Fatty acid synthesis and pyruvate metabolism pathways remain active in dihydroartemisinin induced dormant ring stages of Plasmodium falciparum

Artemisinin (ART) based combination therapy (ACT) is used as the first line treatment of uncomplicated falciparum malaria worldwide. However, despite high potency and rapid action there is a high rate of recrudescence associated with ART monotherapy or ACT long before the recent emergence of ART resistance. ART induced ring stage dormancy and recovery has been implicated as possible cause of recrudescence; however, little is known about the characteristics of dormant parasites including whether dormant parasites are metabolically active. We investigated the transcription of 12 genes encoding key enzymes in various metabolic pathways in P. falciparum during dihydroartemisinin (DHA) induced dormancy and recovery. Transcription analysis showed an immediate down regulation for 10 genes following exposure to DHA, but continued transcription of 2 genes encoding apicoplast and mitochondrial proteins. Transcription of several additional genes encoding apicoplast and mitochondrial proteins, particularly genes encoding enzymes in pyruvate metabolism and fatty acid synthesis pathways, were also maintained. Additions of inhibitors for biotin acetyl CoA carbozylase and enoyl-acyl carrier reductase of the fatty acid synthesis pathways delayed the recovery of dormant parasites by 6 and 4 days, respectively following DHA treatment. Our results demonstrate most metabolic pathways are down regulated in DHA induced dormant parasites. In contrast fatty acid and pyruvate metabolic pathways remain active. These findings highlight new targets to interrupt recovery of parasites from ART-induced dormancy and to reduce the rate of recrudescence following ART treatment.

Resistance to amoebic gill disease (AGD) is characterised by the transcriptional dysregulation of immune and cell cycle pathways

Amoebic gill disease (AGD) is a parasite-mediated proliferative gill disease capable of affecting a range of teleost hosts. While a moderate heritability for AGD resistance in Atlantic salmon has been reported previously, the mechanisms by which individuals resist the proliferative effects remain poorly understood. To gain more knowledge of this commercially important trait, we compared gill transcriptomes of two groups of Atlantic salmon, one designated putatively resistant, and one designated putatively susceptible to AGD. Utilising a 17k Atlantic salmon cDNA microarray we identified 196 transcripts that were differentially expressed between the two groups. Expression of 11 transcripts were further examined with real-time quantitative RT-PCR (qPCR) in the AGD-resistant and AGD-susceptible animals, as well as non-infected naïve fish. Gene expression determined by qPCR was in strong agreement with the microarray analysis. A large number of differentially expressed genes were involved in immune and cell cycle responses. Resistant individuals displayed significantly higher expression of genes involved in adaptive immunity and negative regulation of the cell cycle. In contrast, AGD-susceptible individuals showed higher expression of acute phase proteins and positive regulators of the cell cycle. Combined with the gill histopathology, our results suggest AGD resistance is acquired rather than innately present, and that this resistance is for the most part associated with the dysregulation of immune and cell cycle pathways. © 2008 Elsevier Ltd. All rights reserved.

Model-based analysis and optimization of bioreactor for hematopoietic stem cell cultivation

One of the problems to be solved in attaining the full potentials of hematopoietic stem cell (HSC) applications is the limited availability of the cells. Growing HSCs in a bioreactor offers an alternative solution to this problem. Besides, it also offers the advantages of eliminating labour intensive process as well as the possible contamination involved in the periodic nutrient replenishments in the traditional T-flask stem cell cultivation. In spite of this, the optimization of HSC cultivation in a bioreactor has been barely explored. This manuscript discusses the development of a mathematical model to describe the dynamics in nutrient distribution and cell concentration of an ex vivo HSC cultivation in a microchannel perfusion bioreactor. The model was further used to optimize the cultivation by proposing three alternative feeding strategies in order to prevent the occurrence of nutrient limitation in the bioreactor. The evaluation of these strategies, the periodic step change increase in the inlet oxygen concentration, the periodic step change increase in the media inflow, and the feedback control of media inflow, shows that these strategies can successfully improve the cell yield of the bioreactor. In general, the developed model is useful for the design and optimization of bioreactor operation.

Mathematical modelling of human corneal oxygenation and cell kinetics in corneal epithelial wound healing

Healthy transparent cornea depends upon the regulation of fluid, nutrient and oxygen transport through the tissue to sustain cell metabolism and other critical processes for normal functioning. This research considers the corneal geometry and investigates oxygen distribution using a two-dimensional Monod kinetic model, showing that previous studies make assumptions that lead to predictions of near-anoxic levels of oxygen tension in the limbal regions of the cornea. It also considers the comparison of experimental spatial and temporal data with the predictions of novel mathematical models with respect to distributed mitotic rates during corneal epithelial wound healing.

Caspase-8 and c-FLIPL associate in lipid rafts with NF-kappaB adaptors during T cell activation.

Humans and mice lacking functional caspase-8 in T cells manifest a profound immunodeficiency syndrome due to defective T cell antigen receptor (TCR)-induced NF-kappaB signaling and proliferation. It is unknown how caspase-8 is activated following T cell stimulation, and what is the caspase-8 substrate(s) that is necessary to initiate T cell cycling. We observe that following TCR ligation, a small portion of total cellular caspase-8 and c-FLIP(L) rapidly migrate to lipid rafts where they associate in an active caspase complex. Activation of caspase-8 in lipid rafts is followed by rapid cleavage of c-FLIP(L) at a known caspase-8 cleavage site. The active caspase.c-FLIP complex forms in the absence of Fas (CD95/APO1) and associates with the NF-kappaB signaling molecules RIP1, TRAF2, and TRAF6, as well as upstream NF-kappaB regulators PKC theta, CARMA1, Bcl-10, and MALT1, which connect to the TCR. The lack of caspase-8 results in the absence of MALT1 and Bcl-10 in the active caspase complex. Consistent with this observation, inhibition of caspase activity attenuates NF-kappaB activation. The current findings define a link among TCR, caspases, and the NF-kappaB pathway that occurs in a sequestered lipid raft environment in T cells.

Lifespan, body size and oxygen : aerobic metabolism and oxidative stress in cultured fibroblasts /

The allometric scaling relationship observed between metabolic rate (MR) and species body mass can be partially explained by differences in cellular MR (Porter & Brand, 1995). Here, I studied cultured cell lines derived from ten mammalian species to determine whether cells propagated in an identical environment exhibited MR scaling. Oxidative and anaerobic metabolic parameters did not scale significantly with donor body mass in cultured cells, indicating the absence of an intrinsic MR setpoint. The rate of oxygen delivery has been proposed to limit cellular metabolic rates in larger organisms (West et al., 2002). As such cells were cultured under a variety of physiologically relevant oxygen tensions to investigate the effect of oxygen on cellular metabolic rates. Exposure to higher medium oxygen tensions resulted in increased metabolic rates in all cells. Higher MRs have the potential to produce more reactive oxygen species (ROS) which could cause genomic instability and thus reduced lifespan. Longer-lived species are more resistant to oxidative stress (Kapahi et al, 1999), which may be due to greater antioxidant and/or DNA repair capacities. This hypothesis was addressed by culturing primary dermal fibroblasts from eight mammalian species ranging in maximum lifespan from 5 to 120 years. Only the antioxidant manganese superoxide dismutases (MnSOD) positively scaled with species lifespan (p<0.01). Oxidative damage to DNA is primarily repaired by the base excision repair (BER) pathway. BER enzyme activities showed either no correlation or as in the case of polymerase p correlated, negatively with donor species (p<0.01 ). Typically, mammalian cells are cultured in a 20% O2 (atmospheric) environment, which is several-fold higher than cells experience in vivo. Therefore, the secondary aim of this study was to determine the effect of culturing mammalian cells at a more physiological oxygen tension (3%) on BER, and antioxidant, enzyme activities. Consistently, standard culture conditions induce higher antioxidant and DNA ba.se excision repair activities than are present under a more physiological oxygen concentration. Therefore, standard culture conditions are inappropriate for studies of oxidative stress-induced activities and species differences in fibroblast DNA BER repair capacities may represent differences in ability to respond to oxidative stress. An interesting outcome firom this study was that some inherent cellular properties are maintained in culture (i.e. stress responses) while others are not (i.e. MR).

Cell wall composition regulates cell shape and growth behaviour in pollen tubes

L’une des particularités fondamentales caractérisant les cellules végétales des cellules animales est la présence de la paroi cellulaire entourant le protoplaste. La paroi cellulaire joue un rôle primordial dans (1) la protection du protoplaste, (2) est impliquée dans les mécanismes de filtration et (3) est le lieu de maintes réactions biochimiques nécessaires à la régulation du métabolisme et des propriétés mécaniques de la cellule. Les propriétés locales d’élasticité, d’extensibilité, de plasticité et de dureté des composants pariétaux déterminent la géométrie et la forme des cellules lors des processus de différentiation et de morphogenèse. Le but de ma thèse est de comprendre les rôles que jouent les différents composants pariétaux dans le modelage de la géométrie et le contrôle de la croissance des cellules végétales. Pour atteindre cet objectif, le modèle cellulaire sur lequel je me suis basé est le tube pollinique ou gamétophyte mâle. Le tube pollinique est une protubérance cellulaire qui se forme à partir du grain de pollen à la suite de son contact avec le stigmate. Sa fonction est la livraison des cellules spermatiques à l’ovaire pour effectuer la double fécondation. Le tube pollinique est une cellule à croissance apicale, caractérisée par la simple composition de sa paroi et par sa vitesse de croissance qui est la plus rapide du règne végétal. Ces propriétés uniques font du tube pollinique le modèle idéal pour l’étude des effets à courts termes du stress sur la croissance et le métabolisme cellulaire ainsi que sur les propriétés mécaniques de la paroi. La paroi du tube pollinique est composée de trois composantes polysaccharidiques : pectines, cellulose et callose et d’une multitude de protéines. Pour comprendre les effets que jouent ces différents composants dans la régulation de la croissance du tube pollinique, j’ai étudié les effets de mutations, de traitements enzymatiques, de l’hyper-gravité et de la gravité omni-directionnelle sur la paroi du tube pollinique. En utilisant des méthodes de modélisation mathématiques combinées à de la biologie moléculaire et de la microscopie à fluorescence et électronique à haute résolution, j’ai montré que (1) la régulation de la chimie des pectines est primordiale pour le contrôle du taux de croissance et de la forme du tube et que (2) la cellulose détermine le diamètre du tube pollinique en partie sub-apicale. De plus, j’ai examiné le rôle d’un groupe d’enzymes digestives de pectines exprimées durant le développement du tube pollinique : les pectate lyases. J’ai montré que ces enzymes sont requises lors de l’initiation de la germination du pollen. J’ai notamment directement prouvé que les pectate lyases sont sécrétées par le tube pollinique dans le but de faciliter sa pénétration au travers du style.

Dual-specificity phosphatases in the hypo-osmotic stress response of keratin-defective epithelial cell lines

Although mutations in intermediate filament proteins cause many human disorders, the detailed pathogenic mechanisms and the way these mutations affect cell metabolism are unclear. In this study, selected keratin mutations were analysed for their effect on the epidermal stress response. Expression profiles of two keratin-mutant cell lines from epidermolysis bullosa simplex patients (one severe and one mild) were compared to a control keratinocyte line before and after challenge with hypo-osmotic shock, a common physiological stress that transiently distorts cell shape. Fewer changes in gene expression were found in cells with the severely disruptive mutation (55 genes altered) than with the mild mutation (174 genes) or the wild type cells (261 genes) possibly due to stress response pre-activation in these cells. We identified 16 immediate-early genes contributing to a general cell response to hypo-osmotic shock, and 20 genes with an altered expression pattern in the mutant keratin lines only. A number of dual-specificity phosphatases (MKP-1, MKP-2, MKP-3, MKP-5 and hVH3) are differentially regulated in these cells, and their downstream targets p-ERK and p-p38 are significantly up-regulated in the mutant keratin lines. Our findings strengthen the case for the expression of mutant keratin proteins inducing physiological stress, and this intrinsic stress may affect the cell responses to secondary stresses in patients' skin.

Photodynamic therapy associating Photogem (R) and blue LED on L929 and MDPC-23 cell culture

To evaluate the cytotoxicity of PDT (photodynamic therapy) with Photogem (R) associated to blue LED (light-emitting diode) on L929 and MDPC-23 cell cultures, 30000 cells/cm(2) were seeded in 24-well plates for 48 h, incubated with Photogem (R) (10, 25 or 50 mg/l) and irradiated with an LED source (460 +/- 3 nm; 22 mW/cm(2)) at two energy densities (25.5 or 37.5 J/cm(2)). Cell metabolism was evaluated by the MTT (methyltetrazolium) assay (Dunnet`s post hoc tests) and cell morphology by SEM (scanning electron microscopy). Flow cytometry analysed the type of PDT-induced cell death as well and estimated intracellular production of ROS (reactive oxygen species). There was a statistically significant decrease of mitochondrial activity (90% to 97%) for all Photogem (R) concentrations associated to blue LED, regardless of irradiation time. It was also demonstrated that the mitochondrial activity was not recovered after 12 or 24 h, characterizing irreversible cell damage. PDT-treated cells presented an altered morphology with ill-defined limits. In both cell lines, there was a predominance of necrotic cell death and the presence of Photogem (R) or irradiation increased the intracellular levels of ROS. PDT caused severe toxic effects in normal cell culture, characterized by the reduction of the mitochondrial activity, morphological alterations and induction of necrotic cell death.

beta-Glucan extracted from the medicinal mushroom Agaricus blazei prevents the genotoxic effects of benzo[a]pyrene in the human hepatoma cell line HepG2

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effects of intrapulpal temperature change induced by visible light units on the metabolism of odontoblast-like cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of the activation mode of a self-etch resin-based luting cement upon the metabolism of odontoblast-like cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Trans-enamel and trans-dentinal cytotoxic effects of a 35% H2O2 bleaching gel on cultured odontoblast cell lines after consecutive applications

To evaluate the trans-enamel and trans-dentinal cytotoxic effects of a 35% H2O2 bleaching gel on an odontoblast-like cell lines (MDPC-23) after consecutive applications.Fifteen enamel/dentine discs were obtained from bovine central incisor teeth and placed individually in artificial pulp chambers. Three groups (n = 5 discs) were formed according to the following enamel treatments: G1: 35% H2O2 bleaching gel (15 min); G2: 35% H2O2 bleaching gel (15 min) + halogen light (20 s); G3: control (no treatment). After repeating the treatments three consecutive times, the extracts (culture medium + gel components that had diffused through enamel/dentine discs) in contact with the dentine were collected and applied to previously cultured MDPC-23 cells (50 000 cells cm(-2)) for 24 h. Cell metabolism was evaluated by the MTT assay and data were analysed statistically (alpha = 5%; Kruskal-Wallis and Mann-Whitney U-test). Cell morphology was analysed by scanning electron microscopy.Cell metabolism decreased by 92.03% and 82.47% in G1 and G2 respectively. G1 and G2 differed significantly (P < 0.05) from G3. Regardless of halogen light activation, the application of the bleaching gel on the cultured odontoblast-like cells caused significantly more severe cytotoxic effects than those observed in the nontreated control group. In addition, significant morphological cell alterations were observed in G1 and G2.After three consecutive applications of a 35% H2O2 bleaching agent, the diffusion of the gel components through enamel and dentine caused severe toxic effects to cultured pulp cells.

Photodynamic therapy associating Photogem (R) and blue LED on L929 and MDPC-23 cell culture

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)