A comparison of the association of spermine with duplex and quadruplex DNA by NMR

AbstractThe association of [1′,1″-13C2]spermine ([1′,1″-13C2]N,N′-bis(3-aminopropyl)-1,4-butanediamine) with duplex and quadruplex DNA has been studied by nuclear magnetic resonance spectroscopy. 1D NOESY experiments using two-way selective cross-polarization (ISI-SCP-NOESY) showed spermine intramolecular NOEs are either weakly positive or weakly negative when spermine is complexed to duplex B-DNA and linear four-stranded quadruplex DNA. In contrast, large negative intramolecular NOEs are observed when spermine is complexed to two distinct forms of folded quadruplex DNA suggesting greater immobilization of spermine on these folded DNA quadruplexes. No changes in the quadruplex stem structure are observed but there are minor changes to the loop structure of a two-stranded folded quadruplex on binding spermine

Campus Environment 2008: A National Report Card on Sustainability in Higher Education

Presents survey findings on national and regional trends among colleges in environmental leadership in management, academic courses in sustainability, and conservation efforts in operations. Profiles exemplary programs and notes areas for improvement

Involvement of TGFβ signaling pathway in oxidative stress and diabetic retinopathy

Diabetic Retinopathy (DR) is a leading cause of blindness in the U.S. However, not much is known of underlying molecular mechanism and how oxidative stress contributes to its development. In the present study, we investigated the involvement of TGFβ signaling pathway on the effect of oxidative stress on VEGF secretion and viability of retinal cells. VEGF is the hallmark that exacerbates DR progression in prolonged diabetes. Some major concerns that have arisen are the underlying effects of antioxidants in elevating VEGF secretion in diabetes. In this study, we evaluated how hypoxia (or low oxygen) impacts viability and VEGF secretion using 661W cone photoreceptor cells. Confluent 661W cells were grown in 5.5 mM normal or 30 mM high glucose, as well as subjected to CoCl2 to induce hypoxia. After treatment for 24 hours, conditioned media were collected for ELISA measurement to determine the amount of protein (VEGF) secretion. Viable cell numbers were also recorded. High glucose did not induce significant changes in viable cell number nor VEGF concentration in cell media. However, hypoxia condition resulted in a three-fold decrease in viable cell numbers and a three-fold increase in VEGF concentration. Furthermore, treatment with two TGFβ inhibitors: SMAD 3, SIS (or Inhibitor 1) and TGFβ receptor 1 kinase inhibitor (or Inhibitor 2) resulted in a reversal of hypoxia-induced changes. These results strongly suggest that TGFβ signaling pathway mediates hypoxia-induced retinal cell viability and VEGF secretion. Further translational research studies will provide evidence to identify appropriate and effective pharmaceutical targets in this molecular pathway to mitigate the development of DR

New Frontiers for the NFIL3 bZIP Transcription Factor in Cancer, Metabolism and Beyond

The bZIP transcription factor NFIL3 (Nuclear factor Interleukin 3 regulated, also known as E4 binding protein 4, E4BP4) regulates diverse biological processes from circadian rhythm to cellular viability. Recently, a host of novel roles have been identified for NFIL3 in immunological signal transduction, cancer, aging and metabolism. Elucidating the signaling pathways that are impacted by NFIL3 and the regulatory mechanisms that it targets, inhibits or activates will be critical for developing a clearer picture of its physiological roles in disease and normal processes. This review will discuss the recent advances and emerging issues regarding NFIL3-mediated transcriptional regulation of CEBPb and FOXO1 activated genes and signal transduction

The FOXO1 inhibitor AS1842856 triggers apoptosis in glioblastoma multiforme and basal-like breast cancer cells

Basal-like breast cancer (BBC) and glioblastoma multiforme (GBM) are poor-prognosis cancers that lack effective targeted therapies and harbor embryonic stem gene expression signatures. Recently, our group and others found that forkhead box transcription factor FOXO1 promotes stem gene expression in BBC and GBM cell lines. Given the critical role of cancer stem cells in promoting cancer progression, we examined the impact of FOXO1 inhibition with AS1842856 (a cell-permeable small molecule that directly binds to unphosphorylated FOXO1 protein to block transcriptional regulation) on BBC and GBM cell viability. We treated a set of BBC and GBM cancer cell lines with increasing concentrations of AS1842856 and found reduced colony formation. Treatment of BBC and GBM cancer cells with AS1842856 led to increases in FAS (FAS cell surface death receptor) and BIM (BCL2L11) gene expression, as well as increased positivity for markers for apoptosis such as annexin V and propidium iodide. Treatment with another FOXO1 inhibitor AS1708727 or FOXO1 RNAi also led to FAS induction. This work is the first to show that targeting BBC and GBM with FOXO1 inhibition leads to apoptosis. These novel findings may ultimately expand the repertoire of therapies for poor-prognosis cancers

Oxidative insults disrupt OPA1-mediated mitochondrial dynamics in cultured mammalian cells

Objective: To explore the impact of oxidative insults on mitochondrial dynamics. In mammalian cells, oxidative insults activate stress response pathways including inflammation, cytokine secretion, and apoptosis. Intriguingly, mitochondria are emerging as a sensitive network that may function as an early indicator of subsequent cellular stress responses. Mitochondria form a dynamic network, balancing fusion, mediated by optic atrophy-1 (OPA1), and fission events, mediated by dynamin-related protein-1 (DRP1), to maintain homeostasis. Methods: Here, we examine the impact of oxidative insults on mitochondrial dynamics in 143B osteosarcoma and H9c2 cardiomyoblast cell lines via confocal microscopy, flow cytometry, and protein-based analyses. Results: When challenged with hydrogen peroxide (H2O2), a ROS donor, both cell lines display fragmentation of the mitochondrial network and loss of fusion-active OPA1 isoforms, indicating that OPA1-mediated mitochondrial fusion is disrupted by oxidative damage in mammalian cells. Consistent with this, cells lacking OMA1, a key protease responsible for cleavage of OPA1, are protected against OPA1 cleavage and mitochondrial fragmentation in response to H2O2 challenge. Discussion: Taken together, these findings indicate that oxidative insults damage OPA1-mediated mitochondrial dynamics in mammalian cells via activation of OMA1, consistent with an emerging role for mitochondrial dynamics as an early indicator of cellular stress signaling

Seasonal Flow Rates along the Lower Bear River, UT

The goal of this research is to identify how flow on the Bear River in Cache Valley has changed over the last three years and how flow changes seasonally. Identifying flows is important to manage water resources along the Bear River. We collected and processed water pressure data every 30 minutes using HOBO transducers at two sites in Cache Valley (Morton, just downstream of highway 142, and Confluence which is located at the confluence of the Bear and Cub Rivers) south of the Idaho‐Utah border in 2015. We also measured flow and water stage up to three times per year at each site using an Acoustic Doppler Current profiler and transom survey equipment. We pooled these observations with measurements and data collected by prior undergraduate Bear River Fellow researchers in 2012 and 2013 and used the observations to generate linear regression models to relate water stage to flow at each site. By applying the linear regression model to our pressure measurements, we calculated flow at each time a pressure reading was recorded for its respective location on the lower Bear River. We used the flow rates to determine that very little water is lost or gained between the USGS gage and the Morton site but that during summer months nearly 300cfs is lost between the Morton and Confluence sites. This information can help Bear River pumpers better manage their use

PI3K Pathway Inhibition with NVP-BEZ235 Hinders Glycolytic Metabolism in Glioblastoma Multiforme Cells

Glioblastoma (GBM) is the most lethal primary brain cancer that lacks effective molecular targeted therapies. The PI3K/AKT/mTOR pathway is activated in 90% of all Glioblastoma multiforme (GBM) tumors. To gain insight into the impact of the PI3K pathway on GBM metabolism, we treated U87MG GBM cells with NVP-BEZ235 (PI3K and mTOR a dual inhibitor) and identified differentially expressed genes with RNA-seq analysis. RNA-seq identified 7803 differentially regulated genes in response to NVP-BEZ235. Gene Set Enrichment Analysis (GSEA) identified two glycolysis-related gene sets that were significantly enriched (p \u3c 0.05) in control samples compared to NVP-BEZ235-treated samples. We validated the inhibition of glycolytic genes by NVP-BEZ235 and examined the impact of the FOXO1 inhibitor (AS1842856) on these genes in a set of GBM cell lines. FOXO1 inhibition alone was associated with reduced LDHA expression, but not ENO1 or PKM2. Bioinformatics analyses revealed that PI3K-impacted glycolytic genes were over-expressed and co-expressed in GBM clinical samples. The elevated expression of PI3K-impacted glycolytic genes was associated with poor prognosis in GBM based on Kaplan–Meier survival analyses. Our results suggest novel insights into hallmark metabolic reprogramming associated with the PI3K-mTOR dual inhibition

CRISPR Cas9 Genome Editing in Human Cell Lines with DONOR Vector Made by Gibson Assembly

CRISPR Cas9 genome editing allows researchers to modify genesin a multitude of ways including to obtain deletions, epitope-tagged loci, and knock-in mutations. Within six years of its initial application, CRISPR Cas9 genome editing has become widely employed, but disadvantages to this method, such as low modification efficiencies and off-target effects,need careful consideration. Obtaining custom donor vectors can also be expensive and time consuming. This chapter details strategies to overcome barriers to CRISPR Cas9 genome editing as well as recent developments in employing this technique