Nonredundant mass spectrometry: a strategy to integrate mass spectrometry acquisition and analysis

Protein identification using automated data-dependent tandem mass spectrometry (MS/MS) is now a standard procedure. However, in many cases data-dependent acquisition becomes redundant acquisition as many different peptides from the same protein are fragmented, whilst only a few are needed for unambiguous identification. To increase the quality of information but decrease the amount of information, a nonredundant MS (nrMS) strategy has been developed. With nrMS, data analysis is an integral part of the overall MS acquisition and analysis, and not an endpoint as typically performed. In this nrMS workflow a matrix assisted laser desorption/ionization-time of flight-time of flight (MALDI-TOF/TOF) instrument is used. MS and restricted MS/MS data are searched and identified proteins are used to generate an "exclusion list", after in silico digestion. Peptide fragmentation is then restricted to only the most intense ions not present in the exclusion list. This process is repeated until all peaks are accounted for or the sample is consumed. Compared to nanoLC-MS/MS, nrMS yielded similar results for the analysis of six pooled two-dimensional electrophoresis (2-DE) spots. In comparison to standard data-dependent MALDI-MS/MS for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel band analysis, nrMS dramatically increased the number of identified proteins. It was also found that this new workflow significantly increased sequence coverage by identifying unexpected peptides, which can result from post-translational modifications.     

Age-dependent response to insecticides and enzymatic variation in susceptible and resistant codling moth larvae

Insecticide resistance in the codling moth, Cydia pomonella, partly results from increased metabolic detoxification. The aim of this study was to follow the age variations in larval susceptibility to deltamethrin and teflubenzuron in one susceptible (S) strain, and two resistant (Rv and Rt) ones selected for resistance to deltamethrin and diflubenzuron, respectively. The age variation of the activities of cytochrome P450-dependent monooxygenase (MFO), glutathione S-transferases (GST), and esterases in S and both resistant strains were simultaneously investigated. The highest levels of insecticide resistance were recorded in late instars in both resistant strains, although Rv neonates exhibited enhanced resistance to deltamethrin. The involvement of an additional deltamethrin-specific mechanism of resistance, which could be mainly expressed in early instars, was supported by previous demonstration of a kdr point mutation in the Rv strain. The cross-resistance between deltamethrin and teflubenzuron indicated the involvement of non-specific metabolic pathways in resistance to teflubenzuron, rather than target site modification. A positive correlation between enhanced GST activities and deltamethrin resistance suggested that this mechanism might take place into the adaptive response of C. pomonella to pyrethroids treatments. Enhanced MFO activity was recorded in each instar of the two resistant strains compared to the susceptible one. But these activities were not correlated to the responses to deltamethrin nor to teflubenzuron. In the light of these findings, studying age-dependence of responses to selection is central to the implementation of monitoring tests of resistances, especially if the target instars are difficult to collect in the field.     

Daily melatonin supplementation in mice increases atherosclerosis in proximal aorta

Considerable evidence supports the hypothesis that LDL oxidation plays an important role in atherosclerosis. Even though high melatonin doses inhibit LDL oxidation in vitro, the effect of melatonin on atherosclerosis has never been studied. We have demonstrated that the feeding of hypercholesterolemic mice with an atherogenic diet supplemented with melatonin highly increases the surface of atherosclerotic lesions in the proximal aorta. These observations occur without detectable lipidic or glucidic phenotype alteration. Melatonin treatment increased highly the sensitivity of atherogenic lipoprotein to Cu(2+) and gamma-radiolysis generated oxyradical ex vivo oxidation during the fasting period. Moreover, these altered lipoproteins were less recognized by the LDL receptor metabolic pathway of murine fibroblasts while they transferred many more cholesteryl esters to murine macrophages. This study suggests that caution should be taken as regards high melatonin dosage in hypercholesterolemic patients.     

New species and new records of Ergasilus Nordmann (Copepoda: Ergasilidae) from the gills of grey mullet (Mugilidae)

Four new Ergasilus species are described from five host species of grey mullet, family Mugilidae, held in the fish collections of The Natural History Museum, London. Ergasilus extensus n. sp. was found on Myxus petardi (Castelnau) from Australia, E. sittangenesis n. sp. on Sicamugil hamiltoni (Day) from Burma, E. piriformis n. sp. on Sicamugil cascasia (Hamilton Buchanan) from India, and E. ecuadorensis n. sp. on Mugil curema Valenciennes and M. hospes Jordan & Culver from Ecuador. New records of E. cyanopictus Caravalho, E. magnicornis Yin, E. orientalis Yamaguti and E. rostralis Ho, Jayarajian & Radhakrishnan on grey mullet are also given.     

The Spatiotemporal Program of DNA Replication Is Associated with Specific Combinations of Chromatin Marks in Human Cells

The duplication of mammalian genomes is under the control of a spatiotemporal program that orchestrates the positioning and the timing of firing of replication origins. The molecular mechanisms coordinating the activation of about predicted origins remain poorly understood, partly due to the intrinsic rarity of replication bubbles, making it difficult to purify short nascent strands (SNS). The precise identification of origins based on the high-throughput sequencing of SNS constitutes a new methodological challenge. We propose a new statistical method with a controlled resolution, adapted to the detection of replication origins from SNS data. We detected an average of 80,000 replication origins in different cell lines. To evaluate the consistency between different protocols, we compared SNS detections with bubble trapping detections. This comparison demonstrated a good agreement between genome-wide methods, with 65% of SNS-detected origins validated by bubble trapping, and 44% of bubble trapping origins validated by SNS origins, when compared at the same resolution. We investigated the interplay between the spatial and the temporal programs of replication at fine scales. We show that most of the origins detected in regions replicated in early S phase are shared by all the cell lines investigated whereas cell-type-specific origins tend to be replicated in late S phase. We shed a new light on the key role of CpG islands, by showing that 80% of the origins associated with CGIs are constitutive. Our results further show that at least 76% of CGIs are origins of replication. The analysis of associations with chromatin marks at different timing of cell division revealed new potential epigenetic regulators driving the spatiotemporal activity of replication origins. We highlight the potential role of H4K20me1 and H3K27me3, the coupling of which is correlated with increased efficiency of replication origins, clearly identifying those marks as potential key regulators of replication origins.     

Improvement of β-glucosidase production by co-culture of Aspergillus niger and A. oryzae under solid state fermentation through feeding process

Eleven different Aspergillus strains were evaluated for their ability to produce β-glucosidase using sugar cane bagasse as a sole carbon source under solid state fermentation (SSF). The most potent strains, A. niger NRC 7 (674.6 U/g ds) and A. oryzae NRRL 447 (83 U/g ds), were used in a mixed culture to enhance β-glucosidase production by co-culturing under SSF. In mixed culture, β-glucosidase of the two strains (814 U/g ds) was nearly 1.2- and 9.8-fold than that of monocultures of A. niger NRC 7A and A. oryzae NRRL 447, respectively. Optimization of the culture parameters, initial pH value, moisture content, inoculum size and ratios of the two strains. and incubation time exhibited a significant increase in β-glucosidase production (1,893 U/g ds) than before optimization. Single feeding with citrate-phosphate buffer, succinate buffer, casein. and soybean flour individually after the third day of the fermentation time and controlling the moisture content at 90 % (w/w) induced β-glucosidase production. Maximum enzyme production increased up to 2.1-fold compared to 2,188 U/g ds during normal batch culture. Among nitrogen sources, soybean flour gave the highest β-glucosidase (4,578 U/g ds). while urea reduced β-glucosidase production (1,693 U/g ds). However, the combination of buffers with soybean flour through two fed cycles resulted in a decrease of the enzyme than single fed with buffers or soybean flour alone.     

Isolation of scFv antibody fragments against HER2 and CEA tumor antigens from combinatorial antibody libraries derived from cancer patients

Tumor cells expressing HER-2/neu and CEA antigens are potentially ideal targets for antibody-targeted therapy. In this study, two large human combinatorial libraries have been generated from the lymph nodes of breast cancer patients that express HER2 and CEA antigens in their tumors. These ‘immune’ libraries have been constructed in two different formats of scFv, differing in the length of the peptide linker connecting the two variable VH and VL domains. Libraries derived from these patients may contain a larger pool of anti-tumor antigen antibodies and are useful repertoire for isolating scFvs against any tumor markers. The results of this study showed that we were successful in obtaining human scFvs against HER-2/neu and CEA. For HER-2, cell-panning strategy was performed and resulted in two scFv binders that detected the complete HER-2 receptor on the cell membrane and internalized to the cells. Also, preliminary ELISA data showed that several anti-CEA scFv binders were isolated by panning.     

4-Mercaptoimidazoles derived from the naturally occurring antioxidant ovothiols 1. Antioxidant properties

4-Mercaptoimidazoles derived from the naturally occurring family of antioxidants, the ovothiols, were assayed for their antioxidant properties. These compounds are powerful HOCI scavengers, more potent than the aliphatic thiol N-acetylcysteine. They react slowly with hydrogen peroxide with second order rate constants of 0.13–0.89 M^-1 s^-1. Scavenging of hydroxyl radical occurs at a diffusion-controlled rate (k = 2.0–5.0 × 10¹⁰ M^-1 s^-1) for the most active compounds, which are also able to inhibit copper-induced LDL peroxidation. The combination of radical scavenging and copper chelating properties may explain the inhibitory effects on LDL peroxidation. Two molecules of mercaptoimidazole can chelate a copper ion and form a square planar complex detected by EPR. Compounds bearing an electron-withdrawing group on position 2 of the imidazole ring are the most potent antioxidant molecules in this series.