Subcellular redox responses reveal different Cu-dependent antioxidant defenses between mitochondria and cytosol.

Excess intracellular Cu perturbs cellular redox balance and thus causes diseases. However, the relationship between cellular redox status and Cu homeostasis and how such an interplay is coordinated within cellular compartments has not yet been well established. Using combined approaches of organelle-specific redox sensor Grx1-roGFP2 and non-targeted proteomics, we investigate the real-time Cu-dependent antioxidant defenses of mitochondria and cytosol in live HEK293 cells. The Cu-dependent real-time imaging experiments show that CuCl2 treatment results in increased oxidative stress in both cytosol and mitochondria. In contrast, subsequent excess Cu removal by bathocuproine sulfonate, a Cu chelating reagent, lowers oxidative stress in mitochondria but causes even higher oxidative stress in the cytosol. The proteomic data reveal that several mitochondrial proteins, but not cytosolic ones, undergo significant abundance change under Cu treatments. The proteomic analysis also shows that proteins with significant changes are related to mitochondrial oxidative phosphorylation and glutathione synthesis. The differences in redox behaviors and protein profiles in different cellular compartments reveal distinct mitochondrial and cytosolic response mechanisms upon Cu-induced oxidative stress. These findings provide insights into how redox and Cu homeostasis interplay by modulating specific protein expressions at the subcellular levels, shedding light on understanding the effects of Cu-induced redox misregulation on the diseases.

[Establishment of a microtubule-fluorescent fusion protein mosaically labeled zebrafish motor neuron system].

Motor neurons are an important type of neurons that control movement. The transgenic fluorescent protein (FP)-labeled motor neurons of zebrafish line is disadvantageous for studying the morphogenesis of motor neurons. For example, the individual motor neuron is indistinguishable in this transgenic line due to the high density of the motor neurons and the interlaced synapses. In order to optimize the in vivo imaging methods for the analysis of motor neurons, the present study was aimed to establish a microtubule-fluorescent fusion protein mosaic system that can label motor neurons in zebrafish. Firstly, the promotor of mnx1, which was highly expressed in the spinal cord motor neurons, was subcloned into pDestTol2pA2 construct combined with the GFP-α-Tubulin fusion protein sequence by Gateway cloning technique. Then the recombinant constructs were co-injected with transposase mRNA into the 4-8 cell zebrafish embryos. Confocal imaging analysis was performed at 72 hours post fertilization (hpf). The results showed that the GFP fusion protein was expressed in three different types of motor neurons, and individual motor neurons were mosaically labeled. Further, the present study analyzed the correlation between the injection dose and the number and distribution of the mosaically labeled neurons. Fifteen nanograms of the recombinant constructs were suggested as an appropriate injection dose. Also, the defects of the motor neuron caused by the down-regulation of insm1a and kif15 were verified with this system. These results indicate that our novel microtubule-fluorescent fusion protein mosaic system can efficiently label motor neurons in zebrafish, which provides a more effective model for exploring the development and morphogenesis of motor neurons. It may also help to decipher the mechanisms underlying motor neuron disease and can be potentially utilized in drug screening.

Probing GFP Chromophore Analogs as Anti-HIV Agents Targeting LTR-III G-Quadruplex.

Green fluorescent protein (GFP) chromophore and its congeners draw significant attention mostly for bioimaging purposes. In this work we probed these compounds as antiviral agents. We have chosen LTR-III DNA G4, the major G-quadruplex (G4) present in the long terminal repeat (LTR) promoter region of the human immunodeficiency virus-1 (HIV-1), as the target for primary screening and designing antiviral drug candidates. The stabilization of this G4 was previously shown to suppress viral gene expression and replication. FRET-based high-throughput screening (HTS) of 449 GFP chromophore-like compounds revealed a number of hits, sharing some general structural features. Structure-activity relationships (SAR) for the most effective stabilizers allowed us to establish structural fragments, important for G4 binding. Synthetic compounds, developed on the basis of SAR analysis, exhibited high LTR-III G4 stabilization level. NMR spectroscopy and molecular modeling revealed the possible formation of LTR-III G4-ligand complex with one of the lead selective derivative ZS260.1 positioned within the cavity, thus supporting the LTR-III G4 attractiveness for drug targeting. Selected compounds showed moderate activity against HIV-I (EC50 1.78-7.7 µM) in vitro, but the activity was accompanied by pronounced cytotoxicity.

A Promising Intracellular Protein-Degradation Strategy: TRIMbody-Away Technique Based on Nanobody Fragment.

Most recently, a technology termed TRIM-Away has allowed acute and rapid destruction of endogenous target proteins in cultured cells using specific antibodies and endogenous/exogenous tripartite motif 21 (TRIM21). However, the relatively large size of the full-size mAbs (150 kDa) results in correspondingly low tissue penetration and inaccessibility of some sterically hindered epitopes, which limits the target protein degradation. In addition, exogenous introduction of TRIM21 may cause side effects for treated cells. To tackle these limitations, we sought to replace full-size mAbs with the smaller format of antibodies, a nanobody (VHH, 15 kDa), and construct a new type of fusion protein named TRIMbody by fusing the nanobody and RBCC motif of TRIM21. Next, we introduced enhanced green fluorescent protein (EGFP) as a model substrate and generated αEGFP TRIMbody using a bispecific anti-EGFP (αEGFP) nanobody. Remarkably, inducible expression of αEGFP TRIMbody could specifically degrade intracellular EGFP in HEK293T cells in a time-dependent manner. By treating cells with inhibitors, we found that intracellular EGFP degradation by αEGFP TRIMbody relies on both ubiquitin-proteasome and autophagy-lysosome pathways. Taken together, these results suggested that TRIMbody-Away technology could be utilized to specifically degrade intracellular protein and could expand the potential applications of degrader technologies.

Dynamic patterns of correlated activity in the prefrontal cortex encode information about social behavior.

New technologies make it possible to measure activity from many neurons simultaneously. One approach is to analyze simultaneously recorded neurons individually, then group together neurons which increase their activity during similar behaviors into an "ensemble." However, this notion of an ensemble ignores the ability of neurons to act collectively and encode and transmit information in ways that are not reflected by their individual activity levels. We used microendoscopic GCaMP imaging to measure prefrontal activity while mice were either alone or engaged in social interaction. We developed an approach that combines a neural network classifier and surrogate (shuffled) datasets to characterize how neurons synergistically transmit information about social behavior. Notably, unlike optimal linear classifiers, a neural network classifier with a single linear hidden layer can discriminate network states which differ solely in patterns of coactivity, and not in the activity levels of individual neurons. Using this approach, we found that surrogate datasets which preserve behaviorally specific patterns of coactivity (correlations) outperform those which preserve behaviorally driven changes in activity levels but not correlated activity. Thus, social behavior elicits increases in correlated activity that are not explained simply by the activity levels of the underlying neurons, and prefrontal neurons act collectively to transmit information about socialization via these correlations. Notably, this ability of correlated activity to enhance the information transmitted by neuronal ensembles is diminished in mice lacking the autism-associated gene Shank3. These results show that synergy is an important concept for the coding of social behavior which can be disrupted in disease states, reveal a specific mechanism underlying this synergy (social behavior increases correlated activity within specific ensembles), and outline methods for studying how neurons within an ensemble can work together to encode information.

Systemic AAV6-synapsin-GFP administration results in lower liver biodistribution, compared to AAV1&2 and AAV9, with neuronal expression following ultrasound-mediated brain delivery.

Non-surgical gene delivery to the brain can be achieved following intravenous injection of viral vectors coupled with transcranial MRI-guided focused ultrasound (MRIgFUS) to temporarily and locally permeabilize the blood-brain barrier. Vector and promoter selection can provide neuronal expression in the brain, while limiting biodistribution and expression in peripheral organs. To date, the biodistribution of adeno-associated viruses (AAVs) within peripheral organs had not been quantified following intravenous injection and MRIgFUS delivery to the brain. We evaluated the quantity of viral DNA from the serotypes AAV9, AAV6, and a mosaic AAV1&2, expressing green fluorescent protein (GFP) under the neuron-specific synapsin promoter (syn). AAVs were administered intravenously during MRIgFUS targeting to the striatum and hippocampus in mice. The syn promoter led to undetectable levels of GFP expression in peripheral organs. In the liver, the biodistribution of AAV9 and AAV1&2 was 12.9- and 4.4-fold higher, respectively, compared to AAV6. The percentage of GFP-positive neurons in the FUS-targeted areas of the brain was comparable for AAV6-syn-GFP and AAV1&2-syn-GFP. In summary, MRIgFUS-mediated gene delivery with AAV6-syn-GFP had lower off-target biodistribution in the liver compared to AAV9 and AAV1&2, while providing neuronal GFP expression in the striatum and hippocampus.

Human Bone Marrow Mesenchymal Stem Cells Modified Hybrid Baculovirus-Adeno-Associated Viral Vectors Targeting 131I Therapy of Hypopharyngeal Carcinoma.

Hypopharyngeal carcinoma is one of the most aggressive subtypes of squamous cell carcinoma of the head and neck. Although significant progress has been made in surgical techniques, radiotherapy, and chemotherapy, the prognosis is still poor. Mesenchymal stem cells (MSCs) have attracted substantial attention as tumor-targeted cellular carriers for cancer gene therapy. We have previously shown that recombinant baculovirus-adeno-associated vectors (BV-AAV) possessed high efficiency for multi-gene coexpression in human bone marrow MSCs (BMSCs) and BV-AAV-engineered BMSCs could effectively target hypopharyngeal cancer tissues in vivo. However, it was not clear whether BV-AAV-engineered BMSCs as cellular vehicles, mediating the expression of the sodium iodide symporter (NIS), would be effective in controlling the growth of hypopharyngeal carcinoma by radioiodine therapy. We constructed a hybrid BV-AAV containing the Luc-P2A-eGFP fusion or NIS sequence to modify BMSCs (BMSCs-Bac-Luc-P2A-eGFP or BMSCs-Bac-NIS). The 125I uptake of BMSCs-Bac-NIS was analyzed by an automatic gamma counter in vitro and micro-single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging in vivo. The value of radioiodine therapy for hypopharyngeal carcinoma was evaluated by measuring tumor volume, glucose metabolism (via 2-deoxy-2-[18F] glucose [18F-FDG] positron emission tomography/CT), and proliferation of tumor cells. We demonstrated that 125I uptake of BMSCs-Bac-NIS persists over long-term in vitro (at least 8 h). Radioactive uptake could be detected by SPECT/CT 1 h after 125I injection in the BMSCs-Bac-NIS group, showing that this strategy allows for the tracking of real-time migration and transgene expression of BMSCs. Radioiodine therapy resulted in a significant reduction in tumor growth (386.93 ± 249.23 mm3 vs 816.56 ± 213.87 mm3 in controls), increased survival, and decreased SUVmax of 18F-FDG. The hybrid BV-AAV that can provide a variety of genes and regulatory elements, as a novel gene therapy strategy opens the prospect of NIS-mediated radionuclide therapy of hypopharyngeal carcinoma after MSC-mediated gene delivery.

GAF-CaMP3-sfGFP, An Enhanced Version of the Near-Infrared Genetically Encoded Positive Phytochrome-Based Calcium Indicator for the Visualization of Neuronal Activity.

The first generation of near-infrared, genetically encoded calcium indicators (NIR-GECIs) was developed from bacterial phytochrome-based fluorescent proteins that utilize biliverdin (BV) as the chromophore moiety. However, NIR-GECIs have some main drawbacks such as either an inverted response to calcium ions (in the case of NIR-GECO1) or a limited dynamic range and a lack of data about their application in neurons (in the case of GAF-CaMP2-superfolder green fluorescent protein (sfGFP)). Here, we developed an enhanced version of the GAF-CaMP2-sfGFP indicator, named GAF-CaMP3-sfGFP. The GAF-CaMP3-sfGFP demonstrated spectral characteristics, molecular brightness, and a calcium affinity similar to the respective characteristics for its progenitor, but a 2.9-fold larger DF/F response to calcium ions. As compared to GAF-CaMP2-sfGFP, in cultured HeLa cells, GAF-CaMP3-sfGFP had similar brightness but a 1.9-fold larger DF/F response to the elevation of calcium ions levels. Finally, we successfully utilized the GAF-CaMP3-sfGFP for the monitoring of the spontaneous and stimulated activity of neuronal cultures and compared its performance with the R-GECO1 indicator using two-color confocal imaging. In the cultured neurons, GAF-CaMP3-sfGFP showed a linear DF/F response in the range of 0-20 APs and in this range demonstrated a 1.4-fold larger DF/F response but a 1.3- and 2.4-fold slower rise and decay kinetics, respectively, as compared to the same parameters for the R-GECO1 indicator.

A Mechanistic High-Content Analysis Assay Using a Chimeric Androgen Receptor That Rapidly Characterizes Androgenic Chemicals.

Human health is at risk from environmental exposures to a wide range of chemical toxicants and endocrine-disrupting chemicals (EDCs). As part of understanding this risk, the U.S. Environmental Protection Agency (EPA) has been pursuing new high-throughput in vitro assays and computational models to characterize EDCs. EPA models have incorporated our high-content analysis-based green fluorescent protein estrogen receptor (GFP-ER): PRL-HeLa assay, which allows direct visualization of ER binding to DNA regulatory elements. Here, we characterize a modified functional assay based on the stable expression of a chimeric androgen receptor (ARER), wherein a region containing the native AR DNA-binding domain (DBD) was replaced with the ERα DBD (amino acids 183-254). We demonstrate that the AR agonist dihydrotestosterone induces GFP-ARER nuclear translocation, PRL promoter binding, and transcriptional activity at physiologically relevant concentrations (<1 nM). In contrast, the AR antagonist bicalutamide induces only nuclear translocation of the GFP-ARER receptor (at µM concentrations). Estradiol also fails to induce visible chromatin binding, indicating androgen specificity. In a screen of reference chemicals from the EPA and the Agency for Toxic Substances and Disease Registry, the GFP-ARER cell model identified and mechanistically grouped activity by known (anti-)androgens based on the ability to induce nuclear translocation and/or chromatin binding. Finally, the cell model was used to identify potential (anti-)androgens in environmental samples in collaboration with the Houston Ship Channel/Galveston Bay Texas A&M University EPA Superfund Research Program. Based on these data, the chromatin-binding, in vitro assay-based GFP-ARER model represents a selective tool for rapidly identifying androgenic activity associated with drugs, chemicals, and environmental samples.

A lysosome targeting probe based on fluorescent protein chromophore for selectively detecting GSH and Cys in living cells.

The low-molecular weight biothiols like glutathione (GSH) and cysteine (Cys) play many important roles in various biological processes, the imbalance of biothiols level will lead to many diseases. However, methods that can selectively detect the GSH and Cys was rarely reported because of the similar reactivity and structure. Here, a fluorogenic method was presented to selectively detect the GSH and Cys in vitro using probe ML-FP based on fluorescent protein mimics. Probe ML-FP is a fluorescence turn on probe with lysosome targeting capacity. 2,4-dinitrobenzenesulfonyl serves as fluorescence quench and detection group in probe ML-FP. Differentiating the GSH and Cys was realized benefit from the different reaction time as well as fluorescence response between probe and target species. Low detection limit (4.98 nM for Cys and 4.39 nM for GSH) as well as fast response time was founded in this work. Probe ML-FP possess excellent biocompatibility due to fluorescent protein chromophore and was successfully used for bioimaging in living cells.

Cell-penetrating peptide sequence and modification dependent uptake and subcellular distribution of green florescent protein in different cell lines.

Protein therapy holds great promise for treating a variety of diseases. To act on intracellular targets, therapeutic proteins must cross the plasma membrane. This has previously been achieved by covalent attachment to a variety of cell-penetrating peptides (CPPs). However, there is limited information on the relative performance of CPPs in delivering proteins to cells, specifically the cytosol and other intracellular locations. Here we use green fluorescent protein (GFP) as a model cargo to compare delivery capacity of five CPP sequences (Penetratin, R8, TAT, Transportan, Xentry) and cyclic derivatives in different human cell lines (HeLa, HEK, 10T1/2, HepG2) representing different tissues. Confocal microscopy analysis indicates that most fusion proteins when incubated with cells at 10 µM localise to endosomes. Quantification of cellular uptake by flow cytometry reveals that uptake depends on both cell type (10T1/2 > HepG2 > HeLa > HEK), and CPP sequence (Transportan > R8 > Penetratin≈TAT > Xentry). CPP sequence cyclisation or addition of a HA-sequence increased cellular uptake, but fluorescence was still contained in vesicles with no evidence of endosomal escape. Our results provide a guide to select CPP for endosomal/lysosomal delivery and a basis for developing more efficient CPPs in the future.

Super-resolution Imaging of Structure, Molecular Composition, and Stability of Single Oligonucleotide Polyplexes.

The successful application of gene therapy relies on the development of safe and efficient delivery vectors. Cationic polymers such as cell-penetrating peptides (CPPs) can condense genetic material into nanoscale particles, called polyplexes, and induce cellular uptake. With respect to this point, several aspects of the nanoscale structure of polyplexes have remained elusive because of the difficulty in visualizing the molecular arrangement of the two components with nanometer resolution. This limitation has hampered the rational design of polyplexes based on direct structural information. Here, we used super-resolution imaging to study the structure and molecular composition of individual CPP-mRNA polyplexes with nanometer accuracy. We use two-color direct stochastic optical reconstruction microscopy (dSTORM) to unveil the impact of peptide stoichiometry on polyplex structure and composition and to assess their destabilization in blood serum. Our method provides information about the size and composition of individual polyplexes, allowing the study of such properties on a single polyplex basis. Furthermore, the differences in stoichiometry readily explain the differences in cellular uptake behavior. Thus, quantitative dSTORM of polyplexes is complementary to the currently used characterization techniques for understanding the determinants of polyplex activity in vitro and inside cells.

Luminescent silica mesoparticles for protein transduction.

Unlike silica nanoparticles, the potential of silica mesoparticles (SMPs) (i.e. particles of submicron size) for biological applications in particular the in vitro (let alone in vivo) cellular delivery of biological cargo has so far not been sufficiently studied. Here we examine the potential of luminescent (namely, octahedral molybdenum cluster doped) SMPs synthesised by a simple one-pot reaction for the labelling of cells and for protein transduction into larynx carcinoma (Hep-2) cells using GFP as a model protein. Our data demonstrates that the SMPs internalise into the cells within half an hour. This results in cells that detectably luminesce via conventional methods. In addition, the particles are non-toxic both in darkness and upon photo-irradiation. The SMPs were modified to allow their functionalisation by a protein, which then delivered the protein (GFP) efficiently into the cells. Thus, the luminescent SMPs offer a cheap and trackable alternative to existing materials for cellular internalisation of proteins, such as the HIV TAT protein and commercial protein delivery agents (e.g. Pierce™).

Green Fluorescent Protein Nanovessel Serves as a Nucleolus Targeting Material and Molecule Carrier in Living Cells.

The nucleolus is responsible for RNA transcription, processing, and ribosome assembly, the dysfunction of which is associated with a number of diseases. In this report, a new member of fluorescent protein nanovessels (FPNs), constructed using thioflavin-T (ThT) and bovine serum albumin (BSA) as building blocks, is described. As a popular amyloid specific dye, ThT is nonfluorescent by itself, while its fluorescence can be lighted up upon interacting with amyloid proteins. Herein, ThT is coassembled with the BSA scaffold at high temperature to form T(hT)-FPNs. These green fluorescence emissive bio-abiotic hybrid materials can serve as a novel probe for real-time nucleolus imaging of living cells. Besides, T-FPNs show potential in delivering insoluble and/or impenetrable drugs into living cells, suggesting another role as a molecule carrier.

The effects of elevated fibroblast growth factor 23 on mandibular growth in rats.

OBJECTIVE: The aim of this study is to elucidate the local effects of fibroblast growth factor 23 (FGF23) in on mandibular condylar growth in growing rats. DESIGN: Growing Sprague-Dawley rats received intra-temporomandibular joint injections of phosphate buffer solution (PBS), adenovirus-mediated green fluorescent protein (Ad-GFP) or adenovirus-mediated fibroblast growth factor 23 (Ad-FGF23), which were marked as groups A, B, and C, respectively. In vitro, we treated rat mandibular cartilage chondrocytes with PBS, Ad-GFP, and Ad-FGF23. RESULTS: The mandibular condyles in group C grew smaller sizes than those in the other control groups due to significant differences among the experimental and control groups with the value of C-D, Q-R (P ≤ 0.05), accompanied by diminished bone mass of sub-cartilage condyles via micro CT analysis. Histologically, the length of the hypertrophic zone was diminished and was associated with decreasing chondrocyte proliferation in group C. Quantitative real-time PCR indicated significant decreases in the expression of chondrogenesis marker genes, including Type X collagen (Col X) and SRY-type box 9 (Sox 9). Moreover, elevated Ad-FGF23 suppressed chondrocyte proliferation and the expression of the chondrogenic differentiation markers Col X and Sox 9 of in vitro. CONCLUSIONS: Local injection of FGF23 suppressed the development and decreased the bone mass of condyles through the decreasing the formation of condylar cartilage, specifically by regulating condylar cartilage cell viability and chondrogenesis expression.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans.

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and γ-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.

Antimicrobial peptide Pc-pis: A new cancer cell killer.

The antimicrobial peptide (AMP) Pc-pis, a member of Piscidin family from fish with cationic amphipathic structure, has potent, broad-spectrum antimicrobial activity against bacteria, fungi and parasite, and lower hemolytic activity. Here, we reported that Pc-pis had antitumor activity. Pc-pis killed tumor cells including HeLa cells. Previously, it is reported that AMPs bind to the membrane of bacteria to generate the pores to lyse the target cells, and similarly, the cancer cell incorporate phosphatidyl-serine on the outer leaflet of plasma membrane so that amphipathic AMPs can bind to the membrane to kill it. Our data supported that notion because suitable size osmo-protectant PEG4000 prevented HeLa cells from death induced by Pc-pis. Additionally, Fusion protein GFP-Pc-pis accumulated mainly at the nuclear membranes of HeLa cells and positive net charge in modified Pc-pis intensified but negative net charges eliminated this effect. Thus, positively charged residues were important for its affinity to the membrane. Our work will lay the groundwork of the development of Pc-pis antitumor activity.

N-terminal tagging of human P2X7 receptor disturbs calcium influx and dye uptake.

The P2X7 receptor is a frequently studied member of the purinergic receptor family signalling via channel opening and membrane pore formation. Fluorescent imaging is an important molecular method for studying cellular receptor expression and localization. Fusion of receptors to fluorescent proteins might cause major functional changes and requires careful functional evaluation such as has been done for the rat P2X7 receptor. This study examines fusion constructs of the human P2X7 receptor. We assessed surface expression, channel opening with calcium influx, and pore formation using YO-PRO-1 dye uptake in response to BzATP stimulation in transfected cells. We found that tagging at the N-terminal of the human P2X7 receptor with the enhanced green fluorescent protein (eGFP) disturbed channel opening and pore formation despite intact surface expression. A triple hemagglutinin (3HA) fused to the N-terminal also disrupted pore formation but not channel opening showing that even a small tag alters the normal function of the receptor. Together, this suggests that in contrast to what has been observed for the rat P2X7 receptor, the human P2X7 receptor contains N-terminal motifs important for signalling that prevent the construction of a functionally active fusion protein.

Supercharged green fluorescent protein delivers HPV16E7 DNA and protein into mammalian cells in vitro and in vivo.

Macromolecules including DNA and proteins serve as important human therapeutics but are limited by their general inability to cross cell membranes. Supercharged proteins have been known as potent tools for delivery of macromolecules into mammalian cells. Thus, the use of these delivery systems is important to reduce the human papillomavirus (HPV)-associated malignancies through improvement of vaccine modalities. In this study, we used a supercharged green fluorescent protein (+36 GFP) for delivery of the full-length HPV16 E7 DNA and protein into mammalian cells and evaluated immune responses, and protective/therapeutic effects of different formulations in C57BL/6 tumor mice model. Our results showed that the complexes of E7 DNA/+36 GFP and also E7 protein/+36 GFP form stable nanoparticles through non-covalent binding with an average size of ∼ 200-300 nm. The efficient delivery of E7 DNA or protein by +36 GFP was detected in HEK-293T cell line for 4 h and 24 h post-transfection. Mice immunization with E7 protein/+36 GFP nanoparticles elicited a higher Th1 cellular immune response with the predominant IgG2a and IFN-γ levels than those induced by E7 protein, E7 DNA, E7 DNA/+36 GFP and control groups (p < .05). Moreover, the E7 DNA/+36 GFP and E7 protein/+36 GFP nanoparticles similarly protected mice against TC-1 tumor challenge (∼67%) as compared to E7 DNA and E7 protein (∼33%), respectively. These data suggest that +36 GFP may provide a promising platform to improve protein and DNA delivery in vitro and in vivo.

A New Mutation in FIG4 Causes a Severe Form of CMT4J Involving TRPV4 in the Pathogenic Cascade.

Mutations in FIG4, coding for a phosphoinositol(3,5) bisphosphate 5' phosphatase and involved in vesicular trafficking and fusion, have been shown causing a recessive form of Charcot-Marie-Tooth (CMT). We have identified a novel intronic mutation in the FIG4 in a wheel-chair bound patient presenting with a severe form of CMT4J and provide a longitudinal study. Investigations indicated a demyelinating sensorimotor polyneuropathy with diffuse active denervation and severe axonal loss. Genetic testing revealed that the patient is heterozygous for 2 FIG4 mutations, p.I41T and a T > G transversion at IVS17-10, the latter predicted to cause a splicing defect. FIG4 was severely diminished in patient's fibroblasts indicating loss-of-function. Consistent with FIG4's function in phosphoinositol homeostasis and vesicular trafficking, fibroblasts contained multiple large vacuoles and vesicular organelles were abnormally dispersed. FIG4 deficiency has implications for turnover of membrane proteins. The transient receptor cation channel, TRPV4, accumulated at the plasma membrane of patient's fibroblasts due to slow turnover. Knocking down Fig4 in murine cultured motor neurons resulted in vacuolation and cell death. Inhibiting TRPV4 activity significantly preserved viability, although not correcting vesicular trafficking. In conclusion, we demonstrate a new FIG4 intronic mutation and, importantly, a functional interaction between FIG4 and TRPV4.