CsCl Density Gradient Centrifugation Research Articles

Dominance of phage particles carrying antibiotic resistance genes in the viromes of retail food sources

The growth of antibiotic resistance has stimulated interest in understanding the mechanisms by which antibiotic resistance genes (ARG) are mobilized. Among them, studies analyzing the presence of ARGs in the viral fraction of environmental, food and human samples, and reporting bacteriophages as vehicles of ARG transmission, have been the focus of increasing research. However, it has been argued that in these studies the abundance of phages carrying ARGs has been overestimated due to experimental contamination with non-packaged bacterial DNA or other elements such as outer membrane vesicles (OMVs). This study aims to shed light on the extent to which phages, OMVs or contaminating non-packaged DNA contribute as carriers of ARGs in the viromes. The viral fractions of three types of food (chicken, fish, and mussels) were selected as sources of ARG-carrying phage particles, whose ability to infect and propagate in an Escherichia coli host was confirmed after isolation. The ARG-containing fraction was further purified by CsCl density gradient centrifugation and, after removal of DNA outside the capsids, ARGs inside the particles were confirmed. The purified fraction was stained with SYBR Gold, which allowed the visualization of phage capsids attached to and infecting E. coli cells. Phages with Myoviridae and Siphoviridae morphology were observed by electron microscopy. The proteins in the purified fraction belonged predominantly to phages (71.8% in fish, 52.9% in mussels, 78.7% in chicken sample 1, and 64.1% in chicken sample 2), mainly corresponding to tail, capsid, and other structural proteins, whereas membrane proteins, expected to be abundant if OMVs were present, accounted for only 3.8–21.4% of the protein content. The predominance of phage particles in the viromes supports the reliability of the protocols used in this study and in recent findings on the abundance of ARG-carrying phage particles.

British Society for Gene and Cell Therapy Autumn Conference Friday 23 November 2018 Regent's University Conference Centre, London, UK www.bsgct.org

British Society for Gene and Cell Therapy Autumn Conference Friday 23 November 2018 Regent's University Conference Centre, London, UK www.bsgct.org

Development of in Planta Chemical Cross-Linking-Based Quantitative Interactomics in Arabidopsis.

An in planta chemical cross-linking-based quantitative interactomics (IPQCX-MS) workflow has been developed to investigate in vivo protein-protein interactions and alteration in protein structures in a model organism, Arabidopsis thaliana. A chemical cross-linker, azide-tag-modified disuccinimidyl pimelate (AMDSP), was directly applied onto Arabidopsis tissues. Peptides produced from protein fractions of CsCl density gradient centrifugation were dimethyl-labeled, from which the AMDSP cross-linked peptides were fractionated on chromatography, enriched, and analyzed by mass spectrometry. ECL2 and SQUA-D software were used to identify and quantitate these cross-linked peptides, respectively. These computer programs integrate peptide identification with quantitation and statistical evaluation. This workflow eventually identified 354 unique cross-linked peptides, including 61 and 293 inter- and intraprotein cross-linked peptides, respectively, demonstrating that it is able to in vivo identify hundreds of cross-linked peptides at an organismal level by overcoming the difficulties caused by multiple cellular structures and complex secondary metabolites of plants. Coimmunoprecipitation and super-resolution microscopy studies have confirmed the PHB3-PHB6 protein interaction found by IPQCX-MS. The quantitative interactomics also found hormone-induced structural changes of SBPase and other proteins. This mass-spectrometry-based interactomics will be useful in the study of in vivo protein-protein interaction networks in agricultural crops and plant-microbe interactions.

Rapid Identification of Intact Staphylococcal Bacteriophages Using Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry.

Staphylococcus aureus is a major causative agent of infections associated with hospital environments, where antibiotic-resistant strains have emerged as a significant threat. Phage therapy could offer a safe and effective alternative to antibiotics. Phage preparations should comply with quality and safety requirements; therefore, it is important to develop efficient production control technologies. This study was conducted to develop and evaluate a rapid and reliable method for identifying staphylococcal bacteriophages, based on detecting their specific proteins using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling that is among the suggested methods for meeting the regulations of pharmaceutical authorities. Five different phage purification techniques were tested in combination with two MALDI-TOF MS matrices. Phages, either purified by CsCl density gradient centrifugation or as resuspended phage pellets, yielded mass spectra with the highest information value if ferulic acid was used as the MALDI matrix. Phage tail and capsid proteins yielded the strongest signals whereas the culture conditions had no effect on mass spectral quality. Thirty-seven phages from Myoviridae, Siphoviridae or Podoviridae families were analysed, including 23 siphophages belonging to the International Typing Set for human strains of S. aureus, as well as phages in preparations produced by Microgen, Bohemia Pharmaceuticals and MB Pharma. The data obtained demonstrate that MALDI-TOF MS can be used to effectively distinguish between Staphylococcus-specific bacteriophages.

Charge, size distribution and hydrophobicity of viruses: Effect of propagation and purification methods

Two virus propagation methods (in broth and on double agar overlay) and three purification procedures (PEG precipitation, centrifugal diafiltration and CsCl density gradient centrifugation) were comparatively evaluated using MS2 and P22 bacteriophages as model viruses. The prepared stocks were characterized in terms of electrophoretic mobility as a function of pH, particle size distribution, surface tension components and the overall hydrophobicity of the virus, as well as the percentage of infectious and total virus recovered. The obtained data were used to rank the purification methods according to six criteria of likely practical relevance. Regardless of the purification method applied, virus propagation in broth media resulted in higher purity virus stocks as the growth on double agar overlay introduced difficult-to-remove residual agar. CsCl density gradient centrifugation gave the highest quality bacteriophage suspensions, recovered infectious P22 at least as efficiently as the other two purification methods and selected for intact P22 virions over damaged ones. The impurities remaining in the virus suspension after PEG precipitation and centrifugal diafiltration broadened the size distribution and interfered with electrophoretic mobility measurements. The residual impurities had a major impact on the free energy of virus-virus interfacial interaction (the quantitative measure of virus hydrophobicity/hydrophilicity) leading to an incorrect determination of P22 bacteriophage as hydrophilic. The trends in measured physicochemical properties can be rationalized by considering impurity-coated virions as permeable soft particles.

Fluoroquinolone-Gyrase-DNA Cleaved Complexes.

The quinolones are potent antibacterials that act by forming complexes with DNA and either gyrase or topoisomerase IV. These ternary complexes, called cleaved complexes because the DNA moiety is broken, block replication, transcription, and bacterial growth. Cleaved complexes readily form in vitro when gyrase, plasmid DNA, and quinolone are combined and incubated; complexes are detected by the linearization of plasmid DNA, generally assayed by gel electrophoresis. The stability of the complexes can be assessed by treatment with EDTA, high temperature, or dilution to dissociate the complexes and reseal the DNA moiety. Properties of the complexes are sensitive to quinolone structure and to topoisomerase amino acid substitutions associated with quinolone resistance. Consequently, studies of cleaved complexes can be used to identify improvements in quinolone structure and to understand the biochemical basis of target-based resistance. Cleaved complexes can also be detected in quinolone-treated bacterial cells by their ability to rapidly block DNA replication and to cause chromosome fragmentation; they can even be recovered from lysed cells following CsCl density-gradient centrifugation. Thus, in vivo and cell-fractionation tests are available for assessing the biological relevance of work with purified components.

An optimized method for high quality DNA extraction from microalga Prototheca wickerhamii for genome sequencing

BackgroundThe complex cell wall structure of algae often precludes efficient extraction of their genetic material. The purpose of this study was to design a next-generation sequencing-suitable DNA isolation method for unicellular, achlorophyllous, yeast-like microalgae of the genus Prototheca, the only known plant pathogens of both humans and animals. The effectiveness of the newly proposed scheme was compared with five other, previously described methods, commonly used for DNA isolation from plants and/or yeasts, available either as laboratory-developed, in-house assays, based on liquid nitrogen grinding or different enzymatic digestion, or as commercially manufactured kits.ResultsAll five, previously described, isolation assays yielded DNA concentrations lower than those obtained with the new method, averaging 16.15 ± 25.39 vs 74.2 ± 0.56 ng/µL, respectively. The new method was also superior in terms of DNA purity, as measured by A260/A280 (−0.41 ± 4.26 vs 2.02 ± 0.03), and A260/A230 (1.20 ± 1.12 vs 1.97 ± 0.07) ratios. Only the liquid nitrogen-based method yielded DNA of comparable quantity (60.96 ± 0.16 ng/µL) and quality (A260/A280 = 2.08 ± 0.02; A260/A230 = 2.23 ± 0.26). Still, the new method showed higher integrity, which was best illustrated upon electrophoretic analysis. Genomic DNA of Prototheca wickerhamii POL-1 strain isolated with the protocol herein proposed was successfully sequenced on the Illumina MiSeq platform.ConclusionsA new method for DNA isolation from Prototheca algae is described. The method, whose protocol involves glass beads pulverization and cesium chloride (CsCl) density gradient centrifugation, was demonstrated superior over the other common assays in terms of DNA quantity and quality. The method is also the first to offer the possibility of preparation of DNA template suitable for whole genome sequencing of Prototheca spp.

Recombinant expression of beak and feather disease virus capsid protein and assembly of virus-like particles in Nicotiana benthamiana

BackgroundBeak and feather disease virus (BFDV) is an important disease causing agent affecting psittacines. BFDV is highly infectious and can present as acute, chronic or subclinical disease. The virus causes immunodeficiency and is often associated with secondary infections. No commercial vaccine is available and yields of recombinant BFDV capsid protein (CP) expressed in insect cells and bacteria are yet to be seen as commercially viable, although both systems produced BFDV CP that could successfully assemble into virus-like particles (VLPs). Plants as expression systems are increasingly becoming favourable for the production of region-specific and niche market products. The aim of this study was to investigate the formation and potential for purification of BFDV VLPs in Nicotiana benthamiana.MethodsThe BFDV CP was transiently expressed in N. benthamiana using an Agrobacterium-mediated system and plant expression vectors that included a bean yellow dwarf virus (BeYDV)-based replicating DNA vector. Plant-produced BFDV CP was detected using immunoblotting. VLPs were purified using sucrose cushion and CsCl density gradient centrifugation and visualised using transmission electron microscopy.ResultsIn this study we demonstrate that the BFDV CP can be successfully expressed in N. benthamiana, albeit at relatively low yield. Using a purification strategy based on centrifugation we demonstrated that the expressed CP can self-assemble into VLPs that can be detected using electron microscopy. These plant-produced BFDV VLPs resemble those produced in established recombinant expression systems and infectious virions. It is possible that the VLPs are spontaneously incorporating amplicon DNA produced from the replicating BeYDV plant vector.ConclusionsThis is the first report of plant-made full-length BFDV CP assembling into VLPs. The putative pseudovirions could be used to further the efficacy of vaccines against BFDV.

Expression and Characterization of Capsid Proteins Derived from GII.17 and GII.7 Noroviruses

Objective: Noroviruses (NoVs) are the leading cause of non-bacterial gastroenteritis worldwide and genogroup II, genotype 4 variants have been responsible for the majority of outbreaks reported. In winter 2014-15, the transient burst outbreaks caused by a new GII.17 variant that differed from previous strains was observed in several countries. In this study, major capsid proteins derived from GII.17 and GII.7 NoVs were expressed in sf9 cells and used to produce hyperimmune sera in rabbits for characterization of GII.17 and GII7 NoV Virus like particles (VLPs). Methods: Baculovirus-insect cell expression system was used to assemble NoV VLPs. Salivary HBGA-viruslike particle (VLP) binding and binding blockade assays were performed. Results: High expression levels were observed for both capsid proteins and expression led to successful assembly of VLPs. CsCl density gradient centrifugation indicated presence of two bands for both VLPs and SDS-PAGE analysis of these two bands showed contradictory ratio of the full-length and truncated capsid proteins. Both VLPs bound to salivary HBGAs derived from blood type A, B, AB and O individuals. In vitro HBGA-VLP binding blockade assay using hyperimmune sera against multiple genotypes, including current pandemic Sydney 2012 GII.4, GI.2, GII.7 and GII.17 VLPs demonstrated absence of blocking antibodies against the binding of GII.17 and GII.7 VLPs to salivary HBGAs. Conclusion: The expression of GII.17 and GII.7 capsid proteins and VLP assembling allowed the antigenic comparison of different recombinant NoVs.

Characterization of the Proteins Associated with Caulobacter crescentus Bacteriophage CbK Particles.

Bacteriophage genomes contain an abundance of genes that code for hypothetical proteins with either a conserved domain or no predicted function. The Caulobacter phage CbK has an unusual shape, designated morphotype B3 that consists of an elongated cylindrical head and a long flexible tail. To identify CbK proteins associated with the phage particle, intact phage particles were subjected to SDS-PAGE, and the resulting protein bands were digested with trypsin and analyzed using MALDI mass spectroscopy to provide peptide molecular weights. These peptide molecular weights were then compared with the peptides that would be generated from the predicted amino acid sequences that are coded by the CbK genome, and the comparison of the actual and predicted peptide masses resulted in the identification of single genes that could code for the set of peptides derived from each of the 20 phage proteins. We also found that CsCl density gradient centrifugation resulted in the separation of empty phage heads, phage heads containing material organized in a spiral, isolated phage tails, and other particulate material from the intact phage particles. This additional material proved to be a good source of additional phage proteins, and preliminary results suggest that it may include a CbK DNA replication complex.

An enzyme-linked immuno focus assay for rapid detection and enumeration, and a newborn mouse model for human non-polio enteroviruses associated with acute diarrhea

We have recently reported significant association of non-polio enteroviruses (NPEVs) with acute and persistent diarrhea (18–21% of total diarrheal cases), and non-diarrheal Increased Frequency of Bowel Movements (IFoBM-ND) (about 29% of the NPEV infections) in children and that the NPEV-associated diarrhea was as significant as rotavirus diarrhea. However, their diarrhea-causing potential is yet to be demonstrated in an animal model system. Since the determination of virus titers by the traditional plaque assay takes 4–7 days, there is a need for development of a rapid method for virus titer determination to facilitate active clinical research on enterovirus-associated diarrhea.The goal of this study is to develop a cell-based rapid detection and enumeration method and to demonstrate the diarrhea-inducing potential of purified and characterized non-polio enteroviruses, which were isolated from diarrheic children. Here we describe generation of monoclonal and polyclonal antibodies against purified strains belonging to different serotypes, and development of an enzyme-linked immuno focus assay (ELIFA) for detection and enumeration of live NPEV particles in clinical and purified virus samples, and a newborn mouse model for NPEV diarrhea. Plaque-purified NPVEs, belonging to different serotypes, isolated from children with diarrhea, were grown in cell culture and purified by isopycnic CsCl density gradient centrifugation. By ELIFA, NPEVs could be detected and enumerated within 12h post-infection. Our results demonstrated that Coxsackievirus B1 (CVB1) and CVB5 strains, isolated from diarrheic children, induced severe diarrhea in orally-inoculated 9–12 day-old mouse pups, fulfilling Koch's postulates. The methods described here would facilitate studies on NPEV-associated gastrointestinal disease.

A simplified purification method for AAV variant by polyethylene glycol aqueous two-phase partitioning

Adeno-Associated Virus (AAV) has been widely used for in vivo study and preclinical therapy due to its ability to mediate long-term transgene expression, its lack of pathogenicity and low immunogenicity. It has been found that AAV has more than ten serotypes, which each transfect certain types of cells in the viral infected organ. Current methods for purification of different AAV serotypes utilize CsCl or Iodixanol ultrahigh speed density gradient centrifugation, which is expensive and time consuming. We recently developed a simplified method, PEG/(NH4)2SO4 aqueous two phase partitioning, for purification of AAV serotype 2 and 8. The method does not require ultrahigh speed gradient centrifugation or chromatography. Here we further explore the simplified method for purification of other serotypes of AAV, serotype 6 and 9. This simplified method not only can be used to purify serotype 2 and 8, but also serotype 6 and 9, indicating that a variety of AAV serotypes can be purified by this method.

Purification of white spot syndrome virus by iodixanol density gradient centrifugation

Up to now, only a few brief procedures for purifying white spot syndrome virus (WSSV) have been described. They were mainly based on sucrose, NaBr and CsCl density gradient centrifugation. This work describes for the first time the purification of WSSV through iodixanol density gradients, using virus isolated from infected tissues and haemolymph of Penaeus vannamei (Boone). The purification from tissues included a concentration step by centrifugation (2.5 h at 60,000 g) onto a 50% iodixanol cushion and a purification step by centrifugation (3 h at 80,000 g) through a discontinuous iodixanol gradient (phosphate-buffered saline, 5%, 10%, 15% and 20%). The purification from infected haemolymph enclosed a dialysis step with a membrane of 1,000 kDa (18 h) and a purification step through the earlier iodixanol gradient. The gradients were collected in fractions and analysed. The number of particles, infectivity titre (in vivo), total protein and viral protein content were evaluated. The purification from infected tissues gave WSSV suspensions with a very high infectivity and an acceptable purity, while virus purified from haemolymph had a high infectivity and a very high purity. Additionally, it was observed that WSSV has an unusually low buoyant density and that it is very sensitive to high external pressures.

Aggrecanase cleavage in juvenile idiopathic arthritis patients is minimally detected in the aggrecan interglobular domain but robust at the aggrecan C‐terminus

To investigate aggrecan degradation in juvenile idiopathic arthritis (JIA). The pattern and abundance of aggrecan fragments in synovial fluid (SF) aspirates from JIA patients were analyzed and compared with aggrecan fragments in SF from patients with other arthritides, children with knee injury, and a knee-healthy reference group. Concentrations of sulfated glycosaminoglycan (sGAG) in SF were measured by Alcian blue precipitation assay. Aggrecan fragments were purified by dissociative CsCl density-gradient centrifugation, deglycosylated, and analyzed by Western blot using antibodies specific for either aggrecanase-derived ARGS, SELE, and KEEE neoepitopes or the aggrecan G3 domain. The concentration of sGAG in SF from patients with JIA was significantly lower compared with that in SF from patients with osteoarthritis (OA) (P < 0.001), patients with juvenile knee injury (P = 0.006), and knee-healthy controls (P = 0.022). Western blot analysis revealed KEEE, SELE, and G3 fragments generated by aggrecanase cleavage in the chondroitin sulfate-rich region of aggrecan in patients with JIA. The pattern of aggrecan fragments in JIA patients was not identical to that in pooled OA SF, although there were notable similarities. Surprisingly, aggrecanase-derived ARGS fragments were barely detectable in JIA SF, in marked contrast to levels in OA SF. Aggrecanases appear to cleave minimally in the interglobular domain of aggrecan in JIA patients despite robust levels of cleavage in the chondroitin sulfate-rich region. These results suggest that in JIA, unlike other arthritides, aggrecanase cleavage in the aggrecan interglobular domain might not be a major pathogenic event.

Human CST Has Independent Functions during Telomere Duplex Replication and C-Strand Fill-In

Human CST (CTC1-STN1-TEN1) is an RPA-like complex that is needed for efficient replication through the telomere duplex and genome-wide replication restart after fork stalling. Here, we show that STN1/CST has a second function in telomere replication during G-overhang maturation. Analysis of overhang structure after STN1 depletion revealed normal kinetics for telomerase-mediated extension in S phase but a delay in subsequent overhang shortening. This delay resulted from a defect in C-strand fill-in. Short telomeres exhibited the fill-in defect but normal telomere duplex replication, indicating that STN1/CST functions independently in these processes. Our work also indicates that the requirement for STN1/CST in telomere duplex replication correlates with increasing telomere length and replication stress. Our results provide direct evidence that STN1/CST participates in C-strand fill-in. They also demonstrate that STN1/CST participates in two mechanistically separate steps during telomere replication and identify CST as a replication factor that solves diverse replication-associated problems.

Isolation and Quantitation of Topoisomerase Complexes Accumulated on Escherichia coli Chromosomal DNA

DNA topoisomerases are important targets in anticancer and antibacterial therapy because drugs can initiate cell death by stabilizing the transient covalent topoisomerase-DNA complex. In this study, we employed a method that uses CsCl density gradient centrifugation to separate unbound from DNA-bound GyrA/ParC in Escherichia coli cell lysates after quinolone treatment, allowing antibody detection and quantitation of the covalent complexes on slot blots. Using these procedures modified from the in vivo complexes of enzyme (ICE) bioassay, we found a correlation between gyrase-DNA complex formation and DNA replication inhibition at bacteriostatic (1× MIC) norfloxacin concentrations. Quantitation of the number of gyrase-DNA complexes per E. coli cell permitted an association between cell death and chromosomal gyrase-DNA complex accumulation at norfloxacin concentrations greater than 1× MIC. When comparing levels of gyrase-DNA complexes to topoisomerase IV-DNA complexes in the absence of drug, we observed that the gyrase-DNA complex level was higher (∼150-fold) than that of the topoisomerase IV-DNA complex. In addition, levels of gyrase and topoisomerase IV complexes reached a significant increase after 30 min of treatment at 1× and 1.7× MIC, respectively. These results are in agreement with gyrase being the primary target for quinolones in E. coli. We further validated the utility of this method for the study of topoisomerase-drug interactions in bacteria by showing the gyrase covalent complex reversibility after removal of the drug from the medium, and the resistant effect of the Ser83Leu gyrA mutation on accumulation of gyrase covalent complexes on chromosomal DNA.

N-Glycosylation of Haloferax volcanii Flagellins Requires Known Agl Proteins and Is Essential for Biosynthesis of Stable Flagella

N-glycosylation, a posttranslational modification required for the accurate folding and stability of many proteins, has been observed in organisms of all domains of life. Although the haloarchaeal S-layer glycoprotein was the first prokaryotic glycoprotein identified, little is known about the glycosylation of other haloarchaeal proteins. We demonstrate here that the glycosylation of Haloferax volcanii flagellins requires archaeal glycosylation (Agl) components involved in S-layer glycosylation and that the deletion of any Hfx. volcanii agl gene impairs its swimming motility to various extents. A comparison of proteins in CsCl density gradient centrifugation fractions from supernatants of wild-type Hfx. volcanii and deletion mutants lacking the oligosaccharyltransferase AglB suggests that when the Agl glycosylation pathway is disrupted, cells lack stable flagella, which purification studies indicate consist of a major flagellin, FlgA1, and a minor flagellin, FlgA2. Mass spectrometric analyses of FlgA1 confirm that its three predicted N-glycosylation sites are modified with covalently linked pentasaccharides having the same mass as that modifying its S-layer glycoprotein. Finally, the replacement of any of three predicted N-glycosylated asparagines of FlgA1 renders cells nonmotile, providing direct evidence for the first time that the N-glycosylation of archaeal flagellins is critical for motility. These results provide insight into the role that glycosylation plays in the assembly and function of Hfx. volcanii flagella and demonstrate that Hfx. volcanii flagellins are excellent reporter proteins for the study of haloarchaeal glycosylation processes.

Chondroadherin Fragmentation as a Biochemical Marker for Early Stage Disk Degeneration

Introduction Intervertebral disk (IVD) degeneration has been strongly associated with and named a major cause of back pain. At present, little is known about the molecular mechanisms involved in the degeneration of IVD and how these may differ from normal turnover of the tissue. As a result of this, a biomarker for disk degeneration has not yet been identified and we propose chondroadherin (CHAD) fragmentation as a potential option. CHAD, a protein of the leucine rich repeat (LRR) family, is one of the proteins predominantly expressed in the extracellular matrix of cartilaginous tissue, including that of the IVD. This restricted distribution is unusual among LRR proteins, which commonly show a wide tissue distribution. CHAD is primarily found close to the cells, where it can interact with collagen fibrils of the ECM and molecules at the cell surface, providing a mechanism for regulating cell metabolism and ECM structure. These interactions may also aid in promoting matrix homeostasis, and variation in CHAD abundance or structure might therefore lead to pathological changes in the tissue over time. The aims of this study were to determine whether CHAD fragmentation occurs and is unique to disk degeneration, and to characterize the cleavage site within CHAD at which fragmentation occurs. Materials and Methods Healthy and degenerate lumbar IVDs were obtained through organ donations via Transplant Quebec. IVDs from patients with degenerative disk disease and from patients with scoliosis were obtained at the time of surgery. Punches of 4 mm were taken and disk tissue then extracted using 15 volumes of extraction buffer (4 M GuCl, 10 mM EDTA, COMPLETE, 50 mM NaAc, pH 5.8) on a wet weight per volume basis. Extracted proteins were ethanol precipitated, and CHAD fragmentation was studied using SDS-PAGE and western blotting in combination with specific antibodies. To characterize the CHAD cleavage site, a degenerate disk extract was subjected to CsCl density gradient centrifugation to remove proteoglycans. CHAD was purified from the protein fraction by chromatography through carboxymethyl 52. The CHAD-containing samples were then fractionated on an SDS-PAGE gel and stained with Coomassie Blue. Gel portions containing the CHAD fragment were excised, then lyophilized, reduced and alkylated, and subjected to trypsin digestion. Peptides were then identified by liquid chromatography mass spectrometry. Antineoepitope antibodies specifically recognizing the fragmented CHAD were generated by immunizing rabbits with synthetic peptides conjugated to KLH. The peptides represented the terminal amino acid sequences at the site of CHAD fragmentation. Results Proteolytic fragmentation of CHAD was observed in IVDs from patients with DDD and in some individuals with adolescent idiopathic scoliosis (AIS). Its presence appeared to be related to the degree of degeneration in both cases (Fig. 1). This phenomenon was found to be specific to disk degeneration, as CHAD fragmentation was not observed in healthy adolescent and adult disks from organ donors. Within the degenerate disk, fragmentation was evident in tissue with signs of degeneration but not in tissue that had no signs of degeneration. This same trend was also seen when comparing normal and degenerate disk tissue from different levels of the spine from the same donor. Upon analysis with the antineoepitope antibody, it was apparent that CHAD fragmentation occurred at the same site in degenerate disks from Transplant Quebec donors, surgical samples from adults with DDD, and surgical samples from adolescents with AIS. Normal tissue samples showed no antineoepitope antibody binding, confirming that CHAD fragmentation at this site was not present in the healthy disk. Conclusion CHAD fragmentation is associated with disk degeneration present both in the adult with DDD and the adolescent with scoliosis, and fragmentation is created by cleavage at the same site within both disorders. Fragmentation is not, however, present in normal disk tissue. Thus, CHAD fragmentation may distinguish catabolic processes leading to disk degeneration from those associated with normal turnover of the tissue, and as such serve as a marker of disk degeneration. It is necessary to recognize the biochemical processes that specifically contribute to disk degeneration, if degeneration is to be prevented or retarded, and if novel treatments initiating disk regeneration are to be developed. The mechanism leading to fragmentation and the proteinase involved are currently under investigation. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Chondroadherin Fragmentation as a Biochemical Marker for Early Stage Disk Degeneration

IntroductionIntervertebral disk (IVD) degeneration has been strongly associated with and named a major cause of back pain. At present, little is known about the molecular mechanisms involved in the degeneration of IVD and how these may differ from normal turnover of the tissue. As a result of this, a biomarker for disk degeneration has not yet been identified and we propose chondroadherin (CHAD) fragmentation as a potential option. CHAD, a protein of the leucine rich repeat (LRR) family, is one of the proteins predominantly expressed in the extracellular matrix of cartilaginous tissue, including that of the IVD. This restricted distribution is unusual among LRR proteins, which commonly show a wide tissue distribution. CHAD is primarily found close to the cells, where it can interact with collagen fibrils of the ECM and molecules at the cell surface, providing a mechanism for regulating cell metabolism and ECM structure. These interactions may also aid in promoting matrix homeostasis, and variation in CHAD abundance or structure might therefore lead to pathological changes in the tissue over time.The aims of this study were to determine whether CHAD fragmentation occurs and is unique to disk degeneration, and to characterize the cleavage site within CHAD at which fragmentation occurs.Materials and MethodsHealthy and degenerate lumbar IVDs were obtained through organ donations via Transplant Quebec. IVDs from patients with degenerative disk disease and from patients with scoliosis were obtained at the time of surgery. Punches of 4 mm were taken and disk tissue then extracted using 15 volumes of extraction buffer (4 M GuCl, 10 mM EDTA, COMPLETE, 50 mM NaAc, pH 5.8) on a wet weight per volume basis. Extracted proteins were ethanol precipitated, and CHAD fragmentation was studied using SDS-PAGE and western blotting in combination with specific antibodies.To characterize the CHAD cleavage site, a degenerate disk extract was subjected to CsCl density gradient centrifugation to remove proteoglycans. CHAD was purified from the protein fraction by chromatography through carboxymethyl 52. The CHAD-containing samples were then fractionated on an SDS-PAGE gel and stained with Coomassie Blue. Gel portions containing the CHAD fragment were excised, then lyophilized, reduced and alkylated, and subjected to trypsin digestion. Peptides were then identified by liquid chromatography mass spectrometry.Antineoepitope antibodies specifically recognizing the fragmented CHAD were generated by immunizing rabbits with synthetic peptides conjugated to KLH. The peptides represented the terminal amino acid sequences at the site of CHAD fragmentation.ResultsProteolytic fragmentation of CHAD was observed in IVDs from patients with DDD and in some individuals with adolescent idiopathic scoliosis (AIS). Its presence appeared to be related to the degree of degeneration in both cases (Fig. 1). This phenomenon was found to be specific to disk degeneration, as CHAD fragmentation was not observed in healthy adolescent and adult disks from organ donors. Within the degenerate disk, fragmentation was evident in tissue with signs of degeneration but not in tissue that had no signs of degeneration. This same trend was also seen when comparing normal and degenerate disk tissue from different levels of the spine from the same donor.Upon analysis with the antineoepitope antibody, it was apparent that CHAD fragmentation occurred at the same site in degenerate disks from Transplant Quebec donors, surgical samples from adults with DDD, and surgical samples from adolescents with AIS. Normal tissue samples showed no antineoepitope antibody binding, confirming that CHAD fragmentation at this site was not present in the healthy disk.ConclusionCHAD fragmentation is associated with disk degeneration present both in the adult with DDD and the adolescent with scoliosis, and fragmentation is created by cleavage at the same site within both disorders. Fragmentation is not, however, present in normal disk tissue. Thus, CHAD fragmentation may distinguish catabolic processes leading to disk degeneration from those associated with normal turnover of the tissue, and as such serve as a marker of disk degeneration. It is necessary to recognize the biochemical processes that specifically contribute to disk degeneration, if degeneration is to be prevented or retarded, and if novel treatments initiating disk regeneration are to be developed. The mechanism leading to fragmentation and the proteinase involved are currently under investigation.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declared

Yeast DNA Replication 2D Gel Protocol

Two-dimensional agarose gel electrophoresis (2D gel) analysis is used extensively as a method to detect origins of replication. Here, I present a simplified method for the isolation of yeast genomic DNA for 2D gel analysis from a small number of yeast cells. This DNA isolation method is simpler and less time consuming than the traditional method that involves CsCl density gradient centrifugation. This method could be modified for 2D gel analysis in other organisms as well.