MADS‐box protein named MuMADS1 interacted with an ovate family protein named MaOFP1 to regulate banana fruit ripening. To further investigate the role of MADS1Mu and OFP1Ma in the regulation of fruit quality, a combination of genetic transformation and transcriptional characterization was used. The results indicated that the co‐expression of MADS1Mu and OFP1Ma in the ovate mutant could compensate for fruit shape and inferior qualities relating to fruit firmness, soluble solids and sugar content. The number of differentially expressed genes (DEGs) was 1395 in WT vs. ovate, with 883 up‐regulated and 512 down‐regulated genes, while the numbers of DEGs gradually decreased with the transformation of MADS1Mu and OFP1Ma into ovate. ‘Starch and sucrose metabolism’ constituted the primary metabolic pathway, and the gene numbers in this pathway were obviously different when MADS1Mu and OFP1Ma were integrated into ovate. A series of metabolic genes involved in cell wall biosynthesis were up‐regulated in the WT vs. ovate, which probably resulted in the firmer texture and lower sugar contents in the ovate fruit. These results demonstrate that MADS1Mu and OFP1Ma are coregulators of fruit quality, facilitating the dissection of the molecular mechanisms underlying fruit quality formation.Fruit ripening and quality are common botanical phenomena that are closely linked and strictly regulated by transcription factors. It was previously discovered that a banana  More than three independent transgenic lines confirmed for transgene integration were recovered, and two independent lines for each transformant were chosen for their transgene homozygosity in the T1 generation from the DNA gel blot evaluation and were phenotypically categorized in T3 production , mature green (MG), breaker fruit (BR) and red mature (RM)] of the four transgenic lines  and MaOFP1 expression in various fruit maturation periods of the four transgenic lines  was additionally determined via quantitative real‐time PCR (qRT‐PCR). The outcomes suggested that MuMADS1 and MaOFP1 were greatly expressed in transgenic tomatoes. During the process of fruit development, the gene expression levels gradually increased and peaked at the RM stage. Moreover, MuMADS1 and MaOFP1 were expressed at higher levels in MO transgenic lines than in the M or O transgenic lines  genome was ~80.92%–85.62%, as shown in Table A total of 416.06 million clean reads from the samples were obtained following quality assessment and data filtering. The GC% of sequenced data from the 15 libraries was 42.56%, and the percentage of reads with an average quality score >30 was 88.36%. This indicated that the accuracy and quality of the sequencing data were sufficient for further analysis. The general sequencing statistics are shown in Table MuMADS1 and MaOFP1 in regulating fruit quality, transcriptional analysis was considered. The number of DEGs was 1395 in WT vs. ovate, with 883 up‐regulated and 512 down‐regulated genes  and 904 (500 up and 404 down), respectively  and 342 (135 up and 207 down), respectively  and 45 (8.46%), respectively , 16 (8.65%) and 21 (6.89%), respectively , Swiss‐Prot, Gene Ontology (GO), Clusters of Orthologous Groups (COG), Eukaryotic Orthologous Groups (KOG), evolutionary genealogy of genes: Nonsupervised Orthologous Groups (eggNOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were introduced to annotate the DEGs. Of these, 4252 (99.98%) were matched to the Nr database, while the smallest number of DEGs was found in the KEGG database Table . To furtovate: 10 (0.40%); WT vs. MuMADS1 transformants: eight (0.32%); WT vs. MaOFP1 transformants: eight (0.32%); WT vs. MuMADS1 + MaOFP1 transformants: six (0.24%); ovate vs. MuMADS1 transformants: 0; ovate vs. MaOFP1 transformants: 0; ovate vs. MuMADS1 + MaOFP1 transformants: six (0.24%)], followed by ‘signalling’ . Within the ‘cellular component’ principal category, unigenes  belonged to the ‘extracellular matrix’ category. In the principal category of ‘molecular function’, the most‐represented category was ‘enzyme regulator activity’ .Unigenes that were successfully annotated to the GO database were classified into three principal categories for the seven assemblies, including ‘cellular component’, ‘molecular function’ and ‘biological process’, which were further subdivided into 53 categories Table . Of thesovate, WT vs. MuMADS1 transformants, WT vs. MaOFP1 transformants, WT vs. MuMADS1 + MaOFP1 transformants, ovate vs. MuMADS1 transformants, ovate vs. MaOFP1 transformants and ovate vs. MuMADS1 + MaOFP1 transformants, respectively  DEGs in WT vs. MuMADS1 transformants, 16 (6.32%) DEGs in WT vs. MaOFP1 transformants, five (1.98%) DEGs in WT vs. MuMADS1 + MaOFP1 transformants, one (0.40%) DEG in ovate vs. MuMADS1 transformants, three (1.19%) DEGs in ovate vs. MaOFP1 transformants and four (1.58%) DEGs in ovate vs. MuMADS1 + MaOFP1 transformants, respectively. In the assembly of WT vs. ovate, the second largest term was ‘carbon metabolism’, which included 25 (8.68%) DEGs. In the assemblies of WT v MuMADS1 transformants and WT vs. MuMADS1 + MaOFP1 transformants, the ‘carbon metabolism’ term changed from 14 (4.86) and three (1.04%) DEGs, respectively. This result indicated that ‘starch and sucrose metabolism’ and ‘carbon metabolism’ constituted the primary metabolic pathways that they changed with the integration of exogenous genes and that they might be more active during tomato fruit quality formation.To identify the active pathways in the transformants, the obtained unigenes were mapped to the canonical reference pathways in the KEGG database. KEGG pathway enrichment examination was performed to classify the biological tasks of the DEGs. Specific enrichment of genes was obtained for 98, 85, 88, 47, 22, 61 and 74 pathways for WT vs. ly Table . The topP < 0.01 and |log2 (fold change)| ≥1, the DEGs of the assemblies involved in sugar and cell wall metabolism were identified as being up‐regulated or down‐regulated , sucrose synthase 2‐like (Solyc09g098590.2), bidirectional sugar transporter SWEET14‐like (Solyc03g097560.2), were differentially expressed in the WT vs. ovate assembly, which suggested that these genes might be the targets of OVATE in the regulation of fruit sugar accumulation. The 41 DEGs involved in cell wall metabolism, including cellulose synthase A catalytic subunit 5 (Solyc11g005560.1) and galacturonosyltransferase 8‐like (Solyc12g013560.1), were differentially expressed in the WT vs. ovate assembly, which suggested that these genes might be the putative targets of OVATE in the regulation of fruit firmness , 60 DEGS  and 83 DEGs , respectively. As shown in Figure Considering the compensation of plastidic hexokinase (Solyc04g081400.2), putative beta‐glucosidase 41 (Solyc07g063880.2), UDP–glucose 6‐dehydrogenase 1 (Solyc02g067080.2), UDP–glucuronate 4‐epimerase 1‐like (Solyc12g010540.1) and galacturonosyltransferase (Solyc06g083310.2). The expression levels of the selected DEGs revealed by qRT‐PCR were generally consistent with those from the RNA‐Seq analysis at the three developmental stages, which indicated that the results of the RNA‐Seq analysis showed a high degree of correlation with those of the qRT‐PCR  and 10 (4.74%) in the assembly of WT vs. MuMADS1 transformants and WT vs. MuMADS1 + MaOFP1 transformants, respectively. We have summarized the distribution of DEGs regulated by MuMADS1, MaOFP1, MuMADS1 + MaOFP1 and OVATE based on the RNA‐Seq data as shown in Figure ovate mutation. Either MuMADS1 or MaOFP1 could partially regulate the expression of genes involved in carbohydrate and cell wall metabolism to control fruit sugar accumulation and softening. MuMADS1 is not a homolog of tomato OVATE. However, MuMADS1 shared almost all target genes such as lysosomal beta‐glucosidase‐like and pectate lyase 8 with OVATE, MaOFP1 and MuMADS1 + MaOFP1. Similarly, MaOFP1 shared most target genes with OVATE and MuMADS1 + MaOFP1. MADS‐box genes have been shown to play a role in the formation of fruits  leads to a change in ovary locule shape from a ‘tear drop’ to a more open triangular shape by strongly reducing the cortex layer  was up‐regulated in the WT vs. ovate mutant, indicating that it contributes greatly to fruit cell wall biosynthesis in the ovate mutant, which was highly consistent with the firmer fruit observed in ovate than in WT. This up‐regulation could be suppressed by the co‐expression of MuMADS1 and MaOFP1 in ovate, which suggests that this gene may be regulated by MuMADS1 and MaOFP1. The increased firmness associated with the ovate mutation might be due to its increased cell wall polysaccharide content, which may result in the reduction of soluble carbohydrates  might be the main factor accounting for the increase in fruit firmness and decreased glucose in the ovate mutant. However, this down‐regulation could be compensated for by the co‐expression of MuMADS1 and MaOFP1 in the ovate mutant, suggesting that MuMADS1 and MaOFP1 act synergistically to regulate the expression of β‐glucosidase (Solyc07g063880.2). In this study, another β‐glucosidase named lysosomal beta‐glucosidase‐like (Solyc04g015560.2) displayed a different expression pattern  is a typical cellulase that acts synergistically to hydrolyse cell wall cellulose to glucose  and UDP–glucuronate 4‐epimerase 1‐like (Solyc12g010540.1) in the WT vs. ovate mutant may result in increased cell wall polysaccharide contents, which was consistent with the increased firmness observed in the ovate mutant. In the MuMADS1 and MaOFP1‐transformed ovate mutant, the expressions of UDP–glucose 6‐dehydrogenase (Solyc02g067080.2) and UDP–glucuronate 4‐epimerase 1‐like (Solyc12g010540.1) decreased, which was consistent with the decreased firmness in the MuMADS1 + MaOFP1 transformants   in WT vs. ovate suggested that these genes contribute to the increased fruit firmness in the ovate mutant. This up‐regulatory phenomenon of three galacturonosyltransferases gradually disappeared as MuMADS1 and MaOFP1 transformed into the ovate mutation, which suggests that the expression of these genes is regulated by MuMADS1 and MaOFP1  and wild‐type  were kindly provided by the Tomato Genetics Resource Center (http://tgrc.ucdavis.edu). Plants were raised in a field environment in groups at the Institute of Tropical Bioscience and Biotechnology . Transgenic plants were produced to the T3 line, and just the homozygous transgenic plants were utilized in the quantitative analyses  technique  based on the company's directions. The primers utilized for real‐time RT‐PCR are revealed in Table MuMADS1 and MaOFP1, as well as key genes responding to fruit quality formation in transgenic lines and controls, were determined via qRT‐PCR analysis on a Stratagene Mx3000P Real‐Time PCR system with SYBR® Premix Ex Taq™ . The PCR amplification set up for every one of the reactions was: 10 min at 95 °C, then 40 cycles of 10 s at 95 °C, 15 s at 50 °C and 30 s at 72 °C. The typical expressions of the target genes were quantified by the 2−ΔΔCT method  was used as the internal control to normalize the expression of the target genes in tomato.Transcriptional changes in et al.'s technique  using a Vernier calliper  was used to directly read the % soluble solids (as Brix) of the blended fruit at room temperature  contents were analysed using high‐performance liquid chromatography .MuMADS1 transformants), O (MaOFP1 transformants) and MO (MuMADS1 + MaOFP1 transformants), as well as WT and ovate were collected to extract total RNA using the plant RNeasy extraction kit  for transcriptome analysis. The sequencing was performed with an Illumina GAII following the manufacturer's instructions, with three replicates for each sample. The average sequencing depth was 5.34X. Using the FASTX‐toolkit, adapter sequences in the raw sequence reads were removed. Clean reads were generated after examining the sequence quality and removing low‐quality sequences using FastQC. Using Tophat v. 2.0.10, clean reads were mapped to the tomato genome . The transcriptome assemblies were performed by Cufflinks . FDR <0.01 and |log2 (fold change)| > 1 or <−1 were set as the thresholds for differential gene expression. Fold changes in the expression levels between samples were used as the criteria in the screening process. The unigene sequences were searched against the following public databases: NR database (Deng et al., et al., et al., et al., et al., RM fruits of different transgenic lines including M (The authors declare that they have no conflicts of interest.Figure S1 Identification of transgenic tomatoes.Click here for additional data file.Figure S2 GO classification. The DEGs corresponded to three main categories: “biological process”, “cellular component” and “molecular function”. The left‐hand y‐axes indicate the percentage of genes.Click here for additional data file.Table S1 RNA‐Seq analysis.Table S2 Mapped results of RNA‐Seq and reference genome.Table S3 Annotated number of DEGs.Table S4 Analysis of GO classification.Table S5 Analysis of KEGG classification.Table S6 The expression data of the DEGs in different assemblies.Table S7 The DEGs regulated by OVATE, MuMADS1, MaOFP1 and MuMADS1 + MaOFP1.Table S8 The primer sequences used for qRT‐PCR.Click here for additional data file.


The Movement Disorder Society Progressive Supranuclear Palsy Study Group defined MRI, SPECT/PET, DAT imaging, and tau imaging as biomarkers. Other diagnostic criteria for neurodegenerative disease described neuroimaging findings as only characteristic finding, not as biomarker. In this review, we describe the role of neuroimaging as a potential biomarker for neurodegenerative diseases.It has recently been recognized that neurodegenerative diseases are caused by common cellular and molecular mechanisms including protein aggregation and inclusion body formation. Each type of neurodegenerative disease is characterized by the specific protein that aggregates. In these days, the pathway involved in protein aggregation has been elucidated. These are leading to approaches toward disease-modifying therapies. Neurodegenerative diseases are fundamentally diagnosed pathologically. Therefore, autopsy is essential for a definitive diagnosis of a neurodegenerative disease. However, recently, the development of various molecular brain imaging techniques have enabled pathological changes in the brain to be inferred even without autopsy. Some molecular imaging techniques are described as biomarker in diagnostic criteria of neurodegenerative disease. Magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT), positron emission tomography (PET), and amyloid imaging are described in the diagnostic guidelines for Alzheimer’s disease in the National Institute on Aging-Alzheimer’s Association. MRI, dopamine transporter (DAT) imaging, and  Each neurodegenerative disease type is characterized by the specific protein that aggregates. Recently, extensive research has been performed on disease-modifying therapies for neurodegenerative diseases , which are expected to be developed in the near future. Simple and practical biomarkers specific for each neurodegenerative disease are urgently required for their accurate diagnosis and facilitate the development of disease-modifying interventions. A recent study demonstrated the potential clinical utility of plasma biomarkers in predicting brain amyloid-β burden . HoweverIn many diagnostic criteria for neurodegenerative disease, characteristic findings in neuroimaging are mentioned. Moreover, only few recent diagnostic criteria for neurodegenerative diseases have mentioned neuroimaging techniques as biomarkers that can estimate pathological changes occurring in the brains of neurodegenerative disease patients , 3, 5, 6The current mini-review addresses the roles of neuroimaging techniques described as biomarkers on diagnostic criteria for individual neurodegenerative diseases as research topics. Even though there were numerical findings of neuroimaging technique of neurodegenerative disease, we omitted those of not described in diagnostic criteria. For example, about prion disease, hyperintensity in cortex and basal ganglia on FLAIR and DWI are widely known as characteristic magnetic resonance imaging (MRI) finding of prion disease in daily clinical setting. However, this characteristic MRI finding is not described in diagnostic criteria for prion disease which is widely used .Level A: able to use the research criteria; described as biomarkers in diagnostic criteria.Level B: supportive biomarker of clinical diagnosis; described as supportive biomarkers for research criteria in diagnostic criteria.Level C: supportive of clinical diagnosis; not described as biomarkers but characteristic finding in diagnostic criteria.In this review, we defined the characteristic neuroimaging findings described in diagnostic criteria from Level A and B based on evidence level as follows:Table Magnetic resonance imaging is one of the most widely used neuroimaging techniques for the diagnosis of neurodegenerative diseases. However, among the diagnostic criteria for neurodegenerative diseases, only two diagnostic guidelines, namely, for Alzheimer disease (AD) , 3 and fThe National Institute on Aging-Alzheimer’s Association (NIA-AA) diagnostic guidelines for AD state disproportionate atrophy on structural MRI in the medial, basal, and lateral temporal lobes, as well as the medial parietal cortex, as a biomarker of neuronal degeneration or injury , 3 (LeveThe latest diagnostic guidelines of DLB  state thThe movement disorder society (MDS) clinical diagnostic criteria for progressive supranuclear palsy (PSP) that has recently been established state a characteristic MRI finding, namely, predominant midbrain atrophy relative to the pons, as a supportive feature, but not a biomarker of PSP  Level C.Other diagnostic criteria for neurodegenerative diseases also describe characteristic findings on MRI, but there are no descriptions of biomarkers. Frontotemporal lobar degeneration (FTLD) has three clinical subtypes , which dA second consensus statement on the diagnosis of multiple system atrophy (MSA) mentioned atrophy displayed on MRI of the putamen, middle cerebellar peduncle, pons, or cerebellum as an additional feature suggestive of MSA-P or MSA-C, but not as a biomarker   uptake on PET in the temporoparietal cortex as a biomarker of neuronal degeneration or injury  cardiac scintigraphy described below, occipital hypoperfusion has low sensitivity and specificity. Therefore, it is understandable that it stayed in only one of the supportive biomarker with most resent diagnostic criteria. Relative preservation of the metabolic activities of the posterior cingulate cortex and midcingulate cortex on FDG-PET (the cingulate island sign) has been described in DLB -N-(3-fluoropropyl) nortropane (123I-FP-CIT) is the ligand for DAT-SPECT that is most widely used. 123I-FP-CIT has been used in a large number of trials to identify the in vivo loss of dopamine transporters in the striatum of patients with presynaptic parkinsonism  uptake in the basal ganglia displayed on SPECT or PET as indicative biomarkers  Level A.123I-iodobenzamide (IBZM)-SPECT or 18F-desmethoxyfallypride (DMFP)-PET, is described as a supportive feature rather than as a biomarker  MIBG myocardial scintigraphy as an indicative biomarker  Level A, 29. TheThe recent clinical diagnostic criteria for PD by the MDS described the presence of either olfactory loss or cardiac sympathetic denervation on MIBG myocardial scintigraphy as supportive criteria, but not as a biomarker  , the first and most widely studied PET Aβ ligand, indicated that Aβ imaging may enable the earlier diagnosis of AD , florbetapir , florbetaben , and 18F-AZD4694 (recently renamed NAV4694)  are derived from stilbene. The above 18F-labeled tracers except NAV4694 have been approved by the Food and Drug Administration. Correlations have been shown between levels of biomarker CSF Aβ42 and PET signals using 11C-PiB as a tracer for Aβdeposits in the brain  as a biomarker of PSP-RS  gene. More importantly, the inclusion or exclusion of exon 10 results in either 4 repeats (4R) or 3 repeats (3R) of the microtubule-binding domain being transcribed in the tau protein, respectively (However, tau imaging is expected to become an important biomarker of tau pathology in the future. There are six different isoforms of tau, formed by alternative mRNA splicing of the microtubule-associated protein tau (ectively . The 3R/ectively .18F-FDDNP (18F-AV1451 (11C-PBB3 (18F-THK5351 (The radiotracers used to image tau neurofibrillary tangle deposition in living AD patient brains are 8F-FDDNP , 18F-AV1F-AV1451 , 11C-PBB-THK5351 . These t18F-THK5351 has been confirmed to also bind to monoamine oxidase B (18F-AV1451 is well known to bind to calcifications, iron, melanin, and blood vessels (However, PET tracers used for tau imaging still have some problems that remain to be resolved. These tracers often show “off-target” binding. Recently, it has become clear that these tracers detected the distribution of not only tau but also other proteins . For exaxidase B . Moreove vessels . TherefoIn this review, we introduced the various neuroimaging techniques described in the current diagnostic criteria for neurodegenerative diseases and the possibility of new neuroimaging techniques as biomarkers. We believe that further advances in these neuroimaging techniques will lead to useful biomarkers that can accurately predict the pathological changes occurring in various neurodegenerative diseases.All procedures performed in the studies involving human participants were in accordance with the ethical standards of the institutional or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent: informed consent was obtained from all individual participants included in the study.SS, DH, Hi.H, NT, YK, and YO wrote the manuscript. HS and Ha.H critically reviewed the manuscript.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.


Increasing the variety of better‐tasting and healthier gluten‐free products is important for consumers with gluten‐related disorders. This work aimed to develop a gluten‐free bread formulation containing whole chia flour with acceptable sensory properties. A mixture design for three ingredients and response surface methodology were used to identify the proportions of potato starch, rice flour and whole chia flour to achieve the best physical properties and result in sensory‐accepted products. The physical properties and visual appearance showed that whole chia flour alone is not suitable for bread production. Nevertheless, it is possible to add up to 14% whole chia flour to a rice flour‐based gluten‐free bread formulation while negligibly diminishing the loaf volume, crumb firmness and crumb moisture. The best formulations were prepared from rice flour blends with 5, 10, and 14% whole chia flour, which received overall acceptability scores of 8.7, 8.1 and 7.9 on a 10‐cm scale, respectively, similar to those of their white gluten‐free bread and wheat bread counterparts. Incorporating 5%–14% whole chia flour in the formulation increased the levels of ash, lipid, protein and dietary fiber compared to those of the white gluten‐free bread. These factors are responsible for hampering adherence to the GF diet and for general dissatisfaction  was an important staple food for pre‐Columbian societies in Central America. Following the recent evaluation of their nutritional and functional potential, chia seeds have attracted a great deal of interest in the research community and food and pharmaceutical industries, as well as among consumers  and rice flour (RF) achieving the best physical properties. Subsequently, the physical properties and sensory acceptability scores of the best formulations were compared to those of their white GFB and wheat bread counterparts.22.1® Chia omega‐3) from Santa Cruz Valley, Bolivia was supplied by R & S Blumos Comercial de Produtos Alimentícios Ltda . Whole chia flour was obtained by grinding the seeds into flour with a coffee grinder . The resulting flour was packed in polyethylene bags and stored at room temperature (approximately 25°C) prior to further use.Chia seed  and carboxymethylcellulose  were donated by Eurogerm Brasil Produtos Alimentícios Ltda. . The other ingredients were obtained at the local market.The flour/starch blends consisted of WCF, RF, and PS. The compositions were determined in triplicate by standard methods AOAC . The resThe particle size distributions were 92% 425 μm and 8% 250 μm for WCF; 24% 425 μm, 46% 250 μm, 28% 180 μm, and 2% ≤ 150 μm for RF; and 4% 425 μm, 29% 250 μm, 31% 180 μm and 36% ≤ 150 μm for PS. The particle size distributions were determined according to AOAC method 965.22 AOAC .2.2The GFB formulation consisted of the following, on a % of total flour weight basis (fwb): 100% flour/starch blend, 100% water, 25% whole egg, 10.5% whole milk powder, 6% white cane sugar, 6% soy oil, 2% salt, 0.8% instant dry yeast, 0.3% xanthan gum, and 0.3% carboxymethylcellulose. The flour/starch blend consisted of RF, PS, and WCF in blends summing to 100% (fwb), according to the experimental design.A straight dough process was performed using a stand mixer  with a paddle attachment. All ingredients were mixed at speed 4  for 4 min. The resulting dough (400 g) was then spread into previously greased and floured baking pans (19 × 7.5 × 5 cm) and proofed in a proofing chamber at 40°C and 85% relative humidity for 45 min . Baking was performed in an electric oven at 160°C for 22 min . After baking, the loaves were depanned and cooled for 2 hr on cooling racks at room temperature. The loaves were then stored in polyethylene bags to prevent moisture loss at room temperature (approximately 25°C). All analyses were performed within 3 hr.Six loaves for each of the GFB trials were prepared from one batch. Three random loaves were used for the specific volume and crumb moisture analyses, and three random loaves were used for the crumb texture evaluation and photographs. An extra six loaves from the selected treatments were produced for sensory evaluation.2.31), PS (x2), and WCF (x3) on the physical properties of GFB. The experiment was performed on three centroid point replications and included three axial points, for a total of twelve trials ls Table . The flo2.43]/ weight [g]) and the bake loss were also evaluated in triplicate.After cooling, the loaves were weighed , and the loaf volumes were measured by millet‐seed displacement  according to AACC method 10‐05.01 AACC . The loaplus, Stable Micro Systems, Surrey, UK). Texture measurements  were performed on two bread slices that were taken from the centers of three different loaves.The crumb moisture was evaluated in triplicate, according to AACC method 44‐15A AACC . The cru2.51, β2, β3, β12, β13, β23, and β123 are regression parameters; and x1, x2, and x3 are the proportions of RF, PS, and WCF, respectively, in the flour/starch blend.The bread physical properties were used as response variables for the mixture design regression models. The following Scheffé canonical polynomial models were applied:12, β13, and β23) or the ternary coefficient (β123) indicate synergistic effects, and negative values represent antagonistic effects between the ingredients  recruited from the campus via internal announcements. All the panelists agreed to taste the samples before the tests occurred and attested that they had bread‐consuming habits and did not have any allergy or intolerance to any of the ingredients present in the products. They had no gluten‐related disease and were made aware that they were tasting GFBs. The Human Research Ethics Committee of the Federal University of São Paulo approved the research protocol under number 203.145. Informed consent was obtained from all individuals in the study.The panellists scored the appearance, color, aroma, texture, taste, and overall acceptability of the formulations on a 10‐cm hybrid hedonic scale , R2 values, lack‐of‐fit tests, and diagnostic plots such as normal and residual plots. Data were processed using Statistica 12.0 statistical software .Differences in treatment means were identified by one‐way analysis of variance (ANOVA) and Tukey's test. A simple linear correlation (Pearson correlation coefficient) was also evaluated. The model adequacies were checked by variance analysis  and the higher the crumb firmness . These technological limitations of WCF use were also observed by Pizarro, Almeida, Samman, and Chang , with 72% to 97% of the variation being explained by the models. These well‐adjusted models were used to generate the contour plots for the physical properties of the GFB , and thus a lower loaf‐specific volume results in a greater firmness because of the denser crumb and more compact cells. Figure The loaf‐specific volume was inversely correlated with the crumb firmness (Figure From the regression coefficients shown in Table The focus of this study was to verify the suitability of GFBs containing WCF. Considering that the loaf volume is directly related to the crumb softness and texture acceptance (Capriles & Arêas, The results of the mixture design experiments showed that GFBs with good physical properties could be prepared with 5%, 10% and 14% WCF. These formulations present loaf volumes similar to those of two white GFBs, which were prepared with 100% RF and with 50% RF and 50% PS, but they had slightly higher crumb firmness values (trials 1 and 4 from Table The GFB formulations made from RF blends with 5%, 10% and 14% WCF were accepted, with scores for appearance, color, aroma, texture, taste, and overall acceptability ranging from 7.3 to 8.7 on a 10‐cm hybrid hedonic scale, as shown in Table The GFB formulations made from RF blends with 5%, 10%, and 14% WCF, for which the proximate compositions are presented in Table 4The application of a mixture design allowed finding that it is possible to add up to 14% WCF to an RF‐based GFB formulation while negligibly diminishing the loaf volume, crumb firmness and crumb moisture. The best formulations were prepared from RF blends with 5, 10 and 14% WCF, and they received overall acceptability scores similar to those of their white GFB and standard wheat bread counterparts. Incorporating 5%–14% whole chia flour in the formulation increased the levels of ash, lipid, protein and dietary fiber compared to those of the white GFB.This research highlights the potential of WCF for producing nutrient‐dense and acceptable GFB, which is important for consumers with gluten‐related disorders because those products often lack nutrition content and acceptability.The authors declare that they have no conflicts of interest.


Bordetella pertussis, and it has a critical role in colonization of the respiratory tract and establishment of the disease. ACT targets phagocytes via binding to the CD11b/CD18 integrin and delivers its N-terminal adenylate cyclase (AC) domain directly to the cell cytosol, where it catalyzes unregulated conversion of cytosolic ATP into cAMP upon activation by binding to cellular calmodulin. High cAMP levels disrupt bactericidal functions of the immune cells, ultimately leading to cell death. In spite of its relevance in the ACT biology, the mechanism by which its ≈400 amino acid-long AC domain is transported through the target plasma membrane, and is released into the target cytosol, remains enigmatic. This article is devoted to refresh our knowledge on the mechanism of AC translocation across biological membranes. Two models, the so-called “two-step model” and the recently-proposed “toroidal pore model”, will be considered.Adenylate cyclase toxin (ACT) is one of the principal virulence factors secreted by the whooping cough causative bacterium  Bordetella pertussis secretes several virulence factors, and among them the Adenylate Cyclase Toxin (ACT or CyaA) is crucial in the early steps of colonization of the respiratory tract by the bacterium [CyaCABD operon [2+ ions into the mentioned calcium-binding sites whose affinity constant for the cation is in the mM range [αMβ2, Mac-1, or CR3) expressed on myeloid phagocytes [The whooping cough bacterium acterium  ACT beloacterium . SynthesD operon . Gene prD operon ,5. ACT iD operon . Like otD operon , but alsD operon ,6. The hD operon ,5; a serD operon ,8,9 and D operon ,4  domain. This AC domain catalyses the uncontrolled conversion of cytosolic ATP into cAMP , which cBordetella adenylate cyclase is, together with anthrax toxin edema factor (EF), the prototype of a bacterial protein toxin that attacks the immune response by overboosting the major signalling pathways in the immune response, the cAMP and MAPK pathways, upon activation of its adenylyl cyclase activity by calmodulin [lmodulin ,21,22,23lmodulin : anthraxlmodulin ,25,26. H2-terminal fragment (AC domain) would be translocated. However, this model was rapidly discarded after the publication of a biophysical study of conductance measurements in planar lipid bilayers [+ target cells large heterologous cargo polypeptides of up to 200 residues in length within the AC domain [Almost thirty years have passed since Gordon and colleagues  suggestebilayers  in whichbilayers . Other sbilayers  and, togbilayers . This cobilayers . It is ibilayers ,29. Charbilayers . One woubilayers . Other cbilayers ,31,32,33bilayers , negativbilayers , monomerbilayers , or unfoC domain ,37,38.2+ entry into target cells independently of the ACT pore, and that the translocating AC polypeptide itself would participate in formation of a novel type of membrane path for calcium ions [2+ influx into macrophages is practically null . This is thus a weak point of the two-step model, since it cannot explain in a satisfactory way how the toxin-receptor complex can relocate into lipid rafts at low ACT concentrations, at which cell intoxication by cAMP is particularly relevant for cell intoxication by ACT.Ten years ago it was reported that ACT induced extracellular Caium ions . Three yium ions : in a fiium ions ,40. ThisOther issues await a satisfactory response, such as how can ACT translocate its AC domain into cells that do not express the CD11b/CD18 integrin receptor ,42,43 orThe ACT segment bearing adenylate cyclase activity (AC domain) extends approximately between residues 1 and 390 of the toxin polypeptide chain. The crystal structure of this AC domain in complex with the C-terminal domain of calmodulin was published more than a decade ago . The isoA fundamental premise for any polypeptide segment that has to cross directly the cell membrane to get into the cytosol is that it has to be transiently inserted into the lipid bilayer. The isolated AC domain does not, however, appear to have this ability . A proteThe segment that joins the AC domain with the hydrophobic domain of ACT, which we will refer to as “linker segment” has attracted the attention of several researchers in the last years. The ACT linker segment has no homologs in the other toxins of the RTX family. In Interestingly, it has been recently found that the linker segment appears not only to be involved in AC translocation, but it appears to modulate the pore-forming activity of ACT . It was 2 [606AG sequence (consensus motif GXSXG) and D1079GG sequence (consensus motif DXG/A) have been identified in the toxin sequence and it has been proved that Ser-606 and Asp-1079 are essential catalytic residues in the ACT-PLA1 activity, validating that ACT is a serine acylhydrolase [sn-1 and the sn-2 ester bonds of glycerophospholipid substrates, releasing into the membrane free fatty acids and lysophospholipids. However, it has no lysophospholipase activity; the toxin cleaves with notably greater efficiency the sn-1 ester bond of glycerophospholipid substrates than the sn-2 one, suggesting that ACT most likely acts as a PLA1 on natural membranes [Recently it has been revealed that ACT is a calcium-dependent phospholipase A , sharing2 . Two conydrolase . ACT canembranes . ACT hydembranes . ACT alsembranes .A significant contribution of the mentioned study has been the elucidation of the biological consequence of this novel ACT-PLA activity. It has been demonstrated that ACT-PLA activity is directly linked to the AC translocation across the plasma membrane of target phagocytes . Consist+ target cells large heterologous cargo polypeptides of up to 200 residues in length within the AC domain [The reaction products of ACT-PLA activity on plasma membrane phospholipids are lysophospholipids and free fatty acids . LysophoC domain ,37 sinceC domain ,59.Direct participation in AC domain translocation of an ACT-PLA-generated hydrophilic toroidal tunnel, in which the pore-walls may in part be lined by phospholipid head groups, may also be consistent with previous observations showing a net positive charge requirement for AC domain to be translocated . MoreoveAfter more than thirty years we still do not know what the molecular details of the ACT catalytic domain delivery into the cell cytosol are and how the 400 amino acid-long AC domain crosses the cell membrane exactly. For a long time the determination of very low single-channel conductance values for the pore presumably formed by ACT in lipid bilayers has strongly conditioned the field, prevailing the idea of a direct “non-pore model” for AC translocation. The most recent discovery that ACT is, itself, a phospholipase A and that such lipid hydrolytic activity is critical for AC domain delivery, together with the postulation of a new “toroidal pore model” has opened a debate of pore-yes, pore-not, to explain the AC transport, offering exciting possibilities to investigate new questions that arise: which are the exact protein segments that conform the proteolipidic toroidal pore for AC transport? Is the toroidal pore involved only in AC transport, or is it also responsible of the ACT-induced permeabilization and haemolysis? Might the AC domain itself be capping the lumen of the ACT pore? Could a toroidal pore be also involved in cell membrane permeabilization by other homologous RTX toxins in the family? The new discovery of ACT-PLA activity opens a new window not only to continue investigating the mechanistic and molecular details of AC transport across cell membranes, but also to explore how this ACT-PLA activity can be regulated and, thus, opens new possibilities for the therapeutic control of whooping cough, an infectious disease that is the fifth largest cause of vaccine-preventable death in infants. We envision coming years of exciting research to achieve major progress in our understanding of PLA-based AC transport across biological membranes and how to modulate it.


To gain functional insights into the LNX family, we have characterised the LNX1 interactome using affinity purification and mass spectrometry. This approach identified a large number of novel LNX1-interacting proteins, as well as confirming known interactions with NUMB and ERC2. Many of the novel interactions mapped to the LNX PDZ domains, particularly PDZ2, and many showed specificity for LNX1 over the closely related LNX2. We show that PPFIA1 (liprin-α1), KLHL11, KIF7 and ERC2 are substrates for ubiquitination by LNX1. LNX1 ubiquitination of liprin-α1 is dependent on a PDZ binding motif containing a carboxyl terminal cysteine that binds LNX1 PDZ2. Surprisingly, the neuronally-expressed LNX1p70 isoform, that lacks the RING domain, was found to promote ubiquitination of PPFIA1 and KLHL11, albeit to a lesser extent than the longer RING-containing LNX1p80 isoform. Of several E3-ligases identified in the LNX1 interactome we confirm interactions of LNX1 with MID2/TRIM1 and TRIM27. On this basis we propose a model whereby LNX1p70, despite lacking a catalytic RING domain, may function as a scaffold to promote ubiquitination of its ligands through recruitment of other E3-ligases. These findings provide functional insights into the LNX protein family, particularly the neuronal LNX1p70 isoform.Ligand of Numb protein X1 (LNX1) is an E3 ubiquitin ligase that contains a catalytic RING  domain and four PDZ  domains. LNX1 can ubiquitinate Numb, as well as a number of other ligands. However, the physiological relevance of these interactions  Ligand of Numb protein X1 (LNX1) was first characterised based on its ability to bind to the cell fate determinant protein, NUMB [Really Interesting New Gene) domain, a NUMB-binding motif (NPAY or NPAF) and four carboxyl-terminal PDZ  domains  [in vivo is very incomplete.in, NUMB . LNX1 an domains . Three mem (CNS) , 7. In aem (CNS) . The molem (CNS) , and in em (CNS) . Howeveret al. [et al. [in vivo functions of mammalian LNX proteins.The combination of a RING and one or more PDZ domains is a unique feature of the LNX family . PDZ domet al.  compiled [et al.  identifi [et al. , only a  [et al. , 16, whi [et al. , 17. The [et al. , it is iTo gain further insights into the poorly understood LNX proteins, we sought to examine the molecular interactions of the full length LNX1 protein in mammalian cells under conditions that are more physiologically relevant than previous studies. To that end, we have characterised the LNX1 interactome using affinity purification and mass spectrometry. Our results validate some known LNX interactions and identify a significant number of new ones. We show that several of these novel ligands co-localize with LNX1 in cultured cells and are substrates for ubiquitination by LNX1. In addition, we provide evidence that the neuronal LNX1p70 isoform that lacks the RING domain, may nevertheless promote ubiquitination of interacting proteins possibly through recruitment of other E3 ligases into multi-protein complexes. This proteomic analysis thus provides novel insights into LNX1 protein function.Lnx1 (p80 isoform) and Lnx2 were cloned into the pEGFP-C2 vector (Clontech). Empty pEGFP-C2 vector was used to express EGFP alone. LNX1 sequences in pEGFP-C2, for mapping interactions, encoded the following amino acids (aa) based on the LNX1p80 protein sequence: RING-NPAY motif, aa1-277; PDZ1-4, aa268-728; PDZ1, aa271-384; PDZ2, aa377-470; PDZ3, aa500-598; PDZ4, aa630-725. MID2 constructs based on transcript variant 2 of human MID2 (NM_052817) encoded the following amino acids: full length MID2, aa 1–705; RING domain—BBOX type Zinc finger-2, aa 1–235; Coiled coil–SPRY domain, aa 222–705; Coiled coil-COS domain, aa 222–406; FN3 domain—SPRY domain, aa 395–705. MID1 and TRIM 27 constructs encoded aa 1–667 of human MID1 (NM_000381) and aa 1–513 of human TRIM27 (NM_006510) respectively. Coding sequences for LNX interacting proteins were cloned into the vectors pCMV-N-HA, pCMV-N-FLAG or pDEST27 to produce proteins with amino-terminal HA, FLAG and GST tags respectively. The following antibodies were used: anti-GFP , anti-HA , anti-GST , anti-FLAG , anti-liprin-α1 , anti-liprin-α3 , anti-KIF7 , anti-NUMB , anti-AKAP13 . Horse radish peroxidase, Dylight 488 and Cy3 conjugated secondary antibodies were purchased from Jackson ImmunoResearch, while IR700 and IR800 conjugated secondary antibodies were from Li-Cor Biosciences. All other reagents were from Sigma-Aldrich unless stated otherwise.The coding sequences of mouse ® beads, as per manufacturer’s instructions (ChromoTek). Purified complexes were separated by gel electrophoresis and stained using GelCode Blue Protein Stain (Thermo Scientific Pierce). Each lane was cut into slices for mass spectrometry analysis.HEK (Human Embryonic Kidney) 293 cells (ATTC), cultured under standard conditions, were transfected with GFP and GFP-LNX1 expression constructs using calcium phosphate precipitation, and stable cell pools were selected using G418 antibiotic. To purify GFP-LNX1 and GFP protein complexes, ten confluent 15 cm dishes of cells were harvested and GFP affinity purifications performed using magnetic GFP-TrapProtein digestion, nano-liquid chromatography and MS/MS mass spectrometric analysis was performed at the Fingerprints Proteomics facility at University of Dundee, Scotland, UK. Proteins were identified by searching against the IPI protein database and data analysis was performed as previously described , 19. BriExpression vectors encoding GFP-tagged LNX1 or LNX2 constructs were transfected into HEK 293 cells, either alone or together with constructs encoding a LNX-interacting protein as previously described . BrieflyMCF7 cells (ATTC) were chosen for these experiments because of their flat morphology and good adherence to coverslips during the immunostaining procedure. They were grown under standard conditions on poly-D-lysine coated glass coverslips in a 6 well plate, were transfected with 2 μg of DNA using a calcium phosphate precipitation protocol. 24 hrs post transfection cells were washed with PBS and fixed with 4% PFA for 10 minutes at 4°C. Cells were incubated in blocking buffer  prior to antibody incubation. All antibodies were diluted in 5% goat serum and 2% bovine serum in PBS (as described ). Cells N-Ethylmaleimide (NEM) and 1x Complete protease inhibitors (Roche Applied Sciences), and boiled for 5 min. Following cooling on ice, samples were diluted with 900 μl of ice cold buffer that contained 50 mM Tris (pH 8.0), 150 mM NaCl, 1% Triton, 0.5% sodium deoxycholate, 50 mM sodium fluoride, 0.1 mM sodium orthovanadate and protease inhibitors. Lysates were cleared by centrifugation at 13,000 rpm for 30 min at 4°C. Ubiquitinated proteins were immunoprecipitated overnight at 4°C from cell lysates using an anti-HA antibody. The ubiquitination status of substrate proteins was then revealed by Western blot analysis.Expression vectors encoding LNX proteins, LNX-interacting proteins and a HA epitope-tagged ubiquitin construct were co-transfected into HEK293 cells. Twenty hours after transfection, cells were incubated in fresh medium containing 10 μM MG132 (Merck Millipore). Cells were lysed in 100 μl 1% (w/v) Sodium Dodecyl Sulphate (SDS) supplemented with 15 mM et al. [et al. [et al. [http://david.abcc.ncifcrf.gov/) using default settings [To compare interactions identified here with those from previous studies, lists of interacting proteins were taken from Wolting et al. , Guo et  [et al.  and Leni [et al.  and genesettings .t tests were performed using Microsoft Excel software. Where appropriate, data were analysed by one-way ANOVA, followed by Bonferroni post-hoc test using GraphPad Prism v.6.0 . P values of less than 0.05 were considered significant. Unless stated otherwise, all data are presented as mean ± SEM.Two tailed Student ® magnetic beads. Cells expressing GFP alone were used as a negative control for non-specific binding to either the beads or GFP tag. Purified proteins from both samples were identified by mass spectrometry. Non-specific interactions present in control GFP complexes, and likely false positives or environmental contaminants, were eliminated to generate a list of over 70 proteins specifically identified in affinity purified GFP-LNX1 complexes. The top 30 proteins ranked according to Mascot scores are shown in The majority of LNX-interacting proteins have been identified by yeast-two hybrid assays and protein/peptide arrays , 15. OnlWe chose five of the most reliably identified novel GFP-LNX1 interacting proteins from our proteomics study for further confirmation and analysis . The choice of these particular proteins was based on our ability to obtain cDNAs to make expression constructs or antibodies to detect them. We also examined two proteins (EphA7 and SynGAP1) that we previously demonstrated to interact with both LNX1 and LNX2 in yeast two-hybrid assays [We then attempted to map these interactions to particular regions or individual domains of LNX1. We used amino-terminal (RING-NPAY motif) and carboxyl-terminal (PDZ1-PDZ4) fragments of LNX1, as well as constructs encoding the individual LNX1 PDZ domains, to map these interactions within LNX1 . All theTo further evaluate the relevance of these interactions we sought to assess potential co-localisation of LNX1 with some of these interacting proteins in a cellular context. LNX1p80 was co-expressed with either HA- or FLAG-epitope-tagged interacting proteins in MCF-7 cells and detected by immunofluorescence microscopy. In this analysis we observe partial co-localisation of liprin-α1 with LNX1 in the cytoplasm, particularly in structures in the perinuclear region of co-expressing cells . This stMany PDZ domains bind to carboxyl terminal motifs of their interaction partners, with the amino acids at positions 0 and -2 relative to the carboxyl terminus being particularly crucial for recognition by PDZ domains . Liprin-p < 0.0001, F4,15 = 17.15). Ubiquitination in immunoprecipitates from cells expressing LNX1p80 was significantly stronger than in those from cells transfected with the control vector (p < 0.0001), as revealed by Bonferroni post-hoc test, indicating that LNX1p80 strongly promotes ubiquitination of liprin-α1. Surprisingly, cells expressing LNX1p80 containing the catalytically inactive RING-finger domain, (LNX1p80C48A) [p < 0.05), which lacks the RING finger domain required for ubiquitination, and LNX2 (p < 0.05), compared to the control vector  were identified in the LNX1 interactome we sought to further characterize some of these interactions . We focuWe next sought to confirm the interaction of LNX1 with MID1 and TRIM27. Surprisingly, no interaction with LNX1p80 was seen for transfected epitope-tagged MID1 under the same experimental conditions for which MID2 binding was observed . HoweverGiven the robust interaction of MID2 with the LNX1 PDZ domains we sought to test the idea that the LNX1p70 isoform, while lacking its own RING domain, might target the ubiquitin ligase activity of the MID2 RING domain to proteins that bind the LNX1 PDZ domains. To do this we examined whether MID2, LNX1p70 and the LNX1 ligand liprin-α1 could form a ternary complex . While LIn an analysis of the human PDZ domain-ligand interactions network (PDZNet), LNX1 ranked third in terms of total number of interactions . The larOur goal in characterising the LNX1 interactome was to gain insights into potential functions of LNX proteins and to identify differential functions of LNX1 and LNX2. We used the DAVID Bioinformatics Resource  to identLnx1 and Lnx2 genes arose by gene duplication early in the vertebrate lineage [Lnx1/2-like gene is more similar to Lnx2, indicating that Lnx1 may have may have undergone neofunctionalisation following the gene duplication[The  lineage . The invplication. We idenplication, 15. Theplication and someWe have characterised the interaction with liprin-α1 in more detail by verifying that the interaction is dependent on the carboxyl-terminal–YSC* motif and showing that liprin-α1 is a substrate for ubiquitination by LNX1p80. However, ubiquitination of liprin-α1 by LNX1 did not significantly alter liprin-α1 protein levels. This suggests that LNX1-mediated ubiquitination does not target liprin-α1 for proteasomal  degradation, but perhaps affects some other aspect of liprin-α1 function. While liprin-α1 is widely expressed in many tissues, liprin-αs are best characterised as a component of the presynaptic cytomatrix of the active zone (CAZ) complex, that is involved in synapse maturation in neurons . While Lhttp://www.ebi.ac.uk/intact/) that MID2 can dimerize with TRIM27, and so we cannot completely rule out the possibility that the interaction of TRIM27 with LNX1 might be mediated by endogenous MID2 in our experiments.These findings suggest that the neuronal LNX1p70 isoform may be able to recruit other E3 ligases to mediate ubiquitination of substrates that bind to its PDZ domains. Notably we identified six E3-ubiquitin ligases in our LNX1 interactome  and some were identified in previous studies . MID1/TRTo explore whether MID2 might be responsible for the ability of LNX1p70 to promote ubiquitination of liprin-α1 we tested the ability of these three proteins to form a ternary complex. Robust binding of LNX1p70 to GFP-tagged MID2 was observed, but co-purification of liprin-α1 was not observed in this experimental setup. It may be that binding of MID2 and liprin-α1 to LNX1 is competitive, or that the formation of a ternary complex is very transient. While we were not able to obtain direct evidence for LNX1p70 acting to scaffold an interaction of MID2 with liprin-α1 in this case, it will be interesting to test this hypothesis for other combinations of LNX1-interacting E3 ligases and substrates including TRIM27 and KLHL11. Such a mechanism, if proven, could explain the existence and conservation of the LNX1p70 isoform in diverse vertebrate species despite its lack of catalytic activity .in vivo since ERC2 (and the isoform of ERC1 that has the IWA* motif) are exclusively expressed in the brain [in vivo. Mice lacking LNX2 as well as the neuronal LNX1p70 and p62 isoforms have recently been shown to exhibit decreased anxiety-related behaviour [Another component of the presynaptic CAZ complex that we identified is the known LNX1 interacting protein ERC2/CAST1. ERC2 was previously shown to bind to LNX1-PDZ2 via a carboxyl terminal IWA* motif and to co-localise with LNX1 in neurons . We showhe brain , whereashe brain . Insteadhe brain , is perhehaviour . Notablyehaviour , 34, as ehaviour , while MS1 Table(DOC)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


The human protein disulfide isomerase (hPDI), is an essential four-domain multifunctional enzyme. As a result of disulfide shuffling in its terminal domains, hPDI exists in two oxidation states with different conformational preferences which are important for substrate binding and functional activities. Here, we address the redox-dependent conformational dynamics of hPDI through molecular dynamics (MD) simulations. Collective domain motions are identified by the principal component analysis of MD trajectories and redox-dependent opening-closing structure variations are highlighted on projected free energy landscapes. Then, important structural features that exhibit considerable differences in dynamics of redox states are extracted by statistical machine learning methods. Mapping the structural variations to time series of residue interaction networks also provides a holistic representation of the dynamical redox differences. With emphasizing on persistent long-lasting interactions, an approach is proposed that compiled these time series networks to a single dynamic residue interaction network (DRIN). Differential comparison of DRIN in oxidized and reduced states reveals chains of residue interactions that represent potential allosteric paths between catalytic and ligand binding sites of hPDI. As the first discovered protein-folding catalyst2, hPDI works as both an enzyme and a chaperone in various cellular processes including the oxidative stress, unfolded protein response, apoptosis and viral membrane fusion, using thiol disulfide exchange reactions6. A wide range of multifunctional features of hPDI are tightly associated with its unique molecular architecture. The horseshoe-like structure of hPDI is composed of four thioredoxin-like domains named a, b, b’ and a’  and reduced (red-hPDI) states, respectively10. Conformational transition of red/ox-hPDI is under the influence of a disulfide bond in the a’ domain, which leads to the rearrangements of b’ and a’ domains, followed by switching its enzymatic activities11. Despite the available crystal structures of both states7, different functions of these conformations still conceal behind their dynamical complexities. Some studies have investigated the relations among the inter-domain flexibility and its effects on global domain motions of partial and full human and yeast PDI using limited proteolysis, SAXS, intrinsic fluorescence and NMR spectroscopies15. However, limited differences have been reported between these two redox states in terms of their distinct dynamical functions. From this perspective, identification of the structural determinants that control the dynamical behavior of each state is crucial for better understanding of their functions.The human protein disulfide isomerase (hPDI), is one of the most abundant redox-regulated molecular chaperones accounting for the folding of almost one-third of proteins in cells16. Some tools such as RINerator and RING have been utilized to construct residue interaction networks (RIN) by considering geometrical, physicochemical, evolutionary and energetics of each residue18. Such networks could be used to compare and find hotspot players within the protein structure. However, the application of RIN is limited to the analysis of a single static snapshot of the protein structure obtained from experimental or computational resources. On the other hand, MD simulations are capable of producing a large number of conformations resulting from the time evolution of a protein. In these cases, each snapshot of the MD trajectory could be mapped to a corresponding RIN, and the dynamical analysis of such a huge number of networks would be a challenging task.Differences in the dynamical behaviors of ox- and red-hPDI are the results of complex geometrical and physicochemical interplays of many residues as the structural units. An important question is which residues play more critical roles and through what mechanisms. In recent years, conversion of the 3D structure of proteins into a 2D network of interacting residues has found useful applications in dealing with the complexity of biomolecular structuresIn the present work, we used MD simulations to reproduce the dynamical behaviors of hPDI in its oxidized and reduced states, independently. Regularities in large-scale domain motions of protein were then obtained through principal component analysis. Over the ensemble of the generated configurations, statistical machine learning methods were conducted to extract structural features that are related to the redox-dependent dynamics of proteins. By converting the dynamically sampled configurations to a sequence of residue interaction graphs, an approach was suggested, which provided a network based analysis of the dynamical differences of residue interactions between the two oxidation states.Time series of backbone (root mean square deviation) RMSD values were monitored with respect to the initial structures to check whether the recorded atomic movements were caused by the collective domain motions or irregular random ones. Some trajectories showed RMSD values as large as 15 Å for whole protein backbone while the single domain values were mostly less than 2 Å . The cumulative contribution of principal components (PCs) in total variance is shown in Figure e Figure . The phyIt should be noted that while the hinge motions of a and a’ domains were more highlighted in the first four PCs, there was a minor clockwise and counterclockwise twisting motion in b and b’ domains which increased the amplitude of movements in the terminal domains. A quite different form of collective motion could be found in PC5 and PC6, which was a twisting of terminal domains around ab and a’b’ inter-domain axes, respectively Figure . Althoug20.Projections of free energy landscape (FEL) on two-dimensional spaces spanned by PC1-PC2, PC1-PC3 and PC2-PC3 were plotted in Fig. 21. With application of linear classifiers, we aimed to determine whether some structural features  could discriminate ox and red snapshots of MD simulation by a simple threshold. We also used Support Vector Machine (SVM) with radial-basis kernel as a widely-used non-linear classifier since it could highlight more-complicated discriminative patterns in structural features such as bimodal distributions of domain level features (see Methods for more details).To uncover which structural features of the hPDI could better discriminate between ox and red states, we employed both linear and non-linear classification methodsb’a’ with an accuracy of 93%. The next most discriminative features were the inter-domain angle ѳabb’ and inter-domain distance Rab’ with classification accuracies of 80% and 76%, respectively. Different red/ox behavior of Rb’a’ was also reflected in its separated distribution of values over ox- and red-hPDI trajectories in Fig. abb’ and Rab’ had a considerable Red/Ox overlap but showed that the a and b’ domains could be in closer contact with each other in ox-hPDI. Fully closed conformations as identified on ox-hPDI FEL were thus the result of the elongated distance between b’ and a’ domains, which permits domain a to be in close contact with b’. The wide range of sampled Rb’a’ values in both ox- and red-hPDI was consistent with previous works that report the X-linker as the most flexible region of the protein9. However, it seems that the oxidized state was affected by the flexibility of the X-linker, to a higher extent. The underlying mechanism resulting in different values of Rb’a’ in ox- and red-hPDI systems is discussed below. X-ray structures suggest different restricted values for Raa’ in ox- and red-hPDI but as could be seen in Fig. Accuracy of linear and non-linear classifiers in discrimination of red/ox states, based on different domain level features, were reported in Table dij was presented as a residue-residue discrimination accuracy matrix . This loop had a conserved ω-shaped structure over red-hPDI trajectories that became slightly distorted in ox-hPDI state.Accuracy of the linear method in the case of residue-residue distances rix Fig. . Each el of Fig. . It shoud53,56 and d397,400 were 62% and 99%, respectively, as was evident from their distributions in Fig. 10.Another important emerged from the linear classifier accuracies was the differential red/ox behavior of Cys-Cys distances in domains a and a’. Accuracy of the red/ox classifications based on 22. There are some methods that examine the causality of the perturbations within the allosteric path24. Here, the time evolution of residue interaction networks were compiled to a dynamic residue interaction network named as DRIN. Since the DRIN graph reflected long lasting non-covalent interactions, a differential comparative DRIN graph could highlight central residues that are common or dominated in any of the ox- or red-hPDI systems. This representation was depicted in Fig. hPDI conformational changes are influenced by the allosteric redox regulation of disulfide bonds. Both causality and energetics of the allosteric pathway identify the mechanisms governing the regulation of this actionTo test the importance of the identified hub residues of the DRIN graph on the functional activities of hPDI, we examined the situation of previously reported point mutations in differential DRIN graph Table . Most ofbb’ by considering salt-bridges between D227, D210 and K208 in b domain with K285, K276 and E242 in the b’ domain, respectively. These interactions which were mainly red-dominated locked the Rbb’ around its unimodal distribution of values while their absence in ox-hPDI permitted two domains to rest in two different distances as reflected by bimodal form of Rbb’ distribution  and LB14-B15 (e.g. E322 and M324) of the b’ domain to LB17-H15  and LB18-B19  of the a’ domain through hydrogen bonds, van der Waals and π-π stacking interactions  in the b’ domain to LB19-B20 (R452) in the a’ domain via hydrogen bond and salt-bridge interactions  were connected to each other in the hydrophobic pocket of b’ domain by involvement of LB14-B15 . As mentioned earlier, residues within the H11 and LB14-B15 interacted with a’ domain in red-hPDI through pathway I. In addition, residues around H9 also form red-dominated interactions with the b domain  based on hydrogen bonds, salt-bridge and van der Waals interactions. On the other hand, the H9 position was controlled by the formation of ox-dominated interactions in the oxidized state (e.g. D181—K254). These results highlighted the importance of inter-domain long-lasting interactions in substrate selectivity of oxidized and reduced hPDI.Along with pathways I and II between the b’a’ domains, additional chains of interactions seem to adjust the size of hydrophobic pocket in oxidized and reduced conformations. As can be seen in Fig. 28. Redox-associated structural changes are the key intrinsic feature to determine and control hPDI cross-functioning. For this propose, many studies have focused on finding the central residues that might play critical roles in manipulating different functions of hPDI29. In this study, we aimed to identify the redox-specific structural features of hPDI at residue and domain levels.hPDI plays diverse roles in various pathophysiological conditions such as the immune responses, coagulation, angiogenesis and even viral infection  method were applied35. To accelerate the conformational sampling, an initial 10 ns trajectory was clustered based on backbone RMSD in the VMD 1.9 package36 and twelve representative structures (six for each system) from most populated clusters were used as starting points of 55 ns NPT dynamics. The whole procedure results in a sample of 156,000 structures from a total of 600 ns NPT simulation. More methodological details of MD simulations can be found in the supplemental text.X-ray crystal structures of ox-hPDI (PDB ID: 4EL1) and red-hPDI (PDB ID: 4EKZ) were used as the initial conformations for MD simulations37. The overall rotation and translation of the protein were eliminated by alignment from Cα atoms of domains b and b’, since the bb’ pair of domains acts as a base for domain motions. To identify groups of residues with correlated motions, cross-correlation matrix of the atomic displacements was calculated38 (see SI text for details). To quantitatively address domain motions, some inter-domain geometric variables were calculated over trajectories. Six inter-domain distances denoted as Rij, two inter-domain angles Θabb’ and Θbb’a’, and one torsion angle Φabb’a’ were defined based on the domain centers  Domain level features including Rij, Θabb, Θbb’a’ and Φabb’a,ii) Residue level features including pairwise residue-residue distances dij, defined as the Euclidean distance between Cα atoms of residues i and j  and higher SVM accuracies for five other features , which confirmed our hypothesis and also depicted that SVM models did not overfit to the training data (Table bb’ had the highest elevated accuracy between SVM and linear models (80% vs. 67%), which was due to the bimodal distribution of Rbb’ in ox conformations  was considered. From time series of Aij(t), the maximum interaction life time, ij values between ox- and red-hPDI systems. The elements of the differential DRIN matrix Δ were computed for each type of interaction as equation Supplementary_Info


Our studies therefore support a model wherein substantial avidity gains during assembly-mediated capsid recognition by TRIM5α come in part from tailored spacing of tethered recognition domains.Restriction factors are important components of intrinsic cellular defense mechanisms against viral pathogens. TRIM5α is a restriction factor that intercepts the incoming capsid cores of retroviruses such as HIV and provides an effective species-specific barrier to retroviral infection. The TRIM5α SPRY domain directly binds the capsid with only very weak, millimolar-level affinity, and productive capsid recognition therefore requires both TRIM5α dimerization and assembly of the dimers into a multivalent hexagonal lattice to promote avid binding. Here, we explore the important unresolved question of whether the SPRY domains are flexibly linked to the TRIM lattice or more precisely positioned to maximize avidity. Biochemical and biophysical experiments indicate that the linker segment connecting the SPRY domain to the coiled-coil domain adopts an α-helical fold, and that this helical portion mediates interactions between the two domains. Targeted mutations were generated to disrupt the putative packing interface without affecting dimerization or higher-order assembly, and we identified mutant proteins that were nevertheless deficient in capsid binding  TRIM5α is a cytosolic protein that provides effective protection for mammalian cells against retroviral infection. This anti-viral defense mechanism is an unprecedented example of how the cell can recognize entire capsids, which are large, megadalton-sized particles. TRIM5α achieves this by assembling into a hexagonal scaffold that coats the capsid. An important unresolved question is how the capsid-binding SPRY domain of TRIM5α is positioned to optimize its contact points on the capsid surface. Here, we use a variety of techniques to determine that the SPRY domains in the TRIM lattice are organized in pairs and likely to be stably tethered against the hexagonal scaffold. Such an arrangement maximizes the avidity of capsid binding, and allows TRIM5α to act as a “molecular ruler” that matches the spacings and orientations of the capsid subunits. TRIM5α is a restriction factor that recognizes and binds the incoming cores of retroviruses such as HIV –3, and rin vitro TRIM5α/capsid complexes and crystal structures of individual domains and fragments of TRIM5α and other TRIM proteins [Retroviral capsids are higher-order macromolecular assemblages composed of about 1,500 viral CA protein subunits, which assemble on a hexagonal lattice of several hundred hexamers and 12 pentamers , 19. Accproteins , 22, 23.The L2 linker that connects the SPRY domain to the coiled-coil is likely to facilitate positioning of the SPRY domain. Mutagenesis studies have shown that an intact L2 sequence is required for efficient retroviral restriction , 25, and281PDLKGMLDMFRELTDARRYW300) [In the published crystal structure of the TRIM5 B-box 2/coiled-coil/L2 fragment, both of the subunits in the antiparallel dimer had substantial disorder in their L2 regions, but in one subunit the L2 C-terminus was folded into a short α-helix . CrystalRRYW300) .In a control experiment, we first confirmed that labels appended to the main coiled-coil helix (W196R1) had a single distance distribution peaking at the expected distance–about 3 nm–between the two labels . In contTm) was about 41°C. The SPRY domain alone also displayed a single transition, consistent with its monomeric configuration in isolation, with a Tm of about 50°C (green curve). The CC-L2-SPRY protein (maroon curve) had an intermediate Tm about halfway between CC-L2 and SPRY. Most notably, the CC-L2 profile had significantly elevated signals at the start of the experiment, which is distinct from the flat profiles of CC-L2-SPRY and SPRY  with fortuitously well-resolved sequential proton-amide cross-peaks in an 15N-filtered NOESY spectrum  are indeed helical and appear to pack stably against the main body of the domain . Howeverspectrum . The absGiven the above results and in the absence of an experimentally determined structure as yet, we computed a molecular model of the CC-L2-SPRY dimer  and desiTm = 37°C), whereas the others  had no effect or even slightly increased the apparent Tm compared to the wildtype control . Consistent with the thermal shift data, SEC-MALS showed that D186A , I193A , and E20Tm of 50°C , consistent with the less severe biophysical defects observed in the thermal stability assays. E197A pelleted efficiently with the tubes (61%), although we consider this mutant to be also an intermediate binder because in four independent experiments it reproducibly showed high levels of background pelleting (20–30%), likely due to aggregation. For reference, these levels of residual binding are similar to those observed for the R121E and W117E mutations, which disrupt higher-order interactions mediated by the B-box 2 domain and essentially abolish restriction activity , 15. Amoin vitro [in vitro assays, produced virtually no cytoplasmic bodies . Thus, SPRY/coiled-coil interactions do not appear to be fundamentally essential to recognition, but rather help to optimize avidity and maximize binding efficiency. This is consistent with studies showing that artificial restriction factors can be created by appending exogenous capsid-binding domains to the TRIM5α tripartite motif –50. In tAn interesting counter-example is TRIMCyp, which contains the TRIM5α tripartite motif but harbors a cyclophilin domain for capsid binding instead of SPRY . The L2 133–300 (CC-L2) proteins were expressed and purified as described [133–300 proteins were expressed as a His-SUMO-tagged construct in E. coli BL21(DE3) cells using the autoinduction method [133–497 (CC-L2-SPRY) was made from the TRIM5-21R construct by deleting the RING and B-box 2 domains, expressed in Sf9 cells, and purified in the same manner as the full-length protein. All single point mutations were introduced into the appropriate constructs using the Quikchange method (Agilent) and mutants were expressed and purified in the same way as their respective wildtype constructs. Given the established propensity of TRIM5 proteins to spontaneously aggregate or assemble in vitro, biochemical assays were performed as soon as possible after purification. SPRY constructs lacking the V1 loop were expressed as His-GB1 fusion proteins (derived from a plasmid kindly provided by D. Ivanov) and purified as described [Recombinant TRIM5-21R and TRIM5αescribed –11. Brien method . The tag133–300 harboring the W196C, D288C, E292C, and W300C mutations were briefly incubated with excess dithiothreitol to reduce the exogenous cysteines, then exchanged into labeling buffer  using a desalting column. The proteins were then incubated with excess MTSL methylmethanesulfonothioate) overnight at 4°C. After removing unreacted label with a desalting column, the labeled proteins were concentrated to 20 μM. Deuterated glycerol (Cambridge Isotope Laboratories) at approximately 10% final concentration was added to the samples before freezing. DEER EPR measurements were performed and analyzed as described previously [Purified TRIM5αeviously . In brieeviously  were pereviously . The valeviously .Thermofluor melting assays were performed as previously described , with fi15N and/or 13C by growing transformed bacteria in minimal media supplemented with 15NH4Cl and/or 13C-glucose. Assignments for SPRY292-497 were kindly provided by D. Ivanov [281-497 by using the following experiments: 15N/1H HSQC [15N-edited NOESY-HSQC [SPRYΔV1 constructs for NMR experiments were uniformly labeled with . Ivanov . Resonan/1H HSQC , CBCA.These experiments were performed as described . PurifieIn vitro pull-down assays with disulfide-stabilized HIV-1 CA tubes and purified TRIM5-21R proteins were performed as described [escribed .Assembly and negative stain electron microscopy imaging of the I193A and E201A TRIM5-21R mutants were performed as described .The hemagglutinin (HA)-tagged and/or yellow fluorescent protein (YFP)-labeled rhesus TRIM5α and owl monkey TRIMCyp constructs were generated as previously described . OverlapZ-stack images of each cell line were acquired by using identical acquisition parameters. Deconvolved images were analyzed for fluorescent cytoplasmic bodies by using the Surface Finder function of the Imaris software package (Bitplane).HeLa cells  stably expressing YFP-tagged TRIM5α proteins were plated onto fibronectin-treated coverslips, allowed to adhere, and fixed with 3.7% formaldehyde and stained with DAPI. Images were collected with a DeltaVision microscope (Applied Precision) equipped with a digital camera , using a 1.4 numerical aperture objective lens, and were deconvolved with SoftWoRx deconvolution software (Applied Precision). Vesicular stomatitis virus G protein (VSV-G)-pseudotyped R7ΔEnv HIV-GFP was produced by transfecting HEK293T cells as previously described . Virus iS1 FigLeft panels: Uncorrected DEER traces, V(t)/V(0), for each of the four mutants. The mutant W196R1 that is labeled within the coiled-coil helices produce a noticeable DEER echo, indicating a well-defined distance. Red lines indicate the background form factor. Right panels: Corrected dipolar evolution curves, F(t)/F(0), after subtraction of the background form factor. The red traces represent the best fits to the DEER data, and yield the distributions shown in (TIFF)Click here for additional data file.S2 Fig281-497).Top panel: Residues that undergo the largest chemical shift changes  in comparing the two constructs are encircled and labeled. Bottom panel: Enlarged view of central region containing helical resonances, with complete residue assignments for the longer construct (SPRY(TIFF)Click here for additional data file.S3 Fig(A) Anion exchange chromatography reveals the relative fractions of monomer and dimer species at the start of purification. Wildtype TRIM5-21R elutes as a doublet peak, with the minor monomer fraction eluting early and the major dimer fraction eluting late. Blue curve = UV absorbance trace. Brown curve = conductivity trace arising from application of a linear salt gradient. (B) Size exclusion chromatography of pooled anion exchange fractions allows further separation of contaminating monomers from the desired dimer species. (C) Representative anion exchange profiles of class I mutants indicate significantly elevated monomer fractions. (D) Representative anion exchange profiles of class II mutants.(TIFF)Click here for additional data file.S4 Fig(A-D) Representative images of HeLa cells stably expressing the indicated constructs. Cytoplasmic bodies appear as green puncta. DAPI was used to stain nuclei blue. Scale bars = 10 μ. (E) The number of cytoplasmic bodies was counted in each cell and normalized to the intracellular YFP concentration.(TIFF)Click here for additional data file.


This can be used as a guideline for the understanding of domain processes in lead-free piezoelectric ceramics and provide further insight into the mechanisms of structure–property relationship in these technologically important material families.Recent advances in the development of novel methods for the local characterization of ferroelectric domains open up new opportunities not only to image, but also to control and to create desired domain configurations (domain engineering). The morphotropic and polymorphic phase boundaries that are frequently used to increase the electromechanical and dielectric performance of ferroelectric ceramics have a tremendous effect on the domain structure, which can serve as a signature of complex polarization states and link local and macroscopic piezoelectric and dielectric responses. This is especially important for the study of lead-free ferroelectric ceramics, which is currently replacing traditional lead-containing materials, and great efforts are devoted to increasing their performance to match that of lead zirconate titanate (PZT). In this work, we provide a short overview of the recent progress in the imaging of domain structure in two major families of ceramic lead-free systems based on BiFeO Piezoelectric materials exhibit a unique ability to expand under external electric field or to develop a charge under applied mechanical stress, combining high coupling coefficients with exceptional stability and low cost. They are used in a variety of devices, including resonators, ultrasound generators, and actuators realizing precise nano-motion ,2,3,4,5.0.5Na0.5)TiO3 (BNT), BaTiO3 (BT), BiFeO3 (BFO), KNbO3, NaNbO3, and solid-solutions on their base; (ii) non-perovskites—i.e., bismuth layer structured ferroelectrics and tungsten-bronze-type compounds. However, their piezoelectric properties were found to be inferior compared to those of PZT. It was shown that—similarly to PZT—the properties could be improved by forming solid solutions of KNbO3 with NaNbO3 (KNN), so as to sinter ceramics with a composition close to the region of structural instability known as the polymorphic phase boundary (PPB) regulated by the temperature and the morphotropic phase boundary (MPB) controlled by doping. Similar to PZT, the properties of ceramics are also governed by the domain structure and the grain morphology, which are in turn determined by the defect transport and controlled by the sintering method. Such complex interplay of the physical and chemical properties of lead-free piezoelectric ceramics has been recently rationalized in terms of domain wall conductivity and the diffusion of charged defects . Be. Be89]. Commonly, the influence of rare earth element doping on the domain and phase structure results in: (1) transformation of the periodical domain structure to speckle-like domains with smaller sizes; (2) appearance of non-polar/antipolar phase clusters  demonstrated concerted change of both crystal structure and piezoresponse contrast distribution [R3c–Pbam–Pnma phase transformations. A similar effect of doping was revealed by TEM in Nd- and Sm-doped BFO compositions [R3c symmetry, and exhibited superstructure and orientational and translational domains characteristic of an antiphase-tilted ferroelectric perovskite [3 [TEM and PFM studies of Biribution . The charibution . At 8 moribution . PFM conositions ,95. The 3+ and Dy3+ ions, hampering the formation of homogeneous solid solutions. The coexistence of the regions demonstrating a distinct PFM contrast with the areas showing a zero piezoresponse was observed for the x = 0.15 compound.Local piezoresponse in Dy-substituted BFO is approximately three times weaker than in undoped ceramics, thus pointing to a smaller value of the spontaneous polarization and effective piezocoefficient . This ca0.9Gd0.1FeO3 was approximately two times weaker as compared to undoped BFO ceramics, due to a smaller value of the spontaneous polarization [The piezoresponse in Birization .Domain structure of BFO ceramics with heterovalent substitution by Ca, Sr, Pb, and Ba ions was studied in Reference . The domMn-doped BFO samples exhibit a higher volume density of the domain walls than those of undoped ones, suggesting that the Mn ion can effectively reduce the domain size in BFO . 0.9Pr0.1Fex1−ScxO3 (0.01 ≤ x ≤ 0.07), which had a maximum for about 3% Sc and 2% Pr [intE generated by excess electrons and charge defects like oxygen vacancies. These electrons trapped near the interface with the bottom electrode can form defect dipoles aligned during the formation of domain structure, and result in an internal bias field oriented towards the electrode.Negative self-polarization (or polarization offset) was found in the Pr and Sc co-substituted Bind 2% Pr . Negativ3 represented stripe domains with different orientations [Domain structure in Co-, Ni-, Zn-, Nb-, and W-modified multiferroic BiFeOntations . The domntations . Obtainentations .3-xLaFeO3-0.05La2/3TiO3 ceramics has been separated into domains with different length scales [Domain structure in BiFeOh scales : fine sc33 measured at 1 Hz ranged from 33 pm/V at 24 °C to 118 pm/V at 262 °C. Dielectric permittivity demonstrates a similar trend and reaches a value above 3000 at elevated temperatures and low frequencies. This effect was attributed to Maxwell–Wagner relaxation from the grain boundaries and reversible motion of the conductive non-180° domain walls. The addition 9% of Dy stabilized piezoelectric properties up to 350 °C, while BFO doped with Sm and Gd demonstrated strong decrease of d33 at temperatures above 200 °C [As expected, the values of piezoelectric coefficients in RE-doped BFO ceramics decrease significantly with decreasing temperature ,102. Stae 200 °C .3-based ceramics [Generally, the properties of ferroelectric and piezoelectric ceramics strongly depend on the grain size . The effceramics .The main disadvantage of BFO ceramics for piezoelectric applications is the combination of high electrical conductivity and high coercive field . Due to 2) under stepwise-increasing DC voltage  unAnother approach for the evaluation of the switching properties of doped BFO-based ceramics was demonstrated in Reference . The bi-Bmm2 structure [C direction, and 12 polarization orientations are permissible in the orthorhombic phase 0.935Li0.065NbO3 and (K0.50Na0.50)0.92Li0.08NbO3 ceramics is due to the depoling effect attributed to the formation of 180° domains (The poor stability of piezoelectric properties (aging) found in  texturing of the ceramics results in an excellent path for stress relief by the alignment of polar orientations and probably by increasing the grain size that contributes to better accommodation of stresses.The polymorphic phase boundary typically results in a decrease of polarization rather than in an enhancement of dielectric and piezoelectric properties, as for MPBs in piezoceramics . One of o et al.  were attDomain structure transformation as a function of temperature has been studied by PFM . The tra0.4Na0.6)(Nb0.96Sb0.04)O3-0.04Bi0.5K0.5Zr0.85Sn0.15O3 ceramics [Domain structure transformation with temperature was also studied in 0.96(Kceramics . The proceramics . At high0.02(K0.45Na0.55)0.98NbO3 (LKNN) ceramics demonstrated a clear dependence of domain structure on the grain size [0.5Na0.5)NbO3-0.05BaTiO3 (KNN-BT) ceramics with different secondary milling times, leading to different grain sizes. Reduction of the grain size from 14 to 1 μm increased the maximum dielectric permittivity from 2600 to 5000. This fact was attributed to high internal non-uniformly distributed stress in grains resulting in an easier domain wall motion [Liain size . A decrel motion .Dependence of the local domain switching on the domain structure period was observed in Reference . It was Rapid decrease of the piezoresponse amplitude and remarkable broadening of the local loops were obtained in the tetragonal phase. It was assumed that nucleation of new domains contributed to polarization reversal, meaning higher remnant polarization and maximal switchable piezoresponse. The transition into the tetragonal state was accompanied by a change in local polarization switching kinetics: the domain nucleation under the PFM tip occurred at a larger bias voltage, while propagation of the new domain was faster than in the orthorhombic phase. The less saturated local piezoelectric hysteresis loops and increased difference between the maximal and remnant piezoresponse indicated that the switching was incomplete and unstable. Thus, higher temperature promoted the effect of backswitching of the reversed domains.Domain wall vibrations under the action of applied electric field  ,129,130 Emission of elastic shear waves from ferroelastic domain walls ,134,135.Piezoelectric sound generation by laminar stacks of 180° domains (piezoelectric resonance of domains) ,134,135.Maxwell–Wagner effect of the conductive domain walls .Relation between domain structure and dielectric permittivity in wide frequency range is well known in piezoelectric ceramics ,127,128.However, some experimental data exists related to direct measurements of the influence of domain structure on the resulting properties of ferroelectric ceramics. Strong dielectric dispersion in the MHz to GHz range was found in pristine KNN ceramics, and was attributed to the dynamics of ferroelastic–ferroelectric domain walls . The oscThe nonlinear dielectric response at low frequencies and irreversible extrinsic contribution to dielectric permittivity found in KNN-LTS ceramics were strongly dependent on the domain configuration , determi5+-modified KNN-based ceramics, domain-wall vibrations were found to change from the resonance to relaxation mode due to a substantial increase of damping constant [In the Sbconstant . Doping constant . Sufficiconstant .The role of domain structure and nanodomains appeared close to the PPT region in KNN ceramics remains poorly studied. Significant variation in the electrical and mechanical properties was observed in KNN before and after poling . The hig2+ ions entered in B-positions, resulting in the formation of oxygen vacancies that pinned domain walls and concomitant increase of the coercive field [Hayati et al. found that the addition of nanosized ZnO additive increased the grain size of the KNN ceramics, with a corresponding increase of the domain size. This increase was accompanied with a reduction of the coercive field . For higve field .3 ceramics through 71° and 109° domain wall reorientation was shown and quantified using analysis of X-ray diffraction band intensities [4+ and Fe3+ sites [An exhaustive discussion about indirect measurements of domain wall input to dielectric and piezoelectric properties of BFO was reviewed by Rojac and co-authors . The polensities . The nonensities . The domensities . The higThe domain dynamics plays an important role in the physical properties of lead-free piezoelectric ceramics properties, and, as such, should be rigorously studied. The rapid development of modern microscopic techniques gives the opportunity not only to visualize domain structure, but also to quantitatively determine the orientation and values of spontaneous polarization and piezoelectric coefficient locally. The morphotropic and polymorphic phase boundaries existing both in BFO and KNN are principally coined to the transformation of the domain assemblages and phase structure. Ferroelectric nanodomains and inhomogeneously distributed mixed phases appear due to doping and peculiarities of the sintering process, and are extremely important for future applications. This review is one of the first attempts to describe the domain structure and local piezoelectric properties and their impact on the macroscopic performance of lead-free piezoelectric ceramics based on BFO and KNN.