visually projected texture captured by a binocular stereo camera, capable of measuring areas to within 3.4% of the ground-truth areas. This simple, inexpensive technology shows promise for postoperative monitoring of dysesthesia surrounding surgical scars.

Bilateral asymmetry in Caenorhabditis elegans arises in part from cell lineages that differ on the left and right sides of the animal. The unpaired MI neuron descends from the right side of an otherwise left-right symmetric cell lineage that generates the MI neuron on the right and the e3D epithelial cell on the left. We isolated mutations in three genes that caused left-right symmetry in this normally asymmetric cell lineage by transforming MI into an e3D-like cell. These genes encode the proneural bHLH proteins NGN-1 and HLH-2 and the Otx homeodomain protein CEH-36. We identified the precise precursor cells in which ceh-36 and ngn-1 act, and showed that CEH-36 protein is asymmetrically expressed and is present in an MI progenitor cell on the right but not in its bilateral counterpart. This asymmetric CEH-36 expression promotes asymmetric ngn-1 and hlh-2 expression, which in turn induces asymmetric MI neurogenesis. Our results indicate that this left-right asymmetry is specified within the two sister cells that first separate the left and right branches of the cell lineage. We conclude that the components of an evolutionarily conserved Otx/bHLH pathway act sequentially through multiple rounds of cell division on the right to relay an initial apparently cryptic asymmetry to the presumptive post-mitotic MI neuron, thereby creating an anatomical bilateral asymmetry in the C. elegans nervous system.

Computer assisted surgical interventions and research in joint kinematics rely heavily on the accurate registration of three-dimensional bone surface models reconstructed from various imaging technologies. Anomalous results were seen in a kinematic study of carpal bones using a principal axes alignment approach for the registration. The study was repeated using an iterative closest point algorithm, which is more accurate, but also more demanding to apply. The principal axes method showed errors between 0.35 mm and 0.49 mm for the scaphoid, and between 0.40 mm and 1.22 mm for the pisiform. The iterative closest point method produced errors of less than 0.4 mm. These results show that while the principal axes method approached the accuracy of the iterative closest point algorithm in asymmetrical bones, there were more pronounced errors in bones with some symmetry. Principal axes registration for carpal bones should be avoided.

Triggering of the Hendra virus fusion (F) protein is required to initiate the conformational changes which drive membrane fusion, but the factors which control triggering remain poorly understood. Mutation of a histidine predicted to lie near the fusion peptide to alanine greatly reduced fusion despite wild-type cell surface expression levels, while asparagine substitution resulted in a moderate restoration in fusion levels. Slowed kinetics of six-helix bundle formation, as judged by sensitivity to heptad repeat B-derived peptides, was observed for all H372 mutants. These data suggest that side chain packing beneath the fusion peptide is an important regulator of Hendra virus F triggering.

Paramyxovirus fusion (F) proteins promote both virus-cell fusion, required for viral entry, and cell-cell fusion, resulting in syncytia formation. We used the F-actin stabilizing drug, jasplakinolide, and the G-actin sequestrant, latrunculin A, to examine the role of actin dynamics in cell-cell fusion mediated by the parainfluenza virus 5 (PIV5) F protein. Jasplakinolide treatment caused a dose-dependent increase in cell-cell fusion as measured by both syncytia and reporter gene assays, and latrunculin A treatment also resulted in fusion stimulation. Treatment with jasplakinolide or latrunculin A partially rescued a fusion pore opening defect caused by deletion of the PIV5 F protein cytoplasmic tail, but these drugs had no effect on fusion inhibited at earlier stages by either temperature arrest or by a PIV5 heptad repeat peptide. These data suggest that the cortical actin cytoskeleton is an important regulator of fusion pore enlargement, an energetically costly stage of viral fusion protein-mediated membrane merger., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background: Distraction osteogenesis is a well known and frequently described technique in mandibular deformities. Buried intraoral devices have numerous advantages, but success hinges on precise positioning of the implants. Although computer navigation has repeatedly been described for craniofacial applications, research on navigating the mandibular region is scarce. Navigating the device placement for a mandibular distractor could become a viable method for distraction osteogenesis because of the possibility of certainty in achieving a defined device position., Materials and Methods: A clinical situation was simulated by a mandible model mounted inside a phantom head. The screws were positioned according to a virtual plan through transoral and transbuccal approaches, with and without navigation., Results: Without navigation, the mean deviation from the planned position was 4.9 mm (range, 0.9-10.7 mm), with a clear tendency to position the screws in the easy-to-access regions. With navigation, the mean deviation was significantly lower at 1.5 mm (range, 0.1-3.4 mm)., Conclusions: Computer-assisted surgery can provide a high level of accuracy in the region of the mandibular angle, where precision is crucial for buried intraoral distraction devices.

The goal of this study was to investigate whether individualized templates can provide an accurate and reliable computer-assisted system for femoral component placement during hip resurfacing. A consecutive series of 45 patients were examined. Using a 3-dimensional computer model of the femur, the drill trajectory for the central pin of the stem was planned. A surface-matched plastic drilling template was created using a rapid prototyping machine. This patient-specific drill guide was intraoperatively positioned on the patient anatomy, the central pin was drilled into the femoral neck, and the accuracy of the placement with respect to the planned central pin alignment was measured. With mean deviation between planned and actual central pin alignment of 1.14 degrees in varus and 4.49 degrees in retroversion, individualized templates were as accurate as conventional computer-assisted hip resurfacing., (Copyright 2010 Elsevier Inc. All rights reserved.)

We study the effect of point dependent (heteroscedastic) and identically distributed anisotropic fiducial localization noise on fiducial target registration error (TRE). We derive an analytic expression, based on the concept of mechanism spatial stiffness, for predicting TRE. The accuracy of the predicted TRE is compared to simulated values where the optimal registration transformation is computed using the heteroscedastic errors in variables algorithm. The predicted values are shown to be contained by the 95% confidence intervals of the root mean square TRE obtained from the simulations.

Recent research shows that declining oocyte quality with age is not inevitable in nematodes, and similar signals might regulate reproductive aging in women., (© 2010 BioMed Central Ltd)

The standard workflow in many image-guided procedures, preoperative imaging followed by intraoperative registration, can be a challenging process and is not readily adaptable to certain anatomical regions such as the wrist. In this study we present an alternative, consisting of a preoperative registration calibration and intraoperative navigation using 3D cone-beam CT. A custom calibration tool was developed to preoperatively register an optical tracking system to the imaging space of a digital angiographic C-arm. This preoperative registration was then applied to perform direct navigation using intraoperatively acquired images for the purposes of an in-vitro wrist fixation procedure. A validation study was performed to assess the stability of the registration and found that the mean registration error was approximately 0.3 mm. When compared to two conventional techniques, our navigated wrist repair achieved equal or better screw placement, with fewer drilling attempts and no additional radiation exposure to the patient. These studies suggest that preoperative registration coupled with direct navigation using procedure-specific graphical rendering, is potentially a highly accurate and effective means of performing image-guided interventions.

Ultrasound is known to produce a range of nonlethal responses in cells and tissues. Frequencies in the kilohertz ultrasound range have been shown to produce relaxation in large arteries. The present work explores the effects of insonation at MHz frequencies, representative of those used diagnostically and therapeutically, in an in vitro preparation of the carotid artery. Fresh 12.7 mm wide rings of equine common carotid artery obtained from the abattoir were mounted in a purpose-made myograph. They were immersed in a bath of Krebs-Ringer buffer at 37 degrees C and were positioned at the focus of an ultrasound beam from a weakly focused 3.2 MHz source. Continuous wave insonation produced contraction. The tension increased rapidly over the first 2 min, followed by a slower increase for the duration of the exposure up to 15 min. At a power of 145 mW a maximum contractile stress of 0.04 +/- 0.03 mN/mm(2) (mean +/- SD, n = 77) was measured, which was approximately 4% of the maximum wall stress generated by noradrenaline (0.1 mM). The magnitude of the response was weakly dependent on power in the range 72-145 mW and was not significantly different for pulsed and continuous wave stimulation where time averaged power was constant. The response was unaffected by mechanical removal of the endothelium. The ultrasound beam generated insufficient radiation force to produce a measurable effect and streaming at the vessel surface was very small compared with flow rates known to produce physiologic effects. The temperature rise at the beam focus was approximately 0.3 degrees C and we hypothesise that this contributes to the observed response, probably through changes in ion channel activity in smooth muscle cell membranes. (E-mail: e.martin@exeter.ac.uk).

The paramyxovirus family contains established human pathogens such as the measles virus and human respiratory syncytial virus, as well as emerging pathogens including the Hendra and Nipah viruses and the recently identified human metapneumovirus. Two major envelope glycoproteins, the attachment protein and the fusion protein, promote the processes of viral attachment and virus-cell membrane fusion required for entry. Although common mechanisms of fusion protein proteolytic activation and the mechanism of membrane fusion promotion have been shown in recent years, considerable diversity exists in the family relating to receptor binding and the potential mechanisms of fusion triggering.

Elucidating the molecular mechanisms that created ancient complex traits like insect wings is difficult. Fortunately, some complex traits have arisen recently. For example, hermaphroditic reproduction evolved independently many times during recent nematode evolution. Although C. elegans hermaphrodites require fog-2, which encodes an F box protein that regulates the translation of tra-2 mRNAs, the related species C. briggsae lacks fog-2. We identified a critical regulator of hermaphrodite development in C. briggsae, named she-1. Analysis of double mutants indicates that she-1 acts upstream of tra-2 in C. briggsae, just as fog-2 does in C. elegans. Molecular cloning shows that she-1 encodes a novel F box protein that was created by a recent gene duplication. Whereas FOG-2 acts through GLD-1 in C. elegans, SHE-1 does not bind GLD-1 in C. briggsae. Thus, both species recruited F box genes produced by recent duplication events into the sex-determination pathway to control hermaphrodite development, but these genes have distinct activities. This result implies that some gene families are more likely to give rise to novel regulatory genes than other families. Finally, we note that null mutations of she-1 are temperature sensitive, so C. briggsae might once have been a facultative hermaphrodite.

Although the nematode Caenorhabditis elegans produces self-fertile hermaphrodites, it descended from a male/female species, so hermaphroditism provides a model for the origin of novel traits. In the related species C. remanei, which has only male and female sexes, lowering the activity of tra-2 by RNA interference created XX animals that made spermatids as well as oocytes, but their spermatids could not activate without the addition of male seminal fluid. However, by lowering the expression of both tra-2 and swm-1, a gene that regulates sperm activation in C. elegans, we produced XX animals with active sperm that were self-fertile. Thus, the evolution of hermaphroditism in Caenorhabditis probably required two steps: a mutation in the sex-determination pathway that caused XX spermatogenesis and a mutation that allowed these spermatids to self-activate.

Fos and Jun are components of activator protein-1 (AP-1) and play crucial roles in the regulation of many cellular, developmental, and physiological processes. Caenorhabditis elegans fos-1 has been shown to act in uterine and vulval development. Here, we provide evidence that C. elegans fos-1 and jun-1 control ovulation, a tightly regulated rhythmic program in animals. Knockdown of fos-1 or jun-1 blocks dilation of the distal spermathecal valve, a critical step for the entry of mature oocytes into the spermatheca for fertilization. Furthermore, fos-1 and jun-1 regulate the spermathecal-specific expression of plc-1, a gene that encodes a phospholipase C (PLC) isozyme that is rate-limiting for inositol triphosphate production and ovulation, and overexpression of PLC-1 rescues the ovulation defect in fos-1(RNAi) worms. Unlike fos-1, regulation of ovulation by jun-1 requires genetic interactions with eri-1 and lin-15B, which are involved in the RNA interference pathway and chromatin remodeling, respectively. At least two isoforms of jun-1 are coexpressed with fos-1b in the spermatheca, and different AP-1 dimers formed between these isoforms have distinct effects on the activation of a reporter gene. These findings uncover a novel role for FOS-1 and JUN-1 in the reproductive system and establish C. elegans as a model for studying AP-1 dimerization.

Hendra virus F protein-promoted membrane fusion requires the presence of the viral attachment protein, G. However, events leading to the association of these glycoproteins remain unclear. Results presented here demonstrate that Hendra virus G undergoes slower secretory pathway trafficking than is observed for Hendra virus F. This slowed trafficking is not dependent on the G protein cytoplasmic tail, the presence of the G receptor ephrin B2, or interaction with other viral proteins. Instead, Hendra virus G was found to undergo intrinsically slow oligomerization within the endoplasmic reticulum. These results suggest that the critical F-G interactions occur only after the initial steps of synthesis and cellular transport.

Knowledge of the behavior and mechanics of a total knee replacement (TKR) in an in vivo environment is key to optimizing the functional outcomes of the implant procedure. Computational modeling has shown to be an important tool for investigating biomechanical variables that are difficult to address experimentally. To assist in examining TKR mechanics, a dynamic finite-element model of a TKR is presented. The objective of the study was to develop and evaluate a model that could simulate full knee motion using a physiologically consistent quadriceps action, without prescribed joint kinematics. The model included tibiofemoral (TFJs) and patellofemoral joints (PFJs), six major ligament bundles and was driven by a uni-axial representation of a quadricep muscle. An initial parameter screening analysis was performed to assess the relative importance of 31 different model parameters. This analysis showed that ligament insertion location and initial ligament strain were significant factors affecting simulated joint kinematics and loading, with the contact friction coefficient playing a lesser role and ligament stiffness having little effect. The model was then used to simulate in vitro experiments utilizing a flexed-knee-stance testing rig. General model performance was assessed by comparing simulation results with experimentally measured kinematics and tibial reaction forces collected from two implanted specimens. The simulations were able to reproduce experimental differences observed between the test specimens and were able to accurately predict trends seen in the tibial reaction loads. The simulated kinematics of the TFJ and PFJ were less consistent when compared with experimental data but still reproduced many trends.

An investigation of collagen fiber reorientation, as well as fluid and matrix movement of equine articular cartilage and subchondral bone under compressive mechanical loads, was undertaken using small angle X-ray scattering measurements and optical microscopy. Small angle X-ray scattering measurements were made on healthy and diseased samples of equine articular cartilage and subchondral bone mounted in a mechanical testing apparatus on station ID18F of ESRF, Grenoble, together with fiber orientation analysis using polarized light and displacement measurements of the cartilage matrix and fluid using tracers. At surface pressures of up to approximately 1.5 MPa, there was reversible compression of the tangential surface fibers and immediately subjacent zone. As load increased, deformation in these zones reached a maximum and then reorientation propagated to the radial deep zone. Between surface pressures of 4.8 MPa and 6.0 MPa, fiber orientation above the tide mark rotated 10 deg from the radial direction, with an overall loss of alignment. With further increase in load, the fibers "crimped" as shown by the appearance of subsidiary peaks approximately +/-10 deg either side of the principal fiber orientation direction. Failure at higher loads was characterized by a radial split in the deep cartilage, which propagated along the tide mark while the surface zone remained intact. In lesions, the fiber organization was disrupted and the initial response to load was consistent with early rupture of fibers, but the matrix relaxed to an organization very similar to that of the unloaded tissue. Tracer measurements revealed anisotropic solid and fluid displacement, which depended strongly on depth within the tissue.

Human metapneumovirus (HMPV) is a significant respiratory pathogen classified in the Pneumovirinae subfamily of the paramyxovirus family. Recently, we demonstrated that HMPV F protein-promoted cell-cell fusion is stimulated by exposure to low pH, in contrast to what is observed for other paramyxovirus F proteins. In the present study, we examined the potential role of histidine protonation in HMPV F fusion and investigated the role of low pH in HMPV viral entry. Mutagenesis of the three ectodomain histidine residues of the HMPV F protein demonstrated that the mutation of a histidine in the heptad repeat B linker domain (H435) ablated fusion activity without altering cell surface expression or proteolytic processing significantly. Modeling of the HMPV F protein revealed several basic residues surrounding this histidine residue, and the mutation of these residues also reduced fusion activity. These results suggest that electrostatic repulsion in the heptad repeat B linker region may contribute to the triggering of HMPV F. In addition, we examined the effect of inhibitors of endosomal acidification or endocytosis on the entry of a recombinant green fluorescent protein-expressing HMPV. Interestingly, chemicals that raise the pH of endocytic vesicles resulted in a 30 to 50% decrease in HMPV infection, while the inhibitors of endocytosis reduced infection by as much as 90%. These data suggest that HMPV utilizes an endocytic entry mechanism, in contrast to what has been hypothesized for most paramyxoviruses. In addition, our results indicate that HMPV uses the low pH of the endocytic pathway to enhance infectivity, though the role of low pH likely differs from classically described mechanisms.

This article describes a fully automatic, real-time, freehand ultrasound calibration system. The system was designed to be simple and sterilizable, intended for operating-room usage. The calibration system employed an automatic-error-retrieval and accuracy-control mechanism based on a set of ground-truth data. Extensive validations were conducted on a data set of 10,000 images in 50 independent calibration trials to thoroughly investigate the accuracy, robustness, and performance of the calibration system. On average, the calibration accuracy (measured in three-dimensional reconstruction error against a known ground truth) of all 50 trials was 0.66 mm. In addition, the calibration errors converged to submillimeter in 98% of all trials within 12.5 s on average. Overall, the calibration system was able to consistently, efficiently and robustly achieve high calibration accuracy with real-time performance.

Background: The Taylor spatial frame is a fixation device used to implement the Ilizarov method of bone deformity correction to gradually distract an osteotomized bone at regular intervals, according to a prescribed schedule., Methods: We modify conventional technique by: (a) preoperatively planning a virtual three-dimensional (3D) correction; (b) basing the correction on the actual location of the frame with respect to the anatomy, immediately compensating for frame mounting errors; and (c) calculating the correction based on 3D CT data rather than measurements from radiographs. We have performed a laboratory study using plastic phantoms, and a pilot clinical study involving five patients., Results: In 20 tibial phantom experiments, we achieved average correction errors of < 2 degrees total rotation and < 0.5 mm total lengthening. We observed clinically acceptable corrections with no complications in our pilot clinical study., Conclusions: Our method achieved high accuracy and precision in a laboratory setting, and produced acceptable outcomes in a pilot clinical study.

In women, oocytes arrest development at the end of prophase of meiosis I and remain quiescent for years. Over time, the quality and quantity of these oocytes decreases, resulting in fewer pregnancies and an increased occurrence of birth defects. We used the nematode Caenorhabditis elegans to study how oocyte quality is regulated during aging. To assay quality, we determine the fraction of oocytes that produce viable eggs after fertilization. Our results show that oocyte quality declines in aging nematodes, as in humans. This decline affects oocytes arrested in late prophase, waiting for a signal to mature, and also oocytes that develop later in life. Furthermore, mutations that block all cell deaths result in a severe, early decline in oocyte quality, and this effect increases with age. However, mutations that block only somatic cell deaths or DNA-damage-induced deaths do not lower oocyte quality. Two lines of evidence imply that most developmentally programmed germ cell deaths promote the proper allocation of resources among oocytes, rather than eliminate oocytes with damaged chromosomes. First, oocyte quality is lowered by mutations that do not prevent germ cell deaths but do block the engulfment and recycling of cell corpses. Second, the decrease in quality caused by apoptosis mutants is mirrored by a decrease in the size of many mature oocytes. We conclude that competition for resources is a serious problem in aging germ lines, and that apoptosis helps alleviate this problem., Competing Interests: The authors have declared that no competing interests exist.

A new technique for percutaneous fixation of non-displaced scaphoid fractures is described. The technique used pre-operative planning from computed tomography images, registration to intra-operatively acquired three-dimensional ultrasound images, and intra-operative guidance using an optical tracking system. Two stand-alone software applications were developed. The first one was used to determine the surgical plan pre-operatively and the second one was used to guide the surgeon during screw insertion. Laboratory validation of the technique included measurements of the inter-operator and intra-operator variability in the outcome of scaphoid fixation using the proposed procedure, and also included comparison of the performance of this procedure with the conventional percutaneous fixation technique using fluoroscopy. The results showed that the tight accuracy requirements of percutaneous scaphoid fixation were met and that the consistency was superior to the conventional technique.

The bimolecular fluorescence complementation (BiFC) assay is a powerful tool for visualizing and identifying protein interactions in living cells. This assay is based on the principle of protein-fragment complementation, using two nonfluorescent fragments derived from fluorescent proteins. When two fragments are brought together in living cells by tethering each to one of a pair of interacting proteins, fluorescence is restored. Here, we provide a protocol for a Venus-based BiFC assay to visualize protein interactions in the living nematode, Caenorhabditis elegans. We discuss how to design appropriate C. elegans BiFC cloning vectors to enable visualization of protein interactions using either inducible heat shock promoters or native promoters; transform the constructs into worms by microinjection; and analyze and interpret the resulting data. When expression of BiFC fusion proteins is induced by heat shock, the fluorescent signals can be visualized as early as 30 min after induction and last for 24 h in transgenic animals. The entire procedure takes 2-3 weeks to complete.

Sexual identity is one of the most important factors that determine how an animal will develop. Although it controls many dimorphic tissues in the body, its most ancient role is in the germ line, where it species that some cells become sperm, and others become eggs. In most animals, these two fates occur in distinct sexes. However, certain nematodes like C. elegans produce XX hermaphrodites, which make both types of gametes. In these animals, a core sex-determination pathway regulates the development of both the body and the germ line. However, modifier genes alter the activity of this pathway in germ cells, and these changes are critical for allowing XX animals to produce oocytes and sperm in an otherwise female body. In this review, I focus on (1) the core sex-determination pathway, (2) the activity of the transcription factor TRA-1 and its immediate targets fog-1 and fog-3 in germ cells, (3) how the regulation of tra-2 activity allows XX spermatogenesis, and (4) how the regulation of fem-3 activity maintains the appropriate balance between TRA-2 and FEM-3 in the germ line. Finally, I consider the major questions in this field that are driving new research.

We investigate the effect of the skeletal protein spectrin on the lateral order in dipalmitoyl phosphatidylserine monolayers spread on aqueous surfaces using grazing incidence X-ray diffraction. Without spectrin, the condensed lipid monolayer exhibits two-dimensional hexagonal packing, characterized by monotonic decrease in the d-spacing and increase in the degree of order with increasing surface pressure between 17 and 36 mN/m. Addition of spectrin to the aqueous subphase at high pressures preserves the monolayers structural parameters unchanged from 36 to 25 mN/m. These results demonstrate for the first time that spectrin could participate in sustaining the two-dimensional order in lipid domains through a direct interaction with phosphatidylserine species.

The general framework and experimental validation of a novel navigation system designed for shoulder arthroscopy are presented. The system was designed to improve the surgeon's perception of the three-dimensional space within the human shoulder. Prior to surgery, a surface model of the shoulder was created from computed tomography images. Intraoperatively, optically tracked arthroscopic instruments were calibrated. The surface model was then registered to the patient using tracked freehand ultrasound images taken from predefined landmark regions on the scapula. Three-dimensional models of the surgical instruments were displayed, in real time, relative to the surface model in a user interface. Laboratory experiments revealed only small registration and calibration errors, with minimal time needed to complete the intraoperative tasks.

Hendra virus, like most paramyxoviruses, requires both a fusion (F) and attachment (G) protein for promotion of cell-cell fusion. Recent studies determined that Hendra F is proteolytically processed by the cellular protease cathepsin L after endocytosis. This unique cathepsin L processing results in a small percentage of Hendra F on the cell surface. To determine how the surface densities of the two Hendra glycoproteins affect fusion promotion, we performed experiments that varied the levels of glycoproteins expressed in transfected cells. Using two different fusion assays, we found a marked increase in fusion when expression of the Hendra G protein was increased, with a 1:1 molar ratio of Hendra F:G on the cell surface resulting in optimal membrane fusion. Our results also showed that Hendra G protein levels are modulated by both more rapid protein turnover and slower protein trafficking than is seen for Hendra F.

Objective: To determine regional differences in the orientation of collagen in the articular cartilage of the equine metacarpophalangeal joint as well as describing cartilage orientation in lesions using small angle X-ray scattering (SAXS)., Design: SAXS diffraction patterns were taken at the European Synchrotron Radiation Facility (ESRF), with increasing depth into cartilage and bone cross sections. Results for healthy samples were taken at different regions along the joint which receive different loads and differences in collagen orientation were determined. Results were also taken from diseased samples and the collagen orientation changes from that of healthy samples observed., Results: Regions subject to low loads show a lower degree of orientation and regions exposed to the highest loads possess oriented collagen fibres especially in the radial layer. In early lesions the orientations of the collagen fibres are disrupted. Subchondral bone fibres are twisted in regions where the joint receives shear forces. Changes in fibre orientation are also observed in the calcified cartilage even in regions where the cartilage is intact. In more advanced lesions where there is loss of cartilage the fibres in the calcified layer are realigned tangential to the surface., Conclusions: Regional variations in collagen arrangement show that the highly ordered layers of the articular cartilage are the most important elements in supporting high variable loads. In lesions changes occur in the deep tissue whilst the overlying cartilage appeared normal. We therefore suggest that the interface region is a key element in the early stages of the disease.