Gene silencing in tick cell lines using small interfering or long double-stranded RNA

Gene silencing by RNA interference (RNAi) is an important research tool in many areas of biology. To effectively harness the power of this technique in order to explore tick functional genomics and tick-microorganism interactions, optimised parameters for RNAi-mediated gene silencing in tick cells need to be established. Ten cell lines from four economically important ixodid tick genera (Amblyomma, Hyalomma, Ixodes and Rhipicephalus including the sub-species Boophilus) were used to examine key parameters including small interfering RNA (siRNA), double stranded RNA (dsRNA), transfection reagent and incubation time for silencing virus reporter and endogenous tick genes. Transfection reagents were essential for the uptake of siRNA whereas long dsRNA alone was taken up by most tick cell lines. Significant virus reporter protein knockdown was achieved using either siRNA or dsRNA in all the cell lines tested. Optimum conditions varied according to the cell line. Consistency between replicates and duration of incubation with dsRNA were addressed for two Ixodes scapularis cell lines; IDE8 supported more consistent and effective silencing of the endogenous gene subolesin than ISE6, and highly significant knockdown of the endogenous gene 2I1F6 in IDE8 cells was achieved within 48 h incubation with dsRNA. In summary, this study shows that gene silencing by RNAi in tick cell lines is generally more efficient with dsRNA than with siRNA but results vary between cell lines and optimal parameters need to be determined for each experimental system

Three-Dimensional Characterization of the Vascular Bed in Bone Metastasis of the Rat by Microcomputed Tomography (MicroCT)

BackgroundAngiogenesis contributes to proliferation and metastatic dissemination of cancer cells. Anatomy of blood vessels in tumors has been characterized with 2D techniques (histology or angiography). They are not fully representative of the trajectories of vessels throughout the tissues and are not adapted to analyze changes occurring inside the bone marrow cavities. Methodology/Principal Findings We have characterized the vasculature of bone metastases in 3D at different times of evolution of the disease. Metastases were induced in the femur of Wistar rats by a local injection of Walker 256/B cells. Microfil®, (a silicone-based polymer) was injected at euthanasia in the aorta 12, 19 and 26 days after injection of tumor cells. Undecalcified bones (containing the radio opaque vascular casts) were analyzed by microCT, and a first 3D model was reconstructed. Bones were then decalcified and reanalyzed by microCT; a second model (comprising only the vessels) was obtained and overimposed on the former, thus providing a clear visualization of vessel trajectories in the invaded metaphysic allowing quantitative evaluation of the vascular volume and vessel diameter. Histological analysis of the marrow was possible on the decalcified specimens. Walker 256/B cells induced a marked osteolysis with cortical perforations. The metaphysis of invaded bones became progressively hypervascular. New vessels replaced the major central medullar artery coming from the diaphyseal shaft. They sprouted from the periosteum and extended into the metastatic area. The newly formed vessels were irregular in diameter, tortuous with a disorganized architecture. A quantitative analysis of vascular volume indicated that neoangiogenesis increased with the development of the tumor with the appearance of vessels with a larger diameter. Conclusion This new method evidenced the tumor angiogenesis in 3D at different development times of the metastasis growth. Bone and the vascular bed can be identified by a double reconstruction and allowed a quantitative evaluation of angiogenesis upon time

Silencing of genes involved in Anaplasma marginale-tick interactions affects the pathogen developmental cycle in Dermacentor variabilis

<p>Abstract</p> <p>Background</p> <p>The cattle pathogen, <it>Anaplasma marginale</it>, undergoes a developmental cycle in ticks that begins in gut cells. Transmission to cattle occurs from salivary glands during a second tick feeding. At each site of development two forms of <it>A. marginale </it>(reticulated and dense) occur within a parasitophorous vacuole in the host cell cytoplasm. However, the role of tick genes in pathogen development is unknown. Four genes, found in previous studies to be differentially expressed in <it>Dermacentor variabilis </it>ticks in response to infection with <it>A. marginale</it>, were silenced by RNA interference (RNAi) to determine the effect of silencing on the <it>A. marginale </it>developmental cycle. These four genes encoded for putative glutathione S-transferase (GST), salivary selenoprotein M (SelM), H+ transporting lysosomal vacuolar proton pump (vATPase) and subolesin.</p> <p>Results</p> <p>The impact of gene knockdown on <it>A. marginale </it>tick infections, both after acquiring infection and after a second transmission feeding, was determined and studied by light microscopy. Silencing of these genes had a different impact on <it>A. marginale </it>development in different tick tissues by affecting infection levels, the densities of colonies containing reticulated or dense forms and tissue morphology. Salivary gland infections were not seen in any of the gene-silenced ticks, raising the question of whether these ticks were able to transmit the pathogen.</p> <p>Conclusion</p> <p>The results of this RNAi and light microscopic analyses of tick tissues infected with <it>A. marginale </it>after the silencing of genes functionally important for pathogen development suggest a role for these molecules during pathogen life cycle in ticks.</p

Transmission Electron Microscopy Reveals Distinct Macrophage- and Tick Cell-Specific Morphological Stages of Ehrlichia chaffeensis

Background: Ehrlichia chaffeensis is an emerging tick-borne rickettsial pathogen responsible for human monocytic ehrlichiosis. Despite the induction of an active host immune response, the pathogen has evolved to persist in its vertebrate and tick hosts. Understanding how the organism progresses in tick and vertebrate host cells is critical in identifying effective strategies to block the pathogen transmission. Our recent molecular and proteomic studies revealed differences in numerous expressed proteins of the organism during its growth in different host environments. Methodology/Principal Findings: Transmission electron microscopy analysis was performed to assess morphological changes in the bacterium within macrophages and tick cells. The stages of pathogen progression observed included the attachment of the organism to the host cells, its engulfment and replication within a morulae by binary fission and release of the organisms from infected host cells by complete host cell lysis or by exocytosis. E. chaffeensis grown in tick cells was highly pleomorphic and appears to replicate by both binary fission and filamentous type cell divisions. The presence of Ehrlichia-like inclusions was also observed within the nucleus of both macrophages and tick cells. This observation was confirmed by confocal microscopy and immunoblot analysis. Conclusions/Significance: Morphological differences in the pathogen’s progression, replication, and processing within macrophages and tick cells provide further evidence that E. chaffeensis employs unique host-cell specific strategies in support of adaptation to vertebrate and tick cell environments

The glial growth factors deficiency and synaptic destabilization hypothesis of schizophrenia

BACKGROUND: A systems approach to understanding the etiology of schizophrenia requires a theory which is able to integrate genetic as well as neurodevelopmental factors. PRESENTATION OF THE HYPOTHESIS: Based on a co-localization of loci approach and a large amount of circumstantial evidence, we here propose that a functional deficiency of glial growth factors and of growth factors produced by glial cells are among the distal causes in the genotype-to-phenotype chain leading to the development of schizophrenia. These factors include neuregulin, insulin-like growth factor I, insulin, epidermal growth factor, neurotrophic growth factors, erbB receptors, phosphatidylinositol-3 kinase, growth arrest specific genes, neuritin, tumor necrosis factor alpha, glutamate, NMDA and cholinergic receptors. A genetically and epigenetically determined low baseline of glial growth factor signaling and synaptic strength is expected to increase the vulnerability for additional reductions (e.g., by viruses such as HHV-6 and JC virus infecting glial cells). This should lead to a weakening of the positive feedback loop between the presynaptic neuron and its targets, and below a certain threshold to synaptic destabilization and schizophrenia. TESTING THE HYPOTHESIS: Supported by informed conjectures and empirical facts, the hypothesis makes an attractive case for a large number of further investigations. IMPLICATIONS OF THE HYPOTHESIS: The hypothesis suggests glial cells as the locus of the genes-environment interactions in schizophrenia, with glial asthenia as an important factor for the genetic liability to the disorder, and an increase of prolactin and/or insulin as possible working mechanisms of traditional and atypical neuroleptic treatments

Increased Sensory Processing Atypicalities in Parents of Multiplex ASD Families Versus Typically Developing and Simplex ASD Families

Recent studies have suggested that sensory processing atypicalities may share genetic influences with autism spectrum disorder (ASD). To further investigate this, the adolescent/adult sensory profile (AASP) questionnaire was distributed to 85 parents of typically developing children (P-TD), 121 parents from simplex ASD families (SPX), and 54 parents from multiplex ASD families (MPX). After controlling for gender and presence of mental disorders, results showed that MPX parents significantly differed from P-TD parents in all four subscales of the AASP. Differences between SPX and MPX parents reached significance in the Sensory Sensitivity subscale and also in subsequent modality-specific analyses in the auditory and visual domains. Our finding that parents with high genetic liability for ASD (i.e., MPX) had more sensory processing atypicalities than parents with low (i.e., SPX) or no (i.e., P-TD) ASD genetic liability suggests that sensory processing atypicalities may contribute to the genetic susceptibility for ASD