Identification of LDH-A as a therapeutic target for cancer cell killing via (i) p53/NAD(H)-dependent and (ii) p53 independent pathways

Most cancer cells use aerobic glycolysis to fuel their growth. The enzyme lactate dehydrogenase-A (LDH-A) is key to cancer’s glycolytic phenotype, catalysing the regeneration of nicotinamide adenine dinucleotide (NAD þ ) from reduced nicotinamide adenine dinucleotide (NADH) necessary to sustain glycolysis. As such, LDH-A is a promising target for anticancer therapy. Here we ask if the tumour suppressor p53, a major regulator of cellular metabolism, influences the response of cancer cells to LDH-A suppression. LDH-A knockdown by RNA interference (RNAi) induced cancer cell death in p53 wild-type, mutant and p53-null human cancer cell lines, indicating that endogenous LDH-A promotes cancer cell survival irrespective of cancer cell p53 status. Unexpectedly,however,weuncoveredanovelroleforp53intheregulationofcancercellNADþ anditsreducedformNADH.Thus, LDH-A silencing by RNAi, or its inhibition using a small-molecule inhibitor, resulted in a p53-dependent increase in the cancer cell ratioofNADH:NADþ.Thiseffectwasspecificforp53þ/þ cancercellsandcorrelatedwith(i)reducedactivityofNADþ-dependent deacetylase sirtuin 1 (SIRT1) and (ii) an increase in acetylated p53, a known target of SIRT1 deacetylation activity. In addition, activation of the redox-sensitive anticancer drug EO9 was enhanced selectively in p53 þ / þ cancer cells, attributable to increased activity of NAD(P)H-dependent oxidoreductase NQO1 (NAD(P)H quinone oxidoreductase 1). Suppressing LDH-A increased EO9-inducedDNAdamageinp53þ/þ cancercells,butimportantlyhadnoadditiveeffectinnon-cancercells.Ourresultsidentifya unique strategy by which the NADH/NADþ cellular redox status can be modulated in a cancer-specific, p53-dependent manner and we show that this can impact upon the activity of important NAD(H)-dependent enzymes. To summarise, this work indicates two distinct mechanisms by which suppressing LDH-A could potentially be used to kill cancer cells selectively, (i) through induction of apoptosis, irrespective of cancer cell p53 status and (ii) as a part of a combinatorial approach with redox-sensitive anticancer drugs via a novel p53/NAD(H)-dependent mechanism

Build It—And Advocate for It—And They Will Come: Lessons from a Collaborative Project in Archives Advocacy and Program Development

Libraries at small- and mid-sized academic institutions continue to re-define themselves as journal and monograph collections go online, budgets and staffing remain flat or reduced, and value to student learning and the institutional mission needs to be apparent. This all spells opportunity for archival programs which, with a strong focus on advocacy and daylighting formerly hidden collections of unique content, can re-invigorate the library and spotlight the active role today\u27s service- and user-oriented archives can play in supporting student research, fostering ties with constituents, and ensuring the preservation of an institution\u27s stories and history. A recently-completed National Historical Publications and Records Commission (NHPRC)-funded grant project involving seven private institutions in Washington and Oregon utilized a focus on effective advocacy and consulting archivists to move archival programs to the next level. Despite limited resource levels at most of the institutions, tangible and sustainable progress was made on describing collections, establishing best-practices and policies, and perhaps most importantly, cultivating a strong ethic of persistent, creative, low-cost advocacy and outreach

The Interplay Between Self-Regulated Professional Learning And Teachers’ Work-Practice

This paper explores the relationship between practice and learning in the workplace, by focusing on the case of teachers. It is widely acknowledged that (teacher’s) professional learning is heavily informed by practice, and that an individual’s capacity to self-regulate their learning can improve the quality of learning. However less is known about the precise interplay between practice and self-regulated-learning. This paper integrates existing literature in three areas: professional learning, self-regulated learning and teacher professional development, drawing on recent work describing learning behaviors in informal workplace settings and on Teacher Professional Development. The paper develops a hypothesis on how teachers’ work practice stimulates their learning processes and, at the same time, is informed by their capacity to self-regulate their learning

"Meet people where they are": a qualitative study of community barriers and facilitators to HIV testing and HIV self-testing among African Americans in urban and rural areas in North Carolina.

BACKGROUND: HIV testing programs in the United States aim to reach ethnic minority populations who experience high incidence of HIV, yet 40% of African Americans have never been tested for HIV. The objective of this study is to identify community-based strategies to increase testing among African Americans in both urban and rural areas. METHODS: This study conducted focus group discussions (FGDs) informed by community-based participatory research principles to examine African American's concerns and ideas around HIV testing and HIV self-testing. Participants included highly affected (i.e., PLWH, MSM, PWID, low-income, teens and young adults) populations from African American communities in North Carolina, aged 15 years and older. We digitally transcribed and analyzed qualitative data using MAXQDA and axial coding to identify emergent themes. RESULTS: Fifty-two men and women between 15 to 60 years old living in urban (n=41) and rural (n=11) areas of North Carolina participated in focus group discussions. HIV testing barriers differed by HIV testing setting: facility-based, community-based, and HIV self-testing. In community-based settings, barriers included confidentiality concerns. In facility-based settings (e.g., clinics), barriers included negative treatment by healthcare workers. With HIV self-testing, barriers included improper use of self-testing kits and lack of post-test support. HIV testing facilitators included partnering with community leaders, decentralizing testing beyond facility-based sites, and protecting confidentiality. CONCLUSIONS: Findings suggest that HIV testing concerns among African Americans vary by HIV testing setting. African Americans may be willing to test for HIV at community events in public locations if client confidentiality is preserved and use HIV self-testing kits in private if post-test social support and services are provided. These community-identified facilitators may improve African American testing rates and uptake of HIV self-testing kits

RNA Interference by Single- and Double-stranded siRNA With a DNA Extension Containing a 3′ Nuclease-resistant Mini-hairpin Structure

Selective gene silencing by RNA interference (RNAi) involves double-stranded small interfering RNA (ds siRNA) composed of single-stranded (ss) guide and passenger RNAs. siRNA is recognized and processed by Ago2 and C3PO, endonucleases of the RNA-induced silencing complex (RISC). RISC cleaves passenger RNA, exposing the guide RNA for base-pairing with its homologous mRNA target. Remarkably, the 3' end of passenger RNA can accommodate a DNA extension of 19-nucleotides without loss of RNAi function. This construct is termed passenger-3'-DNA/ds siRNA and includes a 3'-nuclease-resistant mini-hairpin structure. To test this novel modification further, we have now compared the following constructs: (I) guide-3'-DNA/ds siRNA, (II) passenger-3'-DNA/ds siRNA, (III) guide-3'-DNA/ss siRNA, and (IV) passenger-3'-DNA/ss siRNA. The RNAi target was SIRT1, a cancer-specific survival factor. Constructs I-III each induced selective knock-down of SIRT1 mRNA and protein in both noncancer and cancer cells, accompanied by apoptotic cell death in the cancer cells. Construct IV, which lacks the SIRT1 guide strand, had no effect. Importantly, the 3'-DNA mini-hairpin conferred nuclease resistance to constructs I and II. Resistance required the double-stranded RNA structure since single-stranded guide-3'-DNA/ss siRNA (construct III) was susceptible to serum nucleases with associated loss of RNAi activity. The potential applications of 3'-DNA/siRNA constructs are discussed. Molecular Therapy-Nucleic Acids (2014) 2, e141; doi:10.1038/mtna.2013.68; published online 7 January 2014

Recommendations for a culturally relevant Internet-based tool to promote physical activity among overweight young African American women, Alabama, 2010-2011

INTRODUCTION: Innovative approaches are needed to promote physical activity among young adult overweight and obese African American women. We sought to describe key elements that African American women desire in a culturally relevant Internet-based tool to promote physical activity among overweight and obese young adult African American women. METHODS: A mixed-method approach combining nominal group technique and traditional focus groups was used to elicit recommendations for the development of an Internet-based physical activity promotion tool. Participants, ages 19 to 30 years, were enrolled in a major university. Nominal group technique sessions were conducted to identify themes viewed as key features for inclusion in a culturally relevant Internet-based tool. Confirmatory focus groups were conducted to verify and elicit more in-depth information on the themes. RESULTS: Twenty-nine women participated in nominal group (n = 13) and traditional focus group sessions (n = 16). Features that emerged to be included in a culturally relevant Internet-based physical activity promotion tool were personalized website pages, diverse body images on websites and in videos, motivational stories about physical activity and women similar to themselves in size and body shape, tips on hair care maintenance during physical activity, and online social support through social media (eg, Facebook, Twitter). CONCLUSION: Incorporating existing social media tools and motivational stories from young adult African American women in Internet-based tools may increase the feasibility, acceptability, and success of Internet-based physical activity programs in this high-risk, understudied population

NH125 Sensitizes Staphylococcus aureus to Cell Wall-Targeting Antibiotics through the Inhibition of the VraS Sensor Histidine Kinase

Staphylococcus aureus utilizes the two-component regulatory system VraSR to receive and relay environmental stress signals, and it is implicated in the development of bacterial resistance to several antibiotics through the upregulation of cell wall synthesis. VraS inhibition was shown to extend or restore the efficacy of several clinically used antibiotics. In this work, we study the enzymatic activity of the VraS intracellular domain (GST-VraS) to determine the kinetic parameters of the ATPase reaction and characterize the inhibition of NH125 under in vitro and microbiological settings. The rate of the autophosphorylation reaction was determined at different GST-VraS concentrations (0.95 to 9.49 μM) and temperatures (22 to 40°C) as well as in the presence of different divalent cations. The activity and inhibition by NH125, which is a known kinase inhibitor, were assessed in the presence and absence of the binding partner, VraR. The effects of inhibition on the bacterial growth kinetics and gene expression levels were determined. The GST-VraS rate of autophosphorylation increases with temperature and with the addition of VraR, with magnesium being the preferred divalent cation for the metal-ATP substrate complex. The mechanism of inhibition of NH125 was noncompetitive in nature and was attenuated in the presence of VraR. The addition of NH125 in the presence of sublethal doses of the cell wall-targeting antibiotics carbenicillin and vancomycin led to the complete abrogation of Staphylococcus aureus Newman strain growth and significantly decreased the gene expression levels of pbpB, blaZ, and vraSR in the presence of the antibiotics

JWST mirror and actuator performance at cryo-vacuum

The James Webb Space Telescope (JWST) telescope’s Secondary Mirror Assembly (SMA) and eighteen Primary Mirror Segment Assemblies (PMSAs) are each actively controlled in rigid body position via six hexapod actuators. Each of the PMSAs additionally has a radius of curvature actuator. The mirrors are stowed to the mirror support structure to survive the launch environment and then must be deployed 12.5 mm to reach the nominally deployed position before the Wavefront Sensing & Control (WFSC) alignment and phasing process begins. JWST requires testing of the full optical system in a Cryogenic Vacuum (CV) environment before launch. The cryo vacuum test campaign was executed in Chamber A at the Johnson Space Center (JSC) in Houston Texas. The test campaign consisted of an ambient vacuum test, a cooldown test, a cryo stable test at 65 Kelvin, a warmup test, and finally a second ambient vacuum test. Part of that test campaign was the functional and performance testing of the hexapod actuators on the flight mirrors. This paper will describe the testing that was performed on all 132 hexapod and radius of curvature actuators. The test campaign first tests actuators individually then tested how the actuators perform in the hexapod system. Telemetry from flight sensors on the actuators and measurements from external metrology devices such as interferometers, photogrammetry systems and image analysis was used to demonstrate the performance of the JWST actuators. The mirror move commanding process was exercised extensively during the JSC CV test and many examples of accurately commanded moves occurred. The PMSA and SMA actuators performed extremely well during the JSC CV test, and we have demonstrated that the actuators are fully functional both at ambient and cryo temperatures and that the mirrors will go to their commanded positions with the accuracy needed to phase and align the telescope