Improvement of electrophoresis performance by spectral analysis

This paper describes a new design of standard agarose gel electrophoresis procedure for nucleic acids analysis. The electrophoresis was improved by using the real-time spectral analysis of the samples to increase its performance. A laser beam illuminated the analysed sample at wavelength with the highest absorption of gel components. Thus, DNA band is detected well before it may exits a gel plate. A modified horizontal electrophoresis device was designed, developed and tested.Keywords: Horizontal electrophoresis, laser, spectral analysis, agarose ge

Impaired proteoglycan glycosylation, elevated TGF-β signaling, and abnormal osteoblast differentiation as the basis for bone fragility in a mouse model for gerodermia osteodysplastica

<div><p>Gerodermia osteodysplastica (GO) is characterized by skin laxity and early-onset osteoporosis. <i>GORAB</i>, the responsible disease gene, encodes a small Golgi protein of poorly characterized function. To circumvent neonatal lethality of the <i>Gorab</i>^<i>Null</i> full knockout, <i>Gorab</i> was conditionally inactivated in mesenchymal progenitor cells (Prx1-cre), pre-osteoblasts (Runx2-cre), and late osteoblasts/osteocytes (Dmp1-cre), respectively. While in all three lines a reduction in trabecular bone density was evident, only <i>Gorab</i>^Prx1 and <i>Gorab</i>^Runx2 mutants showed dramatically thinned, porous cortical bone and spontaneous fractures. Collagen fibrils in the skin of <i>Gorab</i>^<i>Null</i> mutants and in bone of <i>Gorab</i>^Prx1 mutants were disorganized, which was also seen in a bone biopsy from a GO patient. Measurement of glycosaminoglycan contents revealed a reduction of dermatan sulfate levels in skin and cartilage from <i>Gorab</i>^<i>Null</i> mutants. In bone from <i>Gorab</i>^Prx1 mutants total glycosaminoglycan levels and the relative percentage of dermatan sulfate were both strongly diminished. Accordingly, the proteoglycans biglycan and decorin showed reduced glycanation. Also in cultured <i>GORAB</i>-deficient fibroblasts reduced decorin glycanation was evident. The Golgi compartment of these cells showed an accumulation of decorin, but reduced signals for dermatan sulfate. Moreover, we found elevated activation of TGF-β in <i>Gorab</i>^Prx1 bone tissue leading to enhanced downstream signalling, which was reproduced in <i>GORAB</i>-deficient fibroblasts. Our data suggest that the loss of <i>Gorab</i> primarily perturbs pre-osteoblasts. GO may be regarded as a congenital disorder of glycosylation affecting proteoglycan synthesis due to delayed transport and impaired posttranslational modification in the Golgi compartment.</p></div

Transcriptomic Characterization Using RNA-Seq Data Analysis

Vysoce výkonné sekvenční technologie produkují obrovské množství dat, která mohou odhalit nové geny, identifikovat splice varianty a kvantifikovat genovou expresi v celém genomu. Objem a složitost dat z RNA-seq experimentů vyžadují škálovatelné metody matematické analýzy založené na robustníchstatistických modelech. Je náročné navrhnout integrované pracovní postupy, které zahrnují různé postupy analýzy. Konkrétně jsou to srovnávací testy transkriptů, které jsou komplikovány několika zdroji variability měření a představují řadu statistických problémů. V tomto výzkumu byla sestavena integrovaná transkripční profilová pipeline k produkci nových reprodukovatelných kódů pro získání biologicky interpretovovatelných výsledků. Počínaje anotací údajů RNA-seq a hodnocení kvality je navržen soubor kódů, který slouží pro vizualizaci hodnocení kvality, potřebné pro zajištění RNA-Seq experimentu s analýzou dat. Dále je provedena komplexní diferenciální analýza genových expresí, která poskytuje popisné metody pro testované RNA-Seq data. Pro implementaci analýzy alternativního sestřihu a diferenciálních exonů jsme zlepšili výkon DEXSeq definováním otevřeného čtecího rámce exonového regionu, který se používá alternativně. Dále je popsána nová metodologie pro analýzu diferenciálně exprimované dlouhé nekódující RNA nalezením funkční korelace této RNA se sousedícími diferenciálně exprimovanými geny kódujícími proteiny. Takto je získán jasnější pohled na regulační mechanismus a poskytnuta hypotéza o úloze dlouhé nekódující RNA v regulaci genové exprese.The high-throughputs sequence technologies produce a massive amount of data, that can reveal new genes, identify splice variants, and quantify gene expression genome-wide. However, the volume and the complexity of data from RNA-seq experiments necessitate a scalable, and mathematical analysis based on a robust statistical model. Therefore, it is challenging to design integrated workflow, that incorporates the various analysis procedures. Particularly, the comparative transcriptome analysis is complicated due to several sources of measurement variability and poses numerous statistical challenges. In this research, we performed an integrated transcriptional profiling pipeline, which generates novel reproducible codes to obtain biologically interpretable results. Starting with the annotation of RNA-seq data and quality assessment, we provided a set of codes to serve the quality assessment visualization needed for establishing the RNA-Seq data analysis experiment. Additionally, we performed comprehensive differential gene expression analysis, presenting descriptive methods to interpret the RNA-Seq data. For implementing alternative splicing and differential exons usage analysis, we improved the performance of the Bioconductor package DEXSeq by defining the open reading frame of the exonic regions, which are differentially used between biological conditions due to the alternative splicing of the transcripts. Furthermore, we present a new methodology to analyze the differentially expressed long non-coding RNA, by finding the functional correlation of the long non-coding RNA with neighboring differential expressed protein coding genes. Thus, we obtain a clearer view of the regulation mechanism, and give a hypothesis about the role of long non-coding RNA in gene expression regulation.

Cortical porosity and osteoblast dysfunction in the <i>Gorab</i>^Prx1 model recapitulate the gerodermia osteodysplastica bone phenotype.

<p>(A) Von Kossa / hematoxylin stained cortical bone of twelve week old <i>Gorab</i>^Prx1 mouse. Inset showing magnified view of large pores in the mutant diaphysis. Scale bar = 200 μm. (B) Sections of metaphyseal cortical bone of the tibia of four week old <i>Gorab</i>^Prx1 mice stained with Masson Goldner trichrome. Scale bar = 100μm. (C) μCT reconstructed image of a spontaneously fractured humerus from a four week old <i>Gorab</i>^Prx1 mouse. Arrowhead = fracture site. Scale bar = 1mm. (D) Histomorphometric quantitation number of osteoblast per bone perimeter (N.Ob/B.Pm), (E) number of osteocytes per cortical bone perimeter (N.Ot/cort B.Pm). (F) Mineral apposition rate (MAR) at the endosteum of tibia midshaft in four week old <i>Gorab</i>^<i>Prx1</i> (N = 4). Inset showing calcein double labeling, C = control, M = mutant. (G) osteoid volume (OV/TV) in secondary spongiosa of the proximal tibia in <i>Gorab</i>^Prx1 mutants vs. controls at four weeks of age (N = 4–6). (H) Altered expression of osteoblast lineage marker genes in femoral cortical bone of four week old <i>Gorab</i>^Prx1 mutants (N = 6–8). (I) Immunohistochemical detection of Sp7/osterix, Spp1/osteopontin and Dmp1 expression in cortical bone. Note higher number of osterix + cells in <i>Gorab</i>^Prx1 mutants. Scale bar = 50μm. (J) Number of osteoclasts per bone perimeter (N.Oc/B.Pm). (K) Opg to Rankl expression ratio in four week old <i>Gorab</i>^<i>Prx1</i> mutants (N = 6). P = periosteum, C = cortical bone, E = endosteum.</p

Elevated TGF-β signaling in <i>Gorab</i>-deficient skin, bone, and fibroblasts.

<p>(A) Quantitation of active and total TGF-β in skin lysates from E18.5 <i>Gorab</i>^Null mice (N = 3). (B) Enhanced expression of TGF-β regulated genes in the diaphysis of four week old <i>Gorab</i>^Prx1 mutants (N = 7–8). (C) Immunofluorescence staining for p-SMAD2 in <i>Gorab</i>^Prx1 mutants and controls at four weeks of age. Representative picture of N = 4 per group. Note stronger signals in periosteum in mutants. Scale bar = 50μm. (D) Western blot of p-SMAD2 in confluent fibroblasts from GO patients and healthy controls (N = 3) and quantitative evaluation. (E) Quantitative PCR to measure expression of TGF-β responsive genes in GO patient-derived fibroblasts (N = 4). P = periosteum, C = cortical bone. E = endosteum.</p

Golgi retention and reduced glycanation of decorin in <i>Gorab</i>-deficient fibroblasts.

<p>(A) Western blot analysis of decorin in control and <i>Gorab</i>^Null MEF cell lysates. The size ranges for the fully glycanated form of decorin (DCN) and of its core protein are indicated. (B) Left, western blot analysis of decorin in lysates of the extracellular matrix produced by cultured control and GO fibroblasts. Right, levels of glycanated decorin were quantified against total decorin levels (N = 3). (C) Analysis of intra-Golgi levels of decorin in co-cultured control and GO human skin fibroblasts. Immunofluorescence labeling was performed with anti-decorin, anti-GORAB and anti-GM130 antibodies. GORAB staining was used to distinguish control (yellow arrows) and GO (red arrows) cells. Decorin fluorescence intensity in both cell types was normalized against that of the Golgi marker GM130 (N = 3, >500 cells analyzed per cell line). Scale bar = 10 μm. (D) Analysis of intra-Golgi levels of dermatan sulfate (DS)-modified proteins in co-cultured control (yellow arrows) and GO (red arrows) human skin fibroblasts. Cells were labeled with anti-DS (GD3A12), anti-GORAB and anti-TGN46 antibodies. The intensity of the GD3A12 fluorescence signals were measured relative to that of the Golgi marker TGN46 (N = 3, >500 cells analyzed per cell line). Scale bar = 10 μm.</p

Loss of <i>Gorab</i> resulted in underglycanation of proteoglycans.

<p>(A) Quantitation of dermatan sulfate and chondroitin sulfate in skin, cartilage, and lung samples from E18.5 <i>Gorab</i>^Null mice (N = 3–4). (B) Total amount of GAGs in the cortical bone of femora from four week old <i>Gorab</i>^<i>Prx1</i> mutants and littermate controls (N = 3–4). (C) Percentage of dermatan sulfate in the total amount of glycosaminoglycans (GAGs). (D) Immunoblotting for decorin in skin samples from E18.5 <i>Gorab</i>^Null mice. Loss of the 100kDa band, corresponding to the fully glycanated decorin, and higher intensity the core protein band in mutant lysate indicate a glycanation defect. (E) Immunoblotting for decorin in cortical bone lysates from four week old <i>Gorab</i>^Prx1 mice and littermate controls also showing higher intensity of lower bands in mutant. (F) Immunoblot of decorin in cortical bone lysates from wildtype (WT) mice at different ages: newborn (P0), 5 weeks (5W), 26 weeks (26W) and 2 years (2Y). Note reduction in glycanation with increasing age. (G) Immunofluorescence detection of decorin and (H) biglycan in tibia of four week old <i>Gorab</i>^Prx1 mice. Sections were not pretreated with chondroitinase. Higher staining intensities therefore indicate lower glycanation of the core proteins in the periosteum. P = periosteum, C = cortical bone. Scale bar = 50μm. Experiments (D) to (H) were repeated at least three times with independent biological samples, representative results are shown.</p