Novel Reporter for Faithful Monitoring of ERK2 Dynamics in Living Cells and Model Organisms

Uncoupling of ERK1/2 phosphorylation from subcellular localization is essential towards the understanding of molecular mechanisms that control ERK1/2-mediated cell-fate decision. ERK1/2 non-catalytic functions and discoveries of new specific anchors responsible of the subcellular compartmentalization of ERK1/2 signaling pathway have been proposed as regulation mechanisms for which dynamic monitoring of ERK1/2 localization is necessary. However, studying the spatiotemporal features of ERK2, for instance, in different cellular processes in living cells and tissues requires a tool that can faithfully report on its subcellular distribution. We developed a novel molecular tool, ERK2-LOC, based on the T2A-mediated coexpression of strictly equimolar levels of eGFP-ERK2 and MEK1, to faithfully visualize ERK2 localization patterns. MEK1 and eGFP-ERK2 were expressed reliably and functionally both in vitro and in single living cells. We then assessed the subcellular distribution and mobility of ERK2-LOC using fluorescence microscopy in non-stimulated conditions and after activation/inhibition of the MAPK/ERK1/2 signaling pathway. Finally, we used our coexpression system in Xenopus laevis embryos during the early stages of development. This is the first report on MEK1/ERK2 T2A-mediated coexpression in living embryos, and we show that there is a strong correlation between the spatiotemporal subcellular distribution of ERK2-LOC and the phosphorylation patterns of ERK1/2. Our approach can be used to study the spatiotemporal localization of ERK2 and its dynamics in a variety of processes in living cells and embryonic tissues.     

Impact of exogenous sequences on the characteristics of an epidemic type 2 recombinant vaccine-derived poliovirus

Pathogenic circulating vaccine-derived polioviruses (cVDPVs) have become a major obstacle to the successful completion of the global polio eradication program. Most cVDPVs are recombinant between the oral poliovirus vaccine (OPV) and human enterovirus species C (HEV-C). To study the role of HEV-C sequences in the phenotype of cVDPVs, we generated a series of recombinants between a Madagascar cVDPV isolate and its parental OPV type 2 strain. Results indicated that the HEV-C sequences present in this cVDPV contribute to its characteristics, including pathogenicity, suggesting that interspecific recombination contributes to the phenotypic biodiversity of polioviruses and may favor the emergence of cVDPVs.     

Performance of polyvoltine silkworm Bombyx mori L. on different mulberry plant varieties

Five cultivated mulberry plant varieties, BSRM‐34, BSRM‐56, BSRM‐57, BSRM‐58 and BSRM‐59, were used to evaluate the effects of plant variety on larval and cocoon characteristics of mulberry silkworm Bombyx mori L. Silkworm larvae undergo their entire larval periods on leaves of mulberry varieties. Different larval characteristics of larval weight, larval length and larval breadth were measured in different growth stages. After rearing, the fifth instar larvae were transferred into a mountage (Chandraki) structure for cocoon rearing for spinning and cocoon production. The cocoons were then harvested and different cocoon characteristics like cocoon weight, shell weight and shell percentage; and other characters like pupal weight and effective rate of rearing were measured. The results showed statistically significant differences among different varieties on larval and cocoon characteristics. The highest performance was observed by feeding the variety BSRM‐34 in respect of the average weight of 10 mature larvae (40.5 g), single mature larval length (7.6 cm) and breadth (1.0 cm), single cocoon weight (1.4 g), shell weight (0.2 g) and pupal weight (1.2 g) while the average performance was recorded by feeding varieties BSRM‐57 and BSRM‐59. The poorest performance was showed by feeding the variety BSRM‐56 (in case of larval characters) and BSRM‐58 (in case of cocoon characteristics). These two varieties also showed the poorest performance for silk yield. Through comparing all the above parameters it was concluded that BSRM‐34 is the best mulberry variety and it is recommended that Bangladeshi farmers adopt that variety for better yield of silk.     

Climatic niche and neutral genetic diversity of the six Iberian pine species: a retrospective and prospective view

Quaternary climatic fluctuations have left contrasting historical footprints on the neutral genetic diversity patterns of existing populations of different tree species. We should expect the demography, and consequently the neutral genetic structure, of taxa less tolerant to particular climatic extremes to be more sensitive to long‐term climate fluctuations. We explore this hypothesis here by sampling all six pine species found in the Iberian Peninsula (2464 individuals, 105 populations), using a common set of chloroplast microsatellite markers, and by looking at the association between neutral genetic diversity and species‐specific climatic requirements. We found large variation in neutral genetic diversity and structure among Iberian pines, with cold‐enduring mountain species (Pinus uncinata, P. sylvestris and P. nigra) showing substantially greater diversity than thermophilous taxa (P. pinea and P. halepensis). Within species, we observed a significant positive correlation between population genetic diversity and summer precipitation for some of the mountain pines. The observed pattern is consistent with the hypotheses that: (i) more thermophilous species have been subjected to stronger demographic fluctuations in the past, as a consequence of their maladaptation to recurrent glacial cold stages; and (ii) altitudinal migrations have allowed the maintenance of large effective population sizes and genetic variation in cold‐tolerant species, especially in more humid regions. In the light of these results and hypotheses, we discuss some potential genetic consequences of impending climate change.     

Effects of External Mass Transfer and Product Inhibition on a Simulated Immobilized Enzyme‐Catalyzed Reactor for Lactose Hydrolysis

A mathematical model has been developed for predicting the performance and simulation of a packed bed immobilized enzyme reactor performing lactose hydrolysis, which follows Michaelis‐Menten kinetics with competitive product (galactose) inhibition. The performance characteristics of a packed bed immobilized enzyme reactor have been analyzed taking into account the effects of various diffusional phenomena like axial dispersion and external mass transfer limitations. The model design equations are then solved by Galerkin's method and orthogonal collocation on finite elements. The effects of external mass transfer and axial dispersion have been studied and their effects were shown to reduce the external effectiveness factor. The effects of product inhibition have been investigated at different operating conditions correlated at different regimes using dimensionless moduli (St, γ, θ, Da)¹⁾. The product inhibition was shown to reduce the substrate conversion, and, additionally, to decrease the effectiveness factor when Da > Da_xo, however, it increases the effectiveness factor when Da < Da_xo. The effectiveness factor is found to be independent of the product inhibition at a crossover point at which Da_xo is defined. Effects of St and Pe have been investigated at different kinetic regimes and the results show that their effects have a strong dependency on the kinetic parameters θ, γ (i.e., K_m/K_p), and Da_xo.     

Role of nitric oxide synthase/arginase balance in bronchial reactivity in patients with chronic obstructive pulmonary disease

Competition between nitric oxide synthases (NOSs) and arginases for their common substrate l-arginine could be involved in the regulation of cholinergic airway reactivity and subsequent airway remodeling. The aims of this study were to evaluate the relationships between the expression of this enzymatic balance and the effects of NOS and arginase inhibition on bronchoconstrictive response to acetylcholine of patients without and with early chronic obstructive pulmonary disease (COPD). Twenty-two human bronchi [15 COPD (9 GOLD-0, 6 GOLD-1, -2-A), 7 nonsmokers] were investigated for immunohistochemistry and modulation of acetylcholine-induced airway constriction. Significantly increased expression of NOS2 in immunoblots of bronchial tissue and staining in smooth muscle cells was evidenced in patients with COPD compared with control subjects, whereas no modification of arginase expression was evidenced. Forced expiratory volume in 1 s (FEV1) and NOS2 expression were negatively correlated (rho=-0.54, P=0.027). Pharmacological experiments demonstrated that resting tension was elevated in COPD compared with control subjects (2,243+/-154 vs. 1,574+/-218 mg, P=0.03) and was positively correlated with the expression of NOS2 (rho=0.61, P=0.044), whereas constrictor response to acetylcholine was similar [active tension, sensitivity (-logEC10), and reactivity (slope)]. The sole effect of the specific arginase inhibitor Nomega-hydroxy-nor-L-arginine (1 microM) was to decrease sensitivity in COPD patients, whereas 1 mM NG-nitro-L-arginine methyl ester unexpectedly decreased resting tension because of a non-cGMP-dependent effect. In conclusion, an upregulation of NOS2 expression in COPD patients is involved in airway tone regulation and functional airflow limitation, whereas increased arginase activity is involved in airway sensitivity.     

Detection of quantitative trait loci for teat number and female reproductive traits in Meishan × Large White F2 pigs

A quantitative trait locus (QTL) analysis of female reproductive data from a three-generation experimental cross between Meishan (MS) and Large White (LW) pig breeds is presented. Six F1 boars and 23 F1 sows, progeny of six LW boars and six MS sows, produced 573 F2 females and 530 F2 males. Six traits, i.e. teat number (TN), age at puberty (AP), ovulation rate (OR), weight at mating (WTM), number of viable embryos (NVE) and embryo survival (ES) at 30 days of gestation were analysed. Animals were genotyped for a total of 137 markers covering the entire porcine genome. Analyses were carried out based on interval mapping methods, using a line-cross (LC) regression and a half-full sib (HFS) maximum likelihood test. Genome-wide (GW) highly significant (P < 0.001) QTL were detected for WTM on SSC 7 and for AP on SSC 13. They explained, respectively, 14.5% and 8.9% of the trait phenotypic variance. Other GW significant (P < 0.05) QTL were detected for TN on SSC 3, 7, 8, 16 and 17, for OR on SSC 4 and 5, and for ES on SSC 9. Two additional chromosome-wide significant (P < 0.05) QTL were detected for TN, three for WTM, four for AP, three for OR, three for NVE and two for ES. With the exception of the two above-mentioned loci, the QTL explained from 1.2% to 4.6% of trait phenotypic variance. QTL alleles were in most cases not fixed in the grand-parental populations and Meishan alleles were not systematically associated with higher reproductive performance.