Diaphragmatic function during hypoxemia: neonatal and developmental aspects

The effect of acute hypoxemia on diaphragmatic force output was studied in five young (age 4-8 days, wt 1.3-2.2 kg) and five older (age 16-19 days, wt 2.8-3.3 kg), anesthetized, spontaneously breathing piglets. Diaphragmatic force output was assessed by analysis of the transdiaphragmatic pressure (Pdi) generated during an occluded inspiratory effort, at end-expiratory lung volume, triggered by supramaximal transvenous stimulation of both phrenic nerves at frequencies of 20, 30, 50, and 100 Hz. During pressure measurements, the piglets were fitted with a rigid plaster cast covering the abdomen and lower third of the chest to ensure a consistency in diaphragmatic shortening during phrenic nerve stimulation. Pdi was measured under base-line conditions [inspired O2 fractional concentration (FIO2) = 0.50] and after 10 min of hypoxemia induced by breathing 12-14% FIO2. Pdi was significantly less than base line during acute hypoxemia at all frequencies of stimulation in both young and older piglets. The decline in the older piglets' Pdi during hypoxemia was significantly greater than that seen in younger piglets. We conclude that acute hypoxemia impairs the capacity of the developing piglet diaphragm to generate force. Furthermore, our data suggest that the young piglet is more resistant to the depressant effects of hypoxemia when compared to its older counterpart.     

Allelic Selection of Amplicons in Glioblastoma Revealed by Combining Somatic and Germline Analysis

Cancer is a disease driven by a combination of inherited risk alleles coupled with the acquisition of somatic mutations, including amplification and deletion of genomic DNA. Potential relationships between the inherited and somatic aspects of the disease have only rarely been examined on a genome-wide level. Applying a novel integrative analysis of SNP and copy number measurements, we queried the tumor and normal-tissue genomes of 178 glioblastoma patients from the Cancer Genome Atlas project for preferentially amplified alleles, under the hypothesis that oncogenic germline variants will be selectively amplified in the tumor environment. Selected alleles are revealed by allelic imbalance in amplification across samples. This general approach is based on genetic principles and provides a method for identifying important tumor-related alleles. We find that SNP alleles that are most significantly overrepresented in amplicons tend to occur in genes involved with regulation of kinase and transferase activity, and many of these genes are known contributors to gliomagenesis. The analysis also implicates variants in synapse genes. By incorporating gene expression data, we demonstrate synergy between preferential allelic amplification and expression in DOCK4 and EGFR. Our results support the notion that combining germline and tumor genetic data can identify regions relevant to cancer biology.     

Cardiac fibroblasts influence cardiomyocyte phenotype in vitro

Cardiac fibroblasts impact myocardial development and remodeling through intercellular contact with cardiomyocytes, but less is known about noncontact, profibrotic signals whereby fibroblasts alter cardiomyocyte behavior. Fibroblasts and cardiomyocytes were harvested from newborn rat ventricles and separated by serial digestion and gradient centrifugation. Cardiomyocytes were cultured in 1) standard medium, 2) standard medium diluted 1:1 with PBS, or 3) standard medium diluted 1:1 with medium conditioned 72 h by cardiac fibroblasts. Serum concentrations were held constant under all media conditions, and complete medium exchanges were performed daily. Cardiomyocytes began contracting within 24 h at clonal or mass densities with <5% of cells expressing vimentin. Immunocytochemical analysis revealed progressive expression of -smooth muscle actin in cardiomyocytes after 24 h in all conditions. Only cardiomyocytes in fibroblast-conditioned medium stopped contracting by 72 h. There was a significant, sustained increase in vimentin expression specific to these cultures (means ± SD: conditioned 46.3 ± 6.0 vs. control 5.3 ± 2.9%, P < 0.00025) typically with cardiac myosin heavy chain coexpression. Proteomics assays revealed 10 cytokines (VEGF, GRO/KC, monocyte chemoattractant protein-1, leptin, macrophage inflammatory protein-1, IL-6, IL-10, IL-12p70, IL-17, and tumor necrosis factor-) at or below detection levels in unconditioned medium that were significantly elevated in fibroblast-conditioned medium. Latent transforming growth factor- and RANTES were present in unconditioned medium but rose to higher levels in conditioned medium. Only granulocyte-macrophage colony-stimulating factor was present above threshold levels in standard medium but decreased with fibroblast conditioning. These data indicated that under the influence of fibroblast-conditioned medium, cardiomyocytes exhibited marked hypertrophy, diminished contractile capacity, and phenotype plasticity distinct from the dedifferentiation program present under standard culture conditions. proteomics; myosin heavy chain; vimentin; myofibroblast; primary culture; dedifferentiation; plasticity     

Allele-specific amplification in cancer revealed by SNP array analysis

Amplification, deletion, and loss of heterozygosity of genomic DNA are hallmarks of cancer. In recent years a variety of studies have emerged measuring total chromosomal copy number at increasingly high resolution. Similarly, loss-of-heterozygosity events have been finely mapped using high-throughput genotyping technologies. We have developed a probe-level allele-specific quantitation procedure that extracts both copy number and allelotype information from single nucleotide polymorphism (SNP) array data to arrive at allele-specific copy number across the genome. Our approach applies an expectation-maximization algorithm to a model derived from a novel classification of SNP array probes. This method is the first to our knowledge that is able to (a) determine the generalized genotype of aberrant samples at each SNP site (e.g., CCCCT at an amplified site), and (b) infer the copy number of each parental chromosome across the genome. With this method, we are able to determine not just where amplifications and deletions occur, but also the haplotype of the region being amplified or deleted. The merit of our model and general approach is demonstrated by very precise genotyping of normal samples, and our allele-specific copy number inferences are validated using PCR experiments. Applying our method to a collection of lung cancer samples, we are able to conclude that amplification is essentially monoallelic, as would be expected under the mechanisms currently believed responsible for gene amplification. This suggests that a specific parental chromosome may be targeted for amplification, whether because of germ line or somatic variation. An R software package containing the methods described in this paper is freely available at http://genome.dfci.harvard.edu/~tlaframb/PLASQ.     

SNP arrays in heterogeneous tissue: highly accurate collection of both germline and somatic genetic information from unpaired single tumor samples

SNP arrays provide reliable genotypes and can detect chromosomal aberrations at a high resolution. However, tissue heterogeneity is currently a major limitation for somatic tissue analysis. We have developed SOMATICs, an original program for accurate analysis of heterogeneous tissue samples. Fifty-four samples (42 tumors and 12 normal tissues) were processed through Illumina Beadarrays and then analyzed with SOMATICs. We demonstrate that tissue heterogeneity-related limitations not only can be overcome but can also be turned into an advantage. First, admixture of normal cells with tumor can be used as an internal reference, thereby enabling highly sensitive detection of somatic deletions without having corresponding normal tissue. Second, the presence of normal cells allows for discrimination of somatic from germline aberrations, and the proportion of cells in the tissue sample that are harboring the somatic events can be assessed. Third, relatively early versus late somatic events can also be distinguished, assuming that late events occur only in subsets of cancer cells. Finally, admixture by normal cells allows inference of germline genotypes from a cancer sample. All this information can be obtained from any cancer sample containing a proportion of 40-75% of cancer cells. SOMATICs is a ready-to-use open-source program that integrates all of these features into a simple format, comprehensively describing each chromosomal event.     

Correlation of myosin isoforms with anatomical divisions in equine musculus biceps brachii.

The biceps brachii of horses is subdivided into a lateral and medial head. Electrophoresis of samples from the lateral head revealed three slow-migrating native myosin isoforms, including one that does not correspond to slow myosin isoforms described for other mammalian muscles. In contrast, the medial head contained a single slow isoform. Both the lateral and medial heads contained three fast-migrating isoforms corresponding with the FM-2, FM-3 and FM-4 isoforms reported for other mammalian fast-twitch muscle fibers. Electrophoresis of myosin heavy chains (MHCs) revealed only two MHC bands, one fast-migrating band that comigrates with rat type I MHC and a second slower-migrating band that comigrates with rat type IIa MHC. Quantitation of the histochemical data is correlated with densitometric analysis of MHCs in the medial and lateral heads of biceps brachii and is consistent with previously hypothesized functional specializations of this muscle.