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In JoVE (1)

Other Publications (11)

Articles by Margaret Gibson in JoVE

 JoVE Biology

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

1Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City


JoVE 4393

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

Other articles by Margaret Gibson on PubMed

Orientation Behaviors in Residents Relocated to a Redesigned Dementia Care Unit

This descriptive study took advantage of a scheduled environmental renovation in a secured dementia care unit. A convenience sample of 19 residents who were relocated to the unit completed a performance-based orientation task involving locating their own room. The study included a brief structured interview and tests of psychological function (cognition, depression, and visual-spatial ability) two months after admission. Intrusions (uninvited entry into another resident's room) were tracked for one week. Eighty-four percent of participants were able to find their own rooms during the orientation task. The majority of participants reported use of color (n = 13) and structure (n = 12) as cues for locating their rooms. Thirty-eight percent of those who could find their own rooms also intruded into others' rooms; these intrusions were most commonly related to seeking social interaction. The results attest to the importance of understanding the multiple factors that determine environmental use in this population.

Awareness During Anesthesia in Children: a Prospective Cohort Study

During routine adult anesthesia, the risk of awareness is 0.1%-0.2%. No recent studies have reported the incidence in children. Altered pharmacology and differing anesthesia techniques suggest that the incidence may differ in children. In this prospective cohort study, we determined the incidence of awareness during anesthesia in children. Eight-hundred-sixty-four children aged 5-12 yr who had undergone general anesthesia at The Royal Children's Hospital were interviewed on 3 occasions to determine the incidence of awareness. The awareness assessment was nested within a larger study of behavior change after anesthesia. Reports of suspected awareness were sent to four independent adjudicators. If they all agreed, a case was classified as true awareness. Twenty-eight reports were generated. There were 7 cases of true awareness, for an incidence of 0.8% (95% confidence interval, 0.3%-1.7%). Only one aware child received neuromuscular blockers, compared with 12% in the nonaware group. No aware child reported distress, and no substantial difference was detected in behavior disturbance between aware (20%) and nonaware (16%) children. The data provide some evidence that, like adults, children are also at risk of intraoperative awareness. Although the cause remains unclear, anesthesiologists should be alerted to the possibility of awareness in children.

Pain Reports by Older Adults in Long-term Care: a Pilot Study of Changes over Time

To document self-reported pain descriptions throughout residency in a long-term care facility for a convenience sample of older adults (Canadian war veterans).

Predicting Diagnosed Depression and Anti-depressant Treatment in Institutionalized Older Adults by Symptom Profiles: a Closer Look at Anhedonia and Dysphoria

The purpose of this study was to examine the relationships of diagnosis and treatment of depression with anhedonic and dysphoric symptom presentation, using the Minimum Data Set 2.0. Participants were from two sectors of longterm care: 70 nursing home residents and 92 residents in a Veterans' Care Service. The samples differed in their sex distribution and in cognition. A series of logistic regressions that controlled for demographics, type of facility, and cognition showed that dysphoric symptoms predicted diagnosed depression, whereas anhedonic symptoms predicted anti-depressant medication use without a concomitant diagnosis. The findings are consistent with a hypothesis that, in long-term care settings, anhedonic symptoms contribute less to a diagnosis of depression than do dysphoric symptoms. However, findings that anhedonic symptoms relate to treatment have implications for care-planning protocols.

A Cohort Study of the Incidence and Risk Factors for Negative Behavior Changes in Children After General Anesthesia

Hospitalization and anesthesia can have a substantial psychological impact on children, which may be manifested by negative behavioral change. The primary aims of this study were to estimate the incidence of negative behavior change postanesthesia in a large cohort of children, and to identify the possible risk factors.

Risk Factors for Anxiety at Induction of Anesthesia in Children: a Prospective Cohort Study

In children anxiety at induction of anesthesia is a common and important aspect of the psychological impact of anesthesia and surgery. Previous studies examining risk factors for increased anxiety have found contradictory results. This may be due to using small, or highly selective population samples, or failure to adjust for confounding variables. Results may also be culturally or institutionally specific. The aim of this study was to identify possible risk factors in a large representative cohort of children.

Mutation in Erythroid Specific Transcription Factor KLF1 Causes Hereditary Spherocytosis in the Nan Hemolytic Anemia Mouse Model

KLF1 regulates definitive erythropoiesis of red blood cells by facilitating transcription through high affinity binding to CACCC elements within its erythroid specific target genes including those encoding erythrocyte membrane skeleton (EMS) proteins. Deficiencies of EMS proteins in humans lead to the hemolytic anemia Hereditary Spherocytosis (HS) which includes a subpopulation with no known genetic defect. Here we report that a mutation, E339D, in the second zinc finger domain of KLF1 is responsible for HS in the mouse model Nan. The causative nature of this mutation was verified with an allelic test cross between Nan/+ and heterozygous Klf1(+/-) knockout mice. Homology modeling predicted Nan KLF1 binds CACCC elements more tightly, suggesting that Nan KLF1 is a competitive inhibitor of wild-type KLF1. This is the first association of a KLF1 mutation with a disease state in adult mammals and also presents the possibility of being another causative gene for HS in humans.

RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin

The dysregulation of vascular endothelial cells by thrombin has been implicated in the development of a number of pathologic disorders such as inflammatory conditions, cancer, diabetes, coronary heart disease. However, transcriptional regulation of vascular endothelial cells by thrombin is not completely understood. In the present study, Illumina RNA-seq was used to profile the transcriptome in human pulmonary microvascular endothelial cells (HMVEC-L) treated with thrombin for 6 h to gain insight into thrombin's direct effects on the endothelial function. Out of 100 million total reads from a paired end sequencing assay, 91-94% of the reads were aligned to over 16,000 genes in the reference human genome. Thrombin upregulated 150 known genes and 480 known isoforms, and downregulated 2,190 known genes and 3,574 known isoforms by at least 2 fold. Of note, thrombin upregulated 1,775 previously unknown isoforms and downregulated 12,202 previously unknown isoforms by at least 2 fold. Many genes displayed isoform specific differential expression levels and different usage of transcriptional start sites after the thrombin treatment. The cross comparisons between our RNA-seq data and those of DNA microarray analysis of either 6 h thrombin treated HUVEC or 5 h TNFα treated HMVEC have provided a significant overlapping list of differentially expressed genes, supporting the robust utility of our dataset. Further in-depth follow-up analysis of the transcriptional regulation reported in this study may shed light on molecular pathogenic mechanisms underlying thrombin mediated endothelial dysfunction in various diseases and provide new leads of potential therapeutic targets.

Pleiotropic Functions of Pre-B-cell Colony-enhancing Factor (PBEF) Revealed by Transcriptomics of Human Pulmonary Microvascular Endothelial Cells Treated with PBEFsiRNA

This study profiled transcriptomes of human pulmonary microvascular endothelial cells (HMVEC-L) treated with pre-B-cell colony-enhancing factor (PBEF) siRNA or scrambled RNA to gain insight into transcriptional regulations of PBEF on the endothelial function using the Affymetrix GeneChips HG-U133 plus 2. Several important themes are emerged from this study. First, PBEF affected expressions of multiple genes in the endothelium. Expression of 373 genes was increased and 64 genes decreased by at least 1.3-fold in the PBEFsiRNA-treated HMVEC-L versus the scramble RNA control. Second, the microarray results confirmed previous reports of PBEF-mediated gene expressions in some pathways but provided a more complete repertoire of molecules in those pathways. Third, most of the affected canonical pathways have not previously been reported to be PBEF responsive. Fourth, network analysis supports that PBEF has pleiotropic functions. Our first transcriptome analysis of human pulmonary microvascular endothelial cells treated with PBEFsiRNA has provided important insights into the transcriptional regulation of gene expression in HMVEC-L cells by PBEF. Further in-depth analysis of these transcriptional regulations may shed light on molecular mechanisms underlying PBEF-mediated endothelial functions and dysfunctions in various diseases and provide new leads of therapeutic targets to those diseases.

RNA-seq Analysis of Synovial Fibroblasts Brings New Insights into Rheumatoid Arthritis

ABSTRACT:

A Mark That is No Mark? Queer Women and Violence in HIV Discourse

Lesbian, bisexual and queer women are invisible and ignored in HIV discourse, as epidemiological classifications result in their institutionalised exclusion from risk categories. Simultaneously, these women live with HIV, often in situations of societal exclusion and under threat of violence. In this paper, we consider the connections between discourse and violence to examine how both are reproduced through, applied to and dependent upon people. The ways lesbian, bisexual and queer women do (or do not) appear in HIV discourse tells us much about how people and categories operate in the global pandemic. The fault-lines of lesbian, bisexual and queer women's constrained visibility in HIV discourse can be seen in situations where they are exposed to HIV transmission through homophobic sexual assault. In dominant HIV discursive practices, such homophobic assault leaves Judith Butler's 'mark that is no mark', recording neither its violence nor its 'non-heterosexuality'. Structural violence theory offers a means to understand direct and indirect violence as it pertains to HIV and lesbian, bisexual and queer women. We call for forms of modified structural violence theory that better attend to the ways in which discourse connects with material realities. Our theoretical and epidemiological lens must be broadened to examine how anti-lesbian, bisexual and queer-women bias affects transnational understandings of human worth.

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