51 research outputs found
A refined method to study gene dosage changes in-vitro using CRISPR/Cas9
Aims: Gene dosage can have a major impact on cell biology although, hitherto, is has been difficult to study using in-vitro models. We sought to refine and accelerate the development of “gene dosage” models through using CRISPR/Cas9 (a gene-editing technology) for sequential knockout of gene alleles.
Methods: Our method involved (i) using Cas9 nuclease mRNA rather than expression plasmids, (ii) using a fluorescently labelled FAM-6 tracr complexed with guide RNA and (iii) using High Resolution Melting (HRM) analysis to screen for mutations. HCT116 cells, wild-type for TP53, were transfected with different molarities of FAM-6 tracr labelled and guide RNA targeting different exons of TP53 and selected by Fluorescence Associated Cell Sorting (FACS). Single-cell colonies were then isolated, expanded and tested for mutation in the targeted region by PCR/HRM.
Results: Out of 32 clones tested, 12 have shown aberrant melting by HRM, giving a targetting efficiency of 37.5%. One clone was sequenced and a heterozygous mutation found - in this case comprising a single base deletion in exon 3. mRNA sequencing confirmed the mutation was expressed and western blotting for p53 showed the presence of both wild type and truncated protein bands. Changes in expression of MDM-2 isoforms suggested a functional effect of the induced TP53 mutation.
Conclusions: We have developed an in-vitro model to study TP53 gene dosage effects. The protocol is efficient and applicable to any gene. Importantly, we have used Cas9 mRNA and labelled tracr/guideRNA to isolate likely-mutated cells and HRM for rapid mutation detection
Investigating TNS4 in the Colorectal Tumor Microenvironment Using 3D Spheroid Models of Invasion
TNS4 (Tensin 4 or Cten) is a putative oncogene in colorectal cancer (CRC) with a role in regulating cell adhesion, motility, invasion, and epithelial to mesenchymal transition (EMT). The objective is to study the role of TNS4 in CRC using more realistic models of the tumor microenvironment. CRC cells expressing TdTomato protein and shTNS4/shLUC hairpin oligos are grown in 3D spheroids with and without cancer‐associated fibroblasts (CAFs). Adhesiveness to collagen I and CAFs is assessed in 2D and cell proliferation, volume, and invasion are assessed in 3D conditions. The role of TNS4 knockdown in gefitinib chemosensitivity and epidermal growth factor receptor (EGFR) and Ras protein levels are also tested. In general, TNS4 knockdown increases cell proliferation in cell lines producing compact spheroids. The addition of CAFs in spheroids supports CRC cell proliferation, whereas CAFs themselves do not proliferate, but increases ECM degradation. TNS4 knockdown reduces adhesiveness and 3D invasion and disrupts EGFR signaling which results in increased sensitivity to Gefitinib. In conclusion, in a 3D spheroid model, TNS4 inhibits cell proliferation and promotes cell invasion into the ECM, possibly by adhesion to the ECM and stromal cells. TNS4 knockdown enhances sensitivity to the EGFR inhibitor gefitinib and may be helpful for Kirsten ras oncogene homolog mutant CRC patients
Tensin4 (TNS4) is upregulated by Wnt signalling in adenomas in multiple intestinal neoplasia (Min) mice
ApcMin/+ mice are regarded as a standard animal model of colorectal cancer (CRC). Tensin4 (TNS4 or Cten) is a putative oncogene conferring features of stemness and promoting motility. Our objective was to assess TNS4 expression in intestinal adenomas and determine whether TNS4 is upregulated by Wnt signalling. ApcMin/+ mice (n=11) were sacrificed at approximately 120days old at the onset of anaemia signs. Small intestines were harvested, and Swiss roll preparations were tested for TNS4 expression by immunohistochemistry (IHC). Individual polyps were also separately collected (n=14) and tested for TNS4 mRNA expression and Kras mutation. The relationship between Wnt signalling and TNS4 expression was tested by Western blotting in the human CRC cell line HCT116 after inhibition of β-catenin activity with MSAB or its increase by transfection with a Flag β-catenin expression vector. Overall, 135/148 (91.2%) of the total intestinal polyps were positive for TNS4 expression by IHC, whilst adjacent normal areas were negative. RT-qPCR analysis showed approximately 5-fold upregulation of TNS4 mRNA in the polyps compared to adjacent normal tissue and no Kras mutations were detected. In HCT116, β-catenin inhibition resulted in reduced TNS4 expression, and conversely, β-catenin overexpression resulted in increased TNS4 expression. In conclusion, this is the first report linking aberrant Wnt signalling to upregulation of TNS4 both during initiation of intestinal adenomas in mice and in in vitro models. The exact contribution of TNS4 to adenoma development remains to be investigated, but the ApcMin/+ mouse represents a good model to study this
The APOE ε2 allele increases the risk of Earlier Age at onset in Machado-Joseph disease
Background. Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder of late onset, caused by a (CAG)n expansion at the ATXN3 gene (14q32.1). Variation in age-at-onset is partially explained by the size of the (CAG)n tract in expanded alleles. The remaining variation should be the product of other factors, namely modifier genes. The genotype at the APOE locus has been described as a possible modifier in different neurological disorders, namely Parkinson (PD) and Huntington disease (HD). In the CNS, apolipoprotein E constitutes an important mediator of cholesterol transport/metabolism, which is essential for synaptic integrity and neuronal function.
Objective. To investigate a modulating effect of the APOE polymorphism on age-at-onset of MJD.
Design and Subjects. The APOE polymorphism was typed in a series of 192 MJD patients.
Results. Cases with the ε2/ε3 genotype presented an earlier onset, when compared with those with ε3/ε3 or ε3/ε4. In this series of patients, the presence of an APOE ε2 allele implies a decrease of nearly 5 years in the age-at-onset. When combining, in a general linear model, several other predictors, namely the presence/absence of the APOE ε2 allele, with the size of the (CAG)n in expanded alleles, the model was significantly improved and the explanation of onset variance was raised from 59.8% to 66.5%. Furthermore, the presence of the ε2 allele was associated with an onset below 39 years (OR=5.00; 95% CI: 1.18-21.14).
Conclusions. These findings indicate that the polymorphism at the APOE gene plays a role as a genetic modifier of MJD phenotype.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BPD/63121/2009, SFRH/BPD/38659/2007, M3.1.3/F/004/2009, “Secretaria Regional da Ciência, Tecnologia e Equipamentos”.Fundação para a Ciência e a Tecnologia (FCT) - “Transcriptional variation of the ATXN3 gene as modulator of the clinical heterogeneity in Machado-Joseph disease (MJD)” (PIC/IC/83074/2007)Institute of Biotechnology and Biomedicine (IBBA) - “High prevalence diseases in the Azores Islands” (M2.1.2/I/026/2008
Patterns of mitochondrial DNA damage in blood and brain tissues of a transgenic mouse model of Machado-Joseph disease
BACKGROUND:
Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar ataxia caused by a CAG tract expansions in the ATXN3 gene. Patterns of mitochondrial damage associated with pathological findings of brain tissues could provide molecular biomarkers of this disorder.
OBJECTIVE:
The potential of mitochondrial DNA (mtDNA) damage as a biomarker of MJD progression was investigated using a transgenic mouse model.
METHODS:
DNA was obtained from affected (pontine nuclei) and nonaffected tissues (hippocampus and blood) of transgenic animals of three distinct age groups: 8 weeks, before onset of the phenotype; 16 weeks, at onset, and 24 weeks, at well-established phenotype. Wild-type littermate mice, serving as controls, were analyzed for the same tissues and age groups. mtDNA damage was studied by fluorescence-based quantitative PCR in 84 transgenic and 93 wild-type samples.
RESULTS:
A clear pattern of decrease in mtDNA copy number with age and accumulation of 3,867-bp deletions at the initial stages (both being more pronounced in transgenic mice) was observed. Pontine nuclei, the affected tissue in transgenic mice, displayed 1.5 times less copies of mtDNA than nonaffected brain tissue hippocampus (odds ratio = 1.21). Pontine nuclei displayed the highest percentage of mtDNA deletions (6.05% more in transgenic mice).
CONCLUSION:
These results suggest that mtDNA damage is related to the initiation of the phenotype in transgenic mice; mtDNA 3,867-bp deletions may be a biomarker of the initial stages of the disease.This study was supported by the following grants: DRCT Postdoctoral fellowship to N.K. (M3.1.7/F/002/2008), FCT Postdoctoral fellowship to T.C. (SFRH/BPD/38659/2007) and C.B. (SFRH/BPD/63121/2009), FCT research grants to S.S. (PTDC/SAU-GMG/64076/2006) and A.S.F. (PIC/IC/83013/2007)
Production of poly (3-hydroxybutyrate-co-4-hydroxybutyrate) by Burkholderia sacchari using wheat straw hydrolysates and gamma-butyrolactone
“NOTICE: this is the author’s version of a work that was accepted for publication in International Journal of Biological Macromolecules. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in International Journal of Biological Macromolecules, Vol. 71, Special issue, (November 2014). DOI 10.1016/j.ijbiomac.2014.04.054 ""Burkholderia sacchari DSM 17165 is able to grow and produce poly(3-hydroxybutyrate)
both on hexoses and pentoses. In a previous study, wheat straw lignocellulosic
hydrolysates (WSH) containing high C6 and C5 sugar concentrations were shown to be
excellent carbon sources for P(3HB) production.
Using a similar feeding strategy developed for P(3HB) production based on WSH, fedbatch
cultures were developed aiming at the production of the copolymer P(3HB-co-
4HB) (poly(3-hydroxybutyrate-co-4-hydroxybutyrate)) by B. sacchari. The ability of this strain to synthesize P(3HB-co-4HB) was first shown in shake flasks using gammabutyrolactone
(GBL) as precursor of the 4HB units.
Fed-batch cultures using glucose as carbon source (control) and GBL were developed
to achieve high copolymer productivities and 4HB incorporations. The attained P(3HBco-
4HB) productivity and 4HB molar % were 0.7 g/(L·h) and 4.7 molar %, respectively.
The 4HB incorporation was improved to 6.3 and 11.8 molar % by addition of 2 g/L
propionic and acetic acid, respectively. When WSH were used as carbon source under
the same feeding conditions, the values achieved were 0.5 g/(L·h) and 5.0 molar %,
respectively.
Burkholderia sacchari, a strain able to produce biopolymers based on xylose-rich
lignocellulosic hydrolysates, is for the first time reported to produce P(3HB-co-4HB)
using gamma butyrolactone as precursor."This work has received funding from the European Union 7th Framework Programme
(FP7/2007-2013) under Grant Agreement number 246449 / Project: Bugworkers - New tailor-made PHB-based nanocomposites for high performance applications produced
from environmentally friendly production routes.Financial support: Fundação para a Ciência e Tecnologia, Portugal (fellowships SFRH/BPD/68587/2010 and SFRH/BPD/26678/2006)
Novel candidate blood-based transcriptional biomarkers of Machado-Joseph disease
BACKGROUND:
Machado-Joseph disease (or spinocerebellar ataxia type 3) is a late-onset polyglutamine neurodegenerative disorder caused by a mutation in the ATXN3 gene, which encodes for the ubiquitously expressed protein ataxin-3. Previous studies on cell and animal models have suggested that mutated ataxin-3 is involved in transcriptional dysregulation. Starting with a whole-transcriptome profiling of peripheral blood samples from patients and controls, we aimed to confirm abnormal expression profiles in Machado-Joseph disease and to identify promising up-regulated genes as potential candidate biomarkers of disease status.
METHODS:
The Illumina Human V4-HT12 array was used to measure transcriptome-wide gene expression in peripheral blood samples from 12 patients and 12 controls. Technical validation and validation in an independent set of samples were performed by quantitative real-time polymerase chain reaction (PCR).
RESULTS:
Based on the results from the microarray, twenty six genes, found to be up-regulated in patients, were selected for technical validation by quantitative real-time PCR (validation rate of 81% for the up-regulation trend). Fourteen of these were further tested in an independent set of 42 patients and 35 controls; 10 genes maintained the up-regulation trend (FCGR3B, CSR2RA, CLC, TNFSF14, SLA, P2RY13, FPR2, SELPLG, YIPF6, and GPR96); FCGR3B, P2RY13, and SELPLG were significantly up-regulated in patients when compared with controls.
CONCLUSIONS:
Our findings support the hypothesis that mutated ataxin-3 is associated with transcription dysregulation, detectable in peripheral blood cells. Furthermore, this is the first report suggesting a pool of up-regulated genes in Machado-Joseph disease that may have the potential to be used for fine phenotyping of this disease.Fundação para a Ciência e a Tecnologia (FCT) - project FCOMP-01-0124-FEDER-028753 (PTDC/DTP/PIC/0370/2012)Operational Competitiveness Programme—COMPETEUK Medical Research Council (MRC)Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BPD/33611/2009Fundo Regional para a Ciência (FRC), Governo dos Açores - M3.1.2/F/006/2011; M3.1.7/F/031/2011 ; M3.1.3/F/004/200
Matemáticas en la formación profesional
En este capítulo se presenta una visión amplia de la necesidad y el papel de las matemáticas en la Formación Profesional (FP). Para ello se sitúa la FP ante los retos sociales como una pieza clave de aprendizaje, innovación y transformación. Se recogen ejemplos de la aplicación concreta de contenidos matemáticos en diversas profesiones y se analiza la presencia de asignaturas específicas de matemáticas en esta etapa en distintos países. Se muestra un estudio exploratorio diagnóstico de las matemáticas en la FP española, que pone de manifiesto que las matemáticas que se aprenden en la secundaria obligatoria no cubren las exigencias de los Ciclos Superiores. Por último, se dan una serie de perspectivas y orientaciones centradas en aprovechar el carácter interdisciplinar de los módulos de FP para desarrollar propuestas STEM que permitan tanto promover las competencias del alumnado como mejorar sus afectos hacia las matemáticas
Parkinsonian phenotype in Machado-Joseph disease (MJD/SCA3): a two-case report
Background: Machado-Joseph disease (MJD), or spinocerebellar ataxia type 3 (SCA3), is an autosomal dominant neurodegenerative disorder of late onset, which is caused by a CAG repeat expansion in the coding region of the ATXN3 gene. This disease presents clinical heterogeneity, which cannot be completely explained by the size of the repeat tract. MJD presents extrapyramidal motor signs, namely Parkinsonism, more frequently than the other subtypes of autosomal dominant cerebellar ataxias. Although Parkinsonism seems to segregate within MJD families, only a few MJD patients develop parkinsonian features and, therefore, the clinical and genetic aspects of these rare presentations remain poorly investigated. The main goal of this work was to describe two MJD patients displaying the parkinsonian triad (tremor, bradykinesia and rigidity), namely on what concerns genetic variation in Parkinson's disease (PD) associated loci (PARK2, LRRK2, PINK1, DJ-1, SNCA, MAPT, APOE, and mtDNA tRNAGln T4336C). Case presentation: Patient 1 is a 40 year-old female (onset at 30 years of age), initially with a pure parkinsonian phenotype (similar to the phenotype previously reported for her mother). Patient 2 is a 38 year-old male (onset at 33 years of age), presenting an ataxic phenotype with parkinsonian features (not seen either in other affected siblings or in his father). Both patients presented an expanded ATXN3 allele with 72 CAG repeats. No PD mutations were found in the analyzed loci. However, allelic variants previously associated with PD were observed in DJ-1 and APOE genes, for both patients. Conclusions: The present report adds clinical and genetic information on this particular and rare MJD presentation, and raises the hypothesis that DJ-1 and APOE polymorphisms may confer susceptibility to the parkinsonian phenotype in MJD
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