Severity of Mitral Valve Degeneration Is Associated with Chromosome 15 Loci in Whippet Dogs

Mitral valve degeneration (MVD) is the most common form of heart disease in dogs, frequently leading to left-sided congestive heart failure and cardiac mortality. Although breed-specific disease characteristics and overrepresentation point towards a genetic origin for MVD, a causative mutation and complete molecular pathogenesis are unknown. Whippet dogs are overrepresented in incidence of MVD, suggesting an inherited component in this breed. Expressivity of this condition is variable with some dogs showing evidence of more severe disease at earlier ages than other dogs. This phenomenon makes a traditional case versus control genetic study prone to phenotyping error. This study sought to avoid these common pitfalls by identifying genetic loci associated with severity of MVD in Whippets through a genome-wide association study (GWAS). 138 Whippet dogs were characterized for MVD by echocardiographic examination and a novel disease severity score was developed and adjusted for age in each subject. Single nucleotide polymorphism (SNP) genotype data (170k Illumina CanineHD SnpChip) was obtained for DNA isolated from blood of each study subject. Continuous variable genome wide association was performed after correction for population stratification by efficient mixed model association expedited (EMMAX) in 130 dogs. A genome wide significant association was identified on chromosome 15 (peak locus 57,770,326; P_adj = 0.049) and secondary loci of suggestive association were identified on chromosome 2 (peak locus 37,628,875; P_adj = 0.079). Positional candidate genes were identified within the primary and secondary loci including follistatin-related protein 5 precursor (FSTL5) and Rho GTPase-activating protein 26 (ARHGAP26). These results support the hypothesis that severity of MVD in whippets has a genetic basis and warrants further study by either candidate gene sequencing or next-generation techniques.     

The Effect of Space Flight on Monoclonal Antibody Synthesis in a Hybridoma Mouse Cell Line

The hybridoma cell line, 3G10G5, producing a monoclonal antibody to the major capsid protein VP1 from the avian polyomavirus budgerigar fledgling disease virus, was produced from a Balb/C mouse. This cell line was used to test the effects of microgravity on cellular processes, specifically protein synthesis. A time course study utilizing incorporation of [³⁵S]methionine into newly synthesized monoclonal antibody was performed on STS-77. After 5.5 days, it was observed that cell counts for the samples exposed to microgravity were lower than those of ground-based samples. However, radiolabel incorporation of the synthesized monoclonal antibody was similar in both orbiter and ground control samples. Overall, microgravity does not seem to have an effect on this cell line's ability to synthesize IgG protein.     

The neutralization of acidic coal mine lakes by additions of natural organic matter: a mesocosm test

Cylindrical polyethylene enclosures 3 m in length and 1 m in diameter reaching from the surface to the bottom were constructed in an acid (pH=3.1) lake on a coal surface mine in southern Illinois. Wheat straw was added to the enclosures to test the effects of dissimilatory sulfate reduction on water chemistry. Added straw increased sulfide concentrations, raised pH to 6.5, reduced O₂ and increased acid neutralizing capacity of the enclosed water columns when compared with a control enclosure and with the open lake. Generation of acid neutralizing capacity exceeded the standing stock of sulfide indicating that sulfide was removed either by precipitation of FeS or outgassing of H₂S. The pH and acid neutralizing capacity within the enclosures eventually returned to the level of the surrounding lake because of water exchange around the enclosure walls. Our results show that additions of organic matter to acid surface mine lakes result in the generation of acid neutralizing capacity.     

β2-Microglobulin Amyloid Fibrils Are Nanoparticles That Disrupt Lysosomal Membrane Protein Trafficking and Inhibit Protein Degradation by Lysosomes

Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of β₂-microglobulin (β₂m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented β₂m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented β₂m fibrils did not, however, cause cell death. Instead, fragmented β₂m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway.     

Long-chain Acyl-CoA Dehydrogenase Deficiency as a Cause of Pulmonary Surfactant Dysfunction

Long-chain acyl-CoA dehydrogenase (LCAD) is a mitochondrial fatty acid oxidation enzyme whose expression in humans is low or absent in organs known to utilize fatty acids for energy such as heart, muscle, and liver. This study demonstrates localization of LCAD to human alveolar type II pneumocytes, which synthesize and secrete pulmonary surfactant. The physiological role of LCAD and the fatty acid oxidation pathway in lung was subsequently studied using LCAD knock-out mice. Lung fatty acid oxidation was reduced in LCAD^−/− mice. LCAD^−/− mice demonstrated reduced pulmonary compliance, but histological examination of lung tissue revealed no obvious signs of inflammation or pathology. The changes in lung mechanics were found to be due to pulmonary surfactant dysfunction. Large aggregate surfactant isolated from LCAD^−/− mouse lavage fluid had significantly reduced phospholipid content as well as alterations in the acyl chain composition of phosphatidylcholine and phosphatidylglycerol. LCAD^−/− surfactant demonstrated functional abnormalities when subjected to dynamic compression-expansion cycling on a constrained drop surfactometer. Serum albumin, which has been shown to degrade and inactivate pulmonary surfactant, was significantly increased in LCAD^−/− lavage fluid, suggesting increased epithelial permeability. Finally, we identified two cases of sudden unexplained infant death where no lung LCAD antigen was detectable. Both infants were homozygous for an amino acid changing polymorphism (K333Q). These findings for the first time identify the fatty acid oxidation pathway and LCAD in particular as factors contributing to the pathophysiology of pulmonary disease.     

Fast Approximate Quadratic Programming for Graph Matching

Quadratic assignment problems arise in a wide variety of domains, spanning operations research, graph theory, computer vision, and neuroscience, to name a few. The graph matching problem is a special case of the quadratic assignment problem, and graph matching is increasingly important as graph-valued data is becoming more prominent. With the aim of efficiently and accurately matching the large graphs common in big data, we present our graph matching algorithm, the Fast Approximate Quadratic assignment algorithm. We empirically demonstrate that our algorithm is faster and achieves a lower objective value on over 80% of the QAPLIB benchmark library, compared with the previous state-of-the-art. Applying our algorithm to our motivating example, matching C. elegans connectomes (brain-graphs), we find that it efficiently achieves performance.     

Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.