Cell Growth Kinetics Research Articles

Expression of alternative NADH dehydrogenases (NDH-2) in the phytopathogenic fungus Ustilago maydis.

Type 2 alternative NADH dehydrogenases (NDH‐2) participate indirectly in the generation of the electrochemical proton gradient by transferring electrons from NADH and NADPH into the ubiquinone pool. Due to their structural simplicity, alternative NADH dehydrogenases have been proposed as useful tools for gene therapy of cells with defects in the respiratory complex I. In this work, we report the presence of three open reading frames, which correspond to NDH‐2 genes in the genome of Ustilago maydis. These three genes were constitutively transcribed in cells cultured in YPD and minimal medium with glucose, ethanol, or lactate as carbon sources. Proteomic analysis showed that only two of the three NDH‐2 were associated with isolated mitochondria in all culture media. Oxygen consumption by permeabilized cells using NADH or NADPH was different for each condition, opening the possibility of posttranslational regulation. We confirmed the presence of both external and internal NADH dehydrogenases, as well as an external NADPH dehydrogenase insensitive to calcium. Higher oxygen consumption rates were observed during the exponential growth phase, suggesting that the activity of NADH and NADPH dehydrogenases is coupled to the dynamics of cell growth.

Interference of starch accumulation in microalgal cell growth measurement

Starch accumulation in microalgae is a subject of growing interest in the energy sector since this polysaccharide can be used as feedstock to produce ethanol and biogas. Microalgal cell growth dynamics are commonly monitored by measuring the optical density (OD) of the culture, which then is converted to either biomass dry weight or cell concentration with the aid of standard curves. However, starch accumulation causes changes in the cell morphology, which may impair the use of OD for the measurement of cell growth. Therefore, this study investigated the correlation of OD to cell concentration and dry weight during the cultivation of Chlorella sorokiniana, a starch-accumulating green microalga. A linear correlation between OD and cell concentration was observed only during the exponential and the decelerating growth phases. The OD increase during the stationary phase was related to starch accumulation rather than to cell growth. Similarly, a lack of correlation between OD and biomass dry weight occurred during the stationary growth phase, as the increase of dry weight, due to the pronounced starch accumulation, was greater than the OD changes. Thus, this study shows that the use of OD to measure microalgal cell growth in starch-accumulating cultures can be misleading. The direct measurement of cell concentration and dry weight is advisable for monitoring the full cell growth cycle and accurately calculating biomass productivity.

Abstract 3100: Replacing fetal calf serum by human platelet lysate in cancer research and toxicology

Abstract Experiments with cultured mammalian cells represent a common in vitro alternative to animal experiments. Fetal calf serum (FCS) is the most commonly used medium supplement. FCS contains a mixture of largely undefined growth factors and cytokines. Since FCS is received from unborn calves older than three months, it represents a massive burden for the pregnant cows and their fetuses. A needle is inserted into the heart of the fetus to collect blood. Since the animal is not under anesthesia, it may suffer pain and discomfort. Furthermore, the undefined nature of FCS is a source of experimental variation, undesired immune responses, and possible contaminations. Thus, alternative, defined, valid, and reliable medium supplements should be identified. We follow the principles of the 3Rs (Replacement, Reduction, Refinement). We established a method to receive human platelet lysate (hPL) from human blood platelets. We investigated if hPL represents potential true alternative for FCS in cell culture experiments and thereby the cruel slaughtering of unborn fetuses. hPL is received by freeze and thawing cycles and centrifugation steps. We successfully adapted three human leukemia cell lines (K562, NB4 and MV4-11) and HCT116 colon cancer cells to culture medium containing 1-5% hPL. We analyzed cell morphology by microscopy. Cell growth kinetics were examined by counting and cell cycle distributions were investigated by flow cytometry. Protein expression patterns were determined globally by global scale proteomics. We additionally determined possible FCS- or hPL-dependent reactions of cells to the well-established anti-cancer drugs Irinotecan, Hydroxyurea and Imatinib, and to epigenetic modulators of the HDAC inhibitor family (FK228 and the new, highly selective HDAC6 inhibitor Marbostat-100) by flow cytometry and Western blot. In these tests, we particularly focused on the expression of the pan-leukemic marker and transcription factor Wilms tumor 1 (WT1) and on the tumor suppressor p53. All tested cell lines grow with 5% FCS and 5% hPL. Cell morphology, cell cycle distribution, and protein expression patterns were not affected by such different culture conditions in resting and treated cells. We further show that the class I HDACs HDAC1-3, but not HDAC6, regulate WT1 and p53. Our findings demonstrate that hPL is a promising replacement for animal serum. Citation Format: Oliver H. Krämer. Replacing fetal calf serum by human platelet lysate in cancer research and toxicology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3100.

Abstract 2796: Predicting in vivo efficacy from in vitro data: Quantitative systems pharmacology modeling for an epigenetic modifier drug in cancer

Abstract Epigenetic modulators are increasing in prominence as potential cancer therapies. These drugs achieve their effectiveness by inducing transcriptional changes that can inhibit cancer progression. Here, we focus on a potent and selective covalent small molecule inhibitor of LSD1 (RO7051750/ORY-1001), a lysine-specific histone demethylase enzyme. LSD1 inhibition causes epigenetic reprogramming of cancer cells by downregulating pro-proliferative neuroendocrine genes, inducing a pro-differentiation, cytostatic effect. Furthermore, the durability of this response is enhanced by covalent drug binding and the resulting dependence on target turnover. To explore the impact of dose and scheduling on the pharmacodynamic profiles underlying treatment efficacy, we developed a predictive model of drug effect, which incorporates experimentally-derived pharmacokinetic and pharmacodynamic data. This mechanistic mathematical model was calibrated based on in vitro cell-type specific kinetic data, which possesses high dimensionality across time points and dose ranges. The model describes drug-induced target engagement of LSD1, which acts as a transcriptional regulator of neuroendocrine gene, GRP. GRP levels drive reversible epigenetic “switching” between a proliferating (denoted P) and a quiescent (denoted Q) cell population (low GRP: P to Q; high GRP: Q to P), capturing the cytostatic effects of the drug. The model was trained with in vitro measurements of target engagement, GRP mRNA levels, and cell growth inhibition (including drug-free cell growth) across various time points and dosages from a small cell lung cancer cell line (NCI-H510A). Importantly, GRP mRNA and cell viability dynamics were also captured upon drug withdrawal, which enables the modeling of drug effect durability. The resulting model quantitatively describes the relationship between drug dose, target engagement, biomarker and cell growth dynamics in vitro. To scale to the in vivo setting, we integrated the in vitro-trained pharmacodynamic model with a pharmacokinetic model describing drug distribution and clearance in mouse. This hybrid model accurately predicted in vivo tumor growth dynamics in the NCI-H510A mouse xenograft across a range of doses and schedules. We used the model to explore the target engagement and biomarker profiles underlying treatment efficacy, both in vitro and in vivo. In conclusion, by training our model on key cell-type specific in vitro kinetic data we were able to accurately predict anti-tumor growth effects in vivo. This quantitative modeling framework highlights the biological profiles underlying efficacy for this latest generation of target-selective epigenetic modifying drugs. Citation Format: Mehdi Bouhaddou, Li J. Yu, Serena Lunardi, Spyros K. Stamatelos, Fiona Mack, James M. Gallo, Marc R. Birtwistle, Antje-Christine Walz. Predicting in vivo efficacy from in vitro data: Quantitative systems pharmacology modeling for an epigenetic modifier drug in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2796.

Multi-objective versus single-objective optimization of batch bioethanol production based on a time-dependent fermentation model

Traditional process simulators, such as Aspen Plus, are inadequate for optimizing multiple-objective systems in fermentation-based processes. This work uses a novel integrated platform of the robust genetic algorithm optimization in MATLAB linked with an Aspen Plus unsteady-state batch fermentation simulation to optimize the batch ethanolic fermentation process with respect to initial substrate concentration, fermentation time, and in situ product removal. A time-dependent fermentation model that utilizes both glucose and xylose, the major sugars present in lignocellulosic hydrolysate, with Monod cell growth kinetics, substrate and product inhibitions, is used as a model system. The optimized design variables from the multi-objective optimization (MOO) and single-objective optimization (SOO) suggest the typical concentrations of sugars from lignocellulosic hydrolysate must be concentrated to optimize the performance of the batch fermentation process. Furthermore, time-dependent information from an unsteady-state simulation was used to design an integrated batch fermentation with in situ product recovery, allowing higher initial sugars concentrations to be used in the fermentation process (about 50%, for the best optimal solution in the MOO). This resulted in 15% ethanol productivity, 143% total ethanol produced, and 67% fraction of sugar converted improvements relative to the batch fermentation without product recovery. Unlike the single optimal solution from the SOO, MOO presents many equally optimal solutions that can be used as a decision-support tool to guide the choice of design variables for optimum process performance. This study creates a platform that can be used to optimize integrated biorefinery and refinery processes.

Towards optimal substrate feeding for heterologous protein production in Pichia pastoris (Komagataella spp) fed‐batch processes under PAOX1 control: a modeling aided approach

AbstractBACKGROUNDTo improve the efficiency of a bioprocess, key parameters, such as yield, titer and productivity, must be considered. They are mainly dependent on the specific rates for product (qP) and cell growth (μ), and their correlation determines the most suitable feeding polices that should be applied.RESULTSThe mathematical description of the Pichia pastoris (Komagataella spp) PAOX1‐based system (Mut+) expressing recombinant Rhizopus oryzae lipase (ROL), which includes cell growth, substrate consumption and product formation kinetics, was used to define the optimal profiles regarding methanol feeding‐rate (F), residual methanol concentration (S) and specific growth rate (μ). Optimal trajectories obtained using numerical optimization and algorithms based on S‐profile determination were first applied to manage the multiplicity of S‐states with both μ and qP. Once the S‐profile was determined, the F and μ profiles were obtained in a straightforward manner from mass balances and growth kinetics. From experimental results, the bioprocess efficiency improvement was confirmed in terms of a 2.2‐fold higher final titer and 3.4‐fold higher productivity compared with the best standard pre‐programmed exponential substrate feeding strategy. Additionally, 1.2‐fold higher productivity and 1.1‐fold higher final titer relative to the most promising feedback control of the methanol concentration strategy were obtained.CONCLUSIONThe whole model‐based approach was established as a versatile and simple platform, which would then be transferable to alternative modes of operation and even to produce other proteins of interest. © 2018 Society of Chemical Industry

In Vitro and In Vivo Osteogenesis of Human Orbicularis Oculi Muscle-Derived Stem Cells.

Cell-based therapies for treating bone defects require a source of stem cells with osteogenic potential. There is evidence from pathologic ossification within muscles that human skeletal muscles contain osteogenic progenitor cells. However, muscle samples are usually acquired through a traumatic biopsy procedure which causes pain and morbidity to the donor. Herein, we identified a new alternative source of skeletal muscle stem cells (SMSCs) without conferring morbidity to donors. Adherent cells isolated from human orbicularis oculi muscle (OOM) fragments, which are currently discarded during ophthalmic cosmetic surgeries, were obtained using a two-step plating method. The cell growth kinetics, immunophenotype and capabilities of in vitro multilineage differentiation were evaluated respectively. Moreover, the osteogenically-induced cells were transduced with GFP gene, loaded onto the porous β-tricalcium phosphate (β-TCP) bioceramics, and transplanted into the subcutaneous site of athymic mice. Ectopic bone formation was assessed and the cell fate in vivo was detected. OOM-derived cells were fibroblastic in shape, clonogenic in growth, and displayed phenotypic and behavioral characteristics similar to SMSCs. In particular, these cells could be induced into osteoblasts in vitro evidenced by the extracellular matrix calcification and enhanced alkaline phosphatase (ALP) activity and osteocalcin (OCN) production. New bone formation was found in the cell-loaded bioceramics 6weeks after implantation. By using the GFP-labeling technique, these muscle cells were detected to participate in the process of ectopic osteogenesis in vivo. Our data suggest that human OOM tissue is a valuable and noninvasive resource for osteoprogenitor cells to be used in bone repair and regeneration.

The full-length microRNA cluster in the intron of large latency transcript is associated with the virulence of pseudorabies virus

Pseudorabies virus (PRV), the etiological pathogen of Aujeszky's disease, belongs to the Alphaherpesvirus subfamily. Large latency transcript (LLT), the most abundant PRV transcript, harbors a ~ 4.6 kb microRNA (miRNA) cluster-encoding intron. To investigate the function of the LLT miRNA cluster during the life cycle of PRV, we generated a miRNA cluster mutation virus (PRV-∆miR cluster) and revertant virus. Analysis of the growth kinetics of PRV-ΔmiR cluster-infected cells revealed significantly smaller plaques and lower titers than the wild-type and revertant viruses. The mutation virus exhibited increased IE180 and decreased EP0 expression. The clinical symptoms observed in mice infected with PRV-ΔmiR cluster revealed that the miRNA cluster is involved in the pathogenesis of PRV. Physical parameters, virus shedding assays, and the SN50 titers revealed that the miRNA cluster enhances PRV virulence in pigs. Collectively, our findings suggest that the full-length miRNA cluster is involved in PRV replication and virulence.

Effect of global transcriptional regulators on kinetic behavior of Escherichia coli under anaerobic fermentation conditions.

The phenotype of Escherichia coli is governed by global transcriptional regulators under variable environmental conditions. Fnr, ArcA, IhfA-B, Crp, and Fis are amongst the major global transcription regulators that change their activity across the range of aeration, hence forming the core transcriptional network responsible for survival under changing aeration conditions. Effect of deletion of these global transcription factors on the kinetics of cell growth and mixed acid production under anaerobic fermentation conditions has not been characterized. To quantify the kinetic parameters in the absence of global transcription factors, experiments were performed using single deletion mutants of the above-mentioned global transcription regulators. The absence of global transcription regulators resulted in a relatively higher glucose uptake rate than that required for the observed growth rate. This further resulted in a higher yield of mixed acids per unit biomass in mutants as compared to the parent strain (E. coli BW25113). Thus, the increased channeling of carbon towards mixed acid secretion resulted in a lower growth rate in the mutants.

Neurofibromatosis type 2 tumor suppressor protein is expressed in oligodendrocytes and regulates cell proliferation and process formation.

The neurofibromatosis type 2 (NF2) tumor suppressor protein Merlin functions as a negative regulator of cell growth and actin dynamics in different cell types amongst which Schwann cells have been extensively studied. In contrast, the presence and the role of Merlin in oligodendrocytes, the myelin forming cells within the CNS, have not been elucidated. In this work, we demonstrate that Merlin immunoreactivity was broadly distributed in the white matter throughout the central nervous system. Following Merlin expression during development in the cerebellum, Merlin could be detected in the cerebellar white matter tract at early postnatal stages as shown by its co-localization with Olig2-positive cells as well as in adult brain sections where it was aligned with myelin basic protein containing fibers. This suggests that Merlin is expressed in immature and mature oligodendrocytes. Expression levels of Merlin were low in oligodendrocytes as compared to astrocytes and neurons throughout development. Expression of Merlin in oligodendroglia was further supported by its identification in either immortalized cell lines of oligodendroglial origin or in primary oligodendrocyte cultures. In these cultures, the two main splice variants of Nf2 could be detected. Merlin was localized in clusters within the nuclei and in the cytoplasm. Overexpressing Merlin in oligodendrocyte cell lines strengthened reduced impedance in XCELLigence measurements and Ki67 stainings in cultures over time. In addition, the initiation and elongation of cellular projections were reduced by Merlin overexpression. Consistently, cell migration was retarded in scratch assays done on Nf2-transfected oligodendrocyte cell lines. These data suggest that Merlin actively modulates process outgrowth and migration in oligodendrocytes.

186 - Critical quality attributes in the filling process for iPSCs and MSCs by considering the kinetics of cell death and growth

186 - Critical quality attributes in the filling process for iPSCs and MSCs by considering the kinetics of cell death and growth

Simultaneous saccharification and bioethanol production from corn cobs: Process optimization and kinetic studies

This study investigates the simultaneous saccharification and fermentation (SSF) process for bioethanol production from corn cobs with prehydrolysis (PSSF) and without prehydrolysis (OSSF). Two response surface models were developed with high coefficients of determination (>0.90). Process optimization gave high bioethanol concentrations and bioethanol conversions for the PSSF (36.92 ± 1.34 g/L and 62.36 ± 2.27%) and OSSF (35.04 ± 0.170 g/L and 58.13 ± 0.283%) models respectively. Additionally, the logistic and modified Gompertz models were used to study the kinetics of microbial cell growth and ethanol formation under microaerophilic and anaerobic conditions. Cell growth in the OSSFmicroaerophilic process gave the highest maximum specific growth rate (µmax) of 0.274 h−1. The PSSFmicroaerophilic bioprocess gave the highest potential maximum bioethanol concentration (Pm) (42.24 g/L). This study demonstrated that microaerophilic rather than anaerobic culture conditions enhanced cell growth and bioethanol production, and that additional prehydrolysis steps do not significantly impact on the bioethanol concentration and conversion in SSF process.

Phylogenetic Analysis and Pathogenicity Assessment of the Emerging Recombinant Subgroup K of Avian Leukosis Virus in South China.

In recent years, cases of avian leukosis virus (ALV) infection have become more frequent in China. We isolated 6 ALV strains from yellow feather broiler breeders in south China from 2014 to 2016. Their full genomes were sequenced, compared, and analyzed with other reference strains of ALV. The complete genomic nucleotide sequences of GD150509, GD160403, GD160607, GDFX0601, and GDFX0602 were 7482 bp in length, whereas GDFX0603 was 7480 bp. They shared 99.7% to 99.8% identity with each other. Homology analysis showed that the gag, pol, long terminal repeats (LTRs), and the transmembrane region (gp37) of the env genes of the 6 viruses were well conserved to endogenous counterpart sequences (>97.8%). However, the gp85 genes displayed high variability with any known chicken ALV strains. Growth kinetics of DF-1 cells infected with the isolated ALV showed viral titers that were lower than those infected with the GD13 (ALV-A), CD08 (ALV-B), and CHN06 (ALV-J) on day 7 post-infection. The infected Specific-pathogen-free (SPF) chickens could produce continuous viremia, atrophy of immune organs, growth retardation and no tumors were observed. These subgroup ALVs are unique and may be common in south China. The results suggested that updating the control and eradication program of exogenous ALV for yellow feather broiler breeders in south China needs to be considered because of the emergence of the new subgroup viruses.

Cyanotrophic and arsenic oxidizing activities of Pseudomonas mendocina P6115 isolated from mine tailings containing high cyanide concentration.

Mine tailings and wastewater generate man-made environments with several selective pressures, including the presence of heavy metals, arsenic and high cyanide concentrations, but severe nutritional limitations. Some oligotrophic and pioneer bacteria can colonise and grow in mine wastes containing a low concentration of organic matter and combined nitrogen sources. In this study, Pseudomonas mendocina P6115 was isolated from mine tailings in Durango, Mexico, and identified through a phylogenetic approach of 16S rRNA, gyrB, rpoB, and rpoD genes. Cell growth, cyanide consumption, and ammonia production kinetics in a medium with cyanide as sole nitrogen source showed that at the beginning, the strain grew assimilating cyanide, when cyanide was removed, ammonium was produced and accumulated in the culture medium. However, no clear stoichiometric relationship between both nitrogen sources was observed. Also, cyanide complexes were assimilated as nitrogen sources. Other phenotypic tasks that contribute to the strain's adaptation to a mine tailing environment included siderophores production in media with moderate amounts of heavy metals, arsenite and arsenate tolerance, and the capacity of oxidizing arsenite. P. mendocina P6115 harbours cioA/cioB and aoxB genes encoding for a cyanide-insensitive oxidase and an arsenite oxidase, respectively. This is the first report where P. mendocina is described as a cyanotrophic and arsenic oxidizing species. Genotypic and phenotypic tasks of P. mendocina P6115 autochthonous from mine wastes are potentially relevant for biological treatment of residues contaminated with cyanide and arsenic.

Spaceflight Modifies Escherichia coli Gene Expression in Response to Antibiotic Exposure and Reveals Role of Oxidative Stress Response.

Bacteria grown in space experiments under microgravity conditions have been found to undergo unique physiological responses, ranging from modified cell morphology and growth dynamics to a putative increased tolerance to antibiotics. A common theory for this behavior is the loss of gravity-driven convection processes in the orbital environment, resulting in both reduction of extracellular nutrient availability and the accumulation of bacterial byproducts near the cell. To further characterize the responses, this study investigated the transcriptomic response of Escherichia coli to both microgravity and antibiotic concentration. E. coli was grown aboard International Space Station in the presence of increasing concentrations of the antibiotic gentamicin with identical ground controls conducted on Earth. Here we show that within 49 h of being cultured, E. coli adapted to grow at higher antibiotic concentrations in space compared to Earth, and demonstrated consistent changes in expression of 63 genes in response to an increase in drug concentration in both environments, including specific responses related to oxidative stress and starvation response. Additionally, we find 50 stress-response genes upregulated in response to the microgravity when compared directly to the equivalent concentration in the ground control. We conclude that the increased antibiotic tolerance in microgravity may be attributed not only to diminished transport processes, but also to a resultant antibiotic cross-resistance response conferred by an overlapping effect of stress response genes. Our data suggest that direct stresses of nutrient starvation and acid-shock conveyed by the microgravity environment can incidentally upregulate stress response pathways related to antibiotic stress and in doing so contribute to the increased antibiotic stress tolerance observed for bacteria in space experiments. These results provide insights into the ability of bacteria to adapt under extreme stress conditions and potential strategies to prevent antimicrobial-resistance in space and on Earth.

Kinetic analysis of curdlan production by Alcaligenes faecalis with maltose, sucrose, glucose and fructose as carbon sources

Curdlan has wide-ranging benefits in food and pharmaceutical industries for its unique rheological and thermal gelling properties. To analyze the cell growth and curdlan biosynthesis kinetics of Alcaligenes faecalis, the kinetic properties of the curdlan fermentation under different carbon sources conditions (maltose, sucrose, glucose and fructose) were investigated using Logistic and Luedeking-Piret equations. The results demonstrated that curdlan fermentation is partial growth-associated process. With maltose as the sole carbon source, the highest curdlan production (Pm = 39.3 g/L), the maximum specific growth rate (μm = 0.44/h) and the growth-associated rate constant (α = 2.05 g curdlan/g cell) were achieved. In contrast, the fructose was the less desired carbon source in both the cell growth and curdlan production. Further, the results demonstrated that slow-releasing glucose from maltose boosted cell growth and curdlan production.

Fermentation and kinetics characteristics of a bioflocculant from potato starch wastewater and its application

Potato starch wastewater was used as fermentation medium for Rhodococcus erythropolis to produce bioflocculant. Kinetics of cell growth and bioflocculant production were firstly constructed. After fermentation for 60 h, 0.97 g of bioflocculant with polysaccharides nature was extracted from 1 L of fermentation liquor. Kinetics characteristics showed that cell growth and bioflocculant production could be simulated well with Logistic and Luedeking-Piret equations, respectively. When R. erythropolis was in logarithm growth phase, COD, ammonium, and TP of the potato starch wastewater medium were rapidly down to 1736, 188, and 146 mg/L, respectively, from 7836, 975, and 712 mg/L, while the medium’s exactly pH value was almost not changed. Furthermore, bioflocculant flocculation can be used as an effective pretreatment way for potato starch wastewater, and it was feasible in actual treatment projects in Ronghua Starch Co., Ltd., Sichuan Province.

Leptin regulates the pro-inflammatory response in human epidermal keratinocytes.

The role of leptin in cutaneous wound healing process has been suggested in genetically obese mouse studies. However, the molecular and cellular effects of leptin on human epidermal keratinocytes are still unclear. In this study, the whole-genome-scale microarray analysis was performed to elucidate the effect of leptin on epidermal keratinocyte functions. In the leptin-treated normal human keratinocytes (NHKs), we identified the 151 upregulated and 53 downregulated differentially expressed genes (DEGs). The gene ontology (GO) enrichment analysis with the leptin-induced DEGs suggests that leptin regulates NHKs to promote pro-inflammatory responses, extracellular matrix organization, and angiogenesis. Among the DEGs, the protein expression of IL-8, MMP-1, fibronectin, and S100A7, which play roles in which is important in the regulation of cutaneous inflammation, was confirmed in the leptin-treated NHKs. The upregulation of the leptin-induced proteins is mainly regulated by the STAT3 signaling pathway in NHKs. Among the downregulated DEGs, the protein expression of nucleosome assembly-associated centromere protein A (CENPA) and CENPM was confirmed in the leptin-treated NHKs. However, the expression of CENPA and CENPM was not coupled with those of other chromosome passenger complex like Aurora A kinase, INCENP, and survivin. In cell growth kinetics analysis, leptin had no significant effect on the cell growth curves of NHKs in the normal growth factor-enriched condition. Therefore, leptin-dependent downregulation of CENPA and CENPM in NHKs may not be directly associated with mitotic regulation during inflammation.

Screening of a biological control bacterium to fight avocado diseases: From agroecosystem to bioreactor

Abstract Avocado diseases remain to be a major constraint for the development of this agribusiness, which is largely led by Latin American countries. In this study, a collection of 667 native avocado bacterial isolates was established and screened for antagonistic activity and bioactive secondary metabolite production against two important avocado pathogens (Phytophthora cinnamomi and Colletotrichum gloeosporioides). The isolate ARP5.1 demonstrated the highest potential for in vitro control of the phytopathogens and was identified as Serratia sp. To enhance bioactive metabolite production by ARP5.1, different combinations of agitation speeds and aeration rates were evaluated on a stirred tank bioreactor. The activity of the crude extracts was tested and cell growth kinetics, oxygen and glucose consumption, and production of secondary metabolites were determined at the fermentation conditions of higher inhibitory activity. Results evidenced oxygen as critical factor for the biosynthesis of metabolites of interest. In addition, shear stress and glucose were limiting factors in metabolite production. Prodigiosin, serratamolide and haterumalide NC were identified in the extracts by Liquid Chromatography coupled to High-Resolution Mass Spectrometry. However, the presence of haterumalide NC might explain the high activity of the crude extract against P. cinnamomi. We concluded that this bacterium might be a promising candidate for the control of avocado pathogens in field and a good source of bioactive metabolites.

Comparison of growth kinetics between static and dynamic cultures of human induced pluripotent stem cells

Understanding the fundamental mechanisms that govern the growth kinetics of human induced pluripotent stem cells (hiPSCs) contributes to culture design strategies to improve large-scale production. Two hiPSC lines (Tic and 253G1) were cultured under static and dynamic suspension conditions, and growth kinetics were compared during early (24-48h), middle (48-72h), and late (72-96h) stages. In 2D static culture, similar growth profiles were observed for both hiPSC lines. However, there were significant differences in growth profile patterns and aggregate morphologies between hiPSC lines grown in 3D static and dynamic cultures. Based on immunostaining comparing the two hiPSC lines, surface distribution of collagen type I was observed in aggregates of the Tic line, but not in those of the 253G1 line. Compared to that in 3D static culture, the numbers of cells at 96h were significantly decreased in 3D dynamic culture. The apparent specific growth rate (μapp) of the Tic line was maintained continuously throughout culture, whereas that of the 253G1 line decreased gradually with culture until the late phase, at which time this parameter was reduced to μapp=(0.85±0.71)×10-2h-1. This indicates that during the growth of hiPSCs in 3D dynamic culture, cells were damaged by liquid flow, which disrupted the cell-synthesized extracellular matrix (ECM). These results demonstrate that cell-synthesized ECM is an important factor affecting cell growth and morphology, and that changes to the ECM within aggregates lead to reduced growth abilities in dynamic culture.