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The nematode worm C. elegans is widely used in basic and translational research. The creation of transgenic strains by injecting DNA constructs into the worm's gonad is an essential step in many C. elegans research projects. This paper describes the fabrication and use of a minimalist microfluidic chip for performing microinjections. The worm is immobilized in a tight-fitting microchannel, one sidewall of which is a thin elastomeric membrane through which the injection pipet penetrates to reach the worm. The pipet is neither broken nor clogged by passing through the membrane, and the membrane reseals when the pipet is withdrawn. Rates of survival and transgenesis are similar to those in the conventional method. Novice users found injections using the device easier to learn than the conventional method. The principle of direct penetration of elastomeric membranes is adaptable to microinjections in a wide range of organisms including cells, embryos, and other small animal models. It could, therefore, lead to a new generation of microinjection systems for basic, translational, and industrial applications.
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OBJECTIVES: Sessile serrated adenoma/polyps (SSA/Ps) contribute up to 30% of all colon cancers. There is considerable histological overlap between SSA/Ps and hyperplastic polyps. Inadequate consensus exists among pathologists, and no molecular biomarkers exist to differentiate these lesions with high accuracy. Lack of reliable diagnosis adversely affects clinical care. We previously defined a novel 7-gene panel by RNA sequencing that differentiates SSA/Ps from hyperplastic polyps. Here, we use the 7-gene panel as a molecular approach to differentiate SSA/Ps and HPs with higher sensitivity and specificity in a large sample set from a tertiary health care center. METHODS: Reverse transcription quantitative polymerase chain reaction of the 7-gene panel was performed on 223 formalin-fixed, paraffin-embedded serrated polyp and normal colon samples. We compare the sensitivity and specificity of the 7-gene panel with the BRAF and KRAS mutation incidence in differentiating SSA/Ps and HPs. We also evaluate the clinical data of patients with SSA/Ps showing high and low expression of the gene panel. RESULTS: The 7-gene RNA expression panel differentiates SSA/Ps and HPs with 89.2% sensitivity and 88.4% specificity. The gene panel outperforms BRAF mutation in identification of SSA/Ps. Clinical data suggest that expression of the 7-gene panel correlates with the development of SSA/Ps in the future. DISCUSSION: This study describes a novel 7-gene panel that identifies SSA/Ps with improved accuracy. Our data show that RNA markers of SSA/Ps advance the distinction of serrated lesions and contribute to the study of the serrated pathway to colon cancer.
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Adenoma/diagnóstico , Neoplasias del Colon/prevención & control , Pólipos del Colon/diagnóstico , Perfilación de la Expresión Génica , Adenoma/genética , Biomarcadores/análisis , Neoplasias del Colon/genética , Pólipos del Colon/genética , Colonoscopía , Análisis Mutacional de ADN , Diagnóstico Diferencial , Estudios de Factibilidad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Squamous cell carcinoma (SCC) of the lung is the second most common subtype of lung cancer. With limited treatment options, the 5-year survival rate of SCC is only 15%. Although genomic alterations in SCC have been characterized, identifying the alterations that drive SCC is critical for improving treatment strategies. Mouse models of SCC are currently limited. Using lentiviral delivery of Sox2 specifically to the mouse lung, we tested the ability of Sox2 to promote tumorigenesis in multiple tumor suppressor backgrounds. Expression of Sox2, frequently amplified in human SCC, specifically cooperates with loss of Lkb1 to promote squamous lung tumors. Mouse tumors exhibit characteristic histopathology and biomarker expression similar to human SCC. They also mimic human SCCs by activation of therapeutically relevant pathways including STAT and mTOR. This model may be utilized to test the contribution of additional driver alterations in SCC, as well as for preclinical drug discovery.
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Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción SOXB1/metabolismo , Proteínas Quinasas Activadas por AMP , Animales , Biomarcadores de Tumor/genética , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Proteínas Serina-Treonina Quinasas/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción STAT/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
BACKGROUND: Sessile serrated adenomas/polyps (SSA/Ps) may account for 20-30% of colon cancers. Although large SSA/Ps are generally recognized phenotypically, small (<1 cm) or dysplastic SSA/Ps are difficult to differentiate from hyperplastic or small adenomatous polyps by endoscopy and histopathology. Our aim was to define the comprehensive gene expression phenotype of SSA/Ps to better define this cancer precursor. RESULTS: RNA sequencing was performed on 5' capped RNA from seven SSA/Ps collected from patients with the serrated polyposis syndrome (SPS) versus eight controls. Highly expressed genes were analyzed by qPCR in additional SSA/Ps, adenomas and controls. The cellular localization and level of gene products were examined by immunohistochemistry in syndromic and sporadic SSA/Ps, adenomatous and hyperplastic polyps and controls. We identified 1,294 differentially expressed annotated genes, with 106 increased ≥10-fold, in SSA/Ps compared to controls. Comparing these genes with an array dataset for adenomatous polyps identified 30 protein coding genes uniquely expressed ≥10-fold in SSA/Ps. Biological pathways altered in SSA/Ps included mucosal integrity, cell adhesion, and cell development. Marked increased expression of MUC17, the cell junction protein genes VSIG1 and GJB5, and the antiapoptotic gene REG4 were found in SSA/Ps, relative to controls and adenomas, were verified by qPCR analysis of additional SSA/Ps (nâ=â21) and adenomas (nâ=â10). Immunohistochemical staining of syndromic (n≥11) and sporadic SSA/Ps (n≥17), adenomatous (n≥13) and hyperplastic (n≥10) polyps plus controls (n≥16) identified unique expression patterns for VSIG1 and MUC17 in SSA/Ps. CONCLUSION: A subset of genes and pathways are uniquely increased in SSA/Ps, compared to adenomatous polyps, thus supporting the concept that cancer develops by different pathways in these phenotypically distinct polyps with markedly different gene expression profiles. Immunostaining for a subset of these genes differentiates both syndromic and sporadic SSA/Ps from adenomatous and hyperplastic polyps.
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Adenoma/genética , Adenoma/patología , Pólipos del Colon/genética , Pólipos del Colon/patología , Análisis de Secuencia de ARN/métodos , Adenoma/metabolismo , Anciano , Antígenos de Neoplasias/metabolismo , Análisis por Conglomerados , Colon/metabolismo , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Colonoscopía , Conexinas/metabolismo , Análisis Mutacional de ADN , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Lectinas Tipo C/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Persona de Mediana Edad , Mucinas/metabolismo , Proteínas Asociadas a Pancreatitis , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas B-raf/genéticaRESUMEN
Desulfitobacterium dehalogenans can use chlorinated aromatics including polychlorinated biphenyls as electron acceptors in a process called dehalorespiration. Expression of the cpr gene cluster involved in this process is regulated by CprK, which is a member of the CRP/FNR (cAMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. High affinity interaction of the chlorinated aromatic compound with the effector domain of CprK triggers binding of CprK to an upstream target DNA sequence, which leads to transcriptional activation of the cpr gene cluster. When incubated with oxygen or diamide, CprK undergoes inactivation; subsequent treatment with dithiothreitol restores activity. Using mass spectrometry, this study identifies two classes of redox-active thiol groups that form disulfide bonds upon oxidation. Under oxidative conditions, Cys105, which is conserved in FNR and most other CprK homologs, forms an intramolecular disulfide bond with Cys111, whereas an intermolecular disulfide bond is formed between Cys11 and Cys200. SDS-PAGE and site-directed mutagenesis experiments indicate that the Cys11/Cys200 disulfide bond links two CprK subunits in an inactive dimer. Isothermal calorimetry and intrinsic fluorescence quenching studies show that oxidation does not change the affinity of CprK for the effector. Therefore, reversible redox inactivation is manifested at the level of DNA binding. Our studies reveal a strategy for limiting expression of a redox-sensitive pathway by using a thiol-based redox switch in the transcription factor.
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Proteínas Bacterianas/metabolismo , Cisteína/metabolismo , Desulfitobacterium , Regulación Bacteriana de la Expresión Génica , Proteínas Hierro-Azufre/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desulfitobacterium/genética , Desulfitobacterium/metabolismo , Diamida/metabolismo , Dimerización , Disulfuros/química , Disulfuros/metabolismo , Ditiotreitol/metabolismo , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Oxígeno/metabolismo , Péptidos/genética , Péptidos/metabolismo , Bifenilos Policlorados/química , Bifenilos Policlorados/metabolismo , Estructura Cuaternaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Reactivos de Sulfhidrilo/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Halorespiration is a bacterial respiratory process in which haloorganic compounds act as terminal electron acceptors. This process is controlled at transcriptional level by CprK, a member of the ubiquitous CRP-FNR family. Here we present the crystal structures of oxidized CprK in presence of the ligand ortho-chlorophenolacetic acid and of reduced CprK in absence of this ligand. These structures reveal that highly specific binding of chlorinated, rather than the corresponding non-chlorinated, phenolic compounds in the NH(2)-terminal beta-barrels causes reorientation of these domains with respect to the central alpha-helix at the dimer interface. Unexpectedly, the COOH-terminal DNA-binding domains dimerize in the non-DNA binding state. We postulate the ligand-induced conformational change allows formation of interdomain contacts that disrupt the DNA domain dimer interface and leads to repositioning of the helix-turn-helix motifs. These structures provide a structural framework for further studies on transcriptional control by CRP-FNR homologs in general and of halorespiration regulation by CprK in particular.
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Proteínas Bacterianas/química , Desulfitobacterium/genética , Regulación Bacteriana de la Expresión Génica , Transcripción Genética , Cristalización , Desulfitobacterium/metabolismo , Dimerización , Fenilacetatos/metabolismo , Estructura Secundaria de ProteínaRESUMEN
Desulfomonile, Desulfitobacterium, and Dehalobacter are anaerobic microbes that can derive energy from the reductive dehalogenation of chlorinated organic compounds, many of which are environmental pollutants. There is very little information about how anaerobic dehalorespiration is regulated. An open reading frame within the Desulfitobacterium dehalogenans chlorophenol reductase (cpr) gene cluster (cprK) was proposed to be a transcriptional regulatory protein (Smidt, H., van Leest, M., van der Oost, J., and deVos, W. M. (2000) J. Bacteriol. 182, 5683-5691). We have cloned, actively overexpressed in Escherichia coli, and purified to homogeneity the D. dehalogenans CprK. The results of electrophoretic mobility shift assays, DNA footprinting studies, and promoter-lac fusion experiments indicate that CprK is a transcriptional activator of the cpr gene cluster. CprK binds 3-chloro-4-hydroxyphenylacetate (CHPA) with high affinity (K(d) = 3.5 mum, determined by isothermal titration calorimetry), which promotes its specific interaction with a DNA sequence (TTAAT-N4-ACTAA) located upstream of the -35 and -10 promoter regions of several cpr genes and activates transcription of these genes. Binding to the upstream "box" sequence increases the affinity of CprK for CHPA by approximately 10-fold (K(d) = 0.4 mum, determined by electrophoretic mobility shift assays). Chlorophenylacetate, which lacks the ortho-hydroxy group, and hydroxyphenylacetate, lacking the chlorine group, do not activate transcription or promote DNA binding, even at millimolar concentrations, at least 1000-fold higher than the K(d) value for CHPA. Lacking metals, CprK is oxygen-sensitive. Oxidation by diamide, which converts thiols to the disulfide, inactivates CprK, and reduction of the oxidized protein by dithiothreitol fully restores DNA binding, indicating that CprK is redox-regulated and is active only when reduced. This is the first reported characterization of a transcriptional regulator of anaerobic dehalorespiration.