Although the phototrophic alphaproteobacterium Rhodopseudomonas palustris encodes multiple proteins that can reduce Fd, the FixABCX complex is the only one shown to support nitrogen fixation, and R. palustris Fix– mutants grow poorly under nitrogen-fixing conditions. To investigate how native electron transfer chains (ETCs) can be redirected toward nitrogen fixation, we leveraged the strong selective pressure of nitrogen limitation to isolate a suppressor of an R. palustris ΔfixC strain that grows under nitrogen-fixing conditions. We found two mutations were required to restore growth under nitrogen-fixing conditions in the absence of functional FixABCX. One mutation was in the gene encoding the primary Fd involved in nitrogen fixation, fer1, and the other mutation was in aadN, which encodes a homolog of NAD+-dependent Fd:NADPH oxidoreductase (Nfn). We present evidence that AadN plays a role in electron transfer to benzoyl coenzyme A reductase, the key enzyme involved in anaerobic aromatic compound degradation. Our data support a model where the ETC for anaerobic aromatic compound degradation was repurposed to support nitrogen fixation in the ΔfixC suppressor strain.Nitrogenase is the key enzyme involved in nitrogen fixation and uses low potential electrons delivered by ferredoxin (Fd) or flavodoxin (Fld) to reduce dinitrogen gas (N Ferredoxins (Fds) and flavodoxins (Flds) are small protein electron carriers that transfer a single electron from an electron donor to an electron acceptor. In particular, Fds have specialized over evolutionary time to associate with specific partner proteins, allowing Fds to selectively shuttle electrons to specific pathways , 2. Key 5–Rhodopseudomonas palustris, electron transfer to nitrogenase requires the FixABCX complex, which couples the oxidation of NADH to the reduction of quinone and an Fd or Fld using flavin-based electron bifurcation (FBEB) + reductase   to nitrogenase.One such reaction is biological nitrogen fixation, which is catalyzed by the enzyme nitrogenase. Nitrogenase uses large amounts of ATP and low potential electrons delivered by Fd or Fld to reduce atmospheric dinitrogen into ammonia, producing hydrogen gas as an obligate product of this activity . In the n (FBEB)  10–12).–12.Rhodoeductase –20. R. pRpa4631) . The FldRpa4631) . Since RR. palustris ΔfixC strain, we isolated a suppressor mutant that restored growth of this strain under nitrogen-fixing conditions. We found that two mutations in the suppressor strain were required to restore nitrogenase activity in the absence of a functional FixABCX complex , and we found it is required for anaerobic aromatic compound degradation in R. palustris  has a severe growth defect when grown under nitrogen-fixing conditions, presumably because R. palustris ΔfixC is unable to generate enough reduced electron carrier  to support nitrogen fixation . To determine whether the Fer1 T11I variant was required for the suppressor phenotype, the T11Ifer1 allele in R. palustris ΔfixC* was replaced with either an in-frame deletion in fer1 or a wild-type fer1 allele. Both strains had a significantly lower growth rate than R. palustris ΔfixC* , R. palustris ΔfixC* with the T11I mutation in fer1 repaired (ΔfixC* fer1WT), and R. palustris ΔfixC* with the C38W mutation in aadN repaired (ΔfixC* aadNWT) in minimal medium lacking ammonium sulfate (nitrogen-fixing) with 20 mM acetate provided as a carbon source. The data are averages of three biological replicates, and error bars represent one standard deviation. Download FIG S1, TIFF file, 0.29 MB.Repairing mutations in Copyright © 2023 Lewis et al.2023Lewis et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.02881-22.6TABLE S2Table S2, DOCX file, 0.02 MB.All strains, plasmids, and primers used in this study. Download Copyright © 2023 Lewis et al.2023Lewis et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the T11Ifer1 allele was sufficient to restore growth under nitrogen-fixing conditions in the absence of an intact FixABCX complex, the T11Ifer1 allele was introduced into R. palustris ΔfixC. As shown in T11Ifer1 allele did not restore growth in R. palustris ΔfixC under nitrogen-fixing conditions, indicating other mutations in R. palustris ΔfixC* are required for electron transfer. This finding revealed that the fer1T11I allele was necessary but not sufficient for the suppressor phenotype. Because many of the remaining mutations in R. palustris ΔfixC* had unknown or hypothetical functions  analysis was carried out to compare gene expression changes in R. palustris ΔfixC* compared to wild-type R. palustris under nitrogen-fixing conditions. We found expression of fldA had the highest change In gene expression and was upregulated 23-fold in R. palustris ΔfixC* compared to wild-type R. palustris  versus R. palustris ΔfixC* (ΔfixC*) grown in minimal medium without ammonium sulfate (nitrogen-fixing medium) and 20 mM acetate. Download Data Set S1, XLSX file, 0.03 MB.Gene expression changes in Copyright © 2023 Lewis et al.2023Lewis et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the R. palustris ΔfixC*, we used a random transposon mutagenesis strategy combined with a metronidazole enrichment . In R. palustris ΔfixC*, aadN had a nonsynonymous mutation, encoding a variant of AadN in which cysteine 38 is substituted for tryptophan (C38W) . Me. MeR. pament see . Transpon (C38W) . We founnditions . When ths ΔfixC* . When thncreased . This suncreased .10.1128/mbio.02881-22.2FIG S25 mosaic end. Incubation times are shown in parentheses. Download FIG S2, TIFF file, 2.44 MB.Workflow for transposon mutagenesis with metronidazole enrichment. NFM, nitrogen-fixing medium which is minimal medium lacking ammonium sulfate and 20 mM acetate provided as a carbon source; non-NFM, minimal medium with 20 mM acetate provided as a carbon source; Met, metronidazole; Kan, kanamycin; Tn5 ME, TnCopyright © 2023 Lewis et al.2023Lewis et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the aadNC38W allele into R. palustris ΔfixC was sufficient to allow hydrogen production, but it produced about 50% less hydrogen compared to wild-type R. palustris or R. palustris ΔfixC*  that uses FBEB to balance NADP(H), NAD(H) and Fd pools to conserve energy and maintain redox balance . Pf. PfC38W  balance  28, 29)C38W is r in AadN  30). We. WeC38W identity . This suidentity .aadN is adjacent to genes involved in anaerobic degradation of aromatic compounds such as benzoate and 4-hydroxybenzoate (4-HB)   31). An. AnaadN e (4-HB)  32–34).–34.aadN as KorAB . While tck korAB . If AadNng korAB . This sunot both . The onlompounds .10.1128/mbio.02881-22.3FIG S3R. palustris incorporating the proposed activity of AadN. The activity of AadN is inferred based on its similarity to PfNfnI. The reaction marked by the dotted line represents the uncertainty of whether benzoyl-CoA reductase (BadDEFG) catalyzes a second reduction of the ring structure after the initial de-aromatization step. Download FIG S3, TIFF file, 0.3 MB.Degradation pathway of benzoate, 4-hydroxybenzoate (4-HBA), and cyclohexane carboxylate (CHC) in Copyright © 2023 Lewis et al.2023Lewis et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the PfNfnI, and its importance in electron transfer to nitrogenase in ΔfixC*, we hypothesized that AadN plays a role in electron transfer to benzoyl-CoA reductase. To test this, we looked at the ability of R. palustris ΔaadN to metabolize aromatic compounds. Benzoate and 4-HB are converted to benzoyl-CoA and are reductively de-aromatized by benzoyl-CoA reductase, but cyclohexane carboxylate (CHC) enters the same degradation pathway after this de-aromatization step . Wh. WhPfNfnand 4-HB . We founn source . We also sources , and R. urce see , indicat10.1128/mbio.02881-22.4FIG S4aadNC38W allele is sufficient to support both nitrogen fixation and anaerobic aromatic compound degradation in the absence of functional FixABCX. Growth phenotypes of R. palustris (WT), R. palustris with a deletion in fixC (ΔfixC), R. palustris with a deletion in fixC encoding the aadNC38W allele (ΔfixC aadNC38W), and R. palustris with a deletion in fixC encoding the fer1T11I and aadNC38W allele (ΔfixC fer1T11IaadNC38W) in minimal medium lacking ammonium sulfate (nitrogen-fixing) supplemented with 20 mM acetate (AC) or 5.7 mM benzoate (BA). The image was obtained once all growing cultures reached stationary phase, and the cultures shown are representative of three independent trials. Download FIG S4, TIFF file, 0.69 MB.The Copyright © 2023 Lewis et al.2023Lewis et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the R. palustris ΔfixC is unable to grow under nitrogen-fixing conditions, the ETC for anaerobic aromatic compound degradation and nitrogen fixation are likely insulated from each other. To probe the insulation of these two ETCs, we grew R. palustris strains with an in-frame deletion in fixC or aadN under nitrogen-fixing conditions with benzoate as a sole carbon source. We found that although aadN was required for growth on benzoate under non-nitrogen-fixing conditions  containing 12.5 mM Na2HPO4, 12.5 mM KH2PO4, 7.6 mM (NH4)2SO4, 0.1 mM Na2S2O3·5H2O, 0.015 mM p-aminobenzoic acid, and 1% of a mineral salt solution  as described previously . Where applicable, R. palustris was grown with 100 μg/mL gentamicin and 200 μg/mL kanamycin, and Escherichia coli strains were grown in lysogeny broth at 37°C supplemented with gentamicin (20 μg/mL). For metronidazole enrichment, metronidazole was added to a final concentration of 50 mM.All tion see  43). Mi. MiR. paeviously . Liquid ously . De. De45). li S17-1 . Gene deli S17-1 . All allR. palustris cells were harvested from 10 mL of nitrogen-fixing medium after cultures had grown to an optical density (660 nm) of 0.4. Cells were incubated on ice for 10 min and harvested by centrifugation. The cell pellet was frozen in liquid nitrogen and stored at −80°C. Cell pellets were thawed and resuspended in 1 mL of QIAzol lysis reagent  and homogenized using a BioSpec Products BeadBeater-24  at maximum rpm for 1 min at 4°C and then allowed to cool for 1 min on ice. This cycle was repeated four times. Total RNA was isolated using the miRNAeasy minikit (Qiagen), and DNA was removed with TURBO DNase . RNA was purified and concentrated using the RNeasy MiniElute Cleanup kit (Qiagen). cDNA library construction and library sequencing were performed at GENEWIZ, LLC . rRNA was depleted using the Ribo-Zero rRNA removal kit . cDNA was prepared using the NEBNext Ultra RNA Library Prep kit and sequencing reactions, image analysis, and base calling were performed on an Illumina HiSeq 2500 instrument (Illumina).https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and TrimGalore (v0.6.2) was used to remove adapter sequences, process, and validate all reads using the default parameters. Analysis was performed on the Avadis software package . Reads were aligned to the published genome of R. palustris CGA009 and differentially expressed genes were identified using the DESeq2 package . 1 μg of genomic DNA was digested with the restriction enzyme AatII overnight at 37°C to generate fragments of genomic DNA which were, on average, 1,500 bp (New England Biolabs). The digestion product was then treated with Antarctic phosphatase for 1 h at 37°C (New England Biolabs). PCR products were purified using the Zymogen Clean and Concentrator PCR-cleanup kit , and the recovered DNA fragments were ligated together to form closed circular DNA using T4 DNA ligase (New England Biolabs). The library of circular DNA fragments was used as a PCR template with forward and reverse primers specific to the transposable element and amplified using Phusion High-Fidelity DNA polymerase (New England Biolabs). PCR products were separated by electrophoresis on a 1% agarose gel and purified using the Zymoclean Gel DNA recovery kit . The purified DNA fragments were sequenced using Sanger sequencing (GENEWIZ) using primers in see .2 was quantified using a Shimadzu GC-2014 gas chromatogram equipped with a thermal conductivity detector and a 60/80 molecular sieve 5-Å column . H2 standards were measured in triplicate. Samples of headspace taken from growing cultures were measured in biological triplicate and technical duplicate. Samples were also taken from the headspace of uninoculated tubes containing the same medium used for growing cultures. The amount of H2 found in the headspace of uninoculated tubes was subtracted from the amount of H2 measured in growing cultures. For growing cultures, the headspace was sampled at an optical density of 0.4 to 0.55 at 660 nm. Cultures were vortexed briefly before sampling. H2 production in each sample was normalized to the optical density (660 nm) of the culture at sampling time.HR. palustris BisB5 KorA as a bait sequence. Candidate homologs of korA had >80% amino acid identity to KorA and were adjacent to genes involved with anaerobic benzoate or 4-hydroxybenzoate degradation. AadN was used as a bait sequence to identify homologs among the selected R. palustris strains. Homologs of AadN had >90% amino acid identity and were adjacent to anaerobic benzoate or 4-hydroxybenzoate degradation genes.Specific protein-protein alignments were generated using the Constraint Based Alignment Tool  using the default parameters. Protein domains were identified using InterPro v.86.0 using the default parameters . HomologPANOVA < 0.001). Welch’s t test was used to compare the mean doubling times of individual strains. Similarly, normalized H2 accumulation was compared using ANOVA, followed by a Welch’s t test. All statistical analyses were performed in R version 4.1.1.Doubling times of different strains in each growth experiment were compared using analysis of variance (ANOVA; R. palustris ΔfixC* has been deposited in the NCBI Sequence Read Archive in BioProject PRJNA858464. RNA-seq reads have been deposited on the NCBI Gene Expression Omnibus in BioProject PRJNA858255. KorA and AadN sequences can be found using accession numbers WP_011501953.1 and WP_011156245, respectively.Genome sequencing data for


Short-dough cookies are one of the most popular cereal-based products in the world, but usually they are considered as foods with a low nutritional value. Therefore, the aim of this study was to investigate the possibility of replacing part of the wheat flour (WF) with hulless barley flour (HLBF), in order to improve the functional properties and nutritional value of the cookies. Cookies were prepared from composite flours in the ratios HLBF:WF 0:100, 25:75, 50:50, 75:25, and 100:0. The results show that as the HLBF content in the composite flour increases, the viscosity of the corresponding HLBF:WF slurries also increases, due to the high dietary fiber content , which is significantly higher when HLBF is included in the formulation. The addition of HLBF decreases the spread factor of the cookies, and increases their softness compared to the control cookies (100% WF), but these changes are not statistically significant up to 50% HLBF addition. The color of the HLBF:WF cookies is not significantly affected. A significant increase in total phenolic content (TPC) and increased antioxidant capacity (AOC) are observed in the HLBF:WF cookies. In addition, sensory evaluation confirms that WF can be replaced by up to 50% with HLBF without significant deterioration of the organoleptic properties of the cookies. It can be concluded that hulless barley serves as a promising raw material if the nutritional and functional properties of cereal-based products are to be improved. Hordeum vulgare L.) is one of the oldest cultivated cereals, used for the production of beer, whiskey, yeast, substitute coffee, barley gruel, animal feed, and so on [The quality characteristics of cookies depend on the chemical composition of the flour used and its quality. The process of cookie production depends on many factors: the raw materials used, the baking time, the type of cookies to be produced, etc. The most commonly used flour for cookie production is wheat flour (WF) . Nowadaynd so on . In recend so on ,5, whichnd so on . Many aund so on . β-glucand so on . Chemicand so on . Recentlnd so on ,10. The nd so on ,11.The majority of barley production consists of the cultivation of covered (hulled) varieties used for malt and feed. The problem with using this type of barley in food is its unpalatable hull. Therefore, barley is often pearled, which means that in addition to the outer hull, part of the bran is also removed, reducing the micronutrient and mineral content of the barley flour and, thus, reducing its nutritional value . In viewThe aim of this study was to investigate the effects of using flour from the hulless barley variety “Osvit” in the production of cookies from composite flours containing hulless barley flour (HLBF) and WF at substitution levels of 25, 50, 75, and 100%. The pasting properties of the composite flours, chemical composition (including fiber and β-glucan content) and energy value, dimensions, texture, sensory properties, and AOC of HLBF:WF composite cookies were evaluated to determine the maximum proportion of HLBF that can replace WF so that the physical and sensory properties of the cookies do not deteriorate, while improving their nutritional value and increasing their functional properties.In this study, 100% commercial plain WF (control), 100% whole grain HLBF, and three different HLBF:WF composite flours  were used to produce HLBF:WF composite cookies. To investigate pasting properties of the HLBF:WF slurries, the Brabender Micro Visco-Amylo-Graph was used.p < 0.05), with the increase in the percentage of barley flour in the mixture (from 60.8 °C for 100% WF to 61.6 °C for 100% HLBF). The lowest peak viscosity is observed at 100% WF (897.0 BU). With the increase in HLBF content in the blend, the peak viscosity increases to 1254.3 BU in 100% HLBF. A higher peak viscosity indicates a greater ability of the starch granules to bind water, and reflects the swelling ability of the granules before breakdown [p < 0.05). A similar pattern of pasting properties of composite flours with HLBF and WF is observed in the study by Liu et al. [reakdown . The breu et al. . These ru et al. ,19.p < 0.05), and thickness decreases from 1.28 cm for 100% WF to 1.22 cm for 100% HLBF cookies, but this reduction is not statistically significant (p < 0.05). As the cookie width decreases much more than the cookie thickness, the spread factor of the cookies also decreases by 5% (from 52.80 for 100% WF cookies to 50.14 for 100% HLBF cookies). A similar decrease in cookie spread factor is observed in the study by Elhadidy et al., when 25% of WF is replaced with HLBF [The physical characteristics of cookies are one of the key factors when customers decide whether to buy them or not. ith HLBF ,20,21,22ith HLBF .The reduction in the width of the cookies and, consequently, of the spread factor, can be explained by the higher dough viscosity in samples with increased HLBF content. Mancebo et al. reached a similar conclusion in their study on the effects of different non-wheat flours on the quality of gluten-free cookies . This isp < 0.05) reduced (from 58.30 N/mm for 100% WF to 26.39 N/mm for 100% HLBF cookies), due to a much softer, crumbly texture of the HLBF cookies with increased graininess. These results, showing the softening effect of barley flour in HLBF:WF cookies and the reduced cohesion of these cookies, are in agreement with several other studies that investigated the replacement of WF with barley or barley malt flour [The determination of the textural properties of HLBF:WF composite cookies was performed using the three-point bend/break test. The results are also shown in lt flour ,21,25,26lt flour ,23. All In addition to the amount of sugar prescribed by the recipe, as well as the temperature and baking time, the color of the cookies is mainly influenced by the type of flour used and the amount of pigments it contains.Lab coordinates, is shown in L* (lightness) (from 66.18 for 100% WF to 62.90 for 100% HLBF cookies), a significant increase in a* (redness) (from 5.49 for 100% WF to 7.48 for 100% HLBF cookies), and a decrease in the color parameter b* (yellowness) (from 36.51 for 100% WF to 31.68 for 100% HLBF cookies) (p < 0.05). The same direction of color changes are observed in the studies by Žilić et al. [a* and a decrease in b* values when WF is replaced by HLBF. Increased a* values indicate a stronger red hue and decreased b* values indicate a stronger blue hue, which, in combination, results in a slightly purple coloration of the cookies. Although the differences in CIELab parameters between HLBF:WF composite cookies are mostly statistically significant (p < 0.05), these differences are actually very difficult to detect. This is evident from the calculated color differences (ΔE) with respect to the control cookies with 100% WF. Only the sample with 100% HLBF has ΔE > 5, which is the smallest color difference that an average consumer can perceive [The color of the different cookies, determined by the CIEć et al.  and by Fć et al. , when whć et al. . This isperceive . Images p < 0.05) increases from 6.03 to 7.02%, available carbohydrates decreases from 71.25 to 65.03%, and ash content increases from 0.78 to 1.47% for 100% WF and 100% HLBF cookies, respectively. The fat content does not vary much because the majority of fat originates from added shortening. The decrease in the content of available carbohydrates can be attributed to the fact that the addition of HLBF increases the content of crude fiber. These results are consistent with other studies on the effects of replacing WF with HLBF in cookie formulation [p < 0.05) decreased with increasing HLBF supplementation, and the 100% HLBF cookies have a 4.16% lower energy value than the control 100% wheat cookies. El-Hadidy et al. report that there is no change in the energy value when 25% of WF is replaced with flour of HLBF [The proximate composition of HLBF:WF cookies is presented in mulation ,16. The  of HLBF .Both moisture content and water activity increases when HLBF is incorporated into the cookie formulation . Moisturp < 0.05). An increase in β-glucan content in cookies is also observed in other studies investigating the replacement of WF with barley flour [The highest content of total (TDF), insoluble (IDF), and soluble (SDF) was determined in cookies with complete replacement of WF by HLBF . According to the Annex of Regulation (EC) No. 1924/2006, barley grain fiber contributes to an increase in fecal bulk, and can be considered a “high fiber source” food if it contains more than 6 g/100 g of dietary fiber . The incey flour ,20,37. Rey flour . The amoIn addition to the significant dietary fiber content, the nutritional value of hulless barley is also complemented by the high content of phenolic substances and the associated high AOC . ConsequNarwal et al. also report that as the amount of HLBF in the cookie composition increases, the AOC also increases . The samp < 0.05). This also confirms the results of the instrumental color determination, when only the 100 HLBF cookies have ΔE > 5 (6.16) compared to a control 100 WF cookie, which is very difficult for the average consumer to detect. Only a few evaluators mentioned that this sample has a slightly darker color, which negatively affects the rating. Panelists perceive the cookies with the higher spread factor as more acceptable, and give a significantly lower rating (5.9) for shape to the 100% HLBF cookie (p < 0.05). The most significant differences between the HLBF:WF composite cookies are observed for texture. The sample with 50% HLBF added has a score of 7.1, which is lower than the score for the control 100% WF cookies, but not significantly different (p < 0.05). The panelists note that this sample is softer than the control sample, but this does not significantly affect the score. Samples with 75% and 100% HLBF receive significantly lower ratings due to the highlighted crumbliness. There are no significant differences among samples for odor scores. The sample with 25% HLBF has the highest score for taste (7.7). Only the cookie with 100% HLBF receives a statistically lower score (6.2) compared to the control cookie with 100% WF (7.6) (p < 0.05). Overall acceptability ratings are 7.8, 7.7, and 7.3 for 100% WF, 25:75 HLBF:WF, and 50:50 HLBF:WF composite cookies, respectively. These ratings place these samples between “like very much” and “like moderately” on the nine point hedonic scale. The overall score for cookies with 75% and 100% HLBF addition is 6.7 and 6.2, respectively, which can be described as a score between “like moderately” and “like slightly”.The sensory ratings of the HLBF:WF composite cookies, as determined by sensory analysis using a nine point hedonic scale, are summarized in Sharma et al. reported a similar decrease in sensory ratings of cookies with increasing barley flour addition, but compared to our study, higher scores were obtained for overall acceptability of cookies with the addition of 100% barley flour . This sa3), and sodium chloride (NaCl).In this study, commercial all-purpose WF  from a previous study was used . WF cont−1 was constant throughout the analysis. Pasting and peak temperatures, peak, breakdown, and setback viscosities were recorded. Viscosity parameters were expressed in Brabender units (BU). Three replicate measurements were made for each sample.The Micro Visco-Amylo-Graph  was used to evaluate the pasting properties of composite HLBF:WF flours. A suspension containing 15 g of flour (14% w.b.) and 100 mL of distilled water was added to a mixing bowl of the instrument and heated from 30 to 92 °C at a rate of 5 °C/min, held constant at 92 °C for 5 min, cooled to 50 °C at a rate of 5 °C/min, and finally held at 50 °C for 1 min. The rate of 250 minThe cookies were prepared according to the AACC international method 10–50.05 . The exaThe dimensions of the cookies (width (W) and thickness (T)) were determined using the instructions of given in the AACC international method 10 50.05 . The sprThe TA.XT2i Texture Analyzer  was used to perform the three-point bend/break test. The distance between two lower supports was 40 mm, and the test speed of the knife blade was 1 mm/s. The snapping force (N) and the breaking distance (mm) were obtained from the test curve. By dividing these two parameters, the bending force index (N/mm) was calculated. This index served as an indicator of the hardness/softness of the cookies . Two cooL*a*b* color model was used. The L* value (from 0 to 100) represents brightness (lightness) or luminance, the b* value (−128 to 127) represents blue–yellow, and the a* value (−128 to 127) represents the green–red axes of the color space. The CIE76 color difference equation was used to calculate the total color difference (ΔE) between the composite HLBF:WF cookies and the control cookie (100% WF).The CR-400 chromameter  was used to determine the color of the cookie surface. The CIEMoisture content was determined in accordance with AACC international method 44-15.02  and wateAACC international method 32-07.01 (Megazyme assay procedure K-TDFR) was used for determination of the dietary fiber content , and AACC international method 32-23.01 ) was used for the determination of β-glucan .g for 10 min, and the supernatant was used for further TPC and AOC analyzes. TPC was determined according to the method of Singleton and Rossi [For the determination of TPC and AOC, cookies were ground  and 1 g of the sample was extracted in 10 mL of 80% methanol, vortexed for 1 min, and shaken for 2 h at 150 rpm on an electronic shaker. The mixture was centrifuged at 3000× nd Rossi , and AOCnd Rossi . CalibraA panel of ten evaluators conducted the sensory analysis of the cookie samples. All were staff and students of the Department of Cereal Technology at the Faculty of Food Technology Osijek, Croatia, and all had previous experience with sensory analysis. There were seven women and three men in the panel (median age = 24 years). The inclusion criteria for the evaluators were the absence of health problems that could affect the sensory evaluation , and the usual preference for consuming similar types of cookies. Sensory analysis was performed in a tasting room equipped with individual test booths. The panelists were given a brief introduction to give them an insight into the research and the samples to be tested. All five cookie samples were handed to the evaluators at the same time and they were asked to rinse their mouths with water between tastings. The 9 point hedonic scale was used to assess sensory properties. Scores from 1 to 9 were: extremely dislike (1), very much dislike (2), moderately dislike (3), slightly dislike (4), neither dislike nor like (5), slightly like (6), moderately like (7), very much like (8), and extremely like (9).p < 0.05). The statistical add-in for Microsoft Excel XLSTAT software  was used for statistical analysis of experimental data.Analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) test was used to detect differences between samples , and high TPC and AOC. Therefore, it can be expected that the use of barley flour in the production of cookies increases the nutritional value and improves the functional properties. Since covered (hulled) barley has an unpalatable hull that can deteriorate the properties of cookies when whole grain flour is used or when the barley is pearled before milling into flour, which reduces the nutritional value of barley flour, hulless barley is imposed as an excellent raw material for flour production that can be used in cookie production, and give them both nutritional value and sensory acceptable properties. With the increasing replacement of wheat flour by barley flour, the physicochemical properties of the cookies change significantly. The spread factor and the hardness of the cookies decrease, and the color becomes slightly darker. From a nutritional and health point of view, it is particularly important that the amount of dietary fiber, especially β glucan, increases, as well as the TPC and AOC. The results show that by replacing WF with up to 50% HLBF, there are no significant differences between HLBF:WF composite cookies and 100% WF cookies in terms of physical and sensory properties, but the nutritional and functional properties are improved.


The Scribble (Scrib) protein is a conserved cell polarity regulator with anti-tumorigenic properties. Viruses like the Tick-born encephalitis virus (TBEV) target Scribble to establish a cellular environment supporting viral replication, which is ultimately associated with poor prognosis upon infection. The TBEV NS5 protein has been reported to harbour both an internal as well as a C-terminal PDZ binding motif (PBM), however only the internal PBM was shown to be an interactor with Scribble, with the interaction being mediated via the Scribble PDZ4 domain to antagonize host interferon responses. We examined the NS5 PBM motif interactions with all Scribble PDZ domains using isothermal titration calorimetry, which revealed that the proposed internal PBM did not interact with any Scribble PDZ domains. Instead, the C-terminal PBM of NS5 interacted with Scrib PDZ3. We then established the structural basis of these interactions by determining crystal structures of Scrib PDZ3 bound to the NS5 C-terminal PBM. Our findings provide a structural basis for Scribble PDZ domain and TBEV NS5 interactions and provide a platform to dissect the pathogenesis of TBEV and the role of cell polarity signalling using structure guided approaches. Cell polarity is the fundamental process that dictates the shape and orientation of a cell via the asymmetric distribution of cellular constituents such as proteins, lipids and carbohydrates . The corScribble is part of the LAP (leucine-rich repeats and PDZ domain) protein family, with sixteen LLRs (Leucine-rich repeats), two LAP-specific domains (LAPSDa and LAPSDb) and four PDZ domains ((Post-synaptic density protein (PSD95), Drosophila disc large tumour suppressor (Dlg), and zonula occludens-1 protein (ZO-1)).COOH motif, Class II with an X-ψ-X-ψCOOH motif, and Class III interacting with X-D/E-X-ψCOOH motif [PDZ domains are ∼90 amino acids long and consist of 5–6 β-strands and 2–3 α-helices with a conserved fold comprising a canonical ligand-binding groove formed by the α2 helix and the β2, β4 and β5 strands . In partOH motif , in whicflaviviridae proteins [The zoonotic Tick-born encephalitis virus TBEV encodes for the NS5 protein, which has been shown to interact with Scribble in a PDZ domain dependent manner , 10. Maiproteins . It contproteins .Although NS5 features two recognisable PBMs, only the internal PBM located within the MTase domain was shown by a yeast-two hybrid screen to bind to Scribble via the PDZ4 domain, whereas the conventional C-terminal PBM did not interact with Scribble , 10. IntHere we provide a structural basis for the subversion of the apical-basal polarity Scribble by the TBEV NS5 and demonstrate biochemically and through structural studies a new interaction of the TBEV NS5 C-terminal PBD with the PDZ3 domain of Scribble.KD value of 18.4 ± 1.3 µM. Examination of the thermodynamic binding parameters of NS5_CTN peptides binding to the SCRIB PDZ domains indicated that the SCRIB PDZ domain:PBM interactions were entropy-driven by a more favourable −TΔS, , JEV (PDB: 4K6M) and DENV. For yellow fever, DENV, JEV, ZIKA and WNV NS5, these related NS5 sequences displayed 62–87% sequence identity in the region spanning the putative NS5_INT motif, suggesting that in all cases this motif would be located within an existing β-sheet. We note that the presence of this motif in a β-sheet does not preclude the possibility of exchange with a different conformational state that would be amenable to an interaction with a PDZ domain.To understand the molecular and structural basis of the human Scribble:NS5 interaction, we systematically examined the ability of recombinant human Scribble PDZ domains to bind to peptides encoding for identified PBM motifs in TBEV NS5 . For thile −TΔS, . In contle −TΔS, . With nole −TΔS, . Examinale −TΔS, . The preTo understand the interactions of SCRIB PDZ domain interactions with NS5 PBM sequences in more detail we determined crystal structures of SCRIB PDZ3:NS5_CTN complexes as well as a structure of SCRIB PDZ3 apo , Table 2NS5–Ile1018PDZ3, and single bonds with Ile3414NS5–Leu1014PDZ3, Ile3414NS5–Gly1015PDZ3, Ile3414NS5–Leu1016PDZ3, Glu3410NS5–Gln1072PDZ3 and Ile3409NS5–His1023PDZ3 as well as Ser3412NS5–His1071PDZ3 , with a second SCRIB PDZ3 molecule (Chain B) engaging the same peptide via its β2 strand . In this1071PDZ3 . The non1071PDZ3 .PDZ3–Asn645β-PIX, Ser1026PDZ3–Trp641β-PIX, Ser1039PDZ3–Glu643β-PIX and H1071PDZ3–T644β-PIX. In the Scrib PDZ3:NS5_CTN complex, His1071PDZ3 contacts Ser3412NS5 in the −2 position in the canonical manner observed in many other PDZ domain complex structures [NS5 is able to contact Gln1072PDZ3 whereas in the PDZ3: β-PIX complex the equivalent Asp642β-PIX is too short to reach Gln1072PDZ3. The Ser1039PDZ3–Glu643β-PIX interaction is recapitulated in a more unconventional configuration, with the Ser1039PDZ3 being provided by chain B which binds NS5 in a non-canonical manner.When the SCRIB PDZ3:β-PIX (PDB ID: 5VWI) complex is superimposed on our PDZ3:NS5_CTN complex, an RMSD of 0.78 Å for Chain A and 0.75 Å for Chain B  over theructures . InteresPDZ3. No major changes in B-factor distribution for backbone atoms are observed upon NS5_CTN binding.Binding of NS5_CTN in a non-canonical manner requires no significant change in the SCRIB PDZ3 domain fold, with a superimposition of both SCRIB PDZ3 chains yielding an RMSD of 1.15 Å in Chain A and 1.35 Å in Chain B  over the−1) and SCRIB PDZ3 K1040A:NS5_CTN , suggesting that the SCRIB PDZ3 K1040 position significantly impacts the enthalpic contribution to NS5_CTN binding. However, the KD difference of SCRIB PDZ3:CTN (18.4 ± 1.3 µM) and SCRIB PDZ3 K1040A:CTN (12.5 ± 0.5) is modest. To ensure that the mutants were correctly folded, CD spectroscopy was performed on mutant proteins, which revealed spectra simular to the corresponding wild-type PDZ domain spectra with comparable proportions of secondary structure elements  pLysS cells (BIOLINE) in super broth supplemented with 200 µg/ml ampicillin (AMRESCO) using auto-induction media  D-lactose, 0.05% (w/v) glucose, 0.5% (v/v) glycerol) [600) reached 1.0 before transferring cultures to 16°C for 24 h for protein expression.Human Scribble (SCRIB) (Uniprot accession number: Q14160) PDZ1 (728–815); PDZ2 (860–950); PDZ3 (1002–1094); PDZ4 (1099–1203)) domain proteins as well as mutant SCRIB PDZ domain proteins were subcloned into the bacterial expression vector pGEX-6P1, and expressed and purified as previously described . Brieflylycerol)  at 37°C g for 20 min using an Avanti® J-E (Beckman Coulter). The resulting supernatant was filtered using Millex-GP syringe filter unit 0.22 µM (Merck Millipore) prior to loading onto equilibrated columns for affinity purification. Glutathione-S-transferase (GST) tagged recombinant proteins were captured using glutathione sepharose 4B  in buffer A  and eluted with buffer A supplemented with 20 mM reduced l-glutathione. Recombinant proteins were subsequently dialysed into buffer A to remove glutathione for the pull down assay. GST tagged PDZ domains were cleaved on-column with HRV 3C protease to remove the GST tag. All recombinant PDZ domain proteins were then subjected to size-exclusion chromatography using a Superdex S75 10/300 column mounted on an AKTA Pure and equilibrated in 20 mM HEPES pH 7.2 and 150 NaCl, where they eluted as single peaks. PDZ2, PDZ3 and PDZ4 lacked Tryptophan residues, requiring concentration quantification via their peptide bond absorption at 205 nm using the Scopes method [Bacterial cells were harvested by centrifugation and lysed in the presence of deoxyribonuclease I (Sigma–Aldrich) from bovine pancreas using TS Series 0.75 kw model cabinet (Constant Systems Ltd.) at 25 kPsi or a Fastprep®-24  using lysing matrix B for 20 s. Lysates were clarified by centrifugation at 20 000×s method . The absThe following peptides were commercially synthesised using solid phase synthesis with Fmoc-protection strategy with subsequent trifluoroacetic acid removal (Genscript) and used in this study: TBEV TBEV NS5_INT: EMYYSTAVTGNI (amino acids 2730–2741), TBEV NS5_CTN: LRLESSII (amino acids 3407–3414) (UniProt accession code Q01299) . SuperpeAll proteins were diluted to 0.150 mg/ml in phosphate buffer (50 mM phosphate pH 7.0) and subjected to CD spectroscopy using the AVIV model 420 CD spectrometer at a wavelength from 190–260 nm with sample entry every 1 nm and an averaging time of 4 s at 25°C. Data were processed using the AVIV Biomedical software and plotted using Excel. Deconvolution of spectra to determine secondary structure composition was performed using Bestsel .SCRIB PDZ1 to PDZ4 proteins were prepared in 20 mM HEPES pH 7.2 and 150 NaCl at a final concentration of 75 µM, with peptides prepared in the same buffer at a concentration of 900 µM. Peptide concentrations were calculated based on the dry peptide weight after synthesis, and were commercially synthesised (Genscript).Independent ITC experiments were conducted using a MicroCal iTC200 System  at 25°C at a stirring speed of 750 rounds-per-minute. A total of 20 injections of 2 µl each and a spacing of 180 s were titrated into the 200 µl protein sample (except for injection 1 which was 0.4 µl), as previously described . Each inSCRIB PDZ domain:NS5 peptide complexes were prepared using a protein:peptide molar ratio of 1 : 4 as previously prescribed . Proteina = 69.96 Å, b = 69.96 Å, c = 201.67 Å, α = 90.00°, β = 90.00°, γ = 120° in the trigonal crystal system at a resolution of 2.05 Å (Rwork/Rfree of 0.24/0.29, with 97.35% of residues in the favoured region of the Ramachandran plot and 0% of rotamer outliers.Crystals of PDZ3:NS5_CTN were grown in 0.2 M Sodium nitrate, 0.1 M Bis-Tris propane pH 7.5, 20% w/v PEG 3350 and were flash cooled at −173°C in mother liquor. PDZ3:NS5_CTN complex formed single dome-shaped crystals beloning to the space group R32 with f 2.05 Å . Molecul12121 with a = 36.88 Å, b = 60.89 Å, c = 64.90 Å, α = 90.00°, β = 90.00°, γ = 90.00° in the monoclinic crystal system at a resolution of 1.84 Å (Rwork/Rfree of 0.24/0.29, with 100% of residues in the favoured region of the Ramachandran plot and no rotamer outliers.Crystals of SCRIB PDZ3 were grown in 1.5 M Sodium nitrate, 0.1 M Sodium acetate trihydrate pH 4.6 and were flash cooled at −173°C in mother liquor. Crystals of SCRIB PDZ3 grew as cluster sof plates belonging to the space group P2f 1.84 Å . Molecul


The consumption of cookies is widely extended throughout the world, although their formulas contain ingredients such as saturated fats or refined flours that are considered harmful to health. In addition, cookies are generally made from wheat flour, and nowadays there is a growing concern about gluten intolerance, thus the demand for gluten-free products is increasing. In this regard, the aim of the present study is to reformulate traditional cookies by replacing wheat flour and butter by flours and oils from nuts and seeds. Within these seeds, poppy or chia are not commonly consumed ingredients as they can cause rejection by consumers. Thus, a study was performed to evaluate the neophobia level of consumers and the consumer acceptance for the inclusion of these novel ingredients in cookies. The results have been obtained by measuring physical parameters, proximate composition and consumer evaluation of five batches of cookies. By replacing butter and wheat flour with maize flour, almond, walnut, chia or poppy seed flours and oils, an increase of protein, fat and fiber has been observed as well as a decrease in carbohydrate content; thus, the resultant cookies would be a good source of vegetal protein as well as a source of oleic and linoleic acid with potential benefits on health. The cookies in general have similar physical properties and a positive consumer acceptance in texture, taste and external aspect. The Food Neophobia Scale results suggest that non-neophobic consumers gave higher scores than neophobic consumers in all the parameters. The resultant product would be a functional product able to substitute traditional ones not only directed to celiac people but all type of consumers because of their beneficial composition. Cookies are a very popular baked snack characterized by a formula in which sugar, shortening and wheat flour are the main ingredients . The shoOne of the main objectives of the agri-food industry is to elaborate healthier foods. In the case of cookies, which are one of the most consumed snacks worldwide, especially for children, the elaboration of a healthier alternative to traditional cookies consists of the partial elimination of harmful ingredients such as butter or wheat flour and the introduction of other beneficial ingredients that contain nutrients such as fiber, protein or unsaturated fatty acids that can improve the health of the general population and help to achieve a better cognitive development in children.Within this frame, it is desirable to replace these saturated fats with other oils that have a higher percentage of unsaturated fatty acids in their composition. In this sense, vegetable oils extracted from nuts and other seeds are generally rich in unsaturated fatty acids. This is the case of walnut oil, which is especially rich in linoleic acid , or chiaOn the other hand, cookies are generally made from wheat flour due to the viscoelastic properties of gluten. This protein is responsible for celiac disease, which can be defined as an autoimmune inflammatory disease that causes gastrointestinal disorders resulting in a deficient nutrient absorption . Celiac Partially defatted almond flour is rich in proteins, which could positively influence the structure of the dough when baking, thus it would be an interesting ingredient to replace conventional flours . This flThe main problem with the introduction of these seeds and nuts is that they could affect the external appearance and taste of the cookies compared with cookies elaborated with wheat flour. Since chia and poppy flours are black, it is necessary to add small quantities to avoid excessive changes in the external appearance. In addition, due to their high fiber content, the percentage used should be small in order to not have to limit its consumption. Moreover, in the case of nut flours, walnut flour is characterized by a great astringency, thus its addition must be controlled to prevent the alteration of cookies’ flavors. On the contrary, almond flour color is like wheat flour and the taste and smell is mild and pleasant. However, none of these flours can be added without the addition of maize flour, because it is necessary to be able to manipulate the dough to obtain a softer cookie. Rai et al. , used a Considering the increased incidence and prevalence of diseases closely linked to food, the objectives of the study were to evaluate the effect of the inclusion of nut and seed oils as well as gluten-free flours in the formulation of cookies. These effects must be analyzed for their physical, nutritional and sensory parameters in order to obtain healthier products with an improvement in their nutritive characteristics and to be well accepted by the consumers. Due to the color and/or the flavor of the flours and oils from nuts and seeds, a combination of different nuts and seeds has been used in order to obtain cookies similar to the traditional recipe and to evaluate the possible nutritional improvement, especially in the content of fiber, protein or fat. Thus, the main objective of this work is to improve the nutritional characteristics of a very popular snack, especially for children, by trying to use novel ingredients to change potentially harmful compounds such as saturated fatty acids for unsaturated fatty acids reported to be beneficial for health.Salvia officinalis) and poppy (Papaver somniferum), the eggs, the sugar, the salt, the milk, the almonds and the walnuts were purchased in local markets.The wheat and maize flour, the butter, the seeds of chia  . Chia anThe different defatted flours from almond, walnut, chia and poppy used in the elaboration of cookies were obtained by grinding the press cake resulting from the oil extraction process and sifting through a sieve of 1 mm.As wheat and maize flours were acquired in a local market, the nutritional value was obtained from the label of both flours. Wheat flour is composed of 1.2% fat, 75% carbohydrates and 9% protein. Maize flour is composed of 0.5% fat, 86% carbohydrates, 0.5% protein and 1% fiber.The cookies were elaborated from a dough formed with the following ingredients: 250 g flour, 125 g sugar, 80 g egg, 150 g fat, 15 g milk, 1 g salt. The flour and fat source were changed as shown in The inclusion of oils from nuts and seeds instead of butter can affect the flavor and the palatability of the cookies and can be perceptible by the consumers. In the case of chia, as it is rich in linolenic acid (omega-3), when cooked it can be perceived with a slight fishy taste, thus it was not used in the formula for the cookies. In addition, poppy seed oil presents a strong flavor that masks the flavor of the cookie if added in large quantities, thus that is the main reason to mix it with other nut oils. In the case of nut oils, both present pleasant flavors, but it is interesting to mix the walnut with the almond, because the walnut oil flavor is stronger. Finally, butter and walnut oil have been combined at 50% to make the cookie more similar to the traditional one.The diameter and the thickness were measured in 5 baked cookies from each batch with a digital caliper. For the diameter, the mean was calculated from two perpendicular measures of each cookie. The expansion factor was calculated by dividing the diameter by the thickness.The color was measured by reflection in three points on the surface of the dough and the baked cookies with a colorimeter , using the illuminant D-65. The CIELab chromatic coordinates a* (red–green component) and b* (yellow–blue component) were annotated as recommended by the International Commission on Illumination . The meaTo measure the texture of the cookies, five cookies of each type were cut perpendicularly with a Warner Bratzler blade with a rectangular slot blade at the constant velocity of 2 mm/s, using a TA-XT Plus texture analyzer . The maximum force that was needed to cut the cookie and the deformation before breaking were recorded.The method used to determine the water content consisted of measuring the weight loss after drying at 103 °C until reaching constant weight . The ashCrude fat was estimated gravimetrically by the filter bag technique after the petroleum ether extraction of the dried sample in an Ankom XT10 extraction system . ProteinThe total carbohydrate content was calculated by subtracting the sum of the contents of the crude protein, fat, water and ash of the total weight of the sample , and theTo evaluate the acceptance of the cookies, an affective test was used. It was carried out in the sensory analysis laboratory at the Higher Technical School of Agricultural and Forestry Engineering in Albacete (Spain). A total of 103 consumers were asked to evaluate the external aspect, the texture and the taste of baked cookies using a nine-point scale ranging from −4, least preference (completely dislike), to +4, most preference (completely like) . The senIn addition, consumers fulfilled a questionnaire related to their degree of food neophobia. A 10-item Food Neophobia Scale was used . Consumep < 0.05). In the food neophobia study, the T-test was applied (p < 0.05) to compare the sensory results between the considered neophobic consumers and the non-neophobic consumers. All statistical analyses were carried out using SPSS software .Results are expressed as means and all the parameters were measured at least in triplicate. To determine the statistical differences, the analysis of variance test (ANOVA) at a 5% level of significance was used, as well as the Duncan test . The lowest spread factor was observed in the control sample, which means that this cookie, elaborated with wheat flour, grew less in diameter but more in thickness and may be because of the gluten that forms a web during the baking of cookies that increases viscosity and decreases the flow of mass, which causes a smaller diameter . The diaColor is one of the main attributes in terms of the physical characteristics of food and, in general, one of the first that consumers notice and value. For this reason, it is important to determine the possible changes in color due to the inclusion of new ingredients in order to verify that the final product has similar characteristics. The addition of new ingredients in the formulation can affect the values for a* and b* components.The control sample presented the lowest values of components a* and b* and, in general, the dough presented higher values compared with the baked cookies . HoweverAs regards texture, it is another important characteristic that the consumer values in the cookies. They must be crispy to be well valued by consumers. The analytical evaluation of cookies texture is difficult, since the results obtained are highly variable and difficult to interpret. However, the evaluation of the changes in the textural behavior can be analyzed by measuring the following two parameters: the maximum force, which measures the force needed to break the cookie, and the deformation that occurs in the cookie when the force is applied until it breaks . RegardiThe results of the proximate composition of the different cookies elaborated are shown in The use of almond and walnut flour in the formulation of cookies resulted in a significant increase in the protein content related to the control cookies elaborated with wheat flour or cookie AWB elaborated with 70% maize flour. This was also reported by , who shoThe inclusion of chia defatted flour in the formulation of cookies (AWC), although in a small percentage, significantly increased the crude fiber content of the cookies. Chia seeds have 22.00 g/100 g of dietary fiber , which pCrude fat was higher in the cookies where no butter was added . Yildiz and Gocmen (2020)  showed tIn the case of carbohydrates, the control sample and AWB that contain high amounts of maize flour in their composition present a higher carbohydrate value than cookies reformulated with nuts and seeds. Regarding these data, cookies reformulated with nuts and seeds would be an alternative source of protein and fiber with reduced carbohydrate content and containing fats that are beneficial for human health without hardly raising the energy value.Sensory tests were made to evaluate consumer preference regarding the external appearance of the cookies, the texture and the taste. The external aspects of the different cookies subjected to sensory evaluation are shown in Parallel to the sensory test, another test was carried out to measure the degree of neophobia of the consumers who participated in the sensory test to assess whether the inclusion of novel ingredients such as chia or poppy could determine the preferences of consumers towards cookies that do not contain these ingredients.The average value of the neophobia of the consumers who participated in the sensory analysis was 30.26 on a scale of 10 to 70 and the results are similar to the ones obtained by  that staThe results obtained in the sensory evaluation for the external aspect, the texture and the taste are shown in p < 0.05). Similar results were obtained by [However, regarding the taste and the texture, the inclusion of chia (AWC) and poppy (AWP) flour in the formulation of the cookies were least preferred compared with the control sample, AW and AWB  in a 9-point scale, where −4 implies extremely dislike, 0 neither like nor dislike and +4 extremely like.Regarding the food neophobia results, it should be noted that non-neophobic consumers are the ones who give higher scores to the taste and texture of cookies elaborated using the traditional composition. Neophobic consumers’ aversions to trying new products makes them give even lower scores to the traditional ones, because when they are faced with a product they do not know, these consumers tend to systematically give lower values.Cookies reformulated with almond, walnut, chia and poppy seed oils and flours are a nutritive alternative to traditional cookies that can be consumed also by celiac people as they do not contain any wheat in the formula. From the physical point of view, the reformulated cookies presented darker colors compared with the control sample, although it does not seem to be important for consumer evaluation. The control sample showed a lower expansion factor due to the presence of gluten in the formulation, and the texture of the cookies that contained flours and oils from seeds and nuts presented a softer texture compared with the control, which is linked to a lower acceptability when sensory tests are performed.In nutritional terms, the resultant cookies had an increase in protein, fiber and fat content, with a reduction in the carbohydrate content, which represents an improvement in the nutritional characteristics of cookies. However, the energy value did not change significantly.From the sensory point of view, the addition of poppy and chia flour resulted in lower values for taste and texture, but in general all the batches were well accepted by consumers since all evaluations were in the positive range. The lowest values of all the sensory characteristics evaluated corresponded to the neophobic consumers, who tend to reject foods with new ingredients that they are not used to.The results obtained meet the demands of today’s society, as people are more concerned about the impact of food on health. Thus, it is important to develop new formulas containing functional components such as nuts and seeds capable of positively inducing the control and prevention of certain diseases, in addition to satisfying an increasing demand for gluten-free products. The inclusion of novel ingredients such as seed and nut oils and defatted flours, even in low concentrations, may lead to an increase in the nutrients needed by the human diet.


Domain D score increased by 2.6 points ; this improvement was driven by countries with “low” 2015 domain D scores. In countries with “low” 2015 domain A scores, domain A score increased by 5.5 points . Conclusions: In 2019, improvements in emergency care organization and an increasing frequency of end-of-life practices were observed primarily in countries with previously “low” scores in the corresponding domains of the 2015 questionnaire.Background: In concordance with the results of large, observational studies, a 2015 European survey suggested variation in resuscitation/end-of-life practices and emergency care organization across 31 countries. The current survey-based study aimed to comparatively assess the evolution of practices from 2015 to 2019, especially in countries with “low”  2015 questionnaire domain scores. Methods: The 2015 questionnaire with additional consensus-based questions was used. The 2019 questionnaire covered practices/decisions related to end-of-life care (domain A); determinants of access to resuscitation/post-resuscitation care (domain B); diagnosis of death/organ donation (domain C); and emergency care organization (domain D). Responses from 25 countries were analyzed. Positive or negative responses were graded by 1 or 0, respectively. Domain scores were calculated by summation of practice-specific response grades. Results: Domain A and B scores for 2015 and 2019 were similar. Domain C score decreased by 1 point [95% confidence interval (CI): 1–3;  Data from multinational, observational studies suggest a substantial variation in end-of-life practices across European countries, and an increasing frequency of decisions to limit life-sustaining treatments, especially in southern Europe ,2. End-oIn 2015, we conducted a survey of experts from 31 European countries. We administered a comprehensive questionnaire spanning the following four domains: A: practices/decisions related to end-of-life care; B: access to best available care; C: death diagnosis and organ donation; and D: emergency care organization. Practices and organization of care were scored by numerical summation of positive responses. Results showed substantial variability in country-specific approaches to resuscitation/end-of-life care, indicating the presence of space for evidence-supported progress in all the aforementioned domains [In 2019, we undertook a methodologically improved version of the 2015 survey to address the following questions: (1) How did resuscitation/end-of-life care and emergency care organization evolve over the 2015–2019 period? and (2) Could such evolution, be more marked in countries with “low”  practice/organization scores for 2015?https://www.jmir.org/2004/3/e34/; accessed on 20 July 2019 see The current survey conforms with the Checklist for Reporting Results of Internet E-Surveys  European Resuscitation Council (ERC) National Resuscitation Council representative; and/or member of the European Registry of Cardiac Arrest investigators network or other ERC related clinical research networks ; (2) Established researcher in the field: first, second or last author of published, scholarly articles in this field; and (3) At least 3 years of prior service as lead clinician in emergency and/or intensive care.To reduce the risk of recall/social desirability bias, we aimed for at least three participants from each country. However, this did not constitute an inclusion criterion for country-specific responses. Consequently, responses from countries with just one or two participants were to be anyway included in the data analyses. Following the obtainment of informed consent see , particinever, sometimes, usually and always or between no and yes [never/sometimes and usually/always were categorized as no and yes, respectively. All data were entered in an original, “anonymized” Excel Masterfile. Original responses were received from 1 September 2019, to 25 October 2019. Participants from each country were asked to reconfirm their answers and provide any missing answers, approximately 3 months after the initial email invitation. Participants were also asked whether they agreed or disagreed with answers provided by other participants from the same country. In cases of disagreement, we encouraged resolution through consensus. The process of data finalization lasted from 1 December 2019 to 31 January 2020. Only consensus-based, country-specific responses were ultimately analyzed, besides the case(s) of having to include responses from just one country-specific respondent. This resulted in a final Excel Masterfile that included a single, country-specific response to each one of the survey questions [Respondents chose either among four options, that is,  and yes . Subsequuestions .The 2019 questionnaire was organized into four domains , preciseDomain B, C, and D scores were also calculated by summation of the respective subscores . Domain As further detailed in the footnote of The primary outcome was the presence/absence of statistically significant differences between 2015 and 2019 in domain A to D scores of all participating countries.The secondary outcome was the presence/absence of significant differences between 2015 and 2019 domain A to D scores of countries with “low” domain scores in 2015. “Low” 2015 scores were defined as domain scores equal to or lower than the corresponding, overall mean score values of 2015 ; more spThe tertiary outcome was the presence/absence of significant differences between changes in “low” 2015 domain scores from 2015 to 2019, and changes in “high” 2015 domain scores from 2015 to 2019.In an effort to determine any potential effect of the coronavirus disease-19 (COVID-19), we asked respondents to determine whether the pandemic could have resulted in changes in any of their original responses to the questionnaire . PertineThe study protocol was approved by the Ethics and Scientific Committee of Evaggelismos General Hospital Athens, Greece. The approval was used for study conduct in 32/44 European countries (73%) and Turkey. Countries are listed in the online supplement, along with details for informed consent and personal data protection. The protocol was registered with Clinicaltrials.gov (Identifier: NCT04078815).t-test or the Mann–Whitney U test.The internal consistency of the 2019 and the 2015 questionnaires was assessed by the determination of domain-specific Cronbach’s alpha. The distribution normality of domain scores and subscores was assessed by the Kolmogorov–Smirnov test with Lilliefors significance correction. Data are reported as number, number (percentage) and median (IQR) or mean ± SD unless otherwise specified. Comparisons pertaining to (1) study outcomes; and (2) domain subscores were conducted using an independent samples p < 0.05.Bivariate linear regression was used to explore possible associations between (1) the 2019 domain A and D scores with and without the “new—2019-only” questions ; and (2)A study flow diagram is presented in Domains A (end-of-life care practices/decisions), B (access to best resuscitation/postresuscitation care), C (death diagnosis/organ donation) and D (emergency care organization) of the 2019 questionnaire had Cronbach’s alpha values of 0.94, 0.94, 0.63 and 0.74, respectively. Domains A, B, C and D of the 2015 questionnaire, had Cronbach’s alpha values of 0.94, 0.88, 0.61 and 0.78, respectively. Regarding domain C, deletion of a question regarding “organ donation by opting in” in the 2019 questionnaire , and delResults on the primary and secondary outcomes are summarized in p = 0.02); this change was driven by a reduction in the organ donation subscore (p = 0.035)  . Regardi= 0.035)  and coef= 0.035)  suggest p = 0.047) (p = 0.009)  . The dom= 0.009) , therebyIn the context of a “pragmatic”, practice-level presentation, p ≤ 0.02)  .Responses to questions included only in the 2019 survey are detailed in the 2 = 0.35 to 0.43; p ≤ 0.001; 2 = 0.68, p <0.001; 2 = 0.79, p < 0.001; Linear regression revealed significant associations between 2019 domain A (end-of-life care practices/decisions) and domain D (emergency care organization) scores  of <1% ,44. HoweOrgan transplantation prolongs the life of recipients and improves its quality . An ongoAutomated external defibrillator (AED) availability and use improve survival and neurological outcome after shockable out-of-hospital cardiac arrest ,56,57,58Our results of increased defibrillation availability are consistent with the reported improvements in ambulance/pre-hospital level of care. However, the pertinent key determinant was the reported increase in ambulance advanced life support (ALS) . PrehospAs elsewhere detailed , registrResponses to domain A and D “2019-only” questions suggested variation in end-of-life practices and emergency care organization, respectively. Variable-specific scores for DNACPR and advance directives were predictive of the variable-specific score for ACP, possibly implying increasing integration of advance directives with ACP ,18. OverThe current questionnaire was not specifically designed to detect pandemic-induced changes in resuscitation practices and patient outcomes ,75,76. OThe current survey’s strengths include coverage of multiple aspects of resuscitation and end-of-life practices/care and comparative determination of their time-dependent changes ,31.Limitations (including subjectivity-related bias) exhibit similarities to those acknowledged in our 2015 survey . In 2019As in 2015, we assumed that respondents had a thorough knowledge of multiple aspects of practice/care or access to information concerning survey items . FurtherOur results were derived by simple summation of positive responses and should therefore be interpreted with caution as a higher domain A score may not always indicate better practice. Controversial end-of-life practices such as euthanasia and physician-assisted suicide (PHAD) ,81 shoulFinally, in 5/25 countries , we noted domain A score changes of >10 points from 2015 to 2019 ; pertineThere was a persisting, substantial variation in resuscitation/end-of-life practices across Europe in 2019, indicating a need for progress, preferably through harmonized legislations, governmental policies, and education/training. There was an overall improvement in emergency care organization from 2015 to 2019, driven by countries with “low” 2015 emergency care organization scores. Significant end-of-life practice changes were also noted in countries with “low” 2015 end-of-life practice scores. As in 2015, higher end-of-life practice scores were associated with better emergency care organization in 2019.


Simulation according to the phenomenological theory indicates that the domain-wall ME effect is dramatically enhanced when the separation between the FE domain walls shrinks below the characteristic width of the clamped AFM domain walls during the ferroelectric switching. Experimentally, we show that while the magnetization magnitude remains same for both the positive and the negative saturation polarization states, there is evidence of magnetization reduction at the coercive voltages. These results suggest that the domain-wall ME effect is viable for electrical control of magnetization.Electrical modulation of magnetic states in single-phase multiferroic materials, using domain-wall magnetoelectric (ME) coupling, can be enhanced substantially by controlling the population density of the ferroelectric (FE) domain walls during polarization switching. In this work, we investigate the domain-wall ME coupling in multiferroic h-YbFeO However, over the past decades, ME effects, with various coupling mechanisms between electrical and magnetic properties, were revealed in both composite materials and single-phase materials via the interfaces and the intrinsic interplay of charge, spin, and crystal structures respectively8. Among various ME effects, the electrical control of magnetization is especially appealing for information storage and processing applications due to the scalability and high energy efficiency9.Ferroelectricity and magnetism, originating from different symmetry conditionsFor a single-phase multiferroic materials, the effect of bulk-state polarization reversal on the magnetization, i.e., bulk ME effect, is expected to be weak, since the inversion of crystal structure, as required by polarization reversal, does not change the preferred spin direction necessarily within individual domain. Alternatively, ME effects can be triggered by FE domain walls if they are coupled to the magnetic domain walls. Therefore, by controlling the polarization switching, the domain-wall ME effect may be effective for electric-field modulation of magnetization.RFeO3, R = Y, Sc, Ho-Lu), whose complex interplay between ferroelectricity, magnetism and structural distortion11, especially the spin–lattice coupling, may enable substantial domain-wall ME effects. The crystal structure of h-RFeO3 consists of layers of FeO5 trigonal bipyramids separated by layers of R ions, as shown in Fig. RMnO3)16, h-RFeO3 exhibits improper ferroelectricity with non-centrosymmetric P63cm structure, which are induced by the K3 structural distortion below ~ 1000 K. As illustrated in Fig. 3 structural distortion can be viewed as the collective tilt of the FeO5, which causes buckling of the R layer and induces the spontaneous polarization (c axis18. Below the Neel temperature (~ 150 K)20, h-RFeO3 becomes antiferromagnetic with the Fe spins mostly lying in the basal plane with an 120° order. The tilt of FeO5 in the K3 distortion leads to the canting of Fe spins due to the spin–lattice coupling manifested in single-ion magnetic anisotropy (SIA) and the Dzyaloshinskii-Moriya (DM) interaction21, generating the spontaneous magnetization along the c axis, namely, canted antiferromagnetism22, as shown in Fig. 5 tilt can be represented by an in-plane angle 3+, occupy the R sites, their magnetic moments are aligned by the exchange field from the neighboring Fe spins, leading to the enhancement of magnetization20.Here we focus on multiferroic hexagonal ferrites  during polarization switching. Meanwhile, larger width of the FE walls reduces the domain-wall ME effect, but to a smaller extent. Experimentally, broadened FE walls and related distribution of structural order parameters  substrates by pulsed laser deposition (PLD), in which the LSMO (La2/3Sr1/3MnO3) layer is used as the bottom electrode, and the CFO (CoFe2O4) layer serves as a buffer layer to reduce the lattice mismatch between the LSMO layer and the h-YbFeO3 layer, stabilizing the P63cm structure of h-YbFeO3. To illustrate the structure of the h-YbFeO3 films, x-ray diffraction (XRD), reflection high energy electron diffraction (RHEED), and scanning transmission electron microscopy (STEM) measurements were carried out. Figure θ-2θ XRD scan of h-YbFeO3/CFO/LSMO/STO heterostructure without obvious impurity phases. As shown in Fig. 3 layer indicate smooth surfaces and the in-plane epitaxial relationship between individual layers. Along the STO [–211]/(111) orientation, the intense diffraction streaks separated by the weak streaks in the RHEED pattern of the h-YbFeO3 layer are indicators of the polar P63cm structure. Figure 3 (002) peak, indicating grain size of 10.3 nm according to Scherrer formula. Figure 3/CFO/LSMO/STO heterostructure viewed along the h-YbFeO3[001] direction, where the two arrows indicate the atomically sharp h-YbFeO3/CFO and CFO/LSMO interfaces, respectively. In the annular bright-field (ABF) image of a single ferroelectric domain region in the h-YbFeO3 layer 11; SIA requires 2 magnetic structure in h-RFeO3. As shown in Fig. To illustrate the available switching paths for both bulk and domain-wall ME coupling effects in h-YbFeO3, we start with the modulation of magnetization within a single clamped AFM wall. Across an FE domain wall, S and A are exchange stiffness and anisotropy constant.11 Using the parameters in Table S125, we found To analyze the important factors that determine the domain-wall ME coupling in h-YbFeOmentclass2pt{minimConsidering the larger population of the FE domain walls during the polarization switching, the modulation of average magnetization mentclass2pt{minim30. Since the width mismatch between the FE domain wall and the clamped AFM wall is the key, the broadening of the FE wall is expected to reduce the mismatch between In contrast to the separation of the FE wall  hysteresis loop, measured at 20 K, demonstrates that coercive voltages are around 5 edge with a circularly polarized x-ray in zero magnetic field after applying To examine the enhancement of domain-wall ME coupling effect mentioned above, the magnetization of Yb ions were measured using x-ray absorption spectroscopy (XAS) and x-ray magnetic circular dichroism (XMCD) during in-situ polarization switching, as illustrated by the schematic diagram in Fig. see Fig. b. The maP = 0) with highest density of FE domain walls. The detailed XMCD spectra for individual voltages between 5 edge undergoes observable reduction near the negative coercive voltage (block 2), comparing with the contrast of positive polarization state (block 1). The detailed comparison for the four blocks is given in Fig. The domain-wall ME effect is measured by comparing the XMCD spectra of Yb ions at different stage of polarization switching. As shown in Fig. 3 film is indeed the one for bulk ME decoupling, as indicated by low-barrier switching path in Fig. 3 films by domain-wall ME coupling on the switching path favoring bulk-state ME decoupling.Based on the polarization and magnetization states of each block in Fig. 3 films. In particular, in the heterostructure studied here, reduction of the h-RFeO3 film thickness may promote the multidomain state before reaching the ultrathin limit32. The corresponding higher population density of the FE walls may further enhance the domain-wall ME effect. Since the ME effect relies on both the ferroelectric and the magnetic orders, the temperature range under discussion is limited by TN ~ 120 K for h-YbFeO3. In this range, the ferroelectric properties are not expected to change substantially according to the Curie temperature Tc (~ 1000 K)37. The thermal-driven change for the domain-wall ME effect is expected to come mainly from the variation of magnetic properties such as magnetic anisotropy and exchange stiffness. More in-situ studies of the clamped AFM walls are necessary to reveal the temperature effects.The domain-wall ME effect may be tuned by thickness and temperature in the h-RFeO3 thin films when the distribution of FE domain wall reaches the critical condition  were grown on CoFe2O4 /La2/3Sr1/3MnO3/SrTiO3 (111) and yttrium stabilized zirconia (YSZ) (111) substrates by pulsed laser deposition (PLD) system with a KrF excimer laser (248 nm and 2 Hz repetition rate), at the growth temperature from 650 to 850 °C and oxygen pressure of 10 mTorr. Before thin-film deposition, substrates were pre-annealed at 700 °C for 1 h. The La2/3Sr1/3MnO3 (LSMO) layer (~ 30 nm) was grown at a substrate temperature of 700 °C and oxygen pressure of 80 mTorr on the SrTiO3 (STO) substrate. The CoFe2O4 (CFO) layer (~ 10 nm) was grown at the temperature of 600 °C and the oxygen pressure of 10 mTorr. The film growth was monitored using in-situ reflection high-energy electron diffraction (RHEED). The Au (3–5 nm) top electrodes were evaporated by an AJA sputtering system with 300–400 μm diameter.The h-YbFeO3 film surface. A 20 kV beam with a current of 0.7 nA was used to cut the lift-out. Rough and fine milling were performed at 10 kV and 5 kV with beam currents of 0.07 nA and 0.01 nA respectively. The resulting foil was mounted on a Cu grid, which was baked at 160 ℃ under vacuum prior to the STEM experiments to remove surface contamination.The structural phase of the epitaxial films was determined using X-ray diffraction (XRD) (Rigaku SmartLab). Scanning transmission electron microscopy (STEM) imaging was carried out using the aberration-corrected Nion UltraSTEMTM 200 microscope (operating at 200 kV) at Oak Ridge National Laboratory. An electron transparent thin foil for STEM characterization was prepared using a Hitachi NB5000 focused ion and electron beam system. To protect against the ion beam damage, a 1-μm-thick carbon layer was deposited on top of the h-YbFeOA commercial AFM/MFM  was used to map the topography and magnetic images at 20 K. During the measurement, the MFM was performed in constant height mode (single pass) with PPP-MFMR tip from NANOSENSORS. The lift height is 100 nm. The resolution of the image in supplementary is 60 nm, and the scan speed is 1um/s.The X-ray absorption spectroscopy (XAS) (including X-ray magnetic circular dichroism or XMCD) was studied at the beamline 6.3.1 in the Advanced Light Source at Lawrence Berkeley National Laboratory.The FE polarization was switched by the DC current using a Keithley 236 source meter (the measurements of high-resolution I-V curves) and a Keithley 2450 source meter (in-situ polarization switching during XMCD measurements). The polarization versus electric filed (P–V) loops were measured using a Precision RT66C Ferroelectric Tester.Supplementary Information.


Antisense oligonucleotides (ASOs) represent a new and highly promising class of drugs for personalized medicine. In the last decade, major chemical developments and improvements of the backbone structure of ASOs have transformed them into true approved and commercialized drugs. ASOs target both DNA and RNA, including pre‐mRNA, mRNA, and ncRDA, based on sequence complementary. They are designed to be specific for each identified molecular and genetic alteration to restore a normal, physiological situation. Thus, the characterization of the underpinning mechanisms and alterations that sustain pathology is critical for accurate ASO‐design. ASOs can be used to cure both rare and common diseases, such as orphan genetic alterations and cancer. Through pioneering examples, this review shows the versatility of the mechanisms of action that provide ASOs with the potential capacity to achieve custom treatment, revolutionizing personalized medicine.RNA in Disease and Development > RNA in DiseaseRNA Interactions with Proteins and Other Molecules > Small Molecule–RNA InteractionsThis article is categorized under: ASO functioning towards personalized medicine. Personalized and precision medicine is now becoming essential, alongside more general and sometimes only symptomatic treatment. Personalized medicine is adapted to the genetic, molecular, and phenotypic characteristics of the disease and the patient, by targeting specifically causative components. Importantly, one of the major challenges in the current management of many diseases is predicting the responses to treatment as a function of patient‐intrinsic characteristics. There is, therefore, a genuine revolution underway in patient care.BCR‐ABL). The resulting BCR‐ABL fusion protein has oncogenic properties due to its constitutive tyrosine kinase activity  fusion genes recently led to the development of specific inhibitors  for the management of NTRK‐positive cancers in adults and children  was driven by the understanding of the role of ER in breast cancer and the identification of mechanisms for its blockade. Tamoxifen is now a standard treatment for ER‐positive breast cancers. It acts by competitively inhibiting the binding of estradiol to its receptors Jordan, . MonocloIn some cases, very little or no protein is produced. This is true for insulin, an enzyme that is deficient in patients with insulin‐dependent diabetes (type I). Patients are treated by insulin therapy to faithfully reproduce the effects of the physiological secretion of insulin by administering a substitute protein. The release of the first human insulin protein onto the market in 1982 launched a new paradigm: it became possible to modify the sequence of the hormone protein to match the pharmacokinetic properties to the physiological needs of the patient  have been developed. As for proteins, the first therapeutic attempts were based on global alterations to DNA, through the use of alkylating agents, for example. These agents induce the creation of non‐specific covalent bonds, resulting in the production of DNA‐adducts. They disrupt replication and transcription, explaining their use in cancer therapeutics  in the United States of America and European Medicines Agency (EMA) in Europe). This is the result of many years of basic research and clinical trials. Throughout pioneering examples, this review aims to underscore the versatility of ASOs for the treatment of various diseases through the targeted and personalized RNA‐based approach they allow.1.1ASOs are short (about 12 to 25 nucleotides long) single‐stranded nucleic acid (DNA or RNA) sequences. In their naked form, they cannot permeate the plasma membrane and are highly sensitive to degradation by endonucleases and exonucleases. ASOs have been chemically modified to overcome these problems. On the basis of these modifications, ASOs can be broadly classified into three generations , methyl‐phosphonates, and phosphoramidates, respectively Figure . These mO‐methyl (2′‐OME) and 2′‐O‐methoxyethyl (2′‐MOE) nucleotides  have created new opportunities in chemical therapeutics. Tricyclo‐DNA (tcDNA) belongs to this class of conformationally constrained DNA analogs with enhanced binding properties. They do not elicit RNAse‐H activity but show increased stability and improved cellular uptake, giving them substantial therapeutic advantages over those of ASOs  and the target sequence. The choice of ASO is, therefore, guided by the required molecular effect , by sequence complementarity. This process was first described in plants  approved by the FDA in 1998 and the EMA in 1999 Figure . Fomivir1.2.2β‐thalassemia. They showed that 2′‐O‐methylribooligonucleotides targeting specific sequence elements in mutated human β‐globin pre‐mRNA could eliminate the aberrant splicing pattern of the β‐globin pre‐mRNA  gene. These genetic alterations lead to the absence of SMN protein production. Interestingly, SMN proteins are encoded by two paralogous genes: SMN1 and SMN2. The SMN1 and SMN2 genes are identical (99% homology), except for an 11‐nucleotide sequence in exon 7. This difference alters splicing. The splicing of the SMN2 pre‐mRNA leads to the exclusion of exon 7, generating a truncated, unstable protein that is rapidly degraded. SMN2 cannot, therefore, compensate for the loss of SMN1 expression in SMA. Nusinersen, discovered by Ionis Pharmaceuticals and co‐developed by Biogen (Spinraza®), was designed to correct SMN2 splicing, thus allowing SMN2 to compensate for the loss of expression caused by the SMN1 mutation. This 2′‐O‐methoxyethyl (2′‐OME) phosphorothioate (PS) 18‐mer is a second‐generation ASO, which cannot recruit RNAse H. It targets the intronic splicing silencer (ISS‐N1), promoting the inclusion of exon 7, thereby restoring the production of a stable SMN2 protein  non‐coding RNAs that specifically hybridize to mRNAs to fine‐tune their expression. miRNAs bind to their cognate target RNA through RNA–RNA base‐pairing. The miRNA binding sequence is referred to as a miRNA recognition element (MRE). MREs are complementary to miRNA seed regions Bartel, . PerfectThe overexpression of miRNA in certain diseases, including cancers, can lead to massive pathological degradation of their targets. ASOs binding to specific MREs could thus be used to block such mRNA degradation. In this case, ASOs are specific for mRNA and not miRNA. No therapeutic molecules based on this mode of action are currently available. However, the group of Prevot has demonstrated the efficacy of such an approach  gene , is a clinically heterogeneous neuromuscular disease. DM1 is a triplet‐repeat disease originating from progressive expansion of CTG repeats in the 3′ UTR of the DMPK mRNA with an ASO displaying RNAse H activity (MOE gapmers), the sequestered MBNL1 protein is released and accurate splicing is restored  competes with MBNL binding to the CUG‐repeats. This ASO has been shown to be effective in triggering the release of MBNL and restoring splicing function in mice  in exon 23 of the dystrophin gene, resulting in a premature translation termination codon. They obtained comparable results in a hemophilia mouse model, in which Upf3b‐ASO treatment restored the production of functional factor IX protein  is one of such quality‐control mechanisms for mRNAs. It eliminates mRNAs that include a premature stop codon. This allows the removal of abnormal proteins that are non‐functional or with impaired function, and prevents the production of proteins with dominant‐negative activity . NGD is triggered by obstacles that impede ribosome movement along the mRNA. These altered mRNA molecules are targeted for degradation, initiated by endonucleolytic cleavage in the vicinity of the stalled ribosome  that specifically hybridize to the start codon (uAUG) of such uORFs. By specifically blocking translation of the uORF, the ASO redirects the translation machinery to the pORF. The efficacy and specificity of this approach has been evaluated with several human and mouse model genes (including the human RNAseH1 and sideroflexin 3 [SFXN3] genes). This resulted in a significant increase in the level of the corresponding proteins  to specifically increase the synthesis of the main protein translated from a downstream primary ORF (pORF). They generated ASOs  and in vivo (mouse liver)   by targeting its regulator PSCK9  is one of the first miRNA therapies to be developed and undergo clinical testing. It targets miR‐122 for the treatment of hepatitis C virus infections. miR‐122 is a key player in liver development. This abundant miRNA is produced in a specific manner. It also plays an important role in the replication cycle of the hepatitis C virus (HCV), helping to stabilize the viral genome. Patients infected with HCV have a long‐term risk of developing liver cancer . Like miravirsen, RG101 is an anti‐miR‐122 agent. It differs from miravirsen in its chemical structure. It is an ASO phosphorothioate coupled to an N‐acetylgalactosamine group , to enhance uptake by hepatocytes via the asialoglycoprotein receptor. Despite these significant improvements and the therapeutic effects observed  complementary to the coding region of the ApoB100 mRNA leads to its degradation by RNAse H. Mipomersen use over several weeks induces a progressive decrease of LDL‐cholesterol levels  is an inhibitor of the synthesis of ApoB100   complementary to the non‐coding UBEA3‐ATS RNA caused its degradation after the recruitment of RNAse H. As the paternal copy of UBE3A is no longer repressed, the paternal UBE3A protein is produced, thereby restoring UBE3A function , which, like coding RNAs, may cause disease. Angelman syndrome is a genetic neurological disorder characterized by severe motor and intellectual disability , resulting in wide systemic distribution and possible uptake by target tissues and cells. Unfortunately, these interactions also contribute to the toxicity of ASOs. Chemical modifications designed to minimize these effects and increase the therapeutic index are currently being studied  administration should be favored for systemic effects with the most recent generation of ASOs. This administration route maximizes the bioavailability of ASOs and leads to rapid distribution to highly irrigated organs . By contrast, distribution is weak in the lungs, cardiac muscle, and skin, and negligible in adipose tissue. Nonetheless, iv administration is considered to be the optimal route for the treatment of Duchenne muscular dystrophy   gene. Approximately 100 mutations of this gene have been described, associated with varying degrees of disease severity due to amyloid deposits in tissues and nerves. Such deposits lead to the progressive onset of cardiomyopathy, nephropathy, polyneuropathy, and gastrointestinal disorders  route is an interesting alternative to the iv route, increasing the ease of administration, when possible. Inotersen (Tegsedi®), marketed by Ionis Pharmaceuticals and Akcea Therapeutics, has recently been licensed for sc administration in the United States, Canada, and Europe Figure  Keam, 2. This drhuntingtin gene. In the presence of abnormally expanded CAG repeats, toxic accumulation of the protein occurs  to the eye . This is true for eluforsen in cystic fibrosis patients homozygous for the F508del‐CFTR mutation (cystic fibrosis transmembrane conductance regulator). This molecule restores the activity of the CFTR channel and, therefore, the respiratory function of treated patients   moiety to the ASO. The GalNAc receptor ligand specifically binds to the Asialoglycoprotein Receptor (ASGR), which is highly expressed at the surface of hepatocytes, substantially increasing the targeting of GalNac‐ASO to the liver and its uptake  makes ASOs up to 30 times more potent non‐conjugated ASOs, supporting lower doses and increased safety  that targets lipoprotein(a) mRNA (LPA), showed a significant dose‐dependent reduction in lipoprotein(a) levels in patients with cardiovascular disease . This is a major breakthrough, as APO(a)‐LRX is the first and currently only drug in clinical development designed to selectively and robustly inhibit the production of Lp(a)  and very recently for neuronal ceroid lipofuscinosis 7 (CLN7).DMD is a rare genetic disorder linked to the X chromosome. It affects between 1 in 3,500 and 1 in 6,000 newborn boys worldwide each year  and PRO044  target exon 45 . This strategy is, however, limited by the intrinsic packaging capacity of these viruses, which can hold, at most, only one third of the dystrophin sequence. Recent studies have been conducted with lentiviruses  and an SVA (SINE‐VNTR‐ALU) retrotransposon insertion in the second allele. The SVA insertion affects splicing of the nearby gene, inducing mis‐splicing of exon 6 of CLN7. 22‐nucleotide ASOs  were designed and tested in patient fibroblasts to restore the altered splicing. The most effective were retained for further development. This patient‐customized ASO was named Milasen. The urgency of the patient's clinical state sped up toxicological evaluation. After obtaining FDA authorization and institutional review board approval, the investigators administered the compound intrathecally to the patient in ascending doses, as for nusinersen, which had been safely administrated to infants with SMA , a form of Batten's disease  in BRAF‐V600 mutated metastatic melanoma  was designed to reduce the production of STAT3 . STAT3 plays a key role in transducing signals that promote cell growth and survival, and is thus a potential drug target for several types of cancer  , a phosphorothioate ASO, targets the transforming growth factor beta (TGF‐β) pathway. Trabedersen specifically targets TGF‐β2, which plays a key role in the late stages of tumor development . Several phase I/II clinical trials have evaluated the utility of this ASO to treat high‐grade gliomas. Although unsuccessful, these trials made it possible to evaluate the safety of this ASO and its administration. Promising results have also been obtained for the treatment of advanced pancreatic cancer , a second‐generation ASO, targets the clusterin mRNA. Clusterin is a stress‐dependent chaperone protein. Its anti‐apoptotic activity promotes the development of resistance to anticancer treatments. Clusterin inhibition therefore potentiates the therapeutic activity of conventional chemotherapies , a second‐generation ASO, targets another chaperone protein, heat‐shock protein 27 (Hsp27). Hsp27 levels are very high in some cancers and this is associated with a poor prognosis. The inhibition of Hsp27 decreases tumor growth and sensitizes the tumor to cytotoxic chemotherapies. The preliminary results of a phase II trial assessing docetaxel alone or in combination with apatorsen for the treatment of metastatic urothelial carcinoma showed a slight increase in overall survival in the combination group. These results are promising and require confirmation . Decreasing MALAT‐1 levels with an ASO significantly inhibits tumor growth and metastasis in lung and breast cancers . In vivo, the potential therapeutic value of these gapmers has been demonstrated in PDX models (patient‐derived xenografts)  Figure .tyrosinase‐related protein 1 (TYRP1) mRNA. TYRP1 is specifically expressed in the melanocyte lineage and is highly expressed in melanoma. We identified three non‐canonical MREs for miR‐16 in the 3′ UTR of TYRP1. These non‐canonical MRE‐16s provide a non‐coding function to TYRP‐1 mRNA, which acts as a miRNA‐sponge. Indeed, such non‐canonical binding results in the sequestration of miR‐16, with delayed or no TYRP1 mRNA degradation. As miR‐16 has tumor suppressor activity in cutaneous melanoma, its sequestration promotes tumor growth. In this context, we designed ASOs to prevent such sequestration. They consist of target‐site blockers (TSB): a third‐generation ASO, consisting of a mixmer of DNA and LNA, with no RNAse H activity. These TSBs were designed to be specific for TYRP1 mRNA, hybridizing to TYRP1 in place of miR‐16. As they are perfectly complementary to the TYRP1 mRNA along their entire length, their affinity for TYRP1 mRNA is greater than that of miR‐16. TSB displaces miR‐16, allowing the redirection of miR‐16 to other targets. The tumor suppressor activity of miR‐16 is thus restored. These TSBs were shown to be effective in vivo in a PDX model. This specific and ground‐breaking treatment strategy can restore the activity of miR‐16 exclusively in cells expressing TYRP1. This innovation circumvents the need for a synthetic microRNA, which through its systemic effects, would have consequences throughout the body  and bioavailability and, importantly, their sequence can be adapted to genetic alterations. The fact that ASO can be customized in a sequence‐specific fashion to match patient disorders was recently strongly underscored with the splice‐switch ASO Milasen (J. Kim, Jo, et al., Important chemical developments have been made to diminish the toxicity of ASOs, and the most representative molecules of these chemical classes are well tolerated. However, substantial toxicity may still be observed when they are used chronically. This may be an important drawback that should be lessened by the development of conjugated‐ASOs that promote cell‐specific uptake.ASOs may be used in monotherapy or in combination with conventional approaches. They are therefore both an alternative for diseases with no effective available treatments and an option for use in patients with refractory diseases, as second‐ or third‐line treatments.In conclusion, the phrases “Targeting the Untargetable” and “Treating the Untreatable” summarize the potential of ASO strategies, thus revolutionizing personalized medicine.Anais M. Quemener: Conceptualization; writing‐review and editing. Laura Bachelot: Conceptualization; writing‐review and editing. Anne Forestier: Writing‐review and editing. Emmanuelle Donnou‐Fournet: Writing‐review and editing. David Gilot: Writing‐review and editing. Marie‐Dominique Galibert: Conceptualization; funding acquisition; writing‐review and editing.The authors have declared no conflicts of interest for this article.Natural antisense transcripts in diseases: From modes of action to targeted therapiesAlternative‐splicing defects in cancer: Splicing regulators and their downstream targets, guiding the way to novel cancer therapeuticsSplicing and neurodegeneration: Insights and mechanisms