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

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

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

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