Engineering mammalian cell growth dynamics for biomanufacturing.

Precise control over mammalian cell growth dynamics poses a major challenge in biopharmaceutical manufacturing. Here, we present a multi-level cell engineering strategy for the tunable regulation of growth phases in mammalian cells. Initially, we engineered mammalian death phase by employing CRISPR/Cas9 to knockout pro-apoptotic proteins Bax and Bak, resulting in a substantial attenuation of apoptosis by improving cell viability and extending culture lifespan. The second phase introduced a growth acceleration system, akin to a "gas pedal", based on an abscidic acid inducible system regulating cMYC gene expression, enabling rapid cell density increase and cell cycle control. The third phase focused on a stationary phase inducing system, comparable to a "brake pedal". A tetracycline inducible genetic circuit based on BLIMP1 gene led to cell growth cessation and arrested cell cycle upon activation. Finally, we developed a dual controllable system, combining the "gas and brake pedals", enabling for dynamic and precise orchestration of mammalian cell growth dynamics. This work exemplifies the application of synthetic biology tools and combinatorial cell engineering, offering a sophisticated framework for manipulating mammalian cell growth and providing a unique paradigm for reprogramming cell behaviour for enhancing biopharmaceutical manufacturing and further biomedical applications.

Transfer of tritium released into the marine environment by French nuclear facilities bordering the English Channel.

Controlled amounts of liquid tritium are discharged as tritiated water (HTO) by the nuclear industry into the English Channel. Because the isotopic discrimination between 3H and H is small, organically bound tritium (OBT) and HTO should show the same T/H ratio under steady-state conditions. We report data collected from the environment in the English Channel. Tritium concentrations measured in seawater HTO, as well as in biota HTO and OBT, confirm that tritium transfers from HTO to OBT result in conservation of the T/H ratio (ca. 1 × 10(-16)). The kinetics of the turnover of tritium between seawater HTO, biota HTO, and OBT was investigated. HTO in two algae and a mollusk is shown to exchange rapidly with seawater HTO. However, the overall tritium turnover between HTO and the whole-organism OBT is a slow process with a tritium biological half-life on the order of months. Nonsteady-state conditions exist where there are sharp changes in seawater HTO. As a consequence, for kinetic reasons, the T/H ratio in OBT may deviate transiently from that observed in HTO of samples from the marine ecosystem. Dynamic modeling is thus more realistic for predicting tritium transfers to biota OBT under nonsteady-state conditions.

Iodine uptake in brown seaweed exposed to radioactive liquid discharges from the reprocessing plant of ORANO La Hague.

Iodine-129 is present in controlled liquid radioactive waste routinely released in seawater by the ORANO nuclear fuel reprocessing plant in La Hague (Normandy, France). Brown algae are known for their exceptional ability to concentrate iodine from seawater. They also potentially emit volatile iodine compounds in response to various stresses, such as during emersion at low tide. For these reasons, brown seaweed is routinely collected for radioactivity monitoring in the marine environment (Fucus serratus and Laminaria digitata). Despite the high concentration ratio, the exact mechanism of iodine uptake is still unclear. Chemical imaging by laser desorption/ionization mass spectrometry provided evidence that iodine is stored by kelps as I-. In this study we investigate in vivo iodine uptake in kelps (L. digitata) with an emphasis on seawater iodine chemical speciation. Our results showed that kelp plantlets were able to take up iodine in the forms of both IO3- and I-. We also observed transient net efflux of I- back to seawater but no IO3- efflux. Since the seaweed stores I- but takes up both IO3- and I-, IO3- was likely to be converted into I- at some point in the plantlet. One major outcome of our experiments was the direct observation of the kelp-based biogenic conversion of seawater IO3- into I-. On the basis of both IO3- and I- uptakes by the seaweed, we propose new steps in the possible iodine concentration mechanism used by brown algae.

Concentration factors and biological half-lives for the dynamic modelling of radionuclide transfers to marine biota in the English Channel.

The biokinetics of radionuclide transfers to biota in the marine environment can be modelled using two parameters, specific to both each element/radionuclide and biota. The Concentration Factor (CF) reflects the ratio between the activity concentrations in the biota and the surrounding seawater in steady state. The biological half-life (tb1/2) characterizes depuration kinetics for the radionuclide from the biota. While recommended CF values can be found in the literature, no guidelines actually exist for tb1/2 values. We used available time-series activity concentration measurements in biota in the English Channel, where controlled amounts of liquid radioactive waste are discharged by the ORANO La Hague reprocessing plant. We calculated the corresponding time-series activity concentrations in seawater for each biota dataset using an extensively-validated hydrodynamic model. We derived the values of CF and tb1/2 from seawater and biota data, to model radionuclide transfers between the two compartments. To assess the performance of the model, we analyzed the residual between observed and calculated levels in the biota. Datasets for macroalgae, mollusks, crustaceans and fish yielded parameters (CF, tb1/2) for H-3 (as body water and as organically bound tritium), C-14, Sb-125, Cs-137, I-129, Mn-54, Co-60, Zn-65 and Ru-106. After discussing the results and qualifying the model's reliability, we proposed recommendations for CF and tb1/2 for the purposes of the operational modelling of radionuclide transfers to biota in the marine environment.

Mass spectrometry - based imaging techniques for iodine-127 and iodine-129 detection and localization in the brown alga Laminaria digitata.

129I is one of the main radioisotopes of iodine derived from the nuclear fuel cycle that can be found sustainably in the environment due to its long half-life. In coastal marine environment, brown macroalgae, such laminariales (or kelps), are known to naturally feature highest rates of iodine accumulation, and to be an important source of biogenic volatile iodinated compounds released to the atmosphere. These seaweeds are therefore likely to be significantly marked by but also potential vectors of radioactive iodine. In order to better understand the chemical and isotopic speciation of iodine in brown algal tissues, we combined mass spectrometry-based imaging approaches in natural samples of Laminaria digitata young sporophytes, collected at two different locations along the south coast of the English Channel (Roscoff and Goury). Laser desorption ionization (LDI) and desorption electrospray-ionization techniques (DESI), coupled with mass spectrometry, confirmed the predominance of inorganic I- species on the surface of fresh algae, and a peripheral iodine localization when applied on micro-sections. Moreover, radioactive isotope 129I was not detected on plantlet surface or in stipe sections of algal samples collected near Roscoff but was detected in L. digitata samples collected at Goury, near La Hague, where controlled liquid radioactive discharges from the ORANO La Hague reprocessing plant occur. At the subcellular scale, cryo-fixed micro-sections of algal blade samples from both sites were further analyzed by secondary ion mass spectrometry (nano-SIMS), leading to similar results. Even if the signal detected for 129I was much weaker than for 127I in samples from Goury, the chemical imaging revealed some differences in extracellular distribution between radioactive and stable iodine isotopes. Altogether LDI and nano-SIMS are complementary and powerful techniques for the detection and localization of iodine isotopes in algal samples, and for a better understanding of radioactive and stable iodine uptake mechanisms in the marine environment.

A comprehensive assessment of two-decade radioactivity monitoring around the Channel Islands.

The Channel Islands are located in the Normand-Breton Gulf (NBG), in the mid-part of the English Channel (France, Normandy). In the northern part, off Cap La Hague, controlled amounts of radioactive liquid waste are discharged by the ORANO La Hague nuclear fuel reprocessing plant (RP). Radionuclides were monitored in the NBG to assess the dispersion of radioactive discharges from the RP in the marine environment. The temporal and spatial distribution of the data are consistent with the history of the discharges, with most gamma emitter radionuclide environmental levels being close to or below the current limits of detection. A clear fingerprint of H-3, C-14 and I-129 radionuclides discharged from the RP is measured. The hydrodynamics in the NBG do not yield a simple gradient with linear distance from the outfall of the RP. Modelling tools were used to understand how radioactive discharges spread from the source of input. Dispersion patterns clearly illustrate the different behaviours of soluble and non-soluble radionuclides. The study indicated that the footprint of radioactive liquid discharges by French nuclear facilities was still measurable in species collected from the NBG for the mostly dissolved radionuclides. The less conservative ones, with a high affinity for suspended matter, are potentially influenced by old releases. These pathways could be investigated by dedicated hydrodynamic dispersion models. Overall, in the Channel Islands the levels are low and consistent with the general decrease in liquid radionuclide discharges by the RP since the 1990s.

Phosphoinositide binding and phosphorylation act sequentially in the activation mechanism of ezrin.

Ezrin, a membrane-actin cytoskeleton linker, which participates in epithelial cell morphogenesis, is held inactive in the cytoplasm through an intramolecular interaction. Phosphatidylinositol 4,5-bisphosphate (PIP2) binding and the phosphorylation of threonine 567 (T567) are involved in the activation process that unmasks both membrane and actin binding sites. Here, we demonstrate that ezrin binding to PIP2, through its NH2-terminal domain, is required for T567 phosphorylation and thus for the conformational activation of ezrin in vivo. Furthermore, we found that the T567D mutation mimicking T567 phosphorylation bypasses the need for PIP2 binding for unmasking both membrane and actin binding sites. However, PIP2 binding and T567 phosphorylation are both necessary for the correct apical localization of ezrin and for its role in epithelial cell morphogenesis. These results establish that PIP2 binding and T567 phosphorylation act sequentially to allow ezrin to exert its cellular functions.

ERM proteins in epithelial cell organization and functions.

ERM (Ezrin, Radixin, Moesin) proteins are membrane-cytoskeleton linkers that regulate the structure and the function of specific domains of the plasma membrane. ERM proteins are expressed in all metazoan analyzed so far. Genetic analysis of ERM protein functions has recently been performed simultaneously in three different organisms, mouse, Drosophila melanogaster and C. elegans. These studies have revealed a remarkable conservation of the protein functions through evolution. Moreover they have shed light on the crucial role these proteins play in various physiological processes that occur in epithelial cells.

Morphogenetic degeneracies in the actomyosin cortex.

One of the great challenges in biology is to understand the mechanisms by which morphogenetic processes arise from molecular activities. We investigated this problem in the context of actomyosin-based cortical flow in C. elegans zygotes, where large-scale flows emerge from the collective action of actomyosin filaments and actin binding proteins (ABPs). Large-scale flow dynamics can be captured by active gel theory by considering force balances and conservation laws in the actomyosin cortex. However, which molecular activities contribute to flow dynamics and large-scale physical properties such as viscosity and active torque is largely unknown. By performing a candidate RNAi screen of ABPs and actomyosin regulators we demonstrate that perturbing distinct molecular processes can lead to similar flow phenotypes. This is indicative for a 'morphogenetic degeneracy' where multiple molecular processes contribute to the same large-scale physical property. We speculate that morphogenetic degeneracies contribute to the robustness of bulk biological matter in development.

Identification of cDNAs encoding HSP70 and HSP90 in the abalone Haliotis tuberculata: Transcriptional induction in response to thermal stress in hemocyte primary culture.

Heat-shock proteins are a multigene family of proteins whose expression is induced by a variety of stress factors. This work reports the cloning and sequencing of HSP70 and HSP90 cDNAs in the gastropod Haliotis tuberculata. The deduced amino acid sequences of both HSP70 and HSP90 from H. tuberculata shared a high degree of homology with their homologues in other species, including typical eukaryotic HSP70 and HSP90 signature sequences. We examined their transcription expression pattern in abalone hemocytes exposed to thermal stress. Real-time PCR analysis indicated that both HSP70 and HSP90 mRNA were expressed in control animals but rapidly increased after heat-shock.

Seasonal changes in mRNA encoding for cell stress markers in the oyster Crassostrea gigas exposed to radioactive discharges in their natural environment.

The North Cotentin area (Normandy, France) hosts several nuclear facilities among which the AREVA reprocessing plant of La Hague is responsible for controlled discharges of liquid radioactive wastes into the marine environment. The resulting increase in radioactivity is very small compared to natural radioactivity. However, concerns about environment protection prompted the scientific community to focus on the effects of the chronic exposure to low concentrations of radionuclides in non-human biota. This study contributes to the evaluation of the possible impact of radioactive discharges on the oyster Crassostrea gigas in the field. Real-time polymerase chain reaction was used to quantify the expression levels of genes involved in cell stress in the oyster. They included members of the heat shock protein family (Hsp70, Hsc72, Hsp90), superoxide dismutase (SOD) and metallothionein (MT). Times series measurements were built from periodic samplings in the natural environment in order to characterize the natural variability as well as possible seasonal fluctuations. The genes studied exhibited a general seasonal expression pattern with a peak value in winter. The data inversely correlated with seawater temperature and the nature of the relationship between gene expression and temperature is discussed. In parallel, oysters were collected in four locations on the French shores, exposed or not to radioactive liquid wastes from the nuclear facilities hosted in the North Cotentin. The comparison of data obtained in the reference location on the Atlantic coast (not exposed) and data from oysters of the English Channel (exposed) gave no evidence for any statistical difference. However, because of the complexity of the natural environment, we cannot rule out the possibility that other parameters may have masked the impact of radioactive discharges. This dense set of data is a basis for the use of the expression levels of those genes as biomarkers to address the question of the possible effects of chronic exposure of the oyster to low concentrations of radionuclides in controlled laboratory experimental conditions.

Correction: A dual pathways transfer model to account for changes in the radioactive caesium level in demersal and pelagic fish after the Fukushima Daï-ichi nuclear power plant accident.

[This corrects the article DOI: 10.1371/journal.pone.0172442.].

A dual pathways transfer model to account for changes in the radioactive caesium level in demersal and pelagic fish after the Fukushima Daï-ichi nuclear power plant accident.

The Fukushima Daï-ichi nuclear power plant (FDNPP) accident resulted in radioactive Cs being discharged into the local marine environment. While Cs bioaccumulates in biota and slowly depurates, the Cs concentrated in biota constitutes a source of Cs for animals feeding on each other. The marine biota therefore serves as a pool that recycles Cs, and this recycling process delays depuration in the fish feeding on this biota pool. Because the continental shelf is squeezed between the coast and very deep sea, the demersal marine species are confined to a narrow strip along the coast, close to the source of the radioactive input. Unlike demersal species, however, pelagic species are not restricted to the most contaminated area but instead spend some, if not most, of their time and feeding off-shore, far from the input source. We suggest that the feeding pathway for fish is a box whose size depends on their mobility, and that this feeding box is much larger and less contaminated (because of dilution through distance) for pelagic fish than for demersal fish. The aim of this paper is to test this hypothesis and to propose a simple operational model implementing two transfer routes: from seawater and from feeding. The model is then used to match the observational data in the aftermath of the FDNPP accident.

Iodine transfers in the coastal marine environment: the key role of brown algae and of their vanadium-dependent haloperoxidases.

Brown algal kelp species are the most efficient iodine accumulators among all living systems, with an average content of 1.0% of dry weight in Laminaria digitata, representing a ca. 30,000-fold accumulation of this element from seawater. Like other marine macroalgae, kelps are known to emit volatile short-lived organo-iodines, and molecular iodine which are believed to be a main vector of the iodine biogeochemical cycle as well as having a significant impact on atmospheric chemistry. Therefore, radioactive iodine can potentially accumulate in seaweeds and can participate in the biogeochemical cycling of iodine, thereby impacting human health. From a radioecological viewpoint, iodine-129 (129I, half-life of 1.6 x 10(7) years) is one of the most persistent radionuclide released from nuclear facilities into the environment. In this context, the speciation of iodine by seaweeds is of special importance and there is a need to further understand the mechanisms of iodine uptake and emission by kelps. Recent results on the physiological role and biochemistry of the vanadium haloperoxidases of brown algae emphasize the importance of these enzymes in the control of these processes.

Transfer of radiocarbon liquid releases from the AREVA La Hague spent fuel reprocessing plant in the English Channel.

The recent risk assessment by the North-Cotentin Radioecology Group (, 1999) outlined that (14)C has become one of the major sources of the low dose to man through seafood consumption. It was recommended that more data should be collected about (14)C in the local marine environment. The present study aims to respond to this recommendation. The estimation of (14)C activity in marine species is based on concentration factor values. The values reported here ranged from 1x10(3) to 5x10(3)Bqkg(-1)ww/BqL(-1). A comparison was made between the observed and predicted values. The accuracy of (14)C activity calculations was estimated between underestimation by a factor of 2 and over-estimation by 50% (95% confidence interval). However, the use of the concentration factor parameter is based on the biological and seawater compartments being in steady state. This assumption may not be met at short distances from the point of release of discharges, where rapid changes in seawater concentration may be smoothed out in living organisms due to transfer kinetics. The data processing technique, previously published by Fiévet and Plet (2003. Estimating biological half-lives of radionuclides in marine compartments from environmental time-series measurements. Journal of Environmental Radioactivity 65, 91-107), was used to deal with (14)C transfer kinetics, and carbon half-lives between seawater and a few biological compartments were thus estimated.

Radiation dose estimation for marine mussels following exposure to tritium: Best practice for use of the ERICA tool in ecotoxicological studies.

Accurate dosimetry is critically important for ecotoxicological and radioecological studies on the potential effects of environmentally relevant radionuclides, such as tritium ((3)H). Previous studies have used basic dosimetric equations to estimate dose from (3)H exposure in ecologically important organisms, such as marine mussels. This study compares four different methods of estimating dose to adult mussels exposed to 1 or 15 MBq L(-1) tritiated water (HTO) under laboratory conditions. These methods were (1) an equation converting seawater activity concentrations to dose rate with fixed parameters; (2) input into the ERICA tool of seawater activity concentrations only; (3) input into the ERICA tool of estimated whole organism concentrations (woTACs), comprising dry activity plus estimated tissue free water tritium (TFWT) activity (TFWT volume × seawater activity concentration); and (4) input into the ERICA tool of measured whole organism activity concentrations, comprising dry activity plus measured TFWT activity (TFWT volume × TFWT activity concentration). Methods 3 and 4 are recommended for future ecotoxicological experiments as they produce values for individual animals and are not reliant on transfer predictions (estimation of concentration ratio). Method 1 may be suitable if measured whole organism concentrations are not available, as it produced results between 3 and 4. As there are technical complications to accurately measuring TFWT, we recommend that future radiotoxicological studies on mussels or other aquatic invertebrates measure whole organism activity in non-dried tissues (i.e. incorporating TFWT and dry activity as one, rather than as separate fractions) and input this data into the ERICA tool.

Development of emergency response tools for accidental radiological contamination of French coastal areas.

The Fukushima nuclear accident resulted in the largest ever accidental release of artificial radionuclides in coastal waters. This accident has shown the importance of marine assessment capabilities for emergency response and the need to develop tools for adequately predicting the evolution and potential impact of radioactive releases to the marine environment. The French Institute for Radiological Protection and Nuclear Safety (IRSN) equips its emergency response centre with operational tools to assist experts and decision makers in the event of accidental atmospheric releases and contamination of the terrestrial environment. The on-going project aims to develop tools for the management of marine contamination events in French coastal areas. This should allow us to evaluate and anticipate post-accident conditions, including potential contamination sites, contamination levels and potential consequences. In order to achieve this goal, two complementary tools are developed: site-specific marine data sheets and a dedicated simulation tool (STERNE, Simulation du Transport et du transfert d'Eléments Radioactifs dans l'environNEment marin). Marine data sheets are used to summarize the marine environment characteristics of the various sites considered, and to identify vulnerable areas requiring implementation of population protection measures, such as aquaculture areas, beaches or industrial water intakes, as well as areas of major ecological interest. Local climatological data (dominant sea currents as a function of meteorological or tidal conditions) serving as the basis for an initial environmental sampling strategy is provided whenever possible, along with a list of possible local contacts for operational management purposes. The STERNE simulation tool is designed to predict radionuclide dispersion and contamination in seawater and marine species by incorporating spatio-temporal data. 3D hydrodynamic forecasts are used as input data. Direct discharge points or atmospheric deposition source terms can be taken into account. STERNE calculates Eulerian radionuclide dispersion using advection and diffusion equations established offline from hydrodynamic calculations. A radioecological model based on dynamic transfer equations is implemented to evaluate activity concentrations in aquatic organisms. Essential radioecological parameters (concentration factors and single or multicomponent biological half-lives) have been compiled for main radionuclides and generic marine species (fish, molluscs, crustaceans and algae). Dispersion and transfer calculations are performed simultaneously on a 3D grid. Results can be plotted on maps, with possible tracking of spatio-temporal evolution. Post-processing and visualization can then be performed.

Assessment of growth, genotoxic responses and expression of stress related genes in the Pacific oyster Crassostrea gigas following chronic exposure to ionizing radiation.

Marine organisms are exposed to low doses of anthropogenic contaminants during their entire life. Authorized amounts of radionuclides are discharged in the Channel by nuclear facilities. The Pacific oyster was used to investigate the potential impact of chronic exposure to ionizing radiation. Though we exposed larvae and spat for two weeks to much higher concentrations than those encountered near nuclear facilities, oyster growth and expression of 9 selected stress genes were not significantly changed. To determine potential DNA damage, 2year old oysters were exposed for two weeks to tritiated water. The comet assay was used to evaluate the level of DNA strand breaks in haemocytes, whilst the 'clearance rate' was used as a measure of physiological effects. Whilst other parameters did not alter, DNA damage significantly increased. Our results highlight the significance of the observed DNA damage and their potential consequences at higher levels of biological organization.

Effects of subchronic exposure to glyphosate in juvenile oysters (Crassostrea gigas): From molecular to individual levels.

Glyphosate-based herbicides are extensively used and can be measured in aquatic ecosystems, including coastal waters. The effect of glyphosate on non-target organisms is an issue of worldwide concern. The aim of this study was to investigate the effects of subchronic exposure to glyphosate in juvenile oysters, Crassostrea gigas. Yearling oysters were exposed to three concentrations of glyphosate (0.1, 1 and 100µgL(-1)) for 56days. Various endpoints were studied, from the individual level (e.g., gametogenesis and tissue alterations) to the molecular level (mRNA quantification), including biochemical endpoints such as glutathione-S-transferase (GST) and catalase activities and malondialdehyde content. No mortality and growth occurred during the experiment, and individual biomarkers revealed only slight effects. The levels of gene expression significantly increased in oysters exposed to the highest glyphosate concentration (GST and metallothioneins) or to all concentrations (multi-xenobiotic resistance). These results suggested an activation of defence mechanisms at the molecular level.

Estimating biological half-lifes of radionuclides in marine compartments from environmental time-series measurements.

Modelling radionuclide transfers between seawater and marine species on a short time scale basis requires being able to take into account the transfer kinetics. This means (1) to implement the effect of the biological half-lives of radionuclides together with the concentration factor in the calculation of transfers and (2), to get these kinetic parameters for each element and species. Biological half-lives are usually determined from laboratory labelling experiments with the challenge to match natural environmental conditions. The present work proposes a simple model that implements the effect of kinetic parameters in the calculation of transfers. This model is also used to derive the biological half-life and the concentration factor for 137Cs from time-series measurements of environmental concentrations in seawater and in the brown alga Fucus serratus, as an example. These transfer parameters are finally used to predict the Cs activities in Fucus serratus on the English Channel shores.