Exome sequencing identifies mitochondrial alanyl-tRNA synthetase mutations in infantile mitochondrial cardiomyopathy

Infantile cardiomyopathies are devastating fatal disorders of the neonatal period or the first year of life. Mitochondrial dysfunction is a common cause of this group of diseases, but the underlying gene defects have been characterized in only a minority of cases, because tissue specificity of the manifestation hampers functional cloning and the heterogeneity of causative factors hinders collection of informative family materials. We sequenced the exome of a patient who died at the age of 10 months of hypertrophic mitochondrial cardiomyopathy with combined cardiac respiratory chain complex I and IV deficiency. Rigorous data analysis allowed us to identify a homozygous missense mutation in AARS2, which we showed to encode the mitochondrial alanyl-tRNA synthetase (mtAlaRS). Two siblings from another family, both of whom died perinatally of hypertrophic cardiomyopathy, had the same mutation, compound heterozygous with another missense mutation. Protein structure modeling of mtAlaRS suggested that one of the mutations affected a unique tRNA recognition site in the editing domain, leading to incorrect tRNA aminoacylation, whereas the second mutation severely disturbed the catalytic function, preventing tRNA aminoacylation. We show here that mutations in AARS2 cause perinatal or infantile cardiomyopathy with near-total combined mitochondrial respiratory chain deficiency in the heart. Our results indicate that exome sequencing is a powerful tool for identifying mutations in single patients and allows recognition of the genetic background in single-gene disorders of variable clinical manifestation and tissue-specific disease. Furthermore, we show that mitochondrial disorders extend to prenatal life and are an important cause of early infantile cardiac failure.     

A multi-metabolite analysis of serum by 1H NMR spectroscopy: early systemic signs of Alzheimer's disease

A three-molecular-window approach for ¹H NMR spectroscopy of serum is presented to obtain specific molecular data on lipoproteins, various low-molecular-weight metabolites, and individual lipid molecules together with their degree of (poly)(un)saturation. The multiple data were analysed with self-organising maps, illustrating the strength of the approach as a holistic metabonomics framework in solely data-driven metabolic phenotyping. We studied 180 serum samples of which 30% were related to mild cognitive impairment (MCI), a neuropsychological diagnosis with severely increased risk for Alzheimer’s disease (AD). The results underline the association between MCI and the metabolic syndrome (MetS). Additionally, the low relativeamount of ω-3 fatty acids appears more indicative of MCI than low serum ω-3 or polyunsaturated fatty acid concentration as such. The analyses also feature the role of elevated glycoproteins in the risk for AD, supporting the view that coexistence of inflammation and the MetS forms a high risk condition for cognitive decline.     

Angiogenic effects of dual gene transfer of bFGF and PDGF-BB after myocardial infarction

Therapeutic effects of combination of angiogenic growth factors for the treatment of ischemia after myocardial infarction are largely unknown. Plasmids expressing basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB) or their combination with a 1:1 mass ratio were injected into hearts with 7-day-old myocardial infarction. Hearts were harvested after 1 and 4 weeks after gene transfer. The major findings in this chronic myocardial infarction model were that bFGF, PDGF-BB and their combination all had a more pronounced angiogenic effect on the arteriolar than the capillary level. bFGF stimulated both capillary and arteriolar growth while PDGF-BB preferentially stimulated arterioles. The combination increased the amount of both capillaries and arterioles and in addition gave rise to stable capillaries compared to single factor transfer but did not further enhance angiogenesis. No cardiovascular side effects were observed after gene transfer.     

ANP and BNP but not VEGF are regionally overexpressed in ischemic human myocardium

Angiogenic gene therapy in angina pectoris has been disappointing so far. Reasons might be that the administered genes already are overexpressed in ischemic myocardium, or that atrial and brain natriuretic peptides (ANP and BNP) are overexpressed, as they have anti-angiogenic effects. Five stable angina pectoris patients without heart failure were studied. Left ventricular biopsies were taken during coronary by-pass surgery from a region with stress-inducible ischemia and from a normal region. Both ANP and BNP but not vascular endothelial growth factor (VEGF) and VEGF-receptor 1 and 2 were overexpressed in ischemic regions compared to non-ischemic regions as measured by real-time PCR. The expression of 15 other angiogenic genes measured by oligonucleotide arrays was not consistently increased in ischemic regions. The overexpression of ANP and BNP suggests an anti-angiogenic effect in ischemic heart disease. The lack of overexpression of angiogenic genes supports the concept of therapeutic overexpression of these genes.     

Angiogenic and cardiac functional effects of dual gene transfer of VEGF-A165 and PDGF-BB after myocardial infarction

Therapeutic angiogenesis is a potential treatment modality for myocardial ischemia. phVEGF-A(165), phPDGF-BB, or a combination of the two were injected into the myocardial infarct border zone in rats 7 days after ligation of the coronary left anterior descending artery. Cardiac function was measured by echocardiography. Hearts were harvested 1 and 4 weeks after plasmid injection. phVEGF-A(165) increased capillary density more than phPDGF-BB, and phPDGF-BB preferentially stimulated arteriolar growth. The combination increased both capillaries and arterioles but did not enhance angiogenesis any more than single plasmid treatments did. VEGF-A(165) and the combination of phVEGF-A(165) and phPDGF-BB counteracted left ventricular dilatation after 1 week but did not counteract further deterioration.     

Molecular lipidomics of exosomes released by PC-3 prostate cancer cells

The molecular lipid composition of exosomes is largely unknown. In this study, sophisticated shotgun and targeted molecular lipidomic assays were performed for in-depth analysis of the lipidomes of the metastatic prostate cancer cell line, PC-3, and their released exosomes. This study, based in the quantification of approximately 280 molecular lipid species, provides the most extensive lipid analysis of cells and exosomes to date. Interestingly, major differences were found in the lipid composition of exosomes compared to parent cells. Exosomes show a remarkable enrichment of distinct lipids, demonstrating an extraordinary discrimination of lipids sorted into these microvesicles. In particular, exosomes are highly enriched in glycosphingolipids, sphingomyelin, cholesterol, and phosphatidylserine (mol% of total lipids). Furthermore, lipid species, even of classes not enriched in exosomes, were selectively included in exosomes. Finally, it was found that there is an 8.4-fold enrichment of lipids per mg of protein in exosomes. The detailed lipid composition provided in this study may be useful to understand the mechanism of exosome formation, release and function. Several of the lipids enriched in exosomes could potentially be used as cancer biomarkers.     

Overweight People Have Low Levels of Implicit Weight Bias,but Overweight Nations Have High Levels of Implicit Weight Bias

Although a greater degree of personal obesity is associated with weaker negativity toward overweight people on both explicit (i.e., self-report) and implicit (i.e., indirect behavioral) measures, overweight people still prefer thin people on average. We investigated whether the national and cultural context – particularly the national prevalence of obesity – predicts attitudes toward overweight people independent of personal identity and weight status. Data were collected from a total sample of 338,121 citizens from 71 nations in 22 different languages on the Project Implicit website (https://implicit.harvard.edu/) between May 2006 and October 2010. We investigated the relationship of the explicit and implicit weight bias with the obesity both at the individual (i.e., across individuals) and national (i.e., across nations) level. Explicit weight bias was assessed with self-reported preference between overweight and thin people; implicit weight bias was measured with the Implicit Association Test (IAT). The national estimates of explicit and implicit weight bias were obtained by averaging the individual scores for each nation. Obesity at the individual level was defined as Body Mass Index (BMI) scores, whereas obesity at the national level was defined as three national weight indicators (national BMI, national percentage of overweight and underweight people) obtained from publicly available databases. Across individuals, greater degree of obesity was associated with weaker implicit negativity toward overweight people compared to thin people. Across nations, in contrast, a greater degree of national obesity was associated with stronger implicit negativity toward overweight people compared to thin people. This result indicates a different relationship between obesity and implicit weight bias at the individual and national levels.     

Rhizavidin from Rhizobium etli: the first natural dimer in the avidin protein family

Rhizobium etli CFN42 is a symbiotic nitrogen-fixing bacterium of the common bean Phaseolus vulgaris. The symbiotic plasmid p42d of R. etli comprises a gene encoding a putative (strept)avidin-like protein, named rhizavidin. The amino acid sequence identity of rhizavidin in relation to other known avidin-like proteins is 20-30%. The amino acid residues involved in the (strept)avidin-biotin interaction are well conserved in rhizavidin. The structural and functional properties of rhizavidin were carefully studied, and we found that rhizavidin shares characteristics with bradavidin, streptavidin and avidin. However, we found that it is the first naturally occurring dimeric protein in the avidin protein family, in contrast with tetrameric (strept)avidin and bradavidin. Moreover, it possesses a proline residue after a flexible loop (GGSG) in a position close to Trp-110 in avidin, which is an important biotin-binding residue. [3H]Biotin dissociation and ITC (isothermal titration calorimetry) experiments showed dimeric rhizavidin to be a high-affinity biotin-binding protein. Its thermal stability was lower than that of avidin; although similar to streptavidin, it was insensitive to proteinase K. The immunological cross-reactivity of rhizavidin was tested with human serum samples obtained from cancer patients exposed to (strept)avidin. No significant cross-reactivity was observed. The biodistribution of the protein was studied by SPECT (single-photon emission computed tomography) imaging in rats. Similarly to avidin, rhizavidin was observed to accumulate rapidly, mainly in the liver. Evidently, rhizavidin could be used as a complement to (strept)avidin in (strept)avidin-biotin technology.