There is much interest in virtual consultations using video technology. Randomized controlled trials have shown video consultations to be acceptable, safe, and effective in selected conditions and circumstances. However, this model has rarely been mainstreamed and sustained in real-world settings.The study sought to (1) define good practice and inform implementation of video outpatient consultations and (2) generate transferable knowledge about challenges to scaling up and routinizing this service model.A multilevel, mixed-method study of Skype video consultations (micro level) was embedded in an organizational case study (meso level), taking account of national context and wider influences (macro level). The study followed the introduction of video outpatient consultations in three clinical services  in a National Health Service trust  in London, United Kingdom. Data sources included 36 national-level stakeholders (exploratory and semistructured interviews), longitudinal organizational ethnography , 30 videotaped remote consultations, 17 audiotaped face-to-face consultations, and national and local documents. Qualitative data, analyzed using sociotechnical change theories, addressed staff and patient experience and organizational and system drivers. Quantitative data, analyzed via descriptive statistics, included uptake of video consultations by staff and patients and microcategorization of different kinds of talk .When clinical, technical, and practical preconditions were met, video consultations appeared safe and were popular with some patients and staff. Compared with face-to-face consultations for similar conditions, video consultations were very slightly shorter, patients did slightly more talking, and both parties sometimes needed to make explicit things that typically remained implicit in a traditional encounter. Video consultations appeared to work better when the clinician and patient already knew and trusted each other. Some clinicians used Skype adaptively to respond to patient requests for ad hoc encounters in a way that appeared to strengthen supported self-management. The reality of establishing video outpatient services in a busy and financially stretched acute hospital setting proved more complex and time-consuming than originally anticipated. By the end of this study, between 2% and 22% of consultations were being undertaken remotely by participating clinicians. In the remainder, clinicians chose not to participate, or video consultations were considered impractical, technically unachievable, or clinically inadvisable. Technical challenges were typically minor but potentially prohibitive.Video outpatient consultations appear safe, effective, and convenient for patients in situations where participating clinicians judge them clinically appropriate, but such situations are a fraction of the overall clinic workload. As with other technological innovations, some clinicians will adopt readily, whereas others will need incentives and support. There are complex challenges to embedding video consultation services within routine practice in organizations that are hesitant to change, especially in times of austerity. Outpatient services, particularly for people with long-term conditions, have changed little in recent decades. Yet, in many countries, population demographics, disease epidemiology, and care priorities have changed a great deal, with, for example, an aging population, rising rates of chronic illness and multimorbidity, and an increasing emphasis on multidisciplinary team care and supported self-management. Outpatient nonattendance rates in some patient groups, especially the disadvantaged and those with multiple and complex needs, are high and may be associated with poor disease control, increased use of emergency services, and high costs ,2. PatieThere is a strong policy push in the United Kingdom -9 and elPublished research on video outpatient consultations has been summarized in several recent narrative reviews -15. RandHowever, all these trials were small; publication bias cannot be excluded, uptake rates were not always reported, and the cost of establishing and maintaining the remote service was rarely measured. Although no specific safety issues or critical events were reported in any of the above studies, exclusion or withdrawal rates in some studies were high ,23,25.Notwithstanding the positive findings of randomized trials, audits of actual practice suggest that video outpatient consultations, in common with other forms of telehealth, account for only a tiny fraction of encounters in any specialty ,36. NonaSome authors have queried whether video consultations might be less reliable -43, lessIn short, there is a growing mismatch between the positive evidence base emerging from experimental trials and the variable (mostly negative) experiences of teams who try to introduce video outpatient services in the real world ,36. The Our aims were (1) to define good practice and inform its implementation in relation to video outpatient consultations via Skype and similar media and (2) to generate transferable new knowledge about challenges to scaling up and routinizing this service model in health care organizations.Specifically, our objectives were as follows:At macro level, to build relationships with key national stakeholders, identify from their perspective how to overcome policy and legal barriers, and create a receptive context for our findings.At meso level, to illuminate and explore the sociotechnical system that supports the video consultation at organizational level and identify how organizations can best support the introduction and sustainability of this service model where appropriate.At micro level, to study the clinician-patient interaction in a maximum variety sample of video consultations and a comparator sample of face-to-face consultations, exploring both effective and less effective communication.Our research questions were as follows:Macro level: what is the national-level context for the introduction of video outpatient consultations in the United Kingdom, and what measures might incentivize and make such consultations easier to achieve?Meso level: how can a video consultation service best be established, routinized, and sustained?Micro level: what defines “quality” in a video consultation, and what are the barriers to achieving this?We have published a detailed study protocol previously ; this seThe multilevel, mixed-method study of video outpatient consultations in three hospital departments , embedded in an organizational case study of the introduction and rollout of this new service model (meso level), taking account of the evolving national context (macro level), was used.The research took place from 2015 to 2017 in Barts Health, a large, multisite acute trust in a socioeconomically deprived and multiethnic borough in inner-city London, United Kingdom. The organization was under pressure to deliver services more cost-effectively while responding to rising need and demand; outpatient clinics were crowded, and travel to and between its multiple sites was difficult, time-consuming, and (for patients on low incomes) costly.Clinicians in the diabetes service had been working for several years to establish a remote video option for outpatient consultations to improve accessibility and address high “did not attend” rates. In an early pilot study, we found high acceptance rates by staff and patients for the video option, a significant reduction in “did not attend” rates and small efficiency savings ,48. We sThis study occurred at a time when the trust was seeking to learn from the diabetes pilot and make video outpatient consultations part of business as usual whenever clinically appropriate. We worked mainly with three services on separate sites in east London: diabetes (adult and young adult), which had been piloting the virtual consultation model; antenatal diabetes, which sought to use such consultations to support close (sometimes daily) monitoring of diabetes in pregnancy; and hepatobiliary and pancreatic cancer, a tertiary care service to which patients were sometimes referred from beyond London, requiring a round trip of several hours. By the end of the study period, other specialties at Barts Health  had begun to introduce video outpatient consultations.The study was delivered by a core working group , supported by a 6-monthly independent steering group and a patient advisory group (see below). The steering group had a lay chair and cross-sector stakeholder representation, including patients, National Health Service (NHS) stakeholders, and national-level decision makers ; and a total of 50 patients. Data sources are summarized in We began with individuals charged with delivering information technology (IT) strategy in NHS England, as well as those leading on patient participation. Alongside review of policy documents (from 2000), we used snowball sampling (asking each interviewee to nominate a colleague) to build up a picture of the national context. We invited a maximum variety sample of 39 stakeholders from across government , professional organizations , patient groups , industry , and charitable and third sector organizations , of whom 36 agreed to talk informally with the study team (3 were uncontactable). We then undertook audiotaped, semistructured interviews with a purposive sample of 12 of these stakeholders, ensuring variation in the different institutions, groups, and perspectives represented. Stakeholder details and interview guides are available in The goal of sampling was to map the people, interactions, and organizational routines that support the virtual consultation with a view to building a rich “ecological” picture of the sociotechnical microsystem   with 24We used audio, video, and screen capture to produce rich multimodal data on a total of 30 virtual consultations and made audio recordings of 17 face-to-face recordings matched for clinical condition . Details of these consultations are shown in Specific exclusion criteria were as follows: no 3G access at home, lack of familiarity (by patient or carer) with the relevant technology, clinical inappropriateness , inability to give informed consent, and comorbidity preventing participation . Clinic populations included a high proportion of limited English speakers. In the young adult diabetes clinic, bilingual health advocates were available and trained in the use of remote consulting, so limited English was not an exclusion criterion there. In the antenatal diabetes and cancer clinics, a remote interpreting service was not available, but patients comfortable with a family member interpreter were included.Our micro-level dataset consisted of video recordings of consultations incorporating two video streams: one from the clinic and one from the remote site . We recorded consultations using a small digital camcorder with wide-angle lens and remote control (Sony Handycam DCR-SR72), mounted on a mini tripod. We used a commercially available screen capture software tool (ACA Systems) run directly from an encrypted Universal Serial Bus (USB) stick to record screen images showing on each party’s computer screen as a video file. A researcher started and stopped the recordings but left the room during the consultation. When the patient used a mobile, tablet, or Mac computer (which could not run the encrypted USB device), the researcher positioned a second digital camera to capture the screen. In 12 cases, the consultation was recorded at the clinic end but not at the patient end for logistical or patient preference reasons.Each end of the consultation resulted in 2 digital files, one screen capture and one video. We synchronized these into one file using video editing software (Sony Movie Studio)—allowing us to play the video of the computer screen exactly in parallel with a video of the patient looking at the screen and to view the patient and clinician “ends” in parallel. Face-to-face consultations were audiorecorded using a digital dictaphone. Further details of the informed consent process are given in The micro-level dataset also included contemporaneous field notes from patients’ homes  and the clinic . We also collected demographic data  on patients using the Skype option for remote consulting and (for comparison) on the clinic population as a whole over a 12-month period at each clinic setting.In our original study protocol , we drewshould or must behave in this setting rather than simply by economic or personal concerns, such as maximizing efficiency or pleasure). In such circumstances, behavior is often ritualized . A key question driving our data collection and analysis was how would the technological and material aspects of the remote consultation shape, enable, and constrain the playing out of these institutionalized roles and behaviors.The health care setting is heavily institutionalized  by the technology, and commercial and regulatory considerations. The resulting theoretical framework (NASSS) has recently been published .As described in detail previously , our stuInterviews with national stakeholders were initially analyzed thematically to provide context for the statements, actions, and interpretations made by organizational actors at local level. We also used interpretive policy analysis ,55 to idOur approach to mapping the sociotechnical health care ecosystem  providedWe also drew on the notion of “organizational routines” ,58 definOur initial analysis of micro-level data involved repeated viewing of selected virtual consultations and discussion in our interdisciplinary team , alongside review of interview data with patients and clinicians. This led us to identify a number of questions relating, for instance, to the ways in which the context of the consultation (often involving patients in their home setting and clinician at the clinic) shaped communication; whether the usual format of the medical consultation  might shift when conducted remotely; how talk about technology might reorient patient-clinician interaction; and how sensitive topics (such as breaking bad news) might play out differently.On the basis of these early emerging themes, we sought a methodology to add depth and detail to our findings and identified the Roter interaction analysis system (RIAS), a widely used method for coding medical dialogue . BroadlyRoter’s original taxonomy distinguishes three main categories of talk: “task-focused” , “socioemotional” , and “process” . In this study, we also used a fourth category: “technology talk” , initially introduced by other researchers ,68 and aFollowing a 3-day training course delivered by the team that originally developed RIAS, we applied the RIAS coding scheme to our micro-level data, addressing five questions about the differences between virtual and face-to-face consultations for the same clinical condition:Are remote consultations shorter and more “to the point” than face-to-face ones?How do they differ in the different kinds of (nontechnology-related) talk that occurs?What kind of technology-related talk occurs?What kinds of breaches  of talk occur in virtual consultations, when do such breaches occur, to what extent do they matter, and how might they be reduced?How do interruptions (in the patient’s home and/or in the clinician’s office) affect the flow of talk in the virtual consultation?P<.05). Therefore, Mann-Whitney U tests (nonparametric) were used to compare interactions for virtual and face-to-face consultations.The assumptions for normal distribution of the data were not accepted  who found it difficult to attend face-to-face appointments. As noted above, the VOCAL steering group had a lay chair and patient representation. In addition, we sought ongoing patient feedback on both the research process and the developing video outpatient services at Barts Health via a dedicated patient advisory group, with 12 patients (and one spouse) representing a wide range of ages and ethnic groups and facilitated by an anthropologist with close knowledge of the local community. This group met three times throughout the study, supplemented with ad hoc contact with individual members . In one of the meetings, the patient advisory group was shown (with the written consent of participants) two video clip recordings of virtual consultations as the basis for discussion. In addition, some preexisting lay groups  and a cancer support charity were consulted to extend the range of patient and lay input.The external context for technological innovation in the UK public sector is currently extremely challenging. There is a strong policy push to develop UK’s digital economy -73, digiNotwithstanding these initiatives, constraints imposed by financial austerity—spending plans, for example, indicate a decreasing share of gross domestic product being devoted to the NHS from around 7.3% in 2016 and 2017 to 6.9% by 2022 and 2023 —have meadrive transformation of the NHS. The potential time lag between adoption of technology and realization of productivity gains (if any) was rarely acknowledged either by interviewees or in policy documents.National-level stakeholders and documents depicted technological innovation as imminently poised to deliver efficiency savings in the NHS “at scale” and “at pace” ,93,94, tFew interviewees mentioned low digital literacy and digital access. In the United Kingdom, 9% of all citizens  have never used the Internet . The UK what makes a good quality remote consultation). In contrast, clinicians and policy makers placed high value on RCT evidence generated elsewhere but assumed to be transferable to the current setting Clinician: [Laugh] Um. (0.39) So we will see you again, or touch touch ba-+base—oh yeah you are moving again, now...Nurse: That’s better.Patient: Right.Clinician: We–we’ll make contact again in November or December, after you’ve had another computed tomography (CT) scan and another set of blood tests.Video consultations presented new possibilities for interruption. This included disruptions related to the technology , as well as nontechnological interruptions in the domestic environment . In all cases in our dataset, flow of the consultation resumed readily after such interruptions.U=121.5, P=.43). The extent to which the clinician did more talking (“dominance”) and exerted more control (“directedness”) was similar in both video and face-to-face consultations in each specialty . The one statistically significant difference in clinician dominance was in the diabetes antenatal setting, in which consultations were slightly more clinician-dominated during remote  than face-to-face  consultations . This was probably explained by patients, at the clinician’s request, reading out home blood glucose readings and insulin doses in the video consultations.Findings from our RIAS analysis are summarized in A more fine-grained analysis of the different types of talk, which we will present in a separate publication  likewise confirmed only small and mostly nonstatistically significant differences in categories such as “verbal attentiveness,” “making requests,” “giving information,” and “counseling” .This study has confirmed findings from randomized trials that when clinical, technical, and practical preconditions are met, video consultations are safe, effective, and popular with participating patients and staff. In most cases, video consultations consisted of similar types of talk, in similar proportions, to comparable face-to-face consultations, and differences between different clinical specialties were more striking than those because of the technology. By the end of this study, between 2% and 22% of all consultations were being undertaken via video link by participating clinicians. In the remainder, the video option was considered impractical, technically unachievable, or clinically inadvisable for the patient. Technical challenges were typically minor but potentially prohibitive.Although these findings confirm that video consultations may have an important place in transforming care models, some staff members chose not to participate, and patients for whom video consultations were deemed appropriate represented a fraction of the overall clinic workload in all specialties studied.Notwithstanding policy interest in digital solutions, the reality of establishing video outpatient services in a busy and financially stretched acute trust proved far more complex and time-consuming than anticipated—mainly due to lack of “organizational slack” , disruptAlthough national policy makers viewed video consultations as a driver of change , industry informants viewed this option as low priority because of anticipated (and experienced) challenges of working with the NHS on projects that required complex organizational, policy, and regulatory changes.not adopted or soon abandoned by professionals and/or their patients and clients or else the technology-supported service succeeds as a small-scale demonstration project but fails to scale up locally, spread to other comparable settings, or be sustained over time. The NASSS framework analyses these problems in terms of seven interacting domains: the condition, the technology, the value proposition, individual adopters (staff and patients), the organization, the external  context, and emergence over time [These findings can be theorized using our recently published NASSS framework that was developed to explain why, despite significant investment and high expectations, five problems persist: digital technologies are either ver time . Each dounsuccessful, this can be explained by complexity and unpredictability in the clinical condition; lack of dependability and fitness-for-purpose of the technology; lack of acceptance by staff or patients; limited organizational slack, lack of shared vision, and/or clashes with long-held and difficult-to-change routines; and tricky regulatory or policy issues .To the extent that VOCAL was successful in establishing a video consultation service, this was explained by the various NASSS domains: straightforward, predictable, and low-risk clinical conditions; simple and dependable technology that was fit-for-purpose; clear benefits for both the technology supplier and the patient; acceptance of the technology by staff  and patients (who were able and willing to develop new skills and ways of engaging); alignment with existing—or emerging—organizational routines; and a strong policy push. To the extent that efforts to introduce video consultations were Our study has also illustrated, through detailed multilevel analysis, the interdependence of the different domains in the NASSS framework. For example, our national-level interviews identified a reluctance among major technology vendors in the United Kingdom (not just Microsoft) to make major investments in partnerships with the NHS. This meant that, at the time of writing, the technology being used was an off-the-shelf product that had not been specially adapted for use in video consultations and that this technology was not high priority for support from the local IT department. This partly explains why significant clinician time (and an extension of the clinician role) was needed to complete such tasks as new appointment booking and management of a virtual waiting room. Our findings suggest that proactive codesign between technology suppliers, participating health care organizations, and national policy makers could potentially produce three things: video software that is more fit for purpose, organizational routines that are better aligned to support video consulting, and better incentives for major suppliers to work in a collaborative and ongoing way with health care providers.Although our study was not designed to generate a simple or universal “checklist” for implementing video consultations, it is worth reproducing here the five “key recommendations for practice” aimed at clinicians and managers that were coproduced through action research in this study:Introduce the service slowly and incrementally with direct involvement of the team to ensure compatibility between the technology and existing practiceAllow plenty of time for discussion with staff and patients about how it affects the serviceWork in collaboration with your ICT department and technical support teams to establish roles and processes to assist use of the technologyUse with an understanding of the patients’ lives and how the technology relates to the management of their health conditionSupport flexible use, allowing scope to fit the service around the needs of the patientTo our knowledge, this is the first research study in any clinical field to have taken an in-depth, mixed-methods, and multilevel approach to the study of video outpatient consultations. We succeeded in our goal of collecting rich qualitative data that exposed the “messy reality” of establishing a virtual consultation service and illuminated the pros and cons of using this medium for clinical interaction in different settings. Using action research, we were able to inform and facilitate the work of embedding the new service model and gain detailed insight into organizational complexities and how these changed over time. Working with front-line clinical and technical services, we have developed significant expertise, standard operating procedures, information governance and technical guidance documents, and protocols for setting up and running video outpatient clinics. National policy makers have been engaged from the outset, and the study has attracted interest from other hospitals. A rollout phase continues within the trust, and further work is also ongoing to extend the model to other NHS organizations across the United Kingdom.The main limitation of the study is its focus on a single  health care organization. Barts Health was even more financially stretched than most acute trusts in the United Kingdom; hospitals under less tight financial and staffing pressures may have found the implementation work easier. In addition, the sample size for the detailed analysis of virtual consultations was relatively small.Almost all previous research on video consultations in health care has either addressed the technical detail of the remote connection or undertaken an RCT of virtual vs face-to-face consultations -18,20-34inherent to complex sociotechnical systems. To questions such as “do virtual consultations work?,” “are virtual consultations safe?,” and “are virtual consultations cost effective?,” we suggest the answer will always be “it depends.”Some critics will view it as a limitation that we did not emulate the experimental methodology of previous studies. This was deliberate. Our findings have confirmed that virtual consultations cannot be treated like a drug or even as a complex behavioral intervention to be tested “on” patients. Rather, they are the result of a hugely complex sociotechnical system in which “successful” virtual consulting is contingent on multiple factors at multiple levels. If we appear to have produced ambiguous findings, this is perhaps because ambiguity and tension are In the context of a strong policy push to develop digital alternatives to the traditional consultation, delivering video outpatient services at scale is likely to be far from straightforward, as rollout in any locality will be influenced by (among other things) prevailing organizational culture, financial and human resources and priorities for allocating these, existing organizational and technical infrastructure, the nature and causes of professional resistance, information governance challenges, and the logistics of payment. Video consultations, although safe and effective for selected patients, fundamentally change the nature of outpatient care and require clinician buy-in (which may or may not be forthcoming). Industry, although not opposed to the idea of developing software to support video consultations, appears to view organizationally embedded technical solutions as relatively low priority.The finding that efforts to implement a video consultation service met with multiple challenges in relation to workability and integration aligns with numerous previous studies of other forms of remote care . IndThis study has, in some way, revealed and explored the challenges to establishing video outpatient consultations as a real-world service. Overcoming those challenges will not be easy, but further in-depth case studies in both comparable and contrasting settings are likely to enrich our understanding. As more health care organizations make the strategic decision to introduce video consultation services, research could explore the collaboration and mutual learning that occurs between them and test approaches to supporting that interorganizational interaction.Our macro-level interviews identified a consistent finding from industry informants that the NHS is currently a uniquely difficult setting in which to attempt to introduce technologies that imply major changes in service models. Industry’s preferred model—of long-term partnerships (for technologies plus service support to embed them) rather than one-off procurement contracts—should be introduced in test sites and carefully researched using longitudinal ethnography. The research agenda here is methodological as well empirical; it is founded (we believe) on the notion that technologies and services are continually evolving and mutually shaping; they cannot be fixed in time nor (therefore) be adequately tested using traditional randomized trial designs.One of the most interesting findings of this study was that the technology provided opportunities for clinicians and patients to use the technology adaptively and differently, allowing new modes of consulting to evolve . Further qualitative research could pursue the consequences of such adaptive usage.This study has applied a sociological lens , as well as the recently-published NASSS framework to a real-world empirical study of video outpatient consultations across three contrasting clinical specialties.We found that these consultations appear safe, effective, and convenient for patients in situations where participating clinicians judge them clinically appropriate; however, such patients are a fraction of the overall clinic workload. As with other technological innovations, some clinicians will adopt video consultations readily, whereas others will need incentives and support. There are complex challenges to embedding video consultation services within routine practice in health care organizations that are hesitant to change, especially at a time of austerity.


Together, our findings provide critical insights into the molecular mechanisms underlying coordinated cytoskeletal dynamics during cell movement.Turnover of focal adhesions allows cell retraction, which is essential for cell migration. The mammalian spectraplakin protein, ACF7 (Actin-Crosslinking Factor 7), promotes focal adhesion dynamics by targeting of microtubule plus ends towards focal adhesions. However, it remains unclear how the activity of ACF7 is regulated spatiotemporally to achieve focal adhesion-specific guidance of microtubule. To explore the potential mechanisms, we resolve the crystal structure of ACF7’s NT  domain, which mediates F-actin interactions. Structural analysis leads to identification of a key tyrosine residue at the calponin homology (CH) domain of ACF7, whose phosphorylation by Src/FAK  complex is essential for F-actin binding of ACF7. Using skin epidermis as a model system, we further demonstrate that the phosphorylation of ACF7 plays an indispensable role in focal adhesion dynamics and epidermal migration  The spectraplakin protein ACF7 binds to actin at focal adhesions and targets microtubule plus ends to focal adhesions, promoting their disassembly. Here the authors reveal that ACF7 is phosphorylated by Src/FAK, and this regulates actin binding and focal adhesion dynamics in vitro and in vivo. Cell migration is a fundamental cellular process. The multi-step process of cell migration entails integrated activities of the cytoskeletal networks and cell/extracellular matrix (ECM) adhesions13Drosophila spectraplakin gene leads to various defects that include perturbations in cytoskeletal organization, cell-cell junction and integrin-mediated epidermal attachment to muscleBPAG1 (Bullous Pemphigoid Antigen 1) and ACF7. BPAG1 mutant mice display sensory neuron and muscle degeneration phenotype, and have gross defects in cytoskeletal organization and functionACF7 expression in mice leads to early-embryonic lethality8ACF7 in mice suggest that ACF7 is critically involved in cytoskeletal coordination and cell migration in vivo310111213Evolutionarily conserved in multicellular organisms, spectraplakin proteins such as ACF7 (Actin-Crosslinking Factor 7) can crosslink microtubules and F-actin5While the accumulating evidence underscores the importance of ACF7 in coordinating the cytoskeletal dynamics essential for cell movement, the spatiotemporal regulation of ACF7 remains largely unclear. ACF7 harbours an actin-binding domain (ABD) at its N terminus and a microtubule-binding domain at its C terminus3ECM adhesion signalling is mediated by integrin receptors1920in vitro and in vivo. Taken together, our results illuminate an important molecular mechanism whereby cell adhesion and cytoskeletal coordination are regulated during directional cell movement.In this study, we identify a key tyrosine-phosphorylation site within the ABD of ACF7. Our results show that the FAK/Src-mediated phosphorylation of ACF7 is essential for F-actin binding of ACF7 and coordinated cytoskeletal dynamics at focal adhesions. By disrupting this site, we demonstrate that phosphorylation plays a key role in focal adhesion dynamics and cell motility in vitro are readily available, it remains challenging to develop mammalian models and examine cell movement in vivo. As a surface organ, mammalian skin serves as an ideal candidate for intravital monitoring of cell migration. In addition, the established protocol for culturing skin epidermal stem cells makes it possible to generate functional skin tissue from engineered stem cells, which can carry both fluorescence markers for intravital imaging and defined genetic alterations to investigate the consequence in epidermal migration in vivo. However, engraftment of passaged epidermal stem cells via traditional skin stem cell technique proved unsuccessful. To resolve this issue, we first employed organotypic culture of epidermal keratinocytes in vitro by culturing the cells on top of acellularized dermis (in vitro (Although approaches to exploring cell migration d dermis . Exposurin vitro  (ref. 22in vitro . The grain vitro .ACF7-deficient cells exhibited a significant delay in repairing full-thickness wounds as compared with wild-type (WT) skin (ACF7 null cells (Our previous studies have suggested a role of ACF7 in cell motility and skin-wound repair3WT) skin . Histololl cells . Despitell cells .in vivo, we transduced WT and ACF7 null cells with lentivirus encoding Histone H2B–RFP. Regenerated skins were wounded and then imaged by multiphoton microscopy . By contrast, ACF7 null cells move in a rather uncoordinated manner (P=0.516 by Rayleigh test). Taken together, our results demonstrate that ACF7 plays an essential and multifaceted role in epidermal migration in vivo.To directly monitor epidermal migration croscopy  and 2. Winterval . Quantif of ACF7 . In addiionality . Epidermin vitroACF7 is a microtubule and F-actin crosslinker that is critically involved in regulation of cell/ECM adhesion dynamicsThe two tandem CH domains are present in ABDs of many F-actin-binding proteins242526To illuminate the potential mechanisms of how F-actin binding of ACF7-CH domains is regulated, we resolved crystal structure of ACF7-NT (containing the CH domains and a part of the plakin domain) at 2.6 Å resolution . ACF7-NT28Previous studies indicate that CH domains of utrophin associate with F-actin through an induced-fit mechanism, requiring a transition to an extended conformation between the tandem CH domains on binding with F-actin1626In vivo, FAK and Src form a mutually activated kinase complex that initiates a cascade of phosphorylation events, triggering various signalling pathways downstream to cell adhesion. In particular, both FAK and Src have been shown to be critically involved in cell motility and focal adhesion dynamics1920FAK . Liquid chromatography-tandem MS (LC–MS/MS) analysis identified a prominent phosphorylation site (Y259) within the 2nd CH domain at ACF7-NT . To confin vitro co-sedimentation assays with polymerized F-actin to determine their binding affinity -tagged full-length ACF7, as well as point mutants at Y259. As expected, full-length ACF7 or ACF7-Y259F mutant displayed very weak interaction with F-actin in co-sedimentation assay, whereas the phospho-mimetic mutation dramatically increased F-actin binding . In addiConsistent with biochemical analysis, ACF7-NT or its Y259F mutants exhibited no significant decoration of F-actin filaments when expressed in primary keratinocytes. By contrast, Y259D mutant in ACF7-NT showed marked co-localization with F-actin . TogetheACF7 KO cells  derived from the SAXS data of ACF7-NT is slightly larger than that of the crystal structure of ACF7-NT (Rg=29 Å), suggesting that ACF NT in solution is likely to be more extended than that depicted by the crystal structure . The gel filtration profiles of purified ACF7-NT and Y259D mutant suggest that both proteins are monomeric and monodisperse . We fountructure . Althougtructure . Thus, iStructural analysis demonstrated that tyrosine 259 forms hydrogen bonds with isoleucine 150 and tyrosine 151 in the hinge region of CH domains . These hACF7 in skin keratinocytes impairs cell motility and focal adhesion dynamicsACF7 KO keratinocytes to re-express ACF7 or ACF7 mutant. To achieve stable expression of ACF7, we overcame the technical hurdles of ACF7’s enormous size  and engineered mammalian expression vectors encoding full-length ACF7 or ACF7-Y259F mutant within PiggyBac (PB) transposon elementsACF7 was efficiently integrated into the genomes. After selection, the cell lines expressed ACF7 or its Y259F mutant stably. Exogenous ACF7 and Y259F mutants were expressed comparably with the correct size, and exhibited the expected differential phosphorylation status  or both YF and SD mutations (ACF7-YF/SD mutant). When introduced into ACF7-deficient keratinocytes by microinjection, the SD mutant or YF/SD mutant of ACF7 could not significantly rescue the defects in either cell motility or focal adhesion disassembly or assembly  and phosphor-ACF7 Y259 (dilution range 1:50–1:200) was produced by Covance  and Abmart , respectively. Guinea pig anti-K5 (dilution 1:200–1:500), rabbit anti-K10 (dilution 1:200–1:500) and Loricrin (dilution 1:200–1:500) antibodies were generous gifts from Dr Elaine Fuchs at the Rockefeller Univerisity. Rat monoclonal β4-integrin  was obtained from BD Pharmingen . Human plasma fibronectin, ATP, HA-conjugated Agarose, mouse monoclonal Vinculin  and β-tubulin  Abs were obtained from Sigma . Texas Red-conjugated Phalloidin was obtained from Invitrogen . Mouse monoclonal Abs against Myc , and rabbit polyclonal Abs against HA , His tag , Src  and Ki67  and Src inhibitor pp2 were obtained from Santa Cruz Biotechnology . FAK inhibitor PF-562271 was obtained from AdooQ Bioscience . Mouse monoclonal Antibody against phosphor-tyrosine was purchased from Millipore . Actin-binding protein spin-down kit was obtained from Cytoskeleton . Other chemicals or reagents were obtained from Sigma, unless otherwise indicated.ACF7-NT and ACF7 full length were created by over-lapping PCRs with primers: 5′-CGCCTAGGTTTCCTGTTCTTTGGGCC-3′, 5′-CCGCTCGAGTTATTTGTTTGCAATCTGTAGCAG-3′, 5′-TGTGTCTTCGATTTTTGATGCCTTCCCTA-3′, 5′-TAGGGAAGGCATCAAAAATCGAAGACACA-3′, 5′-TGTGTCTTCGATTGATGATGCCTTCCCTA-3′, 5′-TAGGGAAGGCATCATCAATCGAAGACACA-3′. To create ACF7-YFSD mutant, the same mutagenesis PCR was carried out in ACF7-SD mutant backgroundACF7 N-terminal (residues 1–400), CH domain (residues 1–268), plakin domain (residues 268–400) and their mutants were cloned into mammalian expression vectors pKH3S and pHANS  and prokaryotic expression vector pETDuet1. Tyrosin259 (Y259) mutations to Phenylalanine (F) and Aspartic acid (D) in Escherichia coli BL21(DE3) cells. Briefly, cells were grown in 12 l of LB medium containing 100 μg ml−1 ampicillin at 30 °C. The culture was induced with 0.4 mM isopropyl-β,D-thiogalactopyranoside at an OD600  value of ∼0.5. Cells were harvested within 5 h of induction. The collected bacteria were resuspended in ice-cold buffer containing 20 mM Na–HEPES (pH 7.5) and 100 mM NaCl. The cells were then lysed in a French pressure cell. Cell debris was removed by centrifugation for 1 h at 4 °C and 20,000g. ACF7-NT then purified with Ni2+-affinity and superdex-200 gel filtration  columns to >95% purity and its identity was confirmed by N-terminal sequencing. ACF7-NT mutant (Y259D) was isolated with the same procedure.The ACF7-NT containing a 6 × His tag at the N terminus was overproduced in ACF7in vitro kinase assays, purified ACF7 or ACF7 mutants were incubated with recombinant kinases for 10 min at 37 °C in a total volume of 30 μl of protein kinase assay buffer ±1 μCi of [γ-32P] ATP  (ref. Full-length ACF7 was isolated by affinity chromatography from 293F clone that stably expresses HA-tagged −1) ref. .E. coli or expressed in HEK293T cells 2SO4 and 1 M (NH4)2HPO4. Cryoprotection was achieved by raising the glycerol concentration stepwise to 30% with a 5% increment in each step. The diffraction data sets of ACF7-NT were collected at 100 K at Shanghai Synchrotron Radiation Facility. Diffraction data were processed with DENZO and scaled with SCALEPACK. The ACF7-NT structure was determined by molecular replacement with Phaser in CCP4i  in PBS were collected at room temperature at 12ID-B, APS, ANL using 1 mg mlSkin or wound samples were embedded in OCT, frozen, sectioned and fixed in 4% formaldehyde. For paraffin sections, samples were incubated in 4% formaldehyde at 4 °C overnight, dehydrated with a series of increasing concentrations of ethanol and xylene, and then embedded in paraffin. Paraffin sections were rehydrated in decreasing concentrations of ethanol and subjected to antigen unmasking in 10 mM Citrate, pH 6.0. Sections were subjected to haematoxylin and eosin staining or immunofluorescence staining2+. All experiments were performed using primary cells with <10 passages. FAK KO keratinocytes were kindly provided by Dr Markus Schober from New York University. HEK293 cells were cultured in DMEM medium supplemented with 10% serum. For protein analysis, cells were lysed in lysis buffer and subjected to immunoprecipitation with different antibodies. Whole-cell lysates and immunoprecipitates were examined by western blotting analysis with Odyssey infared imaging system or ECL reagents. Uncropped original scan of western results are shown in Primary mouse keratinocytes were isolated from the epidermis of newborn mice using trypsin, after prior separation of the epidermis from the dermis by an overnight dispase treatment. Keratinocytes were plated on mitomycin C–treated 3T3 fibroblast feeder cells until passage 3. Cells were cultured in E-media supplemented with 15% serum and a final concentration of 0.05 mM CaFor scratch-wound healing assay, keratinocytes were plated on 35-mm tissue culture dish coated with fibronectinTo trace the movement of individual keratinocytes, cells were plated on fibronectin-coated dishes and imaged with an Olympus vivaview microscope (× 20) for 2 h at 1 frame per min and manually tracked in Image J.Keratinocytes were plated on fibronectin-coated dishes and transfected with plasmid encoding DsRed-Zyxin. Time series of images were acquired on a 3i Marianas Yokogawa-type spinning-disc confocal microscope equipped with a × 100 α-plane (1.45 oil) lens and an electron microscopy charge-coupled device camera. The rate constants for focal adhesion assembly and disassembly were obtained by calculating the slope of relative fluorescence intensity decreases of individual focal adhesion on a semilogarithmic scale against time226 cultured keratinocytes were seeded onto the dermis in cell culture insert. After overnight attachment, the skin culture was exposed to air/liquid interphase. To transplant to nude animals, two 1 × 1-cm wounds were introduced to the back skin. After transplantation, the wound edge is sealed with surgical glue. The grafted animals were housed separately and the wound bandages were usually removed 1 week post surgery. At 2 weeks post surgery, full-thickness excisional wounds were made on skin grafts using a 6-mm skin puncher. Mice were housed separately, and no self-induced trauma was observed in control or cKO mice. Tissue was collected 2–6 days after wounding, and wound re-epithelialization was evaluated by histological analyses. Hyperproliferative epidermis (HE) was identified by haematoxylin and eosin staining, and the length of HE that extended into the wounds was measured and quantified.Decelluralized dermis (1 × 1 cm) was prepared by EDTA treatment of newborn mouse skinFor skin explant, the punched skin biopsy was cultured on matrigel-coated coverslip with E-medium added with 0.3 mM CaCl2. Pictures were taken at different time points as indicated. The animal studies were carried out in the ALAAC-accredited animal research facility at the University of Chicago. All the animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Chicago.−1). Acquisition and image analysis were performed with Living Image 4.3.1 software.Optical imaging was performed in the integrated small animal imaging research resource (iSAIRR) at the University of Chicago. Bioluminescence images were acquired on an IVIS Spectrum  after animal was injected with luciferin  versus accumulated distance of cell migration.t-test or analysis of variance was used to assess the statistical significance  of differences between experimental conditions.Statistical analysis was performed using Excel or OriginLab software. Box plots are used to describe the entire population without assumptions on the statistical distribution. A student All relevant data are available from the authors. The SAXS data was deposited to SASBDB with accession code SASDBL3 for ACF7-NT and SASDBN3 for ACF7-NT Y259D. The structure data has been deposited to Protein Data Bank (PDB) with accession code 4Z6G.Accession codes: The SAXS data was deposited to SASBDB with accession code SASDBL3 for ACF7-NT and SASDBN3 for ACF7-NT Y259D. The structure data has been deposited to Protein Data Bank (PDB) with accession code 4Z6G.How to cite this article: Yue, J. et al. In vivo epidermal migration requires focal adhesion targeting of ACF7. Nat. Commun. 7:11692 doi: 10.1038/ncomms11692 (2016).Supplementary Figures 1-8in vivo .Intravital imaging of WT epidermal cell migration ACF7 KO epidermal cell migration in vivo .Intravital imaging of ACF7. KO keratinocytes stably rescued with ACF7 were transfected with plasmids encoding GFP-EB1 , CFP-LifeAct , and tagRFP-Zyxin . Cells were then examined by multi-channel confocal spinning-disc videomicroscopy to record coordinated cytoskeletal dynamics. Images were taken at 1 frame every 1.5 secondsRescue of cytoskeletal coordination by exogenous expression of  ACF7-Y259F mutant. KO keratinocytes stably rescued with ACF7-Y259F mutant were transfected with plasmids encoding GFP-EB1 , CFP-LifeAct , and tagRFP-Zyxin . Cells were then examined by multi-channel confocal spinning-disc videomicroscopy to record coordinated cytoskeletal dynamics. Images were taken at 1 frame every 1.5 secondsRescue of cytoskeletal coordination by exogenous expression ofACF7in vivo .Migration of ACF7 KO cells rescued with WT ACF7in vivo .Migration of ACF7 KO cells rescued with Y259F


The training group showed significantly higher subjective cognitive ability, as well as lower self-reported rumination at posttest in contrast to the control group . On a subsample with cognitive performance data (n = 19) we detected an improvement in executive function (Trail Making Task A and B) in the training compared with the control group. The results show that the fast paced action video game employed in the present study improved Trail Making performance and may reduce rumination and enhance subjective cognitive ability. Future research may focus on the investigation of the precise cognitive profile of effects.Cognitive deficits are common in depression and may persist following the resolution of affective symptoms. However, therapeutic strategies that successfully target cognitive impairments are lacking. Recent work has demonstrated that playing action video games leads to improvements in cognition, in particular executive function, in healthy individuals. We therefore set out to test whether playing video games can reduce symptoms associated with depression. We focussed on depressive symptoms and on rumination, since rumination is a good predictor of depression and may contribute to triggering depression. We recruited 68 clinically depressed individuals  that were randomized into the training group playing a fast paced action video game for 6 weeks or a waitlist control group. Before and after training participants completed online questionnaires and a neuropsychological test battery. Only participants who actually played the game were included in the analysis. The final sample consisted of  Depression is among the major causes of global disease burden  and psychoeducation such as SPARX  with a verified diagnostic status of major depression or dysthymia .At posttest, participants were asked how frequently they engaged in the training . Participants who were assigned to the training group and indicated that they never played the game were excluded. We also collected logfiles of the game play, however only a small subset of participants . The figure is surrounded by a green circle or a red square in the background. The task of the participant is to press the left or right button depending on the hand in which the figure holds the colored shape that is displayed in the background.The spatial reasoning task consists of a display of a 2-D folding template of a 3-D shape. The participants have to choose the correct 3-D shape from four different shapes. The participants were given 5 min time and we counted the number of correct answers in this time frame.The TMT assesses processing speed and executive functioning. Participants were asked to connect 25 circles as fast as possible, but still being as accurate as possible. In version A, the targets were all numbers  and the participant needed to connect them in sequential order. In version B, the points alternated between numbers and letters .http://www.millisecond.com) and we used the tasks as provided online in the Test Library of Inquisit. Completion of the cognitive battery took about 30 min in total. All tasks were performed online on the personal computer of the participants at home. Not all participants took part in this part of the pre- and posttest (n = 19), some because they were not able to access the tests for technical reasons, some because they did not respond to the link  because it can be easily accessed and played on a home computer. In addition, the controls are relatively simple, and can therefore be played by individuals who do not regularly play video games. We collaborated with the game designer Ian MacLarty to obtain a version that enabled us to track game performance parameters. Participants were provided with a link to download the game. Boson X is a fast-paced action game in which the player navigates a running avatar (shown from a 3rd person perspective) through a tunnel. The avatar is navigated by pressing the arrow keys on the computer keyboard . The tunnel consists of various tiles and the player has to avoid letting the avatar fall out of the tunnel. If the player steps on a missing tile, the avatar has to repeat the current level. If the player steps on a normal tile, the avatar successfully continues through the tunnel. If the player navigates the avatar onto highlighted tiles, the avatar collects “energy”  test Little, . Since tWe employed separate ANCOVAs in which we compared the experimental group with the control group on the dependent variables: depression , rumination, subjective cognition, and the cognitive test scores at posttest while controlling for the pretest scores of the respective dependent variable at posttest and habitual video game playing by entering these variables as covariates in the ANCOVA. Sex was entered as a fixed factor, since the sex distribution differed between groups, but most importantly because females are known to play less video games  values as an effect size measure to facilitate the use of it in power analyses to determine the desired sample size for future experiments.In separate analyses we correlated the subjective cognition score with TMT performance using a Pearson correlation coefficient. In a post-hoc test we repeated the ANCOVAs for subscores of the subjective cognition score that capture different cognitive domains. We regard effects below a 2 = 0.69, p = 0.012) and on the PHQ-9 score , which was higher in the control group. Moreover, we found a trend for BDI score differences at pretest with the same direction  and a tendency for more habitual video game playing in the video gaming group . Participants in the video game group on average reported to have played two to three times a week . However, due to the fact that participants were randomly assigned to the groups we assume that the differences in sex and PHQ-9 arose by chance and are not systematically related to the dependent variable which makes it feasible to use ANCOVA as a statistical procedure.Patients' demographic characteristics and clinical parameters at pretest are reported in Table F = 72.65, p = 0.017, ηp2 = 0.123, with the covariate of pretest rumination score being significant, p < 0.001, see Table F = 162.31, p = 0.043, ηp2 = 0.090, with the covariate of pretest subjective cognition score, p < 0.001] and habitual video gaming (p = 0.035) being significant. For the same analysis on BDI scores  and PHQ-9 scores  we did not find a significant group effects at posttest. When correcting for multiple testing by means of Bonferroni correction  none of the analyses presented would survive this correction. The changes from pre- to posttest in subjective cognition, BDI, PHQ9 and rumination were not associated with self-reported playtime at posttest (p > 0.354).Our main focus was on potential changes in depressive symptoms but also on associated symptoms such as rumination and subjective cognition due to our video game intervention. In separate ANCOVAs we compared the video game group against the passive control group at posttest and found a significant effect of group reflecting lower rumination score in the video gaming group . We found no significant effects in the performance data of the other cognitive tests (p > 0.261). The TMT result does not survive when applying Bonferroni correction to adjust for multiple testing. TMT performance data was uncorrelated to the subjective cognition score at pretest  as well as at posttest .When exploring the subsample with data on cognitive performance  = 4.691, p = 0.036, ηp2 = 0.096], and the semantic memory showing a tendency for higher values in the experimental group at posttest .To explore the finding of an increase in subjective cognition in the experimental training group in more detail, we decomposed the SSTICS total score according to the cognitive domains that the items of the SSTICS referred to. While controlling for habitual video game playing and the pretest score and accounting for sex, as described above, we found that the subscore of episodic memory was significantly higher [Within the scope of the present training study we found results suggesting that a 6-week training with a fast paced action video game (Boson X) in depressed patients contributes to lower levels of rumination and higher levels of subjective cognitive ability at posttest in comparison to a control group . On a small subsample, we detected better processing speed and cognitive flexibility (TMT) in the training group at posttest compared with a passive control group when controlling for pretest performance, pointing into the direction of our hypothesis that fast paced action video games may train executive functioning.Although we did not find effects on the measure of depression severity , the observed reduction in rumination is highly relevant with respect to depression. The measure we used to assess rumination is based on the response style theory  are also highly relevant in depression. Although the size of the subsample from which we obtained cognitive data was admittedly fairly small, we did not observe an association between objective performance and subjective assessment of cognition. This is in line with previous research on depressed patients failing to show correlations between objective and subjective cognitive performance data , the speed of processing task (DSST) or the two spatial processing tasks . The absence of an effect on processing speed is in line with a recent study on healthy adults who have been trained with an action video game that likewise found no improvement in a speed task  based on the reasoning that the spatial nature of the tasks may be a proxy of hippocampal functioning and previous studies have demonstrated positive effects of action video gaming on spatial processing. In one study, pre-existing gender differences in spatial processing were eliminated after 10 h of action video game training and in another study, training with action video games was demonstrated to improve performance on a virtual surgery task in medical students (Feng et al., A profound limitation of the study is the overall sample size, as well as the small sample size in the subgroup that completed the neuropsychological test battery before and after the intervention. This may have considerably reduced the power of the present design to detect effects.Another critical point is that the observed effects, in particular on self-reported subjective cognition, may have been caused by the non-blind nature of the intervention and may reflect the participants' expectation that the game improved their cognition. However, at least in Germany, where the study was performed, video games are not very highly valued, which may also have lead participants to believe that video games should impair their cognition, although this was not assessed. Future work may usefully explore how factors such as expectation and motivation may influence the efficacy of video game interventions. Another limitation is that we were not able to evaluate potential associations between performance in the video game to the outcome, since we did not obtain the game logging information from all participants. Future studies should carefully log performance data as it may reveal insights into the particular elements of the game that drive the observed effects. Furthermore, the fact that the testing and training was accomplished by the participants at their home computer is a disadvantage because it is not well-controlled how much participants trained and whether participants engaged in other activities while filling in questionnaires or during the training. Future studies should likewise control the training time to investigate dose-response relationships.Due to the fact that the control group in the present study did not perform any task, we cannot finally exclude that the observed effects are due to distraction in the experimental group that was absent in the control group. However, this is unlikely to be the only mechanism at work here, as we observed the predicted effects of action video games on executive function, similar to previous work in healthy individuals (Green and Bavelier, Taken together, the present results suggest beneficial effects of training with an action video game on executive function, rumination and subjective cognition in a sample of depressed patients, albeit the results do not survive multiple comparison correction. These results pave the way for future studies that may set out to unravel the potentially mediating role of cognitive changes in the beneficial effects of action video game training on rumination in depressed patients. We did not observe significant improvements in affective symptoms following an action video game intervention. Other games, such as puzzle-like video games, might be better suited to improve mood in depression (Russoniello et al., Contribution to the design of the study: SK, SM, and JG; to the acquisition of the data: SK, SM, FB, and TL; to data analysis: SK, SM, and FB; to drafting the manuscript: SK, SM, FB, TL, and JG. All authors take full responsibility for the content of the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite over a decade of research and technological advances, sublingual microcirculatory monitoring has not yet reached clinical utility. Offline analysis is time consuming and occurs away from the patient. A system to assess the microcirculation at the point of care is desirable. We present a novel 5-point grading system (the point of care microcirculation (POEM) scoring system) that can be used at the point of care during non-invasive sublingual microcirculatory monitoring.The POEM score is an ordinal scale from 1 (worst) to 5 (best), based on a composite assessment of flow and heterogeneity of four individual sublingual video-microscopy clips. Thirty-two healthcare professionals were trained in how to assign POEM scores. Following training they assigned scores to five test sequences (each consisting of four video clips). They were blinded to clinical status. Inter-user consistency and agreement were assessed using intra-class correlation coefficient (ICC) analysis. In addition, blinded expert scores for 68 video clips were compared to offline computer analysis using traditional microcirculatory parameters including total vessel density (TVD), perfused vessel density (PVD), proportion of perfused vessels (PPV), microcirculatory flow index (MFI) and microcirculatory heterogeneity index (MHI). The time taken to assign each was recorded.R2 = 0.39; p < 0.05), PPV , MFI , and MHI . POEM scores took less time to assign than conventional offline computer analysis (2 minutes versus 44 minutes).Participants showed good inter-rater consistency  and agreement  for assigned POEM scores. Expert scoring of videos correlated with offline values for PVD  contains supplementary material, which is available to authorized users. As the microcirculation is the anatomical location of oxygen and substrate exchange, its behavior during shock is of interest to those involved in patient resuscitation. The term “hemodynamic coherence” has been used to describe a situation in which resuscitation aimed at restoring systemic hemodynamic parameters (such as cardiac output) also makes a corresponding improvement to the microcirculation . Some paThe use of hand held non-invasive sublingual video-microscopes (such as sidestream dark field (SDF) or incident dark field (IDF) microscopy) has allowed researchers to study microcirculatory flow in vivo, both in experimental models and in patients. However, despite over a decade of detailed investigations and demonstrations of the rationale of monitoring the microcirculation, this technology and associated techniques have still not advanced from the research to clinical domains. A major limitation in the use of current video-microscope technology is that analysis depends on the capture of video clips that require offline analysis. This takes a considerable amount of time . Real-time point of care automated (computerized) analysis has not yet been validated against traditional offline analysis. Even if computerized automated analysis were to yield accurate, validated parameters, the clinical applicability is unlikely to be meaningful without user interpretation and some form of clinical grading system that might determine particular therapeutic pathways based on target readings. This is because such a system may yield parameters of unknown clinical relevance. What is required in real-life clinical practice is a simple grading system that acts as a trigger or guide for the delivery of particular interventions.Here we describe for the first time a 5-point ordinal grading scale of microcirculatory function based on a composite of flow and heterogeneity in vessel segments viewed by sublingual video-microscopy. The score is relatively simple and can be assigned at the point of care. It may be one way of facilitating goal-directed therapy using microcirculatory parameters.A schematic diagram Fig.  summarizFlowNormal: <25 % of vessel segments in view are sluggish/stoppedImpaired: 25–50 % of vessel segments in view are sluggish/stoppedCritically impaired: >50 % of vessel segments in view are sluggish/stoppedHeterogeneityOnly if the user determines that a clip has “normal” overall flow are they prompted to also assign whether heterogeneity is present or absent. This is because during the pilot phase of developing the scoring system Fig.  we foundThe final POEM scoring system is a 5-point ordinal scale that integrates assessments of both flow and heterogeneity Table . It enabEach individual video clip is therefore assigned either: “critically impaired”, “impaired”, “normal flow with heterogeneity”, or “normal flow without heterogeneity”.It is the combination of all four video clips that gives the overall POEM score as per the algorithm in Fig. http://www.POEMscore.com) can be used to perform the calculation for the POEM score based on the scores for all four video clips with minimal effort. An example of an online POEM score calculation is shown in Fig. An online tool . All participants were current intensive care clinicians or nurses and had not previously used sublingual microcirculatory monitoring or interpreted video-microscopy clips.All study participants undertook a standardized 60-minute interactive training session using 18 slides and a pre-selected set of videos of varying microcirculatory dysfunction as examples , each of which consisted of 4 video clips . These videos were taken from the Microshock study , and hadA random selection of 68 individual video clips, acquired by sublingual IDF video-microscopy during the Microshock study from eight patients at 15 time points , and theUsing the assessments from these individual video clips, POEM scores were applied to all 15 time points according to the algorithm in Fig. Inter-user variability for the ordinal 5-point scale was assessed using the intra-class correlation coefficient (ICC) in terms of both consistency (e.g. do participants tend to score item A higher than item B but lower than item C) and agreement . Values are presented with 95 % confidence intervals (CI). Analysis assumed a two-way model . Naïve participant scores were also compared to expert scores by subtracting the true expert score from the observed rater scores and then perform a linear regression on these “error scores”, including rater and video sequence as independent predictors.p value <0.05 was considered significant.Values from computer analysis  were compared to ordinal POEM scores using linear regression. Further comparison was made between computer analysis parameters and both individual video scores and POEM scores as categorical variables . A There were 32 study participants, including 12 consultants, 18 training-grade doctors, and 2 intensive care nurses. All of these participants worked in their hospital ICUs. None had used sublingual video-microscopy before, and all were naïve to IDF video analysis. All participants completed the training and assessment sessions. The expert user (SDH) who conducted the offline computer analysis and POEM scoring has analyzed over 1000 video-microscopy clips.p < 0.001 and p = 0.046, respectively).When the naïve user POEM scores were analyzed to determine the inter-user variability between clips and between each other, the ICC values for consistency and agreement were 0.83  and 0.815 , respectively. From the analysis of variance between users and expert, there was greater agreement for “critically impaired” and “normal” but higher variability for the scores in between; both item and rater were significant predictors of the level of error in the scores when compared to expert , PVD , MFI , and MHI , but not TVD   Fig. .Fig. 4Rep < 0.001).In addition, when offline computer analysis parameters were compared to expert assigned grades for the 68 individual video clips, these scores corresponded well to perfusion (PPV and PVD) and flow (MFI) parameters, but there was no statistically significant relationship to the pure density parameter (TVD)  for the hybrid assessment of microcirculatory flow and heterogeneity. Naïve users can be trained to assess the microcirculation using this system, and have produced scores with relative consistency and agreement even after just one teaching session. Furthermore the scoring is in keeping with offline computer-generated analysis of flow, perfusion and heterogeneity and takes several minutes rather than an hour to perform. We present a user-friendly, straightforward tool that may be used as a point of care test in the observation of patients who are being treated for shock. Clinical validation and further examination of the real-time applicability are required before the 5-point grading system can be introduced into clinical practice, in particular in relation to other parameters such as COThe semi-quantitative “Boerma Method” of assigning MFI values during sublingual video-microscopy  has the Although heterogeneity is usually presented as MHI by calculating the differences in MFI values, the POEM score uses a simplified method that does not require calculation, in order to make it easier to use. The clinician is only required to determine whether there are five vessel segments different to the rest in the field of view, regardless of quadrant. As POEM scores reflect MHI well, this method may not necessarily be objectively precise but appears to be suitable in determining the level of heterogeneity for the purposes of a 5-point ordinal scale as well as full traditional MHI calculations.Recently some investigators have demonstrated that visual inspection by clinicians may have good agreement with more detailed offline computer analysis, making the prospect of point of care microcirculatory analysis more of a realistic prospect . A furthAlthough it has been an aspiration for some time, the real-time monitoring of the microcirculation at the point of care has been more of a futuristic prospect , 17 rathTraditional offline measurements of microcirculatory function will be of continued value in the research context for studies that utilize microcirculatory parameters as resuscitation outcome measures. They will also be important when testing particular therapeutic measures following shock, and in providing diagnostic information (such as type of shock and classification of subtypes), and in tailoring therapy to specific requirements. The benefit of the POEM study in the clinical context is that it is faster to obtain, can more easily be used in real time, and has the potential to guide and direct therapy at the bedside. Rather than replacing traditional numerical values, the POEM score is designed so that it can be used concurrently. One of the potential uses of the POEM score is as an early warning marker for microcirculatory dysfunction and loss of coherence. More detailed assessment of the microcirculation will still be required to determine the precise nature of microcirculatory dysfunction.The POEM score does not replace traditional parameters in their role as research endpoints. Rather, it brings the overall assessment of the microcirculation forward to the point of care, in order to act as guidance of initial therapy. It is not currently known whether this will improve outcomes for patients, but future prospective randomized studies may wish to test the utilization of the POEM score versus standard practice in the management of patients in shock (such as septic or traumatic hemorrhagic shock). Furthermore, it is possible that the clinician might direct resuscitative fluids in a manner that addresses microcirculatory dysfunction when such a phenomenon is detected at the point of care . Recent studies have reported that the delivery of plasma appears to have a restorative effect on the endothelial glycocalyx and ameliorate the endotheliopathy of trauma , 19. CouThe introduction of user-dependency on a point of care test is not without precedence; cardiac echo and ultrasonography are some obvious examples of techniques that require high-quality assessment by expert users for reliable readings. Trained users are accustomed to the practice of a fast, efficient assessment of patients in the Emergency Department (ED) in the case of focused assessment with sonography in trauma (FAST). Quality assurance and regular training, experience and revalidation should be of upmost importance amongst microcirculation monitoring users if their point of care assessment were to enter the domain of clinical utility. The current study did not test the level of skill fade or utility of the scoring system after a time interval, but these are aspects of training and professional development that would require attention if the POEM score were to be used clinically. Similarly, devices and technology would need to come under the scrutiny of clinical point of care governance to ensure safety and consistency in clinical practice.If real-time microcirculatory assessment is to be performed at the bedside, then quality assessment of video clips must also be undertaken before an assessment of flow and heterogeneity can be made. The current study only utilized videos that were high quality; whether poorer quality videos in a real-world scenario might lead to more variability in grading is unknown. The assessment of quality needs to be consistent, and reproducible, and with little inter-user variability. In particular attention should be paid to guarding against pressure artifact, perhaps by ensuring that there are visible flowing venules within the field of view.A decision was made early in the formation of the POEM score to not incorporate an assessment of vessel density, instead favoring assessments of flow and heterogeneity. Assessment of density was not a good discriminator between video clips when testing the early forms of the scoring system. Early pilot testing of the scoring system also showed that density assessment was difficult to apply in a manner that would accurately reflect TVD values. Nevertheless, POEM scores do correspond well to PVD, which is in itself a mixed density and perfusion variable. As the POEM score corresponds well to PPV, PVD, MFI, and MHI, it is the first tool to yield a composite assessment of flow and heterogeneity and also yield potentially meaningful data on perfusion and perfused vessel density.None of the POEM study subjects were physically next to a patient’s bedside when assigning POEM scores. It is unknown whether this physical proximity and patient contact would influence the scoring or performance of the test. Instead, the study subjects were blinded to patient status in order to establish the scoring system without any bias that clinical exposure might bring. Further validation at the patient’s bedside may be warranted in future studies that utilize the POEM score.This study utilized video-microscopy clips of patients with traumatic hemorrhagic shock. Further validation in other clinical scenarios such as sepsis and following major surgery are required in order to determine whether this scoring tool has more generalizable utility.Although there is a good association between POEM assessments and PVD, the POEM scoring system does not take into account pure density parameters of the microcirculation (such as TVD). This may limit its usefulness in determining the etiology of the microcirculatory derangement. Measures to determine density parameters must rely on alternative techniques, which are currently not available at the point of care.A new 5-point ordinal scoring system of microcirculatory flow and heterogeneity has been tested amongst healthcare professionals at two large UK teaching hospitals, and has relatively high consistency and agreement even after just 1 hour of training. POEM scores take a matter of minutes to assign, and correspond well to computer-analysis variables of flow, perfusion and heterogeneity. We present for the first time a point of care microcirculatory assessment tool that is quick, reliable, and gives potentially meaningful clinical parameters that might guide resuscitation. Prospective randomized trials utilizing goal directed therapy targeted at the POEM score are required to test its real-life clinical utility.


Oryza sativa; background Nipponbare) contains nine HKT (high-affinity K+ transport)-like genes encoding membrane proteins belonging to the superfamily of Ktr/TRK/HKT. OsHKTs have been proposed to include four selectivity filter-pore-forming domains homologous to the bacterial K+ channel KcsA, and are separated into OsHKT1s with Na+-selective activity and OsHKT2s with Na+-K+ symport activity. As a member of the OsHKT2 subfamily, OsHKT2;4 renders Mg2+ and Ca2+ permeability for yeast cells and Xenopus laevis oocytes, besides K+ and Na+. However, physiological functions related to Mg2+in planta have not yet been identified. Here we report that OsHKT2;4 from rice  functions as a low-affinity Mg2+ transporter to mediate Mg2+ homeostasis in plants under high-Mg2+ environments. Using the functional complementation assay in Mg2+-uptake deficient Salmonella typhimurium strains MM281 and electrophysiological analysis in X. laevis oocytes, we found that OsHKT2;4 could rescue the growth of MM281 in Mg2+-deficient conditions and induced the Mg2+ currents in oocytes at millimolar range of Mg2+. Additionally, overexpression of OsHKT2;4 to Arabidopsis mutant lines with a knockout of AtMGT6, a gene encoding the transporter protein necessary for Mg2+ adaptation in Arabidopsis, caused the Mg2+ toxicity to the leaves under the high-Mg2+ stress, but not under low-Mg2+ environments. Moreover, this Mg2+ toxicity symptom resulted from the excessive Mg2+ translocation from roots to shoots, and was relieved by the increase in supplemental Ca2+. Together, our results demonstrated that OsHKT2;4 is a low-affinity Mg2+ transporter responsible for Mg2+ transport to aerials in plants under high-Mg2+ conditions.Rice ( Up to now, most studies are focused on identifying the active, high-affinity channels and transporters, which function in K+ and Mg2+ uptake from the nutrient-deficient environments  family has been widely studied and most of its members are characterized as being permeable for specific ions in heterologous expression systems (+ transporter (Trk and Ktr) counterparts in fungi and bacteria form a HKT/Trk/Ktr superfamily  with up to four members, OsHKT2;1, OsHKT2;2, OsHKT2;3, and OsHKT2;4 characterized for the structure, expression, and function  ecotype was used in this study. The T-DNA insertion mutant atmgt6 (SALK_203866) was obtained from the Arabidopsis Biological Resource Center. Homozygous individuals of atmgt6 were screened by PCR using primers listed in Supplementary Table 2+, 1.5 mM Ca2+, 1% sucrose (Sigma) and solidified with 0.8% phytoblend (Caisson Labs). The plates were kept at 4°C for 2 days and then were placed vertically in growth chamber under 90 μmol⋅m-2⋅s-1 light intensity with a 16 h light/8 h dark photoperiod. Three-day-old seedlings were transferred onto media containing various ions as indicated in the figure legends. For hydroponic cultures, 7-day-old seedlings germinated in half-strength MS (1/2 MS) were transferred to one-sixth-strength (1/6 MS) hydroponic medium containing 0.25 mM Mg2+ and 0.5 mM Ca2+ without sucrose for another 7 days. Plants were then transferred to hydroponic 1/6 MS media containing various contents of Mg2+. Plant materials were harvested for further analyses 2 days after treatment.S. typhimurium mutant MM281, which lacks the Mg2+ transporter-CorA, MgtA, and MgtB, is used as a system for functional complementation analysis of candidate Mg2+-transporter genes. MM281 competent cells were transformed with empty pTrc99A vector, AtMGT10-pTrc99A or OsHKT2;4-pTrc99A plasmid by electroporation. Cells were plated onto LB medium containing 10 mM Mg2+ and indicated antibiotics (34 μg⋅mL-1 chloramphenicol and 100 μg⋅mL-1 ampicillin), and incubated at 37°C overnight. The transformants were confirmed by PCR amplification and individual positive ones were grown in liquid LB medium containing 10 mM Mg2+ and antibiotics as indicated above. Fifty micrometer IPTG was applied for the induction of protein expression. The liquid cultures were adjusted to OD600 = 1.0, diluted in a 10-fold series, and spotted 3 μL onto N-minimal medium supplemented with different concentrations of MgSO4 and the antibiotics. Growth of different strains was pictured after incubation at 37°C for 2 days. The growth rate of the three strains in liquid medium was also monitored as previously described . Cells were then adjusted to a final OD600 of 0.001–0.002. The growth of the cultures was monitored and was plotted as a function of growth time.The escribed . After gOsHKT2;4 cDNA fragment was amplified using the primers OsHKT2;4-FC and OsHKT2;4-RC and ligated to the pTrc99A vector. For overexpressing OsHKT2;4 in wild type and the atmgt6 mutant, the genomic fragment  was amplified using primer pair OsHKT2;4-OE-F and OsHKT2;4-OE-R and cloned into pCAMBIA1300 vector. This construct was introduced into Agrobacterium tumefaciens strain GV3101 by electroporation and was selected on 1/2 MS medium containing kanamycin. The selected positive transformant was used to transform developing floral tissues of 4-week-old atmgt6 plants using the flora dip method  was used as the internal reference. Primers used are listed in the Supplementary Table Total RNA was extracted from rosette leaves using the TRizol Reagent (Invitrogen), and the first-strand cDNA was synthesized by M-MLV Reverse Transcriptase (Promega) following the manufacturer’s instructions. The semi-quantitative RT-PCR analysis of gene expression using cDNA of Col-0, tin2 AT3G8780 was in vitro transcription kit (Ambion) according to the manufacturer’s recommendations and stored at -80°C. Stage V to VI X. laevis oocytes were harvested, defolliculated, and cultured in ND96 solution containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 25 μg⋅mL-1 gentamicin, pH 7.4 adjusted with 5 mM HEPES/NaOH. Approximately 50 ng of cRNA, in a total volume of 23 nL, was injected into each X. laevis oocyte. Oocytes of 2 days after injection were used for two-electrode voltage-clamp analysis. The perfusion solution was used as described previously (I-V) curves plot current values at the end of each voltage-clamp episode .cRNA was synthesized from 1 μg linearized DNA template using a mMessage mMachine eviously  with som2+. After 2-day exposure, both the roots and shoots were harvested and sampled for analysis. The dry weight (DW) of the samples was measured after drying for 48 h at 60°C. Subsequently, the samples were digested in 0.5 ml of 70% HNO3 at 100°C for 30 min on a digester . Ion concentration was measured by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) (PerkinElmer NexION 300).Two-week-old hydroponically grown plants were exposed to solution containing different concentrations of Mg2+ transport, a cDNA fragment containing the complete open reading frame with 1530 bases encoded a protein of 509 residues was cloned and expressed in S. typhimurium mutant strain MM281. MM281 is incapable of loading Mg2+ into cellular compartment, as it lacks three functional Mg2+ transporters CorA, MgtA, and MgtB, and its growth is retarded or arrested when the culture medium contains less than 10 mM Mg2+ , also named as AtMRS2-11, as the positive control in the complementation assay of MM281 due to its high affinity in Mg2+ transport in MM281 system , and still grew but to a less extent in the media containing 500 and 100 μM Mg2+, while the control did not grow at these conditions , suggesting that OsHKT2;4 renders the mutant strains more tolerant to Mg2+ deficiency by enhancing the Mg2+ transport activity. However, OsHKT2;4 was less effective to restore the growth of mutant strains compared with AtMGT10 in the media containing insufficient Mg2+. For example, AtMGT10 rescued MM281 growth in medium containing 10 μM Mg2+ , as shown before . These results indicated that although OsHKT2;4 had the Mg2+ transport activity similar to AtMGT10, it might have the kinetic property with lower affinity to Mg2+ in heterologous MM281 system.We used 1 system . As shown before , while OFigure 1A), the bacteria were cultured in the liquid media containing 0.1, 0.5, 1, or 10 mM Mg2+, and their growth curves were established within 24 h after cultured. As shown in Figure 1B, the strain expressing AtMGT10 grew the most rapidly at these Mg2+ concentrations, supporting that AtMGT10 is a high-affinity Mg2+ transporter. In addition, MM281 expressing OsHKT2;4 displayed faster growth than those with empty vector pTrc99A under the conditions in which the Mg2+ concentration was 1 or 10 mM. By contrast, in the presence of 0.1 or 0.5 mM Mg2+, the strains expressing OsHKT2;4 displayed the similar rate of growth to the strains expressing empty vector pTrc99A. These results were consistent with the ones observed on the agar plates, and demonstrated that OsHKT2;4 might mediate low-affinity Mg2+ uptake in vivo.To further verify the complementation of OsHKT2;4 for the growth of MM281 tested in the agar plates . In contrast, OsHKT2;4-expressing oocytes generated the larger currents in the solutions containing 1.2, 6, and 20 mM Mg2+ . The current-voltage relationship displayed the currents from OsHKT2;4-expressing oocytes perfused with 6 or 20 mM Mg2+ were significantly larger than those from OsHKT2;4-expressing oocytes perfused with 0.3 or 1.2 mM Mg2+. It was noteworthy that the currents from OsHKT2;4-expressing oocytes perfused with 0.3 mM Mg2+ were similar to those with 1.2 mM Mg2+, implying they might not be Mg2+ sensitive under low-Mg2+ conditions .To further assess the transporting properties of OsHKT2;4 under different Mg2+. The currents generated from the oocytes expressing OsHKT2;4 perfused with 0.3 or 1.2 mM Mg2+ displayed the similar levels of the amplitudes and reversal potentials, even were similar to those without Mg2+ . These Mg2+ insensitive currents were larger than the currents from the oocytes injected with water , and thus they may result from other ions, such as Na+ or K+ currents generated by OsHKT2;4, as suggested by the previous studies . Thus, these results further supported the hypothesis that OsHKT2;4 exhibits permeability for Mg2+ only under the conditions containing high-Mg2+ concentrations.To test this possibility, we compared the amplitude and reversal potential of the currents generated from the oocytes perfused with 0, 0.3, 1.2, 6, or 20 mM Mg studies . By cont2+ deficiency-induced Mg2+ transporter, mediates directly Mg2+ uptake in roots and is required for plant adaptation to low-Mg2+ environment , in which T-DNA was inserted into the third exon of AtMGT6 gene . Transcript of AtMGT6 in the line SALK_203866 was not detected by RT-PCR , indicating that the T-DNA insertion line was a knockout allele, and referred to as atmgt6 line hereafter. We then expressed the coding region of OsHKT2;4 into atmgt6 line driven by its native promoter . The transformants were screened by hygromycin, and were further analyzed for the expression levels of OsHKT2;4 by RT-PCR. We selected two of them as the representative transgenic lines due to their relatively high OsHKT2;4 expression levels, and referred to as OE29 and OE34, respectively, to perform subsequent experiments .We have shown previously that AtMGT6, a Mgironment . An indenditions . Consideatmgt6, and transgenic lines OE29 and OE34 in Mg2+-depleted medium supplemented with various contents of Mg2+ as indicated in Figure 3D. The mutant atmgt6 exhibited growth defects in the medium containing 0, 0.01, 0.1, or 0.25 mM Mg2+, and had lower fresh weight and shorter roots than those of the Col-0 plants, while the growth retardation could be rescued in the Mg2+-sufficient medium (2 mM Mg2+) , consistent with the idea that AtMGT6 confers low-Mg2+ tolerance for Arabidopsis . These results suggested that OsHKT2;4 might not function in low-Mg2+ conditions in planta.We examined the growth of the Col-0, bidopsis . Howeverin vitro, OsHKT2;4 exhibiting Mg2+ transport activity in both heterologous MM281 system and X. laevis oocytes happened only at high external Mg2+ concentrations . Therefore, we presumed that OsHKT2;4 might mediate Mg2+ transport when plants were cultivated under the Mg2+ abundant conditions, though it was unable to function in low-Mg2+ conditions in planta . To conduct assessment of the sensitivity to high-Mg2+ condition of OsHKT2;4, we used 1/6 MS medium supplemented with several concentrations of Mg2+  for growth assays. After growing on 1/6 MS for 2 weeks, both the root length and fresh weight of transgenic lines OE29 and OE34 were comparable to those of atmgt6 under the normal condition. However, addition of 2 mM Mg2+ resulted in growth arrest of OE29 and OE34 compared with atmgt6. Further increases of extra Mg2+ (up to 10 mM) demonstrated a consistent dosage-dependent inhibitory manner . Quantitative analysis of root length  and fresh weight  indicated that, compared with Col-0 and atmgt6, the aerial parts of OE29 and OE34 exhibited a more severe growth retardation in Mg2+-abundant conditions, while their root length were not altered. Taken together, these results demonstrated that OsHKT2;4 results in Mg toxicity on aerial tissues in high-Mg2+ conditions in Arabidopsis, supporting the idea of low-affinity Mg2+ uptake of OsHKT2;4.As shown in the experiments 2+ toxic phenotype is a consequence of the ectopic expression of OsHKT2;4 in OE lines, we conducted an RT-PCR analysis to verify the expression of OsHKT2;4 under a high-Mg2+ condition supplemented with 6 mM Mg2+. As demonstrated in Supplementary Figure OsHKT2;4 in the shoots was not significantly induced by 6 mM Mg2+, and even decreased after 24 h′ treatment with 6 mM Mg2+. However, expression of OsHKT2;4 in the roots was dramatically induced after 4 h′ treatment with 6 mM Mg2+, and became even stronger after 24 h of this treatment , and 6 mM Mg2+ for another 2 days before the roots and shoots were harvested, respectively, for analysis. As shown in Figure 5A, Mg2+ content was consistently higher in the OE lines than atmgt6 in shoot tissues when plants grown in all Mg2+ regimes tested . In analysis of Mg2+ content of root tissues among different plants, although they exhibited similar and incremental Mg2+ content in the Mg2+-deficient and normal medium, the OE lines contained ∼30% less Mg2+ compared with the atmgt6 plants in 6 mM Mg2+ condition . These results indicated an altered Mg2+ distribution ratio in shoot and root. We thus analyzed the Mg2+ partitioning between shoots and roots in atmgt6 and OE lines, and noticed that as the Mg2+ level elevated, more Mg2+ sequestered in root tissues than in low-Mg2+ condition. In medium containing 6 mM Mg2+, the shoots of atmgt6 accumulated ∼37%, while the OE lines accumulated over ∼50% of the total Mg2+ enclosed in plants , further confirming the critical role of OsHKT2;4 in the Mg2+ allocation between shoots and roots.To probe the reason responsible for the increased sensitivity to high external Mg2+ and Mg2+ compete for the same sites of substrates  on Mg2+-abundant 1/6 MS medium supplemented with different concentrations of excess Ca2+.Due to the similar physical properties, Cabstrates , and theand Mg2+ . To examatmgt6. As the concentration of external Mg2+ increased, OE29 and OE34 started to show a more severe growth arrest than atmgt6 . However, increasing Ca2+ improved the plant growth  of all genotypes, and the improvement was more obvious in the OE29 and OE34 lines , however, when 1 mM Ca2+ was added to this basal medium, the fresh weight of OE lines was restored and reached to ∼80% to that in atmgt6. Moreover, when 3 mM Ca2+ were added to the basal medium, both root length and fresh weight of OE29 and OE34 were recovered to almost an identical level with that of atmgt6 , similar phenotype was observed in atmgt6 and OE lines, suggesting that OsHKT2;4 is not responsible for Ca2+ deficiency. On the contrary, as the increasing content of extra Mg2+  was added to the basal medium, differences of fresh weight between atmgt6 and OE lines started to occur , and OsHKT2;4 is the member of class II HKTs with the conserved Gly residues at the four P-loop filter positions  was also found to result in robust Mg2+ permeability of the oocytes, although to a lesser degree . Similarly, the expression of OsHKT2;4 rescued growth defects of MM281 bacteria cells that are deficient in Mg2+ uptake in the presence of relatively high-Mg2+ concentration, but the rescuing effect was much less than MGT10, the high-affinity Mg2+ transporter . Complementary to the observations in the oocytes and bacteria, the phenotype relating to Mg2+ stress in transgenic OsHKT2;4-overexpressed atmgt6 lines happened at the Mg2+-abundant , but not at Mg2+-deficient conditions . Taken together, our findings showed that OsHKT2;4 has a distinct low-affinity Mg2+ transportation, and confirm Mg2+ permeability of OsHKT2;4 as reported  was used.Rice contains up to nine ositions . OsHKT2;r levels . Triticur degree . OsHKT2;to 50 mM . The prereported . It is wnt study . Due to cessions , the difcessions  and the 2+ is taken up from the soil by the plant root system, which is likely to be mediated by AtMGT6/MRS2-4. AtMGT6/MRS2-4 is located in the plasma membrane or the endoplasmic reticulum and highly expressed in the root epidermal cells, and its disruption resulted in growth retardation of Arabidopsis under the low-Mg2+ condition , fitting the idea of its low-affinity Mg2+ transportation. After satisfying the needs of the roots, the rest of the Mg2+ will be transported to the shoot through the process involving AtMGT6/MRS2-4 activity , indicating the expression level of OsHKT2;4 was high enough to drive transportation of Mg2+ from root to shoot. However, once the expression level of OsHKT2;4 was further enhanced under high-Mg2+ conditions , which is also a supportive evidence for the antagonistic interaction between Ca2+ and Mg2+in planta. However, Mg2+ currents through OsHKT2;4 in oocytes were not inhibited and their reversal potentials were not significantly shifted in the presence of 1.8 mM Ca2+ in the perfusion solution . Rice  is predicted to have nine AtMGT orthologs based on the BLAST search (1 (Supplementary Figure In our previous study, we found that OsHKT2;4 had the diverse expression pattern in rice plants, including leaves, stems and primary/lateral roots, and was highly expressed at xylem and phloem of epidermis . Howevere plants , and conand Ca2+ . The absT search , and Os1CZ, BZ, HL, JG, and WL designed the study. CZ, HL, BZ, and WL performed experiments. CZ, HL, JW, BZ, WW, JG, and WL analyzed and interpreted the data. CZ, HL, BZ, and WL wrote the manuscript. CZ, BZ, HL, SL, JG, and WL revised the manuscript critically. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.