Cell cycle analysis of primary sponge cell cultures

Proliferation of sponge cells is generally measured via cell counts or viability assays. However, more insight into the proliferative state of a sponge cell population can be obtained from the distribution of the cells over the different phases of the cell cycle. Cell cycle distribution of sponge cells was measured via flow cytometry after staining the DNA with propidium iodide. The five sponges studied in this paper all showed a large fraction of cells in G1/G0 compared to G2/M and S, indicating that cells were not actively dividing. In addition, some sponges also showed a large apoptotic fraction, indicating cell death. Additional apoptosis measurements, based on caspase activity, showed that harvesting and dissociation of sponge tissue to initiate a primary cell culture was directly correlated with an increase in apoptotic cells. This indicates that for the development of cell cultures, more attention should be given to harvesting, dissociation, and quality of starting material. Finally, cultivation conditions used were ineffective for proliferation, since after 2 d of cultivating Haliclona oculata cells, most cells shifted towards the apoptotic fraction, indicating that cells were dying. For development of in vitro sponge cell cultures, flow cytometric cell cycle analysis is a useful method to assess the proliferative state of a sponge cell culture and can be used to validate improvements in harvesting and dissociation, to select sponges with good proliferative capacities and to study the influence of culture conditions for stimulating cell growth

Growth and death of animal cells in bioreactors

Animal-cell cultivation is becoming increasingly important especially for the area of hunian- health products. The products range from vaccines to therapeutic proteins and the cells themselves. The therapeutic application of proteins puts high demands upon their quality with respect to purity and structure. For example, a correct folding and glycosylation is of importance for the activity, the in vivo clearance rate and the possible immunogenicity of the protein, and can often only be obtained by production in animal cells. An important class of proteins produced by animal cells is formed by monoclonal antibodies. Monoclonal antibodies are produced by hybridoma cells and have the capacity to bind very specifically to a particular molecular structure (epitope), a quality that makes them suitable for application in in vivo and in vitro diagnostics, in separation technology and for the in vivo targeting of drugs.The occurrence of substantial cell death and the presence of cell debris is a major problem in animal-cell cultivation. It interferes with the attainment of high volumetric productivities and with a proper functioning of the process. In addition, it may affect the quality of the product and cause problems in down-stream processing. Cell death may follow two different pathways, being apoptosis and necrosis, which have very distinct physiological and morphological features. Necrosis is a passive process generally caused by sudden high levels of environmental stress, whereas apoptosis is an active, genetically controlled process induced by mild stress conditions or specific signals from the environment.After the introduction in Chapter 1, the application of a general framework for the construction of segregated models is discussed in Chapter 2 with respect to the behaviour of animal-cell populations. For the construction of segregated models, the physiological state of an animal cell must be specified, which is discussed in this chapter with special attention for the experimental verification of the models. Finally, a number of age-structured, segregated models, which are of importance for animal-cell cultivation are reviewed in this chapter.The required amounts of animal-cell products are expected to be in the order of kilograms or even tonnes on a yearly basis. In order to produce these amounts, scale-up is necessary, which is most easily done in conventional reactor systems like the stirred-tank, bubble-column, and air-lift reactor. A main problem in the scale-up of these reactors is the supply of sufficient oxygen to the culture, which often requires sparging. Hydrodynamic forces associated with sparging cause cell death. In Chapters 3, 4, and 5 the specific death rate of hybridoma cells in bubble-column and air-lift reactors is studied with the hypothetical-killing-volume theory as a central theme. The hypothetical killing volume is a hypothetical volume associated with an air bubble during its lifetime in the reactor in which all cells are killed. The first-order death-rate constant in bubble- column and air-lift reactors can then be derived to be the product of this hypothetical killing volume and the number of bubbles introduced into the reactor per unit time and per unit reactor volume. The specific death rate of the hybridoma cells in the bubble-column and air-lift reactors is shown to be proportional to the gas flow rate and the reciprocal reactor height. Furthermore, in bubble columns the specific death rate is shown to be proportional to the square of the reciprocal reactor diameter. These results are in accordance with the hypothetical-killing-volume theory. The main cause of cell death is found to be bubble breakup at the surface, although detrimental effects at the sparger cannot be excluded. In Chapter 6 the specific death rate of Vero cells immobilized on microcarriers is shown to be proportional to the gas flow rate. Since the height of the reactor is not varied, it cannot be excluded that in this case also the rising of the bubbles or the associated liquid flow cause cell damage.A common method to reduce the detrimental effects of air bubbles is the use of protective additives. In this thesis it is shown that the addition of two such protectants, Pluronic F68 (Chapter 3) and serum (Chapter 4), respectively, reduces the amount of cell death as a consequence of sparging. Furthermore, as demonstrated in Chapter 4, the protective effect of serum has a fast-acting, physical, and a slow-acting, physiological component. In Chapter 5 the effect of the specific growth rate on the specific death rate of cells due to sparging is studied in air-lift loop reactors. Cells with varying specific growth rates are obtained from steady-state continuous cultures run at different dilution rates. Remarkably, the specific death rate of the cells due to sparging decreased as their specific growth rate decreased. Furthermore, in Chapter 6 it is shown that the specific death rate of Vero cells is reduced by immobilisation of the cells inside porous carriers.Below a critical dilution rate in continuous culture as well as towards the end of batch cultures, the specific death rate of hybridoma cells increases rapidly. In this case, the cells mainly die through apoptosis as a consequence of substrate depletion and the accumulation of toxic products. In Chapter 7 an age-structured model is developed to describe the rate of apoptosis as a function of the dilution rate in continuous culture. In this model a critical specific growth rate is introduced below which the cells start becoming apoptotic. In addition to the specific deathand growth rate, the average cell volume of the viable cells and the specific consumption and production rates for glucose, glutamine, lactate and ammonia are calculated. The model can reasonably well describe a set of literature data, with respect to the specific growth- and death rate and the concentrations of viable cells, dead cells, glucose, glutamine, lactate, and ammonia. In Chapter 8 the model is extended with equations concerning two hypotheses for the production of monoclonal antibody being: (1) Passive release of antibody from dead cells. (2) Increased productivity by the apoptotic cells.Both hypotheses can describe the increase in productivity at decreasing dilution rates as observed in Chapter 5. Furthermore, the distribution of cells over the different phases of the cell cycle is calculated and the equations for the average cell volume are rewritten in terms of forward scatter as measured by flow cytometry. Model predictions concerning these variables are compared to results obtained in Chapter 5. The G 1 - and G 2 /M-phase fractions are not predicted correctly, which may be caused by a cell-cycle-phase specificity of apoptosis. In addition, a good prediction of all cell-cycle fractions can be obtained if it is assumed that the duration of the G 2 /M phase is not constant, but increases as the specific growth rate decreases from the maximum to its critical value. Furthermore, the calculated fraction of apoptotic cells is shown to be proportional to the forward scatter.Cell death associated with sparging may be minimised by: (1) Maximizing the amount of oxygen transferred per bubble introduced in the reactor. (2) The addition of a shear-protective agent like Pluronic F68 or serum. (3) The immobilisation of cells inside porous microcarriers.Point one is most easily done by increasing the reactor height and the oxygen tension in the air bubbles. With respect to the second point, it should be mentioned that the additive must be removed from the final product and can cause problems in down-stream processing. This may lead to an increase in the product cost. Finally, in the case of immobilisation of cells inside carriers, transport limitations may occur, which may in turn induce apoptosis. Cell death through apoptosis as caused by low levels of shear, substrate limitation and the presence of toxic products may be reduced through a careful process design and genetic manipulation of the cells

Introduction à la méthode statistique et probabiliste

This manuscript provides an introduction to statistics and probability method. It is structured around three themes developed in five chapters. The first part is an introduction to probability calculations in which we introduce the notions of elementary probability, probabilized spaces, random variables, distribution et density functions. We also review the main probability laws that characterize the common phenomenas of observation. The second part is an introduction to statistical analytical approaches. In this part, we present, one hand, the descriptive and exploratory methods and on other hand the inferential methods. The notions discussed in this section are statistical distributions, links between variables, estimators and hypothesis testing. As for the third part, it provides an introduction to survey techniques centered on the study of sampling protocols and estimations methods for extrapolation

Increased MAPK1/3 Phosphorylation in Luminal Breast Cancer Related with PIK3CA Hotspot Mutations and Prognosis

INTRODUCTION: While mutations in PIK3CA are most frequently (45%) detected in luminal breast cancer, downstream PI3K/AKT/mTOR pathway activation is predominantly observed in the basal subtype. The aim was to identify proteins activated in PIK3CA mutated luminal breast cancer and the clinical relevance of such a protein in breast cancer patients. MATERIALS AND METHODS: Expression levels of 171 signaling pathway (phospho-)proteins established by The Cancer Genome Atlas (TCGA) using reverse phase protein arrays (RPPA) were in silico examined in 361 breast cancers for their relation with PIK3CA status. MAPK1/3 phosphorylation was evaluated with immunohistochemistry on tissue microarrays (TMA) containing 721 primary breast cancer core biopsies to explore the relationship with metastasis-free survival. RESULTS: In silico analyses revealed increased phosphorylation of MAPK1/3, p38 and YAP, and decreased expression of p70S6K and 4E–BP1 in PIK3CA mutated compared to wild-type luminal breast cancer. Augmented MAPK1/3 phosphorylation was most significant, i.e. in luminal A for both PIK3CA exon 9 and 20 mutations and in luminal B for exon 9 mutations. In 290 adjuvant systemic therapy naïve lymph node negative (LNN) breast cancer patients with luminal cancer, high MAPK phosphorylation in nuclei (HR = 0.49; 95% CI, 0.25–0.95; P =.036) and in tumor cells (HR = 0.37; 95% CI, 0.18–0.79; P =.010) was related with favorable metastasis-free survival in multivariate analyses including traditional prognostic factors. CONCLUSION: Enhanced MAPK1/3 phosphorylation in luminal breast cancer is related to PIK3CA exon-specific mutations and correlated with favorable prognosis especially when located in the nuclei of tumor cells

Recent Developments of NEMO: Detection of Solar Eruptions Characteristics

The recent developments in space instrumentation for solar observations and telemetry have caused the necessity of advanced pattern recognition tools for the different classes of solar events. The Extreme ultraviolet Imaging Telescope (EIT) of solar corona on-board SOHO spacecraft has uncovered a new class of eruptive events which are often identified as signatures of Coronal Mass Ejection (CME) initiations on solar disk. It is evident that a crucial task is the development of an automatic detection tool of CMEs precursors. The Novel EIT wave Machine Observing (NEMO) (http://sidc.be/nemo) code is an operational tool that detects automatically solar eruptions using EIT image sequences. NEMO applies techniques based on the general statistical properties of the underlying physical mechanisms of eruptive events on the solar disc. In this work, the most recent updates of NEMO code - that have resulted to the increase of the recognition efficiency of solar eruptions linked to CMEs - are presented. These updates provide calculations of the surface of the dimming region, implement novel clustering technique for the dimmings and set new criteria to flag the eruptive dimmings based on their complex characteristics. The efficiency of NEMO has been increased significantly resulting to the extraction of dimmings observed near the solar limb and to the detection of small-scale events as well. As a consequence, the detection efficiency of CMEs precursors and the forecasts of CMEs have been drastically improved. Furthermore, the catalogues of solar eruptive events that can be constructed by NEMO may include larger number of physical parameters associated to the dimming regions.Comment: 12 Pages, 5 figures, submitted to Solar Physic

Mutations in the M-Gene Segment Can Substantially Increase Replication Efficiency of NS1 Deletion Influenza A Virus in MDCK Cells

Influenza viruses unable to express NS1 protein (delNS1) replicate poorly and induce high amounts of interferon (IFN). They are therefore considered as candidate viruses for live-attenuated influenza vaccines. Their attenuated replication is generally assumed to result from the inability to counter the antiviral host response, as delNS1 viruses replicate efficiently in Vero cells, which lack IFN expression. In this study, delNS1 virus was parallel passaged on IFN competent MDCK cells, which resulted in two strains that were able to replicate to high virus titres in MDCK cells due to adaptive mutations in especially the M-gene segment, but also the NP and NS gene segments. Most notable were clustered U-to-C mutations in the M segment of both strains and clustered A-to-G mutations in the NS segment of one strain, which presumably resulted from host cell mediated RNA editing. The M segment mutations in both strains changed the ratio of M1 to M2 expression, probably by affecting splicing efficiency. In one virus, 2 amino acid substitutions in M1 additionally enhanced virus replication, possibly through changes in the M1 distribution between the nucleus and the cytoplasm. Both adapted viruses induced equal levels of IFN as delNS1 virus. These results show that the increased replication of the adapted viruses is not primarily due to altered IFN induction, but rather related to changes in M1 expression or localization. The mutations identified in this paper may be used to enhance delNS1 virus replication for vaccine production

Metabolic engineering of lactic acid bacteria, the combined approach: kinetic modelling, metabolic control and experimental analysis

Everyone who has ever tried to radically change metabolic fluxes knows that it is often harder to determine which enzymes have to be modified than it is to actually implement these changes. In the more traditional genetic engineering approaches ’bottle-necks’ are pinpointed using qualitative, intuitive approaches, but the alleviation of suspected ’rate-limiting’ steps has not often been successful. Here the authors demonstrate that a model of pyruvate distribution in Lactococcus lactis based on enzyme kinetics in combination with metabolic control analysis clearly indicates the key control points in the flux to acetoin and diacetyl, important flavour compounds. The model presented here (available at http://jjj.biochem.sun.ac.za/wcfs.html) showed that the enzymes with the greatest effect on this flux resided outside the acetolactate synthase branch itself. Experiments confirmed the predictions of the model, i.e. knocking out lactate dehydrogenase and overexpressing NADH oxidase increased the flux through the acetolactate synthase branch from 0 to 75% of measured product formation rates

Cell-free DNA mutations as biomarkers in breast cancer patients receiving tamoxifen

The aim was to identify mutations in serum cell-free DNA (cfDNA) associated with disease progression on tamoxifen treatment in metastatic breast cancer (MBC). Sera available at start of therapy, during therapy and at disease progression were selected from 10 estrogen receptor (ER)-positive breast cancer patients. DNA from primary tumor and normal tissue and cfDNA from minute amounts of sera were analyzed by targeted next generation sequencing (NGS) of 45 genes (1,242 exons). At disease progression, stop-gain single nucleotide variants (SNVs) for CREBBP (1 patient) and SMAD4 (1 patient) and non-synonymous SNVs for AKAP9 (1 patient), PIK3CA (2 patients) and TP53 (2 patients) were found. Mutations in CREBBP and SMAD4 have only been occasionally reported in breast cancer. All mutations, except for AKAP9, were also present in the primary tumor but not detected in all blood specimens preceding progression. More sensitive detection by deeper re-sequencing and digital PCR confirmed the occurrence of circulating tumor DNA (ctDNA) and these biomarkers in blood specimens

Intratumoral heterogeneity of second-harmonic generation scattering from tumor collagen and its effects on metastatic risk prediction

Background: Metastases are the leading cause of breast cancer-related deaths. The tumor microenvironment impacts cancer progression and metastatic ability. Fibrillar collagen, a major extracellular matrix component, can be studied using the light scattering phenomenon known as second-harmonic generation (SHG). The ratio of forward- to backward-scattered SHG photons (F/B) is sensitive to collagen fiber internal structure and has been shown to be an independent prognostic indicator of metastasis-free survival time (MFS). Here we assess the effects of heterogeneity in the tumor matrix on the possible use of F/B as a prognostic tool. Methods: SHG imaging was performed on sectioned primary tumor excisions from 95 untreated, estrogen receptor-positive, lymph node negative invasive ductal carcinoma patients. We identified two distinct regions whose collagen displayed different average F/B values, indicative of spatial heterogeneity: the cellular tumor bulk and surrounding tumor-stroma interface. To evaluate the impact of heterogeneity on F/B’s prognostic ability, we performed SHG imaging in the tumor bulk and tumor-stroma interface, calculated a 21-gene recurrence score (surrogate for OncotypeDX®, or S-ODX) for each patient and evaluated their combined prognostic ability. Results: We found that F/B measured in tumor-stroma interface, but not tumor bulk, is prognostic of MFS using three methods to select pixels for analysis: an intensity threshold selected by a blinded observer, a histogram-based thresholding method, and an adaptive thresholding method. Using both regression trees and Random Survival Forests for MFS outcome, we obtained data-driven prediction rules that show F/B from tumor-stroma interface, but not tumor bulk, and S-ODX both contribute to predicting MFS in this patient cohort. We also separated patients into low-intermediate (S-ODX < 26) and high risk (S-ODX ≥26) groups. In the low-intermediate risk group, comprised of patients not typically recommended for adjuvant chemotherapy, we find that F/B from the tumor-stroma interface is prognostic of MFS and can identify a patient cohort with poor outcomes. Conclusions: These data demonstrate that intratumoral heterogeneity in F/B values can play an important role in its possible use as a prognostic marker, and that F/B from tumor-stroma interface of prim