Recognition of 5-aminouracil (U(#)) in the central strand of a DNA triplex: orientation selective binding of different third strand bases

A necessary feature of the natural base triads for triplex formation is the requirement of a purine (A or G) in the central position, since only these provide sets of two hydrogen bond donors/acceptors in the major groove of the double helix. Pyrimidine bases devoid of this feature have incompatible complementarity and lead to triplexes with lower stability. This paper demonstrates that 5-aminouracil (U^#) (I), a pyrimidine nucleobase analogue of T in which 5-methyl is replaced by 5-amino group, with hydrogen bonding sites on both sides, is compatible in the central position of triplex triad X*U^#·A, where X = A/G/C/T/2-aminopurine (AP), and * and · represent Hoogsteen and Watson–Crick hydrogen bonding patterns respectively. A novel recognition selectivity based on the orientation (parallel/antiparallel) of the third strand purines A, G or AP with A in the parallel motif (A_p*U^#·A), and G/AP in the antiparallel motif (G_ap/AP_ap*U^#·A) is observed. Similarly for pyrimidines in the third strand, C is accepted only in a parallel mode (C_p*U^#·A). Significantly, T is recognised in both parallel and antiparallel modes (T_p/T_ap*U^#·A), with the antiparallel mode being stable compared to the parallel one. The ‘U^#’ triplexes are also more stable than the corresponding control ‘T’ triplexes. The results expand the lexicon of triplex triads with a recognition motif consisting of pyrimidine in the central strand.     

Erdosteine modulates radiocontrast‐induced hepatotoxicity in rat

It has been suggested that reactive oxygen species (ROS) plays an important role in radio contrast media (RCM)‐induced ischemia reperfusion tissue injury although antioxidants may have protective effects on the injury. We investigated the effects of erdosteine as an antioxidant agent on RCM‐induced liver toxicity in rats by evaluation of lipid peroxidation (as TBARS), catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH) and glutathione peroxidase (GSH‐Px) values and histological evaluation. Twenty‐one rats were equally divided into three groups as follows: control, RCM, and RCM plus erdosteine. RCM was intraperitoneally administered for 1 day. Erdosteine was administered orally for 2 days after RCM administration. Liver samples were taken from the rats and they homogenized in a motor‐driven tissue homogenizer. TBARS levels were significantly (p < 0.005) higher in RCM group than in control although SOD activities significantly (p < 0.05) decreased in RCM group. TBARS levels were lower in RCM plus erdosteine group than in control although SOD activity and GSH level increased (p < 0.05) in liver as compared to RCM alone. Erdosteine showed also histopathological protection (p < 0.0001) against RCM induced hepatotoxicity. GSH‐Px and CAT activities were not statistically changed by the erdosteine. According to our results, it can be concluded that radiocontrast media can induce oxidative stress in liver as suggested by previous studies. Erdosteine seems to be protective agent on the radiocontrast media‐induced liver toxicity by inhibiting the production of ROS via the enzymatic antioxidant system. Copyright © 2009 John Wiley & Sons, Ltd.     

First-In-Human,Double-Blind,Placebo-Controlled,Randomized, Dose-Escalation Study of BG00010, a Glial Cell Line-Derived Neurotrophic Factor Family Member,in Subjects with Unilateral Sciatica

ObjectiveTo evaluate the safety, tolerability, and pharmacokinetics of single doses of BG00010 (neublastin, artemin, enovin) in subjects with unilateral sciatica.MethodsThis was a single-center, blinded, placebo-controlled, randomized Phase 1 sequential-cohort, dose-escalation study (ClinicalTrials.gov identifier {"type":"clinical-trial","attrs":{"text":"NCT00961766","term_id":"NCT00961766"}}NCT00961766; funded by Biogen Idec). Adults with unilateral sciatica were enrolled at The Royal Adelaide Hospital, Australia. Four subjects were assigned to each of eleven cohorts (intravenous BG00010 0.3, 1, 3, 10, 25, 50, 100, 200, 400, or 800 μg/kg, or subcutaneous BG00010 50 μg/kg) and were randomized 3:1 to receive a single dose of BG00010 or placebo. The primary safety and tolerability assessments were: adverse events; clinical laboratory parameters and vital signs; pain as measured by a Likert rating scale; intra-epidermal nerve fiber density; and longitudinal assessment of quantitative sensory test parameters. Blood, serum, and plasma samples were collected for pharmacokinetic and pharmacodynamic assessments. Subjects were blinded to treatment assignment throughout the study. The investigator was blinded to treatment assignment until the Data Safety Review Committee review of unblinded data, which occurred after day 28.ResultsBeyond the planned enrollment of 44 subjects, four additional subjects were enrolled into to the intravenous BG00010 200 μg/kg cohort after one original subject experienced mild generalized pruritus. Therefore, a total of 48 subjects were enrolled between August 2009 and December 2011; all were included in the safety analyses. BG00010 was generally well tolerated: in primary analyses, the most common treatment-emergent adverse events were changes in temperature perception, pruritus, rash, or headache; no trends were observed in clinical laboratory parameters, vital signs, intra-epidermal nerve fiber density, or quantitative sensory testing. BG00010 was not associated with any clear, dose-dependent trends in Likert pain scores. BG00010 was rapidly distributed, with a prolonged terminal elimination phase.ConclusionsThese data support the development of BG00010 for the treatment of neuropathic pain.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00961766","term_id":"NCT00961766"}}NCT00961766     

Is Demography Destiny? Application of Machine Learning Techniques to Accurately Predict Population Health Outcomes from a Minimal Demographic Dataset

For years, we have relied on population surveys to keep track of regional public health statistics, including the prevalence of non-communicable diseases. Because of the cost and limitations of such surveys, we often do not have the up-to-date data on health outcomes of a region. In this paper, we examined the feasibility of inferring regional health outcomes from socio-demographic data that are widely available and timely updated through national censuses and community surveys. Using data for 50 American states (excluding Washington DC) from 2007 to 2012, we constructed a machine-learning model to predict the prevalence of six non-communicable disease (NCD) outcomes (four NCDs and two major clinical risk factors), based on population socio-demographic characteristics from the American Community Survey. We found that regional prevalence estimates for non-communicable diseases can be reasonably predicted. The predictions were highly correlated with the observed data, in both the states included in the derivation model (median correlation 0.88) and those excluded from the development for use as a completely separated validation sample (median correlation 0.85), demonstrating that the model had sufficient external validity to make good predictions, based on demographics alone, for areas not included in the model development. This highlights both the utility of this sophisticated approach to model development, and the vital importance of simple socio-demographic characteristics as both indicators and determinants of chronic disease.     

Aptamer-Based Detection of Disease Biomarkers in Mouse Models for Chagas Drug Discovery

Drug discovery initiatives, aimed at Chagas treatment, have been hampered by the lack of standardized drug screening protocols and the absence of simple pre-clinical assays to evaluate treatment efficacy in animal models. In this study, we used a simple Enzyme Linked Aptamer (ELA) assay to detect T. cruzi biomarker in blood and validate murine drug discovery models of Chagas disease. In two mice models, Apt-29 ELA assay demonstrated that biomarker levels were significantly higher in the infected group compared to the control group, and upon Benznidazole treatment, their levels reduced. However, biomarker levels in the infected treated group did not reduce to those seen in the non-infected treated group, with 100% of the mice above the assay cutoff, suggesting that parasitemia was reduced but cure was not achieved. The ELA assay was capable of detecting circulating biomarkers in mice infected with various strains of T. cruzi parasites. Our results showed that the ELA assay could detect residual parasitemia in treated mice by providing an overall picture of the infection in the host. They suggest that the ELA assay can be used in drug discovery applications to assess treatment efficacy in-vivo.     

Model structures of inactive and peptide agonist bound C5aR: Insights into agonist binding,selectivity and activation

C5a receptor (C5aR) is one of the major chemoattractant receptors of the druggable proteome that binds C5a, the proinflammatory polypeptide of complement cascade, triggering inflammation and SEPSIS. Here, we report the model structures of C5aR in both inactive and peptide agonist (YSFKPMPLaR; a=D-Ala) bound meta-active state. Assembled in CYANA and evolved over molecular dynamics (MD) in POPC bilayer, the inactive C5aR demonstrates a topologically unique compact heptahelical bundle topology harboring a β-hairpin in extracellular loop 2 (ECL2), derived from the atomistic folding simulations. The peptide agonist bound meta-active C5aR deciphers the “site2” at an atomistic resolution in the extracellular surface (ECS), in contrast to the previously hypothesized inter-helical crevice. With estimated Ki≈2.75 μM, the meta-active C5aR excellently rationalizes the IC₅₀ (0.1–13 μM) and EC₅₀ (0.01–6 μM) values, displayed by the peptide agonist in several signaling studies. Moreover, with Ki≈5.3×10⁵ μM, the “site2” also illustrates selectivity, by discriminating the stereochemical mutant peptide (YSFkPMPLaR; k=D-Lys), known to be inert toward C5aR, up to 1 mM concentration. Topologically juxtaposed between the structures of rhodopsin and CXCR1, the C5aR models also display excellent structural correlations with the other G-protein coupled receptors (GPCRs). The models elaborated in the current study unravel many important structural insights previously not known for regulating the agonist binding and activation mechanism of C5aR.     

ILCD Handbook Public Consultation Workshop

Introduction

The European Commission is supporting the development of the International Reference Life Cycle Data System (ILCD). This consists primarily of the ILCD Handbook and the ILCD Data Network. This paper gives an insight into the scientific positions of business, governments, consultants, academics, and others that were expressed at this public consultation workshop.

Workshop focus

The workshop focused on four of the topics of the main guidance documents of the ILCD Handbook: (1) general guidance on life cycle assessment (LCA); (2) guidance for generic and average life cycle inventory (LCI) data sets; (3) requirements for environmental impact assessment methods, models and indicators for LCA; and (4) review schemes for LCA.

Workshop participation

This consultation workshop was attended by more than 120 participants during the 4 days of the workshop. Representatives came from 23 countries, from both within and outside the European Union.

Workshop structure

Approximately half of the participants were from business associations or individual companies. Another 20% were governmental representatives. Others came predominantly from consultancies and academia.

Results

This public consultation workshop provided valuable inputs into the overall ILCD Handbook developments as well as for further development. This paper focuses on some of the main scientific issues that were raised.     

Pneumolysin activates the NLRP3 inflammasome and promotes proinflammatory cytokines independently of TLR4

Pneumolysin (PLY) is a key Streptococcus pneumoniae virulence factor and potential candidate for inclusion in pneumococcal subunit vaccines. Dendritic cells (DC) play a key role in the initiation and instruction of adaptive immunity, but the effects of PLY on DC have not been widely investigated. Endotoxin-free PLY enhanced costimulatory molecule expression on DC but did not induce cytokine secretion. These effects have functional significance as adoptive transfer of DC exposed to PLY and antigen resulted in stronger antigen-specific T cell proliferation than transfer of DC exposed to antigen alone. PLY synergized with TLR agonists to enhance secretion of the proinflammatory cytokines IL-12, IL-23, IL-6, IL-1β, IL-1α and TNF-α by DC and enhanced cytokines including IL-17A and IFN-γ by splenocytes. PLY-induced DC maturation and cytokine secretion by DC and splenocytes was TLR4-independent. Both IL-17A and IFN-γ are required for protective immunity to pneumococcal infection and intranasal infection of mice with PLY-deficient pneumococci induced significantly less IFN-γ and IL-17A in the lungs compared to infection with wild-type bacteria. IL-1β plays a key role in promoting IL-17A and was previously shown to mediate protection against pneumococcal infection. The enhancement of IL-1β secretion by whole live S. pneumoniae and by PLY in DC required NLRP3, identifying PLY as a novel NLRP3 inflammasome activator. Furthermore, NLRP3 was required for protective immunity against respiratory infection with S. pneumoniae. These results add significantly to our understanding of the interactions between PLY and the immune system.