Cellular Growth Kinetics Research Articles

Abstract 582: Oncogenic PIK3CA fortifies immune determinants in vascular cancers

Abstract Angiosarcomas are a group of vascular cancers that form malignant blood vessels. These malignancies are seemingly inflamed primarily due to their pathognomonic nature, which consists of irregular endothelium and tortuous blood channels. The malignant vascular spaces host a variety of blood and immune components, where a controlled scavenging system is necessary for tumor maintenance. Oncogenes may convey molecular signals to foster the tumor immune niche, and PIK3CA mutations that induce continuous activation of the PI3K pathway occur in a subset of angiosarcomas. Despite the undoubtedly oncogenic properties of PIK3CA mutations, the mechanistic role of these mutations - particularly the potential for controlling the hemato-endothelial system in angiosarcomas - remains undetermined. In this study, we demonstrate the functional consequences of oncogenic PIK3CA and its molecular properties in angiosarcoma cells. We created isogenic cell clones from two angiosarcoma cell lines engineered to harbor hotspot mutations in PIK3CA using CRISPR/Cas9: two homozygous PIK3CA H1047R cell clones (C8 and C35) and one wild-type (WT) clone were generated from DHSA-1426 angiosarcoma cells; and one homozygous mutant clone was created from COSB cells. We found that PIK3CA mutant cells constitutively activated the PI3K/AKT/mTOR pathway at a greater level than that of WT cells. Bulk- and single cell(sc)-RNA-seq data revealed that mutant cells enriched gene sets associated with PI3K signaling and other oncogenic pathways including KRAS, MAPK, and TGF-beta. Also, mutant cells significantly increased cytokines regulating immune cell migration and chemotaxis such as IL-8 and MCP-1. In addition to the common features, we identified functional and molecular divergence between mutant clones. Noticeable changes in cellular morphology, growth kinetics, and tube-forming capacity were observed between two mutant clones. C35 mutant cells were more sensitive to both pan-PI3K and PI3K-alpha specific inhibitor, and DNA damage was greater than in the C8 clone. C35 cells enhanced activation of IL-6/STAT3 signaling compared to C8, despite the increment of the aforementioned immune cytokines in both mutants. Moreover, our comprehensive analysis of bulk- and sc-RNA-seq, ATAC-seq, and metabolomics data identified molecular signatures associated with metabolic vulnerabilities of C35 cells. In summary, we demonstrate that oncogenic PIK3CA perpetuates PI3K signaling activation and reinforces a supply for immune signaling. Furthermore, our data suggest that PIK3CA mutations contribute to establishing cellular heterogeneity that reveals a spectrum of vascular, immunogenic, and metabolic activities. Our ongoing work involves developing an experimental model of angiosarcoma using induced pluripotent stem cells to examine if oncogenic PIK3CA controls immune determinants during malignant endothelial transformation. Citation Format: Donghee Lee, Emma Kozurek, Md Abdullah, Rong Li, Erin B. Dickerson, Jong Hyuk Kim. Oncogenic PIK3CA fortifies immune determinants in vascular cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 582.

A mechanobiological model for tumor spheroid evolution with application to glioblastoma: A continuum multiphysics approach

Background:Spheroids are in vitro quasi-spherical structures of cell aggregates, eventually cultured within a hydrogel matrix, that are used, among other applications, as a technological platform to investigate tumor formation and evolution. Several interesting features can be replicated using this methodology, such as cell communication mechanisms, the effect of gradients of nutrients, or the creation of realistic 3D biological structures. The main objective of this work is to link the spheroid evolution with the mechanical activity of cells, coupled with nutrient consumption and the subsequent cell dynamics. Method:We propose a continuum mechanobiological model which accounts for the most relevant phenomena that take place in tumor spheroid evolution under in vitro suspension, namely, nutrient diffusion in the spheroid, kinetics of cellular growth and death, and mechanical interactions among the cells. The model is qualitatively validated, after calibration of the model parameters, versus in vitro experiments of spheroids of different glioblastoma cell lines. Results:Our model is able to explain in a novel way quite different setups, such as spheroid growth (up to six times the initial configuration for U-87 MG cell line) or shrinking (almost half of the initial configuration for U-251 MG cell line); as the result of the mechanical interplay of cells driven by cellular evolution. Conclusions:Glioblastoma tumor spheroid evolution is driven by mechanical interactions of the cell aggregate and the dynamical evolution of the cell population. All this information can be used to further investigate mechanistic effects in the evolution of tumors and their role in cancer disease.

In Vitro and In Vivo Studies for the Investigation of γ-Globin Gene Induction by Adhatoda vasica: A Pre-Clinical Study of HbF Inducers for β-Thalassemia.

Fetal hemoglobin (HbF) is a potent genetic modifier, and the γ-globin gene induction has proven to be a sustainable therapeutic approach for the management of β-thalassemia. In this study, we have evaluated the HbF induction ability of A. vasica in vitro and in vivo, and the identification of potential therapeutic compounds through a bioassay-guided approach. In vitro benzidine-Hb assay demonstrated strong erythroid differentiation of K562 cells by A. vasica extracts. Subsequently, an in vivo study with an aqueous extract of A. vasica (100 mg/kg) showed significant induction of the γ-globin gene and HbF production. While in the acute study, the hematological and biochemical indices were found to be unaltered at the lower dose of A. vasica. Following the bioassay-guided approach, two isolated compounds, vasicinol (1) and vasicine (2) strongly enhanced HbF levels and showed prominent cellular growth kinetics with ample accumulation of total hemoglobin in K562 cultures. High HbF levels were examined by immunofluorescence and flow cytometry analysis, concomitant with the overexpression in the γ-globin gene level. Compound 1 (0.1 µM) and compound 2 (1 µM) resulted in a greater increase in F-cells (90 and 83%) with marked up (8-fold and 5.1-fold) expression of the γ-globin gene, respectively. Molecular docking studies indicated strong binding affinities of (1) and (2) with HDAC2 and KDM1 protein that predict the possible mechanism of compounds in inhibition of these epigenetic regulators in the γ-globin gene reactivation. Altogether, these observations demonstrated the therapeutic usefulness of A. vasica for fostering HbF production in clinical implications for blood disorders.

Growth kinetics of caprine bone marrow-derived mesenchymal stem cells at different passages

A study was conducted on cellular growth kinetics of caprine bone marrow mesenchymal stem cells (MSCs) with the help of growth curve, population doubling time (PDT) and colony forming unit fibroblast assay (CFU-f). MSCs were cultured up to 10 passages (from P0 to P10) consequent to their isolation, purification and characterization. After employing statistical analysis it was observed that maximum cellular growth (270.00±4.33 cells/well) and minimum PDT (39.16±4.18hr) were noticed in P3 passage followed by in passage 4 which was 263.33±5.07/well and 39.92±1.36 hr respectively. In a similar fashion maximum number of colonies were also counted inpassages which depicted the maximum proliferation and differentiation ability of cells which was 74.00±3.61 in P3 followed by in P4 (66.67±3.84). The potency was calculated at the samepassages on the basis of CFU-f assay which indicated that cells of P3 and P4 passages are best suited for transplantation, cryopreservation and experimental studies.

Multiparameter Single-Cell Characterization of Ovarian Intratumor Heterogeneity.

Cancer is a complex disease rooted in heterogeneity, which is the phenomenon of individual cells, tissues, or patients having distinct phenotypic and/or genetic characteristics. Observed divergent disease etiology is likely rooted, at least in part, in tumor heterogeneity and the classification of distinct and important subpopulations of cells within the tumor and its associated microenvironment has remained a technical challenge. Standard next-generation sequencing of bulk tumor tissue provides an overall average genetic profile of the sample, and masks contributions from individual cells and minor populations of cells, particularly in heterogeneous samples. Only with the advent of single-cell analysis and sequencing technologies has it become possible to characterize key contributions of cellular subpopulations in order to more comprehensively characterize disease. This chapter describes a method to generate linked phenotypic and genotypic data at single-cell resolution using a real-time single-cell resolved platform. Specifically, the example method provided here is used to link cellular growth kinetics and expression of a prognostic marker protein, CA-125, in cells derived from ovarian cancer patients with their single-cell genomic profiles, but the method is translatable to other cell types and phenotypes of interest.

Population Cellular Kinetics of Lisocabtagene Maraleucel, an Autologous CD19-Directed Chimeric Antigen Receptor T-Cell Product, in Patients with Relapsed/Refractory Large B-Cell Lymphoma.

Background and ObjectivesLisocabtagene maraleucel (liso-cel) is a CD19-directed, defined composition, 4-1BB chimeric antigen receptor (CAR) T-cell product administered at equal target doses of CD8+ and CD4+ CAR+ T cells. Large between-subject variability has been noted with CAR T-cell therapies; patient characteristics might contribute to CAR T-cell expansion variability. We developed a population cellular kinetic model to characterize the kinetics of the liso-cel transgene, via quantitative polymerase chain reaction assessment after intravenous infusion of liso-cel, and to understand covariates that might influence liso-cel kinetics in individual patients.MethodsWe employed nonlinear mixed-effects modeling to develop a population cellular kinetic model for liso-cel. The population cellular kinetic analysis was performed using 2524 post-infusion transgene observations from 261 patients with relapsed/refractory large B-cell lymphoma who were treated with a single dose of liso-cel in TRANSCEND NHL 001. Covariates for the analysis included baseline intrinsic factors such as age, baseline disease characteristics, and liso-cel and coadministration factors.ResultsLiso-cel cellular kinetics were well described by a piecewise model of cellular growth kinetics that featured lag, exponential growth, and biexponential decay phases. Population means (95% confidence interval) of lag phase duration, doubling time, time to maximum levels, initial decline half-life, and terminal half-life were 3.27 (2.71–3.97), 0.755 (0.667–0.821), 9.29 (8.81–9.70), 5.00 (4.15–5.90), and 352 (241–647) days, respectively. The magnitude of effect on liso-cel expansion metrics demonstrated that the covariate associations were smaller than the residual between-subject variability in the population.ConclusionsThe covariates tested were not considered to have a meaningful impact on liso-cel kinetics.Clinical Trial RegistrationNCT02631044.

Polyacrylamide Hydrogels with Rigidity-Independent Surface Chemistry Show Limited Long-Term Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells on Soft Substrates

In general, cells are cultured and adapted to the in vitro rigidities of plastic or glass ranging between 1 and 10 GPa, which is very far from physiological values that are mostly in the kilopascal range. Stem cells however show a high sensitivity to the rigidity of their culture environment, which impacts their differentiation program. Here, we address the impact of rigidity on the long-term maintenance of pluripotency in human induced pluripotent stem cells (hiPSCs) to determine whether soft substrates could provide a new standard for hiPSC expansion and maintenance. To do this, we set up a fabrication process of polyacrylamide-based culture supports with a rigidity-decoupled surface chemistry. Soft elastic substrates with uniform and reproducible physicochemical properties were designed. The maintenance of pluripotency of two hiPSCs lines on substrates with stiffnesses ranging from 3 to 25 kPa was studied with an identical chemical coating consisting of a truncated recombinant vitronectin with defined surface density. Based on the analysis of cellular adhesion, survival, growth kinetics, three-dimensional distribution, and gene and protein expressions, we demonstrate that below 25 kPa hiPSCs do not maintain pluripotency on long-term culture, while pluripotency and self-renewal capacities are maintained above 25 kPa. In contrast to previous studies, no drift toward a specific germ line lineage was revealed. On soft substrates, cell colonies started to grow in three-dimensional (3D), suggesting that softness allows cells to limit contact with the synthetic matrix and to build their own microenvironment. These observations drastically limit the benefit of using standardized soft substrates to expand hiPSCs, at least with the current culture conditions. The development of a robust technology for the design of soft substrates nevertheless opens up perspectives to fine-tune physicochemical properties of the culture environment in addition to or in replacement of soluble growth factors to finely direct cell fate.

A homeostatic mechanism rapidly corrects aberrant nucleocytoplasmic ratios maintaining nuclear size in fission yeast.

ABSTRACTNuclear size scales with cell size across a wide range of cell types. The mechanism by which this scaling is maintained in growing cells remains unclear. Here, we investigate the mechanism of nuclear size homeostasis in the simple eukaryote fission yeast, by monitoring the recovery of aberrant nuclear volume to cell volume (N/C) ratios following perturbation. We demonstrate that both high and low N/C ratios correct rapidly, maintaining nuclear size homeostasis. We assess the kinetics of nuclear and cellular growth and of N/C ratio correction, and demonstrate that nuclear and cellular growth rates are not directly coupled. We propose that the mechanism underlying nuclear size homeostasis involves multiple limiting factors implicated in processes including nucleocytoplasmic transport, lipid biogenesis and RNA processing. We speculate that these link cellular size increases to changes in nuclear contents, which in turn lead to changes in nuclear membrane surface area. Our study reveals that there is rapid nuclear size homeostasis in cells, informing understanding of nuclear size control and size homeostasis of other membrane-bound organelles.

Kinetic characteristics and N2O production of a heterotrophic nitrifying bacterium Pseudomonas putida YH capable of tolerating adverse environmental conditions

AbstractBACKGROUNDHeterotrophic nitrifying bacteria (HNB) have rapid growth rate, high activity and strong anti‐shock ability. These attributes are advantageous in engineering applications, yet the use of HNB is restricted by lack of clarity about the kinetics and metabolic mechanisms involved. Thus, nitrogen conversion characteristics, kinetics and N2O production of the HNB Pseudomonas putida YH were evaluated.RESULTSStrain YH exhibited superior heterotrophic nitrification‐aerobic denitrification (HNAD) ability in treating nitrogenous wastewater, with maximum removal rates of 10.07, 8.86 and 8.78 mg L−1 h−1 for NH4+–N, NO3−–N and NO2−–N, respectively. Mass balance analysis demonstrated that the removed TN was mainly due to bacterial assimilation (53–55%) and conversion into gaseous N2 (34–39%), instead of nitrification intermediate accumulation and N2O emission. In addition, the kinetics of nitrogen degradation and cellular growth of strain YH exactly conformed to the modified Gompertz equation and the Logistic equation, respectively. Moreover, strain YH showed excellent adaptability to variations of temperature, pH, C:N ratio and initial NH4+–N concentration in terms of nitrogen removal and N2O emission. The highest NH4+–N removal rate was obtained at pH 7.0, temperature 30 °C and C:N ratio of 15. Furthermore, the heterotrophic nitrification ability was just partially deteriorated after the addition of inhibitors, which further proved the multiple metabolic pathways of heterotrophic nitrification by strain YH.CONCLUSIONThe results demonstrated that strain YH exhibited excellent abilities of HNAD, N2O emission control and tolerance to adverse conditions, indicating that strain YH can be an effective candidate for treating nitrogenous wastewater without secondary pollution. © 2019 Society of Chemical Industry

Iron-oxide nanoparticles target intracellular HSP90 to induce tumor radio-sensitization

BackgroundNanoparticle-based therapies have emerged as a promising approach to overcome limitations of conventional chemotherapy. Present study investigates the potential of oleic acid-functionalized iron-oxide nanoparticles (MN-OA) to enhance the radiation response of fibrosarcoma tumor and elucidates its underlying mechanism. MethodsVarious cellular and molecular assays (e.g. MTT, clonogenic, cell cycle analysis, cell death, DNA damage/repair) and tumor growth kinetics were employed to investigate the mechanism of MN-OA induced radio-sensitization. ResultsMouse (WEHI-164) and human (HT-1080) fibrosarcoma cells treated with MN-OA and gamma-radiation (2 Gy) showed a significant decrease in the cell proliferation. Combination treatment showed significant decrease in clonogenic survival of WEHI-164 cells and was found to induce cell cycle arrest, apoptosis and mitotic catastrophe. The mechanism of radio-sensitization was found to involve binding of MN-OA with HSP90, resulting in down-regulation of its client proteins, involved in cell cycle progression (Cyclin B1 and CDC2) and DNA-double strand break repair (e.g. RAD51 and BRCA1). Consistently, longer persistence of DNA damage in cells treated with MN-OA and radiation was observed in the form of γ-H2AX foci. The efficacy and mechanism of MN-OA-induced radio-sensitization was also validated in an immuno-competent murine fibrosarcoma model. ConclusionThis study reveals the key role of HSP90 in the mechanism of tumor radio-sensitization by MN-OA. General significancePresent work provides a deeper understanding about the mechanism of MN-OA-induced tumor radiosensitization, highlighting the role of HSP90 protein. In addition to diagnostic and magnetic hyperthermia abilities, present remarkable radiosensitizing activity of MN-OA would further excite the clinicians to test its anti-cancer potential.

Learning the space-time phase diagram of bacterial swarm expansion

Coordinated dynamics of individual components in active matter are an essential aspect of life on all scales. Establishing a comprehensive, causal connection between intracellular, intercellular, and macroscopic behaviors has remained a major challenge due to limitations in data acquisition and analysis techniques suitable for multiscale dynamics. Here, we combine a high-throughput adaptive microscopy approach with machine learning, to identify key biological and physical mechanisms that determine distinct microscopic and macroscopic collective behavior phases which develop as Bacillus subtilis swarms expand over five orders of magnitude in space. Our experiments, continuum modeling, and particle-based simulations reveal that macroscopic swarm expansion is primarily driven by cellular growth kinetics, whereas the microscopic swarming motility phases are dominated by physical cell-cell interactions. These results provide a unified understanding of bacterial multiscale behavioral complexity in swarms.

Influence of Initiation Site and Lamellar Divergency on the Overall Kinetics of Cellular Growth and Coarsening in Aged U-Nb Alloys

Overall kinetics of lamellar overaging reactions in U-5.5Nb and U-7.5Nb were analyzed by Avrami-Arrhenius analyses of volume fractions measured from an extensive temperature-time (T-t) matrix of specimens. The cellular initiation site (grain boundaries, inclusions) and regimes of lamellar divergency-cum-slowing growth rate were explicitly accounted for. Avrami exponents n from T-t regimes of constant-growth rate were consistent with theory (1<n<3); those from divergent T-t regimes were smaller, n~0.7, which is not surprising given their different growth rate behavior. The apparent activation energies Q were similar for grain-boundary and inclusion-nucleated discontinuous precipitation, indicating that their nucleation site does not alter their overall kinetics. Avrami Analysis of Isothermal Aging Kinetics

Multi-scale computational study of the Warburg effect, reverse Warburg effect and glutamine addiction in solid tumors.

Cancer metabolism has received renewed interest as a potential target for cancer therapy. In this study, we use a multi-scale modeling approach to interrogate the implications of three metabolic scenarios of potential clinical relevance: the Warburg effect, the reverse Warburg effect and glutamine addiction. At the intracellular level, we construct a network of central metabolism and perform flux balance analysis (FBA) to estimate metabolic fluxes; at the cellular level, we exploit this metabolic network to calculate parameters for a coarse-grained description of cellular growth kinetics; and at the multicellular level, we incorporate these kinetic schemes into the cellular automata of an agent-based model (ABM), iDynoMiCS. This ABM evaluates the reaction-diffusion of the metabolites, cellular division and motion over a simulation domain. Our multi-scale simulations suggest that the Warburg effect provides a growth advantage to the tumor cells under resource limitation. However, we identify a non-monotonic dependence of growth rate on the strength of glycolytic pathway. On the other hand, the reverse Warburg scenario provides an initial growth advantage in tumors that originate deeper in the tissue. The metabolic profile of stromal cells considered in this scenario allows more oxygen to reach the tumor cells in the deeper tissue and thus promotes tumor growth at earlier stages. Lastly, we suggest that glutamine addiction does not confer a selective advantage to tumor growth with glutamine acting as a carbon source in the tricarboxylic acid (TCA) cycle, any advantage of glutamine uptake must come through other pathways not included in our model (e.g., as a nitrogen donor). Our analysis illustrates the importance of accounting explicitly for spatial and temporal evolution of tumor microenvironment in the interpretation of metabolic scenarios and hence provides a basis for further studies, including evaluation of specific therapeutic strategies that target metabolism.

Monitoring of adherent live cells morphology using the undecimated wavelet transform multivariate image analysis (UWT-MIA).

Cell morphology is an important macroscopic indicator of cellular physiology and is increasingly used as a mean of probing culture state in vitro. Phase contrast microscopy (PCM) is a valuable tool for observing live cells morphology over long periods of time with minimal culture artifact. Two general approaches are commonly used to analyze images: individual object segmentation and characterization by pattern recognition. Single-cell segmentation is difficult to achieve in PCM images of adherent cells since their contour is often irregular and blurry, and the cells bundle together when the culture reaches confluence. Alternatively, pattern recognition approaches such as the undecimated wavelet transform multivariate image analysis (UWT-MIA), allow extracting textural features from PCM images that are correlated with cellular morphology. A partial least squares (PLS) regression model built using textural features from a set of 200 ground truth images was shown to predict the distribution of cellular morphological features (major and minor axes length, orientation, and roundness) with good accuracy for most images. The PLS models were then applied on a large dataset of 631,136 images collected from live myoblast cell cultures acquired under different conditions and grown in two different culture media. The method was found sensitive to morphological changes due to cell growth (culture time) and those introduced by the use of different culture media, and was able to distinguish both sources of variations. The proposed approach is promising for application on large datasets of PCM live-cell images to assess cellular morphology and growth kinetics in real-time which could be beneficial for high-throughput screening as well as automated cell culture kinetics assessment and control applications. Biotechnol. Bioeng. 2017;114: 141-153. © 2016 Wiley Periodicals, Inc.

Cellular characteristics and growth behavior of iron‐limited Microcystis aeruginosa in nutrient‐depleted and nutrient‐replete chemostat systems

Cellular responses of Fe-limited Microcystis aeruginosa were investigated under nutrient-depleted and -replete conditions. Cellular growth, Fe quota and Fe uptake kinetics were examined in chemostat systems using nutrient-replete Fraquil* (where all nutrients except for Fe are present at sufficient level to achieve optimal growth) and nutrient-deplete Fraquil* (where some nutrients in addition to Fe are potentially growth-limiting factors). For both nutrient conditions, cellular Fe quota increased with increasing dilution rate in a manner consistent with Droop theory. However, the Fe quota in nutrient-deplete Fraquil* was determined to be lower, indicating lower cellular Fe requirement in the nutrient-depleted condition. Short-term Fe uptake assays indicated that cells acclimated in nutrient-replete conditions adjust to various degrees of Fe stress by solely increasing maximum Fe uptake rate, consistent with expected negative feedback regulation. In contrast, the maximum Fe uptake rate decreased with increasing degree of Fe limitation in the nutrient-depleted chemostat (particularly nitrate and molybdenum in this study). This non-negative feedback regulation is likely associated with lower Fe requirement for specific functions (e.g., intracellular nitrate reduction). Cellular affinity for Fe uptake and cellular size were independent of degree of Fe stress for both nutrient conditions.

Antiproliferative, Apoptotic, and Autophagic Activity of Ranibizumab, Bevacizumab, Pegaptanib, and Aflibercept on Fibroblasts: Implication for Choroidal Neovascularization.

Purpose. Choroidal neovascularization (CNV) is one of the most common complications of retinal diseases accompanied by elevated secretion of vascular endothelial growth factor (VEGF). Intravitreal anti-VEGFs (ranibizumab, bevacizumab, pegaptanib, and aflibercept) can suppress neovascularization, decrease vascular permeability and CNV size, and, thereby, improve visual function. The antiproliferative, apoptotic, and autophagic effect of anti-VEGF drugs on fibroblasts found in CNVs has not been yet explored. Methods. Concentration-dependent cellular effects of the four anti-VEGFs were examined in L929 fibroblasts over a 5-day period. The cell survival, mitotic and polykaryocytic indices, the level of apoptosis and autophagy, and the cellular growth kinetics were all assessed. Results. The anti-VEGFs could inhibit the survival, mitotic activity, and proliferation as well as increase the cellular heterogeneity, apoptosis, and autophagy of the fibroblasts in a dose-dependent manner. Cellular growth kinetics showed ranibizumab to be less aggressive, but three other anti-VEGFs showed higher antiproliferative and apoptotic activity and expressed negative cellular growth kinetics. Conclusions. The antiproliferative, apoptotic, and autophagic activity of anti-VEGFs upon fibroblasts may explain the cellular response and the etiology of CNV involution in vivo and serve as a good study model for CNV in vitro.

Modeling the Growth Kinetics of Chlorella vulgaris Cultivated in Microfluidic Devices

The third generation biofuels such as algal biodiesel is the future potential source of renewable energy. Recently Dewan et al. (2012) developed a drop-based microfluidics device platform to investigate cellular growth kinetics of single and few cells of Chlorella vulgaris. The results showed the typical asymmetric sigmoidal growth pattern. Since there exists a variety of models for describing the growth profile of microorganism such as logistic, Gompertz, Richards, Schnute, Baranyi-Roberts, Von Bertalanffy, Buchanan three-phase and more recently Huang models, the growth curves exhibit under such conditions would be an excellent study for finding the best model. The Buchanan three-phase model was chose as the best model based on statistical tests such as root-mean-square error (RMSE), adjusted coefficient of determination (R2), bias factor (BF), accuracy factor (AF), corrected AICc (Akaike Information Criterion) and F-test. Parameters obtained from the growth fitting exercise were maximum specific growth rate (mmax), lag time (l) and maximal number of cells achieved per droplet (Ymax) with the values of 1.301 (day-1), 1.861 (day) and 77 (no of cells/droplet), respectively. The parameters obtained from fitting the algae growth curve using this model can bed used for further modeling and optimization exercises for identifying key controlling parameters of the microfluidic devices.

BCR-ABL Cooperates With a “Telomere-Associated Secretory Phenotype” (TASP) To Facilitate Malignant Proliferation Of Hematopoietic Stem Cells

Chronic myeloid leukemia (CML) is a hematopoietic stem cell (HSC) disease caused by the reciprocal translocation t(9;22). Although there is clear evidence that the resulting oncogenic tyrosine kinase BCR-ABL is the key event of leukemia initiation which drives stem cell proliferation and expansion of myeloid progenitors in early chronic phase (CP), the mechanism leading to advanced phases remains elusive. Recently, we could show that telomere attrition correlates with disease stages due to increased leukemic stem cell turnover. Here, we could provide first time evidence that this can functionally contribute to disease progression in CML.In our study we made use of the well-described telomerase knockout mouse model (mTR-/-), lacking the RNA subunit of telomerase and resulting in significant telomere shortening with each generation, and retrovirally introduced BCR-ABL into primary bone marrow cells of different generation. Although all CML-like cultures (hereafter referred to as “CML”) grew exponentially and growth factor independently in vitro, they showed remarkable differences in cellular growth kinetics depending on the generation of mTR-/-mice the cells were derived from. CML-HSCs of generation iG4 (CML-iG4) are functionally impaired with respect to their growth properties and ceased to proliferate due to a robust senescent-like cell cycle arrest. Interestingly, they did not show overt genomic instability, but and are less susceptible to Imatinib-induced apoptosis compared to wildtype cells (CML-WT). In sharp contrast, CML-G2 cells with only pre-shortened telomere lengths grew most rapidly and presented with an impressive proliferation advantage compared to CML-WT and -iG4 cells, while they still retain Imatinib sensitivity. Notably, we uncovered that this growth advantage is related to a “telomere-associated secretory phenotype” (TASP), comprising the upregulation and secretion of chemokines, interleukins and other growth factors, thereby potentiating oncogene-driven growth in an autocrine fashion. In line with those observations, we found that conditioned supernatant of CML-G2 cells markedly enhanced proliferation of CML-WT and pre-senescent CML-iG4 HSCs. To investigate if a TPE (telomere position effect)-related mechanism is responsible for inducing inflammatory gene expression in BCR-ABL positive cells, we mapped selected TASP genes for their chromosomal location. However, although they are frequently found in well-known cluster (e.g. chemokines), TASP genes are not preferentially located close to the (sub-) telomere. This suggests that a yet unknown mechanism controls TASP gene expression upon telomere shortening. Most importantly, a similar inflammatory mRNA expression pattern was found in CML patients of accelerated phase (AP), but not in blast crisis (BC).Taken together, those data support the hypothesis that accelerated telomere shortening contributes to disease progression in BCR-ABL-driven leukemogenesis by the expression of an inflammatory signature, while telomere-induced senescence needs to be bypassed (e.g. by upregulation of telomerase) in order for leukemic cells to be able to progress to blast crisis (BC) CML. Disclosures:Brümmendorf:Pfizer: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Ariad: Consultancy.

Temporal Behavior of Capsule Enlargement by Cryptococcus neoformans

Microbial capsules are important virulence traits that mediate cell-host interactions and provide protection against host immune defense mechanisms. Cryptococcus neoformans is a yeast-like fungus that is capable of synthesizing a complex polysaccharide (PS) capsule that is required for causing disease. Microscopic visualization of capsule enlargement is difficult, because the capsule is a highly hydrated structure with an index of refraction that is very close to that of aqueous medium. In this study, we took advantage of the capsular reaction ("quellung" effect) produced by IgM monoclonal antibody (MAb) 13F1 to increase the refraction index difference between capsule and medium such that we visualized the capsule using differential interference contrast (DIC) microscopy. Time-lapse size measurements allowed us to quantify the growth rate of the capsule relative to that of the cell body. The increase in capsule volume per unit of time was consistent with a logistic variable slope model in which the capsule's final size was proportional to the rate of its growth. The rate of capsule growth (0.3 to 2.5 µm(3)/min) was at least 4-fold faster than the rate of cell body growth (0.1 to 0.3 µm(3)/min), and there was large cell-to-cell variation in the temporal kinetics of capsule and cellular growth. Previous to the first cellular replication event, both the capsule and cell body enlarged simultaneously, and their differences showed monotonic growth, which was affected only by its rate of volume increase per unit of time. Using these results, we provide an updated model for cryptococcal capsule biogenesis.

Cellular Growth Kinetics Distinguish a Cyclophilin Inhibitor from an HSP90 Inhibitor as a Selective Inhibitor of Hepatitis C Virus

During antiviral drug discovery, it is critical to distinguish molecules that selectively interrupt viral replication from those that reduce virus replication by adversely affecting host cell viability. In this report we investigate the selectivity of inhibitors of the host chaperone proteins cyclophilin A (CypA) and heat-shock protein 90 (HSP90) which have each been reported to inhibit replication of hepatitis C virus (HCV). By comparing the toxicity of the HSP90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) to two known cytostatic compounds, colchicine and gemcitabine, we provide evidence that 17-AAG exerts its antiviral effects indirectly through slowing cell growth. In contrast, a cyclophilin inhibitor, cyclosporin A (CsA), exhibited selective antiviral activity without slowing cell proliferation. Furthermore, we observed that 17-AAG had little antiviral effect in a non-dividing cell-culture model of HCV replication, while CsA reduced HCV titer by more than two orders of magnitude in the same model. The assays we describe here are useful for discriminating selective antivirals from compounds that indirectly affect virus replication by reducing host cell viability or slowing cell growth.