Receptor Tyrosine Kinases ror1/2 and ryk Are Co-expressed with Multiple Wnt Signaling Components During Early Development of Sea Urchin Embryos.

AbstractA combination of receptors, co-receptors, and secreted Wnt modulators form protein complexes at the cell surface that activate one or more of the three different Wnt signaling pathways (Wnt/ß-catenin, Wnt/JNK, and Wnt/Ca2+). Two or more of these pathways are often active in the same cellular territories, forming Wnt signaling networks; however, the molecular mechanisms necessary to integrate information from these pathways in these situations are unclear in any in vivo model system. Recent studies have implicated two Wnt binding receptor tyrosine kinases, receptor tyrosine kinase-like orphan receptor (Ror) and related-to-receptor tyrosine kinase (Ryk), in the regulation of canonical and non-canonical Wnt signaling pathways, depending on the context; however, the spatiotemporal expression of these genes in relation to Wnt signaling components has not been well characterized in most deuterostome model systems. Here we use a combination of phylogenetic and spatiotemporal gene expression analyses to characterize Ror and Ryk orthologs in sea urchin embryos. Our phylogenetic analysis indicates that both ror1/2 and ryk originated as single genes from the metazoan ancestor. Expression analyses indicate that ror1/2 and ryk are expressed in the same domains of many Wnt ligands and Frizzled receptors essential for the specification and patterning of germ layers along the early anterior-posterior axis. In addition, both genes are co-expressed with Wnt signaling components in the gut, ventral ectoderm, and anterior neuroectoderm territories later in development. Together, our results indicate that Ror and Ryk have a complex evolutionary history and that their spatiotemporal expression suggests that they could contribute to the complexity of Wnt signaling in early sea urchin embryogenesis.

Outcome of temporary tracheostomy tube-placement following surgery for brachycephalic obstructive airway syndrome in 42 dogs.

OBJECTIVES: To describe the use, complications and outcome of temporary tracheostomy tube placement as part of the management of acute upper airway obstruction in the postoperative period following multi-level airway surgery in patients with brachycephalic obstructive airway syndrome. MATERIALS AND METHODS: Retrospective review of records of dogs surgically treated for brachycephalic obstructive airway syndrome that had a temporary tracheostomy tube placed in the postoperative period. RESULTS: Forty-two dogs were included. Median duration of temporary tracheostomy tube placement was 2 days (range 1 to 7). The major complication rate was 83.3%, minor complication rate was 71.4%, resulting in an overall postoperative complication rate of 95.2%. The most common postoperative complications were tracheostomy tube obstruction (32/42), cough (25/42) and tracheostomy tube dislodgement (16/42). Temporary tracheostomy tube management was classified as successful in 97.6%. Dyspnoea was the most common clinical sign in the short-term postoperative follow-up period, while dyspnoea and increased upper respiratory tract noise were the most common clinical sign in the long term. The median duration of follow-up was 251 days. CLINICAL SIGNIFICANCE: In an appropriate clinical setting, placement of temporary tracheostomy tubes following multi-level airway surgery for brachycephalic obstructive airway syndrome is a useful strategy to manage postoperative airway obstruction, carrying a low mortality rate, and with a complication rate similar to that found in previous reports.

An in-vitro investigation into the efficacy of disinfectants used in the duck industry against Salmonella.

In 2010, 81 confirmed cases of Salmonella Typhimurium DT8 were reported across England and Northern Ireland - an increase of 26% from 2009 and 41% since 2008. Five cases were hospitalized and one death reported, with a strong association found between cases and the consumption of duck eggs. Once present on farms, Salmonella may become persistent and can survive for long periods of time in residual organic matter, increasing risk of infection for follow-on flocks if cleaning and disinfection is not carried out effectively. The aim of this study was to investigate the efficacy of a range of disinfectants used by the duck industry against Salmonella using laboratory models. Sixteen products were selected from seven chemical groups and the Minimum Inhibitory Concentration and Minimum Bactericidal Concentrations determined. Each product was also tested at the recommended general orders (GO) concentration using a faecal suspension model to mimic boot dips and a surface contamination model to simulate contaminated building fabric and equipment. In the faecal suspension model, all products were effective at 2 × GO concentration, and activity was more inconsistent at GO concentration. At 0.5 × GO concentration, iodine-based and quaternary-ammonium-compound-based products were significantly less effective than products within other chemical groups (P < 0.001). Glutaraldehyde-based products were significantly more effective than the other products in the surface contamination tests (P < 0.001). Chlorocresol-based products were found to be most effective for use in boot dips and aldehyde-based products for surface disinfection, although there was variability between products within a chemical group.

Endothelial CXCR7 regulates breast cancer metastasis.

Atypical chemokine receptor CXCR7 (ACKR3) functions as a scavenger receptor for chemokine CXCL12, a molecule that promotes multiple steps in tumor growth and metastasis in breast cancer and multiple other malignancies. Although normal vascular endothelium expresses low levels of CXCR7, marked upregulation of CXCR7 occurs in tumor vasculature in breast cancer and other tumors. To investigate effects of endothelial CXCR7 in breast cancer, we conditionally deleted this receptor from vascular endothelium of adult mice, generating CXCR7(ΔEND/ΔEND) animals. CXCR7(ΔEND/ΔEND) mice appeared phenotypically normal, although these animals exhibited a modest 35±3% increase in plasma CXCL12 as compared with control. Using two different syngeneic, orthotopic tumor implant models of breast cancer, we discovered that CXCR7(ΔEND/ΔEND) mice had significantly greater local recurrence of cancer following resection, elevated numbers of circulating tumor cells and more spontaneous metastases. CXCR7(ΔEND/ΔEND) mice also showed greater experimental metastases following intracardiac injection of cancer cells. These results establish that endothelial CXCR7 limits breast cancer metastasis at multiple steps in the metastatic cascade, advancing understanding of CXCL12 pathways in tumor environments and informing ongoing drug development targeting CXCR7 in cancer.

CXCL12-γ in primary tumors drives breast cancer metastasis.

Compelling evidence shows that chemokine C-X-C motif chemokine ligand 12 (CXCL12) drives metastasis in multiple malignancies. Similar to other key cytokines in cancer, CXCL12 exists as several isoforms with distinct biophysical properties that may alter signaling and functional outputs. However, effects of CXCL12 isoforms in cancer remain unknown. CXCL12-α, -ß and -γ showed cell-type-specific differences in activating signaling through G protein-dependent pathways in cell-based assays, while CXCL12-γ had greatest effects on recruitment of the adapter protein ß-arrestin 2. CXCL12-ß and -γ also stimulated endothelial tube formation to a greater extent than CXCL12-α. To investigate the effects of CXCL12 isoforms on tumor growth and metastasis, we used a mouse xenograft model of metastatic human breast cancer combining CXCR4+ breast cancer cells and mammary fibroblasts secreting an isoform of CXCL12. Altough all CXCL12 isoforms produced comparable growth of mammary tumors, CXCL12-γ significantly increased metastasis to bone marrow and other sites. Breast cancer cells originating from tumors with CXCL12-γ fibroblasts upregulated RANKL (receptor activator of nuclear factor-κB ligand), contributing to bone marrow tropism of metastatic cancer cells. CXCL12-γ was expressed in metastatic tissues in mice, and we also detected CXCL12-γ in malignant pleural effusions from patients with breast cancer. In our mouse model, mammary fibroblasts disseminated to sites of breast cancer metastases, providing another mechanism to increase levels of CXCL12 in metastatic environments. These studies identify CXCL12-γ as a potent pro-metastatic molecule with important implications for cancer biology and effective therapeutic targeting of CXCL12 pathways.

Analysis of 16S rDNA sequences from pathogenic Leptospira serovars and use of single nucleotide polymorphisms for rapid speciation by D-HPLC.

Leptospira have a worldwide distribution and include important zoonotic pathogens yet diagnosis and differentiation still tend to rely on traditional bacteriological and serological approaches. In this study a 1.3 kb fragment of the rrs gene (16S rDNA) was sequenced from a panel of 22 control strains, representing serovars within the pathogenic species Leptospira interrogans, Leptospiraborgpetersenii, and Leptospirakirschneri, to identify single nucleotide polymorphisms (SNPs). These were identified in the 5' variable region of the 16S sequence and a 181 bp PCR fragment encompassing this region was used for speciation by Denaturing High Performance Liquid Chromatography (D-HPLC). This method was applied to eleven additional species, representing pathogenic, non-pathogenic and intermediate species and was demonstrated to rapidly differentiate all but 2 of the non-pathogenic Leptospira species. The method was applied successfully to infected tissues from field samples proving its value for diagnosing leptospiral infections found in animals in the UK.

The EuroPhysiome, STEP and a roadmap for the virtual physiological human.

Biomedical science and its allied disciplines are entering a new era in which computational methods and technologies are poised to play a prevalent role in supporting collaborative investigation of the human body. Within Europe, this has its focus in the virtual physiological human (VPH), which is an evolving entity that has emerged from the EuroPhysiome initiative and the strategy for the EuroPhysiome (STEP) consortium. The VPH is intended to be a solution to common infrastructure needs for physiome projects across the globe, providing a unifying architecture that facilitates integration and prediction, ultimately creating a framework capable of describing Homo sapiens in silico. The routine reliance of the biomedical industry, biomedical research and clinical practice on information technology (IT) highlights the importance of a tailor-made and robust IT infrastructure, but numerous challenges need to be addressed if the VPH is to become a mature technological reality. Appropriate investment will reap considerable rewards, since it is anticipated that the VPH will influence all sectors of society, with implications predominantly for improved healthcare, improved competitiveness in industry and greater understanding of (patho)physiological processes. This paper considers issues pertinent to the development of the VPH, highlighted by the work of the STEP consortium.

Analytical description of dose profile behaviour in Gamma Knife radiosurgery.

Stereotactic Gamma Knife radiosurgery utilizes ionizing beams from (60)Co sources and relies on a combination of collimator sizes, weighting, etc to generate a high-dose region that is conformal with a designated target volume. Dose computation is typically performed by computer, but in this paper, single collimator dose profile behaviour is modelled analytically and then extended to accommodate multiple collimators of different weights with co-located isocentres. The dose profile from a single helmet is derived from a top-hat beam profile approximation and an idealized symmetric distribution of sources is used to represent the 201 sources within a collimating helmet. The results from the analysis are validated by an independent numerical model and also compared with those obtained by other groups using numerical and experimental methods. With respect to multiple collimators, the relationship between the size (full width half maximum) of the irradiated volume and relative collimator weighting is also examined using the simple analytical model. The simplicity of the mathematics clarifies the relationship between beam profile, dose profile and multiple collimator behaviour, and provides data that compare favourably with published literature.

Radiosurgery planning supported by the GEMSS grid.

GEMSS (Grid Enabled Medical Simulation Services IST-2001-37153) is an EU project funded to provide a test bed for Grid-enabled health applications. Its purpose is evaluation of Grid computing in the health sector. The health context imposes particular constraints on Grid infrastructure design, and it is this that has driven the feature set of the middleware. In addition to security, the time critical nature of health applications is accommodated by a Quality of Service component, and support for a well defined business model is also included. This paper documents experience of a GEMSS compliant radiosurgery application running within the Medical Physics department at the Royal Hallamshire Hospital in the UK. An outline of the Grid-enabled RAPT radiosurgery application is presented and preliminary experience of its use in the hospital environment is reported. The performance of the software is compared against GammaPlan (an industry standard) and advantages/disadvantages are highlighted. The RAPT software relies on features of the GEMSS middleware that are integral to the success of this application, and together they provide a glimpse of an enabling technology that can impact upon patient management in the 21st century.

A practical demonstration of improved technique factors in paediatric fluoroscopy.

Dose incurred with fluoroscopic procedures accounts for a significant proportion of medically induced diagnostic exposure. Children are particularly vulnerable and it is therefore important to minimize exposure where practicable. A recent theoretical study has highlighted the potential for X-ray equipment to produce significant dose savings during paediatric fluoroscopy without incurring loss of diagnostic image quality. This is achieved by hardening the beam with additional copper (Cu) filtration (approximately 0.2 mm Cu) and biasing exposure factors towards low tube potential, high tube current output. In practice, this method will have limited applicability because the high powered and programmable generator characteristics required are not commonly available in installations used for paediatric imaging. However, we describe a simple experiment in which our clinical equipment was modified to approximate desired low dose performance by altering the filtration and automatic exposure control characteristics of ordinary clinical equipment in the Sheffield Children's Hospital. This enabled us to obtain significant savings in dose. We performed a comparative study (normal dose vs low dose) using water phantoms to simulate patient attenuation in the age range 0-15 years. The Leeds N2 contrast sensitivity phantom was used to provide a measure of image quality. Dosimetric measurements recorded up to 40% reduction in dose rate with only marginal loss of image quality when 0.1-0.2 mm Cu filtration was used with the modified settings. This is a strong indication that significant dose reduction is achievable on routine clinical equipment without compromising image quality. Such simple and cost effective methods of dose reduction should be considered for wider implementation.

Stable periodic vortex shedding studied using computational fluid dynamics, laser sheet flow visualization, and MR imaging.

Recirculating and detached flow patterns close to the carotid bifurcation are thought to play an important role in the development of carotid stenoses by promoting atherosclerosis. The aim of this study was to investigate a flow regime with strong transient characteristics, including vortex shedding and transport to develop methodologies appropriate to the analysis of carotid stenoses. The existence of a regular periodic vortex street behind a cylindrical flow obstruction was predicted and analysed in detail by Theodore van Karman in the early 20th century. This model was chosen in our study for both ease of phantom construction and of theoretical modelling using finite element computational fluid dynamics (CFD). The results of the theoretical calculations have been compared with two methods of flow visualization-laser sheet imaging and real-time echo planar magnitude MR imaging. Flow was investigated over a range of Reynold's number from 40 through 400 through which vortex shedding is predicted. Good overall agreement was obtained between the theoretical (16 mm-CFD) and experimental (16+/-2 mm-Laser, 17+/-2 mm-MRI) estimates of the Karman Vortex street wavelength for a Reynolds number of 200.

SOCS1 is a critical inhibitor of interferon gamma signaling and prevents the potentially fatal neonatal actions of this cytokine.

Mice lacking suppressor of cytokine signaling-1 (SOCS1) develop a complex fatal neonatal disease. In this study, SOCS1-/- mice were shown to exhibit excessive responses typical of those induced by interferon gamma (IFNgamma), were hyperresponsive to viral infection, and yielded macrophages with an enhanced IFNgamma-dependent capacity to kill L. major parasites. The complex disease in SOCS1-/- mice was prevented by administration of anti-IFNgamma antibodies and did not occur in SOCS1-/- mice also lacking the IFNgamma gene. Although IFNgamma is essential for resistance to a variety of infections, the potential toxic action of IFNgamma, particularly in neonatal mice, appears to require regulation. Our data indicate that SOCS1 is a key modulator of IFNgamma action, allowing the protective effects of this cytokine to occur without the risk of associated pathological responses.

The effects of cardioplegic arrest and reperfusion on the microvasculature of the heart.

OBJECTIVES: Despite laboratory evidence of leucocyte involvement in reperfusion injury, cardiac surgical clinical trials do not support the therapeutic effectiveness of leucocyte filtration. Furthermore, the direct effects of crystalloid cardioplegia and reperfusion on the capillaries of the heart have yet to be elucidated. We tested the effects of cardioplegic arrest and reperfusion both with and without leucocyte depletion, in a model of cardiopulmonary bypass that mimics clinical cardiac surgical conditions. METHODS: Four groups of Landrace pigs were studied. Group A (n = 6) underwent 30 min of hypothermic (28 degrees C) cardiopulmonary bypass. Groups B (n = 6), C (n = 6) and D (n = 6) also had 90 min of cardioplegic arrest. Group C was then reperfused with whole blood, while Group D was reperfused with leucocyte-depleted blood. Microvascular methylmethacrylate corrosion casts were made at the end of the experimental period. Myocardial vascular anatomy was defined by electron microscopy and capillary abundance derived from this and from the weight of casts from representative areas. Leucocyte deposition was assessed using radioisotope-labelled leucocytes. Ischaemic damage to tissues was graded according to light and electron microscopic findings. RESULTS: In Group A the mean (+/- S.D.) vascular cast weight/volume of myocardium (density) was 125 +/- 9 mg/mm3. After cardioplegic arrest (Group B), it fell to 74 +/- 7 mg/mm3 (P < 0.0001) due to absence of capillaries, although arterioles, venules and non-nutritive bypass vessels remained patent. Following reperfusion with whole blood (Group C), capillary numbers partially recovered but luminal diameters were reduced with a cast density of 94 +/- 5 mg/mm3 (P < 0.0001 versus Group A and B). Leucocyte-depleted (87-92%) reperfusion in Group D did not affect cast density (90 +/- 3 mg/mm3; P = 0.17). Coronary vascular resistances in Groups C and D rose slightly, but not significantly, during reperfusion. CONCLUSIONS: Following cardioplegic arrest, microvascular changes are marked. These changes are partially reversed by 30 min reperfusion. Leucocyte depletion does not ameliorate these effects in this model.

Laser profiling: a technique for the study of prosthetic heart valve leaflet motion.

A detailed understanding of the stresses and strains developed in functioning flexible-leaflet valves is necessary if a durable, non-thrombogenic heart valve replacement is to be realized. A new experimental tool, laser profiling, is presented for the study of flexible-leaflet heart valve dynamics. Profiles of moving leaflet surfaces are obtained by projecting parallel sheets of laser light onto valve leaflets as the valves open and close in a mock circulatory loop. Two versions of laser profiling have been developed. In two-dimensional mode multiple profiles are generated on a fixed plane in space but at discrete intervals in time, whereas in three-dimensional mode multiple profiles are generated across the leaflet surface at (effectively) a single instant in time. Highlighted leaflet profiles are captured by camera and transferred to an image processing system for analysis. A simple algorithm permits digitized profiles to be reconstructed within a computer-aided design software package, providing detailed visualization and quantification of valve motion. Extensive validation studies have been performed using the Medtronic-Hall mechanical prosthetic heart valve. Laser profiling enables computer reconstruction of the rigid occluder to an accuracy of +/- 200 microns from a 0.7 ms exposure taken during the period at which the occluder moves with greatest velocity. The technique has been applied to investigate the leaflet dynamics of a bovine pericardial heart valve prosthesis.

Comparisons between three dual-energy X-ray absorptiometers used for measuring spine and femur.

Lunar, Hologic and Norland dual-energy X-ray absorptiometry scanners have been compared for measurements of spine and femur bone density. Precision was not greatly different in realistic phantoms and volunteer subjects. Most clinical therapeutic trials are concerned with measuring changes of bone mineral, and interchangeability in this context was examined using phantoms of spine and femur in which changes of bone mineral density (BMD) were simulated. With each instrument the measured changes were closely linear. For the spine the biggest difference of slope between instruments was 15%. For the femur, in all areas of interest, the differences of slope were less than 10%. It is concluded that the three instruments can be satisfactorily used in multicentre clinical trials to investigate changes in bone mineral. 12 volunteers were measured with each scanner. There were significant mean differences between each pair of instruments, suggesting different calibration criteria. More importantly, those mean differences had appreciable standard deviations (SDs), in proportional terms from 3% to 10%. When the measurements were related to reference ranges and expressed in terms of age-matched normal values the mean biases disappeared, but the SDs did not improve. Results from different manufacturers' apparatus are not interchangeable for studying individual patients. Measurements from the phantoms were used to cross-calibrate the scanners. Those from the variable spine phantom predicted the in vivo ratio within 4%, but this was no better than measurements of the unmodified phantom alone. Results using the European Spine Phantom were less satisfactory. No phantom provided an adequate cross-calibration for femur measurements.

Dehydration: a model for (low-temperature) argon laser tissue bonding.

Despite considerable investigation, the mechanism of laser assisted vascular anastomosis remains unknown. Indications suggest that bonding is the result of thermal action, particularly the thermal denaturation of tissue proteins. However, our own work has led us to conclude that dehydration is an important factor. Hence, we have proposed that laser anastomosis is the result of dehydration at the apposed tissue faces, induced by laser irradiation. This was investigated by comparing the properties of bonds created by dehydration with those created by laser. The bonds were created using parameters consistent with laser anastomoses created in vivo. Results revealed that anastomoses created by dehydration were equivalent to those created by laser, with little difference in strength, histology or response to rehydration. The only significant difference (p < 0.02) was mean bond strength created at temperatures above the denaturation temperature of the tissue (548 g cm(-2) by laser, 994 g cm(-2) by dehydration). Given the similarity of bonds created by the two methods, we conclude that the same mechanism (i.e. dehydration) is likely to be responsible for bonding in both cases and therefore that argon laser bonding is mediated by dehydration.

Association of severe haemophilia A with osteoporosis: a densitometric and biochemical study.

Following a femoral neck fracture and vertebral compression fractures in two patients with severe haemophilia A, bone density and turnover were measured in 19 males with severe haemophilia A (all HIV negative, 18/19 hepatitis C antibody positive) and in 19 age/sex matched controls. Bone density at the lumbar spine (L2-4), measured by dual energy X-ray absorptiometry, was significantly lower in the haemophiliac patients (HPs) at (mean +/- SEM) 1.109 +/- 0.042 g/cm2 vs. 1.234 +/- 0.027 in controls; p = 0.018. Femoral neck density was also lower at 0.877 +/- 0.034 g/cm2 (HPs) vs. 1.067 +/- 0.032; p < 0.0005. No significant differences were evident between the groups for serum calcium, parathyroid hormone, luteinizing hormone, follicle-stimulating hormone or 1,25 dihydroxyvitamin D3, nor for fasting urinary hydroxyproline, pyridinoline or deoxypyridinoline excretion. Serum total alkaline phosphatases was elevated in HPs at 200 +/- 10 U/l vs. 158 +/- 8; p = 0.004. Similarly, gamma-glutamyl transferase was elevated at 42 +/- 7 U/l (HPs) vs. 20 +/- 2; p = 0.007. Serum total testosterone and sex-hormone-binding globulin (SHBG) were higher in HPs at 26 +/- 2.5 nmol/l vs. 17.4 +/- 1.6 (p = 0.009) and 56 +/- 6 nmol/l vs. 27 +/- 3 (p = 0.0005), respectively. Free androgen index, however, was lower in HPs at 44 +/- 5 vs 69 +/- 7; p = 0.008. These results suggest significant osteopenia associated with haemophilia A. This may be partly due to liver dysfunction in HPs, but other factors, e.g. relative immobilization, may also be relevant.

An evaluation of bone density and turnover in premenopausal women with type 1 diabetes mellitus.

Measurement of bone density and turnover was assessed in 20 premenopausal females with Type 1 diabetes mellitus and 27 age-sex-matched controls. Measurement was made of spinal (L2-4) and neck of femur bone density by dual-energy X-ray absorptiometry. L2-4 density was significantly higher in the diabetic patients compared with controls (1.224 +/- 0.021 g cm-2 vs. 1.161 +/- 0.020 g cm-2: p = 0.016). No significant difference was noted between the groups in neck of femur density. Measurement of bone formation was assessed by serum alkaline phosphatase and bone resorption by fasting urinary hydroxyproline/creatinine ratio. Alkaline phosphatase was significantly higher in the diabetic patients (185 +/- 16 Ul-1 vs 135 +/- 10 Ul-1: p < 0.01) as was hydroxyproline/creatinine ratio (0.028 +/- 0.003 vs 0.017 +/- 0.002: p = 0.002). No significant correlation was found between L2-4 density and glycated haemoglobin, duration of diabetes or daily dose of insulin taken. These data suggest that osteopenia is not associated with Type 1 diabetes mellitus; however these patients do have evidence of increased bone turnover and may therefore be at risk of osteoporosis in later life, particularly after the menopause.