CHK1 Inhibition Synergizes with Gemcitabine Initially by Destabilizing the DNA Replication Apparatus.

Combining cell-cycle checkpoint kinase inhibitors with the DNA-damaging chemotherapeutic agent gemcitabine offers clinical appeal, with a mechanistic rationale based chiefly on abrogation of gemcitabine-induced G2-M checkpoint activation. However, evidence supporting this mechanistic rationale from chemosensitization studies has not been consistent. Here we report a systematic definition of how pancreatic cancer cells harboring mutant p53 respond to this combination therapy, by combining mathematical models with large-scale quantitative biologic analyses of single cells and cell populations. Notably, we uncovered a dynamic range of mechanistic effects at different ratios of gemcitabine and CHK1 inhibitors. Remarkably, effective synergy was attained even where cells exhibited an apparently functional G2-M surveillance mechanism, as exemplified by a lack of both overt premature CDK1 activation and S-phase mitotic entry. Consistent with these findings, S-G2 duration was extended in treated cells, leading to a definable set of lineage-dependent catastrophic fates. At synergistic drug concentrations, global replication stress was a distinct indicator of chemosensitization as characterized molecularly by an accumulation of S-phase cells with high levels of hyperphosphorylated RPA-loaded single-stranded DNA. In a fraction of these cells, persistent genomic damage was observed, including chromosomal fragmentation with a loss of centromeric regions that prevented proper kinetochore-microtubule attachment. Together, our results suggested a "foot-in-the-door" mechanism for drug synergy where cells were destroyed not by frank G2-M phase abrogation but rather by initiating a cumulative genotoxicity that deregulated DNA synthesis.This study was funded by Cancer Research UK via Institute Senior Group Leader funding (C14303/A17197) to DI Jodrell, and by Sentinel Oncology through an award from Innovate UK.This is the author accepted manuscript. The final version is available from American Association for Cancer Research via http://dx.doi.org/10.1158/0008-5472.CAN-14-334

Gold nanoparticle interactions with endothelial cells cultured under physiological conditions

PEGylated gold nanoparticles (AuNPs) have an extended circulation time after intravenous injection in vivo and exhibit favorable properties for biosensing, diagnostic imaging, and cancer treatment. No impact of PEGylated AuNPs on the barrier forming properties of endothelial cells (ECs) has been reported, but recent studies demonstrated that unexpected effects on erythrocytes are observed. Almost all studies to date have been with static-cultured ECs. Herein, ECs maintained under physiological cyclic stretch and flow conditions and used to generate a blood–brain barrier model were exposed to 20 nm PEGylated AuNPs. An evaluation of toxic effects, cell stress, the release profile of pro-inflammatory cytokines, and blood–brain barrier properties showed that even under physiological conditions no obvious effects of PEGylated AuNPs on ECs were observed. These findings suggest that 20 nm-sized, PEGylated AuNPs may be a useful tool for biomedical applications, as they do not affect the normal function of healthy ECs after entering the blood stream

Global state and potential scope of investments in watershed services for large cities

Investments in watershed services (IWS) programs, in which downstream water users pay upstream watershed service suppliers for actions that protect drinking water, are increasing in number and scope. IWS programs represent over $170 million of investment in over 4.3 million ha of watersheds, providing water to over 230 million people. It is not yet fully clear what factors contribute to the establishment and sustainability of IWS. We conducted a representative global analysis of 416 of the world’s largest cities, including 59 (14%) with IWS programs. Using random forest ensemble learning methods, we evaluated the relative importance of social and ecological factors as predictors of IWS presence. IWS programs are more likely present in source watersheds with more agricultural land and less protected area than otherwise similar watersheds. Our results suggest potential to expand IWS as a strategy for drinking water protection and also contribute to decisions regarding suitable program locations

Double-Peaked Low-Ionization Emission Lines in Active Galactic Nuclei

We present a new sample of 116 double-peaked Balmer line Active Galactic Nuclei (AGN) selected from the Sloan Digital Sky Survey. Double-peaked emission lines are believed to originate in the accretion disks of AGN, a few hundred gravitational radii (Rg) from the supermassive black hole. We investigate the properties of the candidate disk emitters with respect to the full sample of AGN over the same redshifts, focusing on optical, radio and X-ray flux, broad line shapes and narrow line equivalent widths and line flux-ratios. We find that the disk-emitters have medium luminosities (~10^44erg/s) and FWHM on average six times broader than the AGN in the parent sample. The double-peaked AGN are 1.6 times more likely to be radio-sources and are predominantly (76%) radio quiet, with about 12% of the objects classified as LINERs. Statistical comparison of the observed double-peaked line profiles with those produced by axisymmetric and non-axisymmetric accretion disk models allows us to impose constraints on accretion disk parameters. The observed Halpha line profiles are consistent with accretion disks with inclinations smaller than 50 deg, surface emissivity slopes of 1.0-2.5, outer radii larger than ~2000 Rg, inner radii between 200-800Rg, and local turbulent broadening of 780-1800 km/s. The comparison suggests that 60% of accretion disks require some form of asymmetry (e.g., elliptical disks, warps, spiral shocks or hot spots).Comment: 60 pages, 19 figures, accepted for publication in AJ. For high quality figures and full tables, please see http://astro.princeton.edu/~iskra/disks.htm

Evidence for Reionization at z ~ 6: Detection of a Gunn-Peterson Trough in a z=6.28 Quasar

We present moderate resolution Keck spectroscopy of quasars at z=5.82, 5.99 and 6.28, discovered by the Sloan Digital Sky Survey (SDSS). We find that the Ly Alpha absorption in the spectra of these quasars evolves strongly with redshift. To z~5.7, the Ly Alpha absorption evolves as expected from an extrapolation from lower redshifts. However, in the highest redshift object, SDSSp J103027.10+052455.0 (z=6.28), the average transmitted flux is 0.0038+-0.0026 times that of the continuum level over 8450 A < lambda < 8710 A (5.95<z(abs)<6.16), consistent with zero flux. Thus the flux level drops by a factor of >150, and is consistent with zero flux in the Ly Alpha forest region immediately blueward of the Ly Alpha emission line, compared with a drop by a factor of ~10 at z(abs)~5.3. A similar break is seen at Ly Beta; because of the decreased oscillator strength of this transition, this allows us to put a considerably stronger limit, tau(eff) > 20, on the optical depth to Ly Alpha absorption at z=6. This is a clear detection of a complete Gunn-Peterson trough, caused by neutral hydrogen in the intergalactic medium. Even a small neutral hydrogen fraction in the intergalactic medium would result in an undetectable flux in the Ly Alpha forest region. Therefore, the existence of the Gunn-Peterson trough by itself does not indicate that the quasar is observed prior to the reionization epoch. However, the fast evolution of the mean absorption in these high-redshift quasars suggests that the mean ionizing background along the line of sight to this quasar has declined significantly from z~5 to 6, and the universe is approaching the reionization epoch at z~6.Comment: Revised version (2001 Sep 4) accepted by the Astronomical Journal (minor changes

The FIRST Bright Quasar Survey. II. 60 Nights and 1200 Spectra Later

We have used the VLA FIRST survey and the APM catalog of the POSS-I plates as the basis for constructing a new radio-selected sample of optically bright quasars. This is the first radio-selected sample that is competitive in size with current optically selected quasar surveys. Using only two basic criteria, radio-optical positional coincidence and optical morphology, quasars and BL Lacs can be identified with 60% selection efficiency; the efficiency increases to 70% for objects fainter than magnitude 17. We show that a more sophisticated selection scheme can predict with better than 85% reliability which candidates will turn out to be quasars. This paper presents the second installment of the FIRST Bright Quasar Survey with a catalog of 636 quasars distributed over 2682 square degrees. The quasar sample is characterized and all spectra are displayed. The FBQS detects both radio-loud and radio-quiet quasars out to a redshift z>3. We find a large population of objects of intermediate radio-loudness; there is no evidence in our sample for a bimodal distribution of radio characteristics. The sample includes ~29 broad absorption line quasars, both high and low ionization, and a number of new objects with remarkable optical spectra.Comment: 41 pages plus 39 gifs which contain all quasar spectra. Accepted for publication in the Astrophysical Journal Supplement Serie

The Ensemble Photometric Variability of ~25000 Quasars in the Sloan Digital Sky Survey

Using a sample of over 25000 spectroscopically confirmed quasars from the Sloan Digital Sky Survey, we show how quasar variability in the rest frame optical/UV regime depends upon rest frame time lag, luminosity, rest wavelength, redshift, the presence of radio and X-ray emission, and the presence of broad absorption line systems. The time dependence of variability (the structure function) is well-fit by a single power law on timescales from days to years. There is an anti-correlation of variability amplitude with rest wavelength, and quasars are systematically bluer when brighter at all redshifts. There is a strong anti-correlation of variability with quasar luminosity. There is also a significant positive correlation of variability amplitude with redshift, indicating evolution of the quasar population or the variability mechanism. We parameterize all of these relationships. Quasars with RASS X-ray detections are significantly more variable (at optical/UV wavelengths) than those without, and radio loud quasars are marginally more variable than their radio weak counterparts. We find no significant difference in the variability of quasars with and without broad absorption line troughs. Models involving multiple discrete events or gravitational microlensing are unlikely by themselves to account for the data. So-called accretion disk instability models are promising, but more quantitative predictions are needed.Comment: 41 pages, 21 figures, AASTeX, Accepted for publication in Ap

Depletion of Macrophages Improves Therapeutic Response to Gemcitabine in Murine Pancreas Cancer.

BACKGROUND: The tumor microenvironment (TME) is composed of fibro-inflammatory cells and extracellular matrix (ECM) components. However, the exact contribution of the various TME compartments towards therapeutic response is unknown. Here, we aim to dissect the specific contribution of tumor-associated macrophages (TAMs) towards drug delivery and response in pancreatic ductal adenocarcinoma (PDAC). METHODS: The effect of gemcitabine was assessed in human and murine macrophages, human pancreatic stellate cells (hPSCs), and tumor cells (L3.6pl, BxPC3 and KPC) in vitro. The drug metabolism of gemcitabine was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Preclinical studies were conducted using KrasG12D;p48-Cre and KrasG12D;p53172H;Pdx-Cre mice to investigate gemcitabine delivery at different stages of tumor progression and upon pharmacological TAM depletion. RESULTS: Gemcitabine accumulation was significantly increased in murine PDAC tissue compared to pancreatic intraepithelial neoplasia (PanIN) lesions and healthy control pancreas tissue. In vitro, macrophages accumulated and rapidly metabolized gemcitabine resulting in a significant drug scavenging effect for gemcitabine. Finally, pharmacological TAM depletion enhanced therapeutic response to gemcitabine in tumor-bearing KPC mice. CONCLUSION: Macrophages rapidly metabolize gemcitabine in vitro, and pharmacological depletion improves the therapeutic response to gemcitabine in vivo. Our study supports the notion that TAMs might be a promising therapeutic target in PDAC

Macrophage-Engineered Vesicles for Therapeutic Delivery and Bidirectional Reprogramming of Immune Cell Polarization

Macrophages, one of the most important phagocytic cells of the immune system, are highly plastic and are known to exhibit diverse roles under different pathological conditions. The ability to repolarize macrophages from pro-inflammatory (M1) to anti-inflammatory (M2) or vice versa offers a promising therapeutic approach for treating various diseases such as traumatic injury and cancer. Herein, it is demonstrated that macrophage-engineered vesicles (MEVs) generated by disruption of macrophage cellular membranes can be used as nanocarriers capable of reprogramming macrophages and microglia toward either pro- or anti-inflammatory phenotypes. MEVs can be produced at high yields and easily loaded with diagnostic molecules or chemotherapeutics and delivered to both macrophages and cancer cells in vitro and in vivo. Overall, MEVs show promise as potential delivery vehicles for both therapeutics and their ability to controllably modulate macrophage/microglia inflammatory phenotypes