Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Instructional Models for Course-Based Research Experience (CRE) Teaching

The course-based research experience (CRE) with its documented educational benefits is increasingly being implemented in science, technology, engineering, and mathematics education. This article reports on a study that was done over a period of 3 years to explicate the instructional processes involved in teaching an undergraduate CRE. One hundred and two instructors from the established and large multi-institutional SEA-PHAGES program were surveyed for their understanding of the aims and practices of CRE teaching. This was followed by large-scale feedback sessions with the cohort of instructors at the annual SEA Faculty Meeting and subsequently with a small focus group of expert CRE instructors. Using a qualitative content analysis approach, the survey data were analyzed for the aims of inquiry instruction and pedagogical practices used to achieve these goals. The results characterize CRE inquiry teaching as involving three instructional models: 1) being a scientist and generating data; 2) teaching procedural knowledge; and 3) fostering project ownership. Each of these models is explicated and visualized in terms of the specific pedagogical practices and their relationships. The models present a complex picture of the ways in which CRE instruction is conducted on a daily basis and can inform instructors and institutions new to CRE teaching

Chronic hypoxia promotes pulmonary artery endothelial cell proliferation through H2O2-induced 5-lipoxygenase.

Pulmonary Hypertension (PH) is a progressive disorder characterized by endothelial dysfunction and proliferation. Hypoxia induces PH by increasing vascular remodeling. A potential mediator in hypoxia-induced PH development is arachidonate 5-Lipoxygenase (ALOX5). While ALOX5 metabolites have been shown to promote pulmonary vasoconstriction and endothelial cell proliferation, the contribution of ALOX5 to hypoxia-induced proliferation remains unknown. We hypothesize that hypoxia exposure stimulates HPAEC proliferation by increasing ALOX5 expression and activity. To test this, human pulmonary artery endothelial cells (HPAEC) were cultured under normoxic (21% O2) or hypoxic (1% O2) conditions for 24-, 48-, or 72 hours. In a subset of cells, the ALOX5 inhibitor, zileuton, or the 5-lipoxygenase activating protein inhibitor, MK-886, was administered during hypoxia exposure. ALOX5 expression was measured by qRT-PCR and western blot and HPAEC proliferation was assessed. Our results demonstrate that 24 and 48 hours of hypoxia exposure have no effect on HPAEC proliferation or ALOX5 expression. Seventy two hours of hypoxia significantly increases HPAEC ALOX5 expression, hydrogen peroxide (H2O2) release, and HPAEC proliferation. We also demonstrate that targeted ALOX5 gene silencing or inhibition of the ALOX5 pathway by pharmacological blockade attenuates hypoxia-induced HPAEC proliferation. Furthermore, our findings indicate that hypoxia-induced increases in cell proliferation and ALOX5 expression are dependent on H2O2 production, as administration of the antioxidant PEG-catalase blocks these effects and addition of H2O2 to HPAEC promotes proliferation. Overall, these studies indicate that hypoxia exposure induces HPAEC proliferation by activating the ALOX5 pathway via the generation of H2O2

A Meta-analysis of neuropsychological functioning in first-episode bipolar disorders

Broad neuropsychological deficits have been consistently demonstrated in well-established bipolar disorder. The aim of the current study was to systematically review neuropsychological studies in first-episode bipolar disorders to determine the breadth, extent and predictors of cognitive dysfunction at this early stage of illness through meta-analytic procedures. Electronic databases were searched for studies published between January 1980 and December 2013. Twelve studies met eligibility criteria (N = 341, mean age = 28.2 years), and pooled effect sizes (ES) were calculated across eight cognitive domains. Moderator analyses were conducted to identify predictors of between-study heterogeneity. Controlling for known confounds, medium to large deficits (ES ≥ 0.5) in psychomotor speed, attention and working memory, and cognitive flexibility were identified, whereas smaller deficits (ES 0.20-0.49) were found in the domains of verbal learning and memory, attentional switching, and verbal fluency. A medium to large deficit in response inhibition was only detected in non-euthymic cases. Visual learning and memory functioning was not significantly worse in cases compared with controls. Overall, first-episode bipolar disorders are associated with widespread cognitive dysfunction. Since euthymia was not associated with superior cognitive performance in most domains, these results indicate that even in the earliest stages of disease, cognitive deficits are not mood-state dependent. The current findings have important implications for whether cognitive impairments represent neurodevelopmental or neurodegenerative processes. Future studies need to more clearly characterise the presence of psychotic features, and the nature and number of previous mood episodes.11 page(s

ROS mediate hypoxia-induced increases in endothelial ALOX5 expression and cell proliferation.

<p>Human pulmonary artery endothelial cells (HPAEC) were exposed to 0, 10, 100, and 200 µM hydrogen peroxide (H₂O₂) for 24 hours. Following exposure, supernatants were collected to assess cell toxicity by adenylate kinase release. Results demonstrate no significant changes in cell death as indicated by adenylate kinase release (n = 4–6; data not shown). HPAEC were collected and total RNA was isolated for quantitative real-time PCR gene expression analysis. ALOX5 was normalized to the housekeeping gene β-globin. Relative expression was calculated using the Delta-Delta C_T method and values were expressed as percent of control (A, n = 4–5). * p<0.05 when compared to untreated controls. H₂O₂ exposure stimulates HPAEC ALOX5 protein levels as analyzed by western blot (B, n = 4). PEG-Catalase (10U - 1000 U/ml) administration during the final 24 hours of the 72 hour hypoxia exposure prevents hypoxia-induced elevations in endothelial ALOX5 expression (C, n = 5) and cell proliferation (D, n = 6). * p<0.01 when compared to normoxic groups. ** p<0.05 when compared to untreated hypoxia controls.</p

Hypoxia exposure stimulates endothelial ROS release.

<p>Human pulmonary artery endothelial cells were exposed to normoxic or hypoxic (1% O₂) conditions for 24-, 48- or 72-hours. Following exposure, HPAEC ROS release was assessed by DCF staining (A, n = 3) and Amplex Red assay (C, n = 4). Results demonstrate that prolonged hypoxia exposure significantly increases endothelial ROS production whereas administration with the antioxidants, PEG- catalase or superoxide dismutase reduces these effects (B). Amplex Red Assay indicates that chronic hypoxia exposure promotes H₂O₂ release (C). * p<0.0001 when compared to normoxic controls.</p

Pharmacological inhibition of ALOX5 signaling attenuates hypoxia-induced endothelial proliferation.

<p>ALOX5 blockade by zileuton administration reduces hypoxia-induced endothelial proliferation when measured by MTT Assay (A, n = 4). FLAP inhibition by MK-886 attenuates endothelial proliferation following hypoxia exposure (B, n = 4) Pre-treatment with the cysteinyl leukotriene receptor antagonist, montelukast prevents endothelial proliferation during prolonged hypoxia exposure (C, n = 6) HPAEC were exposed to normoxic or hypoxic conditions for 72 hours. ALOX5 inhibitors, zileuton (10 µM) and MK-886 (0.5 µM) were administered during the final 24 hours of normoxia or hypoxia exposure. Cell proliferation was then assessed by MTT assay. * p<0.05 when compared to normoxic groups. ** p<0.05 when compared to untreated hypoxic groups.</p

Prolonged hypoxia exposure increases endothelial cell proliferation.

<p>Seventy two hours of hypoxia exposure significantly stimulates endothelial cell proliferation when compared to all other groups. Human pulmonary artery endothelial cells (HPAEC) were exposed to normoxic or hypoxic (1% O₂) conditions for 24-, 48-, or 72 hours (n = 4). Following exposure, cell proliferation was assessed by MTT (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098532#pone-0098532-g001" target="_blank">Figure 1A</a>) assay and Trypan Blue Dye Exclusion Assay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098532#pone-0098532-g001" target="_blank">Figure 1B</a>). * p<0.0001.</p

Chronic hypoxia exposure increases endothelial ALOX5 expression.

<p>Seventy two hours of hypoxia exposure significantly stimulates endothelial ALOX5 expression when compared to all other groups. HPAEC were exposed to normoxic or hypoxic conditions for 24-, 48-, or 72 hours. Following exposure, cells were collected, and total RNA and protein were isolated for expression analyses via quantitative real time PCR and Western blot respectively. Results indicate that ALOX5 mRNA levels are significantly increased following hypoxia exposure (A, n = 5). Chronic hypoxia exposure also causes a 3-fold elevation in ALOX5 protein expression levels (B, n = 4). Endothelial FLAP expression is also increased when compared to all other groups (C, n = 5–7). Values are expressed as percent of control. * p<0.001 when compared to all other groups.</p

HIV X4 Variants Increase Arachidonate 5-Lipoxygenase in the Pulmonary Microenvironment and are associated with Pulmonary Arterial Hypertension.

Pulmonary Arterial Hypertension (PAH) is overrepresented in People Living with Human Immunodeficiency Virus (PLWH). HIV protein gp120 plays a key role in the pathogenesis of HIV-PAH. Genetic changes in HIV gp120 determine viral interactions with chemokine receptors; specifically, HIV-X4 viruses interact with CXCR4 while HIV-R5 interact with CCR5 co-receptors. Herein, we leveraged banked samples from patients enrolled in the NIH Lung HIV studies and used bioinformatic analyses to investigate whether signature sequences in HIV-gp120 that predict tropism also predict PAH. Further biological assays were conducted in pulmonary endothelial cells in vitro and in HIV-transgenic rats. We found that significantly more persons living with HIV-PAH harbor HIV-X4 variants. Multiple HIV models showed that recombinant gp120-X4 as well as infectious HIV-X4 remarkably increase arachidonate 5-lipoxygenase (ALOX5) expression. ALOX5 is essential for the production of leukotrienes; we confirmed that leukotriene levels are increased in bronchoalveolar lavage fluid of HIV-infected patients. This is the first report associating HIV-gp120 genotype to a pulmonary disease phenotype, as we uncovered X4 viruses as potential agents in the pathophysiology of HIV-PAH. Altogether, our results allude to the supplementation of antiretroviral therapy with ALOX5 antagonists to rescue patients with HIV-X4 variants from fatal PAH