48 research outputs found
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ACME: Automated Cell Morphology Extractor for Comprehensive Reconstruction of Cell Membranes
The quantification of cell shape, cell migration, and cell rearrangements is important for addressing classical questions in developmental biology such as patterning and tissue morphogenesis. Time-lapse microscopic imaging of transgenic embryos expressing fluorescent reporters is the method of choice for tracking morphogenetic changes and establishing cell lineages and fate maps in vivo. However, the manual steps involved in curating thousands of putative cell segmentations have been a major bottleneck in the application of these technologies especially for cell membranes. Segmentation of cell membranes while more difficult than nuclear segmentation is necessary for quantifying the relations between changes in cell morphology and morphogenesis. We present a novel and fully automated method to first reconstruct membrane signals and then segment out cells from 3D membrane images even in dense tissues. The approach has three stages: 1) detection of local membrane planes, 2) voting to fill structural gaps, and 3) region segmentation. We demonstrate the superior performance of the algorithms quantitatively on time-lapse confocal and two-photon images of zebrafish neuroectoderm and paraxial mesoderm by comparing its results with those derived from human inspection. We also compared with synthetic microscopic images generated by simulating the process of imaging with fluorescent reporters under varying conditions of noise. Both the over-segmentation and under-segmentation percentages of our method are around 5%. The volume overlap of individual cells, compared to expert manual segmentation, is consistently over 84%. By using our software (ACME) to study somite formation, we were able to segment touching cells with high accuracy and reliably quantify changes in morphogenetic parameters such as cell shape and size, and the arrangement of epithelial and mesenchymal cells. Our software has been developed and tested on Windows, Mac, and Linux platforms and is available publicly under an open source BSD license (https://github.com/krm15/ACME)
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Genomic mapping of RNA polymerase II reveals sites of co-transcriptional regulation in human cells
BACKGROUND: Transcription by RNA polymerase II is regulated at many steps including initiation, promoter release, elongation and termination. Accumulation of RNA polymerase II at particular locations across genes can be indicative of sites of regulation. RNA polymerase II is thought to accumulate at the promoter and at sites of co-transcriptional alternative splicing where the rate of RNA synthesis slows. RESULTS: To further understand transcriptional regulation at a global level, we determined the distribution of RNA polymerase II within regions of the human genome designated by the ENCODE project. Hypophosphorylated RNA polymerase II localizes almost exclusively to 5' ends of genes. On the other hand, localization of total RNA polymerase II reveals a variety of distinct landscapes across many genes with 74% of the observed enriched locations at exons. RNA polymerase II accumulates at many annotated constitutively spliced exons, but is biased for alternatively spliced exons. Finally, RNA polymerase II is also observed at locations not in gene regions. CONCLUSION: Localizing RNA polymerase II across many millions of base pairs in the human genome identifies novel sites of transcription and provides insights into the regulation of transcription elongation. These data indicate that RNA polymerase II accumulates most often at exons during transcription. Thus, a major factor of transcription elongation control in mammalian cells is the coordination of transcription and pre-mRNA processing to define exons
Identification of a Mutation Associated with Fatal Foal Immunodeficiency Syndrome in the Fell and Dales Pony
The Fell and Dales are rare native UK pony breeds at risk due to falling numbers, in-breeding, and inherited disease. Specifically, the lethal Mendelian recessive disease Foal Immunodeficiency Syndrome (FIS), which manifests as B-lymphocyte immunodeficiency and progressive anemia, is a substantial threat. A significant percentage (∼10%) of the Fell ponies born each year dies from FIS, compromising the long-term survival of this breed. Moreover, the likely spread of FIS into other breeds is of major concern. Indeed, FIS was identified in the Dales pony, a related breed, during the course of this work. Using a stepwise approach comprising linkage and homozygosity mapping followed by haplotype analysis, we mapped the mutation using 14 FIS–affected, 17 obligate carriers, and 10 adults of unknown carrier status to a ∼1 Mb region (29.8 – 30.8 Mb) on chromosome (ECA) 26. A subsequent genome-wide association study identified two SNPs on ECA26 that showed genome-wide significance after Bonferroni correction for multiple testing: BIEC2-692674 at 29.804 Mb and BIEC2-693138 at 32.19 Mb. The associated region spanned 2.6 Mb from ∼29.6 Mb to 32.2 Mb on ECA26. Re-sequencing of this region identified a mutation in the sodium/myo-inositol cotransporter gene (SLC5A3); this causes a P446L substitution in the protein. This gene plays a crucial role in the regulatory response to osmotic stress that is essential in many tissues including lymphoid tissues and during early embryonic development. We propose that the amino acid substitution we identify here alters the function of SLC5A3, leading to erythropoiesis failure and compromise of the immune system. FIS is of significant biological interest as it is unique and is caused by a gene not previously associated with a mammalian disease. Having identified the associated gene, we are now able to eradicate FIS from equine populations by informed selective breeding
1994 Swinburne Hawthorn Campus Library tour
1994 introductory video to Swinburne University of Technology Hawthorn Campus Library. Mike Bishop takes the viewer through the services of the Library including the catalogue, circulation, audio-visual and recreation
Improved Long-Term Imaging of Embryos with Genetically Encoded α-Bungarotoxin
Rapid advances in microscopy and genetic labeling strategies have created new opportunities for time-lapse imaging of embryonic development. However, methods for immobilizing embryos for long periods while maintaining normal development have changed little. In zebrafish, current immobilization techniques rely on the anesthetic tricaine. Unfortunately, prolonged tricaine treatment at concentrations high enough to immobilize the embryo produces undesirable side effects on development. We evaluate three alternative immobilization strategies: combinatorial soaking in tricaine and isoeugenol, injection of α-bungarotoxin protein, and injection of α-bungarotoxin mRNA. We find evidence for co-operation between tricaine and isoeugenol to give immobility with improved health. However, even in combination these anesthetics negatively affect long-term development. α-bungarotoxin is a small protein from snake venom that irreversibly binds and inactivates acetylcholine receptors. We find that α-bungarotoxin either as purified protein from snakes or endogenously expressed in zebrafish from a codon-optimized synthetic gene can immobilize embryos for extended periods of time with few health effects or developmental delays. Using α-bungarotoxin mRNA injection we obtain complete movies of zebrafish embryogenesis from the 1-cell stage to 3 days post fertilization, with normal health and no twitching. These results demonstrate that endogenously expressed α-bungarotoxin provides unprecedented immobility and health for time-lapse microscopy
Oh Shenandoah
Examines the causes of environmental degradation of the Shenandoah River
Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription
Pre-mRNA processing often occurs in coordination with transcription thereby coupling these two key regulatory events. As such, many proteins involved in mRNA processing associate with the transcriptional machinery and are in proximity to DNA. This proximity allows for the mapping of the genomic associations of RNA binding proteins by chromatin immunoprecipitation (ChIP) as a way of determining their sites of action on the encoded mRNA. Here, we used ChIP combined with high-density microarrays to localize on the human genome three functionally distinct RNA binding proteins: the splicing factor polypyrimidine tract binding protein (PTBP1/hnRNP I), the mRNA export factor THO complex subunit 4 (ALY/THOC4), and the 3′ end cleavage stimulation factor 64 kDa (CSTF2). We observed interactions at promoters, internal exons, and 3′ ends of active genes. PTBP1 had biases toward promoters and often coincided with RNA polymerase II (RNA Pol II). The 3′ processing factor, CSTF2, had biases toward 3′ ends but was also observed at promoters. The mRNA processing and export factor, ALY, mapped to some exons but predominantly localized to introns and did not coincide with RNA Pol II. Because the RNA binding proteins did not consistently coincide with RNA Pol II, the data support a processing mechanism driven by reorganization of transcription complexes as opposed to a scanning mechanism. In sum, we present the mapping in mammalian cells of RNA binding proteins across a portion of the genome that provides insight into the transcriptional assembly of RNA–protein complexes
α-bungarotoxin immobilizes embryos while permitting normal development.
<p><b>(A)</b> Percent of embryos immobile after injection of α-bungarotoxin protein (0.046–4.6ng) into the yolk at 24 hpf. <b>(B)</b> Percent of embryos immobile after injection of α-bungarotoxin mRNA (20–400 pg) into the yolk at 24 hpf. <b>(C)</b> Percent of embryos immobile after injection of of α-bungarotoxin mRNA (5–100 pg) into the 1-cell zygote. <b>(D)</b> Percent control OVD at 72 hpf for injection of α-bungarotoxin mRNA into the 1-cell zygote (green), into the yolk (yellow), and reference anesthetic treatments that permitted long-term immobilization (blue). (*) Not significantly different from control, Mann-Whitney-Wilcoxon two tailed P-value 0.87. (†) Significantly different from control, Mann-Whitney-Wilcoxon two tailed P-value 0.0011. <b>(E, G)</b> Control embryo at 72 hpf that was injected with 50 pg of membrane-citrine mRNA into the 1-cell zygote. <b>(F, H)</b> 72 hpf embryo that was injected with 50 pg of α-bungarotoxin mRNA into the 1-cell zygote. <b>(I)</b> Control larva at 8 days post fertilization (dpf) injected with 50 pg of membrane-citrine mRNA into the 1-cell zygote. <b>(J)</b> 8 dpf larva that was injected with 50 pg of α-bungarotoxin mRNA into the 1-cell zygote.</p
Tricaine and isoeugenol co-operate towards healthier immobilization.
<p><b>(A)</b> Heat map of percent immobile for 48 combinations of tricaine (0–200 μg/ml) and isoeugenol (0–0.003% v/v). Embryos were dechorionated and soaked from 24–27 hpf when they were assayed for immobility. <b>(B)</b> Continuation of treatments from (A), embryos were assayed for immobility at 72 hpf. <b>(C,D)</b> Representative micrographs of control (C) and 200 μg/ml tricaine treated (D) embryos. Arrow in (D) shows failure of semicircular canal projection fusion. Asterisk in (D) shows pericardial edema. <b>(E)</b> Heat map of percent of embryos with pericardial edema at 72 hpf. <b>(F)</b> Percent control otic vesicle diameter (OVD) was calculated by dividing the average of 10–30 experimental embryos by the average of 10–30 control embryos. Heat map of percent control OVD for the combinatorial treatments. OVD was measured at 72 hpf using micrographs like those in (C, D). Percentage is based on normalization to untreated control. <b>(G)</b> Merge of heatmaps from (A, 27 hpf) and (F) that highlights the tradeoffs between embryo immobility and healthy development.</p