Construction, electroporatic transfer and expression of ZpβypGH and ZpβrtGH in zebrafish

Recombinant transformation vectors (ZPβypGH and ZpβrtGH) consisting of fish growth hormone cDNA, and a reporter gene β-galactosidase driven by fish promoter (Zp) were constructed. Freshly fertilized eggs of zebrafish (Brachydanio rerio) were electroporated at optimum conditions (0.07 kV voltage; 25 μF capacitance; ∞ ohm resistance and 2 pulses) in the presence of one of these transformation vectors (100 μg circular DNA/ml). In either cases 72% of the electroporated eggs successfully hatched, in comparison to the 85% hatchability of the control eggs. Genomic DNA extracted from fins of randomly chosen F0 individuals was screened (by Southern blot hybridization); the transgenes were retained in the host genome of all the randomly chosen adult transformants. Fin-positive presumptive founder parents were crossed with control counterparts and the DNA of randomly chosen F1 progenies was screened for germline transformation. Southern analysis of chosen F1 progenies revealed the persistence of ZPβypGH or ZpβrtGH in 53% of the F1 progenies. Southern analyses of chosen F1 progenies and the frequency (53% of F1 ZpβrtGH and 53% of F1 ZP{β}ypGH) of transmission revealed the degree of mosaicism in F0 transformants. Expression was confirmed from the 3-4 times elevated levels of activity of the reporter gene and 30-40% accelerated growth of transgenic F0 and F1 progenies

Identification of inhibitors of the Schistosoma mansoni VKR2 kinase domain

Schistosomiasis is a neglected tropical disease caused by parasitic flatworms. Current treatment relies on just one partially effective drug, praziquantel (PZQ). Schistosoma mansoni Venus Kinase Receptors 1 and 2 (SmVKR1 and SmVKR2) are important for parasite growth and egg production, and are potential targets for combating schistosomiasis. VKRs consist of an extracellular Venus Flytrap Module (VFTM) linked via a transmembrane helix to a kinase domain. Here, we initiated a drug discovery effort to inhibit the activity of the SmVKR2 kinase domain (SmVKR2KD) by screening the GSK published kinase inhibitor set 2 (PKIS2). We identified several inhibitors, of which four were able to inhibit its enzymatic activity and induced phenotypic changes in ex vivoS. mansoni. Our crystal structure of the SmVKR2KD displays an active-like state that sheds light on the activation process of VKRs. Our data provide a basis for the further exploration of SmVKR2 as a possible drug target

Transcriptome Analysis of Zebrafish Embryogenesis Using Microarrays

Zebrafish (Danio rerio) is a well-recognized model for the study of vertebrate developmental genetics, yet at the same time little is known about the transcriptional events that underlie zebrafish embryogenesis. Here we have employed microarray analysis to study the temporal activity of developmentally regulated genes during zebrafish embryogenesis. Transcriptome analysis at 12 different embryonic time points covering five different developmental stages (maternal, blastula, gastrula, segmentation, and pharyngula) revealed a highly dynamic transcriptional profile. Hierarchical clustering, stage-specific clustering, and algorithms to detect onset and peak of gene expression revealed clearly demarcated transcript clusters with maximum gene activity at distinct developmental stages as well as co-regulated expression of gene groups involved in dedicated functions such as organogenesis. Our study also revealed a previously unidentified cohort of genes that are transcribed prior to the mid-blastula transition, a time point earlier than when the zygotic genome was traditionally thought to become active. Here we provide, for the first time to our knowledge, a comprehensive list of developmentally regulated zebrafish genes and their expression profiles during embryogenesis, including novel information on the temporal expression of several thousand previously uncharacterized genes. The expression data generated from this study are accessible to all interested scientists from our institute resource database (http://giscompute.gis.a-star.edu.sg/~govind/zebrafish/data_download.html)

A gas cell for stopping, storing and polarizing radioactive particles

A radioactive beam of 20Na is stopped in a gas cell filled with Ne gas. The stopped particles are polarized by optical pumping. The degree of polarization that can be achieved is studied. A maximum polarization of 50% was found. The dynamic processes in the cell are described with a phenomenological model.Comment: 16 pages, 6 figure

Highly Asynchronous and Asymmetric Cleavage Divisions Accompany Early Transcriptional Activity in Pre-Blastula Medaka Embryos

In the initial phase of development of fish embryos, a prominent and critical event is the midblastula transition (MBT). Before MBT cell cycle is rapid, highly synchronous and zygotic gene transcription is turned off. Only during MBT the cell cycle desynchronizes and transcription is activated. Multiple mechanisms, primarily the nucleocytoplasmic ratio, are supposed to control MBT activation. Unexpectedly, we find in the small teleost fish medaka (Oryzias latipes) that at very early stages, well before midblastula, cell division becomes asynchronous and cell volumes diverge. Furthermore, zygotic transcription is extensively activated already after the 64-cell stage. Thus, at least in medaka, the transition from maternal to zygotic transcription is uncoupled from the midblastula stage and not solely controlled by the nucleocytoplasmic ratio

Gene expression throughout a vertebrate's embryogenesis

Abstract Background Describing the patterns of gene expression during embryonic development has broadened our understanding of the processes and patterns that define morphogenesis. Yet gene expression patterns have not been described throughout vertebrate embryogenesis. This study presents statistical analyses of gene expression during all 40 developmental stages in the teleost Fundulus heteroclitus using four biological replicates per stage. Results Patterns of gene expression for 7,000 genes appear to be important as they recapitulate developmental timing. Among the 45% of genes with significant expression differences between pairs of temporally adjacent stages, significant differences in gene expression vary from as few as five to more than 660. Five adjacent stages have disproportionately more significant changes in gene expression (> 200 genes) relative to other stages: four to eight and eight to sixteen cell stages, onset of circulation, pre and post-hatch, and during complete yolk absorption. The fewest differences among adjacent stages occur during gastrulation. Yet, at stage 16, (pre-mid-gastrulation) the largest number of genes has peak expression. This stage has an over representation of genes in oxidative respiration and protein expression (ribosomes, translational genes and proteases). Unexpectedly, among all ribosomal genes, both strong positive and negative correlations occur. Similar correlated patterns of expression occur among all significant genes. Conclusions These data provide statistical support for the temporal dynamics of developmental gene expression during all stages of vertebrate development

Tiling Histone H3 Lysine 4 and 27 Methylation in Zebrafish Using High-Density Microarrays

BACKGROUND: Uncovering epigenetic states by chromatin immunoprecipitation and microarray hybridization (ChIP-chip) has significantly contributed to the understanding of gene regulation at the genome-scale level. Many studies have been carried out in mice and humans; however limited high-resolution information exists to date for non-mammalian vertebrate species. PRINCIPAL FINDINGS: We report a 2.1-million feature high-resolution Nimblegen tiling microarray for ChIP-chip interrogations of epigenetic states in zebrafish (Danio rerio). The array covers 251 megabases of the genome at 92 base-pair resolution. It includes ∼15 kb of upstream regulatory sequences encompassing all RefSeq promoters, and over 5 kb in the 5' end of coding regions. We identify with high reproducibility, in a fibroblast cell line, promoters enriched in H3K4me3, H3K27me3 or co-enriched in both modifications. ChIP-qPCR and sequential ChIP experiments validate the ChIP-chip data and support the co-enrichment of trimethylated H3K4 and H3K27 on a subset of genes. H3K4me3- and/or H3K27me3-enriched genes are associated with distinct transcriptional status and are linked to distinct functional categories. CONCLUSIONS: We have designed and validated for the scientific community a comprehensive high-resolution tiling microarray for investigations of epigenetic states in zebrafish, a widely used developmental and disease model organism

Integrated annotation and analysis of genomic features reveal new types of functional elements and large-scale epigenetic phenomena in the developing zebrafish

Zebrafish, a popular model for embryonic development and for modelling human diseases, has so far lacked a systematic functional annotation programme akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created the first central repository to store and process zebrafish developmental functional genomic data. Our Data Coordination Center (https://danio-code.zfin.org) combines a total of 1,802 sets of unpublished and reanalysed published genomics data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements in development, including novel classes with distinct features dependent on their activity in time and space. We delineated the distinction between regulatory elements active during zygotic genome activation and those active during organogenesis, identifying new aspects of how they relate to each other. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predict functional relationships between them beyond sequence similarity, extending the utility of zebrafish developmental genomics to mammals

Automatic Detection of User Abilities through the SmartAbility Framework

This paper presents a proposed smartphone application for the unique SmartAbility Framework that supports interaction with technology for people with reduced physical ability, through focusing on the actions that they can perform independently. The Framework is a culmination of knowledge obtained through previously conducted technology feasibility trials and controlled usability evaluations involving the user community. The Framework is an example of ability-based design that focuses on the abilities of users instead of their disabilities. The paper includes a summary of Versions 1 and 2 of the Framework, including the results of a two-phased validation approach, conducted at the UK Mobility Roadshow and via a focus group of domain experts. A holistic model developed by adapting the House of Quality (HoQ) matrix of the Quality Function Deployment (QFD) approach is also described. A systematic literature review of sensor technologies built into smart devices establishes the capabilities of sensors in the Android and iOS operating systems. The review defines a set of inclusion and exclusion criteria, as well as search terms used to elicit literature from online repositories. The key contribution is the mapping of ability-based sensor technologies onto the Framework, to enable the future implementation of a smartphone application. Through the exploitation of the SmartAbility application, the Framework will increase technology amongst people with reduced physical ability and provide a promotional tool for assistive technology manufacturers