Studies on the actin-binding protein HS1 in platelets

<p>Abstract</p> <p>Background</p> <p>The platelet cytoskeleton mediates the dramatic change in platelet morphology that takes place upon activation and stabilizes thrombus formation. The Arp2/3 complex plays a vital role in these processes, providing the protrusive force for lamellipodia formation. The Arp2/3 complex is highly regulated by a number of actin-binding proteins including the haematopoietic-specific protein HS1 and its homologue cortactin. The present study investigates the role of HS1 in platelets using HS1^-/-mice.</p> <p>Results</p> <p>The present results demonstrate that HS1 is not required for platelet activation, shape change or aggregation. Platelets from HS1^-/-mice spread normally on a variety of adhesion proteins and have normal F-actin and Arp2/3 complex distributions. Clot retraction, an actin-dependent process, is also normal in these mice. Platelet aggregation and secretion is indistinguishable between knock out and littermates and there is no increase in bleeding using the tail bleeding assay.</p> <p>Conclusion</p> <p>This study concludes that HS1 does not play a major role in platelet function. It is possible that a role for HS1 is masked by the presence of cortactin.</p

Evaluation of the prophylactic use of mitomycin-C to inhibit haze formation after photorefractive keratectomy in high myopia: a prospective clinical study

BACKGROUND: To study the effect of prophylactic application of mitomycin-C on haze formation in photorefractive keratectomy (PRK) for high myopia. METHODS: Fifty-four eyes of 28 myopic patients were enrolled in this prospective study. All eyes were operated by PRK followed by 0.02% mitomycin-C application for two minutes and washed with 20 ml normal saline afterwards. All eyes were examined thoroughly on the first 7 days and one month after surgery; 48 eyes (88.9%) at 3 and 6 months postoperatively. Hanna grading (in the scale of 0 to 4+) was used for assessment of corneal haze. RESULTS: The mean spherical equivalent refraction (SE) was -7.08 diopters (D) ± 1.11 (SD) preoperatively. Six months after surgery, 37 eyes (77.1%) achieved an uncorrected visual acuity (UCVA) of 20/20 or better, all eyes had a UCVA of 20/40 or better and 45 (93.7%) eyes had an SE within ± 1.00D. One month postoperatively, 2 eyes (3.7%) had grade 0.5+ of haze, while at 3 and 6 months after surgery no visited eye had haze at all. All eyes had a best corrected visual acuity (BCVA) of 20/40 or better and there were no lost lines in BCVA by 6 months after surgery. In spatial frequencies of 6 and 12 cycles per degree contrast sensitivity had decreased immediately after PRK and it had increased 1.5 lines by the 6(th )postoperative month compared to the preoperative data. CONCLUSIONS: The results show the efficacy of mitomycin-C in preventing corneal haze after treatment of high myopia with PRK. This method- PRK + mitomycin-C – can be considered an alternative treatment for myopic patients whose corneal thicknesses are inadequate for laser in situ keratomileusis (LASIK). However, the results should be confirmed in longer follow-ups

Is PTEN loss associated with clinical outcome measures in human prostate cancer?

Inactivating PTEN mutations are commonly found in prostate cancer, resulting in an increased activation of Akt. In this study, we investigate the role of PTEN deletion and protein expression in the development of hormone-refractory prostate cancer using matched hormone-sensitive and hormone-refractory tumours. Fluorescent in situ hybridisation and immunohistochemistry was carried out to investigate PTEN gene deletion and PTEN protein expression in the transition from hormone-sensitive to hormone-refractory prostate cancer utilising 68 matched hormone sensitive and hormone-refractory tumour pairs (one before and one after hormone relapse). Heterogeneous PTEN gene deletion was observed in 23% of hormone sensitive tumours. This increased significantly to 52% in hormone-refractory tumours (P=0.044). PTEN protein expression was observed in the membrane, cytoplasm and the nucleus. In hormone sensitive tumours, low levels of cytoplasmic PTEN was independently associated with shorter time to relapse compared to high levels of PTEN (P=0.028, hazard ratio 0.51 (95%CI 0.27–0.93). Loss of PTEN expression in the nucleus of hormone sensitive tumours was independently associated with disease-specific survival (P=0.031, hazard ratio 0.52, 95%CI 0.29–0.95). The results from this study demonstrate a role for both cytoplasmic and nuclear PTEN in progression of prostate cancer to the hormone-refractory state

Application of Autologous Bone Marrow Derived Mesenchymal Stem Cells to an Ovine Model of Growth Plate Cartilage Injury

Injury to growth plate cartilage in children can lead to bone bridge formation and result in bone growth deformities, a significant clinical problem currently lacking biological treatment. Mesenchymal stem/stromal cells (MSC) offer a promising therapeutic option for regeneration of damaged cartilage, due to their self renewing and multi-lineage differentiation attributes. Although some small animal model studies highlight the therapeutic potential of MSC for growth plate repair, translational research in large animal models, which more closely resemble the human condition, are lacking. Our laboratory has recently characterised MSCs derived from ovine bone marrow, and demonstrated these cells form cartilage-like tissue when transplanted within the gelatin sponge, Gelfoam, in vivo. In the current study, autologous bone marrow MSC were seeded into Gelfoam scaffold containing TGF-β1, and transplanted into a surgically created defect of the proximal ovine tibial growth plate. Examination of implants at 5 week post-operatively revealed transplanted autologous MSC failed to form new cartilage structure at the defect site, but contributed to an increase in formation of a dense fibrous tissue. Importantly, the extent of osteogenesis was diminished, and bone bridge formation was not accelerated due to transplantation of MSCs or the gelatin scaffold. The current study represents the first work that has utilised this ovine large animal model to investigate whether autologous bone marrow derived MSC can be used to initiate regeneration at the injured growth plate

Inducible Cre recombinase activity in mouse mature astrocytes and adult neural precursor cells

Two transgenic mouse lines expressing an inducible form of the Cre recombinase (CreERTM) under the control of the human GFAP promoter have been generated and characterized. In adult mice, expression of the fusion protein is largely confined to astrocytes in all regions of the central nervous system. Minimal spontaneous Cre activity was detected and recombination was efficiently induced by intraperitoneal administration of tamoxifen in adult mice. The pattern of recombination closely mirrored that of transgene expression. The percentage of astrocytes undergoing recombination varied from region to region ranging from 35% to 70% while a much smaller portion (<1%) of oligodendrocytes and neural precursor cells showed evidence of Cre activity. These mouse lines will provide important tools to dissect gene function in glial cells and in gliomagenesis

Gonad shielding in paediatric pelvic radiography: disadvantages prevail over benefit

Objective To re-evaluate gonad shielding in paediatric pelvic radiography in terms of attainable radiation risk reduction and associated loss of diagnostic information. Methods A study on patient dose and the quality of gonad shielding was performed retrospectively using 500 pelvic radiographs of children from 0 to 15 years old. In a subsequent study, 195 radiographs without gonad shielding were included. Patient doses and detriment adjusted risks for heritable disease and cancer were calculated with and without gonad shielding. Results For girls, gonad shields were placed incorrectly in 91% of the radiographs; for boys, in 66%. Without gonad shielding, the hereditary detriment adjusted risk for girls ranged between 0.1?×?10?6 and 1.3?×?10?6 and for boys between 0.3?×?10?6 and 3.9?×?10?6, dependent on age. With shielding, the reduction in hereditary risk for girls was on average 6?±?3% of the total risk of the radiograph, for boys 24?±?6%. Without gonad shielding, the effective dose ranged from 0.008 to 0.098 mSv. Conclusions With modern optimised X-ray systems, the reduction of the detriment adjusted risk by gonad shielding is negligibly small. Given the potential consequences of loss of diagnostic information, of retakes, and of shielding of automatic exposure-control chambers, gonad shielding might better be discontinued.Support TNWApplied Science

Endothelial Expression of TGFβ Type II Receptor Is Required to Maintain Vascular Integrity during Postnatal Development of the Central Nervous System

TGFβ signalling in endothelial cells is important for angiogenesis in early embryonic development, but little is known about its role in early postnatal life. To address this we used a tamoxifen inducible Cre-LoxP strategy in neonatal mice to deplete the TypeII TGFβ receptor (Tgfbr2) specifically in endothelial cells. This resulted in multiple micro-haemorrhages, and glomeruloid-like vascular tufts throughout the cerebral cortices and hypothalamus of the brain as well as in retinal tissues. A detailed examination of the retinal defects in these mutants revealed that endothelial adherens and tight junctions were in place, pericytes were recruited and there was no failure of vascular smooth muscle differentiation. However, the deeper retinal plexus failed to form in these mutants and the angiogenic sprouts stalled in their progress towards the inner nuclear layer. Instead the leading endothelial cells formed glomerular tufts with associated smooth muscle cells. This evidence suggests that TGFβ signalling is not required for vessel maturation, but is essential for the organised migration of endothelial cells as they begin to enter the deeper layers of the retina. Thus, TGFβ signalling is essential in vascular endothelial cells for maintaining vascular integrity at the angiogenic front as it migrates into developing neural tissues in early postnatal life

Knowledge-Driven Multi-Locus Analysis Reveals Gene-Gene Interactions Influencing HDL Cholesterol Level in Two Independent EMR-Linked Biobanks

Genome-wide association studies (GWAS) are routinely being used to examine the genetic contribution to complex human traits, such as high-density lipoprotein cholesterol (HDL-C). Although HDL-C levels are highly heritable (h2∼0.7), the genetic determinants identified through GWAS contribute to a small fraction of the variance in this trait. Reasons for this discrepancy may include rare variants, structural variants, gene-environment (GxE) interactions, and gene-gene (GxG) interactions. Clinical practice-based biobanks now allow investigators to address these challenges by conducting GWAS in the context of comprehensive electronic medical records (EMRs). Here we apply an EMR-based phenotyping approach, within the context of routine care, to replicate several known associations between HDL-C and previously characterized genetic variants: CETP (rs3764261, p = 1.22e-25), LIPC (rs11855284, p = 3.92e-14), LPL (rs12678919, p = 1.99e-7), and the APOA1/C3/A4/A5 locus (rs964184, p = 1.06e-5), all adjusted for age, gender, body mass index (BMI), and smoking status. By using a novel approach which censors data based on relevant co-morbidities and lipid modifying medications to construct a more rigorous HDL-C phenotype, we identified an association between HDL-C and TRIB1, a gene which previously resisted identification in studies with larger sample sizes. Through the application of additional analytical strategies incorporating biological knowledge, we further identified 11 significant GxG interaction models in our discovery cohort, 8 of which show evidence of replication in a second biobank cohort. The strongest predictive model included a pairwise interaction between LPL (which modulates the incorporation of triglyceride into HDL) and ABCA1 (which modulates the incorporation of free cholesterol into HDL). These results demonstrate that gene-gene interactions modulate complex human traits, including HDL cholesterol

High expression of Lewis(y/b )antigens is associated with decreased survival in lymph node negative breast carcinomas

INTRODUCTION: There is sufficient evidence that blood group related Lewis antigens are tumour-associated molecules. The Lewis(y )and Lewis(b )antigens are complex carbohydrates that are over-expressed by breast, lung, colon and ovarian cancers. The SC101 mAb is a unique Lewis(y/b )binding antibody that binds to native and extended Lewis(y )and Lewis(b )haptens, displaying no cross reactivity with H type 1, H type 2, Lewis(x )or normal blood group antigens. METHODS: Immunohistochemical detection of Lewis(y/b )was performed on 660 formalin-fixed, paraffin embedded breast tumour specimens using a streptavidin-biotin peroxidase technique. Tissue from these patients had previously been included in tissue microarrays. This cohort comprises a well characterized series of patients with primary operable breast cancer diagnosed between 1987 and 1992, obtained from the Nottingham Tenovus Primary Breast Carcinoma Series. This includes patients 70 years of age or less, with a mean follow up of 7 years. RESULTS: Of the breast carcinomas, 370 of 660 (56%) were negative for Lewis(y/b )expression, 110 (17%) cases showed a low level of expression (<25% of positive cells) and only 54 cases (8%) showed extensive expression of Lewis(y/b )(>75% of positive cells). We found significant positive associations between histological grade (p < 0.001), Nottingham Prognostic Index (p = 0.016), tumour type (p = 0.007) and the level of Lewis (y/b )expression. There was a significant correlation between the proportion of Lewis(y/b )positive tumour cells and survival in lymph-node negative patients (p = 0.006). CONCLUSION: The unique epitope recognised by SC101 mAb on Lewis(y/b )hapten is over-expressed on breast tumour tissue compared with normal breast. In this large series of invasive breast cancers, higher expression of Lewis(y/b )was more often found in high grade and poor prognosis tumours compared to good prognosis cancers. Moreover, in lymph node negative breast carcinomas, over-expression of Lewis(y/b )hapten was associated with significantly decreased patient survival