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.

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.

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.

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.

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.

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.

Systematic genetic interaction screens uncover cell polarity regulators and functional redundancy.

Although single-gene loss-of-function analyses can identify components of particular processes, important molecules are missed owing to the robustness of biological systems. Here we show that large-scale RNAi screening for suppression interactions with functionally related mutants greatly expands the repertoire of genes known to act in a shared process and reveals a new layer of functional relationships. We performed RNAi screens for 17 Caenorhabditis elegans cell polarity mutants, generating the most comprehensive polarity network in a metazoan, connecting 184 genes. Of these, 72% were not previously linked to cell polarity and 80% have human homologues. We experimentally confirmed functional roles predicted by the network and characterized through biophysical analyses eight myosin regulators. In addition, we discovered functional redundancy between two unknown polarity genes. Similar systematic genetic interaction screens for other biological processes will help uncover the inventory of relevant genes and their patterns of interactions.

Effect of chronic exposure to zinc in young spats of the Pacific oyster (Crassostrea gigas).

The marine coastal environment is exposed to a mixture of environmental pollutants of anthropogenic origin, resulting in chronic low concentrations of contaminants. As a consequence, most coastal marine species are exposed to low doses of such pollutants during their entire life. Many marine species live for years in their natural environment, whereas they do not under laboratory exposure conditions. Using early stages of development in laboratory work allows animals to be chronically exposed from an early age over a reasonable experiment period. In the present study, the authors investigated the effect of chronic exposure to zinc in spats of the Pacific oyster (Crassostrea gigas), from metamorphosis up to 10 weeks. The authors investigated integrated biological endpoints that would account for the apparent general health of the animals as well as molecular markers showing more subtle effects that could potentially go unnoticed at a biologically integrated level. The authors measured in parallel both growth and the transcriptional level of target stress genes. Growth was monitored by image analysis of large samples to avoid high variability and ensure statistical robustness. A dose-response relationship was derived from growth data, yielding a median effective concentration (EC50) of 7.55 µM. Stress genes selected on the basis of available RNA sequences in C. gigas included genes involved in chaperone proteins, oxidative stress, detoxification, and cell cycle regulation. Out of nine stress target genes, only metallothionein displayed overexpression in response to high levels of zinc.

Effect of ionizing radiation on the transcription levels of cell stress marker genes in the Pacific oyster Crassostrea gigas.

In the North-Cotentin (Normandy, France), the marine environment is chronically exposed to liquid releases from the La Hague nuclear fuel recycling plant (Areva NC), resulting in a small increase in radioactivity compared to natural background. The transcriptional expression levels of stress genes were investigated in oysters exposed to ionizing radiation. Adult oysters were kept for 6 weeks in (60)Co-labeled seawater (400 Bq liter(-1)), resulting in a total dose of 6.2 mGy. Transcriptional expression of target genes was monitored by reverse-transcription quantitative polymerase chain reaction. Nine genes were selected for their sensitivity to ionizing radiation based on the literature and available DNA sequences. They included genes encoding chaperone proteins and genes involved in oxidative stress regulation, cell detoxification and cell cycle regulation. Of the nine genes of interest, metallothionein (MT) and multi-drug resistance (MDR) displayed significant overexpression in response to chronic exposure to an internal low dose. For comparison, oysters were acutely exposed to an external high dose for 100 min, resulting in 20 Gy, and the same target gene expression analysis was carried out. As in the case of chronic exposure to the low dose, MT and MDR displayed significant increases. The results suggest that the transcriptional expression levels of cell stress genes may be used as a biosensor of exposure of oysters to ionizing radiation, with a particular focus on the MT and MDR genes. However, the upregulation of these potential players in the cellular response to radiation-induced stress was not correlated with mortality or apparent morbidity. The possible role of these stress genes in the resistance of oysters to ionizing radiation is discussed.

Dealing with non-detect values in time-series measurements of radionuclide concentration in the marine environment.

The attention of scientists in the field of environmental radioactivity is drawn to statistical methods recommended by Dennis Helsel for dealing with datasets including measurements that fall below the detection limits, as often encountered in environmental monitoring programmes. The methods are described by Helsel in his book entitled "Nondetects and Data Analysis: Statistics for Censored Environmental Data" (John Wiley and Sons, New York, 2005, 250p). These methods are applied to a data subset (using data from France) of the Radioactive Substance Committee (OSPAR commission for the protection of the marine environment of the North-East Atlantic), corresponding to time-series measurements of Cs-137 concentration in seaweed in the vicinity of the Areva NC reprocessing plant at La Hague, which is used as an illustrative example. Despite the presence of 163 non-detect values out of 514 measurements, it is possible to estimate descriptive parameters and perform statistical tests to compare concentration levels between two periods of time. Finally, evidence is obtained for an overall decreasing trend with time. The benefits of these statistical methods for data analysis are discussed.

Transcriptional expression levels of cell stress marker genes in the Pacific oyster Crassostrea gigas exposed to acute thermal stress.

During the annual cycle, oysters are exposed to seasonal slow changes in temperature, but during emersion at low tide on sunny summer days, their internal temperature may rise rapidly, resulting in acute heat stress. We experimentally exposed oysters to a 1-h acute thermal stress and investigated the transcriptional expression level of some genes involved in cell stress defence mechanisms, including chaperone proteins (heat shock proteins Hsp70, Hsp72 and Hsp90 (HSP)), regulation of oxidative stress (Cu-Zn superoxide dismutase, metallothionein (MT)), cell detoxification (glutathione S-transferase sigma, cytochrome P450 and multidrug resistance (MDR1)) and regulation of the cell cycle (p53). Gene mRNA levels were quantified by reverse transcription-quantitative polymerase chain reaction and expressed as their ratio to actin mRNA, used as a reference. Of the nine genes studied, HSP, MT and MDR1 mRNA levels increased in response to thermal stress. We compared the responses of oysters exposed to acute heat shock in summer and winter and observed differences in terms of magnitude and kinetics. A larger increase was observed in September, with recovery within 48 h, whereas in March, the increase was smaller and lasted more than 2 days. The results were also compared with data obtained from the natural environment. Though the functional molecule is the protein and information at the mRNA level only has limitations, the potential use of mRNAs coding for cell stress defence proteins as early sensitive biomarkers is discussed.

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.

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.