Novel incorporation of red-stage haematococcus pluvialis wet paste as a colourant and enhancer of the organoleptic and functional properties of filloas †

Haematococcus pluvialis Flotow is a microalga used as a nutraceutical, due to its high content in bioactive compounds, mainly carotenoids, in which astaxanthin stands out [1]. Furthermore, H. plu- vialis has shown a high antioxidant potential, and combined with its intense red colour, this microalga could have dual functionality: as a colourant and a bioactive ingredient [ 2]. The process to obtain this ingredient involves several transformation steps—namely, lyophilisation and saponification—raising the costs to develop and obtain free astaxanthin, which paradoxically presents greater instability and solubility than its esterified counterpart [ 3]. Thus, this study provides an alternative approach for the application of red, astaxanthin-rich, H. pluvialis wet paste as a partial substitute for wheat flour (7% and 13% w/w) in the preparation of filloas (Galician pancakes), a typical dessert from the northwestern region of the Iberian Peninsula. To evaluate its power as a natural pigment, the stability of colour over time (3, 6, and 9 days) was measured, and the results were compared with those of a commercial colourant. At the same time, its physicochemical properties such as the microbiological profile were measured to determine its functionality as a food preservative. As a result, redness stability (a*) of 8% higher than that of the commercial colourant was obtained for the maximum concentration of H. pluvialis analysed. The texture showed a significant response (p < 0.02), improving its properties as the concentration of the microalga increased, which showed a tenacity of 3.23 N and extensibility of 15.10 mm during the first 6 days, i.e., a 52% and 19% improvement, respectively, in relation to the control group. In turn, an enrichment of carotenoids, fatty acids, and phenolic com- pounds, in combination with a potential moderator of microbiological degradation by this unicellular organism, gives added value to this food matrix.This research was funded by project ED431 2020/06 (Galician Competitive Research Groups Xunta de Galicia). This study was supported by project EQC2018-005011-P (Ministry of Science, Innovation and Universities, Spain). All these programmes are co-funded by FEDER (EU). The authors are also grateful to Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020); to FCT for S. Heleno (CEECIND/03040/2017) and L. Barros contracts through the individual and institutional scientific employment programme contract, respectively. This article is based upon work from the Sample Preparation Study Group and Network, supported by the Division of Analytical Chemistry of the European Chemical Societyinfo:eu-repo/semantics/publishedVersio

The generally recognized as safe (GRAS) microalgae Haematococcus pluvialis (wet) as a multifunctional additive for coloring and improving the organoleptic and functional properties of foods

This work proposes the application of astaxanthin-rich H. pluvialis wet paste (HPW) as a partial substitute for wheat flour in the preparation of filloas, a dish that combines the basic ingredients of industrial bakery. The nutritional and color profile of HPW-enriched samples was evaluated by comparative analysis with a mixture of synthetic food dyes. The highest content of carotenoids (798 ± 12 µg g−1) and fatty acids (76 ± 2mgg−1) was obtained for a filloa fortified with H. pluvialis in contrast to a non-significant dye response. Subsequently, the color stability of the fortified filloa was evaluated over time (3, 6 and 9 days), as well as its physicochemical properties and microbiological profile. As a result, HPW provided filloas with a longer shelf life, brightness (*L), and texture, in comparison with a mixture of synthetic dyes. In addition, an inhibitory effect of HPW towards mesophilic aerobic microorganisms in the food was obtained.This research was funded by project ED431 2020/06 (Galician Competitive Research Groups Xunta de Galicia). All these pro- grammes are co-funded by FEDER (EU). The authors are also grateful to the Foundation for Science and Technology (FCT; Portugal) for financial support through national funds FCT/ MCTES (PIDDAC) to CIMO (UIDB/00690/2020 and UIDP/ 00690/2020) and SusTEC (LA/P/0007/2021). Castillo A. acknowl- edges the support of the European Grouping of Territorial Cooperation Galicia-Norte Portugal (GNP, EGTC) under the IACOBUS Program and the MCI of Spain for his contract part of the grant DIN2021-011976 funded by the MCIN/AEI/ 10.13039/501100011033. L. Barros and S. Heleno acknowledge the national funding by FCT, P. I., through the institutional scientific employment program-contract for their contracts, and Filipa A. Fernandes for the PhD grant (SFRH/BD/145467/ 2019).info:eu-repo/semantics/publishedVersio

ESIPT and FRET probes for monitoring nanoparticle polymer coating stability

Coating strategies of inorganic nanoparticles (NPs) can provide properties unavailable to the NP core alone, such as targeting, specific sensing, and increased biocompatibility. Non-covalent amphiphilic NP capping polymers function via hydrophobic interactions with surface ligands and are extensively used to transfer NPs to aqueous media. For applications of coated NPs as actuators (sensors, markers, or for drug delivery) in a complex environment, such as biological systems, it is important to achieve a deep understanding of the factors affecting coating stability and behavior. We have designed a system that tests the coating stability of amphiphilic polymers through a simple fluorescent readout using either polarity sensing ESIPT (excited state intramolecular proton transfer) dyes or NP FRET (Förster resonance energy transfer). The stability of the coating was determined in response to changes in polarity, pH and ionic strength in the medium. Using the ESIPT system we observed linear changes in signal up to ∼20-25% v/v of co-solvent addition, constituting a break point. Based on such data, we propose a model for coating instability and the important adjustable parameters, such as the electrical charge distribution. FRET data provided confirmatory evidence for the model. The ESIPT dyes and FRET based methods represent new, simple tools for testing NP coating stability in complex environments.Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

ESIPT and FRET probes for monitoring nanoparticle polymer coating stability

Coating strategies of inorganic nanoparticles (NPs) can provide properties unavailable to the NP core alone, such as targeting, specific sensing, and increased biocompatibility. Non-covalent amphiphilic NP capping polymers function via hydrophobic interactions with surface ligands and are extensively used to transfer NPs to aqueous media. For applications of coated NPs as actuators (sensors, markers, or for drug delivery) in a complex environment, such as biological systems, it is important to achieve a deep understanding of the factors affecting coating stability and behavior. We have designed a system that tests the coating stability of amphiphilic polymers through a simple fluorescent readout using either polarity sensing ESIPT (excited state intramolecular proton transfer) dyes or NP FRET (Förster resonance energy transfer). The stability of the coating was determined in response to changes in polarity, pH and ionic strength in the medium. Using the ESIPT system we observed linear changes in signal up to ∼20-25% v/v of co-solvent addition, constituting a break point. Based on such data, we propose a model for coating instability and the important adjustable parameters, such as the electrical charge distribution. FRET data provided confirmatory evidence for the model. The ESIPT dyes and FRET based methods represent new, simple tools for testing NP coating stability in complex environments.Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

What potential do mosses have as biomonitors of POPs? A comparative study of hexachlorocyclohexane sorption

Persistent organic pollutants (POPs) pose a significant global threat to human health and the environment, and require continuous monitoring due to their ability to migrate long distances. Active biomonitoring using cloned mosses is an inexpensive but underexplored method to assess POPs, mainly due to the poor understanding of the loading mechanisms of these pollutants in mosses. In this work, Fontinalis antipyretica (aquatic moss) and Sphagnum palustre (terrestrial moss) were evaluated as potential biomonitors of hexachlorocyclohexanes (HCHs: α-, β-, γ-, δ-HCH), crucial POPs. Moss clones, grown in photobioreactors and subsequently oven-dried, were used. Their lipid composition and distribution were characterized through molecular and histochemical studies. Adsorption experiments were carried out in the aqueous phase using the repeated additions method and in the gas phase using an active air sampling technique based on solid-phase extraction, a pioneering approach in moss research. F. antipyretica exhibited greater lipid content in the walls of most cells and higher adsorption capacity for all HCH isomers in both gaseous and liquid environments. These findings highlight the need for further investigation of POP loading mechanisms in mosses and open the door to explore other species based on their lipid contentThis work was supported by the Governments of Spain (PID2019- 107879RB-100; PID2022-140985NB-C22) and Galicia (ED431C 2022/ 40; ED431B 2023/04, ED431C 2020/19) and was co-funded by ERDF (EU

Quantum Dots as Templates for Self-Assembly of Photoswitchable Polymers: Small, Dual-Color Nanoparticles Capable of Facile Photomodulation

A photomodulatable amphiphilic polymer has been synthesized with a backbone of poly[isobutylene-alt-maleic anhydride] and pendant dodecyl alkyl chains, Lucifer Yellow (LY) fluorescent probes, and diheteroarylethenes photochromic (PC) groups. The latter serve as reversible UV-activated FRET acceptors for the LY donors. We characterized the spectral and switching properties of the polymer in an organic solvent (CHCl₃). In an aqueous medium the polymer forms polymersomes, constituting fluorescence probes ∼75 nm in diameter. Self-assembly of the polymer on the surface of a quantum dot (QD) serving as a template creates a dual-color photoswitchable nanoparticle (psNP) with improved properties due to the increase in polymer density and efficiency of PC photoconversion. The psNP exhibits a second QD red emission band that functions as an internal standard requiring only a single excitation wavelength, and is much reduced in size (<20 nm diameter) compared to the polymersomes. The QD template also greatly increases the depth of modulation by photochromic FRET of the LY emission monitored by both steady-state and time-resolved (lifetime) fluorescence (from 20%→70%, and from 12%→55%, respectively).Facultad de Ciencias ExactasInstituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

A New Paradigm for MAPK: Structural Interactions of hERK1 with Mitochondria in HeLa Cells

Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) are members of the MAPK family and participate in the transduction of stimuli in cellular responses. Their long-term actions are accomplished by promoting the expression of specific genes whereas faster responses are achieved by direct phosphorylation of downstream effectors located throughout the cell. In this study we determined that hERK1 translocates to the mitochondria of HeLa cells upon a proliferative stimulus. In the mitochondrial environment, hERK1 physically associates with (i) at least 5 mitochondrial proteins with functions related to transport (i.e. VDAC1), signalling, and metabolism; (ii) histones H2A and H4; and (iii) other cytosolic proteins. This work indicates for the first time the presence of diverse ERK-complexes in mitochondria and thus provides a new perspective for assessing the functions of ERK1 in the regulation of cellular signalling and trafficking in HeLa cells

MicroRNAs expression, chromosomal alterations and immunoglobulin variable Heavy chain hypermutations in Mantle Cell Lymphomas

The contribution of microRNAs (miR) to the pathogenesis of mantle cell lymphoma (MCL) is not well known.We investigated the expression of 86 mature miRs mapped to frequently altered genomic regions in MCL in CD5+/CD5 normal B cells, reactive lymph nodes, and purified tumor cells of 17 leukemic MCL, 12 nodal MCL, and 8MCL cell lines. Genomic alterations of the tumors were studied by single nucleotide polymorphism arrays and comparative genomic hybridization. Leukemic and nodal tumors showed a high number of differentially expressed miRs compared with purified normal B cells, but only some of them were commonly deregulated in both tumor types. An unsupervised analysis of miR expression profile in purified leukemic MCL cells revealed two clusters of tumors characterized by different mutational status of the immunoglobulin genes, proliferation signature, and number of genomic alterations. The expression of most miRs was not related to copy number changes in their respective chromosomal loci. Only the levels of miRs included in the miR-17-92 cluster were significantly related to genetic alterations at 13q31. Moreover, overexpression of miR-17-5p/miR-20a from this cluster was associated with high MYC mRNA levels in tumors with a more aggressive behavior. In conclusion, the miR expression pattern of MCL is deregulated in comparison with normal lymphoid cells and distinguishes two subgroups of tumors with different biological features.Postprint (updated version

Some Secrets of Fluorescent Proteins: Distinct Bleaching in Various Mounting Fluids and Photoactivation of cyan fluorescent proteins at YFP-Excitation

Background&#xd;&#xa;The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins.&#xd;&#xa;&#xd;&#xa;Methodology/Principal Findings&#xd;&#xa;When we applied a commonly used FRET microscopy technique - the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10 - 15% after illumination at the YFP-excitation wavelength &#x2013; a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor.&#xd;&#xa;&#xd;&#xa;Conclusions/Significance&#xd;&#xa;Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions