Collagen Fiber Regulation in Human Pediatric Aortic Valve Development and Disease

Congenital aortic valve stenosis (CAVS) affects up to 10% of the world population without medical therapies to treat the disease. New molecular targets are continually being sought that can halt CAVS progression. Collagen deregulation is a hallmark of CAVS yet remains mostly undefined. Here, histological studies were paired with high resolution accurate mass (HRAM) collagen-targeting proteomics to investigate collagen fiber production with collagen regulation associated with human AV development and pediatric end-stage CAVS (pCAVS). Histological studies identified collagen fiber realignment and unique regions of high-density collagen in pCAVS. Proteomic analysis reported specific collagen peptides are modified by hydroxylated prolines (HYP), a post-translational modification critical to stabilizing the collagen triple helix. Quantitative data analysis reported significant regulation of collagen HYP sites across patient categories. Non-collagen type ECM proteins identified (26 of the 44 total proteins) have direct interactions in collagen synthesis, regulation, or modification. Network analysis identified BAMBI (BMP and Activin Membrane Bound Inhibitor) as a potential upstream regulator of the collagen interactome. This is the first study to detail the collagen types and HYP modifications associated with human AV development and pCAVS. We anticipate that this study will inform new therapeutic avenues that inhibit valvular degradation in pCAVS and engineered options for valve replacement

iQuantitator: A tool for protein expression inference using iTRAQ

<p>Abstract</p> <p>Background</p> <p>Isobaric Tags for Relative and Absolute Quantitation (iTRAQ™) [Applied Biosystems] have seen increased application in differential protein expression analysis. To facilitate the growing need to analyze iTRAQ data, especially for cases involving multiple iTRAQ experiments, we have developed a modeling approach, statistical methods, and tools for estimating the relative changes in protein expression under various treatments and experimental conditions.</p> <p>Results</p> <p>This modeling approach provides a unified analysis of data from multiple iTRAQ experiments and links the observed quantity (reporter ion peak area) to the experiment design and the calculated quantity of interest (treatment-dependent protein and peptide fold change) through an additive model under log transformation. Others have demonstrated, through a case study, this modeling approach and noted the computational challenges of parameter inference in the unbalanced data set typical of multiple iTRAQ experiments. Here we present the development of an inference approach, based on hierarchical regression with batching of regression coefficients and Markov Chain Monte Carlo (MCMC) methods that overcomes some of these challenges. In addition to our discussion of the underlying method, we also present our implementation of the software, simulation results, experimental results, and sample output from the resulting analysis report.</p> <p>Conclusion</p> <p>iQuantitator's process-based modeling approach overcomes limitations in current methods and allows for application in a variety of experimental designs. Additionally, hypertext-linked documents produced by the tool aid in the interpretation and exploration of results.</p

Liver Disease and HPLC Quantification of Disialotransferrin for Heavy Alcohol Use: A Case Series

Rocky outcropping of shoreline near seawall of one of the cottages; Located on Little Harbor, part the outermost island of Casco Bay. Driftwood is the oldest Inn on Bailey Island and has been in continuous operation for over 100 years. Bailey Island is two and a half miles long by one half mile wide. The Inn sits on three acres with several large cottages and cabins

Imbalanced Expression of <i>Vcan</i> mRNA Splice Form Proteins Alters Heart Morphology and Cellular Protein Profiles

<div><p>The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the <i>Vcan</i> null mouse called heart defect (<i>hdf</i>). Total absence of the <i>Vcan</i> gene halts heart development at a stage prior to the heart’s pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican’s expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, <i>Vcan</i>^(tm1Zim), of heart defects that results from deletion of exon 7 in the <i>Vcan</i> gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the <i>Vcan</i> gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of <i>Vcan</i> exon 7. The <i>Vcan</i>^(tm1Zim) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles.</p></div

Targeting protein tyrosine phosphatases for CDK6-induced immunotherapy resistance

Summary: Elucidating the mechanisms of resistance to immunotherapy and developing strategies to improve its efficacy are challenging goals. Bioinformatics analysis demonstrates that high CDK6 expression in melanoma is associated with poor progression-free survival of patients receiving single-agent immunotherapy. Depletion of CDK6 or cyclin D3 (but not of CDK4, cyclin D1, or D2) in cells of the tumor microenvironment inhibits tumor growth. CDK6 depletion reshapes the tumor immune microenvironment, and the host anti-tumor effect depends on cyclin D3/CDK6-expressing CD8+ and CD4+ T cells. This occurs by CDK6 phosphorylating and increasing the activities of PTP1B and T cell protein tyrosine phosphatase (TCPTP), which, in turn, decreases tyrosine phosphorylation of CD3ζ, reducing the signal transduction for T cell activation. Administration of a PTP1B and TCPTP inhibitor prove more efficacious than using a CDK6 degrader in enhancing T cell-mediated immunotherapy. Targeting protein tyrosine phosphatases (PTPs) might be an effective strategy for cancer patients who resist immunotherapy treatment

Three-dimensional reconstructions and quantitative measurements of wild-type and <i>Vcan</i> exon 7 AV associated cushions in E13.5 pc hearts.

<p>The differences found in the mesenchymalized AV cushions, visually apparent in the 3-dimensional comparisons (Panel A, ventral view; B, dorsal view), were also quantified using the AMIRA imaging software to measure the cushion volumes. A significant reduction in volume was measured in the central AV cushion comprising the aortic leaflets (AL in panels A, B; reduced 35%; *p<0.034 n = 3 for each genotype, panel C) and septal (SL in panels A,B; reduced 30% p<0.046 n = 3 for each genotype in panel D). A significant (*p value 0.05 panel G) decrease in volume (0.58x) of the dorsal mesenchymal protrusion (DM) was measured. The other cushions showed no significant difference and the overall size of the E13.5 <i>Vcan</i>^(tm1Zim) and wild-type hearts (measured by tissue weight) was not significantly different (74 mg and 75.6 mg respectively; n = 3 for each genotype). AL-aortic leaflet (red); PL-parietal leaflet (blue); SL-septal leaflet (pink); ML-mural leaflet (green); DM-dorsal mesenchyme (white).</p

Histological comparison of cushions in the <i>Vcan</i>^(tm1Zim).

<p>Hematoxylin/eosin stained sections of postnatal day 1 (B, D) and <i>Vcan</i>^(tm1Zim) (A, C) hearts were compared. Boxed region in A and B of the cushion is shown higher magnification in C and D. Note the smaller cushions in the <i>Vcan</i>^(tm1Zim) heart. Panel A and B are the same magnification as are C and D; magnification bars = 200 µm.</p

Normal rotation and integration of the outflow tract (cardiac outlet) into the fused atrioventricular (AV) cushion in the final stages of looping and septal alignment.

<p>Upper panels are cross section dorsal views showing left and right AV canals, aorta and pulmonary trunks (Ao & P). Lower panels are depictions of the sagittal view. The dotted line indicates approximate section shown in upper panels. The myocardium (*inner curvature) between the outlet and AV cushion is removed and myocardial cells invade the proximal outlet cushion as in E12.5. P-pulmonary artery, Ao-aorta, LAV & RAV, left & right atrioventricular canals, M-mitral valve, T-tricuspid valve.</p

Western blot comparison of four proteins with altered abundance by itraq in <i>Vcan</i>^(tm1Zim) mutant and wild-type hearts.

<p>Proteins that showed different levels of altered abundance in the <i>Vcan</i>^(tm1Zim) hearts are shown in the blot. Annexin A6 and Stathmin are proteins expressed in the heart that showed a relative decrease (0.85x and 0.73x respectively) in abundance in the <i>Vcan</i>^(tm1Zim) mutant by iTRAQ and serphin1 (Hsp 47) that by iTRAQ showed increased abundance (1.55x). Additionally, Desmin that did not change with any significance between mutant (Mut) and wild-type (Wt) by iTRAQ, showed no significant change by western blot. Results of average relative density measurements of separately analyzed hearts are shown in the graph for each protein (for Stmn, Hsp47, Desmin n = 3; p<0.01; for Annexin A6 n = 2; p<0.0045).</p