Pseudoperoxidase investigations of hydroperoxides and inhibitors with human lipoxygenases

AbstractUnderstanding the mode of action for lipoxygenase (LOX) inhibitors is critical to determining their efficacy in the cell. The pseudoperoxidase assay is an important tool for establishing if a LOX inhibitor is reductive in nature, however, there have been difficulties identifying the proper conditions for each of the many human LOX isozymes. In the current paper, both the 234nM decomposition (UV) and iron-xylenol orange (XO) assays are shown to be effective methods of detecting pseudoperoxidase activity for 5-LOX, 12-LOX, 15-LOX-1 and 15-LOX-2, but only if 13-(S)-HPODE is used as the hydroperoxide substrate. The AA products, 12-(S)-HPETE and 15-(S)-HPETE, are not consistent hydroperoxide substrates since they undergo a competing transformation to the di-HETE products. Utilizing the above conditions, the selective 12-LOX and 15-LOX-1 inhibitors, probes for diabetes, stroke and asthma, are characterized for their inhibitory nature. Interestingly, ascorbic acid also supports the pseudoperoxidase assay, suggesting that it may have a role in maintaining the inactive ferrous form of LOX in the cell. In addition, it is observed that nordihydroguaiaretic acid (NDGA), a known reductive LOX inhibitor, appears to generate radical species during the pseudoperoxidase assay, which are potent inhibitors against the human LOX isozymes, producing a negative pseudoperoxidase result. Therefore, inhibitors that do not support the pseudoperoxidase assay with the human LOX isozymes, should also be investigated for rapid inactivation, to clarify the negative pseudoperoxidase result

Strong Gravitational Lensing and Dark Energy Complementarity

In the search for the nature of dark energy most cosmological probes measure simple functions of the expansion rate. While powerful, these all involve roughly the same dependence on the dark energy equation of state parameters, with anticorrelation between its present value w_0 and time variation w_a. Quantities that have instead positive correlation and so a sensitivity direction largely orthogonal to, e.g., distance probes offer the hope of achieving tight constraints through complementarity. Such quantities are found in strong gravitational lensing observations of image separations and time delays. While degeneracy between cosmological parameters prevents full complementarity, strong lensing measurements to 1% accuracy can improve equation of state characterization by 15-50%. Next generation surveys should provide data on roughly 10^5 lens systems, though systematic errors will remain challenging.Comment: 7 pages, 5 figure

Intravitreal Moxifloxacin: Retinal Safety Study with Electroretinography and Histopathology in Animal Models

PURPOSE. To determine whether moxifloxacin can be used safely as an intraocular antibiotic, retinal safety of intravitreal moxifloxacin was studied with electroretinography (ERG) and histopathology in animal models. METHODS. Moxifloxacin was injected into mouse eyes at intravitreal concentrations of 5 to 500 g/mL and into rabbit eyes at 150 g/mL. As the control, the vehicle was injected into the fellow eyes of each animal. Four weeks after injection, ERG recordings were performed, and animal eyes were processed for histologic examination. RESULTS. ERG studies showed no significant difference between control and moxifloxacin-injected eyes at any dose in either the mouse or rabbit model. Histologic examination revealed no retinal abnormality in mice at 5 to 100 g/mL or in rabbits at 150 g/mL intravitreal moxifloxacin. In mice at 500 g/mL, occasional focal retinal necroses were observed, suggesting isolated retinal toxicity at this concentration of moxifloxacin. CONCLUSIONS. Intravitreal moxifloxacin, up to 100 g/mL in mice or 150 g/mL in rabbits, caused no ERG or retinal histologic abnormality. These results indicate that moxifloxacin is a safe intravitreal antibiotic in mouse and rabbit animal models. If proven safe and efficacious by further study in humans, intravitreal injection of moxifloxacin could be considered as an alternative to currently used antibiotics in selected patients with resistance or allergy to the more traditional antibiotics

High-content siRNA screening of the kinome identifies kinases involved in Alzheimer's disease-related tau hyperphosphorylation

<p>Abstract</p> <p>Background</p> <p>Neurofibrillary tangles (NFT), a cardinal neuropathological feature of Alzheimer's disease (AD) that is highly correlated with synaptic loss and dementia severity, appear to be partly attributable to increased phosphorylation of the microtubule stabilizing protein tau at certain AD-related residues. Identifying the kinases involved in the pathologic phosphorylation of tau may provide targets at which to aim new AD-modifying treatments.</p> <p>Results</p> <p>We report results from a screen of 572 kinases in the human genome for effects on tau hyperphosphorylation using a loss of function, high-throughput RNAi approach. We confirm effects of three kinases from this screen, the eukaryotic translation initiation factor 2 α kinase 2 (EIF2AK2), the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A), and the A-kinase anchor protein 13 (AKAP13) on tau phosphorylation at the 12E8 epitope (serine 262/serine 356). We provide evidence that EIF2AK2 effects may result from effects on tau protein expression, whereas DYRK1A and AKAP13 are likely more specifically involved in tau phosphorylation pathways.</p> <p>Conclusions</p> <p>These findings identify novel kinases that phosphorylate tau protein and provide a valuable reference data set describing the kinases involved in phosphorylating tau at an AD-relevant epitope.</p

The Carnegie Supernova Project: First Near-Infrared Hubble Diagram to z~0.7

The Carnegie Supernova Project (CSP) is designed to measure the luminosity distance for Type Ia supernovae (SNe Ia) as a function of redshift, and to set observational constraints on the dark energy contribution to the total energy content of the Universe. The CSP differs from other projects to date in its goal of providing an I-band {rest-frame} Hubble diagram. Here we present the first results from near-infrared (NIR) observations obtained using the Magellan Baade telescope for SNe Ia with 0.1 < z < 0.7. We combine these results with those from the low-redshift CSP at z <0.1 (Folatelli et al. 2009). We present light curves and an I-band Hubble diagram for this first sample of 35 SNe Ia and we compare these data to 21 new SNe Ia at low redshift. These data support the conclusion that the expansion of the Universe is accelerating. When combined with independent results from baryon acoustic oscillations (Eisenstein et al. 2005), these data yield Omega_m = 0.27 +/- 0.0 (statistical), and Omega_DE = 0.76 +/- 0.13 (statistical) +/- 0.09 (systematic), for the matter and dark energy densities, respectively. If we parameterize the data in terms of an equation of state, w, assume a flat geometry, and combine with baryon acoustic oscillations, we find that w = -1.05 +/- 0.13 (statistical) +/- 0.09 (systematic). The largest source of systematic uncertainty on w arises from uncertainties in the photometric calibration, signaling the importance of securing more accurate photometric calibrations for future supernova cosmology programs. Finally, we conclude that either the dust affecting the luminosities of SNe Ia has a different extinction law (R_V = 1.8) than that in the Milky Way (where R_V = 3.1), or that there is an additional intrinsic color term with luminosity for SNe Ia independent of the decline rate.Comment: 44 pages, 23 figures, 9 tables; Accepted for publication in the Astrophysical Journa

Multimessenger astronomy with the Einstein Telescope

Gravitational waves (GWs) are expected to play a crucial role in the development of multimessenger astrophysics. The combination of GW observations with other astrophysical triggers, such as from gamma-ray and X-ray satellites, optical/radio telescopes, and neutrino detectors allows us to decipher science that would otherwise be inaccessible. In this paper, we provide a broad review from the multimessenger perspective of the science reach offered by the third generation interferometric GW detectors and by the Einstein Telescope (ET) in particular. We focus on cosmic transients, and base our estimates on the results obtained by ET's predecessors GEO, LIGO, and Virgo.Comment: 26 pages. 3 figures. Special issue of GRG on the Einstein Telescope. Minor corrections include

Randomized Phase IIb Study of Brimonidine Drug Delivery System Generation 2 for Geographic Atrophy in Age-Related Macular Degeneration

Purpose: To evaluate the safety and efficacy of repeat injections of Brimonidine Drug Delivery System (Brimo DDS) Generation 2 (Gen 2) containing 400-μg brimonidine in patients with geographic atrophy (GA) secondary to age-related macular degeneration (AMD). Design: A phase IIb, randomized, multicenter, double-masked, sham-controlled, 30-month study (BEACON). Participants: Patients diagnosed with GA secondary to AMD and multifocal lesions with total area of &gt; 1.25 mm2 and ≤ 18 mm2 in the study eye. Methods: Enrolled patients were randomized to treatment with intravitreal injections of 400-μg Brimo DDS (n = 154) or sham procedure (n = 156) in the study eye every 3 months from day 1 to month 21. Main Outcome Measures: The primary efficacy endpoint was GA lesion area change from baseline in the study eye, assessed with fundus autofluorescence imaging, at month 24. Results: The study was terminated early, at the time of the planned interim analysis, because of a slow GA progression rate (∼ 1.6 mm2/year) in the enrolled population. Least squares mean (standard error) GA area change from baseline at month 24 (primary endpoint) was 3.24 (0.13) mm2 with Brimo DDS (n = 84) versus 3.48 (0.13) mm2 with sham (n = 91), a reduction of 0.25 mm2 (7%) with Brimo DDS compared with sham (P = 0.150). At month 30, GA area change from baseline was 4.09 (0.15) mm2 with Brimo DDS (n = 49) versus 4.52 (0.15) mm2 with sham (n = 46), a reduction of 0.43 mm2 (10%) with Brimo DDS compared with sham (P = 0.033). Exploratory analysis showed numerically smaller loss over time in retinal sensitivity assessed with scotopic microperimetry with Brimo DDS than with sham (P = 0.053 at month 24). Treatment-related adverse events were usually related to the injection procedure. No implant accumulation was observed. Conclusions: Multiple intravitreal administrations of Brimo DDS (Gen 2) were well tolerated. The primary efficacy endpoint at 24 months was not met, but there was a numeric trend for reduction in GA progression at 24 months compared with sham treatment. The study was terminated early because of the lower-than-expected GA progression rate in the sham/control group. Financial Disclosure(s): Proprietary or commercial disclosures may be found after the references

Structure-Function Correlation of the Human Central Retina

The impact of retinal pathology detected by high-resolution imaging on vision remains largely unexplored. Therefore, the aim of the study was to achieve high-resolution structure-function correlation of the human macula in vivo.To obtain high-resolution tomographic and topographic images of the macula spectral-domain optical coherence tomography (SD-OCT) and confocal scanning laser ophthalmoscopy (cSLO), respectively, were used. Functional mapping of the macula was obtained by using fundus-controlled microperimetry. Custom software allowed for co-registration of the fundus mapped microperimetry coordinates with both SD-OCT and cSLO datasets. The method was applied in a cross-sectional observational study of retinal diseases and in a clinical trial investigating the effectiveness of intravitreal ranibizumab in macular telangietasia type 2. There was a significant relationship between outer retinal thickness and retinal sensitivity (p<0.001) and neurodegeneration leaving less than about 50 µm of parafoveal outer retinal thickness completely abolished light sensitivity. In contrast, functional preservation was found if neurodegeneration spared the photoreceptors, but caused quite extensive disruption of the inner retina. Longitudinal data revealed that small lesions affecting the photoreceptor layer typically precede functional detection but later cause severe loss of light sensitivity. Ranibizumab was shown to be ineffective to prevent such functional loss in macular telangietasia type 2.Since there is a general need for efficient monitoring of the effectiveness of therapy in neurodegenerative diseases of the retina and since SD-OCT imaging is becoming more widely available, surrogate endpoints derived from such structure-function correlation may become highly relevant in future clinical trials

A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants.

This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/ng.3448Advanced age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with limited therapeutic options. Here we report on a study of >12 million variants, including 163,714 directly genotyped, mostly rare, protein-altering variants. Analyzing 16,144 patients and 17,832 controls, we identify 52 independently associated common and rare variants (P < 5 × 10(-8)) distributed across 34 loci. Although wet and dry AMD subtypes exhibit predominantly shared genetics, we identify the first genetic association signal specific to wet AMD, near MMP9 (difference P value = 4.1 × 10(-10)). Very rare coding variants (frequency <0.1%) in CFH, CFI and TIMP3 suggest causal roles for these genes, as does a splice variant in SLC16A8. Our results support the hypothesis that rare coding variants can pinpoint causal genes within known genetic loci and illustrate that applying the approach systematically to detect new loci requires extremely large sample sizes.We thank all participants of all the studies included for enabling this research by their participation in these studies. Computer resources for this project have been provided by the high-performance computing centers of the University of Michigan and the University of Regensburg. Group-specific acknowledgments can be found in the Supplementary Note. The Center for Inherited Diseases Research (CIDR) Program contract number is HHSN268201200008I. This and the main consortium work were predominantly funded by 1X01HG006934-01 to G.R.A. and R01 EY022310 to J.L.H