Characterization of copy-number variants in a large cohort of patients with von Willebrand disease reveals a relationship between disrupted regions and disease type

BACKGROUND: Genetic analysis for von Willebrand disease (VWD) commonly utilizes DNA sequencing to identify variants in the von Willebrand factor ( OBJECTIVES: This study aimed to characterize CNVs in a large sample of VWF mutation-negative VWD patients. METHODS: To determine the role of CNVs in VWD, a VWF high-resolution comparative genomic hybridization array was custom-designed to avoid multiple sequence variations, repeated sequences, and the RESULTS: Among the 204 patients, 7 unique CNVs were found, with a total of 24 CNVs (12%). Of the 7 unique CNVs, 1 was novel, 1 was found in a VWF database, and 5 were previously reported. All patients with type 1C VWD and a CNV had the same exon 33 and 34 in-frame deletion. Certain clinical variables were also significantly different between those with and without CNVs. CONCLUSION: The in-frame deletion in patients with type 1C VWD exactly matches the D4N module of the D4 domain, a region where mutations and deletions are known to affect clearance. We observed significantly higher VWF-to-ristocetin cofactor levels in patients with type 1C VWD and a CNV than in patients without a CNV, suggesting a relationship between CNVs and the increased clearance observed in patients with type 1C VWD. Glycoprotein IbM activity was significantly lower in patients with type 1 VWD and a CNV than in patients without a CNV, suggesting that platelet binding is more affected by CNVs than single base pair mutations. This work elucidates some of the underlying genetic mechanisms of CNVs in these patients

Re-establishment of VWF-dependent Weibel-Palade bodies in VWD endothelial cells

Type 3 von Willebrand disease (VWD) is a severe hemorrhagic defect in humans. We now identify the homozygous mutation in the Chapel Hill strain of canine type 3 VWD that results in premature termination of von Willebrand factor (VWF) protein synthesis. We cultured endothelium from VWD and normal dogs to study intracellular VWF trafficking and Weibel-Palade body formation. Weibel-Palade bodies could not be identified in the canine VWD aortic endothelial cells (VWD-AECs) by P-selectin, VWFpp, or VWF immunostaining and confocal microscopy. We demonstrate the reestablishment of Weibel-Palade bodies that recruit endogenous P-selectin by expressing wild-type VWF in VWD-AECs. Expression of mutant VWF proteins confirmed that VWF multimerization is not necessary for Weibel-Palade body creation. Although the VWF propeptide is required for the formation of Weibel-Palade bodies, it cannot independently induce the formation of the granule. These VWF-null endothelial cells provide a unique opportunity to examine the biogenesis of Weibel-Palade bodies in endothelium from a canine model of type 3 VWD

Validation of a clinical-grade assay to measure donor-derived cell-free DNA in solid organ transplant recipients

[Abstract] The use of circulating cell-free DNA (cfDNA) as a biomarker in transplant recipients offers advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection and immunosuppression optimization. However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant recipient plasma is low and challenging to quantify. Previously reported methods to measure dd-cfDNA require donor and recipient genotyping, which is impractical in clinical settings and adds cost. We developed a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms to accurately quantify dd-cfDNA in transplant recipients without separate genotyping. Analytical performance of the assay was characterized and validated using 1117 samples comprising the National Institute for Standards and Technology Genome in a Bottle human reference genome, independently validated reference materials, and clinical samples. The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across the linear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision. Application of the assay to clinical samples from heart transplant recipients demonstrated increased levels of dd-cfDNA in patients with biopsy-confirmed rejection and decreased levels of dd-cfDNA after successful rejection treatment. This noninvasive clinical-grade sequencing assay can be completed within 3 days, providing the practical turnaround time preferred for transplanted organ surveillance

The Power of Play: A Pediatric Role in Enhancing Development in Young Children

Children need to develop a variety of skill sets to optimize their development and manage toxic stress. Research demonstrates that developmentally appropriate play with parents and peers is a singular opportunity to promote the social-emotional, cognitive, language, and self-regulation skills that build executive function and a prosocial brain. Furthermore, play supports the formation of the safe, stable, and nurturing relationships with all caregivers that children need to thrive

Advanced Land Imager Assessment System

The Advanced Land Imager Assessment System (ALIAS) supports radiometric and geometric image processing for the Advanced Land Imager (ALI) instrument onboard NASA s Earth Observing-1 (EO-1) satellite. ALIAS consists of two processing subsystems for radiometric and geometric processing of the ALI s multispectral imagery. The radiometric processing subsystem characterizes and corrects, where possible, radiometric qualities including: coherent, impulse; and random noise; signal-to-noise ratios (SNRs); detector operability; gain; bias; saturation levels; striping and banding; and the stability of detector performance. The geometric processing subsystem and analysis capabilities support sensor alignment calibrations, sensor chip assembly (SCA)-to-SCA alignments and band-to-band alignment; and perform geodetic accuracy assessments, modulation transfer function (MTF) characterizations, and image-to-image characterizations. ALIAS also characterizes and corrects band-toband registration, and performs systematic precision and terrain correction of ALI images. This system can geometrically correct, and automatically mosaic, the SCA image strips into a seamless, map-projected image. This system provides a large database, which enables bulk trending for all ALI image data and significant instrument telemetry. Bulk trending consists of two functions: Housekeeping Processing and Bulk Radiometric Processing. The Housekeeping function pulls telemetry and temperature information from the instrument housekeeping files and writes this information to a database for trending. The Bulk Radiometric Processing function writes statistical information from the dark data acquired before and after the Earth imagery and the lamp data to the database for trending. This allows for multi-scene statistical analyses

Nitric Oxide Mediates Stretch-Induced Ca2+ Release via Activation of Phosphatidylinositol 3-Kinase-Akt Pathway in Smooth Muscle

Hollow smooth muscle organs such as the bladder undergo significant changes in wall tension associated with filling and distension, with attendant changes in muscle tone. Our previous study indicated that stretch induces Ca(2+) release occurs in the form of Ca(2+) sparks and Ca(2+) waves in urinary bladder myocytes. While, the mechanism underlying stretch-induced Ca2+ release in smooth muscle is unknown.We examined the transduction mechanism linking cell stretch to Ca(2+) release. The probability and frequency of Ca(2+) sparks induced by stretch were closely related to the extent of cell extension and the time that the stretch was maintained. Experiments in tissues and single myocytes indicated that mechanical stretch significantly increases the production of nitric oxide (NO) and the amplitude and duration of muscle contraction. Stretch-induced Ca(2+) sparks and contractility increases were abrogated by the NO inhibitor L-NAME and were also absent in eNOS knockout mice. Furthermore, exposure of eNOS null mice to exogenously generated NO induced Ca(2+) sparks. The soluble guanylyl cyclase inhibitor ODQ did not inhibit SICR, but this process was effectively blocked by the PI3 kinase inhibitors LY494002 and wortmannin; the phosphorylation of Akt and eNOS were up-regulated by 204+/-28.6% and 258+/-36.8% by stretch, respectively. Moreover, stretch significantly increased the eNOS protein expression level.Taking together, these results suggest that stretch-induced Ca2+ release is NO dependent, resulting from the activation of PI3K/Akt pathway in smooth muscle

Gene expression profiling of alveolar soft-part sarcoma (ASPS)

<p>Abstract</p> <p>Background</p> <p>Alveolar soft-part sarcoma (ASPS) is an extremely rare, highly vascular soft tissue sarcoma affecting predominantly adolescents and young adults. In an attempt to gain insight into the pathobiology of this enigmatic tumor, we performed the first genome-wide gene expression profiling study.</p> <p>Methods</p> <p>For seven patients with confirmed primary or metastatic ASPS, RNA samples were isolated immediately following surgery, reverse transcribed to cDNA and each sample hybridized to duplicate high-density human U133 plus 2.0 microarrays. Array data was then analyzed relative to arrays hybridized to universal RNA to generate an unbiased transcriptome. Subsequent gene ontology analysis was used to identify transcripts with therapeutic or diagnostic potential. A subset of the most interesting genes was then validated using quantitative RT-PCR and immunohistochemistry.</p> <p>Results</p> <p>Analysis of patient array data versus universal RNA identified elevated expression of transcripts related to angiogenesis (ANGPTL2, HIF-1 alpha, MDK, c-MET, VEGF, TIMP-2), cell proliferation (PRL, IGFBP1, NTSR2, PCSK1), metastasis (ADAM9, ECM1, POSTN) and steroid biosynthesis (CYP17A1 and STS). A number of muscle-restricted transcripts (ITGB1BP3/MIBP, MYF5, MYF6 and TRIM63) were also identified, strengthening the case for a muscle cell progenitor as the origin of disease. Transcript differentials were validated using real-time PCR and subsequent immunohistochemical analysis confirmed protein expression for several of the most interesting changes (MDK, c-MET, VEGF, POSTN, CYP17A1, ITGB1BP3/MIBP and TRIM63).</p> <p>Conclusion</p> <p>Results from this first comprehensive study of ASPS gene expression identifies several targets involved in angiogenesis, metastasis and myogenic differentiation. These efforts represent the first step towards defining the cellular origin, pathogenesis and effective treatment strategies for this atypical malignancy.</p

Equity Premium Predictions with Adaptive Macro Indexes

Fundamental economic conditions are crucial determinants of equity premia. However, commonly used predictors do not adequately capture the changing nature of economic conditions and hence have limited power in forecasting equity returns. To address the inadequacy, this paper constructs macro indexes from large data sets and adaptively chooses optimal indexes to predict stock returns. I find that adaptive macro indexes explain a substantial fraction of the short-term variation in future stock returns and have more forecasting power than both the historical average of stock returns and commonly used predictors. The forecasting power exhibits a strong cyclical pattern, implying the ability of adaptive macro indexes to capture time-varying economic conditions. This finding highlights the importance of using dynamically measured economic conditions to investigate empirical linkages between the equity premium and macroeconomic fundamentals