Calcium-dependent protein kinase OsCPK10 mediates both drought tolerance and blast disease resistance in rice plants

OsCPK10 mediates drought tolerance and blast disease resistance in rice plants by enhancing their reactive oxygen species scavenging capacity and so reducing oxidative damage associated with both stresses. Plant growth and productivity is negatively affected by different stresses. Most stresses trigger calcium signals that initiate acclimation responses in plants. The multigene family of plant calcium-dependent protein kinases (CPKs) functions in multiple stress responses by transducing calcium signals into phosphorylation events. This work reports that the OsCPK10 isoform positively mediates tolerance to different stresses in rice plants by enhancing their antioxidant capacity and protecting them from reactive oxygen species (ROS) damage, with the uncontrolled generation of ROS being a common feature of these stresses. Here, we show that the constitutive accumulation of an HA-tagged OsCPK10 full-length protein enhances the hydrogen peroxide detoxifying capacity of rice plants during desiccation. This is achived by modulating the accumulation of catalase proteins, which reduces the extent of lipid peroxidation and protects the integrity of cell membranes, resulting in drought tolerance. OsCPK10HA accumulation also confers blast disease resistance by interfering with fungal necrotrophic growth via a reduction in the accumulation of hydrogen peroxide. Furthermore, we show by bimolecular complementation assays that OsCPK10 is a plasma membrane protein that physically interacts in vivo with catalase A. OsCPK10 therefore appears to be a good molecular target to improve tolerance to abiotic stresses as well as to blast disease, which limit rice crop productivity

Recommendation system to determine suitable and viable hiking routes: a prototype application in Sierra de las Nieves Nature Reserve (southern Spain)

This paper describes a system for recommending hiking routes to help manage hiking activities in a protected area. The system proposes various routes, based on five criteria that maximize some aspects of hikers’ requirements (by analyzing the viability and difficulty of the trails) and also those of protected areas managers (by proposals to relieve congestion in areas already used for hiking and to promote awareness of new ones, as a contribution to environmental education). The recommendation system uses network analysis, multi-criteria decision analysis and geographic information system by free software tools: PgRouting, PostgreSQL and PostGIS. This system has been tested in Sierra de las Nieves Nature Reserve (Andalusia, Spain). Of the 182 routes obtained by the system, 62 (34%) are considered viable for hikers in Sierra de las Nieves, taking into account the type of user most likely to visit this protected area. Most routes have a high difficulty level, which is coherent with the mountainous character of the protected area.This study is a contribution to the Research Projects SEJ2007-67690, funded by the Spanish Ministry of Science and Innovation, and P07HUM-03049, funded by the Ministry of Innovation, Science and Enterprise of the Andalusian Regional Government. This work was carried out within the framework of research group HUM 776 (Geographic Analysis) of the Andalusian Regional Government

Carrot discard as a promising feedstock to produce 2,3-butanediol by fermentation with P. polymyxa DSM 365

Producción CientíficaThe valorization of fruit and vegetable residues (such as carrot discard) and their microbial conversion into 2,3-butanediol (BDO) can be considered as a very interesting way to reduce food waste and sustainably originate high value-added products. This work analyzes the valorization of carrot discard as feedstock for 2,3-butanediol (BDO) production by Paenibacillus polymyxa DSM 365. The influences of stirring and the presence of tryptone (nitrogen source) are studied. Furthermore, in order to evaluate the influence of the pre-culture medium (nitrogen source, nutrients, and pH) and the substrate, fermentation assays in simple and mixture semi-defined media (glucose, fructose, and/or galactose) were also carried out. As a result, 18.8 g/L BDO, with a BDO yield of 0.43 g/g (86% of its theoretical value), could be obtained from carrot discard enzymatic hydrolysate at 100 rpm, no tryptone, and pre-culture Häßler medium. No hydrothermal pre-treatment was necessary for BDO production from carrot discard, which increases the profitability of the process. Therefore, 18.8 g BDO, as well as 2.5 g ethanol and 2.1 g acetoin by-products, could be obtained from 100 g of carrot discard (dry matter).Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación (AEI)/10.13039/501100011033 - (project PID2020- 115110RB-I00,Junta de Castilla y León - (grant CLU 2017-09, CL-EI-2021-07, UIC 320

Efficient production and characterization of the novel and highly active antifungal protein AfpB from Penicillium digitatum

Filamentous fungi encode distinct antifungal proteins (AFPs) that offer great potential to develop new antifungals. Fungi are considered immune to their own AFPs as occurs in Penicillium chrysogenum, the producer of the well-known PAF. The Penicillium digitatum genome encodes only one afp gene (afpB), and the corresponding protein (AfpB) belongs to the class B phylogenetic cluster. Previous attempts to detect AfpB were not successful. In this work, immunodetection confirmed the absence of AfpB accumulation in wild type and previous recombinant constitutive P. digitatum strains. Biotechnological production and secretion of AfpB were achieved in P. digitatum with the use of a P. chrysogenum-based expression cassette and in the yeast Pichia pastoris with the α-factor signal peptide. Both strategies allowed proper protein folding, efficient production and single-step purification of AfpB from culture supernatants. AfpB showed antifungal activity higher than the P. chrysogenum PAF against the majority of the fungi tested, especially against Penicillium species and including P. digitatum, which was highly sensitive to the self-AfpB. Spectroscopic data suggest that native folding is not required for activity. AfpB also showed notable ability to withstand protease and thermal degradation and no haemolytic activity, making AfpB a promising candidate for the control of pathogenic fungi

Dinuclear CoIIYIII vs Tetranuclear Co2IIY2III complexes: The effect of increasing molecular size on magnetic anisotropy and relaxation dynamics

The authors acknowledge financial support from Ministerio de Economía y Competitividad (MINECO) for Project CTQ2014-56312-P. The authors also acknowledge a Maria de Maeztu grant (MDM-2017-0767) and thank Ministerio de Educación, Cultura y Deporte for projects PGC2018-093863-B-C21 and PGC2018-102052-B-C21, the Junta de Andalucía (FQM-195) and the Project of excellence P11-FQM-7756, the University of Granada and the Generalitat de Catalunya (SGR2017-1289). M. A. P. thanks MINECO for a Juan de la Cierva Incorporation contract (IJCI-2014-19485). E. R. thanks the Generalitat de Catalunya for an ICREA Academia fellowship and CSUC for computational resources. S. G.-C. thanks Generalitat de Catalunya for a Beatriu de Pinós Fellowship (2017 BP 00080). The HFEPR studies were supported by the NHMFL, which is funded by the National Science Foundation (Cooperative Agreement DMR 1157490) and the State of Florida. The authors thank Dr. A. Ozarowski (NHMFL) for his EPR software SPIN.A new CoII2YIII2 complex with the formula [{Co(u-L)Y(NO3)}2(u-CO3)2]·2CH3OH·2H2O (where H2L = N,N’,N”-trimethyl-N,N”-bis(2-hydroxy-3-methoxy-5-methylbenzyl) diethylenetriamine) has been prepared and its structure solved by single-crystal X-Ray diffraction. The tetranuclear structure is formed by the connection of two [Co(μ-L)Y(NO3)] dinuclear units through two carbonate bridging ligands, which exhibit a tetradentate coordination mode. The CoII ion exhibits a slightly distorted octahedral CoN3O3 coordination environment. From direct-current magnetic data a large and positive axial anisotropy parameter was extracted (D = +82.62 cm-1) and its sign unambiguously confirmed by HFEPR spectra and ab initio calculations. The extracted D value is rather larger than those previously reported for the analogous CoIIYIII dinuclear complexes, which agrees with the fact that the CoII ion in the CoII2YIII2 complex exhibits the lower distortion from the octahedral geometry in this family of CoIInYIIIn complexes. Dynamic ac magnetic measurements show that the reported compound presents field-induced slow relaxation for magnetization reversal, through a combination of direct and Raman processes. Magnetic measurements on the diluted magnetic counterpart (Zn/Co = 10/1) show the persistence of these processes, pointing out their single-ion origin. The Raman relaxation process for the Co2Y2 complex is faster that those observed for the CoY dinuclear counterparts. This fact and the existence of the persistent direct process at low temperature could be because the former molecule is larger and flexible than the latter ones.Maria de Maeztu grant MDM-2017-0767Ministerio de Educación, Cultura y Deporte PGC2018-093863-B-C21, PGC2018-102052-B-C21Junta de Andalucía FQM-195, P11-FQM-7756University of GranadaMinisterio de Economía y Competitividad CTQ2014-56312-P, IJCI-2014-19485Generalitat de Catalunya SGR2017-1289, 2017 BP 00080National Science Foundation DMR 115749

Waste-to-fuel technologies for the bioconversion of carrot discards into biobutanol

Producción CientíficaCarrot discard was evaluated as a raw material for acetone-butanol-ethanol (ABE) fermentation. Different strategies based on hydrothermal pretreatment and/or enzymatic hydrolysis were compared for biobutanol production from carrot discard pulp. In addition, the use of different types of enzymes and diverse enzyme mixtures were evaluated. In this way, total sugar recoveries of up to 76%, and butanol and ABE concentrations of 7.4 and 11.5 g/L, respectively (74 g butanol and 115 g ABE/kg carrot pulp), were achieved when the carrot discard pulp was enzymatically hydrolyzed, without pretreatment, using a mixture of enzymes of Cellic CTec2 and Viscozyme L at a dosage of 0.1 and 0.2 g/g, respectively. When a hydrothermal pretreatment was applied, a total sugar recovery of 88%, 6.9 g/L butanol and 10.1 g/L ABE (69 g butanol and 101 g ABE/kg carrot pulp) were attained using the same mixture of enzymes. In this way, no hydrothermal pretreatment would be necessary to produce ABE from carrot discard, which is very interesting for the profitability of the process. Furthermore, the carrot discard juice yielded 6.4 and 9 g/L butanol and ABE, respectively, showing that all the carrot discards could be used for ABE production.Ministerio de Ciencia, Innovación y Universidades (project PID2020-115110RB-I00/AEI/10.13039/501100011033))Junta de Castilla y León (projects UIC 320, VAG028G19, CLU-2017-09 and CL-EI-2021-07

Influence of freeze-drying and γ-irradiation in preclinical studies of flurbiprofen polymeric nanoparticles for ocular delivery using d-(+)-trehalose and polyethylene glycol.

This study investigated the suspension of poly(ε-caprolactone) nanoparticles as an ocular delivery system for flurbiprofen (FB-PεCL-NPs) in order to overcome the associated problems, such as stability, sterility, tolerance, and efficacy, with two different FB-PεCL-NP formulations. The formulations were stabilized with poloxamer 188 (1.66% and 3.5%) and submitted individually for freeze-drying and γ-irradiation with polyethylene glycol 3350 (PEG3350) and d-(+)-trehalose (TRE). Both formulations satisfied criteria according to all physicochemical parameters required for ocular pharmaceuticals. The FB-PεCL-NP formulations showed non-Newtonian behavior and sustained drug release. Ex vivo permeation analysis using isolated ocular pig tissues suggested that the presence of PEG3350 results in a reduction of FB transcorneal permeation. Moreover, TRE improved the penetration of FB across the cornea, especially after γ-irradiation. In addition, both formulations did not show a significant affinity in increasing FB transscleral permeation. Both formulations were classified as nonirritating, safe products for ophthalmic administration according to hen's egg test-chorioallantoic membrane and Draize eye test. Furthermore, an in vivo anti-inflammatory efficacy test showed that irradiated FB-PεCL-NPs prepared with PEG3350 (IR-NPsPEG) have longer anti-inflammatory effects than those presented with irradiated FB-PεCL-NPs prepared with TRE (IR-NPsTRE). IR-NPsPEG showed a suitable physical stability after an aqueous reconstitution over .30 days. This study concludes that both formulations meet the Goldman's criteria and demonstrate how irradiated nanoparticles, with innovative permeation characteristics, could be used as a feasible alternative to a flurbiprofen solution for ocular application in clinical trials

Autonomic nervous system measurement in hyperbaric environments using ECG and PPG signals

The main aim of this work was to characterise the Autonomic Nervous System (ANS) response in hyper- baric environments using electrocardiogram (ECG) and pulse- photoplethysmogram (PPG) signals. To that end, 26 subjects were introduced into a hyperbaric chamber and five stages with different atmospheric pressures (1 atm; descent to 3 and 5 atm; ascent to 3 and 1 atm) were recorded. Respiratory information was extracted from the ECG and PPG signals and a combined respiratory rate was studied. This information was also used to analyse Heart Rate Variability (HRV) and Pulse Rate Variability (PRV). The database was cleaned by eliminating those cases where the respiratory rate dropped into the low frequency band (LF: 0.04-0.15 Hz) and those in which there was a discrepancy between the respiratory rates estimated using the ECG and PPG signals. Classical temporal and frequency indices were calculated in such cases. The ECG results showed a time-related depen- dency, with the heart rate and sympathetic markers (normalised power in LF and LF/HF ratio) decreasing as more time was spent inside the hyperbaric environment. A dependency between the atmospheric pressure and the parasympathetic response, as reflected in the high frequency band power (HF: 0.15-0.40 Hz), was also found, with power increasing with atmospheric pressure. The combined respiratory rate also reached a maximum in the deepest stage, thus highlighting a significant difference between this stage and the first one. The PPG data gave similar findings and also allowed the oxygen saturation to be computed, therefore we propose the use of this signal for future studies in hyperbaric environments

Biotechnological production of the cell penetrating antifungal PAF102 peptide in pichia pastoris

Antimicrobial peptides (AMPs) have potent and durable antimicrobial activity to a wide range of fungi and bacteria. The growing problem of drug-resistant pathogenic microorganisms, together with the lack of new effective compounds, has stimulated interest in developing AMPs as anti-infective molecules. PAF102 is an AMP that was rationally designed for improved antifungal properties. This cell penetrating peptide has potent and specific activity against major fungal pathogens. Cecropin A is a natural AMP with strong and fast lytic activity against bacterial and fungal pathogens, including multidrug resistant pathogens. Both peptides, PAF102 and Cecropin A, are alternative antibiotic compounds. However, their exploitation requires fast, cost-efficient production systems. Here, we developed an innovative system to produce AMPs in Pichia pastoris using the oleosin fusion technology. Oleosins are plant-specific proteins with a structural role in lipid droplet formation and stabilization, which are used as carriers for recombinant proteins to lipid droplets in plant-based production systems. This study reports the efficient production of PAF102 in P. pastoris when fused to the rice plant Oleosin 18, whereas no accumulation of Cecropin A was detected. The Ole18-PAF102 fusion protein targets the lipid droplets of the heterologous system where it accumulates to high levels. Interestingly, the production of this fusion protein induces the formation of lipid droplets in yeast cells, which can be additionally enhanced by the coexpression of a diacylglycerol transferase gene that allows a three-fold increase in the production of the fusion protein. Using this high producer strain, PAF102 reaches commercially relevant yields of up to 180 mg/l of yeast culture. Moreover, the accumulation of PAF102 in the yeast lipid droplets facilitates its downstream extraction and recovery by flotation on density gradients, with the recovered PAF102 being biologically active against pathogenic fungi. Our results demonstrate that plant oleosin fusion technology can be transferred to the well-established P. pastoris cell factory to produce the PAF102 antifungal peptide, and potentially other AMPs, for multiple applications in crop protection, food preservation and animal and human therapies