Life tables and predation activity of Orius laevigatus and O. albidipennis at three constant temperatures

Effects of three constant temperatures (15, 25, and 35 -C) on development and reproduction of Orius laevigatus (Fieber) and O. albidipennis (Reuter) (Heteroptera: Anthocoridae) and on their predation activity against the western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) were investigated in the laboratory. Small rooted plants of Spanish pepper (Capsicum annuum L. cv. Creta, long red) served as oviposition substrate and moisture source. Survival of eggs and nymphs of both species was high at 25 and 35 °C. At 15 °C, none of the eggs of O. albidipennis hatched and the number of nymphs completing the immature stage was extremely low. Developmental time of nymphs was not significantly different between species at 15 °C, but at 25 and 35 °C nymphs of O. laevigatus took significantly longer to develop than those of O. albidipennis. Females of O. albidipennis lived longer than those of O. laevigatus at 15 and 35 °C, but no differences were observed at 25 °C. Fecundity of O. albidipennis was greatly reduced at 15 °C, whereas a temperature of 35 °C was close to the upper reproduction threshold of O. laevigatus. Fecundity was highest at 25 °C for both species. At 15 °C, the intrinsic rate of increase (r_m) reached a minimum for both species. For O. albidipennis, the r_m-value increased with temperature (0.121 at 25 °C and 0.202 at 35 °C), whereas for O. laevigatus it peaked at 25 °C (0.105) but decreased at 35 °C (0.051). At 15 and 25 °C, adults of O. laevigatus consumed more F. occidentalis adults during their total lifespan than those of O. albidipennis, but the latter showed a better predation activity at 35 °C; in all treatments, however, adults of O. laevigatus consumed more prey per day than did those of O. albidipennis. The performance of both anthocorids at the different temperatures is discussed in relation to their practical use in integrated pest control programmes.     

Improvements in the chain-termination method of DNA sequencing through the use of 7-deaza-2'-deoxyadenosine.

Significant improvements in the quality of DNA sequencing data have been shown when deoxyadenosine triphosphate (dATP) is replaced by 7-deaza-2'-deoxyadenosine triphosphate (c7dATP). The use of c7dATP in conjunction with 7-deaza-2'-deoxyguanosine triphosphate (c7dGTP) further decreases anomalies in electrophoretic mobility which are caused by compressions involving G and/or A residues. This effect is observed for both isotope-based and fluorescence-based sequencing approaches. Replacing dATP with c7dATP also results in a higher degree of uniformity in the frequency of chain termination reactions, when such terminations involve the incorporation of fluorescence-labeled dideoxynucleotides by T7 polymerase. These improvements in the gel-resolution and distribution of chain-terminated DNA products result in higher accuracy in both manual and automated base assignment.     

Characterization of three novel pathogenic SLC40A1 mutations and genotype/phenotype correlations in 7 Italian families with type 4 hereditary hemochromatosis

Mutations of SLC40A1 encoding ferroportin (Fpn), the unique cellular iron exporter, severely affect iron homeostasis causing type 4 hereditary hemochromatosis, an autosomal dominant iron overload condition with variable phenotypic manifestations. This disease can be classified as type 4A, better known as “ferroportin disease”, which is due to “loss of function” mutations that lead to decreased iron export from cells, or as type 4B hemochromatosis, which is caused by “gain of function” mutations, conferring partial or complete resistance to hepcidin-mediated Fpn degradation.In this work, we discuss clinical and molecular findings on a group of patients in whom a SLC40A1 single copy missense variant was identified. Three novel variants, p.D181N, p.G204R and p.R296Q were functionally characterized. Fpn D181N and R296Q mutants can be classified as full or partial loss of function, respectively. Replacement of G204 with arginine appears to cause a more complex defect with impact both on iron export function and hepcidin sensitivity. This finding confirms the difficulty of predicting the effect of a mutation on the molecular properties of Fpn in order to provide an exhaustive explanation to the wide variability of the phenotype in type 4 hereditary hemochromatosis.     

Fermentative production of superoxide dismutase with <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

This work sought to develop a fermentative process for the microbial production of superoxide dismutase (SOD), to overcome extraction from animal tissues. Twenty-eight wild-type yeast strains were screened for SOD productivity. Kluyveromyces marxianus L3 showed the highest SOD activity (62 U mg⁻¹) and was used for process development. Oxidative stress conditions and parameters affecting oxygen transfer rate were exploited to improve production. The effects of dilution rate (0.067 vs 0.2 h⁻¹), aeration pressure (0.3 vs 1.2 bar) and H₂O₂ (0 vs 50 mM) were studied during chemostat experiments. Low dilution rate, high pressure and H₂O₂ resulted in an increase in CuZn–SOD up to 475 U mg⁻¹. When a regulation of oxygen saturation was applied during batch cultures, CuZn–SOD was progressively higher at 60, 80 and 90% dissolved oxygen tension (DOT) (250, 330 and 630 U mg⁻¹, respectively). Furthermore, the highest growth rate and biomass yield were achieved at 90% DOT, this being therefore the best DOT condition for high overall productivity. Growth and productivity on different carbon sources were compared. Specific activity was higher on glycerol than on lactose or glucose (496, 454 and 341 U mg⁻¹, respectively). The highest biomass yield was achieved on lactose. It may be therefore the best substrate for SOD production.     

Cost-effectiveness of exome sequencing: an Italian pilot study on undiagnosed patients

Recent advances in genomic sequencing and their implementation in clinical practice are widely recognized as diagnostic milestones, and are influencing considerably medical decision making in term of patients’ management. The cost-effectiveness of genomic analysis as first-tier tests has been documented. However, only a few studies have assessed systematically the economic impact of a revised diagnostic trajectory based on exome sequencing in the health system for undiagnosed patients. We report on the assessment of diagnostic costs referred to a large cohort of patients enrolled in the Bambino Gesù Children’s Hospital’s “Undiagnosed Patients Program”, supporting the cost-effectiveness of exome sequencing in a universalistic health care service compared to the traditional multi-step diagnostic workup. Our data provide evidence that revision of health policy to promote genomic sequencing of patients with suspected Mendelian disorders would allow reallocation of resources for rare diseases from diagnostics to patient care. At a social level, diagnosis is crucial to receive the social “sick role” and establish an effective doctor-patient relationship. The application of genomic sequencing as first-tier diagnostic test does improve this process speeding up the diagnosis and management of undiagnosed patients.     

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers^1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer^3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)^1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis⁹. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity^10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids¹². This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites¹³.Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.     

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.     

Effect of the feeding system on the fatty acid composition,expression of the Δ<Superscript>9</Superscript>-desaturase,Peroxisome Proliferator-Activated Receptor Alpha,Gamma, and Sterol Regulatory Element Binding Protein 1 genes in the semitendinous muscle of light lambs of the Rasa Aragonesa breed

Background  

Conjugated linoleic acids (CLAs) are receiving increasing attention because of their beneficial effects on human health, with milk and meat products derived from ruminants as important sources of CLA in the human diet. SCD gene is responsible for some of the variation in CLA concentration in adipose tissues, and PPARγ, PPARα and SREBP1 genes are regulator of SCD gene. The aim of this work was to evaluate the effect of the feeding system on fatty acid composition, CLA content and relative gene expression of Δ⁹-desaturase (SCD), Peroxisome Proliferator-Activated Receptor Gamma (PPARγ), Peroxisome Proliferator-Activated Receptor Alpha, (PPARα) and Sterol Regulatory Element Binding Protein (SREBP1) in Rasa Aragonesa light lambs in semitendinous muscle. Forty-four single-born male lambs were used to evaluate the effect of the feeding system, varying on an intensity gradient according to the use of concentrates: 1. grazing alfalfa, 2. grazing alfalfa with a supplement for lambs, 3. indoor lambs with grazing ewes and 4. drylot.