Population dynamics of stable flies Stomoxys calcitrans (Diptera: Muscidae) at an organic dairy farm in Denmark based on mark-recapture with destructive sub-sampling

A population of stable flies, Stomoxys calcitrans (L.), was studied on a Danish cattle farm in two successive years. Flies were captured monthly by sweep nettings and marked with fluorescent dust. Absolute population size, dilution rate, loss rate, and adult longevity were estimated by means of a modified version of Bailey's triple catch method. In both years, the abundance of flies peaked in July. Using a statistical model, we were able to explain 86.6% of the variation in the per capita growth rate r as a function of current temperature, precipitation, and population size. Omitting precipitation from the model, it still explained 69.3%. The model predicts that stable flies have a temperature optimum at 21.8°C, and that no development will take place when temperatures inside the stable are below 10.2°C or above 33.5°C. At the optimal temperature the intrinsic rate of natural increase is 0.070 d(-1). The per capita dilution rate increased with temperature and decreased with population size, whereas no effect of these factors on the per capita loss rate could be shown. Mean adult survival time was estimated to 6.3 d with 95% CL ranging from 4.3 to 11.1 d. The study points at the possibility of developing predictive models as tools for achieving better, and more environmentally sound, control of stable flies.     

Monitoring moderate Cu and Cd toxicity by chlorophyll fluorescence and P700 absorbance in pea leaves

We investigated the effect of moderate Cu²⁺ and Cd²⁺ stress by applying chlorophyll (Chl) fluorescence and P₇₀₀ absorbance measurements to monitor the photosynthetic electron transport activity of 3-week-old Pisum sativum L. cv. Petit Proven?al plants grown in a modified Hoagland solution containing 50 ??M CuSO₄ or 5 ??M CdCl₂. Both heavy metals caused a slight inhibition in PSII photochemistry as indicated by the decrease in the effective quantum efficiency of PSII (??_PSII), the maximum electron transport capacity (ETR_max), and the maximum quantum yield for electron transport (??). PSI photochemistry was also affected by these heavy metals. Cu²⁺ and Cd²⁺ decreased the quantum efficiency of PSI (??_PSI) as well as the number of electrons in the intersystem chain, and the Cu²⁺ treatment significantly reduced the number of electrons from stromal donors available for PSI. These results indicate that PSII and PSI photochemistry of pea plants are both sensitive to moderate Cu²⁺ and Cd²⁺ stress, which in turn is easily detected and monitored by Chl fluorescence and P₇₀₀ absorbance measurements. Therefore, monitoring the photochemistry of pea plants with these noninvasive, yet sensitive techniques offers a promising strategy to study heavy metal toxicity in the environment.     

Chemokines regulate small leucine-rich proteoglycans in the extracellular matrix of the pressure-overloaded right ventricle

Chemokines have been suggested to play a role during development of left ventricular failure, but little is known about their role during right ventricular (RV) remodeling and dysfunction. We have previously shown that the chemokine (C-X-C motif) ligand 13 (CXCL13) regulates small leucine-rich proteoglycans (SLRPs). We hypothesized that chemokines are upregulated in the pressure-overloaded RV, and that they regulate SLRPs. Mice with RV pressure overload following pulmonary banding (PB) had a significant increase in RV weight and an increase in liver weight after 1 wk. Microarray analysis (Affymetrix) of RV tissue from mice with PB revealed that CXCL10, CXCL6, chemokine (C-X3-C motif) ligand 1 (CX3CL1), chemokine (C-C motif) ligand 5 (CCL5), CXCL16, and CCL2 were the most upregulated chemokines. Stimulation of cardiac fibroblasts with these same chemokines showed that CXCL16 increased the expression of the four SLRPs: decorin, lumican, biglycan, and fibromodulin. CCL5 increased the same SLRPs, except decorin, whereas CX3CL1 increased the expression of decorin and lumican. CXCL16, CX3CL1, and CCL5 were also shown to increase the levels of glycosylated decorin and lumican in the medium after stimulation of fibroblasts. In the pressure-overloaded RV tissue, Western blotting revealed an increase in the total protein level of lumican and a glycosylated form of decorin with a higher molecular weight compared with control mice. Both mice with PB and patients with pulmonary stenosis had significantly increased circulating levels of CXCL16 compared with healthy controls measured by enzyme immunoassay. In conclusion, we have found that chemokines are upregulated in the pressure-overloaded RV and that CXCL16, CX3CL1, and CCL5 regulate expression and posttranslational modifications of SLRPs in cardiac fibroblasts. In the pressure-overloaded RV, protein levels of lumican were increased, and a glycosylated form of decorin with a high molecular weight appeared.     

Regulatory processes of hunger motivated behavior

While food intake and body weight are under homeostatic regulation, eating is a highly motivated and reinforced behavior that induces feelings of gratification and pleasure. The chemical senses (taste and odor) and their evaluation are essential to these functions. Brainstem and limbic glucose-monitoring (GM) neurons receiving neurochemical information from the periphery and from the local brain milieu are important controlling hunger motivation, and brain gut peptides have a modulatory role on this function. The hypothalamic and limbic forebrain areas are responsible for evaluation of reward quality and related emotions. They are innervated by the mesolimbic dopaminergic system (MLDS) and majority of GM neurons are also influenced by dopamine. Via dopamine release, the MLDS plays an essential role in rewarding-reinforcing processes of feeding and addiction. The GM network and the MLDS in the limbic system represent essential elements in the neural substrate of motivation.     

Plasmid vectors for proteomic analyses in Giardia: purification of virulence factors and analysis of the proteasome

In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide-glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG-tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes.     

��-Microseminoprotein Endows Post Coital Seminal Plasma with Potent Candidacidal Activity by a Calcium- and pH-Dependent Mechanism

The innate immune factors controlling Candida albicans are mostly unknown. Vulvovaginal candidiasis is common in women and affects approximately 70–75% of all women at least once. Despite the propensity of Candida to colonize the vagina, transmission of Candida albicans following sexual intercourse is very rare. This prompted us to investigate whether the post coital vaginal milieu contained factors active against C. albicans. By CFU assays, we found prominent candidacidal activity of post coital seminal plasma at both neutral and the acid vaginal pH. In contrast, normal seminal plasma did not display candidacidal activity prior to acidification. By antifungal gel overlay assay, one clearing zone corresponding to a protein band was found in both post coital and normal seminal plasma, which was subsequently identified as β-microseminoprotein. At neutral pH, the fungicidal activity of β-microseminoprotein and seminal plasma was inhibited by calcium. By NMR spectroscopy, amino acid residue E₇₁ was shown to be critical for the calcium coordination. The acidic vaginal milieu unleashed the fungicidal activity by decreasing the inhibitory effect of calcium. The candidacidal activity of β-microseminoprotein was mapped to a fragment of the C-terminal domain with no structural similarity to other known proteins. A homologous fragment from porcine β-microseminoprotein demonstrated calcium-dependent fungicidal activity in a CFU assay, suggesting this may be a common feature for members of the β-microseminoprotein family. By electron microscopy, β-microseminoprotein was found to cause lysis of Candida. Liposome experiments demonstrated that β-microseminoprotein was active towards ergosterol-containing liposomes that mimic fungal membranes, offering an explanation for the selectivity against fungi. These data identify β-microseminoprotein as an important innate immune factor active against C. albicans and may help explain the low sexual transmission rate of Candida.     

Language Lateralization in Children Aged 10 to 11 Years: A Combined fMRI and Dichotic Listening Study

Objective

The aims of this study were to develop and assess a method to map language networks in children with two auditory fMRI protocols in combination with a dichotic listening task (DL). The method is intended for pediatric patients prior to epilepsy surgery. To evaluate the potential clinical usefulness of the method we first wanted to assess data from a group of healthy children.

Methods

In a first step language test materials were developed, intended for subsequent implementation in fMRI protocols. An evaluation of this material was done in 30 children with typical development, 10 from the 1^st, 4^th and the 7^th grade, respectively. The language test material was then adapted and implemented in two fMRI protocols intended to target frontal and posterior language networks. In a second step language lateralization was assessed in 17 typical 10–11 year olds with fMRI and DL. To reach a conclusion about language lateralization, firstly, quantitative analyses of the index data from the two fMRI tasks and the index data from the DL task were done separately. In a second step a set of criteria were applied to these results to reach a conclusion about language lateralization. The steps of these analyses are described in detail.

Results

The behavioral assessment of the language test material showed that it was well suited for typical children. The results of the language lateralization assessments, based on fMRI data and DL data, showed that for 15 of the 17 subjects (88%) a conclusion could be reached about hemispheric language dominance. In 2 cases (12%) DL provided critical data.

Conclusions

The employment of DL combined with language mapping using fMRI for assessing hemispheric language dominance is novel and it was deemed valuable since it provided additional information compared to the results gained from each method individually.     

Detection of Giardia duodenalis Assemblages A and B in Human Feces by Simple, Assemblage-Specific PCR Assays

The flagellated protozoan Giardia duodenalis is a common gastrointestinal parasite of mammals, including humans. Molecular characterizations have shown the existence of eight genetic groups (or assemblages) in the G. duodenalis species complex. Human infections are caused by assemblages A and B, which infect other mammals as well. Whether transmission routes, animal reservoirs and associations with specific symptoms differ for assemblage A and assemblage B is not clear. Furthermore, the occurrence and clinical significance of mixed (A+B) infections is also poorly understood. To date, the majority of PCR assays has been developed to identify all G. duodenalis assemblages based on the use of primers that bind to conserved regions, yet a reliable identification of specific assemblages is better achieved by ad hoc methods. The aim of this work was to design simple PCR assays that, based on the use of assemblage-specific primers, produce diagnostic bands of different lengths for assemblage A and B. We first generated novel sequence information from assemblage B, identified homologous sequences in the assemblage A genome, and designed primers at six independent loci. Experiments performed on DNA extracted from axenic cultures showed that two of the six assays can detect the equivalent of a single cyst and are not negatively influenced by disproportions between DNA of each assemblage, at least up to a 9∶1 ratio. Further experiments on DNAs extracted from feces showed that the two assays can detect both assemblages in single tube reactions with excellent reliability. Finally, the robustness of these assays was demonstrated by testing a large collection of human isolates previously typed by multi-locus genotyping.     

Metabolic engineering of Saccharomyces cerevisiae for bioconversion of D-xylose to D-xylonate

An NAD(+)-dependent D-xylose dehydrogenase, XylB, from Caulobacter crescentus was expressed in Saccharomyces cerevisiae, resulting in production of 17 ± 2 g D-xylonate l(-1) at 0.23 gl(-1)h(-1) from 23 g D-xylose l(-1) (with glucose and ethanol as co-substrates). D-Xylonate titre and production rate were increased and xylitol production decreased, compared to strains expressing genes encoding T. reesei or pig liver NADP(+)-dependent D-xylose dehydrogenases. D-Xylonate accumulated intracellularly to ～70 mgg(-1); xylitol to ～18 mgg(-1). The aldose reductase encoding gene GRE3 was deleted to reduce xylitol production. Cells expressing D-xylonolactone lactonase xylC from C. crescentus with xylB initially produced more extracellular D-xylonate than cells lacking xylC at both pH 5.5 and pH 3, and sustained higher production at pH 3. Cell vitality and viability decreased during D-xylonate production at pH 3.0. An industrial S. cerevisiae strain expressing xylB efficiently produced 43 g D-xylonate l(-1) from 49 g D-xylose l(-1).     

MicroRNAs in Amoebozoa: Deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs

The RNA interference machinery has served as a guardian of eukaryotic genomes since the divergence from prokaryotes. Although the basic components have a shared origin, silencing pathways directed by small RNAs have evolved in diverse directions in different eukaryotic lineages. Micro (mi)RNAs regulate protein-coding genes and play vital roles in plants and animals, but less is known about their functions in other organisms. Here, we report, for the first time, deep sequencing of small RNAs from the social amoeba Dictyostelium discoideum. RNA from growing single-cell amoebae as well as from two multicellular developmental stages was sequenced. Computational analyses combined with experimental data reveal the expression of miRNAs, several of them exhibiting distinct expression patterns during development. To our knowledge, this is the first report of miRNAs in the Amoebozoa supergroup. We also show that overexpressed miRNA precursors generate miRNAs and, in most cases, miRNA* sequences, whose biogenesis is dependent on the Dicer-like protein DrnB, further supporting the presence of miRNAs in D. discoideum. In addition, we find miRNAs processed from hairpin structures originating from an intron as well as from a class of repetitive elements. We believe that these repetitive elements are sources for newly evolved miRNAs.