Zonal Centrifugation Research Articles

Towards improved nepovirus detection and identification in Xiphinema nematodes

Several Xiphinema nematode species are vectors of regulated nepoviruses. The aim of this study was to develop an optimized method to detect virus-carrying Xiphinema specimens. The study compared various techniques for nematode extraction from soil (Automated Zonal Centrifuging and Flegg’s Modified Cobb method), nematode cuticle disruption (slicing, bead beating, and bead beating with collagenase), RNA extraction (RNeasy Plant Mini Kit, CTAB, and KingFisher MagMAX RNA Isolation Kit), and nepovirus detection (nepovirus generic subgroup and species-specific RT-(q)PCR assays, as well as shotgun sequencing with Oxford Nanopore Sequencing technology using a MinION device). Based on this comparison, a diagnostic procedure is proposed including the best performing methods: nematode extraction using automated zonal centrifugation, physical disruption of nematode cuticle using bead beating or slicing, and RNA extraction using either the KingFisher MagMAX or the RNeasy Plant Mini Kit method. While existing generic nepovirus subgroup RT-PCR assays lacked sensitivity, species-specific RT-(q)PCR assays successfully detected ArMV, GFLV, and ToRSV in X. diversicaudatum, X. index, and X. americanum s.s., respectively. The reliability of nepovirus detection was higher with adults compared to juveniles. The minimum nematode quantity required varied depending on the specific nepovirus and the detection method used.

Size-classifiable quantification of nanoplastic by rate zonal centrifugation coupled with pyrolysis-gas chromatography-mass spectrometry

Particle size is an important indicator to evaluate the environmental risk and biotoxicity of nanoplastic (NP, particle diameter <1000 nm). The methods available to determine size classes of NP in environmental samples are few and are rare to achieve efficient separation and recycling of NP with close particle sizes. Here, we show that rate-zonal centrifugation (RZC) can quickly and efficiently collect NP of different sizes based on their sedimentation coefficients. When combined with cloud-point extraction (CPE) and pyrolysis−gas chromatography−mass spectrometry (Py-GC-MS), our method can quantify three NP particle-size classes separately (including 100 nm, 300 nm, and 600 nm) in aqueous samples with high recovery (81.4 %–89.4 %), limits of detections (LODs, 33.5–53.4 μg/L), and limits of quantifications (LOQs, 110.6–167.2 μg/L). Compared with the conventional sample pretreatment process, our method can effectively extract and determine the NP with different sizes. Our approach is highly scalable and can be effectively applied to NP in a wide range of aquatic environments. Meanwhile, our approach is highly scalable to incorporate diverse assays to study the environmental behaviours and ecological risks of NP.

Application of bio-layer interferometry for the analysis of ribosome-protein interactions.

The ribosome, a ribonucleoprotein complex, performs the function of protein translation. While ribosomal RNA catalyzes polypeptide formation, several proteins assist the ribosome throughout the translation process. Studying the biochemical and kinetic properties of these proteins interacting with the ribosome is vital for elucidating their roles. Various techniques, such as zonal centrifugation, pull-down assays, dynamic light scattering (DLS), fluorescence polarization, and surface plasmon resonance (SPR) are employed for this purpose, each presenting unique advantages and limitations. We add to the repertoire of techniques by using Bio-Layer Interferometry (BLI) to examine interactions between the ribosome and translation factors. Our findings demonstrate that BLI can detect interactions of Escherichia coli ribosomes with two proteins: E. coli initiation factor 2 (IF2) and P. falciparum translation enhancing factor (PTEF). A protein (Green Fluorescent Protein; GFP) known not to bind to E. coli ribosomes, shows no binding in the BLI assay. We show that BLI could be used to study the ribosome-protein interactions as it has key advantages like label-free procedures, ease of assay performance, and ribosome sample reuse. Our results highlight the comprehensive use of BLI in studying the ribosome-protein interactions, in addition to studying protein-protein and protein-ligand interactions.

Size Distribution Analysis of the Adeno-Associated Virus Vector by the c(s) Analysis of Band Sedimentation Analytical Ultracentrifugation with Multiwavelength Detection

Adeno-associated virus (AAV) vector is a promising platform for in vivo gene therapy. The accurate assessment of distribution state of particles contained in AAV vector samples is one of the most important and challenging matters and is necessary because the product-related impurities with the capsid structure (empty particles, intermediate particles, and aggregates) could be a possible cause of reducing the therapeutic efficacy and enhancing the unfavorable immune response. In this study, we report an effective approach for size distribution analysis with component identification. A small amount of AAV vectors were used by the analytical zone centrifugation c(s) analysis of band sedimentation analytical ultracentrifugation (BS-AUC) with multiwavelength detection. Using PBS/H218O, the concentration of each component could be determined in BS-AUC with high resolution. Compared with the sedimentation velocity AUC (SV-AUC), which generally requires 2 × 1012 vg of AAV vectors, BS-AUC could be performed with about 1/25 of the AAV vector amount at 260 nm detection and ideally with about 1/50 of the AAV vector amount at 230 nm detection (4 × 1010 vg), depending on the extinction coefficient of the AAV sample at each wavelength. According to the limit of quantification of this BS-AUC, 6.3 × 1011 cp mL−1 of empty particle (EP) and 4.4 × 1011 vg mL−1 of full particle (FP) could be quantified for 4 × 1010 vg in 15 µL of AAV8-CMV-EGFP. These results demonstrated that proposed BS-AUC approach we established here can compensate for the drawback in terms of the sample amount of SV-AUC.

Gated Ethidium- and Bleomycin-Loading in Phage T4 That Is Subsequently Purified Leak-Free

Chemotherapy-inhibiting tumor cell evolution to drug-resistance is potentially suppressed by using a drug delivery vehicle (DDV) that has gating. Gating would be used to increase tumor-selectivity of delivery of DDV packaged drug. Tumor-selectivity increase would make possible increase in tumor-delivered drug dose, which would suppress opportunities to evolve drug resistance. Currently used DDVs do not have gating but gating is a natural feature of some bacteriophages (phages). Phage T4, which has recently been found highly persistent in murine blood, is a potential gated DDV. Thus, here, we proceed towards a T4-DDV by developing (1) improved procedure for generating high concentrations and amounts of phage T4, (2) elevated temperature-driven gate-opening and ethidium- and bleomycin-loading, and (3) purification of loaded T4 by rate zonal centrifugation. We test for loading by native agarose gel electrophoresis (AGE) with fluorescence detection. We observe loading in both phage T4 and T4 (tail-free) heads. The loaded particles have an openable, closed gate. Stored, mature T4 phages and phage heads do not release ethidium during at least a month at 4 °C and 6 days at 37 and 42 °C. Tumor-specific T4 phage delivery is projected via both the EPR effect and high T4 persistence.

Rate zonal centrifugation can partially separate platelets from platelet‐derived vesicles

BackgroundCentrifugation is commonly used as a first step to enrich biomarkers from blood. Biomarkers are separated on the basis of density and/or diameter. However, the centrifugation protocol affects the yield and purity of biomarkers, for example, isolation of platelets results in co‐isolation with extracellular vesicles (EVs). ObjectiveTo assess the ability of rate zonal centrifugation (RZC) to separate platelets from co‐isolated EVs. MethodsUsing a linear Optiprep gradient, RZC was able to separate a mixture of beads with different diameters but similar density. Next, RZC was applied to samples containing both platelets and platelet‐derived EVs (n = 3). After RZC, all fractions were collected and stained with anti‐CD61‐Alexa 488 to measure the concentrations of platelets and platelet‐derived EVs by flow cytometry. ResultsWe confirm that RZC separates polystyrene beads with diameters of 140 nm, 380 nm and 1,000 nm. Next, we show that the majority of platelets occur in fractions 8‐19, whereas the majority of platelet‐derived EVs are detectable in fractions 1‐7. Furthermore, each fraction contains a different diameter range of platelets, which suggests that separation is indeed diameter based. ConclusionRZC can partially separate platelets from EVs.

Purification of rabies virus produced in Vero cells grown in serum free medium

Rabies is a viral zoonosis caused by negative-stranded RNA viruses of the Lyssavirus genus. It can affect all mammals including humans. Dogs are the main source of human rabies deaths, contributing up to 99% of all rabies transmissions to humans. Vaccination against rabies is still the sole efficient way to fight against the disease.Cell culture vaccines are recommended by World Health Organization (WHO) for pre and post exposure prophylaxis; among them Vero cell rabies vaccines which are used worldwide. In this work we studied the purification of inactivated rabies virus produced in Vero cells grown in animal component free conditions, using different methods. Cells were grown in VP-SFM medium in stirred bioreactor, then infected at an MOI of 0.05 with the LP2061 rabies virus strain. Collected harvests were purified by zonal centrifugation, and by chromatography supports, namely the Capto Core 700 and the monolithic CIM-QA column. Generated data were compared in terms of residual DNA level, host cell proteins (HCP) level and the overall recovery yield.Rabies virus purification using the monolithic column resulted in the highest antigen recovery yield, equal to 94%. Capto Core 700 showed a lower yield, about 84%; whereas the purification yield by zonal centrifugation was equal to 60%. In terms of host cell residual DNA removal, zonal centrifugation was the most efficient method; the removal yield was equal to 88.5%; elimination of host cell DNA was slightly lower when using the monolithic CIM-QA (equal to 73%). Whereas Capto Core 700 showed the lowest level (49.2%). Host cell protein removal varied between 92.6% for the monolithic column and 78.6% for the zonal centrifugation. Capto Core 700 eliminated 86.5% of HCP.

Biochemical characterization of the Helicobacter pylori bactofilin-homolog HP1542.

The human pathogen Helicobacter pylori is known for its colonization of the upper digestive system, where it escapes the harsh acidic environment by hiding in the mucus layer. One factor promoting this colonization is the helical cell shape of H. pylori. Among shape determining proteins are cytoskeletal elements like the recently discovered bactofilins. Bactofilins constitute a widespread family of polymer-forming bacterial proteins whose biology is still poorly investigated. Here we describe the first biochemical analysis of the bactofilin HP1542 of H. pylori reference strain 26695. Purified HP1542 forms sheet-like 2D crystalline assemblies, which clearly depend on a natively structured C-terminus. Polymerization properties and protein stability were investigated. Additionally, we also could demarcate HP1542 from amyloid proteins that share similarities with the bactofilin DUF domain. By using zonal centrifugation of total H. pylori cell lysates and immunfluorescence analysis we revealed peripheral membrane association of HP1542 mostly pronounced near mid-cell. Interestingly our results indicate that H. pylori bactofilin does not contribute to cell wall stability. This study might act as a starting point for biophysical studies of the H. pylori bactofilin biology as well as for the investigation of bactofilin cell physiology in this organism. Importantly, this study is the first biochemical analysis of a bactofilin in a human pathogen.

Zonal rotor centrifugation revisited: new horizons in sorting nanoparticles.

Density gradient centrifugation is an effective method for the isolation and purification of small particles. Hollow rotors capable of hosting density gradients replace the need for centrifuge tubes and therefore allow separations at large scales. So far, zonal rotors have been used for biological separations ranging from the purification of whole cells down to serum proteins. We demonstrate that the high-resolution separation method opens up exciting perspectives apart from biology, namely in sorting mixtures of synthetic nanoparticles. Loading and unloading, while the rotor is spinning, avoids perturbations during acceleration and deceleration periods, and thus makes a vital contribution to sorting accuracy. Nowadays one can synthesize nanoscale particles in a wide variety of compositions and shapes. A prominent example for this are “colloidal molecules” or, generally speaking, defined assemblies of nanoparticles that can appear in varying aggregation numbers. Fractionation of such multimodal colloids plays an essential role with regard to their organization into hierarchical organized superstructures such as films, mesocrystals and metamaterials. Zonal rotor centrifugation was found to be a scalable method of getting “colloidal molecules” properly sorted. It allows access to pure fractions of particle monomers, dimers, and trimers, just as well as to fractions that are essentially rich in particle tetramers. Separations were evaluated by differential centrifugal sedimentation, which provides high-resolution size distributions of the collected nanoparticle fractions. The performance achieved in relation to resolution, zone widths, sorting efficiencies and recovery rates clearly demonstrate that zonal rotor centrifugation provides an excellent solution to the fractionation of nanoparticle mixtures.

PM 7/28 (2) Synchytrium endobioticum

Specific scopeThis Standard describes a diagnostic protocol for Synchytrium endobioticum.This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.Specific approval and amendmentApproved in 2003‐09. Revision approved in 2017‐06.

Purification of avian influenza virus (H9N2) from allantoic fluidby size-exclusion chromatography

Avian influenza viruses represent a rising threat of pandemic influenza. Allantoic fluid including influenza virus H9N2 was run on downstream processing to purify the virus. To do this, allantoic fluid was clarified and concentrated. It was applied to size exclusion chromatography. Different parameters such as ovalbumin, protein content, and hemagglutinin activity were determined to confirm the purity. The results may suggest that this method is quite efficient for achieving a purified H9N2 influenza virus and further reveal that size-exclusion chromatography is a practical method for purifying avian influenza viruses. In conclusion, this procedure can be used in vaccine production and antiserum preparation as an alternative to traditional methods and also can be considered in the purifying of other viruses when there is no facility of ultracentrifuge or zonal centrifuge.

Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280-320-kDa oligomeric structure consisting of ∼6-7 subunits.

Efficient chromatographic reduction of ovalbumin for egg-based influenza virus purification

Vaccination is the most effective prevention strategy to avoid influenza infection and for protection of large populations. The vast majority of influenza vaccines are still produced with allantoic fluid from fertilized chicken eggs. The presence of ovalbumin, which can constitute over 60% of the total protein content in allantoic fluid, can result in severe allergies. Consequently, efficient reduction of ovalbumin is critical during egg based vaccine manufacturing. Here we present Capto Core 700, a novel core bead chromatographic flow through mode resin for removal of ovalbumin and compare it to sucrose zonal gradient ultracentrifugation, which is the industry standard for egg-based vaccine production. The results demonstrate that core bead chromatography is fully comparable to zonal centrifugation in removing ovalbumin to meet regulatory requirements. Furthermore, the scalability and the shorter process times of this method have the potential to significantly improve the productivity and economy for industrial production compared to zonal centrifugation.

Extraction techniques of plant-parasitic nematodes: a practical comparison

The efficiency of various nematode extraction methods differs with the time period, equipments used, soil type, moisture content of the soil, different nematode genera with different life stages, and basically upon the performance of the individual (s) who is carrying out the extraction. The present study was conducted in the Institute for Agriculture and Fisheries Research (ILVO), Gent University, Belgium to study the efficiency of different nematode extraction methods and to extract nematodes from soil, root and soil with root samples. From the present study, it was observed that the Seinhorst method is better for extracting both lighter and heavier cysts nematodes even though it needs more expensive equipments and much expert to run as compared to stirring method. Automotive Zonal Centrifugation (AZC) method was found more efficient and rapid technique than Cobb's method in extracting mobile and non mobile nematodes from soil. Baermann funnel is suitable for the extraction of the mobile nematodes and found not to be suitable to explore for quantitative research.

Hepatitis C Virus p7 is Critical for Capsid Assembly and Envelopment

Hepatitis C virus (HCV) p7 is a membrane-associated ion channel protein crucial for virus production. To analyze how p7 contributes to this process, we dissected HCV morphogenesis into sub-steps including recruitment of HCV core to lipid droplets (LD), virus capsid assembly, unloading of core protein from LDs and subsequent membrane envelopment of capsids. Interestingly, we observed accumulation of slowly sedimenting capsid-like structures lacking the viral envelope in cells transfected with HCV p7 mutant genomes which possess a defect in virion production. Concomitantly, core protein was enriched at the surface of LDs. This indicates a defect in core/capsid unloading from LDs and subsequent membrane envelopment rather than defective trafficking of core to this cellular organelle. Protease and ribonuclease digestion protection assays, rate zonal centrifugation and native, two dimensional gel electrophoresis revealed increased amounts of high-order, non-enveloped core protein complexes unable to protect viral RNA in cells transfected with p7 mutant genomes. These results suggest accumulation of capsid assembly intermediates that had not yet completely incorporated viral RNA in the absence of functional p7. Thus, functional p7 is necessary for the final steps of capsid assembly as well as for capsid envelopment. These results support a model where capsid assembly is linked with membrane envelopment of nascent RNA-containing core protein multimers, a process coordinated by p7. In summary, we provide novel insights into the sequence of HCV assembly events and essential functions of p7.

An ultra scale‐down characterization of low shear stress primary recovery stages to enhance selectivity of fusion protein recovery from its molecular variants

Fusion proteins offer the prospect of new therapeutic products with multiple functions. The primary recovery is investigated of a fusion protein consisting of modified E2 protein from hepatitis C virus fused to human IgG1 Fc and expressed in a Chinese hamster ovary (CHO) cell line. Fusion protein products inevitably pose increased challenge in preparation and purification. Of particular concerns are: (i) the impact of shear stress on product integrity and (ii) the presence of product-related contaminants which could prove challenging to remove during the high resolution purification steps. This paper addresses the use of microwell-based ultra scale-down (USD) methods to develop a bioprocess strategy focused on the integration of cell culture and cell removal operations and where the focus is on the use of operations which impart low shear stress levels even when applied at eventual manufacturing scale. An USD shear device was used to demonstrate that cells exposed to high process stresses such as those that occur in the feed zone of a continuous non-hermetic centrifuge resulted in the reduction of the fusion protein and also the release of glycosylated intracellular variants. In addition, extended cell culture resulted in release of such variants. USD mimics of low shear stress, hydrohermetic feed zone centrifugation and of depth filtration were used to demonstrate little to no release during recovery of these variants with both results verified at pilot scale. Furthermore, the USD studies were used to predict removal of contaminants such as lipids, nucleic acids, and cell debris with, for example, depth filtration delivering greater removal than for centrifugation but a small (~10%) decrease in yield of the fusion protein. These USD observations of product recovery and carryover of contaminants were also confirmed at pilot scale as was also the capacity or throughput achievable for continuous centrifugation or for depth filtration. The advantages are discussed of operating a lower yield cell culture and a low shear stress recovery process in return for a considerably less challenging purification demand.

The added value of proficiency tests: choosing the proper method for extracting Meloidogyne second-stage juveniles from soil

To gain insight into the different extraction processes used in laboratories in various European countries and the effect these methods might have on the number of Meloidogyne second-stage juveniles (J2) extracted from soil samples, a ring test with 2 elements was conducted. The first element used the Baermann funnel as a method performance test. The second element was a proficiency test, in which the laboratory’s standard technique was compared with the Baermann method. Chilled samples of 100 cm3 (10 per extraction method) were sent to participants in insulated containers. In the nematode counts distinction was made between Meloidogyne J2, other plant-parasitic nematodes and/or saprophytes. A total number of 18 participants took part in the method performance test and 13 institutes took part in the proficiency test using 11 different extraction techniques. The initial population density in the soil was 2025 J2 (100 cm3 soil)−1, determined by Oostenbrink elutriator and 4 weeks incubation. In the method performance test the institutes found Meloidogyne J2 numbers varied between 0 and 705 J2 (100 cm3)−1. Using the Baermann extraction method the median number per institute ranged from 0 to 377 J2 (100 cm3)−1. In the proficiency test comparison amongst the different methods showed an increase of more than 100-fold, with the highest median of 3733 J2 (100 cm3)−1 and the lowest of 34 J2 (100 cm3)−1; the difference was caused mainly by the incubation effect. For the other plant-parasitic nematodes and the saprophytes, the differences between the most and least efficient extraction methods were much smaller. The variance of the Meloidogyne counts was the highest for the standard Baermann extraction technique; other similar techniques also had high variances. The automated zonal centrifuge had the lowest variance. The Baermann method is not advisable for survey purposes.

Analysis of Alternative Purification of Beta-Propiolactone Inactivated, Tangential Flow Filtration Concentrated Vero Cell Derived Rabies Vaccine

Rabies virus is a single stranded negative sense RNA virus that belongs to the genus lyssavirus of the rhabdoviridae family which causes an acute disease of vertebrate animals. In the production of vaccines, purification plays a crucial role in the vaccine efficacy. Viral inactivation and removal steps are critical parts in mammalian cell culture derived biotechnology products. Regulatory agencies are concerned with the presence of endogenous or adventitious agent in the cell lines or raw materials employed to manufacture pharmaceuticals protein from cell culture. Due to this, numerous techniques are affianced in the downstream processing of viral vaccines manufacturing especially chromatography. Advances in vaccine manufacturing have created an increasing demand for large volumes of highly purified viral antigens. Cellufine sulfate Chromatography which has been used for other viral vaccines of both animal and human, was used in this study for optimal recovery of valuable rabies viral protein and optimal removal of impurities such as residual cellular DNA and host cellular BSA successfully as a replacement of the existing zonal centrifuge method.

Improved real-time PCR assay for detection of the quarantine potato pathogen, Synchytrium endobioticum, in zonal centrifuge extracts from soil and in plants

Real-time PCR was used for quantitative detection of the potato pathogen, Synchytrium endobioticum, in different substrates: zonal centrifuge extracts, warts and different plant parts of potato. Specific primers and a TaqMan probe, designed from the internal transcribed spacer region of the multi-copy rDNA gene were tested in extracts from artificially and naturally infested soil. Co-amplification of target DNA along with an internal competitor DNA fragment made the diagnostic assay more reliable by guarding against false negative results. A calibrations curve was created by spiking zonal centrifuge fractions of clean soil samples with a dilution series of winter spores. The Taqman assay was also performed on infected potato plant material (stolons) along with the detection of the cytochrome oxidase gene as a potato endogenous control. Sensitivity of the TaqMan assay was improved at least 100-fold and proved to be reliable for accurate diagnosis of the disease.

Proteomic profiling of human plasma exosomes identifies PPARγ as an exosome-associated protein

Exosomes are nanovesicles that are released from cells as a mechanism of cell-free intercellular communication. Only a limited number of proteins have been identified from the plasma exosome proteome. Here, we developed a multi-step fractionation scheme incorporating gel exclusion chromatography, rate zonal centrifugation through continuous sucrose gradients, and high-speed centrifugation to purify exosomes from human plasma. Exosome-associated proteins were separated by SDS–PAGE and 66 proteins were identified by LC–MS/MS, which included both cellular and extracellular proteins. Furthermore, we identified and characterized peroxisome proliferator-activated receptor-γ (PPARγ), a nuclear receptor that regulates adipocyte differentiation and proliferation, as well as immune and inflammatory cell functions, as a novel component of plasma-derived exosomes. Given the important role of exosomes as intercellular messengers, the discovery of PPARγ as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors.