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A family, with two affected identical twins with early-onset recessive inherited retinal degeneration, was analyzed to determine the underlying genetic cause of pathology. Exome sequencing revealed a rare and previously reported causative variant (c.1923_1969delinsTCTGGG; p.Asn643Glyfs*29) in the PDE6B gene in the affected twins and their unaffected father. Further investigation, using genome sequencing, identified a novel ~7.5-kb deletion (Chr 4:670,405-677,862del) encompassing the ATP5ME gene, part of the 5' UTR of MYL5, and a 378-bp (Chr 4:670,405-670,782) region from the 3' UTR of PDE6B in the affected twins and their unaffected mother. Both variants segregated with disease in the family. Analysis of the relative expression of PDE6B, in peripheral blood cells, also revealed a significantly lower level of PDE6B transcript in affected siblings compared to a normal control. PDE6B is associated with recessive rod-cone degeneration and autosomal dominant congenital stationary night blindness. Ophthalmic evaluation of these patients showed night blindness, fundus abnormalities, and peripheral vision loss, which are consistent with PDE6B-associated recessive retinal degeneration. These findings suggest that the loss of PDE6B transcript resulting from the compound heterozygous pathogenic variants is the underlying cause of recessive rod-cone degeneration in the study family. [ABSTRACT FROM AUTHOR]

PPAR[gamma] activators such as rosiglitazone (RSG) stimulate adipocyte differentiation and increase subcutaneous adipose tissue mass. However, in addition to preadipocyte differentiation, adipose tissue expansion requires neovascularization to support increased adipocyte numbers. Paradoxically, endothelial cell growth and differentiation is potently inhibited by RSG in vitro, raising the question of how this drug can induce an increase in adipose tissue mass while inhibiting angiogenesis. We find that adipose tissue from mice treated with RSG have increased capillary density. To determine whether adipose tissue angiogenesis was stimulated by RSG, we developed a novel assay to study angiogenic sprout formation ex vivo. Angiogenic sprout formation from equally sized adipose tissue fragments, but not from aorta rings, was greatly increased by obesity and by TZD treatment in vivo. To define the mechanism involved in RSG-stimulated angiogenesis in adipose tissue, the expression of proangiogenic factors by adipocytes was examined. Expression of VEGFA and VEGFB, as well as of the angiopoietin-like factor-4 (ANGPTL4), was stimulated by in vivo treatment with RSG. To define the potential role of these factors, we analyzed their effects on endothelial cell growth and differentiation in vitro. We found that ANG L4 stimulates endothelial cell growth and tubule formation, albeit more weakly than VEGF. However, ANGPTL4 mitigates the growth inhibitory actions of RSG on endothelial cells in the presence or absence of VEGF. Thus, the interplay between VEGF and ANGPTL4 could lead to a net expansion of the adipose tissue capillary network, required for adipose tissue growth, in response to PPAR[gamma], activators. neovascularization; endothelial cell; insulin resistance; diabetes; adipokines

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.020 Byline: Laurence Covassin (a), Julio D. Amigo (a), Kana Suzuki (b), Viktor Teplyuk (b), Juerg Straubhaar (b), Nathan D. Lawson (a) Keywords: Zebrafish; Hematopoietic; Vascular; Endothelial; Microarray Abstract: In this study, we utilize fluorescent activated cell sorting (FACS) of cells from transgenic zebrafish coupled with microarray analysis to globally analyze expression of cell type specific genes. We find that it is possible to isolate cell populations from Tg(fli1:egfp).sup.y1 zebrafish embryos that are enriched in vascular, hematopoietic and pharyngeal arch cell types. Microarray analysis of GFP.sup.+ versus GFP.sup.- cells isolated from Tg(fli1:egfp).sup.y1 embryos identifies genes expressed in hematopoietic, vascular and pharyngeal arch tissue, consistent with the expression of the fli1:egfp transgene in these cell types. Comparison of expression profiles from GFP.sup.+ cells isolated from embryos at two different time points reveals that genes expressed in different fli1.sup.+ cell types display distinct temporal expression profiles. We also demonstrate the utility of this approach for gene discovery by identifying numerous previously uncharacterized genes that we find are expressed in fli1:egfp-positive cells, including new markers of blood, endothelial and pharyngeal arch cell types. In parallel, we have developed a database to allow easy access to both our microarray and in situ results. Our results demonstrate that this is a robust approach for identification of cell type specific genes as well as for global analysis of cell type specific gene expression in zebrafish embryos. Author Affiliation: (a) Program in Gene Function and Expression, University of Massachusetts Medical School, Lazare Research Building, Room 617, 364 Plantation Street, Worcester, MA 01605, USA (b) Diabetes and Endocrinology Research Center, University of Massachusetts Medical School, Biotech 2, Suite 218, 373 Plantation Street, Worcester, MA 01605, USA Article History: Received 27 April 2006; Revised 28 July 2006; Accepted 6 August 2006

Abstract Background DNA-protein interactions in mature brain are increasingly recognized as key regulators for behavioral plasticity and neuronal dysfunction in chronic neuropsychiatric disease. However, chromatin assays typically lack single cell resolution, and therefore little is known about chromatin regulation of differentiated neuronal nuclei that reside in brain parenchyma intermingled with various types of non-neuronal cells. Results Here, we describe a protocol to selectively tag neuronal nuclei from adult brain – either by (anti-NeuN) immunolabeling or transgene-derived histone H2B-GFP fusion protein – for subsequent fluorescence-activated sorting and chromatin immunoprecipitation (ChIP). To illustrate an example, we compared histone H3 lysine 4 and 9 methylation marks at select gene promoters in neuronal, non-neuronal and unsorted chromatin from mouse forebrain and human cerebral cortex, and provide evidence for neuron-specific histone methylation signatures. Conclusion With the modifications detailed in this protocol, the method can be used to collect nuclei from specific subtypes of neurons from any brain region for subsequent ChIP with native/un-fixed or crosslinked chromatin preparations. Starting with the harvest of brain tissue, ChIP-ready neuronal nuclei can be obtained within one day.

Virus-specific CD8+ T cells expand dramatically during acute EBV infection, and their persistence is important for lifelong control of EBV-related disease. To better define the generation and maintenance of these effective CD8+ T cell responses, we used microarrays to characterize gene expression in total and EBV-specific CD8+ T cells isolated from the peripheral blood of 10 individuals followed from acute infectious mononucleosis (AIM) into convalescence (CONV). In total CD8+ T cells, differential expression of genes in AIM and CONV was most pronounced among those encoding proteins important in T cell activation/differentiation, cell division/metabolism, chemokines/cytokines and receptors, signaling and transcription factors (TF), immune effector functions, and negative regulators. Within these categories, we identified 28 genes that correlated with CD8+ T cell expansion in response to an acute EBV infection. In EBV-specific CD8+ T cells, we identified 33 genes that were differentially expressed in AIM and CONV. Two important TF, T-bet and eomesodermin, were upregulated and maintained at similar levels in both AIM and CONV; in contrast, protein expression declined from AIM to CONV. Expression of these TF varied among cells with different epitope specificities. Collectively, gene and protein expression patterns suggest that a large proportion, if not a majority of CD8+ T cells in AIM are virus specific, activated, dividing, and primed to exert effector activities. High expression of T-bet and eomesodermin may help to maintain effector mechanisms in activated cells and to enable proliferation and transition to earlier differentiation states in CONV. [ABSTRACT FROM AUTHOR]