A population-based association study of SNPs of GSTP1, MnSOD, GPX2 and Barrett's esophagus and esophageal adenocarcinoma

Oxidative stress appears to be important in the pathogenesis of Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). Single-nucleotide polymorphisms (SNPs) of antioxidant enzyme genes may play a part in determining individual susceptibility to these diseases. The Factors Influencing the Barrett's Adenocarcinoma Relationship (FINBAR) study is a population-based, case-control study of BE and EAC in Ireland. DNA from EAC (n = 207), BE (> or =3 cm BE at endoscopy with specialized intestinal metaplasia on biopsy, n = 189) and normal population controls (n = 223) were analyzed. Several SNPs spanning the genes for glutathione S-transferase P1 (GSTP1), manganese superoxide dismutase (MnSOD) and glutathione peroxidase 2 (GPX2) were genotyped using multiplex polymerase chain reaction and SNaPshottrade mark. The chi(2) test was used to compare genotype and allele frequencies between case and control subjects. Linkage disequilibrium between SNPs was quantified using Lewontin's D' value and haplotype frequency estimates obtained using Haploview. Eleven SNPs were genotyped (six for GSTP1, three for MnSOD and two for GPX2); all were in Hardy-Weinberg equilibrium. None was significantly associated with EAC or BE even before Bonferroni correction. Odds ratios for EAC for individual SNPs ranged from 0.68 [95% confidence interval (CI) 0.43-1.08] to 1.25 (95% CI 0.73-2.16), and for BE from 0.84 (95% CI 0.52-1.30) to 1.30 (95% CI 0.85-1.97). SNPs in all three genes were in strong linkage disequilibrium (D' > 0.887) but haplotype analysis did not show any significant association with EAC or BE. SNPs involving the GSTP1, MnSOD and GPX2 genes were not associated with BE or EAC. Further studies aimed at identifying susceptibility genes should focus on different antioxidant genes or different pathways.

Outer membrane differences between pathogenic and environmental Yersinia enterocolitica biogroups probed with hydrophobic permeants and polycationic peptides

Sensitivities to polycationic peptides and EDTA were compared in Yersinia enterocolitica pathogenic and environmental biogroups. As shown by changes in permeability to the fluorescent hydrophobic probe N-phenylnaphthylamine (NPN), the outer membranes (OMs) of pathogenic and environmental strains grown at 26 degrees C in standard broth were more resistant to poly-L-lysine, poly-L-ornithine, melittin, cecropin P1, polymyxin B, and EDTA than Escherichia coli OMs. At 37 degrees C, OMs of pathogenic biogroups were resistant to EDTA and polycations and OMs of environmental strains were resistant to EDTA whereas E. coli OMs were sensitive to both EDTA and polycations. Similar results were found when testing deoxycholate sensitivity after polycation exposure or when isogenic pairs with or without virulence plasmid pYV were compared. With bacteria grown without Ca++ available, OM permeability to NPN was drastically increased in pathogenic but not in environmental strains or E. coli. Under these conditions, OMs of pYV+ and pYV- cells showed small differences in NPN permeability but differences in polycation sensitivity could not be detected by fluorimetry. O:1,6 (environmental type) lipopolysaccharide (LPS), but not O:3 or O:8 LPS, was markedly rough at 37 degrees C, and this could explain the differences in polycation sensitivity. LPSs from serotypes O:3 and O:8 grown at 37 degrees C were more permeable to NPN than O:1,6 LPS, and O:8 LPS was resistant to polycation-induced permeabilization. These data suggest that LPSs relate to some but not all the OM differences described. It is hypothesized that the different OM properties of environmental and pathogenic biogroups reflect the adaptation of the latter biogroups to pathogenicity.

Vasoactivity of Rucaparib, a PARP-1 Inhibitor, is a Complex Process that Involves Myosin Light Chain Kinase, P2 Receptors, and PARP Itself

Therapeutic inhibition of poly(ADP-ribose) polymerase (PARP), as monotherapy or to supplement the potencies of other agents, is a promising strategy in cancer treatment. We previously reported that the first PARP inhibitor to enter clinical trial, rucaparib (AG014699), induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK) 10-fold more potently than its commercially available inhibitor ML-9. Moreover, rucaparib produces additive relaxation above the maximal degree achievable with ML-9, suggesting that MLCK inhibition is not solely responsible for dilation. Inhibition of nitric oxide synthesis using L-NMMA also failed to impact rucaparib's activity. Rucaparib contains the nicotinamide pharmacophore, suggesting it may inhibit other NAD+-dependent processes. NAD+ exerts P2 purinergic receptor-dependent inhibition of smooth muscle contraction. Indiscriminate blockade of the P2 purinergic receptors with suramin abrogated rucaparib-induced vasodilation in rat arterial tissue without affecting ML-9-evoked dilation, although the specific receptor subtypes responsible have not been unequivocally identified. Furthermore, dorsal window chamber and real time tumor vessel perfusion analyses in PARP-1-/- mice indicate a potential role for PARP in dilation of tumor-recruited vessels. Finally, rucaparib provoked relaxation in 70% of patient-derived tumor-associated vessels. These data provide tantalising evidence of the complexity of the mechanism underlying rucaparib-mediated vasodilation.

Comet C/2012 S1 (ISON): Observations of the Dust Grains from SOFIA and of the Atomic Gas from NSO Dunn and McMath-Pierce Solar Telescopes (Invited)

Comet C/2012 S1 (ISON) is unique in that it is a dynamically new comet derived from the Oort cloud reservoir of comets with a sun-grazing orbit. Infrared (IR) and visible wavelength observing campaigns were planned on NASA's Stratospheric Observatory For Infrared Astronomy (SOFIA) and on National Solar Observatory Dunn (DST) and McMath-Pierce Solar Telescopes, respectively. We highlight our early results. SOFIA (+FORCAST [1]) mid- to far-IR images and spectroscopy (~5-35 μm) of the dust in the coma of ISON are to be obtained by the ISON-SOFIA Team during a flight window 2013 Oct 21-23 UT (r_h≈1.18 AU). Dust characteristics, identified through the 10 μm silicate emission feature and its strength [2], as well as spectral features from cometary crystalline silicates (Forsterite) at 11.05-11.2 μm, and near 16, 19, 23.5, 27.5, and 33 μm are compared with other Oort cloud comets that span the range of small and/or highly porous grains (e.g., C/1995 O1 (Hale-Bopp) [3,4,5] and C/2001 Q4 (NEAT) [6]) to large and/or compact grains (e.g., C/2007 N4 (Lulin) [7] and C/2006 P1 (McNaught) [8]). Measurement of the crystalline peaks in contrast to the broad 10 and 20 μm amorphous silicate features yields the cometary silicate crystalline mass fraction [9], which is a benchmark for radial transport in our protoplanetary disk [10]. The central wavelength positions, relative intensities, and feature asymmetries for the crystalline peaks may constrain the shapes of the crystals [11]. Only SOFIA can look for cometary organics in the 5-8 μm region. Spatially resolved measurements of atoms and simple molecules from when comet ISON is near the Sun (r_h<0.4 AU, near Nov-20--Dec-03 UT) were proposed for by the ISON-DST Team. Comet ISON is the first comet since comet Ikeya-Seki (1965f) [12,13] suitable for studying the alkalai metals Na and K and the atoms specifically attributed to dust grains including Mg, Si, Fe, as well as Ca. DST's Horizontal Grating Spectrometer (HGS) measures 4 settings: Na I, K, C2 to sample cometary organics (along with Mg I), and [O I] as a proxy for activity from water [14] (along with Si I and Fe I). State-of-the-art instruments that will also be employed include IBIS [15], which is a Fabry-Perot spectral imaging system that concurrently measures lines of Na, K, Ca II, or Fe, and ROSA (CSUN/QUB) [16], which is a rapid imager that simultaneously monitors Ca II or CN. From McMath-Pierce, the Solar-Stellar Spectrograph also will target ISON (320-900 nm, R~21,000, r_h

Smac-derived Aza-peptide As an Aminopeptidase-resistant XIAP BIR3 Antagonist

The peptidic nature of anti-IAPs N-terminus Smac-derived peptides precludes their utilization as potential therapeutic anticancer agents. Recent advances in the development of novel Smac-derived peptidomimetics and non-peptidic molecules with improved anti-IAPs activity and resistance to proteolytic cleavage have been reported and led to a number of candidates that are currently in clinical trials including LCL-161, SM-406/AT-406, GDC-0512/GDC-0917, and birinapant. As an attempt to improve the proteolytic stability of Smac peptides, we developed the Aza-peptide AzaAla-Val-Pro-Phe-Tyr-NH2 (2). Unlike unmodified peptide Ala-Val-Pro-Phe-Tyr-NH2 (1), analogue (2) exhibited resistance towards proteolytic cleavage by two aminopeptidases; LAP and DPP-IV, while retaining its IAP inhibitory activity. This was due to the altered planar geometry of the P1 residue side chain. Our findings showed that using aza-isosteres of bioactive peptide sequences imbue the residue with imperviousness to proteolysis; underscoring a potential approach for developing a new generation of Smac-derived Aza-peptidomimetics.

Cosmological Constraints from Measurements of Type Ia Supernovae Discovered During the First 1.5 Yr of the Pan-STARRS1 Survey

We present griz_P1 light curves of 146 spectroscopically confirmed Type Ia supernovae (SNe Ia; 0.03 < z < 0.65) discovered during the first 1.5 yr of the Pan-STARRS1 Medium Deep Survey. The Pan-STARRS1 natural photometric system is determined by a combination of on-site measurements of the instrument response function and observations of spectrophotometric standard stars. We find that the systematic uncertainties in the photometric system are currently 1.2% without accounting for the uncertainty in the Hubble Space Telescope Calspec definition of the AB system. A Hubble diagram is constructed with a subset of 113 out of 146 SNe Ia that pass our light curve quality cuts. The cosmological fit to 310 SNe Ia (113 PS1 SNe Ia + 222 light curves from 197 low-z SNe Ia), using only supernovae (SNe) and assuming a constant dark energy equation of state and flatness, yields w = -1.120^+0.360_-0.206(Stat)^+0.269_0.291(Sys). When combined with BAO+CMB(Planck)+H₀, the analysis yields Ω_M = 0.280^+0.013_0.012 and w = -1.166^+0.072_-0.069 including all identified systematics. The value of w is inconsistent with the cosmological constant value of -1 at the 2.3σ level. Tension endures after removing either the baryon acoustic oscillation (BAO) or the H₀ constraint, though it is strongest when including the H₀ constraint. If we include WMAP9 cosmic microwave background (CMB) constraints instead of those from Planck, we find w = -1.124^+0.083_-0.065, which diminishes the discord to <2σ. We cannot conclude whether the tension with flat ΛCDM is a feature of dark energy, new physics, or a combination of chance and systematic errors. The full Pan-STARRS1 SN sample with ∼three times as many SNe should provide more conclusive results.

Rapidly Evolving and Luminous Transients from Pan-STARRS1

In the past decade, several rapidly evolving transients have been discovered whose timescales and luminosities are not easily explained by traditional supernovae (SNe) models. The sample size of these objects has remained small due, at least in part, to the challenges of detecting short timescale transients with traditional survey cadences. Here we present the results from a search within the Pan-STARRS1 Medium Deep Survey (PS1-MDS) for rapidly evolving and luminous transients. We identify 10 new transients with a time above half-maximum (t_1/2) of less than 12 days and -16.5 > M > -20 mag. This increases the number of known events in this region of SN phase space by roughly a factor of three. The median redshift of the PS1-MDS sample is z = 0.275 and they all exploded in star-forming galaxies. In general, the transients possess faster rise than decline timescale and blue colors at maximum light (g_P1-rP1 ≲ -0.2). Best-fit blackbodies reveal photospheric temperatures/radii that expand/cool with time and explosion spectra taken near maximum light are dominated by a blue continuum, consistent with a hot, optically thick, ejecta. We find it difficult to reconcile the short timescale, high peak luminosity (L > 10⁴³erg s^-1), and lack of UV line blanketing observed in many of these transients with an explosion powered mainly by the radioactive decay of ⁵⁶Ni. Rather, we find that many are consistent with either (1) cooling envelope emission from the explosion of a star with a low-mass extended envelope that ejected very little (<0.03 M) radioactive material, or (2) a shock breakout within a dense, optically thick, wind surrounding the progenitor star. After calculating the detection efficiency for objects with rapid timescales in the PS1-MDS we find a volumetric rate of 4800-8000 events yr^-1Gpc^-3(4%-7% of the core-collapse SN rate at z = 0.2).

PS1-10afx at z = 1.388: Pan-STARRS1 Discovery of a New Type of Superluminous Supernova

We present the Pan-STARRS1 discovery of PS1-10afx, a unique hydrogen-deficient superluminous supernova (SLSN) at redshift z = 1.388. The light curve peaked at z P1 = 21.7 mag, making PS1-10afx comparable to the most luminous known SNe, with Mu = -22.3 mag. Our extensive optical and near-infrared observations indicate that the bolometric light curve of PS1-10afx rose on the unusually fast timescale of ~12 days to the extraordinary peak luminosity of 4.1 × 1044 erg s-1 (M bol = -22.8 mag) and subsequently faded rapidly. Equally important, the spectral energy distribution is unusually red for an SLSN, with a color temperature of ~6800 K near maximum light, in contrast to previous hydrogen-poor SLSNe, which are bright in the ultraviolet (UV). The spectra more closely resemble those of a normal SN Ic than any known SLSN, with a photospheric velocity of ~11, 000 km s-1 and evidence for line blanketing in the rest-frame UV. Despite the fast rise, these parameters imply a very large emitting radius (gsim 5 × 1015 cm). We demonstrate that no existing theoretical model can satisfactorily explain this combination of properties: (1) a nickel-powered light curve cannot match the combination of high peak luminosity with the fast timescale; (2) models powered by the spindown energy of a rapidly rotating magnetar predict significantly hotter and faster ejecta; and (3) models invoking shock breakout through a dense circumstellar medium cannot explain the observed spectra or color evolution. The host galaxy is well detected in pre-explosion imaging with a luminosity near L*, a star formation rate of ~15 M ⊙ yr-1, and is fairly massive (~2 × 1010 M ⊙), with a stellar population age of ~108 yr, also in contrast to the young dwarf hosts of known hydrogen-poor SLSNe. PS1-10afx is distinct from known examples of SLSNe in its spectra, colors, light-curve shape, and host galaxy properties, suggesting that it resulted from a different channel than other hydrogen-poor SLSNe.

Design, synthesis and validation of an inhibitor of CGRP degradation

Introduction: In addition to their afferent role in detection and signalling noxious stimuli, neuropeptide-containing sensory nerves may initiate and maintain chronic inflammation in diseases such as periodontitis by an efferent process known as neurogenic inflammation. Neuropeptides are susceptible to cleavage by peptidases, and therefore, the exact location and level of expression of peptidases are major determinants of neuropeptide action. Previous studies in our laboratory showed that enzyme components of gingival crevicular fluid (GCF) from periodontitis sites selectively inactivated the neuropeptide calcitonin gene-related peptide (CGRP), known to have a role in inhibiting osteoclastic bone resorption. Objectives: The aim of this study was to design and synthesise a specific inhibitor to prevent the degradation of CGRP by components of GCF. Methods: A hydroxamate-based inhibitor with a biotinylated tag was designed to ensure selectivity for CGRP and ease of use for future purification strategies. The biotinylated peptide hydroxamate contained the P1-P4 amino acid sequence of the potential CGRP cleavage site and was synthesised by solid-phase methods using standard Fmoc chemistry. Inhibition of CGRP metabolism by GCF was determined by MALDI-mass spectrometry (MALDI-MS) using pooled GCF samples from periodontitis patients as a crude source of the CGRP-degrading enzyme. Results: MALDI-MS analysis of CGRP degradation showed almost complete inhibition in the presence of the biotinylated inhibitor. Our results showed that the rate-limiting step in the cleavage of CGRP is endopeptidase cleavage, followed by carboxypeptidase attack. Conclusion: This study demonstrates that the enzyme component of GCF responsible for the degradation of CGRP can be inhibited by a biotinylated hydroxamate modelled on a potential endopeptidase cleavage site. The biotin tag on the inhibitor will facilitate our future purification of the CGRP-cleavage enzyme using a streptavidin-agarose column.

CGRP-cleavage enzyme detection using a biotinylated hydroxymate affinity probe

Introduction: Neuropeptides contribute to the pathophysiology of peripheral inflammation and a neurogenic component has been described for many inflammatory diseases, including periodontitis. Neuropeptides are susceptible to cleavage by peptidases and therefore the exact location and level of expression of peptidases are major determinants of neuropeptide action. Previous studies by our research group suggested that levels of the neuropeptide calcitonin gene-related peptide (CGRP) may be regulated by peptidases present in gingival crevicular fluid (GCF). Objectives: The aim of this work was to purify and partially characterize the GCF enzyme responsible for CGRP degradation using a biotinylated hydroxymate affinity probe (based on the P1-P4 amino acid sequence of the observed cleavage site) which we previously showed to inhibit CGRP degradation. Methods: Pooled healthy and pooled periodontitis GCF samples were subject to a pre-clear step with magnetic streptavadin beads. Healthy and diseased samples were incubated with the biotinylated hydroxymate probe (20 uM) after which biotinylated proteins were purified from the sample using magnetic streptavadin beads. Bound proteins were subjected to SDS-PAGE and western blotting. Biotin incorporated proteins were disclosed using a streptavadin horse radish peroxidase conjugate. Results: A band was disclosed in the periodontitis pooled sample at a molecular weight of approximately 60 kDa. The band was absent in the pooled healthy samples. As expected, when periodontitis samples were pre-boiled to denature proteins before the addition of the hydroxymate probe, no biotin incorporated band was present. Conclusions: This work demonstrates the purification and disclosure of a protein found specifically in periodontitis which binds to the specific biotinylated hydroxymate affinity probe based on the cleavage site of CGRP only when in its native form. We intend to scale up the sample size thus allowing the identification of the putative CGRP degrading peptidase using MALDI-mass spectrometry.
Funded by an IADR/GlaxoSmithKline Innovation in Oral Care Award