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Find video protocols related to scientific articles indexed in Pubmed.
Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material.
Hum. Gene Ther.
PUBLISHED: 10-03-2014
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Abstract Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. The rAAV8RSM was produced using transient transfection, and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to 16 laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50×10(11) pt/ml; CI, 4.26×10(11) to 6.75×10(11) pt/ml), vector genomes (mean, 5.75×10(11) vg/ml; CI, 3.05×10(11) to 1.09×10(12) vg/ml), and infectious units (mean, 1.26×10(9) IU/ml; CI, 6.46×10(8) to 2.51×10(9) IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time when the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.
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Epigenetic modification at Notch responsive promoters blunts efficacy of inducing notch pathway reactivation after myocardial infarction.
Circ. Res.
PUBLISHED: 08-11-2014
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The Notch pathway plays a key role in stimulating mammalian cardiomyocyte proliferation during development and in the early postnatal life; in adult zebrafish, reactivation of this pathway is also essential to drive cardiac regeneration after injury.
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Adeno-associated virus vectors as therapeutic and investigational tools in the cardiovascular system.
Circ. Res.
PUBLISHED: 05-24-2014
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The use of vectors based on the small parvovirus adeno-associated virus has gained significant momentum during the past decade. Their high efficiency of transduction of postmitotic tissues in vivo, such as heart, brain, and retina, renders these vectors extremely attractive for several gene therapy applications affecting these organs. Besides functional correction of different monogenic diseases, the possibility to drive efficient and persistent transgene expression in the heart offers the possibility to develop innovative therapies for prevalent conditions, such as ischemic cardiomyopathy and heart failure. Therapeutic genes are not only restricted to protein-coding complementary DNAs but also include short hairpin RNAs and microRNA genes, thus broadening the spectrum of possible applications. In addition, several spontaneous or engineered variants in the virus capsid have recently improved vector efficiency and expanded their tropism. Apart from their therapeutic potential, adeno-associated virus vectors also represent outstanding investigational tools to explore the function of individual genes or gene combinations in vivo, thus providing information that is conceptually similar to that obtained from genetically modified animals. Finally, their single-stranded DNA genome can drive homology-directed gene repair at high efficiency. Here, we review the main molecular characteristics of adeno-associated virus vectors, with a particular view to their applications in the cardiovascular field.
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Notch1 activity in the olfactory bulb is odour-dependent and contributes to olfactory behaviour.
Eur. J. Neurosci.
PUBLISHED: 05-06-2014
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Notch signalling plays an important role in synaptic plasticity, learning and memory functions in both Drosophila and rodents. In this paper, we report that this feature is not restricted to hippocampal networks but also involves the olfactory bulb (OB). Odour discrimination and olfactory learning in rodents are essential for survival. Notch1 expression is enriched in mitral cells of the mouse OB. These principal neurons are responsive to specific input odorants and relay the signal to the olfactory cortex. Olfactory stimulation activates a subset of mitral cells, which show an increase in Notch activity. In Notch1cKOKln mice, the loss of Notch1 in mitral cells affects the magnitude of the neuronal response to olfactory stimuli. In addition, Notch1cKOKln mice display reduced olfactory aversion to propionic acid as compared to wildtype controls. This indicates, for the first time, that Notch1 is involved in olfactory processing and may contribute to olfactory behaviour.
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The COP9 signalosome is a repressor of replicative stress responses and polyploidization in the regenerating liver.
Hepatology
PUBLISHED: 01-16-2014
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Aberrant DNA replication induced by deregulated or excessive proliferative stimuli evokes a "replicative stress response" leading to cell cycle restriction and/or apoptosis. This robust fail-safe mechanism is eventually bypassed by transformed cells, due to ill-defined epistatic interactions. The COP9 signalosome (CSN) is an evolutionarily conserved regulator of cullin ring ligases (CRLs), the largest family of ubiquitin ligases in metazoans. Conditional inactivation of the CSN in several tissues leads to activation of S- or G2-phase checkpoints resulting in irreversible cell cycle arrest and cell death. Herein we ablated COPS5, the CSNs catalytic subunit, in the liver, to investigate its role in cell cycle reentry by differentiated hepatocytes. Lack of COPS5 in regenerating livers causes substantial replicative stress, which triggers a CDKN2A-dependent genetic program leading to cell cycle arrest, polyploidy, and apoptosis. These outcomes are phenocopied by acute overexpression of c-Myc in COPS5 null hepatocytes of adult mice.
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Carbon nanotubes instruct physiological growth and functionally mature syncytia: nongenetic engineering of cardiac myocytes.
ACS Nano
PUBLISHED: 06-11-2013
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Myocardial tissue engineering currently represents one of the most realistic strategies for cardiac repair. We have recently discovered the ability of carbon nanotube scaffolds to promote cell division and maturation in cardiomyocytes. Here, we test the hypothesis that carbon nanotube scaffolds promote cardiomyocyte growth and maturation by altering the gene expression program, implementing the cell electrophysiological properties and improving networking and maturation of functional syncytia. In our study, we combine microscopy, biological and electrophysiological methodologies, and calcium imaging, to verify whether neonatal rat ventricular myocytes cultured on substrates of multiwall carbon nanotubes acquire a physiologically more mature phenotype compared to control (gelatin). We show that the carbon nanotube substrate stimulates the induction of a gene expression profile characteristic of terminal differentiation and physiological growth, with a 2-fold increase of ?-myosin heavy chain (P < 0.001) and upregulation of sarcoplasmic reticulum Ca(2+) ATPase 2a. In contrast, markers of pathological hypertrophy remain unchanged (?-myosin heavy chain, skeletal ?-actin, atrial natriuretic peptide). These modifications are paralleled by an increase of connexin-43 gene expression, gap junctions and functional syncytia. Moreover, carbon nanotubes appear to exert a protective effect against the pathologic stimulus of phenylephrine. Finally, cardiomyocytes on carbon nanotubes demonstrate a more mature electrophysiological phenotype of syncytia and intracellular calcium signaling. Thus, carbon nanotubes interacting with cardiomyocytes have the ability to promote physiological growth and functional maturation. These properties are unique in the current vexing field of tissue engineering, and offer unprecedented perspectives in the development of innovative therapies for cardiac repair.
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MiR-378 controls cardiac hypertrophy by combined repression of mitogen-activated protein kinase pathway factors.
Circulation
PUBLISHED: 04-26-2013
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Several microRNAs (miRs) have been shown to regulate gene expression in the heart, and dysregulation of their expression has been linked to cardiac disease. miR-378 is strongly expressed in the mammalian heart but so far has been studied predominantly in cancer, in which it regulates cell survival and tumor growth.
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Arteriogenic therapy based on simultaneous delivery of VEGF-A and FGF4 genes improves the recovery from acute limb ischemia.
PUBLISHED: 01-01-2013
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Gene therapy stimulating the growth of blood vessels is considered for the treatment of peripheral and myocardial ischemia. Here we aimed to achieve angiogenic synergism between vascular endothelial growth factor-A (VEGF-A, VEGF) and fibroblast growth factor 4 (FGF4) in murine normoperfused and ischemic limb muscles.
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Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer.
FASEB J.
PUBLISHED: 11-17-2011
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Crigler-Najjar type I (CNI) syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by uridine diphosphoglucuronosyltransferase 1A1 (UGT1A1) deficiency. The disease is lethal due to bilirubin-induced neurological damage unless phototherapy is applied from birth. However, treatment becomes less effective during growth, and liver transplantation is required. To investigate the pathophysiology of the disease and therapeutic approaches in mice, we generated a mouse model by introducing a premature stop codon in the UGT1a1 gene, which results in an inactive enzyme. Homozygous mutant mice developed severe jaundice soon after birth and died within 11 d, showing significant cerebellar alterations. To rescue neonatal lethality, newborns were injected with a single dose of adeno-associated viral vector 9 (AAV9) expressing the human UGT1A1. Gene therapy treatment completely rescued all AAV-treated mutant mice, accompanied by lower plasma bilirubin levels and normal brain histology and motor coordination. Our mouse model of CNI reproduces genetic and phenotypic features of the human disease. We have shown, for the first time, the full recovery of the lethal effects of neonatal hyperbilirubinemia. We believe that, besides gene-addition-based therapies, our mice could represent a very useful model to develop and test novel technologies based on gene correction by homologous recombination.
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Stimulation of cardiac apoptosis in ovariectomized hypertensive rats: potential role of the renin-angiotensin system.
J. Hypertens.
PUBLISHED: 05-05-2011
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The mechanisms underlying the increased cardiovascular risk after menopause are poorly understood. Estrogens modulate the cardiac renin-angiotensin system (RAS) and influence cardiac adaptation to afterload. To investigate whether the loss of the natural inhibition of the RAS by estrogen may be linked to an increase of cardiac apoptosis, we studied 17?-estradiol (E2) and/or angiotensin-converting enzyme (ACE) inhibitor treatment effects on cardiomyocyte survival in ovariectomized spontaneously hypertensive rats (SHRs).
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Intravenous gene therapy with PIM-1 via a cardiotropic viral vector halts the progression of diabetic cardiomyopathy through promotion of prosurvival signaling.
Circ. Res.
PUBLISHED: 04-07-2011
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Studies in transgenic mice showed the key role of (Pim-1) (proviral integration site for Moloney murine leukemia virus-1) in the control of cardiomyocyte function and viability.
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Vascular endothelial growth factor-B gene transfer exacerbates retinal and choroidal neovascularization and vasopermeability without promoting inflammation.
Mol. Vis.
PUBLISHED: 02-14-2011
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The role of vascular endothelial growth factor (VEGF)-B in the eye is poorly understood. The present study was conducted to evaluate the effect of overexpression of VEGF-B via adeno-associated virus (AAV) gene transfer on ocular angiogenesis, inflammation, and the blood-retinal barrier (BRB).
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Sustained expression of vascular endothelial growth factor and angiopoietin-1 improves blood-spinal cord barrier integrity and functional recovery after spinal cord injury.
J. Neurotrauma
PUBLISHED: 10-28-2010
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Spinal cord injury (SCI) results in immediate disruption of the spinal vascular network, triggering an ischemic environment and initiating secondary degeneration. Promoting angiogenesis and vascular stability through the induction of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1), respectively, provides a possible therapeutic approach in treating SCI. We examined whether supplementing the injured environment with these two factors, which are significantly reduced following injury, has an effect on lesion size and functional outcome. Sustained delivery of both VEGF(165) and Ang-1 was realized using viral vectors based on the adeno-associated virus (AAV), which were injected directly into the lesion epicenter immediately after injury. Our results indicate that the combined treatment with VEGF and Ang-1 resulted in both reduced hyperintense lesion volume and vascular stabilization, as determined by magnetic resonance imaging (MRI). Western blot analysis indicated that the viral vector expression was maintained into the chronic phase of injury, and that the use of the AAV vectors did not exacerbate infiltration of microglia into the lesion epicenter. The combined treatment with AAV-VEGF and AAV-Ang-1 improved locomotor recovery in the chronic phase of injury. These results indicate that combining angiogenesis with vascular stabilization may have potential therapeutic applications following SCI.
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Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material.
Hum. Gene Ther.
PUBLISHED: 09-16-2010
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A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18?x?10¹¹ particles/ml; 95% confidence interval [CI], 7.89?x?10¹¹ to 1.05?x?10¹² particles/ml), vector genomes ({X}, 3.28?x?10¹? vector genomes/ml; 95% CI, 2.70?x?10¹? to 4.75?x?10¹? vector genomes/ml), transducing units ({X}, 5.09?x?10? transducing units/ml; 95% CI, 2.00?x?10? to 9.60?x?10? transducing units/ml), and infectious units ({X}, 4.37?x?10? TCID?? IU/ml; 95% CI, 2.06?x?10? to 9.26?x?10? TCID?? IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed.
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Intramyocardial VEGF-B167 gene delivery delays the progression towards congestive failure in dogs with pacing-induced dilated cardiomyopathy.
Circ. Res.
PUBLISHED: 04-29-2010
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Vascular endothelial growth factor (VEGF)-B selectively binds VEGF receptor (VEGFR)-1, a receptor that does not mediate angiogenesis, and is emerging as a major cytoprotective factor.
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The renaissance of competitive PCR as an accurate tool for precise nucleic acid quantification.
Methods Mol. Biol.
PUBLISHED: 03-20-2010
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Here, we report a detailed procedure for the exact quantification of minute amounts of nucleic acids by competitive PCR. This technique entails the co-amplification of a target DNA or cDNA in a biological sample together with a known quantity of a target-specific standard, the competitor, which is added exogenously to the sample and is almost identical to the DNA region to be amplified. Competitive PCR offers the advantage to render the PCR reaction independent of the number of amplification cycles, since any intra-assay variation has the same effect on both target and competitor. Since the final ratio between target and competitor amplification products exactly reflects the initial ratio between the two species, and being the amount of added competitor known, competitive PCR allows the determination of the exact number of molecules of target, with an accuracy that is still unsurpassed by any other alternative procedure, including real-time PCR. The protocols described in this chapter cover most of the possible applications of competitive PCR, including the quantification of an mRNA transcript and the simultaneous determination of multiple targets.
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Insulin downregulates SIRT1 and AMPK activation and is associated with changes in liver fat, but not in inflammation and mitochondrial oxidative capacity, in streptozotocin-diabetic rat.
Clin Nutr
PUBLISHED: 01-17-2010
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Involvement of insulin in diabetes-associated liver triglyceride deposition and its potential pathways remain incompletely defined. SIRT1 may negatively modulate lipogenesis and liver triglyceride accumulation, involving AMP-activated protein kinase (AMPK) activation. In streptozotocin-diabetic rats, we hypothesized that insulin negatively modulates liver SIRT1 and activates AMPK-inhibited lipogenic mediators leading to triglyceride accumulation. The impact of insulin deprivation (INS-) and replacement (INS+) on liver inflammation and mitochondrial oxidative capacity (also potentially regulating triglyceride deposition) was also measured.
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Cardiomyocyte VEGFR-1 activation by VEGF-B induces compensatory hypertrophy and preserves cardiac function after myocardial infarction.
FASEB J.
PUBLISHED: 12-17-2009
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Mounting evidence indicates that the function of members of the vascular endothelial growth factor (VEGF) family extends beyond blood vessel formation. Here, we show that the prolonged intramyocardial expression of VEGF-A(165) and VEGF-B(167) on adeno-associated virus-mediated gene delivery determined a marked improvement in cardiac function after myocardial infarction in rats, by promoting cardiac contractility, preserving viable cardiac tissue, and preventing remodeling of the left ventricle (LV) over time. Consistent with this functional outcome, animals treated with both factors showed diminished fibrosis and increased contractile myocardium, which were more pronounced after expression of the selective VEGF receptor-1 (VEGFR-1) ligand VEGF-B, in the absence of significant induction of angiogenesis. We found that cardiomyocytes expressed VEGFR-1, VEGFR-2, and neuropilin-1 and that, in particular, VEGFR-1 was specifically up-regulated in hypoxia and on exposure to oxidative stress. VEGF-B exerted powerful antiapoptotic effect in both cultured cardiomyocytes and after myocardial infarction in vivo. Finally, VEGFR-1 activation by VEGF-B was found to elicit a peculiar gene expression profile proper of the compensatory, hypertrophic response, consisting in activation of alphaMHC and repression of betaMHC and skeletal alpha-actin, and an increase in SERCA2a, RYR, PGC1alpha, and cardiac natriuretic peptide transcripts, both in cultured cardiomyocytes and in infarcted hearts. The finding that VEGFR-1 activation by VEGF-B prevents loss of cardiac mass and promotes maintenance of cardiac contractility over time has obvious therapeutic implications.
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Semaphorin 3A is an endogenous angiogenesis inhibitor that blocks tumor growth and normalizes tumor vasculature in transgenic mouse models.
J. Clin. Invest.
PUBLISHED: 08-06-2009
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Tumor growth and progression rely upon angiogenesis, which is regulated by pro- and antiangiogenic factors, including members of the semaphorin family. By analyzing 3 different mouse models of multistep carcinogenesis, we show here that during angiogenesis, semaphorin 3A (Sema3A) is expressed in ECs, where it serves as an endogenous inhibitor of angiogenesis that is present in premalignant lesions and lost during tumor progression. Pharmacologic inhibition of endogenous Sema3A during the angiogenic switch, the point when pretumoral lesions initiate an angiogenic phase that persists throughout tumor growth, enhanced angiogenesis and accelerated tumor progression. By contrast, when, during the later stages of carcinogenesis following endogenous Sema3A downmodulation, Sema3A was ectopically reintroduced into islet cell tumors by somatic gene transfer, successive waves of apoptosis ensued, first in ECs and then in tumor cells, resulting in reduced vascular density and branching and inhibition of tumor growth and substantially extended survival. Further, long-term reexpression of Sema3A markedly improved pericyte coverage of tumor blood vessels, something that is thought to be a key property of tumor vessel normalization, and restored tissue normoxia. We conclude, therefore, that Sema3A is an endogenous and effective antiangiogenic agent that stably normalizes the tumor vasculature.
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Inducible adeno-associated virus vectors promote functional angiogenesis in adult organisms via regulated vascular endothelial growth factor expression.
Cardiovasc. Res.
PUBLISHED: 05-14-2009
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Members of the vascular endothelial growth factor (VEGF) family are among the most promising cytokines to induce neovascularization of ischaemic tissues; however, their unregulated expression often results in major undesired effects. Here, we describe the properties of inducible vectors based on the adeno-associated virus (AAV), allowing precise control of VEGF expression, and exploit these vectors to define the kinetics of the angiogenic response elicited by the factor.
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Development of antibody fragments for immunotherapy of prion diseases.
Biochem. J.
PUBLISHED: 03-17-2009
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Prions are infectious proteins responsible for a group of fatal neurodegenerative diseases called TSEs (transmissible spongiform encephalopathies) or prion diseases. In mammals, prions reproduce themselves by recruiting the normal cellular protein PrP(C) and inducing its conversion into the disease-causing isoform denominated PrP(Sc). Recently, anti-prion antibodies have been shown to permanently cure prion-infected cells. However, the inability of full-length antibodies and proteins to cross the BBB (blood-brain barrier) hampers their use in the therapy of TSEs in vivo. Alternatively, brain delivery of prion-specific scFv (single-chain variable fragment) by AAV (adeno-associated virus) transfer delays the onset of the disease in infected mice, although protection is not complete. We investigated the anti-prion effects of a recombinant anti-PrP (D18) scFv by direct addition to scrapie-infected cell cultures or by infection with both lentivirus and AAV-transducing vectors. We show that recombinant anti-PrP scFv is able to reduce proteinase K-resistant PrP content in infected cells. In addition, we demonstrate that lentiviruses are more efficient than AAV in gene transfer of the anti-PrP scFv gene and in reducing PrP(Sc) content in infected neuronal cell lines. Finally, we have used a bioinformatic approach to construct a structural model of the D18scFv-PrP(C) complex. Interestingly, according to the docking results, Arg(PrP)(151) (Arg(151) from prion protein) is the key residue for the interactions with D18scFv, anchoring the PrP(C) to the cavity of the antibody. Taken together, these results indicate that combined passive and active immunotherapy targeting PrP might be promising strategies for therapeutic intervention in prion diseases.
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Functional screening identifies miRNAs inducing cardiac regeneration.
Nature
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In mammals, enlargement of the heart during embryonic development is primarily dependent on the increase in cardiomyocyte numbers. Shortly after birth, however, cardiomyocytes stop proliferating and further growth of the myocardium occurs through hypertrophic enlargement of the existing myocytes. As a consequence of the minimal renewal of cardiomyocytes during adult life, repair of cardiac damage through myocardial regeneration is very limited. Here we show that the exogenous administration of selected microRNAs (miRNAs) markedly stimulates cardiomyocyte proliferation and promotes cardiac repair. We performed a high-content microscopy, high-throughput functional screening for human miRNAs that promoted neonatal cardiomyocyte proliferation using a whole-genome miRNA library. Forty miRNAs strongly increased both DNA synthesis and cytokinesis in neonatal mouse and rat cardiomyocytes. Two of these miRNAs (hsa-miR-590 and hsa-miR-199a) were further selected for testing and were shown to promote cell cycle re-entry of adult cardiomyocytes ex vivo and to promote cardiomyocyte proliferation in both neonatal and adult animals. After myocardial infarction in mice, these miRNAs stimulated marked cardiac regeneration and almost complete recovery of cardiac functional parameters. The miRNAs identified hold great promise for the treatment of cardiac pathologies consequent to cardiomyocyte loss.
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Neuropilin-1 identifies a subset of bone marrow Gr1- monocytes that can induce tumor vessel normalization and inhibit tumor growth.
Cancer Res.
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Improving tumor perfusion, thus tempering tumor-associated hypoxia, is known to impair cancer progression. Previous work from our laboratory has shown that VEGF-A165 and semaphorin 3A (Sema3A) promote vessel maturation through the recruitment of a population of circulating monocytes expressing the neuropilin-1 (Nrp1) receptor (Nrp1-expressing monocytes; NEM). Here, we define the characteristics of bone marrow NEMs and assess whether these cells might represent an exploitable tool to induce tumor vessel maturation. Gene expression signature and surface marker analysis have indicated that NEMs represent a specific subset of CD11b+ Nrp1+ Gr1- resident monocytes, distinctively recruited by Sema3A. NEMs were found to produce several factors involved in vessel maturation, including PDGFb, TGF-?, thrombospondin-1, and CXCL10; consistently, they were chemoattractive for vascular smooth muscle cells in vitro. When directly injected into growing tumors, NEMs, isolated either from the bone marrow or from Sema3A-expressing muscles, exerted antitumor activity despite having no direct effects on the proliferation of tumor cells. NEM inoculation specifically promoted mural cell coverage of tumor vessels and decreased vascular leakiness. Tumors treated with NEMs were smaller, better perfused and less hypoxic, and had a reduced level of activation of HIF-1?. We conclude that NEMs represent a novel, unique population of myeloid cells that, once inoculated into a tumor, induce tumor vessel normalization and inhibit tumor growth.
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Inhibition of choroidal and corneal pathologic neovascularization by Plgf1-de gene transfer.
Invest. Ophthalmol. Vis. Sci.
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Ocular neovascularization (NV), the primary cause of blindness, typically is treated via inhibition of VEGF-A activity. However, besides VEGF-A, other proteins of the same family, including VEGF-B and placental growth factor (PlGF, all together VEGFs), have a crucial role in the angiogenesis process. PlGF and VEGF, which form heterodimers if co-expressed, both are required for pathologic angiogenesis. We generated a PlGF1 variant, named PlGF1-DE, which is unable to bind and activate VEGFR-1, but retains the ability to form heterodimer. PlGF1-DE acts as dominant negative of VEGF-A and PlGF1wt through heterodimerization mechanism. The purpose of our study was to explore the therapeutic potential of Plgf1-de gene in choroid and cornea NV context.
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Capsid protein expression and adeno-associated virus like particles assembly in Saccharomyces cerevisiae.
Microb. Cell Fact.
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The budding yeast Saccharomyces cerevisiae supports replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses) and has provided means for up-scalable, cost- and time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus). We have recently demonstrated that S. cerevisiae can form single stranded DNA AAV2 genomes starting from a circular plasmid. In this work, we have investigated the possibility to assemble AAV capsids in yeast.
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Terminal differentiation of cardiac and skeletal myocytes induces permissivity to AAV transduction by relieving inhibition imposed by DNA damage response proteins.
Mol. Ther.
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Gene therapy vectors based on the adeno-associated virus (AAV) are extremely efficient for gene transfer into post-mitotic cells of heart, muscle, brain, and retina. The reason for their exquisite tropism for these cells has long remained elusive. Here, we show that upon terminal differentiation, cardiac and skeletal myocytes downregulate proteins of the DNA damage response (DDR) and that this markedly induces permissivity to AAV transduction. We observed that expression of members of the MRN complex (Mre11, Rad50, Nbs1), which bind the incoming AAV genomes, faded in cardiomyocytes at ~2 weeks after birth, as well as upon myoblast differentiation in vitro; in both cases, withdrawal of the cells from the cell cycle coincided with increased AAV permissivity. Treatment of proliferating cells with short-interfering RNAs (siRNAs) against the MRN proteins, or with microRNA-24, which is normally upregulated upon terminal differentiation and negatively controls the Nbs1 levels, significantly increased permissivity to AAV transduction. Consistently, delivery of these small RNAs to the juvenile liver concomitant with AAV markedly improved in vivo hepatocyte transduction. Collectively, these findings support the conclusion that cellular DDR proteins inhibit AAV transduction and that terminal cell differentiation relieves this restriction.
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Brain delivery of AAV9 expressing an anti-PrP monovalent antibody delays prion disease in mice.
Prion
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Prion diseases are caused by a conformational modification of the cellular prion protein (PrP (C)) into disease-specific forms, termed PrP (Sc), that have the ability to interact with PrP (C) promoting its conversion to PrP (Sc). In vitro studies demonstrated that anti-PrP antibodies inhibit this process. In particular, the single chain variable fragment D18 antibody (scFvD18) showed high efficiency in curing chronically prion-infected cells. This molecule binds the PrP (C) region involved in the interaction with PrP (Sc) thus halting further prion formation. These findings prompted us to test the efficiency of scFvD18 in vivo. A recombinant Adeno-Associated Viral vector serotype 9 was used to deliver scFvD18 to the brain of mice that were subsequently infected by intraperitoneal route with the mouse-adapted scrapie strain RML. We found that the treatment was safe, prolonged the incubation time of scrapie-infected animals and decreased the burden of total proteinase-resistant PrP (Sc) in the brain, suggesting that scFvD18 interferes with prion replication in vivo. This approach is relevant for designing new therapeutic strategies for prion diseases and other disorders characterized by protein misfolding.
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Semaphorin 3A overcomes cancer hypoxia and metastatic dissemination induced by antiangiogenic treatment in mice.
J. Clin. Invest.
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Cancer development, progression, and metastasis are highly dependent on angiogenesis. The use of antiangiogenic drugs has been proposed as a novel strategy to interfere with tumor growth, but cancer cells respond by developing strategies to escape these treatments. In particular, animal models show that antiangiogenic drugs currently used in clinical settings reduce tumor tissue oxygenation and trigger molecular events that foster cancer resistance to therapy. Here, we show that semaphorin 3A (Sema3A) expression overcomes the proinvasive and prometastatic resistance observed upon angiogenesis reduction by the small-molecule tyrosine inhibitor sunitinib in both pancreatic neuroendocrine tumors (PNETs) in RIP-Tag2 mice and cervical carcinomas in HPV16/E2 mice. By improving cancer tissue oxygenation and extending the normalization window, Sema3A counteracted sunitinib-induced activation of HIF-1?, Met tyrosine kinase receptor, epithelial-mesenchymal transition (EMT), and other hypoxia-dependent signaling pathways. Sema3A also reduced tumor hypoxia and halted cancer dissemination induced by DC101, a specific inhibitor of the VEGF pathway. As a result, reexpressing Sema3A in cancer cells converts metastatic PNETs and cervical carcinomas into benign lesions. We therefore suggest that this strategy could be developed to safely harnesses the therapeutic potential of the antiangiogenic treatment.
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Carbon nanotubes promote growth and spontaneous electrical activity in cultured cardiac myocytes.
Nano Lett.
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Nanoscale manipulations of the extracellular microenvironment are increasingly attracting attention in tissue engineering. Here, combining microscopy, biological, and single-cell electrophysiological methodologies, we demonstrate that neonatal rat ventricular myocytes cultured on substrates of multiwall carbon nanotubes interact with carbon nanotubes by forming tight contacts and show increased viability and proliferation. Furthermore, we observed changes in the electrophysiological properties of cardiomyocytes, suggesting that carbon nanotubes are able to promote cardiomyocyte maturation.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.