Weissella cibaria MG1 (BR-MG1), Leuconostoc citreum TR116 (BR-TR116), Lactiplantibacillus plantarum FST1.7 (BR-FST1.7), Lactobacillus amylovorus FST2.11 (BR-FST2.11), and Limosilactobacillus reuteri R29 (BR-R29) in bread. The antifungal compounds in BR ingredients and the impact of BR on dough rheology, gluten development, and dough mixing properties were analysed. Additionally, their effects on the techno-functional characteristics, in vitro starch digestibility, and sensory quality of bread were determined. BR-UnF showed dough viscoelastic properties and bread quality comparable to the baker’s flour (BF). BR-MG1 inclusion ameliorated bread specific volume and reduced crumb hardness. Breads containing BR-TR116 had comparable bread quality to BF, while the inclusion of BR-R29 substantially slowed microbial spoilage. Formulations containing BR-FST2.11 and BR-FST1.7 significantly reduced the amounts of sugar released from breads during a simulated digestion and resulted in a sourdough-like flavour profile. This study highlights how BR fermentation can be tailored to achieve desired bread characteristics.The process of upcycling and incorporating food by-products into food systems as functional ingredients has become a central focus of research. Barley rootlets (BR) are a by-product of the malting and brewing industries that can be valorised using lactic acid bacteria fermentation. This research investigates the effects of the inclusion of unfermented (BR-UnF), heat-sterilised (BR-Ster), and five fermented BR ingredients (using  In recent years, by-product rejuvenation has become a pivotal point of food research to align with the sustainability goals of the future. Spent barley rootlets (BR) are the main by-products produced during the malting process and can be generated in volumes that equate to up to 5% of the total malt weight ,2. Due tAlthough limited studies exist in the literature regarding the application of BR in food matrices, the implementation of BR into the food chain has promising potential from a nutritional perspective, which can increase the protein and fibre values of foods . HoweverLactic acid bacteria (LAB) fermentation has become a process of interest in the enhancement of side-stream products, with numerous studies showcasing their successes ,16,17,18Weissella cibaria MG1, Lactiplantibacillus plantarum FST1.7, Limosilactobacillus reuteri R29, Leuconostoc citreum TR116, and Lactobacillus amylovorus FST2.11) on BR. Following this, the current study aimed to investigate the effects of the inclusion of fermented BR ingredients in a bread matrix. Fermented BR ingredients were incorporated into bread recipes at a 5% replacement level based on bakers’ wheat flour. The effect of BR addition on dough quality, techno-functional characteristics, microbial shelf-life, sensory experience, and nutritional characteristics (release of reducing sugars during starch digestion) of bread were monitored. Formulations containing bakers’ wheat flour (BF), unfermented BR (BR-UnF), and heat-sterilised BR (BR-Ster) were used as controls.Neylon et al.  performew/v) of BR-UnF were added to water to a final volume of 800 mL and transferred to a 1 L batch bioreactor on a DASGIP Bioblock equipped with DASGIP TC4SC4 and DASGIP PH8 modules . A total of 40 g (5% w/v) of the appropriate sugar source was incorporated in the rootlet/water mixture. A sterilisation process of the rootlet/water/sugar mixture was performed at 90 °C for 30 min, cooled to 30 °C, and the selected LAB strain was inoculated . The BR fermentation was performed at 30 °C for 96 h using a continuous mixing (400 rpm) process. After completion of fermentation, BR mixtures were pasteurised  and finally freeze-dried. Along with BR, bread recipes also included instantaneous freeze-dried yeast ; granulated sugar ; salt ; sunflower oil ; and regular faucet water. The compositional analyses of the BF and BR-UnF are illustrated in Ingredients incorporated into bread recipes included bakers’ flour (BF) ; spent barley rootlets (UnF-BR) supplied by Anheuser-Busch InBev ; a sterilised, freeze-dried BR ingredient (BR-Ster); and five fermented, freeze-dried BR ingredients . The fer®  was utilised to adjust the amount of water required for each recipe using an automatic water dosing system . Recipes for a total volume of 300 g were adjusted to a target dough consistency of 500 ± 20 FU with the mixing chamber temperature set to 30 °C. A 5% addition (based on flour) of the respective BR ingredient was included in bread recipes by replacing BF. The bread recipes used for analysis are included in The Farinograph-TSBread doughs were produced by a straight dough process . FirstlyThe viscoelastic properties of the doughs were evaluated using a Rheometer Physica MCR 301 . Bread doughs were prepared as outlined above, but with the yeast excluded. A serrated plate method was employed for the analysis, with the plates set up in parallel geometry. The lower plate was held at 35 °C and was assembled with an upper plate of 50 mm in diameter. The linear viscoelastic region was evaluated using an amplitude sweep , and fre2 retention coefficient (%), and CO2 volume generated during fermentation (mL) were analysed.The bread dough quality was analysed using a Rheofermentometer . Briefly, 300 g of bread dough was prepared, positioned in the fermentation chamber, and a 1500 g cylindrical weight was placed on top of the dough. The fermentation chamber was sealed, and the BR doughs were left to ferment for 3 h at 30 °C. The maximum dough height (Hm) in mm, CO® values  according to the method detailed by Rosell et al. . Briefly® values  to reachThe gluten network aggregation was evaluated using GlutoPeak . The flour blends  were preFor each recipe outlined in Bake loss was calculated to account for the amount of water lost during the baking process. The bake loss of four loaves per batch was analysed and calculated according to the below formulas:The specific volume of each bread loaf was determined using the Volscan Profiler  and expressed as mL/g. Four bread loaves were analysed per batch.The C-Cell Imaging System  was used to analyse the cell diameter of the breads. Four bread loaves per batch were cut into slices of 20 mm thickness (three slices per loaf), with crust slices omitted from the analysis.The crumb texture of bread slices was measured using the TA-XT2i Texture Analyser . The TA-XT2i Texture Analyser, together with a 25 kg load cell, conducted a two-compression test with a strain of 40%, a test speed of 5 mm/s, and a trigger force of 0.05 N. A waiting time of 5 s between the two compressions was used. A 20 mm cylindrical probe was attached for the analysis. Bread slices of 20 mm thickness were analysed for their hardness and resilience after the breads had cooled. The crumb hardness was determined as the maximum force of the first compression. Breadcrumb resilience was determined by dividing the energy required during the upstroke action of compression one by the energy required during the downstroke action of compression one.2O along with 10 mL of ethyl acetate containing 0.1% (v/v) formic acid. The samples were mixed thoroughly using a vortex shaker. Following this, 4 g of MgSO4 and 1g of NaCl were added, and the samples were shaken by hand for exactly 1 min. Afterwards, samples were centrifuged . The supernatant was placed in solid-phase extraction tubes . A vortex shaker was used to distribute the contents of the supernatant in the tubes, which were then centrifuged . Directly after, a total of 5 mL of supernatant was combined with 100 µL of dimethlsulfoxide. In a vacuum centrifuge , solvents present in samples were allowed to evaporate for two hours at 500 rpm and 45 °C before reconstitution with 400 µL of ultrapure H2O/acetonitrile . A syringe-driven filter (0.2 µm) was used to clarify the samples. Evaluation of the antifungal compound profiles of BR was performed according to chromatographic test parameters detailed by Hoehnel et al. [A total of 15 known antifungal phenolic compounds were tested for their presence in BR ingredients , includil et al. . AntifunThe microbial shelf-life of the breads was measured using the mould environmental challenge method ,44,52 wi2O. The absorbance of the samples at 546 nm was measured, and the reducing sugar release % (RSR) over time was determined using the below formula [The release of reducing sugars was investigated by means of an in vitro digestion assay using an enzymatic degradation of starch. The method was carried out as previously described by Brennan & Tudorica , which w formula .RSR %= from the Food Science department of the University College Cork. Panellists evaluated the intensity of the odour attributes ‘overall intensity, ‘citrus’, ‘vegetables, ‘cereals/grains’, the flavour attributes ‘overall intensity’, ‘muddy/earthy’, ‘fruity’, ‘vegetables’, ‘aftertaste’, the taste attribute ‘sour’, and the texture attributes ‘hardness’, ‘chewiness’ on a scale from 0, ‘not present’ to 10, ‘extremely present’. Panellists were also asked to rank the overall acceptability of the breads. Sensory analysis of all breads was performed in duplicate, and breadcrumb samples were analysed on the same day they were baked. The questionnaire used during sensory analysis is available in the The sensory properties of the breadcrumbs were investigated by performing a descriptive analysis test using an experienced sensory panel  were completed using SPSS statistical software  in order to determine significant differences between sample groups. A Welch test correction and Games Howell post hoc test (p < 0.05) were performed on instances where equal variances could not be assumed. A Kruskal-Wallis test (p < 0.05) was utilised when data did not conform to normal distribution.Experimental analysis was carried out in triplicate unless stated otherwise. In the case of normally distributed data, a one-way ANOVA and post-hoc Tukey test  and the time taken for gluten development (PMT) are shown in The Mixolab analyses were conducted to investigate the effects observed in dough mixing characteristics and pasting properties under mechanical and thermal stresses .The shortest DDT was observed for the BF formulation (1.32 ± 0.27 min), with significantly higher DDT observed in the BR-UnF (7.04 ± 0.23 min) and BR-Ster (7.49 ± 0.32 min) formulations. Significant reductions in DDT (3.66–5.61 min) were determined with the inclusion of most fermented BR ingredients. The BR-MG1 formulation was the exception to this trend, with no significant difference in DDT noted when compared to the BF, BR-UnF, and BR-Ster formulations.The BF control recipe resulted in the highest C2 value (0.427 ± 0.01 Nm), with a significant reduction in C2 values recorded with the inclusion of both BR-UnF (0.33 ± 0.00 Nm) and BR-Ster (0.38 ± 0.03 Nm). Comparing fermented formulations, the C2 values for doughs containing BR-MG1 (0.36 ± 0.00 Nm) and BR-TR116 (0.35 ± 0.01 Nm) were the highest. C2 values for BR-MG1 and BR-TR116 recipes were significantly lower than BF and BR-Ster, however, but were significantly higher than the BR-UnF formulation. The inclusion of BR-R29 reduced the C2 value to 0.34 ± 0.00 Nm, with an even greater reduction observed with doughs containing BR-FST2.11 (0.31 ± 0.01 Nm) and BR-FST1.7 (0.30 ± 0.01 Nm).BF had the highest C3 value recorded (1.63 ± 0.01 Nm), which was significantly higher than the BR-Ster value (1.52 ± 0.01 Nm). In contrast, the BR-UnF formulation had the lowest C3 value (1.24 ± 0.02 Nm). C3 values for fermented formulations were in the range of 1.53–1.63 Nm.Comparing C4 values, BR-UnF had the lowest value recorded (0.71 ± 0.03 Nm). A significant increase in C4 was observed for the BR-Ster formulation (1.38 ± 0.01 Nm), with an even greater increase reported for BF (1.50 ± 0.01 Nm). C4 values for BR-MG1 (1.41 ± 0.01 Nm) were comparable to the BR-Ster formulation. No significant difference was found between BR-MG1 and BR-TR116 (1.44 ± 0.01 Nm). The BR-R29 (1.47 ± 0.01 Nm), BR-FST1.7 (1.50 ± 0.01 Nm), and BR-FST2.11 (1.51 ± 0.01 Nm) C4 values were comparable to the BF recipe.Trends for C5 values mirrored those reported for C4 values. BR-UnF doughs had the lowest C5 value recorded (1.12 ± 0.03 Nm), while significant increases were observed in C5 values for doughs containing BR-Ster (2.23 ± 0.03 Nm) and BF (2.55 ± 0.03 Nm). C5 values for fermented formulations were in the range of 2.28–2.65 Nm.The fermentation capacity of each dough was analysed to provide information on the yeast fermentation process .2 recorded varied between recipes. Firstly, comparing controls, the lowest amount of CO2 produced was recorded in the BF recipe (2045 ± 73 mL), while significant increases were observed in the volume of CO2 produced with BR-UnF (2451 ± 30 mL) and BR-Ster (2399 ± 100 mL) inclusion. The incorporation of BR-MG1 produced the highest amount of CO2 during the analysis (2547 ± 68 mL) and was comparable to the BR-UnF and BR-Ster controls. Similarly, the BR-FST2.11 and BR-FST1.7 formulations maintained high amounts of CO2 production, resulting in values of 2318 ± 131 mL and 2302 ± 172 mL, respectively. A slight drop in the level of CO2 produced was observed in the BR-TR116 recipe (2029 ± 69 mL), which was more comparable to the BF and BR-FST1.7 formulations. The lowest amount of CO2 produced was noted in the BR-R29 recipe (1919 ± 59 mL), which was similar to the volume of CO2 produced in the BF fermentation process. Finally, the CO2 retention coefficient for all recipes was comparable, with no significant difference found between them.Of the formulations tested, BF had the highest Hm (50.93 ± 2.48 mm). The inclusion of BR-UnF maintained the dough height (46.40 ± 1.31 mm), with no significant difference observed compared to the BF control. A greater reduction in Hm was observed when BR-Ster was included (40.43 ± 0.57 mm). Comparing fermented BR formulations, the highest Hm was recorded with BR-MG1 inclusion (44.87 ± 1.52 mm) and was comparable to all control recipes. The inclusion of BR-TR116 resulted in a slight decrease of Hm (43.17 ± 3.26 mm), but was not significantly different from the BR-MG1 Hm results. Compared to BF and BR-UnF, the addition of BR-FST2.11, BR-R29, and BR-FST1.7 reduced Hm significantly, with values of 37.00 ± 0.30 mm, 36.57 ± 2.53 mm, and 34.97 ± 3.10 mm, respectively. However, these values were not found to be statistically different from the Hm of the BR-Ster recipe. The volume of COTo investigate the dough’s elastic (solid) and viscous (liquid) parts, oscillation measurements were conducted, and the damping factor (DF) was determined. A material is considered ideal and elastic if the damping factor is 0, implying that the material contains no viscous elements. An increase in the DF signifies an increase in the viscous parts of the dough system.As shown in A visual representation of the final baked bread products is illustrated in Bake loss (BL) was determined to analyse the extent of water loss during the bake. Results from this analysis are depicted in The specific volume of each bread was analysed as an important parameter to investigate the rise and expansion of the bread. The results from the specific volume analysis are presented in Concerning the fermented ingredients, the formulation including BR-MG1 resulted in the highest specific volume recorded (3.80 ± 0.13 mL/g), which was comparable to BF and significantly higher than the BR-UnF and BR-Ster controls. BR-TR116 had a specific volume of 3.68 ± 0.22 mL/g, which was comparable to BF and BR-UnF. A significant reduction in specific volume was observed in BR-R29 (3.35 ± 0.29 mL/g). The lowest specific volumes recorded were in BR-FST2.11 (2.80 ± 0.15 mL/g) and BR-FST1.7 (2.63 ± 0.19 mL/g) formulations, which were significantly lower than BF, BR-UnF, and BR-Ster control recipes.The cell diameter was determined in order to provide information on the cell structure of the crumb. Results are depicted in The largest cell diameter was determined in the BF control bread (2.93 ± 0.15 mm). A reduction in cell diameter was observed in BR-UnF (2.16 ± 0.15 mm) and BR-Ster (1.94 ± 0.18 mm) bread compared to the BF.Amongst the fermented formulations, BR-MG1 had the highest cell diameter (2.05 ± 0.16 mm), which was not significantly different from the BR-UnF and BR-Ster controls. The BR-TR116 formulation had a cell diameter of 1.95 ± 0.17 mm, followed by BR-FST2.11 (1.83 ± 0.22 mm), BR-R29 (1.83 ± 0.15 mm), and BR-FST1.7 (1.63 ± 0.16 mm).The breadcrumb texture was determined to provide information on the breadcrumb quality. Results of crumb texture parameters hardness and resilience are represented in Among the control recipes, the softest crumb was noted in the BR-UnF formulation (1.77 ± 0.26 N). A significant increase in breadcrumb hardness was observed in BF (2.33 ± 0.32 N), with an even larger increase recorded in the BR-Ster (3.14 ± 0.42 N).The lowest breadcrumb hardness was found in BR-MG1 (1.61 ± 0.22 N), followed by BR-TR116 (1.96 ± 0.30 N), with both recipes having comparable crumb hardness to the BR-UnF formulation. An increase in crumb hardness was found in the BR-R29 formulation (2.39 ± 0.35 N), which was more comparable with the BF control recipe. In the BR-FST2.11 and BR-FST1.7 recipes, a significant increase in crumb hardness was observed, with hardness values of 3.59 ± 0.53 N and 4.11 ± 0.55 N recorded, which were aligned with the BR-Ster hardness value.The highest breadcrumb resilience was noted in the BF control (0.56 ± 0.01 N), with some reductions in breadcrumb strength found in BR-UnF (0.53 ± 0.02 N) and BR-Ster (0.55 ± 0.01 N). Comparing fermented ingredients, the inclusion of BR-MG1 resulted in the highest breadcrumb resilience (0.56 ± 0.01 N). A reduction in breadcrumb strength was observed with BR-TR116 (0.55 ± 0.02 N) and BR-R29 inclusion (0.54 ± 0.01 N), aligning with BR-Ster and BR-UnF values. BR-FST2.11 (0.52 ± 0.02 N) and BR-FST1.7 (0.53 ± 0.01 N) inclusion had the greatest reduction in crumb strength, which was significantly different from BF, BR-UnF, and BR-Ster formulations.The BR ingredients used in the study were screened for the presence of 15 known phenolic-type antifungal compounds to investigate naturally present antifungal compounds as well as the extent of those produced during LAB fermentation. Results from the analysis are presented in Hydroxyphenyllactic acid was not detected in quantifiable amounts in BR-UnF, BR-Ster, or BR-MG1. The highest amount of hydroxyphenyllactic acid was determined in BR-FST1.7 (9.136 ± 0.104 g/100 g D.M.). In comparison to BR-FST1.7, a significant reduction in hydroxyphenyllactic was observed in BR-FST2.11 (2.483 ± 0.247 g/100 g D.M.) and BR-R29 (2.239 ± 0.087 g/100 g D.M.), with the lowest amount recorded in BR-TR116 (0.637 ± 0.091 g/100 g D.M.). Concerning 4-hydroxybenzoic acid, the highest amount detected was in BR-Ster (1.215 ± 0.190 g/100 g D.M.). No significant differences were found in levels of 4-hydroxybenzoic acid between BR-Ster and BR-UnF (0.928 ± 0.006 g/100 g D.M.). Among the fermented ingredients, relatively similar amounts of 4-hydroxybenzoic acid were determined. BR-TR116 (1.177 ± 0.036 g/100 g D.M.) contained the highest amount of 4-hydroxybenzoic acid, followed sequentially by BR-MG1 (0.881 ± 0.101 g/100 g D.M.), BR-FST2.11 (0.864 ± 0.078 g/100 g D.M.), and BR-FST1.7 (0.861 ± 0.004 g/100 g D.M.) with amounts comparable to BR-UnF and BR-Ster. The lowest amount of 4-hydroxybenzoic acid was recorded in BR-29 (0.653 ± 0.034 g/100 g D.M.). With regards to vanillic acid levels, no significant differences were found between samples as contents ranged between 1.144–1.551 g/100 g D.M., except in the case of BR-FST1.7, which had a lower amount (0.553 ± 0.006 g/100 g D.M.). Phenyllactic acid was not detected in BR-UnF and BR-Ster. Among fermented ingredients, BR-R29 and BR-FST1.7 contained the highest amounts of phenyllactic acid, with values of 15.645 ± 0.589 g/100 g D.M. and 13.387 ± 0.074 g/100 g D.M. quantified, respectively. A significantly lower amount of phenyllactic acid was determined in BR-TR116 (5.254 ± 0.090 g/100 g D.M.) and BR-FST2.11 (5.223 ± 0.740 g/100 g D.M.), with the lowest amount of phenyllactic acid quantified in BR-MG1 (0.562 ± 0.078 g/100 g D.M.). Hydroferulic acid was detected only in BR-MG1 (0.924 ± 0.050 g/100 g D.M.), BR-FST2.11 (0.973 ± 0.132 g/100 g D.M.), and BR-FST1.7 (0.815 ± 0.019 g/100 g D.M.), with no significant differences found between samples. Coumaric acid was quantified in BR-Ster (0.537 ± 0.090 g/100 g D.M.) and BR-TR116 (0.842 ± 0.057 g/100 g D.M.), with the amount quantified in BR-TR116 being significantly higher than the amount determined in BR-Ster. BR-UnF contained 1.008 ± 0.095 g/100 g D.M. of ferulic acid and was the lowest amount detected among ingredients. A significantly higher amount of ferulic acid was noted in BR-Ster (1.656 ± 0.197 g/100 g D.M.). A significant increase in ferulic acid content was measured in BR-TR116 (2.220 ± 0.079 g/100 g D.M.), with the highest amount recorded in BR-R29 (4.068 ± g/100 g D.M.). Ferulic acid was not quantified in BR-MG1, BR-FST2.11, and BR-FST1.7.The extent of mould growth on the breadcrumb was analysed to investigate the shelf-life of the bread and determine the kinetics of bread mould growth over time. The results of the microbial shelf-life analysis are illustrated in The first mould growth on the BF was noted after two days, which was similar to the BR-UnF formulation. Relatively similar kinetics were observed in the BF and BR-UnF recipes. The incorporation of BR-Ster prolonged the microbial shelf-life of the bread by one day, with the kinetics of the mould growth also being slightly slower compared to control recipes. Similar to the BR-Ster recipe, formulations containing BR-MG1 and BR-FST1.7 remained mould-free until day 3. However, the extent of mould growth over time in the BR-MG1 and BR-FST1.7 recipes was much slower. A further increase in the number of mould-free days (+1 day) was observed with BR-TR116 and BR-FST2.11 addition, with a considerable delay in mould growth over time compared to BR-Ster, BR-UnF, and BF controls. The inclusion of BR-R29 had the most substantial effect on microbial shelf-life extension, with no mould growth observed until day 4 and little to no increase in mould growth for up to 8 days.The starch digestibility was examined to provide insight into the nutritional characteristics of the bread. The results of the release of reducing sugars (RSR) over time are illustrated in Bread with supplementation of BR-UnF resulted in the highest RSR curve. A slight reduction in the RSR value was noted in the BF and BR-TR116 bread formulations compared to BR-UnF. A further reduction in RSR release was also observed in BR-Ster, BR-MG1, and BR-R29 compared to BR-UnF, but was not statistically significant in BF and BR-TR116 formulations. Bread formulations with the inclusion of BR-FST2.11 and BR-FST1.7 slowed the release of reducing sugars to the greatest extent, particularly in the case of BR-FST1.7, where the lowest RSR was recorded, which was significantly lower than control formulations BF, BR-UnF, and BR-Ster.A sensory analysis of the BR bread was conducted to determine the effect of BR inclusion on the overall sensory experience of the bread and to determine its palatability. Results are represented in Overall, the odour perception of all BR bread was acceptable and comparable to the BF control. No significant differences were found between the bread samples regarding any of the odour attributes. Concerning the taste parameters analysed, some significant differences were found between BR breads. The lowest sourness was perceived in the BR-Ster (0.72 ± 0.83) and BF (0.78 ± 1.26) controls. An increase in sourness was noted in the BR-UnF bread (1.61 ± 1.42); however, this was not significantly different from the BR-Ster and BF formulations. The inclusion of fermented BR increased sourness. The BR-TR116, BR-MG1, and BR-R29 breads had sourness values of 2.17 ± 1.86, 2.22 ± 1.66, and 2.89 ± 2.14, respectively, which were comparable to the BR-UnF formulation but significantly higher than the BF and BR-Ster formulations. An even greater increase in sourness was recorded in BR-FST2.11 (4.22 ± 2.34) and BR-FST1.7 (4.33 ± 2.83), which were significantly higher than all control recipes as well as fermented samples BR-TR116 and BR-MG1. No significant difference was noted in flavour intensity, aftertaste, or the muddy/earthy flavour parameters; however, significant differences were detected between samples for fruity and vegetable flavour compounds in BR bread. Fruity flavours were lowest in BF (0.44 ± 0.51) and BR-Ster (0.61 ± 0.70). An increase in fruity flavours was noted in the BR-UnF (1.56 ± 1.76), BR-TR116 (1.39 ± 1.38), and BR-MG1 (1.56 ± 1.50) compared to the BF and BR-Ster. The values for fruity flavours found in BR-R29 (2.39 ± 1.42) and BR-FST1.7 (2.89 ± 2.49) were slightly higher than those observed in the other fermented samples; however, this was not found to be significant. The highest perception of fruity flavours was detected in the BR-FST2.11 formulations, which were comparable to the BR-R29 and BR-FST1.7 formulations but significantly higher than all control samples. Regarding the vegetable notes in the rootlet breads, BF had the lowest value (1.06 ± 0.94), whereas the inclusion of BR-Ster resulted in the highest perception of vegetable flavours (3.06 ± 1.55). The BR-TR116 addition also resulted in an elevated vegetable flavour that was similar to the BR-Ster sample. Among the remaining samples , no difference in vegetable flavour was detected between samples, with values ranging from 1.89–2.50. Hardness and chewiness values ranged between 3.33–4.00 and 4.50–5.22, respectively, with no significant differences observed between samples. In addition, all breads analysed were ranked highly in overall acceptability (<7), with no significant differences found among the breads tested.The introduction of food by-products into the food chain has become an important part of research to address various aspects of sustainability goals for the future. Fortification of staple foods with by-products poses difficulties, with deterioration in food prototype quality observed, particularly at higher levels of inclusion ,13,54,55The addition of BR, regardless of the processing applied, significantly reduced the strength of the gluten network, suggesting that BR interferes with the formation of the gluten network. The reductions in gluten network strength are likely a contribution of the changes in minerals and protein and fibre interactions with BR addition. BR contain a variety of minerals , which cp < 0.05) to the damping factor from rheological measurements, indicating that the time taken for gluten network development was influential in determining viscoelastic properties. The inclusion of BR-UnF maintained the same viscoelastic properties as BF, which could be linked with the alpha-amylase activity of BR-UnF. Alpha amylases degrade starch molecules into small chain dextrins, which reduce dough viscosity [The time taken for gluten network development also correlated positively , indicating that viscoelastic properties facilitate dough expansion during the fermentation. Interestingly, the BR-MG1 formulation achieved a high Hm, which may be explained by the exopolysaccharides present in the BR-MG1 ingredient after fermentation. W. cibaria MG1 is capable of producing exopolysaccharides of the dextran type and gluco-oligosaccharide type from sucrose [W. cibaria MG1 in sourdough attributed the enhanced loaf volume and reduced crumb hardness to the dextran exopolysaccharides [2 with the inclusion of W. cibaria MG1 in sourdough, suggesting the increased production was a result of an enhanced level of sugars present in the sourdough, favouring yeast fermentation. Results from Vtotal CO2 values in this study complement these findings. The BR-MG1 ingredient (as well as BR-FST2.11 and BR-FST1.7) contains higher levels of residual glucose and fructose [2 produced during proofing. However, it is important to note that the inclusion of BR regardless of the processing applied enhanced CO2 production, indicating BR did not interfere negatively with yeast metabolism but rather improved it. This suggests BR is likely a suitable substrate for inclusion in yeast-leavened products. Thus, the adverse effects on dough rise observed are likely more connected to the observations made in DF values previously discussed. However, in contrast to this trend was the BR-R29 formulation, which behaved differently during the yeast fermentation with a reduced amount of CO2 produced. This could be linked to the levels of antifungals present (discussed in later sections) as well as the probable reuterin present [2 produced were relatively similar to amounts produced in the BF control, indicating that the negative impacts observed were not of major significance.The DF also influenced the maximum dough rise (Hm) achieved during the yeast fermentation , indicating the height reached during yeast fermentation affected bread volumes. BR-MG1 bread had an improved specific volume, reaching volumes higher than those observed for the BF wheat control, suggesting fermentation of BR with W. cibaria MG1 had a positive influence on bread quality. The enhancement observed can be attributed to the more stabilised dough structure due to the presence of exopolysaccharides. Exopolysaccharides have been described to perform similarly to hydrocolloids in a bread system [L. citreum TR116 can also produce exopolysaccharides [p < 0.01). The addition of BR-UnF produced a soft crumb, likely due to the alpha-amylase activity present in BR-UnF [W. cibaria MG1 and L. citreum TR116 also positively influenced crumb hardness, as even lower crumb hardness (compared to BF) was observed, highlighting further enhancements to bread quality using BR-MG1 and BR-TR116 inclusion. Although variations in crumb hardness were observed, overall, relatively similar trends in crumb resilience suggest inclusion of BR does not negatively impact the crumb’s integrity. Dough characteristics such as Hm and DF also had an impact on crumb cell structure, with significant positive correlations observed with cell diameter values . Thus, variations in cell diameters observed with BR inclusion are a result of the restrictions imposed on the viscoelastic properties constricting the cell diameter size.Hm values correlated positively to bread specific volume  with fermented BR addition can be linked with the antifungal metabolites imparted on the BR ingredients during fermentation. Sourdough technology with numerous LAB strains has been previously shown to provide a natural antimicrobial effect due to the synergistic effect of the variety of antifungal metabolites and organic acids produced during fermentation ,80. BR-Me effect . Previouentation ,42, compbstrates ,32,44,81ulations ,83.L. plantarum FST1.7 and L. amylovorus FST2.11 might be more beneficial for the engineering of products for consumers with diabetes. The higher levels of acid in BR-FST1.7 and BR-FST2.11 could also have led to changes observed in the sensory perception of these bread prototypes, with enhancements noted in sour taste and fruity flavours. Increases in fruity and sour tastes are typically found in wheat sourdough bread [L. plantarum FST1.7 and L. amylovorus FST2.11. The high comparability of BF bread and BR breads across all sensory attributes indicates that a 5% BR inclusion level in a bread system is highly acceptable and complements the bread sensory results previously seen [Due to the high fibre content of the BR ingredient, its incorporation into a bread formulation will enhance the fibre content of the bread , thus engh bread , indicatsly seen ,5.To date, the inclusion of BRs and investigation of their effects in bread making have been limited. However, this study shows a promising future for BR in the baking industry, particularly in yeast-leavened products. Furthermore, upcycling processes such as LAB fermentation prove to be viable processing tools for BR, with unique fermentation characteristics being translated to the bread matrix. The addition of BR into the bread system at a 5% addition level was found to decrease the strength of the gluten network, however, the introduction of the fermented ingredients amplified the speed required for gluten development. Concerning the unfermented BR formulation, positive impacts were also observed in the viscoelastic properties of the dough, which were mainly attributed to the enzymatic activity of the unprocessed ingredient. The addition of the BR-MG1 and BR-TR116 ingredients showcased their potential to improve bread quality with high specific volumes and softer crumbs. Moreover, the inclusion of BR-R29 illustrates its power to reduce the microbial growth rate to a significant extent through the natural production of high amounts of antifungal compounds. The incorporation of BR-FST1.7 and BR-FST2.11 shows promising potential to further improve the nutritional characteristics of the BR with a slower release of sugars over time during starch digestion. Furthermore, the inclusion of BR-FST1.7 and BR-FST2.11 altered the sensory experience of wheat bread, creating a bread with flavours that might compare more similarly to those of conventional sourdough. Hence, this study showcases how the LAB fermentation of BR can be tailored based on desired requirements in the bread application at a later stage. Thus, future work in this area indicates that a co-fermentation of BR with multifunctional LAB starter cultures might functionalise BR to an even greater extent and endorse its inclusion in leavened cereal products.


Placopsis contortuplicata, Ochrolechia frigida, and Umbilicaria antarctica, their antioxidant activity, and enzymatic inhibition through in vitro and molecular docking analysis. In total phenol content, FRAP, ORAC, and DPPH assays, the extracts showed significant antioxidant activity, and in in vitro assays for the inhibition of pancreatic lipase, α-glucosidase, and α-amylase enzymes, together with in silico studies for the prediction of pharmacokinetic properties, toxicity risks, and intermolecular interactions of compounds, the extracts evidenced inhibitory potential. A total of 13 compounds were identified by UHPLC-ESI-QTOF-MS in P. contortuplicata, 18 compounds in O. frigida, and 12 compounds in U. antarctica. This study contributes to the knowledge of the pool of bioactive compounds present in lichens of temperate and polar distribution and biological characteristics that increase interest in the discovery of natural products that offer alternatives for treatment studies of diseases related to oxidative stress and metabolic syndrome.The objective of this research was to characterize the chemical composition of ethanolic extracts of the lichen species  Ficus, Artemisia, Solanum, Terminalia, and Euphorbia (Diabetes mellitus (DM), with a 3% age-standardized mortality rate, is one of the metabolic diseases that have increased in prevalence worldwide. It is characterized by the insufficient production of insulin by the pancreas or the ineffective degradation of the insulin produced . One of uphorbia . The comuphorbia .O. frigida, characterized by a crustose, white thallus and lecanorine apothecia with a thin taline edge; P. contortuplicata with crustose thallus, marginal lobes separated by thin cracks or folded with a verrucous central part, and subspherical cephalodia generally orange–brown; and U. antarctica with foliose thallus attached to the substrate by a central umbilicus, gray-to-brown dorsal surface, black ventral surface with abundant simple ricines, and absent apothecia , algae (phycobiont), and/or cyanobacteria (cyanobiont) , and thepothecia   Lynge (Ochrolechiaceae), P. contortuplicata I. M. Lamb (Trapeliaceae), and U. antarctica Frey & I. M. Lamb (Umbilicariaceae) was collected by A.T.-B., M.J.S., and N.J.-P. on Ardley Island, King George Island, South Shetland Archipelago (Maritime Antarctica), in February and December 2021. Specimens were determined by botanist Alfredo Torres-Benítez and deposited at the Natural Products Laboratory of the Universidad Austral , with identification numbers HL-01192021 (O. frigida), HL-01202021 (P. contortuplicata), and HL-01162021 (U. antarctica).A total of 100 g of the lichen species With each sample of lichen species, a total of 5 g was weighed and macerated using an analytical ethanol solvent three times, for 20 min per cycle, using ultrasound equipment at 35°C. Each extract solution was filtered and concentrated under reduced pressure at 38°C, obtaining three gummy extracts.The analysis for the identification of bioactive compounds present in the lichen ethanolic extracts was performed on a UHPLC-ESI-QTOF-MS system equipped with UHPLC UltiMate 3000 RS using Chromeleon 6.8 software  and Bruker maXis ESI-QTOF-MS using the software Data Analysis 4.0 . 5 mg of each extract was dissolved in 2 mL of methanol for analysis and filtered using a polytetrafluoroethylene (PTFE) filter, and 10 μL of the extract was injected into the equipment. The chromatographic equipment consisted of a quaternary pump, an autosampler, a thermostated column compartment, and a photodiode array detector. Elution was performed using a binary gradient system with eluent (A) 0.1% formic acid in water and eluent (B) 0.1% formic acid in acetonitrile: 1% B isocratic (0–2 min), 1%–5% B , 5% B isocratic (3–5 min), 5%–10% B (5–8 min), 10%–30% B (8–30 min), 30%–95% B (30–38 min), and 1% B isocratic (38–50 min). Separation was carried out using a Thermo 5 μm C18 80 Å column (150 mm × 4.6 mm) at a flow rate of 1.0 mL/min. ESI-QTOF-MS experiments were recorded in the negative ion mode, and the scan range was between 100 and 1,200 m/z. Electrospray ionization (ESI) conditions included a capillary temperature of 200°C, a capillary voltage of 2.0 kV, a dry gas flow rate of 8 L/min, and a nebulizer pressure of 2 bar, and the experiments were performed in an automatic MS/MS mode. The structural characterization of secondary metabolites was based on HR full MS, fragmentation patterns, and comparisons with the bibliography.R2 = 0.9992.The content of total phenolic compounds was determined according to the Folin–Ciocalteu assay with each of the working solutions and was quantified spectrophotometrically based on a standard curve of gallic acid . The anaR2 = 0.9945.Trolox was used as standard and a stock concentration (1 mg/mL) diluted in ethanol was prepared, and the concentrations 125, 250, 500, and 1,000 μg/mL were obtained. Subsequently, 290 μL of working solution and 10 μL of each sample were mixed in a microplate well, and after 5 min, they were measured at 593 nm in a microplate reader . The anaR2 = 0.9890.Trolox standard was prepared at the concentrations of 10, 20, 40, 40, 50, 60, 60, 80, 90, and 100 μM to make the calibration curve. Subsequently, 75 mM phosphate buffer at pH 7.0, fluorescein solution preincubated for 15 min at 37°C in microplate wells, compound 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH), and working solutions were used. The measurement was performed every 2 min for 30 min with excitation and emission wavelength at 485 and 530 nm, respectively . The ana50 with a stock solution of 250 μg/mL gallic acid in absolute methanol and diluted to prepare solutions with the following concentrations: 1.525, 3.125, 6.225, 12.05, 25, 50, and 100 μg/mL. Subsequently, a methanolic solution of 400 μM DPPH was prepared, 50 μL of each sample was incorporated into the wells of the microplate, and 150 μL of 400 μM DPPH solution was added to each well, allowed to rest for 30 min, and measured at 515 nm in a microplate reader  was added, and absorbance was measured at 405 nm every 1 min for 20 min using a microplate reader . For ethanolic extracts, solutions at concentrations between 1,000 and 7.8 μg/mL  were prepared from a stock solution of 1 mg/mL in phosphate buffer . 50 μL of each of the working solutions were taken, and 50 μL of 5.0 mM pNPG was added and incubated for 5 min at 37°C. Subsequently, 100 μL of e reader . The anaR2 = 0.9368.The standard used was orlistat, with which a calibration curve was prepared using concentrations ranging from 80 to 0.5 μg/mL in 70% ethanol . For ethanolic extracts, solutions were prepared at concentrations between 1,000 and 7.8 μg/mL  from a stock solution of 1 mg/mL in Tris-HCl buffer . With each working solution, 25 μL was taken with 50 μL of 5.0 mM NPC and 25 μL of pancreatic lipase enzyme (10 mg/mL) and incubated for 5 min at 37°C. Subsequently, measurement at 410 nm using a microplate reader was performed . The anaO. frigida, P. contortuplicata, and U. antarctica were evaluated; to determine whether the compounds obtained are favorable as inhibitors of α-amylase, α-glucosidase, and human pancreatic lipase, the pharmacokinetic properties were calculated using the Osiris DataWarrior (v 5.5.0) computational tool. Compounds were evaluated based on Lipinski’s rule, which states that an orally administered drug must have a molecular weight <500 Da, the partition coefficient (cLogP) must be <5, the number of bond donors of hydrogen must be <5, the number of hydrogen bond acceptors must be <10, and the number of spin bonds must be <10. The topological polar surface area and the percentage absorption (% ABS)  Eq. :% ABS=1To determine the toxicological behavior of the phytochemicals obtained from the lichen, the Osiris DataWarrior computational tool was used. The toxicological risks that were evaluated were mutagenicity, tumorigenicity, irritation, and reproductive effect .https://avogadro.cc, accessed on 06 June 2023) to optimize the geometry of the ligands using the MMFF94 force field function  were prepared using the ChemDraw 8.0 program . Subsequently, the chemical structures of the ligands were imported into the Avogadro program , α-glucosidase  (PDB: 2QMJ), and human pancreatic lipase (PDB:1LPB) were downloaded from the PDB database (http://www.rcsb.org/pdb). These crystal structures are crystallized with the acarbose ligand used as a reference inhibitor for α-amylase and α-glucosidase, while the MUP and orlistat ligands were used for human pancreatic lipase , molecular docking was performed using the rigid crystalline enzyme structures and the flexible ligands where twist angles were identified (for 10 independent urns per ligand). Grid parameters (α-amylase and α-glucosidase and MUP and orlistat for human pancreatic lipase). Each ligand was coupled with the enzymes separately, and the final energy  of the ligand–enzyme interaction was obtained after each coupling. The evaluation of the interactions was carried out using the BIOVIA Discovery Studio program   and the target proteins  .p ˂ 0.05) was calculated using GraphPad Prism 8 software.Three measurements were performed with each sample solution, and the results were expressed as mean values ±standard deviation using Microsoft Excel 2019 software. For comparison of means, a one-way analysis of variance  in the negative mode allowed the identification of 18 compounds including aromatics, carbohydrates, acids, lipids, and depsides  used as the internal standard. Peak 3 was identified as citric acid (C6H7O7).Acid derivatives: Peak 1 corresponded to Na formate (C6H13O6), peak 9 as undecyl glucoside (C17H33O6) with diagnostic peaks at m/z 193.0056 and 181.0059, and peak 15 as tetradecyl glucoside (C20H39O6).Carbohydrates: Peak 3 was identified as mannitol (C9H15O4) and peak 12 as 3,6,9,12-tetraoxapentacosanoic acid (C21H41O6). Peaks 16 and 17 were identified as 18-hydroxy linoleic acid (C18H31O3) and 18-hydroxy linolenic acid (C18H29O3), respectively.Lipids: Peak 4 was identified as azelaic acid (C10H9O5), prephenic acid (C10H9O6), orsellinic acid (C13H9O5), 3,4-dihydroxybenzoic acid (C7H5O4), wedelolactone (C16H9O7), and o-(4-biphenylylcarbonyl) benzoic acid (C20H13O3), respectively. Peak 18 was identified as diospyrol (C22H17O4) with diagnostic peaks at m/z 230.9846 and 167.0309.Aromatic derivatives: Peaks 5, 6, 7, 8, 10, and 14 were identified as hypoxyphenone (C16H13O7), and peak 11 was identified as an unknown compound (C26H16O4).Depsides: Peak 13 was identified as lecanoric acid  in the negative mode allowed the identification of 13 compounds including aromatics, carbohydrates, acids, and depsides  used as the internal standard.Acid derivatives: Peak 1 corresponded to Na formate (C6H13O6), and peak 10 was identified as stearyl glucose (C24H47O6) with diagnostic peaks at m/z 289.0750 and 149.0276.Carbohydrates: Peak 2 was identified as mannitol (C7H5O4) ([M-H]- ion at m/z 153.01953 and diagnostic peak at m/z 109.03217), peak 4 as phthalic acid (C8H5O4) ([M-H]- ion at m/z 165.01933 and diagnostic peaks at m/z 139.0379 and 123.05044), and peak 5 as a 3,4-dihydroxybenzoic acid isomer (C7H5O4) ([M-H]- ion at m/z 153.0169 and diagnostic peak at m/z 109.0285). Peak 6, with a molecular anion at m/z 357.06159 and diagnostic peaks at m/z 124.05044 and 165.06600, was identified as succinyldisalicylic acid (C18H13O8), while peak 7 was identified as cyperine (C15H15O4) with an [M-H]- ion at m/z 259.09758 and diagnostic peaks at m/z 135.04781 and 241.01395 and peak 8 as octadecyl beta-maltoside (C30H57O11) with an [M-H]- ion at m/z 593.38985 and diagnostic peaks at m/z 389.29418, 259.00562, and 241.0077. Peak 11 was identified as crustinic acid (C24H20O11) with a molecular anion at m/z 483.0932 and diagnostic peaks at m/z 431.3434 and 333.03409. Peak 13, with a molecular anion at m/z 497.07255 and diagnostic peaks at m/z 347.04615 and 317.05815, was identified as tetrafucol A (C24H17O12).Aromatic derivatives: Peak 3 was identified as 3,4-dihydroxybenzoic acid (C16H13O7) and peak 12 as a lecanoric acid isomer with diagnostic peaks at m/z 167.0378 and 167.03772, respectively.Depsides: Peak 9 was identified as lecanoric acid  in the negative mode allowed the identification of 12 compounds including aromatics, carbohydrates, acids, and depsides  used as the internal standard.Acid derivatives: Peak 1 corresponded to Na formate (C6H13O6).Carbohydrates: Peak 2 was identified as mannitol (C10H9O5), prephenic acid (C10H9O6), 2,5-dihydroxyterephthalic acid (C8H5O4), phthalic acid (C8H5O4), 3,4-dihydroxybenzoic acid (C7H5O4), orsellinic acid (C8H7O4), rhein (C15H8O6), and 4-hydroxyisophthalaldehyde (C8H5O3), respectively.Aromatic derivatives: Peaks 3, 4, 5, 6, 7, 8, 10, and 12 were identified as hypoxyphenone (C16H13O7) and peak 11 as a lecanoric acid isomer with diagnostic peaks at m/z 167.0342 and 167.0326, respectively.Depsides: Peak 9 was identified as lecanoric acid  showed the highest values, followed by P. contortuplicata (561.2 ± 0.009 mg GAE) with medium values and U. antarctica with the lowest values (245 ± 0.011 mg GAE). In ORAC and FRAP, the results for the three extracts are comparable to the reported phenolic concentration, showing in O. frigida the optimum values 525.11 ± 0.135 and 45.004 ± 0.066 µmol Trolox/g, respectively. As for the DPPH analysis, the three extracts evidenced an IC50 value much higher than the gallic acid standard, suggesting a medium–low inhibition capacity of the study concentrations used; however, the extract of O. frigida continued to show better antioxidant activity than the extracts of P. contortuplicata and U. antarctica .in vitro and in vivo models, yielding positive results regarding cell viability, protection against induced oxidative stress, decrease in reactive oxygen species, improvement of mitochondrial function, and suppression of signaling pathways that induce inflammatory response in astrocytes (These results correlate positively with the reports of extracts and compounds isolated from other lichen species that show highly effective phenolic concentration and antioxidant activity through colorimetric techniques  and electrocytes .O. frigida presented the best activity (16 ± 0.015 μg/mL) with a lower IC50 compared to the standard acarbose; as for the extracts of P. contortuplicata and U. antarctica, the values obtained indicated a low inhibition of the enzyme . For pancreatic lipase, the three extracts showed low inhibition of the enzyme compared to the orlistat standard, with values similar to each other for O. frigida and U. antarctica  and an even higher value for P. contortuplicata (394.7333 ± 0.028 μg/mL). For α-amylase, the extracts show low inhibition of the enzyme compared to the standard orlistat; however, the best result was obtained in the extract of P. contortuplicata with an IC50 of 308.856 ± 0.036 μg/mL, followed by U. antarctica with an IC50 of 607.531 ± 0.038 μg/mL and O. frigida with the least efficient value of inhibition .C. gracilis and C. chlorophaea , with less effective values against the acarbose standard, except for O. frigida, which showed the highest inhibitory potential , X. parietina (IC50 = 0.6 ± 0.0 μg/mL), Parmotrema dilatatum (IC50 = 17.5 μg/mL), and compounds isolated from Parmotrema tsavoense (IC50 = 10.7–17.6 μg/mL)  presented concentrations similar to those found in O. frigida, P. contortuplicata, and U. antarctica; however, in methanolic extracts of species such as X. elegans, X. parietina, and Xanthoria candelaria , better inhibition effectiveness was observed , show effective values of enzyme suppression compared to those obtained in P. contortuplicata, U. antarctica and even greater difference in O. frigida  . As for observed . For enz frigida .α-amylase, α-glucosidase, and human pancreatic lipase. In the same way, the toxicological analysis of all the compounds obtained from lichens was carried out using the Osiris DataWarrior computational tool .The pharmacokinetic properties of the phytochemicals obtained from the lichen species were evaluated using the Osiris DataWarrior computational tool . For a cnal tool  where itnal tool . As theyin silico analysis to observe the behavior against these enzymes, comparing them with known inhibitors such as acarbose, orlistat, and MUP  . This pa and MUP .in silico molecular sugar analysis to investigate their behavior and binding properties at the α-amylase catalytic site  compared to the inhibitor acarbose; this is mainly because the binding was carried out of the catalytic site of in silico analysis of the phytochemicals and α-glucosidase enzyme are shown in π-anion-type interactions with the amino acids Asp197 and Asp536. These interactions presented by the compound diospyrol caused its affinity and geometry in the catalytic site to be more stable compared to the inhibitor acarbose . Of the compounds that presented lower affinities than acarbose, the one that presented important interactions in the catalytic site of α-glucosidase was 2.5DHA , 8A. Thrin silico analysis of the phytochemicals, the cocrystallized ligand (MUP) of the human pancreatic lipase enzyme was used, which was used as a reference compound for coupling and to determine the coordinates of the catalytic site of the enzyme to the compound orlistat as a reference inhibitor since it is the only drug approved by the FDA that acts on pancreatic lipase. The binding affinity results showed that the compounds cyperine, diospyrol, lecanoric acid, SDA, and O4BBA presented binding affinities  higher than those of the reference inhibitors orlistat and MUP . This behavior is mainly because the geometry adopted by these compounds in the catalytic site of the enzyme  benzoic acid, the catalytic sites of α-glucosidase, α-amylase, and pancreatic lipase enzymes were detected. In this research, the potential of extracts and compounds of these species for the promising treatment of metabolic diseases such as diabetes mellitus and those related to oxidative damage such as Alzheimer’s, Parkinson’s and amyotrophic lateral sclerosis is highlighted. Furthermore, the prospects for the near future in this type of research is the biodirected study of ethanolic extract fractions, the isolation, elucidation, and biological assays of major compounds, and the testing of extracts, active fractions, and compounds in murine models with the aim of further strengthening the pharmacological, nutritional, and biomedical potential of the chemical world of lichenized mushrooms.The extracts of the lichenized fungi species


There were no changes in Ca2+ influx through the voltage-gated Ca2+ channels (VGCCs) or inhibitory postsynaptic activity in medial prefrontal cortex neurons from young Alzheimer’s disease mice compared to controls. Additionally, acute exposure to Aβ42 potentiated medial prefrontal cortex neuronal hyperactivity in young Alzheimer’s disease mice but had no effects on controls. These findings indicate that the hyperactivity of medial prefrontal cortex pyramidal neurons at early-stage Alzheimer’s disease is induced by an abnormal increase in presynaptic glutamate release and postsynaptic NMDA receptor activity, which initiates neuronal Ca2+ dyshomeostasis. Additionally, because accumulated Aβ forms unconventional but functional Ca2+ channels in medial prefrontal cortex neurons in the late stage of Alzheimer’s disease, our study also suggests an exacerbated Ca2+ dyshomeostasis in medial prefrontal cortex pyramidal neurons following overactivation of such VGCCs.The normal function of the medial prefrontal cortex (mPFC) is essential for regulating neurocognition, but it is disrupted in the early stages of Alzheimer’s disease (AD) before the accumulation of Aβ and the appearance of symptoms. Despite this, little is known about how the functional activity of medial prefrontal cortex pyramidal neurons changes as Alzheimer’s disease progresses during aging. We used electrophysiological techniques (patch-clamping) to assess the functional activity of medial prefrontal cortex pyramidal neurons in the brain of 3xTg-Alzheimer’s disease mice modeling early-stage Alzheimer’s disease without Aβ accumulation. Our results indicate that firing rate and the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) were significantly increased in medial prefrontal cortex neurons from young Alzheimer’s disease mice  compared to age-matched control mice. Blocking ionotropic glutamatergic NMDA receptors, which regulate neuronal excitability and Ca Howevermbalance . Previoumbalance . Imagingmbalance ; which bmbalance .2+ channels (L-channels)  and voltage-gated L-type Cahannels) , but als-channel . These d-channel  in gluta-channel , which m-channel . However-channel , in causderstood .The earliest accumulation of Aβ in the brain occurs primarily in the orbital/medial PFC (o/mPFC) and HIPP, brain regions that are anatomically connected and functionally interplay to regulate neurocognition early onset/familial AD gene mutations, such as presenilin 1/2 and amyloid precursor protein, can be detected in the carriers at very early-stage AD when cognitive abilities are still intact , dysfunction of neurons in the PFC/HIPP also occurs before fibrillar Aβ deposition and the clinical symptoms of AD   , but noted-old#) . Howevered-old#) .Previous findings from early-stage AD study  align wiad libitum. Animal care and use procedures were conducted in accordance with NIH, USDA and institutional guidelines, and approved by the Institutional Animal Care.Young male 3xTg-AD and non-Tg B6 (WT) mice (The Jackson Laboratory) at the age of 4–5 months  were group-housed at the Rush University Medical Center animal facility on a 12 h light/dark cycle. Food and water were available 2.6H2O, 2 CaCl2.2H2O, and 15 glucose; pH 7.4–7.45, with 305–315 mOsm) containing 1 mM ascorbic acid.The procedure was as descripted elsewhere . Briefly2.6H2O, 3 Na2ATP, 0.3 NaGTP, and 0.1 EGTA; pH: 7.3–7.35; osmolarity: 280–285 mOsm; For sEPSC and sIPSC recording (in mM): 135 CsOH, 5 CsCl, 10 HEPES, 2 MgCl2.6H2O, 3 Na2ATP, 0.3 NaGTP; pH: 7.30; osmolarity: 280–285 mOsm. For recording of VGCC activity (in mM): 140 CsOH, 10 HEPES, 2 MgCl2.6H2O, 3 Na2ATP, 0.3 NaGTP; pH: 7.30; osmolarity: 280–285 mOsm. The slices were constantly perfused with oxygenated aCSF of the same composition as in the holding chamber, with the exception while assessing spontaneous excitatory and inhibitory postsynaptic currents , in which Mg2+ was excluded to remove external blockade of NMDA receptors by Mg2+.After ∼1 h incubation, slices were moved to a recording chamber perfused with oxygenated aCSF. Glass electrodes were pulled and filled with an internal solution which had a resistance of 4–6 MΩ and were used for patch-clamp recording. Different pipette solutions were used optimized for the activity of interest. For evoking action potentials (in mM): 120 K-gluconate, 10 HEPES, 20 KCl, 2 MgClVm) rebound following the end of Vm hyperpolarization was assessed to determine functional activity of low voltage-gated Ca2+ channels in neurons (2+ influx via high voltage-activated (HVA) Ca2+ channels (VGCCs) were evoked with a 40 ms rheobase  in the presence of 0.5 µM tetrodotoxin (TTX), 20 mM tetraethyl ammonium (TEA), 2.5 mM kynurenic acid, and 100 µM picrotoxin to block intrinsic/synaptic activity that could affect Ca2+ influx.Glutamatergic pyramidal neurons in layer V-VI of the mPFC were visually identified using Nikon ECLIPSE E600FN microscope. The acquisition system consisted of Axon Digidata 1,550 analog-digital converter and Multiclamp 700B amplifier. Signals were low-pass filtered at 10 kHz except for the sEPSC/sIPSCs which were filtered at 3 kHz. All neurons from non-Tg B6 (WT) and 3xTg-AD mice met the criteria of the RMP that was equal or more hyperpolarized than −60 mV, and the amplitude of action potential  was ≥60 mV. The protocol for evoking action potentials included a series of 500 ms current pulses with the intensity ranging from −400 to +400 pA with a 25-pA increment. The sEPSCs and sIPSCs were assessed at −70 mV and 0 mV gap-free respectively in the voltage-clamp mode. We also blocked ionotropic glutamatergic AMPA and NMDA receptors using 10 µM NBQX and 50 µM D-AP5, respectively. The membrane potential  was applied in the bath. A previous study demonstrated that the levels of Aβ42 in the cerebrospinal fluid (CSF) of AD patients at the earliest stage are in the range of 136–234 ng/L (up to proximately 50 pM) . Althoug2+ spikes were analyzed using Clampfit 11. The sEPSC/sIPSC properties were assessed using the template method in Axograph, where post-event detection criteria  were used to filter out non-characteristic events. Prism 7  was used for statistical analysis. Two-way repeated measures (rm) ANOVA were used to analyze the spike number vs. current relationship followed by Sidak’s post hoc test, to determine the effects of genotype on firing. Membrane properties, sEPSC/sIPSC properties, and Ca2+ spike features were analyzed using Student’s t-test. One-way ANOVA was used to compare the effects of specific glutamatergic AMPA and NMDA receptor antagonists in each genotype followed by Tukey’s post hoc test. Two sample Kolmogorov-Smirnov (K-S) tests were used to compare the cumulative fraction distribution of the amplitudes and inter-event intervals (IEI) of sEPSCs and sIPSCs in neurons. Statistical significance was set at p < 0.05. The outlier(s) was excluded if the value of which was greater than 2-fold of the standard deviation from the mean. All data were expressed as mean ± standard error (SE).We used Clampex 11 to acquire all electro-physiological data. The features of action potentials and Can = 13 neurons/9 mice vs. 14 neurons/9 mice; Two-way rm ANOVA: genotype effect: F = 7.713, p = 0.0102; current effect: F = 135.8, p < 0.0001; interaction: F = 2.943, p = 0.0011; Sidak’s post hoc test: *,**p < 0.05, 0.01. We first assessed evoked action potentials of mPFC pyramidal neurons in WT or 3xTg-AD mice at young age  using positive current step pulses  that mimicked excitatory inputs. We found that firing  of mPFC neurons was significantly increased in 3xTg-AD mice compared to those from WT mice , K+ and Ca2+ channels; and ultimately generates action potentials (firing). To determine if excitatory synaptic inputs to the mPFC alter neuronal activity in the early stages AD, we evaluated the spontaneous excitatory postsynaptic currents (sEPSCs) in such hyperactive mPFC neurons.Glutamatergic mPFC pyramidal neurons receive excitatory inputs from the ipsilateral and contralateral mPFC regions, as well as hippocampus and some other regions that are critical for neurocognition . These eVh = −70 mV, we found no significant change in the amplitudes of sEPSCs in mPFC neurons from young 3xTg-AD mice compared to those from WT mice  events , in combn = 11 neurons/7 mice vs. 12 neurons/8 mice, 0.20 ± 0.04 Hz vs. 0.36 ± 0.12 Hz; unpaired t-test: t21 = 1.177, p = 0.2522. n = 12/6 mice vs. 11/3 mice, 0.12 ± 0.03 Hz vs. 0.24 ± 0.07 Hz; unpaired t-test: t21 = 1.566, p = 0.1323; n = 12 neurons/7 mice vs. 8 neurons/4 mice, 0.05 ± 0.02 vs. 0.12 ± 0.03; unpaired t-test: t18 = 1.868, p = 0.0781. F=2.17, p = 0.0421; Tukey’s post hoc test: *p = 0.0464, F = 8.89, ****p < 0.0001; Tukey’s post hoc test: ****p < 0.0001, To determine if the increased excitatory synaptic activity was mediated by presynaptic release of glutamate and postsynaptic glutamatergic receptors, we blocked AMPARs and/or NMDARs and evaluated the sEPSCs thereafter. We found that blocking AMPARs  and NMDARs  significantly reduced the previously-increased sEPSC frequency in neurons from 3xTG-AD mice compared to WT mice  in postsynaptic mPFC neurons during a 300s long gap-free protocol.Neuronal excitability in a brain network is defined by its excitation/inhibition balance. Thus, we also analyzed the inhibitory presynaptic inputs to mPFC neurons and the responses of them during the early stage of AD. Using a holding potential of n = 15 neurons/8 mice vs. 14 neurons/6 mice, 38.70 ± 2.20 pA vs. 38.22 ± 1.67 pA; unpaired t-test: t27 = 0.1712, p = 0.8654. n = 15 neurons/8 mice vs. 14 neurons/6 mice, 0.70 ± 0.13 Hz vs. 0.51 ± 0.08 Hz; unpaired t-test: t27 = 1.230, p = 0.2293. n = 15 neurons/8 mice vs. 14 neurons/6 mice, 0.67 ± 0.11 s vs. 0.70 ± 0.09 s; unpaired t-test: t27 = 0.2626, p = 0.7949. The amplitude of the sIPSCs were found to be very similar in mPFC neurons from 3xTg-AD mice as compared to those of WT mice  plays a critical role. Besides NMDARs, VGCCs (including high and low voltage-activated Ca2+ channels) also contribute to maintaining [Ca2+]in. VGCC dysfunction (including Aβ-formed Ca2+ channels) underlies several neurodegenerative diseases, including but not limited to AD , K+ channel blockers , glutamate receptors , and GABAA receptors , respectively. The total area and duration of the resulting Ca2+ spikes  for ∼10 min.The formation of Aβ42 plaque is thought to be a hallmark of AD beginning with Aβ42 deposition in the extracellular matrix around neurons in the AD brain in its late stage. As depicted in n = 10 neurons/8 mice, Two-way rm ANOVA: dose effect: F = 2.221, p = 0.1374; current effect: F = 333.1, p < 0.0001; interaction: F = 1.626, p = 0.0690. n = 10 neurons/7 mice, Two-way rm ANOVA: dose effect: F = 5.766, p = 0.0116; current effect: F = 140.2, p < 0.0001; interaction: F = 1.027, p = 0.4316; Sidak’s post hoc test: 25 nM vs. control: *,**,***p < 0.05, 0.01, 0.001; 50 nM vs. control: ^^^p < 0.001; 25 nM vs. 50 nM: ##,###p < 0.01, 0.001. We found that the number of spikes evoked in mPFC neurons in young WT mice was not altered by 25 and 50 nM Aβ42 compared to basal control with vehicle perfusion  . The exact Aβ levels in the mPFC, or the effects of Aβ on living cortical neurons in the brain, are challenging to determine because Aβ levels changes dynamically during the progression of AD, decreasing in CSF but increasing in the brain tissues at late-stage AD . Despitemulators ], young e of AD) , Aβ has e of AD) .early AD, our study has focused on testing acute Aβ42 effects, but we do not exclude the possibility that the tau mutation may also play a role in altering the functional activity of mPFC pyramidal neurons at early-stage and/or later-stage AD.3xTg-AD mice carry mutations in the Amyloid Precursor Protein (APP), Microtubule-Associated Protein Tau (MAPT) and presenilin (PSEN-1) genes. In the 3xTg-AD mice, the APP and PSEN-1 mutations assist extracellular Aβ deposits in the cortex by 6 months while tau tangles, owing to MAPT mutation, become evident only after 12 months of age. With the aim to detect cortical changes associated with 42 in disturbing the normal functional activity of cortical pyramidal neurons in young healthy brain, and its capability in worsening cortical neuron dysfunction at the early stage of AD. Because 1) the earliest Aβ accumulation occurs in the orbital/mPFC and HIPP of elder patients at later stage AD  Aβ istage AD , and 3) nd aging , our finnd aging .2+ influx via VGCCs in young mPFC pyramidal neurons in early-stage AD, likely due to the absence of Aβ plaques and lack of Aβ-formed Ca2+ channels, in such early stage of AD. Based upon this finding, we predict that the hyperactivity and overexpression of L-channels, including those formed by Aβ, will occur at the late stage of AD, which will interplay with overactive NMDARs to exacerbate [Ca2+]in dyshomeostasis in pyramidal neurons, thereby inducing neurotoxicity  has modest effects in improving clinical conditions at early-stage AD, likely due to decreasing excessive Castage AD . Given t neurons ; 2022 in neurons .o also causes desensitization and internalization of AMPARs and NMDARs into pyramidal neurons  is reduced in hippocampal pyramidal neurons in 3xTg-AD mice in later-stage AD with older age   and [Ca2+]in dysregulation, which are mediated by dysfunction of L-channels and K+ channels . The novel findings from the present study will help direct future studies focusing on female 3xTg-AD in early- and late-stage AD.2+ channels. Such hyperactivity is mediated by overactive AMPARs and NMDARs. Naïve mPFC pyramidal neurons in young brain are insensitive to Aβ42. But Aβ42 worsens hyperactivity of cortical neurons in early-stage AD. Collectively, our novel findings suggest that Aβ is not a causative factor that triggers AD; rather, it is one of the consequential effects of cortical/hippocampal pyramidal neuron dysfunction, which, in turn, exacerbates neuronal dysfunction and [Ca2+]in dyshomeostasis in the brain during the late stage of AD and aging. Additionally, our findings also suggest a dysfunction of the L-channel (including those formed by Aβ) during the late stage of AD and aging, along with a disrupted interaction between neurons and astrocytes in the brain. These novel findings could advance our understanding of the mechanism that underlies AD, which may also provide insights for developing novel therapeutic approaches.In summary, the present study demonstrates that pyramidal neurons in the mPFC become hyperactive in early-stage AD, in the absence of Aβ plaques and Aβ-formed Ca


Microcystis aeruginosa PCC7806 ΔmcyB mutant strain. This genome is 5,103,923 bp long, with a GC content of 42.07%. Compared to the published wild-type genome (Microcystis aeruginosa PCC7806SL), there is evidence of accumulated mutations beyond the inserted chloramphenicol resistance marker.Here we report the complete, closed genome of the non-toxic  Microcystis aeruginosa PCC7806 ΔmcyB is a non-toxic strain available at the Pasteur Culture Collection  for the last 7 years after an aliquot was transferred from Potsdam (Germany) to Knoxville (USA), has accumulated mutations relative to the published 7806SL genome—we have added the “UTK” annotation to signify our specific isolate. Of note, our mutant strain has an additional putative transposase gene (tpnA), not seen in the 7806SL wild type, inserted in an SLC13-family permease gene. There is also a region of the genome that assembled in a manner suggesting a large inversion (~2.5 Mbp) has occurred. This paper serves as a cautionary tale for labs to routinely re-sequence strains.llection . This mullection –15. ExMicrocystis aeruginosa PCC7806 ΔmcyB (UTK) were maintained in 50 mL of modified CT medium (B-glycerophosphate was replaced with 1 mL of Na2HPO4 (23.19 mg/mL) and grown under a 14-h/10-h day/night cycle (~35 µmol photons/m2/s). Late-log phase cells were pelleted  and resuspended in 400 µL TE buffer. RNase (0.6 µL of 10 mg/mL) and 120 µL of lysozyme (20 mg/mL) were added, and cells were incubated at 37°C for 30 min. Then, 6 µL of proteinase K (20 mg/mL) in 3 mM CaCl2 and 200 mM Tris buffer and 39.5 µL of 20% SDS were added before incubation at 55° C for 2 h. DNA was extracted via a phenol-chloroform method (50 = 17.55 kb). For Illumina sequencing, DNA was sent to SeqCenter . Sample libraries were prepared using an Illumina DNA prep kit and IDT’s 10-bp unique dual indices and were processed on an Illumina NextSeq 2000, generating 8,278,500 151-bp paired-end reads. Demultiplexing, quality control, and adapter trimming were performed with bcl-convert using default parameters (v.3.9.3) (Illumina)  .de novo using Unicycler (v.0.4.9b), using a predicted length of 5 Mbp  , with thof 5 Mbp . A circuof 5 Mbp .


Visual acuity, manifest refraction, topography, specular microscopy, and corneal optical coherence tomography (OCT) were evaluated at baseline and at the last visit. Progression was defined as an increase in the maximum topographic keratometry (Kmax) of 1D. Results: 302 eyes of 241 patients with a mean age of 25.2 ± 7.5 years were included from 2012 to 2019: 231 and 71 eyes in the A-CXL and I-CXL groups, respectively. The mean follow-up was 27.2 ± 13.2 months (maximum: 85.7 months). Preoperatively, the mean Kmax was 51.8 ± 4.0D, with no differences between groups. Mean topographic measurements and spherical equivalent remained stable during the follow-up. At the last visit, CXL failure was reported in 60 eyes (19.9%): 40 (14.7%) versus 20 (28.2%) in A-CXL versus I-CXL, respectively, p = 0.005. The likelihood of progression after CXL was significantly higher following I-CXL: RR = 1.62, CI95 = [1.02 to 2.59], p = 0.04. Demarcation line presence at 1 month was positively correlated with higher efficacy of CXL, p = 0.03. No endothelial damage was reported, especially in 51 thin corneas (range = 342–399 µm). Conclusions: A-CXL seems more effective than I-CXL in stabilizing keratoconus; this is to be taken into account when a therapeutic indication is posed according to the aggressiveness of the keratoconus.Purpose: To investigate the outcomes of accelerated (A-CXL) and iontophoresis (I-CXL) corneal crosslinking in a large retrospective cohort with progressive keratoconus. Methods: This retrospective observational cohort study included consecutive patients treated by A-CXL (9 mW/5.4 J/cm Keratoconus (KC) is a noninflammatory progressive degenerative corneal disorder characterized by progressive corneal thinning, protrusion, and visual impairment . CornealThe aim of this large retrospective cohort study was to describe outcomes following A-CXL and I-CXL protocols for progressive keratoconus.This non-randomized retrospective study with consecutive recruitment included patients who were treated with A-CXL or I-CXL for progressive KC in the Quinze-Vingts National Ophthalmology Hospital  from 1 January 2012, to 31 March 2019. The inclusion criteria were patients with progressive keratoconus defined as a maximal keratometry progression (Kmax) superior to 1.0 D and a clear cornea. The exclusion criteria were a follow-up period <12 months, ocular or systemic disease that may affect the cornea, intracorneal ring segment implantation (ICRS), and pregnancy or breastfeeding. CXL failure was defined as an increase in Kmax of 1.00 D or more in less than 12 months. Indication of A-CXL or I-CXL was assessed in all cases by a cornea specialist. The choice of the CXL protocol was based on the clinician’s appreciation. The tenets of the Declaration of Helsinki were respected, and this study was approved by the Ethics Committee of the French Society of Ophthalmology . Informed consent was obtained from patients before surgery.After epithelial debridement, 0.1% riboflavin solution  is applied for 15 min. Next, the cornea is irradiated with a 9 mW/cm² UV-A light  for 10 min at a 5 cm distance.A 1.0 mA electric current is applied for 5 min using the iontophoresis device  to allow the penetration of riboflavin . Then, the cornea is irradiated with a 9 mW/cm² UV-A light  for 10 min at a 5 cm distance.At the preoperative visit and last visit, the following data were collected: refraction, best-corrected visual acuity (BCVA) with glasses, corneal topography using Scheimpflug imaging , corneal pachymetry, and anterior segment optical coherence tomography (AS-OCT)  outcomes such as the presence of a postoperative demarcation line (DL), endothelial cell density (ECD) (cells/mm²) measured by no-contact specular microscopy, and any adverse event linked to the CXL procedure.t-test for paired data was used to compare preoperative and postoperative continuous parameters. Measurements between groups were compared using an independent sample t-test. The Snellen BCVA was converted to a logarithm of the minimum angle of resolution (logMAR) units for statistical analysis. Additionally, a Pearson correlation analysis was conducted looking at predictive factors of higher Kmax change from baseline to last visit. A 2-tailed probability of 0.05 or less was considered statistically significant. Statistical analysis was performed using SPSS for Windows version 20.0  and Microsoft Excel 2011 .Descriptive statistics were reported as mean ± standard deviation (SD) for continuous variables and as a percentage for categorical variables. We used the d’Agostino–Pearson test to assess the normal distribution of our data. For binary outcomes, the chi-square test was used for intergroup comparisons of proportions. Student’s p = 0.15. Preoperative pachymetry (MCT) was significantly lower in I-CXL: 395.1 ± 40.9 µm versus 436.3 ± 44.4 µm for I-CXL and A-CXL, respectively, p < 0.001. This study included 302 eyes (241 patients) from 1 January 2012, to 31 March 2019: 231 A-CXL and 71 I-CXL. The overall gender ratio was 2:1 . The mean follow-up was 27.2 ± 13.2 months (range 12.0 to 85.7 months). Preoperatively, the mean maximum K value (Kmax) was 51.8 ± 4.0 D with 51.6 ± 4.1 D in A-CXL versus 52.4 ± 3.4 D in I-CXL, 95%= [−0.17–1.20 D]: −0.19 ± 2.62 D in A-CXL versus 0.47 ± 3.06 D in I-CXL, p = 0.01. Kmax value remained stable throughout follow-up in the total population and in A-CXL and I-CXL groups. At the last visit, progression of Kmax equal or above 1 diopter was reported in 60 eyes (19.9%): 40 (17.3%) in group A-CXL and 20 (28.2%) in group I-CXL, p = 0.005 .The presence of a demarcation line (DL) was reported at 1 month postoperatively and at the last visit and was more frequent in A-CXL: 56.3% versus 31.0% for A-CXL versus I-CXL, respectively, 2 = 0.02, p = 0.77), Kmin , and Km values  as well as a lower age  and initial pachymetry  and visual acuity  were not associated with higher Kmax change from baseline to last visit. The main predictors of CXL failure were the I-CXL protocol (p = 0.005) and the absence of a DL at 1 month (p = 0.03), as the DL was reported in 132 eyes in the CXL success subgroup versus 23 eyes in the CXL failure subgroup.Higher Kmax (rp = 0.92). Two eyes required deep lamellar anterior keratoplasty after A-CXL and I-CXL procedure for aggressive KC . In the overall analysis, no significant difference in ECD was reported from baseline to last visit (3.3 ± 263.5 mm/cm2) and within groups. In a subgroup analysis of 51 eyes with pachymetry below 400 µm , including 30 and 21 eyes in the A-CXL and I-CXL groups, respectively, no significant difference in ECD was reported from baseline to last visit nor within groups.We report one (1.4%) case of recurrent corneal erosion syndrome following I-CXL and three (1.1%) cases of corneal abscesses following A-CXL procedure (caused by Streptococcus bovis and Staphylococcus aureus). The mean BCVA difference from baseline to the last visit was 0.01 ± 0.17 logMAR with no difference between groups  versus I-CXL (31.0%) and less frequent in eyes with CXL failure (38.3% versus 54.0%). The DL was deeper after I-CXL (203.0 ± 53.3 µm) versus A-CXL (179.1 ± 37.8 µm). The presence of a DL in one-third of I-CXL cases reported in our study is similar to other similar studies about I-CXL protocol .3% versu. In a preactions ,25.The main limitation of this study is its retrospective design. However, this study was designed to collect real-world, long-term data to explore the long-term effectiveness and safety of these two CXL protocols with a large number of patients. Finally, both A-CXL and I-CXL protocols showed good efficacy in slowing the progression of KC with a high safety level. However, A-CXL seemed to be more effective than I-CXL in stabilizing keratoconus progression. The absence of a demarcation line on anterior segment OCT imaging at 1 month was a predictor of CXL failure. A lower threshold for minimal corneal thickness boundaries could be considered as CXL has been used in thin corneas with a high safety level. Further developments will certainly enhance iontophoresis CXL efficacy.


The utilization of heterosis based on three-line system is an effective strategy in crop breeding. However, cloning and mechanism elucidation of restorer genes for cytoplasmic male sterility (CMS) in upland cotton have yet been realized.Gossypium harknessii (D2-2) and restorer line R186. The offspring of 2074A × R186 were used to conduct genetic analysis. The fertility mechanism of 2074A can be speculated to be governed by multiple genes, since neither the single gene model nor the double genes model could be used. The bulked segregant analysis (BSA) for (2074A × R186) F2 determined the genetic interval of restorer genes on a region of 4.30 Mb on chromosome D05 that contains 77 annotated genes. Four genes were identified as candidates for fertility restoration using the RNA-seq data of 2074A, 2074B, and R186. There are a number of large effect variants in the four genes between 2074A and R186 that could cause amino acid changes. Evolutionary analysis and identity analysis revealed that GH_D05G3183, GH_D05G3384, and GH_D05G3490 have high identity with their homologs in D2-2, respectively. Tissue differential expression analysis revealed that the genes GH_D05G3183, GH_D05G3384, and GH_D05G3490 were highly expressed in the buds of the line R186. The predicted results demonstrated that GH_D05G3183, GH_D05G3384 and GH_D05G3490 might interact with GH_A02G1295 to regulate orf610a in mitochondria.This research is based on CMS line 2074A with the cytoplasm from Our study uncovered candidate genes for fertility restoration in the restorer line R186 and predicted the possible mechanism for restoring the male fertility in 2074A. This research provided valuable insight into the nucleoplasmic interactions.The online version contains supplementary material available at 10.1186/s12870-023-04185-z. The cytoplasm of Gossypium harknessii Brandegee (D2-2) was transferred into AD1 nuclear background, which led to the production of CMS-D2-2 [2-2 male sterile line 2074A and identified 28 ORFs specific to CMS 2074A by mitochondrial genome analysis.Plant male sterility mainly includes three types: cytoplasmic male sterility (CMS), cytoplasmic nuclear interaction male sterility, and nuclear male sterility (GMS) . CMS is orf . Steriliorf , 5, bursorf , inducinorf  and retrorf . CMS-A2 orf [2 , CMS-B1 rf [2 [1 , CMS-D2- [1 [2-2 , CMS-D8  [2-2 [8  and CMS--2 [8 [1  constituCMS-D2-2 . Li [13,CMS-D2-2  construcRestorer of fertility (Rf) genes rescue the fertility of CMS cytoplasm plants by interacting with CMS gene products and alleviating or eliminating the adverse effects on mitochondria of CMS gene products at DNA [Rf genes are also not fixed. More than half of cloned restorer genes encode pentatricopeptide repeat (PPR) proteins [Rf genes and can also restore the fertility of plants with CMS genes.s at DNA , transcrs at DNA , post-trs at DNA , translas at DNA –19, posts at DNA , and mets at DNA  levels. proteins . There aproteins , acyl-caproteins , glycineproteins , peptidaproteins , bHLH trproteins  and tranproteins  are desi2-2 cotton [2-2 cytoplasm is conditioned by a single dominance locus at sporophytical level. The gene located at the single dominant locus is designated as Rf1. CMS-D8 was bred by combining cytoplasm of Gossypium trilobum (DC) Skovst (D8) and AD1 nucleus [Rf2 gene can gametophytically dominate fertility of cotton plant with CMS-D8 cytoplasm [8 cotton can be remedied by Rf1 and Rf2 while fertility restoration of CMS-D2-2 cotton only depends on the function of the Rf1 gene [Although some reports found that multiple genes can rescue sterility of CMS-D2 cotton , Weaver 2 cotton  and Zhan2 cotton  confirme nucleus . A singlytoplasm . The steRf1 gene .Rf1 and Rf2 are located in the 0.93 cM interval of same chromosome [Rf genes is not linked to any morphological markers with known chromosomal locations in cotton [Rf genes in cotton, such as RAPD [Rf1 gene on the long arm of chromosome 4 (A subgenome). The mapping interval of Rf1 locus was delimited to 100 kb and was between 081-05 K and 052-01N BAC clones [Rf1 and Rf2 were delimited to a 1.4 cM genetic distance on chromosome D05 with assistant of four SSR markers. The locus of Rf gene was located on 1.35 Mb of chrD05 by the technology of BSA with SLAF-seq [Rf2 on a 1.48 Mb interval of chromosome D05, based on BSA with high-throughput SNP genotyping. While much efforts has been devoted to the genetic mapping of Rf loci, valuable Rf candidates have rarely been identified in cotton. Upgrading of sequencing technology, reduction of costs, and continuous release of high-quality cotton reference genomes [Rf genes.The tightly linked romosome . Each ofn cotton . A varie as RAPD –34, SSR  as RAPD , 34, STS as RAPD , AFLP [3 as RAPD , SNP [37 as RAPD  and InDe as RAPD , 40. Liu as RAPD  mapped tC clones  found thSLAF-seq  determin genomes –44 proviRf candidates that dominate the fertility of CMS-D2-2 cotton were mapped by BSA and analyzed for genetics, expression, sequence similarity, and evolution. First, strong restorer lines were screened with a fertility survey of F1 hybrids produced by crossing the CMS-D2-2 line with the restorer line. Genetic analysis of the F2 and BC1F1 populations originating from the CMS-D2-2 line and the strong restorer lines proved that the fertility of CMS-D2-2 cotton is controlled neither by a single locus nor by two loci. Then, the genetic interval of the Rf genes was determined using the BSA technology. Next, RNA-seq and sequence variation information supported the identification of four Rf candidate genes. The homologous proteins of four candidate proteins in Gossypium herbaceum (A1) [Gossypium arboreum (A2) [G. hirsutum (AD1) [GH_D05G3183, GH_D05G3384, and GH_D05G3490 were identified as candidate fertility restorer genes by evolutionary and sequence similarity analysis. Real-time quantitative PCR (qRT-PCR) proved that GH_D05G3183, GH_D05G3384, and GH_D05G3490 genes were highly expressed in R186 buds. Protein interaction analysis revealed that GH_D05G3183, GH_D05G3384, and GH_D05G3490 may interact with GH_A02G1295 to regulate orf160a and thus restore male fertility in cotton. From the above evidence, we can conclude that GH_D05G3183, GH_D05G3384, and GH_D05G3490 genes are likely Rf genes of CMS-D2-2. This research lays the foundation for the subsequent identification of restoration genes, the elucidation of restoration mechanisms, and the breeding of strong restorer lines with excellent agronomic traits.In this research, the key eum (A1) , Gossypieum (A2) , G. hirsum (AD1)  and all um (AD1) –47 were Rf loci can rescue the fertility of 2074A with CMS-D2-2 cytoplasm sporophytically [2 in three environments and the BC1F1 population (2074A × (2074A × R186) in two environments were conducted  F1 genome . A totalRf genes in BSA. The Rf genes from R186 were delimited on 34,943,848–35,280,626 bp, 37,694,536–38,093,258 bp, 38,227,690–38,918,070 bp, 43,648,410–43,742,747 bp, 44,220,658–44,835,843 bp, 45,653,643–45,811,858 bp, 46,818,876–47,801,436 bp, 49,315,266–50,052,334 bp, 51,046,570–51,244,308 bp and 51,557,591–51,715,302 bp of chromosome D05 using Δ(SNP-index) algorithm  and ED algorithms, including intervals 34,943,848–35,280,626 bp, 37,694,536–38,088,236 bp, 38,266,076–38,918,070 bp, 43,648,410–43,742,747 bp, 44,220,658–44,834,728 bp, 45,654,955–45,811,858 bp, 46,834,875–47,798,159 bp, 49,315,266–50,052,334 bp, 51,046,570–51,244,308 bp and 51,563,873–51,715,302 bp on chromosome D05, which contain a total of 77 genes  and euclidean distance (ED) algorithms were used to locate intervals of GH_D05G3176, GH_D05G3177, GH_D05G3263, GH_D05G3269, GH_D05G3270, GH_D05G3273, GH_D05G3328, GH_D05G3386, GH_D05G3461, GH_D05G3467, GH_D05G3468, GH_D05G3469 and GH_D05G3491 genes  analysis Fig. a. TwentyGH_D05G3465 gene, was identified in the 4.30 Mb interval, based on the reference AD1 genome [2-2 was only 22.96%  and 1.5–9.0 mm (stage of pollen abortion) were performed. The total DEGs of R186_2 vs 2074A_2 and R186_2 vs 2074B_2 were 12,166 and 21,483 while the up-regulated DEGs in restorer lines were 8191 and 10,798 in R186_2 vs 2074A_2 and R186_2 vs 2074B_2, respectively  and R186 (fertile bulk) at SNP_D05_35158622 are T and A, respectively, which changes the amino acid from K to M  F1  with CMS-D8 cytoplasm were mapped on a 1.48 Mb interval of chromosome D05 [Rf genes for CMS line (6001A) derived from the crossing progenies of G. thurberi (D1) and AD1 were located in the interval of 2.05 Mb  [Rf genes from R186 was delimited in a 4.30 Mb interval of chromosome D05. From our BSA results, it can be determined that the Rf genes for 2074A were mapped on chromosome D05, which was consistent with the location of the Rf genes for CMS lines with AD1, D1 and D8 abortive cytoplasm.Many types of molecular markers, such as SNP , 38, InDfile Rf1 . However-2. Wang  delimiters. Zhao  found th,586 bp) . In thisTriticum timopheevii (T)-type CMS wheat are dominated by multiple genes [1 and the abortive cytoplasm of D2-2. Weaver [2-2 cotton can be rescued by multiple genes. In this research, chi-square analysis results showed that multiple nuclear genes dominate the fertility of 2074A. The discovery is consistent with the views of Gao [orf610a in R186 buds with abortive cytoplasm was extremely significantly lower than that of 2074A, suggesting that the Rf genes may restore male fertility by reducing the expression level of orf610a in mitochondria  rice [Triticum timopheevii (T)-type CMS wheat [pcf petunia [GH_D05G3465, is localized in a 4.30 Mb interval on chromosome D05. However, the identity between GH_D05G3465 protein and its homologous proteins in D2-2 is low  rice , CMS-HonBT) rice , 55, CMSBT) rice , TriticuMS wheat , Ogura  F2 was planted in the winter of 2019 at Sanya Base of Cotton Research Institute of Chinese Academy of Agricultural Sciences   and in the summer of 2020, 2021 at Hejian Guoxin Cotton Base. 2074A × (2074A × R186) was planted in the winter of 2019 at Sanya Base and in the summer of 2020 at Hejian Guoxin Cotton Base.The F2:3 was planted in the summer of 2020 at Hejian Guoxin Cotton Base. The F2:3 population, together with (2074A × R186) F2 planted in the winter of 2019 at Sanya Base, was used for fertility survey and BSA sampling. 2074A and R186 were planted in the summer of 2019 at Hejian Guoxin Cotton Base and used for RNA-seq sampling.(2074A × R186) FThe morphological standard of flower fertility is divided into three levels, such as fully fertile (full pollen), partially fertile (less pollen) and completely sterile (no pollen). There are also three types for fertility of plant individuals including fully fertile individuals , partially fertile individuals  and completely sterile individuals .2 population were collected for DNA extraction and BSA, based on the fertility survey results of (2074A × R186) F2 and (2074A × R186) F2:3 population. The fertility of the F2:3 lines from the extremely fertile F2 individuals must be fully fertile. The DNA of the extremely fertile and extremely sterile individuals were used to construct of the BSA extremely fertile and sterile bulks, respectively. Fresh leaves of 2074A and R186 were also used to extract DNA and were taken as the parents for BSA.The fresh leaves of 19 extremely fertile individuals and 30 extremely sterile individuals from (2074A × R186) FThe buds of 2074A, R186, E5903 (restorer line), R144 (restorer line) and R245 (restorer line) with diameters of 0–1.5 mm (the earlier stage of pollen abortion) and 1.5–9 mm (stage of pollen abortion)  were col2, 2074A and R186 was isolated by cetyltrimethylammonium bromide (CTAB) method [1 genome [1 genome. In order to analyze the variants between samples, the original mutations were screened based on the following criterion: (i) The sequencing depth of the parent is not less than 5; (ii) The sequencing depth of the bulks is not less than 10; (iii) The parents are all homozygous and there are polymorphisms among the parents; (iv) The SNP-index value of bulks cannot be more than 0.8 or less than 0.2 at the same time. ANNOVAR software (2016Feb1) [Rf locus in BSA, including Δ(SNP-index) and ED. The ED algorithm, also called MMAPPR, calculates the frequency distance of each mutant between different bulks, and uses the distance difference to reflect the linkage strength between marker and target interval [The DNA of cotton leaves including extremely fertile and extremely sterile individuals of (2074A × R186) F) method . When th1 genome  and the 016Feb1)  was usedinterval . DeepBSAinterval . The LEV−1. The inserting size of library was then evaluated using the Agilent Bioanalyzer 2100 system. The Q-PCR method was used to accurately quantify the effective concentration of the library (the effective concentration of the library > 2 nM) to ensure the quality of the library after insert size of library meeting expectations. Sequencing was performed on an Illumina Hiseq platform when different libraries were pooled according to the requirements of library effective concentration and the target off-machine data volume. Raw data obtained from HiSeq sequencing was transformed into clean data by a three-step data processing. (i) Removing reads with adapters; (ii) Removing reads with more than 10% uncertain bases; (iii) Removing low-quality reads. At the same time, the Q20, Q30, and GC content of the clean data were calculated. High-quality clean data was the basis for all the downstream analyses. STAR was used to align paired-end clean reads to the reference cotton genome [P-value of 0.05 and |log2(Fold change)|≥ 1.The total RNA was extracted from buds by CTAB-ammonium acetate method with slight modifications . The detn genome . The lenn genome . DESeq RRNA-seq data of the maintainer line 2074B were referred to Nie .http://www.geneontology.org/).Functional enrichment analysis of candidate genes was performed at online website GO  was the internal reference gene in all qRT-PCR experiments. 2−ΔΔCt method was adopted to calculate the relative expression level of each gene [PrimeScript™ RT reagent Kit with gDNA Eraser  was used to complete the reverse transcription of RNA. The experiment of qRT-PCR was executed with the assistance of PrimeScript™ RT reagent Kit . The primers used in the experiments are shown in Additional file ach gene .1, A2, AD1 and all D genome cotton were searched and downloaded from the CottonGen (https://www.cottongen.org) [The homologous proteins sequence of the candidate proteins in Agen.org) . The ideGossypium raimondii L. using STRING V11.5 [Protein interaction analysis of candidate proteins with reported mitochondrial abortive orf610a in cotton  were preNG V11.5 . The minwith the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora, we confirm that the plant materials used in the present study does not involve any species at risk of extinction. All methods performed are in accordance with the relevant institutional, national, and international guidelines and legislation.Complying Gossypium harknessii and were released since 1970s [The cytoplasmic male sterile lines DES-HAMS277, DES-HAMS16 and the restorer lines DES-HAF277 and DES-HAF16 are originated from ce 1970s . All thece 1970s , 68, 71.Additional file 1. Supplemental figure 1Additional file 2. Supplemental figure 2Additional file 3. Supplemental table 1-4Additional file 4. Supplemental table 5-6Additional file 5. Supplemental table 7-8Additional file 6. Supplemental table 9Additional file 7. Supplemental table 10Additional file 8. Supplemental table 11