Relation between cell disruption conditions, cell debris particle size, and inclusion body release

The efficiency of physical separation of inclusion bodies from cell debris is related to cell debris size and inclusion body release and both factors should be taken into account when designing a process. In this work, cell disruption by enzymatic treatment with lysozyme and cellulase, by homogenization, and by homogenization with ammonia pretreatment is discussed. These disruption methods are compared on the basis of inclusion body release, operating costs, and cell debris particle size. The latter was measured with cumulative sedimentation analysis in combination with membrane-associated protein quantification by SDS-PAGE and a spectrophotometric pepticloglycan quantification method. Comparison of the results obtained with these two cell debris quantification methods shows that enzymatic treatment yields cell debris particles with varying chemical composition, while this is not the case with the other disruption methods that were investigated. Furthermore, the experiments show that ammonia pretreatment with homogenization increases inclusion body release compared to homogenization without pretreatment and that this pretreatment may be used to control the cell debris size to some extent. The enzymatic disruption process gives a higher product release than homogenization with or without ammonia pretreatment at lower operating costs, but it also yields a much smaller cell debris size than the other disruption process. This is unfavorable for centrifugal inclusion body purification in this case, where cell debris is the component going to the sediment and the inclusion body is the floating component. Nevertheless, calculations show that centrifugal separation of inclusion bodies from the enzymatically treated cells gives a high inclusion body yield and purity. (C) 2004 Wiley Periodicals, Inc.

Centrifugal processing of cell debris and inclusion bodies from recombinant Escherichia coli

The settling characteristics of cell debris and inclusion bodies prior to, and following, fractionation in a disc-stack centrifuge were measured using Cumulative Sedimentation Analysis (CSA) and Centrifugal Disc photosedimentation (CDS). The impact of centrifuge feedrate and repeated homogenisation on both cell debris and inclusion body collection efficiency was investigated. Increasing the normalised centrifuge feedrate (Q/Sigma) from 1.32 x 10(-9) m s(-1) to 3.97 x 10(-9) m s(-1) leads to a 36% increase in inclusion body paste purity. Purity may also be improved by repeated homogenisation. Increasing the number of homogeniser passes results in smaller cell debris size whilst leaves inclusion body size unaltered. At a normalised centrifuge feedrate of 2.65 x 10(-9) m s(-1), increasing the number of homogeniser passes from two (2) to ten (10) improved overall inclusion body paste purity by 58%. Grade-efficiency curves for both the cell debris and inclusion bodies have also been generated in this study. The data are described using an equation developed by Mannweiler (1989) with parameters of k = 0.15-0.26 and n = 2.5-2.6 for inclusion bodies, and k = 0.12-0.14 and n = 2.0-2.2 for cell debris. This is the first accurate experimentally-determined grade efficiency curve for cell debris. Previous studies have simply estimated debris grade efficiency curves using an approximate debris size distribution and grade efficiency curves determined with 'ideal particles' (e.g. spherical PVA particles). The findings of this study may be used to simulate and optimise the centrifugal fractionation of inclusion bodies from cell debris.

Cumulative sedimentation analysis of Escherichia coli debris size

A new method to measure Escherichia coil cell debris size after homogenization is presented. It is based on cumulative sedimentation analysis under centrifugal force, coupled with Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis of sedimented proteins. The effects that fermentation and homogenization conditions have on the resulting debris distributions were investigated using this method. Median debris size decreased significantly from approximately 0.5 mu m to 0.3 mu m as the number of homogenization passes increased from 2 to 10. Under identical homogenization conditions, uninduced host cells in stationary phase had a larger debris size than exponential cells after 5 homogenizer passes. This difference was not evident after 2 or in passes, possibly because of confounding intact cells and the existence of a minimum debris size for the conditions investigated. Recombinant cells containing protein inclusion bodies had the smallest debris size following homogenization. The method was also used to measure the size distribution of inclusion bodies. This result compared extremely well with an independent determination using centrifugal disc photosedimentation (CDS), thus validating the method. This is the first method that provides accurate size distributions of E. coli debris without the need for sample pretreatment, theoretical approximations (e.g. extinction coefficients), or the separation of debris and inclusion bodies prior to analysis. (C) 1997 John Wiley & Sons, Inc.

A mathematical model for Escherichia coli debris size reduction during high pressure homogenisation based on grinding theory

Experimental data for E. coli debris size reduction during high-pressure homogenisation at 55 MPa are presented. A mathematical model based on grinding theory is developed to describe the data. The model is based on first-order breakage and compensation conditions. It does not require any assumption of a specified distribution for debris size and can be used given information on the initial size distribution of whole cells and the disruption efficiency during homogenisation. The number of homogeniser passes is incorporated into the model and used to describe the size reduction of non-induced stationary and induced E. coil cells during homogenisation. Regressing the results to the model equations gave an excellent fit to experimental data ( > 98.7% of variance explained for both fermentations), confirming the model's potential for predicting size reduction during high-pressure homogenisation. This study provides a means to optimise both homogenisation and disc-stack centrifugation conditions for recombinant product recovery. (C) 1997 Elsevier Science Ltd.

Pilot-scale extraction of PHB from recombinant E-coli by homogenization and centrifugation

A new method of poly-beta-hydroxybutyrate (PHB) extraction from recombinant E. coli is proposed, using homogenization and centrifugation coupled with sodium hypochlorite treatment. The size of PHB granules and cell debris in homogenates was characterised as a function of the number of homogenization passes. Simulation was used to develop the PHB and cell debris fractionation system, enabling numerical examination of the effects of repeated homogenization and centrifuge-feedrate variation. The simulation provided a good prediction of experimental performance. Sodium hypochlorite treatment was necessary to optimise PHB fractionation. A PHB recovery of 80% at a purity of 96.5% was obtained with the final optimised process. Protein and DNA contained in the resultant product were negligible. The developed process holds promise for significantly reducing the recovery cost associated with PHB manufacture.

Physicochemical characteristics of LR3-IGF1 protein inclusion bodies: Electrophoretic mobility studies

A knowledge of the physicochemical properties of inclusion bodies is important for the rational design of potential recovery processes such as flotation and precipitation. In this study, measurement of the size and electrophoretic mobility of protein inclusion bodies and cell debris was undertaken. SDS-PAGE analysis of protein inclusion bodies subjected to different cleaning regimes suggested that electrophoretic mobility provides a qualitative measure of protein inclusion body purity. Electrophoretic mobility as a function of electrolyte type and ionic strength was investigated. The presence of divalent ions produced a stronger effect on electrophoretic mobility compared with monovalent ions. The isoelectric point of cell debris was significantly lower than that for the inclusion bodies. Hence, the contaminating cell debris may be separated from inclusion bodies using flotation by exploiting this difference in isoelectric points. Separation by this method is simple, convenient, and a possible alternative to the conventional route of centrifugation.

Production of recombinant protein hLIF in static and dynamic systems of animal cell culture

Thesis for the Degree of Master of Science in Biotechnology Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Rapid detection by transmission electron microscopy of mycoplasma contamination in sera and cell cultures

Transmission electon microscopy has been employed for the rapid detection of mycoplasma in sera and cell cultures. High speed centrifugation of sera or low speed centrifugation of cell debris, followed by negative staining of the resuspended pellet, detected mycoplasma contamination more frequently than a culture method followed by direct fluorescence (DAPI), which was used as a control procedure. The appearance of the mycoplasma cell border and content gives some information about particle viability.

The Endothelial Sample Size Analysis in Corneal Specular Microscopy Clinical Examinations

Purpose: To evaluate endothelial cell sample size and statistical error in corneal specular microscopy (CSM) examinations. Methods: One hundred twenty examinations were conducted with 4 types of corneal specular microscopes: 30 with each BioOptics, CSO, Konan, and Topcon corneal specular microscopes. All endothelial image data were analyzed by respective instrument software and also by the Cells Analyzer software with a method developed in our lab(US Patent). A reliability degree (RD) of 95% and a relative error (RE) of 0.05 were used as cut-off values to analyze images of the counted endothelial cells called samples. The sample size mean was the number of cells evaluated on the images obtained with each device. Only examinations with RE<0.05 were considered statistically correct and suitable for comparisons with future examinations. The Cells Analyzer software was used to calculate the RE and customized sample size for all examinations. Results: Bio-Optics: sample size, 97 +/- 22 cells; RE, 6.52 +/- 0.86; only 10% of the examinations had sufficient endothelial cell quantity (RE<0.05); customized sample size, 162 +/- 34 cells. CSO: sample size, 110 +/- 20 cells; RE, 5.98 +/- 0.98; only 16.6% of the examinations had sufficient endothelial cell quantity (RE<0.05); customized sample size, 157 +/- 45 cells. Konan: sample size, 80 +/- 27 cells; RE, 10.6 +/- 3.67; none of the examinations had sufficient endothelial cell quantity (RE>0.05); customized sample size, 336 +/- 131 cells. Topcon: sample size, 87 +/- 17 cells; RE, 10.1 +/- 2.52; none of the examinations had sufficient endothelial cell quantity (RE>0.05); customized sample size, 382 +/- 159 cells. Conclusions: A very high number of CSM examinations had sample errors based on Cells Analyzer software. The endothelial sample size (examinations) needs to include more cells to be reliable and reproducible. The Cells Analyzer tutorial routine will be useful for CSM examination reliability and reproducibility.

Analysis of tape tether survival in LEO against orbital debris

The low earth orbit (LEO) environment contains a large number of artificial debris, of which a significant portion is due to dead satellites and fragments of satellites resulted from explosions and in-orbit collisions. Deorbiting defunct satellites at the end of their life can be achieved by a successful operation of an Electrodynamic Tether (EDT) system. The effectiveness of an EDT greatly depends on the survivability of the tether, which can become debris itself if cut by debris particles; a tether can be completely cut by debris having some minimal diameter. The objective of this paper is to develop an accurate model using power laws for debris-size ranges, in both ORDEM2000 and MASTER2009 debris flux models, to calculate tape tether survivability. The analytical model, which depends on tape dimensions (width, thickness) and orbital parameters (inclinations, altitudes) is then verified with fully numerical results to compare for different orbit inclinations, altitudes and tape width for both ORDEM2000 and MASTER2009 flux data.

Bare-tape scaling laws for de-orbit missions in a space debris environment

El entorno espacial actual hay un gran numero de micro-meteoritos y basura espacial generada por el hombre, lo cual plantea un riesgo para la seguridad de las operaciones en el espacio. La situación se agrava continuamente a causa de las colisiones de basura espacial en órbita, y los nuevos lanzamientos de satélites. Una parte significativa de esta basura son satélites muertos, y fragmentos de satélites resultantes de explosiones y colisiones de objetos en órbita. La mitigación de este problema se ha convertido en un tema de preocupación prioritario para todas las instituciones que participan en operaciones espaciales. Entre las soluciones existentes, las amarras electrodinámicas (EDT) proporcionan un eficiente dispositivo para el rápido de-orbitado de los satélites en órbita terrestre baja (LEO), al final de su vida útil. El campo de investigación de las amarras electrodinámicas (EDT) ha sido muy fructífero desde los años 70. Gracias a estudios teóricos, y a misiones para la demostración del funcionamiento de las amarras en órbita, esta tecnología se ha desarrollado muy rápidamente en las últimas décadas. Durante este período de investigación, se han identificado y superado múltiples problemas técnicos de diversa índole. Gran parte del funcionamiento básico del sistema EDT depende de su capacidad de supervivencia ante los micro-meteoritos y la basura espacial. Una amarra puede ser cortada completamente por una partícula cuando ésta tiene un diámetro mínimo. En caso de corte debido al impacto de partículas, una amarra en sí misma, podría ser un riesgo para otros satélites en funcionamiento. Por desgracia, tras varias demostraciones en órbita, no se ha podido concluir que este problema sea importante para el funcionamiento del sistema. En esta tesis, se presenta un análisis teórico de la capacidad de supervivencia de las amarras en el espacio. Este estudio demuestra las ventajas de las amarras de sección rectangular (cinta), en cuanto a la probabilidad de supervivencia durante la misión, frente a las amarras convencionales (cables de sección circular). Debido a su particular geometría (longitud mucho mayor que la sección transversal), una amarra puede tener un riesgo relativamente alto de ser cortado por un único impacto con una partícula de pequeñas dimensiones. Un cálculo analítico de la tasa de impactos fatales para una amarra cilindrica y de tipo cinta de igual longitud y masa, considerando el flujo de partículas de basura espacial del modelo ORDEM2000 de la NASA, muestra mayor probabilidad de supervivencia para las cintas. Dicho análisis ha sido comparado con un cálculo numérico empleando los modelos de flujo el ORDEM2000 y el MASTER2005 de ESA. Además se muestra que, para igual tiempo en órbita, una cinta tiene una probabilidad de supervivencia un orden y medio de magnitud mayor que una amarra cilindrica con igual masa y longitud. Por otra parte, de-orbitar una cinta desde una cierta altitud, es mucho más rápido, debido a su mayor perímetro que le permite capturar más corriente. Este es un factor adicional que incrementa la probabilidad de supervivencia de la cinta, al estar menos tiempo expuesta a los posibles impactos de basura espacial. Por este motivo, se puede afirmar finalmente y en sentido práctico, que la capacidad de supervivencia de la cinta es bastante alta, en comparación con la de la amarra cilindrica. El segundo objetivo de este trabajo, consiste en la elaboración de un modelo analítico, mejorando la aproximación del flujo de ORDEM2000 y MASTER2009, que permite calcular con precisión, la tasa de impacto fatal al año para una cinta en un rango de altitudes e inclinaciones, en lugar de unas condiciones particulares. Se obtiene el numero de corte por un cierto tiempo en función de la geometría de la cinta y propiedades de la órbita. Para las mismas condiciones, el modelo analítico, se compara con los resultados obtenidos del análisis numérico. Este modelo escalable ha sido esencial para la optimización del diseño de la amarra para las misiones de de-orbitado de los satélites, variando la masa del satélite y la altitud inicial de la órbita. El modelo de supervivencia se ha utilizado para construir una función objetivo con el fin de optimizar el diseño de amarras. La función objectivo es el producto del cociente entre la masa de la amarra y la del satélite y el numero de corte por un cierto tiempo. Combinando el modelo de supervivencia con una ecuación dinámica de la amarra donde aparece la fuerza de Lorentz, se elimina el tiempo y se escribe la función objetivo como función de la geometría de la cinta y las propietades de la órbita. Este modelo de optimización, condujo al desarrollo de un software, que esta en proceso de registro por parte de la UPM. La etapa final de este estudio, consiste en la estimación del número de impactos fatales, en una cinta, utilizando por primera vez una ecuación de límite balístico experimental. Esta ecuación ha sido desarollada para cintas, y permite representar los efectos tanto de la velocidad de impacto como el ángulo de impacto. Los resultados obtenidos demuestran que la cinta es altamente resistente a los impactos de basura espacial, y para una cinta con una sección transversal definida, el número de impactos críticos debidos a partículas no rastreables es significativamente menor. ABSTRACT The current space environment, consisting of man-made debris and tiny meteoroids, poses a risk to safe operations in space, and the situation is continuously deteriorating due to in-orbit debris collisions and to new satellite launches. Among these debris a significant portion is due to dead satellites and fragments of satellites resulted from explosions and in-orbit collisions. Mitigation of space debris has become an issue of first concern for all the institutions involved in space operations. Bare electrodynamic tethers (EDT) can provide an efficient mechanism for rapid de-orbiting of defunct satellites from low Earth orbit (LEO) at end of life. The research on EDT has been a fruitful field since the 70’s. Thanks to both theoretical studies and in orbit demonstration missions, this technology has been developed very fast in the following decades. During this period, several technical issues were identified and overcome. The core functionality of EDT system greatly depends on their survivability to the micrometeoroids and orbital debris, and a tether can become itself a kind of debris for other operating satellites in case of cutoff due to particle impact; however, this very issue is still inconclusive and conflicting after having a number of space demonstrations. A tether can be completely cut by debris having some minimal diameter. This thesis presents a theoretical analysis of the survivability of tethers in space. The study demonstrates the advantages of tape tethers over conventional round wires particularly on the survivability during the mission. Because of its particular geometry (length very much larger than cross-sectional dimensions), a tether may have a relatively high risk of being severed by the single impact of small debris. As a first approach to the problem, survival probability has been compared for a round and a tape tether of equal mass and length. The rates of fatal impact of orbital debris on round and tape tether, evaluated with an analytical approximation to debris flux modeled by NASA’s ORDEM2000, shows much higher survival probability for tapes. A comparative numerical analysis using debris flux model ORDEM2000 and ESA’s MASTER2005 shows good agreement with the analytical result. It also shows that, for a given time in orbit, a tape has a probability of survival of about one and a half orders of magnitude higher than a round tether of equal mass and length. Because de-orbiting from a given altitude is much faster for the tape due to its larger perimeter, its probability of survival in a practical sense is quite high. As the next step, an analytical model derived in this work allows to calculate accurately the fatal impact rate per year for a tape tether. The model uses power laws for debris-size ranges, in both ORDEM2000 and MASTER2009 debris flux models, to calculate tape tether survivability at different LEO altitudes. The analytical model, which depends on tape dimensions (width, thickness) and orbital parameters (inclinations, altitudes) is then compared with fully numerical results for different orbit inclinations, altitudes and tape width for both ORDEM2000 and MASTER2009 flux data. This scalable model not only estimates the fatal impact count but has proved essential in optimizing tether design for satellite de-orbit missions varying satellite mass and initial orbital altitude and inclination. Within the frame of this dissertation, a simple analysis has been finally presented, showing the scalable property of tape tether, thanks to the survivability model developed, that allows analyze and compare de-orbit performance for a large range of satellite mass and orbit properties. The work explicitly shows the product of tether-to-satellite mass-ratio and fatal impact count as a function of tether geometry and orbital parameters. Combining the tether dynamic equation involving Lorentz drag with space debris impact survivability model, eliminates time from the expression. Hence the product, is independent of tether de-orbit history and just depends on mission constraints and tether length, width and thickness. This optimization model finally led to the development of a friendly software tool named BETsMA, currently in process of registration by UPM. For the final step, an estimation of fatal impact rate on a tape tether has been done, using for the first time an experimental ballistic limit equation that was derived for tapes and accounts for the effects of both the impact velocity and impact angle. It is shown that tape tethers are highly resistant to space debris impacts and considering a tape tether with a defined cross section, the number of critical events due to impact with non-trackable debris is always significantly low.

Phloem sap proteins from Cucurbita maxima and Ricinus communis have the capacity to traffic cell to cell through plasmodesmata

In angiosperms, the functional enucleate sieve tube system of the phloem appears to be maintained by the surrounding companion cells. In this study, we tested the hypothesis that polypeptides present within the phloem sap traffic cell to cell from the companion cells, where they are synthesized, into the sieve tube via plasmodesmata. Coinjection of fluorescently labeled dextrans along with size-fractionated Cucurbita maxima phloem proteins, ranging in size from 10 to 200 kDa, as well as injection of individual fluorescently labeled phloem proteins, provided unambiguous evidence that these proteins have the capacity to interact with mesophyll plasmodesmata in cucurbit cotyledons to induce an increase in size exclusion limit and traffic cell to cell. Plasmodesmal size exclusion limit increased to greater than 20 kDa, but less than 40 kDa, irrespective of the size of the injected protein, indicating that partial protein unfolding may be a requirement for transport. A threshold concentration in the 20–100 nM range was required for cell-to-cell transport indicating that phloem proteins have a high affinity for the mesophyll plasmodesmal binding site(s). Parallel experiments with glutaredoxin and cystatin, phloem sap proteins from Ricinus communis, established that these proteins can also traffic through cucurbit mesophyll plasmodesmata. These results are discussed in terms of the requirements for regulated protein trafficking between companion cells and the sieve tube system. As the threshold value for plasmodesmal transport of phloem sap proteins falls within the same range as many plant hormones, the possibility is discussed that some of these proteins may act as long-distance signaling molecules.

Factors determining the size frequency distribution of β-amyloid (Aβ) deposits in Alzheimer's disease

The size frequency distributions of discrete β-amyloid (Aβ) deposits were studied in single sections of the temporal lobe from patients with Alzheimer's disease. The size distributions were unimodal and positively skewed. In 18/25 (72%) tissues examined, a log normal distribution was a good fit to the data. This suggests that the abundances of deposit sizes are distributed randomly on a log scale about a mean value. Three hypotheses were proposed to account for the data: (1) sectioning in a single plane, (2) growth and disappearance of Aβ deposits, and (3) the origin of Aβ deposits from clusters of neuronal cell bodies. Size distributions obtained by serial reconstruction through the tissue were similar to those observed in single sections, which would not support the first hypothesis. The log normal distribution of Aβ deposit size suggests a model in which the rate of growth of a deposit is proportional to its volume. However, mean deposit size and the ratio of large to small deposits were not positively correlated with patient age or disease duration. The frequency distribution of Aβ deposits which were closely associated with 0, 1, 2, 3, or more neuronal cell bodies deviated significantly from a log normal distribution, which would not support the neuronal origin hypothesis. On the basis of the present data, growth and resolution of Aβ deposits would appear to be the most likely explanation for the log normal size distributions.

Loxosceles gaucho Venom-Induced Acute Kidney Injury - In Vivo and In Vitro Studies

Background: Accidents caused by Loxosceles spider may cause severe systemic reactions, including acute kidney injury (AKI). There are few experimental studies assessing Loxosceles venom effects on kidney function in vivo. Methodology/Principal Findings: In order to test Loxosceles gaucho venom (LV) nephrotoxicity and to assess some of the possible mechanisms of renal injury, rats were studied up to 60 minutes after LV 0.24 mg/kg or saline IV injection (control). LV caused a sharp and significant drop in glomerular filtration rate, renal blood flow and urinary output and increased renal vascular resistance, without changing blood pressure. Venom infusion increased significantly serum creatine kinase and aspartate aminotransferase. In the LV group renal histology analysis found acute epithelial tubular cells degenerative changes, presence of cell debris and detached epithelial cells in tubular lumen without glomerular or vascular changes. Immunohistochemistry disclosed renal deposition of myoglobin and hemoglobin. LV did not cause injury to a suspension of fresh proximal tubules isolated from rats. Conclusions/Significance: Loxosceles gaucho venom injection caused early AKI, which occurred without blood pressure variation. Changes in glomerular function occurred likely due to renal vasoconstriction and rhabdomyolysis. Direct nephrotoxicity could not be demonstrated in vitro. The development of a consistent model of Loxosceles venom-induced AKI and a better understanding of the mechanisms involved in the renal injury may allow more efficient ways to prevent or attenuate the systemic injury after Loxosceles bite.