Congener specific determination of polychlorinated naphthalenes in sediment and biota by gas chromatography high resolution mass spectrometry

© 2016 Elsevier B.V. An isotope dilution congener-specific method for the determination of the most abundant and most toxic polychlorinated naphthalenes (PCNs) was developed using gas chromatography with high resolution mass spectrometry (GC-HRMS). The method was used to determine the concentration of 24 target congeners and total PCN concentrations in fish and sediment samples. Tissue samples were extracted using pressurized liquid extraction (PLE) and sediment samples were extracted using Soxhlet extraction. Sample extracts were cleaned up using either a manual two-stage open column procedure or an automated FMS Power Prep System with multi-analyte and multi-sample capability using a three-column cleanup procedure. Sediment extracts were cleaned up with a dual open column cleanup technique involving the use of both a multi-layered silica (silver nitrate/acid/base/neutral silica) column followed by column containing carbon-activated silica. Fish tissue extracts were cleaned up on the automated system involving the use of a high capacity ABN (acid/base/neutral column), carbon celite column, and a basic alumina column. The method is capable of producing instrument detection limits (IDLs) between 0.06 and 0.13 pg for each PCN (on column), with method detection limits (MDLs) for the fish extracts ranging from 1.3 to 3.4 pg/g (wet weight) and 0.46 to 1.2 pg/g (dry weight) for sediments. The average accuracy of 34 spiked fish samples analysed over a period of several months was 100% with a precision (%RSD) of 12%. Similarly, the average accuracy for 28 spiked sediment samples was 104% with a precision (%RSD) of 12%. The application of the method to environmental samples was demonstrated through the analysis of sediment and fish samples obtained from Lake Ontario, Canada. The method is used both for the determination of 24 PCNs and to perform non-targeted screening for the remaining 51 PCN congeners, which are included in the total PCN quantification result. It is currently one of the most comprehensive and accurate congener-specific methods available and was developed from the existing techniques used for the determination of polychlorinated dioxins and furans to produce high quality data with only minor modifications in the clean-up procedure. It can therefore be readily adopted by other laboratories performing dioxin and POP analyses

Overexpression of human kynurenine-3-monooxygenase protects against 3-hydroxykynurenine-mediated apoptosis through bidirectional non-linear feedback

Kynurenine 3-monooxygenase (KMO) is a critical regulator of inflammation. The preferred KMO substrate, kynurenine, is converted to 3-hydroxykynurenine (3HK), and this product exhibits cytotoxicity through mechanisms that culminate in apoptosis. Here, we report that overexpression of human KMO with orthotopic localisation to mitochondria creates a metabolic environment during which the cell exhibits increased tolerance for exogenous 3HK-mediated cellular injury. Using the selective KMO inhibitor Ro61-8048, we show that KMO enzyme function is essential for cellular protection. Pan-caspase inhibition with Z-VAD-FMK confirmed apoptosis as the mode of cell death. By defining expression of pathway components upstream and downstream of KMO, we observed alterations in other key kynurenine pathway components, particularly tryptophan-2,3-dioxygenase upregulation, through bidirectional nonlinear feedback. KMO overexpression also increased expression of inducible nitric oxide synthase (iNOS). These changes in gene expression are functionally relevant, because siRNA knockdown of the pathway components kynureninase and quinolinate phosphoribosyl transferase caused cells to revert to a state of susceptibility to 3HK-mediated apoptosis. In summary, KMO overexpression, and importantly KMO activity, have metabolic repercussions that fundamentally affect resistance to cell stress

Transport of Anthocyanins and other Flavonoids by the Arabidopsis ATP-Binding Cassette Transporter AtABCC2

Flavonoids have important developmental, physiological, and ecological roles in plants and are primarily stored in the large central vacuole. Here we show that both an ATP-binding cassette (ABC) transporter(s) and an H+-antiporter(s) are involved in the uptake of cyanidin 3-O-glucoside (C3G) by Arabidopsis vacuolar membrane-enriched vesicles. We also demonstrate that vesicles isolated from yeast expressing the ABC protein AtABCC2 are capable of MgATP-dependent uptake of C3G and other anthocyanins. The uptake of C3G by AtABCC2 depended on the co-transport of glutathione (GSH). C3G was not altered during transport and a GSH conjugate was not formed. Vesicles from yeast expressing AtABCC2 also transported flavone and flavonol glucosides. We performed ligand docking studies to a homology model of AtABCC2 and probed the putative binding sites of C3G and GSH through site-directed mutagenesis and functional studies. These studies identified residues important for substrate recognition and transport activity in AtABCC2, and suggest that C3G and GSH bind closely, mutually enhancing each other’s binding. In conclusion, we suggest that AtABCC2 along with possibly other ABCC proteins are involved in the vacuolar transport of anthocyanins and other flavonoids in the vegetative tissue of Arabidopsis

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

Higher Level Phylogeny and the First Divergence Time Estimation of Heteroptera (Insecta: Hemiptera) Based on Multiple Genes

Heteroptera, or true bugs, are the largest, morphologically diverse and economically important group of insects with incomplete metamorphosis. However, the phylogenetic relationships within Heteroptera are still in dispute and most of the previous studies were based on morphological characters or with single gene (partial or whole 18S rDNA). Besides, so far, divergence time estimates for Heteroptera totally rely on the fossil record, while no studies have been performed on molecular divergence rates. Here, for the first time, we used maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) with multiple genes (18S rDNA, 28S rDNA, 16S rDNA and COI) to estimate phylogenetic relationships among the infraorders, and meanwhile, the Penalized Likelihood (r8s) and Bayesian (BEAST) molecular dating methods were employed to estimate divergence time of higher taxa of this suborder. Major results of the present study included: Nepomorpha was placed as the most basal clade in all six trees (MP trees, ML trees and Bayesian trees of nuclear gene data and four-gene combined data, respectively) with full support values. The sister-group relationship of Cimicomorpha and Pentatomomorpha was also strongly supported. Nepomorpha originated in early Triassic and the other six infraorders originated in a very short period of time in middle Triassic. Cimicomorpha and Pentatomomorpha underwent a radiation at family level in Cretaceous, paralleling the proliferation of the flowering plants. Our results indicated that the higher-group radiations within hemimetabolous Heteroptera were simultaneously with those of holometabolous Coleoptera and Diptera which took place in the Triassic. While the aquatic habitat was colonized by Nepomorpha already in the Triassic, the Gerromorpha independently adapted to the semi-aquatic habitat in the Early Jurassic

An Inserted α/β Subdomain Shapes the Catalytic Pocket of Lactobacillus johnsonii Cinnamoyl Esterase

Microbial enzymes produced in the gastrointestinal tract are primarily responsible for the release and biochemical transformation of absorbable bioactive monophenols. In the present work we described the crystal structure of LJ0536, a serine cinnamoyl esterase produced by the probiotic bacterium Lactobacillus johnsonii N6.2.We crystallized LJ0536 in the apo form and in three substrate-bound complexes. The structure showed a canonical α/β fold characteristic of esterases, and the enzyme is dimeric. Two classical serine esterase motifs (GlyXSerXGly) can be recognized from the amino acid sequence, and the structure revealed that the catalytic triad of the enzyme is formed by Ser(106), His(225), and Asp(197), while the other motif is non-functional. In all substrate-bound complexes, the aromatic acyl group of the ester compound was bound in the deepest part of the catalytic pocket. The binding pocket also contained an unoccupied area that could accommodate larger ligands. The structure revealed a prominent inserted α/β subdomain of 54 amino acids, from which multiple contacts to the aromatic acyl groups of the substrates are made. Inserts of this size are seen in other esterases, but the secondary structure topology of this subdomain of LJ0536 is unique to this enzyme and its closest homolog (Est1E) in the Protein Databank.The binding mechanism characterized (involving the inserted α/β subdomain) clearly differentiates LJ0536 from enzymes with similar activity of a fungal origin. The structural features herein described together with the activity profile of LJ0536 suggest that this enzyme should be clustered in a new group of bacterial cinnamoyl esterases

Acute effects of intracranial hypertension and ARDS on pulmonary and neuronal damage: a randomized experimental study in pigs

Abstract PURPOSE: To determine reciprocal and synergistic effects of acute intracranial hypertension and ARDS on neuronal and pulmonary damage and to define possible mechanisms. METHODS: Twenty-eight mechanically ventilated pigs were randomized to four groups of seven each: control; acute intracranial hypertension (AICH); acute respiratory distress syndrome (ARDS); acute respiratory distress syndrome in combination with acute intracranial hypertension (ARDS + AICH). AICH was induced with an intracranial balloon catheter and the inflation volume was adjusted to keep intracranial pressure (ICP) at 30-40 cmH2O. ARDS was induced by oleic acid infusion. Respiratory function, hemodynamics, extravascular lung water index (ELWI), lung and brain computed tomography (CT) scans, as well as inflammatory mediators, S100B, and neuronal serum enolase (NSE) were measured over a 4-h period. Lung and brain tissue were collected and examined at the end of the experiment. RESULTS: In both healthy and injured lungs, AICH caused increases in NSE and TNF-alpha plasma concentrations, extravascular lung water, and lung density in CT, the extent of poorly aerated (dystelectatic) and atelectatic lung regions, and an increase in the brain tissue water content. ARDS and AICH in combination induced damage in the hippocampus and decreased density in brain CT. CONCLUSIONS: AICH induces lung injury and also exacerbates pre-existing damage. Increased extravascular lung water is an early marker. ARDS has a detrimental effect on the brain and acts synergistically with intracranial hypertension to cause histological hippocampal damage

Quantifying unpredictability: A multiple-model approach based on satellite imagery data from Mediterranean ponds.

Fluctuations in environmental parameters are increasingly being recognized as essential features of any habitat. The quantification of whether environmental fluctuations are prevalently predictable or unpredictable is remarkably relevant to understanding the evolutionary responses of organisms. However, when characterizing the relevant features of natural habitats, ecologists typically face two problems: (1) gathering long-term data and (2) handling the hard-won data. This paper takes advantage of the free access to long-term recordings of remote sensing data (27 years, Landsat TM/ETM+) to assess a set of environmental models for estimating environmental predictability. The case study included 20 Mediterranean saline ponds and lakes, and the focal variable was the water-surface area. This study first aimed to produce a method for accurately estimating the water-surface area from satellite images. Saline ponds can develop salt-crusted areas that make it difficult to distinguish between soil and water. This challenge was addressed using a novel pipeline that combines band ratio water indices and the short near-infrared band as a salt filter. The study then extracted the predictable and unpredictable components of variation in the water-surface area. Two different approaches, each showing variations in the parameters, were used to obtain the stochastic variation around a regular pattern with the objective of dissecting the effect of assumptions on predictability estimations. The first approach, which is based on Colwell's predictability metrics, transforms the focal variable into a nominal one. The resulting discrete categories define the relevant variations in the water-surface area. In the second approach, we introduced General Additive Model (GAM) fitting as a new metric for quantifying predictability. Both approaches produced a wide range of predictability for the studied ponds. Some model assumptions-which are considered very different a priori-had minor effects, whereas others produced predictability estimations that showed some degree of divergence. We hypothesize that these diverging estimations of predictability reflect the effect of fluctuations on different types of organisms. The fluctuation analysis described in this manuscript is applicable to a wide variety of systems, including both aquatic and nonaquatic systems, and will be valuable for quantifying and characterizing predictability, which is essential within the expected global increase in the unpredictability of environmental fluctuations. We advocate that a priori information for organisms of interest should be used to select the most suitable metrics estimating predictability, and we provide some guidelines for this approach

Downregulation of drug transport and metabolism in mice bearing extra-hepatic malignancies

There is increasing evidence of a systemic inflammatory response associated with malignancy, which may have an impact on both drug disposition and resistance to cytotoxic therapy. The impact of inflammation on drug disposition was studied in mice bearing a number of common tumour xenografts. C57BL/6 mice were inoculated with tumour xenografts. Hepatic expressions of Cyp3a and drug transporters were analysed at the mRNA, protein and functional levels (Cyp3a only). Circulating serum cytokines and the hepatic expression of acute phase proteins (APPs) were measured. Intratumoral levels of multidrug resistance genes were determined. Tumour xenografts elicited an inflammatory response that coincided with repression in hepatic Cyp3a11 activity and the expression of a number of hepatic drug transporters. With tumour growth, a progressive reduction in hepatic Cyp3a11 mRNA expression was seen. Conversely, an increase in the hepatic APP expression and circulating interleukin (IL)-6 levels was observed. Furthermore, a correlation was seen between increased intratumoral expression of the multidrug resistance gene, Mdr1a, and levels of circulating IL-6. Malignancy results in reduced hepatic drug disposition that correlates with an associated inflammatory response. Reduction of inflammation may improve the clinical outcome for patients receiving chemotherapeutic agents that undergo hepatic metabolism

Stridulations Reveal Cryptic Speciation in Neotropical Sympatric Ants

The taxonomic challenge posed by cryptic species underlines the importance of using multiple criteria in species delimitation. In the current paper we tested the use of acoustic analysis as a tool to assess the real diversity in a cryptic species complex of Neotropical ants. In order to understand the potential of acoustics and to improve consistency in the conclusions by comparing different approaches, phylogenetic relationships of all the morphs considered were assessed by the analysis of a fragment of the mitochondrial DNA cytochrome b. We observed that each of the cryptic morph studied presents a morphologically distinct stridulatory organ and that all sympatric morphs produce distinctive stridulations. This is the first evidence of such a degree of specialization in the acoustic organ and signals in ants, which suggests that stridulations may be among the cues used by these ants during inter-specific interactions. Mitochondrial DNA variation corroborated the acoustic differences observed, confirming acoustics as a helpful tool to determine cryptic species in this group of ants, and possibly in stridulating ants in general. Congruent morphological, acoustic and genetic results constitute sufficient evidence to propose each morph studied here as a valid new species, suggesting that P. apicalis is a complex of at least 6 to 9 species, even if they present different levels of divergence. Finally, our results highlight that ant stridulations may be much more informative than hitherto thought, as much for ant communication as for integrative taxonomists