A Trichodorus (Triplonchida: Trichodoridae) Nematode from Thrips (Thysanoptera: Panchaetothripinae)

A thrips insect Caliothrips sp. (Thysanoptera: Panchaetothripinae) from persimmon fruit (Ebenaceae: Diospyros sp.) from an unknown origin, possibly Asia, was intercepted in a passenger bag in November 2012 at the Peace Arch Border Crossing from Canada to Blaine, WA, by a USDA-APHIS-PPQ port inspector. Nematodes were attached to the abdomen of the female insect and sent to us in saline. Seven nematodes (five females, two males) were measured and these and others were processed for permanent slides. An adult female and a female juvenile were prepared for PCR. Morphologically these nematodes belonged to the Trichodorus sparsus group, and the 28S rDNA D2-D3 sequence showed greatest similarity to Trichodorus paragiennensis (94%) and T. giennensis (93%), with greatest morphological similarity to the latter species. Among other morphological differences, the innermost uterus width is wider than in related species. Trichodorus spp. are normally found in soil, so this is the first population seen in the atypical habitat of an insect. Morphological and molecular characteristics of Trichodorus sp. are presented, but a putative new species name is not currently advisable because of relatively poor condition of specimens. Ecological associations are also discussed.     

PTPepsilon has a critical role in signaling transduction pathways and phosphoprotein network topology in red cells

Protein tyrosine phosphatases (PTPs) are crucial components of cellular signal transduction pathways. Here, we report that red blood cells (RBCs) from mice lacking PTPepsilon (Ptpre(-/-)) exhibit (i) abnormal morphology; (ii) increased Ca(2+)-activated-K(+) channel activity, which was partially blocked by the Src family kinases (SFKs) inhibitor PP1; and (iii) market perturbation of the RBC membrane tyrosine (Tyr-) phosphoproteome, indicating an alteration of RBC signal transduction pathways. Using the signaling network computational analysis of the Tyr-phosphoproteomic data, we identified seven topological clusters. We studied cluster 1 containing Fyn, SFK, and Syk another tyrosine kinase. In Ptpre(-/-)mouse RBCs, the activity of Fyn was increased while Syk kinase activity was decreased compared to wild-type RBCs, validating the network computational analysis, and indicating a novel signaling pathway, which involves Fyn and Syk in regulation of red cell morphology.     

Biological nitrogen and phosphorus removal in membrane bioreactors: model development and parameter estimation

Membrane bioreactors (MBR) are being increasingly used for wastewater treatment. Mathematical modeling of MBR systems plays a key role in order to better explain their characteristics. Several MBR models have been presented in the literature focusing on different aspects: biological models, models which include soluble microbial products (SMP), physical models able to describe the membrane fouling and integrated models which couple the SMP models with the physical models. However, only a few integrated models have been developed which take into account the relationships between membrane fouling and biological processes. With respect to biological phosphorus removal in MBR systems, due to the complexity of the process, practical use of the models is still limited. There is a vast knowledge (and consequently vast amount of data) on nutrient removal for conventional-activated sludge systems but only limited information on phosphorus removal for MBRs. Calibration of these complex integrated models still remains the main bottleneck to their employment. The paper presents an integrated mathematical model able to simultaneously describe biological phosphorus removal, SMP formation/degradation and physical processes which also include the removal of organic matter. The model has been calibrated with data collected in a UCT-MBR pilot plant, located at the Palermo wastewater treatment plant, applying a modified version of a recently developed calibration protocol. The calibrated model provides acceptable correspondence with experimental data and can be considered a useful tool for MBR design and operation.     

Biomonitoring of primary aluminium industry workers: detection of micronuclei and repairable DNA lesions by alkaline SCGE

The genetic effects of occupational exposure to low polycyclic aromatic hydrocarbon (PAH) concentrations were investigated in primary aluminium industry workers. The study subjects were employed in a plant that uses pre-baked anode cells, and has relatively low PAH contamination. Forty-two male workers belonging to different job categories (anode fabrication, baking, rodding, electrolysis, maintenance), together with 16 male local residents with no occupational exposure to PAHs were selected for the analysis of micronuclei and DNA lesions in peripheral lymphocytes. The incidence of micronuclei determined in 1000 cytokinesis-blocked cells in each subject was not significantly different between workers and controls (8.5+/-5.4 per thousand versus 9.7+/-4.9 per thousand, respectively), nor between smokers and non-smokers (8.3+/-5.8 per thousand versus 9.2+/-5.1 per thousand), but was significantly (P<0.05) related to the subjects' age. Also the analysis of DNA damage in unstimulated and mitogen-stimulated lymphocytes by single cell gel electrophoresis (SCGE) did not show significant differences between the studied groups (average tail moment values were 0.53+/-0.53 and 0.49+/-0.45 microm in exposed subjects and controls, respectively). However, when lymphocytes were cultured in the presence of cytosine arabinoside (Ara-C, 1 microg/ml for 16h) the SCGE analysis revealed a significant (P=0.018) difference in tail moment values between aluminium workers and controls (1.73+/-1.05 microm versus 0.93+/-0.88 microm, respectively). This difference may highlight an excess of relatively stable DNA lesions, that do not affect strand integrity, and are expressed as intermediates of excision repair in stimulated cells, when gap refilling is inhibited by cytosine arabinoside (Ara-C).     

Protein/RNA coextraction and small two-dimensional polyacrylamide gel electrophoresis for proteomic/gene expression analysis of renal cancer biopsies

A small amount of bioptic tissue ( approximately 5-10mg of fresh tissue) usually does not contain enough material to extract protein and RNA separately, to obtain preparative two-dimensional polyacrylamide gel electrophoresis (2-DE), and to identify a large number of separated proteins by MS. We tested a method, on small renal cancer specimens, for the coextraction of protein and RNA coupled with 2-DE and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) or quadrupole time-of-flight (Q-TOF) analysis. We coextracted 0.28+/-0.05mg of proteins and 2.5+/-0.33microg of RNA for each 10mg of renal carcinoma tissue. Small and large 2-DE gels were compared: they showed a similar number of spots, and it was possible to match each other; using small format gels, one-fifth of the protein amount was required to identify, by Q-TOF analysis, the same number of proteins identifiable in large-format gel using MALDI-TOF analysis. Quality of RNA coextracted with the proteins was tested by real-time PCR on a set of housekeeping genes. They were quantified with high amplification efficiency and specificity. In conclusion, using 5 to 10mg of fresh tissue, it was possible to perform comprehensive parallel proteomic and genomic analysis by high-resolution, small-format 2-DE gels, allowing approximately 300 proteins identification and 1000 genes expression analysis.     

Plant regeneration from seeds responds to phylogenetic relatedness and local adaptation in Mediterranean Romulea (Iridaceae) species

Seed germination is the most important transitional event between early stages in the life cycle of spermatophytes and understanding it is crucial to understand plant adaptation and evolution. However, so far seed germination of phylogenetically closely related species has been poorly investigated. To test the hypothises that phylogenetically related plant species have similar seed ecophysiological traits thereby reflecting certain habitat conditions as a result of local adaptation , we studied seed dormancy and germination in seven Mediterranean species in the genus Romulea (Iridaceae). Both the across‐species model and the model accounting for shared evolutionary history showed that cool temperatures (≤ 15°C) were the main factor that promoted seed germination. The absence of embryo growth before radicle emergence is consistent with a prompt germination response at cool temperatures. The range of temperature conditions for germination became wider after a period of warm stratification, denoting a weak primary dormancy. Altogether these results indicate that the studied species exhibit a Mediterranean germination syndrome, but with species‐specific germination requirements clustered in a way that follows the phylogenetic relatedness among those species. In addition, species with heavier seeds from humid habitats showed a wider range of conditions for germination at dispersal time than species from dry habitats possessing lighter seeds. We conclude that while phylogenetically related species showed very similar germination requirements, there are subtle ecologically meaningful differences, confirming the onset of adaptation to local ecological factors mediated by species relatedness.     

Free energy of DNA duplex formation on short oligonucleotide microarrays

DNA/DNA duplex formation is the basic mechanism that is used in genome tiling arrays and SNP arrays manufactured by Affymetrix. However, detailed knowledge of the physical process is still lacking. In this study, we show a free energy analysis of DNA/DNA duplex formation these arrays based on the positional-dependent nearest-neighbor (PDNN) model, which was developed previously for describing DNA/RNA duplex formation on expression microarrays. Our results showed that the two ends of a probe contribute less to the stability of the duplexes and that there is a microarray surface effect on binding affinities. We also showed that free energy cost of a single mismatch depends on the bases adjacent to the mismatch site and obtained a comprehensive table of the cost of a single mismatch under all possible combination of adjacent bases. The mismatch costs were found to be correlated with those determined in aqueous solution. We further demonstrate that the DNA copy number estimated from the SNP array correlates negatively with the target length; this is presumably caused by inefficient PCR amplification for long fragments. These results provide important insights into the molecular mechanisms of microarray technology and have implications for microarray design and the interpretation of observed data.     

The rate of interleukin-1beta secretion in different myeloid cells varies with the extent of redox response to Toll-like receptor triggering

Human myeloid cells activate the NLRP3 inflammasome and secrete interleukin (IL)-1β in response to various Toll-like receptor (TLR) ligands, but the rate of secretion is much higher in primary human monocytes than in cultured macrophages or THP-1 cells. The different myeloid cells also display different redox status under resting conditions and redox response to TLR activation. Resting monocytes display a balanced redox state, with low production of reactive oxygen species (ROS) and antioxidants. TLR engagement induces an effective redox response with increased ROS generation followed by a sustained antioxidant response, parallelled by efficient IL-1β secretion. Drugs blocking ROS production or the antioxidant response prevent the secretion of mature IL-1β but not the biosynthesis of pro-IL-1β, indicating that redox remodeling is responsible for IL-1β processing and release. Unlike monocytes, THP-1 cells and cultured macrophages have up-regulated antioxidant systems that buffer the oxidative hit provided by TLR triggering and suppress the consequent redox response. This aborted redox remodeling is paralleled by low efficiency IL-1β processing and secretion. High doses (5 mM) of H(2)O(2) overcome the high antioxidant capacity of THP-1 cells, restore an efficient redox response, and increase the rate of IL-1β secretion. Together these data indicate that a tightly controlled redox homeostasis in resting cells is a prerequisite for a robust redox response to TLR ligands, in turn necessary for the efficient inflammasome activation. Inflammasome activation by bacterial DNA is not modulated by redox responses, suggesting that redox-dependent regulation of IL-1β secretion is restricted to some inflammasomes including NLRP3 but excluding AIM-2.     

Neurosteroids enhance spontaneous glutamate release in hippocampal neurons. Possible role of metabotropic sigma1-like receptors

Pregnenolone sulfate (PREGS), one of the most abundantly produced neurosteroids in the mammalian brain, improves cognitive performance in rodents. The mechanism of this effect has been attributed to its allosteric modulatory actions on glutamate- and gamma-aminobutyric acid-gated ion channels. Here we report a novel effect of PREGS that could also mediate some of its actions in the nervous system. We found that PREGS induces a robust potentiation of the frequency but not the amplitude of miniature excitatory postsynaptic currents (mEPSCs) mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors in cultured hippocampal neurons. PREGS also decreased paired pulse facilitation of autaptic EPSCs evoked by depolarization, indicating that it modulates glutamate release probability presynaptically. PREGS potentiation of mEPSCs was mimicked by dehydroepiandrosterone sulfate and (+)-pentazocine but not by (-)-pentazocine, the synthetic (-)-enantiomer of PREGS or the inactive steroid isopregnanolone. The sigma receptor antagonists, haloperidol and BD-1063, blocked the effect of PREGS on mEPSCs, as did pertussis toxin and the membrane-permeable Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl) ester. These results suggest that PREGS increases spontaneous glutamate release via activation of a presynaptic G(i/o)-coupled sigma receptor and an elevation in intracellular Ca2+ levels. We postulate that presynaptic actions of neurosteroids have a role in the maturation and/or maintenance of synaptic networks and the processing of information in the central nervous system.     

Asymmetric synthesis with immobilized yeast in organic solvents: equilibrium conversion and effect of reactant partitioning on whole cell biocatalysis

A newly isolated strain of the yeast Saccharomyces cerevisiae is investigated for the biocatalytic reduction of ketones and the oxidation of alcohols in organic solvents. The yeast cells are immobilized by entrapment within calcium alginate beads and are found to possess the ability to stereoselectively reduce prochiral ketones and oxidize chiral alcohols to equilibrium conversions. The effect of reactant partitioning on the initial rate of the reactions is also investigated. The observed initial rates are found to vary inversely with reactant partitioning between the organic solvent and the biocatalyst beads. A kinetic model is developed to describe the initial reaction rate of hexanone reduction as a function of substrate concentration within the alginate beads.