Leptin modulates energy metabolism and lung development. We hypothesize that the effects of leptin on postnatal lung development are volume dependent from 2 to 10 wk of age and are independent of hypometabolism associated with leptin deficiency. To test the hypotheses, effects of leptin deficiency on lung maturation were characterized in age groups of C57BL/6J mice with varying Lep ob genotypes. Quasi-static pressure-volume curves and respiratory impedance measurements were performed to profile differences in respiratory system mechanics. Morphometric analysis was conducted to estimate alveolar size and number. Oxygen consumption was measured to assess metabolic rate. Lung volume at 40-cmH2O airway pressure (V40) increased with age in each genotypic group, and V40 was significantly ( P < 0.05) lower in leptin-deficient ( ob/ ob) mice beginning at 2 wk. Differences were amplified through 7 wk of age relative to wild-type (+/+) mice. Morphometric analysis showed that alveolar surface area was lower in ob/ ob compared with +/+ and heterozygote ( ob/+) mice beginning at 2 wk. Unlike the other genotypic groups, alveolar size did not increase with age in ob/ ob mice. In another experiment, ob/ ob at 4 wk received leptin replacement (5 μg·g−1·day−1) for 8 days, and expression levels of the Col1a1, Col3a1, Col6a3, Mmp2, Tieg1, and Stat1 genes were significantly increased concomitantly with elevated V40. Leptin-induced increases in V40 corresponded with enlarged alveolar size and surface area. Gene expression suggested a remodeling event of lung parenchyma after exogenous leptin replacement. These data support the hypothesis that leptin is critical to postnatal lung remodeling, particularly related to increased V40 and enlarged alveolar surface area.

The approach of this study is to identify gene expression profiles that provide genomic mechanisms underlying the pathophysiology of the aging lung. Choice of B6 and D2 inbred strains is based on their differences in aging physiology, lung morphometry, and natural longevity. Lungs were harvested from B6 mice at 2, 18, and 26 months and from D2 mice at 2 and 18 months. Purified RNA was put to oligonucleotide microarray analyses using Affymetrix genechips. Biochemical networks were deciphered using Ingenuity Pathways analysis. Genes from these networks were cross-referenced to single nucleotide polymorphism (SNP) databases for these two strains, and candidate aging-related SNPs were validated by sequencing entire genes. Approximately 67–89% of expressed genes were upregulated with aging in both strains. Genes in aging D2 mouse lung were uniquely upregulated in xenobiotic detoxification cascades, whereas genes in aging B6 lung were downregulated for heat shock response and upregulated for T cell receptor signaling. IGF and PTEN signaling were differentially expressed between strains at 2 months of age. Several polymorphisms were identified in the regulatory and untranslated regions of the Mmp14 gene. In sum, lungs of B6 and D2 mice age differently at the gene expression level, which when integrated with strain-dependent SNP associations, leads to a blueprint of regulated aging. NIA AG-21057 and NHLBI HL-010342

Previous studies from our laboratories showed lung development differences between inbred strains of mice. In the present study, the C57BL/6J (B6) and DBA/2J (D2) strains were examined for senescent-dependent differences with respect to the lung structure and function. Specifically, we hypothesize that senescent changes in lung vary between strains due to identifiable gene expression differences. Quasi-static pressure-volume curves and respiratory impedance measurements were performed on 2- and 20-mo-old B6 and D2 mice. Lung volume at 30 cm H(2)O (V(30)) pressure was significantly (P < 0.01) increased with age in both strains, but the increase was proportionally greater in D2 (68%) than in B6 (40%) mice. In addition, decreased elastic recoil pressure at 50% of V(30) and a reduction in airway resistance as a function of positive end-expiratory pressure were observed in 20-mo-old D2 mice but not in B6 mice. Morphometric analysis of lung parenchyma showed significant decreases in elastic fiber content with age in both strains, but the collagen content was significantly (P < 0.01) increased with age in D2 but not B6 mice at 20 mo. Furthermore, using quantitative RT-PCR methods, gene expression differences between strains suggested that D2 mice significantly (P < 0.05) downregulated the expressions of elastin (Eln) and procollagen I, III, and VI (Col1a1, Col3a1, and Col6a3) in lung tissue at 20 mo of age. These age-dependent changes were accompanied by an increased gene expression in matrix metalloproteinase 9 (Mmp9) in D2 and an increase in tissue inhibitor of matrix metalloproteinase (Timp1 and Timp4) in B6 mice. In conclusion, the results from the present study demonstrate that lung mechanics of both strains show significant age-dependent changes. However, changes in D2 mice are accelerated relative to B6 mice. Moreover, gene expression differences appear to be involved in the strain-specific changes of lung mechanic properties.

Evidence is accumulating about the difficulties that users have in managing their work using contemporary graphical user interfaces. Current designs offer a hierarchy of folders containing documents and taskbar operations to launch/exit applications. We propose a Personal Role Management strategy that emphasizes management of the multiple roles users have in their professional and personal lives. Each role involves coordination with groups of people and accomplishment of tasks within a schedule. We define Personal Role Management and summarize our earlier work that led to this strategy. This current project focused on understanding how Personal Role Management might improve email for college students. College students often assume distinct and predictable roles. Their student role is structured by the rhythm and interactions of classes, projects and exams. In both their family role and their work role for local companies, they deal with separate groups of people. We describe scenarios of use of a role-based email system, an interface mockup and user reactions. This research suggests that using those roles as a driving component for designing an email interface might address problems identified in our surveys and interviews of college students. To appear in: Kaptelinin, V., Czerwinski, M. (Eds) Integrated Digital Work Environments: Beyond the Desktop, MIT Press 1

This study identified gene expression profiles that provided evidence for genomic mechanisms underlying the pathophysiology of aging lung. Aging lungs from C57BL/6 (B6) and DBA/2 (D2) mouse strains differ in physiology and morphometry. Lungs were harvested from B6 mice at 2, 18, and 26 mo and from D2 mice at 2 and 18 mo of age. Purified RNA was subjected to oligonucleotide microarray analyses, and differential expression analyses were performed for comparison of various data sets. A significant majority of differentially expressed genes were upregulated with aging in both strains. Aging D2 lungs uniquely exhibited upregulation in stress-response genes including xenobiotic detoxification cascades. In contrast, aging B6 lungs showed downregulation of heat shock-response genes. Age-dependent downregulation of genes common to both B6 and D2 strains included several collagen genes (e.g., Col1a1 and Col3a1). There was a greater elastin gene ( Eln) expression in D2 mice at 2 mo, and Eln was uniquely downregulated with age in this strain. The matrix metalloproteinase 14 gene ( Mmp14), critical to alveolar structural integrity, was also downregulated with aging in D2 mice only. Several polymorphisms in the regulatory and untranslated regions of Mmp14 were identified between strains, suggesting that variation in Mmp14 gene regulation contributes to accelerated aging of lungs in D2 mice. In summary, lungs of B6 and D2 mice age with variable rates at the gene expression level, and these quantifiable genomic differences provide a template for understanding the variability in age-dependent changes in lung structure and function.

The lung architecture of different inbred strains of mice is highly variable. However, little is known about the lung gene expression differences that lead to this strain variation. In this study, gene expression profiles from the lungs of C3H/HeJ (C3), C57BL/6J (B6) and A/J mice as well as offspring of C3 and B6 progenitors, B6C3F1 (F1), were generated by microarray analysis to find which genes might influence why B6 and C3 mice have such distinct lung structure from one another and to understand gene heritability patterns. Replicate samples (two animals per strain) of five-week-old mouse lungs were collected, prepared and hybridized onto Affymetrix Mouse MOE430A gene chips. Stringent analysis of the data revealed common and unique gene expression patterns among B6, C3, and F1 mice with A/J used as a reference. To confirm the data, real-time PCR analysis was performed on genes shown to be specific to B6 or C3 mice. Several of the structural or transcription factor genes including decorin (Dcn), elastin (Eln), wnt1-inducible signaling protein 1 (Wisp1), and SRY-box containing gene 7 (Sox7) have been linked to Wnt signaling in previous studies, implicating the role of the Wnt pathway in the variation among mouse strains in lung structure.

Background Nuclear factor erythroid-2 related factor 2 (NRF2) is a redox-sensitive transcription factor that positively regulates the expression of genes encoding antioxidants, xenobiotic detoxification enzymes, and drug efflux pumps, and confers cytoprotection against oxidative stress and xenobiotics in normal cells. Kelch-like ECH-associated protein 1 (KEAP1) negatively regulates NRF2 activity by targeting it to proteasomal degradation. Increased expression of cellular antioxidants and xenobiotic detoxification enzymes has been implicated in resistance of tumor cells against chemotherapeutic drugs.Methods and Findings Here we report a systematic analysis of the KEAP1 genomic locus in lung cancer patients and cell lines that revealed deletion, insertion, and missense mutations in functionally important domains of KEAP1 and a very high percentage of loss of heterozygosity at 19p13.2, suggesting that biallelic inactivation of KEAP1 in lung cancer is a common event. Sequencing of KEAP1 in 12 cell lines and 54 non-small-cell lung cancer (NSCLC) samples revealed somatic mutations in KEAP1 in a total of six cell lines and ten tumors at a frequency of 50% and 19%, respectively. All the mutations were within highly conserved amino acid residues located in the Kelch or intervening region domain of the KEAP1 protein, suggesting that these mutations would likely abolish KEAP1 repressor activity. Evaluation of loss of heterozygosity at 19p13.2 revealed allelic losses in 61% of the NSCLC cell lines and 41% of the tumor samples. Decreased KEAP1 activity in cancer cells induced greater nuclear accumulation of NRF2, causing enhanced transcriptional induction of antioxidants, xenobiotic metabolism enzymes, and drug efflux pumps.Conclusions This is the first study to our knowledge to demonstrate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC. Loss of KEAP1 function leading to constitutive activation of NRF2-mediated gene expression in cancer suggests that tumor cells manipulate the NRF2 pathway for their survival against chemotherapeutic agents.

Host genetic factors that regulate innate immunity determine susceptibility to sepsis. Disruption of nuclear factor-erythroid 2-related factor 2 (Nrf2), a basic leucine zipper transcription factor that regulates redox balance and stress response, dramatically increased the mortality of mice in response to endotoxin- and cecal ligation and puncture-induced septic shock. LPS as well as TNF-alpha stimulus resulted in greater lung inflammation in Nrf2-deficient mice. Temporal analysis of pulmonary global gene expression after LPS challenge revealed augmented expression of large numbers of proinflammatory genes associated with the innate immune response at as early as 30 minutes in lungs of Nrf2-deficient mice, indicating severe immune dysregulation. The expression profile indicated that Nrf2 has a global influence on both MyD88-dependent and -independent signaling. Nrf2-deficient mouse embryonic fibroblasts showed greater activation of NF-kappaB and interferon regulatory factor 3 in response to LPS and polyinosinic-polycytidylic acid [poly(I:C)] stimulus, corroborating the effect of Nrf2 on MyD88-dependent and -independent signaling. Nrf2's regulation of cellular glutathione and other antioxidants is critical for optimal NF-kappaB activation in response to LPS and TNF-alpha. Our study reveals Nrf2 as a novel modifier gene of sepsis that determines survival by mounting an appropriate innate immune response.

The carotid body (CB) is the primary hypoxic chemosensory organ. Its hypoxic response appears to be genetically controlled. We have hypothesized that: 1) genes related to CB function are expressed less in the A/J mice (low responder to hypoxia) compared with DBA/2J mice (high responder to hypoxia); and 2) gene expression levels of morphogenic and trophic factors of the CB are significantly lower in the A/J mice than DBA/2J mice. This study utilizes microarray analysis to test these hypotheses. Three sets of CBs were harvested from both strains. RNA was isolated and used for global gene expression profiling (Affymetrix Mouse 430 v2.0 array). Statistically significant gene expression was determined as a minimum six counts of nine pairwise comparisons, a minimum 1.5-fold change, and P <or= 0.05. Our results demonstrated that 793 genes were expressed less and that 568 genes were expressed more in the A/J strain vs. the DBA/2J strain. Analysis of individual genes indicates that genes encoding ion channels are differentially expressed between the two strains. Genes related to neurotransmitter metabolism, synaptic vesicles, and the development of neural crest-derived cells are expressed less in the A/J CB vs. the DBA/2J CB. Through pathway analysis, we have constructed a model that shows gene interactions and offers a roadmap to investigate CB development and hypoxic chemosensing/chemotransduction processes. Particularly, Gdnf, Bmp2, Kcnmb2, Tph1, Hif1a, and Arnt2 may contribute to the functional differences in the CB between the two strains. Bmp2, Phox2b, Dlx2, and Msx2 may be important for the morphological differences.

Disruption of NF-E2-related factor (Nrf2), a redox-sensitive basic leucine zipper transcription factor, causes early-onset and more severe emphysema due to chronic cigarette smoke. Nrf2 determines the susceptibility of lungs to cigarette smoke-induced emphysema in mice through the transcriptional induction of numerous antioxidant genes. The lungs of Nrf2-/- mice have higher oxidative stress as evident from the increased levels of lipid peroxidation (4-hydroxy-2-nonenal) and oxidative DNA damage (7,8-dihydro-8-Oxo-2'deoxyguanosine) in response to cigarette smoke. Glutathione peroxidases (GPX) are the primary antioxidant enzymes that scavenge hydrogen peroxide and organic hydroperoxides. Among the five GPX isoforms, expression of GPX2 was significantly induced at both mRNA and protein levels in the lungs of Nrf2+/+ mice, in response to cigarette smoke. Activation of Nrf2 by specific knock down of the cytosolic inhibitor of Nrf2, Keap1, by small inhibitory RNA (siRNA) upregulated the expression of GPx2, whereas Nrf2 siRNA down-regulated the expression of GPX2 in lung epithelial cells. An ARE sequence located in the 5' promoter-flanking region of exon 1 that is highly conserved between mouse, rat, and human was identified. Mutation of this ARE core sequence completely abolished the activity of promoter-reporter gene construct. The binding of Nrf2 to the GPX2 antioxidant response element was confirmed by chromatin immunoprecipation, electrophoretic mobility shift assays, and site-directed mutagenesis. This study shows that GPX2 is the major oxidative stress-inducible cellular GPX isoform in the lungs, and that its basal as well as inducible expression is dependent on Nrf2.

Nat. Methods 2, 345–349 (2005). The GEO accession number for the array data is GSE2521.

Microarray technology is a powerful tool for measuring RNA expression for thousands of genes at once. Various studies have been published comparing competing platforms with mixed results: some find agreement, others do not. As the number of researchers starting to use microarrays and the number of cross-platform meta-analysis studies rapidly increases, appropriate platform assessments become more important. Here we present results from a comparison study that offers important improvements over those previously described in the literature. In particular, we noticed that none of the previously published papers consider differences between labs. For this study, a consortium of ten laboratories from the Washington, DC–Baltimore, USA, area was formed to compare data obtained from three widely used platforms using identical RNA samples. We used appropriate statistical analysis to demonstrate that there are relatively large differences in data obtained in labs using the same platform, but that the results from the best-performing labs agree rather well.

Introduction: Identification of novel genes involved in early Crohn's disease (CD) may lead to a better understanding of disease pathogenesis. We evaluated gene expression profiles in colonic mucosa of children with newly diagnosed CD. Methods: Two colonic biopsies were obtained from patients (pts) with GI symptoms undergoing colonoscopy. Total RNA was isolated from 5 pts with histopathologically confirmed moderate to severe CD and from 6 children with normal histology (controls). Pts had not been exposed to any CD medications. RNA purification and amplification were performed prior to microarray analysis using Affymetrix Human Genome U133 Plus 2.0 Array,containing ~31,836 unique entries. Data analysis for comparison between pts and controls using dChip Analyzer, included signal difference ≥100, fold change ≥1.5, “present call” >50%, t test, p value < 0.05. Results: Mean ages of pts and controls were 14.2 y and 14.6 y. Mean ESR was 42 mm/hr in pts and 3 mm/hr in controls. 66 genes were differentially up-regulated in CD including lipocalin 2 (LCN2), S100 calcium binding protein, TGM2, CXCL2, CXCL3, IFITM, B-factor properdin, dual oxidase 2 (DUOX2), SPINK4, SLC2A3, TIMP1, NNMT, SDCCAG33, RIS1, COL6A3 with functions involved in inflammation, immune response, metabolism, cell cycle regulation, transcription, oncogenesis and remodeling. 27 genes, mainly involved in metabolism and inflammation, were down-regulated in CD including prostaglandin D2 receptor (PTGDR), SLC16A9, HSD17B2, APOBEC3A, SLC38A4, TRPM7, ZNF124, ATP-binding cassette B1 (MDR). Conclusion: Microarray analysis of colonic biopsies from newly diagnosed children with CD revealed multiple differentially expressed genes. Examination of the gene patterns expressed in early CD may provide useful information for improved disease characterization, development of diagnostic markers and for future therapeutic targets.

In order to accommodate the increasing diversity of email users, applications have evolved in both functionality and user interface. In this study, we attempt to determine whether email user interfaces can be improved to serve a specific target population: college students. We present our results from college campus surveys that examine email usage patterns and subjective experiences among college students. From our survey feedback and related research, we conclude that email overload and feature intimidation are the greatest hindrances to email communication on campus. To address these problems, we propose employing role management to organize messages calendar and contacts in an email program for students, using school, work and family roles. We describe a prototype and user reactions. Our conclusion is that role management, integrated into email software, may help college students manage their email more effectively.

In order to accommodate the increasing diversity of email users, applications have evolved in both functionality and user interface. In this study, we attempt to determine whether email user interfaces can be improved to serve a specific target population: college students. We present our results from college campus surveys that examine email usage patterns and subjective experiences among college students. From our survey feedback and related research, we conclude that email overload and feature intimidation are the greatest hindrances to email communication on campus. To address these problems, we propose employing role management to organize messages calendar and contacts in an email program for students, using school, work and family roles. We describe a prototype and user reactions. Our conclusion is that role management, integrated into email software, may help college students manage their email more effectively.