20 research outputs found
Cassiosomes are stinging-cell structures in the mucus of the upside-down jellyfish Cassiopea xamachana
This work is licensed under a Creative Commons Attribution 4.0 International License.Snorkelers in mangrove forest waters inhabited by the upside-down jellyfish Cassiopea xamachana report discomfort due to a sensation known as stinging water, the cause of which is unknown. Using a combination of histology, microscopy, microfluidics, videography, molecular biology, and mass spectrometry-based proteomics, we describe C. xamachana stinging-cell structures that we term cassiosomes. These structures are released within C. xamachana mucus and are capable of killing prey. Cassiosomes consist of an outer epithelial layer mainly composed of nematocytes surrounding a core filled by endosymbiotic dinoflagellates hosted within amoebocytes and presumptive mesoglea. Furthermore, we report cassiosome structures in four additional jellyfish species in the same taxonomic group as C. xamachana (Class Scyphozoa; Order Rhizostomeae), categorized as either motile (ciliated) or nonmotile types. This inaugural study provides a qualitative assessment of the stinging contents of C. xamachana mucus and implicates mucus containing cassiosomes and free intact nematocytes as the cause of stinging water
Bimodal Gold Nanoparticle Therapeutics for Manipulating Exogenous and Endogenous Protein Levels in Mammalian Cells
A new advance in cell transfection protocol using a bimodal
nanoparticle
agent to selectively manipulate protein expression levels within mammalian
cells is demonstrated. The nanoparticle based transfection approach
functions by controlled release of gene regulatory elements from a
6 nm AuNP (gold nanoparticle) surface. The endosomal release of the
regulatory elements from the nanoparticle surface results in endogenous
protein knockdown simultaneously with exogenous protein expression
for the first 48 h. The use of fluorescent proteins as the endogenous
and exogenous signals for protein expression enables the efficiency
of codelivery of siRNA (small interfering RNA) for GFP (green fluorescent
protein) knockdown and a dsRed-express linearized plasmid for induction
to be optically analyzed in CRL-2794, a human kidney cell line expressing
an unstable green fluorescent protein. Delivery of the bimodal nanoparticle
in cationic liposomes results in 20% GFP knockdown within 24 h of
delivery and continues exhibiting knockdown for up to 48 h for the
bimodal agent. Simultaneous dsRed expression is observed to initiate
within the same time frame with expression levels reaching 34% after
25 days although cells have divided approximately 20 times, implying
daughter cell transfection has occurred. Fluorescence cell sorting
results in a stable colony, as demonstrated by Western blot analysis.
The simultaneous delivery of siRNA and linearized plasmid DNA on the
surface of a single nanocrystal provides a unique method for definitive
genetic control within a single cell and leads to a very efficient
cell transfection protocol
Anti-GITR therapy promotes immunity against malignant glioma in a murine model
vi, 42 hlm.: ils,; 25 c
A Gold Nanoparticle Pentapeptide: Gene Fusion To Induce Therapeutic Gene Expression in Mesenchymal Stem Cells
Mesenchymal stem
cells (MSC) have been identified as having great
potential as autologous cell therapeutics to treat traumatic brain
injury and spinal injury as well as neuronal and cardiac ischemic
events. All future clinical applications of MSC cell therapies must
allow the MSC to be harvested, transfected, and induced to express
a desired protein or selection of proteins to have medical benefit.
For the full potential of MSC cell therapy to be realized, it is desirable
to systematically alter the protein expression of therapeutically
beneficial biomolecules in harvested MSC cells with high fidelity
in a single transfection event. We have developed a delivery platform
on the basis of the use of a solid gold nanoparticle that has been
surface modified to produce a fusion containing a zwitterionic, pentapeptide
designed from Bax inhibiting peptide (Ku70) to enhance cellular uptake
and a linearized expression vector to induce enhanced expression of
brain-derived neurotrophic factor (BDNF) in rat-derived MSCs. Ku70
is observed to effect >80% transfection following a single treatment
of femur bone marrow isolated rat MSCs with efficiencies for the delivery
of a 6.6 kbp gene on either a Au nanoparticle (NP) or CdSe/ZnS quantum
dot (QD). Gene expression is observed within 4 d by optical measurements,
and secretion is observed within 10 d by Western Blot analysis. The
combination of being able to selectively engineer the NP, to colocalize
biological agents, and to enhance the stability of those agents has
provided the strong impetus to utilize this novel class of materials
to engineer primary MSCs
Plasmid Transfection in Mammalian Cells Spatiotemporally Tracked by a Gold Nanoparticle
Recent advances in cell transfection have suggested that delivery of a gene on a gold nanoparticle (AuNP) can enhance transfection efficiency. The mechanism of transfection is poorly understood, particularly when the gene is appended to a AuNP, as expression of the desired exogenous protein is dependent not only on the efficiency of the gene being taken into the cell but also on efficient endosomal escape and cellular processing of the nucleic acid. Design of a multicolor surface energy transfer (McSET) molecular beacon by independently dye labeling a linearized plasmid and short duplex DNA (sdDNA) appended to a AuNP allows spatiotemporal profiling of the transfection events, providing insight into package uptake, disassembly, and final plasmid expression. Delivery of the AuNP construct encapsulated in Lipofectamine2000 is monitored in Chinese hamster ovary cells using live-cell confocal microscopy. The McSET beacon signals the location and timing of the AuNP release and endosomal escape events for the plasmid and the sdDNA discretely, which are correlated with plasmid transcription by fluorescent protein expression within the cell. It is observed that delivery of the construct leads to endosomal release of the plasmid and sdDNA from the AuNP surface at different rates, prior to endosomal escape. Slow cytosolic diffusion of the nucleic acids is believed to be the limiting step for transfection, impacting the time-dependent expression of protein. The overall protein expression yield is enhanced when delivered on a AuNP, possibly due to better endosomal escape or lower degradation prior to endosomal escape
Controlled Payload Release by Magnetic Field Triggered Neural Stem Cell Destruction for Malignant Glioma Treatment.
Stem cells have recently garnered attention as drug and particle carriers to sites of tumors, due to their natural ability to track to the site of interest. Specifically, neural stem cells (NSCs) have demonstrated to be a promising candidate for delivering therapeutics to malignant glioma, a primary brain tumor that is not curable by current treatments, and inevitably fatal. In this article, we demonstrate that NSCs are able to internalize 2 μm magnetic discs (SD), without affecting the health of the cells. The SD can then be remotely triggered in an applied 1 T rotating magnetic field to deliver a payload. Furthermore, we use this NSC-SD delivery system to deliver the SD themselves as a therapeutic agent to mechanically destroy glioma cells. NSCs were incubated with the SD overnight before treatment with a 1T rotating magnetic field to trigger the SD release. The potential timed release effects of the magnetic particles were tested with migration assays, confocal microscopy and immunohistochemistry for apoptosis. After the magnetic field triggered SD release, glioma cells were added and allowed to internalize the particles. Once internalized, another dose of the magnetic field treatment was administered to trigger mechanically induced apoptotic cell death of the glioma cells by the rotating SD. We are able to determine that NSC-SD and magnetic field treatment can achieve over 50% glioma cell death when loaded at 50 SD/cell, making this a promising therapeutic for the treatment of glioma
Recommended from our members
Controlled Payload Release by Magnetic Field Triggered Neural Stem Cell Destruction for Malignant Glioma Treatment
Stem cells have recently garnered attention as drug and particle carriers to sites of tumors, due to their natural ability to track to the site of interest. Specifically, neural stem cells (NSCs) have demonstrated to be a promising candidate for delivering therapeutics to malignant glioma, a primary brain tumor that is not curable by current treatments, and inevitably fatal. In this article, we demonstrate that NSCs are able to internalize 2 μm magnetic discs (SD), without affecting the health of the cells. The SD can then be remotely triggered in an applied 1 T rotating magnetic field to deliver a payload. Furthermore, we use this NSC-SD delivery system to deliver the SD themselves as a therapeutic agent to mechanically destroy glioma cells. NSCs were incubated with the SD overnight before treatment with a 1T rotating magnetic field to trigger the SD release. The potential timed release effects of the magnetic particles were tested with migration assays, confocal microscopy and immunohistochemistry for apoptosis. After the magnetic field triggered SD release, glioma cells were added and allowed to internalize the particles. Once internalized, another dose of the magnetic field treatment was administered to trigger mechanically induced apoptotic cell death of the glioma cells by the rotating SD. We are able to determine that NSC-SD and magnetic field treatment can achieve over 50% glioma cell death when loaded at 50 SD/cell, making this a promising therapeutic for the treatment of glioma
HIF-1α Is a Metabolic Switch between Glycolytic-Driven Migration and Oxidative Phosphorylation-Driven Immunosuppression of Tregs in Glioblastoma
Summary: The mechanisms by which regulatory T cells (Tregs) migrate to and function within the hypoxic tumor microenvironment are unclear. Our studies indicate that specific ablation of hypoxia-inducible factor 1α (HIF-1α) in Tregs results in enhanced CD8+ T cell suppression versus wild-type Tregs under hypoxia, due to increased pyruvate import into the mitochondria. Importantly, HIF-1α-deficient Tregs are minimally affected by the inhibition of lipid oxidation, a fuel that is critical for Treg metabolism in tumors. Under hypoxia, HIF-1α directs glucose away from mitochondria, leaving Tregs dependent on fatty acids for mitochondrial metabolism within the hypoxic tumor. Indeed, inhibition of lipid oxidation enhances the survival of mice with glioma. Interestingly, HIF-1α-deficient-Treg mice exhibit significantly enhanced animal survival in a murine model of glioma, due to their stymied migratory capacity, explaining their reduced abundance in tumor-bearing mice. Thus HIF-1α acts as a metabolic switch for Tregs between glycolytic-driven migration and oxidative phosphorylation-driven immunosuppression. : Miska et al. demonstrate that regulatory T cell (Treg)-specific depletion of HIF-1α promotes enhanced immune suppression at the cost of migration under hypoxic conditions. Within the hypoxic brain-tumor environment, Tregs are uniquely able to metabolize extracellular free fatty acids to promote their immunosuppressive functionality, which can be targeted in vivo. Keywords: regulatory T cell, glioblastoma, migration, glycolysis, fatty acid oxidation, oxidative phosphorylation, immunosuppressio
A: MTT assay of HB1.F3.CD cells. 10, 20, and 50 SD particles per cell were added into HB1.F3.CD cells for 24 hours. MF treatment consists of applying a 1T field rotating at 20 Hz for 30 min. B: Representative optical microscopy images of HB1.F3.CD cells before and after MF treatment.
<p>The top panel contains SD and the bottom panels are control without SD.</p
SD uptake and MF effect on glioma cells, U87.
<p>The cells are treated with 50SD/cell and allowed to incubate for 24h. Afterwards, the cells are exposed to MF treatment for 30 seconds and then stained with 7-AAD. The cells that receive SD + no MF do not have compromised membranes. The cells with SD and MF stain red.</p