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Find video protocols related to scientific articles indexed in Pubmed.
Site-specific reference person parameters and derived concentration standards for the Savannah River Site.
Health Phys
PUBLISHED: 03-27-2014
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The U.S. Department of Energy Order 458.1 states that the compliance with the 1 mSv annual dose constraint to a member of the public may be demonstrated by calculating dose to the maximally exposed individual (MEI) or to a representative person. Historically, the MEI concept was used for dose compliance at the Savannah River Site (SRS) using adult dose coefficients and adult male usage parameters. For future compliance, SRS plans to use the representative person concept for dose estimates to members of the public. The representative person dose will be based on the reference person dose coefficients from the U.S. DOE Derived Concentration Technical Standard and on usage parameters specific to SRS for the reference and typical person. Usage parameters and dose coefficients were determined for inhalation, ingestion and external exposure pathways. The reference intake for air, water, meat, dairy, freshwater fish, saltwater invertebrates, produce (fruits and vegetables), and grains for the 95th percentile are 17.4 m d, 2.19 L d, 220.6 g d, 674 cm d, 66.4 g d, 23.0 g d, 633.4 g d (448.5 g dand 631.7 g d) and 251.3 g d, respectively. For the 50th percentile: 13.4 m d, 0.809 L d, 86.4 g d, 187 cm d, 8.97 g d, 3.04 g d, 169.5 g d (45.9 g d and 145.6 g d), 101.3 g d, respectively. These parameters for the representative person were used to calculate and tabulate SRS-specific derived concentration standards (DCSs) for the pathways not included in DOE-STD-1196-2011.
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In vitro Inactivation of Latent HSV by Targeted Mutagenesis Using an HSV-specific Homing Endonuclease.
Mol Ther Nucleic Acids
PUBLISHED: 02-06-2014
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Following acute infection, herpes simplex virus (HSV) establishes latency in sensory neurons, from which it can reactivate and cause recurrent disease. Available antiviral therapies do not affect latent viral genomes; therefore, they do not prevent reactivation following therapy cessation. One possible curative approach involves the introduction of DNA double strand breaks in latent HSV genomes by rare-cutting endonucleases, leading to mutagenesis of essential viral genes. We tested this approach in an in vitro HSV latency model using the engineered homing endonuclease (HE) HSV1m5, which recognizes a sequence in the HSV-1 gene UL19, encoding the virion protein VP5. Coexpression of the 3'-exonuclease Trex2 with HEs increased HE-mediated mutagenesis frequencies up to sixfold. Following HSV1m5/Trex2 delivery with adeno-associated viral (AAV) vectors, the target site was mutated in latent HSV genomes with no detectable cell toxicity. Importantly, HSV production by latently infected cells after reactivation was decreased after HSV1m5/Trex2 exposure. Exposure to histone deacetylase inhibitors prior to HSV1m5/Trex2 treatment increased mutagenesis frequencies of latent HSV genomes another two- to fivefold, suggesting that chromatin modification may be a useful adjunct to gene-targeting approaches. These results support the continuing development of HEs and other nucleases (ZFNs, TALENs, CRISPRs) for cure of chronic viral infections.Molecular Therapy-Nucleic Acids (2014) 3, e1; doi:10.1038/mtna.2013.75; published online 4 February 2014.
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DNA cleavage enzymes for treatment of persistent viral infections: recent advances and the pathway forward.
Virology
PUBLISHED: 01-31-2014
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Treatment for most persistent viral infections consists of palliative drug options rather than curative approaches. This is often because long-lasting viral DNA in infected cells is not affected by current antivirals, providing a source for viral persistence and reactivation. Targeting latent viral DNA itself could therefore provide a basis for novel curative strategies. DNA cleavage enzymes can be used to induce targeted mutagenesis of specific genes, including those of exogenous viruses. Although initial in vitro and even in vivo studies have been carried out using DNA cleavage enzymes targeting various viruses, many questions still remain concerning the feasibility of these strategies as they transition into preclinical research. Here, we review the most recent findings on DNA cleavage enzymes for human viral infections, consider the most relevant animal models for several human viral infections, and address issues regarding safety and enzyme delivery. Results from well-designed in vivo studies will ideally provide answers to the most urgent remaining questions, and allow continued progress toward clinical application.
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AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.
PLoS ONE
PUBLISHED: 01-01-2014
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Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy.
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Kinetics of CH2OO reactions with SO2, NO2, NO, H2O and CH3CHO as a function of pressure.
Phys Chem Chem Phys
PUBLISHED: 11-29-2013
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Kinetics of CH2OO Criegee intermediate reactions with SO2, NO2, NO, H2O and CH3CHO and CH2I radical reactions with NO2 are reported as a function of pressure at 295 K. Measurements were made under pseudo-first-order conditions using flash photolysis of CH2I2-O2-N2 gas mixtures in the presence of excess co-reagent combined with monitoring of HCHO reaction products by laser-induced fluorescence (LIF) spectroscopy and, for the reaction with SO2, direct detection of CH2OO by photoionisation mass spectrometry (PIMS). Rate coefficients for CH2OO + SO2 and CH2OO + NO2 are independent of pressure in the ranges studied and are (3.42 ± 0.42) × 10(-11) cm(3) s(-1) (measured between 1.5 and 450 Torr) and (1.5 ± 0.5) × 10(-12) cm(3) s(-1) (measured between 25 and 300 Torr), respectively. The rate coefficient for CH2OO + CH3CHO is pressure dependent, with the yield of HCHO decreasing with increasing pressure. Upper limits of 2 × 10(-13) cm(3) s(-1) and 9 × 10(-17) cm(3) s(-1) are placed on the rate coefficients for CH2OO + NO and CH2OO + H2O, respectively. The upper limit for the rate coefficient for CH2OO + H2O is significantly lower than has been reported previously, with consequences for modelling of atmospheric impacts of CH2OO chemistry.
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CH2OO Criegee biradical yields following photolysis of CH2I2 in O2.
Phys Chem Chem Phys
PUBLISHED: 10-12-2013
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Yields of CH2OO and CH2IO2 from the reaction of CH2I radicals with O2 are reported as a function of total pressure, [N2] and [O2] at T = 295 K using three complementary methods. Results from the three methods are similar, with no observed additional dependence on [O2]. The CH2I + O2 reaction has a yield of ~18% CH2OO at atmospheric pressure.
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Complex rectovaginal fistula repair with non-cross-linked porcine graft augmentation.
Int Urogynecol J
PUBLISHED: 07-20-2013
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Our aim was to demonstrate a unique approach to repairing a complex rectovaginal fistula using a non-cross-linked porcine dermal graft.
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Predictors of hepatitis B cure using gene therapy to deliver DNA cleavage enzymes: a mathematical modeling approach.
PLoS Comput. Biol.
PUBLISHED: 07-01-2013
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Most chronic viral infections are managed with small molecule therapies that inhibit replication but are not curative because non-replicating viral forms can persist despite decades of suppressive treatment. There are therefore numerous strategies in development to eradicate all non-replicating viruses from the body. We are currently engineering DNA cleavage enzymes that specifically target hepatitis B virus covalently closed circular DNA (HBV cccDNA), the episomal form of the virus that persists despite potent antiviral therapies. DNA cleavage enzymes, including homing endonucleases or meganucleases, zinc-finger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPR-associated system 9 (Cas9) proteins, can disrupt specific regions of viral DNA. Because DNA repair is error prone, the virus can be neutralized after repeated cleavage events when a target sequence becomes mutated. DNA cleavage enzymes will be delivered as genes within viral vectors that enter hepatocytes. Here we develop mathematical models that describe the delivery and intracellular activity of DNA cleavage enzymes. Model simulations predict that high vector to target cell ratio, limited removal of delivery vectors by humoral immunity, and avid binding between enzyme and its DNA target will promote the highest level of cccDNA disruption. Development of de novo resistance to cleavage enzymes may occur if DNA cleavage and error prone repair does not render the viral episome replication incompetent: our model predicts that concurrent delivery of multiple enzymes which target different vital cccDNA regions, or sequential delivery of different enzymes, are both potentially useful strategies for avoiding multi-enzyme resistance. The underlying dynamics of cccDNA persistence are unlikely to impact the probability of cure provided that antiviral therapy is given concurrently during eradication trials. We conclude by describing experiments that can be used to validate the model, which will in turn provide vital information for dose selection for potential curative trials in animals and ultimately humans.
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Targeted gene disruption to cure HIV.
Curr Opin HIV AIDS
PUBLISHED: 03-13-2013
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Recent clinical research suggests that an HIV-infected patient with lymphoma who was transplanted with bone marrow homozygous for a disrupted mutant CCR5 allele has no remaining HIV replication and is effectively cured of HIV. Here, we discuss the approaches of disrupting host and viral genes involved in HIV replication and pathogenesis with the aim of curing patients with HIV.
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Exploring the structure and properties of transition metal templated {VM17(VO4)2} Dawson-like capsules.
Inorg Chem
PUBLISHED: 07-27-2011
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Vanadate(V)-templated Dawson-type capsules {V(IV)M(VI)(17)(VO(4))(2)} (M = Mo, W; 1-2) have been synthesized and investigated by electrochemical methods in aqueous and organic media using spectroscopic techniques, EPR, UV-vis/NIR, IR, and CSI-MS (cryospray ionization mass spec.), and the clusters have been examined in the solid state by magnetic studies. The collision-induced dissociation (CID-MS) studies confirmed the solution structures as well as helped pinpoint the position of the vanadium ion on the {VM(17)}-type shell, which was corroborated by EPR and theoretical studies.
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Intravitreal injection of AAV2 transduces macaque inner retina.
Invest. Ophthalmol. Vis. Sci.
PUBLISHED: 01-01-2011
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Adeno-associated virus serotype 2 (AAV2) has been shown to be effective in transducing inner retinal neurons after intravitreal injection in several species. However, results in nonprimates may not be predictive of transduction in the human inner retina, because of differences in eye size and the specialized morphology of the high-acuity human fovea. This was a study of inner retina transduction in the macaque, a primate with ocular characteristics most similar to that of humans.
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Nox2 redox signaling maintains essential cell populations in the brain.
Nat. Chem. Biol.
PUBLISHED: 03-16-2010
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Reactive oxygen species (ROS) are conventionally classified as toxic consequences of aerobic life, and the brain is particularly susceptible to ROS-induced oxidative stress and damage owing to its high energy and oxygen demands. NADPH oxidases (Nox) are a widespread source of brain ROS implicated in seizures, stroke and neurodegeneration. A physiological role for ROS generation in normal brain function has not been established, despite the fact that mice and humans lacking functional Nox proteins have cognitive deficits. Using molecular imaging with Peroxyfluor-6 (PF6), a new selective fluorescent indicator for hydrogen peroxide (H(2)O(2)), we show that adult hippocampal stem/progenitor cells (AHPs) generate H(2)O(2) through Nox2 to regulate intracellular growth signaling pathways, which in turn maintains their normal proliferation in vitro and in vivo. Our results challenge the traditional view that brain ROS are solely deleterious by demonstrating that controlled ROS chemistry is needed for maintaining specific cell populations.
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Ferromagnetic Ni(II) discs.
Chemistry
PUBLISHED: 09-17-2009
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A family of planar disc-like hexa-, octa- and decametallic Ni(II) complexes exhibit dominant ferromagnetic exchange. The deca- and octametallic clusters [Ni(II) (10)(tmp)(2)(N(3))(8)(acac)(6)(MeOH)(6)] (1, H(3)tmp=1,1,1-tris(hydroxymethyl)propane; acac=acetylacetonate) and [Ni(II) (8)(thme)(2)(O(2)CPh)(4)(Cl)(6)(MeCN)(6)(H(2)O)(2)] (2, H(3)thme=1,1,1-tris(hydroxymethyl)ethane) represent rare examples of Ni(II)-based single-molecule magnets, and [Ni(II) (10)] (1) possesses the largest barrier to magnetisation reversal of any Ni(II) single-molecule magnet to date.
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Tropospheric OH and HO2 radicals: field measurements and model comparisons.
Chem Soc Rev
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The hydroxyl radical, OH, initiates the removal of the majority of trace gases in the atmosphere, and together with the closely coupled species, the hydroperoxy radical, HO(2), is intimately involved in the oxidation chemistry of the atmosphere. This critical review discusses field measurements of local concentrations of OH and HO(2) radicals in the troposphere, and in particular the comparisons that have been made with numerical model calculations containing a detailed chemical mechanism. The level of agreement between field measurements of OH and HO(2) concentrations and model calculations for a given location provides an indication of the degree of understanding of the underlying oxidation chemistry. We review the measurement-model comparisons for a range of different environments sampled from the ground and from aircraft, including the marine boundary layer, continental low-NO(x) regions influenced by biogenic emissions, the polluted urban boundary layer, and polar regions. Although good agreement is found for some environments, there are significant discrepancies which remain unexplained, a notable example being unpolluted, forested regions. OH and HO(2) radicals are difficult species to measure in the troposphere, and we also review changes in detection methodology, quality assurance procedures such as instrument intercomparisons, and potential interferences.
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Targeted DNA mutagenesis for the cure of chronic viral infections.
J. Virol.
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Human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), and herpes simplex virus (HSV) have been incurable to date because effective antiviral therapies target only replicating viruses and do not eradicate latently integrated or nonreplicating episomal viral genomes. Endonucleases that can target and cleave critical regions within latent viral genomes are currently in development. These enzymes are being engineered with high specificity such that off-target binding of cellular DNA will be absent or minimal. Imprecise nonhomologous-end-joining (NHEJ) DNA repair following repeated cleavage at the same critical site may permanently disrupt translation of essential viral proteins. We discuss the benefits and drawbacks of three types of DNA cleavage enzymes (zinc finger endonucleases, transcription activator-like [TAL] effector nucleases [TALENs], and homing endonucleases [also called meganucleases]), the development of delivery vectors for these enzymes, and potential obstacles for successful treatment of chronic viral infections. We then review issues regarding persistence of HIV-1, HBV, and HSV that are relevant to eradication with genome-altering approaches.
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Colloids as mobile substrates for the implantation and integration of differentiated neurons into the mammalian brain.
PLoS ONE
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Neuronal degeneration and the deterioration of neuronal communication lie at the origin of many neuronal disorders, and there have been major efforts to develop cell replacement therapies for treating such diseases. One challenge, however, is that differentiated cells are challenging to transplant due to their sensitivity both to being uprooted from their cell culture growth support and to shear forces inherent in the implantation process. Here, we describe an approach to address these problems. We demonstrate that rat hippocampal neurons can be grown on colloidal particles or beads, matured and even transfected in vitro, and subsequently transplanted while adhered to the beads into the young adult rat hippocampus. The transplanted cells have a 76% cell survival rate one week post-surgery. At this time, most transplanted neurons have left their beads and elaborated long processes, similar to the host neurons. Additionally, the transplanted cells distribute uniformly across the host hippocampus. Expression of a fluorescent protein and the light-gated glutamate receptor in the transplanted neurons enabled them to be driven to fire by remote optical control. At 1-2 weeks after transplantation, calcium imaging of host brain slice shows that optical excitation of the transplanted neurons elicits activity in nearby host neurons, indicating the formation of functional transplant-host synaptic connections. After 6 months, the transplanted cell survival and overall cell distribution remained unchanged, suggesting that cells are functionally integrated. This approach, which could be extended to other cell classes such as neural stem cells and other regions of the brain, offers promising prospects for neuronal circuit repair via transplantation of in vitro differentiated, genetically engineered neurons.
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What is Visualize?

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.