Advancing the deer calcaneus model for bone adaptation studies: ex vivo strains obtained after transecting the tension members suggest an unrecognized important role for shear strains.

Sheep and deer calcanei are finding increased use as models for studies of bone adaptation, including advancing understanding of how the strain (deformation) environment influences the ontogenetic emergence of biomechanically relevant structural and material variations in cortical and trabecular bone. These artiodactyl calcanei seem ideal for these analyses because they function like simply loaded short-cantilevered beams with net compression and tension strains on the dorsal and plantar cortices, respectively. However, this habitual strain distribution requires more rigorous validation because it has been shown by limited in vivo and ex vivo strain measurements obtained during controlled ambulation (typically walking and trotting). The conception that these calcanei are relatively simply and habitually loaded 'tension/compression bones' could be invalid if infrequent, though biologically relevant, loads substantially change the location of the neutral axis (NA) that separates 'compression' and 'tension' regions. The effect on calcaneus strains of the tension members (plantar ligament and flexor tendon) is also not well understood and measuring strains after transecting them could reveal that they significantly modulate the strain distribution. We tested the hypothesis that the NA location previously described during simulated on-axis loads of deer calcanei would exhibit limited variations even when load perturbations are unusual (e.g. off-axis loads) or extreme (e.g. after transection of the tension members). We also examined regional differences in the predominance of the three strain modes (tension, compression, and shear) in these various load conditions in dorsal, plantar, medial, and lateral cortices. In addition to considering principal strains (tension and compression) and maximum shear strains, we also considered material-axis (M-A) shear strains. M-A shear strains are those that are aligned along the long axis of the bone and are considered to have greater biomechanical relevance than maximum shear strains because failure theories of composite materials and bone are often based on stresses or strains in the principal material directions. We used the same load apparatus from our prior study of mule deer calcanei. Results showed that although the NA rotated up to 8° medially and 15° laterally during these off-axis loads, it did not shift dramatically until after transection of all tension members. When comparing results based on maximum shear strain data vs. M-A shear strain data, the dominant strain mode changed only in the plantar cortex - as expected (in accordance with our a priori view) it was tension when M-A shear strains were considered (shear : tension = 0.2) but changed to dominant shear when maximum shear strain data were considered (shear : tension = 1.3). This difference leads to different conclusions and speculations regarding which specific strain modes and magnitudes most strongly influence the emergence of the marked mineralization and histomorphological differences in the dorsal vs. plantar cortices. Consequently, our prior simplification of the deer calcaneus model as a simply loaded 'tension/compression bone' (i.e. plantar/dorsal) might be incorrect. In vivo and in finite element analyses are needed to determine whether describing it as a 'shear-tension/compression' bone is more accurate. Addressing this question will help to advance the artiodactyl calcaneus as an experimental model for bone adaptation studies.

The Evolutionary Biology, Ecology and Epidemiology of Coccidia of Passerine Birds.

Coccidia are intracellular parasites of the phylum Apicomplexa that cause a range of pathologies collectively termed coccidiosis. Species of coccidia of commercial importance have been well studied, with the effect of other species on passerine birds receiving increasing attention. In this chapter, we review the literature on coccidia in passerines, with a particular focus on wild populations. The taxonomy and life cycle of passerine coccidia are covered, as is their impact on the health of passerines, their epidemiology and their role in parasite-mediated natural and sexual selection. Coccidia can pose a significant threat to the health of wild passerine populations, and high rates of mortality have been observed in some studies. We examine some of the genetic factors that influence host resistance to coccidia and discuss how these parasites may be important in relation to sexually selected traits. General patterns are beginning to emerge with regard to the epidemiology of the parasites, and the influence of different aspects of the host's ecology on the prevalence and intensity of coccidia is being revealed. We examine these, as well exceptions, in addition to the phenomenon of diurnal oocyst shedding that can bias studies if not accounted for. Finally, we discuss potential future directions for research on coccidia in passerines and the importance of understanding parasite ecology in the management of threatened species.

Super-resolution microscopy in the diagnosis of platelet granule disorders.

INTRODUCTION: Platelet granule deficiencies lead to bleeding disorders, but their specific diagnosis typically requires whole mount transmission electron microscopy, which is often not available and has a number of important limitations. We recently proposed the use of advanced forms of fluorescence microscopy - the so-called 'super-resolution' microscopies - as a possible solution. Areas covered: In this special report, we review the diagnosis of platelet granule deficiencies, and discuss how recent developments in fluorescence microscopy may be useful in improving diagnostic approaches to these and related disorders. In particular, we conclude that super-resolution fluorescence microscopy may have advantages over transmission electron microscopy in this application. Expert commentary: The value of the super-resolution microscopies has been amply demonstrated in research; however, their potential in diagnostic applications is ripe for further exploration. Hematology is a field particularly likely to benefit because of the relative simplicity of sample preparation. We anticipate that the costs of the necessary instrumentation will continue to fall rapidly, making these technologies widely accessible to clinicians.

Frequency of Shoulder Corticosteroid Injections for Pain and Stiffness After Shoulder Surgery and Their Potential to Enhance Outcomes with Physiotherapy: A Retrospective Study.

INTRODUCTION: What is the rate of injecting patients with shoulder corticosteroid injections to alleviate excessive stiffness and pain within 6 months after shoulder surgery? METHODS: Retrospective 10-year review of a shoulder surgeon's practice. Participants included 754 patients who had 945 non-arthroplasty shoulder surgeries. Outcome measures included the rate of injections, diagnoses, patient characteristics, and efficacy via questionnaire. RESULTS: Approximately one in five patients received a subacromial and/or glenohumeral corticosteroid injection. Over 95% of patients stated that the injections helped reduce shoulder pain and increased function 6 weeks post-injection. Twenty-two percent of cases (208/945) received glenohumeral and/or subacromial injections. The 208 injected cases had these diagnoses: rotator cuff tear (28% of injected patients), subacromial impingement (20%), glenohumeral instability (16%), subacromial impingement with acromioclavicular osteoarthritis (10%), adhesive capsulitis (7%), SLAP lesion (5%), biceps tendinopathy (3%), glenohumeral instability with subacromial impingement (3%), proximal humerus fracture (2%), calcific tendinitis (2%), and less common conditions (4%). Diagnoses among those with the highest rates of injected patients per diagnosis included: SLAP lesions (40%), calcific tendinitis (40%), adhesive capsulitis (29%), subacromial impingement (28%), proximal humerus fracture (24%), rotator cuff tear (19%), and glenohumeral instability (16%). Significant differences (p < 0.03) were found between patients who did and did not receive injections with respect to age (more likely younger patients with cuff tear) and sex (more likely female with subacromial impingement and instability) but not for diabetes or arthroscopic vs. open procedures. CONCLUSION: This is the first study to establish the rates of postoperative shoulder corticosteroid injections within the first 6 months after various non-arthroplasty shoulder surgeries for patients with high pain/stiffness. These data will be useful for establishing guidelines for using corticosteroid injections along with physiotherapy.

Epithelial-mesenchymal transition, IP3 receptors and ER-PM junctions: translocation of Ca2+ signalling complexes and regulation of migration.

Disconnection of a cell from its epithelial neighbours and the formation of a mesenchymal phenotype are associated with profound changes in the distribution of cellular components and the formation of new cellular polarity. We observed a dramatic redistribution of inositol trisphosphate receptors (IP3Rs) and stromal interaction molecule 1 (STIM1)-competent endoplasmic reticulum-plasma membrane junctions (ER-PM junctions) when pancreatic ductal adenocarcinoma (PDAC) cells disconnect from their neighbours and undergo individual migration. In cellular monolayers IP3Rs are juxtaposed with tight junctions. When individual cells migrate away from their neighbours IP3Rs preferentially accumulate at the leading edge where they surround focal adhesions. Uncaging of inositol trisphosphate (IP3) resulted in prominent accumulation of paxillin in focal adhesions, highlighting important functional implications of the observed novel structural relationships. ER-PM junctions and STIM1 proteins also migrate to the leading edge and position closely behind the IP3Rs, creating a stratified distribution of Ca(2+) signalling complexes in this region. Importantly, migration of PDAC cells was strongly suppressed by selective inhibition of IP3Rs and store-operated Ca(2+) entry (SOCE), indicating that these mechanisms are functionally required for migration.

Radiographic morphometry and densitometry predict strength of cadaveric proximal humeri more reliably than age and DXA scan density.

Methods are needed for identifying poorer quality cadaver proximal humeri to ensure that they are not disproportionately segregated into experimental groups for fracture studies. We hypothesized that measurements made from radiographs of cadaveric proximal humeri are stronger predictors of fracture strength than chronological age or bone density values derived from dual-energy x-ray absorptiometry (DXA) scans. Thirty-three proximal humeri (range: 39-78 years) were analyzed for: (1) bone mineral density (BMD, g/cm(2)) using DXA, (2) bulk density (g/cm(3)) using DXA and volume displacement, (3) regional bone density in millimeters of aluminum (mmAl) using radiographs, and (4) regional mean (medial+lateral) cortical thickness and cortical index (CI) using radiographs. The bones were then fractured simulating a fall. Strongest correlations with ultimate fracture load (UFL) were: mean cortical thickness at two diaphyseal locations (r = 0.71; p < 0.001), and mean mmAl in the humeral head (r = 0.70; p < 0.001). Weaker correlations were found between UFL and DXA-BMD (r = 0.60), bulk density (r = 0.43), CI (r = 0.61), and age (r = -0.65) (p values <0.01). Analyses between UFL and the product of any two characteristics showed six combinations with r-values >0.80, but none included DXA-derived density, CI, or age. Radiographic morphometric and densitometric measurements from radiographs are therefore stronger predictors of UFL than age, CI, or DXA-derived density measurements.

Reusing cadaveric humeri for fracture testing after testing simulated rotator cuff tendon repairs.

The financial cost of using human tissues in biomedical testing and surgical reconstruction is predicted to increase at a rate that is disproportionately greater than other materials used in biomechanical testing. Our first hypothesis is that cadaveric proximal humeri that had undergone monotonic failure testing of simulated rotator cuff repairs would not differ in ultimate fracture loads or in energy absorbed to fracture when compared to controls (i.e., bones without cuff repairs). Our second hypothesis is that there can be substantial cost savings if these cadaveric proximal humeri, with simulated cuff repairs, can be re-used for fracture testing. Results of fracture tests (conducted in a backwards fall configuration) and cost analysis support both hypotheses. Hence, the bones that had undergone monotonic failure tests of various rotator cuff repair techniques can be re-used in fracture tests because their load-carrying capacity is not significantly reduced.

Temporary sternoclavicular plating for an unusual double clavicle fracture (medial nonunion, lateral acute) complicated by an intraoperative pneumothorax.

Double (segmental) clavicle fractures, involving both the medial and lateral aspects of the clavicle, are very uncommon. Even less common is an asynchronous double fracture with one of the fractures being a nonunion. We report the case of a 30-year-old healthy male patient who had an unusual double clavicle fracture (medial nonunion, lateral acute) that occurred in separate traumatic events during motocross (motorcycle) racing. His fractures were treated surgically in two stages. In the first stage a long reconstruction plate was used that spanned onto the sternum and two transcortical screws were placed into the manubrium to enhance purchase for the deficient bone of the medial clavicle. In accordance with the preoperative plan, the medial one-third of the plate and the medial four screws (of the total 13 used) were removed. Although our patient had an excellent final result, he did have an intraoperative pneumothorax that was treated uneventfully with a chest tube. Medial clavicle fractures are difficult to treat, especially if they are nonunions and surgical complication rates can be high. Our case is one of the few that has been described where temporary sternoclavicular plating was successful in achieving an excellent long-term outcome.

Flat clathrin lattices: stable features of the plasma membrane.

Clathrin-mediated endocytosis (CME) is a fundamental property of eukaryotic cells. Classical CME proceeds via the formation of clathrin-coated pits (CCPs) at the plasma membrane, which invaginate to form clathrin-coated vesicles, a process that is well understood. However, clathrin also assembles into flat clathrin lattices (FCLs); these structures remain poorly described, and their contribution to cell biology is unclear. We used quantitative imaging to provide the first comprehensive description of FCLs and explore their influence on plasma membrane organization. Ultrastructural analysis by electron and superresolution microscopy revealed two discrete populations of clathrin structures. CCPs were typified by their sphericity, small size, and homogeneity. FCLs were planar, large, and heterogeneous and present on both the dorsal and ventral surfaces of cells. Live microscopy demonstrated that CCPs are short lived and culminate in a peak of dynamin recruitment, consistent with classical CME. In contrast, FCLs were long lived, with sustained association with dynamin. We investigated the biological relevance of FCLs using the chemokine receptor CCR5 as a model system. Agonist activation leads to sustained recruitment of CCR5 to FCLs. Quantitative molecular imaging indicated that FCLs partitioned receptors at the cell surface. Our observations suggest that FCLs provide stable platforms for the recruitment of endocytic cargo.

Dilemma of high rate of conversion from knee arthroscopy to total knee arthroplasty.

We tried to reduce our rate of conversion from index knee arthroscopy to total knee arthroplasty (TKA) for degenerative pathology (primarily meniscal) in the setting of coexisting arthritis in patients 50 years or older. We hypothesized that, by using a 2-surgeon independent evaluation method, we could reduce the rate to less than 10% by 3-year follow-up. Forty-two consecutive patients were initially evaluated by the knee replacement surgeon to determine if they were TKA candidates. They were then independently evaluated by another surgeon regarding the need for TKA and the possibility of arthroscopic debridement. The data showed a tendency: The under-10% target rate was nearly reached in patients younger than 65 years (12%; 2/17) but not in patients older than 65 years (36%; 9/25). The overall rate of conversion to TKA was 26%. The 2 main groups (arthroscopy only, arthroscopy-plus-TKA) did not differ in all measured characteristics. Failure of our method to achieve better outcomes demonstrates that conventional criteria are poor in predicting which patients with meniscal pathology, which is believed to be relatively more symptomatic than coexisting arthritis, should avoid arthroscopy and go straight to TKA.

A two-tier Golgi-based control of organelle size underpins the functional plasticity of endothelial cells.

Weibel-Palade bodies (WPBs), endothelial-specific secretory granules that are central to primary hemostasis and inflammation, occur in dimensions ranging between 0.5 and 5 µm. How their size is determined and whether it has a functional relevance are at present unknown. Here, we provide evidence for a dual role of the Golgi apparatus in controlling the size of these secretory carriers. At the ministack level, cisternae constrain the size of nanostructures ("quanta") of von Willebrand factor (vWF), the main WPB cargo. The ribbon architecture of the Golgi then allows copackaging of a variable number of vWF quanta within the continuous lumen of the trans-Golgi network, thereby generating organelles of different sizes. Reducing the WPB size abates endothelial cell hemostatic function by drastically diminishing platelet recruitment, but, strikingly, the inflammatory response (the endothelial capacity to engage leukocytes) is unaltered. Size can thus confer functional plasticity to an organelle by differentially affecting its activities.

TestSTORM: Simulator for optimizing sample labeling and image acquisition in localization based super-resolution microscopy.

Localization-based super-resolution microscopy image quality depends on several factors such as dye choice and labeling strategy, microscope quality and user-defined parameters such as frame rate and number as well as the image processing algorithm. Experimental optimization of these parameters can be time-consuming and expensive so we present TestSTORM, a simulator that can be used to optimize these steps. TestSTORM users can select from among four different structures with specific patterns, dye and acquisition parameters. Example results are shown and the results of the vesicle pattern are compared with experimental data. Moreover, image stacks can be generated for further evaluation using localization algorithms, offering a tool for further software developments.

Test samples for optimizing STORM super-resolution microscopy.

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.

Correcting chromatic offset in multicolor super-resolution localization microscopy.

Localization based super-resolution microscopy techniques require precise drift correction methods because the achieved spatial resolution is close to both the mechanical and optical performance limits of modern light microscopes. Multi-color imaging methods require corrections in addition to those dealing with drift due to the static, but spatially-dependent, chromatic offset between images. We present computer simulations to quantify this effect, which is primarily caused by the high-NA objectives used in super-resolution microscopy. Although the chromatic offset in well corrected systems is only a fraction of an optical wavelength in magnitude (<50 nm) and thus negligible in traditional diffraction limited imaging, we show that object colocalization by multi-color super-resolution methods is impossible without appropriate image correction. The simulated data are in excellent agreement with experiments using fluorescent beads excited and localized at multiple wavelengths. Finally we present a rigorous and practical calibration protocol to correct for chromatic optical offset, and demonstrate its efficacy for the imaging of transferrin receptor protein colocalization in HeLa cells using two-color direct stochastic optical reconstruction microscopy (dSTORM).

Scaling of Haversian canal surface area to secondary osteon bone volume in ribs and limb bones.

Studies of secondary osteons in ribs have provided a great deal of what is known about remodeling dynamics. Compared with limb bones, ribs are metabolically more active and sensitive to hormonal changes, and receive frequent low-strain loading. Optimization for calcium exchange in rib osteons might be achieved without incurring a significant reduction in safety factor by disproportionally increasing central canal size with increased osteon size (positive allometry). By contrast, greater mechanical loads on limb bones might favor reducing deleterious consequences of intracortical porosity by decreasing osteon canal size with increased osteon size (negative allometry). Evidence of this metabolic/mechanical dichotomy between ribs and limb bones was sought by examining relationships between Haversian canal surface area (BS, osteon Haversian canal perimeter, HC.Pm) and bone volume (BV, osteonal wall area, B.Ar) in a broad size range of mature (quiescent) osteons from adult human limb bones and ribs (modern and medieval) and various adult and subadult non-human limb bones and ribs. Reduced major axis (RMA) and least-squares (LS) regressions of HC.Pm/B.Ar data show that rib and limb osteons cannot be distinguished by dimensional allometry of these parameters. Although four of the five rib groups showed positive allometry in terms of the RMA slopes, nearly 50% of the adult limb bone groups also showed positive allometry when negative allometry was expected. Consequently, our results fail to provide clear evidence that BS/BV scaling reflects a rib versus limb bone dichotomy whereby calcium exchange might be preferentially enhanced in rib osteons.

The artiodactyl calcaneus as a potential 'control bone' cautions against simple interpretations of trabecular bone adaptation in the anthropoid femoral neck.

For over a century, the arched trabecular patterns of the human proximal femur have been considered to resemble tension and compression stress trajectories produced by stereotypical bending loads. This reflects conventional modeling of the human femoral neck-head region as a short cantilevered beam. Although this conception is the foundation of many biomechanical, clinical, paleontological, and comparative morphological studies of trabecular bone in various species, attempts have not been made to contrast these data to a bone that could be considered a 'control' for simple/stereotypical bending. We quantified trabecular architectural characteristics in sheep and deer calcanei as a first step in potentially establishing them as 'controls' in this context because they have arched trabecular patterns that resemble tension/compression stress trajectories, and have been shown by strain gauge measurements to be relatively simply loaded in bending. Using micro-computed tomography, calcanei from adult domesticated sheep and wild deer were analyzed where in the dorsal 'compression' and plantar 'tension' trabecular tracts they begin to separate and bending is less complex (mid-shaft), and where trabeculae extensively interconnect and loading is more complex (distal shaft). Of the eight trabecular architectural characteristics evaluated, only one (trabecular number, Tb.N) showed a probable mechanically relevant dorsal/plantar difference. However, this was paradoxically opposite in the sheep calcanei. Aside from Tb.N, the architectural characteristics showed little, if any, evidence of habitual bending. The non-uniformity of the stresses between the trabecular tracts in these bones might be reduced by load-sharing functions of their robust cortices and the nearby ligament and tendon, which might account for the similar morphologies between the tracts. These findings may help to explain why in many cases regional trabecular architectural variations seem to lack sufficient sensitivity and specificity for interpreting habitual bending in other bone regions. This cautions against simple interpretations of trabecular bone adaptation in the anthropoid femoral neck.

Do regional modifications in tissue mineral content and microscopic mineralization heterogeneity adapt trabecular bone tracts for habitual bending? Analysis in the context of trabecular architecture of deer calcanei.

Calcanei of mature mule deer have the largest mineral content (percent ash) difference between their dorsal 'compression' and plantar 'tension' cortices of any bone that has been studied. The opposing trabecular tracts, which are contiguous with the cortices, might also show important mineral content differences and microscopic mineralization heterogeneity (reflecting increased hemi-osteonal renewal) that optimize mechanical behaviors in tension vs. compression. Support for these hypotheses could reveal a largely unrecognized capacity for phenotypic plasticity - the adaptability of trabecular bone material as a means for differentially enhancing mechanical properties for local strain environments produced by habitual bending. Fifteen skeletally mature and 15 immature deer calcanei were cut transversely into two segments (40% and 50% shaft length), and cores were removed to determine mineral (ash) content from 'tension' and 'compression' trabecular tracts and their adjacent cortices. Seven bones/group were analyzed for differences between tracts in: first, microscopic trabecular bone packets and mineralization heterogeneity (backscattered electron imaging, BSE); and second, trabecular architecture (micro-computed tomography). Among the eight architectural characteristics evaluated [including bone volume fraction (BVF) and structural model index (SMI)]: first, only the 'tension' tract of immature bones showed significantly greater BVF and more negative SMI (i.e. increased honeycomb morphology) than the 'compression' tract of immature bones; and second, the 'compression' tracts of both groups showed significantly greater structural order/alignment than the corresponding 'tension' tracts. Although mineralization heterogeneity differed between the tracts in only the immature group, in both groups the mineral content derived from BSE images was significantly greater (P < 0.01), and bulk mineral (ash) content tended to be greater in the 'compression' tracts (immature 3.6%, P = 0.03; mature 3.1%, P = 0.09). These differences are much less than the approximately 8% greater mineral content of their 'compression' cortices (P < 0.001). Published data, suggesting that these small mineralization differences are not mechanically important in the context of conventional tests, support the probability that architectural modifications primarily adapt the tracts for local demands. However, greater hemi-osteonal packets in the tension trabecular tract of only the mature bones (P = 0.006) might have an important role, and possible synergism with mineralization and/or microarchitecture, in differential toughening at the trabeculum level for tension vs. compression strains.