Modeling tobacco mosaic virus proliferation in protoplasts

The tobacco mosaic virus (TMV) is one of the most studied viruses. It is frequently used as a model in the research of virus-host interactions. The interest in understanding the mechanism of its proliferation stems basically from the field of agriculture, due to the detrimental effect this virus has on several crops. In addition to this direct application, virus-mediated protein expression systems, which are well established for the synthesis of foreign proteins in animal cell cultures, are now being applied to plants and plant cells by means of plant viral vectors. The use of transformed roots for the propagation of viral vectors has also been proposed. This work presents a mechanistic model describing the transient process of TMV multiplication in a protoplast (a wall-deprived cell). It aims to be a mathematical tool able to simulate the transient behavior of the main molecular pools taking part in the process, which will be useful for exploring, understanding and predicting the dynamics of a host-virus system. The variables considered are the pools of the main molecules taking part in the viral replication process. The basic balance equations for the cellular pools are presented and a satisfactory fit of the model to the experimental data is shown. The presented model is a necessary step toward the formulation of a basic mechanistic model for the systemic propagation of the virus in a plant tissue. It may be extended in many directions as to the optimization of a system for the production of a foreign protein, to the simulation of manipulation of the virus-cell interaction by external factors, to the mechanism of gene silencing or to the prediction of co-infection dynamics.Fil: Merchuk, Jose C. Ben-Gurion University of the Negev; IsraelFil: Sánchez-Mirón, Asterio. Universidad de Almería; EspañaFil: Asurmendi, Sebastian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Shacham, Mordechai. Ben-Gurion University of the Negev; Israe

Método para la cuantificación fluoirimétrica de la enzima LDH en disolución

Número de publicación: ES2527796 A1 (29.01.2015) También publicado como: ES2527796 B1 (10.11.2015) Número de Solicitud: Consulta de Expedientes OEPM (C.E.O.) P201300775 (29.07.2013)La invención está dirigida al desarrollo de un método y creación de un kit para la cuantificación del efecto citotóxico y/o viabilidad celular ante agentes químicos (elemento, compuesto, mezcla o fármaco en cualquier de sus estados) y/o físicos (i.e. iluminación, temperatura, turbulencia, fluidodinámica, etc.) que puedan producir rotura de la membrana celular. El kit mide la liberación de la enzima citoplasmática lactato deshidrogenasa (LDH) proveniente de células muertas y/o lisadas. Esta enzima cataliza la oxidación del lactato, presente en el kit, a piruvato. Durante la reacción, el NAD+, también presente en el kit, es reducido a NADH. La concentración de LDH, se cuantifica a través de la fluorescencia del NADH formado. Las mediciones indirectas de la enzima LDH se realizan en el sobrenadante de cultivos celulares. El método es aplicable tanto en medios inorgánicos para microalgas como en medios comerciales para células animales y de insecto.Universidad de Almerí

Simultaneous Effect of Temperature and Irradiance on Growth and Okadaic Acid Production from the Marine Dinoflagellate Prorocentrum belizeanum

Benthic marine dioflagellate microalgae belonging to the genus Prorocentrum are a major source of okadaic acid (OA), OA analogues and polyketides. However, dinoflagellates produce these valuable toxins and bioactives in tiny quantities, and they grow slowly compared to other commercially used microalgae. This hinders evaluation in possible large-scale applications. The careful selection of producer species is therefore crucial for success in a hypothetical scale-up of culture, as are appropriate environmental conditions for optimal growth. A clone of the marine toxic dinoflagellate P. belizeanum was studied in vitro to evaluate its capacities to grow and produce OA as an indicator of general polyketide toxin production under the simultaneous influence of temperature (T) and irradiance (I0). Three temperatures and four irradiance levels were tested (18, 25 and 28 °C; 20, 40, 80 and 120 µE·m−2·s−1), and the response variables measured were concentration of cells, maximum photochemical yield of photosystem II (PSII), pigments and OA. Experiments were conducted in T-flasks, since their parallelepipedal geometry proved ideal to ensure optically thin cultures, which are essential for reliable modeling of growth-irradiance curves. The net maximum specific growth rate (µm) was 0.204 day−1 at 25 °C and 40 µE·m−2·s−1. Photo-inhibition was observed at I0 > 40 μEm−2s−1, leading to culture death at 120 µE·m−2·s−1 and 28 °C. Cells at I0 ≥ 80 µE·m−2·s−1 were photoinhibited irrespective of the temperature assayed. A mechanistic model for µm-I0 curves and another empirical model for relating µm-T satisfactorily interpreted the growth kinetics obtained. ANOVA for responses of PSII maximum photochemical yield and pigment profile has demonstrated that P. belizeanum is extremely light sensitive. The pool of photoprotective pigments (diadinoxanthin and dinoxanthin) and peridinin was not able to regulate the excessive light-absorption at high I0-T. OA synthesis in cells was decoupled from optimal growth conditions, as OA overproduction was observed at high temperatures and when both temperature and irradiance were low. T-flask culture observations were consistent with preliminary assays outdoors

Procedimiento para obtener extracto de pulpo, el producto obtenido y su aplicación como suplemento para el cultivo y criopreservación de tejido y de células en suspensión de invertebrados marinos

Número de publicación: ES2261054 A1 (01.11.2006) También publicado como: ES2261054 B1 (16.11.2007) Número de Solicitud: Consulta de Expedientes OEPM (C.E.O.)P200403171 (31.12.2004)Extracto de pulpo como suplemento para el cultivo y criopreservación de tejido y de células en suspensión de invertebrados marinos. La presente invención se refiere a un procedimiento para obtención de un suplemento para el cultivo in vitro y criopreservación de tejido y de células en suspensión de invertebrados marinos a base de extracto acuoso de pulpo homogeneizado y la utilización de dicho suplemento preparación de medios para la criopreservación de células en suspensión y de tejidos de organismos invertebrados marinos.Universidad de Almerí

Production of extracts with anaesthetic activity from the culture of Heterosigma akashiwo in pilot-scale photobioreactors

The shear-sensitive microalga Heterosigma akashiwo is known to produce brevetoxin-like compounds that are active in voltage-dependent sodium channels. In this work, we present a study on the production of anaesthetic extracts from H. akashiwo biomass obtained in low-shear bioreactors at different growth phases. The photobioreactors (PBRs) used had specific configurations and were operated in such a way as to avoid cellular damage by hydrodynamic stress. Cultures were developed in small static-control flasks and PBRs with volumes ranging from 1.5 L to 200 L. The bioactivity of the produced extracts was assessed in vitro (Neuro-2a cell-based assay) and in vivo (Zebra fish model). Bioactivity depended slightly on the growth phase and culture system, with greater toxicity with the Neuro-2a model when stationary-phase extracts were used. Interestingly, extracts were not cytotoxic against other human cell lines. Steady production of anaesthetic bioactives was observed. In vivo anaesthetic efficacy, assessed with the Zebra fish model, was similar to that of commercial products, and without any observed mortality at 24-h post exposure

Sublethal effect of the toxic dinoflagellate Karlodinium veneficum on early life stages of zebrafish (Danio rerio)

Dinoflagellates of the genus Karlodinium are ichthyotoxic species that produce toxins including karlotoxins and karmitoxins. Karlotoxins show hemolytic and cytotoxic activities and have been associated with fish mortality. This study evaluated the effect of toxins released into the environment of Karlodinium veneficum strain K10 (Ebro Delta, NW Mediterranean) on the early stages of Danio rerio (zebrafish). Extracts of the supernatant of K10 contained the mono-sulfated KmTx-10, KmTx-11, KmTx-12, KmTx-13, and a di-sulfated form of KmTx-10. Total egg mortality was observed for karlotoxin concentration higher than 2.69 μg L−1. For 1.35 μg L−1, 87% of development anomalies were evidenced (all concentrations were expressed as KmTx-2 equivalent). Larvae of 8 days postfertilization exposed to 1.35 µg L−1 presented epithelial damage with 80% of cells in the early apoptotic stage. Our results indicate that supernatants with low concentration of KmTxs produce both lethal and sublethal effects in early fish stages. Moreover, apoptosis was induced at concentrations as low as 0.01 μg L−1. This is of great relevance since detrimental long-term effects due to exposure to low concentrations of these substances could affect wild and cultured fish

Bioactives Overproduction through Operational Strategies in the Ichthyotoxic Microalga Heterosigma akashiwo Culture

The red tide-forming microalga Heterosigma akashiwo has been associated with massive events of fish deaths, both wild and cultured. Culture conditions are responsible for the synthesis or accumulation of some metabolites with different interesting bioactivities. H. akashiwo LC269919 strain was grown in a 10 L bubble column photobioreactor artificially illuminated with multi-coloured LED lights. Growth and production of exopolysaccharides, polyunsaturated fatty acids (PUFAs), and carotenoids were evaluated under different culture modes (batch, fed-batch, semicontinuous, and continuous) at two irradiance levels (300 and 700 µE·s−1·m−2). Continuous mode at the dilution rate of 0.2·day−1 and 700 µE·s−1·m−2 provided the highest production of biomass, PUFAs (132.6 and 2.3 mg·L−1·day−1), and maximum fucoxanthin productivity (0.16 mg·L−1·day−1). The fed-batch mode accumulated exopolysaccharides in a concentration (1.02 g·L−1) 10-fold over the batch mode. An extraction process based on a sequential gradient partition with water and four water-immiscible organic solvents allowed the isolation of bioactive fucoxanthin from methanolic extracts of H. akashiwo. Metabolites present in H. akashiwo, fucoxanthin and polar lipids (i.e., eicosapentaenoic acid (EPA)), or probably such as phytosterol (β-Sitosterol) from other microalgae, were responsible for the antitumor activity obtained.This research was funded by the General Secretariat of Universities, Research and Technology of the Andalusian Government (grant: P18-RT-2477) and the State Research Agency (grants PID2019-109476RB-C22) of the Spanish Ministry of Science, Innovation and Universities

Isolation and Structural Elucidation of New Amphidinol Analogues from Amphidinium carterae Cultivated in a Pilot-Scale Photobioreactor

The demand for valuable products from dinoflagellate biotechnology has increased remarkably in recent years due to their many prospective applications. However, there remain many challenges that need to be addressed in order to make dinoflagellate bioactives a commercial reality. In this article, we describe the technical feasibility of producing and recovering amphidinol analogues (AMs) excreted into a culture broth of Amphidinium carterae ACRN03, successfully cultured in an LED-illuminated pilot-scale (80 L) bubble column photobioreactor operated in fed-batch mode with a pulse feeding strategy. We report on the isolation of new structurally related AMs, amphidinol 24 (1, AM24), amphidinol 25 (2, AM25) and amphidinol 26 (3, AM26), from a singular fraction resulting from the downstream processing. Their planar structures were elucidated by extensive NMR and HRMS analysis, whereas the relative configuration of the C-32®C-47 bis-tetrahydropyran core was confirmed to be antipodal in accord with the recently revised configuration of AM3. The hemolytic activities of the new metabolites and other related derivatives were evaluated, and structure–activity conclusions were established. Their isolation was based on a straightforward and high-performance bioprocess that could be suitable for the commercial development of AMs or other high-value compounds from shear sensitive dinoflagellates

Production of extracts with anaesthetic activity from the culture of Heterosigma akashiwo in pilot-scale photobioreactors

The shear-sensitive microalga Heterosigma akashiwo is known to produce brevetoxin-like compounds that are active in voltage-dependent sodium channels. In this work, we present a study on the production of anaesthetic extracts from H. akashiwo biomass obtained in low- shear bioreactors at different growth phases. The photobioreactors (PBRs) used had specific configurations and were operated in such a way as to avoid cellular damage by hydrodynamic stress. Cultures were developed in small static-control flasks and PBRs with volumes ranging from 1.5 L to 200 L. The bioactivity of the produced extracts was assessed in vitro (Neuro- 2a cell-based assay) and in vivo (Zebra fish model). Bioactivity depended slightly on the growth phase and culture system, with greater toxicity with the Neuro-2a model when stationary-phase extracts were used. Interestingly, extracts were not cytotoxic against other human cell lines. Steady production of anaesthetic bioactives was observed. In vivo anaesthetic efficacy, assessed with the Zebra fish model, was similar to that of commercial products, and without any observed mortality at 24-h post exposure

CFD-aided optimization of a laboratory-scale centrifugation for a shear-sensitive insect cell line

A study has been conducted to assess cell damage during centrifugation and to optimize this operation for the insect cell line Se301. Experiments were carried out in a discontinuous centrifuge using Falcon tubes of different sizes as containers. The cells were easily recovered from the cell suspension and the time required for complete sedimentation was found to be independent of tube size or design. Cell damage was observed once the cells were sedimented, therefore this process depends on both the residence time of the cells in the pellet and on the acceleration applied. The sensitivity of the cells to this operation was higher than for other naked microalgae or for very sensitive red blood cells. Indeed, excessive treatment produced cell damage that reduced the productivity of subsequent cultures. CFD simulations were carried out in 2D and a good agreement was found between the CFD and experimental values in terms of the time required for full sedimentation. In addition, this technique allowed the calculation of shear stress, a key variable in the study of cell sensitivity to flow