Axis Determination Research Articles

Effectiveness of Treatment for Distal Tibial Nonunions With Associated Complex Deformities Using a Hexapod External Fixator

To determine the efficacy of a hexapod external fixator (TSF) and its ability to perform 6-axis correction in restoring deformities associated with distal tibial nonunions. Consecutive retrospective case review. St. Louis University Hospital. Level 1 Trauma Center. Thirty-seven patients with distal tibial nonunion with greater than 5 degrees of deformity in any plane. Patients were excluded who were less than 18 years of age, had tibial deformities less than 5 degrees, had proximal or middle tibial deformities, or had less than 1 year of follow-up. Patients with distal tibial nonunions with combined deformities were treated by the senior author with a hexapod device. Preoperative mechanical axis deviation and union status were assessed. Postoperative parameters evaluated included anatomic and mechanical axis determination and adequacy of union. Metrics include long alignment films with routine clinical follow-up. Assessment of deformity correction in 6 axes, restoration to union, and mechanical axis correction. Average combined preoperative deformity was greater than 17.7 degrees. Average mechanical axis was restored within 5 degrees of the desired goal in all categories except in patients with severe preoperative valgus deformities. The complex nonunion group had a 94% union rate. Average time in the hexapod was 106.7 days. Average follow-up time was 2 years. Hexapod external fixators can be used as an accurate modality to heal complex distal tibia nonunions with multifocal deformities and significant mechanical axis deviation. These difficult reconstructions are accomplished with minimum complications using these devices. Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

On the magnetization process and the associated probability in anisotropic cubic crystals

We present a theoretical method to calculate specific magnetic properties, e.g. magnetization curves, magnetic susceptibility and probability landscapes along the [100], [110] and [111] crystallographic directions of a crystal of cubic symmetry. The probability landscape displays the evolution of the most probable angular orientation of the magnetization vector, for selected temperatures and magnetic fields. Our method is based on the premises of classical statistical mechanics. The energy density, used in the partition function, is the sum of magnetic anisotropy and Zeeman energies, however no other energies e.g. elastic or magnetoelastic terms are considered in the present work. Model cubic systems of diverse anisotropies are analyzed first, and subsequently material magnetic systems of cubic symmetry; namely iron, nickel and Cox Fe100−x compounds, are discussed. We highlight a correlation between magnetization curves and the associated probability landscapes. In addition, determination of easiest axes of magnetization, using energy consideration, is done and compared with the results of the present method.

Cell lineage of timed cohorts of Tbx6-expressing cells in wild-type and Tbx6 mutant embryos.

ABSTRACTTbx6 is a T-box transcription factor with multiple roles in embryonic development as evidenced by dramatic effects on mesoderm cell fate determination, left/right axis determination, and somite segmentation in mutant mice. The expression of Tbx6 is restricted to the primitive streak and presomitic mesoderm, but some of the phenotypic features of mutants are not easily explained by this expression pattern. We have used genetically-inducible fate mapping to trace the fate of Tbx6-expressing cells in wild-type and mutant embryos to explain some of the puzzling features of the mutant phenotype. We created an inducible Tbx6-creERT2 transgenic mouse in which cre expression closely recapitulates endogenous Tbx6 expression both temporally and spatially. Using a lacZ-based Cre reporter and timed tamoxifen injections, we followed temporally overlapping cohorts of cells that had expressed Tbx6 and found contributions to virtually all mesodermally-derived embryonic structures as well as the extraembryonic allantois. Contribution to the endothelium of major blood vessels may account for the embryonic death of homozygous mutant embryos. In mutant embryos, Tbx6-creERT2-traced cells contributed to the abnormally segmented anterior somites and formed the characteristic ectopic neural tubes. Retention of cells in the mutant tail bud indicates a deficiency in migratory behavior of the mutant cells and the presence of Tbx6-creERT2-traced cells in the notochord, a node derivative provides a possible explanation for the heterotaxia seen in mutant embryos.

Formation of the germ-disc in spider embryos by a condensation-like mechanism.

BackgroundDetermination of the embryonic body axes is a crucial developmental process in all animals. The establishment of the embryonic axes of spiders has been best studied in the common-house-spider Parasteatoda tepidariorum. Here, anteroposterior (AP) polarity arises during germ disc formation; the centre of the germ-disc marks the future posterior pole, and the rim of the disc the future anterior pole of the spider embryo. The centre of the germ disc is also needed for the formation of the cumulus, a group of migratory cells needed to establish dorsoventral (DV) polarity. Thus, both body axes depend on proper germ disc formation and patterning. However, these processes have not been fully analysed at the cellular and molecular level.ResultsHere I present new techniques to stain the cell membranes/outlines in live and fixed spider embryos. I show that the germ-disc is formed from a regular and contiguous blastoderm and that co-ordinated cell shape changes, rather than migration of single cells, are required to drive germ-disc formation in P. tepidariorum embryos. Furthermore, I show that the rate of cell divisions within the embryonic and extra-embryonic region is not involved in the rapid establishment of the germ-disc. Finally, I show that the process of germ-disc formation is dependent on the initiation of zygotic transcription.ConclusionsThe presented data provide new insights in to the formation of the germ-disc in spider embryos. The establishment of the germ-disc in Parasteatoda embryos is a highly dynamic process that involves wide scale cell-shape changes. While most of the blastodermal cells become cuboidal to form the dense germ-disc, the remaining blastodermal cells stay squamous and develop into huge extra-embryonic, yolk rich cells. In addition, this study shows that the onset of zygotic transcription is needed to establish the germ-disc itself, and that the mid-blastula transition of Parasteatoda tepidariorum embryos is prior to any overt axis establishment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12983-016-0166-9) contains supplementary material, which is available to authorized users.

Identifying the position of the right atrium to align pressure transducer for CVP : Spirit level or 3D electromagnetic positioning?

The central venous pressure, CVP, is an important variable in the management of selected perioperative and intensive care cases and in clinical decision support systems, CDSS. In current routine, when measuring CVP the health care provider may use anatomical landmarks and a spirit level, SL, to adjust the pressure transducer to the level of the tricuspid valve, i.e. the phlebostatic axis. The aim of the study was to assess the agreement in the postoperative setting between the SL method and electromagnetic 3D positioning (EM). CVP was measured with patients in positions dictated by nursing routines. The staff members measured CVP using SL to position the transducer at the perceived phlebostatic level. This position was compared to coordinates based on an electromagnetic field with external sensors at anatomical landmarks and an internal sensor in the CV catheter for 3D determination of the phlebostatic axis. An electronic survey took bearing on the accepted error in measurement among colleagues at the department. There was a clinically relevant difference between the CVP measured by the staff members and the CVP based on the 3D EM positioning. The limits of agreement extended in excess of ±8mmHg and half of the measurements had deviations outside an accepted error range of ±2.5mmHg. There was a large variation in CVP measurements when assessing the agreement with the current method. This may indicate the need for improvement in accuracy, e.g. using the electromagnetic field positioning system, in association with routine monitoring and clinical decision support systems.

ANKS3 is mutated in a family with autosomal recessive laterality defect.

Laterality defects are heterogeneous groups of congenital malformations that arise from perturbed asymmetrical development of visceral organs. The central role of the motile cilia-generated nodal flow in breaking early embryonic symmetry is reflected in the large contribution of ciliary genes to the etiology of these disorders. In a consanguineous multiplex family with a laterality defect that resembles situs inversus totalis, and complex congenital heart disease, we combined autozygome and exome analysis to identify a novel homozygous variant in ANKS3. ANKS3 encodes a recently described ciliary protein with known interaction with other ciliary proteins, and deficiency of its zebrafish ortholog causes laterality defects. Consistent with the proposed role of the ANKS3 variant in the pathogenesis of the reported family's phenotype, we show that the mutant RNA failed to rescue the laterality defect in anks3 morphants compared to wild-type RNA. Furthermore, we describe a new mutant anks3 line that also displays laterality defect in the homozygous state. Our study suggests a role for ANKS3 in right-left axis determination in humans.

Determination of the reference symmetry axis of a generally anisotropic medium which is approximately transversely isotropic

Abstract For a given stiffness tensor (tensor of elastic moduli) of a generally anisotropic medium, we estimate to what extent the medium is transversely isotropic (uniaxial) and determine the direction of its reference symmetry axis expressed in terms of the unit reference symmetry vector. If the medium is exactly transversely isotropic (exactly uniaxial), we obtain the direction of its symmetry axis. We can also calculate the first–order and second–order spatial derivatives of the reference symmetry vector which may be useful in tracing the reference rays for the coupling ray theory. The proposed method is tested using various transversely isotropic (uniaxial) and approximately transversely isotropic (approximately uniaxial) media.

Patronin Takes a Shot at Polarity

To specify the anterior-posterior axis of Drosophila embryos, noncentrosomal microtubules grow out from cortical regions of the oocyte and help transport axis determinants. In this issue of Developmental Cell, Nashchekin etal. (2016) report a Shot- and Patronin-dependent mechanism by which the oocyte cortex produces polarized microtubule arrays.

Patronin/Shot Cortical Foci Assemble the Noncentrosomal Microtubule Array that Specifies the Drosophila Anterior-Posterior Axis

SummaryNoncentrosomal microtubules play an important role in polarizing differentiated cells, but little is known about how these microtubules are organized. Here we identify the spectraplakin, Short stop (Shot), as the cortical anchor for noncentrosomal microtubule organizing centers (ncMTOCs) in the Drosophila oocyte. Shot interacts with the cortex through its actin-binding domain and recruits the microtubule minus-end-binding protein, Patronin, to form cortical ncMTOCs. Shot/Patronin foci do not co-localize with γ-tubulin, suggesting that they do not nucleate new microtubules. Instead, they capture and stabilize existing microtubule minus ends, which then template new microtubule growth. Shot/Patronin foci are excluded from the oocyte posterior by the Par-1 polarity kinase to generate the polarized microtubule network that localizes axis determinants. Both proteins also accumulate apically in epithelial cells, where they are required for the formation of apical-basal microtubule arrays. Thus, Shot/Patronin ncMTOCs may provide a general mechanism for organizing noncentrosomal microtubules in differentiated cells.

Cilia gene mutations cause atrioventricular septal defects by multiple mechanisms.

Atrioventricular septal defects (AVSDs) are a common severe form of congenital heart disease (CHD). In this study we identified deleterious non-synonymous mutations in two cilia genes, Dnah11 and Mks1, in independent N-ethyl-N-nitrosourea-induced mouse mutant lines with heritable recessive AVSDs by whole-exome sequencing. Cilia are required for left/right body axis determination and second heart field (SHF) Hedgehog (Hh) signaling, and we find that cilia mutations affect these requirements differentially. Dnah11avc4 did not disrupt SHF Hh signaling and caused AVSDs only concurrently with heterotaxy, a left/right axis abnormality. In contrast, Mks1avc6 disrupted SHF Hh signaling and caused AVSDs without heterotaxy. We performed unbiased whole-genome SHF transcriptional profiling and found that cilia motility genes were not expressed in the SHF whereas cilia structural and signaling genes were highly expressed. SHF cilia gene expression predicted the phenotypic concordance between AVSDs and heterotaxy in mice and humans with cilia gene mutations. A two-step model of cilia action accurately predicted the AVSD/heterotaxyu phenotypic expression pattern caused by cilia gene mutations. We speculate that cilia gene mutations contribute to both syndromic and non-syndromic AVSDs in humans and provide a model that predicts the phenotypic consequences of specific cilia gene mutations.

Determination of the optical axis in Z-cut uniaxial crystals by a polarization-based method

A polarization method is proposed for determination of the orientation of the optical axis in a Z-cut uniaxial crystal. The applicability of the method for controlling the deflection angles of the optical axis from the normal to the crystal surface ~0.01° was shown using the example of lithium niobate crystal with a thickness of 514 μm. Peculiarities of the practical implementation of this method and approaches for increasing its accuracy and throughput are considered.

SU‐D‐201‐06: Remote Dosmetric Auditing of VMAT Deliveries for Clinical Trials Using EPID

Purpose:To develop a method for remote dosimetric auditing the delivery of VMAT using EPID which allows for simple, inexpensive and time efficient dosimetric credentialing for clinical trials.Methods:Remote centers are provided with CT datasets and planning guidelines to produce VMAT plans for a head and neck and a post‐prostatectomy treatment. Plans are transferred in the planning system to two virtual water equivalent phantoms, one flat and one cylindrical. Cine images are acquired during VMAT delivery to the EPID in air with gantry angle recorded in image headers. Centers also deliver provided calibration plans to enable EPID signal to dose conversion, determination of the central axis, and correction of EPID sag prior to analysis. EPID images and planned doses are sent to the central site. EPID cine images are converted to dose in the virtual phantoms using an established backprojection method (King et al., Med.Phys. 2012) with EPID backscatter correction. Individual arcs (with gantry angles collapsed to zero) are evaluated at 10 cm depth in the flat phantom using 2D gamma, and total doses are evaluated in the cylindrical phantom using 3D gamma. Results are reported for criteria of 3%,3mm, 3%,2mm and 2%,2mm for all points greater than 10% of global maximum.Results:The pilot study for Varian centers has commenced, and three centers have been audited for head and neck plans and two for post‐prostatectomy plans to date. The mean pass rate for arc‐by‐arc 2D analysis at 3%,3mm is 99.5% and for 3D analysis is 95.8%. A method for Elekta linacs using an inclinometer for gantry angle information is under development.Conclusion:Preliminary results for this new method are promising. The method takes advantage of EPID equipment available at most centers and clinically established software to provide a feasible, low cost solution to credentialing centers for clinical trials.Funding has been provided from Calvary Mater Newcastle Department of Radiation Oncology, TROG Cancer Research and the University of Newcastle. Kimberley Legge is the recipient of an Australian Postgraduate Award. Narges Miri is a recipient of a University of Newcastle postgraduate scholarship.

SLC7 family transporters control the establishment of left-right asymmetry during organogenesis in medaka by activating mTOR signaling

The precise government of the left-right (LR) specification of an organ is an essential aspect of its morphogenesis. Multiple signaling cascades have been implicated in the establishment of vertebrate LR asymmetry. Recently, mTOR signaling was found to critically regulate the development of LR asymmetry in zebrafish. However, the upstream factor(s) that activate mTOR signaling in the context of LR specification are as yet unknown. In this study, we identify the SLC7 amino acid transporters Slc7a7 and Slc7a8 as novel regulators of LR asymmetry development in the small fish medaka. Knockdown of Slc7a7 and/or Slc7a8 in medaka embryos disrupted LR organ asymmetries. Depletion of Slc7a7 hindered left-sided expression of the southpaw (spaw) gene, which is responsible for LR axis determination. Work at the cellular level revealed that Slc7a7 coordinates ciliogenesis in the epithelium of Kupffer's vesicle and thereby the generation of the nodal fluid flow required for LR asymmetry. Interestingly, knockdown of Slc7a7 depressed mTOR signaling activity in medaka embryos. Treatment with rapamycin, an inhibitor of mTOR signaling, together with Slc7a7 knockdown synergistically perturbed spaw expression, indicating an interaction between Slc7a7 and mTOR signaling affecting gene expression required for LR specification. Taken together, our results demonstrate that Slc7a7 governs the regulation of LR asymmetry development via the activation of mTOR signaling.

Cell biology: Calcium contradictions in cilia.

Organelles called primary cilia that protrude from cells have been thought to sense the surrounding environment through calcium-channel proteins that respond to force. Two scientists discuss the implications for developmental biology and kidney disease of a study that challenges this hypothesis. See Letter p.656 The primary cilium is a single organelle protruding from the cell surface. Given this antenna-like structure, its function has been assumed to be sensing of the surrounding environment. Specifically, the main assumption has been that calcium-permeable ion channels within the cilium sense and respond to mechanical forces — a hypothesis that has been used to explain a large range of biological responses, from control of left–right axis determination during embryonic development to adult progression of polycystic kidney disease and some cancers. David Clapham and colleagues, however, present evidence that refutes this hypothesis. They measured Ca2+ signals in primary cilia following a mechanical force in mice engineered to express a sensor protein that fluoresces in response to Ca2+. They find no evidence of force-driven Ca2+ influx in primary cilia and conclude that if mechanosensation originates in primary cilia, it is not via calcium signalling.

Primary cilia are not calcium-responsive mechanosensors.

Primary cilia are solitary, generally non-motile, hair-like protrusions that extend from the surface of cells between cell divisions. Their antenna-like structure leads naturally to the assumption that they sense the surrounding environment, the most common hypothesis being sensation of mechanical force through calcium-permeable ion channels within the cilium1. This Ca2+- Responsive MechanoSensor (CaRMS) hypothesis for primary cilia has been invoked to explain a large range of biological responses, from control of left-right axis determination in embryonic development to adult progression of polycystic kidney disease and some cancers2,3. Here, we report the complete lack of mechanically induced calcium increases in primary cilia, in tissues upon which this hypothesis has been based. First, we developed a transgenic mouse, Arl13b-mCherry-GECO1.2, expressing a ratiometric genetically encoded calcium indicator (GECI) in all primary cilia. We then measured responses to flow in primary cilia of cultured kidney epithelial cells, kidney thick ascending tubules, crown cells of the embryonic node, kinocilia of inner ear hair cells, and several cell lines. Cilia-specific Ca2+ influxes were not observed in physiological or even highly supraphysiological levels of fluid flow. We conclude that mechanosensation, if it originates in primary cilia, is not via calcium signaling.

A Comparative Study of Estimation of lower Limb alignment by Physical Examination versus Lower Leg Radiography Measurements

Aims and Objectives:Alignment of the lower limb is a key parameter in the diagnostics and treatment concepts of osteoarthritis, in the treatment of cartilage defects and in ligamentous instabilities. The purpose of this study was to evaluate the relevance of the physical examination in direct comparison to radiographic examination in determination of the mechanical axis of the lower extremities.Materials and Methods:A total of 92 Patient with different pathologies of the knee joint have been included. (Male: n=60, Female: n=32; overall BMI 26,7 kg/m2). The physical Examination of the mechanical axis was determined by two independent examiners (orthopaedic surgeons) by clinical estimation using a goniometer. The radiographic measurement of the mechanical axis was performed on full weight-bearing whole Radiographs accordning to the recommendations given by Pauwels and Moreland. All variables failed test for normal distribution, so that Spearman’s ratio and for inter observer reliability Kendall’s tau was performed. For all statistical tests, SPSS was used for statistics. P-values < 0.05 were considered statistical significant.Results:The physical examination has a relevant variance to radiographic determination of the lower limb axis. In comparison to the control (radiographic evaluation), a misinterpretation of 3 degrees or more has been found in 30 % of the physical measurements in the overall study group, while in the subgroup of obese patients (BMI > 30kg/m2) the number of miscalculations increased to 44 %(p<0,05). In addition, a moderate correlation was found between the two independent investigators (r=0,61; p<0,001). Resulting in a low inter observer reliability. Higher degree of malalignment was found to correlate with inferior correlation factors between clinical and radiographic evaluation, while best correlation factors were found in the subgroup of patients without significant malalignment (Percentage of Misinterpretation 5 %). This percentage was significantly higher in valgus (40%) and varus (36%) deformities.Conclusion:In conclusion the physical examination has a large insecurity compared with the lower leg radiography. Reliability depends on BMI and failure probability increases in the subgroup of obese patients. With regard to the results of the present study and against the background of the great importance of lower limb alignment in the context of surgical treatments for cartilage defects, osteoarthritis and chronic ligamentous insufficiency, the indication for radiographic evaluation of alignment of the lower limb using full weight bearing AP radiographs should be made generously.

The handedness-associated PCSK6 locus spans an intronic promoter regulating novel transcripts.

We recently reported the association of the PCSK6 gene with handedness through a quantitative genome-wide association study (GWAS; P < 0.5 × 10−8) for a relative hand skill measure in individuals with dyslexia. PCSK6 activates Nodal, a morphogen involved in regulating left–right body axis determination. Therefore, the GWAS data suggest that the biology underlying the patterning of structural asymmetries may also contribute to behavioural laterality, e.g. handedness. The association is further supported by an independent study reporting a variable number tandem repeat (VNTR) within the same PCSK6 locus to be associated with degree of handedness in a general population cohort. Here, we have conducted a functional analysis of the PCSK6 locus combining further genetic analysis, in silico predictions and molecular assays. We have shown that the previous GWAS signal was not tagging a VNTR effect, suggesting that the two markers have independent effects. We demonstrated experimentally that one of the top GWAS-associated markers, rs11855145, directly alters the binding site for a nuclear factor. Furthermore, we have shown that the predicted regulatory region adjacent to rs11855415 acts as a bidirectional promoter controlling the expression of novel RNA transcripts. These include both an antisense long non-coding RNA (lncRNA) and a short PCSK6 isoform predicted to be coding. This is the first molecular characterization of a handedness-associated locus that supports the role of common variants in non-coding sequences in influencing complex phenotypes through gene expression regulation.

Postoperative Increased Loading Leads to an Alteration in the Radiological Mechanical Axis After Total Knee Arthroplasty

BackgroundStanding long-leg radiographs allow assessment of the mechanical axis in the frontal plane before and after total knee arthroplasty (TKA). An alteration in loading, and hence in the forces acting on the knee joint, occurs postoperatively. We therefore postulated that the mechanical axis measured in the long-leg standing radiograph would change within the first year after TKA. MethodsStanding long-leg radiographs of 156 patients were performed 7 days, 3 months, and 12 months after TKA with determination of mechanical axis of the lower limb. ResultsSeven days after surgery, the mechanical axis amounted 0.8° ± 1.7° valgus. Three months after the operation, at 1.3° ± 1.3° varus, it was significantly different (P < .001) from the primary measurement. No further alteration in the mechanical axis occurred during the first year after TKA. This difference was even more pronounced (P < .001) in patients with a postoperative lack of complete extension. Seven days after surgery, they had a valgus axis deviation of 1.6° ± 1.6°; after 3 months, the measurement amounted 1.2° ± 1.3° varus. ConclusionMeasured by a standing long-leg radiograph, the frontal mechanical axis after TKA changes over time. The predictive power of a standing long-leg radiograph in the first week after surgery is limited because limb loading is altered because of pain and is therefore nonphysiological. The actual mechanical axis resulting after TKA can only be assessed in a standing long-leg radiograph at physiological loading.

Assessment of housekeeping genes as internal references in quantitative expression analysis during early development of oyster.

The early development of mollusks exhibits important characteristics from the developmental and evolutionary perspective. With the increasing number of genome-wide studies, accurate analyses of quantitative gene expression during development are impeded by the lack of validated reference genes. To improve the situation, in this study, we analyzed the expression stability of seven candidate housekeeping genes during early development of the Pacific oyster Crassostrea gigas: actin, glyceraldehyde-3-phosphate dehydrogenase (gapdh), α subunit of elongation factor 1 (elf1α), adp-ribosylation factor 1 (arf1), heterogeneous nuclear ribonucleoprotein q, ubiquitin-conjugating enzyme e2d2 and ribosomal protein s18. We focused on 11 stages from oocyte to D-veliger, which include crucial developmental processes such as axis determination, gastrulation and shell formation. Gene expression stabilities were assessed with the three commonly used programs geNorm, NormFinder and BestKeeper. Although the results obtained with the three programs varied to some extent, in general, arf1, elf1α and gapdh were highly ranked and actin was poorly ranked. This analysis also indicated that multiple genes should be used for normalization, and we concluded that arf1-elf1α-gapdh should be used as internal references. The findings of this study will help researchers to obtain accurate results in future quantitative gene expression analysis of development in bivalve mollusks.

Visualization and Manipulation of Cilia and Intraciliary Calcium in the Zebrafish Left-Right Organizer.

Cilia play a key role in the determination of the left-right axis in vertebrates by generating and sensing flow of extraembryonic fluid at the left-right organizer (LRO). Perception of cilia-directed flow triggers a calcium signaling cascade which originates within the cilium itself and then is relayed into the surrounding mesendodermal tissue, thereby directing organ situs via the nodal pathway. Two types of cilia, motile and immotile, function simultaneously to coordinate and direct asymmetric intraciliary calcium signaling cues in the LRO. Here, we describe tools, reagents, and methodologies for the visualization and manipulation of both cilia types as well as intraciliary calcium signaling in the LRO of zebrafish.