TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita

Patients with dyskeratosis congenita (DC), a heterogeneous inherited bone marrow failure syndrome, have abnormalities in telomere biology, including very short telomeres and germline mutations in DKC1, TERC, TERT, or NOP10, but approximately 60% of DC patients lack an identifiable mutation. With the very short telomere phenotype and a highly penetrant, rare disease model, a linkage scan was performed on a family with autosomal-dominant DC and no mutations in DKCI, TERC, or TERT. Evidence favoring linkage was found at 2p24 and 14q11.2, and this led to the identification of TINF2 (14q11.2) mutations, K280E, in the proband and her five affected relatives and TINF2 R282H in three additional unrelated DC probands, including one with Revesz syndrome; a fifth DC proband had a R282S mutation. TINF2 mutations were not present in unaffected relatives, DC probands with mutations in DKC1, TERC, or TERT or 298 control subjects. We demonstrate that a fifth gene, TINF2, is mutated in classical DC and, for the first time, in Revesz syndrome. This represents the first shelterin complex mutation linked to human disease and confirms the role of very short telomeres as a diagnostic test for DC.

Rotational atherectomy followed by drug-eluting stent implantation (Rota-DES): a rational approach for complex calcified coronary lesions

Rotational atherectomy has been regaining interest over the last couple of years after it almost has disappeared from most interventional catheterization laboratories for several years due to failure to prove its original concept of improving long term results of percutaneous coronary interventions (PCI) as was repeatedly shown in studies in the 1990s. Its revival coupled the introduction of drug-eluting stents (DES); these devices have led to treating much more complex lesions and high-risk patients by PCI. However, real-world experience suggested that off-label use of DES is associated with a higher rate of early and late stent thrombosis. Therefore, more attention is now being paid to the initial implantation technique of DES including aggressive lesion preparation to facilitate stent delivery and expansion. The limited studies with rot-ablation and DES showed promising results with no long term safety concerns. In these studies, a subtle observation was made suggesting that rot-ablation prior to DES implantation in such lesions may have an add-on effect on long term outcome compared to DES alone. An ongoing multicenter study is investigating such effect among complex calcified coronary lesions. Even if this additive benefit does not prove true, rot-ablation remains an efficient tool for preparing certain lesions to facilitate effective and safe DES implantation. Therefore, interventional training programs should focus on this difficult technique to bridge the gap of experience which resulted from neglecting it for several years. In this regard, dedicated courses at experienced sites as well as medical simulation may be appropriate.

Off-pump extraanatomic aortic bypass for the treatment of complex aortic coarctation and hypoplastic aortic arch

BACKGROUND: Despite advances in surgical and interventional techniques, the optimal surgical treatment of severe aortic (re) coarctation and hypoplastic aortic arch is still controversial. Anatomic repair may require extensive dissection, cardiopulmonary bypass, and deep hypothermic circulatory arrest with their inherent risks. The aim of this study was to analyze the outcome of off-pump extraanatomic aortic bypass as a surgical alternative to local repair. METHODS: From February 2000 to December 2005, ten consecutive patients (median age 20 years; range, 11 to 38 years) with severe aortic (re) coarctation (n = 4) and (or) hypoplastic aortic arch (n = 7) underwent off-pump extraanatomic aortic bypass through median sternotomy. All but three patients had undergone previous surgery for coarctation and angioplasty or stenting. Three patients underwent concomitant replacement of the ascending aorta because of an aneurysm using cardiopulmonary bypass. RESULTS: Postoperative hospital course was uneventful in all patients. There was no perioperative mortality or significant morbidity. During a mean follow-up of 48 +/- 22 months no patient required additional procedures. All patients were free of symptoms; no patient showed signs of heart failure after follow-up. At last follow-up, no patient presented with claudication, nor any patient experienced orthostatic problems due to a steal phenomenon. During follow-up, hypertension resolved in all patients with residual mild hypertension in two patients. CONCLUSIONS: Off-pump extraanatomic aortic bypass is an attractive treatment option for complex aortic (re) coarctation and hypoplastic aortic arch. Perioperative risks are minimized, hypertension is influenced favorably, and midterm survival is event-free.

Numerical modeling of the failure mechanisms in Si thin film anode for Li-ion batteries

In recent times, the demand for the storage of electrical energy has grown rapidly for both static applications and the portable electronics enforcing the substantial improvement in battery systems, and Li-ion batteries have been proven to have maximum energy storage density in all rechargeable batteries. However, major breakthroughs are required to consummate the requirement of higher energy density with lower cost to penetrate new markets. Graphite anode having limited capacity has become a bottle neck in the process of developing next generation batteries and can be replaced by higher capacity metals such as Silicon. In the present study we are focusing on the mechanical behavior of the Si-thin film anode under various operating conditions. A numerical model is developed to simulate the intercalation induced stress and the failure mechanism of the complex anode structure. Effect of the various physical phenomena such as diffusion induced stress, plasticity and the crack propagation are investigated to predict better performance parameters for improved design.

Heart failure gene therapy: the path to clinical practice

Gene therapy, aimed at the correction of key pathologies being out of reach for conventional drugs, bears the potential to alter the treatment of cardiovascular diseases radically and thereby of heart failure. Heart failure gene therapy refers to a therapeutic system of targeted drug delivery to the heart that uses formulations of DNA and RNA, whose products determine the therapeutic classification through their biological actions. Among resident cardiac cells, cardiomyocytes have been the therapeutic target of numerous attempts to regenerate systolic and diastolic performance, to reverse remodeling and restore electric stability and metabolism. Although the concept to intervene directly within the genetic and molecular foundation of cardiac cells is simple and elegant, the path to clinical reality has been arduous because of the challenge on delivery technologies and vectors, expression regulation, and complex mechanisms of action of therapeutic gene products. Nonetheless, since the first demonstration of in vivo gene transfer into myocardium, there have been a series of advancements that have driven the evolution of heart failure gene therapy from an experimental tool to the threshold of becoming a viable clinical option. The objective of this review is to discuss the current state of the art in the field and point out inevitable innovations on which the future evolution of heart failure gene therapy into an effective and safe clinical treatment relies.

A-kinase anchoring protein Lbc coordinates a p38 activating signaling complex controlling compensatory cardiac hypertrophy

In response to stress, the heart undergoes a remodeling process associated with cardiac hypertrophy that eventually leads to heart failure. A-kinase anchoring proteins (AKAPs) have been shown to coordinate numerous prohypertrophic signaling pathways in cultured cardiomyocytes. However, it remains to be established whether AKAP-based signaling complexes control cardiac hypertrophy and remodeling in vivo. In the current study, we show that AKAP-Lbc assembles a signaling complex composed of the kinases PKN, MLTK, MKK3, and p38α that mediates the activation of p38 in cardiomyocytes in response to stress signals. To address the role of this complex in cardiac remodeling, we generated transgenic mice displaying cardiomyocyte-specific overexpression of a molecular inhibitor of the interaction between AKAP-Lbc and the p38-activating module. Our results indicate that disruption of the AKAP-Lbc/p38 signaling complex inhibits compensatory cardiomyocyte hypertrophy in response to aortic banding-induced pressure overload and promotes early cardiac dysfunction associated with increased myocardial apoptosis, stress gene activation, and ventricular dilation. Attenuation of hypertrophy results from a reduced protein synthesis capacity, as indicated by decreased phosphorylation of 4E-binding protein 1 and ribosomal protein S6. These results indicate that AKAP-Lbc enhances p38-mediated hypertrophic signaling in the heart in response to abrupt increases in the afterload.

ΔNp63 REGULATES A COMPLEX NETWORK OF TARGET GENES IN LIMB AND EPIDERMAL DEVELOPMENT

The skin is composed of two major compartments, the dermis and epidermis. The epidermis forms a barrier to protect the body. The stratified epithelium has self-renewing capacity throughout life, and continuous turnover is mediated by stem cells in the basal layer. p63 is structurally and functionally related to p53. In spite of their structural similarities, p63 is critical for the development and maintenance of stratified epithelial tissues, unlike p53. p63 is highly expressed in the epidermis and previously has been shown to play a critical role in the development and maintenance of the epidermis. The study of p63 has been complicated due to the existence of multiple isoforms: those with a transactivation domain (TAp63) and those lacking this domain (ΔNp63). Mice lacking p63 cannot form skin, have craniofacial and skeletal defects and die within hours after birth. These defects are due to the ability of p63 to regulate multiple processes in skin development including epithelial stem cell proliferation, differentiation, and adherence programs. To determine the roles of these isoforms in skin development and maintenance, isoform specific p63 conditional knock out mice were generated by our lab. TAp63-/- mice age prematurely, develop blisters, and display wound-healing defects that result from hyperproliferation of dermal stem cells. That results in premature depletion of these cells, which are necessary for wound repair, that indicates TAp63 plays a role in dermal/epidermal maintenance. To study the role of ΔNp63, I generated a ΔNp63-/- mouse and analyzed the skin by performing immunofluorescence for markers of epithelial differentiation. The ΔNp63-/- mice developed a thin, disorganized epithelium but differentiation markers were expressed. Interestingly, the epidermis from ΔNp63-/- mice co-expressed K14 and K10 in the same cell suggesting defects in epidermal differentiation and stratification. This phenotype is reminiscent of the DGCR8fl/fl;K14Cre and Dicerfl/fl;K14Cre mice skin. Importantly, DGCR8-/- embryonic stem cells (ESCs) display a hyperproliferation defect by failure to silence pluripotency genes. Furthermore, I have observed that epidermal cells lacking ΔNp63 display a phenotype reminiscent of embryonic stem cells instead of keratinocytes. Thus, I hypothesize that genes involved in maintaining pluripotency, like Oct4, may be upregulated in the absence of ΔNp63. To test this, q-RT PCR was performed for Oct4 mRNA with wild type and ΔNp63-/- 18.5dpc embryo skin. I found that the level of Oct4 was dramatically increased in the absence of ΔNp63-/-. Based on these results, I hypothesized that ΔNp63 induces differentiation by silencing pluripotency regulators, Oct4, Sox2 and Nanog directly through the regulation of DGCR8. I found that DGCR8 restoration resulted in repression of Oct4, Sox2 and Nanog in ΔNp63-/- epidermal cells and rescue differentiation defects. Loss of ΔNp63 resulted in pluripotency that caused defect in proper differentiation and stem cell like phenotype. This led me to culture the ΔNp63-/- epidermal cells in neuronal cell culture media in order to address whether restoration of DGCR8 can transform epidermal cells to neuronal cells. I found that DGCR8 restoration resulted in a change in cell fate. I also found that miR470 and miR145 play a role in the induction of pluripotency by repressing Oct4, Sox2 and Nanog. This indicates that ΔNp63 induces terminal differentiation through the regulation of DGCR8.

Prevalence of Premature Ovarian Failure in Women with Tuberous Sclerosis

Tuberous Sclerosis Complex (TSC) is an autosomal dominant tumor suppressor disorder characterized by hamartomas, or benign growths, in various organ systems. Inactivating mutations in either the TSC1 or the TSC2 gene cause most cases of TSC. Recently, the use of ovarian specific conditional knock-out mouse models has demonstrated a crucial role of the TSC genes in ovarian function. Mice with complete deletion of Tsc1 or Tsc2 showed accelerated ovarian follicle activation and subsequent premature follicular depletion, consistent with the human condition premature ovarian failure (POF). POF is defined in women as the cessation of menses before the age of 40 and elevated levels of follicle stimulating hormone (FSH). The prevalence of POF is estimated to be 1%, affecting a substantial number of women in the general population. Nonetheless, the etiology of most cases of POF remains unknown. Based on the mouse model results, we hypothesized that the human TSC1 and TSC2 genes are likely to be crucial for ovarian development and function. Moreover, since women with TSC already have one inactivated TSC gene, we further hypothesized that they may show a higher prevalence of POF. To test this hypothesis, we surveyed 1000 women with TSC belonging to the Tuberous Sclerosis Alliance, a national support organization. 182 questionnaires were analyzed for information on menstrual and reproductive function, as well as TSC. This self-reported data revealed 8 women (4.4%) with possible POF, as determined by menstrual history report and additional supportive data. This prevalence is much higher than 1% in the general population. Data from all women suggested other reproductive pathology associated with TSC such as a high rate of miscarriage (41.2%) and menstrual irregularity of any kind (31.2%). These results establish a previously unappreciated effect of TSC on women’s reproductive health. Moreover, these data suggest that perturbations in the cellular pathways regulated by the TSC genes may play an important role in reproductive function.

Immune responses against major histocompatibility complex and interleukin-2 gene transfected K1735 murine melanoma: Potential vaccines for the immunotherapy of cancer

Tumor specific immunity is mediated by cytotoxic T lymphocytes (CTL) that recognize peptide antigen (Ag) in the context of major histocompatibility complex (MHC) class I molecules and by helper T (Th) lymphocytes that recognize peptide Ag in the context of MHC class II molecules. The purpose of this study is (1) to induce or augment the immunogenicity of nonimmunogenic or weakly immunogenic tumors by genetic modification of tumor cells, and (2) to use these genetically altered cells in cancer immunotherapy. To study this, I transfected a highly tumorigenic murine melanoma cell line (K1735) that did not express constitutively either MHC class I or II molecules with syngeneic cloned MHC class I and/or class II genes, and then determined the tumorigenicity of transfected cells in normal C3H mice. K1735 transfectants expressing either $\rm K\sp{k}$ or $\rm A\sp{k}$ molecules alone produced tumors in normal C3H mice, whereas most transfectants that expressed both molecules were rejected in normal C3H mice but produced tumors in nude mice. The rejection of K1735 transfectants expressing $\rm K\sp{k}$ and $\rm A\sp{k}$ Ag in normal C3H mice required both $\rm CD4\sp+$ and $\rm CD8\sp+$ T cells. Interestingly, the $\rm A\sp{k}$ requirement can be substituted by IL-2 because transfection of $\rm K\sp{k}$-positive/A$\sp{\rm k}$-negative K1735 cells with the IL-2 gene also resulted in abrogation of tumorigenicity in normal C3H mice but not in nude mice. In addition, 1735 $(\rm I\sp+II\sp+)$ transfected cells can function as antigen presenting cells (APC) since they could process and present native hen egg lysozyme (HEL) to HEL specific T cell hybridomas. Furthermore, the transplantation immunity induced by K1735 transfectants expressing both $\rm K\sp{k}$ and $\rm A\sp{k}$ molecules completely cross-protected mice against challenge with $\rm K\sp{k}$-positive transfectants but weakly protected them against challenge with parental K1735 cells or $\rm A\sp{k}$-positive transfectants. Finally, I demonstrated that MHC $(\rm I\sp+II\sp+)$ or $\rm K\sp{k}$-positive/IL-2-positive cells can function as anti-cancer vaccines since they can abrogate the growth of established tumors and metastasis.^ In summary, my results indicate that expression of either MHC class I or II molecule alone is insufficient to cause the rejection of K1735 melanoma in syngeneic hosts and that both molecules are necessary. In addition, my data suggest that the failure of $\rm K\sp{k}$-positive K1735 cells to induce a primary tumor-rejection response in normal C3H mice may be due to their inability to induce the helper arm of the anti-tumor immune response. Finally, the ability of MHC $(\rm I\sp+II\sp+)$ or $\rm K\sp{k}$-positive/IL-2-positive cells to prevent growth of established tumors or metastasis suggests that these cell lines can serve as potential vaccines for the immunotherapy of cancer. (Abstract shortened by UMI.) ^

Von Willebrand Factor Improves Risk Prediction in Addition to N-Terminal Pro-B-type Natriuretic Peptide in Patients Referred to Coronary Angiography and Signs and Symptoms of Heart Failure and Preserved Ejection Fraction.

BACKGROUND Heart failure with preserved ejection fraction (HFpEF) represents a growing health burden associated with substantial mortality and morbidity. Consequently, risk prediction is of highest importance. Endothelial dysfunction has been recently shown to play an important role in the complex pathophysiology of HFpEF. We therefore aimed to assess von Willebrand factor (vWF), a marker of endothelial damage, as potential biomarker for risk assessment in patients with HFpEF. METHODS AND RESULTS Concentrations of vWF were assessed in 457 patients with HFpEF enrolled as part of the LUdwigshafen Risk and Cardiovascular Health (LURIC) study. All-cause mortality was observed in 40% of patients during a median follow-up time of 9.7 years. vWF significantly predicted mortality with a hazard ratio (HR) per increase of 1 SD of 1.45 (95% confidence interval, 1.26-1.68; P<0.001) and remained a significant predictor after adjustment for age, sex, body mass index, N-terminal pro-B-type natriuretic peptide (NT-proBNP), renal function, and frequent HFpEF-related comorbidities (adjusted HR per 1 SD, 1.22; 95% confidence interval, 1.05-1.42; P=0.001). Most notably, vWF showed additional prognostic value beyond that achievable with NT-proBNP indicated by improvements in C-Statistic (vWF×NT-proBNP: 0.65 versus NT-proBNP: 0.63; P for comparison, 0.004) and category-free net reclassification index (37.6%; P<0.001). CONCLUSIONS vWF is an independent predictor of long-term outcome in patients with HFpEF, which is in line with endothelial dysfunction as potential mediator in the pathophysiology of HFpEF. In particular, combined assessment of vWF and NT-proBNP improved risk prediction in this vulnerable group of patients.

Favourable mid-term outcome after heart transplantation for late Fontan failure.

OBJECTIVES Fontan failure (FF) represents a growing and challenging indication for paediatric orthotopic heart transplantation (OHT). The aim of this study was to identify predictors of the best mid-term outcome in OHT after FF. METHODS Twenty-year multi-institutional retrospective analysis on OHT for FF. RESULTS Between 1991 and 2011, 61 patients, mean age 15.0 ± 9.7 years, underwent OHT for failing atriopulmonary connection (17 patients = 27.8%) or total cavopulmonary connection (44 patients = 72.2%). Modality of FF included arrhythmia (14.8%), complex obstructions in the Fontan circuit (16.4%), protein-losing enteropathy (PLE) (22.9%), impaired ventricular function (31.1%) or a combination of the above (14.8%). The mean time interval between Fontan completion and OHT was 10.7 ± 6.6 years. Early FF occurred in 18%, requiring OHT 0.8 ± 0.5 years after Fontan. The hospital mortality rate was 18.3%, mainly secondary to infection (36.4%) and graft failure (27.3%). The mean follow-up was 66.8 ± 54.2 months. The overall Kaplan-Meier survival estimate was 81.9 ± 1.8% at 1 year, 73 ± 2.7% at 5 years and 56.8 ± 4.3% at 10 years. The Kaplan-Meier 5-year survival estimate was 82.3 ± 5.9% in late FF and 32.7 ± 15.0% in early FF (P = 0.0007). Late FF with poor ventricular function exhibited a 91.5 ± 5.8% 5-year OHT survival. PLE was cured in 77.7% of hospital survivors, but the 5-year Kaplan-Meier survival estimate in PLE was 46.3 ± 14.4 vs 84.3 ± 5.5% in non-PLE (P = 0.0147). Cox proportional hazards identified early FF (P = 0.0005), complex Fontan pathway obstruction (P = 0.0043) and PLE (P = 0.0033) as independent predictors of 5-year mortality. CONCLUSIONS OHT is an excellent surgical option for late FF with impaired ventricular function. Protein dispersion improves with OHT, but PLE negatively affects the mid-term OHT outcome, mainly for early infective complications.

Linking megathrust earthquakes to brittle deformation in a fossil accretionary complex

Seismological data from recent subduction earthquakes suggest that megathrust earthquakes induce transient stress changes in the upper plate that shift accretionary wedges into an unstable state. These stress changes have, however, never been linked to geological structures preserved in fossil accretionary complexes. The importance of coseismically induced wedge failure has therefore remained largely elusive. Here we show that brittle faulting and vein formation in the palaeo-accretionary complex of the European Alps record stress changes generated by subduction-related earthquakes. Early veins formed at shallow levels by bedding-parallel shear during coseismic compression of the outer wedge. In contrast, subsequent vein formation occurred by normal faulting and extensional fracturing at deeper levels in response to coseismic extension of the inner wedge. Our study demonstrates how mineral veins can be used to reveal the dynamics of outer and inner wedges, which respond in opposite ways to megathrust earthquakes by compressional and extensional faulting, respectively.

Quantitative multi-slice computed tomography assessment of the mitral valvular complex for transcatheter mitral valve interventions part 1: systematic measurement methodology and inter-observer variability

AIMS Transcatheter mitral valve replacement (TMVR) is an emerging technology with the potential to treat patients with severe mitral regurgitation at excessive risk for surgical mitral valve surgery. Multimodal imaging of the mitral valvular complex and surrounding structures will be an important component for patient selection for TMVR. Our aim was to describe and evaluate a systematic multi-slice computed tomography (MSCT) image analysis methodology that provides measurements relevant for transcatheter mitral valve replacement. METHODS AND RESULTS A systematic step-by-step measurement methodology is described for structures of the mitral valvular complex including: the mitral valve annulus, left ventricle, left atrium, papillary muscles and left ventricular outflow tract. To evaluate reproducibility, two observers applied this methodology to a retrospective series of 49 cardiac MSCT scans in patients with heart failure and significant mitral regurgitation. For each of 25 geometrical metrics, we evaluated inter-observer difference and intra-class correlation. The inter-observer difference was below 10% and the intra-class correlation was above 0.81 for measurements of critical importance in the sizing of TMVR devices: the mitral valve annulus diameters, area, perimeter, the inter-trigone distance, and the aorto-mitral angle. CONCLUSIONS MSCT can provide measurements that are important for patient selection and sizing of TMVR devices. These measurements have excellent inter-observer reproducibility in patients with functional mitral regurgitation.

Out-of-Body Experiences and Other Complex Dissociation Experiences in a Patient with Unilateral Peripheral Vestibular Damage and Deficient Multisensory Integration

Out-of-body experiences (OBEs) are illusory perceptions of one's body from an elevated disembodied perspective. Recent theories postulate a double disintegration process in the personal (visual, proprioceptive and tactile disintegration) and extrapersonal (visual and vestibular disintegration) space as the basis of OBEs. Here we describe a case which corroborates and extends this hypothesis. The patient suffered from peripheral vestibular damage and presented with OBEs and lucid dreams. Analysis of the patient's behaviour revealed a failure of visuo-vestibular integration and abnormal sensitivity to visuo-tactile conflicts that have previously been shown to experimentally induce out-of-body illusions (in healthy subjects). In light of these experimental findings and the patient's symptomatology we extend an earlier model of the role of vestibular signals in OBEs. Our results advocate the involvement of subcortical bodily mechanisms in the occurrence of OBEs.

Disruption of the CD4–major histocompatibility complex class II interaction blocks the development of CD4+ T cells in vivo

The experiments presented in this report were designed to specifically examine the role of CD4–major histocompatibility complex (MHC) class II interactions during T cell development in vivo. We have generated transgenic mice expressing class II molecules that cannot interact with CD4 but that are otherwise competent to present peptides to the T cell receptor. MHC class II expression was reconstituted in Aβ gene knock-out mice by injection of a transgenic construct encoding either the wild-type I-Aβb protein or a construct encoding a mutation designed to specifically disrupt binding to the CD4 molecule. We demonstrate that the mutation, EA137 and VA142 in the β2 domain of I-Ab, is sufficient to disrupt CD4–MHC class II interactions in vivo. Furthermore, we show that this interaction is critical for the efficient selection of a complete repertoire of mature CD4+ T helper cells as evidenced by drastically reduced numbers of conventional CD4+ T cells in animals expressing the EA137/VA142 mutant I-Ab and by the failure to positively select the transgenic AND T cell receptor on the mutated I-Ab. These results underscore the importance of the CD4–class II interaction in the development of mature peripheral CD4+ T cells.