T-cadherin signaling in endothelial cells

The cadherin superfamily comprises transmembrane glycoproteins that mediate calciumdependent homophilic cell-cell adhesion. In addition to their pivotal role in mechanical adhesion between cells, cadherins have multiple functions in tissue morphogenesis, cell recognition and sorting, regulated cell motility and the induction and maintenance of tissue/cell polarity. T-cadherin (T-cad) is unusual member of cadherin superfamily; while possessing the Nterminal tandem cadherin repeat structure (EC domain), it lacks both transmembrane and cytoplasmic domains, and is bound to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. T-cad has five EC domains and a propeptide in its precursor form (130kDa), which upon cleavage gets converted to mature form (105kDa). A role for T-cad in tissue organization was first demonstrated in the avian embryonic nervous system where the protein influenced the pattern of neural crest cell migration and maintained somite polarity. Many cancer cell lines (e.g. breast, colon, lung, inter alia) display allelic loss of T-cad which is correlated with tumor progression, and hence T-cad has been described as tumor suppressor gene. T-cad is widely expressed in the vasculature and is upregulated in proliferative vascular disorders such as atherosclerosis and restenosis. GPI-anchored T-cad is not localized at adherent junctions but rather distributed globally over the cell surface. T-cad is localized within lipid rafts. In vitro data supports participation of T-cad in many cellular processes such as vascular differentiation, migration and proliferation of smooth muscle cells (SMC) and endothelial cells (EC) and angiogenesis. Adenoviral mediated overexpression of T-cad in EC and SMC results in cell cycle progression and a concomitant promotion of proliferation. T-cad exhibits deadhesive functions upon homophilic ligation with antibody against T-cad or with immobilized recombinant protein. Homophilic ligation of T-cad induces polarization and migration of endothelial cells in a RhoA/ROCK and Rac dependent fashion. T-cad stimulates in-gel outgrowth of endothelial sprouts in 3-dimensional EC-spheroid and heart tissue models of angiogenesis. In vivo, myoblast-mediated delivery of recombinant soluble T-cad to mouse skeletal muscle facilitates VEGF-induced angiogenesis, supporting a physiological role for Tcad as a proangiogenic protein. The present thesis is focused on the regulation of T-cad expression and the signaling mechanisms whereby T-cad affects vascular cell behavior. The conditions of proliferative vascular cell disorders in which T-cad is upregulated are associated with oxidative stress and cell survival/cell death. We observed an elevation in T-cad levels under condition of oxidative stress induced by serum-deprivation and H2O2; this response was normalized upon inclusion of an antioxidant, N-acetyl cysteine or NADPH oxidase inhibitor diphenyleneiodonium, suggesting T- cad induction by reactive oxygen species is NADPH oxidase dependent. Adenoviral mediated overexpression of T-cad in EC facilitated EC survival upon induction of apoptosis by serumdeprivation and various apoptosis-inducing pharmacologicals. Western blot analysis of lysates infected with Empty –adenovirus (E-EC) and T-cad adenovirus (T-cad+-EC) resulted in hyperactivity of anti-apoptotic proteins (Akt and mTOR target p70S6 kinase) and diminished activity of pro-apoptotic proteins (p38MAPK and active caspase3). PI3 kinase inhibitor, wortmannin, and mTOR inhibitor, rapamycin, normalized anti-apoptotic effects of T-cad; these data suggest that upregulation of T-cad in response to oxidative stress functions to protect EC by concomitant induction of PI3K/Akt/mTOR pathway and suppression of p38/caspase3 pathways. Subsequently we focused on identifying downstream targets of Akt and candidate proximal molecular mediators for T-cad. T-cad+-EC exhibited hyperphosphorylation of glycogen synthase kinase β (GSK3β) and concomitant nuclear accumulation of active β-catenin, a transcription factor regulating cell cycle proteins. Using various GSK3β-carrying adenovectors (kinase mutant, dominant negative or wild type) we demonstrated that T-cad induced nuclear accumulation of β-catenin is GSK3β-dependent. siRNA mediated knockdown of T-cad resulted in decreased phosphorylation of Akt and GSK3β and also in reduced nuclear accumulation of β- catenin. T cell factor (TCF) and Leukocyte enhancer factor (LEF) are co-factors for β-catenin; we found that luciferase (reporter) activity of TCF/LEF elements in T-cad+-EC was markedly increased as compared to E-EC. Cyclin D1, one of the important regulators of the cell cycle is a target of β-catenin/TCF/LEF transcription machinery; T-cad+-EC showed increased mRNA and protein levels of cyclin D1 and increased cell proliferation. In searching for molecular mediators of T-cad we considered Integrin linked kinase (ILK) as a putative candidate because both the proteins are located in rafts and ILK acts upstream of Akt and GSK3β in a PI3K-dependent fashion. T-cad+-EC exhibited increased ILK “kinase” activity in a pull-down assay. RNAimediated knockdown of ILK abrogated effects of T-cad on both phosphorylation of Akt and GSK3β and the nuclear accumulation β-catenin, suggesting involvement of ILK in T-cad signaling. Confocal microscopy studies revealed colocalisation of T-cad and ILK in EC which was most prominent within leading edges of migratory cells and at focal adhesions. Anti-ILK immunoprecipitates contained T-cad indicating the existence of T-cad/ILK complexes, and supporting our hypothesis that ILK can function as a proximal molecular mediator for T-cadelicited PI3K/Akt/GSK3β signaling. Transcriptional regulation of T-cad in endothelial cells is poorly understood. To characterize the minimal promoter region of T-cad, we cloned serially deleted fragments of Tcad promoter stretches into luciferase reporter vector (pGL3). Reporter gene analysis exhibited basal levels of luciferase activity within -285bps suggesting existence of minimal promoter region within -285bps from translational start site. Oxidative stress elevated reporter activity of -285 bps construct, suggesting the minimal promoter region might be responsible for the redox sensitivity of T-cad expression. To identify regulatory elements (transcription factors) responsible for T-cad regulation gel shift assays were performed using nuclear extracts of EC and various oligos designed from T-cad promoter region from -1 to -284 bps We identified specific binding of regulatory protein(s) between -156 to -203 bps. Nuclear extracts from serumdeprived EC exhibited increased binding to -156 to -203 bps oligo, suggesting that the identified nucleoprotein complex could function to induce T-cad expression under conditions of oxidative stress. To identify transcription factor(s) within the identified nucleoprotein complex we performed pull-down assay using nuclear extracts of EC, biotinylated -156 to -203 bps and streptavidin agarose beads. Proteins pulled down were subjected to microsequencing by mass spectrometry. Interestingly thioredoxin (TRX1) was found to be present. TRX1 is a 12kDa protein induced by NADPH oxidase under stress and it acts as an antioxidant by facilitating the reduction of other proteins by cysteine thiol-disulfide exchange. Following its translocation to the nucleus TRX1 reduces transcription factors, enabling their binding to regulatory elements. Preliminary data using RNAi-mediated knockdown of TRX1 abrogates oxidative stress-induced upregulation of T-cad in EC, suggesting that NADPH dependent-induction of T-cad involves nuclear translocation of TRX1. These data may explain the observations of upregulation of Tcad on vascular cells in atherosclerotic lesions where oxidative stress plays a key pathogenic role

Sperm mitochondrial mutations as a cause of low sperm motility

We report the unique case of a 28-year-old man who, in spite of having a varicocele and a sperm concentration of 5 million/mL, of which 10% were motile and 20% had normal forms (oligoasthenoteratozoospermia [OAT]), was fertile. This was confirmed by paternity testing using 16 autosomal and 6 Y-chromosomal short tandem repeat (STR) loci. An analysis of mitochondrial genes that included cytochrome oxidase I (COI), cytochrome oxidase II (COII), adenosine triphosphate synthase6 (ATPase6), ATPase8, transfer ribonucleic acid (tRNA) serine I, tRNA lysine, and NADH dehydrogenase3 (ND3) revealed, for the first time, 9 missense and 27 silent mutations in the sperm's mitochondrial DNA (mtDNA) but not in the DNA from the blood cells. There was a 2-nucleotide deletion in the mitochondrial COII genes, introducing a stop codon, which might be responsible for low sperm motility

Microsatellite-based phylogeny of Indian domestic goats

<p>Abstract</p> <p>Background</p> <p>The domestic goat is one of the important livestock species of India. In the present study we assess genetic diversity of Indian goats using 17 microsatellite markers. Breeds were sampled from their natural habitat, covering different agroclimatic zones.</p> <p>Results</p> <p>The mean number of alleles per locus (NA) ranged from 8.1 in Barbari to 9.7 in Jakhrana goats. The mean expected heterozygosity (He) ranged from 0.739 in Barbari to 0.783 in Jakhrana goats. Deviations from Hardy-Weinberg Equilibrium (HWE) were statistically significant (P < 0.05) for 5 loci breed combinations. The D_Ameasure of genetic distance between pairs of breeds indicated that the lowest distance was between Marwari and Sirohi (0.135). The highest distance was between Pashmina and Black Bengal. An analysis of molecular variance indicated that 6.59% of variance exists among the Indian goat breeds. Both a phylogenetic tree and Principal Component Analysis showed the distribution of breeds in two major clusters with respect to their geographic distribution.</p> <p>Conclusion</p> <p>Our study concludes that Indian goat populations can be classified into distinct genetic groups or breeds based on the microsatellites as well as mtDNA information.</p

T-cadherin attenuates insulin-dependent signalling, eNOS activation, and angiogenesis in vascular endothelial cells

Aims T-cadherin (T-cad) is a glycosylphosphatidylinositol-anchored cadherin family member. Experimental, clinical, and genomic studies suggest a role for T-cad in vascular disorders such as atherosclerosis and hypertension, which are associated with endothelial dysfunction and insulin resistance (InsRes). In endothelial cells (EC), T-cad and insulin activate similar signalling pathways [e.g. PI3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR)] and processes (e.g. angiogenesis). We hypothesize that T-cad is a regulatory component of insulin signalling in EC and therefore a determinant of the development of endothelial InsRes. Methods and results We investigated T-cad-dependent effects on insulin sensitivity using human EC stably transduced with respect to T-cad overexpression or T-cad silencing. Responsiveness to insulin was examined at the level of effectors of the insulin signalling cascade, EC nitric oxide synthase (eNOS) activation, and angiogenic behaviour. Overexpression and ligation of T-cad on EC attenuates insulin-dependent activation of the PI3K/Akt/mTOR signalling axis, eNOS, EC migration, and angiogenesis. Conversely, T-cad silencing enhances these actions of insulin. Attenuation of EC responsiveness to insulin results from T-cad-mediated chronic activation of the Akt/mTOR-dependent negative feedback loop of the insulin cascade and enhanced degradation of the insulin receptor (IR) substrate. Co-immunoprecipitation experiments revealed an association between T-cad and IR. Filipin abrogated inhibitory effects of T-cad on insulin signalling, demonstrating localization of T-cad-insulin cross-talk to lipid raft plasma membrane domains. Hyperinsulinaemia up-regulates T-cad mRNA and protein levels in EC. Conclusion T-cad expression modulates signalling and functional responses of EC to insulin. We have identified a novel signalling mechanism regulating insulin function in the endothelium and attribute a role for T-cad up-regulation in the pathogenesis of endothelial InsRe

Phylogeography and origin of Indian domestic goats

The Indian subcontinent contains 20 well-characterized goat breeds, which vary in their genetic potential for the production of milk, meat, and fibre; disease resistance; heat tolerance; and fecundity. Indian goats make up 20% of the world's goat population, but there has been no extensive study of these economically important animals. Therefore, we have undertaken the present investigation of 363 goats belonging to 10 different breeds from different geographic regions of India using mtDNA sequence data from the HVRI region. We find evidence for population structure and novel lineages in Indian goats and cannot reconcile the genetic diversity found within the major lineage with domestication starting 10,000 years ago from a single mtDNA ancestor. Thus, we propose a more complex origin for domestic goats

T-cadherin is present on endothelial microparticles and is elevated in plasma in early atherosclerosis

Aims The presence of endothelial cell (EC)-derived surface molecules in the circulation is among hallmarks of endothelial activation and damage in vivo. Previous investigations suggest that upregulation of T-cadherin (T-cad) on the surface of ECs may be a characteristic marker of EC activation and stress. We investigated whether T-cad might also be shed from ECs and in amounts reflecting the extent of activation or damage. Methods and results Immunoblotting showed the presence of T-cad protein in the culture medium from normal proliferating ECs and higher levels in the medium from stressed/apoptotic ECs. Release of T-cad into the circulation occurs in vivo and in association with endothelial dysfunction. Sandwich ELISA revealed negligible T-cad protein in the plasma of healthy volunteers (0.90 ± 0.90 ng/mL, n = 30), and increased levels in the plasma from patients with non-significant atherosclerosis (9.23 ± 2.61 ng/mL, n = 63) and patients with chronic coronary artery disease (6.93 ± 1.31 ng/mL, n = 162). In both patient groups there was a significant (P = 0.043) dependency of T-cad and degree of endothelial dysfunction as measured by reactive hyperaemia peripheral tonometry. Flow cytometry analysis showed that the major fraction of T-cad was released into the EC culture medium and the plasma as a surface component of EC-derived annexin V- and CD144/CD31-positive microparticles (MPs). Gain-of-function and loss-of-function studies demonstrate that MP-bound T-cad induced Akt phosphorylation and activated angiogenic behaviour in target ECs via homophilic-based interactions. Conclusion Our findings reveal a novel mechanism of T-cad-dependent signalling in the vascular endothelium. We identify T-cad as an endothelial MP antigen in vivo and demonstrate that its level in plasma is increased in early atherosclerosis and correlates with endothelial dysfunctio

DRAMMS: deformable registration via attribute matching and mutual-saliency weighting

A general-purpose deformable registration algorithm referred to as ”DRAMMS” is presented in this paper. DRAMMS adds to the literature of registration methods that bridge between the traditional voxel-wise methods and landmark/feature-based methods. In particular, DRAMMS extracts Gabor attributes at each voxel and selects the optimal components, so that they form a highly distinctive morphological signature reflecting the anatomical context around each voxel in a multi-scale and multi-resolution fashion. Compared with intensity or mutual-information based methods, the high-dimensional optimal Gabor attributes render different anatomical regions relatively distinctively identifiable and therefore help establish more accurate and reliable correspondence. Moreover, the optimal Gabor attribute vector is constructed in a way that generalizes well, i.e., it can be applied to different registration tasks, regardless of the image contents under registration. A second characteristic of DRAMMS is that it is based on a cost function that weights different voxel pairs according to a metric referred to as ”mutual-saliency”, which reflects the uniqueness (reliability) of anatomical correspondences implied by the tentative transformation. As a result, image voxels do not contribute equally to the optimization process, as in most voxel-wise methods, or in a binary selection fashion, as in most landmark/feature-based methods. Instead, they contribute according to a continuously-valued mutual-saliency map, which is dynamically updated during the algorithm’s evolution. The general applicability and accuracy of DRAMMS are demonstrated by experiments in simulated images, inter-subject images, single-/multi-modality images, and longitudinal images, from human and mouse brains, breast, heart, and prostate

Selected MicroRNAs Define Cell Fate Determination of Murine Central Memory CD8 T Cells

During an immune response T cells enter memory fate determination, a program that divides them into two main populations: effector memory and central memory T cells. Since in many systems protection appears to be preferentially mediated by T cells of the central memory it is important to understand when and how fate determination takes place. To date, cell intrinsic molecular events that determine their differentiation remains unclear. MicroRNAs are a class of small, evolutionarily conserved RNA molecules that negatively regulate gene expression, causing translational repression and/or messenger RNA degradation. Here, using an in vitro system where activated CD8 T cells driven by IL-2 or IL-15 become either effector memory or central memory cells, we assessed the role of microRNAs in memory T cell fate determination. We found that fate determination to central memory T cells is under the balancing effects of a discrete number of microRNAs including miR-150, miR-155 and the let-7 family. Based on miR-150 a new target, KChIP.1 (K + channel interacting protein 1), was uncovered, which is specifically upregulated in developing central memory CD8 T cells. Our studies indicate that cell fate determination such as surface phenotype and self-renewal may be decided at the pre-effector stage on the basis of the balancing effects of a discrete number of microRNAs. These results may have implications for the development of T cell vaccines and T cell-based adoptive therapies

Single amino acid change in gp41 region of HIV-1 alters bystander apoptosis and CD4 decline in humanized mice

<p>Abstract</p> <p>Background</p> <p>The mechanism by which HIV infection leads to a selective depletion of CD4 cells leading to immunodeficiency remains highly debated. Whether the loss of CD4 cells is a direct consequence of virus infection or bystander apoptosis of uninfected cells is also uncertain.</p> <p>Results</p> <p>We have addressed this issue in the humanized mouse model of HIV infection using a HIV variant with a point mutation in the gp41 region of the Env glycoprotein that alters its fusogenic activity. We demonstrate here that a single amino acid change (V38E) altering the cell-to-cell fusion activity of the Env minimizes CD4 loss in humanized mice without altering viral replication. This differential pathogenesis was associated with a lack of bystander apoptosis induction by V38E virus even in the presence of similar levels of infected cells. Interestingly, immune activation was observed with both WT and V38E infection suggesting that the two phenomena are likely not interdependent in the mouse model.</p> <p>Conclusions</p> <p>We conclude that Env fusion activity is one of the determinants of HIV pathogenesis and it may be possible to attenuate HIV by targeting gp41.</p

The management and outcome for patients with chronic subdural hematoma: a prospective, multicenter, observational cohort study in the United Kingdom

Symptomatic chronic subdural hematoma (CSDH) will become an increasingly common presentation in neurosurgical practice as the population ages, but quality evidence is still lacking to guide the optimal management for these patients. The British Neurosurgical Trainee Research Collaborative (BNTRC) was established by neurosurgical trainees in 2012 to improve research by combining the efforts of trainees in each of the United Kingdom (UK) and Ireland's neurosurgical units (NSUs). The authors present the first study by the BNTRC that describes current management and outcomes for patients with CSDH throughout the UK and Ireland. This provides a resource both for current clinical practice and future clinical research on CSDH