The Human Connectome Project: a data acquisition perspective.

The Human Connectome Project (HCP) is an ambitious 5-year effort to characterize brain connectivity and function and their variability in healthy adults. This review summarizes the data acquisition plans being implemented by a consortium of HCP investigators who will study a population of 1200 subjects (twins and their non-twin siblings) using multiple imaging modalities along with extensive behavioral and genetic data. The imaging modalities will include diffusion imaging (dMRI), resting-state fMRI (R-fMRI), task-evoked fMRI (T-fMRI), T1- and T2-weighted MRI for structural and myelin mapping, plus combined magnetoencephalography and electroencephalography (MEG/EEG). Given the importance of obtaining the best possible data quality, we discuss the efforts underway during the first two years of the grant (Phase I) to refine and optimize many aspects of HCP data acquisition, including a new 7T scanner, a customized 3T scanner, and improved MR pulse sequences.

Revolution through genomics in investigative and discovery toxicology.

The remarkable technologic and methodologic advances spurred on by the Human Genome Project are being applied throughout the life sciences. In the field of toxicology, high-resolution assays now make it possible to discover virtually all the differences in gene expression brought on by exposure to a particular xenobiotic. There are 2 principal approaches used to build a catalog of changes in gene expression: hybridization microarrays and gel-based methods, such as differential display and AFLP-based mRNA finger-printing. The power of such approaches is exemplified by the identification of more than 300 genes that differ in expression level by at least 2-fold in response to the nongenotoxic rodent liver carcinogen phenobarbital.

Phenobarbital does not promote hepatic tumorigenesis in a twenty-six-week bioassay in p53 heterozygous mice.

The tumorigenic potential of phenobarbital was examined in a 26-wk carcinogenesis bioassay using p53 heterozygous mice and wild-type controls. Fifteen mice/sex/genotype were exposed to either 500 or 1,000 ppm phenobarbital in the diet. Dietary administration of 3,750 ppm p-cresidine, a transspecies mutagenic carcinogen, to both heterozygous and wild-type mice served as a positive control. Phenobarbital treatment caused increases in liver:body weight ratios and histologic evidence of centrilobular hepatocellular hypertrophy. No tumors were observed in any phenobarbital-treated mice. Mice given p-cresidine exhibited a moderate reduction in body weight gain over the course of the study. Heterozygous mice treated with p-cresidine exhibited a high incidence of urinary bladder tumors. Similar tumors were also present in a small number of p-cresidine-treated wild-type mice. Our results demonstrate the lack of a hepatic tumor response to phenobarbital, a compound that is a potent and potent and prototypic hepatic microsomal enzyme inducer, a nongenotoxic rodent carcinogen, and a human noncarcinogen. This finding supports the continued utility of this model as an alternative to the mouse bioassay for human carcinogenic safety assessment of potentially genotoxic carcinogenes because it did not produce a false-positive response to this potent nongenotoxic agent.

Unusual molecular evolution of an Adh pseudogene in Drosophila.

The Adh locus in Drosophila species which are members of the repleta group contains products of one or two duplication events. In all species examined to date one of the Adh genes is now a pseudogene, since mutations have rendered these genes incapable of being translated into a functional alcohol dehydrogenase. These pseudogenes contain introns in the standard Adh gene position; hence, their origin is not by retrotransposition. Comparison of the sequences of the Adh-psi from representatives of each of the subgroups of the repleta group reveal that the Adh pseudogene is present in each subgroup and that mutations at codon 2 and a deletion in the region immediately 5' to Adh-psi are common to all species. Therefore, it is likely that the translational inactivation event that resulted in a pseudogene occurred before the divergence of the species that make up the repleta group. We have investigated the transcription of Adh-psi of D. hydei and have found that the transcription has a developmental profile dissimilar from any known Adh gene, does not utilize an Adh promoter, and is initiated at a point almost 12 kb upstream. Comparison of sequence divergence of Adh-psi within species of the repleta group reveals that rates of evolution of the exons of Adh-psi are substantially slower than intergenic regions and are only slightly faster than those of exons of functional Adh genes. Second, retention of codon bias is found in the Adh-psi of most species, and substitution at synonymous coding positions substantially exceeds substitution at nonsynonymous coding positions. Comparison of the evolution of other putative pseudogenes with repleta group Adh pseudogenes suggests that at least some pseudogene sequences in Drosophila may be evolving through mechanisms and/or under influences not presently understood.

Delineation of cis-acting sequences required for expression of Drosophila mojavensis Adh-1.

The control of expression of the Adh-1 gene of Drosophila mojavensis has been analyzed by transforming ADH null Drosophila melanogaster hosts with P element constructs which contain D. mojavensis Adh-1 having deletions of different extent in the 5' and 3' ends. Adh-1 expression in the D. melanogaster hosts is qualitatively similar to expression in D. mojavensis, although expression is quantitatively lower in transformants. Deletions of the 5' end indicate that information required for normal temporal and tissue expression in larvae is contained within 70 bp of the transcription start site. However, deletion constructs to -70 are deficient in ovarian nurse cell expression, whereas the additional upstream sequences present in constructs containing deletions to -257 do support expression in the ovary. Comparison of the nucleotide sequence in the -257 to -70 region of Adh-1 of four species: D. mojavensis and Drosophila arizona, which express Adh-1 in the ovary, and Drosophila mulleri and Drosophila navojoa, which do not, has led to the identification of regions of sequence similarity that correlate with ovary expression. One of these bears a striking similarity to a conserved sequence located upstream of the three heat shock genes that have constitutive ovarian expression and may be an ovarian control element. We have identified an aberrant aspect of Adh-1 expression. In transformants which carry an Adh-1 gene without a functional upstream Adh-2 gene Adh-1 expression continues into the adult stage instead of ceasing at the onset of metamorphosis. In transformants with a functional Adh-2 gene, Adh-1 expression ceases in the third larval instar stage and aberrant expression in the adult stage does not occur.

The regulation of lung elastin synthesis.

The important role that elastin plays in the development and proper function of lung has long been recognized. Also, the intimate connection between pulmonary emphysema and the destruction of alveolar elastin has been well established. Understanding the mechanisms regulating pulmonary elastin synthesis is crucial to fully understanding these normal and pathological processes. In this article, we review recent literature on elastin structure, the elastin gene and its multiple RNA transcripts, and the different tropoelastin isoforms that are translated from these mRNAs. The similarity of lung and aortic elastin and the cellular origin of lung elastin are also discussed. We next examine the few studies addressing regulation of elastin expression during lung development, maturation, and aging. The search for modulators of pulmonary elastogenesis, which has yielded mostly negative results, is then reviewed. Finally, we present a cell culture model that has been developed to study the molecular basis of lung injury in pulmonary emphysema.

Chick tropoelastin isoforms. From the gene to the extracellular matrix.

Studies from several laboratories have demonstrated the existence of multiple tropoelasting mRNAs and protein isoforms. The present study was designed to examine the developmental expression of a specific tropoelastin mRNA, its encoded isoform, and the fate of that isoform in the extracellular matrix. A chick genomic DNA library was screened with a chick tropoelastin cDNA. Seven unique, overlapping clones spanning 39 kilobases were isolated. A synthetic oligonucleotide complementary to a variable tropoelastin mRNA sequence was used to identify a 1.5-kilobase PstI-BamHI genomic fragment. Nucleotide sequence data revealed that the putative exon was surrounded by intron sequences possessing canonical splice sites at the exon/intron borders. Using both immunologic and molecular probes specific to the tropoelastin isoform and mRNA, quantitative protein and RNA analyses were performed. Results demonstrate that total tropoelastin mRNAs increased significantly during aortic embryogenesis whereas the amount of mRNA containing the variable exon remained relatively constant. The amount of total tropoelastins within the same developmental period reflect the level of total tropoelastin mRNA. The amount of the tropoelastin isoform containing the variable exon essentially mirrored the corresponding mRNA with the exception that a decrease in the isoform at day 15 was not seen in the mRNA level. Immunoelectron micrographs of 13-day chick aortic tissue using both total and isoform-specific antisera showed ultrastructural localization to definable elastic fibers. Antibodies to the variable tropoelastin isoform occurred preferentially at sites where elastic fiber microfibril structures were evident.