scholarly journals The Spherical Nucleic Acids mRNA Detection Paradox

2016 ◽  
Author(s):  
Marie Held ◽  
Raphael Levy
Keyword(s):  
Gold Nanoparticles ◽  
Mrna Level ◽  
Membrane Vesicles ◽  
Apparent Paradox ◽  
Intracellular Vesicle ◽  
Intracellular Sensing ◽  
The 1950S ◽  
Spherical Nucleic Acids ◽  
New Applications ◽  
Endocytic Compartments

<p>From the 1950s onwards, our understanding of the formation and intracellular trafficking of membrane vesicles was informed by experiments in which cells were exposed to gold nanoparticles and their uptake and localisation, studied by electron microscopy.&nbsp; In the last decade, building on progress in the synthesis of gold nanoparticles and their controlled functionalisation with a large variety of biomolecules (DNA, peptides, polysaccharides), new applications have been proposed, including the imaging and sensing of intracellular events. Yet, as already demonstrated in the 1950s, uptake of nanoparticles results in confinement within an intracellular vesicle which in principle should preclude sensing of cytosolic events. To study this apparent paradox, we focus on a commercially available nanoparticle probe that detects mRNA through the release of a fluorescently-labelled oligonucleotide (unquenching the fluorescence) in the presence of the target mRNA. Using electron, fluorescence and photothermal microscopy, we show that the probes remain in endocytic compartments and that they do not report on mRNA level. We suggest that the validation of any nanoparticle-based probes for intracellular sensing should include a quantitative and thorough demonstration that the probes can reach the cytosolic compartment.</p>

scholarly journals The Spherical Nucleic Acids mRNA Detection Paradox

2015 ◽  
Author(s):  
Raphael Levy ◽  
Marie Held ◽  
David Mason ◽  
Joan Comenge ◽  
Gemma Carolan ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Mrna Level ◽  
Membrane Vesicles ◽  
Apparent Paradox ◽  
Intracellular Vesicle ◽  
Intracellular Sensing ◽  
The 1950S ◽  
Spherical Nucleic Acids ◽  
New Applications ◽  
Endocytic Compartments

<p>From the 1950s onwards, our understanding of the formation and intracellular trafficking of membrane vesicles was informed by experiments in which cells were exposed to gold nanoparticles and their uptake and localisation, studied by electron microscopy.&nbsp; In the last decade, building on progress in the synthesis of gold nanoparticles and their controlled functionalisation with a large variety of biomolecules (DNA, peptides, polysaccharides), new applications have been proposed, including the imaging and sensing of intracellular events. Yet, as already demonstrated in the 1950s, uptake of nanoparticles results in confinement within an intracellular vesicle which in principle should preclude sensing of cytosolic events. To study this apparent paradox, we focus on a commercially available nanoparticle probe that detects mRNA through the release of a fluorescently-labelled oligonucleotide (unquenching the fluorescence) in the presence of the target mRNA. Using electron, fluorescence and photothermal microscopy, we show that the probes remain in endocytic compartments and that they do not report on mRNA level. We suggest that the validation of any nanoparticle-based probes for intracellular sensing should include a quantitative and thorough demonstration that the probes can reach the cytosolic compartment.</p>

scholarly journals Controllable Synthesis and Catalytic Performance of Gold Nanoparticles with Cucurbit[n]urils (n = 5–8)

Nanomaterials ◽  
2018 ◽  
Vol 8 (12) ◽  
pp. 1015 ◽  
Author(s):  
Liangfeng Zhang ◽  
Simin Liu ◽  
Yuhua Wang ◽  
Haijun Zhang ◽  
Feng Liang
Keyword(s):  
Aqueous Solution ◽  
Gold Nanoparticles ◽  
Direct Relationship ◽  
Surface Coverage ◽  
Catalytic Performance ◽  
Ring Size ◽  
Controllable Synthesis ◽  
New Applications ◽  
Nanoparticle Sizes

A series of gold nanoparticles (AuNPs) was prepared in situ with different cucurbit[n]urils (CB[n]s) in an alkaline aqueous solution. The nanoparticle sizes can be well controlled by CB[n]s (n = 5, 6, 7, 8) with different ring sizes. The packing densities of CB[5–8] and free surface area on AuNPs were determined. A direct relationship was found between the ring size and packing density of CB[n]s with respect to the AuNP-catalyzed reduction of 4-nitrophenol in the presence of NaBH4. The larger particle size and higher surface coverage of bigger CB[n]-capped AuNPs significantly decreased the catalytic activity. Furthermore, this work could lead to new applications that utilize AuNPs under an overlayer of CB[n]s for catalysis, sensing, and drug delivery.

scholarly journals Intracellular fusion of sequentially formed endocytic compartments.

1988 ◽  
Vol 106 (4) ◽  
pp. 1083-1091 ◽  
Author(s):  
N H Salzman ◽  
F R Maxfield
Keyword(s):  
Cho Cells ◽  
Culture Medium ◽  
Chinese Hamster ◽  
Fluid Phase ◽  
Vesicle Traffic ◽  
Intracellular Vesicle ◽  
Surface Time ◽  
Golgi Region ◽  
Fluid Phase Endocytosis ◽  
Endocytic Compartments

A polyclonal anti-fluorescein antibody (AFA) which quenches fluorescein fluorescence has been used to distinguish between two models of intracellular vesicle traffic. These models address the question of whether sequentially endocytosed probes will mix intracellularly or whether they are carried through the cell in a sequential, isolated manner. Using transferrin (Tf) as a recycling receptor marker, we incubated Chinese hamster ovary (CHO) cells with fluorescein-Tf (F-Tf) which is rapidly endocytosed. After the F-Tf was completely cleared from the surface, AFA was added to the incubation medium and entered endocytic compartments by fluid phase endocytosis. Fusion of a vesicle containing AFA with the compartment containing F-Tf results in binding of AFA to fluorescein and the quenching of fluorescein fluorescence. When AFA was added to the culture medium 2 min after clearance of F-Tf from the surface, time dependent fluorescence quenching occurred. After 20 min, 67% saturation of F-Tf with AFA was observed. When the interval between F-Tf clearance and AFA addition was increased to 5 min only 41% saturation of F-Tf was found. These data indicate that there are some compartments which are accessible for mixing with subsequently endocytosed molecules, but the efficiency of mixing falls off rapidly as the interval between pulses is increased. In CHO cells Tf swiftly segregates to a collection of vesicles or tubules in the para-Golgi region, and at steady state most of the F-Tf is in this compartment. Using digital image analysis to quantify quenching in this region, we have found that F-Tf/AFA mixing is occurring either within this compartment or before transferrin enters it.

scholarly journals Tuning DNA–nanoparticle conjugate properties allows modulation of nuclease activity

Nanoscale ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 4956-4970
Author(s):  
Jeff C. Hsiao ◽  
Tomas Buryska ◽  
Eunjung Kim ◽  
Philip D. Howes ◽  
Andrew J. deMello
Keyword(s):  
Gold Nanoparticles ◽  
Nucleic Acids ◽  
Systematic Study ◽  
Nuclease Activity ◽  
Modular Units ◽  
Spherical Nucleic Acids

A systematic study of the interactions between nucleases and oligonucleotide-coated gold nanoparticles (spherical nucleic acids, SNAs) demonstrates that the modular units of SNAs can be leveraged to either accelerate or suppress nuclease kinetics.

scholarly journals Alcohol Promotes Exosome Biogenesis and Release via Modulating Rabs and miR-192 Expression in Human Hepatocytes

2022 ◽  
Vol 9 ◽  
Author(s):  
Shashi Bala ◽  
Mrigya Babuta ◽  
Donna Catalano ◽  
Aman Saiju ◽  
Gyongyi Szabo
Keyword(s):  
Liver Disease ◽  
Mrna Level ◽  
Alcohol Treatment ◽  
Membrane Vesicles ◽  
Alcohol Exposure ◽  
Human Hepatocytes ◽  
Transcriptional Level ◽  
Rab Proteins ◽  
Human Hepatoma Cells ◽  
Exosome Biogenesis

Exosomes are membrane vesicles released by various cell types into the extracellular space under different conditions including alcohol exposure. Exosomes are involved in intercellular communication and as mediators of various diseases. Alcohol use causes oxidative stress that promotes exosome secretion. Here, we elucidated the effects of alcohol on exosome biogenesis and secretion using human hepatocytes. We found that alcohol treatment induces the expression of genes involved in various steps of exosome formation. Expression of Rab proteins such as Rab1a, Rab5c, Rab6, Rab10, Rab11, Rab27a and Rab35 were increased at the mRNA level in primary human hepatocytes after alcohol treatment. Rab5, Rab6 and Rab11 showed significant induction in the livers of patients with alcohol-associated liver disease. Further, alcohol treatment also led to the induction of syntenin, vesicle-associated membrane proteins (VAMPs), and syntaxin that all play various roles in exosome biogenesis and secretion. VAMP3, VAMP5, VAPb, and syntaxin16 mRNA transcripts were increased in alcohol treated cells and in the livers of alcohol-associated liver disease (ALD) patients. Induction in these genes was associated with increases in exosome secretion in alcohol treated hepatocytes. We found that hepatocyte enriched miR-192 and miR-122 levels were significantly decreased in alcohol treated hepatocytes whereas their levels were increased in the cell-free supernatant. The primary transcripts of miR-192 and miR-122 were reduced in alcohol treated hepatocytes, suggesting alcohol partially affects these miRNAs at the transcriptional level. We found that miR-192 has putative binding sites for genes involved in exosome secretion. Inhibition of miR-192 in human hepatoma cells caused a significant increase in Rab27a, Rab35, syntaxin7 and syntaxin16 and a concurrent increase in exosome secretion, suggesting miR-192 regulates exosomes release in hepatocytes. Collectively, our results reveal that alcohol modulates Rabs, VAMPs and syntaxins directly and partly via miR-192 to induce exosome machinery and release.

scholarly journals Electroporation of outer membrane vesicles derived from Pseudomonas aeruginosa with gold nanoparticles

2019 ◽  
Vol 1 (12) ◽  
Author(s):  
Zeineb Ayed ◽  
Luana Cuvillier ◽  
Garima Dobhal ◽  
Renee V. Goreham
Keyword(s):  
Electron Microscopy ◽  
Drug Delivery ◽  
Gold Nanoparticles ◽  
Outer Membrane ◽  
Drug Delivery System ◽  
Delivery System ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Novel Drug Delivery System ◽  
Novel Drug

Abstract Since their discovery, extracellular vesicles have gained considerable scientific interest as a novel drug delivery system. In particular, outer membrane vesicles (OMVs) play a critical role in bacteria–bacteria communication and bacteria–host interactions by trafficking cell signalling biochemicals (i.e. DNA, RNA, proteins). Although previous studies have focused on the use of OMVs as vaccines, little work has been done on loading them with functional nanomaterials for drug delivery. We have developed a novel drug delivery system by loading OMVs with gold nanoparticles (AuNPs). AuNPs are versatile nanoparticles that have been extensively used in disease therapeutics. The particles were loaded into the vesicles via electroporation, which uses an electric pulse to create a short-lived electric field. The resulting capacitance on the membrane generates pores in the lipid bilayer of the OMVs allowing AuNPs (or any nanoparticle under 10 nm) inside the vesicles. Closure of the pores of the lipid membrane of the OMVs entraps the nanoparticles as cargo. Transmission electron microscopy was used to confirm the loading of AuNPs inside the OMVs and dynamic light scattering (DLS) and cryogenic scanning electron microscopy (cryo-SEM) verified the size and integrity of the OMVs. This is the first report to load nanoparticles into OMVs, demonstrating a potential method for drug delivery. Graphic abstract

Epistemic Anxiety

2018 ◽  
pp. 686-708
Author(s):  
Michael Rustin
Keyword(s):  
Social Research ◽  
Melanie Klein ◽  
Contemporary Society ◽  
Educational Settings ◽  
Wilfred Bion ◽  
The 1950S ◽  
New Applications

This chapter develops a concept of epistemic anxiety and explores its particular relevance to educational settings and to wider contexts in contemporary society. The idea brings into conjunction two significant psychoanalytical ideas. The first is that of the epistemophilic instinct proposed, following Freud’s reflections on anxiety, by Melanie Klein and developed by Wilfred Bion into a theory of thinking and its relations to love and hate. The second is the theory of unconscious social defences against anxiety which first evolved within the Tavistock tradition of psychoanalytic social research in the 1950s and has recently been subject to reappraisal and new applications. Its argument is that epistemic anxiety frequently arises in learning situations, and that learning and thinking can be facilitated if this is understood.

Regulation of expression of the SN1 transporter during renal adaptation to chronic metabolic acidosis in rats

2002 ◽  
Vol 283 (5) ◽  
pp. F1011-F1019 ◽  
Author(s):  
Anne M. Karinch ◽  
Cheng-Mao Lin ◽  
Christopher L. Wolfgang ◽  
Ming Pan ◽  
Wiley W. Souba
Keyword(s):  
Metabolic Acidosis ◽  
Amino Acid ◽  
Mrna Level ◽  
Membrane Vesicles ◽  
Fold Increase ◽  
Amino Acid Transporter ◽  
Regulation Of Expression ◽  
Chronic Metabolic Acidosis ◽  
Acid Transporter ◽  
Glutamine Uptake

During chronic metabolic acidosis, renal glutamine utilization increases markedly. We studied the expression of the system N1 (SN1) amino acid transporter in the kidney during chronic ammonium chloride acidosis in rats. Acidosis caused a 10-fold increase in whole kidney SN1 mRNA level and a 100-fold increase in the cortex. Acidosis increased Na+-dependent glutamine uptake into basolateral and brush-border membrane vesicles (BLMV and BBMV, respectively) isolated from rat cortex (BLMV, 219 ± 66 control vs. 651 ± 180 pmol · mg−1 · min−1 acidosis; BBMV, 1,112 ± 189 control vs. 1,652 ± 148 pmol · mg−1 · min−1 acidosis, both P < 0.05). Na+-independent uptake was unchanged by acidosis in BLMV and BBMV. The acidosis-induced increase in Na+-dependent glutamine uptake was eliminated by histidine, confirming transport by system N. SN1 protein was detected only in BLMV and BBMV from acidotic rats. After recovery from acidosis, SN1 mRNA and protein and Na+-dependent glutamine uptake activity rapidly returned to control levels. These data provide evidence that regulation of expression of the SN1 amino acid transporter is part of the renal homeostatic response to acid-base imbalance.

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