Starting small: Transcriptomics with single and small populations of cells

Studying the transcriptome of organisms allows for the characterization of different aspects of development and disease progression. Studies of this kind are usually done with large numbers of cells, giving an expression profile that serves as an overview of that cell type or tissue. In this thesis, two different transcriptomic studies were done with small populations of cells and with single cells. To examine the immune response of a hSOD1G93A Amytrophic Lateral Sclerosis (ALS) mouse model, small populations of cells were analyzed by microarray using a protocol modified from one developed by Trimarchi et al (2007) to study single cells. Multiple genes were found to be significantly differentially expressed between mutant and wild-type ALS mice that with further study will lead to a better understanding of the immune response\u27s role in this disease. To study the development of the enteric nervous system in zebrafish, single enteric neurons were isolated from 7 day post fertilization (7 dpf) and 48 hour post fertilization (48 dpf) zebrafish larvae and prepared for RNA sequencing analysis. There are no sequencing results yet, but preliminary data suggests that different subtypes of enteric neuron precursors were isolated from 48 hpf larvae, and that both differentiated enteric neurons and enteric neurons undergoing differentiation were isolated from 7 dpf larvae. This study should expand our knowledge of signaling pathways and other genes involved in migration, proliferation, and differentiation of the ENS, as well as identify specific markers of precursors and enteric neurons

Plasma-seq: a novel strategy for metastatic prostate cancer analysis

Personalized genomics will only be useful for monitoring the prognosis of patients with cancer when it becomes much more cost-effective and quicker to apply. A recent study brings this closer to reality with the development of plasma-seq, a rapid, low-cost method that sequences the circulating DNA present in the peripheral blood of patients with cancer. The power of this technique is demonstrated with the examination of tumor genomes from patients with prostate cancer

Individual limb mechanical analysis of gait following stroke

The step-to-step transition of walking requires significant mechanical and metabolic energy to redirect the center of mass. Inter-limb mechanical asymmetries during the step-to-step transition may increase overall energy demands and require compensation during single-support. The purpose of this study was to compare individual limb mechanical gait asymmetries during the step-to-step transitions, single-support and over a complete stride between two groups of individuals following stroke stratified by gait speed (≥0.8 m/s o

Starting small: Transcriptomics with single and small populations of cells

Studying the transcriptome of organisms allows for the characterization of different aspects of development and disease progression. Studies of this kind are usually done with large numbers of cells, giving an expression profile that serves as an overview of that cell type or tissue. In this thesis, two different transcriptomic studies were done with small populations of cells and with single cells. To examine the immune response of a hSOD1G93A Amytrophic Lateral Sclerosis (ALS) mouse model, small populations of cells were analyzed by microarray using a protocol modified from one developed by Trimarchi et al (2007) to study single cells. Multiple genes were found to be significantly differentially expressed between mutant and wild-type ALS mice that with further study will lead to a better understanding of the immune response's role in this disease. To study the development of the enteric nervous system in zebrafish, single enteric neurons were isolated from 7 day post fertilization (7 dpf) and 48 hour post fertilization (48 dpf) zebrafish larvae and prepared for RNA sequencing analysis. There are no sequencing results yet, but preliminary data suggests that different subtypes of enteric neuron precursors were isolated from 48 hpf larvae, and that both differentiated enteric neurons and enteric neurons undergoing differentiation were isolated from 7 dpf larvae. This study should expand our knowledge of signaling pathways and other genes involved in migration, proliferation, and differentiation of the ENS, as well as identify specific markers of precursors and enteric neurons.</p

Plasma-seq: a novel strategy for metastatic prostate cancer analysis

Personalized genomics will only be useful for monitoring the prognosis of patients with cancer when it becomes much more cost-effective and quicker to apply. A recent study brings this closer to reality with the development of plasma-seq, a rapid, low-cost method that sequences the circulating DNA present in the peripheral blood of patients with cancer. The power of this technique is demonstrated with the examination of tumor genomes from patients with prostate cancer.This article is from Genome Medicine 5 (2013): 35, doi: 10.1186/gm439. Posted with permission.</p

Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment

Background: Motility and the coordination of moving food through the gastrointestinal tract rely on a complex network of neurons known as the enteric nervous system (ENS). Despite its critical function, many of the molecular mechanisms that direct the development of the ENS and the elaboration of neural network connections remain unknown. The goal of this study was to transcriptionally identify molecular pathways and candidate genes that drive specification, differentiation and the neural circuitry of specific neural progenitors, the phox2b expressing ENS cell lineage, during normal enteric nervous system development. Because ENS development is tightly linked to its environment, the transcriptional landscape of the cellular environment of the intestine was also analyzed. Results: Thousands of zebrafish intestines were manually dissected from a transgenic line expressing green fluorescent protein under the phox2b regulatory elements [Tg(phox2b:EGFP)w37]. Fluorescence-activated cell sorting was used to separate GFP-positive phox2b expressing ENS progenitor and derivatives from GFP-negative intestinal cells. RNA-seq was performed to obtain accurate, reproducible transcriptional profiles and the unbiased detection of low level transcripts. Analysis revealed genes and pathways that may function in ENS cell determination, genes that may be identifiers of different ENS subtypes, and genes that define the non-neural cellular microenvironment of the ENS. Differential expression analysis between the two cell populations revealed the expected neuronal nature of the phox2b expressing lineage including the enrichment for genes required for neurogenesis and synaptogenesis, and identified many novel genes not previously associated with ENS development. Pathway analysis pointed to a high level of G-protein coupled pathway activation, and identified novel roles for candidate pathways such as the Nogo/Reticulon axon guidance pathway in ENS development. Conclusion: We report the comprehensive gene expression profiles of a lineage-specific population of enteric progenitors, their derivatives, and their microenvironment during normal enteric nervous system development. Our results confirm previously implicated genes and pathways required for ENS development, and also identify scores of novel candidate genes and pathways. Thus, our dataset suggests various potential mechanisms that drive ENS development facilitating characterization and discovery of novel therapeutic strategies to improve gastrointestinal disorders.This article is published as Roy-Carson, Sweta, Kevin Natukunda, Hsien-chao Chou, Narinder Pal, Caitlin Farris, Stephan Q. Schneider, and Julie A. Kuhlman. "Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment." BMC genomics 18 (2017): 290. doi: 10.1186/s12864-017-3653-2.</p

Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment

Abstract Background Motility and the coordination of moving food through the gastrointestinal tract rely on a complex network of neurons known as the enteric nervous system (ENS). Despite its critical function, many of the molecular mechanisms that direct the development of the ENS and the elaboration of neural network connections remain unknown. The goal of this study was to transcriptionally identify molecular pathways and candidate genes that drive specification, differentiation and the neural circuitry of specific neural progenitors, the phox2b expressing ENS cell lineage, during normal enteric nervous system development. Because ENS development is tightly linked to its environment, the transcriptional landscape of the cellular environment of the intestine was also analyzed. Results Thousands of zebrafish intestines were manually dissected from a transgenic line expressing green fluorescent protein under the phox2b regulatory elements [Tg(phox2b:EGFP) w37 ]. Fluorescence-activated cell sorting was used to separate GFP-positive phox2b expressing ENS progenitor and derivatives from GFP-negative intestinal cells. RNA-seq was performed to obtain accurate, reproducible transcriptional profiles and the unbiased detection of low level transcripts. Analysis revealed genes and pathways that may function in ENS cell determination, genes that may be identifiers of different ENS subtypes, and genes that define the non-neural cellular microenvironment of the ENS. Differential expression analysis between the two cell populations revealed the expected neuronal nature of the phox2b expressing lineage including the enrichment for genes required for neurogenesis and synaptogenesis, and identified many novel genes not previously associated with ENS development. Pathway analysis pointed to a high level of G-protein coupled pathway activation, and identified novel roles for candidate pathways such as the Nogo/Reticulon axon guidance pathway in ENS development. Conclusion We report the comprehensive gene expression profiles of a lineage-specific population of enteric progenitors, their derivatives, and their microenvironment during normal enteric nervous system development. Our results confirm previously implicated genes and pathways required for ENS development, and also identify scores of novel candidate genes and pathways. Thus, our dataset suggests various potential mechanisms that drive ENS development facilitating characterization and discovery of novel therapeutic strategies to improve gastrointestinal disorders

Additional file 4: Figure S3. of Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment

Top 50 significant GO terms from the biological process category in the neurons. A bar graph illustrating the first significant 50 biological processes GO term nodes based on the p-value. The y-axis shows the top 50 GO terms for the biological processes, while the x-axis shows the corresponding –log(p-value). The specific values can be found in the additional file 21. (JPG 82 kb

[Matbuat Umum Müdürü Vedat Nedim Tör tarafından gönderilen yazı]

Taha Toros Arşivi, Dosya No: 145-Vedat Nedim Tör. Not: Ekte ilgili yazının Osmanlıca sureti ve konu ile ilgili bir başka yazışmanın müsveddesi bulunmaktadır.Unutma İstanbul projesi İstanbul Kalkınma Ajansı'nın 2016 yılı "Yenilikçi ve Yaratıcı İstanbul Mali Destek Programı" kapsamında desteklenmiştir. Proje No: TR10/16/YNY/010