Evaluation of candidate F-box genes for the pollen S of gametophytic self-incompatibility in the Pyrinae (Rosaceae) on the basis of their phylogenomic context

The recent analysis of the S-locus region of apple and Japanese pear, two species of Pyrinae (Rosaceae), suggested multiple and different F-box genes (called SFBBs) as candidates for the male determinant (pollen S) of RNase-based gametophytic self-incompatibility in these two species. Here, we followed a phylogenetic approach to take advantage of the pattern of molecular evolution of the S-locus of Pyrinae in characterizing SFBB homologs belonging to S-haplotypes of apple and three species of Pyrus (European, Japanese, and Chinese pears). Our results suggested that the S-locus region of Pyrinae contains no less than six SFBB members and that its structure seems to be rather conserved between apple and pear species. In accordance with the prevailing theory on S-haplotype evolution, the pollen S is expected to have coevolved with the S-RNase and to show some common features derived from the long-term evolution under frequency-dependent balancing selection, i.e., high sequence diversity, evidence of positive selection, and shared ancestral polymorphisms. Using this conceptual framework, we present evidence that some SFBB genes may be better candidates for pollen S in Pyrinae than others. Overall, the SFBB genes analyzed exhibited much lower sequence diversity than their associated S-RNases; likewise, they showed little or no evidence of positive selection. However, evidence of coevolution with the S-RNase clearly emerged for two of them. Altogether our results suggested different evolutionary histories for different SFBBs putatively derived from their distinct involvement in self-incompatibility.     

Reliability of surface EMG matrix in locating the innervation zone of upper trapezius muscle

The identification of the motor unit (MU) innervation zone (IZ) using surface electromyographic (sEMG) signals detected on the skin with a linear array or a matrix of electrodes has been recently proposed in the literature. However, an analysis of the reliability of this procedure and, therefore, of the suitability of the sEMG signals for this purpose has not been reported.The purpose of this work is to describe the intra and inter-rater reliability and the suitability of surface EMG in locating the innervation zone of the upper trapezius muscle.Two operators were trained on electrode matrix positioning and sEMG signal analysis. Ten healthy subjects, instructed to perform a series of isometric contractions of the upper trapezius muscle participated in the study. The two operators collected sEMG signals and then independently estimated the IZ location through visual analysis.Results showed an almost perfect agreement for intra-rater and inter-rater reliability. The constancy of IZ location could be affected by the factors reflecting the population of active MUs and their IZs, including: the contraction intensity, the acquisition period analyzed, the contraction repetition. In almost all cases the IZ location shift due to these factors did not exceed 4 mm. Results generalization to other muscles should be made with caution.     

Randomized trial on 14 versus 7 days of esomeprazole, moxifloxacin, and amoxicillin for second-line or rescue treatment of Helicobacter pylori infection

Background: Triple therapy with a proton pump inhibitor, moxifloxacin, and amoxicillin has been proven effective in first‐line treatment of Helicobacter pylori infection. Aim: To explore 1, the value of triple therapy with esomeprazole, moxifloxacin, and amoxicillin in second‐line or rescue treatment of Caucasian patients and 2, the impact of treatment duration on eradication success. Methods: H. pylori‐infected patients with at least one previous treatment failure were randomized to oral esomeprazole 20 mg b.i.d., moxifloxacin 400 mg o.d., and amoxicillin 1000 mg b.i.d. for either 7 (EMA‐7) or 14 days (EMA‐14). Eradication was confirmed by 13C urea breath test. Antimicrobial susceptibility testing was performed in all patients at baseline and in patients who failed treatment. Results: Eighty patients were randomized, and 60% had ≥2 previous treatment failures. Pretreatment resistance against clarithromycin and metronidazole was found in 70.5 and 61.5% of cases, respectively. The intention‐to‐treat eradication rate was significantly higher after EMA‐14 compared with EMA‐7 (95.0 vs 78.9%, p = .036). No independent risk factor for treatment failure could be identified. There were no serious adverse events. Five of the EMA‐14 patients (12.5%) compared with none of the EMA‐7 patients discontinued prematurely because of adverse events (p = .031). Post‐treatment resistance against moxifloxacin was found in one of seven patients with isolated organisms (14.3%). Conclusion: Second‐line/rescue H. pylori eradication therapy with esomeprazole, moxifloxacin, and amoxicillin is very effective and well tolerated. Fourteen days of treatment significantly increase the eradication rate but also the rate of adverse events.     

Unbalanced oxidant-antioxidant status and its effects in pediatric diseases

Oxidative stress results from a disparity between the generation of reactive oxygen species and the antioxidant ability of the organism. The alteration of the oxidant-antioxidant system brings in adults an effective state of imbalance, which may influence the pathogenesis of many diseases. Oxidative stress also plays a pivotal role in the progression of various pathologies in childhood, through a manipulation of regulatory proteins. In fact, several studies have demonstrated that an unbalanced oxidant-antioxidant status is able to determine toxic effects even during infancy. Therefore, the aim of this review was to summarize current knowledge about the dynamic relationship between oxidative stress and systemic diseases during childhood. In order to better understand these complex mechanisms, a comprehensive review of the literature was done, focusing mainly on pre-pubertal children. In fact, this age-group offers a unique opportunity to exclude confounding factors, especially those related to the metabolic effects induced by puberty. Early identification of these very young patients should be aimed at minimizing the degree of oxidative damage. Only by achieving early diagnosis, will it be possible to identify those children who could benefit from specific therapeutic approaches targeting oxidative stress.     

Distinct regulatory programs establish widespread sex-specific alternative splicing in Drosophila melanogaster

In Drosophila melanogaster, female-specific expression of Sex-lethal (SXL) and Transformer (TRA) proteins controls sex-specific alternative splicing and/or translation of a handful of regulatory genes responsible for sexual differentiation and behavior. Recent findings in 2009 by Telonis-Scott et al. document widespread sex-biased alternative splicing in fruitflies, including instances of tissue-restricted sex-specific splicing. Here we report results arguing that some of these novel sex-specific splicing events are regulated by mechanisms distinct from those established by female-specific expression of SXL and TRA. Bioinformatic analysis of SXL/TRA binding sites, experimental analysis of sex-specific splicing in S2 and Kc cells lines and of the effects of SXL knockdown in Kc cells indicate that SXL-dependent and SXL-independent regulatory mechanisms coexist within the same cell. Additional determinants of sex-specific splicing can be provided by sex-specific differences in the expression of RNA binding proteins, including Hrp40/Squid. We report that sex-specific alternative splicing of the gene hrp40/squid leads to sex-specific differences in the levels of this hnRNP protein. The significant overlap between sex-regulated alternative splicing changes and those induced by knockdown of hrp40/squid and the presence of related sequence motifs enriched near subsets of Hrp40/Squid-regulated and sex-regulated splice sites indicate that this protein contributes to sex-specific splicing regulation. A significant fraction of sex-specific splicing differences are absent in germline-less tudor mutant flies. Intriguingly, these include alternative splicing events that are differentially spliced in tissues distant from the germline. Collectively, our results reveal that distinct genetic programs control widespread sex-specific splicing in Drosophila melanogaster.     

Cloning and optimization of induction conditions for mature PsaA (pneumococcal surface adhesin A) expression in Escherichia coli and recombinant protein stability during long-term storage

The gene corresponding to mature PsaA from Streptococcus pneumoniae serotype 14 was cloned into a plasmid with kanamycin resistance and without a purification tag in Escherichia coli to express high levels of the recombinant protein for large-scale production as a potential vaccine candidate or as a carrier for polysaccharide conjugation at Bio-Manguinhos/Fiocruz. The evaluation of induction conditions (IPTG concentration, temperature and time) in E. coli was accomplished by experimental design techniques to enhance the expression level of mature recombinant PsaA (rPsaA). The optimization of induction process conditions led us to perform the recombinant protein induction at 25°C for 16 h, with 0.1mM IPTG in Terrific Broth medium. At these conditions, the level of mature rPsaA expression obtained in E. coli BL21 (DE3) Star by pET28a induction with IPTG was in the range of 0.8 g/L of culture medium, with a 10-fold lower concentration of inducer than usually employed, which contributes to a less expensive process. Mature rPsaA expressed in E. coli BL21 (DE3) Star accounted for approximately 30-35% of the total protein. rPsaA purification by ion exchange allowed the production of high-purity recombinant protein without fusion tags. The results presented in this work confirm that the purified recombinant protein maintains its stability and integrity for long periods of time in various storage conditions (temperatures of 4 or -70°C using different cryoprotectors) and for at least 3 years at 4 or -70°C in PBS. The conformation of the stored protein was confirmed using circular dichroism. Mature rPsaA antigenicity was proven by anti-rPsaA mouse serum recognition through western blot analysis, and no protein degradation was detected after long periods of storage.     

FhuA deletion variant Δ1-159 overexpression in inclusion bodies and refolding with Polyethylene-Poly(ethylene glycol) diblock copolymer

Membrane protein isolation is a challenging problem. In fact especially their extraction from the respective membrane is difficult and often goes along with losses in yield. Usually expensive detergents are needed to extract the target protein from the membrane. Therefore finding an efficient overexpression and extraction method and an alternative to detergents is desirable. In this study we describe a new and fast method to express, extract and purify an engineered variant of the FhuA protein (FhuA Δ1-159) that acts as passive diffusion channel, using a diblock copolymer as an alternative to detergents like octyl-POE (n-octylpolyoxyethylene). The N-terminal leader sequence, facilitating the protein's transport to the outer membrane was deleted (FhuA Δ1-159 Δsignal), resulting in protein accumulation in easy to isolate inclusion bodies. Urea was used to solubilise the unfolded protein and dialysis against phosphate-buffer containing the commercially available diblock copolymer PE-PEG[Polyethylene-Poly(ethyleneglycol)] lead to protein refolding. Circular dichroism spectroscopy revealed a high β-sheet percentage within the refolded protein secondary structure indicating the successful reconstitution of FhuA Δ1-159 Δsignal native state. Furthermore the channel functionality of FhuA Δ1-159 Δsignal was verified by measuring the in and out-flux through the protein when inserted into liposome membrane, using the HRP/TMB (HRP=Horse Radish Peroxidase, TMB=3,3',5,5'-tetramethylbenzidine) assay system.     

Biooxidation of monoterpenes with bacterial monooxygenases

Biotechnological monoterpene oxidation has a considerable economic potential as an alternative route to natural monoterpenoid compounds with desirable organoleptic and pharmaceutical properties. Bacterial cytochrome P450 monooxygenases (CYPs) constitute ideal catalysts for monoterpene oxidation due to their pronounced selectivities, comparably high activities and ease of recombinant expression. Research activities of the recent decades resulted in the identification and characterization of many monoterpene oxidizing bacterial CYPs, often together with their electron transfer partners. To the authors’ knowledge, no industrial process of bacterial monoterpene oxidation has been established up to date. However, the last decade has seen movement away from small scale test tube sized reactions to research activities focusing on more sophisticated processes in larger volumes and in bioreactors. These research activities successfully combined improvements on all levels of a biotransformation process. Activity, selectivity and stability of bacterial CYPs were enhanced by rational protein design, substrate and product toxicity was counteracted with the development of feeding strategies and in situ product removal techniques. The disadvantage of costly cofactors was bypassed by the application of cofactor regeneration systems and by electrochemical substitution of cofactors.