Secretion induces cell pH dynamics impacting assembly-disassembly of the fusion protein complex: A combined fluorescence and atomic force microscopy study.

A wide range of cellular activities including protein folding and cell secretion, such as neurotransmission or insulin release, are all governed by intracellular pH homeostasis, underscoring the importance of pH on critical life processes. Nano- scale pH measurements of cells and biomolecules therefore hold great promise in understanding a plethora of cellular functions, in addition to disease detection and therapy. In the current study, a novel approach using cadmium telluride quantum dots (CdTeQDs) as pH sensors, combined with fluorescent imaging, spectrofluorimetry, atomic force microscopy (AFM), and Western blot analysis, enabled the study of intracellular pH dynamics at 1 milli-pH sensitivity and 80nm pixel resolution, during insulin secretion. Additionally, the pH-dependent interaction between membrane fusion proteins, also called the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE), was determined. Glucose stimulation of CdTeQD-loaded insulin secreting Min-6 mouse insulinoma cell line demonstrated the initial (5-6min) intracellular acidification reflected as a loss in QD fluorescence, followed by alkalization and a return to resting pH in 10min. Analysis of the SNARE complex in insulin secreting Min-6 cells demonstrated an initial gain followed by loss of complexed SNAREs in 10min. Stabilization of the SNARE complex at low intracellular pH is further supported by results from studies utilizing both native and AFM measurements of liposome-reconstituted recombinant neuronal SNAREs, providing a molecular understanding of the role of pH during cell secretion.

Human Platelet Vesicles Exhibit Distinct Size and Proteome.

In the past 50 years, isolated blood platelets have had restricted use in wound healing, cancer therapy, and organ and tissue transplant, to name a few. The major obstacle for its unrestricted use has been, among others, the presence of ultrahigh concentrations of growth factors and the presence of both pro-angiogenic and anti-angiogenic proteins. To overcome this problem requires the isolation and separation of the membrane bound secretory vesicles containing the different factors. In the current study, high-resolution imaging of isolated secretory vesicles from human platelets using atomic force microscopy (AFM) and mass spectrometry enabled characterization of the remaining vesicles size and composition following their immunoseparation. The remaining vesicles obtained following osmotic lysis, when subjected to immunoseparation employing antibody to different vesicle-associated membrane proteins (VAMPs), demonstrate for the first time that VAMP-3-, VAMP-7-, and VAMP-8-specific vesicles each possesses distinct size range and composition. These results provide a window into our understanding of the heterogeneous population of vesicles in human platelets and their stability following both physical manipulation using AFM and osmotic lysis of the platelet. This study further provides a platform for isolation and the detailed characterization of platelet granules, with promise for their future use in therapy. Additionally, results from the study demonstrate that secretory vesicles of different size found in cells reflect their unique and specialized composition and function.

Proteome of the porosome complex in human airway epithelia: interaction with the cystic fibrosis transmembrane conductance regulator (CFTR).

The surface of the airways is coated with a thin film of mucus composed primarily of mucin, which is under continuous motion via ciliary action. Mucin not only serves to lubricate the airways epithelia, but also functions as a trap for foreign particles and pathogens, thereby assisting in keeping the airways clean and free of particulate matter and infections. Altered mucin secretion especially increased mucin viscosity, results in mucin stagnation due to the inability of the cilia to propel them, leading to infections and diseases such as cystic fibrosis (CF). Since porosomes have been demonstrated to be the secretory portals at the cell plasma membrane in cells, their presence, structure, and composition in the mucin-secreting human airway epithelial cell line Calu-3 expressing CF transmembrane receptor (CFTR), were investigated. Atomic force microscopy (AFM) of Calu-3 cells demonstrates the presence of approximately 100nm in diameter porosome openings at the plasma membrane surface. Electron microscopy confirms the AFM results, and tandem mass spectrometry and immunoanalysis performed on isolated Calu-3 porosomes, reveal the association of CFTR with the porosome complex. These new findings will facilitate understanding of CFTR-porosome interactions influencing mucous secretion, and provide critical insights into the etiology of CF disease. BIOLOGICAL SIGNIFICANCE: In the present study, the porosome proteome in human airway epithelia has been determined. The interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) and the porosome complex in the human airway epithelia is further demonstrated. The possible regulation by CFTR on the quality of mucus secretion via the porosome complex at the cell plasma membrane is hypothesized. These new findings will facilitate understanding of CFTR-porosome interactions influencing mucous secretion, and provide critical insights into the etiology of CF disease.

Dendrimer-enabled transformation of Chlamydia trachomatis.

Lack of a system for genetic manipulation of Chlamydia trachomatis has been a key challenge to advancing understanding the molecular genetic basis of virulence for this bacterial pathogen. We developed a non-viral, dendrimer-enabled system for transformation of this organism and used it to characterize the effects of inserting the common 7.5 kbp chlamydial plasmid into strain L2(25667R), a C. trachomatis isolate lacking it. The plasmid was cloned in pUC19 and the clone complexed to polyamidoamine dendrimers, producing ∼83 nm spherical particles. Nearly confluent McCoy cell cultures were infected with L2(25667R) and reference strain L2(434). At 16 h post-infection, medium was replaced with dendrimer-plasmid complexes in medium lacking additives (L2(25667R)) or with additive-free medium alone (L2(434)). Three h later complexes/buffer were removed, and medium was replaced; cultures were harvested at various times post-transformation for analyses. Real time PCR and RT-PCR of nucleic acids from transformed cultures demonstrated plasmid replication and gene expression. A previous report indicated that one or more plasmid-encoded product govern(s) transcription of the glycogen synthase gene (glgA) in standard strains. In L2(25667R) the gene is not expressed, but transformants of that strain given the cloned chlamydial plasmid increase glgA expression, as does L2(434). The cloned plasmid is retained, replicated, and expressed in transformants over at least 5 passages, and GFP is expressed when transformed into growing L2(25667R). This transformation system will allow study of chlamydial gene function in pathogenesis.

Dendrimer-enabled DNA delivery and transformation of Chlamydia pneumoniae.

The chlamydiae are important human pathogens. Lack of a genetic manipulation system has impeded understanding of the molecular bases of virulence for these bacteria. We developed a dendrimer-enabled system for transformation of chlamydiae and used it to characterize the effects of inserting the C. trachomatis plasmid into C. pneumoniae, which lacks any plasmids. The plasmid was cloned into modified yeast vector pEG(KG) and the clone complexed to polyamidoamine dendrimers, producing 50-100 nm spherical particles. HEp-2 cell cultures were infected with C. pneumoniae strain AR-39. Twenty-four hours later, medium was replaced for 3 hours with dendrimer-plasmid complexes, then removed and the medium replaced. Cultures were harvested at various times post-transformation. Real-time PCR and RT-PCR of nucleic acids from transformed cultures demonstrated plasmid replication and gene expression. The cloned plasmid was replicated and expressed in transformants over 5 passages. This system will allow study of chlamydial gene function, allowing development of novel dendrimer-based therapies. FROM THE CLINICAL EDITOR: This team of investigators developed a dendrimer-enabled system for transformation of chlamydiae and successfully utilized it to characterize the effects of inserting the C. trachomatis plasmid into C. pneumonia. This system will allow study of chlamydial gene function, allowing development of novel dendrimer-based therapies.

A supra-monolayer nanopattern for organic nanoparticle array deposition.

Nanopatterns have applications in many areas including sensors, optoelectronics, and crystallization screening. Particle lithography is a convenient method to manufacture nanoring nanopatterns based on organosilane surface chemistry. The pattern thickness is generally limited to the monolayer thickness. This work is focused on the chemical vapor deposition conditions that yield nanopatterns with multilayer thickness. The supra-monolayer n-octadecyltrichlorosilane (OTS) nanoring patterns are made using polystyrene particle lithography. The supra-monolayer nanopatterns are used as "nano-flasks" to deposit and nucleate nanoparticles of small organic molecules including n-docosane, aspirin, and clarithromycin. The supra-monolayer OTS nanopattern is an effective template for nanoparticle array deposition of all three chemicals with high degree of fidelity to the substrate pattern. The nanoparticle size is varied by solution concentration. The preferential deposition of the organic molecules inside the nanoring is attributed to the dewetting of the liquid film on the nanopattern. The dewetting process effectively distributes the liquid film among the "nano-flasks" so that millions of solution experiments can be carried out in isolated droplets with droplet volume as small as 1×10(-10) nL. The research demonstrates a method to manufacture "nano-flask" arrays for high-throughput nanoparticle deposition trials and manufacture of monodisperse organic/drug nanoparticles through self-assembly.

Aquaporin-assisted and ER-mediated mitochondrial fission: a hypothesis.

It is well established that the status of the endoplasmic reticulum (ER) and mitochondria, and the interactions between them, is critical to numerous cellular functions including apoptosis. Mitochondrial dynamics is greatly influenced by cell stress, and recent studies implicate ER in mitochondrial fission. Although a number of proteins have been identified to participate in ER-induced mitochondrial fission, the molecular mechanism of the process is little understood. In the current study, we confirm the involvement of ER in mitochondrial fission and hypothesize the involvement of water channels or aquaporins (AQP) in the process. Previous studies demonstrate the presence of AQP both in the ER and mitochondrial membranes. Mitochondrial swelling has been observed following mitochondrial calcium overload, and studies report that chelation of cytosolic calcium induces extensive mitochondrial division at ER contact sites. Based on this information, the involvement of ER in mitochondrial division, possibly via water channels, is hypothesized. Utilizing a multi-faceted imaging approach consisting of atomic force microscopy on aldehyde-fixed and semi-dry cells, transmission electron microscopy, and immunofluorescence microscopy on live cells, the physical interactions between the two organelles are demonstrated. Mitochondrial fission following ER stress was abrogated with exposure of cells to the AQP inhibitor mercuric chloride, suggesting the involvement of AQP(s) especially AQP8 and AQP9 known to be present in the mitochondrial membrane, in mitochondrial fission.

3D organization and function of the cell: Golgi budding and vesicle biogenesis to docking at the porosome complex.

Insights into the three-dimensional (3D) organization and function of intracellular structures at nanometer resolution, holds the key to our understanding of the molecular underpinnings of cellular structure-function. Besides this fundamental understanding of the cell at the molecular level, such insights hold great promise in identifying the disease processes by their altered molecular profiles, and help determine precise therapeutic treatments. To achieve this objective, previous studies have employed electron microscopy (EM) tomography with reasonable success. However, a major hurdle in the use of EM tomography is the tedious procedures involved in fixing, high-pressure freezing, staining, serial sectioning, imaging, and finally compiling the EM images to obtain a 3D profile of sub-cellular structures. In contrast, the resolution limit of EM tomography is several nanometers, as compared to just a single or even sub-nanometer using the atomic force microscope (AFM). Although AFM has been hugely successful in 3D imaging studies at nanometer resolution and in real time involving isolated live cellular and isolated organelles, it has had limited success in similar studies involving 3D imaging at nm resolution of intracellular structure-function in situ. In the current study, using both AFM and EM on aldehyde-fixed and semi-dry mouse pancreatic acinar cells, new insights on a number of intracellular structure-function relationships and interactions were achieved. Golgi complexes, some exhibiting vesicles in the process of budding were observed, and small vesicles were caught in the act of fusing with larger vesicles, possibly representing either secretory vesicle biogenesis or vesicle refilling following discharge, or both. These results demonstrate the power and scope of the combined engagement of EM and AFM imaging of fixed semi-dry cells, capable of providing a wealth of new information on cellular structure-function and interactions.

Electrospun polyvinyl alcohol-collagen-hydroxyapatite nanofibers: a biomimetic extracellular matrix for osteoblastic cells.

The failure of prosthesis after total joint replacement is due to the lack of early implant osseointegration. In this study polyvinyl alcohol-collagen-hydroxyapatite (PVA-Col-HA) electrospun nanofibrous meshes were fabricated as a biomimetic bone-like extracellular matrix for the modification of orthopedic prosthetic surfaces. In order to reinforce the PVA nanofibers, HA nanorods and Type I collagen were incorporated into the nanofibers. We investigated the morphology, biodegradability, mechanical properties and biocompatibility of the prepared nanofibers. Our results showed these inorganic-organic blended nanofibers to be degradable in vitro. The encapsulated nano-HA and collagen interacted with the PVA content, reinforcing the hydrolytic resistance and mechanical properties of nanofibers that provided longer lasting stability. The encapsulated nano-HA and collagen also enhanced the adhesion and proliferation of murine bone cells (MC3T3) in vitro. We propose the PVA-Col-HA nanofibers might be promising modifying materials on implant surfaces for orthopedic applications.

Lysophosphatidylcholine inhibits membrane-associated SNARE complex disassembly.

In cells, N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors called SNAREs are involved in membrane fusion. In neurons, for example, target membrane proteins SNAP-25 and syntaxin called t-SNAREs present at the pre-synaptic membrane, and a synaptic vesicle-associated membrane protein (VAMP) or v-SNARE, is part of the conserved protein complex involved in neurotransmission. Cholesterol and LPC (L-α-lysophosphatidylcholine) are known to contribute to the negative and positive curvature respectively of membranes. In this study, using purified recombinant neuronal membrane-associated SNAREs, we demonstrate for the first time that membrane-curvature-influencing lipids profoundly influence SNARE complex disassembly. Exposure of cholesterol-associated t-SNARE and v-SNARE liposome mixtures to NSF-ATP results in dissociated vesicles. In contrast, exposure of LPC-associated t-SNARE and v-SNARE liposome mixtures to NSF-ATP, results in inhibition of t-/v-SNARE disassembly and the consequent accumulation of clustered vesicles. Similarly, exposure of isolated rat brain slices and pancreas to cholesterol or LPC, also demonstrates LPC-induced inhibition of SNARE complex disassembly. Earlier studies demonstrate a strong correlation between altered plasma LPC levels and cancer. The altered plasma LPC levels observed in various cancers may in part contribute to defects in SNARE assembly-disassembly and membrane fusion, consequently affecting protein maturation and secretion in cancer cells.

Cyclodextrin-erythromycin complexes as a drug delivery device for orthopedic application.

BACKGROUND: Erythromycin, a hydrophobic antibiotic used to treat infectious diseases, is now gaining attention because of its anti-inflammatory effects and ability to inhibit osteoclasts formation. The aim of this study was to explore a cyclodextrin-erythromycin (CD-EM) complex for sustained treatment of orthopedic inflammation. METHODS AND RESULTS: Erythromycin was reacted with ß-cyclodextrin to form a nonhost-guest CD-EM complex using both kneading and stirring approaches. Physiochemical measurement data indicated that erythromycin and cyclodextrin formed a packing complex driven by intermolecular forces instead of a host-guest structure due to the limited space in the inner cavity of ß-cyclodextrin. The CD-EM complex improved the stability of erythromycin in aqueous solution and had a longer duration of bactericidal activity than free erythromycin. Cytotoxicity and cell differentiation were evaluated in both murine MC3T3 preosteoblast cells and RAW 264.7 murine macrophage cells. The CD-EM complex was noncytotoxic and showed significant inhibition of osteoclast formation but had little effect on osteoblast viability and differentiation. CONCLUSION: These attributes are especially important for the delivery of an adequate amount of erythromycin to the site of periprosthetic inflammation and reducing local inflammation in a sustained manner.

Multifunctional Dendrimer-templated Antibody Presentation on Biosensor Surfaces for Improved Biomarker Detection.

Dendrimers, with their well-defined globular shape and a high density of functional groups, are ideal nanoscale materials for templating sensor surfaces. This work exploits dendrimers as a versatile platform for capturing biomarkers with improved sensitivity and specificity. Synthesis, characterization, fabrication, and functional validation of the dendrimer-based assay platform are described. Bifunctional hydroxyl/thiol functionalized G4-polyamidoamine (PAMAM) dendrimer is synthesized and immobilized on to the polyethylene-glycol (PEG)-functionalized assay plate by coupling PEG-maleimide and dendrimer thiol groups. Simultaneously, part of the dendrimer thiol groups are converted to hydrazide functionalities. The resulting dendrimer-modified surface is coupled to the capture antibody in the Fc region of the oxidized antibody. This preserves the orientation flexibility of the antigen binding region (Fv) of the antibody. To validate the approach, the fabricated plates are further used as a solid phase for developing a sandwich type ELISA to detect IL-6 and IL-1ß, important biomarkers for early stages of chorioamnionitis. The dendrimer-modified plate provides assays with significantly enhanced sensitivity, lower nonspecific adsorption, and a detection limit of 0.13 pg ml-1 for IL-6 luminol detection and 1.15 pg ml-1 for IL-1ß TMB detection, which are significantly better than those for the traditional ELISA. The assays were validated in human serum samples from normal (non-pregnant) woman and pregnant women with pyelonephritis. The specificity and the improved sensitivity of the dendrimer-based capture strategy could have significant implications for the detection of a wide range of cytokines and biomarkers since the capture strategy could be applied to multiplex microbead assays, conductometric immunosensors and field effect biosensors.

Surfactant-free synthesis of hyperbranched monoclinic bismuth vanadate and its applications in photocatalysis, gas sensing, and lithium-ion batteries.

Hyperbranched monoclinic BiVO(4) (h-BiVO(4)) has been synthesized on a large scale and with good uniformity by a surfactant-free hydrothermal route. h-BiVO(4) consists of four trunks with branches distributed on opposite sides. From observation of the intermediates at an early stage of the reaction process, it can be seen that during formation h-BiVO(4) has different growth rates along the a, b, and c axes. Based on crystal structure analysis and experimental results, h-BiVO(4) shows preferential growth along the [100] direction, and subsequently, along the [010] and [001] directions. As-synthesized h-BiVO(4) exhibits excellent photocatalytic ability in the photodegradation reaction of an aqueous solution of RB under visible light. Electrochemical measurements predict that h-BiVO(4) possesses high sensitivity to formaldehyde and ethanol gases, favorable discharge capacity, and capacity retention, which indicate potential applications in the fields of sensing devices and lithium-ion batteries.