RESUMEN
Endometriosis is a common inflammatory estrogen-dependent gynecological disorder, associated with pelvic pain and reduced fertility in women. Several aspects of this disorder and its cellular and molecular etiology remain unresolved. We have analyzed the global gene expression patterns in the endometrium, peritoneum and in endometriosis lesions of endometriosis patients and in the endometrium and peritoneum of healthy women. In this report, we present the EndometDB, an interactive web-based user interface for browsing the gene expression database of collected samples without the need for computational skills. The EndometDB incorporates the expression data from 115 patients and 53 controls, with over 24000 genes and clinical features, such as their age, disease stages, hormonal medication, menstrual cycle phase, and the different endometriosis lesion types. Using the web-tool, the end-user can easily generate various plot outputs and projections, including boxplots, and heatmaps and the generated outputs can be downloaded in pdf-format.Availability and implementationThe web-based user interface is implemented using HTML5, JavaScript, CSS, Plotly and R. It is freely available from https://endometdb.utu.fi/ .
Asunto(s)
Endometriosis/genética , Endometrio/metabolismo , Expresión Génica , Peritoneo/metabolismo , Endometrio/patología , Femenino , Humanos , Peritoneo/patologíaRESUMEN
Preoperative diagnostics of ovarian neoplasms rely on ultrasound imaging and the serum biomarkers CA125 and HE4. However, these markers may be elevated in non-neoplastic conditions and may fail to identify most non-serous epithelial cancer subtypes. The objective of this study was to identify histotype-specific serum biomarkers for mucinous ovarian cancer. The candidate genes with mucinous histotype specific expression profile were identified from publicly available gene-expression databases and further in silico data mining was performed utilizing the MediSapiens database. Candidate biomarker validation was done using qRT-PCR, western blotting and immunohistochemical staining of tumor tissue microarrays. The expression level of the candidate gene in serum was compared to the serum CA125 and HE4 levels in a patient cohort of prospectively collected advanced ovarian cancer. Database searches identified REG4 as a potential biomarker with specificity for the mucinous ovarian cancer subtype. The specific expression within epithelial ovarian tumors was further confirmed by mRNA analysis. Immunohistochemical staining of ovarian tumor tissue arrays showed distinctive cytoplasmic expression pattern only in mucinous carcinomas and suggested differential expression between benign and malignant mucinous neoplasms. Finally, an ELISA based serum biomarker assay demonstrated increased expression only in patients with mucinous ovarian cancer. This study identifies REG4 as a potential serum biomarker for histotype-specific detection of mucinous ovarian cancer and suggests serum REG4 measurement as a non-invasive diagnostic tool for postoperative follow-up of patients with mucinous ovarian cancer.
Asunto(s)
Adenocarcinoma Mucinoso/metabolismo , Lectinas Tipo C/metabolismo , Neoplasias Ováricas/metabolismo , Adenocarcinoma Mucinoso/sangre , Adenocarcinoma Mucinoso/patología , Biomarcadores de Tumor/metabolismo , Antígeno Ca-125/sangre , Bases de Datos Factuales , Femenino , Humanos , Lectinas Tipo C/sangre , Neoplasias Ováricas/sangre , Neoplasias Ováricas/patología , Proteínas Asociadas a Pancreatitis , Proteínas/metabolismo , Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAPRESUMEN
Here we demonstrate a novel homogeneous one-step immunoassay, utilizing a pair of recombinant antibody antigen-binding fragments (Fab), that is specific for HT-2 toxin and has a positive readout. Advantages over the conventional competitive immunoassay formats such as enzyme-linked immunosorbent assay (ELISA) are the specificity, speed, and simplicity of the assay. Recombinant antibody HT2-10 Fab recognizing both HT-2 and T-2 toxins was developed from a phage display antibody library containing 6 × 10(7) different antibody clones. Specificity of the immunoassay was introduced by an anti-immune complex (IC) antibody binding the primary antibody-HT-2 toxin complex. When the noncompetitive immune complex assay was compared to the traditional competitive assay, an over 10-fold improvement in sensitivity was observed. Although the HT2-10 antibody has 100% cross-reactivity for HT-2 and T-2 toxins, the immune complex assay is highly specific for HT-2 alone. The assay performance with real samples was evaluated using naturally contaminated wheat reference material. The half-maximal effective concentration (EC50) value of the time-resolved fluorescence resonance energy transfer (TR-FRET) assay was 9.6 ng/mL, and the limit of detection (LOD) was 0.38 ng/mL (19 µg/kg). The labeled antibodies can be predried to the assay vials, e.g., microtiter plate wells, and readout is ready in 10 min after the sample application.
Asunto(s)
Inmunoensayo , Toxina T-2/análogos & derivados , Anticuerpos Monoclonales/inmunología , Transferencia Resonante de Energía de Fluorescencia , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Conformación Molecular , Toxina T-2/análisis , Toxina T-2/inmunologíaRESUMEN
New molecular information on potential therapeutic targets or tools for noninvasive diagnosis for endometriosis are important for patient care and treatment. However, surprisingly few efforts have described endometriosis at the protein level. In this work we enumerate the proteins in patient endometrium and ovarian endometrioma by extensive and comprehensive analysis of minute amounts of cryosectioned tissues in a three-tiered mass spectrometric approach. Quantitative comparison of the tissues revealed 214 differentially expressed proteins in ovarian endometrioma and endometrium. These proteins are reported here as a resource of SRM (selected reaction monitoring) assays that are unique, standardized, and openly available. Pathway analysis of the proteome measurements revealed a potential role for Transforming growth factor ß-1 in ovarian endometriosis development. Subsequent mRNA microarray analysis further revealed clear ovarian endometrioma specificity for a subset of these proteins, which was also supported by further in silico studies. In this process two important proteins emerged, Calponin-1 and EMILIN-1, that were additionally confirmed in ovarian endometrioma tissues by immunohistochemistry and Western blotting. This study provides the most comprehensive molecular description of ovarian endometriosis to date and researchers with new molecular methods and tools for high throughput patient screening using the SRM assays.
Asunto(s)
Endometriosis/metabolismo , Espectrometría de Masas/métodos , Proteínas/análisis , Proteínas/metabolismo , Proteómica/métodos , Western Blotting , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Simulación por Computador , Endometriosis/genética , Femenino , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Enfermedades del Ovario/genética , Enfermedades del Ovario/metabolismo , Proteínas/genética , Reproducibilidad de los Resultados , Factor de Crecimiento Transformador beta1/metabolismo , CalponinasRESUMEN
Glandular epithelial cells differentiate into complex multicellular or acinar structures, when embedded in three-dimensional (3D) extracellular matrix. The spectrum of different multicellular morphologies formed in 3D is a sensitive indicator for the differentiation potential of normal, non-transformed cells compared to different stages of malignant progression. In addition, single cells or cell aggregates may actively invade the matrix, utilizing epithelial, mesenchymal or mixed modes of motility. Dynamic phenotypic changes involved in 3D tumor cell invasion are sensitive to specific small-molecule inhibitors that target the actin cytoskeleton. We have used a panel of inhibitors to demonstrate the power of automated image analysis as a phenotypic or morphometric readout in cell-based assays. We introduce a streamlined stand-alone software solution that supports large-scale high-content screens, based on complex and organotypic cultures. AMIDA (Automated Morphometric Image Data Analysis) allows quantitative measurements of large numbers of images and structures, with a multitude of different spheroid shapes, sizes, and textures. AMIDA supports an automated workflow, and can be combined with quality control and statistical tools for data interpretation and visualization. We have used a representative panel of 12 prostate and breast cancer lines that display a broad spectrum of different spheroid morphologies and modes of invasion, challenged by a library of 19 direct or indirect modulators of the actin cytoskeleton which induce systematic changes in spheroid morphology and differentiation versus invasion. These results were independently validated by 2D proliferation, apoptosis and cell motility assays. We identified three drugs that primarily attenuated the invasion and formation of invasive processes in 3D, without affecting proliferation or apoptosis. Two of these compounds block Rac signalling, one affects cellular cAMP/cGMP accumulation. Our approach supports the growing needs for user-friendly, straightforward solutions that facilitate large-scale, cell-based 3D assays in basic research, drug discovery, and target validation.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Procesamiento de Imagen Asistido por Computador/métodos , Línea Celular Tumoral , Células Epiteliales/citología , Matriz Extracelular/metabolismo , HumanosRESUMEN
We present a high-throughput roll-to-roll (R2R) manufacturing process for foil-based polymethyl methacrylate (PMMA) chips of excellent optical quality. These disposable, R2R hot embossed microfluidic chips are used for the identification of the antibiotic resistance gene mecA in Staphylococcus epidermidis. R2R hot embossing is an emerging manufacturing technology for polymer microfluidic devices. It is based on continuous feeding of a thermoplastic foil through a pressurized area between a heated embossing cylinder and a blank counter cylinder. Although mass fabrication of foil-based microfluidic chips and their use for biological applications were foreseen already some years ago, no such studies have been published previously.
Asunto(s)
Proteínas Bacterianas/genética , Electroforesis en Gel de Agar , Técnicas Analíticas Microfluídicas/instrumentación , Staphylococcus epidermidis/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/análisis , Farmacorresistencia Bacteriana/genética , Técnicas Analíticas Microfluídicas/métodos , Microscopía Confocal , Polimetil Metacrilato/químicaRESUMEN
BACKGROUND: Type 1 diabetes mellitus results from destruction of the pancreatic insulin-producing beta cells by a chronic autoimmune process. Methods are needed for the detection of circulating autoantibodies to glutamic acid decarboxylase (GAD65), a major marker of this process. METHODS: Streptavidin-coated microtiter plates were incubated with biotinylated GAD65, and after incubation with serum samples from patients with type 1 diabetes mellitus and control individuals, europium-labeled GAD65 was added. After washing steps, the delayed fluorescence was measured in duplicate in a fluorometer. Samples collected from 100 patients with newly diagnosed type 1 diabetes mellitus and 100 healthy controls were measured by the new assay and by a radiobinding assay. RESULTS: The detection limit of the new assay was 1.49 WHO units/mL, the calibration curve was linear to 4140 WHO units/mL, and no hook effect was observed up to 41,400 WHO units/mL. The intraassay CV was 2.1-6.3% over the calibration range. For patient serum samples, the intraassay, interassay, and total CVs were 5.4-7.0%, 9.8-13%, and 12-14%, respectively. Compared with conventional radioimmunologic methods, the analytical range was broader and the analysis time required to perform the measurements was shorter. At a cutoff with 99% specificity, the new assay and the radiobinding assay were positive in 71 and 67 patients, respectively. CONCLUSIONS: The new assay provides a rapid and sensitive nonradioactive method applicable for large-scale screening for beta-cell autoimmunity. It has a broad linear analytical range, is easy to perform and automate, and has sensitivity and specificity comparable to those for the conventional radioisotope assay.