Feasibility of Haralick's Texture Features for the Classification of Chromogenic In-situ Hybridization Images

This paper presents a proof of concept for the usefulness of second-order texture features for the qualitative analysis and classification of chromogenic in-situ hybridization whole slide images in high-throughput imaging experiments. The challenge is that currently, the gold standard for gene expression grading in such images is expert assessment. The idea of the research team is to use different approaches in the analysis of these images that will be used for structural segmentation and functional analysis in gene expression. The article presents such perspective idea to select a number of textural features that are going to be used for classification. In our experiment, natural grouping of image samples (tiles) depending on their local texture properties was explored in an unsupervised classification procedure. The features are reduced to two dimensions with fuzzy c-means clustering. The overall conclusion of this experiment is that Haralick features are a viable choice for classification and analysis of chromogenic in-situ hybridization image data. The principal component analysis approach produced slightly more "understandable" from an annotator's point of view classes.Comment: 4 pages, 1 figur

Deep convolutional neural networks for annotating gene expression patterns in the mouse brain

Abstract Background Profiling gene expression in brain structures at various spatial and temporal scales is essential to understanding how genes regulate the development of brain structures. The Allen Developing Mouse Brain Atlas provides high-resolution 3-D in situ hybridization (ISH) gene expression patterns in multiple developing stages of the mouse brain. Currently, the ISH images are annotated with anatomical terms manually. In this paper, we propose a computational approach to annotate gene expression pattern images in the mouse brain at various structural levels over the course of development. Results We applied deep convolutional neural network that was trained on a large set of natural images to extract features from the ISH images of developing mouse brain. As a baseline representation, we applied invariant image feature descriptors to capture local statistics from ISH images and used the bag-of-words approach to build image-level representations. Both types of features from multiple ISH image sections of the entire brain were then combined to build 3-D, brain-wide gene expression representations. We employed regularized learning methods for discriminating gene expression patterns in different brain structures. Results show that our approach of using convolutional model as feature extractors achieved superior performance in annotating gene expression patterns at multiple levels of brain structures throughout four developing ages. Overall, we achieved average AUC of 0.894 ± 0.014, as compared with 0.820 ± 0.046 yielded by the bag-of-words approach. Conclusions Deep convolutional neural network model trained on natural image sets and applied to gene expression pattern annotation tasks yielded superior performance, demonstrating its transfer learning property is applicable to such biological image sets.http://deepblue.lib.umich.edu/bitstream/2027.42/111637/1/12859_2015_Article_553.pd

Deep Convolutional Neural Networks for Annotating Gene Expression Patterns in the Mouse Brain

Background: Profiling gene expression in brain structures at various spatial and temporal scales is essential to understanding how genes regulate the development of brain structures. The Allen Developing Mouse Brain Atlas provides high-resolution 3-D in situ hybridization (ISH) gene expression patterns in multiple developing stages of the mouse brain. Currently, the ISH images are annotated with anatomical terms manually. In this paper, we propose a computational approach to annotate gene expression pattern images in the mouse brain at various structural levels over the course of development. Results: We applied deep convolutional neural network that was trained on a large set of natural images to extract features from the ISH images of developing mouse brain. As a baseline representation, we applied invariant image feature descriptors to capture local statistics from ISH images and used the bag-of-words approach to build image-level representations. Both types of features from multiple ISH image sections of the entire brain were then combined to build 3-D, brain-wide gene expression representations. We employed regularized learning methods for discriminating gene expression patterns in different brain structures. Results show that our approach of using convolutional model as feature extractors achieved superior performance in annotating gene expression patterns at multiple levels of brain structures throughout four developing ages. Overall, we achieved average AUC of 0.894 ± 0.014, as compared with 0.820 ± 0.046 yielded by the bag-of-words approach. Conclusions: Deep convolutional neural network model trained on natural image sets and applied to gene expression pattern annotation tasks yielded superior performance, demonstrating its transfer learning property is applicable to such biological image sets

Localizing Genes to Cerebellar Layers by Classifying ISH Images

Gene expression controls how the brain develops and functions. Understanding control processes in the brain is particularly hard since they involve numerous types of neurons and glia, and very little is known about which genes are expressed in which cells and brain layers. Here we describe an approach to detect genes whose expression is primarily localized to a specific brain layer and apply it to the mouse cerebellum. We learn typical spatial patterns of expression from a few markers that are known to be localized to specific layers, and use these patterns to predict localization for new genes. We analyze images of in-situ hybridization (ISH) experiments, which we represent using histograms of local binary patterns (LBP) and train image classifiers and gene classifiers for four layers of the cerebellum: the Purkinje, granular, molecular and white matter layer. On held-out data, the layer classifiers achieve accuracy above 94 % (AUC) by representing each image at multiple scales and by combining multiple image scores into a single gene-level decision. When applied to the full mouse genome, the classifiers predict specific layer localization for hundreds of new genes in the Purkinje and granular layers. Many genes localized to the Purkinje layer are likely to be expressed in astrocytes, and many others are involved in lipid metabolism, possibly due to the unusual size of Purkinje cells

Perspectives On Ethanol Toxicity In Medaka Embryogenesis And Possible Protection By Asian Ginseng (Panax Ginseng): Histological And Biochemical Analysis

Alcohol is recognized as a classic teratogen capable of inducing a wide range of developmental abnormalities. Alcohol consumption during pregnancy may produce permanent brain damage in the fetus and is associated with the development of a life-long behavioral, social, and cognitive disorder now known as fetal alcohol spectrum disorder (FASD). The most clinically recognizable form of FASD is fetal alcohol syndrome (FAS) which is characterized by specific features including facial dysmorphogenesis, mental dysfunction, growth retardation, and cardiovascular and limb defects. Due to ethical constraints, human studies of FASD are very limited. Therefore our current understanding of FASD is mainly based on several animal models (vertebrate and invertebrate). The fish embryo, especially zebrafish (Danio rerio) and Japanese medaka ( Oryzias latipes), are long-established models for research in developmental biology and have been used to explore alcohol\u27s effect on neurogenesis, cardiogenesis, intracellular signaling, neurobehavioral aspects, and apoptosis. These two fish models have proven utility for studying alcohol\u27s damaging effects during morphogenesis. In this study we have demonstrated that exposing Japanese medaka embryos to alcohol during development shophenotypic features and biochemical parameters that are comparable to FASD phenotypes observed in humans. Asian ginseng (PG) root extract has been shown to have anti alcoholic activities in a number of studies. In this study we have used medaka embryos to induce FASD like neurological defects. We have also investigated the ability of Asian ginseng (PG) to function as a preventive agent of these defects. Our findings suggest that medaka embryogenesis, can be a very useful model for investigating the molecular and morphological endpoints of FASD, and that PG root extract has a protective effect against alcohol induced toxicity in medaka embryogenesis

Cerebellar degeneration-related proteins in anti-Yo associated paraneoplastic cerebellar degeneration

Background: Paraneoplastic neurological syndromes (PNS) are rare immune-mediated diseases triggered by cancer, and characterized by circulating onconeural antibodies directed against antigens expressed by neurons and tumor cells. One of the most common forms of PNS is paraneoplastic cerebellar degeneration (PCD). In patients with PCD and ovarian or breast cancer, the dominant onconeural antibody is anti-Yo, detected in serum and cerebrospinal fluid (CSF). Anti-Yo targets two intracellular antigens, cerebellar degeneration-related protein 2 (CDR2) and CDR2-like (CDR2L), expressed in the nucleus and cytoplasm of Purkinje neurons in the cerebellum, respectively. The interaction between anti-Yo and CDR proteins is thought to mediate Purkinje neuron dysfunction and death, leaving the patients in a severely disabled state. The pathomechanisms underlying PCD, including the cellular functions and molecular mechanisms driven by the CDR proteins, are limited. About ten years ago, CDR2L was identified as a new target associated with PCD, and its specific location and neuronal functions remains elusive. Herein, we aim to draw a clearer picture of the anti-Yo associated neurodegeneration by addressing which onconeural antigen is the major target of Yo antibodies, identifying the subcellular localization of CDR2 and CDR2L and interaction partners. Finally, we evaluate the possibility of including CDR2L as a diagnostic marker when diagnosing patients with PCD. Aims: The overarching aim of this work was to gain in depth knowledge about CDR2 and CDR2L in anti-Yo associated PCD. Materials and methods: In paper I, anti-Yo reactivity towards CDR2 and CDR2L was addressed by staining cerebellar sections and cancer cells, and performing immunoprecipitation and fluorescent immunoblotting analysis. HepG2 cells were transfected to investigate whether Yo antibodies detect recombinant forms of the proteins. In paper II, mass spectrometry based proteomics was used to determine antibody specificity, and to identify CDR2 and CDR2L protein binding partners. Findings were further investigated by co-immunoprecipitation, proximity ligation assay and co-localization studies using super-resolution microscopy. Immunochemistry and super-resolution microscopy enabled determination of the subcellular localizations of CDR2 and CDR2L. In paper III, the potential of including CDR2L as a diagnostic marker in PCD was assessed by developing two in-house techniques, cell-based assay for CDR2L and western blot analysis of recombinant CDR2 and CDR2L. Results: We show that CDR2L is the major Yo antigen and that including CDR2L as a marker in the diagnosis of PCD greatly improves test specificity. Furthermore, the subcellular location of CDR2L is found to be in the cell cytoplasm, more specifically in direct contact with the ribosomal subunit protein, rpS6. CDR2 localizes to the nucleus in contact with nuclear speckle proteins, SON and eIF4A3. Conclusions: Identifying CDR2L as the major Yo antigen is an important finding in the work of increasing test specificity for PCD diagnosis, and is essential for further investigation of PCD pathogenesis. However, a central role of CDR2 in PCD can not be excluded. By determining the subcellular locations of the proteins and binding partners, we are one step closer in understanding the functions of CDR2 and CDR2L in anti-Yo associated PCD.Doktorgradsavhandlin

Prognostic Factors In Breast Cancer. With Special Reference to Cyclins A, B1, D1 and E, MMP-1 and Decorin

Breast cancer is a highly heterogenous malignancy, which despite of the similar histological type shows different clinical behaviour and response to therapy. Prognostic factors are used to estimate the risk for recurrence and the likelihood of treatment effectiveness. Because breast cancer is one of the most common causes of cancer death in women worldwide, identification of new prognostic markers are needed to develop more specific and targeted therapies. Cancer is caused by uncontrolled cell proliferation. The cell cycle is controlled by specific proteins, which are known as cyclins. They function at important checkpoints by activating cyclin-dependent kinase enzymes. Overexpression of different cyclins has been linked to several cancer types and altered expression of cyclins A, B1, D1 and E has been associated with poor survival. Little is known about the combined expression of cyclins in relation to the tumour grade, breast cancer subtype and other known prognostic factors. In this study cyclins A, B1 and E were shown to correlate with histological grade, Ki-67 and HER2 expression. Overexpression of cyclin D1 correlated with receptor status and non-basal breast cancer suggesting that cyclin D1 might be a marker of good prognosis. Proteolysis in the surrounding tumour stroma is increased during cancer development. Matrix metalloproteinases (MMPs) are proteolytic enzymes that are capable of degrading extracellular matrix proteins. Increased expression and activation of several MMPs have been found in many cancers and MMPs appear to be important regulators of invasion and metastasis. In this study MMP-1 expression was analysed in breast cancer epithelial cells and in cancer associated stromal cells. MMP-1 expression by breast cancer epithelial cells was found to carry an independent prognostic value as did Ki-67 and bcl-2. The results suggest that in addition to stromal cells MMP-1 expression in tumour cells control breast cancer progression. Decorin is a small proteoglycan and an important component of the extracellular matrix. Decorin has been shown to inhibit growth of tumour cells and reduced decorin expression is associated with a poor prognosis in several cancer types. There has been some suspicion wheather different cancer cells express decorin. In this study decorin expression was shown to localize only in the cells of the original stroma, while breast cancer epithelial cells were negative for decorin expression. However, transduction of decorin in decorin-negative human breast cancer cells markedly modulated the growth pattern of these cells. This study provides evidence that targeted decorin transduction to breast cancer cells could be used as a novel adjuvant therapy in breast malignancies.Siirretty Doriast

The Role of Prostaglandin E2 in the Expression of Hemoglobin During Prenatal and Postnatal Brain Development - Potential Connection to Autism Spectrum Disorders

Abnormal levels of the fetal hemoglobin isoform Hbb- and the adult isoforms Hbb- and Hba- have been found in the prenatal male brain of our mouse model lacking prostaglandin E2 (PGE2) producing enzyme cyclooxygenase-2 (COX2-knockout). Dysregulations of the COX2/PGE2 pathway has also been linked to autism. In this study, we use quantitative Real-Time PCR, western blots, and immunohistochemistry to investigate the expression of Hba-, Hbb-, and Hbb- in the brain of COX2-knockin (COX2-KI) mice. Overall, gene expression was affected in a sex-dependent manner and by deficits of the COX2/PGE2 pathway. Protein expression analysis also revealed sex-dependent changes and differences for the COX2-KI animals. Interestingly, regional expression analysis showed a distinct pattern of Hbb- in the white matter, whereas Hba- and Hbb- were expressed in the grey matter. These results add new knowledge about the involvement of COX2/PGE2 pathway in the regulation of hemoglobin in the brain with implications for autism

Identificación of ERBB4 and SOX1 role in central nervous system tumors. Implication in medulloblastoma and glioblastoma.

223 p.Los tumores cerebrales, como el glioblastoma (GBM) en adultos y el meduloblastoma (MB) en niños, representan uno de los retos más importantes de la medicina actual, ya que resisten a las terapias disponibles, recurren y se diseminan. Dichos tumores presentan una alta heterogeneidad, ya que albergan una subpoblación de células madre tumorales (CSCs), responsables de la metástasis, resistencia a tratamientos y recurrencia tumoral. Además, la desregulación de aquellos genes relacionados con el desarrollo embrionario y el mantenimiento de las células madre, como ERBB4 y SOX1, parece ser crítica para el desarrollo y la progresión del fenotipo canceroso. La presente tesis doctoral tiene como objetivo principal determinar la función de ambos genes en el GBM y el MB, así como establecer su relación con las CSCs específicas de cada tumor (GSCs y MBSCs, respectivamente). En primer lugar, observamos que ERBB4 tiene una función esencial en el desarrollo del cerebelo, controlando la población de las células progenitoras y su proceso de migración. Además, determinamos que ERBB4 se encuentra altamente expresado en los MBs del Grupo 4 y SOX1 en los del Grupo SHH, asociándose en ambos casos a una peor supervivencia. Al inhibir la expresión de ERBB4 y SOX1, observamos una reducción de la viabilidad celular, capacidad de autorrenovación, y de iniciación y progresión tumoral. Asimismo, identificamos un enriquecimiento de la expresión de SOX1 y ERBB4 en las MBSCs. También establecimos una relación entre la elevada expresión de SOX1 y una peor supervivencia en GBM, además del enriquecimiento de su expresión en las GSCs. Por último, al inhibir la expresión de SOX1 en GBM, observamos una disminución de la capacidad de proliferación, iniciación y progresión tumoral. En conjunto, nuestros resultados sugieren un papel oncogénico hasta ahora no descrito de ERBB4 y SOX1 en MB y GBM. Además, este trabajo propone la inhibición de ERBB4 y SOX1 como una estrategia terapéutica prometedora para atacar las CSCs y así combatir la resistencia a las terapias

Identificación of ERBB4 and SOX1 role in central nervous system tumors. Implication in medulloblastoma and glioblastoma.

223 p.Los tumores cerebrales, como el glioblastoma (GBM) en adultos y el meduloblastoma (MB) en niños, representan uno de los retos más importantes de la medicina actual, ya que resisten a las terapias disponibles, recurren y se diseminan. Dichos tumores presentan una alta heterogeneidad, ya que albergan una subpoblación de células madre tumorales (CSCs), responsables de la metástasis, resistencia a tratamientos y recurrencia tumoral. Además, la desregulación de aquellos genes relacionados con el desarrollo embrionario y el mantenimiento de las células madre, como ERBB4 y SOX1, parece ser crítica para el desarrollo y la progresión del fenotipo canceroso. La presente tesis doctoral tiene como objetivo principal determinar la función de ambos genes en el GBM y el MB, así como establecer su relación con las CSCs específicas de cada tumor (GSCs y MBSCs, respectivamente). En primer lugar, observamos que ERBB4 tiene una función esencial en el desarrollo del cerebelo, controlando la población de las células progenitoras y su proceso de migración. Además, determinamos que ERBB4 se encuentra altamente expresado en los MBs del Grupo 4 y SOX1 en los del Grupo SHH, asociándose en ambos casos a una peor supervivencia. Al inhibir la expresión de ERBB4 y SOX1, observamos una reducción de la viabilidad celular, capacidad de autorrenovación, y de iniciación y progresión tumoral. Asimismo, identificamos un enriquecimiento de la expresión de SOX1 y ERBB4 en las MBSCs. También establecimos una relación entre la elevada expresión de SOX1 y una peor supervivencia en GBM, además del enriquecimiento de su expresión en las GSCs. Por último, al inhibir la expresión de SOX1 en GBM, observamos una disminución de la capacidad de proliferación, iniciación y progresión tumoral. En conjunto, nuestros resultados sugieren un papel oncogénico hasta ahora no descrito de ERBB4 y SOX1 en MB y GBM. Además, este trabajo propone la inhibición de ERBB4 y SOX1 como una estrategia terapéutica prometedora para atacar las CSCs y así combatir la resistencia a las terapias