L), which has evolved to fold rapidly and very stably to serve as a catalyst for the folding of the heavy chain CH1 domain. The first hurdle is the reduction of the disulfide bond in the CL domain, which is required for retrotranslocation to the cytosol. In spite of being reduced, the CL domain retains structure, giving rise to the second rate-limiting step, the unfolding of this domain at the proteasome, which results in a stalled degradation intermediate.Antibody monomers are produced from two immunoglobulin heavy chains and two light chains that are folded and assembled in the endoplasmic reticulum This process is assisted and monitored by components of the endoplasmic reticulum quality control machinery; an outcome made more fraught by the unusual genetic machinations employed to produce a seemingly unlimited antibody repertoire. Proper functioning of the adaptive immune system is as dependent on the success of this operation, as it is on the ability to identify and degrade those molecules that fail to reach their native state. In this study, two rate-limiting steps were identified in the degradation of a non-secreted κ light chain. Both focus on the constant domain (C H), Diversity (DH), and Joining (JH), and two each for the κ and λ light chains . One DNA segment from each of these heavy and light chain gene families must be successfully recombined, and non-templated nucleotides are added to the ends of the DNA segments prior to their relegation to produce a heavy chain and light chain variable region. In addition, the assembled variable region is subjected to hypermutation to increase antigen affinity  domain is unique in that it remains reduced  system assists and monitors the maturation of nascent proteins. Monomeric IgG antibodies are covalently assembled from two identical γ heavy chains and two identical either κ or λ light chains, which possess four and two Ig domains, respectively, whereas pentameric IgM antibodies are covalently assembled from ten μ heavy chains consisting of five Ig domains, ten light chains, and a J or joining chain. Each Ig domain is approximately 100 amino acids in length and folds into an anti-parallel β barrel structure that is secured with a disulfide bond between highly conserved cysteines in strands 2 and 6 . Antibodturation . These sturation , the fir reduced  and unst reduced  prior tomediates . Contactsecreted . In the elopment . Thus, cGiven the unusual mechanisms needed to produce the vast antibody repertoire, as well as the extremely high biosynthetic rate achieved in plasma cells, it is to be expected that some genetic manipulations, and even random mistakes during normal synthesis, will produce antibody subunits that fail to fold or assemble properly. The presence of a single unfolded domain requires that the heavy or light chain be targeted for degradation, which occurs through the actions of the ER associated degradation (ERAD) system . Once thL) that forms its disulfide bond but folds unstably and a well-folded, oxidized constant domain (CL). We found that although the VL domain must be reduced  to minimize adverse effects on the natural folding of this domain. A cytosolically expressed NS1 (ΔssNS1) was engineered by removing the ER targeting signal sequence, and replacing methionines present at positions 4, 11, 13 with alanines to prevent possible alternative translation initiation sites. Primers used to create the amino acid changes in these constructs are found in L or CL domains with serine codons. The PDIA6 construct in the pCDNA3 vector was generously provided by Dr. Neil Bulleid , and ERdj5, also in pCDNA3, was a kind gift from Dr. Kazuhiro Nagata . Wild type p97 and the ATP hydrolysis-defective mutant p97QQ, each in the pcDNA3 vector, were considerate gifts from Dr. Yihong Ye . The Hrd1 mutant deficient in ubiquitin ligase activity (Hrd1 C291S in pcDNA3) was kindly supplied by Dr. Yuval Reiss .The non-secreted murine NS1 κ LC  and the LSTK-  were clo2. 293T cells were plated 24 h prior to transfection, which was performed using GeneCellin  according to the manufacturer’s protocol. For all analysis, 1 μg of each indicated ERAD substrate was used per p60 dish. When p97WT, p97QQ or Hrd1C291S was co-expressed, 1.5 μg of each plasmid was used and an equal amount of empty pcDNA3.1 vector was used in the control samples. When PDIA6 or ERdj5 was co-expressed, 2 μg of the plasmid was used. The P3U.1 murine plasmacytoma cells, which naturally synthesize the NS1 κ LC, were grown in complete DMEM supplemented with 55 µM 2-mercaptoethanol  at 37°C and 5% CO2. The Ag8.653 chain-loss variant cell line was grown in the same conditions and was used as a negative control for experiments with P3U.1 cells.293T human embryonic kidney cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% (v/v) heat-inactivated fetal bovine serum , 2 mM L-glutamine , and a 1% (v/v) antibiotic-antimycotic solution  at 37°C and 5% CO−/Met−) , which was supplemented with 1% L-glutamine and 10% FBS dialyzed against PBS to deplete amino acids. Cells were labeled with Express [35S] Labeling Mix  for 15 min. After labeling, culture dishes were placed on ice, washed once with 2 ml cold DPBS , and cells were re-incubated in 2 ml of chase media  and cysteine . For the NS1-N157 deglycosylation experiments, cells were pre-incubated in 10 µm MG132 for 2 h and 15 min before starving. The starving, labeling and chase media all contained 10 µm MG132. Zero time points were lysed immediately in Nonidet P-40 lysis buffer , and all other plates were returned to 37°C for the indicated time points. Lysates were clarified by centrifugation at 18,000 g and incubated with goat anti-mouse κ (SouthernBiotechonology) overnight at 4°C on a rotator. Protein A agarose beads  were added for an hour the following morning. After washing the beads 3 times with Nonidet P-40 wash buffer (NP-40 lysis buffer with 400 mM NaCl), proteins were eluted with 2x sample buffer at 95°C. 30% of immunoprecipitated proteins were electrophoresed on polyacrylamide gels and transferred to PVDF membranes. Dry membranes were exposed to BAS storage phosphor screens  and scanned using a phosphor imager  to detect signals. Signals were quantified with the ImageQuant TL software (Cytiva). Non-transfected cells were used to subtract any background signal from the samples.Twenty-four hours after transfection, cells were pre-incubated at 37°C for 30 min in 2 ml media consisting of 1x DMEM without Cys and Met (CysClarified NP40-solubilized lysates were mixed with reducing sample buffer (1:1), and samples were separated on 13% SDS-polyacrylamide gels for nearly all analyses. Exceptions include studies to determine the oxidation status of our substrates in which 15% gels were used, and the Proteinase K digestion and pulse-chase experiments where 12% gels were used. Post-electrophoresis, proteins were transferred to PVDF membranes . Membranes were fixed with methanol, blocked with gelatin wash buffer, and incubated overnight with the indicated primary immune reagents in blocking buffer followed by species-specific secondary reagents, also in blocking buffer. Western blots with HRP-conjugated secondary antibodies were developed using the Pierce™ ECL , and for quantitative analysis they were scanned with the LI-COR Fc Odyssey scanner . When secondary antibodies conjugated to IRDye680 or IRDye800 (Li-COR) were employed, protein bands were detected using the LI-COR Odyssey CLx Imager (LI-COR Biosciences). Analysis and quantification were performed with the Image Studio Lite software. For experiments where the oxidation status of clients was studied, cells were lysed in complete NP-40 buffer additionally supplemented with 20 mM NEthylmaleimide .6 P3U.1 (κ LC expressing) or Ag8.653 (Ig−) cells were used. To detect association of proteins, cells were incubated with a membrane-permeable cross-linking agent prior to lysing as described previously : goat anti-mouse IgG (925–32210), goat anti-rabbit IgG (925–68071), and donkey anti-goat IgG (926–68074).The following antibodies were used for blotting: polyclonal goat anti-mouse κ LC ; mouse anti-GAPDH ; mouse anti-Hsc70 (B-6) , mouse anti-VCP/p97 , mouse anti-20S proteasome subunit alpha (C8) , rabbit anti-PSMC2 , mouse anti-actin , rabbit anti-PDIA6 . HRP-conjugated antibodies were used at 1:10,000 dilution and include goat anti-rabbit (sc-2054), donkey anti-goat (sc-2020), and goat anti-mouse (sc-2031) all purchased from Santa Cruz Biotechnology. The rabbit anti-ERdj3 antibody was produced in our lab . IRdye® For cycloheximide (CHX) and/or MG132 chase experiments, cells were treated with 10 μΜ ΜG132 or 100 μg/ml CHX for the indicated times. The cells were lysed in 0.5 ml of NP40 lysis buffer, clarified by centrifugation at 18,000 × g for 15 min, and 1.5% of the resulting lysate was analyzed by western blotting. To remove N-linked glycans from the NS1 glyco-mutant constructs, we treated samples with Endo H  according to the manufacturer’s protocol. Samples were mixed with 2x Laemmli sample buffer and analyzed by western blotting.To monitor the oxidation status of the various proteins, cells expressing the constructs of interest were washed twice with PBS containing 20 mM NEM  and lysed in 0.5 ml NP-40 supplemented with 0.1 mM PMSF, 1X complete protease inhibitor tablets without EDTA and 20 mM NEM.2, 1 mM MgCl2, 1 mM MnCl2, 0.1 mM PMSF and 20 mM NEM. Clarified lysates were incubated overnight at 4°C with 2:1 v/v Con A bead slurry. Beads were centrifuged (500 × g for 5 min) and the supernatant (unbound) was collected and mixed with an equal volume of 2x non-reducing Laemmli buffer for analysis of the non-glycosylated proteins. The glycosylated proteins (bound) were eluted from the beads with a volume of 1x non-reducing Laemmli buffer equal to the total volume of the non-bound sample. The resulting pools were analyzed by western blotting.Concanavalin A (Con A) beads  were used to separate glycosylated and non-glycosylated species. 293T cells were grown in p60 dishes and transfected with the mono-glycosylated NS1 client (NS1-N100). After 24 h, the cells were incubated with 10 μM MG132 in DMSO or in an equal volume of DMSO alone as a control for 3.5 h and were then lysed in 0.5 ml of NP-40 lysis buffer supplemented with 1 mM CaClStability of indicated proteins was assessed by partial proteolysis experiments. Cells expressing the indicated constructs were lysed in 0.5 ml NP-40 devoid of protease inhibitors and PMSF. Proteinase K  was added to a final concentration of 20 μg/ml. Digestion was performed on ice for 25 min and was followed by a 5 min incubation with PMSF  (Sigma) to inactivate the Proteinase K. An equal volume of 2x Laemmli sample buffer was added to samples, which were heated to 95°C for 5 min and immediately loaded onto SDS-PAGE gels for western blotting. To determine the glycosylation status of the Proteinase K resistant fragments, the samples were treated with Endo H. At the end of this reaction 2x Laemmli buffer was added, and samples were immediately analyzed by western blotting.6 of each) were incubated with 10 μM MG132 or DMSO for 3.5 h and lysed in NP40 lysis buffer. LCs were isolated by immunoprecipitated overnight with anti-mouse-κλ-conjugated agarose beads  and washed 3 times in NP40 lysis buffer made to 400 mM NaCl. Beads were resuspended in 20 μL of 2x Lammeli buffer with 2-β mercaptoethanol and heated to 95°C for 5 min. Solubilized proteins were loaded on a precast 4–15% gradient gel (BioRad) and run 1 cm into the gel. The gel was then incubated for 1 h in GelCode-Blue (Thermo Scientific), destained overnight in ultrapure H2O at room temperature, and transferred to our Center for Proteomics and Metabolomics core for proteome profiling by spectral counting.P3U.1 and Ag8.653 cells  and eluted by a gradient (∼60 min). The eluted peptides were ionized by electrospray ionization and detected by an inline mass spectrometer (Thermo Elite). The MS spectra were collected first, and the 20 most abundant ions are sequentially isolated for MS/MS analysis. This process is cycled over the entire liquid chromatography gradient. Database searches were performed using the Sequest search engine in our in-house SPIDERS software package. All matched MS/MS spectra were filtered by mass accuracy and matching scores to reduce protein false discovery rate to ∼1%. Finally, all proteins identified in one gel lane were combined.L domain that does not fold stably. However, its CL domain folds well, forms its intrachain disulfide bond, and can stimulate the folding of the CH1 domain of a γ heavy chain leading to the secretion of a properly antibody , one at the boundary between the two domains (N100), and one in the CL domain (N129). Co-expression of a domain negative Hrd1 mutant (C291S) inhibited deglycosylaton of the NS1-N28 protein ; the well-folded CL domain is left intact and is not ubiquitinated (L domain (N28), and a separate one was introduced near the C-terminus of this domain (N100). In this case, inhibition of the proteasome did not lead to the appearance of a significant amount of the deglycosylated form of either NS1-VL-STK- construct , which is consistent with previously reported data , and a partially oxidized (ox1) form, in which only the Cide bond . The preide bond . To deteted data . Howeverted data . This rereatment . To detereatment , demonstL domain and in the CL domain separately by mutating the two cysteines in each domain to serines  alone or co-expressed it with either PDIA6 or ERdj5, another PDI family member with reductase activity, and performed pulse-chase experiments  responsible for reducing the disulfide bonds in NS1, we used the P3U.1 mouse plasmacytoma cells, which is the source of the NS1 construct used in our studies thus far  and the eriments . PDIA6 heriments  or by wed NS1 LC . Somewhain vitro study found that reduction of a recombinant CL domain did not significantly alter its structure (L domain in which the unstable VL domain had been digested (L domain (red asterisk). Proteinase K treatment of the lysate from MG132-treated cells resulted in the disappearance of this band and the appearance of an ∼12 kDa band that co-migrated with the band generated in the Proteinase K treated cells in which the proteasome had not been inhibited.A previous tructure . To detedigested . When thL band arose only from the ox1 form, which would have been in the ER lumen possessing the intramolecular disulfide bond in the CL domain, or if it also included the retrotranslocated and fully reduced ox0 isoform. To distinguish between these possibilities, we similarly examined lysates from two of the NS1 constructs possessing a single N-linked glycan; the NS1-N100 with an engineered glycan very near the C-terminus of the VL domain and the NS1-N129 with the glycan position in a loop in the CL domain. For the NS1-N100 construct, treatment of the control (no MG132) sample with Proteinase K produced two anti-κ-reactive fragments; both of which must originate from ER localized, glycosylated protein, as this is the only form present without proteasome inhibition , whereas the fastest migrating fragment in this sample was unglycosylated and thus represented the retrotranslocated protein that had been deglycosylated by N-glycanase and was digested by Proteinase K to an isolated CL domain. Thus, it appeared that the glycan engineered just 9 amino acids from the VL:CL boundary partially interfered with complete proteolysis of the VL domain (indicated with two red arrows on the cartoon) giving rise to the doublet in the Proteinase K and Endo H treated sample.To determine the source of the various fragments generated in L domain (green downward arrow) after the glycan was removed. In this case the position of the engineered glycan did not interfere with complete proteolysis of the VL domain. Furthermore, the glycosylated, isolated CL domain (green upward arrow) was shown to co-migrate with the MG132-generated fragment (red asterisk). Cartoon depictions of the composition of the various bands generated as deduced from the combinations of treatments are included in the figure and L domain of the deglycosylated ox0 isoform generated after MG132 treatment still possessed significant structure in the cytosol, even though the disulfide bond had been reduced. Based on the molecular weight of the MG132-generated, non-glycosylated fragment recognized by anti-κ LC antisera we deduced that it must include ∼30 amino acids of the VL domain in addition to the 105 amino acid CL domain and represents an intermediate in proteasomal degradation.When the NS1-N129 construct was similarly examined , we agaiL domain, which would be sufficient to reach the proteolytic active site, indicating that unfolding of the structured CL domain by the ATPase ring of the proteasome represented a second rate-limiting step in the degradation of NS1 κ LCs (To identify proteins that the retrotranslocated NS1 protein interacted with, we examined the spectral count data that was obtained with κ LC immunoprecipitated from the P3U.1 cells obtained with and without inhibition of the proteasome . We idenS1 κ LCs .H1 domain ensures the free HC are not secreted. Subsequently, LC rearrangement is initiated to produce a single LC that must induce folding of the CH1 domain and release BiP, so the assembled Ig protein can be transported to the cell surface or secreted. This represents a critical checkpoint in both B cell development and in Ig assembly, and as such, LC genes have evolved to produce CL domains that fold very stably. The failure of a LC to rescue the free HC can initiate another round of LC gene rearrangements, necessitating the disposal of the LC produced first. Even once a compatible LC partner has been selected, the extremely high rate of antibody synthesis observed in plasma cells is likely to result in some failures in proper Ig maturation resulting in the need to target improperly folded subunits for degradation. The NS1 LC is an example of such a protein and provides an excellent model to ask what the requirements and checkpoints for degrading a protein when one domain fails to fold, and the other domain folds very stably and secured with a disulfide bond. Our studies revealed two checkpoints or rate-limiting steps in the degradation of this non-secreted LC (L domain. Although the VL domain of NS1 obtains a disulfide bond initially, it is readily reduced, due to its unstable fold giving rise to the ER resident ox1 form of NS1. Although we observed PDIA6 associated with NS1, over-expression of this reductase only modestly increased the turnover of the Vmut construct in which the only disulfide bond present was in the CL domain. PDIA6 over-expression had even less of an effect on the half-life of the Cmut that possessed only the VL domain disulfide bond. The interpretation of this result is complicated by the fact that genetically ablating the disulfide bond in the VL domain is not rate-limiting for NS1 turnover. Thus, it remains formally possible that PDIA6 plays a role in reducing the disulfide bond in the VL domain. Although we were unable to detect endogenous ERdj5 association with NS1, perhaps because its association is too transient to detect in these types of experiments, over-expression of ERdj5 readily increased the turnover of the Vmut construct arguing that it is responsible for reducing the disulfide in the CL domain and thus executing this rate-limiting step. Upon reduction of the CL domain, this LC is retrotranslocated and degraded so rapidly that a completely reduced, ox0 form is never observed unless proteasomal activity is inhibited. These data argue that accessibility of this PDI family member to the disulfide bond in the CL domain is restricted by the very stable fold of this domain.Ig HC are expressed first in pre-B cells during B cell development, and the association of BiP with the unstructured Creted LC . The firThe extraction of the NS1 LC from the ER lumen required the activity of the AAA-ATPase p97 but was not dependent on proteasomal function. A group of papers from the Sitia lab has examined requirements for degradation of Ig μ heavy chain multimers  and μ heL-STK- mutant that cannot be ubiquitinated (Our data on the requirements for ERAD components in the extraction of NS1 from the ER are contrary to a study using proximity biotinylation, which found that this client was modified by cytosolically expressed BirA even in the presence of the p97QQ mutant, as was the NS1 Vitinated . Althougitinated .L domain either retains or re-obtains structure in the cytosol. Indeed, previous studies from our lab revealed that light chain constant domains fold so rapidly in the ER that BiP trap mutants are unable to bind and prevent their folding and are more resistant to heat/chemical denaturation in vitro than the molecular chaperone BiP (2 microglobulin. They found that addition of methotrexate to stabilize the DHFR moiety in a fully folded state did not impede retrotranslocation (Somewhat less expected was our finding that, in spite of being reduced for retrotranslocation, the Crone BiP . Howeverrone BiP . In anotlocation . The narlocation , which wlocation . It is cL domain together with an additional ∼30 amino acids of the VL domain. It has been estimated that the distance from the outside of the 20S proteasome to the proteolytic active site is ∼70Å (L domain stalling ∼10 amino acids from the 20S proteasome particle. This argues that the CL domain represented an impediment to entry into the proteasome core, consistent with this domain retaining structure, until it was unfolded by the AAA-ATPases found in the base of the proteasome lid (The continued presence of a structured domain while this ERAD client was in the cytosol provided the second rate-limiting step in its turnover. Intriguingly, our studies detected a very sharp, anti-κ reactive intermediate migrating at ∼15–16 kDa when MG132 was used without the addition of a protease. This fragment is a minor population and small enough that it runs near the dye front and is readily lost if the samples are electrophoresed to long. Based on the apparent molecular weight of this species together with the actual sequence of the NS1 protein, we calculated that this fragment was comprised of the ∼12 kDa C is ∼70Å , which isome lid . This inL domain must be broken for the LC to be transported to the cytosol. In spite of becoming reduced, this domain retains or re-obtains structure in the cytosol. This structured domain represents the second rate-limiting step, and it must be unfolded by the AAA-ATPases that are a component of the proteasome regulatory particle. Since all κ LC share the CL domain, these checkpoints are valid for any LC that fails to achieve its native state. Although our studies focused on a non-secreted κ LC, the Ig fold is the second most common structural motif found in metazoan proteins and is particularly abundant in cell surface receptors and secreted proteins (In summary, our studies revealed two checkpoints in the degradation of an Ig LC. First, the disulfide bond in the well-folded Cproteins . This du


This Research Topic was designed to explore the role of microbiota in neurodegenerative diseases. This topic is critical since it aims to shed light on the very first stages of neurodegenerative diseases. Understanding the multifaceted roles of microbiota in the development of these diseases enables the discovery of novel therapeutic targets. There are two major reasons why so many drugs for the treatment of neurodegenerative diseases have failed in the last few years . The uniqueness of bacterial extracellular DNA and LPS is that they can reach the central nervous system (CNS) either through systemic circulation if the blood-brain barrier is impaired or can be released by microorganisms directly located inside the brain   in particular, as well as extracellular DNA- and RNA-based bacterial TezR receptors or lipopolysaccharides (LPS), have been highlighted as novel and highly specific triggering factors for beta-amyloid and Tau prionogenic aggregation .Overall, the articles published in this current issue cover the critical topic for the role of microbiota in neurodegenerative diseases. Together with previous data, they pave the way for using brain-localized bacteria and fungi, as well as those located in the gut, as novel potential therapeutic targets for the treatment of these devastating disorders.GT analyzed and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The physical, textural, and especially nutritional characteristics of the optimal WF-AF bread were enhanced. The sensory evaluation results revealed high scores (8.70) for the acceptability of optimal bread with a medium particle size as compared to wheat bread (8.25). The protein and ash content of the optimal breads with large, medium, and small AF particle sizes, respectively, increased significantly, from 8.92 to 10.58%, and 0.82 to 0.99%, respectively, relative to the wheat flour bread . The mineral content was up to two times higher in the optimal breads compared to wheat flour bread. The findings generated from this study are helpful in bakery industry for designing products with enhanced nutritional properties and for introducing new products to the market.Amaranth is an underutilized pseudocereal that can be used to supplement wheat flour (WF) in order to improve the nutritional quality of bread. Bread digestibility is impacted by particle size which produces different nutritional properties. This research aims to evaluate the baking characteristics of optimal wheat–amaranth composite flour for each studied amaranth flour (AF) particle size at doses previously established based on an optimization process and to characterize from a physical, textural, nutritional, and sensorial point of view the obtained bread. The results revealed that the optimal wheat–amaranth composite flour with medium and small particle size, respectively showed a slightly lower α-amylase activity, while dough development time was significantly higher compared to the WF. A significant ( Bread is one of the most frequently consumed foods, being the main source of energy supplied for the body , but refInterest in pseudocereals’ use in food technologies has increased in recent years, because they offer a valuable nutritional composition, having a high-quality protein content amino acid composition point of view . AmarantAmaranthus cruentus whole meal in gradual doses up to 40% to wheat flour remarkably increased the protein, lipid, ash, and dietary fiber contents of white bread [The use of protein-rich and gluten-free amaranth flour mixed with cereal flour had a negative impact on the dough’s rheological properties. The addition of amaranth flour at different doses  in refined wheat flour or whole wheat flour determined an increase in dough tenacity and a decrease in its extensibility . In anotte bread . An incrte bread . AlthougEnhancing nutritional aspects when using wheat–amaranth composite flour with different particle sizes of amaranth in bread-making requires an adapted formulation. A desirability function has previously been applied to find the optimal amount of AF added in WF, depending on the AF particle size, to enhance dough and bread properties . These oSaccharomyces cerevisiae yeast from Rompak , and salt from SanoVita .Amaranth seeds were purchased from Solaris Plant , wheat flour (WF) (flour yield 65%) from Mopan , fresh The wheat flour type 650 with a wet gluten content of 30.00%, dry gluten content of 9.75%, gluten deformation index of 6 mm, and FN index of 312 s was used in the research. The bread evaluated in this study was made from wheat–amaranth composite flour, formulated based on the optimal amounts obtained for each particle size in previous research . The proSaccharomyces cerevisiae baker’s yeast (3%). The biphasic procedure for the preparation of dough samples consisted in mixing half of the composite flour amount with the entire amount of water and yeast in order to form a leaven 30 ± 2 °C and 85% relative air humidity (RH). The leaven thus formed was left to ferment for two hours in a fermentation chamber . After the leaven had fermented for two hours, the other part of optimal composite flour and salt were incorporated, kneaded for 10 min using a Kitchen Aid mixer , and allowed to complete its process of sugar fermentation for another hour at 30 ± 2 °C and 85% RH [Bread dough samples were prepared from wheat flour and optimal composite flours using the indirect method, at the optimum water absorption capacity determined at Mixolab , according to the method of Coțovanu and Mironeasa . For thed 85% RH . The douThe bread samples were analyzed for moisture content according to the gravimetric method; the protein content was determined using a rapid Kjeldahl device with digestion and steam distillation , and the conversion factor was 6.25 for WFB and 5.53 for composite flour bread. The ash content was determined in a muffle oven by incineration at 900 °C, per the standard protocols  of the International Association for Cereal Chemistry , while t3 69% and brought to the mark (50 mL) with deionized water. As a control sample, deionized water was used following the same procedure as for the analyzed sample. The spectrophotometric determination involved the following steps: activating the cathode lamp corresponding to the elements , adjusting the operational parameters, activating and adjusting the flame, and establishing the curve standard by absorbing four working standard solutions of different concentrations. In the flame system used, the nebulizer and the atomizer play a decisive role: the nebulizer aspirates a liquid sample with a controlled flow and the atomizer creates a fine aerosol and mixes the aerosol with the oxidizing gas. The mineral elements are expressed as mg/100 g of flour and were calculated with Equation (1):E—mineral element concentration, mg/100 g;C—the concentration measured on the calibration curve, mg/L;F—dilution factor;V—sample volume, mL;M—sample mass taken in the analysis, g.Atomic absorption spectrometry (AAS) was used to identify chemical elements through the absorption of optical radiation by a population of free atoms in the gaseous state. For the determination of macro- and micro-elements from wheat flour, amaranth fractions, and bread, the method described in the SR EN 14082:2003 standard was used. The analysis of the sample involved two stages: the mineralization of the sample and the metal dosage by spectrophotometry. During mineralization, the organic matter in the sample (5.00 ± 0.001 g) was destroyed by carbonization and combustion in the calciner, with the temperature gradually increasing from 250 °C to 450 °C, up to 900 °C, for 8 h. A total of 5 mL HCl 6 mol/L (STAS 13013/1-91) was added to the ash obtained, and then the acid was evaporated using a sand bath. The residue was dissolved with 730 µL HNOIn order to determine the amino acid (AA) content of wheat flour, amaranth fractions, and bread, first the samples (3.70 ± 0.5 g) were mixed with 30 mL of trichloroacetic acid (TCA) 15%. Next, the pH of the solution was adjusted to 2.2 with sodium hydroxide solution and further diluted with 50 mL of 15% TCA. After centrifugation for 5 min at 3000 rpm, the supernatant was filtered through a 0.45 µm filter . Seven emax), and creep-recovery compliance  [A Thermo-HAAKE, MARS 40  with parallel plate-plates geometry was used to determine the dough dynamic rheological behavior in terms of elastic (G′) and viscous modulus (G″), viscosity factor (tan δ), maximum gelatinization temperature  .The falling number index (FN) of the wheat flour and optimal composite flour related to each of the studied particle size of AF was determined using a Falling Number device  .The Mixolab Chopin equipment  was used for a complete rheological test of wheat flour and optimal wheat–amaranth composite flour formulations following the ICC 173, AACC 54–60.01 method . The MixThe biaxial extension of dough in terms of dough tenacity (P), dough extensibility (L), dough strength (W), and alveograpic ratio (P/L), was analyzed with an Alveograph Chopin equipment  following the ICC 121 method .2 production (VT), the volume of the gas retained in the dough at the end of the test (VR), and retention coefficient (CR), was performed with a rheofermentometer device , as described in a previous study [Dough fermentation testing, in terms of maximum height of the gas release curve (H’m), the total volume of COus study , followius study .The physical characteristics of bread samples made from wheat flour and optimal wheat–amaranth composite flour, bread volume (BV), specific volume, porosity, and elasticity was determined according to the Romanian standard SR 90: 2007 .A TVT-6700 texture analyzer  was the equipment used for analyzing bread firmness, elasticity, cohesiveness, gumminess, resilience, and masticability, by using the working setting shown in a previous study , and theL*), redness/greenness (a*), and yellowness/blueness (b*) of the crust and crumb bread samples were measured with a CR-700 colorimeter .The CIELAB colour coordinates for the lightness . The method consisted of identifying the most suitable terms to describe the general acceptability, appearance, crust and crumb structure, taste, and smell. The terms proposed in the sensory evaluation were those mentioned in the SR ISO 11035:2007 standard , which dp < 0.05 was used to test the relationships among the studied parameters. A principal component analysis (PCA) was applied to observe the relationships between the wheat–amaranth dough, bread proximate composition, textural characteristics, and acceptability, and to visualize similarities or dissimilarities between them.All the data in the tables and figures were presented as a mean with standard deviation, and the determinations were performed at least in duplicate. SPSS software 25.0   was used for statistical analysis of the data. Statistically significant differences between samples were determined by one-way ANOVA. Tukey’s test was used to test the differences between the characteristics of the optimal and control samples. Pearson’s coefficient at The physicochemical characterization of the composition of wheat flour and amaranth flour fractions was reported in a previous study . In addiThe baking characteristics of the optimal wheat–amaranth formulated composite flours and the quality parameters of the bread compared to wheat dough and bread are shown in According to the obtained results, the optimal wheat–amaranth composite flours with large fractions presented α-amylase activity, quantified by the falling number (FN) index, similar to that of the control sample, whereas the optimal composite flour with medium and small fraction presented a slightly lower α-amylase activity.Dough stability (ST), protein thermal weakening (C1-2), and starch gelatinization (C3-2) also showed values very close to those of the control sample, while the dough development time (DT) and the stability of the hot gel (C3-4) were considerably higher than the control sample, whilst starch retrogradation (C5-4) presented significantly higher values for the optimal composite flour than for wheat flour.Alveographic parameters’ values presented differences between samples, the highest values of dough tenacity being observed for the optimal dough with small AF particle size, while the lowest extensibilty value was obtained for the optimal composite dough with a large particle size.max) decreased compared to the control sample. A lower resistance to deformation indicated by an increase in the creep compliance (Jcmax) value was obtained for the samples with the optimal addition of small sizes of amaranth flour compared to the control sample.The optimal dough samples with large, medium, and small particle sizes had a higher maximum height (H’m), volume of gas total (VT) and retained (VR), and gas retention capacity (CR) compared to wheat flour dough. The elasticity (G′) and viscosity (G″) modulus showed higher values than the control for all of the optimal dough samples, which indicated the viscoelastic nature of the dough, the results being expected. Remarkable differences were obtained between the optimal wheat–amaranth composite flour dough samples with large fraction sizes (O_AL) and those with medium and small sizes (O_AM and O_AS). The maximum gelatinization temperature , the intensity of the red (a*) or green (−a*), and the yellow (b*) or blue (−b*) shade—for the crust and the crumb of the breads made from the optimal composite flours varied according to the related optimal dose , medium (M), and small (S) particle sizes compared to bread from refined wheat flour are presented in p < 0.05) were observed between the firmness of bread crumb with AF fractions compared to the control. A gradual increase in firmness was observed in bread with particle size reduction, while the bread sample with large AF particles showed lower firmness values. The elasticity of the bread samples with the optimal dose of amaranth decreased in the following order: M < L < S, with significant differences between the optimal samples and the control bread, but no significant differences were recorded between the samples with medium and small particles.According to the experimental data obtained from the texture analysis of the bread samples, significant differences  between the samples and compared to the control sample. The results indicated an improvement in the protein, lipid, and ash contents of bread obtained from the optimal composite flour, especially in the case of large and medium fractions of amaranth flour, while the carbohydrate content and energy values decreased  content in bread samples with the optimal dose (O) of amaranth flour addition corresponding to large (L), medium (M), and small (S) particle sizes and in comparison with bread made from refined wheat flour is presented in Six essential amino acids were identified in bread samples formulated with the optimal doses of amaranth flour, for each individual particle size, a, represThe results of the sensory analysis in terms of overall acceptability, appearance, crumb structure, taste, and smell for the optimal bread formulations compared to the control can be observed in Relationships between the variables were highlighted by applying a multivariate technique, principal components analysis (PCA). The application of PCA provided information about the similarities and/or differences between the evaluated variables, in terms of chemical composition, dough dynamic rheological parameters, bread physical, texture, and sensory properties .p < 0.05, between some of the determined characteristics.From the analysis of Pearson correlation matrix, direct or indirect correlations were observed, significant at Considering the impact of the nutritional characteristics of wheat flour and amaranth flour fractions on the final product, the results of the mineral and amino-acids contents are discussed in relation to the nutritional properties of bread obtained from optimal wheat–amaranth composite flour specific to each particle size and wheat bread. Alencar et al. (2015)  found thmax) of optimal wheat–amaranth composite flour decreased in all of the optimal wheat–amaranth composite flours, a fact that may be due to the insoluble amylose–lipid complexes formation that occurred during the heating of starch slurries, which reduces and delays the swelling of starch granules [The increase in the FN index, of the optimal formulations with medium and small AF particle sizes, leads to a decrease in α-amylase activity, which can be correlated with the presence of calcium ions in amaranth grains . Dough dgranules . Creep-rgranules . The difgranules , mineralBread obtained from optimal composite flour determined a slight decrease in volume. This behavior can be explained by the intake of dietary fiber from the large fractions, which interact with wheat glutenin through disulfide bonds, not diluting the gluten network very much . The besRegarding bread textural properties, an increase of crumb firmness in bread formulations was observed when AF particle size decreased. This fact can be explained by the dietary fiber content of the large particle, which contains albumin that can interact with wheat glutenin through disulfide bonds and will act as a surface active agent and thus will maintain the gluten matrix ,39. SimiGenerally, both colour of the crust and of the bread crumb impact consumer acceptability. The colour of the optimal bread formulations varied depending on the fraction used in optimal bread, this fact being possible due to the intense Maillard-type reaction that occurs in the samples with the addition of protein-rich fractions. In addition, the decrease in colour lightness of the optimal bread may also be explained by the higher amount of phenolic and flavonoids compounds of the AF ,41The increase in the nutritional value of bread may be due to the high content of amaranth flour fractions in these nutrients , and theThe results obtained were consistent with those reported by Sanz-Penella et al.  and MiraMiranda-Ramos et al.  replacedThe bread made from optimal wheat–amaranth composite flour was enhanced with essential and nonessential amino acids compared to wheat flour bread. Methionine, phenylalanine, and threonine were the essential amino acids found in higher amounts in the optimal bread for all amaranth fractions. The variation in the amino acids content can be explained due to the presence of the AA from the amaranth fractions  that areOne of the main challenges in wheat flour bread production replaced by non-gluten amaranth flour is the level of consumer acceptance. This is crucial considering that the appearance, aroma, taste, and texture of food play a key role in the acquisition process. Compared with the control sample, an improvement of the organoleptic characteristics of the optimal bread formulations, excepting optimal bread with AF small particle size, was found. The optimal bread with medium and large particle sizes presented similar or better sensorial characteristics than that of the control bread. The respondent’s acceptance can be explained by the soft crumbs due to the presence of natural emulsifiers in amaranth flours. Our results are in agreement with those found by other authors when they made bread from amaranth flour .p < 0.05. The protein content of bread was directly correlated with the viscosity modulus (r = 0.95) and inversely with the gumminess and chewiness of the bread (r = −0.97). Positive correlations were obtained between the carbohydrate content of bread and the gumminess (r = 0.99) and chewiness (r = 0.99), while these texture parameters are negatively associated with the viscosity modulus . Regarding bread firmness, an inverse correlation was found with the viscosity factor (r = −0.97), while bread elasticity was directly associated with the maximum gelatinization temperature (r = 0.98). Bread cohesiveness was negatively associated with bread crumb porosity (r = −0.99). The appearance of the bread was negatively correlated with the bread volume (r = −0.96), while the taste of the bread was positively correlated with the viscous modulus (r = 0.98). Some authors found a direct correlation between dough elasticity and bread firmness and chewiness [The use of principal component analysis (PCA) highlighted a plot of the features and type of the sample on the two principal components . The firhewiness ,55.The bread produced from optimal wheat–amaranth composite flour specific to each particle size presented an improvement in volume, porosity, and elasticity, while the crust and crumb lightness, firmness, resilience, and chewiness decreased in bread with small particle sizes compared to wheat-flour bread. From a nutritional point of view, bread made with amaranth flour at an established addition dose depending on particle size showed a significant increase in protein, lipid, and ash content, with higher content of potassium, calcium, magnesium, zinc, and iron compared to the wheat flour bread values, while the carbohydrate content and energy value decreased. The highest protein content was found in bread with medium and small particle sizes, an increase of 26.7%, and 15.8%, respectively, being observed. There was an increase in the mineral content by over 37% in the bread samples obtained with composite flour with medium and small particles, and an improvement in protein by more than 26% in the optimal bread with large particles compared to wheat flour bread. Phenylalanine, threonine, and valine were the essential amino acids, while aspartic and glutamic acids were the nonessential amino acids that were identified in higher amounts in the studied samples. An increase in the total amino acid content of 19.5% and 8.7% was observed in the optimal bread with small and medium amaranth flour, respectively. The results of the sensory analysis revealed an increase in the overall acceptability score, appearance, crumb structure, taste, and smell for the bread samples obtained from the optimal composite flours containing medium and large particles of amaranth flour. The bread made from composite flour with medium particles was very well accepted with a score higher than 8.5 for the general acceptability.As a result, it can be concluded that the optimal composite bread with AF medium particle size presents the highest nutritional, physical, and sensorial values, followed by the optimal composite bread made with AF small particle size.