Coomassie Staining Research Articles

Protease resistance of food proteins: a mixed picture for predicting allergenicity but a useful tool for assessing exposure

BackgroundSusceptibility to pepsin digestion of candidate transgene products is regarded an important parameter in the weight-of-evidence approach for allergenicity risk assessment of genetically modified crops. It has been argued that protocols used for this assessment should better reflect physiological conditions encountered in representative food consumption scenarios.AimTo evaluate whether inclusion of more physiological conditions, such as sub-optimal and lower pepsin concentrations, in combination with pancreatin digestion, improved the performance of digestibility protocols used in characterization of protein stability.MethodsFour pairs of established allergens and their related non/weakly-allergenic counterparts (seed albumins, muscle tropomyosins, plant lipid transfer proteins [LTP] and collagens) plus fish parvalbumin, were subjected to nine combinations of pH (1.2–2.5–4.0) and pepsin-to-protein ratio (PPR: 10–1–0.1 U/µg) for pepsin digestion, followed by pancreatin digestion in the presence of bile salts. Digestion was monitored by SDS-PAGE in conjunction with Coomassie staining and immunoblotting using rabbit antisera and human IgE.ResultsAt pH 4.0 and at PPR 0.1 most proteins, both allergen and non-allergen, were highly resistant to pepsin. Under conditions known to favor pepsin proteolysis, the established major allergens Ara h 2, Pru p 3 and Pen a 1 were highly resistant to proteolysis, while the allergen Cyp c 1 was not. However, this resistance to pepsin digestion only made Ara h 2 and to a lesser extent Pen a 1 and Pru p 3 stand out compared to their non-allergenic counterparts. Largely irrespective of preceding pepsin digestion conditions, pancreatin digestion was very effective for all tested proteins, allergens and non-allergens, except for Cyp c 1 and bovine collagen.ConclusionsSub-optimal pH, low pepsin-to protein ratio, and sequential pepsin and pancreatin digestion protocols do not improve the predictive value in distinguish allergens from non-allergens. Digestion conditions facilitating such distinction differ per protein pair.

PO-012 Ferulic acid increases ABT-888 sensitivity in breast cancer cells

IntroductionThe MRE11-RAD50-NBS1 (MRN) protein complex mediates DNA damage signalling and double-strand break repair. MRE11 can be regulated by histone modification, resulting in variable expression levels. We previously found that high MRE11 expression was associated with improved survival following radiotherapy, contrary to our expectations in terms of DNA DSB repair efficiency. We also discovered a C-terminally truncated form of MRE11 (TR-MRE11) in patients’ bladder tumour samples. To understand these findings further, we looked for the cleavage site of TR MRE11, and its functional impact on DNA damage repair.Material and methodsRT112 bladder carcinoma cells were lysed for immunoprecipitation with MRE11 antibody. After Coomassie staining, bands corresponding to full-length (FL) or TR-MRE11 were excised and in-gel digestion carried out with trypsin or elastase. Samples were analysed on an Orbitrap Fusion Lumos Mass Spectrometer. We then inserted point mutations into the DNA sequence of MRE11 corresponding to exons 15 to 20, which resulted in a premature stop codon, in an attempt to investigate the core functional MRE11 sequences which contribute to cell survival after radiation. Then various types of DNA damage by several methods such as overgrowth, starvation, irradiation or drug treatments to assess the effects on TR-MRE11 expression.Results and discussionsFL and TR MRE11 mass spectrometry narrowed down the cleavage site position to between αα 538 and 592. Cells transfected with MRE11 plasmids containing different lengths of c-terminal truncation showed two major patterns: low and high survival after irradiation. The c-terminal truncations occurring before exon 16 showed higher sensitivity to irradiation than c-terminal truncations after exon 16. These two patterns suggest that an important role in DNA damage repair exists in MRE11 after exon 16. Among various cells subjected to DNA damage, the cells overgrown consistently showed significantly increased TR-MRE11. This work is ongoing, and we plan to use molecules related to overgrowth in functional assays to determine the exact cleavage site.ConclusionIf lack of the c-terminal has functional impact in terms of DNA repair efficiency, this could explain our ‘paradoxical’ MRE11 IHC results seen in primary bladder cancer samples. Furthermore, further investigation of TR-MRE11 induction and cleavage sites will enable us to use this data for clinical benefit.

PO-009 Investigating the putative cleavage site of C-terminally truncated MRE11 using mass spectrometry and its function on DNA damage repair

IntroductionThe MRE11-RAD50-NBS1 (MRN) protein complex mediates DNA damage signalling and double-strand break repair. MRE11 can be regulated by histone modification, resulting in variable expression levels. We previously found that high MRE11 expression was associated with improved survival following radiotherapy, contrary to our expectations in terms of DNA DSB repair efficiency. We also discovered a C-terminally truncated form of MRE11 (TR-MRE11) in patients’ bladder tumour samples. To understand these findings further, we looked for the cleavage site of TR MRE11, and its functional impact on DNA damage repair.Material and methodsRT112 bladder carcinoma cells were lysed for immunoprecipitation with MRE11 antibody. After Coomassie staining, bands corresponding to full-length (FL) or TR-MRE11 were excised and in-gel digestion carried out with trypsin or elastase. Samples were analysed on an Orbitrap Fusion Lumos Mass Spectrometer. We then inserted point mutations into the DNA sequence of MRE11 corresponding to exons 15 to 20, which resulted in a premature stop codon, in an attempt to investigate the core functional MRE11 sequences which contribute to cell survival after radiation. Then various types of DNA damage by several methods such as overgrowth, starvation, irradiation or drug treatments to assess the effects on TR-MRE11 expression.Results and discussionsFL and TR MRE11 mass spectrometry narrowed down the cleavage site position to between αα 538 and 592. Cells transfected with MRE11 plasmids containing different lengths of c-terminal truncation showed two major patterns: low and high survival after irradiation. The c-terminal truncations occurring before exon 16 showed higher sensitivity to irradiation than c-terminal truncations after exon 16. These two patterns suggest that an important role in DNA damage repair exists in MRE11 after exon 16. Among various cells subjected to DNA damage, the cells overgrown consistently showed significantly increased TR-MRE11. This work is ongoing, and we plan to use molecules related to overgrowth in functional assays to determine the exact cleavage site.ConclusionIf lack of the c-terminal has functional impact in terms of DNA repair efficiency, this could explain our ‘paradoxical’ MRE11 IHC results seen in primary bladder cancer samples. Furthermore, further investigation of TR-MRE11 induction and cleavage sites will enable us to use this data for clinical benefit.

Stable Protein Gel Storage in Acetonitrile for Mass Spectrometric Analysis.

Long-term storage of protein samples for transportation is a challenge in the field of mass spectrometric analysis because the low temperature condition is not easy to be maintained. Here we introduce a simple method to preserve proteins at room temperature for at least one month. In this method, the protein sample is run shortly into a polyacrylamide gel which is then excised after Coomassie staining. The protein gel band is then dehydrated by 100% acetonitrile three times and kept in 100% acetonitrile for storage at room temperature. By the TMT 10-plex based quantitative proteomic analyses, we have found that these proteins are stable in their levels and modifications (phosphorylation, oxidation, and ubiquitination) for 30 days. Further analysis has revealed this storage method also well preserves proteins even at 45°C. We therefore recommend to use acetonitrile to dehydrate and store protein gel pieces as an effective alternative for sample shipping over days. Therefore, it might facilitate worldwide collaborations in the mass spectrometry-based proteomic research.

Back Cover: Optimized two‐dimensional gel electrophoresis in an alkaline pH range improves the identification of intracellular CFDA‐cisplatin‐protein adducts in ovarian cancer cells

DOI: 10.1002/elps.201700377 The cover picture shows the visualization of CFDA-cisplatin-protein adducts through 2DE. Human ovarian carcinoma cell lines A2780 and A2780cis cells were treated with CFDA-cisplatin and separated by 2DE. The use of fluorescent CFDA-cisplatin enables the visualization of CFDA-cisplatin-protein adducts. For the image analysis the fluorescence scans from CFDA-cisplatin-protein adducts and the Coomassie staining were fused into one image. The identification of nine previously unknown intracellular binding partners of CFDA-cisplatin by mass spectrometry provides new insights into the cellular processing of cisplatin.

Structural characteristics of the serum endonuclease Dnase1L3

Abstract Systemic Lupus Erythematosus (SLE) is a debilitating autoimmune disease with poly- and mono-genic etiologies. One cause of monogenic SLE is the disruption of Dnase1L3. Dnase1L3 is a serum endonuclease that degrades chromatin in apoptotic microparticles. Dnase1L3 could represent a novel therapy for some cases of SLE. However, the structure of Dnase1L3 is predicted by the 42% identical protein Dnase1. To better understand the structure-function of Dnase1L3, we set out to solve the crystal structure. To solve the crystal structure, we first generated a recombinant His6-Maltose Binding Protein-Dnase1L3 fusion protein. We developed a protocol for purifying Dnase1L3. We isolated Dnase1L3 from the cell lysate using Nickel-NTA beads in maltose buffer, removed purification tags on Dnase1L3 with Tobacco Etch Virus protease, and purified Dnase1L3 on a negatively charged ion exchange column. We then purified Dnase1L3 by size exclusion chromatography to 96.7% final purity. We verified purity at each step by Coomassie staining and, confirmed identity of the final product by Western blot. Recombinant Dnase1L3 robustly degraded plasmid DNA, with a specific activity of 1.83 x 104 U/mg. We found Dnase1L3 had a propensity for small mosaic crystals in a Tris and MgCl2 crystallization buffer. However, larger crystals were grown at 7 °C, compared to 22 °C. Overall, we have developed a new purification method for generating active Dnase1L3 useful for crystallography and other high-purity applications. These applications will further our understanding of Dnase1L3 structure and its function in SLE.

Rapid Protein Analysis and Western Blotting by Capillary Gel Electrophoresis

Capillary gel electrophoresis has been used since the early 1980s to separate analytes based upon their mass to charge ratio. However, optical limitations prevented high sensitivity instruments from being developed, limiting functionality. As manufacturing capabilities have improved the high‐throughput, hands‐off nature of parallel capillary gel electrophoresis has made it a desirable technique for analysis of DNA, RNA, and protein. Traditionally, protein analysis and Western blotting have been accomplished using SDS‐PAGE followed by staining of proteins in the gel or by downstream transfer of proteins to a blotting membrane, respectively. The trademark feature of these methods is their labor‐intensive nature with many hours or days passing between sample loading and results. Additionally, manual analysis prevents processing of large numbers of samples.Advanced Analytical Technologies, Inc, (AATI) has developed a high‐throughput capillary electrophoresis instrument capable of running 12 samples in parallel with analysis of a complete 96‐well plate without user intervention. This method relies on the rapid fluorescent labeling (~5 min hands on, 15 min total) of total protein sample, with a linear labeling dynamic range of 1 ng − 1000 ng protein input, followed by heat denaturing of the sample in an SDS buffer. The samples are then separated electrophoretically through a proprietary gel matrix (~30 min run time). Sizing of samples is provided using internal markers and comparison to a standardized ladder, allowing for sizing of samples from 14 kDa to greater than 200 kDa with an accuracy of ≤5% and precision of less ≤5%. Relative quantification of proteins is accomplished by comparing the corrected peak area of sample peaks with a precision of ≤10%. Absolute quantification of proteins can be accomplished through a calibration curve using the same protein as the unknown, as each unique protein species will label at varying efficiencies.Capillary gel electrophoresis coupled with total protein labeling can be used to replace traditional Western blots by analyzing and quantifying immunoprecipitations. Analysis of immunoprecipitations by SDS‐PAGE and Coomassie staining are limited due to sensitivity and the copious amounts of antibody present in the sample. By using an unlabeled antibody coupled with labeled total protein, the capillary electrophoresis instrument will only detect the labeled protein of interest isolated by the immunoprecipitation. This provides relative concentration and size information analogous to that provided by the traditional western in a fraction of the time. The AATI protocol eliminates signal bleeding which allows for a greater dynamic range than film. A pull down of endogenous GAPDH demonstrated a 3‐fold dynamic response (0.01 μg – 10 μg protein input).By harnessing capillary gel electrophoresis, AATI has developed a high sensitivity SDS‐PAGE/Western blot replacement with a 3‐fold dynamic range, allowing for rapid analysis of total and purified protein. Please contact the authors at AATI‐us.com if you have interest in this technology.Support or Funding InformationThis work was funded by Advanced Analytical Technologies, Inc.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of drug-neutralizing antibodies in patients with Fabry disease during infusion

Characterization of drug-neutralizing antibodies in patients with Fabry disease during infusion

Oxidative Stress in Autistic Children Alters Erythrocyte Shape in the Absence of Quantitative Protein Alterations and of Loss of Membrane Phospholipid Asymmetry.

Red blood cells (RBCs) from people affected by autism spectrum disorders (ASDs) are a target of oxidative stress. By scanning electron microscopy, we analyzed RBC morphology from 22 ASD children and show here that only 47.5 ± 3.33% of RBC displayed the typical biconcave shape, as opposed to 87.5 ± 1.3% (mean ± SD) of RBC from 21 sex- and age-matched healthy typically developing (TD) controls. Codocytes and star-shaped cells accounted for about 30% of all abnormally shaped ASD erythrocytes. RBC shape alterations were independent of the anticoagulant used (Na2-EDTA or heparin) and of different handling procedures preceding glutaraldehyde fixation, thus suggesting that they were not artefactual. Incubation for 24 h in the presence of antioxidants restored normal morphology in most erythrocytes from ASD patients. By Coomassie staining, as well as Western blotting analysis of relevant proteins playing a key role in the membrane-cytoskeleton organization, we were unable to find differences in RBC ghost composition between ASD and normal subjects. Phosphatidylserine (PS) exposure towards the extracellular membrane domain was examined in both basal and erythroptosis-inducing conditions. No differences were found between ASD and TD samples except when the aminophospholipid translocase was blocked by N-ethylmaleimide, upon which an increased amount of PS was found to face the outer membrane in RBC from ASD. These complex data are discussed in the light of the current understanding of the mode by which oxidative stress might affect erythrocyte shape in ASD and in other pathological conditions.

Microfluidic device-assisted etching of p-HEMA for cell or protein patterning.

The construction of biomaterials with which to limit the growth of cells or to limit the adsorption of proteins is essential for understanding biological phenomena. Here, we describe a novel method to simply and easily create thin layers of poly (2-hydroxyethyl methacrylate) (p-HEMA) for protein and cellular patterning via etching with ethanol and microfluidic devices. First, a cell culture surface or glass coverslip is coated with p-HEMA. Next, a polydimethylsiloxane (PDMS) microfluidic is placed onto the p-HEMA surface, and ethanol is aspirated through the device. The PDMS device is removed, and the p-HEMA surface is ready for protein adsorption or cell plating. This method allows for the fabrication of 0.3 µm thin layers of p-HEMA, which can be etched to 10 µm wide channels. Furthermore, it creates regions of differential protein adhesion, as shown by Coomassie staining and fluorescent labeling, and cell adhesion, as demonstrated by C2C12 myoblast growth. This method is simple, versatile, and allows biologists and bioengineers to manipulate regions for cell culture adhesion and growth. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:243-248, 2018.

Abolishment of proximal tubule albumin endocytosis does not affect plasma albumin during nephrotic syndrome in mice

The megalin/cubilin receptor complex is required for proximal tubular endocytosis and degradation of filtered albumin. An additional high-capacity retrieval pathway of intact albumin for the recovery of large amounts of filtered albumin has been proposed, possibly involving cooperation between megalin/cubilin and the neonatal Fc receptor. To clarify the potential role of such a pathway, we examined the effects of megalin/cubilin gene inactivation on tubular albumin uptake and plasma albumin levels in nephrotic, podocin knockout mice. Immunofluorescence microscopy of megalin/cubilin/podocin knockout mouse kidneys demonstrated abolishment of proximal tubule albumin uptake, in contrast to the excessive albumin accumulation observed in podocin knockout mice compared to controls. Correspondingly, urinary albumin excretion was increased 1.4 fold in megalin/cubilin/podocin compared to podocin knockout mice (albumin/creatinine: 226 vs. 157 mg/mg). However, no difference in plasma albumin levels was observed between megalin/cubilin/podocin and podocin knockout mice, as both were reduced to approximately 40% of controls. There were no differences in liver albumin synthesis by mRNA levels and protein abundance. Thus, megalin/cubilin knockout efficiently blocks proximal tubular albumin uptake in nephrotic mice but plasma albumin levels did not differ as a result of megalin/cubilin-deficiency, suggesting no significance of the megalin/cubilin-pathway for albumin homeostasis by retrieval of intact albumin.

Coomassie staining provides routine (sub)femtomole in-gel detection of intact proteoforms: Expanding opportunities for genuine Top-down Proteomics.

Modified colloidal Coomassie Brilliant Blue (cCBB) staining utilising a novel destain protocol and near-infrared fluorescence detection (nIRFD) rivals the in-gel protein detection sensitivity (DS) of SYPRO Ruby. However, established DS estimates are likely inaccurate in terms of 2DE-resolved proteoform 'spots' since DS is routinely measured from comparatively diffuse protein 'bands' following wide-well 1DE. Here, cCBB DS for 2DE-based proteomics was more accurately determined using narrow-well 1DE. As precise estimates of protein standard monomer concentrations are essential for accurate quantitation, coupling UV absorbance with gel-based purity assessments is described. Further, as cCBB is compatible with both nIRFD and densitometry, the impacts of imaging method (and image resolution) on DS were assessed. Narrow-well 1DE enabled more accurate quantitation of cCBB DS for 2DE, achieving (sub)femtomole DS with either nIRFD or densitometry. While densitometry offers comparative simplicity and affordability, nIRFD has the unique potential for enhanced DS with Deep Imaging. Higher-resolution nIRFD also improved analysis of a 2DE-resolved proteome, surpassing the DS of standard nIRFD and densitometry, with nIRFD Deep Imaging further maximising proteome coverage. cCBB DS for intact proteins rivals that of mass spectrometry (MS) for peptides in complex mixtures, reaffirming that 2DE-MS currently provides the most routine, broadly applicable, robust, and information-rich Top-down approach to Discovery Proteomics.

In vivo Assembly in Escherichia coli of Transformation Vectors for Plastid Genome Engineering.

Plastid transformation for the expression of recombinant proteins and entire metabolic pathways has become a promising tool for plant biotechnology. However, large-scale application of this technology has been hindered by some technical bottlenecks, including lack of routine transformation protocols for agronomically important crop plants like rice or maize. Currently, there are no standard or commercial plastid transformation vectors available for the scientific community. Construction of a plastid transformation vector usually requires tedious and time-consuming cloning steps. In this study, we describe the adoption of an in vivo Escherichia coli cloning (iVEC) technology to quickly assemble a plastid transformation vector. The method enables simple and seamless build-up of a complete plastid transformation vector from five DNA fragments in a single step. The vector assembled for demonstration purposes contains an enhanced green fluorescent protein (GFP) expression cassette, in which the gfp transgene is driven by the tobacco plastid ribosomal RNA operon promoter fused to the 5′ untranslated region (UTR) from gene10 of bacteriophage T7 and the transcript-stabilizing 3′UTR from the E. coli ribosomal RNA operon rrnB. Successful transformation of the tobacco plastid genome was verified by Southern blot analysis and seed assays. High-level expression of the GFP reporter in the transplastomic plants was visualized by confocal microscopy and Coomassie staining, and GFP accumulation was ~9% of the total soluble protein. The iVEC method represents a simple and efficient approach for construction of plastid transformation vector, and offers great potential for the assembly of increasingly complex vectors for synthetic biology applications in plastids.

The Susceptibilities of Respiratory Syncytial Virus to Nucleolin Receptor Blocking and Antibody Neutralization are Dependent upon the Method of Virus Purification.

Respiratory Syncytial Virus (RSV) that is propagated in cell culture is purified from cellular contaminants that can confound experimental results. A number of different purification methods have been described, including methods that utilize fast protein liquid chromatography (FPLC) and gradient ultracentrifugation. Thus, the constituents and experimental responses of RSV stocks purified by ultracentrifugation in sucrose and by FPLC were analyzed and compared by infectivity assay, Coomassie stain, Western blot, mass spectrometry, immuno-transmission electron microscopy (TEM), and ImageStream flow cytometry. The FPLC-purified RSV had more albumin contamination, but there was less evidence of host-derived exosomes when compared to ultracentrifugation-purified RSV as detected by Western blot and mass spectrometry for the exosome markers superoxide dismutase [Cu-Zn] (SOD1) and the tetraspanin CD63. Although the purified virus stocks were equally susceptible to nucleolin-receptor blocking by the DNA aptamer AS1411, the FPLC-purified RSV was significantly less susceptible to anti-RSV polyclonal antibody neutralization; there was 69% inhibition (p = 0.02) of the sucrose ultracentrifugation-purified RSV, 38% inhibition (p = 0.03) of the unpurified RSV, but statistically ineffective neutralization in the FPLC-purified RSV (22% inhibition; p = 0.30). The amount of RSV neutralization of the purified RSV stocks was correlated with anti-RSV antibody occupancy on RSV particles observed by immuno-TEM. RSV purified by different methods alters the stock composition and morphological characteristics of virions that can lead to different experimental responses.

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with "heavy" amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.HCV-infected/"light" amino acids and uninfected control/"heavy" amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Exopolymer production as a function of cell permeability and death in a diatom (Thalassiosira weissflogii) and a cyanobacterium (Synechococcus elongatus).

Exopolymer particles are found throughout the ocean and play a significant biogeochemical role in carbon cycling. Transparent exopolymer particles (TEP) are composed of acid polysaccharides, and Coomassie staining particles (CSP) are proteins. TEPs have been extensively studied in the ocean, while CSP have been largely overlooked. The objective of this research was to determine the role of stress and cell permeability in the formation of TEP and CSP. The diatom Thalassiosira weissflogii and cyanobacterium Synechococcus elongatus were grown in batch cultures and exposed to hydrogen peroxide (0, 10, and 100μM) as an environmental stressor. There was no correlation between TEP and CSP concentrations, indicating that they are different populations of particles rather than different chemical components of the same particles. CSP concentrations were not affected by hydrogen peroxide concentration and did not correlate with indicators of stress and cell death. In contrast, TEP concentrations in both taxa were correlated with a decrease in the effective quantum yield of photosystem II, increased activity of caspase-like enzymes, and an increase in the proportion of the population with permeable cell membranes, indicating that TEP production was associated with the process of cell death. These data show that different environmental factors and physiological processes affected the production of TEP and CSP by phytoplankton. TEP and CSP are separate populations of exopolymer particles with potentially different biogeochemical roles in the ocean.

Chm7 and Heh1 collaborate to link nuclear pore complex quality control with nuclear envelope sealing

The integrity of the nuclear envelope barrier relies on membrane remodeling by the ESCRTs, which seal nuclear envelope holes and contribute to the quality control of nuclear pore complexes (NPCs); whether these processes are mechanistically related remains poorly defined. Here, we show that the ESCRT-II/III chimera, Chm7, is recruited to a nuclear envelope subdomain that expands upon inhibition of NPC assembly and is required for the formation of the storage of improperly assembled NPCs (SINC) compartment. Recruitment to sites of NPC assembly is mediated by its ESCRT-II domain and the LAP2-emerin-MAN1 (LEM) family of integral inner nuclear membrane proteins, Heh1 and Heh2. We establish direct binding between Heh2 and the "open" forms of both Chm7 and the ESCRT-III, Snf7, and between Chm7 and Snf7. Interestingly, Chm7 is required for the viability of yeast strains where double membrane seals have been observed over defective NPCs; deletion of CHM7 in these strains leads to a loss of nuclear compartmentalization suggesting that the sealing of defective NPCs and nuclear envelope ruptures could proceed through similar mechanisms.

Protein and Carbohydrate Exopolymer Particles in the Sea Surface Microlayer (SML)

Exchanges of matter and energy between ocean and atmosphere occur through the sea surface microlayer (SML). The SML is the thin surface layer of the ocean at the ocean-atmosphere interface that has distinctive physical, chemical and biological properties compared with the underlying water. We measured the concentration of two types of exopolymer particles in the SML and underlying water in the Pacific Ocean off the coast of Oregon (United States) during July 2011. Transparent exopolymer particles (TEP) are defined by their acidic polysaccharide content, whereas Coomassie staining particles (CSP) are composed of protein. TEP and CSP were ubiquitous in the SML. TEP were not significantly enriched in the SML compared with the underlying water. CSP were significantly enriched in the SML, with an enrichment factor (EF) of 1.4 to 2.4. The distribution of exopolymer particles in the water and microscopic imaging indicated that TEP and CSP are distinct populations of particles rather than different chemical components of the same particles. Dissolved polysaccharides were not enriched in the SML, whereas monosaccharides had an EF of 1.2 to 1.8. Sampling occurred during the collapse of a diatom bloom, and diatoms were found both in the water column and SML. While there were living diatoms in the samples, most of the diatoms were dead and there were abundant empty frustules covered in layer of TEP. The collapsing diatom bloom was probably the source of exopolymer particles to both the SML and underlying water. Exopolymer particles are a component of the SML that may play a significant role in the marine carbon and nitrogen cycles, and the exchange of material between ocean and atmosphere.

Assessment and Quantification of Noncollagenic Matrix Proteins Released from Human Dentin Powder Incorporated into a Silated Hydroxypropylmethylcellulose Biomedical Hydrogel

IntroductionThe dentin extracellular matrix is a reservoir of bioactive molecules sequestered into dentin during dental initial development. They can be released under pathological conditions but also by controlled demineralization with bioactive materials. The purpose of this study was to investigate the ability of a biomedical hydrogel to extract and release these proteins from smashed dentin. MethodsSmashed dentin was obtained with 2 different kinds of grinders: a blade mill and a zirconia mortar grinder. The particle size was measured by scanning electron microscopy. Dentin powder was incorporated into a silated hydroxypropylmethylcellulose hydrogel. Several types of mixtures with variable parameters were tested. The mixtures were immersed into phosphate-buffered saline. The supernatants were collected, and the total released proteins were quantified by gel shift migration and Coomassie staining. The presence of transforming growth factor beta 1 was investigated by Western blot analysis and the ELISA. ResultsThe mixture dentin powder/hydrogel released proteins (from 49.1 μg/mL–137.9 μg/mL according to the mixtures). The release kinetics was growing and started from the first day until stabilization at 14 days. The quantity of released proteins was directly related to the size of the particles and the weight of the powder incorporated into the hydrogel. Gel shift with direct revelation by ultraviolet and Western blot analyses confirmed the presence of transforming growth factor beta 1 using ELISA. ConclusionsWe showed that silated hydroxypropylmethylcellulose hydrogel was able to extract dentin matrix proteins from smashed dentin powder. This mixture could be considered a new way of dental treatment for the dentin-pulp complex and bone regeneration.

94. A Scalable Recombinant HSV-Based Manufacturing System for Production of Highly Potent Adeno-Associated Type 9 Vectors

Introduction: Adeno-associated virus vectors (AAV) have been utilized as a gene delivery tool to treat a multitude of diseases. As the potential uses for these vectors expand, the need for improved manufacturing methods becomes essential for large-scale implementation. Recombinant Herpes Simplex Virus (rHSV)-based method delivering AAV capsid and transgenes for recombinant AAV (rAAV) production has been shown to be a robust and scalable method for producing multiple rAAV serotypes for pre-clinical and clinical studies. Here we will present the first scalable production and purification method for rAAV serotype 9 utilizing the rHSV system. Methods: Recombinant rAAV9-GFP stocks were produced by transfection or rHSV co-infection on HEK 293. Vector genome titers were assessed by quantitative PCR, digital droplet PCR and dot blot. Infectivity was assessed by an infectious center assay, flow cytometry, and green cell transduction assay (GCA). Coomassie SDS-PAGE, or western blot analyses were performed to evaluate identity and purity of rAAV samples and electron microscopy to assess full versus empty ratio for each method. Results: rAAV vectors produced by the HSV method showed a 7 and 10 fold increase in total vector genomes and transducing units per CellStack®, respectively. Increased infectivity was observed in rHSV-made vectors as demonstrated by an increasing in transducing unit titers by GCA and infectious center assay. Flow cytometry of C12 cells infected with equal vector genome amounts of both transfection and rHSV made rAAV showed a 30% increase in GFP expressing cells. Coomassie staining and western blot analysis showed a 20-32% reduction in total capsid proteins in rAAV made by rHSV co-infection. Electron microscopy confirmed this reduction in total capsid protein was due to a decrease in empty particles in HSV-made rAAV samples. Overall, this method produced greater than 1×1014 rAAV9 vector genomes per liter equivalent of 293 cells of a final, purified product. Conclusions: Our data demonstrated the significant improvement in rAAV9 manufacturing by utilization of the rHSV based manufacturing methods. An improvement in both vector titer and infectivity was observed in rHSV made rAAV vectors compared to transfection-based production. Importantly, this scalable purification method is readily suitable for pre-clinical GLP studies and GMP manufacturing in our laboratory. Further improvements will be presented specifically evaluating rHSV production in a suspension-adapted HEK 293 cell system in view of a large scale manufacturing protocol of rAAV9 vectors for clinical studies.