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PDF file - 114K, Supplemental Figure 5 Cellular distribution of HIF-2alpha under normal oxygen conditions. (a) Representative confocal microscopy images (magnification 400x) showing HIF-2α or Nox4 immunofluorescense, DAPI nuclear staining, or merged images. b) Control (NS) or Nox4 shRNA (KD) cells were imaged following exposure to TEMPOL (0.25 mM), DTT (1mM). c) 786-0 NS cells imaged following transduction with Ad-GFP or Ad-MnSOD. d) 786-0 cells expressing pcDNA-Nox4 imaged with or without treatment with TEMPOL (0.25 mM).

PDF file - 376K, Supplemental Figure 2. Characterization of 786-0 cells stably transfected with the human, full-length Nox4 cDNA. (a) Morphology of 786-0 cells following exogenous expression of Nox4 reveals smaller, denser, more-rounded appearance relative to parental 786-0 cells. (b) Cell viability assay. Equal numbers of untransfected cells or 786-0 Nox4 cells were plated in 96-well plates. After attachment, assay reagent was added, incubated at 37{degree sign}C, and the fluorescent signal read at 590 nm.

PDF file - 224K, Supplemental Figure 1. Quantitative RT-PCR (a) and semi-quantitative RT-PCR (b) for detection of Nox1 in 786-0, RCC4 or LNCaP prostate cancer cells using two published primer pairs(14, 17),. Error bar represents +/- SE.

PDF file - 183K, Supplemental Figure 4. Cytotoxicity was measured by CellTiter-Blue (Promega, Madison, WI). 786-0 NS cells were plated at 1 x 104 cells per well in 96-well plates. After 24 hours, dithiothreitol was added as indicated in 5 replicate wells. 20 �l of the CellTiter-Blue reagent was added to each well and viability quantitated by spectrofluorometer (Spectra Max Plus384, Molecular Devices) and expressed as percentage of viability of media-only controls. Error bars indicate +/- S.E. mean.

Most sporadically occurring renal tumors include a functional loss of the tumor suppressor von Hippel Lindau (VHL). Development of VHL-deficient renal cell carcinoma (RCC) relies upon activation of the hypoxia-inducible factor-2α (HIF2α), a master transcriptional regulator of genes that drive diverse processes, including angiogenesis, proliferation, and anaerobic metabolism. In determining the critical functions for HIF2α expression in RCC cells, the NADPH oxidase NOX4 has been identified, but the pathogenic contributions of NOX4 to RCC have not been evaluated directly. Here, we report that NOX4 silencing in VHL-deficient RCC cells abrogates cell branching, invasion, colony formation, and growth in a murine xenograft model RCC. These alterations were phenocopied by treatment of the superoxide scavenger, TEMPOL, or by overexpression of manganese superoxide dismutase or catalase. Notably, NOX4 silencing or superoxide scavenging was sufficient to block nuclear accumulation of HIF2α in RCC cells. Our results offer direct evidence that NOX4 is critical for renal tumorigenesis and they show how NOX4 suppression and VHL re-expression in VHL-deficient RCC cells are genetically synonymous, supporting development of therapeutic regimens aimed at NOX4 blockade. Cancer Res; 74(13); 3501–11. ©2014 AACR.

PDF file - 98K, Supplemental Figure 3. Lucigenin chemiluminescence assay for superoxide detection performed as described in materials and methods on a) isolated membrane fractions from parental 786-0, RCC4 and Caki-1 cells or isolated cell fractions from 786-0 (b) or RCC4 (c) cells. RLU: relative light units.

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PDF file - 446K, Supplemental Figure 6 Immunohistochemistry for Nox4 and HIF-2alpha in 786-0 NS and KD xenograft explants confirms re-expression of Nox4 in KD tumors with corresponding elevated HIF-2alpha expression. Magnification 200X.

OBJECTIVE/BACKGROUND: Approximately 400,000 adolescents give birth in the US annually. Although one-half experience depressive symptoms, less than 25% comply with referrals for depression evaluation and treatment. The current study tested the effectiveness of an internet based depression intervention on seeking depression treatment. METHODS: Based upon the Theory of Planned Behavior (TPB), the intervention included vignettes, questions and answers, and resources. Before the intervention, immediately after the intervention, and two-weeks later the adolescent mothers (n=151) answered questions related to TPB variables and depression treatment. Data were compared to adolescent mothers (n=138) in the control group. Data were collected in community organizations or home visits for the control group. Adolescent mothers in the intervention group answered questions and completed the intervention from a computer of their choice. RESULTS: The adolescents were primarily African American (89.2%); less than high school educated (51.7%); had given birth in last year (97.1%); with a mean age 18.2 years. The intervention led to significant changes in attitude, perceived control, intention to seek mental health treatment, and actually seeking depression treatment. CONCLUSIONS/DISCUSSION: Untreated postpartum depression dramatically impacts a mother’s relationships with her child, her functioning at work and school, health care seeking behaviors, mothering skills, and her development as well as the development of her child. An Internet based depression intervention is an inexpensive method to increase rates of depression treatment.

Problem Little is known about how adolescent mothers use social media and the Internet, especially to access health information. Methods In this cross-sectional, descriptive study, adolescent mothers were recruited from an academic medical center after the birth of their child (n = 94) or from a state-funded, home visitation program during the first year after birth (n = 91). They completed the Pew Internet Survey: 37 questions related to use of social media and Internet, particularly in regard to obtaining health information. Findings All adolescent mothers used a computer and almost all went online. Most accessed the Internet by cell phone (67.4%) and used social media. The health topics searched most frequently were pregnancy/birth control (85.8%), sexually transmitted diseases (n = 134, 72.6%) and HIV (66.3%). Response to survey questions differed between the two groups (adolescent mothers surveyed after birth from academic medical center and adolescent mothers surveyed in the first postpartum year in the community). Conclusions Adolescent mothers spend significant time on the Internet including searching for health information. Cell phones are their preferred methods for accessing the Internet, and they use social media. Thus, social media and the Internet are potentially feasible and acceptable vehicles to deliver health interventions to adolescent mothers.

Most sporadically occurring renal tumors include a functional loss of the tumor suppressor von Hippel Lindau (VHL). Development of VHL-deficient renal cell carcinoma (RCC) relies upon activation of the hypoxia-inducible factor-2α (HIF2α), a master transcriptional regulator of genes that drive diverse processes, including angiogenesis, proliferation, and anaerobic metabolism. In determining the critical functions for HIF2α expression in RCC cells, the NADPH oxidase NOX4 has been identified, but the pathogenic contributions of NOX4 to RCC have not been evaluated directly. Here, we report that NOX4 silencing in VHL-deficient RCC cells abrogates cell branching, invasion, colony formation, and growth in a murine xenograft model RCC. These alterations were phenocopied by treatment of the superoxide scavenger, TEMPOL, or by overexpression of manganese superoxide dismutase or catalase. Notably, NOX4 silencing or superoxide scavenging was sufficient to block nuclear accumulation of HIF2α in RCC cells. Our results offer direct evidence that NOX4 is critical for renal tumorigenesis and they show how NOX4 suppression and VHL re-expression in VHL-deficient RCC cells are genetically synonymous, supporting development of therapeutic regimens aimed at NOX4 blockade. Cancer Res; 74(13); 3501–11. ©2014 AACR.

Cell adhesion molecules (CADMs) comprise a protein family whose functions include maintenance of cell polarity and tumor suppression. In this report, we show that the CADM2 gene is repressed in human clear renal cell carcinoma by DNA promoter hypermethylation and/or loss of heterozygosity. Moreover, the loss of CADM2 expression is associated with a higher tumor pathology stage (p

Background The excision repair cross complementing (ERCC1) gene product plays a vital role in the nucleotide excision repair and DNA interstrand crosslink repair pathways, which protect the genome from mutations and chromosomal aberrations, respectively. Genetic deletion of Ercc1 in the mouse causes dramatically accelerated aging. We examined the effect of Ercc1 deletion in the development of prostate cancer in a prostate recapitulation model as Ercc1 deficient mice die within four weeks of birth.

The early growth response gene 1, EGR1, is an important transcriptional regulator and acts as the convergent point between a variety of extracellular stimuli and activation of target genes. Unlike other tumor types, prostate tumors express high levels of EGR1 relative to normal tissues. However, the mechanism of EGR1 regulation in prostate tumor cells is unknown. As EGR1 expression and epidermal growth factor (EGF) signaling are frequently upregulated in prostate tumors, we tested the hypothesis that EGF induces EGR1 expression in prostate cancer cells. Using RT-PCR to quantify EGR1 transcripts, we found that EGF induced EGR1 expression in a dose- and time-dependent manner and the ERK pathway inhibitor, PD98059, abrogated the EGF-mediated EGR1 response in LNCaP and PC3 cells. Analysis of the EGR1 promoter using deletion constructs identified an EGF-responsive region in the proximal promoter (-771 to -245 bp) containing 3 potential serum response element (SRE) sites. In vivo chromatin immunoprecipitation assays demonstrated that Elk-1 binding at the SRE sites of the EGR1 promoter was enhanced by EGF treatment in PC3 cells. Overexpression of Elk-1 was sufficient to activate the EGF-responsive region of EGR1 promoter in PC3 cells and, similarly, a dominant-negative Elk-1 suppressed EGR1 promoter activity. Taken together, these results demonstrate for the first time that EGR1 expression in PC3 cells is mediated through an EGF-ERK-Elk-1 signaling cascade.

Significant public investments in rural broadband internet service authorized by the American Recovery and Reinvestment Act raise new questions regarding the impacts of broadband on rural residents and communities. The results of a natural field experiment involving broadband internet projects funded by the Community Connect program of the Rural Utilities Service are reviewed here to provide a context for future evaluations of the effects of broadband infrastructure deployment and efforts to stimulate sustainable broadband use in rural areas. Based on pre–post surveys conducted in four communities in 2005 and 2008, the implementation of broadband infrastructure grants increased broadband adoption, although penetration also increased where a grant was awarded but later terminated. Public education efforts in a community participating in the ConnectKentucky initiative had an incremental effect on broadband adoption by positively affecting residents' perceptions of broadband service. Impacts on individual economic development activities and community satisfaction were not found. Implications for rural broadband initiatives and future evaluations of their effects are drawn.

Cell volume is one of the basic characteristics of a cell and is being extensively studied in relationship to a variety of processes, such as proliferation, apoptosis, fertility, or locomotion. At the same time, its measurement under a microscope has not been well developed. The method we propose uses negative transmission contrast rendered to cells by a strongly absorbing dye present in the extracellular medium. Cells are placed in a shallow compartment, and a nontoxic and cell-impermeant dye, such as acid blue 9, is added to the medium. Transmission images are collected at the wavelength of maximum dye absorption (630 nm). Where the cell body displaces the dye, the thickness of the absorbing layer is reduced; thus, an increase in cell thickness produces brighter images and vice versa. The absolute values for cell thickness and volume can be easily extracted from the image by computing the logarithm of intensity and dividing it by the absorption coefficient. The method is fast, impervious to instability of the light source, and has a high signal-to-noise ratio; it can be realized either on a laser scanning or a conventional microscope equipped with a bandpass filter. For long-term experiments, we use a Bioptechs perfusion chamber fitted with a 0.03-mm spacer and an additional port to enable rapid switching of solutions. To show possible applications of this method, we investigated the kinetics of the cell volume response to a hypotonic buffer and to the apoptotic agents staurosporine and ionomycin.

Objectives: To identify ethical dilemmas in communication presented in My Sister's Keeper, a novel by Jodi Picoult. To examine how one's position/perspective shapes how one interprets and evaluates...

Even as geographic disparities in high speed Internet access narrow, an urban-rural broadband gap persists, pointing to the importance of individual differences in motivations to adopt broadband as the key to closing the gap. Diffusion of innovation is reconceptualized through contemporary perspectives of the digital divide and social cognitive theory and tested on a survey sample drawn from four rural communities. Path analysis was used to examine the factors that cause broadband Internet service adoption in rural communities. Prior experience with the Internet, the expected outcomes of broadband usage, direct personal experience with broadband, and self-efficacy had direct effects on broadband intentions. Age and income, but not education or ethnicity, also had direct impacts. Efforts that promote the personal benefits of broadband and advanced ICT literacy skills among Internet users are recommended.

In 1990 the United States Congress approved the Television Decoder Circuitry Act, which mandated that all television sets 13 inches or larger for sale in the United States be manufactured with caption‐decoding microchips. This legislation allowed millions of deaf and hearing‐impaired people throughout the US access to captions on commercials and television programs. Access to technology is one determinant of who can participate in the social, cultural, political and economic facets of a society. Scholars recognize that communication processes in the public sphere often are unbalanced. Access to media outlets creates a gap between those with media power and those without. Using a contextual analysis framework supported by a social model of disability, this paper defines the roles of parties involved in the passage of closed‐captioning legislation and highlights how social forces were successful in passing legislation beneficial to the Deaf and Hearing‐impaired community.

Purpose This article describes testing of a prototype Web site for adolescent mothers with postpartum depression; providing proof of concept. Methods Participants ( N =138) were recruited from a public school-based program for adolescent parents and completed the Mental Health Acceptability Scale, Stigma Scale for Receiving Psychological Help, and Attitudes Towards Seeking Professional Psychological Help Scale before, and after, the Web site intervention. They also provided feedback on the usability of the Web site. Results Attitudes related to depression and treatment (ATSPPH) improved after viewing the Web site ( p =.023). Feedback on the Web site indicated that it was easy to use (77%), reflecting highly acceptable score for product usability. Conclusions The data provide the foundation for the launch of the Web site from prototype to product and more comprehensive testing. The creation and testing of informational text messages will be added to the Web site to increase the interactivity and dose of the intervention.

Background The prostate gland represents a multifaceted system in which prostate epithelia and stroma have distinct physiological roles. To understand the interaction between stroma and glandular epithelia, it is essential to delineate the gene expression profiles of these two tissue types in prostate cancer. Most studies have compared tumor and normal samples by performing global expression analysis using a mixture of cell populations. This report presents the first study of prostate tumor tissue that examines patterns of differential expression between specific cell types using laser capture microdissection (LCM). Methods LCM was used to isolate distinct cell-type populations and identify their gene expression differences using oligonucleotide microarrays. Ten differentially expressed genes were then analyzed in paired tumor and non-neoplastic prostate tissues by quantitative real-time PCR. Expression patterns of the transcription factors, WT1 and EGR1, were further compared in established prostate cell lines. WT1 protein expression was also examined in prostate tissue microarrays using immunohistochemistry. Results The two-step method of laser capture and microarray analysis identified nearly 500 genes whose expression levels were significantly different in prostate epithelial versus stromal tissues. Several genes expressed in epithelial cells (WT1, GATA2, and FGFR-3) were more highly expressed in neoplastic than in non-neoplastic tissues; conversely several genes expressed in stromal cells (CCL5, CXCL13, IGF-1, FGF-2, and IGFBP3) were more highly expressed in non-neoplastic than in neoplastic tissues. Notably, EGR1 was also differentially expressed between epithelial and stromal tissues. Expression of WT1 and EGR1 in cell lines was consistent with these patterns of differential expression. Importantly, WT1 protein expression was demonstrated in tumor tissues and was absent in normal and benign tissues. Conclusions The prostate represents a complex mix of cell types and there is a need to analyze distinct cell populations to better understand their potential interactions. In the present study, LCM and microarray analysis were used to identify novel gene expression patterns in prostate cell populations, including identification of WT1 expression in epithelial cells. The relevance of WT1 expression in prostate cancer was confirmed by analysis of tumor tissue and cell lines, suggesting a potential role for WT1 in prostate tumorigenesis.

Today, fashion items such as rubber wristbands in various colors, pink ribbons, and red dresses represent different health-related causes and can be seen frequently across demographic groups. Complete with pithy slogans (e.g., “Go Red for Women”), these items are part of a larger “health fashion” trend—one that involves wearing, using, and displaying health-cause clothing and accessories. In this article, the authors explore recent interest in “health fashion,” examining in particular its origins, effectiveness, and implications.

Treatment for head and neck cancer precipitates a myriad of distressing symptoms. Patients may be isolated both physically and socially and may lack the self-efficacy to report problems and participate as partners in their care. The goal of this project was to design a telehealth intervention to address such isolation, develop patient self-efficacy, and improve symptom management during the treatment experience. Participatory action research and a review of the literature were used to develop electronically administered symptom management algorithms addressing all major symptoms experienced by patients undergoing treatment for head and neck cancers. Daily questions and related messages were then programmed into an easy-to-use telehealth messaging device, the Health Buddy(R). Clinician and patient acceptance, feasibility, and technology issues were measured. Using participatory action research is an effective means for developing electronic algorithms acceptable to both clinicians and patients. The use of a simple tele-messaging device as an adjunct to symptom management is feasible, affordable, and acceptable to patients. This telehealth intervention provides support and education to patients undergoing treatment for head and neck cancers.

Prior research on instructional media effects suggested that an audiographic approach to World Wide Web based courses would optimize educational effectiveness along with cost effectiveness, although with a possible loss of teacher immediacy that could adversely affect student attitudes. An introductory telecommunication course was converted to an audiographic Web telecourse in which students listened to pre-recorded audio classroom interactions while viewing a detailed course outline and illustrative sites over the World Wide Web. Forty-nine subjects were recruited from a live lecture class and randomly assigned to either the experimental (Web course) group or a control group that took the class in a traditional lecture section. Analysis of covariance (ANCOVA) showed that the experimental group had test scores and student attitude and teacher immediacy ratings equal to those of the control group after controlling for student gender, class level, grade point average and attendance. Open-ended interviews were also conducted to assess qualitative dimensions of student satisfaction. The results supported the audiographic telecourse model as a potentially cost-effective approach to distributing courses over the Web. New directions in research on instructional media effects and teacher immediacy were formulated from an analysis of the unique characteristics of the World Wide Web as an instructional medium.

The early growth response gene 1, EGR1, is an important transcriptional regulator and acts as the convergent point between a variety of extracellular stimuli and activation of target genes resulting in diverse responses such as cell growth, proliferation, and apoptosis. Unlike other tumor types, in prostate tumor tissues EGR1 expression is elevated relative to normal tissues. However, the mechanism(s) of regulation of EGR1 in prostate tumor cells are unknown. As EGR1 expression and epidermal growth factor (EGF) signaling are frequently upregulated in prostate tumors, we tested the hypothesis that EGF induces EGR1 expression in prostate cancer cells. Using real-time PCR to quantify EGR1 transcripts, EGF induced EGR1 expression levels in a dose and time-dependent manner; and kinase inhibitors, PD98059 and wortmannin, abrogated the EGF-mediated EGR1 response. Analysis of the EGR1 promoter using reporter constructs identified an EGF-responsive region in the proximal promoter containing three potential serum response element (SRE) sites. In vivo chromatin immunoprecipitation assays demonstrated that EGF enhanced Elk-1 binding at the SRE sites of the EGR1 promoter in PC3 cells, and Elk-1 also bound in the absence of stimulus. Overexpression of Elk-1 enhanced activation of the EGF-responsive region indicating that Elk-1 is sufficient to activate EGR1 in PC3 cells. Similarly, a dominant-negative Elk-1 suppressed EGR1 promoter activity indicating that Elk-1 is necessary for EGF-induced EGR1 transcription in both PC3 and LNCaP cells. Taken together, these results demonstrate that EGR1 expression in prostate cancer cells is mediated through an EGF-ERK-Elk-1 signaling cascade. Since elevated EGF and EGF receptor are associated with prostate cancer progression, these results may explain elevated EGR1 in high-grade prostate tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4013. doi:10.1158/1538-7445.AM2011-4013