Development and characterization of self-assembling nanoparticles using a bio-inspired amphipathic peptide for gene delivery

The design of a non-viral gene delivery vehicle capable of delivering and releasing a functional nucleic acid cargo intracellularly remains a formidable challenge. For systemic gene therapy to be successful a delivery vehicle is required that protects the nucleic acid cargo from enzymatic degradation, extravasates from the vasculature, traverses the cell membrane, disrupts the endosomal vesicles and unloads the cargo at its destination site, namely the nucleus for the purposes of gene delivery. This manuscript reports the extensive investigation of a novel amphipathic peptide composed of repeating RALA units capable of overcoming the biological barriers to gene delivery both in vitro and in vivo. Our data demonstrates the spontaneous self-assembly of cationic DNA-loaded nanoparticles when the peptide is complexed with pDNA. Nanoparticles were < 100 nm, were stable in the presence of serum and were fusogenic in nature, with increased peptide α-helicity at a lower pH. Nanoparticles proved to be non-cytotoxic, readily traversed the plasma membrane of both cancer and fibroblast cell lines and elicited reporter-gene expression following intravenous delivery in vivo. The results of this study indicate that RALA presents an exciting delivery platform for the systemic delivery of nucleic acid therapeutics.

Graphene Nanoribbons as a Drug Delivery Agent for Lucanthone Mediated Therapy of Glioblastoma Multiforme

We report use of PEG-DSPE coated oxidized graphene nanoribbons (O-GNR-PEG-DSPE) as agent for delivery of anti-tumor drug Lucanthone (Luc) into Glioblastoma Multiformae (GBM) cells targeting base excision repair enzyme APE-1 (Apurinic endonuclease-1). Lucanthone, an endonuclease inhibitor of APE-1, was loaded onto O-GNR-PEG-DSPEs using a simple non-covalent method. We found its uptake by GBM cell line U251 exceeding 67% and 60% in APE-1-overexpressing U251, post 24 h. However, their uptake was ~ 38% and 29% by MCF-7 and rat glial progenitor cells (CG-4), respectively. TEM analysis of U251 showed large aggregates of O-GNR-PEG-DSPE in vesicles. Luc-O-GNR-PEG-DSPE was significantly toxic to U251 but showed little/no toxicity when exposed to MCF-7/CG-4 cells. This differential uptake effect can be exploited to use O-GNR-PEG-DSPEs as a vehicle for Luc delivery to GBM, while reducing nonspecific cytotoxicity to the surrounding healthy tissue. Cell death in U251 was necrotic, probably due to oxidative degradation of APE-1.

Graphical abstract

We used O-GNR-PEG-DSPE as a reliable, non-toxic vehicle for delivery of APE-1 inhibiting Lucanthone into GBM tumor cell lines. LUC-O-GNR-PEG-DSPE particles showed 60% or more uptake by CMV/U251 and A1-5/CMV/U251 where as the uptake by MCF7 and normal CG4 glial cells was much lower (38% and 29% respectively). Different concentrations of Luc (5–80 μM) loaded onto O-GNR-PEG-DSPE showed lower toxicity in the exposed cells compared to the free drug, due to possible slow release of the drug from this particle, which ensures minimum non-specific release of the drug from the particle once it is injected in vivo.
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Dendrons with spermine surface groups as potential building blocks for nonviral vectors in gene therapy

This paper investigates a series of dendrons based on the Newkome dendritic scaffold that displays a naturally occurring polyamine (spermine) on their surface. These dendrons have previously been shown to interact with DNA in a generation dependent manner with the more highly branched dendrons exhibiting a strong multivalency effect for the spermine surface groups. In this paper, we investigate the ability of these dendrons to transfect DNA into cells (human breast carcinoma cells, MDA-MB-231, and murine myoblast cells, C2C12) as determined by the luciferase assay. Although the dendrons are unable to transfect DNA in their own right, they are capable of delivering DNA in vitro when administered with chloroquine, which assists with escape from endocytic vesicles. The cytotoxicity of the dendrons was determined using the XTT assay, and it was shown that the dendrons were nontoxic either alone or in the presence of DNA. However, when administered with DNA and chloroquine, the most highly branched dendron did exhibit some cytotoxicity. This paper elucidates the relationship between in vitro transfection efficiency and toxicity. While transfection efficiencies are modest, the low toxicity of the dendrons, both in their own right, and in the presence of DNA, provides encouragement that this type of building block, which has a relatively high affinity for DNA, will provide a useful starting point for the further synthetic development of more effective gene transfection agents.

A Microscopic Study of Iron and Manganese Oxide Distribution in Soils from East Tennessee (U.S.A.)

Soils and saprolites developed from interbedded shales and limestones of the Conasauga Group are widespread in the Valley and Ridge Province of East Tennessee. Thin sections from four soil profiles were examined by petrographic and scanning electron microscopy including backscatter electron and energy-dispersive X-ray analyses. Iron and manganese released by weathering had migrated differentially downward and precipitated as crystalline and noncrystalline oxides. Oxides were observed as nodules, granular particulates, pore fillings, and coatings on other minerals, packing voids, vesicles, channels, and chambers. Iron oxides formed predominantly as coatings on packing-void walls and on laminated clays in vesicles and channels. Manganese oxides occurred as an early replacement phase of packing voids and of fracture-filling carbonate minerals. Iron oxides were dominant in moderately well-drained and oxidized horizons of the soil solum, whereas manganese oxides were abundant in the oxidized and moderately leached saprolite zone where the water table fluctuates seasonally. Therefore, a manganese enrichment zone, on a bulk soil basis, occurred generally below the iron oxide zone in the soil profile. Such differential migration and accumulation of iron and manganese have been controlled by localized soil microenvironments. Micromorphologic features observed in this study are important in land-use evaluation for hazardous waste disposal. © 1990.

Measurement of the Electrogenicity of Bile Salt/H+ Antiport in Escherichia coli

The transmembrane proton gradient (ΔpH) is the primary source of energy exploited by secondary active substrate/H+ antiporters to drive the electroneutral transport of substrates across the Escherichia coli (E. coli) inner membrane. Such electroneutral transport results in no net movement of charges across the membrane. The charge on the transported substrate and the stoichiometry of the exchange reaction, however, can result in an electrogenic reaction which is driven by both the ΔpH and the electrical (∆Ψ) components of the proton electrochemical gradient, resulting in a net movement of electrical charges across the membrane. We have shown that the major facilitator superfamily transporter MdtM - a multidrug efflux protein from E. coli that functions physiologically in protection of bacterial cells against bile salts - imparts bile salt resistance to the bacterial cell by coupling the exchange of external protons (H+) to the efflux of bile salts from the cell interior via an electrogenic antiport reaction (Paul et al., 2014). This protocol describes, using fluorometry, how to detect electrogenic antiport activity of MdtM in inverted membrane vesicles of an antiporter-deficient strain of E. coli TO114 cells by measuring transmembrane ∆Ψ. The method exploits changes that occur in the intensity of the fluorescence signal (quenching and dequenching) of the probe Oxonol V in response to changes in membrane potential due to the MdtM-catalysed sodium cholate/H+ exchange reaction. The protocol can be adapted to detect activity of any secondary active antiporter that couples the electrogenic translocation of H+ across a biological membrane to that of its counter-substrate, and may be used to unmask otherwise camouflaged transport activities and physiological roles.

Measurement of proton-driven antiport in Escherichia coli

Secondary active transport of substrates across the inner membrane is vital to the bacterial cell. Of the secondary active transporter families, the ubiquitous major facilitator superfamily (MFS) is the largest and most functionally diverse (Reddy et al., 2012). Recently, it was reported that the MFS multidrug efflux protein MdtM from Escherichia coli (E. coli) functions physiologically in protection of bacterial cells against bile salts (Paul et al., 2014). The MdtM transporter imparts bile salt resistance to the bacterial cell by coupling the exchange of external protons (H+) to the efflux of bile salts from the cell interior via an antiport reaction. This protocol describes, using fluorometry, how to detect the bile salt/H+ antiport activity of MdtM in inverted membrane vesicles of an antiporter-deficient strain of E. coli TO114 cells by measuring transmembrane ∆pH. This method exploits the changes that occur in the intensity of the fluorescence signal (quenching and dequenching) of the pH-sensitive dye acridine orange in response to changes in [H+] in the vesicular lumen. Due to low levels of endogenous transporter expression that would normally make the contribution of individual transporters such as MdtM to proton-driven antiport difficult to detect, the method typically necessitates that the transporter of interest be overexpressed from a multicopy plasmid. Although the first section of the protocol described here is very specific to the overexpression of MdtM from the pBAD/Myc-His A expression vector, the protocol describing the subsequent measurement of bile salt efflux by MdtM can be readily adapted for measurement of antiport of other substrates by any other antiporter that exchanges protons for countersubstrate.

Ultrastructure of head organs (anterior adhesive apparatus) and posterior secretory systems of Caballeria liewi Lim, 1995 (Monogenea, Ancyrocephalidae)

Caballeria liewi Lim, 1995, uses adhesive secretions from the head organs and posterior secretory systems to assist in locomotion and attachment. Ultrastructural investigations show that the head organs of C. liewi consist of three pairs of antero-lateral pit-like openings bearing microvilli and ducts leading from two types of uninucleated gland cells (located lateral to the pharynx), one type producing rod-like (S1) bodies with an electron-dense matrix containing less electron-dense vesicles and the second type producing oval (S2) bodies with a homogeneous electron-dense matrix. Interlinking band-like structures are observed between S1 bodies and between S2 bodies. S1 body is synthesised in the granular endoplasmic reticulum, transported to a Golgi complex to be packaged into vesicles and routed into ducts for exudation. The synthesis of the S2 body is unresolved. Haptoral secretions manifested externally as net-like structures are derived from dual electron-dense (DED) secretory body produced in the peduncular gland cells. The DED body consists of a less electron-dense oval core in a homogeneous electron-dense matrix. On exocytosis into the pyriform haptoral reservoir, DED bodies are transformed into a secretion with two types of inclusions (less electron-dense oval and electron-dense spherical inclusions) in an electron-dense matrix. The secretions are further transformed (as small, oval, electron-dense bodies) when transported to the superficial anchor grooves, and on exudation into the gill tissues, the secretions become an electron-dense matrix. Secretory bodies associated with uniciliated structures, anchor sleeves and marginal hooks are also observed.

Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

Autophagic flux involves formation of autophagosomes and their degradation by lysosomes. Autophagy can either promote or restrict viral replication. In the case of Dengue virus (DENV) several studies report that autophagy supports the viral replication cycle, and describe an increase of autophagic vesicles (AVs) following infection. However, it is unknown how autophagic flux is altered to result in increased AVs. To address this question, and gain insight into the role of autophagy during DENV infection, we established an unbiased, image-based flow cytometry approach to quantify autophagic flux under normal growth conditions and in response to activation by nutrient deprivation or the mTOR inhibitor Torin1. We found that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Early after infection, basal and activated autophagic flux was enhanced. However, during established replication, basal and Torin1-activated autophagic flux was blocked, while autophagic flux activated by nutrient deprivation was reduced, indicating a block to AV formation and reduced AV degradation capacity. During late infection AV levels increased as a result of inefficient fusion of autophagosomes with lysosomes. Additionally, endo-lysosomal trafficking was suppressed, while lysosomal activities were increased. We further determined that DENV infection progressively reduced levels of the autophagy receptor SQSTM1/p62 via proteasomal degradation. Importantly, stable over-expression of p62 significantly suppressed DENV replication suggesting a novel role for p62 as viral restriction factor. Overall our findings indicate that in the course of DENV infection, autophagy shifts from a supporting to an anti-viral role, which is countered by DENV.

IMPORTANCE: Autophagic flux is a dynamic process starting with the formation of autophagosomes and ending with their degradation after fusion with lysosomes. Autophagy impacts the replication cycle of many viruses. However, thus far the dynamics of autophagy in case of Dengue virus (DENV) infections has not been systematically quantified. Therefore, we employed high-content, imaging-based flow cytometry to quantify autophagic flux and endo-lysosomal trafficking in response to DENV infection. We report that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Further, lysosomal activity was increased, but endo-lysosomal trafficking was suppressed confirming the block of autophagic flux. Importantly, we provide evidence that p62, an autophagy receptor, restrict DENV replication and was specifically depleted in DENV-infected cells via increased proteasomal degradation. These results suggest that during DENV infection autophagy shifts from a pro- to an antiviral cellular process, which is counteracted by the virus.

Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly

Adaptor protein complex 2 alpha and beta-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of beta-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the beta-appendage (the "top" and "side" sites) that bind motifs distinct from those previously identified on the alpha-appendage. We solved the structure of the beta-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor beta-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the beta-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability ("matricity"). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as beta-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.

Evolving nature of the AP2 alpha-appendage hub during clathrin-coated vesicle endocytosis

Clathrin-mediated endocytosis involves the assembly of a network of proteins that select cargo, modify membrane shape and drive invagination, vesicle scission and uncoating. This network is initially assembled around adaptor protein (AP) appendage domains, which are protein interaction hubs. Using crystallography, we show that FxDxF and WVxF peptide motifs from synaptojanin bind to distinct subdomains on alpha-appendages, called 'top' and 'side' sites. Appendages use both these sites to interact with their binding partners in vitro and in vivo. Occupation of both sites simultaneously results in high-affinity reversible interactions with lone appendages (e.g. eps15 and epsin1). Proteins with multiple copies of only one type of motif bind multiple appendages and so will aid adaptor clustering. These clustered alpha(appendage)-hubs have altered properties where they can sample many different binding partners, which in turn can interact with each other and indirectly with clathrin. In the final coated vesicle, most appendage binding partners are absent and thus the functional status of the appendage domain as an interaction hub is temporal and transitory giving directionality to vesicle assembly.

COP and clathrin-coated vesicle budding:different pathways, common approaches

Vesicle and tubule transport containers move proteins and lipids from one membrane system to another. Newly forming transport containers frequently have electron-dense coats. Coats coordinate the accumulation of cargo and sculpt the membrane. Recent advances have shown that components of both COP1 and clathrin-adaptor coats share the same structure and the same motif-based cargo recognition and accessory factor recruitment mechanisms, which leads to insights on conserved aspects of coat recruitment, polymerisation and membrane deformation. These themes point to the way in which evolutionarily conserved features underpin these diverse pathways.

BAR domains as sensors of membrane curvature:the amphiphysin BAR structure

The BAR (Bin/amphiphysin/Rvs) domain is the most conserved feature in amphiphysins from yeast to human and is also found in endophilins and nadrins. We solved the structure of the Drosophila amphiphysin BAR domain. It is a crescent-shaped dimer that binds preferentially to highly curved negatively charged membranes. With its N-terminal amphipathic helix and BAR domain (N-BAR), amphiphysin can drive membrane curvature in vitro and in vivo. The structure is similar to that of arfaptin2, which we find also binds and tubulates membranes. From this, we predict that BAR domains are in many protein families, including sorting nexins, centaurins, and oligophrenins. The universal and minimal BAR domain is a dimerization, membrane-binding, and curvature-sensing module.

EpsinR an AP1/clathrin interacting protein involved in vesicle trafficking

EpsinR is a clathrin-coated vesicle (CCV) enriched 70-kD protein that binds to phosphatidylinositol-4-phosphate, clathrin, and the gamma appendage domain of the adaptor protein complex 1 (AP1). In cells, its distribution overlaps with the perinuclear pool of clathrin and AP1 adaptors. Overexpression disrupts the CCV-dependent trafficking of cathepsin D from the trans-Golgi network to lysosomes and the incorporation of mannose-6-phosphate receptors into CCVs. These biochemical and cell biological data point to a role for epsinR in AP1/clathrin budding events in the cell, just as epsin1 is involved in the budding of AP2 CCVs. Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 microM, respectively. Thus, potentially, two AP1 complexes can bind to one epsinR. This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in gamma-synergin and use to predict that an uncharacterized EF-hand-containing protein will be a new gamma binding partner.

Curvature of clathrin-coated pits driven by epsin

Clathrin-mediated endocytosis involves cargo selection and membrane budding into vesicles with the aid of a protein coat. Formation of invaginated pits on the plasma membrane and subsequent budding of vesicles is an energetically demanding process that involves the cooperation of clathrin with many different proteins. Here we investigate the role of the brain-enriched protein epsin 1 in this process. Epsin is targeted to areas of endocytosis by binding the membrane lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)). We show here that epsin 1 directly modifies membrane curvature on binding to PtdIns(4,5)P(2) in conjunction with clathrin polymerization. We have discovered that formation of an amphipathic alpha-helix in epsin is coupled to PtdIns(4,5)P(2) binding. Mutation of residues on the hydrophobic region of this helix abolishes the ability to curve membranes. We propose that this helix is inserted into one leaflet of the lipid bilayer, inducing curvature. On lipid monolayers epsin alone is sufficient to facilitate the formation of clathrin-coated invaginations.

GTPase activity of dynamin and resulting conformation change are essential for endocytosis

Dynamin is a large GTPase with a relative molecular mass of 96,000 (Mr 96K) that is involved in clathrin-mediated endocytosis and other vesicular trafficking processes. Although its function is apparently essential for scission of newly formed vesicles from the plasma membrane, the nature of dynamin's role in the scission process is still unclear. It has been proposed that dynamin is a regulator (similar to classical G proteins) of downstream effectors. Here we report the analysis of several point mutants of dynamin's GTPase effector (GED) and GTPase domains. We show that oligomerization and GTP binding alone, by dynamin, are not sufficient for endocytosis in vivo. Rather, efficient GTP hydrolysis and an associated conformational change are also required. These data argue that dynamin has a mechanochemical function in vesicle scission.