Influence of benzo[a]pyrene diol epoxide chirality on solution conformations of DNA covalent adducts: the (-)-trans-anti-[BP]G.C adduct structure and comparison with the (+)-trans-anti-[BP]G.C enantiomer.

Benzo[a]pyrene (BP) is an environmental genotoxin, which, following metabolic activation to 7,8-diol 9,10-epoxide (BPDE) derivatives, forms covalent adducts with cellular DNA. A major fraction of adducts are derived from the binding of N2 of guanine to the C10 position of BPDE. The mutagenic and carcinogenic potentials of these adducts are strongly dependent on the chirality at the four asymmetric benzylic carbon atoms. We report below on the combined NMR-energy minimization refinement characterization of the solution conformation of (-)-trans-anti-[BP]G positioned opposite C and flanked by G.C base pairs in the d(C1-C2-A3-T4-C5-[BP]G6-C7-T8-A9-C10-C11).d(G12-G13-T14++ +-A15-G16-C17- G18-A19-T20-G21-G22) duplex. Two-dimensional NMR techniques were applied to assign the exchangeable and non-exchangeable protons of the benzo[a]pyrenyl moiety and the nucleic acid in the modified duplex. These results establish Watson-Crick base pair alignment at the [BP]G6.C17 modification site, as well as the flanking C5.G18 and C7.G16 pairs within a regular right-handed helix. The solution structure of the (-)-trans-anti-[BP]G.C 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bounds deduced from NOE buildup curves as constraints in energy minimization computations. The BP ring spans both strands of the duplex in the minor groove and is directed toward the 3'-end of the modified strand in the refined structure. One face of the BP ring of [BP]G6 stacks over the C17 residue across from it on the partner strand while the other face is exposed to solvent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Building a Better Fragment Library for De Novo Protein Structure Prediction

Fragment-based approaches are the current standard for de novo protein structure prediction. These approaches rely on accurate and reliable fragment libraries to generate good structural models. In this work, we describe a novel method for structure fragment library generation and its application in fragment-based de novo protein structure prediction. The importance of correct testing procedures in assessing the quality of fragment libraries is demonstrated. In particular, the exclusion of homologs to the target from the libraries to correctly simulate a de novo protein structure prediction scenario, something which surprisingly is not always done. We demonstrate that fragments presenting different predominant predicted secondary structures should be treated differently during the fragment library generation step and that exhaustive and random search strategies should both be used. This information was used to develop a novel method, Flib. On a validation set of 41 structurally diverse proteins, Flib libraries presents both a higher precision and coverage than two of the state-of-the-art methods, NNMake and HHFrag. Flib also achieves better precision and coverage on the set of 275 protein domains used in the two previous experiments of the the Critical Assessment of Structure Prediction (CASP9 and CASP10). We compared Flib libraries against NNMake libraries in a structure prediction context. Of the 13 cases in which a correct answer was generated, Flib models were more accurate than NNMake models for 10. “Flib is available for download at: http://www.stats.ox.ac.uk/research/proteins/resources”.     

Mechanosensitive Adaptation of E-Cadherin Turnover across adherens Junctions

In the natural and technological world, multi-agent systems strongly depend on how the interactions are ruled between their individual components, and the proper control of time-scales and synchronization is a key issue. This certainly applies to living tissues when multicellular assemblies such as epithelial cells achieve complex morphogenetic processes. In epithelia, because cells are known to individually generate actomyosin contractile stress, each individual intercellular adhesive junction line is subjected to the opposed stresses independently generated by its two partner cells. Contact lines should thus move unless their two partner cells mechanically match. The geometric homeostasis of mature epithelia observed at short enough time-scale thus raises the problem to understand how cells, if considered as noisy individual actuators, do adapt across individual intercellular contacts to locally balance their time-average contractile stress. Structural components of adherens junctions, cytoskeleton (F-actin) and homophilic bonds (E-cadherin) are quickly renewed at steady-state. These turnovers, if they depend on forces exerted at contacts, may play a key role in the mechanical adaptation of epithelia. Here we focus on E-cadherin as a force transducer, and we study the local regulation and the mechanosensitivity of its turnover in junctions. We show that E-cadherin turnover rates match remarkably well on either side of mature intercellular contacts, despite the fact that they exhibit large fluctuations in time and variations from one junction to another. Using local mechanical and biochemical perturbations, we find faster turnover rates with increased tension, and asymmetric rates at unbalanced junctions. Together, the observations that E-cadherin turnover, and its local symmetry or asymmetry at each side of the junction, are mechanosensitive, support the hypothesis that E-cadherin turnover could be involved in mechanical homeostasis of epithelia.     

6-Mercaptopurine reduces cytokine and Muc5ac expression involving inhibition of NFκB activation in airway epithelial cells

Background

Mucus hypersecretion and excessive cytokine synthesis is associated with many of the pathologic features of chronic airway diseases such as asthma. 6-Mercaptopurine (6-MP) is an immunosuppressive drug that is widely used in several inflammatory disorders. Although 6-MP has been used to treat asthma, its function and mechanism of action in airway epithelial cells is unknown.

Methods

Confluent NCI-H292 and MLE-12 epithelial cells were pretreated with 6-MP followed by stimulation with TNFα or PMA. mRNA levels of cytokines and mucins were measured by RT-PCR. Western blot analysis was performed to assess the phosphorylation of IκBα and luciferase assays were performed using an NFκB reporter plasmid to determine NFκB activity. Periodic Acid Schiff staining was used to assess the production of mucus.

Results

6-MP displayed no effect on cell viability up to a concentration of 15 μM. RT-PCR analysis showed that 6-MP significantly reduces TNFα- and PMA-induced expression of several proinflammatory cytokines in NCI-H292 and MLE-12 cells. Consistent with this, we demonstrated that 6-MP strongly inhibits TNFα-induced phosphorylation of IκBα and thus attenuates NFκB luciferase reporter activity. In addition, 6-MP decreases Rac1 activity in MLE-12 cells. 6-MP down-regulates gene expression of the mucin Muc5ac, but not Muc2, through inhibition of activation of the NFκB pathway. Furthermore, PMA- and TNFα-induced mucus production, as visualized by Periodic Acid Schiff (PAS) staining, is decreased by 6-MP.

Conclusions

Our data demonstrate that 6-MP inhibits Muc5ac gene expression and mucus production in airway epithelial cells through inhibition of the NFκB pathway, and 6-MP may represent a novel therapeutic target for mucus hypersecretion in airway diseases.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0236-0) contains supplementary material, which is available to authorized users.     

Prospection and Evaluation of (Hemi) Cellulolytic Enzymes Using Untreated and Pretreated Biomasses in Two Argentinean Native Termites

Saccharum officinarum bagasse (common name: sugarcane bagasse) and Pennisetum purpureum (also known as Napier grass) are among the most promising feedstocks for bioethanol production in Argentina and Brazil. In this study, both biomasses were assessed before and after acid pretreatment and following hydrolysis with Nasutitermes aquilinus and Cortaritermes fulviceps termite gut digestome. The chemical composition analysis of the biomasses after diluted acid pretreatment showed that the hemicellulose fraction was partially removed. The (hemi) cellulolytic activities were evaluated in bacterial culture supernatants of termite gut homogenates grown in treated and untreated biomasses. In all cases, we detected significantly higher endoglucanase and xylanase activities using pretreated biomasses compared to untreated biomasses, carboxymethylcellulose and xylan. Several protein bands with (hemi) cellulolytic activity were detected in zymograms and two-dimensional gel electrophoresis. Some proteins of these bands or spots were identified as xylanolytic peptides by mass spectrometry. Finally, the diversity of cultured cellulolytic bacterial endosymbionts associated to both Argentinean native termite species was analyzed. This study describes, for the first time, bacterial endosymbionts and endogenous (hemi) cellulases of two Argentinean native termites as well as their potential application in degradation of lignocellulosic biomass for bioethanol production.     

Post-Prandial Protein Handling: You Are What You Just Ate

Background

Protein turnover in skeletal muscle tissue is highly responsive to nutrient intake in healthy adults.

Objective

To provide a comprehensive overview of post-prandial protein handling, ranging from dietary protein digestion and amino acid absorption, the uptake of dietary protein derived amino acids over the leg, the post-prandial stimulation of muscle protein synthesis rates, to the incorporation of dietary protein derived amino acids in de novo muscle protein.

Design

12 healthy young males ingested 20 g intrinsically [1-¹³C]-phenylalanine labeled protein. In addition, primed continuous L-[ring-²H₅]-phenylalanine, L-[ring-²H₂]-tyrosine, and L-[1-¹³C]-leucine infusions were applied, with frequent collection of arterial and venous blood samples, and muscle biopsies throughout a 5 h post-prandial period. Dietary protein digestion, amino acid absorption, splanchnic amino acid extraction, amino acid uptake over the leg, and subsequent muscle protein synthesis were measured within a single in vivo human experiment.

Results

55.3±2.7% of the protein-derived phenylalanine was released in the circulation during the 5 h post-prandial period. The post-prandial rise in plasma essential amino acid availability improved leg muscle protein balance (from -291±72 to 103±66 μM·min^-1·100 mL leg volume^-1; P<0.001). Muscle protein synthesis rates increased significantly following protein ingestion (0.029±0.002 vs 0.044±0.004%·h^-1 based upon the muscle protein bound L-[ring-²H₅]-phenylalanine enrichments (P<0.01)), with substantial incorporation of dietary protein derived L-[1-¹³C]-phenylalanine into de novo muscle protein (from 0 to 0.0201±0.0025 MPE).

Conclusion

Ingestion of a single meal-like amount of protein allows ~55% of the protein derived amino acids to become available in the circulation, thereby improving whole-body and leg protein balance. About 20% of the dietary protein derived amino acids released in the circulation are taken up in skeletal muscle tissue following protein ingestion, thereby stimulating muscle protein synthesis rates and providing precursors for de novo muscle protein synthesis.

Trial Registration

trialregister.nl 3638     

On the Effect of Prevalent Carbazole Homocoupling Defects on the Photovoltaic Performance of PCDTBT:PC71BM Solar Cells

The photophysical properties and solar cell performance of the classical donor–acceptor copolymer PCDTBT (poly(N‐9′‐heptadecanyl‐2,7‐carbazole‐alt ‐5,5‐(4′,7′‐di‐2‐thienyl‐2′,1′,3′‐benzothiadiazole))) in relation to unintentionally formed main chain defects are investigated. Carbazole–carbazole homocouplings (Cbz hc) are found to significant extent in PCDTBT made with a variety of Suzuki polycondensation conditions. Cbz hc vary between 0 and 8 mol% depending on the synthetic protocol used, and are quantified by detailed nuclear magnetic resonance spectroscopy including model compounds, which allows to establish a calibration curve from optical spectroscopy. The results are corroborated by extended time‐dependent density functional theory investigations on the structural, electronic, and optical properties of regularly alternating and homocoupled chains. The photovoltaic properties of PCDTBT:fullerene blend solar cells significantly depend on the Cbz hc content for constant molecular weight, whereby an increasing amount of Cbz hc leads to strongly decreased short circuit currents J_SC. With increasing Cbz hc content, J_SC decreases more strongly than the intensity of the low energy absorption band, suggesting that small losses in absorption cannot explain the decrease in J_SC alone, rather than combined effects of a more localized LUMO level on the TBT unit and lower hole mobilities found in highly defective samples. Homocoupling‐free PCDTBT with optimized molecular weight yields the highest efficiency up to 7.2% without extensive optimization.     

Exome sequencing identifies a nonsense mutation in <Emphasis Type="Italic">Fam46a</Emphasis> associated with bone abnormalities in a new mouse model for skeletal dysplasia

We performed exome sequencing for mutation discovery of an ENU (N-ethyl-N-nitrosourea)-derived mouse model characterized by significant elevated plasma alkaline phosphatase (ALP) activities in female and male mutant mice, originally named BAP014 (bone screen alkaline phosphatase #14). We identified a novel loss-of-function mutation within the Fam46a (family with sequence similarity 46, member A) gene (NM_001160378.1:c.469G>T, NP_001153850.1:p.Glu157*). Heterozygous mice of this mouse line (renamed Fam46a ^E157*Mhda) had significantly high ALP activities and apparently no other differences in morphology compared to wild-type mice. In contrast, homozygous Fam46a ^E157*Mhda mice showed severe morphological and skeletal abnormalities including short stature along with limb, rib, pelvis, and skull deformities with minimal trabecular bone and reduced cortical bone thickness in long bones. ALP activities of homozygous mutants were almost two-fold higher than in heterozygous mice. Fam46a is weakly expressed in most adult and embryonic tissues with a strong expression in mineralized tissues as calvaria and femur. The FAM46A protein is computationally predicted as a new member of the superfamily of nucleotidyltransferase fold proteins, but little is known about its function. Fam46a ^E157*Mhda mice are the first mouse model for a mutation within the Fam46a gene.