HER-1 and HER-2 are tumor-associated antigens overexpressed in several epithelial tumors, and successfully targeted by therapeutic approaches against cancer. Vaccination with their recombinant extracellular domains has had encouraging results in the pre-clinical setting. As complex humoral responses targeting multiple epitopes within each antigen are the ultimate goal of such active immunotherapy strategies, molecular dissection of the mixture of antibody specificities is required. The current work exploits phage display of antigenic versions of HER-1 and HER-2 domains to accomplish domain-level epitope mapping. Recognition of domains I, III and IV of both antigens by antibodies of immunized mice was shown, indicating diverse responses covering a broad range of antigenic regions. The combination of phage display and site-directed mutagenesis allowed mutational screening of antigen surface, showing polyclonal antibodies’ recognition of mutated receptor escape variants known to arise in patients under the selective pressure of the anti-HER-1 antibody cetuximab. Phage-displayed HER domains have thus the potential to contribute to fine specificity characterization of humoral responses during future development of anti-cancer vaccines. These molecules have an extracellular domain (ECD) that interacts with ligands, a transmembrane domain, and a cytoplasmic domain involved in signal transduction1. The ECD is composed by four shorter domains with unique structural and functional features: domains I and III, with inherent ligand binding ability, domain II including a protruding arm for homo- or hetero-dimerization between HER molecules, and membrane-proximal domain IV2. Deregulation of the expression and function of HER family members is linked to cancer development, and these molecules are well established as tumor-associated clinically relevant targets3. HER-related malignancies comprise epithelial tumors of ovary, prostate, breast, head and neck, colon and lung10. Signaling cascades activated by HERs support proliferation, apoptosis evasion, angiogenesis, migration and metastasis of cancer cells11.The family of Human Epidermal growth factor Receptors (HERs) includes four members: EGF-R/HER-1/ErbB1, HER-2/ErbB2, HER-3/ErbB3 and HER-4/ErbB4, which are transmembrane proteins involved in proliferation, survival and differentiation of epithelial cells12. Powerful anti-tumor effects of these antibodies as ligand binding inhibitors and/or mediators of immune effector mechanisms have been shown. Experiments showing the advantages of combinations of anti-HER antibodies over the use of single antibodies indicated the importance of targeting more than one epitope17. Multi-specific polyclonal responses to vaccination of patients with whole HER ECDs would allow simultaneous targeting of a plethora of epitopes on the same antigen, thus producing natural antibody cocktails able to mediate different biological mechanisms, which should result in increased anti-tumor potency. HER-based vaccines are indeed being developed with promising results in animal models20. Optimized vaccination protocols could guarantee sustained levels of functional circulating antibodies, but could also be an attractive technical solution to circumvent treatment resistance, an already described limitation of mAb therapy. Tumors can evolve to the resistant state due to their intrinsic heterogeneity and the occurrence of in vivo selection of cells displaying loss-of-epitope mutated target variants that are no longer recognized by a given antibody24. The likelihood of tumor escape from diverse polyclonal responses should be diminished as compared to mono-specific mAb selective pressure.Monoclonal antibodies (mAbs) against HER family members such as cetuximab, nimotuzumab, panitumumab, trastuzumab and pertuzumab have achieved outstanding results in the clinical setting26 or libraries of phage-displayed random peptides27 that mimic antigen regions, have more chances of being useful to identify linear epitopes than conformational ones. Many relevant HER epitopes are conformation-sensitive and their formation is strongly dependent on the correct folding of large antigenic regions constrained by the presence of multiple disulfide bonds within each domain28. Recapitulation of isolated HER domains for epitope mapping has been achieved using yeast display, with the advantages associated to biosynthesis in an eukaryotic host29. Phage display, the oldest and most extended display platform30, has also been used for similar purposes. Phage display of large protein fragments, 100 amino acids (aa) long, has been proposed as an alternative to conventional peptide libraries in studies aimed at identifying conformational epitopes of HER-231. Antigenic HER-1 domain III has been successfully displayed in our laboratory and used to map fine specificity of mAbs28.Therefore, the evaluation of the outcomes of HER immunization procedures should not be limited to global antibody levels titration. Showing the diversity of polyclonal responses is mandatory, and the development of efficient methods to do that is a pre-requisite for development of such vaccines. Common epitope mapping techniques, based on short synthetic peptides reproducing the primary sequence of the antigenThe current work extended previous experiences to the display of antigenic versions of domains of both HER-1 and HER-2. These tools were useful to show the diversity of polyclonal antibody responses to HER-1 and HER-2 in a mice vaccination model. Domains I, III and IV of each antigen were recognized. The combination of phage display and site-directed mutagenesis allowed mutational screening of antigen surface, showing polyclonal antibodies’ recognition of mutated receptor escape variants known to arise in patients under the selective pressure of the anti-HER-1 antibody cetuximab. The phage display platform developed here for domain-level epitope mapping could aid to the characterization of humoral responses during future development of HER-based vaccines.c-myc tag fused to displayed proteins in our system, showed the presence of every individual HER-1/HER-2 domain on phage surface after rescue with M13KO7 helper phage  on the basis of 9E10 binding using a standard curve of phage displaying 2-Fc format) named D1, previously constructed at our laboratory through single-chain Fv library screening against HER-1 whole ECD, showed specific recognition of domain IV  was indeed observed for HER-1 domain III . The N-terminal regions of Cys-rich domains (II and IV) were appended to domains I and III respectively in our phage-displayed versions, due to their tight packaging with such domains in both native structures. The addition of the N-terminal region of HER-1 domain IV had been previously shown to improve domain III expression and folding39. There was minimal overlap between domains (only 14/13 aa were shared between phage-displayed domains III and IV of HER-1/HER-2) to allow segregating the humoral responses against each of them.The choice of a pVIII-based multivalent display system27 contrasts with our previous experience  and its rationale was aimed at facilitating detection of subdominant antibody specificities under-represented within the mixture through avidity effects.HER family antigens have complex tertiary structures stabilized by multiple disulfide bonds38. As ligand binding site involves both domain I and III40, antibodies against domain I can have similar functions. No ligands have been described yet for HER-2, which is in the extended conformation without the need for ligand binding2. The effects of antibodies to HER-2 domains I and III are thus more difficult to envision, although by virtue of their binding specificity they can theoretically affect this natural dimerization-prone conformation. HER recognition by antibodies, besides direct effects on ligand binding, can recruit immune effectors to attack tumor cells directly. Antibodies against the membrane-proximal domain IV of both antigens  can be particularly important for that effect, as it has been reported that membrane proximity of the target epitope can have a positive influence on antibody-dependent cell cytotoxicity41.Detection of reactivity against domains I, III and IV of every immunizing antigen has implications to define the landscape of potential biological effects of vaccination. HER-1 domain III is indeed the target of very effective therapeutic mAbs that work by blocking receptor-ligand interactions  and precluding the receptor to adopt the active dimerization-ready conformation42 which are much closer to a natural biosynthesis context of human antigens than phage production. The presence of disulfide bonds, although can clearly contribute to display difficulties as it has been reported for other proteins43, does not rule out every possibility of successful display. Domains IV, very similar to domains II in structure and cysteine-rich composition (eight disulfide bonds each), were displayed in antigenic form, as shown through strong recognition by both monoclonal and polyclonal antibodies. The chosen display system was designed to achieve a quick co-translational secretion  to the periplasmic compartment where disulfide bridge formation can readily occur44. It is possible that, besides proper disulfide bonds’ formation, structural stabilization by flanking domains is required for correct folding of domains II. In fact, fusion to domains I or III has been used in previous studies to achieve domain II expression45. Refining domain II display by testing alternative fusion protein designs, other expression conditions and the use of chaperones47 remains as a relevant goal, as dimerization arm , is located in this region. Recognition of HER-2 domain II is indeed the basis of therapeutic effect of pertuzumab. Detecting antibodies against domain II, if they exist, is thus necessary to complete the functional landscape of possible biological effects of vaccination. Additional experiments are thus required to distinguish between a technical display failure and the inability to elicit antibodies against domain II with the experimental immunization protocols we used, which is also a valid possibility.Failure of polyclonal antibodies to recognize domains II of any of the antigens can result from an aberrant protein folding of the displayed proteins. The designed domains II of HER-1 and HER-2 are rich in cysteines and require the formation of nine disulfide bridges each to stabilize the structure formed by loops and random coils. This can complicate their expression as recombinant proteins. Recombinant HER-2 domain II have had severe antigenicity problems even when produced in mammalian cell-based systems24. One of the potential advantages of vaccination responses targeting multiple epitopes is the ability to circumvent resistance against a single mono-specific agent. Recognition of a panel of phage-displayed HER-1 domain III variants having mutations in a cluster of residues located in the cetuximab binding interface22  provided experimental support to this idea.Even though the use of phage-displayed domains was aimed at dissecting antibody specificity at domain-level against whole domains rather than revealing fine details of individual epitopes, mutational screening of the displayed domains opens the possibility to answer relevant questions regarding mutated receptor variants. Growing body of evidence supports the notion of tumors evolving in vivo under the selective pressure of therapeutic antibodies targeting a given epitope. This kind of selection ends with treatment resistant tumors that sometimes lose expression of the target antigen, but can also display mutated antigen variants able to mediate the original biological functions without keeping the specific target epitope. This phenomenon has been well documented during the course of HER-1-directed therapiesThe usefulness of phage-displayed HER domains developed here was assessed with available mAbs and with polyclonal antibodies derived from a simple experimental immunization protocol with model antigens in Freund’s adjuvant. Once the antigenicity of several domains was shown, they are available as tools for the molecular dissection of humoral responses with optimized vaccination strategies, in both pre-clinical and clinical scenario.Soluble versions of recombinant HER-1 (aa 1–621) and HER-2 (aa 1–623) ECDs were provided by the group of Therapeutic Vaccines from the Center of Molecular Immunology (CIM), Cuba.Nimotuzumab  and itolizumab  were obtained from CIMAB S.A., Cuba.Trastuzumab (Herceptin), pertuzumab (Perjeta), cetuximab (Erbitux) and panitumumab (Vectibix) were also obtained from commercial sources .9E10 anti-c-myc tag monoclonal antibody was purchased from CIGB .D1 fusion protein comprising an anti-HER-1 domain IV scFv fused to a human Fc domain was produced at the Protein Engineering laboratory of the Center of Molecular Immunology, Cuba.Reactivity of monoclonal antibodies against HER-1/HER-2 ECDs was verified by indirect ELISA on antigen-coated microtitration plates.6 cells/animal) were inoculated in the peritoneal cavity five days later. After ten days, ascitic fluid was collected. Ascites was also obtained from five non-immunized animals with the same characteristics (control group). IgG antibodies from either immunized or non-immunized animals ascitic fluid were purified by Protein-A affinity chromatography.Two goups of five female SPF BALB/c Ann healthy mice each , provided by the National Center for Laboratory Animals Production , were immunized four times every two weeks with HER-1 ECD  or of HER-2 . Antigens were emulsified in Complete Freund’s adjuvant for the first dose and Incomplete Freund’s adjuvant (IFA) for the subsequent immunizations. Induction of specific antibodies was shown by ELISA on microtitration plates coated with the immunizing antigen. Mice were intraperitoneally primed with IFA two weeks after the last immunization, and mouse P3X63-Ag8.653  myeloma cells . This was considered the minimal suitable number of animals to characterize the antibody responses against the immunizing antigens, given the individual variability of immune responses. Mice were allocated to each group by simple randomization. There were five mice/cage. Before the experiments, mice were kept for seven days at CIM animal care unit (acclimatization period). The animals were maintained all the time at CIM animal care unit at 20–25 °C, 60 ± 5% relative humidity, with light/darkness cycles of 12 h. Food and water were administered ad libitum. At the end of the experiments, the animals were sacrificed.c-myc phagemid vector  were amplified by PCR and cloned into pc89-48. Sequence correctness was confirmed by DNA sequencing . Full DNA and protein sequences corresponding to each mutated genetic construct  are shown in supplemental information. Phage particles displaying mutated variants of the domains were rescued as described above.Mutated variants of genes coding for HER domains were generated by Kunkel mutagenesis using the above described genetic constructs as templates, and antisense mutagenic oligonucleotides c-myc tag  on 9E10-coated microtiter plates, using established procedures48. A standard curve of phages displaying c-myc alone was taken as reference, assuming the presence of 100 arbitrary display units/mL in the undiluted preparation. The relative display levels of HER domains in each preparation were calculated by interpolation from the standard curve.The amounts of phage-displayed domains were determined by ELISA, measuring recognition of Polyvinyl chloride microtiter plates were coated overnight at 4 °C with 10 μg/mL of monoclonal or polyclonal antibodies diluted in phosphate buffered saline (PBS). Plates were blocked 1 h at room temperature (RT) with 4% (w:v) skim milk in PBS (M-PBS). Purified phages displaying HER-1/HER-2 domains or its mutated variants (diluted in M-PBS) were added to the plates and incubated during 1 h at RT. Plates were washed with 0.1% Tween 20 (v:v) in PBS (PBS-T). An anti-M13 mAb conjugated to horseradish peroxidase , appropriately diluted in M-PBS, was added. Plates were incubated 1 h at RT and washed as described with PBS-T. Substrate solution  was added. The reaction was stopped 15 min later with sulfuric acid (2.5 mol/L). The absorbance at 490 mm was determined with a microplate reader. In order to normalize the amounts of different phage-displayed domains within each experiment, equivalent concentrations  were used. For some experiments, samples were normalized on the bases of the total amount of phage particles .To perform phage competition ELISA, 20 µg/mL of HER-1/HER-2 soluble ECD were mixed with the diluted phage samples before incubation on coated/blocked plates.Protocols involving living animals were approved by the Institutional Committee of Laboratory Animal Care and Use (CICUAL) at the Center of Molecular Immunology. Animal experiments were performed in accordance with the relevant national and international guidelines and regulations. These experiments are reported here according to ARRIVE guidelines.Supplementary Information.


Most existing methods for unsupervised domain adaptation (UDA) only involve two domains, i.e., source domain and the target domain. However, such trained adaptive models have poor performance when applied to a new domain without learning. Moreover, using UDA methods to adapt from the source domain to the new domains will lead to catastrophic forgetting of the previous target domain. To handle these issues, inspired by the ability to balance the maintenance of old knowledge and learning new knowledge of the human brain, in this article, we propose a new incremental learning framework for domain-incremental cases, which can harmonize the memorability and discriminability of the existing and the novel domains. By this means, the model can imitate the learning process of the human brain and, thus, improve its adaptability. To evaluate the effectiveness of the proposed methods, we conduct two groups of experiments, including virtual-to-real and diverse-weather cases. The experimental results demonstrate that our approach can avoid catastrophic forgetting, mitigate performance degradation in the previous domains, and improve the object detection accuracy of the novel target domain significantly. The safety of autonomous driving depends on the perceptual models of self-driving cars. With the detection results, the vehicles can plan a reasonable trajectory to avoid traffic accidents. In which, object detection is a fundamental and essential task for autonomous driving. It is similar to humans walking on the streets and crossing the roads. Each person has a learning mechanism to observe the positions of incoming cars and pedestrians. When building self-driving vehicles, to improve the robustness of the object detection algorithms, researchers used to train them with numerous labeled datasets containing as many situations as possible, which have achieved quite promising results. However, the relevant disadvantages are also prominent. For example, dataset annotation is expensive and requires a huge burden of work, i.e., the KITTI dataset includes 15,000 images containing over 80,000 objects. Moreover, it is well known that different datasets have different data distributions. For example, the data distribution of the Rainy-Cityscapes dataset is different from the typical Cityscapes dataset because of the raindrop imprint imposed. Therefore, in terms of the practical applications, Domain adaption becomes a promising research direction to overcome these problems by transferring knowledge from the unlabeled (the source domain) to the labeled (the target domain) data.The actual driving scenarios are complex and various. But the aforementioned existing domain-adaption methods can only adapt to two scenarios (one labeled and one unlabeled) and is difficult to cover all possible cases, which differs from the learning mechanisms of the human brain. The brains are capable to learn all cases progressively to deal with all kinds of emergency situations without forgetting previous cases. Moreover, the original purpose of building a self-driving vehicle is to endow a normal car with the intelligence of human brains. Thus, improving the generalization capability of domain adaption models is key to solving this issue. One possible way is to train a model for each target domain and select the most suitable one based on predefined rules. However, normally the size of the model parameters is huge and could cost much computation resources for the parameter storage, loading, and switching. Another way is to simply re-train the model by using another target domain, which may lead to a forgetting issue of the previously learned domains. To solve these problems, in this study, we propose that all target domains should be conducted sequentially instead of simultaneously so that they cannot be blended into a whole during the training process of the detectors.To this end, as shown in The highlight of the contributions: We discuss why general incremental learning cannot apply to multiple domain scenes. After analyzing the research gap in the existing methods, we propose a domain-incremental learning framework and design a domain tree to decide the domain adaptation order. In the framework, we introduce “Domain-Mix” and design patch-based adversarial learning to refine the quality of pseudo labels, thus enhancing the discriminability on two domains without extra computational cost. Experiments and comparison results demonstrate that our approach achieves the best performance in domain-incremental adaptive object detection problems. To the best of our knowledge, we are the first of reporting incremental adaptation results from a virtual domain to multiple actual domains.The remainder of the article is organized as follows. Section 2 briefly introduces some related studies. In Section 3, we address the problem formulation and single-step domain adaption method. In Section 4, we present the proposed domain-incremental adaptation algorithm. Experimental results are shown in Section 5. Section 6 concludes the article.via regularization , to acquire three-level features fl for pixel-level adaptation. Besides, we denote the two fully-connected layers after the ROI-Align module as F4 and the output of F4 as f4 for instance-level adaptation. Before adaptation, all of the fl pass Gradient Reversal Layers (GRLs) for extracting domain-invariant features by using adversarial training. Three pixel-level domain classifiers Cins with a fully-convolutional structure, are constructed to discriminate which domain each pixel (instance) of the features fl is from. The optimization objectives of four domain classifiers are to output corresponding domain maps, 0 for the source domain and 1 for the target domain.As shown in l-th layer features from source domain data where C and B as the abbreviation of all domain classifiers and remaining networks, respectively. Combining supervised loss Denote Because only the source images have ground-truth labels, the supervised loss is only performed on the source domain C and minimize that of other modules B. In this way, B aims to confuse the discrimination of C and generate domain-invariant features. Thus, we can obtain the adapted model M from the source domain Given the loss function shown in Equation 4, the optimization objective is to maximize the gradient from Based on the single-step domain adaption in Section 3, the framework of domain-incremental adaption can be further constructed in this section. To overcome the computation intensive and under-fitting problems, different from the traditional methods which either train a model for each pair of source-target domains or connect target domains in one, we propose a domain-incremental adaptation framework that adapts from a mixed source domain to the new target domain. We refer to the process of the human learning, dividing the whole incremental learning framework into two main parts: the “Recall” and “Adapt” stages. The former is akin to reviewing the previously learned knowledge (previous domains). The latter is similar to studying new knowledge (the novel target domain), which is performed by finding the common point between the old knowledge and the new knowledge. After training through the proposed framework, the final model can maintain a balance between the memorability of previous domains and the discriminability of the new domains. Details will be given in this section.m≠n.First, we provide some preliminaries. Compared with single-step domain adaptation, domain-incremental adaptation contains multiple unlabeled target domains instead of one, i.e., In single-step domain adaptation, which only adapts from one domain Here, we provide a simple example for illustration purposes. Assume that Cityscapes and Foggy-Cityscapes are the source domain M1 completes the first domain adaptation task from M1 can be continually adapted from Suppose that the model However, the above approach is susceptible to the inaccurate labels in i et al.  which feThe disadvantage of simply combining two datasets (domains) into one is obvious. On the one hand, if one domain has ground-truth labels while the other only has unreliable pseudo labels, training the model with these inaccurate labels will decrease the performance of the latest target domain. On the other hand, sampling images randomly from a hybrid dataset can cause inconsistent data distribution of two consecutive inputs. This case will reduce the model generalization capabilities and increase the difficulties in model fitting, thus obtaining sub-optimal results.To solve these problems, inspired by Ramamonjison et al. , we propMk. Coarse pseudo labels of Mk, coming into p represents a target domain with pseudo labels. In the first stage named “Recall,” we group Mk with the above “Self-Training” strategy. Stage “Recall” aims to raise the quality of pseudo labels and reduce the degree of knowledge forgetting, which will be further demonstrated in the later ablation study section. In the second stage “Adapt,” we update the labels of via the refined pseudo labels of On the basis of the aforesaid “Self-Training” strategy, we propose a two-stage domain-incremental learning framework. Suppose a model trained from the source domain Sim10K  is observed and the total precision is calculated. (ii) Virtual-to-Real. SIM10K → Cityscapes → Foggy-Cityscapes → Rainy-Cityscapes. Due to the category space limitation of the Sim10K, this series of experiments only validate the “Car” models.Given these datasets, we design two series of experiments to evaluate our model in domain-incremental adaptation: (i) via random flipping. During each iteration of training, one source-domain image and one target-domain image are input successively. For the primary domain adaptation task, from the source domain to the first target domain, we follow a regular training strategy with a learning rate of 0.001 for 50k iterations and then decrease by a factor of 10 for the last 20k iterations. For the other adaptation tasks, e.g., from the first target domain to another new target domain, for instance, we maintain a lower learning rate of 0.0001 and report the model which performs best on the Rainy-Cityscapes dataset. When generating pseudo labels, we use a confidence threshold of 0.5 and 0.7 for Experiments Group I and II, respectively. At the validation phase, we demonstrate the performance of our model by the mean average precision (mAP) of all categories with a widely-used intersection over union (IoU) threshold of 0.5. Supposed that APi is the precision of category i, the mAP is calculated by the means of all APi. Finally, we use the PyTorch framework to implement our domain-incremental learning framework.We build our detector with ResNet-101 .via trained models of the last adaptation task and then utilizes them to perform the next adaptation period. Besides, we also specify the lower-bound and upper-bound bounds. “Source only” trains a detector only with the source domains and evaluates it on target domains without adaptation to new target domains. Other than “PFT,” “SFT” fine-tune the last trained model from the old target domain with ground-truth labels to new domains, while “SMFT” has access to the ground-truth source and the last target domain. Moreover, “SSFT” also adopts a two-stage training strategy but utilizes ground-truth labels of the last target domain in the second stage. The degree of forgetting during the incremental learning process is marked via a number with the symbol “↓.” It is calculated by subtracting MAPpre from MAPaft where MAPpre is the accuracy before adapting to the new target domain while MAPaft means the precision after adapting. We choose “ILB”  are shown as “Base-line” in To the best of our knowledge, we are the first to survey incremental adaptation results from virtual datasets to real-world ones. In this section, the experiment includes three tasks: a) an initial task: adaptation from the source domain (Sim10K) to the first target domain (Cityscapes); b) the first incremental task: adaptation to the second target domain (Foggy-Cityscapes); c) the second incremental task: adaptation to the third target domain (Rainy-Cityscapes). On account of the increasing number of datasets, we display experimental results in the form of histograms rather than tables. Moreover, due to the availability of only one class in the source domain (Sim10K dataset), we only evaluate the performance of “Car.” The related experimental results can be found in Mk−1 adapted from the source domain DS to the last target domain DS as a new source domain and adapt toward To verify the impact of the “Recall” stage, when we obtain model We consider “HTCN” .The authors declare 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 discovery of coding variants in genes that confer risk of intellectual disability (ID) is an important step toward understanding the pathophysiology of this common developmental disability.Drosophila melanogaster and mice with targeted interference of the orthologous gene were used. Behavioral, electrophysiological, and structural magnetic resonance imaging analyses were conducted for phenotypic testing.Homozygosity mapping, whole-exome sequencing, and cosegregation analyses were used to identify gene variants responsible for syndromic ID with autistic features in two independent consanguineous families from the Arabian Peninsula. For in vivo functional studies of the implicated gene’s function in cognition, PDZD8, encoding an endoplasmic reticulum–anchored lipid transfer protein, showed cosegregation with syndromic ID in both families. Drosophila melanogaster with knockdown of the PDZD8 ortholog exhibited impaired long-term courtship-based memory. Mice homozygous for a premature termination codon in Pdzd8 exhibited brain structural, hippocampal spatial memory, and synaptic plasticity deficits.Homozygous premature termination codons in PDZD8 in a neurodevelopmental cognitive disorder. Model organisms with manipulation of the orthologous gene replicate aspects of the human phenotype and suggest plausible pathophysiological mechanisms centered on disrupted brain development and synaptic function. These findings are thus consistent with accruing evidence that synaptic defects are a common denominator of ID and other neurodevelopmental conditions.These data demonstrate the involvement of homozygous loss-of-function mutations in  SEE VIDEO CONTENT ONLINEIntellectual disability (ID) refers to a heterogeneous group of neurodevelopmental disorders affecting 2% to 3% of the general population, characterized by significant impairment in cognitive ability and adaptive behaviors. It is usually subdivided into nonsyndromic and syndromic forms, depending on the manifestation of additional physical, neurologic, and/or metabolic abnormalities. Typically identified in childhood because of delayed developmental milestones, affected individuals struggle with memory, problem solving, language, and visual comprehension, reflected by an IQ score of <70 .ID has high phenotypic variability and etiologic diversity. Based on the IQ score, ID can be classified as mild (50–69), moderate 35–49), severe 20–34), or profound (under 20) , a gene of five exons located at 10q25.3-q26.11, encoding a 1154 aa endoplasmic reticulum (ER) transmembrane (TM) protein.Herein, we report the clinical features and molecular diagnosis of two independent consanguineous families affected by syndromic ID from the Arabian Peninsula. Through the application of homozygosity mapping and whole-exome sequencing (WES), we report that all affected individuals are homozygous for premature termination codons (PTCs) in 2+) uptake by mitochondria following synaptic transmission–induced Ca2+ release from the ER  Act 1986 under UK Home Office licenses and approved by institutional Animal Welfare and Ethical Review Bodies.Homozygosity mapping and WES were conducted as described previously . SegregaCG10362-RNAi  line  that inhibits expression of the target CG10362 gene via RNA interference. Behavioral testing was performed as described previously Wtsi mice were generated by replacing an 835-bp sequence including exon 3 with a lacZ expression cassette, which created a frameshift that changed the phenylalanine (F) and isoleucine (I) at positions 333 and 334 to an asparagine (N) and a termination codon (∗) (p.F333Nfs1∗)  and heterozygous and wild-type (WT) littermates for phenotypic testing.C57BL/6NTac-33Nfs1∗) , C 21)..Pdzd8tm1Behavioral testing of early adults over 8 weeks of age was performed as described previously , 24, 25.Extracellular field recordings using transverse hippocampal slices prepared from 4- to 6-week-old mice were performed as described previously . Three d3) and relative volume of every region independently and to every voxel independently in the brains of Pdzd8tm1b and WT mice, with a false discovery rate threshold of 1%.For high-resolution structural magnetic resonance imaging, 16-week-old mice were terminally anesthetized and intracardially perfused. Samples were processed, imaged, and analyzed as described previously . A lineaFamily A consists of 3 affected  and 2 unaffected (A.IV.3 and A.IV.4) siblings born to consanguineous parents (first cousins) (A.III.1 and A.III.2) within an extended Omani pedigree . ClinicaFamily B consists of 1 affected (B.IV.2) and 3 unaffected  siblings born to first cousin parents (B.III.1 and B.III.2) within an Emirati pedigree . On clinThe pedigree structures of families A and B suggested autosomal recessive transmission of a homozygous mutant allele from a common ancestor as the most likely explanation for syndromic ID in each family. In family A, homozygosity mapping using single nucleotide polymorphism array data in affected males A.IV.1 and A.IV.2 and variants extracted from WES in affected female A.IV.5 identified homozygous regions on chromosomes 6 (2.57 Mb), 10 (28.28 Mb), 13 (5.20 Mb), and 17 (8.38 Mb) shared by all three .ANKRD2  and PDZD8  remained, both within the 28.28-Mb region on chromosome 10  (The  and PTC  absent fubjects) , and cauubjects) , predictubjects) ,30 (Tablubjects) . In a stubjects) . Both vaANKRD2 is upregulated in congenital myopathies (Because very little ANKRD2 (UniProtKB: Q9GZV1) is detected in the human brain  and primopathies , p.] , with filtering for predicted pathogenic variants and segregation analysis, revealed a homozygous nonsense variant in (Y298∗)]  as the mariation .PDZD8 encodes an integral ER protein (UniProtKB: Q8NEN9) anchored to the membrane by an N-terminal TM helical domain (2–24 aa), which is followed by a synaptotagmin-like mitochondrial lipid-binding domain (91–294 aa), a PDZ (PSD-95/DlgA/ZO-1-like) domain (366–449 aa), a proline-rich region (551–626 aa), a C1 (phorbol-ester/diacylglycerol binding) domain (840–891 aa), and a coiled-coil domain (1028–1063 aa) (PDZD8 is highly expressed throughout the human brain (PDZD8 expression is relatively stable from 8 weeks after conception to early adulthood (23 years) across regions of the developing human brain (PDZD8 a strong candidate for involvement in ID.1063 aa) ,34,35. I) domain 6–449 aa,1063 aa) . PDZD8 ian brain , includian brain  , encodes a 1037 aa protein  that has a similar domain structure but relatively low amino acid conservation compared with human PDZD8 (24% identity). Drosophila PDZD8 has an N-terminal TM domain followed by a synaptotagmin-like mitochondrial lipid-binding domain, a PDZ domain, and a C1 domain but, unlike mammalian PDZD8, lacks a C-terminal coiled-coil domain  tissue  males upon comparison with UAS-RNAi and Act5C-Gal4 control animals . In. InD. me) tissue . RNA int animals .Figure 2CG10362-KD flies to memorize a novel association between an odor and mechanical shock for 30 seconds or 30 minutes was unaltered . These results suggest that KD of CG10362 impairs long-term courtship-based memory but does not affect learning, short-term memory, or basal sexual behavior.In the aversive olfactory conditioning assay, the ability of naltered . In the d female . UncondiPdzd8tm1b mouse line, generated in the EUCOMM  program  that has 87% aa conservation with, and a similar domain structure to, human PDZD8. Real-time quantitative reverse transcription polymerase chain reaction confirmed the absence of Pdzd8 mRNA including exon 3 in Pdzd8tm1b mouse brain  and p.(S733∗) in d by NMD . Becaused by NMD , it is pThe lack of PDZD8 protein in human blood limits our ability to detect truncated PDZD8 proteins . If prodPDZD8. Although PDZD8 is one of multiple genes occasionally hemizygously lost in distal 10q deletion syndrome, in which ID and dysmorphic features are common (ZFYVE27) has previously been identified in spastic paraplegia (RAB7A) have been identified in Charcot-Marie-Tooth type 2B neuropathy  exhibited decreased preweaning viability. Surviving Pdzd8tm1b mice showed spontaneous repetitive hindlimb jumping, a stereotyped motor behavior with potential relevance to lower-order human motor stereotypies, such as hand flapping, common in ASD  receptors enriched in hippocampal CA1 pyramidal cells  in family A. The relative expansion of the hippocampus is comparable to that observed in other mouse models of neurodevelopmental disorders exhibiting cognitive deficits. The heterozygous Arid1b knockout mouse model of Coffin-Siris syndrome, a syndromic ID, exhibits body weight and growth rate deficits, reduced total brain volume but hippocampal enlargement, and deficits in novel object recognition (Chd8 mutant mouse model of ASD exhibits an increase in hippocampal volume that is correlated with deficits in contextual fear conditioning (Adult ognition . The hetitioning . Deciphe2+ uptake following synaptic transmission–induced Ca2+ release from ER stores (PDZD8 in syndromic ID. This knowledge will benefit affected families through genetic counseling and carrier screening and facilitate the genetic diagnosis of other patients. Disrupting the orthologous gene resulted in long-term memory deficits in fruit flies and brain structural alterations, long-term memory deficits, and impaired hippocampal neurophysiology in mice, replicating aspects of the human phenotype and demonstrating a critical role for PDZD8 in brain development and synaptic plasticity. We can use these models to decipher precisely how PDZD8 disruption affects neurodevelopment and synapse function, thus providing insight to the pathophysiological mechanism and potential treatment of this lifelong disability.To summarize, PDZD8 is an ER TM protein required for the extraction of lipids from the ER to late endosomes and lysosomes ,49 and mR stores . Our dat


The microbial loads seemed somewhat acceptable as overall acceptability favoured sample WDFb . The acceptability of cookies baked at 160 °C over those baked at 180 °C suggests the need for further studies to determine the energy requirements, and long-term environmental implications such (baking) temperatures would pose.Studying the effects of temperature on cookies are necessary especially if the impact on quality attributes are to be deduced. More so, blending wheat flour, date flour, and pineapple juice could improve the nutrient quality required in modern-day cookies. This current study investigated the quality attributes of date and wheat flour pineapple juice blended cookies as affected by different baking temperatures. With pineapple juice serving as water substitute, the formulated date, and wheat flour blends adhered to the following ratios: 100:0, 90:10, 80:20, and 70:30. Baking cookies involved two different temperatures (160 °C and 180 °C) with constant time (30 min). Quality attributes determined proximate composition, micronutrients, physical and functional properties, and microbial and sensory qualities. Cookies proximate results showed moisture (6.89–7.40%), protein (8.73–10.22%), fat (14.37–15.99%), fiber (1.02–1.11%), ash (0.77–1.20%) and carbohydrate (64.85–67.71%). Various ranges appeared, from energy values , minerals , vitamins , physical (weight = 7.4–7.75 g; diameter = 3.50–4.01 cm; thickness = 0.99–1.20 cm; volume = 3.11–3.77 cm The pulp with pericarp was then oven dried at 75 °C for 6–8 h and subsequently milled using a hand milling machine and sieved through a 0.35 mm mesh sieve to obtain fine homogenized particles . The pinA two-component augmented mixture design was used to assemble the blends of wheat and date flours by sample ratio, which were as follows: WFa (100:0) representing 100% wheat flour, 0% date flour; WDFb (90:10) representing 90% wheat flour, 10% date flour; WDFcm (80:20) representing 80% wheat flour, 20% date flour; and WDFd (70:30) representing 70% wheat flour, 30% date flour. This enabled the study design to produce four mixtures, with proportions for each ingredient applicable to each treatment combination. Additionally, the recipe for cookies production involved the following ingredients with their respective quantities: flour = 500 g, sugar = 100 g, baking fat = 180 g, salt = 2 g, baking powder = 2.5 g, baking soda = 2 g, water, = 80 mL and pineapple juice = 80 mL. The ingredients were weighed and mixed thoroughly into a dough. The dough was rolled on a board (with flour spread on it to avoid sticking) using a rolling pin. The rolled batter was cut into shapes and arranged on a greased baking tray and were placed in an electric oven facility . According to Proximate compositions, which included moisture content, crude fibre, crude fat, crude ash, crude protein, and carbohydrate, together with energy values of samples were determined using the official methods of analysis .Functional properties involved bulk density, and water and oil absorption capacities. The bulk density of the flour blends was determined according to the method of The bulk density (BD) was calculated as:3); W1 = Weight of empty cylinder (g); W2 = Volume of cylinder + sample (g); V = Volume of cylinder occupied by the sample (cm3).g for 30 min. The volume of free water or oil (supernatant) was read directly from the graduated centrifuge tube. The absorption capacity was expressed as a gram of oil or water absorbed (or retained) per gram of sample.The water and oil absorption capacities of the formulated samples were determined using the method described by 3), and density (g/cm3) values according to the Approved Methods of the American Association of Cereal Chemists , thickness (cm), and average weight (g) of the cookies, which helped to calculate the speed ratio (SR), volume . The averages of the colonies from duplicates were determined.Microbial load determinations involved total viable count (TVC) and mould count (MC). With respect to TVC, the method of −1) was pipetted into duplicate Petri dishes. Also, 15 mL of SDA (Sabaroud and Dextrose Agar) was pipetted aseptically into the Petri dishes. These Petri dishes were swirled in order to mix the content thoroughly, allowed to solidify and turned upside down thereafter. Petri dishes were incubated for 48 h at room temperature. The mould counts were expressed as cfu/g (colony forming unit per gram) of the samples.With respect to mould count, method described by The cookies samples were evaluated for color, flavour, taste after taste, texture, mouthfeel, crispness and overall acceptability on a nine-point Hedonic scale where nine (like extremely) was the highest and one (dislike extremely) was the lowest score. The evaluation was done by 20 semi-trained panelists selected from the students of the Department of Food Science and Technology, University of Nigeria, Nsukka. The panelists during their studies had undertaken sensory training, and therefore were familiar regards various evaluation criteria specific to the cookies. Participation was voluntary, verbal consent was taken, and no information regards the panelists were taken. The samples were coded and presented without further treatment in coded plastic plates to the panelists randomly during the day. The order of presentation of the samples was randomized. Clean water was presented to the panelist to rinse their mouth in between samples, to ensure the previous sample tested did not influence the new, adhering to report of p < 0.05. Statistical Product for Service Solution (SPSS) version 25.0 was used to run the data.Results from two determinations independently measured from different samples were submitted to analysis of variance (ANOVA). Duncan’s new multiple range tests (DNMRT) were used to compare the treatment means. Statistical significance was accepted at d (6.89%) and WDFc (7.40%) having the least and highest moisture content, respectively. Significant (p < 0.05) differences were observed between all the samples. Increasing the baking temperature from 160–180 °C steadily decrease in moisture content, like samples WDFb (7.26–6.61%), WDFc (7.58–7.23%) and others. Besides samples with the same formulation baked at two temperatures produced different moisture content effects, there was significant interaction (p < 0.05) between the formulation and baking temperature, seemingly to agree with data of In a (10.22%) with 100% wheat flour as peak, and sample WDFd (8.73%) with 70% wheat and 30% date flour as least protein content. An increased substitution of wheat with date flour in the formulation led decreased protein content in the cookies. According to b (9.57%), WDFc (9.42%) and others. This steady increase could be that at high temperatures, proteins are denatured and anti-nutrients would be bound to proteins being liberated from the sample leading to available proteins at high temperatures (In eratures .p < 0.05) in protein occurred across all the samples, like with sample WFa with 100% wheat flour. Also, a significant (p < 0.05) interaction term between the formulation and the baking temperature seemed apparent. More so, the quality and nutritional content of food are a simultaneous function of both formulation and processing methods (a (15.99%) with 100% wheat flour having the highest and sample WDFd (14.37%) with 70% wheat and 30% date flour having the lowest fat content. Probably, fat content reduction across the formulations occurred because of date flour. Baking temperatures increased with fat content for samples with resembling formulation baked at different temperatures as seen in samples WDFb (15.34– 15.45%), WDFc (14.43–14.55%) and in other samples. Moreover, the lowering of moisture resulted to fat concentration in the formulated cookies. Notably, fats can withstand high-temperature processing with little changes (p < 0.05) cross-interaction seemed apparent between the formulation and baking temperature.Significant differences ( methods . Table 1 changes . Additioa (1.02%) as a minimum, and WDFd (1.11%) as the maximum. The inclusion of date flour during formulation steadily increased with fibre content, similarly reported by p > 0.05) difference between sample WDFb (1.06%) and WDFc (1.07%). Besides, the ash content ranged from 0.77–1.20% with sample WFa (0.77%) and WDFd (1.20%) having the lowest and highest, respectively (b (0.60–1.22%), WDFc (0.52–1.31%) and in other samples. Date palm fruits according to p < 0.05) interaction was also observed, although there was no significant (p > 0.05) difference between sample WDFb (0.91%) and WDFc (0.92%).In ectively . The adda (64.85%) as minimum and WDFd (67.71%) as maximum. Significant differences (p < 0.05) were observed between all the samples. The substitution of date flour in the flour blends increased with the carbohydrate content of the cookies. The results obtained in carbohydrate content were similar to the results of a  as the maximum, and sample WDFc  as a minimum. Between the formulation and temperature, there was no significant (p > 0.05) difference between sample WFa  and WDFb. . The energy value reduction could be due to higher protein and fat contents in wheat flour (control) above those used in formulations supplemented with date flour. Also, the concentration of fats, carbohydrates, and proteins could be a result of moisture loss during formulation at high temperatures (160–180 °C) with an increase in energy content.In a (33.18 mg/100 g) with 100% wheat flour and sample WDFd (62.45 mg/100 g) with 30% substitution of date flour respectively. An increased inclusion of date flour in the formulation resulted in an increase in the Ca content of the cookies with significant (p < 0.05) differences observed between all the samples as seen in samples WDFb (53.32 mg/100 g), WDFc (58.62 mg/100 g) and in other samples. Compared to wheat, the Ca content increased with inclusion of date fruit flour. This happened probably due to high amount of calcium present in date fruit  and sample WDFd (5.75 mg/100 g), respectively. The iron content increased in the formulation with the increased inclusion of date flour as seen in samples WDFb (2.26 mg/100 g), WDFc (5.38 mg/100 g) and in other samples. This could be due to the high iron content of date flour compared to wheat flour. Significant (p < 0.05) differences were observed in the iron content with sample WDFd (5.75 mg/100 g) with 30% substitution significantly higher than the other samples. The two baking temperatures employed in the production did not significantly affect (p > 0.05) the iron content of the cookies. The results obtained for the iron content were higher than the values (1.40–2.76 mg/100 g) reported by In a (100.07 mg/100 g) and sample WDFd (358.63 mg/100 g), respectively. An increased inclusion of date flour in the formulation resulted in an increase in the potassium content of the cookies with significant (p < 0.05) differences observed between all the samples as seen in samples WDFb (312.34 mg/100 g), WDFc (321.74 mg/100 g) and in other samples. An increase in the potassium content with increased inclusion of date flour could be due to the high amount of potassium reported to be present in date flour compared to wheat. A change in baking temperature did not significantly affect the potassium content of the cookies.In d  with 30% substitution of date flour was higher than the other samples. This could be due to the high amount of vitamins A and C concentrated in date flour. The vitamins A and C contents were higher for samples with the same formulation and baked at 160 °C compared to samples baked at 180 °C. Vitamins A and C are sensitive to temperature changes and degrade at high temperatures. This could explain the reason why an increase in baking temperature reduced the vitamin A and C contents of the cookies as seen in samples WDFb (4.07–4.06 mg/100 g), WDFc (4.47–4.35 mg/100 g) and in other samples. Also, a significant interaction between the formulation and baking temperature suggests these vitamins in cookies simultaneously function with the formulation/treatment given to the sample  and WFa (7.75 g), respectively (p > 0.05) differences were observed between samples WDFc (7.49 g) and WDFd (7.49 g) with 20% and 30% substitution of wheat flour with date flour. Sample WFa (7.75 g) with 100% wheat flour was significantly (p < 0.05) higher than all the samples in terms of average mass. The high average mass could be due to the fact that nutrients and matter are more concentrated in wheat flour compared to date flour. Results for average mass were lower than the values of The average mass of the cookies ranged from 7.49–7.75 g for samples WDFectively . No signa (3.50 cm) with 100% wheat flour and sample WDFd (4.01 cm) substituted with 30% date flour, respectively (p < 0.05) differences as seen in samples WDFb (3.80 cm), WDFc (3.98 cm) and in other samples. The diameter increased with inclusion of date flour, probably because of reduced protein gluten. Moreover, substituting wheat decreased dough viscosity and resulted in a high flow rate leading to increased diameter of the cookies  and WFa (1.20 cm), respectively (b (1.11 cm), WDFc (0.99 cm) and in other samples. For samples with the same formulation and baked at different temperatures, there was no significant (p > 0.05) difference in thickness. Such a decrease in the thickness could be due to the dilution of wheat gluten by date fruit (The thickness of the cookies ranged from 0.99–1.20 cm for samples WDFectively . Increaste fruit . The higte fruit . Resultste fruit .3 for sample WDFd (3.11 cm3) with 30% date flour and sample WFa (3.77 cm3) with 100% wheat flour, respectively (p < 0.05) differences observed among the samples except for samples WDFc (3.11 cm3) and WDFd (3.11 cm3). Samples with the same formulation despite being baked at different temperatures show no significant (p > 0.05) difference by volume. Reduction in volume with increased substitution of wheat flour with date flour could associate with the high fibre content of date fruit flour. Fiber can help reduce dough spread during baking, which strengthens its resistance to deformation (The volume of the cookies ranged from 3.11–3.77 cmectively . An incrormation . However3 for sample WFa (2.06 g/cm3) with 100% wheat flour and sample WDFd (2.41 g/cm3) with 30% date flour, respectively (b (2.14–2.15 g/m3), WDFc (2.40–2.41 g/cm3) and in other samples. Sample WDFd (2.41 g/cm3) with 30% substitution was significantly (p < 0.05) higher than the other samples. No significant (p > 0.05) difference happened between samples with resembling formulation and baked at the two baking temperatures (160 and 180 °C). Probably, the increase in density of the cookies could arise from the addition of fibrous matter coming from date fruit flour.The density of the cookies ranged from 2.06–2.41 g/cmectively . Resultsa (2.92) with 100% wheat flour and sample WDFd (4.05) with 30% date flour, respectively (d (4.05) significantly (p < 0.05) higher than the other samples. The increase in spread ratio with the increased addition of date flour could be due to the restriction of dough viscosity. Cookie spread represents the ratio of diameter to thickness. Cookies’ spread ratio appears to be controlled by dough viscosity; dough with low viscosity causes cookies to spread more rapidly compared those with high viscosity (The spread ratio of the cookies ranged from 2.92–4.05 for sample WFectively . The resiscosity . Cookiesiscosity . Cookiesa (1.18 g/g) with 100% wheat flour and sample WDFd (1.14 g/g) with 30% date fruit flour substitution, respectively. Increased addition of date flour in the formulation reduced the WAC with sample WFa significantly (p < 0.05) higher than the other samples. High WAC in sample WFa (1.18 g/g) could be due to protein and starch molecules in wheat flour creating hydrogen bonds and hydrophilic interactions with the added water molecules in the flour. Water molecules hydrate the gluten-forming proteins gliadin and glutenin, as well as damaged starch and other ingredients (b (1.15 g/g), WDFc (1.14 g/g), and WDFd (1.14 g/g) could therefore be due to a dilution effect of wheat starch and protein by date flour. Values obtained for water absorption capacity were in the range of values (1.00 to 1.60) reported by In redients . Lower va (1.31 g/g) with 100% wheat flour and sample WDFd (1.33 g/g) with 30% date fruit flour substitution, respectively. The oil absorption capacity as observed increased with an increased substitution of wheat flour with date flour. Fat content of the samples could also be attributed to the margarine added. Sample WFa (1.31 g/g) was significantly (p < 0.05) lower than the other samples, with no significant (p > 0.05) difference seen between sample WDFc (1.33 g/g) and WDFd (1.33 g/g). Oil absorption in starch relies predominantly on the physical entrapment of oil within the starch structures. This is because starch does not have nonpolar sites, compared to those found in proteins (In proteins . The incd (0.74 g/mL) with 30% date fruit flour substitution and sample WFa (0.76 g/mL) with 100% wheat flour and respectively (a (0.76 g/mL) was significantly (p < 0.05) higher than all the other samples but no significant (p > 0.05) difference was observed for the different substitution levels. Bulk density is the mass per unit volume of food. It is also indicative of how porous the food product is, and how it can be packed together. Values obtained for bulk density were within the range of values earlier reported by The bulk density of the flour samples ranged from 0.74–0.76 g/mL for sample WDFectively . The res4 to 7.7 × 104 CFU/g) reported by etc.In b (6.23) and WDFd (7.45), respectively. No significant (p > 0.05) difference was observed across samples WDFd (7.45), WDFc (7.10), and WDFa (6.95), despite the highly rated colour for WDFd, probably due to the desirable brown date flour colour in the formulation. Generally, samples baked at 160 °C appeared more appealing compared to samples baked at 180 °C for the sample formulation. Baking temperatures as well as formulation significantly (p < 0.05) affected the colour of the blend samples. Besides Lagenaria siceraria) seed cake fortified biscuit occurred with increased baking temperature(s). Lower values could be due to the intense brown colour of the cookies that resulted from a caramelization reaction. The color results of cookies herein seems to agree data reported by In b (6.83) and WDFd (7.08), respectively. There were no significant (p > 0.05) differences in flavour between samples baked at 160 °C and samples baked at 180 °C, although samples baked at 180 °C were having lower values. This could be attributed to the fact that at higher baking temperatures, the flavouring volatiles in the samples was more prone to lose. Also, the interaction between the two treatments (formulation and temperature) did not significantly (p > 0.05) affect the flavour as perceived by the panelist. Results obtained in flavour were higher than the values reported by b (6.85) and WDFc (7.30), respectively. Significant (p > 0.05) differences were not observed in terms of taste for samples produced with different formulations, although an increase in baking temperature reduced scores for the taste. This agreed with the results reported by p > 0.05) interaction effect was observed. Further, the taste results of the cookies seem to agree with the data reported by In b (6.63) and WDFd (6.85), respectively. There were resembling (p > 0.05) among the samples. Temperature variation did not significantly affect the aftertaste despite the lower values at samples baked at 180 °C. High ratings for aftertaste may associate with processing temperatures unable to burn the samples. Moreover, the interaction between the processing temperature and samples obtained a significant effect (p < 0.05) specific to the aftertaste, which tends to reflect the opinion of a (6.95) and WDFc (7.33), respectively. Thus, the increased substitution of wheat flour with date fruit flour occurred with texture, even though the mean scores across the samples resembled (p > 0.05). More so, the interaction between the processing temperature and the formulation also resembled (p > 0.05) by texture as rated by the panelists.The sensory scores in the aftertaste of the cookies ranged from 6.63–6.85 for sample WDFa (6.93) and WDFb (7.35), respectively. Significant (p > 0.05) differences were not observed between samples. The cookies baked at 160 °C were generally preferred by the panelist compared to the cookies baked at 180 °C. The mouthfeel is a sensation acquired when food is placed in the mouth and it is very important in sensory ratings for overall acceptability. High ratings by the panelist could be indicating a pleasant sensation of the cookies in the mouth. The interaction between the mixture formulation and the baking temperature was insignificant (p > 0.05). p < 0.05) between samples WFa (6.85) and WDFb (7.63). Both crispness/crunchiness are textural attributes often associated with freshness/firmness of natural manufactured foods (In ed foods . The crued foods . An incla (7.00) as a minimum, and WDFb (7.30) as a maximum. There was no significant (p > 0.05) difference between the treatment temperatures on the overall acceptability even though panelists gave higher values on cookies baked at 160 °C compared to samples baked at 180 °C. An increase in the baking temperature led to a decrease in the overall acceptability of the samples, which seemed to agree with the results observed by Lagenaria siceraria) seed cake fortified biscuit. Formulation and processing temperature appeared not to affect overall acceptability (p > 0.05). Values for the overall acceptability were higher than the values reported by b (7.30) was more preferred followed by sample WDFc (7.28), then sample WDFd (7.15), and WDFa (7.00), respectively.In Promising quality cookies can be produced from blends of wheat and date flour with pineapple juice. Date flour probably accounted for increased the ash, fibre, mineral, and carbohydrate content of the cookies. Cookies baked with 70% wheat and 30% date flour with pineapple juice had the highest nutrient retention. Cookies having a higher spread ratio could be more desirable, especially those baked with 90% wheat, and 10% date flour with pineapple juice. Also, cookies baked at 160 °C appeared more acceptable compared to the cookies baked at 180 °C. Studying the influence of different baking temperatures has a direct link with moisture/water, and therefore energy. Considering that the food, water, and energy nexus demands a broad scope, which should not be seen only to directly relate to climatic context, but should also be seen to indirectly relate to climatic context, the acceptability of cookies baked at 160 over those baked at 180 °C points to why the results of current work serve as a foundation further studies to specifically determine the energy requirements, and long-term environmental implications that this baking (temperature) position would pose.10.7717/peerj.14876/supp-1Supplemental Information 1Click here for additional data file.


We have tested simultaneous tracking of extracellular superoxide by 15N-CAT1H and intramitochondrial superoxide by conventional 14N-containing spin probe mitoTEMPO-H in immune cells isolated from spleen, splenocytes, under basal conditions or stimulated with inflammatory cytokines IL-17A and TNFα, NADPH oxidase activator PMA, or treated with inhibitors of mitochondrial complex I rotenone or complex III antimycin A. 15N-CAT1H provides two-fold increase in sensitivity and improves detection since EPR spectrum of 15N-CAT1 nitroxide does not overlap with biological radicals. Furthermore, concurrent use of cell impermeable 15N-CAT1H and mitochondria-targeted 14N-mitoTEMPO-H allows simultaneous detection of extracellular and mitochondrial superoxide. Analysis of IL-17A- and TNFα-induced superoxide showed parallel increase in 15N-CAT1 and 14N-mitoTEMPO signals suggesting coupling between phagocytic NADPH oxidase and mitochondria. The interplay between mitochondrial superoxide production and activity of phagocytic NADPH oxidase was further investigated in splenocytes isolated from Sham and angiotensin II infused C57Bl/6J and Nox2KO mice. Angiotensin II infusion in wild-type mice increased the extracellular basal splenocyte superoxide which was further enhanced by complex III inhibitor antimycin A, mitochondrial uncoupling agent CCCP and NADPH oxidase activator PMA. Nox2 depletion attenuated angiotensin II mediated stimulation and inhibited both extracellular and mitochondrial PMA-induced superoxide production. These data indicate that splenocytes isolated from hypertensive angiotensin II-infused mice are “primed” for enhanced superoxide production from both phagocytic NADPH oxidase and mitochondria. Our data demonstrate that novel 15N-CAT1H provides high sensitivity superoxide measurements and combination with mitoTEMPO-H allows independent and simultaneous detection of extracellular and mitochondrial superoxide. We suggest that this new approach can be used to study the site-specific superoxide production and analysis of important sources of oxidative stress in cardiovascular conditions.Superoxide radical plays an important role in redox cell signaling and physiological processes; however, overproduction of superoxide or insufficient activity of antioxidants leads to oxidative stress and contributes to the development of pathological conditions such as endothelial dysfunction and hypertension. Meanwhile, the studies of superoxide in biological systems represent unique challenges associated with short lifetime of superoxide, insufficient reactivity of the superoxide probes, and lack of site-specific detection of superoxide. In this work we have developed  Nitroxide radicals TEMPOL and mitoTEMPO were purchased from Enzo Life Sciences . All other reagents were obtained from Sigma . Preparation of mitoTEMPO-H, 1-Hydroxy-2,2,6,6-tetra(2H3)methyl-4-trimethylammonio piperidine chloride hydrochloride was prepared from 4-oxo-2,2,6,6-tetra(2H3)methyl piperidine   with iodomethane in analogy to literature  compounds prior to application to isotope-enriched material.A-D,15N)  accordinA-D,15N)  with somterature  affordedterature , to giveterature . Each pr4-Dimethylamino-2,2,6,6-tetramethylpiperidine (1). A mixture of triacetonamine , dimethylamine hydrochloride , sodium cyanoborohydride , and methanol was stirred at room temperature for 48 h. The inorganic precipitate was filtered off, methanol was distilled off in vacuum. Saturated solution of NaCl was added to the residue, the mixture was basified with NaOH (0.5 g) and extracted with diethyl ether (3 ×10 mL). The extract was dried with Na2CO3 and ether was distilled off in vacuum to give 1 , colorless liquid, IR (neat), νmax cm−1: 2955, 2931, 2862, 2820, 2771, 1456, 1375, 1365, 1325, 1242, 1219, 1164, 1103, 1030, 856; 1H NMR : 0.78 , 0.93 (6H s), 0.99 (6H s), 1.53 , 2.07 (6H s), 2.44 ; 13C{1H} NMR : 28.26 and 34.95 (Me), 40.65 (CH2), 40.84 (NMe), 50.58 (CMe2), 55.84 (CH); cf methylpiperidine  was prepared similarly from TAA-N, 15D , νmaxcm−1: 2931, 2862, 2818, 2771, 2212, 2123, 2069, 1470, 1456, 1441, 1267, 1209, 1196, 1169, 1093, 1053, 993, 856, 654, 613; 1H NMR : 2.11 (6H s), 2.46 (1H m); 13C{1H} NMR : 27.36  and 34.00 , 40.2 , 40.95 (NMe), 50.14 , 55.71 .A-N, 15D  in CH3OD2,2,6,6-Tetramethyl-4-trimethylammoniopiperidine iodide (2) was prepared using the procedure described in literature  in diethyl ether (15 mL). The mixture was allowed to stand at room temperature for 24 h. The precipitate was filtered and washed with diethyl ether to give 2 monohydrate , colorless crystals, mp 216–217°C (i-PrOH – MeOH). Elemental analysis, found: C, 41.88; H, 8.23; N, 8.14; I, 36.57; calcd. for C12H29N2OI: C, 41.86; H, 8.49; N, 8.14, I, 36.86 %. IR (KBr), νmax cm−1: 3508, 3354, 3296, 3248, 3198, 2971, 2870, 1645, 1492, 1481, 1390, 1371, 1340, 1261, 1230, 1194, 972, 885, 866, 804; 1H NMR : 0.99 (6H s), 1.00 (6H s), 1.24 , 1.80 , 2.91 (9H s), 3.73 ; 13C{1H} NMR : 27.03 and 33.06 (Me), 36.78 (CH2), 50.73, 50.76 and 50.79 (NMe), 51.60 (CMe2), 69.06 (CH); cf (terature . Methyl (CH); cf .2,2,6,6-Tetra(2H3)methyl-4-trimethylammoniopiperidine iodide  was prepared similarly from 1-D,15N. IR (KBr), νmax cm−1: 3506, 3354, 3239, 3194, 3012, 2946, 2224, 2125, 2071, 1645, 1491, 1479, 1417, 1241, 1200, 1161, 958, 879; 1H NMR : 2.89 (9H s), 3.79 (1H m); 13C{1H} NMR : 26.54  and 32.47 , 36.5 (m CH2), 50.39, 50.44 and 50.48 (NMe), 51.26 (m CMe2), 69.18 (m CH).2,2,6,6-Tetramethyl-4-trimethylammoniopiperidine chloride hydrochloride (3). A solution of 2 monohydrate  in H2O (7 mL) was acidified to pH 1 with conc. hydrochloric acid, and hydrogen peroxide (30%)  was added carefully drop-by-drop. The precipitate of crystalline iodine was filtered off and a new very small portion of hydrogen peroxide was added to verify complete oxidation of iodide. If no precipitation of iodine occurred, the solution was extracted with benzene several times to remove iodine and evaporated to dryness in vacuum to give 3 dihydrate , colorless crystals, mp 314-315 °C (i-PrOH – MeOH). Elemental analysis, found: C, 46.69; H, 10.22; N, 9.14; Cl, 23.45; calcd. for C12H32N2Cl2O2: C, 46.90; H, 10.50; N, 9.12; Cl, 23.07 %; IR (KBr), νmax cm−1: 3479, 3398, 3282, 3009, 2966, 2821, 2766, 2611, 2493, 2114, 1633, 1608, 1587, 1487, 1445, 1394, 1384, 1356, 1256, 1188, 1107, 957, 903; 1H NMR : 1.41 (6H s), 1.45 (6H s), 2.04 (4H m), 3.04 (9H s), 4.24 (1H m); cf methyl-4-trimethylammoniopiperidine chloride hydrochloride  was prepared similarly from 2-D,15N. IR (KBr), νmax cm−1: 3456, 3406, 3284, 2976, 2889, 2511, 2227, 2079, 1633, 1606, 1481, 1419, 1248, 1157, 1126, 1051, 947, 887; 1H NMR : 2.94 (9H s), 4.11 (1H m); 13C{1H} NMR : 22.70  and 27.97 , 33.20 (m CH2), 50.48 (NMe), 56.37 (m CMe2), 64.79 (m CH).2,2,6,6-Tetramethyl-4-trimethylammoniopiperidine-1-oxyl chloride (CAT1). A solution of 3 dihydrate  in water (3 mL) was basified with KHCO3 to pH 10. Sodium tungstate (15 mg) and hydrogen peroxide (1 mL) were added to the solution and the reaction mixture was allowed to stand in dark place for 14 days. The orange solution was evaporated in vacuum to dryness, the residue was triturated with hot i-PrOH – MeOH mixture, the colorless inorganic precipitate was filtered off and the solvent was removed in vacuum to give CAT1 (solvate CAT1-Cl × H2O × (CH3)2CHOH) , orange crystals, mp 250 dec. (i-PrOH). Elemental analysis, found: C, 54.83; H, 11.08; N, 8.55; Cl 10.88; calcd. for C12H26NO2Cl × H2O × (CH3)2CHOH: C, 54.94; H, 11.07; N, 8.54; Cl, 10.81 %; IR (KBr), νmaxcm−1: 3426, 3315, 3240, 3017, 2995, 2969, 1633, 1498, 1482, 1448, 1359, 1346, 1270, 1244, 1183, 1135, 1107, 975, 961, 947, 899, 670, 572.2,2,6,6-Tetra(2H3)methyl-4-trimethylammoniopiperidine-1-oxyl chloride (15N-CAT1) was prepared similarly from 3-D,15N. IR (KBr), νmax cm−1: 3425, 3242, 3018, 2966, 2245, 2229, 2125, 2075, 1500, 1479, 1383, 1344, 1184, 1136, 1049, 958, 893, 821, 617, 513.1-Hydroxy-2,2,6,6-tetramethyl-4-trimethylammoniopiperidine chloride (14N-CAT1H). The nitroxide CAT1  was dissolved in a mixture of isopropanol (1 mL), ethanol (1 mL) and conc. hydrochloric acid (1 mL). The mixture was stirred overnight and dehydrated in vacuum to dryness. The residue was crystallized from a mixture i-PrOH – MeOH 10:1 to give CAT1H (solvate CAT1H-Cl × (CH3)2CHOH) , mp 270-273°C. Elemental analysis, found: C, 51.80; H, 10.35; N, 8.29; Cl, 20.16; calcd. for C12H28N2OCl2 × (CH3)2CHOH: C, 54.94; H, 11.07; N, 8.54; Cl, 10.81%; IR (KBr), νmax cm−1: 1643, 1479, 1401, 1392, 1355, 1261, 1244, 1138, 1105, 1065, 955, 908, 855; 1H NMR : 1.58 (6H s), 1.68 (6H s), 2.54 (4H m), 3.26 (9H s), 4.29 (1H m), i-PrOH: 1.18 , 3.95 ; 13C{1H} NMR : 20.35 and 28.17 (Me), 36.63 (CH2), 52.10 (N-CH3), 65.41 (CH), 69.38 (CMe2), cf methyl-4-trimethylammoniopiperidine chloride (15N-CAT1H) was prepare similarly from 15N-CAT1. IR (KBr), νmax cm−1: 3261, 3020, 2969, 2915, 2723, 2486, 2375, 2231, 2046, 1583, 1489, 1471, 1377, 1298, 1244, 1159, 1132, 1051, 955, 887, 820; 1H NMR : 3.25 (9H s), 4.21 (1H m), i-PrOH: 1.18 , 3.95 .1-Hydroxy-2,2,6,6-tetramethyl-4-[2-(triphenylphosphonio)acetamido]piperidinium dichloride (mitoTEMPO-H) was prepared via catalytic hydrogenation of mitoTEMPO on Pd/C in analogy to previously described procedure ((mitoTEMPO-H×HCl×3 H2O). Yield 80%, colorless crystals, mp 188-191°C (reprecipitated from isopropanol with diethyl ether). 1H NMR (CDCl3) δ= 1.22 , 1.42 , 1.86 , 2.00 , 3.85 , 4.94 , 7.59 , 7.72 , 9.54 , 11.09 . 13C NMR (CDCl3) 20.4, 27.9 (CH3); 32.3 , 40.5 (CH), 40.7 (CH2); 68.1 (CMe2), 118.1 , 130.2 , 133.9 , 134.9 , 162.4 . IR (KBr) 1668 (C=O), 1553, 1439, 1388, 1335, 1112, 996, 746, 720, 690, 516. Anal. Found: C, 57.81; H, 6.95; N, 4.66; Cl, 11.50; P, 5.38. Calcd. For C29H37Cl2N2O2P ×3 H2O: C, 57.90; H, 7.21; N, 4.66; Cl, 11.79; P, 5.15.rocedure  and isolWild-type C57BL/6J and mice lacking the gp91 phox catalytic subunit of phagocytic NADPH oxidase (Nox2KO) were obtained from Jackson Laboratories. Some animals were infused with low-suppressor dose of angiotensin II (0.3 mg/kg/day) for 14 days as described previously . BP was ATP channel diazoxide (0.1 μM) specifically inducing mitochondrial superoxide  isolated from Sham and angiotensin II infused C57Bl/6J and Nox2KO mice were treated acutely with antimycin A, CCCP, PMA, rotenone or Cu,Zn-SOD and then incubated with spin probes.In initial experiments splenocytes were treated with either vehicle , TNFα (1 ng/ml) or IL-17A (10 ng/ml) for 3-h at 37C. To induce superoxide production as a “positive control,” unstimulated splenocytes were treated acutely with NADPH oxidase activator PMA (10 μM)  or openeperoxide . To inve7/ml) were incubated with 15N-CAT1H (500 μM) and/or 14N-mitoTEMPO-H (25 μM) for 30 min at 37°C prior to EPR analysis. Extracellular O2• was measured by accumulation of 15N-CAT1 nitroxide following low field component of EPR spectra. Mitochondrial O2• production was measured by accumulation of 14N-mitoTEMPO nitroxide following central field component of EPR spectra. The potential contamination of 14N-mitoTEMPO signal with extracellular superoxide did not exceed 5%, since the amount of 14N-mitoTEMPO-H is 20-fold lower than 15N-CAT1H spin probe (25 μM and 500 μM accordingly), and these probes have similar reaction rate constant with superoxide. This was confirmed experimentally in the xanthine oxidase superoxide system containing 14N-mitoTEMPO-H (25 μM) and 15N-CAT1H (500 μM) spin probes  and xanthine oxidase (2 mUnits/ml) superoxide generating system. Specificity of superoxide detection was confirmed by blocking the reaction with Cu,Zn-SOD (50 U.ml). In cellular experiments, splenocytes .EPR measurements were performed in 50 μL glass capillary tubes at room temperature using the Bruker EMX spectrometer. Spectrometer settings were as follows: field sweep, 60 G; microwave frequency, 9.82 GHz; microwave power, 20 mW; modulation amplitude, 1 G; conversion time, 164 ms; time constant 328 ms; sweep time, 168 s; receiver gain, 1 ×10escribed . The conpost-hoc test and the analysis of variance (ANOVA). P levels < 0.05 were considered significant.Experiments were analyzed using the Student Neuman Keuls 15N-CAT1H spin probe was studied in xanthine oxidase superoxide generating system using field scan and time scan modes. Incubation of 0.2 mM 15N-CAT1H with xanthine oxidase and xanthine leads to robust accumulation of 15N-CAT1H nitroxide as measured by appearance of doublet EPR spectra (15N-hyperfine splitting constant aN = 23.5 G) . Interestingly, intensity of EPR signal of 15N-CAT1H nitroxide was about 2-fold higher compared with CAT1 due to distribution of EPR intensity into 2-line signal vs. 3-line and narrowing of CAT1 signal due to replacement of hydrogen to deuterium  and 14N-containing CAT1 (aN = 16.7 G) nitroxides in equimolar ratio. EPR spectra of 15N- and 14N-containing CAT1 nitroxides had a very good separation allowing to track accumulation of these nitroxides independently  as well as mitochondrial superoxide   or inducer of mitochondrial superoxide diazoxide (0.1 μM) as a “positive control” for increased superoxide production. Interestingly, PMA increased not only extracellular superoxide production by phagocytic NADPH oxidase as measured by increased itoTEMPO . ParadoxtoTEMPO) . The pot results . AnalysioTEMPO-H . Althougin vivo. Following 14-days of angiotensin II infusion (0.3 mg/kg/day) mice were sacrificed and spleens were isolated for splenocytes preparation.The interplay between mitochondrial superoxide and activity of phagocytic NADPH oxidase was further investigated in splenocytes isolated from Sham and angiotensin II infused C57Bl/6J and Nox2KO mice. Angiotensin II is known to stimulate NADPH oxidase activity via AT1 receptor. Sub-pressor dose of angiotensin II does not significantly raise blood pressure in wild-type mice  but prom15N-CAT1H signal) which was further enhanced by complex III inhibitor antimycin A, mitochondrial uncoupling agent CCCP and NADPH oxidase agonist PMA  showed substantial increase in wild-type splenocytes but not in Nox2KO  vs. three lines (14N). Replacement of 1H to 2H (deuterium) decreases the observed linewidth because of much smaller hyperfine-coupling constant on deuterium nuclei compared with protons which results in EPR line narrowing leading to further increase in the intensity of EPR spectral lines. The utility of simultaneous use of 15N- and 14N-labeled nitroxides in cellular/tissue distribution studies have been shown previously,  improved sensitivity in superoxide detection; ii) independent detection of 15N- and 14N- spin probes in distinct cellular compartments; iii) EPR signal of 15N-spin probe does not overlap with spectra of biological radicals and paramagnetic background  can increase mitochondrial superoxide production via Hchanisms . The patInflammation and oxidative stress play an important role in pathogenesis of cardiovascular disease. Development of new treatments targeting these pathways could improve the treatment of many pathological conditions. Blocking inflammatory pathways is hindered by the off-target effects on the immune system and non-discriminatory antioxidant treatment can impair redox cell signaling pathways. Understanding the precise regulations of immune cells and oxidant production can help in the development of new therapies targeting specific inflammatory and oxidative stress mechanisms. In this work we described the interplay between phagocytic NADPH oxidase activity and mitochondrial superoxide production in immune cells stimulated by pro-inflammatory cytokines, mitochondrial agents or angiotensin II. This crosstalk between NADPH oxidase and mitochondria in inflammatory cell can promote inflammatory injury and end-organ-damage. Indeed, metabolic conditions are associated with both mitochondrial dysfunction and upregulation of pro-inflammatory pathways. We suggest that specific targeting Nox2 or mitochondrial function can break this pathophysiological vicious cycle , reduce The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Vanderbilt Institutional Animal Care and Use Committee.SD conceived, designed research, drafted manuscript, and edited and revised manuscript. AD performed animal and cellular studies and analyzed data. IK synthesized the spin probes. AD and IK interpreted results of experiments and approved final version of manuscript. All authors contributed to the article and approved the submitted version.This work was supported by funding from National Institutes of Health (R01HL144943 and RO1HL157583) and American Heart Association Transformational Project Award (19TPA34910157).The authors declare 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 aim of this study was to test the suitability of three different ancient wheat varieties  to produce spontaneously fermented sourdough bread and to evaluate the impact on the dough rheological properties, ultrastructure and baking quality. Modern wheat sourdough bread and bakery yeast fermented bread were used as controls. Sourdoughs produced from modern and ancient wheats exerted different effects on dough viscoelastic properties, bread specific volume, texture, firming rate, colour and sensory properties, while there was no influence on bread water activity. Both khorasan sourdough, being characterised with the highest dough strength and dense gluten protein matrix, and emmer sourdough, with loose and thin gluten strands of low strength, yielded breads characterised by low specific volume and hard crumb texture. Spelt and modern wheat sourdough were characterised by foam-like dough structures with entrapped gas cells leading to breads of similar specific volume and texture. Although the yeast-fermented wheat flour exerted a higher specific volume and the lowest firmness, the sourdough wheat flour bread had a lower firming rate. A comparison of sourdough bread prepared with modern and ancient wheats revealed that breads based on ancient varieties possess a less noticeable sour taste, odour and flavour, thus contributing to more sensory-appealing sourdough bread. The paradigm of agricultural development focusing narrowly on increasing crop yields has caused clear-cut homogeneity in the global cereal supply and obvious dominance of three major cereal crops: wheat, rice and maize. While great strides have been made toward breeding new wheat varieties, genetic evolution has caused the creation of more productive plants with modified chemical compositions and other quality attributes. Paradoxically, improvements in grain yield, as well as some technological quality attributes of flour, has caused an increase in starch content and has consequently led to the reduction of other grain components, especially minerals . The curSourdough fermentation, a mixture of water and flour fermented with lactic acid bacteria and yeasts, is a specific process where developed microbial communities are bound by a range of competitive and cooperative interactions that, through complex biochemical changes, result in the transformation of ingredients. These transformations, caused by complex microbiome consortiums, lead to positive nutritional implications in terms of increasing mineral, protein and free amino acid bioavailability, reducing anti-nutrients  and reducing starch digestibility, followed by a decrease in the glycaemic index of baked goods . Among tThere are a certain number of publications concerning the use of sourdough fermentation for the production of ancient wheat flour-based products ,11,12. HThree flours from three different ancient wheat varieties—emmer, spelt and khorasan—were purchased from a small local producer , whereas flour from a modern wheat variety was obtained from a milling company Danubius d.o.o. . All wheat samples were cultivated in the same production year and at the same location. After harvesting, grains were stored for six months. Subsequently, they were dehulled by Heger’s large-scale friction de-huller  and milled by a large-scale stone mill Osttiroler Getreidemühlen . The obtained flour samples were as follows: modern wheat flour , spelt flour , emmer flour  and khorasan flour . The salt and bakery yeast used in the breadmaking were purchased from the local market.w/w)) with a resulting dough yield of 200 and incubated at 25 °C for 24 h in a laboratory incubator . After this first fermentation, back-slopping steps were further carried out by using the same condition of fermentation (25 °C for 24 h), where a portion of fermented dough from a previous day was used as an inoculum for subsequent back-slopping, mixed with flour and demineralised water in a 1:2:2 ratios (fermented dough:flour:water). Mature sourdough that was prepared as described above served as a “starter” instead of bakery yeast in the breadmaking procedure.Sourdough fermentation of modern wheat, emmer, spelt and khorasan flours was carried out without using starter cultures or baker’s yeast, through a back-slopping procedure . Doughs were prepared by mixing flour and demineralised water . Frequency sweep measurements were carried out at 25 °C using PP35 S serrated parallel plate measuring geometry  to eliminate sample slippage during the tests. The excess of dough sample was removed and the edges were covered with paraffin oil to prevent the dough from drying during the measurement procedure. After that, samples were left to rest between the plates for 10 min, allowing residual stresses to relax. Mechanical spectra were recorded over the frequency range 1–10 Hz at a constant shear stress of 0.5 Pa, which was within the previously determined linear viscoelastic region. All tests were performed in triplicate.Dough ultrastructure characterisation was carried out using a JEOL JSM 6460LV scanning electron microscope . All samples were previously lyophilised using an Alpha 1-2 LDplus freeze dryer . Before scanning electron microscopy, powdered samples were mounted on scanning electron microscope stubs using double-sided adhesive tape and afterward coated with gold using Sputter Coater SC 005 . Consequently, the ultrastructure was analysed under high vacuum conditions at an accelerating voltage of 25 kV at a magnification of 1000×.Control bread formulation consisted of 300 g of modern wheat flour, demineralised water up to 400 BU consistency, 7.5 g of bakery yeast and 5.4 g of salt. In doughs prepared with sourdough as a leavening agent, 25% of flour was replaced with the appropriate type of sourdough in order to keep the total flour amount at 300 g. Subsequently, 5.4 g of salt and demineralised water in an amount to achieve 400 BU consistency were added. Mixing was conducted in a Farinograph mixing bowl  for seven minutes. Subsequently, dough samples were left for fermentation in a cabinet at 30 °C for 30 min and then punched down. This step was repeated for an additional 3 times and then the amount of 120 g of dough samples was hand-moulded and placed into Teflon pans (L × W × H: 80 mm × 50 mm × 50 mm). The proofing procedure was performed at 30 °C and a relative humidity of 85% up to the optimum volume increase (~3 h for samples containing sourdough and 65 min for control bread with bakery yeast). Consequently, the baking procedure was carried out in a modular deck oven  at 220 °C until a mass loss of 8%. Finally, bread samples were left to cool down for 2 h at room temperature and sealed in polyethylene bags for further analysis. Two batches of each bread sample were prepared.The specific volume of bread samples was measured on 3 loaves per batch by a Volscan Profiler . w) of bread crumb samples (5 mm thickness) taken from the middle of each bread loaf after 24 h of storage was measured by a LabSwift-aw instrument .Water activity  equipped with a 30-kg load cell. Measurements were performed using the modified standard method for the determination of bread firmness AACC (74-09) . The modL*—lightness; a*—redness to greenness, positive to negative values, respectively; b*—yellowness to blueness, positive to negative values, respectively).Bread crust and bread crumb colour was evaluated in six replicates per loaf, 24 after baking using a Minolta Chroma Meter CR-400 colorimeter  (8 mm Ø contact area). Prior to the measurements, the instrument was calibrated by a standard light white reference tile and the tests were performed under standard illuminant D65. The obtained results were expressed according to the CIELab colour system . The descriptive terminology of the products was created in a pre-trial session using a control and bread from emmer flour. After two 1 h pre-trial sessions, the descriptors, together with their definitions and reference samples, were agreed upon by the panellists. The final list encompassed five appearance attributes , three taste attributes , eight odour attributes , eight flavour attributes  and five textural attributes . Once the descriptors had been selected, panellists were trained by measuring the intensities of selected descriptors perceived in breads using 10-cm unstructured scales. By the end of the pre-trial sessions, all panellists were able to discriminate among samples with good repeatability between sessions and to reach agreement with other assessors in the panel. Sensory profiling was performed on bread samples through quantitative descriptive analysis QDA  by a FINThe sensory analysis was conducted in individual sensory booths. The bread samples (one slice per sample) were served at room temperature in closed odourless plastic containers coded with 3-digit random numbers following a complete balance block design. All panellists evaluated all samples in a sequential monadic manner. The sensory profiling was done in two repetitions. Water was used for rinsing between samples.A consumer test was performed with 72 consumers  recruited from students and academic staff from the University of Novi Sad based on bread consumption frequency (at least 3 times per week) and without food allergies, especially celiac and gluten sensitivity. Consumers were asked to rate how much they liked the bread samples in terms of overall acceptability, the taste, the texture of the crumb and the texture of the crust using the 9-point hedonic scale with the extremes labelled 1 = dislike very much and 9 = like very much. The bread samples were distributed in closed odourless plastic containers coded with 3-digit random numbers. The order of sample presentation was randomised across consumers. The evaluation was performed in individual sensory booths. This study was approved by the Ethics Committee of the Institute of Food Technology in Novi Sad, University of Novi Sad, Serbia (No. 175/I/7-3).All measurements were performed in replicate and the mean values ± standard deviations were reported. One-way analysis of variance (ANOVA) followed by Tukey’s minimum square difference test was carried out for the evaluation of statistical differences between tested samples using Statistica 10.0 , whereas XLSTAT 2022.1.2 (Addinsoft) was used for sensory and consumer data analysis. The significance of the difference between groups was indicated at the 95% confidence level.The effect of wheat variety on the viscoelastic properties of spontaneously fermented sourdough was assessed using dynamic oscillatory measurements. According to obtained mechanical spectra , storageSourdough ultrastructure is illustrated in 2 bubbles. These results are in accordance with results of rheological characterisation, where khorasan sourdough exhibited the highest dough strength and elasticity  and the wet gluten value of khorasan flour in comparison with other ancient wheat varieties [2 produced in the fermentation step. Moreover, khorasan sourdough was characterised by high stiffness  factor is a valuable tool for predicting microbial growth and metabolic activity [w values that did not significantly differ among themselves. The obtained aw values are in accordance with the findings of Neylon et al., [w values of tested bread crumb samples in the range of 0.95–0.97.Water represents one of the most important parameters influencing microbial spoilage of foods; the water activity  lighter colour of crumb and crust and smaller sized uniform pores with a more compact and hard crumb texture than all the other samples. Moreover, although overall odour and flavour of this bread were mild, it was specific and intense enough to allow a clear distinction of this sample in terms of cheese, flour and vinegar odour and flavour notes. The bread with emmer was similar to the khorasan bread in terms of cheesy odour and flavour, while, in terms of textural attributes, significant differences among them were observed for hardness and crumb compactness. Moreover, the bread with emmer possessed a significantly (p < 0.05) darker crust colour, odour on malt, bitter taste and overall flavour intensity in comparison with all the other samples. The control bread stood out from the other samples with the more noticeable sharpness and moisture absorption, odour and flavour on yeast, as well as flavour persistence, which is consistent with the recipes and the experimental design. All the sourdough bread samples were more sour than the control bread, with the exception of emmer bread, which was perceived as equally sour as the control. The sourdough bread made with modern wheat flour was described as significantly (p < 0.05) more sour, with a very intense odour and flavour on vinegar. A similar finding was reported by Coda et al., [According to ANOVA, almost all selected sensory attributes help to discriminate sensory profiles among the samples . The mai et al., , who fouTo create a visual representation of the sensory acceptability  of the bread samples given by the participants involved in the consumer study, a spider plot was created  by plottThe suitability of three different ancient wheat varieties  to produce spontaneously fermented sourdough bread was evaluated by comparing their dough rheological properties, ultrastructure and breadmaking quality with modern wheat bread produced from sourdough, as well as with bread produced using bakery yeast without sourdough addition. A khorasan sourdough and an emmer sourdough yielded breads with low specific volume and hard crumb texture. On the contrary, spelt sourdough, characterised by a foam-like dough structure and balanced dough viscoelasticity, produced bread of similar specific volume and texture as modern wheat sourdough. Although the bread prepared from bakery yeast had a superior quality in terms of specific volume and texture, the sourdough bread from the modern wheat flour was characterised by a lower firming rate. The comparison between modern and ancient wheat varieties revealed that sourdough bread from ancient wheats was perceived less sour in taste, odour and flavour, thus producing a more sensory appealing product than modern wheat. In addition, consumer tests indicated that overall acceptability was the highest for the yeast-fermented and spelt sourdough bread, followed by khorasan, wheat and emmer sourdough bread, respectively. In general, the obtained results indicated that it is possible to produce technologically and sensory acceptable sourdough bread from ancient wheat varieties and thus contribute to both cereal and microbial biodiversity protection. Future work can be oriented towards investigations of the potential of mixtures of different ancient wheat varieties for sourdough bread production and quality improvement.