This article was migrated. The article was marked as recommended.Introduction: Advance Care Planning (ACP) is a part of comprehensive palliative care but there are challeges for its implementation. In Thailand, undergraduate medical curriculum also has not implemented palliative care and ACP as a core teaching topic for the medical students yet. Life Unlocking Card Game is an end-of-life conversation card game that aims to bridge this gap.Objective: To assess second year medical students’ attitude of death by using Life Unlocking Card Game and its effectiveness to teach about death and dying.Methods: Non-equivalent quasi-experimental design with convenience sampling method. All (48) of second year medical students participated in an end-of-life conversation game . After that, each group formed an after-game focus group interview. Seven students also joined individual semi-structured interviews. We used content analysis approach along with investigator triangulation and methodological triangulation methods for the qualitative analysis.Results: Participants (n = 48) were second-year preclinical medical students. 26 of them were male (54.2%), 22 were female (45.8%), with the mean age of 20 years (SD 0.6). Five primary themes regarding the card game emerged: 1) Safe space to disclose personal issues 2) Seeing the world through different views 3) Surprise elements 4) Death distant closure 5) Changed behaviour.Conclusions: Life Unlocking Card Game proves to be an effective tool to teach death and dying issues and also ACP in second year medical students. Further study in clinical year students or postgraduate students are recommended. The purpose of ACP is to reach a mutual agreement between the health care team, patients, and family if the patient become incapacitated (Advance Care Planning (ACP) is one of the essential elements to deliver comprehensive palliative care, an approach that aims to improve patient and family’s quality of life in incurable diseases WHO | WHO Definition of Palliative Care [Internet]. Who.int. 2016 [cited 30 November 2016]. Available from:citated . Apart fcitated . Howevercitated .http://www.isaanlawyers.com/living-will-thailand/. This Act enables any Thai citizen to write a living will and clarify their needs. However, living wills are still not widely recognized. The prevalence of living wills uptake in Thailand is still unknown. Social context that perceived death as a sensitive subject is another challenge that make it harder to talk about ACP  consisted of 45 questions grouped into three subcategories; 1) Emotional respite, 2) Death and Dying Issues and 3) ACP. The card game was conceived with the objective to use the gamification element to talk about hard and sensitive topics such as ACP and death and dying issue เกมไพ่ ไขชีวิต (no date) เกมไพ่ ไขชีวิต | โครงการเผชิญความตายอย่างสงบ. Available at:http://boonbudnet.com/sunset/node/403 . It aims to raise the awareness of death and dying issues and the importance of ACP. Each session consisted of 4-10 players with one facilitator to guide the group  What are your worries about playing this game? 2) What is your expectation from playing this game? After that, players then shuffled the deck, drew the top card, and read the question out loud. Once a question card was drawn, each of them would think or write their answers on a piece of paper. They then took turns explaining and discussing their answers within the group. Participants could give “like” card to players that made insightful comments or to show their support and gratitude to that player. After everybody took turns in answering the question, another player drew a card, and the cycle went on. The session lasted for 90 to 120 minutes. The game concluded with two final questions; 1) What did you learn from this game? 2) What would you do after this game?Non-equivalent quasi-experimental design with convenience sampling method. We used O’Brien’s Standard for Reporting Qualitative Research as a framework to report our findings  their experience and attitude towards the card game 2) its practicability in understanding the concepts of death and ACP 3) their opinion of this teaching method. Seven students also joined the semi-structured interviews afterwards. All interviews were audio recorded and transcribed verbatim. TP wrote field notes and audit trails immediately after each interview session. Data were gathered until we reached saturation, the point where no new concepts emerged. Demographic data were reported as frequencies and percentages for categorical variables with means and standard deviations (SDs) for continuous variables. For the qualitative analysis, we used investigator triangulation and methodological triangulation methods. Each researcher created individual codes and discussed any discrepancies before making the final codes using content analysis approach. Data from participants also came from multiple sources; focus group interview, semi-structured interview, students’ feedback, and their last wish card. Atlas.Ti 8.0 was used for the coding process.After three months, TP made follow-up phone calls and asked if the students changed their behaviour according to what they wrote in their last wish card and assessed students’ behaviour using Prochaska’s transtheoretical model (TTM). TTM is a model presumed that behavioural change can occur in 5 stages of change overtime and that proper intervention is needed at each stage to make participants change their behaviour . For thiWe performed eight games in total. The games lasted for 98-120 minutes. After-game focus group interviews lasted 19-35 minutes while semi-structured interviews took 23-38 minutes. All participants (48) are second year medical students from Walailak University School of Medicine. 26 (54.17%) of them were male and 22 (45.83%) were female with the average age of 20 years old. Twenty students (41.7%) recently lost someone close to them within one-year period. Six of them (30%) stated that their loved one’s death had changed their perspective of death .As for the students’ bahavioural change using TTM model assessment, before the card game session, 42 (87.5%) of the students were in pre-contemplation stage while six (12.5%) of them were in their contemplation stage. After the game, 15 (31.25%) of the students changed their behaviour to contemplation stage while 33 (68.75%) shifted to preparation stage. After three months, follow-up phone calls revealed that none of them reverted to precontemplation stage while 12 (35%) and 32 (66.7%) of them were in contemplation and preparation stage respectively. Four (8.3%) of them went into action stage .For the content analysis relating to the card game, five primary themes with eleven subthemes emerged .Theme1: Death is a distant phenomenonBefore the game, majority of the students (42) did not think of their death at all. They viewed the concept of death as a vague and distant event that could not relate to them in anyway.Magic of being young and healthyThey also expressed that it is due to their youth that make it further away from their thought.“I am still young and healthy. It [death] won’t come to me soon.” Student 4Not my cup of tea talkOthers (8) stated that talking or even thinking about it make them feel uncomfortable and rather avoiding the subject.“It just feels weird talking or thinking about it while we can talk about something else.” Student 2/ Focus group 2Theme 2: Fear of deathFear of the unknownAnother thought that make students feel uncomfortable talking about death is that they never know what they would meet.“I fear it [death]. Because we never know what could happen after that. It could be heaven or hell up to your religious belief or nothing at all. I guess “not knowing” is what terrifies me, not the death itself.” Student 6Unexpected nature of death12 of them also mentioned the fear of talking about death due to its uncertainty nature.“It could be any moment [death]. Tomorrow, next year, that’s why it is so terrifying.” Student 4/Focus group 8These findings coincide with students’ TTM that 87.5% of them were in pre-contemplation stage and had no intention of thinking nor preparing about ACP at all. However, after the game, their attitudes changed drastically.Theme 3: Safe space to disclose personal issuesAll students (48) praised the card game for making a safe space for them. They could express their inner thought freely and safely without fear of being judged thanks to the facilitators and their classmates.Friendly facilitators“I feel safe to talk about anything during the game. Because she [facilitator] teaches me and we are very close.” “Me too. She is friendly and down-to-earth so we can talk it out without fear of being judged.” Focus group 7Bonding with classmates“I feel like I know my friend better. She is always a cheerful girl but I never knew that she has been through a lot at home.” Student 2Theme 4: Seeing the world through different viewsConstructive views35 students mentioned about how this session make them see certain issues differently such as death.“There can be several answers to a question. For example, when we talked about our place of death. I want to die at home but my friend did not want to. He said that those who were left behind would be devastated. And both of us aren’t wrong.” Student 1/Focus group 2Shared learning experiencesThey also liked the shared learning element of the card game, especially experiences from facilitators.“Some questions we could only see them from only one angle” “A kid’s view.” “Yeah. And he [facilitator] opened our eyes.” “Right? Like when we talked about our last meal. I never knew that dying people could not eat much.” Focus group 3Theme 5: Surprise elementsApart from that, they were also fond of the surprise elements in the game. It helped to stimulate situations they had never thought of before.Surprised questions“Most questions are what I had never thought of before. Like, what would you do if you were to die tomorrow? I never thought of that and there are lots of things to prepare.” Student 7/Focus group 1Simulated situation“I prefer this card game over regular lectures. I can be totally into it; exchanging ideas with friends as to what would I do in certain situation unlike lectures class where we can only learn the principles.” Student 2/ Focus group 6Theme 6: Death distant closure48 of them viewed death as closer to them than they had thought, reflecting the change in TTM into contemplation and preparation stages in all students.Death is closer than I thought“I can see now that death is closer to us than I thought. I thought that we will die at 80s or 90s but after this game, I know that that is not always the case. You could pass away just mere moments later. *Poof*. Just like that.” Student 6Impact of your death to othersThey also realised that one’s death brought larger impact to people, especially their family.“Death involves so many people. For example, if I die, my parents would be heartbroken. And who’s going to take care of my cats too? My parents are one thing but my cats depend on me solely.” Student 5/ Focus group 2Theme 7: Changed behaviourWhile only 15 of them changed into preparation stage of change after the game, 33 of them changed to contemplation stage, rethought about death, and also considering changing their behaviour.Mindfulness and self-reflectionThough they have not formed any concrete plan yet, 15 students considered the need to prepare for one’s death.“I feel like I need to prepare for it [death]. Like I need to do more about it.” “Yeah, me too. ‘cos we never know.” Focus group 1“I always drive very fast, passing through the red light and stuff but I will drive slowly and more carefully now. Because we never know what could happened.” Students 3/ Focus group 1Getting more connected with loved onesMajority of the students (31) also expressed the need to be more connected to their loved ones.“[last wish card] I will be a better daughter and talk with my grandma more often.”Advance Care Planning (ACP)4 students stated their plan to make ACP with their family afterward.“[last wish card] I will make a will, save money for my cats, and ask my parents about questions in the cards.”ACP is one of the key elements of comprehensive palliative care. However, previous studies showed lack of knowledge and confidence to initiate one among doctors  students reflected on theme 3 that the card game created an engaging and safe environment for a serious topic such as death and dying. It also made them see the different perspective from other players, as shown in theme 4. Apart from that, the majority (45) of them preferred this teaching method over the conventional, didactic lecture. Results from this study showed that the Life Unlocking Card game is an effective tool to initiate an active learning method for serious topics such as death and dying due to its safe environment for participants and surprising elements. Still, this pilot study only demonstrates second-year medical students’ attitude of death and its effectiveness to teach about death and dying. Further study is needed to determine whether students, who have early exposure to end of life experiences, have a better understanding of end-of-life care or not  because they are of the same age group so their views of death are not much differed. Hence, the need for facilitators to give them a broader perspective from adults who have been through losses.The Life Unlocking Card Game proves to be a useful tool to teach second-year medical students in death and dying and ACP issues. We found that students have a better understanding of the concept of death, changed their behaviour, and have positive experiences during the game. Further study in clinical year students or postgraduate students is recommended.•It is possible to teach sensitive issues such as death and dying in pre-clinical year medical students.•Key elements are using a safe environment, a proper-tool, and a good facilitation.Dr Tharin Phenwan is a Family Medicine lecturer at Walailak University School of Medicine. His research focuses on palliative care, qualitative research, interprofessional, and medical students assessment.Tanida Apichanakulchai is one of the leading members of Buddhika Foundation, a non-profit organisation that aims to raise death awareness and also the concept of peaceful death. Her work focuses on social engagement, volunteerisms, and mindfulness.Ekkapop Sittiwantana is also one of the members of Buddhika Foundation, a non-profit organisation that aims to raise death awareness and also the concept of peaceful death. His works focus on building social awareness. This review has been migrated. The reviewer awarded 3 stars out of 5 I think the paper highlights the use of ‘gamification’ to explore student attitudes and experiences relating to death in a non-threatening and accessible way. Some of the focus group themes around uncertainty and the appreciation of no right and wrong personal attitudes to death are really insightful. The use of similarly styled ‘games’ as learning and discussion tools can be transferable regarding topic and perhaps to promote interdisciplinary discussion. I found this paper really useful and accessible, and the authors appropriately acknowledge the limitations of their sampling/population. I am definitely interested in exploring these ideas in future curriculum development.Reviewer Expertise:NANo decision status is available This review has been migrated. The reviewer awarded 4 stars out of 5 I also enjoyed this. I presume that the cards produce a randomness to the process which enables people to talk more openly. It would be interesting to know if those who had been through this process found it easier to talk to patients about death when they were further on in the course.Reviewer Expertise:NANo decision status is available This review has been migrated. The reviewer awarded 3 stars out of 5 A really interesting paper to read that uses a relatively simple card game to bring about active learning in the areas of personal values / beliefs about death and dying. I though this was pitched at an appropriate level for relatively new students, but I can easily see its potential for the more advanced clinical students. I feel that the paper can be improved by:- giving an example of the writings / questions on the cards- perhaps exploring how the students might perceive the effect of this new learning on future patients  The authors have rightly explored the limitations of this paper but should be encouraged to explore this research further- on a greater number of students and taking it through to a clinical perspectiveA paper of interest to anyone involved with curriculum planning.Reviewer Expertise:NANo decision status is available


Cells in every epithelium can be roughly divided in three compartments: stem cell (SC) compartment, transient amplifying cell (TA) compartment and terminally differentiated (TD) compartment. Maturation of stem cells is characterized by epithelial stromal interaction and sequential maturational movement of stem cell’s progeny through those compartments. In this work we hypothesize that providing an artificial stroma, which murine breast cancer metastatic cells can infiltrate, will induce their differentiation.6 isogenic 4T1 breast cancer cells labeled with GFP. After 20 days primary tumors were removed, and artificial ε-PCL implants were implanted on the contralateral side. After 10 more days mice were sacrificed and implants along with lung tissue were harvested. Mice were divided in four groups: tumor removal with sham implantation surgery (n = 5), tumor removal with ε-PCL implant (n = 5), tumor removal with VEGF enriched ε-PCL implant (n = 7) and mice without tumor with VEGF enriched ε-PCL implant (n = 3). Differentiational status of GFP + cells was assessed by Ki67 and activated caspase 3 expression, thus dividing the population in SC like cells (Ki67+/dim aCasp3−), TA like cells (Ki67+/dim aCasp3+/dim) and TD like cells (Ki67− aCasp3+/dim) on flow cytometry.BALB/c female mice were injected with 10.Lung metastatic load was reduced by 33% in mice with simple ε-PCL implant when compared to tumor bearing group with no implant. Mice with VEGF enriched implants had 108% increase in lung metastatic load in comparison to tumor bearing mice with no implants. Likewise, amount of GFP + cells was higher in simple ε-PCL implant in comparison to VEGF enriched implants. Differentiation-wise, process of metastasizing to lungs reduces the average fraction of SC like cells when compared to primary tumor. This effect is made more uniform by both kinds of ε-PCL implants. The opposite process is mirrored in TA like cells compartment when it comes to averages. Effects of both types of implants on TD like cells were negligible. Furthermore, if gene expression signatures that mimic tissue compartments are analyzed in human breast cancer metastases, it turns out that TA signature is associated with increased survival probabilityε-PCL implants without VEGF can reduce metastatic loads in lungs, after primary tumor removal. Both types of implants cause lung metastasis differentiation by shifting cancer cells from SC to TA compartment, leaving the TD compartment unaffected.The online version contains supplementary material available at 10.1186/s12885-023-10813-6. Every epithelial tissue is in a constant process of turnover , 2. RegaOver the last 70 years, a body of evidence that interruption of stromal – epithelial interactions can lead to the development of carcinoma has been accumulated and united to a certain extent under the theoretical framework know as Tissue Organization Field Theory (TOFT) of cancer ; a very Mathematical modeling of stem cell differentiation by maturational movement trough epithelial compartments points out that differentiating tumor stem cells might be more efficacious than killing them . Further6 isogenic 4T1 murine breast cancer cells labeled with green fluorescent protein  in right mammary line, under isoflurane anesthesia. Cells were grown in RPMI media with 10% FBS without antibiotics in atmosphere supplemented to 5% CO2 at 37 °C. After one or two passages they were considered to be fit for injection into mice. After 20 days, mice were again anesthetized with isoflurane and primary tumors were excised en-block. Furthermore, artificial ε-PCL implants (3D Biotek Polycaprolactone (PCL) scaffold inserts, fiber diameter ≈300 μm, ≈300 μm spacing, Merk KGaA, Darmstadt, Germany) were implanted subcutaneously on the contralateral side. One eight (1/8) of the original scaffold volume was implanted. After 10 more days mice were sacrificed and implants along with the lung tissue were harvested for flow cytometry. Based on intervention, mice were divided in four groups: tumor excision with sham implantation surgery , tumor excision with ε-PCL implant (n = 5), tumor excision with vascular endothelial growth factor (VEGF)—enriched ε-PCL implant (n = 7) and mice without tumor with VEGF—enriched ε-PCL implant (n = 3)  from E. coli  dissolved in PBS and dried in laminar hood for 40 min. After this, the scaffolds were immersed in 1 ml of PBS at 4 °C for overnight before implantation to remove freely diffusible VEGF and to reduce the possible in vivo burst release of VEGF.Lungs and ε-PCL implants were explanted on day 30 of experiment during the necropsy, whereas primary tumor was removed on day 20 by the surgical en block excision. After that, the tissues were disintegrated mechanically by mincing and digested enzymatically for 1 h on 37 °C in 10 mg/mL Collagenase type I  solution in RPMI, 5 ml of the solution was applied per 100 mg of tissue. Single cells solutions of tissues were fixed in 0.1% paraformaldehyde solution for 15 min followed by permeabilization in 0.1% solution of TritonX in PBS, also for 15 min. Next, cells were stained by two antibody panels. First one was composed of anti-GFP , ant- Ki-67  and anti-activated caspase 3  antibodies. Second panel included PI (BD Biosciences) and anti GFP antibody (Santa Cruz Biotechnology). Samples were analyzed by Accuri C6 (BD Biosciences) flow cytometer. Gating controls were prepared with Fluorescence Minus One (FMO) stains of tumor cells and appropriate biological controls . Three methods were used for drawing gates (i.e. discriminating positive and negative events): for FMO controls based gates 3% background method was used; for comparison of an experimental sample with biological control, percentile by percentile histogram subtraction method was used if cumulative probability curve of sample was uniformly shifted to the higher florescence level in comparison to biological control, if the latter was not the case, then the maximum difference method was used. The described gating methods were used either in one or two dimensions. Detail description of these methods can be found in paper by W. R. Overton  and textMKI-67 and Caspase 3 genes in certain gene ontology categories was done by PANTHER software with Fisher’s Exact Test without correction for multiple comparisons, on Gene Ontology website [Data for transcriptomic analysis were obtained from Gene Expression Omnibus (GEO) . They we website –30.2 or η2, evidence ratio (ER) based on difference in Akaiake Information criterion (ΔAIC) [p values were used. Standard errors  were used as an uncertainty measure of the estimates, they were estimated by maximum likelihood or parametric (percentile) bootstrap method. When it comes to notation convention used throughout the paper, ER < 1 and ΔAIC < 0 favor the null model, a number following ± sign is the standard error, not the range nor any other descriptive statistic used to describe the spread of data . P values were calculated by extra sums of squares F test if not stated otherwise. They are interpreted according to ASA statement on p values [Depending on distribution, continuous data are presented as either arithmetic mean or mode and range (range = max. – min.) or 0.5% trimmed range. These were estimated by fitting the normal, uniform, triangular, exponential, or power law distribution (n (ΔAIC)  and p vap values . Model dp values .2 = 71.13%, ΔAIC = 14.65, ER≈1.52·103, p = 0.0002) that can be described as follows: in relative difference terms ε-PCL implants decreased the lung metastatic load, on average, by 33 ± 6%; whereas, unexpectedly, enriching the ε-PCL implants with VEGF increased metastatic load by 108 ± 25%, when compared to control group .Implanting ε-PCL scaffolds with and without VEGF enrichment resulted in parabolic (quadratic) trend . There, also, seems to be influence of VEGF on heterogeneity of effect. Furthermore, when the relationship between metastatic loads in the lungs and ε-PCL implant without VEGF is considered, it turns out that those two are inversely proportional, i.e., increase in GFP + cells in the implants is associated with the decrease in lung metastatic load   in all phases of mammary growth and differentiation  expression  and milk lipids (SREBP) also follow the same temporal expression pattern as caspase 3, during the differentiation phases , milk mucins (Glycam 1) and glucose transporter (GLUT 1), which is involved in lactose synthesis.  like, those that were Ki67+/dim aCasp3+/dim were considered to be transient amplifying cell (TA) like. The nonproliferating subpopulation of GFP + cells  was further divided into terminally differentiated or apoptotic like (TD) cells (Ki67− aCasp3+/dim) and quiescent stem cell like (qSC) cells (Ki67− Casp3−).To study differentiation of tumor cells, based on cell turnover patterns in highly proliferative tissues such as hematopoietic tissue , 43 and − Casp3+/dim) in primary tumors was 5.64 ± 0.85%, whilest in metastases from mice that were only treated with primary tumor excision average was 0 ± 0.05%. Furthermore, mice treated with both kinds of ε-PCL implants also had an average of 0 ± ≈0%. This can be described as decreasing exponential trend  with the average levels of qSC fraction being the same, i.e., 0% in all metastases regardless of treatment , with the range of observed data being the greatest in primary tumors (32.4 ± 3.35%) and then decreasing in mice that had surgery only (11.2 ± 0.7%), with its minimum (0 ± ≈0%) in group that was treated with implantation of simple ε-PCL implant and then raising again in group treated with ε-PCL implant enriched with VEGF (17.54 ± 1.07%)  follows the same exponential trend as quiescent cells in terms of average response  , with the range being the greatest in primary tumors (97.8 ± 0.01%) and surgery only group (80.58 ± 4.4%), decreasing to 20.7 ± 1.7% in mice treated with surgery and ε-PCL implant. Furthermore, the most homogeneous, i.e., the most uniform, effects were observed in mice treated with surgery and VEGF enriched implants; the range was 14.35 ± 0.54%  cell like fraction showed relationships that are the mirror image of those observed in pSC fraction . Influence of the factors on heterogeneity of response is similar to the one observed in pSC like cells.The TA .Terminally differentiated or apoptotic (TD) like cells had the highest average fraction in primary tumors (6.07 ± 2.5%), followed by all other metastases regardless of treatment (≈0 ± 0%)  and Cell population proliferation for MKI-67. Based on GO terms two lists of genes associated with those terms in The Molecular Signatures Database were selected and their expression was analyzed by single sample GSEA. Based on ssGSEA score, the samples were divided by quartiles (Q1 – 4) of expression with the Q1 having the lowest and Q4 the highest expression. To mimic tissue compartments with genetic signatures samples were divided in groups that parallel those used on mice  . Patients whose metastases expressed TA or TD like signatures exhibited survival plateaus that were well above 0%. More detailly, patients with TA signature plateaued at 22.2 ± 4.7% of survival probability and patients with TD signature had a survival probability tail at 37.4 ± 4.5%. If survival plateaus from all gene signatures are ordered in a manner that fallows usual tissue differentiation process (i.e. starting with qSC to pSC to TA and ending with TD) a rising exponential trend can be noticed .Stratifying patients according to gene signatures that mimic tissue compartment was found to be more accurate and parsimonious model then the one which ignores such information  Fig. . FurtherWe speculated that purpose of metastatic process might be providing an additional stroma which might be able to differentiate or induce quiescence in incoming metastatic cells. Additionally, we provided an artificial stroma in form of an implant in hope that this will augment differentiation or quiescence processes. Mice that went through a primary tumor removal and simultaneous implantation of ε-PCL implants without VEGF showed a relative decrease of lung metastatic load by 30% when compared to mice that had primary tumor removal only. The decrease in lung metastatic load was associated with an increase in proportion of tumor cells in the implant.The ability of ε-PCL implants to attract breast cancer cells and decrease metastatic load to other organs was previously demonstrated by Azarin et al. . FurtherIf the cancer lethality is considered from general systems theory point of view a following conjecture can be made: since the cause of tumor lethality is either an acute local complication  or chronic systemic complication  , physiolWe analyzed differentiation by quantifying proportions of different tissue compartments, through which cells pass during the maturational process. aCasp3, along with the Ki—67, was used as differentiation marker. The use of activated caspase 3 for such purpose is quite unusual, but the body of evidence from previous research , 50 and When it comes to terminally differentiated or apoptotic like (TD) cells we can notice that both metastatic process and implants did not increase their proportions, therefore the effects of these processes on differentiation are only partial when it comes to differentiation, i.e., they only manage to differentiate cells to TA stage. Potential consequences of partial differentiation are twofold. Firstly, TA cells are also proliferating cells and therefore they increase the tumor load of an organism and eventually can contribute to lethal outcome. Secondly, due to the increased levels of aCasp3 this population should be more chemo or hormonal sensitive and slow growing then the stem cell populations and therefore in a setting of chemo or hormonal therapy, such as in metastatic breast cancer, might be the cause of prolonged survival.If quiescent stem cell like (qSC) cells and VGEF enriched implants are considered the data are more puzzling. These cells were the most abundant in primary tumors, whereas mice treated with non-enriched implants had levels that were below detection threshold on flow cytometry. A group of mice treated with tumor excision only and the group that was treated with VEGF enriched implants, were not uniform when it comes to qSC cells. Roughly speaking, maximal proportions of qSC cells in these group were half the size of the maximal proportion of qSC cells in primary tumors. qSC tissue compartment signature was associated with the worst survival in metastatic breast patient’s cohort. This is not surprising since the mainstay therapy of patients with metastatic breast cancer is chemo and/or hormonal therapy . QuiesceOn the other hand, long term survival is sometimes associated with quiescent or dormant tumor metastases . This obThe purpose of implanting VEGF-enriched implants was to establish a link between circulatory system and the implant sooner, so that more metastases might be attracted in the implant and thus an effect of tumor load reduction in lungs maximized. However, it turned out that the effect was the opposite, i.e., mice with VEGF-enriched implants had the greatest metastatic load in lungs. One reason for this might be a leak of loosely bound VEGF from the implant, although this is unlikely because the implants were washed in PBS for 24 h before implantation. Another reason might be a gradual release of bounded VEGF from the implant since ε-PCL is biodegradable material. Third and more complex reason might lie in a fact that ε-PCL once implanted gets infiltrated with various cells of myeloid origin, what could be more completely described as granulation or wound healing tissue . This kiWhen it comes to limitations of the study two issues come to mind. The first one is marker or markers of cell differentiation and the way how different tissue compartments were defined in flow cytometric analysis. The former has been addressed earlier in the Results and Discussion sections, regarding the latter we used simple logic that tumor cells which do not express differentiation marker (aCasp 3) were considered stem cells, that could be further divided based on proliferation marker expression. Following the same logic cells that express differentiation marker and proliferate were considered as transient amplifying cells. Finally, tumor cells that were not proliferating and were expressing aCasp3 were either considered terminally differentiated or apoptotic.The second issue is a 10 days of time lag between the primary tumor excision and the harvest of metastatic tissue in lungs. This means that, in a strict sense, the differences between these two tissues must be interpreted as effects of time, metastatic process and the implants. In alternative design we could have made an excision of primary tumor and harvest of metastatic tissue in a synchronous manner, however in this case it would be impossible to study the effect of the implants on metastases since, the implants need time to interact with metastases. The question of whether the differences between primary tumors and lung metastases in terms of differentiation can be attributed to the time and not to different biology (i.e. microenvironment) of primary and metastatic tumors, seems to be resolved in favor of microenvironment given the overwhelming evidence in literature .When it comes to VEGF enrichment of the implants, this study would definitely benefit if dose response relationship between different VEGF concentrations and effects of implants on metastases could be elucidated.2 [Finally, when thinking of the future experimentation, given what has already been said in previous subsections, a next logical step is to induce complete instead of only partial differentiation in residual metastases. Theoretically, this might establish nongrowing tissue kinetics  in leftover cancer. Off note, Fig. S2  or iL6 a2 ) after tε-PCL implants without VEGF can reduce metastatic loads in lungs. The metastatic process reduces the average fraction of pSC and qSC like cells when compared to primary tumor. This effect is made more uniform by both types of ε-PCL implants.Additional file 1.Additional file 2.Additional file 3.Additional file 4.


The superior flexibility and piezoelectric properties are attributed to the electric polarization and mechanical load transfer paths formed by the ceramic skeleton, and dielectric mismatch mitigation between ceramic fillers and elastomer matrix by the dielectric transition layer. The synergistic fusion of ultrahigh piezoelectric properties and superior flexibility in these polymer composites is expected to drive emerging applications in flexible smart electronics.Flexible piezoelectric materials capable of withstanding large deformation play key roles in flexible electronics. Ferroelectric ceramics with a high piezoelectric coefficient are inherently brittle, whereas polar polymers exhibit a low piezoelectric coefficient. Here we report a highly stretchable/compressible piezoelectric composite composed of ferroelectric ceramic skeleton, elastomer matrix and relaxor ferroelectric-based hybrid at the ceramic/matrix interface as dielectric transition layers, exhibiting a giant piezoelectric coefficient of 250 picometers per volt, high electromechanical coupling factor  Through structure design and interface modifications, this work addressed the dielectric as well as mechanical mismatch between ceramic and polymer in composites, leading to soft composites with ultrahigh piezoelectric performances comparable to piezoelectric ceramics. Piezoel−1 [−1 , where p−1 [−1 , lead ma−1 [−1  or bariu−1 [−1  have beeThe other major challenge for piezoelectric composites is the mismatching of electrical and mechanical properties between the ceramic fillers and polymer matrices, which, over the past decades, have severely hindered their synergistic fusion into stretchable piezoelectric composites with high piezoelectric properties. The large difference in dielectric constant between ferroelectric ceramic and polymer leads to a discontinuity in electric polarization at the filler/matrix interfaces and forms an electric double-layer that shields the dielectric response of ceramic fillers. The dielectric difference also leads to severe concentration of the local electric field in polymer matrix, hence substantially reducing the local electric field in the ferroelectric ceramic filler, thereby compromising the overall piezoelectric response of the composites. Analogous to the dielectric constant difference, the large difference in elastic modulus between ceramic and polymer also results in the concentration of local stress fields in the polymer matrix. Therefore, the mechanical load may not be efficiently transferred to the ceramic filler in order to induce a high piezoelectric response. Numerous strategies have been employed to establish transfer paths for both electric polarization and mechanical load in polymer matrix to provide robust composites, such as reinforcing polymer in 3 dimensions by low magnetic field , using f33*d of 250 pm V−1 which is even comparable to commercial piezoelectric ceramics. Of particular importance, the composites can be stretched and compressed to a high elastic strain of 50% without sacrificing the piezoelectric properties, due to their low Young's modulus of <30 MPa. In addition, the composites exhibit a very low acoustic impedance of ∼3 MRayl, one order of magnitude lower than that of PZT ceramic, being beneficial to the design of ultrasonic transducers considering the complex acoustic impedance matching layers in ceramic-based transducers [Here we propose a combination approach to simultaneously address both dielectric and mechanical mismatch issues in order to achieve stretchable polymer composites with giant piezoelectric properties. We first assemble PZT particles into an interconnected skeleton inside the polydimethylsiloxane (PDMS) polymer matrix to form ‘3–3’ type composites, in which both PZT and PDMS form global networks as illustrated in Fig. nsducers .mP of 0.75 μC cm−2 and a coercive electric field of ∼15 kV cm−1  and PDMS (rε of 4) by introducing P(VDF-TrFE-CFE) terpolymer with moderate dielectric constant (rε of ∼50) as a dielectric transition layer at the PZT/PDMS interfaces. To further mitigate the dielectric mismatch, we incorporate CNTs into the terpolymer to increase dielectric constant of the dielectric transition layers. With ultralow volume fraction of terpolymer/CNT-terpolymer layer uniformly deposited on the PZT skeleton (mP (2 μC cm−2), being 50% higher than that of the ‘3–3–3’ composites (mP ∼1.3 μC cm−2). No significant increase in dielectric loss or electrical conductivity is observed for CNT@‘3–3–3’ composites owing to the ultralow volume content of CNT in composites . When considering a terpolymer dielectric transition layer, the average local electric field in PZT of ‘3–3–3’ composite increases to 68 kV cm−1  of PZT skeleton coated with terpolymer layer are enhanced by ∼40% and 300% respectively. We calculate the electric polarization at different angles between the polarization vector and the poling direction (<001>) in the PZT skeleton. As shown in the insets of Fig. The results of phase-field simulation also indicate that the CNT-terpolymer dielectric transition layers complement the polarization path in open pores of the PZT skeleton, hence facilitating electric polarization switching of the PZT skeleton in CNT@‘3–3–3’ composite through interfacial coupling . As showS-E) curves in all composites with PZT skeletons. As shown in Fig. −1, which is in line with the enhanced polarization as observed in P-E loops and the phase-field simulation. Yet, even with 20 vol.% of PZT particles, the ‘0–3’ composite still exhibits a slight negative electric-field-induced strain, a characteristic of an electrostrictive elastomer. We calculate the converse piezoelectric strain coefficient (33*d) from the S-E curves. The highest 33*d of ∼250 pm V−1 is observed for CNT@‘3–3–3’ composite, which is 50% higher than that of ‘3–3–3’ composites (170 pm V−1).We measure the electric-field-induced strain and observe typical butterfly strain-electric field (33d) measured by a quasi-static 33dmeter, where 33d increases monotonically with increasing volume fraction of the PZT skeleton for all composites. The ‘3–3–3’ composites consistently exhibit higher 33d than their ‘3–3’ counterparts due to the presence of a terpolymer transition layer, while the CNT-terpolymer transition layer induces the highest 33d of 120 pC N−1 for CNT@‘3–3–3’ composites. Images of piezo-response force microscopy  is substantially lower than the converse piezoelectric strain coefficients (33*d ∼250 pm V−1). To elucidate this discrepancy, we perform a finite-element simulation on the stress/strain distribution in the composites. As shown in Fig. 33d of the composites than that of bulk ceramics, due to the smaller dynamic stress applied to the PZT skeleton during quasi-static measurement. In stark contrast, the CNT-terpolymer dielectric transition layer induces significantly enhanced local electric field (∼80 kV cm−1) in the PZT skeleton close to the overall applied electric field (∼100 kV cm−1), leading to a substantially increased electric-field-induced strain, i.e. the converse piezoelectric strain coefficient 33*d. The intrinsic piezoelectric contribution obtained from Rayleigh analysis is ∼129 pm V−1 under electrical clamping conditions , the CNT@‘3–3–3’ composites also exhibit low transverse piezoelectric coefficient (31d). As shown in Fig. 2e, 33dmonotonically increases with increasing PZT volume fraction in CNT@‘3–3–3’ composites, while the consistently lower 31d is insensitive to the PZT volume fraction. Results of the finite-element simulation indicate that PDMS with low Young's modulus acts as a mechanical damping medium and hinders the lateral load transfer in the composite (31d. The anisotropic piezoelectric response gives rise to high hydrostatic piezoelectric charge coefficient hd of ∼110 pC N−1 for the CNT@‘3–3–3’ composite (h ∼d100 pC N−1). The high hd and moderate rε of CNT@‘3–3–3’ composite (rε ∼97) also lead to an ultrahigh hydrostatic sensitivity coefficient (h · ghd) of 14.3 pm2 N−1  of ∼0.65  close to water is achieved in the CNT@‘3–3–3’ composite because of its ultralow bulk density of 1.9 g cm−3. The high coupling factor and ultralow acoustic impedance are expected to greatly benefit the application of ultrasonic transducers, whose sensitivity, power efficiency and bandwidth are closely associated with the coupling factor, while the low acoustic impedance is expected to simplify the design of acoustic impedance matching layers [We summarize and compare the piezoelectric properties of our composites with four types of piezoelectric materials Table . As seenof ∼0.65 , which ig layers .in-situ compression strain -based transducers fabricated under the same condition . The outn strain .in-situ microscopic uniaxial compression cycling measurements on the CNT@‘3–3–3’ composites. As shown in Fig. To understand the superior flexibility and elucidate the origin of the mechanical compliance of the piezoelectric composites, we perform both macroscopic and in-situ cyclic uniaxial compression tests using SEM to observe changes in the structure of CNT@‘3–3–3’ composite during dynamic compression. PDMS matrix distributed in the PZT skeleton exhibits excellent resilience as the macroscopic deformation of the sample gradually increases (We then perform ncreases . Due to ncreases , accompancreases . The PZTncreases . The smoncreases . On the ncreases . We alsoncreases  by reducin-situ Micro-CT scanning on CNT@‘3–3–3’ composite with 17 vol.% of PZT under uniaxial stretch. The cross-section CT images reveal that the diameters of the corresponding two groups of pores increase from 172 μm and 134 μm at the initial 0% strain state (inset I of Fig. We also characterize the mechanical properties of the CNT@‘3–3–3’ composite under stretching conditions. As shown in Fig. 33d of 114 pC N−1 to be obtained after stretching the composite to 30% strain (Fig. S-E loop measured at 30 kV cm−1 shows that the electric-field-induced strain of CNT@‘3–3–3’ composite does not change substantially after 0%–50% strain compression cycles (Fig. 6 cycles of bending or stretching tests, where the 33d maintains the same value during the bending test and only decreases by ∼6% during the stretching test.The collective uniform deformation of the PZT skeleton enables the CNT@‘3–3–3’ composite with stable piezoelectric properties under tensile strain, e.g. ain Fig. . The sliles Fig. . The ele6 cycles. Together with the facile fabrication process, the highly flexible composites with excellent piezoelectric properties provide a new design paradigm for smart structures and electromechanical devices, such as intravascular ultrasonic transducers, wearable self-powered devices with sensing and actuation functions, and soft robotics for intelligent functions.In summary, we design and demonstrate highly stretchable/compressible polymer composites featuring ‘3–3–3’ connectivity with dielectric transition layer. The established electric polarization/load-transfer pathways and the mitigated local electric field concentration in the CNT@‘3–3–3’ composite lead to unprecedented properties that are not achievable in individual ceramic or polymer, including the high piezoelectric coefficients, high hydrostatic sensitivity, high electromechanical coupling factor and ultralow acoustic impedance close to water. These composites also exhibit high stability and reliability under giant elastic strain up to 50% and bending tests up to 2The details of preparation and characterization of piezoelectric composites are shown in the nwad177_Supplemental_FilesClick here for additional data file.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility, virulence and immune escape abilities have heavily altered the COVID-19 pandemic’s course. Deciphering local and global transmission patterns of those variants is thus key in building a profound understanding of the virus’ spread around the globe. In the present study, we investigate SARS-CoV-2 variant epidemiology in Côte d’Ivoire, Western sub-Saharan Africa. We therefore generated 234 full SARS-CoV-2 genomes stemming from Central and Northern Côte d’Ivoire. Covering the first and second pandemic wave the country had been facing, we identified 20 viral lineages and showed that in Côte d’Ivoire the second pandemic wave in 2021 was driven by the spread of the Alpha (B.1.1.7) and Eta (B.1.525) variant. Our analyses are consistent with a limited number of international introductions of Alpha and Eta into Côte d’Ivoire, and those introduction events mostly stemmed from within the West African subregion. This suggests that subregional travel to Côte d’Ivoire had more impact on local pandemic waves than direct intercontinental travel. The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is driven by the emergence and spread of virus variants with clinically relevant mutations that lead to increased transmissibility, virulence and immune escape abilities ).The virus has spread on the entire African continent in multiple pandemic waves. The third and fourth pandemic waves were essentially driven by the uniform expansion of Delta and Omicron, but the first and second waves were respectively coined by the co-circulation of multiple SARS-CoV-2 lineages (mostly PANGO B.1 viruses) and the Alpha (B.1.1.7) and Beta B.1.351) variants 51 varian. The sechttps://www.worldometers.info). Here, we set out to shed light on the evolution and spread of SARS-CoV-2 and its VOC/VOI in Côte d’Ivoire during the first and second pandemic waves, by sequencing viral genomes and subsequently using them for in-depth phylogeographic analyses.A country where comprehensive investigation of variant epidemiology at a national scale is still lacking, is Côte d’Ivoire in the Southern coastal region of West Africa. By September 2022, Côte d’Ivoire had observed 86,821 cases of COVID-19, with 819 deaths recorded , following manufacturer’s instructions. Tiled amplicons of each about 400 bp in length were generated by two multiplex PCRs, using primer scheme V3 and following reaction and cycling conditions of the ARTIC protocol . The twohttps://clades.nextstrain.org), those with missing sampling dates, those with < 90% coverage, those with > 40 SNPs, those with > 10 ambiguous base-calls per genome, and those with clustered SNPs. Of the 461 specimen we had attempted to sequence, 234 high quality complete or near-complete SARS-CoV-2 genomes were retrieved and deposited on GISAID, the Global Initiative on Sharing All Influenza Data [Bases were called with the MinKNOW software, while we followed the ARTIC bioinformatics protocol  for demuWe assigned the Côte d'Ivoire SARS-CoV-2 genomes to virus lineages defined in the dynamic nomenclature of SARS-CoV-2 lineages  via panWe retrieved respective sequence data sets compiled by Emma Hodcroft and Richard Neher (Neherlab) for Alpha (4886 sequences) and Eta (4965 sequences) variants from Nextstrain  on Septe–4 nucleotide substitutions per site per year, with a standard deviation of 4.0 × 10–4, in TreeTime v0.8.6 [We next produced a time scaled phylogenetic tree based on sampling dates, using a fixed rate of 8.0 × 10e v0.8.6 . Prior tThe dated phylogenetic tree was used to fit a mugration model, which treats locations (in our case countries) as discrete traits that evolve through the phylogeny. Mapping countries to tips and internal nodes of the tree allows to estimate the number of viral transmission events for the Alpha and Eta lineage between Côte d’Ivoire and the rest of the world, which was done via a Python script developed by the authors . We performed a sensitivity test to examine the robustness of this introduction analysis towards which time-scaled phylogenetic tree is used as starting point for subsequent analyses. For this, we replicated the inference of a maximum likelihood phylogenetic tree in IQ-TREE ten times, starting with different seeds and re-ran the entire workflow from each tree to reconstruct ancestral states and infer introduction events. We plotted average number of introductions into Côte d’Ivoire with standard errors over time. Plots and phylogenetic trees were visualized using R ggplot2 .tested = 828, [Between May 23rd 2020 and May 31th 2021, 4071 naso/oropharyngeal specimens from COVID-19 suspect cases had been received by the Centre Hospitalier et Universitaire (CHU) de Bouaké, of which 719 specimens tested positive for SARS-CoV-2. An additional 8 SARS-CoV-2 positive nucleic acids were obtained from a running surveillance study on acute respiratory infections in the Bouaké region and Western Côte d’Ivoire  dominated through to December 2020  in February 2021 (Fig. % in FebrIndeed, VOC Alpha and VOI Eta quickly rose to being the most prevalent variants in Côte d’Ivoire during the second pandemic wave the country was facing. From January 2021 on, both variants increased in frequency through to May 2021, right after the peak of A.27, and presented overall 22.9% and 31.3% of all genomes sampled since January 2021, respectively. Alpha is characterized by 21 lineage defining mutations or deletions, including eight changes within the viral Spike gene which are linked to increased ACE-2 receptor binding affinity and innate and adaptive immune evasion . The varUnlike Alpha, which was originally introduced into West Africa multiple times from mainly Europe , Eta likIn order to shed light on the origins of Alpha and Eta circulation in Côte d’Ivoire, we generated time scaled phylogenies Fig A and C aTaken together, our analyses are consistent with a limited number of international introductions of Alpha and Eta into Côte d’Ivoire. Importantly, these introduction events mostly stemmed from within the West African subregion and this, irrespective of the origin of the variant, suggests that subregional travel to Côte d’Ivoire had more impact on local pandemic waves than direct intercontinental travel to the country. The subsequent rapid propagation of both variants within Côte d’Ivoire seeded the second wave of the pandemic and might have been facilitated by founder effects at a time when case numbers had dropped significantly. An important limitation of our study is it being geographically limited to Central and Northern Côte d’Ivoire, while further investigations in the coastal region, where Abidjan acts as the country’s hub for intercontinental travel, will be needed to fully understand SARS-CoV-2 dynamics within Côte d’Ivoire.Monitoring the spread and possibly local emergence of virus variants provides information guiding governmental measures towards pandemic control. Hence, reinforcing genomic surveillance on the African continent remains an important regional and global task.


Al-doped ZnO combines high transparency and conductivity with abundant and non-toxic elements; making it suitable for optoelectronic devices with large-scale applications. In order to check the quality of the material deposited over large areas, spectroscopic ellipsometry is a powerful technique that allows the determination of various optical and electrical parameters by applying suitable oscillator models. This technique is used here to obtain sheet resistance and visible transmittance data at several equidistant points of Al:ZnO thin films deposited using DC sputtering on 15 cm × 15 cm glass substrates. Independent measurements using other optical (spectrophotometry) and electrical (four point probe) methods show analogous visible transmittance but somewhat higher resistance values than those obtained with ellipsometry, which is explained by the contribution of grain-boundary scattering compared to in-grain properties provided using ellipsometry. However, the mapping of the data gives a similar spatial distribution to the different types of measurement; therefore, proving the capacity of ellipsometry to study with a single tool the uniformity of the optical and electrical characteristics of large areas. V = 80–90% and a sheet resistance of sR = 10–30 Ω/sq [V10/sR, which is used to compare different TCOs.ZnO is a transparent conductive oxide (TCO), showing high optical transmittance in the visible range and low electrical resistivity in its native structure . The inc–30 Ω/sq , with a –30 Ω/sq  φ = TV10AZO thin films have been prepared using various chemical and physical techniques: electrodeposition , sol-gelAnother important technical challenge is to achieve high uniformity of the AZO characteristics over a large area. Typical characterization requires profilometry to determine surface roughness and film thickness; spectrophotometry to determine optical transmittance; and four point probe (FPP) electrical measurements to obtain sheet resistance . SpectroIn this work, AZO thin films have been deposited using DC sputtering on unheated glass with a 15 cm × 15 cm area, and their uniformity has been analyzed with variable-angle spectroscopic ellipsometry performed on several points throughout the glass substrate. The optical constants  and electrical parameters  have been accurately obtained using a combination of the Drude oscillator model and Bruggeman effective medium approximations, including the simulated substrate and a rough top layer in the optical model, as reported by other authors ,15. The 2O3. After the chamber was evacuated below 1 × 10−4 Pa, high purity Ar and O2 were introduced until they reached a process pressure of 4 × 10−1 Pa. Then, a DC power was applied to the target (set to 1.7 W/cm2) for 7 min to obtain a film thickness of 0.80 ± 0.04 μm, as was measured after deposition with a Dektak 3030 profilometer . These preparation parameters were selected according to previous works [V~85%) and sheet resistance (sR~20 Ω/sq).AZO thin films were prepared on 15 cm × 15 cm × 2 mm soda lime glasses (SLG) with DC magnetron sputtering at room temperature, using a homemade vacuum deposition system. The substrate was placed in a vertical stainless steel frame and moved in front of a rectangular target  consisting of 98 wt% ZnO and 2 wt% Alus works ,13, in oOptical transmittance was measured with unpolarized light at normal incidence, using the spectrophotometer Avantes AS-5216 . Sheet resistance was obtained with a Signatone FPP head, combined with a Keithley 2450  Source-Measurement Unit. These optical and electrical data are compared with the information extracted from a Semilab SE-2000 Spectrocopic Ellipsometer  (SemilabpR/sR = tan(Ψ)exp(iΔ), were acquired in the wavelength range λ = 300–2100 nm at three incident angles Φ0 = 55, 60 and 65°. In order to extract useful information, the structure of the sample and the corresponding optical dispersions must be modeled, whilst taking into account that the ellipsometric model must distinguish the glass substrate from the AZO features, and AZO depth heterogeneities can be considered by introducing different sublayers in the simulation [jd and complex refractive index jN = jn − jik, with jn as the real part of the refractive index and jk the extinction coefficient. During the simulation, all theoretically calculated spectra (pR/sR)cal originate from the measured ratios (pR/sR)meas, which depend on the incident angle Φ0, the photon energy E = hc/λ, and implicitly on the characteristics of the phases used in the model: tan(ψ)exp(iΔ) = f. The model parameters are thus obtained by minimizing the error function [The ellipsometric parameters , defined as the ratio of the reflection coefficients for p- and s-polarizations , Rp/Rs =mulation . The modfunction :(1)G=∑Φ0r2) [The quality of the fit is obtained using the coefficient of determination (r2) , which msR/pR ratio, below the Brewster angle at which pR reaches zero [SLG and kSLG values to feed into the simulation of the SLG/AZO system. Next, the AZO characteristics were modeled, assuming there was a compact layer and rough top layer, with a combination of the Drude oscillator model (for the AZO compact layer) [r2 = 91%. Subsequently, the same experimental data were simulated assuming some depth heterogeneity of the AZO characteristics, with a first compact layer close to the substrate (AZO1), a second compact layer (AZO2) and the rough top layer (AZO2 + void), which gave a better r2 = 96%. Although a somewhat higher coefficient of determination could be achieved with a four-layer model (r2 = 98%), the results do not make physical sense due to abnormally high conductivity values (σ > 106 S/cm) at some points. Therefore, the three-layer model is considered optimum. It should be noted that, the simulated spectra start at λ = 400 nm because these models do not take into account the fundamental absorption that occurs at wavelengths below the semiconductor bandgap, which is located around 350 nm [hes zero . Previout layer)  and the t layer) . The coed 350 nm .The optical parameters derived from the three-layer model are illustrated in j) are formulated as follows [PE (eV units) and EΓ (eV units) are the plasma energy and the broadening, which is in connection with the scattering frequency.The Drude oscillator is used to describe the electrical conduction of free carriers in semiconductor materials . From th follows ,17:(3)εjε0 (55.263 × 106 e/Vm) is the free-space permittivity, −16 eV s) is the reduced Planck constant, e is the electron charge and m* denotes the scalar effective mass of carriers, which is assumed to be 0.25 me (me = 0.511 × 106 eV/c2 the electron rest mass) for AZO according to the literature [jd), the sheet resistance (Ω units) is calculated as follows:The electrical conductivity, free carrier concentration and mobility are obtained using:terature . Further−2/3, and the conductivity proportional to N1/3, which corresponds to scattering by ionized impurities [The evolution of conductivity and mobility with the carrier concentration is represented in purities . This ispurities .For each measured point in the sample, the ellipsometric fit for the three-layer model gives the thickness values that are plotted in Taking the conductivity and thickness data determined by the optical dispersion model, the overall sheet resistance is calculated assuming the parallel connection of the conductive phases :(9)Rop=(elR) at the different points of the sample. Higher resistances are observed at the horizontal edges, in relation to the greater thickness of AZO2 and the top layer (AZO2 + void), which have a lower conductivity than AZO1, based on the data in It is mapped in opR and elR differ due to a discrepancy in the conductivities determined using optical or electrical measurements. σop = opR/dtotal and σel = elR/dtotal) as a function of the total film thickness. It is noted that both conductivities tend to decrease as the film thickness increases, which is related to the increase in roughness  evidenced in σop/σel~1.6) are consistent with those found by other authors, indicating a similar ratio of RDL~1.6 [μL) and the resistance to electron transport due to defects such as grain boundaries and neutral impurities (1/μD). This is because the change in conductivity is mainly related to the carrier mobility [σop/σel~RDL = μL/μD.At the same sample point, the values of  RDL~1.6 , which imobility ,28, so σs) [m), as illustrated in s = Tm), as expected when using the Drude oscillator model, which optimally reproduces the behavior of the material in the zone of optical absorption by free carriers [s < Tm), but in any case both increase or decrease proportionally when moving from one point to another on the sample.Regarding optical transmittance, the global spectrum at each sample point has been simulated (Ts)  and compcarriers . In the Vs and measured TVm) has been calculated as the average value in the range λ = 400–800 nm from the respective SLG/AZO spectra and discounting the SLG substrate. These values are mapped in For each sample point, the AZO visible transmittance (both simulated Ts = TVs10/opR) and from independent electrical and spectrophotometric measurements (φm = TVm10/elR), which are represented in s > φm) because the optically determined resistance is lower (opR < elR). In fact, the ratio between the maximum values φs/φm = 0.022/0.014 = 1.6 is analogous to that established for the respective conductivities (σop/σel~1.6) in −1, higher than that reported for other TCOs grown at high substrate temperature [Finally, the figure of merit defined by Haacke  has beenperature ,31,32.σop), free carrier concentration and mobility indicate scattering by ionized impurities, being somewhat higher than the electrical conductivity (σel) determined using four point probe measurements. This corroborates that optical measurements provide in-grain properties, and the ratio σop/σel~1.6 obtained at different points of the sample gives the relation between the resistance to electron transport inside the lattice and the resistance to electron transport due to grain boundaries and neutral impurities.Ellipsometric data acquired for AZO thin films deposited using DC sputtering on SLG substrates show an optimal fit to a three-layer model consisting of a first compact layer that is close to the substrate (AZO1), a second compact layer (AZO2) and a rough top layer (AZO2 + void). The values obtained using optical simulation for the conductivity  at these edges. In any case, the variation in thickness is only 6% over the whole area and the figure of merit (TV10/sR) is always above 0.010 Ω−1, proving that high-quality AZO thin films are obtained using DC sputtering at room temperature on large areas.The maps of sheet resistance and visible transmittance performed on the 15 cm × 15 cm sample area show the same spatial distribution whether they are obtained from ellipsometry or from independent spectrophotometry and electrical measurements. Slightly higher


The lavas associated with mantle plumes may sample domains throughout Earth’s mantle and probe its dynamics. However, plume studies are often only able to take snapshots in time, usually of the most recent plume activity, leaving the chemical and geodynamic evolution of major convective upwellings in Earth’s mantle poorly constrained. Here, we report the geodynamically key information of how the lithology and density of a plume change from plume head phase to tail. We use iron stable isotopes and thermodynamic modeling to show that the Galápagos plume has contained small, nearly constant, amounts of dense recycled crust over its 90-million-year history. Despite a temporal evolution in the amount of recycled crust-derived melt in Galápagos-related lavas, we show that this can be explained by plume cooling alone, without associated changes in the plume’s mantle source; results are also consistent with a plume rooted in a lower mantle low-velocity zone also sampling primordial components. The Galápagos plume has contained small amounts of pyroxenite over its history, decreasing its buoyancy through time. The nature and temporal evolution of mantle material sampled by hot upwellings (plumes) is enigmatic. Many mantle plumes are thought to carry lower mantle material, which may be associated with large low–shear velocity provinces (LLSVPs) present at the core-mantle boundary , which produces steady-state OIB volcanism  than mantle peridotite, either because of heavier [mid-ocean ridge basalt (MORB)–like] bulk Fe isotope composition than peridotite  of 1700° to 1800°C proposed in the Tortugal suite (Costa Rica) and present-day plume tail estimates of Tp = 1360° to 1490°C  history are recorded in erupted basalts either in the Pacific, in terranes accreted onto Central America , and Gorgona Island (Colombia)], 60- to 70-Ma head-tail transitional accreted terranes [Quepos (Costa Rica) and Azuero Peninsula (Panama)], and modern (<2 Ma) steady-state plume . We analyzed samples from five southwestern and western Galápagos volcanoes, which span multiple isotopic domains of the recent archipelago .To address this problem, we measured δ57Femeasured that are different from δ57Feprimary , we correct our data back to an estimated primary liquid composition (Supplementary Text and data S3). As the plume ages and cools, δ57Feprimary is observed to increase from plume head to tail  (57Femelt−source and create heavier Fe isotopic compositions in the melt (57Fe flattens for Tp below 1500°C (because the maximum mantle-melt isotopic fractionation is approached), whereas the data suggest a continuing increase in δ57Feprimary from Azuero and Quepos (60 to 70 Ma) to modern Galápagos (present day). The gradual increase in δ57Fe as the plume cools in this model is driven both by decreasing average melt fraction and by increasing Fe3+/FeT of the melt, as the average pressure of melting decreases with decreasing Tp (57Fe (fig. S4), although a contribution from pyroxenite melting is required for the isotopically heaviest Galápagos Spreading Center samples (The increase in δ57Fe than peridotite (largely because they inherit a high δ57Fe from their MORB protolith) . We consider a range of bulk δ57Fe values for the pyroxenite , which span the range of average MORB data for each mid-ocean ridge segment given the likely origin of mantle pyroxenite as recycled crust and includes the δ57Fe = 0.15‰ proposed as average MORB (Supplementary Text)  because of its possible formation by hybridization with mantle peridotite  processed through column chemistry, giving values in agreement with previous studies  of Fe (2018–2019) or 8 ppm of Fe (2021–2022) in wet plasma and medium resolution mode. Samples were introduced using a quartz cyclonic spray chamber, and instrumental mass bias was corrected for by sample standard bracketing. Sample and standard iron beam intensities (typically 35 to 45 V of


To determine if the transcriptomic effects of the klf9−/− mutation vary with time of day, we performed bulk RNA-seq on 5 dpf zebrafish embryos sampled at three timepoints encompassing the predawn peak and midmorning nadir of klf9 expression. We found that while the major effects of the klf9−/− mutation that we reported previously are robust to time of day, the mutation has additional effects that manifest only at the predawn time point. We used a published single-cell atlas of zebrafish development to associate the effects of the klf9−/− mutation with different cell types and found that the mutation increased mRNA associated with digestive organs  and decreased mRNA associated with differentiating neurons and blood. Measurements from confocally-imaged larvae suggest that overrepresentation of liver mRNA in klf9−/− mutants is due to development of enlarged livers.Krüppel-like factor 9 (Klf9) is a ubiquitously expressed transcription factor that is a feedforward regulator of multiple stress-responsive and endocrine signaling pathways. We previously described how loss of Klf9 function affects the transcriptome of zebrafish larvae sampled at a single time point 5 days post-fertilization (dpf). However,  Klf9 is also known to regulate both developmental cell differentiation and physiology in multiple tissue types and organ systems, including the central nervous system11, blood2, adipose tissue12, and liver13, and to interact with and regulate the circadian clock15. As is the case with many ubiquitously expressed transcription factors, the function of Klf9 depends on context, both spatially and temporally. While this limits broad generalizations about Klf9 function, evidence from various systems suggests that, like other KLFs, Klf9 often functions to regulate and/or fine-tune cellular responses to signal-responsive transcriptional activators, and that it often does so in incoherent feedforward loops, i.e., as a target whose activation functions to repress other targets17. Incoherent feedforward regulation is widely used in biological systems to optimize responsivity and mediate biochemical adaptation to signal strength23.Krüppel-like factor 9 (Klf9), a ubiquitously expressed vertebrate member of the Krüppel-like family of transcription factors, has recently emerged as a critical regulator of multiple environmental- and stress-response pathways, including those mediated by the glucocorticoid receptor (GR), the thyroid receptor (TR), aryl hydrocarbon receptor (AHR), the redox homeostasis/oxidative stress factor Nrf2, and the proteostasis/ER-stress factor Xbp1klf9 is transcriptionally upregulated by GR signaling and that the transcriptomic response to chronic cortisol treatment measured by bulk RNA-seq of zebrafish larvae is significantly altered by loss of klf9 function25. We subsequently showed that klf9 mRNA expression is oscillatory, with a diurnal rhythm that peaks just before dawn and then rapidly declines to a mid-morning nadir, a pattern also displayed by mRNA of the GR (and Klf9) target gene fkbp5, but not by several other GR target genes17. The diurnal oscillation of klf9 transcript levels depends on a functional GR and is thus probably at least in part due to the diurnal oscillation in circulating cortisol levels17. Our previous RNA-seq experiment assessing the transcriptomic effects of the klf9−/− mutation was carried out on pooled zebrafish larvae collected at a single mid-morning timepoint1, when klf9 mRNA expression is at its nadir, leaving open the possibility that the observed effects were specific to that time of day.We previously showed that zebrafish klf9−/− mutation depend on the time of day. As in our previous experiment, we sampled zebrafish larvae on day 5 post-fertilization, a full day after the hypothalamus-pituitary-interrenal  axis has developmentally matured and klf9 expression has developmentally plateaued17. Samples were taken at three different time points: − 3, 0, and + 2.5 h with respect to the time when the lights come on , encompassing the klf9 expression peak and nadir (respectively at ~ ZT0 and ZT+2.5). Four biological replicates each of wildtype (WT) and klf9−/− larvae were sampled at each time point and subjected to bulk RNA-seq. We used the unbiased approach of Principal Component Analysis (PCA) to identify patterns of variance in the data, which were then mapped to the experimental design variables and other metadata to identify the sources of the variation. This analysis showed (unsurprisingly) that the transcriptome varies substantially with time of day, and that this dynamic is affected by the klf9−/− mutation, such that a subset of the mutation’s effects manifests only at a specific time of day. Nevertheless, many effects of the mutation manifest independently of the time of day, including the major effects that we reported previously1. Using a published zebrafish larva single-cell transcriptome atlas26 we mapped the gene expression effects of the klf9−/− mutation identified in our bulk RNA-seq experiments to putative cell and tissue type, which revealed potential functions of Klf9 in regulating the development of liver and other organ systems.Here we report the results of another bulk RNA-seq experiment that we carried out to assess the extent to which the transcriptomic effects of the klf9−/−) at each of three time points   and the circadian regulator cry1a (negatively correlated)  and hmgrca (negatively correlated)  and fkbp5 (negatively correlated)  of the RNA-seq data was performed to identify patterns of variance in the transcriptomes among four biological replicate samples taken from two genotypes (wildtype and .5) Fig. . Surpris.5) Fig. . This wa.5) Fig. . PC1 was.5) Fig.  includeded) Fig. . Notablylf9 Fig.  includeded) Fig. . PC4 caped) Fig.  includeded) Fig. .Figure 1klf9−/− mutant samples both for the overall experiment  and independently at each time point (Supplementary Table klf9−/− mutants (937 vs. 366), reinforcing previous findings that suggested that Klf9 functions more often as a repressor than as an activator of transcription28.Differential gene expression DESeq2) analysis was performed to specifically assess how loss of Klf9 function affects gene expression, comparing wildtype and  analysis17. Our previous study1 found that, compared to wildtype controls, klf9−/− mutants overexpress genes associated with the complement system, an effect that was reproduced here, although only a subset of complement genes are overexpressed  were downregulated in klf9−/− mutants, which as noted above was also reproduced here. However, DESeq2 analysis of all genes associated with sterol biosynthesis showed that some are overexpressed in the mutants, indicating that the mutation has complex effects on that biological process , but which also showed modest correlation with PCs 3 and 4  were differentially expressed in the mutants. Examples are hmgcra, which as noted above is significantly downregulated in klf9−/− mutants but specifically associated with the liver (hepatocyte) cluster that overall is overrepresented, and thy1, which is significantly upregulated in klf9−/− mutants but associated with four clusters  that do not change in overall representation , or that they have more liver cells, or both. Either scenario would be expected to manifest as enlarged livers in klf9−/− mutants compared to wildtype controls. To determine how the klf9−/− mutant affects liver size we stained fixed 5 dpf larvae with fluorescent streptavidin, which labels liver, intestinal epithelium, and yolk32, and imaged the stained larvae confocally. Since the livers can be distinguished morphologically from the other stained structures, this allowed us to estimate liver volumes in images of the liver surface reconstructed from confocal z-stacks  given our recent finding that klf9 expression oscillates diurnally17, we sought to determine the extent to which the transcriptomic effects observed in our klf9−/− mutant depend on the time of day the samples were obtained; and (2) given the availability of a recently published single cell atlas of zebrafish development26, we sought to map the transcriptomic effects of the klf9−/− mutation to specific cell types.RNA-seq is commonly used to comprehensively assess changing patterns in gene activity associated with development and/or physiology under different genetic and/or environmental conditions. Originally the technique was applied to whole tissues, organs, or organisms (“bulk” RNA-seq), but more recently single-cell RNA-seq (scRNA-seq) methods have allowed investigators to assess effects of experimental variables on specific cells and thus the overall effect on different cell populations. A limitation of scRNA-seq compared to bulk RNA-seq is that the depth of sequencing is much lower, and thus less sensitive to subtle transcriptomic effects. Recently methods have been developed that allow bulk RNA-seq results to be mapped to cell types resolved in scRNA-seq atlaseswithin each experimental condition , and Zeitgeber (the onset of the light phase in the incubator) , as shown by the consistent differences in klf9−/− and WT seen along those PCs  analysis to parse the variance in the bulk RNA-seq data irrespective of the experimental design variables, an unbiased approach that allows the variance to be correlated with those variables and other experimental metadata post hoc. Based on previous results  indicating that the acute stress of sample collection can introduce a stress-response gene expression signature, we took great care in this experiment to eliminate acute stress as a variable between serially collected biological replicates see "". Despitor) Fig. . As we wPCs Fig. . This suthy1 and hmgcra  is the rate-limiting enzyme in cholesterol biosynthesis30. While the biological significance of thy1 upregulation and hmgcra downregulation in klf9−/− mutants is not clear, we can surmise that it may relate to Klf9 function in regulating development. In various contexts, Klf9 has been shown to be important for terminal cell differentiation and concomitant cessation of cell proliferation, a role that is consistent with studies that indicate that it often functions as a tumor suppressor38. Thus, one might expect klf9−/− mutants to show signs of underdevelopment such as impeded cell differentiation, and perhaps also loss of growth regulation. Indeed, the results of the single cell integration analysis , whereas loss of growth regulation is suggested by higher representation of genes associated with endodermal derivatives dedicated to metabolism   Fig. . The obsklf9−/− mutants of genes associated with that organ is due to its overgrowth is supported by measurements obtained of the volumes of fluorescently stained and confocally imaged livers  target31. The fact that per1a is overexpressed in klf9−/− mutants only at the predawn timepoint may simply reflect that that is its normal expression peak, which coincides with that of klf917. Notably however, klf9−/− mutants also overexpress a battery of genes encoding Crystallin proteins (cry genes) at ZT−3 but not ZT0 and ZT+2.5  role for Klf9 is also suggested by the fact that many genes were either over- or under-expressed in specific cell or tissue types that were not on the whole over- or under-represented in the klf9−/− mutants  correlated with PC4 suggests that its temporal dynamic is entrained by the Zeitgeber. Given our previous results17, Klf9 may contribute to this regulation. The observation that many more genes are upregulated at ZT0 in Klf9 mutants than at the other two time points , we cannot exclude the formal possibility that the overrepresentation of those genes in klf9−/− mutants is due to their ectopic expression in cell types that normally don’t express them. Further experiments using in situ hybridization are required to test that possibility. Nevertheless, the results depicted in Fig. klf9−/− mutants is correct. A second limitation is that the measurements depicted in Fig. Although the results depicted in Fig. klf9−/− mutants derived from the AB strain1, and wildtype controls derived from the same grandparents. Husbandry and procedures were as described previously25. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the MDI Biological Laboratory, and all methods were performed in accordance with the relevant guidelines and regulations. The study is reported in accordance with ARRIVE guidelines.Zebrafish were 1. Briefly, fertilized eggs were collected in the morning, disinfected, and at ~ 4 h post fertilization placed in dishes with embryo media. Embryos developed in a 28.5 °C incubator with a 14/10 light/dark cycle synchronized with the core fish room. Media was changed daily.Embryo culture and cortisol treatments were performed as previously describedklf9−/− mutant and wildtype larvae were collected as follows. Each biological replicate from each genotype and timepoint consisted of six larvae in a 30 mm dish in 4 ml of media. Larvae were collected from the dish into Eppendorf tubes, media was removed, and larvae immediately snap frozen in liquid nitrogen. Collection of the 8 samples at each time point occurred over ~ 10 min. Frozen samples were stored at − 80° C. RNA was prepared using the Qiagen RNeasy Plus mini kit (Qiagen 74134). For each set of four biological replicate samples, RLT Plus lysis buffer  was added to each sample immediately upon its retrieval from the freezer, and the sample was then immediately homogenized with a motorized pestle and placed on ice before the next sample was retrieved from the freezer and subjected to the same procedure. Following pestle homogenization replicate samples received simultaneous processing—i.e., passed through Qiashredder columns (Qiagen 79654), bound, washed, and eluted per kit instructions. RNA was quantified and stored at – 80 °C.At − 3, 0, and + 2.5 h zeitgeber time (onset of the light phase in the incubator) on day 5 post-fertilization, four biological replicates of For follow-up experiments using quantitative reverse transcription and polymerase chain reaction (qRT-PCR) and Nanostring, RNA was prepared either by the column method as described above or by the Trizol method, and for Nanostring analysis RNA was prepared by the Trizol method. The Trizol method includes a controlled incubation time of 5 min in Trizol immediately after homogenization. This step was not included in the RNeasy plus method described above, which had variable times in the homogenization buffer . We therefore tested whether this variable might be a source of variance in the data by controlling the incubation time in RLT+ buffer at 5 min per sample and found that this significantly reduced the variance between samples.47 with options: “--fastqc_args ‘--noextract’”, “--illumina”, “--length_1 35”, “--length_2 35”, “--stringency 1”, “--length 20”, and “--quality 20”. Gene and isoform expression were estimated with RSEM Version 1.3.348 using Ensembl release 10149 of the Danio rerio genome for alignment and using command “rsem-calculate-expression” with options: “--keep-intermediate-files”, “--star-gzipped-read-file”, “-p 64”, “--estimate-rspd”, “--append-names”, “--output-genome-bam”, and “--star”. RSEM estimates of sample-specific expression were merged using R (version 4.0.3) package tximport version 1.0.350.Frozen RNA was sent to the Oklahoma State Genomics Facility for Illumina library preparation and sequencing. RNA-seq libraries were generated with Illumina-compatible KAPA libraries and sequenced as single-end 75 bp reads on an Illumina NextSeq 500 High Output sequencer. Quality and adapter trimming was applied to fastq formatted read files with Trim Galore Version 0.6.5For downstream analysis, it is more valuable to refer to genes by their name to estimate expression. There were 581 genes that were associated with more than one Ensembl ID, so the count matrix collapsed these distinct transcript counts by taking the sum of all transcript counts that correspond to the same gene symbol (retaining the earliest EnsemblID), effectively removing gene duplicates from the count matrix.klf9−/− samples in the count matrix with DESeq2, version 1.30.151, using R version 4.0.3. DESeq2 was also used to generate a rlog-matrix which was subsequently Z-transformed  to normalize each gene across all samples. The Z-transformed rlog matrix was then loaded into JMP version 16 and used for PCA (using the “Wide Method”). As described below, the first PC in all analyses was attributed to a technical effect, therefore the PC coordinate was subsequently used as a covariate in DESeq2 differential expression analysis. Four DESeq2 runs were performed: klf9−/− vs WT samples at each of the three time points, and then overall with time as a covariate to determine the time-independent effect of klf9. Gene Ontology Term Enrichment analysis was performed using GOrilla52, and only included genes in the top 90% of baseMean counts and bottom 85% of logfoldchangeSE. Gene ontology treemaps of the GOrilla results were generated using REViGO53.Differential Expression Analysis was performed between wildtype and 17, using custom PlexSet probes designed by Nanostring , then dehydrated through a methanol/PBST series (20/80 → 50/50 → 80/20 → 100% methanol), and stored in methanol at 4 °C. Fixed larvae were rehydrated through a series of methanol/PBST solutions in the reverse order as described above, then bleached with 10% H2O2/0.5× SSC/0.5% formamide, for 12 min at room temperature (RT). After blocking with a solution of PBST containing 10% bovine serum albumin (BSA) for 1 h at RT, the larvae were incubated with Alexafluor568-Streptavadin in the dark for 2 h at RT, then washed 3 × with PBST and stored in 80% glycerol at 4 °C.Staining and imaging of livers was carried out following the protocol of Sadler et al.For imaging, 5 fixed and stained larvae were mounted in 2% low melt agarose on coverslips affixed to the bottom of 3.5 mm plastic Petri dishes, on their left side such that the liver was oriented toward the objective of an inverted microscope.Images were acquired using a CSU-W1 spinning-disk confocal  with 25 µm Disk, Aperture of 10 and speed 4000 rpm, mounted on an inverted Nikon Ti-Eclipse  equipped with a Nikon Plan Apo 10×/0.45NA objective lens .Alexafluor568 fluorescence was excited with the 561 nm laser line from a multi–Laser Unit (LUN-F) and collected by using the ET605/52 band pass emission filter  and the DM405/488/532/647 dichroic mirror .Images were acquired with an Andor Zyla  controlled with NIS-Elements  software, with binning 1 × 1, at resolution of 2048 × 2048 pixels, in 16-bit, dual Gain 1/4, and saved in Nd2 file format.Z-stack images of the liver were collected with a step size of 2.5 µm with the Motorized Scanning Stage  and the Ti ZDrive Device. The Z-stack range was manually adjusted for each sample to yield the best possible detection.klf9−/−. After randomization, using Imaris, the images in nd2 format were converted to ims format for segmentation and analysis. The liver was segmented manually by outlining the liver’s Alexafluor568 fluorescence signal, by navigation through the Z-stack and using the surface tool. The following sequence of commands and steps were used: (1) ‘Create a new surface’, (2) ‘Turn off the volume’, (3) check ‘skip auto creation’, (4) under ‘draw’ click ‘visibility (none)’. The segmented rendered surface was used as a mask and used to obtain the liver volume.Prior to image analysis in Imaris software , each file was renamed with a random designation so that the analysis was performed blind to the identity of the sample. After the measurements were made the file names were decoded to identify the samples as wildtype of For statistical analysis, we combined the results of two sets of measurements made independently by different individuals. Preliminary analysis of these data revealed a slight systematic difference between the sizes measured by each analyst. We calculated the average value for each set of measurements and found that after each subset was zero-centered, the resulting distributions were statistically equivalent, and therefore they were joined into one set for comparative analysis.Supplementary Figures.Supplementary Legends.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.Supplementary Table S5.Supplementary Table S6.Supplementary Table S7.