MUC4/Y can be a model of MUC4  in pancreatic cancer. More importantly, based on MUC4/Y with the appropriate length of gene sequence, it is easy to construct the unique domain-lacking models of MUC4/Y (MUC4) for research. The present study focuses on investigation of the respective role of the unique NIDO, AMOP, and vWD domain or their synergistic effect on MUC4(MUC4/Y)-mediated functions and mechanisms by series of in vitro assays, sequence-based transcriptome analysis, validation of qRT-PCR & Western blot, and systematic comparative analysis. Our results demonstrate: 1) NIDO, AMOP, and vWD domain or their synergy play significant roles on MUC4/Y-mediated malignant function of pancreatic cancer, downstream of molecule mechanisms, particularly MUC4/Y-triggered malignancy-related positive feedback loops, respectively. 2) The synergistic roles of three unique domains on MUC4/Y-mediated functions and mechanisms are more prominent than the respective domain because the synergy of three domain plays the more remarkable effects on MUC4/Y-mediated signaling hub. Thus, to improve reversed effects of domain-lacking and break the synergism of domains will contribute to block MUC4/Y(MUC4) triggering various oncogenic signaling pathways.MUC4 mucin is well known as an important potential target to overcome pancreatic cancer. Three unique domains  with unclear roles only present in MUC4 but are not found in other membrane-bound mucins. Our previous studies first reported that its splice variant,  As a high-molecular-weight member of the transmembrane mucin family , 2, MUC4abbr. FL-MUC4. NCBI Reference Sequence: NM_018406.6) contains 26 exons, which encode various functional domains from the amino end to carboxyl end, in order as follows: a 27 residue signal peptide, serine & threonine-enriched imperfect repetition motifs, a centrally located large tandem repeat (TR) domain, nidogen (NIDO)-like domain, adhesion-associated domain , von Willebrand factor , and three EGF-like domains  and [MUC4/Y over-expressing groups vs control group] revealed that the expression levels  of most DEGs of MUC4/Y triggering were reversed afforded by domain-lacking to a variable extent. Altered gene expression was then confirmed by carrying out qRT–PCR and Western blotting as follows, which results in variation trends were consistent with sequence-based transcriptome analysis.Altogether, the above enrichment results and analyses of GO function and the KEGG pathway show the global function triggered by NIDO(N), or AMOP(A), or vWD(V), or synergism of NIDO, AMOP and vWD domain(NAV) of MUC4/Y triggering can be reversed by the absence of unique MUC4/Y domains, we selected a batch of DEG molecules of MUC4/Y triggering for QPCR validation, and we focused on MUC4/Y-mediated key mechanisms. Additional File 4  on MUC4/Y-mediated mechanisms, altered gene expression of the lacking-domains groups  vs. control group (MUC4/Y-overexpression) was validated by independent qRT-PCR test. The results showed the expression levels of most DEGs of MUC4/Y triggering were reversed by the absence of unique MUC4/Y domains. And the expression levels of representative 68 molecules were all reversed in V△ group (vs Y). Only small number of molecules expression levels were not reversed, as follows: 1) N△vs Y: 8 molecules which failed to reverse expression were RAC2, MAPK10, FGF5, ITGB8, MMP25, MMP28, TSPAN8, CEACAM6  have roles in MUC4/Y-mediated downstream of molecule mechanisms.Altogether, these results show that MUC4/Y’s domains  on MUC4/Y-mediated main signaling pathways, the expression changes of the key nodes in the signal path, especially in protein phosphorylation, were verified and compared between the lacking-domains groups  and control group (MUC4/Y-overexpression) by western blotting and measurement of optical density value.To investigate the effect of MUC4/Y, which include MUC4/Y-dependent increase of ERBB2 and ERBB3 phosphorylation activation, paralleled by the upregulation of key molecules , as either total or phosphorylated proteins, in the downstream signaling pathways.Additional File 4  also shoMUC4/Y-mediated signal path in the majority. And the expression levels of representative 20 molecules were all reversed in NAV△ group (vs Y). Non- reverse effects were in the minority, as follows: 1) N△vs Y: Molecules which failed to lower 6 proteins expression were ERBB2, Src, FAK, ERK, Phospho-ERK, Phospho-c-JUN. 2) A△vs Y: Molecules which failed to reduce 2 proteins expression were ERBB3, Phospho-NFκB. 3) V△vs Y: Molecules which failed to down- regulate 2 proteins expression were ERBB3, c-JUN. So, these results indicate that MUC4/Y’s unique domains have effects on MUC4/Y-mediated downstream of signaling pathways, consistently with sequence-based transcriptome analysis and QPCR validation.As shown in Figure MUC4/Y’s unique domains weakened the roles of MUC4/Y on malignant activities, and impaired expression levels of downstream effector molecules of MUC4/Y triggering, demonstrating that MUC4/Y’s unique domains have significant roles in MUC4/Y-mediated malignant function of pancreatic cancer, downstream of molecule mechanisms, including signaling pathways, respectively. To illustrate the separate feature  and reverse-effect rate or extent  to MUC4/Y functional categories, systematic comparative analysis were carried out, as shown in Figure As mentioned above, on the whole, loss of MUC4/Y overexpression in PANC-1 cells focuses on 18 functional categories [P-values, ≤0.05), as follows: cell projection, neuron projection, membrane, extracellular region, integral to membrane, extracellular region part, membrane part, intrinsic to membrane, cytokine activity, lipopolysaccharide binding, signaling, signal transmission, signaling process, cell communication, MAPK signaling pathway, Chemokine signaling pathway, Cytokine-cytokine receptor interaction, TGF-beta signaling pathway.Figure tegories  , A△vs Y, V△vs Y , NAV△vs Y .Compared with P-values of the above18 functional categories (of MUC4/Y) showed that the least P-values of “extracellular region”and “extracellular region part” were acquired by A△vs Y, suggesting that the two functional categories of MUC4/Y are the most highly correlated to the function or role of AMOP domain.The comparison of FDR-corrected P-values of “cell projection”, “neuron projection”, “membrane”, “membrane part”, “intrinsic to membrane”, “Chemokine signaling pathway”, and “Cytokine-cytokine receptor interaction” were acquired by V△vs Y, suggesting that the seven functional categories of MUC4/Y are the most highly correlated to the function or role of vWD domain.The least P-values of “signaling”, “signal transmission”, “signaling process”, “MAPK signaling pathway” were acquired by NAV△vs Y, suggesting that the four functional categories are the most highly correlated to the synergistic role of unique domains.The least MUC4/Y were classified as twelve feature subsets, i.e. group1-12. Group1-5 can be incorporated as a feature subset of crucial factors involved in signaling hub. Group 6 is a feature subset of extracellular growth factors & membrane receptors. Group7-9 can be incorporated as a feature subset of crucial factors involved in oncogenic function. Group10-12 can be incorporated as a feature subset of crucial factors involved in energy metabolism  and protein synthesis & modification including glycosylated modification (Golgi function). Figure △, A△, V△, NAV△, as follows: 1) Group1-5: 95.45% (21/22), 95.45% (21/22), 100% (22/22), 100% (22/22), respectively. 2) Group 6: 84.61% (11/13), 100% (13/13), 100% (13/13), 92.31% (12/13), respectively. 3) Group7-9: 73.91% (17/23), 95.65% (22/23), 100% (23/23), 91.30% (21/23), respectively. 4) Group10-12: 100% (18/18), 100% (18/18), 100% (18/18), 100% (18/18), respectively. Figure △, A△, V△, NAV△, as follows: 1) Group1-5: 63.64% (14/22), 72.73% (16/22), 86.36% (19/22), 95.45% (21/22), respectively. 2) Group 6: 46.15% (6/13), 61.54% (8/13), 61.54% (8/13), 84.62% (11/13), respectively. 3) Group7-9: 56.53% (13/23), 69.57% (16/23), 82.61% (19/23), 91.30% (21/23), respectively. 4) Group10-12: 55.56% (10/18), 77.78% (14/18), 72.22% (13/18), 77.78% (14/18), respectively.Figure MUC4/Y were detected. Figure △, A△, V△, NAV△ was 70% (14/20), 90% (18/20), 90% (18/20), 100% (20/20), respectively. Figure △, A△, V△, NAV△ was 10% (2/20), 20% (4/20), 40% (8/20), 60% (12/20), respectively.Figure △, A△, V△) plays the definite effects on reversing MUC4/Y-mediated malignant function and downstream of molecule mechanisms, among which N△ has the weakest effects on that. Notably, the simultaneous lack of three unique domains (NAV△) plays the most significant effects on reversing MUC4/Y-mediated downstream of signal pathways than the respective lack groups , which is consistent with above mentioned that four functional categories of MUC4/Y  were the most highly correlated to the synergistic role of unique domains.Altogether, these results indicate that the lack of three unique domains (NMUC4/Y(MUC4)-mediated functions and mechanisms. Based on MUC4/Y, we initially constructed homologous genes lacking unique domains present in MUC4, i.e., MUC4/Y-NIDO△, MUC4/Y-AMOP△, MUC4/Y-vWD△, MUC4/Y-NIDO△-AMOP△-vWD△. We also constructed series of stable PANC-1 cell strains transfected by the MUC4/Y gene without or with domain-lacking, which with consistent forced gene expression rate and subcellular localization, for comparison of different quality caused by domain-lacking.In the present study, we focus on investigate the respective role of the unique NIDO, AMOP, and vWD domain or their synergistic effect on in vitro, sequence-based transcriptome analysis and confirmatory testing were similar in variation trends, as follows: 1) Domain-lacking weakened the roles of MUC4/Y on malignant activities of PANC-1 cell in vitro, which include the significant decrease of cell proliferation and DNA replication, consistently followed by the cell cycle arrest at G0/G1 phase, significant increase of apoptosis & necrosis rate under stress from low nutritional status, which also include significant decrease of the capabilities of migration and invasion. 2) Domain-lacking reversed the expression levels of the most of differentially expressed genes (DEGs) caused by MUC4/Y over-expressing PANC-1 cells compared with the blank and negative controls. Firstly, sequence-based transcriptome analysis revealed that among 1575 DEGs of MUC4/Y-overexpression triggered, the expression levels  of 932 genes were reversed 2 folds or more than by NIDO-lacking triggered, 990 genes by AMOP-lacking triggered, 1033 genes by vWD-lacking triggered, 1214 genes by three domains-simultaneously-lacking triggered, as shown in Additional File 2  revealed that the expression levels of representative 68 molecules were all reversed by vWD-lacking with 100% reversed rate, NIDO-lacking with 88.24% (60/68) reversed rate, AMOP-lacking with 97.06% (66/68) reversed rate, three domains-simultaneously-lacking with 98.53% (67/68) reversed rate. Thirdly, WB validation of the expression changes (causesd by domain-lacking) of the key nodes on MUC4/Y-mediated main signaling pathways revealed that the expression levels of representative 20 molecules were all reversed by three domains-simultaneously-lacking with 100% reversed rate, NIDO-lacking with 70% (14/20) reversed rate, AMOP-lacking with 90% (18/20) reversed rate, vWD-lacking with 90% (18/20) reversed rate. Altogether, these results demonstrate that MUC4/Y’s unique domains have significant roles in MUC4/Y-mediated malignant function of pancreatic cancer, downstream of molecule mechanisms, particularly MUC4/Y-triggered malignancy-related positive feedback loops, respectively.On the whole, the results of function assays l File 2 . Secondl△) had the most significant effects on reversing MUC4/Y-mediated functions in vitro , as shown in Figure △ group ranked the highest overall on positive proportion of greater than 50% reversal rate . Coincidentally, the above two points are consistent with the results of systematic comparative analysis, which revealed that the four enriched functional categories of MUC4/Y  were the most highly correlated to the synergy of three unique domains (NAV). Thus, we conclude that the synergistic roles of NIDO, AMOP and vWD domains on MUC4/Y-mediated functions and mechanisms are more prominent than the respective domain because the synergy of three domain plays the more remarkable effects on MUC4/Y-mediated signaling hub.Notably, the simultaneous lack of three unique domains (NAV△) weakened the roles of MUC4/Y on malignant activities of PANC-1 cell in vitro, including cancer metastasis-related capabilities. And NAV△ reversed batches of crucial factors involved in oncogenic function . Thus, finding paths to break the synergy of three unique domains (NAV) will be helpful to weaken capabilities of pancreatic cancer metastasis, specially to the patients with MUC4 positive expression.In addition, the results and analyses of GO function and the KEGG pathway show that the functional category “GTPase regulator activity” was enriched or triggered only by the synergy of three unique domains (NAV). The functional category “GTPase regulator activity” plays roles in the growth control, regulating the organization and remodelling of the actin cytoskeleton, regulating cell migration, regulating cancer metastasis via modulation of GTPases and GTP hydrolysis , 35. ConMUC4/Y (MUC4)-mediated mechanisms were with complex features, even the overlapping in different dimension, in accordance with MUC4/Y (MUC4)-mediated mechanisms.In contrast to previous studies , 40–44, MUC4/Y (MUC4), which is with remarkable features, as follows: 1) The malignancy-related positive feedback loops work on the ring circuit path, i.e., triggering- activating- transmitting(endonuclear transcription factors)- producing or upregulating- activating- transmitting(endonuclear transcription factors)- sustained upregulating . 2) Producing or upregulating crucial factors involved in oncogenic function is for malignant activities of pancreatic cancer. 3) Producing or upregulating crucial factors involved in energy metabolism, protein synthesis & modification is for supplying energy and survial material. 4) Producing or upregulating cytokines, growth factors, and adhesion molecules is for pancreatic cancer cell to affect the tumor milieu by cell–ECM and cell–cell interplay. 5) Complex interplays between several signaling pathways form network. So, MUC4 overexpression correlates significantly with poor prognosis of PDAC, not only because it plays important roles in the carcinogenesis and malignant progression of human pancreatic cancer, but also its triggered malignancy-related positive feedback loops are the root of resistance to chemotherapy and molecular targeted therapy.Our previous studies  have detMUC4/Y (MUC4)-mediated complex mechanisms, batches of MUC4/Y triggering DEG molecules which can represent above mentioned five remarkable features were selected for QPCR and western blotting validation of changes afforded by different domains-lacking, followed by systematic comparison to effect extent of different domains-lacking on two levels. Importantly, the present study is the first to demonstrate that NIDO, AMOP, and vWD domain or their synergy play significant roles on MUC4/Y (MUC4)- triggered malignancy-related positive feedback loops of pancreatic cancer to a variable extent. Excitingly, we find and verify that the absence of the unique domains  respectively or simultanously contributes to weaken MUC4-mediated malignant activities, cut off MUC4/Y-triggered malignancy-related positive feedback loops, and down-regulating transcription of the cascading downstream effectors in pancreatic cancer. These mean that in order to defeat the refractory and drug-resistant pancreatic cancer with MUC4 expression, some pathways can be explored besides repressing MUC4 transcription [Thus, in light of cription , 46, as MUC4/Y, i.e., MUC4 or MUC4/Y is anchored to the cell surface by hydrophobic transmembrane region, which locates carboxyl terminal of NIDO, AMOP and vWD domains, so the three domains protrude from the cell surface. The common characters of A, V, NAV were enriched in two function-sets “membrane” and “signaling”, suggesting that A, V, NAV have the more significant role on MUC4/Y-mediate membrane-related molecules expression and signal activation and transmission, which contribute to MUC4/Y-triggered malignancy-related positive feedback loops. The common characters of A, V were enriched in two function-sets “extracellular region” and “neuron projection”, suggesting that both of the domains play more prominent roles in cell-cell interaction, adhesion to the extracellular matrix, and specialized features of neuron. Furthermore, systematic comparative analysis revealed that the two enriched functional categories of MUC4/Y  were the most highly correlated to AMOP domain.Additionally, in this paper, the results of sequence-based functional annotation for global mRNA analysis also are in agreement with the results of QPCR validation, and are helpful for illustrating the universality and individuality of the role of these three unique domains. For example, the enrichment analyses of GO function  and the MUC4/Y  were the most highly correlated to vWD domain. Altogether, these individuality of vWD domain suggests MUC4-vWD domain may be involved in various biological function owing to its structure characteristics, i.e., vWD carry the putative GDPH cleavage site in its N-terminal region [Interestingly, GO function and the KEGG pathway show that the function on “nervous system generation, development, differentiation” and “regulation of multicellular organismal development” and “effect on Immune system”was enriched or triggered only by vWD domain, which is consistent with systematic comparative analysis results that the seven enriched functional categories of l region  and adjal region , 23, 31,MUC4/Y with domain-lacking stably, major workflow as follows: 1) Series of cDNA fragments encoding the MUC4/Y gene with domain-lacking were designed , including the lack of NIDO, or AMOP, or vWD, or the simultaneous lack of NIDO, AMOP and vWD domain, named MUC4/Y-NIDO△, MUC4/Y-AMOP△, MUC4/Y-vWD△, MUC4/Y-NIDO△-AMOP△-vWD△ correspondingly. A Kozak sequence (GCCACC) before the ATG initiation codon for optimal translation and with unique restriction sites present in the multiple clone site (MCS) of the lentiviral vector but absent from the target cDNA sequence was contained, respectively. These target sequences were synthesized and cloned in a pUC57vector (GenScript). 2) The series of target cDNA sequences were subcloned into the lentiviral vector pCDH-CMV-MCS-EF1-Puro  respectively, and lentiviral supernatant were produced by 293T cells which were transiently transfected with pCDH-CMV-MCS-EF1-Puro/ target-gene and the pPACKH1 Lentivector Packaging Kit  with Lipofectamine 2000 (Invitrogen LifeTechnology) according to the manufacturer’s instructions. 3) PANC-1 cells were transfected by series of target genes by carrying out at 20 multiplicity of infection with the lentivirus and using polybrene  to augment infection efficiency. Stable clones were then selected in medium containing puromycin . 4) As described previously [MUC4/Y gene were designated PANC-1-MUC4/Y(abbr. Y). Correspondingly, stable transfected PANC-1 cells overexpressing the series of MUC4/Y with domain-lacking genes AMOP were designated PANC-1- MUC4/Y-NIDO△(abbr. N△), PANC-1- MUC4/Y-AMOP△(abbr. A△), PANC-1- MUC4/Y-vWD△(abbr. V△), PANC-1- MUC4/Y-NIDO△-AMOP△-vWD△(abbr. NAV△), respectively.The PANC-1 pancreatic cancer cell line was obtained from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. As described earlier , we estaeviously , stable To verify these stable clones and identify the characteristic of expression and localization of target genes, a variety of detection methods were used, including immunofluorescence (IF), flow cytometry (FCM), quantitative real-time PCR (QPCR), and western blotting (WB).http://www.abcam.com/muc4-antibody-ab60720.html), which is existing consistently in the sequence of MUC4/Y, MUC4/Y-NIDO△, MUC4/Y-AMOP△, MUC4/Y-vWD△, MUC4/Y-NIDO△-AMOP△-vWD△, respectively. Detailed methods of IF, FCM and WB have been described earlier [We used anti-MUC4 mouse monoclonal antibody  to detect specific protein expression because this specific antibody is against N-terminal amino acids 79-189 of Human MUC4  cell proliferation assay was performed according to the manufacturer’s instructions. Cells were grown in low-serum (1% FBS) medium as described earlier . Cell grCells were treated with trypsin at 72 hours after incubation with low-serum (1% FBS) medium and fixed in 70% ethanol for 2 hours at 4°C. After being washed twice with phosphate-buffered saline, the cells were incubated with 0.1 mg/mL RNase A  at 37°C for 30 minutes. The cells were then resuspended in 0.05 mg/mL of propidium iodide  at 4°C for 30 minutes while being protected from light. Finally, the processed cells were analyzed in a FACSort flow cytometer . Evaluation of the data was performed by CellQuest software (BD Biosciences). The experiments were repeated thrice independently.Following 48-h treatment with low-serum (1% FBS) medium, PANC-1–derived clones were collected in PBS for apoptosis assay and flow cytometric analysis as described previously . The expin vitro wound-healing assay, a cell-free area of the culture medium was wounded by scratching with a 200-μL pipette tip. Cell migration into the wound area was monitored in serum-free medium and photographed under a fluorescence microscope at 0 and 24 h. The experiments were repeated thrice independently.We used modified 24-well Boyden chambers for the cell migration and invasion assays as described earlier . For theMUC4/Y, PANC-1-MUC4/Y-N△, PANC-1-MUC4/Y-A△, PANC-1-MUC4/Y-V△, PANC-1-MUC4/Y-NAV△, same protocol was carried out, as described previously [P ≤ 0.05 for GO and pathways analysis.For the five groups of cells: PANC-1-eviously , mainly vs. MUC4/Y-overexpression group were validated by Quantitative real-time PCR (QPCR), which was performed using standard procedures using a SYBR Premix Ex Taq Kit  with specific primers as described previously [18S gene and a relative quantitative method (ΔΔCt) was used to evaluate quantitative variation. The relative expression level (defined as the fold change) of target genes were calculated to the relative expression detected in the corresponding control cells, which was defined as 1.0.Altered gene expression of the lacking- domains groups eviously . cDNA waAltered expression of crucial factors involved in signaling hub in different groups were validated by western blotting (WB), which was performed using standard procedures as described previously . Brieflyn independent measurements unless noted otherwise. Statistical analysis was performed with one-way ANOVA for multiple groups and the unpaired Student t-test for individual groups. P<0.05 was considered statistically significant.Statistical analysis was performed using SPSS 20.0 (IBM SPSS Inc.). The results were confirmed by conducting at least three independent experiments in the present study. All data presented are the mean ± standard deviation (SD) of


Simulations of the depolarization fields under various boundary conditions support the experimental observations. The employed approach can be expanded to other low-dimensional nano-scale ferroelectric systems.Strain engineering is a widespread strategy used to enhance performance of devices based on semiconductor thin films. In ferroelectrics strain engineering is used to control the domain pattern: When an epitaxial film is biaxially compressed, e.g. due to lattice mismatch with the substrate, the film displays out-of-plane, often strongly enhanced polarization, while stretching the film on the substrate results in in-plane polarization. However, this strategy is of a limited applicability in nanorods because of the small rod/substrate contact area. Here we demonstrate another strategy, in which the polar axis direction is controlled by charge screening. When charge screening is maintained by bottom and top metallization, the nanorods display an almost pure  In ferroelectrics, the orientation, size and distribution of the domains affect the dielectric, ferroelectric, and piezoelectric response2; therefore, the ability to control the domain structure is essential. However, available strategies for domain control in ferroelectrics are limited. In the case of thin films, a prominent approach is to control the crystal orientation and strain of the films by the selection of appropriate substrates20. The control of the cooling rate across a phase transition is used too for domain structure control21.The manipulation of domains in ferroic materials is a key in emerging functionalities of nano-scale devices22. For example, epitaxial PbTiO3 thin films grown on SrTiO3 (STO) substrates show a fine 180° domain structure, such that overall, the bound charges self-compensate23. On the contrary, when a conductive substrate is used, a monodomain structure is maintained24.Electrostatic boundary conditions also affect the ferroelectric domain profoundly. They are the major reason for 180° domain formation: Without perfect charge screening, depolarizing field arising due to the polarization discontinuity at the surface of the film counteracts the non-screened polarization, resulting in antiparallel (180°) domain formation that reduces the energy of the depolarizing field25, or linear arrays of <10 nm wide domains26. This is a topic of increasing research interest in thin films. In nanostructures, the subject is much less developed. Indeed, the polarization and domain patterns in nanostructures have been modeled intensively in recent years. An interesting work27 has demonstrated by phase-field modeling that adjusting surface charge screening can provide an efficient way to gain control of vortex domain structure in ferroelectric nano dots. However, experimental results rely mainly on the pioneering work of Schilling et al.28 who showed indeed morphological control of polar direction in ferroelectric nanowires, where changing the aspect ratio of nanowires, or changing their lateral dimensions locally resulted in local variation of the polarization direction.In thin films, the interplay between electrostatics, elasticity, surface energy, and self-energy of domain walls can be utilized to obtain non-trivial patterns such as reconfigurable charged domain wallsc- or a-type domain orientation without the need of poling.The present study is focused on the role of charge screening in the control of domain pattern in nanorods and shows experimentally how to obtain 0.35Ti0.65)O3 (PZT) films and the domain orientations in focused ion beam (FIB)-fabricated nanorods made of these films, which were grown on Si and Nb-doped SrTiO3 (Nb-STO)  Figs  and 3. Dlts Fig.  confirmi0.5Sr0.5)CoO3 (LSCO)/CeO2/yttria-stabilized zirconia with 8 mol % Y2O3 (YSZ) and on Nb-STO. The 2θ/ω scans showed that both PZT were grown with (100) and (001)-orientations, indicating the a/c-domain structure as normally observed in {100}-oriented PZT films. The ϕ scans for PZT (101), Si (202), and Nb-STO (101) indicated that the deposited PZT films have epitaxial relationships of PZT 〈100〉 || Si 〈110〉 and PZT 〈100〉 || Nb-STO 〈100〉, respectively. As can be seen in Fig. P-E hysteresis curves, though the measured remnant polarization values 37 μC/cm2 and 50 μC/cm2 for the films on LSCO/CeO2/YSZ/Si and Nb-STO were smaller than the theoretical spontaneous polarization 74 μC/cm2 of a single crystal due to the coexistence of a- and c-domains.Figure a/c-domain structure of tetragonal ferroelectrics, the out-of-plane axes of a- and c-domains can be slightly tilted off the normal direction to the substrate surface. This allowed us to measure the diffraction intensities for the out-of-plane lattice spacing in a- and c-domains as functions of tilt angles, ω and ψ, with the rotation axes along Si [110] and [ω-ψ maps for PZT (300) and (003), representing the volumes of the a- and c-domains in the film and the rods with 2 μm- and 1 μm-widths on LSCO/CeO2/YSZ/Si. As can be seen in Fig. c-domain fraction is 0.19, which is less than the expected 1/3 for equilibrium-oriented domains without external fields and forces. Thus, the a-domain is dominant in the film grown on LSCO/CeO2/YSZ/Si. The reason for the preferred formation of a-domain is the mismatch of TEC between PZT and Si. Since TEC of PZT is larger than that of Si, PZT sustains increasing tensile stress during cooling down from the deposition temperature, which preferably generates the a-domain in order to minimize the elastic energy of the system. A similar domain structure of the films on Si has been also reported in the literature15.In order to clarify the domain structure of FIB-fabricated PZT nanorods, synchrotron micro XRD was employed. As illustrated in Fig. c-domain fraction is 0.71 and 0.95 for 2 μm- and 1 μm-wide rods, respectively. It is thus observed clearly that while in the PZT film a-domains are predominant (>80%), the PZT rods fabricated from the film, have a predominantly c-domain structure.The domain fraction was drastically varied by forming the rod structure. As can be seen in Fig. c-domain oriented, contrary to the >80% a-orientation in the films on the LSCO/CeO2/YSZ/Si substrate which were under tension. However, our previous study29 on the rods fabricated by FIB from PZT film grown on Nb-STO also showed similar features to the rods on the LSCO/CeO2/YSZ/Si substrate described above, with c-domains ratio increasing upon reduction of rod width, as shown in Fig. 2/YSZ/Si and those on Nb-STO precludes that the change in mechanical clamping plays an important role in the drastic change in domain orientation upon reduction of the rod width and is a key in the demonstration of the role of charge screening in domain orientation control.PZT films grown on Nb-STO were under compression and hence were predominantly (80–90%) 31. Similar post-treatments have been also reported for recovering from the FIB damage in nanostructured ferroelectrics33. This is further confirmed by our finding that self-assembled PZT nanorods grown on SrRuO3/STO(100) by pulsed laser deposition (PLD) without using any post-structuring showed as well the complete c-domain structure. As can be seen in Fig. 002 peak from the c-domain but no PZT 200 peak from the a-domain. This confirms that a FIB damage is not the driving force for the drastic change in domain fraction that occurred by the rod structure formation.We can also exclude a damage by the FIB process as the origin of the drastic change in domain fraction upon forming of the rod structures. Indeed, although we carefully etched the PZT with low acceleration voltages of ion beam, especially for finalizing the FIB process, a reduction of the piezo-response was observed due to the Ga injection and amorphousization at the vicinity of the surface. However, this was recovered by annealing the fabricated structure at the temperature ≥550 °Ca-domain is prohibited. Indeed, the self-assembled PZT nanorods, whose average aspect ratio is much larger than one, had only the c-domain  increased by the metallization of sidewall. Although the change in domain fraction is not easily recognized by the naked eyes for the rod on LSCO/CeO2/YSZ/Si, the rod on Nb-STO clearly shows the appearance of a-domain in the sidewall metalized rod relative to the complete c-domain structure before the sidewall metallization. Figure c-domain fraction before the sidewall metallization, and the increased amount of a-domain fraction by the sidewall metallization as a function of the inverse of the rod width. As can be seen in Fig. a-domain is accelerated with decreasing rod width and is most appreciable when the rod width is 1 μm . However, the narrower rods showed a lesser impact of the sidewall metallization. Indeed, the domain fraction was not obviously changed by sidewall metallization for the 200 nm-width rod on Nb-STO. The smaller impact of the depolarizing field on domain fraction in such a narrow rod is due to the appreciable depolarizing field perpendicular to the rod resulting from the finite screening length at the metal/ferroelectric interfaces perpendicular to the rod axis34.To further investigate the role of charge screening as the major driving force in the control of the domain pattern, we deposited 10 nm-thick platinum layer on the sidewalls of the same rods that were measured by XRD. The rods were then heated above c-domain fraction than those on Nb-STO due to the biaxial tensile stress. However, the c-domain fraction is always much larger than 1/3 when top and sidewall surfaces of the rods are not metalized  as the depolarizing field perpendicular to the rod weakens O3 films of 1200 nm thickness were grown epitaxially on two type of substrates, (a) Si(100) substrates buffered with LSCO/CeO2/YSZ epitaxial triple layers, and (b) Nb-STO (100) substrates. All layers were deposited by PLD with KrF excimer laser (λ = 248 nm). The epitaxial triple layer template on Si consisted of 10 nm-thick YSZ, 20 nm-thick CeO2, and 20 nm-thick LSCO, whose deposition conditions can be found in ref. 2 pressure of 200 mTorr and temperature of 625 °C. To achieve the stoichiometric lead content in the deposited PZT films, a ceramic Pb (Zr0.35Ti0.65)O3 containing 5% excess PbO, sintered by spark-plasma sintering, was used as the target.Tetragonal-phase (100)/(001) PbZr0.Ti0.65O3 37. 80–100 μm squared area surrounding the rod was etched away to avoid XRD from the film. To shape its vertical walls, the roughly etched rod was irradiated by a FIB inclined from the normal to the PZT film by 5°. Rods of widths 200 nm, 500 nm, 1 μm, 2 μm, and 4 μm were fabricated . The detailed structure was reported in refs Following the PZT film deposition explained above, circular platinum pads with 200 μm diameter and thickness 50 nm were deposited by electron beam evaporation, to serve as a protective layer for the FIB treatment which followed. The PZT film under the circular platinum pad was then etched into a rectangular columnar rod shape by the FIB, in a similar procedure to that reported in the literature3/STO(100) by PLD at elevated O2 pressure (2 Torr), which enhances the shadowing effect38 for PLD species due to their numerous scattering during the flight from target to substrate, resulting in the epitaxial growth of nanorods. The details of the growth and properties of the self-assembled nanorod will be described elsewhere.In order to verify that the domain structure formed in FIB-rods did not originate from a damage during FIB process, self-assembled epitaxial nanorods with similar thickness to FIB-rods were also prepared. These nanorods were grown on SrRuOKα1 X-rays  and synchrotron X-rays . Their ferroelectric properties were measured with a ferroelectric tester . The microstructure of the fabricated PZT rods was observed by scanning electron microscopy (SEM). To investigate the domain structure of FIB-PZT rods, synchrotron micro XRD  was employed. In these measurements, the X-ray beam was focused onto a single PZT rod centered on the square etched area using the 2D focusing lens system (Forschungszentrum Karlsruhe). We used X-rays with 12.4 keV photon energy (wavelength 0.1 nm) for these measurements. Due to the long duration of the measurements, the experiments were restricted to a single set of samples with different sizes. However, the contentious change of the domain fraction O3 from ref. Phase-field simulations were performed and compared with the experimentally observed domain patterns. A two-dimensional phase-field model was adopted from ref. The authors declare that the data supporting the findings of this study are available within the paper.


We show unconventional magnetoelectric couplings such as the magnetic-field-driven reversal of ferroelectric polarization with no change of spin-helicity, and present a phenomenological theory that successfully explains the magnetoelectric coupling. Our measurements establish Mn2GeO4 as a conceptually simple multiferroic in which the magnetic-field-driven flop of conical spin spirals leads to the simultaneous reversal of magnetization and electric polarization.Despite remarkable progress in developing multifunctional materials, spin-driven ferroelectrics featuring both spontaneous magnetization and electric polarization are still rare. Among such ferromagnetic ferroelectrics are conical spin spiral magnets with a simultaneous reversal of magnetization and electric polarization that is still little understood. Such materials can feature various multiferroic domains that complicates their study. Here we study the multiferroic domains in ferromagnetic ferroelectric Mn 2GeO4 using neutron diffraction and develop a phenomenological theory to explain their results.The coexistence of ferromagnetism and ferroelectricity in a multiferroic material often arises from complex magnetoelectric couplings. Here, the authors observe switching of domains in Mn M and electric polarization P simultaneously, that is, multiferroics in the original definition, are not common. One of the few systems exhibiting both spontaneous M and P is a spiral magnet with the so-called transverse conical spin structure, which consists of a cycloidal spiral spin component and a ferromagnetic component along the spin rotation axis of the cycloid3Q structure in which two distinct magnetic propagation vectors Q coexist. In the framework of the spin–current or inverse Dzyaloshinskii–Moriya (DM) mechanismP in the direction perpendicular to both the spin rotation axis and the modulation wave vector. This means that P in transverse conical systems develops in the direction perpendicular to M. CoCr2O4 134562O4 refs , 10 and 2GeO4 was reported to be a rare multiferroic below 5.5 K, where both spontaneous M and P develop in the same direction182GeO4  octahedrally coordinated by O2− anions, and form sawtooth-like chains along the b axis1819Q magnetic order with both commensurate (C) and incommensurate (IC) components for the multiferroic state. The C component characterized by the propagation vector of (0 0 0) is described by a combination of two irreducible representations (irreps) Γ1 and Γ3 and allows finite M along the c axis (Mc). The IC component forms a spin spiral with the propagation vector Qic=(qhqk 0), where qh=0.136 and qk=0.211. This structure is described by two irreps D1 and D2, and contains a cycloidal component which can be responsible for P along the c axis (Pc). To the best of our knowledge, Mn2GeO4 is the only multiferroic with the M||P configuration where P can be understood as generated by a spiral spin order. Phenomenological symmetry analysis on the respective C and IC components well explained not only the simultaneous appearance of finite |M| and |P| but also the M||P configurationM–P coupling has been provided to date. In addition, as shown in M accompanies that of P in the multiferroic state of Mn2GeO4, indicating a synchronized switching of both ferromagnetic and ferroelectric domains. This coupled switching mechanism still remains an unsolved issue, both microscopically and phenomenologically.Recently, an olivine-type compound Mnn182GeO4  is descr2GeO4 by the application of electric and/or magnetic fields, and reveal one-to-one phenomenological correspondence relations between certain C and Q domains, which arise from the C and IC components in the multi-Q magnetic order, respectively. From this we show that the multiferroic state of Mn2GeO4 can be regarded as canted conical spin spirals, as illustrated in P but also with M via the C,Q domain switching, and its sign can be reversed by a magnetic field in Mn2GeO4. We also clarify the mechanism of both the M||P configuration and the simultaneous reversal of M and P observed in this unique multiferroic compound.Here we report on the evolution of these domains in Mn2GeO4, we investigated the effects of electric and magnetic fields on domains by means of unpolarized and polarized neutron scattering measurements on single crystal samples. Both the ferromagnetic and ferroelectric domains in this compound involve several types of magnetic domains, which are ascribed to the complex multi-Q magnetic order with both C and IC components. As illustrated in A domain for ±Γ1⊕±Γ3 and CB domain for ±Γ1⊕±Γ3). Each C domain possesses its time-reversal counterpart with the opposite sign of Mc (compare upper and lower panels of A and QB domains for the propagation vectors (qhqk 0) and (qh−qk 0), respectively). Each Q domain possesses its space-inversion counterparts with the opposite sign of Pc (or the opposite sign of spin-helicity h) , time-reversed domains [C(+Mc)↔C(−Mc)], configuration (or Q) domains (QA↔QB) and so-called chirality domains [Q(+h)↔Q(−h)].To elucidate the coupling mechanism between the ferromagnetism and ferroelectricity in Mn2GeO4 (see Methods). To describe the couplings seven order parameters are identified: ferroelectric order Pz, two complex order parameters 1 and D2, respectively, for each IC Q domain, Q=QA or QB , and two C order parameters X1 and X3, belonging to irreps Γ1 and Γ3, respectively. 1 and D2 for the space group Pnma and Qic=(qhqk 0) (ref. z axis (mz), and mz. X1 describes 180° domains of the dominant C antiferromagnetic component while X3 corresponds to the ferromagnetic component with magnetization along the z axis (Mz). Here the z axis corresponds to the c axis in Mn2GeO4. First, we present a phenomenological theory that describes the possible coupling terms between the ferroelectric and magnetic order parameters in the multiferroic state of Mn 0) ref. , for exas, s′, r, and r′ are real numbers. U describes the coupling between the spin-helicity and the electric polarization, and is equivalent to the trilinear coupling term developed for Ni3V2O8  define strict relationships between the order parameters. X3 (=Mz) allows not only a concomitant reversal of Pz but also a switching between the QA and QB domains. We will provide experimental evidence for the presence of all three coupling terms.Here 2O8 ref.  and TbMn. 3 ref. . The spiU coupling term ) at TASP, Paul Scherrer Institut (PSI). Details of the experiments are fully described in Methods. The intensity profiles of the (±2∓qh 1−qk 0) peaks were measured in both the non-spin-flip  and spin-flip  polarization channels. From the difference in the intensities observed in each polarization channel, the relative proportions of the two helicity domains within each Q domain can be determined directly (see Methods). These measurements were done in zero electric field at 2 K, after having cooled the specimen from 10 to 2 K under various Ecool along the c axis. In A domain peak (2−qh 1−qk 0) after cooling under Ecool=±0.5 and ±1.5 MV m−1. The results show a strong sensitivity to both the neutron polarization state and size of Ecool, with the difference between the non-spin-flip and spin-flip scattering being larger in the data for Ecool=±1.5 MV m−1. Furthermore, by switching the sign of Ecool the polarization-dependent magnitude of the spin-flip and non-spin-flip intensities becomes reversed. Similar data are also obtained for the QB domain. Importantly, these results show that the Pzx elements of the polarization matrix (see Methods) can be made finite, with both the magnitude and sign tunable by the electric-field-cooling condition. This confirms the magnetic structure in the multiferroic state of Mn2GeO4 to include a spin spiral component with a spin-helicity that can be selected by an electric field.To prove the existence of the ing term , we studA and QB domains (Methods). Using both the MuFIT programh domains) within each of the QA and QB domains, and for various Ecool. h domain fractions (p(−h) and p(+h) in the QA and QB domains, respectively), as a function of Ecool. For comparison, the Ecool dependence of Pc measured in the same condition is also plotted in h domain fraction in each Q domain behaves in the same way, namely increasing in proportion with positive Ecool up to 2 MV m−1 and saturating above 2 MV m−1. The Ecool dependence of the domain fraction matches up nicely with that of Pc. Thus, the QA and QB domains coexist even after the electric-field cooling and contribute to the ferroelectric polarization, with their spin-helicity (or h domain fraction) equivalently tuned by Ecool. These observations, in particular the opposite sign of h in the two domains, are entirely consistent with the presence of the U coupling term. It is also worth pointing out that large Ecool (>2 MV m−1) is required to obtain single spin-helicity in each Q domain. In most spin spiral ferroelectrics in single crystal form, an order of magnitude smaller Ecool is enough to pole specimens .A↔CB) and the configuration domains (QA↔QB)Ecool=+3 MV m−1 and Bcool=+1.5 T (magnetoelectric (ME) cooling). These cooling fields are large enough to fully pole both the ferromagnetic and ferroelectric domains, which is confirmed by measurements of M and P or by checking the chiral scattering terms of the polarization matrix obtained from SNP experiments , (−3 −1 0), (−2+qh −1+qk 0) and (−2+qh 1−qk 0) reflections are shown in B was swept to −1.5 T and then set to zero at 4.5 K. This B-sweeping procedure causes the reversal of M and P. Then the same measurements were done and the integrated intensity data of the same magnetic peaks were obtained again. Red symbols in θ-scan profiles of the above-mentioned reflections after the reversal of M and P. By comparing the data before and after the reversal of M and P, it is apparent that the intensities of the (−3 1 0) and (−2+qh −1+qk 0) reflections are suppressed after the reversal while those of the (−3 −1 0) and (−2+qh 1−qk 0) reflections are enhanced. In fact, as shown in qh 1+qk 0) magnetic reflections is observed at the magnetic field where the reversal of M and P occurs.Next, we show the results of unpolarized neutron scattering measurements done at TriCS, PSI. Note that the unpolarized neutron scattering technique cannot distinguish a pair of time-reversed 180° domains or a pair of helicity domains. From this technique, however, we obtain quantitative information about the orientation domains (Cents see  and 2e. p(CA) : p(CB)=0.60(1) : 0.40(1) and p(QA) : p(QB)=0.600(3) : 0.400(3) before the reversal while those after the reversal were p(CA) : p(CB)=0.33(1) : 0.67(1) and p(QA) : p(QB)=0.371(2) : 0.629(2). Thus, the domain fractions of the CA and CB domains are nearly the same with those of the QA and QB domains, respectively. This result suggests that the C and IC domains are strongly entangled, and that the CA (CB) domain is always coupled with the QA (QB) domain. Furthermore, the domain fraction relationships between the domains A and B are reversed in both the C and Q domains after the reversal of M and P. These coupling features were observed in all the data for various field cooling conditions, as summarized in A and CB domains but also between QA and QB domains when the simultaneous reversal of M and P takes place. For a given sign of X1, CA and CB domains are distinguished by the sign of X3, and the presence of coupling term V  measurements at D3, ILL (see Methods). As listed in variant, , so that2GeO4 is of the double-Q type, being namely a superposition of the CA and QA domains (or the CB and QB domains). This means that only the following eight-types of C, Q domain combinations are possible: , , , and  (the double sign applies in the same order as written). The sign of X1 determines whether M is parallel or antiparallel to P. In Q domains. The resulting magnetic structures can be simplified by considering them to include two types of conical spin chain (α and β) formed by Mn1 moments. The simplified structure is illustrated in || vector sum of the Mn moments in each chain) making a finite angle with the a, b, and c axes. The coupling between the conical chains α and β is canted antiferromagnetic. Though the net moments along the a and b axes are cancelled out, the c axis component remains finite. As a result, a net magnetization Mnet develops along the c axis, as schematically illustrated in P in the direction perpendicular to both the component of the propagation vector ||b, and the spin rotation axis through the inverse DM mechanismP in the conical chains α and β is aligned along the c axis, and a net electric polarization Pnet develops along the c axis  whose sign is opposite to that of Mc. As mentioned above, the results of unpolarized neutron scattering experiments suggest that domain switching occurs between the  and  domains when the reversal of M and P takes place. Schematics of the change in a cone axis upon the reversal of M and P (or domain switching) are illustrated in A(+Mc), QA(+Pc)) domain with positive Mc and Pc (Top left panel of Mc will lead to the domain switching into the (CB(−Mc), QB(−Pc)) domains with negative Mc and Pc  leads to switching of the Q domains (from QA to QB or vice versa). The coupling term W dictates that the two different Q domains couple with the same helicity to both magnetization and ferroelectricity. Experimentally, we established that a reversal of the magnetization leads to a conservation of the spin-helicity and to a switch of the Q domains, which leaves the term in W describing the magnetic structure invariant under a reversal of the magnetization. The term W describes thus a direct coupling between the ferroelectric polarization and the magnetization—consistent with the microscopic scenario explained above.Phenomenologically, the g term W , as we werm W  and incommensurate (IC) magnetic peak positions.Polarized neutron diffraction (PND) measurements were carried out using both the cold neutron triple-axis spectrometer TASP (λ=3.189 Å) equipped with the MuPAD devicec axis. The sample space at the end of the stick was indium-sealed and covered by an Al vacuum can. The sample space was evacuated in order to avoid electric-field breakdown across the sample during the measurements. The high voltage stick with pumped sample space was then installed into a standard Orange cryostat (base temperature of 2 K), which itself was installed into the MuPAD device at the TASP beamline. Polarization matrix measurements were carried out at 2 K, after either zero-field cooling or electric-field cooling through the multiferroic transition at 5.5 K. When at the base temperature, the electric field could be removed for the SNP measurements. We observed no effect on the resulting data due to the electric field removal in the multiferroic phase.For the TASP experiment, a 10 nm layer of Cr and 50 nm layer of Au were sequentially evaporated onto the surfaces of the plate-like sample for making electrical connections. The sample was then oriented and mounted on an aluminium spacer positioned at the end of a bespoke sample stick designed for the application of high voltages (up to 5 kV) at cryogenic temperaturesc axis is achieved by a potential difference applied across two parallel Al plates. The sample was fixed to one plate by a silver epoxy, and the other plate positioned close to, but not touching the other sample surface. Thus the actual electric field across the sample was not exactly the same as the applied voltage. By comparing between similar electric-field cooling measurements as done at TASP we could estimate the absolute electric field scale in the D3 experiments. Similarly as for the TASP experiments, the sample space of the sample stick used at D3 was also indium-sealed by an Al can and the sample space evacuated. The pumped sample stick was then installed into a ‘thin tail' Orange cryostat (base temperature of 3.5 K), which could then be installed into the CryoPAD device at the D3 beamline. SNP measurements were done after either zero-field cooling or electric-field cooling through the multiferroic transition, similarly as at TASP.For the D3 experiments, a plate-like sample (without pre-deposited electrodes) was installed onto a different bespoke 5 kV high voltage sample stick. On this stick the application of electric fields along the c axis, in addition to electric fields. Thus at D3 we could investigate the effects of magnetoelectric cooling (simultaneous magnetic field- and electric field-cooling) into the multiferroic phase, and also magnetic field sweeping from positive to negative magnetic fields within the multiferroic phase. Since the electromagnet cannot be integrated with the CryoPAD device, each sample state preparation involving the magnetic field was done ‘offline;' by using the beamline crane we could seamlessly interchange the high voltage stick and cryostat ensemble between the CryoPAD device and the electromagnet stationed nearby. Thus, once the sample had been either poled to 3.5 K through the multiferroic transition under simultaneously applied electric- and magnetic-fields, or the magnetic field was swept between ±1.2 T at the slightly elevated temperature of 4.5 K, all fields were then removed before the cryostat and sample stick were installed back into the CryoPAD device for the SNP measurements of the newly prepared state.In addition at D3, an electromagnet (maximum applied field ±1.2 T) was available. By installing the thin-tail cryostat into the electromagnet, vertical magnetic fields could be applied along the Both of the MuPAD and CryoPAD devices allowed for SNP measurements of magnetic Bragg scattering at TASP and D3, respectively. SNP is a technique that, given an incident neutron beam polarized along an arbitrary direction, enables the full measurement of the direction of neutron spin polarization after the scattering process. The results are expressed as a polarization matrixσ is the Pauli operator for the neutron spin, and q. Here M(q) is the magnetic structure factor. Full details of the calculation of M(q) for the different magnetic structures in Mn2GeO4, are given in the Supplement of ref. i and outgoing j spin polarizations.where the ket 2GeO4, and for a perfectly efficient polarized neutron scattering setup, a general polarization matrix can be written for a magnetic Bragg peak probed at q:The SNP technique is a particularly powerful probe of complex magnetic structures and magnetic domain distributions. For the IC spiral structure in the multiferroic phase of Mnx is defined as always parallel to q, z perpendicular to the scattering plane, and y orthogonal to both x and z. Consequently at each q, Here F and G parameters of G are the so-called ‘chiral' scattering elements of 2GeO4. The measured value of G not only depends on the magnetic structure in the sample, but it is also sensitive to the populations of the possible domains of the magnetic structure. In the multiferroic phase of Mn2GeO4, the spin spiral magnetic structure of each Q domain has a helicity degree-of-freedom (±h) the sign of which is intimately related to the direction of ferroelectric polarization along either +c or −c. After zero-field cooling, equal populations of opposite helicity domains are realized, which will give either G<0 or G>0. Thus for equal helicity domain populations the so-called ‘chiral' scattering averages out and the measured value of G=0, as would be observed for a collinear structure. On the other hand, as we demonstrate in our experiments at TASP, by applying an electric field along the ferroelectric c axis while cooling, the helicity domain population within each Q domain can be controlled, and finite values of G are observed. Thus, not only does measuring a finite value of G allow the determination of the relative fraction of spiral domains of opposite handedness , but it further confirms the IC phase of Mn2GeO4 to be symmetry-breaking complex magnetic order with out-of-phase modes.The F and G parameters further depends on the exact orientation of the magnetic scattering vector q with respect to the real-space spin distribution. If the measured q is exactly perpendicular to the spin rotation plane of the non-collinear spiral structure, then one obtains G=±1 for a mono-handedness spiral state and F=0. Typically however, most of our polarization matrix measurements were done at IC q vectors which lie at angles away from perpendicular to the spin spiral rotation plane. Consequently, even for a Q domain containing mono-handed spiral order, the magnitude of G becomes <1, and the magnitude of F larger than 0. Nevertheless, when armed with a full model for the magnetic structure, the measured values for F and G at any q allow for a full determination of the spiral-handedness domain populations. We used the MuFIT programThe full calculation of the expected 2GeO4. As expected for these simple q=0 antiferromagnetically modulated structures, the measured matrices only had finite diagonal elements, and the G parameters were always measured to be 0 within uncertainty.We also briefly mention that in our SNP experiments we measured some polarization matrices of Bragg peaks due to the C magnetic orders in the various phases of Mn2GeO4 sample was mounted on the same 5 kV high voltage stickc axis. The horizontal scattering plane at TriCS contained the [100]−[010] directions. The high voltage stick was installed into the variable temperature insert (VTI) of a 6 T vertical field cryomagnet (base temperature 2 K), so that both electric and magnetic fields could be applied simultaneously along the crystal c axis. Unlike at D3, the cryomagnet apparatus could be installed on the beamline, and the electric- and/or magnetic-field cooling from high temperature into the multiferroic phase done ‘online'. After field-cooling the sample through the multiferroic transition, all fields were then removed before data collection at 2 K. In the case of magnetic-field sweeping an already poled sample, the magnetic field sweeps were done after carefully elevating the temperature to 4.5 K. We found that at this slightly higher temperature the effect of the magnetic field sweeping on the magnetic domain populations was more pronounced than when sweeping was done at 2 K. After the sweeps the field was removed and the temperature lowered back to 2 K for the data collection.Unpolarized neutron diffraction measurements were carried out using the TriCS diffractometer at PSI (λ =1.178 Å). Similarly as for the SNP experiments described above at TASP, a plate-like MnA and CB C domain types, and the QA and QB IC domain types for each prepared state (with these domain types introduced in the main text), and consequently the relationships between them based on the response of their relative populations to the applied electric and magnetic fields.After each multiferroic state preparation, either by electric- and/or magnetic-field cooling, or by magnetic field sweeping, we studied both the C and IC magnetic structure components that exist in this state. Typical data sets constituted integrated intensity measurements for 50 C magnetic peaks and 70 IC magnetic peaks. Refinements of the integrated intensity data and magnetic domain populations were done using the FullProf programThe data supporting the findings of this study are available from the corresponding authors on request.How to cite this article: Honda, T. et al. Coupled multiferroic domain switching in the canted conical spin spiral system Mn2GeO4. Nat. Commun.8, 15457 doi: 10.1038/ncomms15457 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figure, Supplementary Tables, Supplementary Note and Supplementary References


Gene3D has previously featured in the Database issue of NAR and here we report a significant update to the Gene3D database. The current release, Gene3D v16, has significantly expanded its domain coverage over the previous version and now contains over 95 million domain assignments. We also report a new method for dealing with complex domain architectures that exist in Gene3D, arising from discontinuous domains. Amongst other updates, we have added visualization tools for exploring domain annotations in the context of other sequence features and in gene families. We also provide web-pages to visualize other domain families that co-occur with a given query domain family.Gene3D ( Sequence and structural databases continue to expand rapidly, with the current version of UniProtKB v2017_08) containing nearly 90 million sequences  ( (4) to bThe CATH resource provides different domain sequence clusterings  using complete-linkage clustering where the number represents the minimum sequence identity that all members of a cluster must share (hence S95 clusters are finer grained than S30 clusters). We have updated our v16 pipeline to build HMM’s for all S95 models, compared to using only S35 HMMs in v14, resulting in an increase in the number of HMMs (from 21 000 to over 59 000).Z-score parameter set), we find that the number of sequences covered by at least one domain annotation has grown by 5% in Gene3D v16 relative to Gene3D v14. Most important to the increase in sequence coverage is the CATH update from v4.1 to v4.2, expanding the number of S35 domain sequences from 21 155 to over 32 000.Despite the more conservative approach in building the HMMs, Gene3D v16 has improved sequence coverage of domain assignments compared to v14. Mapping between releases, using a shared set of Ensembl genomes sequences and scaling to the equivalent search space . As mentioned in the introduction, predicting domains for a query protein involves scanning the protein sequence against profile-HMM libraries and then resolving the candidate matches to obtain a final set of non-overlapping domain assignments . The scans typically assign a score  to the candidate matches and this can be used to prioritise strong matches in cases of domain overlaps. CRH uses dynamic programming instead of a more computationally expensive graph-based method. It is faster, more flexible and at least as accurate as the previous DomainFinder3 method (http://cath-tools.readthedocs.io/). The new Gene3D annotation pipeline is easy to run, memory efficient and can be downloaded along with the HMMs from the FTP site (ftp://orengoftp.biochem.ucl.ac.uk/gene3d/CURRENT_RELEASE/gene3d_hmmsearch.tar.gz).Apart from the improvements to the HMM-building pipeline, we have also updated the domain resolution algorithm, to a new method we have developed called 3 method . CRH is As in the previous release we added CATH-FunFam annotations produced by the FunFHMMer pipeline . In thisIn the following sections we discuss some of the new visualization features that make use of the database updates described above. As an example, in the protein page view, we have added the ProtVista tool  which alThe protein sequence viewer now also optionally displays CATH-PDB domain architectures (i.e. direct CATH choppings of the PDB mapped onto the protein sequence by SIFTS ), allowihttp://www.phyloxml.org) to download or access via web-services. For a given protein coding gene from Ensembl, we can now visualise its globular domains relative to those of other genes from the same Ensembl Compara , a regulator of kinase activity, along with other genes from its Ensembl Compara gene family. By default, Gene3D filters domain annotations with a HMMER independent e-value < 0.001. However HMMER also provides domain predictions measured by a conditional e-value and we allow these domains to be optionally displayed in the tree by adjusting the domain confidence filter bar, and display these low confidence predictions in a lighter color . These co-occurrence matrices, can get quite large, but the website offers various filters and the clustergrammer library allows the user to zoom in for details. Also, mousing-over individual rows and cells produces a popup with more detailed information. As an example, in the Gene3D website, we can search for domains that co-occur with the SH2 domain family (superfamily: 3.30.505.10) in a set of specified Eukaryotic and Metazoan genomes. The resulting page, shown in Figure The Domain Architecture page also includes a table showing co-occurring domain families, grouped across different genomes. A table detailing counts for the query domain family with other domain families in specific taxonomic lineages can be found by scrolling down to the ‘Domain Partners’ section of the page. To look at how these co-occurring domain families compare across specific genomes, we have added another visualisation tool to the Gene3D website. This visualization tool displays the co-occurring domain families, for a given query domain family in a heatmap/matrix style, using the clustergrammer JavaScript software (ftp://orengoftp.biochem.ucl.ac.uk/gene3d/CURRENT_RELEASE/). We provide a number of genome annotations (e.g. UniProt Reference Proteomes) and can make bespoke datasets (e.g. a newly sequenced genome not in Gene3D) for download upon request. The package for scanning HMMs is provided along with instructions. Some example searches can be found by following the examples link (http://gene3d.biochem.ucl.ac.uk/examples).We provide the Gene3D data and the domain assignment pipeline at the FTP site (Improvements in techniques such as Cryo-EM , will leSupplementary DataClick here for additional data file.


This paper is concerned with an explicit value of the embedding constant from  NeverthNq/(N−q) . It was nt}Lp(Ω) . Moreoveomains Ω –27. In te, e.g.,  and 29]\documentWe propose four theorems Theorem  to 3.4 fmentclass2pt{minimTheorems The remainder of this paper is organized as follows. In Section m, that is, The following notation is used throughout this paper. For any bounded domain For two domains In the following theorem, we provide a formula for obtaining an explicit value of Letbe a bounded domain, and letpandqsatisfySuppose that there exists a finite number of bounded domainssatisfying ). From  and 7),,\documenFor When 1p. From  and 7),,\documenf and g defined byLet Γ be the gamma function, that is, p(Ωi) in  for bounLetbe a bounded convex domain. Forand any pointwe haveA proof of Lemma Forwe putIfholds valid forwhere this is the best constant in Suppose thatandForandwe havewithThe constantis the best constant in (mentclasspt{minimap, q, and N are imposed on the assumptions listed in Table The following Theorems mentclass2pt{minimLetbe a bounded convex domain. Assume thatsatisfiesifandifForsuch thatwe havewithLet Letbe a bounded convex domain. Assume thatifandifFor allwe havewithLet 1p. From , it follfined in  with \doLetbe a bounded convex domain. Suppose thatifandifThen we havewithwhereforandFirst, we prove J is defined byTherefore,Next, we show . For \doSince Letbe a bounded convex domain, and letThen we havewithwhereVis defined in Theorem J is defined in the proof of Theorem First, we show ψ is denoted in the proof of Theorem Next, we prove . Let \doIn this section, we present numerical examples where explicit values of First, we select domains entclass1pt{minimatisfying . For allV (in Theorems For the first example, we select the case in which mentclass2pt{minimn are shown in the corresponding parentheses. Moreover, these values are plotted in Figure Table p’s. The estimates derived by Theorem Theorems We also show the values of V is the regular hexagon displayed in Figure For the second example, we select the case in which Ω is a regular triangle with the vertices n are shown in the corresponding parentheses. Moreover, these values are plotted in Figure p satisfying Table We also show the values of The values of mentclass2pt{minimV is also a cube with the side length For the third example, we select the case in which n are shown in the corresponding parentheses. The minimum value for each p is written in bold. We also show the values of Table We proposed several theorems that provide explicit values of Sobolev type embedding constant mentclass2pt{minimethod in . This ac


Two domains of the protein, the N-terminal BTB-POZ domain and the RD2 domain are responsible for recruitment of co-repressor molecules and histone deacetylases. The BTB-POZ domain is found in a large and diverse range of proteins that play important roles in development, homeostasis and neoplasia. Crystal structures of several BTB-POZ domains, including BCL6 have been determined. The BTB-POZ domain of BCL6 not only mediates dimerisation but is also responsible for recruitment of co-repressors such as SMRT, NCOR and BCOR. Interestingly both SMRT and BCOR bind to the same site within the BCL6 BTB-POZ domain despite having very different primary sequences. Since both peptides and small molecules have been shown to bind to the co-repressor binding site it would suggest that the BTB_POZ domain is a suitable target for drug discovery. Here we report near complete backbone  BCL6 is a zinc finger transcription factor and regulator of lymphocyte differentiation. The enforced expression of BCL6 prevents terminal B-cell differentiation to plasma cells in vitro and in vivo is sufficient to promote lymphomagenesis in mice  translocation was associated with improved prognosis. Gene expression studies have shown that relatively high BCL6 mRNA expression is a feature of the germinal centre (GC) type of high grade B-cell lymphoma, and is again associated with good prognosis, whereas the activated B-cell (ABC) type of lymphoma has relatively low BCL6 expression and poorer clinical outlook.BCL6 is a transcriptional repressor and the N-terminal BTB-POZ domain is responsible for the recruitment of co-repressor molecules and histone deacetylases  Rosetta. Uniformly 15N, 13C/15N and 2H/13C/15N labelled samples were grown in modified Spizizen’s media with 1.0 g/L of 15N ammonium chloride and 4 g/L of 13C-glucose  as the sole nitrogen and carbon sources. Bacterial cultures were grown at 37 °C to an optical density of ~ 0.5, whereupon the temperature of the culture was reduce to 20 °C and protein expression was induced by addition of IPTG to a final concentration of 0.225 mM. Cultures were grown for a further 16 h  or 40 h (2H/13C/15N labelling). Cells were harvested and then lysed into 50 mM Tris, pH 8.5. Initial purification (~70%) was achieved by affinity chromatography using Ni–NTA resin. After TEV cleavage of the affinity tag and dialysis into 50 mM phosphate buffer pH 6.0, 300 mM NaCl, 1 mM DTT, the protein was further purified by size exclusion chromatography using a Superdex S200 column (GE Lifesciences). The protein was concentrated and its purity assessed by Coomassie stained SDS–PAGE.The BCL6-BTB/POZ domain  was cloned into an expression vector CACB, HN(CO)CA, HNCA . TypicaAll NMR data sets were non-uniform sampled to 20–25% of the full data size, and were then reconstructed and processed using hmsIST . Chemical shift differences in the 15N-TROSY experiments recorded in the two buffer systems were relatively minor  with the following exceptions: V18, R40, Q64, E99, E115, I125, K126 & A127 which showed significant chemical shift changes between “high” and “low” salt buffer systems.Backbone amide resonance assignments were obtained for all residues in the N-terminal domain except for T12, R13, I36, 86, 87, 88, 91, T92, G100, H116 (92%) and for all Cα and Cβ resonances apart from T12, M90, 91, E99 T108 (96%). The comprehensive Analysis of the backbone chemical shifts of the BCL6-BTB/POZ domain using Talos+ (Shen et al.


Thermus thermophilus. A structural perspective and impeding challenges in further elucidating the mechanism of TH are discussed.Nicotinamide nucleotide transhydrogenase (TH) is an enzyme complex in animal mitochondria and bacteria that utilizes the electrochemical proton gradient across membranes to drive the production of NADPH. The enzyme plays an important role in maintaining the redox balance of cells with implications in aging and a number of human diseases. TH exists as a homodimer with each protomer containing a proton-translocating transmembrane domain and two soluble nucleotide binding domains that mediate hydride transfer between NAD(H) and NADP(H). The three-domain architecture of TH is conserved across species but polypeptide composition differs substantially. The complex domain coupling mechanism of TH is not fully understood despite extensive biochemical and structural characterizations. Herein the progress is reviewed, focusing mainly on structural findings from 3D crystallization of isolated soluble domains and more recently of the transmembrane domain and the holo-enzyme from  Nicotinamide nucleotide transhydrogenase (TH) is a key enzyme residing in mitochondrial inner membrane and bacterial cytoplasmic membrane that helps to maintain cellular redox balance through the following reaction:+, resulting in the generation of NADPH under most physiological conditions  of domain II are encoded by one gene α, whereas in T. thermophilus, two separate genes (α1 and α2) encode domain I and the first three TM helices (TM 2–4) of domain II; the remaining TM helices (TM 6–14) of domain II together with domain III in both species are encoded by gene β. Of note, domain II differs in the number of TM helices in different species, for example, with 14 for human, 13 for E. coli and 12 for T. thermophilus. To date, most functional and structural characterizations have been performed on TH from lower organisms  for NAD+ is 100–500 μM and that for NADH is 50 μM, comparable to values for the isolated domain I  is orders of magnitude weaker: the Kd values are 16 μM for NADP+ and 0.87 μM for NADPH  binding such as open, intermediate, and closed forms have been demonstrated, both in domain I structures and the apo structures   and NADP(H) binding sites Figure . In thesT. thermophilus TH domain II construct (containing two hydrophobic polypeptide chains: subunit α2 and a truncated version of subunit β from which the soluble domain III was removed from the C-terminus), using 1-(8Z-pentadecenoyl)-rac-glycerol (MAG 8.7) as the host lipid. Compared to the commonly used monoolein (MAG 9.9), MAG 8.7 has a shorter alkyl chain and tends to form a more fluidic cubic phase.Structural determination of the transmembrane domain of TH has recently been achieved by the use of lipidic cubic phase (LCP) crystallization  form an extended H-bonded network together with two bound water molecules in the cytoplasmic half of the channel, and this connection likely provides a proton conduit to the cytoplasmic side. A central role for His42α2 in proton translocation by TH is consistent with extensive biochemical characterizations; the central histidine is conserved in other TH species although its position may shift  simulations, allows a clearer interpretation of the proton translocation channel and its key functional residues Figure  Padayat. A clustβ on the cytosolic end of TM13 and Arg254β at the start of the linker region connecting domain II and domain III. This linkage is positioned on the membrane surface adjacent to the entrance of the proton channel. The Asp-Arg salt bridge at equivalent positions in E. coli TH (Asp213β and Arg265β) has been postulated and implicated as important for both proton translocation in domain II and NADP(H) binding in domain III  binding site faces down to approach the channel surface of domain II. The two orientations of domain III are supported by disulfide cross-linking of inserted cysteine pairs on separate domains  that connects domain II and domain III, and which is supposedly important to mediate the two domain interactions and the rotation of domain III as well. Modeling of human TH structure based on homologs is challenging given the significant sequence disparity, especially for domain II because of the presence of extra TM segments in the human enzyme (Metherell et al., The mechanistic understanding of mitochondria TH has benefited from extensive structural and biochemical characterizations of isolated domains and homolog proteins, particularly those from lower organisms. The use of stably expressed and purified TH from All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Testin has been proposed to have an open and a closed conformation based on the observation that its N-terminal half and C-terminal half directly interact. Here we extend the testin conformational model by demonstrating that testin can also form an antiparallel homodimer. In support of this extended model we determined that the testin region (amino acids 52–233) harbouring the PET domain interacts with the C-terminal LIM1-2 domains  TES gene (Unigene Hs.592286), is a modular scaffold protein consisting of a cysteine rich (CR) region, a central PET  domain and three LIM  domains together constituting the C-terminal half of testin (Tes knockout mice have an enhanced susceptibility to develop drug-induced gastric carcinomas . L. LTES ge56.1, encrcinomas .Although a role for testin in p38-signalling has been suggested , previouin vitro and in cells, we show that the region 52–233 containing the PET domain directly interacts with the LIM1-2 domains and contributes to the inter- or intramolecular interaction. This assigns a function to the PET domain and its flanking regions as an additional protein interaction module within testin. Because testin is capable of forming a monomer or a dimer, we conclude that the conformational repertoire of testin is more extensive than the open/closed monomeric transition previously described  is plotted versus wavelength (nm). Spectra of PET52-233 and PET92-199 evidence these fragments of testin are structured.CD spectra of PET(TIF)Click here for additional data file.S4 Fig52-233 myc/mito was used as bait (A-F). Note the dotted appearance of the (red) bait signal consistent with targeting to the mitochondria via the mito-tag. When used as prey, LIM1-2-GFP was recruited by PET52-233 and GFP-tagged (prey) testin constructs in HeLa cells. Myc signal (red), GFP signal (green) are shown for each condition. PET(TIF)Click here for additional data file.


P], Final [F] setback [S] viscosity, pasting temperature [PT] and time [T]) and alveogram  properties of flour from the pretreated bananas and its composite with wheat flour (WF) were examined. The baking (water absorption [WA] and specific volume [SV]) and sensory properties of bread produced from the flour were also examined. Protein, gluten, and alpha‐amylase activity ranged from 4.75 ± 0.07%, 30.25 ± 0.05%, and 4.00 ± 0.05 min to 13.75 ± 0.06%, 35.64 ± 0.06%, and 39.61 ± 1.18 min with WF:PTBF/95:05, WF:CTBF/00:100, WF:BBF/80:20, WF:100 and WF:CTBF/00:100, WF:PTBF/95:05, WF:100, WF:PTBF/00:100 having lowest and highest values, respectively. P, F, S viscosities, PT and T ranged from 186.17 ± 0.71, 217.08 ± 1.41, 38.92 ± 5.42 RVU, 84.70 ± 0.28°C, 5.04 ± 0.05 min to 461.0 ± 5.07, 348.5 ± 8.84, 88.83 ± 0.24 RVU, 87.20 ± 0.00°C, 6.24 ± 0.05 min, respectively. E, MI, P/L, and EI ranged from 141.50 ± 0.71 × 10−4J, 15.35 ± 0.07, 0.59 ± 0.83 and 35.85 ± 0.07 to 325.00 ± 1.4 × 10−4J, 22.55 ± 0.07, 2.75 ± 0.07, and 70.50 ± 0.71, respectively. WA and SV were 48.12 ± 0.07 to 52.60 ± 0.14 and 2.850 ± 0.07 to 5.635 ± 0.18 with the WF having significantly (P < 0.05) higher values than other blends and the most acceptable in terms of appearance and taste.Effects of chemical and heat pretreatments on the protein, gluten, and alpha‐amylase activity, pasting (Peak [ Consumption of wheat‐based products is not likely to decrease, considering the growth in population and urbanization Bakare  and the Several studies have reported the prospects of composite flour technology as a means of reducing the dependence on wheat for the production of bakery products  are highly perishable crop produced in the tropical and subtropical countries of the world where they are mostly grown for either home consumption or for the local markets. It is the world's most popular fruit and one of the world's most important staple foods, after rice, wheat, and maize. About 107 million metric tons of bananas were produced in more than 130 countries in 2011 representing a total trade value of US$9 billion . Browning is usually initiated by the enzymatic oxidation of monophenols into Banana is susceptible to enzymatic browning during processing  bunches were purchased from an Agriculture produce market in Abeokuta South Local Government area of Ogun State, Nigeria. WF was obtained from the Nigeria Eagle Flour Mills Ltd, Ibadan, Oyo State while other ingredients such as sugar, iodized salt, yeast, fat, and ascorbic acid were purchased in the retail markets.Banana was processed as described by Bakare et al.  with somμm mesh sieve .The banana slices were placed on a foil paper placed on drying racks to avoid sticking together; it was then dried in a cross‐flow Gallenkamp oven  at 70°C for 18 h. The dried chips were milled to flour in a Disc mill  at 8800 rpm and sieved through a 250 All analyses were performed in triplicates. Proximate, gluten, and water absorption of WF and blends were analyzed with official methods AACC  using a Pasting characteristics of the treated flour were determined according to ICC No 162 method. Rapid visco analyser (RVA) series 4  with the aid of thermocline for Windows  was used for the analysis. The 12‐min profile was used for all the analyses. It consists of idle temp of 50°C for 1 min, then 50–95°C in 3 min 45 sec, held at 95°C for 2 min 30 sec, cooled to 50°C over 3 min 45 sec, finally, held at 50°C for 2 min. Two paddle speeds of 960 rpm for the first 10 sec followed by 150 rpm were employed for the remaining duration of the test cycle. The weight per sample used for each analysis was calculated from the formula:A = weight of flour sample: using the RVA manual as a guide.where S = corrected sample weight.M = actual moisture content of the sample.W = volume of water used.L) of the alveogram curve, energy input (Joules) required for the mechanical deformation of the dough (W), inflation required for maximum development (G) and the elastic resistance (Ie) of the measured dough samples.The Alveograph (Chopin NG France) was used to measure AACC  characteFlour sample (250 g) of known moisture content was placed into the mixer, sodium chloride solution (2.5%) was added through a buret  and mixed for 7 min. The dough was forced through the extrusion gate in the form of a thin strip on to a small oiled steel plate. Five extruded dough pieces of designated length were cut off, rolled with an oiled rolling pin to a uniform thinness, cut into a circular disk, transferred to an oiled steel plate and subjected to a brief rest period in a tempered compartment of the alveograph for 15 min. Each circular dough test pieces were removed from the compartment and inserted between two metal plates that held it securely in position. The air valve was opened to supply air pressure to the held dough through an orifice. The electrically driven recording manometer was simultaneously activated to record the air pressure inside the dough bubble against time.Bread loaves were produced according to AACC  with sliThe bread samples were produced in the laboratory of Nigerian Eagle Flour Mills Ltd, Ibadan‐Lagos Expressway, Ibadan, Oyo State, Nigeria. The dough was mixed using Chaplain spiral mixer , fermented (proofed) at initial and final fermentation time of 15 and 28 min and at ambient condition of 28 ± 2°C and 85 ± 12% relative humidity and baked in an oven  at 220°C for 30 min. The bread samples were cooled for 1 h, then placed in low‐density polyethylene bags and kept at 24 ± 2°C.Weights of bread loaves were measured with a Mettler Toledo (A204) digital weighing scale. Volume was measured by millet seed displacement method AACC  with minThe weight loss of the bread was determined as described by Kim et al. . The douwhere:A = weight of dough.B = weight of baked bread.Thirty people were selected from a pool of volunteers comprising of officers, lecturers, and students of tertiary institutions. The panelists were selected after an oral interview conducted on the basis of a criteria checklist that included: good health, nonsmoker, nonallergic to wheat/banana, willingness to participate, and passion/likeness for the consumption of bread.Quantitative acceptance test was used to assess consumers’ liking for the product. The thirty untrained panelists rated their liking or otherwise for the bread samples produced from the blends on a 9‐point hedonic scale .All experiments and analyses were conducted in triplicates. Data obtained from different aspects of the study were subjected to analysis of variance and the Duncan multiple range test was used to separate the means Duncan . StatistMoisture content determination is an important index of quality for flour generally, and WF in particular. Moisture content above 14% affects the storage quality of flour and at more higher content, it may increase the microbial content and may also predispose the flour to infestation by insects. It may also cause the flour to agglomerate, become wet, and create production problem. The moisture content of the flour (Table P < 0.05) affected the protein content  had relatively lower protein content (4.64 ± 0.06–5.46 ± 0.00%) than the WF (11.00 ± 0.14). The pretreatment appeared to have significantly  lower than that of the treated flour, except for CTBF. The fat content of the PTBF containing blends was higher in the 5% and 10% blend, but lower at subsequent substitution levels except at 20%. In all, the fat contents of the blends ranged from 1.08 ± 0.01 to 1.46 ± 0.01 with the WF:CTBF/95:05 and WF:BBF/80:20 having the highest and lowest values, respectively.The fat content of the WF was significantly (P < 0.05) increase in the ash content of the blends at all levels of substitution except that of WF:CTBF/85:15.Flour from the treated banana had significantly higher ash contents than the WF and this resulted in significant (P < 0.05) lower than the value reported by Zuwariah and Aziah  is one such few enzymes. α‐amylases are endoenzymes that randomly hydrolyze the a‐‐linkages in the starch polymers, generating low molecular weight dextrin ‐bonds of starch, alpha‐ amylase activity in flour is measured in Falling number values. Flour from the pretreated banana had significantly (P < 0.05) higher Falling number values (27.78 ± 0.11–39.61 ± 1.18) than the WF (4.0.00 ± 0.05). However, the effect of this was not pronounced in the composite blends suggesting that the flour either possesses little or no endogenous alpha‐amylase, the long outer amylopectin chains in banana starch  had more tendency toward retrogradation than the chemically treated sample, but both were better than the WF in this regard.Peak time was the time at which the peak viscosity was attained in minutes. The WF had a significantly higher peak time than flour from the treated banana.The alveograph is an important dough testing instrument used to evaluate the quality of WFs for bread and biscuit and cookie making  indicated the resistance that the dough offered to deformation and it is related to the tensile strength or stability that the dough exhibited during the proofing stage of bread making  indicated the extensibility of the dough. The L values ranged from 46.50 ± 0.71 to 103.00 ± 1.41 mm with the WF:PTBF/95:05 and WF:BBF/80:20 having the highest and least extensibility, respectively. The L values decreased significantly with substitution levels with the WF:BBF being the least extensible at 5% level while at subsequent levels the WF:CTBF was the least extensible.The length (P/L (configuration ratio) ranged from 0.59 ± 0.83 to 2.75 ± 0.07 with the WF:BBF/95:05 and WF:BBF/80:20 having the lowest and highest values, respectively. The P/L values increased as the WF was replaced with flour from each of the treated banana, respectively. There were no significant (P < 0.05) differences between the WF and the WF:PTBF (5–20%), WF:CTBF (5%), and WF:BBF (5–10%). The curve configuration ratio (P/L) is an index of gluten behavior. However, the strength of composite flour are influenced by considerations other than gluten behavior. Obviously, the significantly higher P/L values of WF:CTBF/90:10, WF:CTBF/85:15, WF:CTBF/80:20, WF:BBF/85:15, and WF:BBF/80:20 relative to other blends and the WF could not have been due to the effect of gluten as shown in Table The WF:BBF 5–0%. The c−4J to 337.50 × 10−4J WF:BBF/95:05 and WF:PTBF/80:20 having the highest and lowest values, respectively. Except for WF:BBF/95:05, baking strength decreased as WF was replaced with flour from the treated banana. The energy required to expand the dough for each of the three categories of blends decreased significantly with levels of substitution.The area under the curve represents the energy required to expand the dough and is related to the baking strength of the flour. This area is generally much larger for hard WFs than for soft WFs. It ranged from 141.50 × 10G values (Table The es Table  depictedIe) may be used to characterize the dough on the basis of the elastic resistance that they offer during their biaxial deformation  different from the blends. Actual water absorption decreased as the WF was replaced by each of the flour from the treated bananas and there was no significant difference between the blends at each of the substitution ratios.The quantity of water used to form the dough for baking Table  ranged f3/g with the WF:PTBF/95: 05 having significantly higher specific volume than other blends. The values decreased as flour from the treated banana replaces the WF in the blends but the 5% WF:PTBF was significantly (P > 0.05) better than the WF.Loaf volume is used as a criterion to measure the quality of fresh bread in research quality control in industry and by consumers  higher Falling number values implying that the pretreatments may have (since the study design did not include untreated banana flour) reduced the alpha‐ amylase activity of the treated flour thereby decreasing the extent of liquefaction and diastatic activity of their starches. This observation reflected in the blends as the proportion of the flour from the treated banana was increased.Flour from the pretreated banana had significantly , which is an index of the rheological attributes of the dough, showed that composite flour containing the potassium‐treated flour (WF:PTBF) was not significantly different from the WF at all the levels of inclusion (5–20%) in this study, while those containing the blanched sample (WF:BBF) and the citric acid‐treated (CTBF) sample were only comparable to the WF at 5% and up to 10% levels of inclusions, respectively. The specific volume of WF:PTBF bread was significantly (P > 0.05) better than the bread produced from the WF and had better scores for appearance, texture, and taste than bread samples from other blends.The rheological attributes of the composite flour in which they were included was also affected in different ways at various levels of inclusion in their composite with WF. For instance, the curve configuration ratio (None declared.