A versatile, automated and high-throughput drug screening platform for zebrafish embryos

Zebrafish provide a unique opportunity for drug screening in living animals, with the fast developing, transparent embryos allowing for relatively high-throughput, microscopy-based screens. However, the limited availability of rapid, flexible imaging and analysis platforms has limited the use of zebrafish in drug screens. We have developed an easy-to-use, customisable automated screening procedure suitable for high-throughput phenotype-based screens of live zebrafish. We utilised the WiScan® Hermes High Content Imaging System to rapidly acquire brightfield and fluorescent images of embryos, and the WiSoft® Athena Zebrafish Application for analysis, which harnesses an Artificial Intelligence-driven algorithm to automatically detect fish in brightfield images, identify anatomical structures, partition the animal into regions, and exclusively select the desired side-oriented fish. Our initial validation combined structural analysis with fluorescence images to enumerate GFP-tagged haematopoietic stem and progenitor cells in the tails of embryos, which correlated with manual counts. We further validated this system to assess the effects of genetic mutations and x-ray irradiation in high content using a wide range of assays. Further, we performed simultaneous analysis of multiple cell types using dual fluorophores in high throughput. In summary, we demonstrate a broadly applicable and rapidly customisable platform for high-content screening in zebrafish

Somatostatin treatment attenuates proteinuria and prevents weight loss in NZB/W F1 mice

Somatostatin, a naturally occurring neuropeptide, is an immunomodulator which inhibits humoral and cell mediated immunity as well as secretion of proinflammatory cytokines. The objective of this study was to examine the effects of a somatostatin analogue on the severity of glomerulonephritis in the female NZB/W F1 murine model of systemic lupus erythematosus (SLE). Twenty female NZB/W F1 mice were treated at 23 weeks of age with 10 mg/kg of the somatostatin analogue Sandostatin-LAR, IM every four weeks. Ten control mice received IM injection of vehicle. Mice were assessed at fourweek intervals for weight change, proteinuria, anti-DNA antibodies and splenocyte cytokine profile. The mice were sacrificed at age 34.5 weeks. Kidneys were collected and evaluated by light and immunofluorescence (IF) microscopy. Spleens were collected and splenocyte intracellular cytokines were measured by FACS analysis. In the treatment group significantly less proteinuria was observed four weeks after the second somatostatin analogue injection (dipstik scale: ϩ2.07 Ϯ 0.95 versus. ϩ3.5 Ϯ 1.08, P ϭ 0.0002). The treated mice did not lose weight while the control group lost weight over time (P ϭ 0.016). No differences were noted between the groups in anti-DNA antibody titres, cytokine profile or the severity of lupus nephritis as assessed by light and IF microscopy. Somatostatin analogue treatment attenuated proteinuria and prevented weight loss in NZB/W F1 mice, suggesting a possible beneficial effect on renal parameters and systemic manifestations of the disease. Further studies will be needed to assess the value of somatostatin analogue treatment in lupus nephritis, utilizing higher doses, at different stages of the disease, for longer periods. Lupus (2006) 15, 526-531

Co-ordinated regulation of flowering time, plant architecture and growth by FASCICULATE: the pepper orthologue of SELF PRUNING

Wild peppers (Capsicum spp.) are either annual or perennial in their native habitat and their shoot architecture is dictated by their sympodial growth habit. To study shoot architecture in pepper, sympodial development is described in wild type and in the classical recessive fasciculate (fa) mutation. The basic sympodial unit in wild-type pepper comprises two leaves and a single terminal flower. fasciculate plants are characterized by the formation of floral clusters separated by short internodes and miniature leaves and by early flowering. Developmental analysis of these clusters revealed shorter sympodial units and, often, precocious termination prior to sympodial leaf formation. fa was mapped to pepper chromosome 6, in a region corresponding to the tomato SELF-PRUNING (SP) locus, the homologue of TFL1 of Arabidopsis. Sequence comparison between wild-type and fa plants revealed a duplication of the second exon in the mutants' orthologue of SP, leading to the formation of a premature stop codon. Ectopic expression of FASCICULATE complemented the Arabidopsis tfl1 mutant plants and as expected, stimulated late flowering. In agreement with the major effect of FASCICULATE imposed on sympodial development, the gene transcripts were localized to the centre of sympodial shoots but could not be detected in the primary shoot. The wide range of pleiotropic effects on plant architecture mediated by a single ‘flowering’ gene, suggests that it is used to co-ordinate many developmental events, and thus may underlie some of the widespread variation in the Solanaceae shoot architecture

Successful medical management of emphysematous gastritis with concomitant portal venous air: a case report

<p>Abstract</p> <p>Introduction</p> <p>The causes of diffuse abdominal pain following pelvic surgery are numerous. We present a rare case of acute abdominal pain in a woman in the post-partum period.</p> <p>Case presentation</p> <p>A 25-year-old Caucasian woman with neurofibromatosis type 1 presented to our hospital with diffuse abdominal pain immediately after a cesarean section. The patient was acutely ill and toxic with a fever of 38.8°C, a pulse of 120 beats per minute and a distended abdomen with absent bowel sounds. A computed tomography scan showed air in the wall of the stomach and portal venous system. The patient was successfully treated with intravenous antibiotics, bowel rest and total parenteral nutrition.</p> <p>Conclusion</p> <p>It is rare for a case of emphysematous gastritis associated with portal venous air to be treated successfully without surgery. To the best of our knowledge, to date there has been no reported association of emphysematous gastritis with neurofibromatosis.</p

Harnessing anthocyanin-rich fruit: a visible reporter for tracing virus-induced gene silencing in pepper fruit

BACKGROUND: Virus-induced gene silencing (VIGS) has become a powerful tool for post-genomic technology in plant species. This is important, especially in select plants, such as the pepper plant, that are recalcitrant to Agrobacterium-mediated transformation. Although VIGS in plants has been widely employed as a powerful tool for functional genomics, scattering phenotypic effects by uneven gene silencing has been implemented in order to overcome challenges in experiments with fruit tissues. RESULTS: We improved the VIGS system based on the tobacco rattle virus (TRV) containing the An2 MYB transcription factor, which is the genetic determinant of purple colored- or anthocyanin-rich pepper. Silencing of endogenous An2 in the anthocyanin-rich pepper with the modified TRV vector for ligation-independent cloning (LIC) lacked purple pigment in its leaves, flowers, and fruits. Infection with TRV–LIC containing a tandem construct of An2 and phytoene desaturase (PDS) resulted in a typical photobleaching event in leaves without the purple pigment, whereas silencing of PDS led to the presence of photobleached and purple-colored leaves. Cosilencing of endogenous An2 and capsaicin synthase in fruits resulted in decreased levels of capsaicin and dihydrocapsaicin as assessed by high performance liquid chromatography analysis coupled with the absence of the purple pigment in fruits. CONCLUSIONS: VIGS with tandem constructs harboring An2 as a visible reporter in anthocyanin-rich pepper plants can facilitate the application of functional genomics in the study of metabolic pathways and fruit biology

Drosophila S2 Cells Are Non-Permissive for Vaccinia Virus DNA Replication Following Entry via Low pH-Dependent Endocytosis and Early Transcription

Vaccinia virus (VACV), a member of the chordopox subfamily of the Poxviridae, abortively infects insect cells. We have investigated VACV infection of Drosophila S2 cells, which are useful for protein expression and genome-wide RNAi screening. Biochemical and electron microscopic analyses indicated that VACV entry into Drosophila S2 cells depended on the VACV multiprotein entry-fusion complex but appeared to occur exclusively by a low pH-dependent endocytic mechanism, in contrast to both neutral and low pH entry pathways used in mammalian cells. Deep RNA sequencing revealed that the entire VACV early transcriptome, comprising 118 open reading frames, was robustly expressed but neither intermediate nor late mRNAs were made. Nor was viral late protein synthesis or inhibition of host protein synthesis detected by pulse-labeling with radioactive amino acids. Some reduction in viral early proteins was noted by Western blotting. Nevertheless, synthesis of the multitude of early proteins needed for intermediate gene expression was demonstrated by transfection of a plasmid containing a reporter gene regulated by an intermediate promoter. In addition, expression of a reporter gene with a late promoter was achieved by cotransfection of intermediate genes encoding the late transcription factors. The requirement for transfection of DNA templates for intermediate and late gene expression indicated a defect in viral genome replication in VACV-infected S2 cells, which was confirmed by direct analysis. Furthermore, VACV-infected S2 cells did not support the replication of a transfected plasmid, which occurs in mammalian cells and is dependent on all known viral replication proteins, indicating a primary restriction of DNA synthesis

Identification of Novel Pro-Migratory, Cancer-Associated Genes Using Quantitative, Microscopy-Based Screening

Background: Cell migration is a highly complex process, regulated by multiple genes, signaling pathways and external stimuli. To discover genes or pharmacological agents that can modulate the migratory activity of cells, screening strategies that enable the monitoring of diverse migratory parameters in a large number of samples are necessary. Methodology: In the present study, we describe the development of a quantitative, high-throughput cell migration assay, based on a modified phagokinetic tracks (PKT) procedure, and apply it for identifying novel pro-migratory genes in a cancer-related gene library. In brief, cells are seeded on fibronectin-coated 96-well plates, covered with a monolayer of carboxylated latex beads. Motile cells clear the beads, located along their migratory paths, forming tracks that are visualized using an automated, transmitted-light screening microscope. The tracks are then segmented and characterized by multi-parametric, morphometric analysis, resolving a variety of morphological and kinetic features. Conclusions: In this screen we identified 4 novel genes derived from breast carcinoma related cDNA library, whose over-expression induces major alteration in the migration of the stationary MCF7 cells. This approach can serve for high throughput screening for novel ways to modulate cellular migration in pathological states such as tumor metastasis and invasion

Identification of the Er1 resistence gene and RNase S-alleles in Malus prunifolia var. ringo rootstock

Woolly apple aphid (WAA; Eriosoma lanigerum Hausm.) is a major insect pest that has significant economic impact on apple growers worldwide. Modern breeding technologies rely on several molecular tools to help breeders select genetic determinants for traits of interest. Consequently, there is a need for specific markers linked to the genes of interest. Apple scions and rootstocks have an additional barrier to the introduction of pest resistance genes due to the presence of self-incompatibility S-RNase alleles. The genetic characterization and early identification of these alleles can amplify the contribution of a breeding program to the selection of resistant genitors that are as compatible as possible. In this study, we identified the Er1 gene involved in the resistance to WAA in Malus prunifolia var. ringo, also known as ‘Maruba Kaido’ rootstock, and we analyzed the inheritance pattern of the WAA resistance Er1 gene in a segregant population derived from Malus pumila ‘M.9’ and ‘Maruba Kaido’ rootstocks. The self-incompatibility of S-RNase alleles S6S26 of ‘Maruba Kaido’ were also identified along with their inheritance pattern. We also confirmed the identification of the S1S3 alleles in the ‘M.9’ rootstock. To the best of our knowledge, this is the first study to characterize WAA resistance and RNase S-alleles in ‘Maruba Kaido’. Furthermore, we discuss the potential use of the genetic markers for these genes and their potential impact on apple breeding programs

The construction of a Solanum habrochaites LYC4 introgression line population and the identification of QTLs for resistance to Botrytis cinerea

Tomato (Solanum lycopersicum) is susceptible to grey mold (Botrytis cinerea). Partial resistance to this fungus has been identified in accessions of wild relatives of tomato such as Solanum habrochaites LYC4. In a previous F2 mapping study, three QTLs conferring resistance to B. cinerea (Rbcq1, Rbcq2 and Rbcq4a) were identified. As it was probable that this study had not identified all QTLs involved in resistance we developed an introgression line (IL) population (n = 30), each containing a S. habrochaites introgression in the S. lycopersicum cv. Moneymaker genetic background. On average each IL contained 5.2% of the S. habrochaites genome and together the lines provide an estimated coverage of 95%. The level of susceptibility to B. cinerea for each of the ILs was assessed in a greenhouse trial and compared to the susceptible parent S. lycopersicum cv. Moneymaker. The effect of the three previously identified loci could be confirmed and seven additional loci were detected. Some ILs contains multiple QTLs and the increased resistance to B. cinerea in these ILs is in line with a completely additive model. We conclude that this set of QTLs offers good perspectives for breeding of B. cinerea resistant cultivars and that screening an IL population is more sensitive for detection of QTLs conferring resistance to B. cinerea than the analysis in an F2 population