Down-regulation of 8-oxoguanine DNA glycosylase 1 expression in the airway epithelium ameliorates allergic lung inflammation

Allergic airway inflammation is characterized by increased expression of pro-inflammatory mediators, inflammatory cell infiltration, mucus hypersecretion, and airway hyperresponsiveness, in parallel with oxidative DNA base and strand damage, whose etiological role is not understood. Our goal was to establish the role of 8-oxoguanine (8-oxoG), a common oxidatively damaged base, and its repair by 8-oxoguanine DNA glycosylase 1 (Ogg1) in allergic airway inflammatory processes. Airway inflammation was induced by intranasally administered ragweed (Ambrosia artemisiifolia) pollen grain extract (RWPE) in sensitized BALB/c mice. We utilized siRNA technology to deplete Ogg1 from airway epithelium; 8-oxoG and DNA strand break levels were quantified by Comet assays. Inflammatory cell infiltration and epithelial methaplasia were determined histologically, mucus and cytokines levels biochemically and enhanced pause was used as the main index of airway hyperresponsiveness. Decreased Ogg1 expression and thereby 8-oxoG repair in the airway epithelium conveyed a lower inflammatory response after RWPE challenge of sensitized mice, as determined by expression of Th2 cytokines, eosinophilia, epithelial methaplasia, and airway hyperresponsiveness. In contrast, 8-oxoG repair in Ogg1-proficient airway epithelium was coupled to an increase in DNA single-strand break (SSB) levels and exacerbation of allergen challenge-dependent inflammation. Decreased expression of the Nei-like glycosylases Neil1 and Neil2 that preferentially excise ring-opened purines and 5-hydroxyuracil, respectively, did not alter the above parameters of allergic immune responses to RWPE. These results show that DNA SSBs formed during Ogg1-mediated repair of 8-oxoG augment antigen-driven allergic immune responses. A transient modulation of OGG1 expression/activity in airway epithelial cells could have clinical benefits.     

Naphthalenyl derivatives for hitting P-gp/MRP1/BCRP transporters

Substituted naphthalenyl derivatives bearing oxazole, or thiazole or furyl heteronuclei have been carried out as bioisosters of aryl-oxazoles and -thiazoles derivatives previously reported in order to investigate the role of the hindrance on the activity towards P-gp/BCRP/and MRP1 transporters. In addition, the role of naphthalenyl group to modulate P-gp intrinsic activity of these compounds was ascertained.The results demonstrated that all naphthalenyl derivatives displayed comparable P-gp activity with respect to lead compounds previously characterized in our SAR studies but were less active towards BCRP and MRP1 pumps. In terms of intrinsic activity, the replacement of aryl with naphthalenyl moiety led to P-gp inhibitors, unambiguous or ambiguous substrates on the base of the heteronucleus and the substituent on the naphthalenyl fragment. Indeed, oxazole derivatives were: inhibitors (R = H, F, OH), unambiguous substrates (R = OCH₃), or ambiguous substrate (R = Br); thiazole derivatives were: unambiguous substrates (R = OCH₃, Br), or ambiguous substrates (R = H, F). Finally furyl derivatives were ambiguous substrates.     

Nicotine: the link between cigarette smoking and the progression of renal injury?

Cigarette smoke (CS) is the most important source of preventable morbidity and mortality in the United States. Recent clinical studies have suggested that, in addition to being a major cardiovascular risk factor, CS promotes the progression of kidney disease. The mechanisms by which CS promotes the progression of chronic kidney disease have not been elucidated. Here we demonstrate for the first time that human mesangial cells (MCs) are endowed with the nicotinic ACh receptors (nAChRs) alpha4, alpha5, alpha7, beta2, beta3, and beta4. Studies performed in other cell types have shown that these nAChRs are ionotropic receptors that function as agonist-regulated Ca(2+) channels. Nicotine induced MC proliferation in a dose-dependent manner. At 10 (-7) M, a concentration found in the plasma of active smokers, nicotine induced MC proliferation [control, 1,328 +/- 50 vs. nicotine, 2,761 +/- 90 counts/minute (cpm); P < 0.05] and increased the synthesis of fibronectin (50%), a critical matrix component involved in the progression of chronic kidney disease. We and others have shown that, in response to PKC activation, MC synthesize reactive oxygen species (ROS) via NADPH oxidase. In the current studies we demonstrate that PKC inhibition as well as diphenyleneiodonium and apocynin, two inhibitors of NADPH oxidase, prevented the effects of nicotine on MC proliferation and fibronectin production, hence establishing ROS as second messengers of the actions of nicotine. Furthermore, nicotine increased the production of ROS as assessed by 2',7'-dichlorofluorescein diacetate fluorescence [control, 184.4 +/- 26 vs. nicotine, 281.5 +/- 26 arbitrary fluorescence units (AFU); n = 5 experiments, P < 0.05]. These studies unveil previously unrecognized mechanisms that indict nicotine, a component of CS, as an agent that may accelerate and promote the progression of kidney disease.     

New tissue microarray technology for analyses of gene expression in frozen pathological samples

Tissue microarrays (TMAs) are widely used to analyze gene expression in multiple pathological samples on a single slide. Currently, most TMA slides are made by coring paraffin-embedded tissues and arraying them into a paraffin block, from which TMA sections are cut. However paraffin-based TMA technology may not be compatible with frozen clinical tissue samples, which have a higher quality of RNAs and proteins for preparing TMAs than paraffin-embedded tissue samples. In this study, we developed an alternative TMA technology that is applicable to a broader range of frozen tissue samples. Our method takes advantage of a newly developed array recipient block that can be used to array small tissue cores. After arraying tissue cores, the tissue block can be immediately sectioned on a cryostat microtome to make TMA slides. TMAs made using this method have well-defined array configurations and good tissue/cell morphology. Immunohistochemistry and in situ hybridization study also revealed well-preserved proteins and mRNAs on TMA slides. Our method significantly simplifies TMA preparation and assembly when frozen pathological tissues are used. Our technology provides an alternative tool for creating high-quality TMAs for the general research community to study gene expressions in pathological samples.     

Design,Synthesis, Lipophilic Properties,and Binding Affinities of Potential Ligands in Positron Emission Tomography (PET) for Visualization of Brain Dopamine D4 Receptors

We report the synthesis of compounds structurally related to the high‐affinity dopamine D₄ receptor ligand N‐{2‐[4‐(3‐cyanopyridin‐2‐yl)piperazin‐1‐yl]ethyl}‐3‐methoxybenzamide ( 1e ). All compounds were specifically designed as potential PET radioligands for brain D₄ receptor visualization, having lipophilicity within a range for brain uptake and weak non‐specific binding (0.75<cLogP<3.15) and bearing a substituent for easy access to labeling with the positron emitter isotope ¹¹C or ¹⁸F. The best compound of the series, N‐{2‐[4‐(4‐chlorophenyl)piperazin‐1‐yl]ethyl}‐6‐fluoropyridine‐3‐carboxamide ( 7a ), displayed excellent selectivity over D₂ and D₃ receptors (>100‐fold), but its D₄ receptor affinity was suboptimal for imaging of brain D₄ receptors (K_i=30 nM ).     

Bryophyte-Cyanobacteria Associations during Primary Succession in Recently Deglaciated Areas of Tierra del Fuego (Chile)

Bryophyte establishment represents a positive feedback process that enhances soil development in newly exposed terrain. Further, biological nitrogen (N) fixation by cyanobacteria in association with mosses can be an important supply of N to terrestrial ecosystems, however the role of these associations during post-glacial primary succession is not yet fully understood. Here, we analyzed chronosequences in front of two receding glaciers with contrasting climatic conditions (wetter vs drier) at Cordillera Darwin (Tierra del Fuego) and found that most mosses had the capacity to support an epiphytic flora of cyanobacteria and exhibited high rates of N₂ fixation. Pioneer moss-cyanobacteria associations showed the highest N₂ fixation rates (4.60 and 4.96 µg N g⁻¹ bryo. d⁻¹) very early after glacier retreat (4 and 7 years) which may help accelerate soil development under wetter conditions. In drier climate, N₂ fixation on bryophyte-cyanobacteria associations was also high (0.94 and 1.42 µg N g⁻¹ bryo. d⁻¹) but peaked at intermediate-aged sites (26 and 66 years). N₂ fixation capacity on bryophytes was primarily driven by epiphytic cyanobacteria abundance rather than community composition. Most liverworts showed low colonization and N₂ fixation rates, and mosses did not exhibit consistent differences across life forms and habitat (saxicolous vs terricolous). We also found a clear relationship between cyanobacteria genera and the stages of ecological succession, but no relationship was found with host species identity. Glacier forelands in Tierra del Fuego show fast rates of soil transformation which imply large quantities of N inputs. Our results highlight the potential contribution of bryophyte-cyanobacteria associations to N accumulation during post-glacial primary succession and further describe the factors that drive N₂-fixation rates in post-glacial areas with very low N deposition.     

Estimation of genealogical coancestry in plant species using a pedigree reconstruction algorithm and application to an oil palm breeding population

Key message

Explicit pedigree reconstruction by simulated annealing gave reliable estimates of genealogical coancestry in plant species, especially when selfing rate was lower than 0.6, using a realistic number of markers. Genealogical coancestry information is crucial in plant breeding to estimate genetic parameters and breeding values. The approach of Fernández and Toro (Mol Ecol 15:1657–1667, 2006) to estimate genealogical coancestries from molecular data through pedigree reconstruction was limited to species with separate sexes. In this study it was extended to plants, allowing hermaphroditism and monoecy, with possible selfing. Moreover, some improvements were made to take previous knowledge on the population demographic history into account. The new method was validated using simulated and real datasets. Simulations showed that accuracy of estimates was high with 30 microsatellites, with the best results obtained for selfing rates below 0.6. In these conditions, the root mean square error (RMSE) between the true and estimated genealogical coancestry was small (<0.07), although the number of ancestors was overestimated and the selfing rate could be biased. Simulations also showed that linkage disequilibrium between markers and departure from the Hardy–Weinberg equilibrium in the founder population did not affect the efficiency of the method. Real oil palm data confirmed the simulation results, with a high correlation between the true and estimated genealogical coancestry (>0.9) and a low RMSE (<0.08) using 38 markers. The method was applied to the Deli oil palm population for which pedigree data were scarce. The estimated genealogical coancestries were highly correlated (>0.9) with the molecular coancestries using 100 markers. Reconstructed pedigrees were used to estimate effective population sizes. In conclusion, this method gave reliable genealogical coancestry estimates. The strategy was implemented in the software MOLCOANC 3.0.     

Lunar and Seasonal Rhythms and Childhood Mortality

The aim of the present study was to assess the effect of the lunar cycle phases, the lunar apogee and perigee, and the geophysical factors (humidity, temperature, and rainfall) on the childhood mortality. We designed a retrospective study by reviewing the clinical charts of the population of patient deceases during the lunar pre-phase (a day before), phase (day of lunar change of phase), and post-phase (a day after), between the years 1991-1996 in a children's hospital. From the 44,982 discharges from the hospital in the interval between 1991 and 1996, 2003 corresponded to mortal cases, and 522 died within the 3 days we considered as representative for each phase. The number of deaths was approximately the same in between phases. The percentage of male deaths was higher during phase days than interphase ones. Male deceases exhibited a statistically significant predominance during lunar first quarter, full moon, and new moon. A discreet increase of mortality was present in the months of March, June, August, September, October, and November, as well as during summer and fall, but these increments were not significant. Minimum and maximum temperatures exhibited no relation to this behavioral pattern for deceases, nor the humidity index. Early lactating infants presented a significant higher mortality during November, December, and March. We suggest to investigate the causes of the increase of deceases in males in relation to the circalunar rhythms, as well as the lift up of deceases in neonates and early lactating children during November and December.     

Synovial effusion and synovial fluid biomarkers in psoriatic arthritis to assess intraarticular tumor necrosis factor-α blockade in the knee joint

Introduction

The purpose of this study was theevaluation of synovial effusion (SE), synovial fluid (SF) and synovial tissue (ST) biomarkers in relation to disease activity indexes to assess the response to intraarticular (IA) tumor necrosis factor (TNF)-α blockers in psoriatic arthritis (PsA).

Methods

Systemic and local disease activity indexes (disease activity score (DAS); the Ritchie articular index (mRAI), erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP); Thompson articular (THOMP) and joint articular (KJAI)-Index ) and ST samples were assessed at baseline, throughout treatment, and during the follow-up in 14 patients affected with PsA who underwent IA injections (0.5 ml to 12.5 mg) in the knee joint of etanercept (E) or placebo (P) once every two weeks for a 10-week period. Total SF white blood cell (WBC) counts (WBC/μl) and SF cytokine/chemokine (CK/CCK) levels were measured before IA-E at baseline, after IA-E, and as long as there were adequate amounts of SF for knee aspiration (post). Characterization of synovial mononuclear cell infiltration and synovial vessels was carried out in 8 out of 14 knees by staining serial sections of synovial tissue biopsies for CD45, CD3, CD68, CD31 and CD105.

Results

At baseline, CRP and/or ESR were significantly correlated with SF-CK (interleukin- (IL-)1β, IL-1Ra, IL-6, IL-8) and CCK (CCL3). Post-IA injections, there was a decrease in SE in the knees in which aspiration following IA-E injection was possible as well as a significant reduction in SF WBC/μl and in SF-CK (IL-1β, IL-1Ra, IL-6 and IL-22). Pre- and post-IA-E injections, there were significant correlations between ST markers and SF-CK (IL-1β with CD45; IL-1β and IL-6 with CD31) and between SF-CCK (CCL4 and CCL3 with CD3). At the end of the study, there was a significant reduction in disease activity indexes (CRP, DAS, RAI, THOMP, KJAI) as well as in the ST markers (CD45; CD3).

Conclusions

Synovial effusion regression is a reliable indicator of the response to IA TNF-α blockers in PsA patients as it is confirmed by the correlation between SF biomarkers to disease activity and synovial tissue inflammation.