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Find video protocols related to scientific articles indexed in Pubmed.
Characterization of circulating microparticle-associated CD39 family ecto-nucleotidases in human plasma.
Purinergic Signal.
PUBLISHED: 08-28-2014
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Phosphohydrolysis of extracellular ATP and ADP is an essential step in purinergic signaling that regulates key pathophysiological processes, such as those linked to inflammation. Classically, this reaction has been known to occur in the pericellular milieu catalyzed by membrane bound cellular ecto-nucleotidases, which can be released in the form of both soluble ecto-enzymes as well as being associated with exosomes. Circulating ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1/CD39) and adenylate kinase 1 (AK1) activities have been shown to be present in plasma. However, other ecto-nucleotidases have not been characterized in depth. An in vitro ADPase assay was developed to probe the ecto-enzymes responsible for the ecto-nucleotidase activity in human platelet-free plasma, in combination with various specific biochemical inhibitors. Identities of ecto-nucleotidases were further characterized by chromatography, immunoblotting, and flow cytometry of circulating exosomes. We noted that microparticle-bound E-NTPDases and soluble AK1 constitute the highest levels of ecto-nucleotidase activity in human plasma. All four cell membrane expressed E-NTPDases are also found in circulating microparticles in human plasma, inclusive of: CD39, NTPDase 2 (CD39L1), NTPDase 3 (CD39L3), and NTPDase 8. CD39 family members and other ecto-nucleotidases are found on distinct microparticle populations. A significant proportion of the microparticle-associated ecto-nucleotidase activity is sensitive to POM6, inferring the presence of NTPDases, either -2 or/and -3. We have refined ADPase assays of human plasma from healthy volunteers and have found that CD39, NTPDases 2, 3, and 8 to be associated with circulating microparticles, whereas soluble AK1 is present in human plasma. These ecto-enzymes constitute the bulk circulating ADPase activity, suggesting a broader implication of CD39 family and other ecto-enzymes in the regulation of extracellular nucleotide metabolism.
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NAD+ protects against EAE by regulating CD4+ T-cell differentiation.
Nat Commun
PUBLISHED: 01-09-2014
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CD4(+) T cells are involved in the development of autoimmunity, including multiple sclerosis (MS). Here we show that nicotinamide adenine dinucleotide (NAD(+)) blocks experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, by inducing immune homeostasis through CD4(+)IFN?(+)IL-10(+) T cells and reverses disease progression by restoring tissue integrity via remyelination and neuroregeneration. We show that NAD(+) regulates CD4(+) T-cell differentiation through tryptophan hydroxylase-1 (Tph1), independently of well-established transcription factors. In the presence of NAD(+), the frequency of T-bet(-/-) CD4(+)IFN?(+) T cells was twofold higher than wild-type CD4(+) T cells cultured in conventional T helper 1 polarizing conditions. Our findings unravel a new pathway orchestrating CD4(+) T-cell differentiation and demonstrate that NAD(+) may serve as a powerful therapeutic agent for the treatment of autoimmune and other diseases.
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Role of endothelial progenitor cells and inflammatory cytokines in healing of diabetic foot ulcers.
PLoS ONE
PUBLISHED: 01-01-2013
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To evaluate changes in endothelial progenitor cells (EPCs) and cytokines in patients with diabetic foot ulceration (DFU) in association with wound healing.
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Signal sequence is still required in genes downstream of "autocleaving" 2A peptide for secretary or membrane-anchored expression.
Anal. Biochem.
PUBLISHED: 11-17-2009
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2A Peptide sequences are now being widely used to construct multicistronic expression vectors. It is suggested that when only the first 2A-linked protein bears a signal sequence, the signal-less protein(s) downstream of 2A can also be translocated into the mammalian endoplasmic reticulum system through a "slipstreaming" mechanism. By using flow cytometry and cell surface CD90 as a localization indicator, we show here that slipstreaming translocation does not occur in mammalian cells; that is, the second protein downstream of 2A still requires signal sequence for secretary or membrane-anchored expression.
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Alternative methods for characterization of extracellular vesicles.
Front Physiol
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Extracellular vesicles (ECVs) are nano-sized vesicles released by all cells in vitro as well as in vivo. Their role has been implicated mainly in cell-cell communication, but also in disease biomarkers and more recently in gene delivery. They represent a snapshot of the cell status at the moment of release and carry bioreactive macromolecules such as nucleic acids, proteins, and lipids. A major limitation in this emerging new field is the availability/awareness of techniques to isolate and properly characterize ECVs. The lack of gold standards makes comparing different studies very difficult and may potentially hinder some ECVs-specific evidence. Characterization of ECVs has also recently seen many advances with the use of Nanoparticle Tracking Analysis, flow cytometry, cryo-electron microscopy instruments, and proteomic technologies. In this review, we discuss the latest developments in translational technologies involving characterization methods including the facts in their support and the challenges they face.
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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.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

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.