1,721,099 research outputs found
Viral infection protocols
No full textThis chapter describes the protocol for preparation of recombinant adenoviruses and infection of target cells to express transiently G-protein-coupled receptors or other proteins of interest. Adenoviruses are nonenveloped viruses containing a linear double-stranded DNA genome. Their life cycle does not normally involve integration into the host genome, rather they replicate as episomal elements in the nucleus of the host cell and consequently there is no risk of insertional mutagenesis. The wild-type adenovirus genome is approx 35 kb, of which up to 30 kb can be replaced by foreign DNA. Adenoviral vectors are very efficient at transducing the gene of interest in target cells in vitro and in vivo and can be produced at high titers (>10(11)/mL). The viral infection has a number of useful features: (1) the efficiency of gene transduction is very high (up to 100% in sensitive cells). (2) The infection is easy and does not alter physically the cell membrane for gene transduction. (3) It is possible to infect cells that are resistant to transfection with plasmids (including nondividing cells)
Viral Infection for GPCR Expression in Eukaryotic Cells.
No full textThis chapter describes the protocol for the preparation of recombinant adenoviruses and infection of target cells to transiently express G protein-coupled receptors (GPCRs) or other proteins of interest. Adenoviruses are non-enveloped viruses containing a linear double-stranded DNA genome. Their life cycle does not normally involve integration into the host genome, rather they replicate as episomal -elements in the nucleus of the host cell, and consequently there is no risk of insertional mutagenesis. Up to 30 kb out of the 35 kb of the wild-type adenovirus genome can be replaced by foreign DNA. Adenoviral vectors are very efficient in transducing target cells in vitro and in vivo and can be produced at high titers (>1011/mL). The viral infection has a number of useful features: (1) the efficiency of gene transduction is very high (up to 100% in sensitive cells); (2) the infection is easy and does not physically alter the cell membrane for gene transduction; (3) it is possible to infect cells that are resistant to transfection with plasmids (including nondividing cells); and (4) the viral vectors can be used for infection in vivo (including gene therapy) and can potentially be targeted cell-specifically
First attempt to measure adult neurogenesis in a lophotrocozoan (Octopus vulgaris) brain using flow-cytometry technique
No full textIn the past decades, researchers provided an amount of significant information about the anatomical, molecular and functional mechanisms underlying neurogenesis in the adult brain.
Adult neurogenesis consists in proliferation, migration and differentiation of newborn cells that will be functionally integrated into the existing neural circuitry of adult brain. This process plays a crucial role in adaptation to the environmental challenges.
It occurs in animals with complex and centralized nervous system exhibiting cognitive capabilities and sophisticated behavioral repertoires, such as mammals, including humans, non-mammals vertebrates and, among invertebrates, it has been demonstrated in ecdisozoan taxa such as insects and crustaceans too. Octopus vulgaris is considered an “advanced invertebrate” for the size of its brain, the largest of any invertebrates. Evolved from the basal molluscan plan of tetraneury and characterized by a hierarchical organization. Octopus central nervous system is located around the esophagus, in a cartilaginous “cranium” between the eyes, and consists in a supra-esophageal and sub-esophageal masses connected to two optic lobes.
Octopus shows complex behaviours and unusual cognitive skills, as learning and memory, problem solving, individual personality and capabilities to play. For these reasons, it seems to be the most likely candidate for the neurogenic process among lophotrocozoans.
In our previous works, we found cell proliferation in specific areas of octopus brain involved in learning, memory and processing sensory information. Moreover, we demonstrated, using specific markers as PCNA, PARP1 and Oct-elav1 gene, that enriched environment increases proliferation and synaptogenesis.
Given that, we developed a protocol for flow-cytometry analysis to measure the proliferative activity in a faster and more reliable manner then the classical immunohistochemistry.
Dissociated cells from proliferating areas of octopus brain, vertical frontal system and optic-olfactory lobes were exposed to BrdU and subjected to PI staining and FACS analysis. Bivariate distributions of BrdU content vs DNA content were analyzed and the G1/S subpopulation was determined. Univariate analysis of DNA content was used to determine the percentage of G2/S cells. Using this technique, we accurately quantified the effective number of proliferating cells in each areas, in order to determine the magnitude of the neurogenic process in octopus brain
Viral Infection for G Protein-Coupled Receptor Expression in Eukaryotic Cells
No full textThis chapter describes the protocol for the preparation of recombinant adenoviruses and infection of target cells to transiently express G protein-coupled receptors (GPCRs) or other proteins of interest. Adenoviruses are non-enveloped viruses containing a linear double-stranded DNA genome. Their life cycle does not normally involve integration into the host genome, rather they replicate as episomal elements in the nucleus of the host cell, and consequently there is no risk of insertional mutagenesis. Up to 30 kb out of the 35 kb of the wild-type adenovirus genome can be replaced by foreign DNA. Adenoviral vectors are very efficient in transducing target cells in vitro and in vivo and can be produced at high titers (>1011/ml). The viral infection has a number of useful features: (1) the efficiency of gene transduction is very high (up to 100 % in sensitive cells); (2) the infection is easy and does not physically alter the cell membrane for gene transduction; (3) it is possible to infect cells that are resistant to transfection with plasmids (including nondividing cells); and (4) the viral vectors can be used for infection in vivo (including gene therapy) and can potentially be targeted to specific cells or tissues
Mechanism of retinoic acid-induced transcription: epigenetic changes, DNA oxidation and formation of chromatin loops
No full text
Lattice Corneal Dystrophy: a report of two cases in twin si- sters due to 3 mutations (T1620C, C1416T, A1924G) in the TGFBI (BIGH3) gene
No full textPhenotypic characteristics associated with mutations in the tran- sforming growth factor beta-induced (TGFBI) gene in two twin sisters suffering from lattice corneal dystrophy are reported. Genomic DNA was extracted from peripheral blood and 3 new mutations in association with exons 11-12-14 of the TGFBI gene were found
Role of p85a PI3K mutants on the insulin regulation of estrogen receptor positive MCF-7 cell growth and motility
No full textKRAS is a molecular determinant of platinum responsiveness in glioblastoma
Full text linkBackground: KRAS is the undisputed champion of oncogenes, and despite its prominent role in oncogenesis as mutated gene, KRAS mutation appears infrequent in gliomas. Nevertheless, gliomas are considered KRAS-driven cancers due to its essential role in mouse malignant gliomagenesis. Glioblastoma is the most lethal primary brain tumor, often associated with disturbed RAS signaling. For newly diagnosed GBM, the current standard therapy is alkylating agent chemotherapy combined with radiotherapy. Cisplatin is one of the most effective anticancer drugs and is used as a first-line treatment for a wide spectrum of solid tumors (including medulloblastoma and neuroblastoma) and many studies are currently focused on new delivery modalities of effective cisplatin in glioblastoma. Its mechanism of action is mainly based on DNA damage, inducing the formation of DNA adducts, triggering a series of signal-transduction pathways, leading to cell-cycle arrest, DNA repair and apoptosis. Methods: Long-term cultures of human glioblastoma, U87MG and U251MG, were either treated with cis-diamminedichloroplatinum (cisplatin, CDDP) and/or MEK-inhibitor PD98059. Cytotoxic responses were assessed by cell viability (MTT), protein expression (Western Blot), cell cycle (PI staining) and apoptosis (TUNEL) assays. Further, gain-of-function experiments were performed with cells over-expressing mutated hypervariable region (HVR) KRASG12V plasmids. Results: Here, we studied platinum-based chemosensitivity of long-term cultures of human glioblastoma from the perspective of KRAS expression, by using CDDP and MEK-inhibitor. Endogenous high KRAS expression was assessed at transcriptional (qPCR) and translational levels (WB) in a panel of primary and long-term glioblastoma cultures. Firstly, we measured immediate cellular adjustment through direct regulation of protein concentration of K-Ras4B in response to cisplatin treatment. We found increased endogenous protein abundance and involvement of the effector pathway RAF/MEK/ERK mitogen-activated protein kinase (MAPK) cascade. Moreover, as many MEK inhibitors are currently being clinically evaluated for the treatment of high-grade glioma, so we concomitantly tested the effect of the potent and selective non-ATP-competitive MEK1/2 inhibitor (PD98059) on cisplatin-induced chemosensitivity in these cells. Cell-cycle phase distribution was examined using flow cytometry showing a significant cell-cycle arrest in both cultures at different percentage, which is modulated by MEK inhibition. Cisplatin-induced cytotoxicity increased sub-G1 percentage and modulates G2/M checkpoint regulators cyclins D1 and A. Moreover, ectopic expression of a constitutively active KRASG12V rescued CDDP-induced apoptosis and different HVR point mutations (particularly Ala 185) reverted this phenotype. Conclusion: These findings warrant further studies of clinical applications of MEK1/2 inhibitors and KRAS as 'actionable target' of cisplatin-based chemotherapy for glioblastoma
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