Search

Congenital heart disease (CHD) is the most common congenital anomaly, with an overall incidence of approximately 1% in the United Kingdom. Exome sequencing in large CHD cohorts has been performed to provide insights into the genetic aetiology of CHD. This includes a study of 1891 probands by our group in collaboration with others, which identified three novel genes—CDK13, PRKD1, and CHD4, in patients with syndromic CHD. PRKD1 encodes a serine/threonine protein kinase, which is important in a variety of fundamental cellular functions. Individuals with a heterozygous mutation in PRKD1 may have facial dysmorphism, ectodermal dysplasia and may have CHDs such as pulmonary stenosis, atrioventricular septal defects, coarctation of the aorta and bicuspid aortic valve. To obtain a greater appreciation for the role that this essential protein kinase plays in cardiogenesis and CHD, we have analysed a Prkd1 transgenic mouse model (Prkd1em1) carrying deletion of exon 2, causing loss of function. High‐resolution episcopic microscopy affords detailed morphological 3D analysis of the developing heart and provides evidence for an essential role of Prkd1 in both normal cardiac development and CHD. We show that homozygous deletion of Prkd1 is associated with complex forms of CHD such as atrioventricular septal defects, and bicuspid aortic and pulmonary valves, and is lethal. Even in heterozygotes, cardiac differences occur. However, given that 97% of Prkd1 heterozygous mice display normal heart development, it is likely that one normal allele is sufficient, with the defects seen most likely to represent sporadic events. Moreover, mRNA and protein expression levels were investigated by RT‐qPCR and western immunoblotting, respectively. A significant reduction in Prkd1 mRNA levels was seen in homozygotes, but not heterozygotes, compared to WT littermates. While a trend towards lower PRKD1 protein expression was seen in the heterozygotes, the difference was only significant in the homozygotes. There was no compensation by the related Prkd2 and Prkd3 at transcript level, as evidenced by RT‐qPCR. Overall, we demonstrate a vital role of Prkd1 in heart development and the aetiology of CHD. [ABSTRACT FROM AUTHOR]

Cardiogenesis is influenced by both environmental and genetic factors, with blood flow playing a critical role in cardiac remodelling. Perturbation of any of these factors could lead to abnormal heart development and hence the formation of congenital heart defects. Although abnormal blood flow has been associated with a number of heart defects, the effects of abnormal pressure load on the developing heart gene expression profile have to date not clearly been defined. To determine the heart transcriptional response to haemodynamic alteration during development, outflow tract (OFT) banding was employed in the chick embryo at Hamburger and Hamilton stage (HH) 21. Stereological and expression studies, including the use of global expression analysis by RNA sequencing with an optimised procedure for effective globin depletion, were subsequently performed on HH29 OFT‐banded hearts and compared with sham control hearts, with further targeted expression investigations at HH35. The OFT‐banded hearts were found to have an abnormal morphology with a rounded appearance and left‐sided dilation in comparison with controls. Internal analysis showed they typically had a ventricular septal defect and reductions in the myocardial wall and trabeculae, with an increase in the lumen on the left side of the heart. There was also a significant reduction in apoptosis. The differentially expressed genes were found to be predominately involved in contraction, metabolism, apoptosis and neural development, suggesting a cardioprotective mechanism had been induced. Therefore, altered haemodynamics during development leads to left‐sided dilation and differential expression of genes that may be associated with stress and maintaining cardiac output. [ABSTRACT FROM AUTHOR]

Survivin (also known as BIRC5) is a cancer-associated protein that exists in several locations in the cell. Its cytoplasmic residence in interphase cells is governed by CRM1 (also known as XPO1)- mediated nuclear exportation, and its localisation during mitosis to the centromeres and midzone microtubules is that of a canonical chromosomal passenger protein. In addition to these wellestablished locations, survivin is also a mitochondrial protein, but how it gets there and its function therein is presently unclear. Here, we show that the first ten amino acids at the N-terminus of survivin are sufficient to target GFP to the mitochondria in vivo, and ectopic expression of this decapeptide decreases cell adhesion and accelerates proliferation. The data support a signalling mechanism in which this decapeptide regulates the tyrosine kinase Src, leading to reduced focal adhesion plaques and disruption of F-actin organisation. This strongly suggests that the N-terminus of survivin is a mitochondrial-targeting sequence that regulates Src, and that survivin acts in concert with Src to promote tumorigenesis. [ABSTRACT FROM AUTHOR]

Purpose: The authors describe a design for prepatient region of interest attenuators (ROIAs) to reduce dose area product (DAP) for clinical use. The authors describe a model to predict DAP values from x-ray technique parameters recorded during a clinical procedure for image sequences obtained in the presence or absence of ROIAs. The model was developed primarily to determine what the DAP to a patient undergoing cardiac catheterization with a ROIA would have been if no ROIA had been used allowing a determination of DAP reduction. Methods: Copper ROIAs with thicknesses that vary gradually so as not to cause significant image artifacts were constructed. X-ray image sequences were acquired on a clinical catheterization system with and without ROIAs with varying x-ray technique parameters. DAP values were measured for all said exposures using an ionization chamber and compared to a model the authors developed. Results: The model can predict DAP values within 3.5% on average with or without ROIAs when compared to ionization chamber measurements. Conclusions: The proposed experimental design is adequate for measuring DAP reductions on the order of 1.5-3.5 that are expected when introducing a ROIA during patient catheterization imaging. [ABSTRACT FROM AUTHOR]

Objective Cognitive impairment and depressive symptoms are common among the geriatric population but the co-occurrence of both is rarely studied. The purpose of this study was to identify and compare the factors associated with three groups of elderly people: those assessed with cognitive impairment alone (COG), depressive symptoms alone (DEP) or co-existence of both (COG-DEP). Methods The cross-sectional study included 600 community-dwellers ages 65 and older. All participants underwent a comprehensive evaluation. Global cognition was measured by the Mini-Mental State Examination (MMSE) and depressive symptoms were defined by the Geriatric Depression Scale (GDS). Specific chronic illnesses relevant to the Charlson comorbidity index (CCI) were self-reported. Functional status was evaluated by the Katz' basic (ADL) and Lawton's instrumental (IADL) activities of daily living scales. Results COG-DEP was explained by IADL dependence (OR: 11.9, 95% CI: 4.59-30.78), ADL dependence (OR: 11.5, 95% CI: 5.59-23.69), cerebrovascular disease (OR: 3.6, 95% CI: 1.48-8.68), congestive heart failure (OR: 3.4, 95% CI: 1.77-6.59) and diabetes (OR: 2.6, 95% CI: 1.30-5.18), but it was best predicted by functional limitations in the adjusted model. Being functionally dependent and medically ill with shorter life expectancy was shown to significantly increase the odds of being DEP. Functional limitation in IADL was without distinction associated to COG, DEP and COG-DEP. Conclusion The present results on COG, DEP and COG-DEP show the particular relevance of certain medical comorbidities and functional limitations to those three distinct groups of elderly people. Copyright © 2010 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: This retrospective study determines the role of cognitive decline as a predictor of functional dependence. In a representative 600 community-dwellers aged 65 or older, we examined using a logistic regression model, the association between cognitive status (taking into account age and educational level) and dependence on basic and instrumental activities of daily living (ADL and IADL, resp.), controlling for socio-demographic variables and health conditions. The Mini-Mental State Examination (MMSE) scores were compared in participants with functional disability to perform basic and instrumental activities. Cognitive status influenced functional dependence on activities of daily living, basic (OR=4.1, 95%CI=2.7–6.1) and instrumental (OR=5.7, 95%CI=3.5–9.3), independently of gender, age, educational level and health conditions. Besides, cognitive impairment was associated with the dependence on certain basic (e.g., bathing, toileting) and instrumental (e.g., using the telephone, taking medications, and handling finances) activities. This was a gradual relationship, the highest cognitive decline implied the highest loss of ability at carrying out activities, with a larger impact on basic activities. These findings suggest that cognitive decline can be a predictor for functional dependence, independently of other variables, and turn into a very useful tool indicating the need for support. [Copyright &y& Elsevier]

We have developed a polypeptide lysostaphin FRET (fluorescence resonance energy transfer) substrate (MV11F) for the endopeptidase activity of lysostaphin. Site-directed mutants of lysostaphin that abolished the killing activity against Staphylococcus aureus also completely inhibited the endopeptidase activity against the MV11 FRET substrate. Lysostaphin-producing staphylococci are resistant to killing by lysostaphin through incorporation of serine residues at positions 3 and 5 of the pentaglycine cross-bridge in their cell walls. The MV11 FRET substrate was engineered to introduce a serine residue at each of four positions of the pentaglycine target site and it was found that only a serine residue at position 3 completely inhibited cleavage. The introduction of random, natural amino acid substitutions at position 3 of the pentaglycine target site demonstrated that only a glycine residue at this position was compatible with lysostaphin cleavage of the MV11 FRET substrate. A second series of polypeptide substrates (decoys) was developed with the GFP (green fluorescent protein) domain of MV11 replaced with that of the DNase domain of colicin E9. Using a competition FRET assay, the lysostaphin endopeptidase was shown to bind to a decoy peptide containing a GGSGG cleavage site. The MV11 substrate provides a valuable system to facilitate structure/function studies of the endopeptidase activity of lysostaphin and its orthologues.