ABSTRACT
In situ transgenesis methods such as viruses and electroporation can rapidly create somatic transgenic mice but lack control over copy number, zygosity, and locus specificity. Here we establish mosaic analysis by dual recombinase-mediated cassette exchange (MADR), which permits stable labeling of mutant cells expressing transgenic elements from precisely defined chromosomal loci. We provide a toolkit of MADR elements for combination labeling, inducible and reversible transgene manipulation, VCre recombinase expression, and transgenesis of human cells. Further, we demonstrate the versatility of MADR by creating glioma models with mixed reporter-identified zygosity or with "personalized" driver mutations from pediatric glioma. MADR is extensible to thousands of existing mouse lines, providing a flexible platform to democratize the generation of somatic mosaic mice. VIDEO ABSTRACT.
Subject(s)
Brain Neoplasms/genetics , Disease Models, Animal , Gene Targeting/methods , Genetic Loci/genetics , Glioma/genetics , Mutagenesis, Insertional/methods , Transgenes/genetics , Animals , Cell Line, Tumor , Female , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neural Stem Cells/metabolism , Recombinases/metabolism , TransfectionABSTRACT
Small vessel strokes (SVS) and intracerebral haemorrhages (ICH) are acute outcomes of cerebral small vessel disease (SVD). Genetic studies combining both phenotypes have identified three loci associated with both traits. However, the genetic cis-regulation at the protein level associated with SVD has not been studied before. We performed a proteome-wide association study (PWAS) using FUSION to integrate a genome-wide association study (GWAS) and brain proteomic data to discover the common mechanisms regulating both SVS and ICH. Dorsolateral prefrontal cortex (dPFC) brain proteomes from the ROS/MAP study (N = 376 subjects and 1443 proteins) and the summary statistics for the SVS GWAS from the MEGASTROKE study (N = 237,511) and multi-trait analysis of GWAS (MTAG)-ICH−SVS from Chung et al. (N = 240,269) were selected. We performed PWAS and then a co-localization analysis with COLOC. The significant and nominal results were validated using a replication dPFC proteome (N = 152). The replicated results (q-value < 0.05) were further investigated for the causality relationship using summary data-based Mendelian randomization (SMR). One protein (ICA1L) was significantly associated with SVS (z-score = −4.42 and p-value = 9.6 × 10−6) and non-lobar ICH (z-score = −4.8 and p-value = 1.58 × 10−6) in the discovery PWAS, with a high co-localization posterior probability of 4. In the validation PWAS, ICA1L remained significantly associated with both traits. The SMR results for ICA1L indicated a causal association of protein expression levels in the brain with SVS (p-value = 3.66 × 10−5) and non-lobar ICH (p-value = 1.81 × 10−5). Our results show that the association of ICA1L with SVS and non-lobar ICH is conditioned by the cis-regulation of its protein levels in the brain.
Subject(s)
Proteome , Stroke , Cerebral Hemorrhage/complications , Cerebral Hemorrhage/genetics , Genome-Wide Association Study , Humans , Proteome/genetics , Proteomics , Stroke/etiologyABSTRACT
Introduction: Obesity promotes hypothalamic inflammation and local morphological changes in astrocytes, including the increased expression of the astrocytic biomarker glial fibrillary acidic protein (GFAP), which is seen as a sign of neuroinflammation.Objective: This study aimed to observe the astrocytic expression of GFAP in different brain areas from female rats that received a hypercaloric (HD) or a normocaloric (ND) diet during puberty (F0 generation) as well as in their male pups (F1 generation).Methods: Female rats received highly palatable HD (Ensure®) or ND from postnatal day (PND) 23-65. On PND90-95, some were euthanized for the immunohistochemical study and some were mated to obtain the F1 generation. Male pups were immunochallenged on PND50 with lipopolysaccharide (LPS, 100â µg/kg) or 0.9% saline solution (1â mL/kg) intraperitoneal injection. Body weight (BW) and retroperitoneal fat weight (RFW) were recorded on PND95 for F0 generation and on PND50 for F1 generation. GFAP expression for both generations was assessed by morphometry in the parietal/frontal cortex, corpus callosum, nucleus accumbens, arcuate/periventricular nuclei of hypothalamus, pons, molecular/granular layers of cerebellum.Results: Female rats fed with HD presented a significant increase in the GFAP expression in all evaluated areas as well as in the RFW. Male rats born from mothers that received HD showed decreased GFAP expression, BW and RFW when treated with LPS in relation to those from mothers fed with ND.Discussion: HD induced astrogliosis in several brain areas in females from F0 generation and an adaptive phenotypic change of decreased GFAP expression in males from F1 generation after LPS challenge.
Subject(s)
Astrocytes/metabolism , Brain/metabolism , Glial Fibrillary Acidic Protein/metabolism , Obesity/metabolism , Animals , Encephalitis/metabolism , Energy Intake , Female , Gliosis/metabolism , Lipopolysaccharides , Male , Rats, WistarABSTRACT
Chronic inflammation and metabolic reprogramming have been proposed as hallmarks of cancer development. Currently, many of the functional clues between these two phenomena are studied under the integrative view of functional stroma-epithelia interaction. It has been proposed that stromal cells, due to their abundance and avidity for glucose, are able to modify the metabolic behavior of an entire solid tumor. In the present study, using a mammary stromal cell line derived from healthy tissue subjected to long-term culture in low (5 mM) or high (25 mM) glucose, we found that the hyperglycemic condition favors the establishment of a pro-inflammatory and pro-oxidant environment characterized by the induction of the COX-2/PGE2 axis. In this condition, epithelial migration was stimulated. Moreover, we also found that stromal-derived PGE2, acting as a stimulator of IL-1 epithelial expression was one of the factors that promote the acquisition of motile properties by epithelial cells and the maintenance of a COX-2/PGE2-dependent inflammatory condition. Overall, our work provides experimental evidence that glucose stimulates a tumor inflammatory environment that, as a result of a functional cross-talk between stroma and epithelia, may be responsible for tumor progression. J. Cell. Biochem. 118: 994-1002, 2017. © 2016 Wiley Periodicals, Inc.
Subject(s)
Breast Neoplasms/metabolism , Cyclooxygenase 2/metabolism , Dinoprostone/pharmacology , Glucose/pharmacology , Interleukin-1/metabolism , Stromal Cells/cytology , Breast Neoplasms/immunology , Cell Culture Techniques , Cell Line, Tumor , Cell Movement , Cells, Cultured , Female , Humans , MCF-7 Cells , Reactive Oxygen Species/metabolism , Stromal Cells/drug effects , Stromal Cells/metabolism , Tumor MicroenvironmentABSTRACT
Potocki-Lupski syndrome (PTLS) is a genomic disorder associated with an â¼3 Mb duplication in 17p11.2. Clinical features include leanness, intellectual disability, autistic features and developmental deficits. RAI1 gene dosage is associated with the PTLS phenotypes. To understand where and when Rai1 overexpression is detrimental, we generated a mouse that over-expresses Rai1 conditionally in forebrain neurons (I-Rai1). Phenotypic characterization of I-Rai1 mice showed significant underweight, hyperactivity and impaired learning and memory ability compared with wild-type littermates. Doxycycline administration can turn off the transgene expression allowing the restoration of Rai1 normal expression levels. When the transgene was turned off from conception to 3 months of age, no phenotypic differences were observed between I-Rai1 and their wild-type littermates. Surprisingly, we found that turning off the transgene expression before the onset of the phenotypes (1-3 months) or after the onset of the phenotypes (3-5 months) cannot prevent nor reverse the phenotypic outcomes. Our results indicate that Rai1 dosage in forebrain neurons is critical during the development and is related to body weight regulation, activity levels and learning and memory.
Subject(s)
Gene Dosage/genetics , Hyperkinesis/genetics , Maze Learning/physiology , Prosencephalon/metabolism , Trans-Activators/genetics , Abnormalities, Multiple , Animals , Anti-Bacterial Agents/pharmacology , Body Weight/genetics , Chromosome Disorders , Chromosome Duplication , Disease Models, Animal , Doxycycline/pharmacology , Intellectual Disability/genetics , Memory/physiology , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , Motor Activity/genetics , Smith-Magenis Syndrome/genetics , Trans-Activators/biosynthesis , Transgenes/drug effects , Transgenes/geneticsABSTRACT
A large fraction of genome variation between individuals is comprised of submicroscopic copy number variation of genomic DNA segments. We assessed the relative contribution of structural changes and gene dosage alterations on phenotypic outcomes with mouse models of Smith-Magenis and Potocki-Lupski syndromes. We phenotyped mice with 1n (Deletion/+), 2n (+/+), 3n (Duplication/+), and balanced 2n compound heterozygous (Deletion/Duplication) copies of the same region. Parallel to the observations made in humans, such variation in gene copy number was sufficient to generate phenotypic consequences: in a number of cases diametrically opposing phenotypes were associated with gain versus loss of gene content. Surprisingly, some neurobehavioral traits were not rescued by restoration of the normal gene copy number. Transcriptome profiling showed that a highly significant propensity of transcriptional changes map to the engineered interval in the five assessed tissues. A statistically significant overrepresentation of the genes mapping to the entire length of the engineered chromosome was also found in the top-ranked differentially expressed genes in the mice containing rearranged chromosomes, regardless of the nature of the rearrangement, an observation robust across different cell lineages of the central nervous system. Our data indicate that a structural change at a given position of the human genome may affect not only locus and adjacent gene expression but also "genome regulation." Furthermore, structural change can cause the same perturbation in particular pathways regardless of gene dosage. Thus, the presence of a genomic structural change, as well as gene dosage imbalance, contributes to the ultimate phenotype.
Subject(s)
Disease Models, Animal , Gene Dosage , Smith-Magenis Syndrome/genetics , Abnormalities, Multiple , Animals , Chromosome Disorders , Chromosome Duplication , Gene Expression , Mice , Phenotype , RNA, Messenger/genetics , Recombination, GeneticABSTRACT
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent responsible for the coronavirus disease 2019 (COVID-19). The high rate of mutation of this virus is associated with a quick emergence of new viral variants that have been rapidly spreading worldwide. Several mutations have been documented in the receptor-binding domain (RBD) of the viral spike protein that increases the interaction between SARS-CoV-2 and its cellular receptor, the angiotensin-converting enzyme 2 (ACE2). Mutations in the spike can increase the viral spread rate, disease severity, and the ability of the virus to evade either the immune protective responses, monoclonal antibody treatments, or the efficacy of current licensed vaccines. This review aimed to highlight the functional virus classification used by the World Health Organization (WHO), Phylogenetic Assignment of Named Global Outbreak (PANGO), Global Initiative on Sharing All Influenza Data (GISAID), and Nextstrain, an open-source project to harness the scientific and public health potential of pathogen genome data, the chronological emergence of viral variants of concern (VOCs) and variants of interest (VOIs), the major findings related to the rate of spread, and the mutations in the spike protein that are involved in the evasion of the host immune responses elicited by prior SARS-CoV-2 infections and by the protection induced by vaccination.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Mutation , Phylogeny , Protein Binding , SARS-CoV-2/genetics , Spike Glycoprotein, CoronavirusABSTRACT
Cimicifuga foetida, an Asian Cimicifuga species, has been employed as a cooling and detoxification agent in traditional Chinese medicine since ancient times. For this herb, two cycloartane triterpene glycosides isolated from the rhizomes have demonstrated cytotoxicity on rat tumor and human cancer cell lines. Since human Hsp27 is increased in various human cancers and exhibits cytoprotective activity that affects tumorigenesis and the susceptibility of tumours to cancer treatment, the purpose of this research was to study the expression of this protein in MCF-7 breast cancer cells. To accomplish this aim, MCF-7 cells were exposed to different concentrations of Cimicifuga foetida extract showing a reduction in cell number measured by the sulforhodamine assay. In addition, the expression of Hsp-27 mRNA detected by RT-PCR and Hsp-27 protein detected by immnofluorescence was present in all conditions, except when using the highest concentration of Cimicifuga foetida extract (2,000 jig /L). We conclude that Hsp 27 expression at 2,000 jig /L Cimicifuga foetida extract is diminished. This is the first report showing the Hsp-27 expression after exposure to Cimicifuga foetida extract in MCF-7 cells.
Subject(s)
Antineoplastic Agents, Phytogenic/therapeutic use , Breast Neoplasms/drug therapy , Cimicifuga/chemistry , HSP27 Heat-Shock Proteins/metabolism , Phytotherapy , Plant Extracts/therapeutic use , Adenocarcinoma/drug therapy , Adenocarcinoma/metabolism , Adult , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Blotting, Western , Breast Neoplasms/metabolism , Cattle , Female , Fluorescent Antibody Technique , Humans , MCF-7 Cells , Plant Extracts/analysis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: Smith-Magenis Syndrome is a contiguous gene syndrome in which the dosage sensitive gene has been identified: the Retinoic Acid Induced 1 (RAI1). Little is known about the function of human RAI1. RESULTS: We generated the full-length cDNA of the wild type protein and five mutated forms: RAI1-HA 2687delC, RAI1-HA 3103delC, RAI1 R960X, RAI1-HA Q1562R, and RAI1-HA S1808N. Four of them have been previously associated with SMS clinical phenotype. Molecular weight, subcellular localization and transcription factor activity of the wild type and mutant forms were studied by western blot, immunofluorescence and luciferase assays respectively. The wild type protein and the two missense mutations presented a higher molecular weight than expected, localized to the nucleus and activated transcription of a reporter gene. The frameshift mutations generated a truncated polypeptide with transcription factor activity but abnormal subcellular localization, and the same was true for the 1-960aa N-terminal half of RAI1. Two different C-terminal halves of the RAI1 protein (1038aa-end and 1229aa-end) were able to localize into the nucleus but had no transactivation activity. CONCLUSION: Our results indicate that transcription factor activity and subcellular localization signals reside in two separate domains of the protein and both are essential for the correct functionality of RAI1. The pathogenic outcome of some of the mutated forms can be explained by the dissociation of these two domains.
Subject(s)
Mutation , Smith-Magenis Syndrome/genetics , Transcription Factors/genetics , Animals , Cell Line , Chromosome Mapping , DNA Mutational Analysis , Gene Dosage , Genes, Reporter , Humans , Mice , Molecular Sequence Data , Smith-Magenis Syndrome/physiopathology , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/metabolismABSTRACT
The Potocki-Lupski syndrome (PTLS) is associated with a microduplication of 17p11.2. Clinical features include multiple congenital and neurobehavioral abnormalities and autistic features. We have generated a PTLS mouse model, Dp(11)17/+, that recapitulates some of the physical and neurobehavioral phenotypes present in patients. Here, we investigated the social behavior and gene expression pattern of this mouse model in a pure C57BL/6-Tyr(c-Brd) genetic background. Dp(11)17/+ male mice displayed normal home-cage behavior but increased anxiety and increased dominant behavior in specific tests. A subtle impairment in the preference for a social target versus an inanimate target and abnormal preference for social novelty (the preference to explore an unfamiliar mouse versus a familiar one) was also observed. Our results indicate that these animals could provide a valuable model to identify the specific gene(s) that confer abnormal social behaviors and that map within this delimited genomic deletion interval. In a first attempt to identify candidate genes and for elucidating the mechanisms of regulation of these important phenotypes, we directly assessed the relative transcription of genes within and around this genomic interval. In this mouse model, we found that candidates genes include not only most of the duplicated genes, but also normal-copy genes that flank the engineered interval; both categories of genes showed altered expression levels in the hippocampus of Dp(11)17/+ mice.
Subject(s)
Autistic Disorder/physiopathology , Disease Models, Animal , Gene Expression , Animals , Autistic Disorder/genetics , Behavior, Animal , Brain/growth & development , Female , Gene Expression Profiling , Humans , Male , Mice , Mice, Inbred C57BL , Organ Size , Phenotype , Species SpecificityABSTRACT
Acenocoumarol is an oral anticoagulant with significant interindividual dose variations. Variants in CYP2C9 and VKORC1 have been associated with acenocoumarol maintenance dose. We analysed whether any of the 49 polymorphisms in CYP2C9 and VKORC1 previously associated with acenocoumarol maintenance dose in a Genome-Wide Association study (GWAs) in Dutch population are associated with stroke recurrence, intracranial haemorrhage (ICH) and acenocoumarol maintenance dose in a Spanish population. We performed a GWAs using Human Core Exome-chip (Illumina) in 78 patients stroke patients treated with acenocoumarol for secondary prevention enrolled as part of the prospective investigator-initiated study (IIS) SEDMAN Study. Patients were followed-up a median of 12.8 months. Three and eight patients had recurrent stroke and ICH events, respectively. We found 14 of the 49 published variants associated with acenocoumarol maintenance dose (p < 0.05). Six polymorphisms were associated with stroke recurrence and four variants with ICH (p < 0.05). In conclusion, variants in VKORC1 and CYP2C9 are associated with acenocoumarol maintenance dose, stroke recurrence and ICH in a Spanish cohort. These results highlight the relevance of studying pharmacogenetics associated with efficacy and safety of anticoagulant drugs and justify studies with larger sample size and different ethnic populations.
Subject(s)
Acenocoumarol , Anticoagulants , Cytochrome P-450 CYP2C9/genetics , Stroke/drug therapy , Vitamin K Epoxide Reductases/genetics , Acenocoumarol/administration & dosage , Aged , Aged, 80 and over , Anticoagulants/administration & dosage , Female , Genome-Wide Association Study , Humans , Male , Pharmacogenetics , Polymorphism, Single Nucleotide , Prospective Studies , SpainABSTRACT
Aerobic metabolism brings inexorably the production of reactive oxygen species (ROS), which are counterbalanced by intrinsic antioxidant defenses avoiding deleterious intracellular effects. Redox balance is the resultant of metabolic functioning under environmental inputs (i.e. diet, pollution) and the activity of intrinsic antioxidant machinery. Monitoring of intracellular hydrogen peroxide has been successfully achieved by redox biosensor advent; however, to track the intrinsic disulfide bond reduction capacity represents a fundamental piece to understand better how redox homeostasis is maintained in living cells. In the present work, we compared the informative value of steady-state measurements and the kinetics of HyPer, a H2O2-sensitive fluorescent biosensor, targeted at the cytosol, mitochondrion and endoplasmic reticulum. From this set of data, biosensor signal recovery from an oxidized state raised as a suitable parameter to discriminate reducing capacity of a close environment. Biosensor recovery was pH-independent, condition demonstrated by experiments on pH-clamped cells, and sensitive to pharmacological perturbations of enzymatic disulfide reduction. Also, ten human cell lines were characterized according their H2O2-pulse responses, including their capacity to reduce disulfide bonds evaluated in terms of their migratory capacity. Finally, cellular migration experiments were conducted to study whether migratory efficiency was associated with the disulfide reduction activity. The migration efficiency of each cell type correlates with the rate of signal recovery measured from the oxidized biosensor. In addition, HyPer-expressing cells treated with N-acetyl-cysteine had accelerated recovery rates and major migratory capacities, both reversible effects upon treatment removal. Our data demonstrate that the HyPer signal recovery offers a novel methodological tool to track the cellular impact of redox active biomolecules.
Subject(s)
Antioxidants/isolation & purification , Biosensing Techniques , Hydrogen Peroxide/chemistry , Reactive Oxygen Species/metabolism , Antioxidants/metabolism , Endoplasmic Reticulum/metabolism , Humans , Mitochondria/metabolism , Oxidation-ReductionABSTRACT
INTRODUCCIÓN: El síndrome de intestino irritable (SII) es un trastorno funcional digestivo frecuente en personal de salud, con importantes repercusiones en la calidad de vida, en el trabajo, en el sistema de salud y la sociedad. El objetivo del presente estudio fue determinar la frecuencia de reporte del SII y sus subtipos en los historiales médicos del personal de enfermería de un hospital en Cuenca - Ecuador. MATERIALES Y MÉTODOS: Estudio descriptivo, transversal, en una muestra aleatoria de trabajadores del área de enfermería del José Carrasco Arteaga de Cuenca-Ecuador, entre junio 2017-abril 2018. De acuerdo con los criterios de Roma III, se valoraron los historiales médicos de los trabajadores para determinar la frecuencia de SII y sus subtipos. Finalmente, se describieron los factores sociodemográficos, laborales y psicológicos del personal de enfermería. RESULTADOS: Fueron evaluados los historiales médicos de 214 miembros del personal de enfermería (media de edad: 38.7±10.4 años, sexo: 95.3% de mujeres). Se encontró diagnóstico de SII en el 7.0% (15/214) del personal evaluado, siendo los subtipos indeterminado (7/15) y mixto (4/15) los más frecuentes. El 20% del personal del área de enfermería con SII tenía trastornos mentales (ansiedad, depresión y/o distimia) y el mismo porcentaje trabajaban por turnos CONCLUSIÓN: Los resultados obtenidos permiten evidenciar que el SII reportado en historiales médicos de personal de enfermería, en la ciudad de Cuenca-Ecuador, es menor a lo esperado (7%). Los subtipos de SII más frecuentes fueron el subtipo indeterminado, seguido del subtipo mixto.
BACKGROUND: Irritable bowel syndrome (IBS) is a common functional-digestive disorder in health personnel, with significant repercussions on life quality, work, healthcare system and society. The aim of this study was to determine the frequency of reported IBS and its subtypes in the nursing staffs' medical records of a public hospital, in Cuenca - Ecuador. METHODOLOGY: A cross-sectional, descriptive study, with a randomized sample of nursing workers of Hospital Jose Carrasco Arteaga, Cuenca- Ecuador; between June 2017 and April 2018. According to Rome III criteria, medical records were assessed looking for IBS diagnosis and the subtype. Finally, sociodemographic, occupational and psychological factors among the nursing staff was also described. RESULTS: Two-hundred and fourteen medical records were evaluated (average age: 38.7 ± 10.4 years, 95.3% women). A diagnosis of IBS was found in 7.0% (15/214) of the personnel evaluated. The most frequent IBS subtypes were undetermined (46.7%, 7/15) and mixed (26.7%, 4/15). Twenty percent of the nursing staff with IBS, had mental disorders diagnosis (anxiety, depression and/or dysthymia), the same percent worked shifts. CONCLUSION: The diagnosis of IBS reported in the nursing staff medical records, in Cuenca- Ecuador was lower than expected (7%). The most frequent IBS subtypes were undetermined subtype, followed by mixed subtype.
Subject(s)
Humans , Male , Female , Adult , Middle Aged , Quality of Life , Health Systems/trends , Irritable Bowel Syndrome/nursing , Gastrointestinal Diseases/diagnosisABSTRACT
Precise methods for transgene regulation are important to study signaling pathways and cell lineages in biological systems where gene function is often recycled within and across lineages. We engineered a genetic toolset for flexible transgene regulation in these diverse cellular contexts. Specifically, we created an optimized piggyBac transposon-based system, allowing for the facile generation of stably transduced cell lineages in vivo and in vitro. The system, termed pB-Tet-GOI (piggyBac-transposable tetracycline transactivator-mediated flexible expression of a genetic element of interest), incorporates the latest generation of tetracycline (Tet) transactivator and reverse Tet transactivator variants--along with engineered mutants--in order to provide regulated transgene expression upon addition or removal of doxycycline (dox). Altogether, the flexibility of the system allows for dox-induced, dox-suppressed, dox-resistant (i.e., constitutive), and dox-induced/constitutive regulation of transgenes. This versatile strategy provides reversible temporal regulation of transgenes with robust inducibility and minimal leakiness.
Subject(s)
Cell Lineage/genetics , DNA Transposable Elements , Gene Expression , Genetic Vectors/genetics , Stem Cells/metabolism , Transgenes , Animals , Cell Line , Gene Expression Regulation , Gene Order , Genes, Reporter , Humans , Mice , Plasmids/geneticsABSTRACT
As the list of putative driver mutations in glioma grows, we are just beginning to elucidate the effects of dysregulated developmental signaling pathways on the transformation of neural cells. We have employed a postnatal, mosaic, autochthonous glioma model that captures the first hours and days of gliomagenesis in more resolution than conventional genetically engineered mouse models of cancer. We provide evidence that disruption of the Nf1-Ras pathway in the ventricular zone at multiple signaling nodes uniformly results in rapid neural stem cell depletion, progenitor hyperproliferation, and gliogenic lineage restriction. Abolishing Ets subfamily activity, which is upregulated downstream of Ras, rescues these phenotypes and blocks glioma initiation. Thus, the Nf1-Ras-Ets axis might be one of the select molecular pathways that are perturbed for initiation and maintenance in glioma.
Subject(s)
Brain Neoplasms/pathology , Glioma/pathology , Proto-Oncogene Proteins c-ets/metabolism , ras Proteins/metabolism , Animals , Brain/metabolism , Brain/pathology , Brain Neoplasms/metabolism , DNA-Binding Proteins/metabolism , Genes, Reporter , Glioma/metabolism , Immunohistochemistry , Mice , Microscopy, Confocal , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neurofibromin 1/antagonists & inhibitors , Neurofibromin 1/genetics , Neurofibromin 1/metabolism , Proto-Oncogene Proteins c-ets/genetics , RNA Interference , RNA, Small Interfering/metabolism , Signal Transduction , Transcription Factors/metabolism , Up-RegulationABSTRACT
Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.
Subject(s)
Electroporation/methods , Pia Mater/physiology , Animals , Cell Lineage , Electroporation/instrumentation , Mice , Pia Mater/cytologyABSTRACT
Mutations in the TRPC6 calcium channel (Transient receptor potential channel 6) gene have been associated with familiar forms of Focal and Segmental Glomerulosclerosis (FSGS) affecting children and adults. In addition, acquired glomerular diseases are associated with increased expression levels of TRPC6. However, the exact role of TRPC6 in the pathogenesis of FSGS remains to be elucidated. In this work we describe the generation and phenotypic characterization of three different transgenic mouse lines with podocyte-specific overexpression of the wild type or any of two mutant forms of Trpc6 (P111Q and E896K) previously related to FSGS. Consistent with the human phenotype a non-nephrotic range of albuminuria was detectable in almost all transgenic lines. The histological analysis demonstrated that the transgenic mice developed a kidney disease similar to human FSGS. Differences of 2-3 folds in the presence of glomerular lesions were found between the non transgenic and transgenic mice expressing Trpc6 in its wild type or mutant forms specifically in podocytes. Electron microscopy of glomerulus from transgenic mice showed extensive podocyte foot process effacement. We conclude that overexpression of Trpc6 (wild type or mutated) in podocytes is sufficient to cause a kidney disease consistent with FSGS. Our results contribute to reinforce the central role of podocytes in the etiology of FSGS. These mice constitute an important new model in which to study future therapies and outcomes of this complex disease.
Subject(s)
Gene Expression , Glomerulosclerosis, Focal Segmental/metabolism , Podocytes/metabolism , TRPC Cation Channels/genetics , TRPC Cation Channels/metabolism , Animals , Cell Line , Disease Models, Animal , Female , Glomerulosclerosis, Focal Segmental/genetics , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , Mutation , Organ Specificity , TRPC6 Cation ChannelABSTRACT
Los tricobezoares son una condición poco común que usualmente ocurre en mujeres jóvenes con una historia de tricotilomanía y tricofagia. En la actualidad, la mayoría de los casos se presentan en pacientes con antecedentes de cirugía gástrica, o con alteraciones en la función pilórica; clínicamente pueden cursar asintomáticos durante meses o años o presentarse como una entidad aguda acompañada por sus complicaciones. En el presente artículo se presenta el caso clínico de una paciente embarazada a quien se le realizó diagnóstico de tricobezoar gastroduodenal - síndrome de Rapunzel (una variedad compleja de este bezoar que involucra estómago, duodeno e intestino); se presenta una revisión de la literatura al respecto.
The presence of trichobezoars is a rare condition which usually occurs in young women who have histories of trichotillomania and trichophagia. Nowadays, the majority of cases occur in patients with a history of gastric surgery, or pyloric function alteration. They may be clinically asymptomatic for months or years or may present acute symptoms accompanied by severe complications.This article presents the case of a pregnant patient who was diagnosed with a case of Rapunzel syndrome a is presented. This complex variety of gastroduodenal trichobezoar involves the stomach, duodenum and intestine. The article also reviews the literature about the Rapunzel syndrome.
Subject(s)
Humans , Female , Young Adult , Bezoars , Pregnant WomenABSTRACT
Cimicifuga foetida, an Asian Cimicifuga species, has been employed as a cooling and detoxification agent in traditional Chinese medicine since ancient times. For this herb, two cycloartane triterpene glycosides isolated from the rhizomes have demonstrated cytotoxicity on rat tumor and human cancer cell lines. Since human Hsp27 is increased in various human cancers and exhibits cytoprotective activity that affects tumorigenesis and the susceptibility of tumours to cancer treatment, the purpose of this research was to study the expression of this protein in MCF-7 breast cancer cells. To accomplish this aim, MCF-7 cells were exposed to different concentrations of Cimicifuga foetida extract showing a reduction in cell number measured by the sulforhodamine assay. In addition, the expression of Hsp-27 mRNA detected by RT-PCR and Hsp-27 protein detected by immnofluorescence was present in all conditions, except when using the highest concentration of Cimicifuga foetida extract (2,000 jig /L). We conclude that Hsp 27 expression at 2,000 jig /L Cimicifuga foetida extract is diminished. This is the first report showing the Hsp-27 expression after exposure to Cimicifuga foetida extract in MCF-7 cells.