Sequence alignment showed that the major residues contributing to the new HBGA binding interface are conserved among most members of the GII.21, as well as a closely related GII.13 genotype. In addition, we found that glycerol inhibits OIF binding to HBGAs, potentially allowing production of cheap antivirals against human NoVs. Taken together, our results reveal a new evolutionary lineage of NoVs selected by HBGAs, a finding that is important for understanding the diversity and widespread nature of NoVs.Norovirus (NoV) causes epidemic acute gastroenteritis in humans, whereby histo-blood group antigens (HBGAs) play an important role in host susceptibility. Each of the two major genogroups (GI and GII) of human NoVs recognizes a unique set of HBGAs through a distinct binding interface that is conserved within a genogroup, indicating a distinct evolutionary path for each genogroup. Here, we characterize a Lewis a (Le Human norovirus (huNoV) has diverged into two major lineages (GI and GII) selected by the host histo-blood group antigens (HBGAs). Both lineages further diverge into various sub-lineages (genotypes) that recognize different ABH and Lewis antigens through a common HBGA binding interface shared among strains within each genogroup. In this study, through X-ray crystallography of the P domain of a GII.21 huNoV (OIF) we identified a unique lineage in GII consisting of GII.13 and GII.21 genotypes that recognize HBGAs through a binding interface distinct from the GII conventional binding interface. While the mechanism remains unknown, our finding raises an alert on future emergence of new lineages by the same way via developing new receptor binding interfaces, as well as further divergence of this new lineage into more sub-lineages recognizing different HBGAs, which may impact future epidemiology and strategies for disease control and prevention against huNoVs. Norovirus genus in the family Caliciviridae. NoVs are genetically diverse, containing six genogroups (GI to GVI) with over 35 genetic genotypes. NoVs exhibit wide host tropisms causing diseases in various mammalians including human. Human NoVs (huNoVs), consisting of mainly GI and GII NoVs, are the most important viral cause of epidemic acute gastroenteritis in humans   was dissolved in double distilled water and prepared as 20 mM solution, and then mixed with an equal volume of native P domain protein (26 mg/mL), and incubated at 4°C for 1 hour before crystallization. The final reservoir for the growth of complex crystals contained 0.25 M (NH4)2SO4, 18% (w/v) PEG 3350. Micro-seeding technique was used to aid the growth of complex crystal with native crystal seeds 16 hours after the setting of crystallization droplets. OIF P domain-Lea complex crystals were harvested in three days.While performing the crystallization experiments, we observed that the presence of glycerol inhibited the co-crystallization of the OIF P domain-Lea complex were the corresponding reservoir solutions complemented with 15% (v/v) PEG 400. Crystals were briefly soaked in the cryo-protectant for 5 seconds before being mounted for diffraction test. The diffraction data for native crystal were collected at the beamline 41U of SPRING8 (Japan) at a wavelength of 1.0000 Å, while the Lea complex data were collected at rotating-anode X-ray source MicroMax-007/Satun 944 HG/Varimax HF  at the wavelength of 1.5418 Å. Diffraction data were processed, scaled, and merged using the HKL-2000 program package [The cryo-protectants of crystals of unliganded P domain and P domain-Le package . Data coa complex were solved using the final refined structure of native P domain protein as model. Structure refinement statistics are summarized in The native crystal structures were solved by molecular replacement method using Phaser of CCP4 suite  and the E. coli system (BL21) as described elsewhere [Single residue mutations were introduced to the HBGA binding site of the OIF P domain by site-directed mutagenesis using the expression plasmid of the wild type OIF P domain as template. Mutagenesis was carried out using the QuickChange Site-Directed Mutagenesis Kit  and corresponding primer pairs containing the mutation sites. After confirmation of mutations, the P proteins were expressed as P particles and purified using an lsewhere , 50, 51.a, Leb, Lex, Ley, sialyl Lea, and sialyl Lex as well as the disaccharides representing the type 1 and type 2 precursors of HBGAs at 2 μg/mL were coated on microtiter plates at 4°C overnight. After blocking with 5% (w/v) non-fat milk, the coated oligosaccharides were incubated with the affinity-column purified OIF P particles (10 ng/μL) for 60 minutes at 37°C. The bound OIF P particles were detected as described previously, using an in-house hyperimmune rabbit serum against various huNoVs [a-tri-PAA (polyacrylamide) conjugates (2 μg/mL). The bound OIF P particles were detected as described above.HBGA assays were carried out as described previously . Brieflys huNoVs . For glya trisaccharide have been deposited in the Protein Data Bank (www.pdb.org) with the pdb accession codes of 4RLZ and 4RM0, respectively. The GenBank accession numbers of the sequences that were used in Figs The cDNA sequences encoding the P domain of the OIF virus has been submitted to GenBank previously with an accession number of AY675554. Coordinates and structure factors of the native OIF P protein and the complex with Le


Despite the long research history of this enzyme, three-dimensional structural data remain unavailable for its ligand-binding mode. Here we characterize PDI substrate recognition using α-synuclein (αSN) as the model ligand. Our nuclear magnetic resonance (NMR) data revealed that the substrate-binding domains of PDI captured the αSN segment Val37–Val40 only in the oxidized form. Furthermore, we determined the crystal structure of an oxidized form of the b′–a′ domains in complex with an undecapeptide corresponding to this segment. The peptide-binding mode observed in the crystal structure with NMR validation, was characterized by hydrophobic interactions on the b′ domain in an open conformation. Comparison with the previously reported crystal structure indicates that the a′ domain partially masks the binding surface of the b′ domain, causing steric hindrance against the peptide in the reduced form of the b′–a′ domains that exhibits a closed conformation. These findings provide a structural basis for the mechanism underlying the redox-dependent substrate binding of PDI.Protein disulfide isomerase (PDI) is a multidomain enzyme, operating as an essential folding catalyst, in which the  Cumulative biochemical data indicate that the b′ and a′ domains are primarily responsible for substrate recognition313b′ domain provides the principal peptide binding site in PDIa′ domain is oxidized by the flavoprotein Ero1 and thereby acts as a disulfide donor for the PDI substrates17In the endoplasmic reticulum (ER) of eukaryotic cells, a number of molecular chaperones and folding enzymes assist the proper folding of newly synthesized polypeptide chains. Protein disulfide isomerase (PDI) is a major ER protein that operates as a molecular chaperone and a folding enzyme by catalyzing the formation, cleavage, and rearrangement of the disulfide bonds of unfolded or misfolded proteins125678101213b′–a′ domains depending on the redox states of the a′ active site13a′ domain. This conformational transition is supposed to be associated with the redox-dependent substrate binding of PDI. However, no three-dimensional structural data have yet been reported for PDI ligand binding despite the long history of research on this topic. In view of this situation, we attempted to provide the structural basis of PDI substrate recognition by using an appropriate model ligand.One unique property of this modular enzyme is that it undergoes conformational rearrangement of the b′–a′ domains of PDI.It has been reported that molecular chaperones actively contribute to the suppression of toxic aggregate formation of various amyloidogenic proteins associated with neurodegenerative disorders1915N-labeled αSN and observed the heteronuclear single-quantum correlation (HSQC) spectral changes induced upon addition of the PDI b′–a′ domains. The results indicate that the oxidized b′–a′ domains caused significant perturbations in the HSQC peaks originating from the αSN segments Val37–Val40 and Val48–Gly51 b′ and a′ domains, respectively. Concerning the bound αSN undecapeptide, all residues were clearly visible in the electron density map (The PDI ed state . Due to sity map . Interesb′–a′ domains and the αSN peptide (-b′) was mediated through the b′ domain surface proximal to the a′ domain with a peptide-binding area of 391.6 Å2. The other interaction mode (termed contact-a′) gave a smaller interface area with 242.9 Å2 exclusively on the a′ domain. In contact-b′, the αSN peptide was recognized through several hydrophobic interactions involving Leu38 and Val40 of αSN and Ile213, Tyr218, Met222, and Phe267 of PDI , b′ domain (residues 208–335), and a′ domain (residues 334–449) from Humicola insolens were performed as previously described121315N-labeled αSN were performed as previously describedExpression and purification of the PDI 2O using an AVANCE800 spectrometer (Bruker Biospin) equipped with a 5 mm triple-resonance cryogenic probe. To prepare the reduced form of the PDI proteins, 10 mM d-DTT was added to the buffer. The 1H-15N HSQC spectra were recorded at a 1H observation frequency of 800.32 MHz with 256 (t1) × 2048 (t2) complex points. The spectral data of the PDI-derived proteins (at a concentration of 0.05 mM) were acquired at 303 K in the presence and absence of 0.2 mM full-length αSN or 0.2 mM αSN peptide. The spectral assignments of the PDI b′–a′ domains, b′ domain, and a′ domain have been described previously15N-labeled full-length αSN (at a concentration of 0.05 mM) were measured at 283 K in the presence and absence of 0.01–0.05 mM PDI b′–a′ domains. The NMR assignments of αSN have been described previouslyδN2 + ΔδH2)1/2, where ΔδH and ΔδN are the observed chemical shift changes for 1H and 15N, respectively. The NMR data were processed and analyzed using TOPSPIN-2.1 (Bruker Biospin) and SPARKY1H-15N HSQC peaks could not be observed because of peak overlapping and/or broadening were shown by asterisks.NMR measurements were performed in 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM KCl, and 10% (v/v) Db′–a′ domains (10 mg/ml) complexed with αSN peptide (1:5 molar ratio) were grown in 0.1 M HEPES buffer (pH 7.5) containing 25% (w/v) PEG3350 for a week at 293 K. The crystals were directly transferred into the reservoir solution and flash-cooled in liquid nitrogen. The diffraction data set was collected using synchrotron radiation at BL44XU of SPring-8 (Japan), and was scaled and integrated using HKL2000The crystals of the PDI b′–a′ domains complexed with the αSN peptide was solved by molecular replacement using the program MOLREPb′ and a′ domain coordinates derived from the crystal structure of H. insolens PDI b′–a′ domain http://www.pymol.org/).The 1.60-Å resolution crystal structure of the PDI Accession codes: The coordinate and structural factor of the crystal structure of the PDI b′–a′ domains complexed with αSN peptide has been deposited in the Protein Data Bank under the accession numbers 5CRW.How to cite this article: Yagi-Utsumi, M. et al. Structural basis of redox-dependent substrate binding of protein disulfide isomerase. Sci. Rep.5, 13909; doi: 10.1038/srep13909 (2015).


Facilitators are known to be influential in the implementation of evidence-based health care (EBHC). However, little evidence exists on what it is that they do to support the implementation process. This research reports on how knowledge transfer associates (KTAs) working as part of the UK National Institute for Health Research ‘Collaboration for Leadership in Applied Health Research and Care’ for Greater Manchester (GM CLAHRC) facilitated the implementation of EBHC across several commissioning and provider health care agencies.A prospective co-operative inquiry with eight KTAs was carried out comprising of 11 regular group meetings where they reflected critically on their experiences. Twenty interviews were also conducted with other members of the GM CLAHRC Implementation Team to gain their perspectives of the KTAs facilitation role and process.There were four phases to the facilitation of EBHC on a large scale: (1) Assisting with the decision on what EBHC to implement, in this phase, KTAs pulled together people and disparate strands of information to facilitate a decision on which EBHC should be implemented; (2) Planning of the implementation of EBHC, in which KTAs spent time gathering additional information and going between key people to plan the implementation; (3) Coordinating and implementing EBHC when KTAs recruited general practices and people for the implementation of EBHC; and (4) Evaluating the EBHC which required the KTAs to set up (new) systems to gather data for analysis. Over time, the KTAs demonstrated growing confidence and skills in aspects of facilitation: research, interpersonal communication, project management and change management skills.The findings provide prospective empirical data on the large scale implementation of EBHC in primary care and community based organisations focusing on resources and processes involved. Detailed evidence shows facilitation is context dependent and that ‘one size does not fits all’. Co-operative inquiry was a useful method to enhance KTAs learning. The evidence shows that facilitators need tailored support and education, during the process of implementation to provide them with a well-rounded skill-set. Our study was not designed to demonstrate how facilitators contribute to patient health outcomes thus further prospective research is required. Facilitation to support research utilization for the benefit of patients and clients has grown in significance over the past twenty years. This is in contrast to passive methods of disseminating research, which are seen as insufficient in influencing its uptake . HoweverThe concept of facilitation in the context of evidence based health care (EBHC) has been defined as: ‘a technique by which one person makes things easier for others’ p152 ,4. A quaTo our knowledge, there is one qualitative research study that explicitly focused on the facilitation of large-scale EBHC implementation, which consists of retrospective interviews with external facilitators of a central change agency, the Quality Enhancement Research Initiative . The finFacilitation may be employed as both a noun, that is, ‘facilitator’ and as a verb, ‘to facilitate’ . This isAs indicated, there are many competing concepts that employ facilitative approaches including change agents, opinion leaders, KTAs, knowledge brokers. Several of the more common roles for the implementation of EBHC are presented in Table There are also numerous models and frameworks providing an overview and explanation of the process of the implementation of EBHC. The older frameworks tend to draw on a rationalistic approach defining research implementation as a technical one-way process . More reA popular definition of EBHC is ‘the conscientious use of current best evidence in making decisions about the care of individual patients or the delivery of health services’ . From thThe Greater Manchester ‘Collaboration for Leadership in Applied Health Research and Care’ (GM CLAHRC) is i) funded by the Department of Health National Institute for Health Research (NIHR) and ii) by matched funding from local National Health Service (NHS) partners in the UK . The GM When the GM CLAHRC began, the KTAs had little direct knowledge of how to implement EBHC, or of the clinical conditions to which their projects pertained except for one KTA who was a nurse by background. Several approaches were employed to assist the KTAs to help them understand and deal with the complexities and difficulties of implementing EBHC including formal teaching sessions and a co-operative inquiry. This paper focuses on the co-operative inquiry. The aims of the co-operative inquiry were: first, that through the process of co-operative inquiry the KTAs would understand better how to implement EBHC, and, second, to provide a qualitative descriptive account of how they facilitated, at both a commissioner and provider level, the implementation of EBHC.The KTAs needed to understand how to facilitate the implementation of EBHC across many organisations and commissioning agencies. ‘Co-operative inquiry’ was selected as the research design because it is a way of researching with people who have related experiences, and who wish to examine with others how they might extend and deepen their understanding of their situation, and to learn how to improve their actions . As in oThe initiating researcher, HW, played the role of facilitator who enabled candid and non-judgmental discussions among the KTAs and assisted them in developing a critical attitude to their reflections. She was deemed a co-researcher alongside the KTAs as she too, although she was not working directly to facilitate EBHC, wished to improve the process of facilitation. HW agreed to carry out interviews and do some of the data analysis with two research assistants because the KTAs could not afford the time. HW has twenty-two years’ experience of action research.The KTAs and HW were both researchers and participants in a transformative research process so the usual notion of researcher neutrality and objectivity could not apply. A reflexive approach was therefore chosen to understand how past and on-going personal and professional experiences influenced the research . During Basically, the KTAs came together for reflective meetings to share what they did on a daily basis, to explore their successes and problems in detail, and to make sense of their experiences. After they would return to their projects and return a month later for further discussions and so on. The intensely reflective method of co-operative inquiry was considered by the KTAs to be pertinent to their educational needs and was an important reason why co-operative inquiry was chosen. Other critical collaborative research methods, such as critical ethnography, could have been chosen but participant self-development in co-operative inquiry was the crucial reason for its selection .Co-operative inquiry has an extended epistemology of four types of knowing: practical (how to do something), experiential (direct encounter), propositional (theory) and presentational (stories) knowledge . During How do KTAs facilitate the implementation of EBHC in the NHS to improve patient/client care?How do the GM CLAHRC managers (Ms), clinical leads (CLs) and academic leads (ALs) perceive KTAs facilitate the implementation of EBHC?Research questions were negotiated and agreed by the KTAs:Phase 1: Preliminary meetings to agree a plan for the inquiryPhase 2: Provisional sense making by the KTAs as co-subjectsPhase 3: KTAs immerse and engage with their action and experiencePhase 4: The original plan and sense making are reviewed by KTAs in light of phases 2 and 3A voluntary co-operative inquiry group was formed that consisted of the KTAs and HW. The inquiry passed through the following cyclical phases :Phase 1:Repeat phases 2 and 3 and so on until agreement to finish.Phase 1: Over six months, the KTAs agreed the research design, who would be involved, and the method of data collection and analysis. The agreed approach to data collection was qualitative because the focus of the study was to describe and understand how KTAs facilitate the implementation of EBHC An additional layer of complexity was added by GM CLAHRC itself, as it was not perceived to be an established part of the NHS. This resulted in NHS staff tending to be suspicious of requests for information and data, and who then delayed or refrained from providing requested information. This was despite the GM CLAHRC being a formal collaboration between a university and the NHS, with KTAs employed by a host NHS organisation. One KTA explains the process and frustrations in trying to get information:The apparent inefficiency was said to slow down initial progression.During this time, KTAs increased their knowledge of the evidence base about the disease condition and existing relevant guidelines on the topic of proposed projects. However, sometimes ideas for projects would change and KTAs would need to begin again collecting new evidence. There were several reasons for changes in the proposed topics, for example, if it were not a priority for influential clinicians and health care organisations.… so we had these meetings and asked people, we didn’t have any patients there but we had people involved in [disease area] from primary and secondary care and we asked them for their opinions on what kind of work we should do. So from that we had just like the first draft of ideas of where we should go with our project… [KTA 4]At this time, different types of meetings were held with key groups. The purpose of these meetings were to: discuss the need for EBHC and potential implementation areas, share the evidence collected to date, suggest potential EBHC opportunities, garner support, further information, and identify drawbacks:Generally, the KTAs led these meetings alone then fed back to the team. In effect, the KTAs function was to pull disparate strands of information and people together to assist the CL and other stakeholders to make or confirm decisions about the focus of the project.When the topic for implementation was agreed, the KTAs then gathered further information on the specifics of the proposed implementation. Information built on that collected in the previous phase and included finding out: (a) who and what competencies are needed to deliver the EBHC, (b) Which settings to select, (c) The resource implications, (d) Whether the project might fall under the auspices of clinical audit, (e) How they might determine the likely viability of the proposed EBHC in actual practice, (f) What support and formal education is required to deliver the EBHC, (g) What other agencies need to be engaged, (h) The degree of variation permissible in the application of the EBHC and (i) How the projects should be evaluated. The KTAs found that their contacts did not have the information that was now required so they had to extend their networks. In this phase, there was a great deal of ‘to-ing and fro-ing’ as knowledge and ideas were exchanged and decisions about the detail were made iteratively between KTAs, CLs, ALs and Ms and in partnership with stakeholders. They found this was time-consuming and painstaking.They (KTAs) went to other projects, they dug out you know already evidenced material if you like, they looked at policy documents but they looked at clinical documentation so they didn’t just read the evidence base that was offered to them if you like from the first PCTs [health commissioning agencies] setting up, they looked at other versions of this and what that meant was that when they went out to talk to the remaining, … so when they went out to the other three PCTs it meant that they didn’t only have to talk about the [name of PCT] experience which some people would have just rejected, they were able to go with a wider evidence base to talk about what other people had done, to talk about what kind of evidence there was, what kind of evaluation that was. They built their own credibility in part by being able to talk more broadly about that… [AL2]During this time, the KTAs gathered a range of different types of evidence a) Research based articles, b) Reports from other implementation studies, c) Government policy or guidance documents, d) Interview/questionnaire data and e) Health databases. The KTAs and CLs were responsible for considering how these different types of evidence may be used to improve patient care. Apart from the instrumental use of evidence, reports, for example, were employed to inform conceptual ideas for the justification or necessity of projects :They , specialist nurses, district nurses, receptionists and practice managers were mostly new.The approach to recruitment of general practices was systematic to get a representative sample of the local population. In one project, recruitment was assisted by a local opinion leader who sent a letter to all potential participants. This opinion leader’s views were valued by his peers. In another case, an opinion leader actually recruited on behalf of a KTA. Word of mouth also helped in the recruitment of general practices.I have improved my skills of negotiating with people, because like again in an informal way we have had, it has kind of trying to get people on board, so maybe communication skills in that respect have improved like dealing with people and working out what motivates them, what you need to say to them to try and get them to do the work for you and things like that. [KTA 10]Also significant in recruiting people, the KTAs negotiation and communication skills improved throughout the project:The data shows that KTAs acquired four key areas of knowledge that helped them to present reasoned arguments to people in an effort to recruit them. For example, they often referred to key stakeholder support to gain interest Table . Buildin…and then once we had got practices interested, going and meeting with them and making sure that they were happy to be part of it, getting them to sign up and then it kicked off the real work of running workshops, going into practices and discussing the …work with them, collecting data, analysing data. Various things we did along the way with practices like assessing their progress against various different frameworks and scorecards and using that to help us think what we can do with them to help them improve more. [KTA 10]All the tasks were project managed by the KTAs, CLs, ALs and Ms. However, each of the projects had different approaches to how they implemented EBHC in the clinical areas. The variation occurred in the formality of the approach, the level of prescription of the EBHC, what needed to change to achieve implementation and the amount of time KTAs spent in each setting where the EBHC was implemented. For example, in one project, an invitation about a planned audit was sent to general practices. Following the audit, written and oral guidance on how clinicians could implement EBHC was offered. This approach was more ‘hands off’ compared to another project in which a link person or team from each practice was identified through which the EBHC was implemented. The involved KTA describes the processes:In this approach, the KTAs spent a varied amount of time with each practice depending on how much support was required to implement the EBHC.Some participants were able to implement the EBHC without much assistance but others found it difficult to move forward. These situations were a challenge for the KTAs. They, in consultation with CLs, ALs and Ms, took the line that they were there to help staff to implement the EBHC for which they had ‘signed up’. They were not in the position to tell them what to do or deliver tasks for them, but they tried to provide as much support as possible. The KTAs also found that honesty, directness and focussing on what needed to be done were effective in helping staff to persist. The KTAs tried to work in partnership with staff, maintaining good relationships with them, to secure commitment and loyalty to the project.Contextual problems encountered by the KTAs included: (a) Poor internal communication channels, (b) Poor leadership, (c) Rejection of the value of using evidence to improve practice, and (d) Poor administrative/management systems. The KTAs, in discussion with the project team, took different approaches to the analysis and actions to overcome these difficulties. Some identified and acted on these issues informally, for example, by noting and addressing them where possible at subsequent meetings. In contrast, others took a formal approach, for example, through supporting staff to use specific tools that enabled them to review and change their situation to enable the implementation of EBHC . AltogetWe have had huge amounts of problems trying to get accurate data, even on some of them. There are some things that the practices measure for QOF [quality and outcomes framework] which is how they get paid, so they have got all their measures accurate for that but there are quite a few things which are measures we just said we would like to look at, and some of them we are still not getting accurate. We have been working on it for eight months and we still don’t get accurate data on it… and the computer systems don’t work and they won’t allow you. [KTA 5]While the evaluation of EBHC projects did not unfold sequentially after the implementing and co-ordinating phase, it was the concluding work of facilitation for all the KTAs before the projects coalesced and/or transformed into other projects. The KTAs often set up new systems to collect data because the information already gathered by general practices was not always appropriate for the purposes of evaluating the project. One KTA reports on the difficulties they have had getting suitable data:The KTAs had to enhance their understanding of databases to obtain this type of information. All the KTAs had access to academic advice about the evaluation of EBHC and drew upon research skills, although, they were not doing research, during this time.KTAs analysed patient outcomes, and evaluated patient and staff experiences by individual or focus group interviews. In all but one project, with the benefit of hindsight, more and different types of data, for example, clinical data, could have been collected. The KTAs also collected data and information to develop general guides to support the spread of the project to new sites.Our research draws on the experiences of 8 KTAs who were responsible for the facilitation of the implementation of EBHC across six projects in the GM area over one year. It is of sufficient scale to provide indicative findings of the process, methods and styles of facilitation that were employed. Figure Although, the activities of facilitators appear similar, the findings show that in practice variations existed between projects in the length of time of each phase, who was involved, level of prescription of the method for the management of change and fidelity to the interventions i.e. how much adaption occurred in each context. The reasons for this depended on the EBHC to be implemented, for example, whether it was a technology or administrative process, available resources, personal preferences of the project team and participants and the context in which it was to be implemented. This means that only a skeletal process framework for facilitation of the implementation of EBHC can be offered, that is, ‘no one size fits all’.The findings also support the conceptualisation of the purpose of facilitation as a continuum ranging from a task oriented approach where the focus is on achievement of a goal to a holistic approach where the emphasis is on helping and supporting individuals and/or teams to improve their way of working . These dUnderlying assumptions about the process of implementation tend to be portrayed from either a rational-technical or interactive social perspective . HoweverThe interactive approach was also apparent when practitioners did not just implement EBHC because of the strength of the research argument, but needed to situate it in their own context to suit their and their patients’ needs . Thus, rOur projects also had elements that map onto a rational-linear approach to EBHC. Rational-linear proponents tend to assume there is a unidirectional process from research to implementation without local adaptation. Within this approach, there is a tendency to assume passive dissemination will lead directly to implementation . A ratioFigure In our study, the KTAs lacked contextual knowledge of the NHS which was not helped by the lack of uniformity between NHS Trusts and general practices in terms of how they were structured, managed and the services they provided. They had to discover this to understand what was likely to be feasible and how to gain agreement about the EBHC. The acquirement of contextual knowledge through networking and gaining information was a significant and explicit part of defining and planning for a realistic and successful implementation of EBHC. In local, insider implementation studies, the need for networking and understanding organisational processes and culture is still important but may be more likely to be known to those facilitating the project and, therefore, be on a lesser scale and less likely to be reported.As our study was large-scale, and centrally driven by the KTAs, CLs, ALs, and Ms , one actAnother recruitment strategy of some KTAs was to employ personal or financial incentives. This strategy of recruitment echoes Roger’s notion of relative advantage where potential adoptees of an innovation assess whether to uptake an innovation according to whether it is perceived as bringing better rewards economically or in terms of social prestige than the current arrangement . IncentiA clear understanding of the process and possessing the right skills are requisites for successful facilitation ,40. In oThe KTAs were perceived as coming from a ‘new and strange organisation’ to the NHS participants with whom they had to work and all except one began with experience external to the clinical area. Latterly, practitioners from primary or acute sector were seconded to either be KTAs for specific projects or to provide specialist clinical knowledge. The gain is thought to be in three ways: i) as they will develop their skills and knowledge base in facilitating EBHC, ii) the GM CLAHRC will benefit from their additional clinical skills and knowledge and understanding of local context and iii) it serves to raise awareness of GM CLAHRC and how it works with the NHS. Their involvement will also increase capability in the local workforce regarding the implementation of EBHC.The co-operative inquiry provided a forum in which the KTAs could discuss openly their experiences and reflect critically upon the process of facilitation. This provided a learning environment that expanded their experience and deepened their understanding about facilitation, which they were able to articulate clearly to others. This was in addition to the formal education they received. The co-operative inquiry itself ran relatively smoothly; this was because the KTAs negotiated time away from daily activities to attend. In comparison, the actual process of implementation, as shown, was messy, complicated and difficult to plan far ahead.Direct observations of the KTAs in their daily work would have provided a more detailed picture of how they facilitated the implementation of EBHC. However, in doing the co-operative inquiry, they ‘observed’ themselves and while they could be accused of bias, we deliberately took a critical position with individual accounts during the group meetings. A strength of co-operative inquiry is the personal development which occurs from participants’ direct involvement in the research but at the same time this feature may be construed as a disadvantage as biases may go unnoticed even though the process is critical. In our study, to counter this effect, preliminary data analysis from the interviews with the CLs, ALs and Ms were fed back to the KTAs and then in turn the results of the overarching analysis were fed back to CLs, ALs and Ms during the second round of interviews and some of them were involved in the writing of the paper (RB and GH) with the KTAs.Two KTAs left their employment, which could have disrupted the research and affected the quality of data collected. However, the disruption was minimal because the new KTA took over early in the process during phase 2. The allocation of KTA work also meant that they overlapped one another having a primary and secondary role in at least two of the projects so that no project was left bereft of a KTA even when KTAs left.The study was not designed to demonstrate whether facilitators of the implementation of EBHC contribute to improved patient or health outcomes. Further research is required to investigate the link between the two and indeed if different facilitation approaches have different impacts on outcomes, within the knowledge that context is a key influence on the facilitation process itself.Our research is important because it articulates insider accounts of the actual process of facilitation and the activities involved in the implementation of EBHC at both commissioning and provider levels. The KTAs who were external to the institution in which EBHC was implemented, were embedded in an organisation (GM CLAHRC) and supported by a team of people who had different amounts of input and expertise. A diverse and flexible approach to the implementation of EBHC is required depending on the context where it is to take place. There was considerable lack of uniformity in the organisation of the health care systems, which meant, for example, that one method of communicating with staff in one setting might not be appropriate in another. The iterative processes in the Results also provide evidence that contextual analyses are required to work successfully with stakeholders. Insider knowledge of structures, interactions between stakeholders and patient services made facilitation easier. If the KTA did not already possess this knowledge then time was spent acquiring it. Altogether this research demonstrates in some detail how no single method of facilitation will fit all circumstances. Further investigation is required of how personal and financial incentives may play a part in gaining ‘buy-in’ of participants for the implementation of EBHC. The focus of EBHC is about the implementation of research, which is usually externally generated, but in this study the KTAs were also responsible for generating evidence to show there was an issue or to solve a problem. The findings are relevant to policy makers as they suggest that facilitation in this capacity is a dedicated role especially since the scope and skills required are extensive. Formal education and support systems are required to prepare a cadre of facilitators capable of operating at this level. Further prospective research is required to demonstrate how facilitation of the implementation of EBHC affects patient health outcomes.


The Ag-(DHQ)2AQN and Ag-(DHQD)2AQN complex nanocrystals were formed at the liquid-liquid interfaces and characterized by using UV-vis absorption and fluorescence spectroscopy and X-ray photoelectron spectroscopy, as well as by using scanning electron microscopy. Screw-like nanocrystals were formed at the initial 30 min after the interfacial coordination reaction started, then they grew into nanorods after several days, and finally became cubic microcrystals after 2 weeks. The pure ligand showed two emission bands centered at about 363 and 522 nm in the methanol solution, the second one of which was quenched and shifted to about 470 nm in the Ag-complex nanocrystals. Two couples of reversible redox waves were recorded for the Ag-complex nanocrystals; one centered at about -0.25 V (vs. Ag/AgCl) was designated to one electron transfer process of Ag - (DHQ)2AQN and Ag - (DHQ)2AQN+, and the other one centered at about 0.2 V was designated to one electron transfer process of Ag - (DHQ)2AQN and Ag+ - (DHQ)2AQN.Immiscible liquid-liquid interfaces provide unique double phase regions for the design and construction of nanoscale materials. Here, we reported Ag(I)-directed growth of metal-organic complex nanocrystals by using AgNO Self-assembly of nanostructural materials at the fluid interfaces has recently received growing attention because the interface regions have a double phase thickness of tens of nanometers depending on the nature of the solvents and species within them, a dimension of which is comparable to that of the nanostructural materials. That is3 and its enantiomer of (DHQD)2AQN as linkers. Our previous work has revealed that AgNO3 could direct formation of chiral coordination polymers (CPs) at the air-water interface[In the present work, Ag(I)-directed metal-organic complex nanocrystals were fabricated at the water-chloroform interface by using AgNOnterface. Atomic 2AQN and its enantiomer (DHQD)2AQN . All chemicals were used as received without further purification. Ultrapure water (18.2 ΩM cm) was prepared with a RephiLe filtration unit .Chiral ligand of hydroquinine anthraquinone-1,4-diyl diether of (DHQ)2AQN or Ag-(DHQD)2AQN complex nanocrystals was performed as follows: 20 mL 10 mM AgNO3 aqueous solution was slowly added onto the surface of 30 mL (DHQ)2AQN or (DHQD)2AQN chloroform solution in a beaker. The reaction system was left undisturbed at room temperature from several minutes to 2 weeks. As a control experiment, interfacial phenomenon of (DHQ)2AQN or (DHQD)2AQN at the interface of pure water and ligand chloroform solution was also investigated.Interfacial self-assembly of the Ag-(DHQ)2AQN or Ag-(DHQD)2AQN complex nanocrystals grown at the interface were transferred onto substrate surfaces with the use of a dipper from KSV 5000 minitrough  or from JML04C2 trough . The substrate was firstly immersed into the liquid-liquid interface before the interfacial reaction started, and after a given time waiting for the coordination reaction and formation of Ag-complex nanocrystals, the substrate was vertically withdrawn from the interface. The dipping rate was kept at 1 mm/min.Layers of Ag-(DHQ)UV-vis spectra were measured with the use of a Shimadzu UV-2550 UV-vis spectrophotometer . Steady-state fluorescence spectra were recorded by using a Shimadzu RF-5300PC spectrophotometer.2/7, and Cu 2p2/3 binding energy. Pass energy of 70 eV and step size of 1 eV were chosen when taking spectra. In the analysis, chamber pressures of 1 ~ 2 × 10-7 Pa were routinely maintained. The binding energies obtained in the XPS analysis were corrected by referencing the C1s peak to 284.60 eV.XPS spectra were recorded by using a VG ESCALAB MKII multifunction spectrometer , with nonmonochromatized Mg-Kα X-rays as the excitation source. The system was carefully calibrated by Fermi edge of nickel, Au 4fScanning electron microscopic (SEM) measurements were performed on a Philips XL30 electron microscope . The samples were deposited on the Si substrate surface. High-resolution transmission electron microscope image was acquired on a JEOL JEM-2010 transmission electron microscope  operating at an accelerating voltage of 200 kV. The sample was deposited onto a 230-mesh copper grid covered with Formvar.2AQN or Ag-(DHQD)2AQN nanocrystals was used as the working electrode with a 10 mmol/L HClO4 solution as the electrolyte. For the CV measurement of the ligand redox reaction, an initial potential of -0.5 V was applied for 2 s, followed by cyclic scans to a final potential of 0 V. For the measurement of the Ag(I) redox reaction, the initial potential of -0.1 V was applied for 2 s, followed by cyclic scans to the final potential of 0.5 V. All CV measurements were done for 10 cycles under an Ar atmosphere at room temperature.Cyclic voltammogram (CV) was measured by using an electrochemical analyzer . A Pt wire and Ag/AgCl electrode were used as the auxiliary and reference electrodes, respectively, and the indium tin oxide (ITO) electrode covered with layers of Ag-(DHQ)2ANQ or (DHQD)2ANQ occurred quickly. During experiments, we found that the reaction rate was closely dependent on the concentration of AgNO3 in water and that of the ligand in chloroform as well as the temperature. Similar to those reported in the literature[Interfacial reaction between the silver ion and ligand of (DHQ)terature, stronge2AQN complex nanocrystals formed at the water-chloroform interface from the initial 5 min to 2 weeks, which revealed the following features.Morphologies of the Ag-directed complex nanocrystals were characterized by using SEM technique. These nanocrystals were deposited on the freshly cleaned Si substrate surface by vertical dipping method. Similar morphologies were obtained for the two ligands, so as an example, Figure Firstly, many screw-like nanocrystals were formed at the initial 30 min with a length of several hundred nanometers and diameters of tens of nanometers Figure A,B. ThesSecondly, the screw-like nanocrystals transformed into nanorods after the interfacial reaction time increased to about 30 min. As shown in Figure + ions were tetrahedrally coordinated with bidentate ligand of (DHQD)2AQN, so screw-like or nanorods were firstly formed, then they grew into larger cubic particles. A comparison of the size of the nanorods with that of the cubic particles could further find that the cubic length was shorter than that of the nanorods; this phenomenon may be attributed to a slow kinetic process of the crystal growing at the earlier stage, then to a thermodynamic process after several days. The other possible reason may be the lowest surface active energy of the cubic crystals; that is, the nanocubes may be more stable than the nanorods.Thirdly, when the interfacial reaction continued up to several days, the nanorods further grew into cubic macrocrystals with the length of a side about hundreds of nanometers Figure E,F. This2AQN complex nanocrystals, but the screw-like nanocrystals formed at the initial time were not very stable under the high beam energy of TEM. As an example, Figure Similar TEM images were observed for the Ag-(DHQ)2AQN complex nanocrystals, which revealed four peaks in the binding energy from 100 to 600 eV except for the Si element from substrate surface. The binding energy of these four peaks was as follows: 284.6, 368.6/374.4, 399.4 ~ 403, and 532.6 eV, which could be assigned to the elements of C(1 s), Ag(3d), N(1 s), and O(1 s), respectively. The C, part of N, and O elements were from the ligand of (DHQ)2AQN, while the elements of Ag, part of N, and O were from AgNO3. Thus, these XPS data confirmed formation of Ag-(DHQ)2AQN complex nanocrystals[Element compositions for the Ag-directed complex nanocrystals were detected by using the XPS technique. Also as an example, Figure crystals.2AQN nanocrystals transferred from the water-chloroform interface at different reaction times, together with a spectrum of the ligand in the dilute methanol solution. Three absorption bands were recorded and appeared at about 230 ~ 238, 323 ~ 334, and 416 nm for the ligand of (DHQ)2AQN in the solution, which can be designated to the electron transition of quinuclidine, quinine, and anthraquinone substituents. Our previous work has revealed that these peaks shifted to about 240, 337, and 416 nm in its Langmuir-Blodgett (LB) film[+ ions to form the LB film of Ag-(DHQ)2AQN coordination polymers, these absorption bands appeared at about 245, 342, and 413 nm, respectively[+ ions.Absorption and emission features of the Ag-complex nanocrystals as-prepared were investigated on the quartz surfaces. As an example, Figure LB) film. When thectively. That is2AQN complex nanocrystals, a broad absorption band was recorded with the maximum at about 270 ~ 296 nm. But it is hard to distinguish each peak as those in the solutions and LB films[2AQN complex nanocrystals was not. The detected light was strongly scattered by the nanoparticles, resulting in a broaden band from 200 to nearly 600 nm. Moreover, with increasing the reaction time, the average sizes of the nanocrystals increased LB films. This di3+ and Tb3+ as luminophores, sensors, and organic light-emitting diodes[2AQN complex nanocrystals were investigated after they were transferred on the quartz substrate surfaces.Based on the chemical structure of the chiral ligands used, we can find that they contain both quinine and anthraquinone substituents, both of them are important light-harvesting units. They can not only absorb ultraviolet light but also give off emission in the near ultraviolet and visible region,31, so tg diodes,33. Here2AQN complex nanocrystals on the quartz substrate surface, together with an emission spectrum of the ligand in the methanol solution. The excited wavelength was 317 nm. These emission spectra revealed the following features. Firstly, two broad emission peaks were recorded and centered at about 363 and 520 to 530 nm for the ligand (DHQ)2AQN in the methanol solution. The first peak may be designated to the emission from the hydroquinine substituents and the second one to that of the anthraquinone[2AQN complex nanocrystals showed also two broad emission peaks; the first one appeared at about 360 nm (very similar to that in the methanol solution), while the second one ‘blue’ shifted to about 470 nm. Previously, we have found that the fluorescent emission features for the ligands in the casting films were similar to those in the LB films; that is, the first one appeared at about 365 nm while the second one slightly red shifted to the range of 530 ~ 560 nm. This redshift has been attributed to a closely packed arrangement of the molecules in the organized ultrathin films as often observed for the macrocyclic molecules such as porphyrins and inorganic complexes[Figure omplexes-12.2ANQ and Pd-(DHQD)2ANQ coordination polymers[2AQN and Pd-(DHQD)2ANQ complexes. During experiments, we measured the emission spectra for the mixtures of (DHQ)2ANQ and AgNO3 at the molar ratios from 1:0 to 1:10 in the methanol solution. As shown in Figure 3. However, the emission at 525 nm gradually weakened when the AgNO3 solution was added. When the molar ratios of AgNO3 relative to the ligand increased to 10, a weak emission peak was observed at approximately 470 nm, which was in agreement with that observed in the Ag-(DHQ)2AQN complex nanocrystals. These results suggested that the blueshift and quenching of the second emission peak may be attributed to the formation of metal-ligand complexes in the nanocrystals[The blueshift phenomenon of the second emission band was also observed in the layer-by-layer multilayers of Pd-(DHQ)polymers, which wcrystals.2AQN and (DHQD)2AQN are also electroactive compounds because they contain the anthraquinone substituents, which have been widely used as electroactive materials either for the fundamental researches on electrochemistry or for the potential applications in the sensors, electrochromism, and organic batteries[Besides the interesting optical and chiral behaviors, (DHQ)atteries,33. Here2AQN complex nanocrystals in the 0.01 mol/L HClO4 electrolyte solutions in the potential range of -0.5 to 0 V at the scan rates from 0.05 to 0.6 V/s. One couple of redox wave was recorded with the cathodic (Epc) and anodic (Epa) potentials at around -0.27 ~ -0.29 and -0.22 ~ -0.20 V vs. Ag/AgCl, respectively. Based on the literature[2AQN and (DHQ)2AQN-. The potential difference ΔE (ΔE = Epa – Epc) was 0.05 V when the scan rate was 0.05 V/s, which slightly increased to 0.09 V when the scan rate was 0.6 V/s. The reduction current intensity was about 15.6 μA, which was also close to that of the oxidation current intensity (12.6 μA) when the scan rate was 0.6 V/s. These CV features suggested that the redox process of the ligands in the nanocrystals was reversible.Figure terature, this re2AQN nanocrystals in the 0.01 mol/L HClO4 electrolyte solutions in the potential range of -0.1 to 0.5 V at the scan rates from 0.05 to 0.6 V/s. One couple of redox wave was recorded with the cathodic and anodic potentials at around -0.13 ~ 0.07 and 0.28 ~ 0.32 V, which was attributed to the redox reaction of the connector of Ag+ ions[2AQN and Ag+-(DHQ)2AQN. The potential difference ΔE was 0.35 V when the scan rate was 0.05 V/s, which increased to 0.45 V when the scan rate was 0.6 V/s.Figure Ag+ ions, with th2AQN → Ag-(DHQ)2AQN- to the scan rate and the root of the scan rate for the ITO electrode modified by the film of Ag-(DHQ)2AQN nanocrystals. Based on these data, we can find that the current intensity was proportional to the root of the scan rates rather than that of the scan rates, which indicated that the electroactive thickness of the nanocrystals was thicker than that of the diffusion layer. This was reasonable because the film was composed of irregular Ag-(DHQ)2AQN complex nanocrystals with the sizes in the range of hundreds of nanometers 2AQN → Ag-(DHQ)2AQN to the scan rate and the root of the scan rate. Different from that observed in Figure Figure 3 as a connector and chiral bidentate ligands as linkers at the water-chloroform interface. Screw-like Ag-directed complex nanocrystals formed at the initial reaction time; then, they grew into nanorods and finally became cubic nano- or microcrystals. The as-prepared Ag-complex nanocrystals showed strong luminescent emissions as well as reversible redox properties, which may have potential interests in the fundamental researches and applications in the fields of chemically modified electrodes and optoelectronic devices.Silver(I)-directed metal-organic complex nanocrystals have been prepared using AgNOThe authors declare that they have no competing interests.YT, HTW, MC, and LZ carried out the synthesis and characterizations of the materials and drafted the manuscript. ML and DJQ contributed in the design and discussion of this work and in the revision of the manuscript. All authors read and approved the final manuscript.


Human epidermal growth factor receptors (HERs or ErbBs) play crucial roles in numerous cellular processes. ErbB2 is a key member of ErbB family, and its overexpression is recognized as a frequent molecular abnormality. In cancer, this overexpression correlates with aggressive disease and poor patient outcomes. Dimer-dependent phosphorylation is a key event for the signal transduction of ErbBs. However, the molecular mechanism of the dimerization of ErbB2 remains elusive. In the present work, we report the homodimer architecture of the ErbB2 extracellular domain (ECD) which is unique compared with other dimer-models of ErbBs. The structure of the ErbB2 ECD homodimer represents a “back to head” interaction, in which a protruding β-hairpin arm in domain II of one ErbB2 protomer is inserted into a C-shaped pocket created by domains I–III of the adjacent ErbB2 protomer. This dimerized architecture and its impact on the phosphorylation of ErbB2 intracellular domain were further verified by a mutagenesis study. We also elucidated the different impacts of two clinically administered therapeutic antibodies, trastuzumab and pertuzumab, on ErbB2 dimerization. This information not only provides an understanding of the molecular mechanism of ErbBs dimerization but also elucidates ErbB2-targeted therapy at the molecular level. The epidermal growth factor receptor (EGFR) family includes four members: the human epidermal growth factor receptor 1-4. The overexpression and dysfunction of ErbBs result in cancer, diabetes, immune deficiencies, cardiovascular diseases, and other human diseases . The curP1 , which helps to stabilize ErbB2 ECD without affecting the dimerization arm in domain II, and solved its structure at 3.1 Å resolution in the space group There are two ErbB2 ECD-Fab complex molecules in one asymmetric unit. The overall architecture of ErbB2 ECD in this complex is very similar to other reported structures of ErbB2 ECD with a root-mean-square deviation (r.m.s.d) of 1.4 Å for the Cα atoms of all residues in the ErbB2 ECD. However, an obvious shift can be observed in the dimerization arms (residues 245 to 266) in the two ErbB2 molecules.Interestingly, the dimerization arm of the ErbB2 protomer B [ErbB2(B)] is well stabilized Fig.  and accoErbB2 dimerization was facilitated mostly by interactions between domain II(B) of ErbB2(B) and domains I(A) and III(A) of ErbB2(A). The dimerization arm formed a β-hairpin in the middle, which protrudes from the domain II(B) globule. Six residues (Y252 to F257) at the tip of the dimerization arm served as a “hand” to interact with the groove made by domain I(A) and domain III(A) Fig. , and S1AA set of “face to face” parallel hydrophobic π interactions can be observed: the first is between F257 in domain II(B) and H447 in domain III(A), and the second involves the phenol ring of Y252 in domain II(B) and the imidazole ring of H448 in domain III(A) . AdditioAnother set of “face to face” parallel hydrophobic π bonds can be observed in the interactions between H245 in domain II(B) and H451 in domain III(A) and T450 in domain III(A) with A248 in domain II(B). These interactions properly fixed the position of the imidazole ring of H451 in domain III(A) such that it was able to form a side-chain to main-chain hydrogen bond through its ε2 nitrogen atom and the oxygen atom of C246 in domain II(B). A hydrogen interaction from S429 and G245 in domain III(A) also strengthened this binding interface. Other interactions, such as the hydrophobic interactions made by P398 and W430 in domain III(A) and L244 in domain II(B), the hydrophilic interactions made by E481 in domain III(A) and S288 in domain II(B), and the side-chain to side-chain hydrogen bond between Q156 in domain I(B) and H490 in domain III(A), are also observed, but these interactions did not seem critical to the interface  hydrophobically interact with F257, T254, and L243 of ErbB2(B) and are positioned at the mouth of a C-shaped pocket. The replacement of these residues with phenylalanines is likely to narrow the C-shaped pocket and cause steric hindrance in the area. Consistent with this hypothesis, the G425F mutation caused the almost complete loss of dimerization and subsequent phosphorylation, and the A15F and S429F mutations also obviously impacted the formation of ErbB2 tyrosine-phosphorylated homodimers. H447 and H448 of ErbB2(A) interact with Y252 and F257 of ErbB2(B). The mutant H447A and double mutant Y252A/F257A significantly attenuated ErbB2 dimerization and phosphorylation. In contrast, the H448A, Y252A, and F257A single mutations exhibited only slight effects on ErbB2 dimerization and phosphorylation. H451 of the ErbB2(A) protomer makes a “face to face” parallel hydrophobic π bond and a hydrogen bond to the H245 and C246 of ErbB2(B) protomer. Substitution of H451 and H245 with alanines abolished the dimerization and autophosphorylation of ErbB2. Alanine mutants of residues Q156 and H490, which form an additional hydrogen bond in ErbB2 dimer, had no detectable impact on ErbB2 dimerization and phosphorylation, indicating their minor role in ErbB2 dimerization. From these results, G425, H447, the Y252/F257 pair, H451, and H245 were revealed to play the most essential roles in ErbB2 dimerization. Interestingly, Y252 and F257 in domain II(B) may facilitate heterodimerization with other ErbB molecules, perhaps even with ErbB monomers from different species, through hydrophobic interactions. In these heterodimers, Y252 and F257 of ErbB2 could interact with the corresponding residues in other monomers, such as ErbB1 residues Y246 and Y251 or dEGFR residues Y242 and Y247 , 18 Fig.Pertuzumab and trastuzumab are two clinically administered anti-ErbB2 antibodies with variable mechanisms of action. Pertuzumab binds at the dimerization arm , and traIt is widely accepted that the homo and/or heterodimerization of ErbBs , 20 switThe ligand-independent activation and self-association of ErbB2 occurs when it is overexpressed at the plasma membrane , and itsAnother interesting observation is that domain II of the two protomers adopts a unique orientation caused by the homodimer interaction. Upon forming the homodimeric complex, domain II(B) “wedges” into the C-shaped pocket between domains I(A) and III(A), pushes them slightly apart, and then causes a substantial reorientation of domain II(A), which connects domain I(A) and III(A) Fig. . AnalogoPrevious study indicated that trastuzumab does not decrease ErbB2 homodimers despite its strong potency to drive ErbB2-overexpressing cells into quiescence, whereas pertuzumab is able to prevent ErbB2 homodimerization . The strThe human breast cancer cell lines SK-BR-3 and African green monkey SV40-transformed kidney fibroblast cell line (COS-7) were obtained from the American Type Culture Collection . The cells were cultured in DMEM medium supplemented with 15% fetal calf serum in 5% CO2 at 37.8°C in a humidified incubator. Trastuzumab and Pertuzumab antibody was purchased from Roche Ltd.Residues 1–624 of the extracellular domain of ErbB2(ErbB2-ecd) was prepared as described previously , except The Fab fragment of the human anti ErbB2 domain I antibody for the crystallographic investigation was obtained through a papain digestion of an antibody. The digested protein sample was loaded onto a Protein A Sepharose 4 FF column . The Fab fragment eluted in the flow through was separated from the Fc fragment and further purified through ion-exchange chromatography using a Q-Sepharose FF column . The protein sample was concentrated to 20 mg/mL and then exchanged to a stock buffer containing 20 mM Tris–HCl (pH 8.0) and 300 mM NaCl. ErbB2-ecd was subsequently mixed with an excess of H218 Fab, and the complex was purified through gel-filtration chromatography . This complex was dialyzed against 10 mM Tris–HCl (pH 8.0) and 50 mM NaCl, and was concentrated to 25 mg/ml.3 with the best diffraction within two weeks. The crystals were then cryo-protected through soaking in a cryo-protectant comprising the reservoir solution and 10% glycol. The cryo-protected crystals were subsequently flash-cooled in liquid nitrogen, and then transferred into a dry nitrogen stream at 100 K for X-ray data collection.Crystallization was performed at 291 K through the hanging-drop vapor-diffusion technique. The crystals were obtained by mixing 1 μl of the protein solution with an equal volume of a reservoir solution. The mixture drop was equilibrated against 500 μl of the reservoir solution. The crystals were obtained with a reservoir solution containing 0.1 M Ammonium acetate, 0.1 M BIS-TRIS pH 5.7, 13% w/v Polyethylene glycol 10,000, which reached the final dimensions of 100 × 100 × 100 μma = 84.7 Å, b = 104.2 Å, c = 116.7 Å, α = 107°, β = 99°, γ = 111°. The statistics of all data collections and structure refinements are summarized in Table The diffraction data for the ErbB2-Fab complex was collected at beamline BL17A  with a resolution of 3.1 Å. The data were processed, integrated, and scaled using the HKL2000 package. The crystals belong to space group P1 with cell parameters Fo–Fc and 2Fo–Fc electron density maps. The residues were refined in PHENIX, and the respective working R-factor and R-free decreased from 0.44 and 0.45 to 0.23 and 0.27, respectively, for all data from 50.0 Å to 3.1 Å. The refinement was monitored by calculating freeR based on a subset containing 5% of the total reflections.The ErbB2-Fab structure was solved through the molecular replacement method, which employs the crystal structures of ErbB2 and Pertuzumab Fab (PDB code: 1S78) as the initial searching model by using the program PHASER. The clear solutions in both the rotation and translation functions indicated the presence of two complex molecule, including two ErbB2 and two Fab molecules, in one asymmetric unit. This result is consistent with the Matthews coefficient and solvent content. The inconsistent residues were manually rebuilt in the program Coot under the guidance of the 6 cells/10 cm dish) were transfected with 1 ug of ErbB2-wild-type-flag, ErbB2-mutant-flag DNA with or without ErbB2-wild-type-V5 DNA using Lipofectamine according to the manufacturer's recommendations (Invitrogen). We also use The human breast cancer cell lines SK-BR-3 cells as a positive control. Twenty-four hours post transfection, cells were serum starved overnight in 0.1% fetal bovine serum/DMEM at 37°C prior to treatment.ErbB2 Site-directed mutants  were created through PCR. All wild-type and ErbB2 mutant constructs used in this study were epitope tagged at the C terminus with a FLAG sequence or a V5 sequence. All mutations were confirmed by automated DNA sequencing. COS-7 cells . Homodimers were immunoprecipitated from 500 ul of lysate using mouse anti-V5 antibody(AbD Serotec) covalently coupled to agarose  at 4°C for 2 h. Complexes were washed twice in lysis buffer and resuspended in SDS sample buffer and boiled. Samples were separated on a 4%–12% polyacrylamide gel (Novex) and electro-blotted onto nitrocellulose membranes. Blots were blocked in 10% BSA/TBST and probed with an anti-Flag antibody to detect ErbB2 followed by a peroxidase-conjugated anti-mouse secondary antibody (Amersham) To verify and normalize for expression of transfected constructs between experimental conditions, 50ug of cell lysate was checked by western blotting with anti-V5 antibody (AbD Serotec) or anti-Flag antibody(Sigma). Anti-mouse HRP-conjugated secondary antibody was used for visualization by enhanced chemiluminescence .Homodimer of ErbB2 was aslo evaluate in a coimmunoprecipitation assay, as describe , 16, 17.Ligand independent activation of ErbB2 or ErbB2 mutants was detected by a tyrosine phosphorylation assay using an anti phospho-ErbB2-Tyr1221/1222 antibody


We find that due to the charge carrier accumulation, the static conductivity may drastically increase at the domain wall by 3 – 4 orders of magnitude in comparison with conductivity through the bulk of the material. Also, a two-dimensional degenerated gas of highly mobile charge carriers could be formed at the wall. The high values of conductivity at domain walls and interfaces explain high efficiency in organometallic solution-processed perovskite films which contains lots of different point and extended defects. These results could suggest new routes to enhance the performance of this promising class of novel photovoltaic materials.The past several years has witnessed a surge of interest in organometallic trihalide perovskites, which are at the heart of the new generation of solid-state solar cells. Here, we calculated the static conductivity of charged domain walls in  Single crystalline semiconductors such have been reported to achieve high power conversion efficiencies (25.6% for Si and 28.8% for GaAs)233 perovskites have been studied in great detail891011β-MAPbI3 belongs to the I4/mcm space group, and therefore, it is expected to be ferroelectric1314β-MAPbI3 film may act as small internal p–n junctions, aiding the separation of photoexcited electron and hole pairs which reduces recombination16β-MAPbI3 thin films were investigated using piezoforce microscopy (PFM)The structural, optical, and electronic properties of MAPbI3NH3PbX3  materials were recently investigated by density functional theory based methodsβ-MAPbI3 thin films used in solar cells are often mixed together with different meso- scale scaffolding materialsA presence of ferroelectric domains may explain some phenomena including the hysteresis in the current–voltage response13193NH3PbI3p- or n-type semiconductors3 (0.036 and 0.022 cm2/s) obtained from fitting the decay dynamics of photoluminescence spectra to the diffusion modelμe ~ 1.5 cm2/(V·s) for the electron mobility which is significantly lower than the value of μe ~ 66 cm2/(V·s) obtained from combined resistivity and Hall effect experimental dataRecently, it was shown that both electron and hole diffusion lengths are at least 100 nm in solution processed CHn- and p- doped β-MAPbI3 thin films using the set of parameters taken from different experiments and existing first-principles calculations. MAPbI3 is the organometallic trihalide material which has been investigated for a long time starting 1980s. Although other similar materials with the formula (RNH3)MX3  could also be perspective for solar cells, existing experimental and theoretical data for them are incomplete and sometimes contradictory. In tetragonal β-MAPbI3 one should expect two types of domain walls β-MAPbI3 thin films are clearly indicated by the complete 180° phase-contrastIn this context, we have investigated charge accumulation at ferroelectric domain walls in 3 film (with sub-micron thickness) which forms two interfaces with two solid electrodes (collectors for electrons and holes). There are inevitable lattice mismatch between the perovskite and materials of the electrodes, i.e., an interfacial stress is applied to the perovskite film at its both surfaces. This interfacial strain generates domain walls of different configurations (charged and uncharged) in the film depending on the direction and value of the strain, the temperature and other factors  used in perovskite solar cellsIn reality, the considered system is more sophisticated and we have a thin MAPbIn-type impurity (for p- type doping the results are similar). A sketch of the charged walls is shown in φ(ξ) and the ferroelectric polarization component Pz(ξ) (order parameter) should be determined from the coupled Poisson and LGD equations (Supplementary), with boundary conditions of the potential vanishing far from the domain wall and the absolute value of Pz(ξ) reaching the value of PS. Several equations for concentration of different charged species , for generation of the electron-hole pairs, and carriers recombination were also added.Following Ref.Then the static conductivity can be calculated as (ions are less mobile species so their contribution to the conductivity is neglected),q is the electron charge, n(ξ) and p(ξ) are electrons and holes concentrations at a distance ξ from the wall, μe and μp – the electron and hole mobility. To perform numerical calculations for domain walls, a significant number of different parameters is needed including dielectric function at different frequencies and polarizations, effective masses, coherence length, spontaneous polarization, etc. Fortunately, for the most investigated organometallic trihalide MAPbI3, one has a significant amount of experimentally measured and calculated parameters which are sufficient for the LGD based modeling. A detailed and accurate description of all the used parameters is provided in Supplementary. In particular, we found that typical concentrations of charge carriers injected by solar light do not exceed 1013 – 1014 cm−3 which is lower than typical concentrations of impurities in doped semiconductors.where n(ξ) and charged donors Nd+(ξ) as a function of the distance from the wall plane ξ (measured in the units of the correlation radius rc), calculated for the inclined head-to-head domain walls with different slope angles θ and two different concentrations of donor impurities are shown in θ = π/2); it decreases with the bound charge decrease (θ decrease) and vanishes at θ = 0. The net electric field “repulses” ionized donors  and forms ionized donor depletion region.The concentration of electrons n- doped material, the concentration of charged donors is depleted in the wall region because the probability for a charged donor defect to become neutral increases with the increase of concentration of free electrons. Calculations also indicated that the hole concentration in the vicinity of the wall is much lower than the concentration of electrons . The electron attraction to this region naturally stops when the electron concentration reaches a saturation level which depends only on the value of spontaneous polarization field. For the head-to-head domain walls and rons see , i.e., obulk. As a result of electron accumulation near the head-to-head domain wall, the static conductance drastically increases at the wall – up to 2 – 3 orders of magnitude for the dopant level of Nd0 = 1018 cm−3 and up to 4 orders of magnitude for Nd0 = 1017 cm−3. This is not surprising because the “saturated” value of the electron concentration at the wall does not depend on Nd0 while the electron concentration of the bulk is completely defined by Nd0 . Also, conductance in the direction parallel to the wall is maximal for a perpendicular wall (θ = π/2). For angle θ = 0, there is no current across the film because this domain wall does not accumulate any charge. It is apparent that the ratio goes up for smaller Nd0 reaching the value of ~104 for Nd0 = 1017 cm−3 and θ = π/2.n- doped semiconductors, the electric field potential at the tail-to-tail wall shows a very narrow and deep peak surrounded by a smooth, slowly growing “background” with the width strongly depending on Nd0  and negatively (head-to-head) charged domain walls, the highest charge carrier transport in both cases occurs in a thin layer near the wall. Also, each wall conducts only one type of charge carriers (electrons or holes), i.e., a significant spatial separation of charge carriers of different types takes place and should reduce the recombination of injected carriers. Holes accumulation near the tail-to-tail domain wall also drastically increases the static conductance at the wall  and the thickness of the wall is d ~ 15 rc, we find that the concentration of electrons at the wall cannot exceed the maximal value,Results presented in L ~ 100 nmrc ~ 1.4 nmnmax ~ 5n∞, i.e., the concentration of accumulated electrons at the walls should be significantly lower than values of 103 – 104n∞ obtained in an assumption that domains are very large and depletion may be neglected. However, even if we do not reach the regime of degenerated two-dimensional charge carrier gas at the domain walls , we still benefit from charge separation and thus spatially separated charge accumulation. This effect suppresses recombination of free charge carriers and is favorable for the device efficiency.It corresponds to the case when all electrons from the bulk of the domain moved to the domain walls. For MAPbI3  the situation is even worse. The holes concentration generated by natural solar light does not exceed 1013 – 1014 cm−3. Therefore, in order to reach the quantum degenerated regime at the tail-to-tail wall with hole concentration of 1020 – 1021 cm−3  and still maintain a significant concentration of accumulated carriers at the domain walls to exhibit significant conductance. For undoped samples where most of the charge carriers are generated by solar light, and their concentrations are rather low, cleaner structures with larger domain sizes should be preferable.3 and a spinel γ-Al2O3 epitaxial film with compatible oxygen ions sublattices5 cm2/Vs at 2 K. Similar effects were observed at ferroelectric domain walls where the steady metallic-type conductivity reached ~109 times that of the parent matrixCharge carrier accumulation at the domain walls is one of the factors that increase the conductance near the domain walls. However, in addition to carrier densities one should take into account the carrier mobility at the walls  which isThe electronic structure within the confinement was calculated by solving the Schroedinger equation for the plane-perpendicular direction in the potential shown in 29θ and donor concentration Nd0 = 1017 cm−3. The electron mobility at the head-to-head walls is extremely high and is comparable to the record electron mobility numbers obtained in a specially grown state of the art heterostructure (3.2 × 107 cm2/Vs) at low temperatures3 perovskite may be very high.n- doped material. These numbers for the mobility were obtained in an assumption that the electron (hole) gas at the domain wall reaches a quantum state and becomes degenerated. However, it may happen that degeneracy won’t be reached. The main factor which regulates the maximal accumulated electron density is the relation between the average size of ferroelectric domain and the thickness of the wall which separates a domain from a neighboring domain and/or from a grain of other material used for scaffolding .If one suggests that array of charged domain walls is dense and consists of alternating sequence of head-to-head and tail-to-tail walls, and if all charged carriers initially positioned between the walls are collected at corresponding walls, the total electron and hole charges at the walls will be the same as they were in the bulk. The difference, however, is that these charge carriers are completely separated, i.e., electrons and holes do not recombine and reach corresponding electrode. If, however, the carrier concentration at the walls reaches the 2DEG regime, the mobility of charge carriers could increase even further which makes the transport even more efficient. This picture, however, is quite approximate, and to simulate these processes, the electron and hole densities should be calculated self-consistently.n- and p- doped organometallic uniaxial ferroelectric semiconductor perovskite CH3NH3PbI3, in which ferroelectric domains were observed directly using piezoforce microscopy. We showed that the carriers diffuse to domain walls and accumulate there. Due to this accumulation, the static conductance at the domain walls may increase by 3 – 4 orders of magnitude in comparison with conductance through the bulk of the material. These results support the transport mechanism suggested in Ref.8In conclusion, we calculated the static conductance of charged head-to-head and tail-to-tail domain walls with different incline angles with respect to the spontaneous polarization vector in the How to cite this article: Rashkeev, S. N. et al. Domain Walls Conductivity in Hybrid Organometallic Perovskites and Their Essential Role in CH3NH3PbI3 Solar Cell High Performance. Sci. Rep.5, 11467; doi: 10.1038/srep11467 (2015).


Kanadaptin is a nuclear protein of unknown function that is widely expressed in mammalian tissues. The crystal structure of the forkhead-associated (FHA) domain of human kanadaptin was determined to 1.6 Å resolution. The structure reveals an asymmetric dimer in which one monomer is complexed with a phosphopeptide mimic derived from a peptide segment from the N-terminus of a symmetry-related molecule as well as a sulfate bound to the structurally conserved phosphothreonine recognition cleft. This structure provides insights into the molecular recognition features utilized by this family of proteins and represents the first evidence that kanadaptin is likely involved in a phosphorylation-mediated signaling pathway. These results will be of use for designing experiments to further probe the function of kanadaptin. The FHA domain of kanadaptin was crystallized using the nanodroplet vapor diffusion method 12121 using the FHA domain of Pml1p subunit of the yeast precursor mRNA retention and splicing complex (PDB ID 3els) cryst of 17.6% and an Rfree of 19.8%. The final model has good geometry and compares favorably to other structures at similar resolution, with an overall MolProbity score The FHA domain of kanadaptin was cloned and expressed in In common with other FHA domains, the FHA domain of kanadaptin adopts a β-sandwich fold consisting of 11 β-strands  and the peptide  are both well-ordered with excellent electron density (2). The backbone atoms of the bound N-terminal portion of the FHA forms multiple hydrogen bonds to Arg193, Arg208, and His240 of the pThr recognition site, while the sulfate group hydrogen bonds with Ser207, Thr239, Arg193, and Arg208 , L4–5, and L6–7 , has pos density ; their ad Arg208 . All thed Arg208  and thusd Arg208 . TherefoIn contrast, monomer B represents a ligand-free state, with its binding site occupied by waters. Nevertheless, the conformation of the phosphopeptide binding site is very similar to that of monomer A , in agreArabidopsis thaliana Dawdle FHA domain (PDB ID 3vpy) The structure of the FHA domain of kanadaptin is very similar to that of other FHA domains. For example, it aligns with the 2 of solvent accessible area per monomer. The two β-sheets of opposing monomers pack in a face-to-face arrangement with the phosphopeptide binding sites on the outer surfaces, distal from the dimerization interface. The dyad axis is approximately parallel to the β-strands. The last β-strand is buried in the dimer interface. The dimerization interface involves a core set of hydrophobic residues in the center of β-sheet1 , and additional residues at the perimeter involved in backbone hydrogen bonding . Thus, the physiological relevance of the kanadaptin-FHA dimer observed in the crystal is currently unclear. However, we postulate that such a dimer may mimic a phosphopeptide-bound state (see below), and could possibly represent a physiologically relevant state (e.g. activated). Indeed, phosphopeptide-mediated FHA dimerization appears to be a common strategy utilized by many FHA-regulated signaling pathways The ubiquity of kanadaptin in mammals suggests that it should have an important physiological function. The structure of the kanadaptin-FHA domain supports current hypotheses that kanadaptin participates in cell signaling pathways via its FHA domain. FHA-containing proteins generally possess one or more “functional” modules, whose activity is regulated by phosphopeptide binding. In kanadaptin, the “functional” module is potentially the predicted dsRBD , which sSecondary structure predictions indicate full-length kanadaptin contains two helical regions between the FHA domain and the dsRBD domain (residues 290–327), and after the dsRBD domain . The resulting DNA was inserted into the expression vector pNIC28-Bsa4 using LIC. The expression vector encodes an amino-terminal tobacco etch virus (TEV) protease-cleavable expression and purification tag (MHHHHHHSSGVDLGTENLYFQ/S). The DNA insert and the vector were both prepared for LIC by treatment with restriction enzyme digestion and T4 DNA polymerase. Escherichia coli MachI (Invitrogen) competent cells were transformed with the treated DNA insert and vector and dispensed on to selective LB-agar plates. The success of cloning was confirmed by DNA sequencing.Clones were generated using Ligation Independent Cloning (LIC). The gene encoding the FHA domain of kanadaptin  was amplified by polymerase chain reaction (PCR) from the Invitrogen Ultimate collection using Phusion DNA polymerase (NEB) and forward primer, E. coli expression strain BL21 Rosetta2 (DE3) R3 T1. 50 ml of TB media containing 50 µg/ml kanamycin and 25 µg/ml chloramphenicol was inoculated with cells from a glycerol stock. The overnight culture was grown at 37°C and used the following morning to inoculate 4.5 L of TB media containing 50 µg/ml kanamycin. The expression culture was grown at 37°C to an OD600 = 1.65. The temperature was then reduced to 18°C, and expression induced by adding IPTG to a final concentration of 0.5 mM. The cells were harvested 19 hours after induction by centrifugation at 4000× g for 10 minutes.Protein expression was carried out using The cell pellets were resuspended in lysis buffer  supplemented with Complete Inhibitor cocktail (EDTA Free) and Benzonase (750 U/100 ml) and the cells were lysed by three passes through a high pressure homogenizer at 1000 Bar (D20 Avestin). The lysate was centrifuged at 18500× g for 40 minutes and the supernatant filtered through a 0.22 µm PES filter. The filtrate was collected for purification. The proteins were initially purified using a two-step affinity and size exclusion chromatography using an ÄKTAxpress system . The affinity chromatography column (1 ml HiTrap Chelating) was equilibrated in binding buffer  and the sample loaded onto the column. The column was washed . The protein was eluted using a step gradient of elution buffer  and fractions collected for further purification. A second purification step was carried out using a Superdex 75 PG 16/60 column pre-equilibrated with running buffer . Fractions were collected and the purification tag was cleaved off by overnight incubation with TEV protease (1∶100 molar ratio) at 4°C. The cleaved purification tag and the protein were separated by an additional pass over the affinity column. The protein was buffer exchanged into the final crystallization buffer  using a PD-10 column  and finally concentrated to 8.0 mg/ml for crystallization trials. The identity of the protein was confirmed by electrospray ionization mass spectrometry (ESI-MS) of the intact protein.12121.The FHA domain of kanadaptin was crystallized using the nanodroplet vapor diffusion method cryst of ∼20% and good completeness, and was confirmed by manual inspection of the corresponding density maps. Further model completion and refinement were performed manually with COOT http://www.pymol.org/). Electrostatic potentials were calculated using the program Delphi http://www.rcsb.org) with PDB codes 4h87.Native data were collected at wavelength 0.97932 Å at 100 K using a Pilatus 6M detector (DECTRIS) at SSRL beamline BL11-1. The data were processed by an automation script Identification of domains and definition of domain boundaries were carried out using PFAM


Phaseolus vulgaris L.) are rich nutritious and affordable by vulnerable groups, thus a good choice for biofortification to address malnutrition. However, increasing micronutrients content of beans, without improving micronutrients bioavailability will not improve the micronutrients status of consumers. Effect of different processing methods on the physicochemical characteristics of biofortified bean flour was determined. Processing methods used in this study were malting (48 h), roasting (170°C/45 min), and extrusion cooking using a twin screw extruder with three heating sections, the first set at 60°C, the second at 130°C, and the last one at 150°C. The screw was set at a speed of 35 Hz (123g) and bean flour moisture content was 15%. Mineral extractability, in vitro protein digestibility, pasting properties, and sensory acceptability of porridge and sauce from processed flour were determined. All processing methods significantly increased (P < 0.05) mineral extractability, iron from 38.9% to 79.5% for K131 and from 40.7% to 83.4% for ROBA1, in vitro protein digestibility from 58.2% to 82% for ROBA1 and from 56.2% to 79% for K131. Pasting viscosities of both bean varieties reduced with processing. There was no significant difference (P < 0.05) between sensory acceptability of porridge or sauce from extruded biofortified bean flour and malted/roasted biofortified bean flour. Acceptability was also not affected by the bean variety used. Mineral bioavailability and in vitro protein digestibility increased more for extruded flour than for malted/roasted flours. Sauce and porridge prepared from processed biofortified bean flour had lower viscosity (extruded flour had the lowest viscosity), thus higher nutrient and energy density than those prepared from unprocessed biofortified bean flour. Estimated nutritional contribution of sauce and porridge made from processed ROBA1 flour to daily requirement of children below 5 years and women of reproductive age found to be high. These results show that processing methods enhanced nutritional value of biofortified bean flour and that processed biofortified bean flour can be used to prepare nutrient and energy‐dense gruel to improve on nutritional status of children under 5 years and women of reproductive age.Common beans ( Micronutrient malnutrition especially iron deficiency anemia and zinc deficiency affect at least half of the world's population  are rich in protein (20–28%) and micronutrients (Fe = 34–89 mg/kg and Zn = 21–54 mg/kg)  was purchased from Community Enterprise Development Organization (CEDO), a local nongovernmental organization (NGO) based in Rakai District. K131 beans were purchased from the Bean program of the National Crops Resource Research Institute (NaCRRI) in Namulonge, Uganda. K131 bean variety was selected because it is high yielding but takes long to cook due to a hard seed coat  was soaked in distilled water at 1:2 (w/v) for 24 h. The soaked beans were germinated for 48 h in a dark place on a wet cloth. Germinated beans were then roasted at 170°C for 45 min in an oven  . Amaranth, rice, and bean flours were mixed at a ratio of 30:30:40, respectively, a proportion reported to be highly acceptable  by heating until clear white precipitates settled at the bottom. The digested samples were dissolved in double distilled water and filtered through Whatman # 42 filter paper. The filtrate was made to 50 mL with double distilled water and used for determination of iron and zinc contents using Atomic Absorption Spectroscopy (AAS) .Iron and zinc contents of raw, extruded, and malted/roasted bean flours of both K131 and ROBA1 bean varieties were analyzed by the method described by Duhan et al. . One graIron and zinc extractability were determined using a method by Duhan et al. . The minProtein content was determined by Kjeldahl method #46‐12 . Approxi2SO4. The end point was determined by the sample solution turning from blue‐green to pink. The volume of the acid used (titer) was noted. The percentage crude protein (% cP) in the sample was calculated using the following formula:Flasks were cooled, after which the digests were transferred quantitatively to 50 mL volumetric flasks, made to volume with distilled water, and mixed immediately. Distillation of samples was done by pipetting 50 mL of sample into the distillation chamber and slowly adding approximately 50% NaOH solution to raise the pH. A conical flask containing 10 mL of 2% boric acid solution plus two drops of bromocresol green methyl red indicator was placed under the condenser stem to collect the distillate. The distillation was allowed to proceed for 7.5 min after which the receiving flasks were lowered so that the distillate could wash any remaining ammonia from the tip of the condensing unit. Titration was done using a burette filled with 0.05 N HT: titer; 14: atomic weight of N; b: normality of acid which was approximate 0.05 N; Wt: sample weight; 6.25: conversion factor for protein from % nitrogen.2SO4 (10 mL), 1 g of potassium sulfate, and 1 mL of 10% copper sulfate solution were added. Digestion, distillation, and titration were done to determine the protein content.This was determined using the method of Mertz et al. . ApproxiA is % protein in sample before digestion and B is % protein in sample after pepsin digestionwhere Pasting properties were determined using the Rapid Visco Analyzer  with Thermocline for windows software . Four grPorridge was prepared from both extruded and malted/roasted bean flours of both the ROBA1 and K131 bean varieties. The flour (250 g) was mixed with 750 mL of cold water. The mixture was brought to the boil while stirring to avoid lumping then cooked for 10 min after which 50 g of sugar was added.Sauce was prepared from both extruded and malted bean flours of both the ROBA1 and K131 bean varieties. Ingredients  were all chopped. The onions and green pepper were shallow fried in 50 mL of vegetable cooking oil. About 2 g of curry powder and 10 g of salt were added. The bean flour (250 g) was stirred into the mixture, 1200 mL of water was added gradually with constant stirring to avoid lumping, and the sauce was cooked for 10 min.Porridge and sauce acceptability were determined by 50 panelists using a 9‐point hedonic scale   using Statistix software (version 9.0) at The iron and zinc contents of ROBA1 beans were higher than that of K131 Table . ROBA1 iThe results of iron and zinc contents obtained were in the same range as that reported by Blair et al.  between Processing by germination/malting followed by roasting resulted in a slight and statistically significant decrease in mineral content of both the ROBA1 and K131 bean varieties compared to the mineral content of raw flour. This can be attributed to leaching of iron and zinc ions into the soaking water  of the ROBA1 and K131 bean varieties studied in this work. Processing malting/roasting and extrusion also significantly (P < 0.05) affected the iron and zinc contents of flour compared to raw beans flour.There were significant differences in both zinc and iron contents  was observed between the mineral extractability of processed bean flour samples and unprocessed bean flour samples for both the ROBA1 and K131 bean varieties. Flour of extruded beans had the highest iron and zinc extractabilities. Similar results were reported for the effect of processing on mineral bioavailability  results in enzymatic degradation of phytates and polyphenols  after both malting/roasting and extrusion cooking, and for both varieties compared to raw samples. Extruded samples had a significantly higher in vitro protein digestibility than samples processed by malting and roasting. There was also a significant difference (P < 0.05) between the in vitro protein digestibility of the two bean varieties with ROBA1 bean flour having a higher in vitro protein digestibility. Similar results were reported by Shimelis and Rakshit  were observed in the different pasting characteristics of flours from both processing methods and varieties (Table P < 0.05) for extruded flour compared to raw (unprocessed) and malted/roasted flours. Pasting temperature of raw flour was higher than reported by Akinjayeju and Ajayi (Pennisetum glaucum) did not observe any effect on pasting temperature due to roasting or germination of pearl millet. Similarly, malting/roasting of beans in this study had no effect on pasting temperature. High pasting temperature may be an indication of the presence of resistant starch, to swelling and rupturing  higher breakdown viscosity (270.6 cP for extruded K131 flour and 749.6 cP for extruded ROBA1 flour) than the malted and unprocessed flours. Pastes from flours with low final viscosity were reported to be less stable when cooked and thus to commonly have high values of breakdown viscosity , while the lowest was observed for extruded flour (399.6 cP for K131 flour and 488.3 cP for ROBA1 flour). Peak viscosity is the maximum viscosity attained by gelatinized starch during heating in water. It indicates the water binding capacity of the starch granules   than those prepared from pure malted/roasted ROBA1 and K131 flours. There was no significant difference in the acceptability of composite porridges prepared from K131 and extruded pure ROBA1 (P < 0.05). The color of composite malted/roasted K131 porridge and extruded pure K131 porridge were significantly different and less acceptable than the colors of the other porridges. Moreover, for thickness and texture of extruded pure K131 and malted/roasted pure K131 porridges were also significantly different from other porridges (P < 0.05). Similarly, the appearances of the extruded composite K131and extruded pure K131 porridges were less acceptable than the other porridges (P < 0.05). For other attributes no significant differences were observed.The overall acceptability was highest for porridges prepared from the composite flour of both extruded and malted/roasted ROBA1 beans which scored 7 on the 9‐point hedonic scale Table . The comP < 0.05). The flavor of sauces made from extruded pure K131 and extruded composite ROBA1 was significantly less liked than the flavor of the other sauces (P < 0.05). The color, thickness, and appearance of sauces made from extruded pure K131 flour, malted/roasted composite ROBA1, and malted/roasted composite K131 flour were less accepted than for other sauces (P < 0.05). No difference was found for smell among all sauces.Sauces prepared from extruded and malted/roasted pure ROBA1 and K131 flours had higher overall acceptability than the composite sauces Table . The tasThe composite flour of ROBA1 made the most acceptable porridge after both processing methods . Similarly, the sauce made from the pure ROBA1 flour was the most acceptable after extrusion processing, while for malting/roasting method, both K131 and ROBA1 pure sauce were equally acceptable. Sensory scores of all products were higher than 5, with overall acceptability scoring higher than 6 . The high acceptability may be attributed to the elimination of the beany flavor, which characterizes all legumes. Products prepared from beans are likely to have this flavor which is considered unpleasant to most consumers Enwere . ProcessOverall, when the acceptability of the sauces and porridges is compared, porridge from the composite flour was more acceptable than that from the pure bean flour while sauce from the pure bean flour was more acceptable than that from the composites. Jackson et al.  also repRaw (unprocessed), malted/roasted, and extruded beans flour from ROBA1 were used to determine the flour rate which resulted in spoonable consistency (2500–3000 cP)  attained the maximum viscosity (3000 cP) at a lower flour concentration compared to extruded and malted/roasted flours Fig. . Malted/Energy and nutrient density of both sauce and porridge were computed based on their flour rates. The highest flour rates within the range were selected for comparison Table . Sauce pWomen of reproductive age and young children are the most affected by malnutrition, especially micronutrient deficiency (iron and zinc). The contribution of sauces to the recommended dietary intake Table  was calcFor children (1–5 years), the lowest nutritional contribution for iron, zinc, protein, and energy were 38.6%, 37.0%, 85.4%, and 25%, respectively , while the highest nutritional contribution were 192.9%, 111.6%, 270.4%, and 89.9%, respectively . For women of reproductive age (15–50 years), the lowest nutritional contribution for iron, zinc, protein, and energy were 23.8%, 23.7%, 38.1%, and 26.2%, respectively, while highest nutritional contribution were 250.1%, 69.7%, 127.4%, and 62.7%, respectively, all from extruded flour. Based on these results, it showed that processed biofortified bean flour can highly improve nutritional status of consumers.Malting/roasting and extrusion processes improve nutritional and physicochemical characteristics of biofortified beans. In the present study it is was found that both processing methods increase the mineral bioavailability and improve in vitro protein digestibility of bean flour. Extruded products had higher mineral bioavailability and in vitro protein digestibility than malted/roasted samples. The pasting properties of bean flour were modified by processing, with extruded flour exhibiting lowest pasting viscosities and pasting temperature compared to malted/roasted samples, and malted/roasted samples exhibiting lower pasting viscosities than raw samples. Products from conventional bean flour (K131) and products from biofortified bean flour (ROBA1) are equally acceptable by consumers. Gruel or sauce prepared from processed biofortified beans exhibited higher nutrients and energy density than gruel/sauce prepared from unprocessed biofortified bean flour. It can be thus concluded that processed biofortified bean flour can be utilized to prepare highly acceptable nutrient and energy dense sauce or gruel suitable for feeding nutritional vulnerable populations.None declared.