This study reports findings from formative research conducted to assess the feasibility and inform the design and implementation of the Addressing Hidden Hunger with Agronomy (AHHA) trial. The AHHA trial was a randomised, controlled trial conducted in rural Malawi, in which participants were given maize flour biofortified with selenium or control flour not biofortified with selenium for a period of 10 weeks, after which blood samples were taken to measure selenium status.Formative research was conducted in villages near to the AHHA trial study site 1 year before the planned intervention. A short questionnaire with adult women (n = 50), focus group discussions with male (n groups = 3) and female (n groups = 3) community members, and in-depth key informant interviews (n = 7) were conducted to understand community practices and perceptions.Meals were typically cooked and eaten at home in this community, while participants reported that maize flour would be less readily sold than maize grain – important considerations for the design of the trial. Regarding intervention delivery, we identified potential concerns around effects on fertility, links between blood sampling and witchcraft, and the potential for social stigma if community members considered participants lazy for receiving free flour. Participants reported that involvement of the Malawi government partners including health extension workers would increase trust.Following the formative research, the AHHA trial appeared feasible. However, community sensitisation would be essential to address potential fears and concerns; effective sensitisation would support recruitment and treatment adherence, and would protect the safety and wellbeing of participants and researchers. People in positions of authority and trust including village headmen, religious leaders, health and agriculture extension workers, and community care groups should be involved in community sensitisation.The online version contains supplementary material available at 10.1186/s40814-021-00877-1. What uncertainties existed regarding the feasibility?What are the key feasibility findings?Would participant households in the AHHA trial be willing to receive and consume the trial maize flour in place of their own flour? How could the trial team improve acceptability and reduce the risk of households sharing or selling the trial flour? Would participants be willing to provide blood samples?What are the implications of the feasibility findings for the design of the main study?Households would be willing to receive and consume the trial flour in place of their own, and limiting sharing with other households is also feasible. However, clear communication of the importance of consuming the flour within the household will be required. Also, community sensitisation will be required to improve acceptability and address potential fears, concerns and rumours regarding consumption of the flour and provision of blood samples.A trial design with households randomised to treatment (rather than a cluster design) was considered feasible. Extensive community sensitisation would be required to ensure the acceptability of the trial intervention and blood sampling. The sensitisation process should involve influential and trusted members of the community including health extension workers and village head men. Members of the community should also be invited to see the maize under production and processing, to increase trust in the flour.Addressing Hidden Hunger with Agronomy (AHHA) trial  because this will be specific for a purpose. Probably we can give the maize flour that we grow.” (FGD M1)Several FGD participants raised the issue of guests, and that it would not be culturally appropriate to cook a different type of flour for guests:“It is wrong to cook and eat for the family and prepare another for the visitor. The visitor will think we have poisoned the food. The visitor will not be comfortable to eat the nsima unless we eat together.” (FGD F2)“…when we have the visitor, they will have to eat the same flour.” (FGD F1)There was a risk that participants would simply sell the trial flour. However, the FGD participants noted:“Selling things that you have been given it’s not good. We sell things that we have worked for.” (FGD M1)“It’s better we sell what is ours, than what we are receiving because that may compromise the study.” (FGD F1)The qualitative results supported the provision of maize flour rather than maize grain because maize flour is less likely to be sold than grain.“…people here will be surprised to see you selling the flour. It’s in town where we see people selling flour.” (FGD M2)“The only year when I saw people selling maize flour was the year when there was hunger the whole country. Then people were indeed selling maize flour. There’s no one who can bring maize flour on the market and expect people to buy.” (FGD M3)However, distribution of flour rather than grain would not eliminate the risk of selling:“It’s better off to give them flour unlike the actual grains, but still we should anticipate some maybe going that far of selling, because I have seen even some people selling the flour they have gotten from the hospital, the therapeutic flour for their child under the CMAM [Community Management of Acute Malnutrition] programme.” “They [the men who sell flour] put the flour in their trousers and when they get to the beer hall, they exchange with beer.” (FGD F3)“Sometimes it is us women, we have a problem, if our neighbour has no maize flour and she has money, we steal from our house and sell the maize, while the husband is away.” (FGD F3)“The maize flour we put in a bucket when we are going to draw water. Our husband thinks the bucket is empty yet there is maize flour which we are going to sell at the borehole to another woman.” (FGD F3)Involving the village chiefs in the process would potentially reduce risk of selling flour:“In our village it is not possible [to sell maize] because when we harvest, we are called by the chief who addresses us. He advises us not to sell the maize because, like this year we did not harvest well so he would tell us not to sell the maize and to care for the food. Because of that we do not sell.” (FGD F3)Focus group discussion findings revealed the participants had mixed perceptions of the proposed trial. Most community members perceived potential benefits and motivation of the intervention .“We will eat the trial maize flour because we don’t have adequate maize flour in our houses”. (FGD F2)“They will give us free maize flour and we will know our nutritional status after blood test”. (FGD M1)Similar observations were made by key informants:“The motivation [to participate] is food. If you give them food, they will be happy. That’s the motivation.” Participation in previous research also seemed to influence their perception of the proposed trial:“Most of the farmers have positive experience with research because they know that after the research those things will be implemented for their own benefit, so they welcome that.” On the other hand, some participants had reservations. The main areas of concern were (i) lack of trust in what will be added to the AHHA trial maize or maize meal, (ii) fears over the blood sampling routine, and (iii) concerns regarding negative social stigma.Community perceptions of blood sampling were critical, because blood samples would be required in the AHHA trial to assess the intervention’s impact on selenium status. Regarding blood sampling, some respondents felt that sampled blood might be used to assess the prevalence of HIV in the area, whereas others were concerned about the amount of blood that would be collected. One of the respondents said:How much blood will be collected? Won’t they test other diseases?” (FGD M1)“Another respondent said:will provide blood samples once we know the required amount.” (FGD F2)“We Other concerns related to whether the blood samples would be used for non-health–related purposes. One of the respondents said:Maybe blood sampling has to do with Satanism.” (FGD F2)“Another participant said:Once the child is sick, many will say; you are in the camp of Satanist; they fed you well to become fat so that they can kill you.” (FGD F3)“On the other hand, most respondents indicated they were not concerned with the sampling of blood, as illustrated by the following quote:Blood sampling will not be a problem since you said you will only test nutrients in the blood.” (FGD M1)“Another respondent said:Blood sampling will not be a problem since small amount of blood will be sampled.” (FGD M3)“Community perceptions of the quality/safety of the flour and the researchers’ intent in providing trial flour is critical because participants and other household members must be happy to consume the allocated flour. One of the major obstacles noted by participants was trust in the trial flour.“…people will relate to anything they receive to be not real things, they will think that it is sort of fake” Male  FGD respondents identified the risk that maize meal would be perceived as a family planning tool. In response to whether children would be allowed to eat the maize meal enriched with selenium, one respondent said:If the maize meal can make our children infertile then children will not be allowed to eat. But if it will make them healthy as indicated, then children will eat since they eat what parents eat.” (FGD M3)“The observation was also noted by key informants:“…they might think you are involving them in family planning.” Previous health-related programmes have identified similar issues when free items were distributed in the community:“I will give you an example of the mosquito nets. So, government distributes mosquito nets to the people, and then the people say that these mosquito nets are there to make them…not to make them have babies…they affect fertility.” Micronutrient programmes in Kasungu District have faced similar challenges:“People were saying just don’t go to the hospital because they have asked you to go…don’t just give your children vitamin A, which is aimed at making future generations impotent.” (District-level Nutrition Officer)Despite these concerns, most respondents indicated that they would be willing to eat the maize meal provided if it was endorsed by government officials:Other people will eat the maize meal because they trust the recommendations by government.” (FGD F1)“We can eat the maize meal because we feel it is good for us since the government has approved it.” (FGD F1)“Previous food distribution schemes caused upset in communities because so few households benefitted:“There’s been an outcry of course, and I remember at one point in time, the chiefs could not even manage to allow the partner to distribute the food, just because it has come little compared to the number that is supposed to benefit from that.” When it was proposed that everyone will be receiving the flour, including the village chief, participants reported that this would likely reduce fear and suspicions:“This is something very good because there will be no people complaining. If that is the case, then most people will be consuming the flour. Because they know that each and every one is consuming this flour. It’s not strange flour.” The timing of the flour distribution was also discussed:“If you change the months, maybe starting in the lean period, it will also be taken as relief food.” Concerns were raised about the social stigma associated with receiving free food. Some community members felt people outside the study area would stigmatise the beneficiaries :Community members talk a lot about free food. Sometime back, people received free maize; they nicknamed them as lazy people.” (FGD M1)“A similar observation was noted by key informants based on their experience of food distribution programmes:“Of course, the ones who were not receiving [food distributions], they were maybe just jealous…so that’s why they were calling them ‘Manjalende’ [lazy people who can’t feed themselves].” And:“[Those who did not receive the food distribution] were calling them ‘Manjalende’, it’s like the people were just waiting for free food.” However, this concern was not universally shared:“…the neighbouring village would be talking, saying ‘oh those people they are very lucky’… they cannot talk negatively, this is food. They would just admire. They can just appreciate that their friends are very lucky.” The results of the formative research underscored the importance of community sensitisation. Good sensitisation—appropriate communication conducted by the right people—would be essential to overcome negative perceptions.“…every new thing it’s received with some doubts. People talk because there’s no information. If people have information and have everything, it clears out those misconceptions.” Sensitisation would be important to garner community support for the trial:“The first thing is sensitisation. You can sensitise them, there after we shall be working hand-in-hand together with them.” With good sensitisation, participants would be more likely to consume the flour:“I don’t think rejecting [the flour] can happen, first of all there has to be strong community mobilisation, people they have to be sensitised fully, why that is being done.” Sensitisation was important to address negative perceptions and fears related to blood sampling.“They can’t raise issues if people can be sensitised before…They must know that our friends are going there for this, we were told about this, so that’s why these women are going over there.” “…because blood sometimes can be a delicate thing in our African culture, but if they understand why you are doing that, I don’t think there can be a problem.” Several key informants noted the importance of involving Health Surveillance Assistants in the sensitisation process. Their support was important to gain trust in blood collection:“…people are [used to] giving blood samples to detect malaria. You know the Health Surveillance Assistants, they go and collect samples of blood… so the HSAs should be there to tell the people and answer their questions.” The health surveillance assistants (HSAs) also provide an important point of contact with the communities, helping to direct sensitisation efforts and addressing particular concerns:“The HSA is the one who works directly with the communities… I think the HSA is the first person who gets information from the household.” The other key individuals identified for the sensitisation process were those in positions of authority or influence:“…the chiefs, they are also the ones who can help, because they respect them, so what the chiefs may say to the villagers, the villagers understand very well that our chief is supporting this.” .“The church has to be involved because a large number of people gather there, so it’s very easy to distribute the information.” An important lesson from the gender segregated FGDs was that men, in this patrilocal society, placed more trust in their village headman, whereas women placed more trust in the HSAs with whom they had frequent interactions through antenatal clinics.The message on feeding trials will be underrated (anthu akanyozera), if it is disseminated by the village headman alone.” (FGD F1)“Participants noted the importance of involving groups who can represent the community during the sensitisation process. The community care group volunteers were cited as an appropriate network:They [the community care group volunteers] are the first people I would use…to disseminate information.” .“The formative research provided valuable information on the feasibility of the AHHA study and guided subsequent decisions on the AHHA trial design and implementation, as follows:Prior to the formative research, two possible designs were being considered: first, a design where households are randomly allocated to treatment and, second, a cluster-randomised design with villages randomised to treatment. The first design would only be appropriate if households did not share the trial flour with other households. If sharing were frequent and unavoidable, a cluster-randomised design  might be more appropriate since this might reduce ‘contamination’ between treatments; however, a cluster-randomised trial would necessitate a larger sample size than an individually randomised design, which is disadvantageous for ethical and resourcing reasons.Based on the findings from the formative research, it was decided that randomisation of treatment at the household level would be feasible and appropriate. To reduce the risk of contamination through sharing of flour, we would clearly explain the need to consume the AHHA trial flour at home and to source gifts from their own flour production. To minimise sharing, we decided to provide flour to all households in the study area, not only to those participating in the trial. Specifically, we would purchase control-equivalent, non-biofortified flour and provide it to non-trial households in the same quantities and at the same distribution time points as the trial households. Providing flour to non-participating households would also help disassociate the trial from food distribution programmes that target assistance to impoverished households, which would reduce potential negative social stigma.Additionally, the timing of the flour distribution was chosen to avoid the ‘lean’ or ‘hungry’ season (i.e. December–March), when food aid programmes typically distribute relief food.granmill due to participants reporting this as their preference for gifts. As found in the FGDs, their preference was likely due to the ability to use granmill for porridge  in addition to nsima . This was advantageous from the trial perspective as it would likely increase adherence.The formative research findings informed the decision to distribute flour rather than grain, mainly to reduce the risk of selling. The type of flour selected was capita-1 day-1 of maize flour was provided for all family members including for children, which was rounded up to the nearest 5 kg at each 2-week distribution time point.To reduce the risk of running out of flour and to allow households to cater for guests, a decision was made to provide slightly more flour than was required to meet a typical family’s energy needs. Specifically, 330 g Selling and sharing of flour was a risk, so it was decided to monitor adherence every 2 weeks among participant households, and to discourage selling and sharing of AHHA trial maize flour through community engagement.Following the formative research findings, trial sensitisation activities were conducted from ~ 8 months prior to recruitment until completion of the endline survey. The formative research findings influenced the decision on which community members to engage in the sensitisation (including HSAs and village headmen), and how best to work with them to conduct community briefings and disseminate information about the trial. Based on the formative research findings, the sensitisation activities also reached villages neighbouring the study area to clearly explain what the trial involved and why they were not selected to participate.To help address any potential mistrust in the flour, the trial team arranged for community members from participating villages to visit the maize production site during crop production, and at a later date to view the milling and packaging process. A community tasting session was also conducted in the study area prior to the first distribution of flour, with community members and those involved in sensitisation invited to participate. These activities all increased trust in the flour and mitigated risks of negative rumours. In addition, the trial maize flour was packaged in sacks similar to those used for packaging commercial maize meal. These sacks were clearly labelled with the logo for Lilongwe University of Agriculture and Natural Resources, the lead implementing partner.To ensure trust in blood sampling, the project sensitised the community regarding the amount of blood to be collected at baseline and endline surveys (6 ml) and nurses from the Ministry of Health were recruited to collect the samples. During recruitment for the AHHA trial, research assistants took with them example blood collection vials to demonstrate the volume of blood that would be required. Mobile, tented clinics were set up in public places, which had been identified though community consultation; this was deemed less likely to raise suspicions related to witchcraft compared with collecting blood samples in private houses. The community members were informed that blood samples would only be used to assess micronutrient status, and HIV/other disease status would not be measured.Sensitisation continued throughout the survey and intervention periods, with members of the trial team engaging regularly with the community, extension workers and volunteers to answer questions, allay concerns or address negative misconceptions. Establishing close and trusted bonds with communities is recognised as an important process for ensuring the successful and ethical conduct of community-based trials , and wasStrengths of the present study included the mixed-methods approach with questionnaire, FGD and KII components, which gave the study team a rich understanding of potential challenges in delivery of the AHHA trial as well as valuable guidance on addressing these. The separation of male and female FGDs was valuable, and identification of KIs through snowball sampling provided an effective way to gather input from a variety of sources. Conducting the research in the local language with trained research assistants was essential. Finally, conducting the formative research ~ 12 months prior to the implementation of the trial provided sufficient time to incorporate learnings into decisions on trial delivery and design, including the initiation of sensitisation activities ~ 8 months prior to participant recruitment.Limitations of the study include the relatively small number of FGDs, although the study team found that thematic saturation was reached. The formative research was conducted near to the AHHA study area, with the aim of providing a relevant population while avoiding influencing potential trial participants. However, it was possible that the views and perceptions of participants in the formative research would differ from participants in the AHHA trial.The formative research findings provided valuable information on the feasibility of the AHHA trial, a community-based randomised, controlled trial conducted in rural Malawi, and the findings were used to inform and guide the design and delivery of the trial. The formative research was essential to inform the community sensitisation process for the AHHA trial, since participant fears could be anticipated (e.g. that the flour might affect fertility or that blood sampling may be linked to witchcraft) and the appropriate local agents could be engaged for sensitisation activities .Additional file 1. Household survey questionnaireAdditional file 2. Focus Group Discussions: Facilitator’s manualAdditional file 3. Structured Interview for Key InformantsAdditional file 4. The TIDieR (Template for Intervention Description and Replication) Checklist*


Saccharomyces cerevisiae. Examination of the telomere functions of Rap1 is complicated by the fact that it also acts as a transcriptional regulator of hundreds of genes and is encoded by an essential gene. In this study, we disrupt Rap1 telomere association by expressing a mutant telomerase RNA subunit (tlc1-tm) that introduces mutant telomeric repeats. tlc1-tm cells grow similar to wild-type cells, although depletion of Rap1 at telomeres causes defects in telomere length regulation and telomere capping. Rif2 is a protein normally recruited to telomeres by Rap1, but we show that Rif2 can still associate with Rap1-depleted tlc1-tm telomeres, and that this association is required to inhibit telomere degradation by the MRX complex. Rif2 and the Ku complex work in parallel to prevent tlc1-tm telomere degradation; tlc1-tm cells lacking Rif2 and the Ku complex are inviable. The partially redundant mechanisms may explain the rapid evolution of telomere components in budding yeast species.Rap1 is the main protein that binds double-stranded telomeric DNA in  Telomeres, nucleoprotein complexes located at the ends of eukaryotic chromosomes, protect chromosome ends from degradation, from telomere-telomere fusion events, and from being recognized as double-stranded DNA breaks . In mostSaccharomyces cerevisiae  that introduces [(TG)0–4TGG]nATTTGG mutant telomeric repeats instead of wild-type (TG)0-6TGGGTGTG(G)0-1 repeats  containing an invariable CCC core 0-6TGGGTGTG(G)0-1 telomeric repeats 0–4TGG]nATTTGG mutant telomeric repeats  ends with a telomere that consists entirely of either wild-type or tlc1-tm telomeric sequence  was longer and more heterogeneous than the wild-type control telomere (VII-L-WT)  . We asseII-L-WT) . Rap1 issequence .S. cerevisiae  assay  of the telomeres being extended during a single-cell cycle  assay . At wildX) assay . At tlc1ll cycle . This obtlc1-tm telomeres is much more dramatic than the increase seen in rif1∆, rif2∆, and pif1-m2 cells . Ablation of Rap1 leads to readily detectable telomere degradation within a few hours , and performed senescence assays with the haploid meiotic progeny. In the presence of wild-type telomeres but absence of telomerase (est2Δ and tlc1Δ), telomeres shortened until the cells senesced after 60–70 population doublings, as expected (est2Δ tlc1-tm* and tlc1Δ*). We examined the telomere length of cells that had undergone ~30 population doublings after isolation of the haploid spores and found that, upon loss of telomerase, mutant telomeres became extremely heterogeneous and degraded , which kills cells expressing URA3  . Reducedtm cells . Rap1, ttm cells . The VIIing URA3 . Strainsilencing , were usilencing , we alsoilencing . In bothRIF1, RIF2, and SIR4 would have on telomere length in tlc1-tm cells. Deletion of SIR4 results in a small decrease in telomere length in TLC1 cells, as expected  and homologous recombination (HR), we tested whether the rif2∆ tlc1-tm telomere profile would change by deleting DNL4 or RAD52, which are required for NHEJ and HR, respectively , which is responsible for recruiting Tel1 to telomeres  show a telomere profile reminiscent of type II survivors, which maintain their telomeres in a telomerase-independent manner that relies on recombination-mediated amplification of telomeric sequence . However, Rad50 is required for type II survivor formation  and Yku (Yku70-Yku80) complexes are also important for inhibiting nucleolytic degradation at telomeres , and theelomeres . The cdceric DNA , and is eric DNA . cdc13-1tm cells . Indepenof RAD52 . cdc13-1y poorly , suggesttm cells . Telomert viable , indicatrif2∆ tlc1-tm cells has also been observed in other tlc1 template mutants, such as the tlc1-476A mutant  and assessed Rif2 association by ChIP-qPCR. We find that Rif2 associates with the wild-type ITS, but not the tlc1-tm ITS . For detection of the artificial VII-L telomere, a similar procedure was performed, except that 6 µg yeast genomic DNA was digested with EcoRV and the membrane was hybridized at 42°C with a digoxigenin-labeled probe complementary to URA3 sequence. Unless otherwise mentioned  oligonucleotides containing representative sequences; Mut-1 5'-GTCATACGTCACACTGTGGTGTGTGGTGTGGTGTGTGGTGGTGGTGTGTGT-3' (37 bp) and Mut-2 5'-GTCATACGTCACACTGTGGTGTGTGTGGTGTGTGGATTTGGTGTGTGG-3' (34 bp). The respective G-rich forward strands and C-rich reverse strands were annealed in 1 mM Tris-HCl, pH 8.0, 0.1 mM MgCl2 by boiling for 2 min and slowly cooling down to room temperature. The correct annealing was guided by the 14 nt non-telomeric region indicated in italics. A ds-oligonucleotide with wild-type telomeric sequence was used as the positive binding control: 5'-TGTGGTGTGTGGGTGTGTG-3' (19 bp). The 5' ends of ds-probes were radioactively labeled with [γ-32P]-ATP using T4 polynucleotide kinase (New England Biolabs), purified on Illustra Microspin G-25 columns (GE), and diluted in 10 mM Tris-HCl, pH 7.5. In binding assays, 10 fmol of labeled probe was mixed with 1.5 µg competitor mix  in 1× binding buffer , and varying amounts (~0.1–0.4 µg) of Rap1 protein extract, in a total of 15 µl. The binding reaction was incubated at 25°C for 15 min and then loaded onto 4% polyacrylamide gels (29:1 acrylamide:bis-acrylamide) and run in 1× TBE , 150 V at 4°C. Radioactive signals from dried gels were visualized with a Typhoon FLA 9500 biomolecular imager (GE Life Sciences). Quantification of signals in the respective lanes was performed using the ImageQuant TL software. Bands were automatically detected with the settings: minimum slope 15, median filter 10, % maximum peak 0. Background subtraction was performed using the ‘rolling ball’ method with a radius of 200. The fraction of bound probe (%) was calculated in each lane as /.The recombinant escribed . The abiGALL-EST1 strain contained the tlc1-tm allele. This strain was then transformed with a PCR product containing the wild-type TLC1 allele and immediately grown on media containing glucose, which shut off expression of EST1. Successfully transformed cells were cultured in YPD media and eventually arrested in late G1 phase by the addition of alpha factor (Sigma-Aldrich). Cells were washed and resuspended in YPGal to induce the expression of wild-type telomerase, allowing the extension of tlc1-tm mutant telomeres during a single cell division.iSTEX was performed essentially as previously described , except Senescence assays in liquid culture were performed essentially as previously described .TERRA levels were measured essentially as previously described , while U The study clarifies the role of Rif2 in telomere homeostasis and how cells can extend telomeres and control senescence in the absence of the Rap1 binding to telomeres. The possibility of coping with telomere sequence modification through flexibility and redundancy of capping proteins is of general interest in terms of telomere evolution. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:Saccharomyces cerevisiae" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Jessica Tyler as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Stéphane Marcand (Reviewer #2).Thank you for submitting your article "Rif2 protects Rap1-depleted telomeres from MRX-mediated degradation in The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:– It seems clear that "recruitment" to MRX, as shown in rad50∆ mutants, is necessary for Rif2 protection in tlc1-tm mutants, but if Rif2 is able to bind DNA, is it possible to generate a DNA-binding-compromised mutant of Rif2, to see whether it can still fulfill its biological function? This will allow you to determine up to which point Rif2 binding to MRX is occurring at the DNA end to which Rif2 is being recruited independently. Alternatively, you could check whether Rif2 co-IPs with MRX .– To complement tlc1-tm rif2∆ cells with a Rif2 mutant specifically unable to interact with Rap1 , but still able to inhibit MRX would validate the model that Rif2 recruitment is Rap1-independent.–You did not investigate Sir3, even though in principle it is an additional player able to bind to Rap1-less telomeres. Could you explain at least what would be the expectation, or better yet, perform experiments with sir3 mutants to check whether its behavior is similar to that of Rif2?– Figure 6B : cdc13-1 tlc1-tm cells do not exhibit the smear observed in rif2 TLC1-tm but instead show a typical profile of type II-like recombination that suggests that Cdc13 has indeed a role in capping TLC1-tm telomeres. Are cdc13-1 tlc1-tm cells viable in the absence of Rad52?– Figure 7D: How much of the reduction in Rif2 ChIP signal in rad50∆ cells is due to telomere shortening? Adding tel1∆ and tel1∆ tlc1-tm cells as additional controls can address this issue.Other comments:– Emphasize what is how your conclusions differ from prior work, considering the large amount of information existing on this topic and the redundancy of Rap1 function with other proteins.– Figure 5 is referred to on p. 8 out of order.– Size markers are missing in Figure 1B.– Figure 1E, Rap1 still binds the mutant telomeric sequences. An estimation of the remaining affinity and its comparison with scramble sequences would help to interpret this result.– page 12, line 272-273: the comparison with type II survivors is a bit obscure, since type II survivors are MRX-dependent. This paragraph can be improved or simplified.– The sentence "We find that Rap1, Rif2, and the Ku complex work in parallel to prevent telomere degradation, and absence of all three at telomeres causes lethality" should be amended. Did you refer to WT or tlc1-tm cells ? In WT cells, Rif2 is rather a regulator of the telomerase than a capping protein and the concerted role of Rap1, KU and Cdc13 is well documented. In tlc1-tm cells, the decreased availability of Rap1 reveals (or creates?) a capping function of Rif2 that acts in parallel with KU and Cdc13.– Rif2 interacts with DNA in vitro. Is this relevant for Rif2 recruitment in the absence of Rap1? Can this hypothesis be explored? And is there a remaining telomere specificity in this recruitment?– A question emerging is whether Rif2 would have a role on DSB repair in general. There are standard NHEJ and HR assays using HO to see whether rif2- cells show DSB repair defects. Wouldn't this be a prediction if Rif2 apparently interacts and work together with non-specific telomeric proteins such as the Yku complex or MRX? Alternatively they can check Rad52 foci accumulation as an indirect measurement. Authors should at least discuss this possibility and future directions in this context.– The term "degradation" used to describe telomeres in rif2 tlc1-tm cells does not seem appropriate in the absence of further characterization. The retention of DNA in the well could be also related to the presence of replication intermediates or telomere entanglement. The smear is indeed partially abrogated by the deletion of Rad52. What are the effects of deletion of EXO1?– The use of 6Y' in the ChIP experiments makes comparison with TELVII-Lmut difficult. Another X- telomere would have been more appropriate. Explain minimally why it was not used.– Line 399-400: The reference to Bonetti et al., 2009 is not appropriate. On the contrary, this study was suggesting a role for Sae2 in the formation of the 3'-overhang, a role that was later questioned by Geli's lab .– The vocabulary used to describe the decreased binding of Rap1 to tlc1-tm telomeric repeats should be standardized taking into account that (1) Rap1 binding is not totally abolished (2) telomeres (except TELVII-L mut) retain RAP1 binding sites in their most proximal part as well as on interstitial telomeric repeats in Y' telomeres. It seems therefore more correct to speak of a decreased level of Rap1 rather than loss or lack of Rap1.– The role of recombination in the homeostasis of tlc1-tm telomeres should be discussed because stochastic recombination events or processing of intermediates could be at the origin of variations of the telomere profiles between isogenic tlc1-tm clones. In this line, the figure 5-Figure Supp2 shows a clear difference in the telomere profiles of tlc1-tm and tlc1-tm rad52 cells but this is not commented in the text. Similarly, the large heterogeneity of X-telomeres is not mentioned though it could be due to de novo acquisition of Y' sequences at least in some cases. Finally, Rad52 is required for the growth of rif2 tlc1-tm cells. Essential revisions:– It seems clear that "recruitment" to MRX, as shown in rad50∆ mutants, is necessary for Rif2 protection in tlc1-tm mutants, but if Rif2 is able to bind DNA, is it possible to generate a DNA-binding-compromised mutant of Rif2, to see whether it can still fulfill its biological function? This will allow you to determine up to which point Rif2 binding to MRX is occurring at the DNA end to which Rif2 is being recruited independently. Alternatively, you could check whether Rif2 co-IPs with MRX .It would indeed be very interesting to test a DNA-binding-compromised mutant of Rif2, but none has been described, nor can be easily predicted by sequence analysis. We feel that an extensive search for such a mutant would take months of work (at minimum), and is therefore beyond the scope of our current study. Regarding the co-IP experiment, we are unclear as to what it would accomplish because Rif2 is already known to physically interact with both the Rad50 and Xrs2 subunits of the MRX complex.– To complement tlc1-tm rif2∆ cells with a Rif2 mutant specifically unable to interact with Rap1 , but still able to inhibit MRX would validate the model that Rif2 recruitment is Rap1-independent.RIF2 or rif2-L44R,V45E,E347R under the control of the RIF2 promoter into the pRS315 plasmid. We transformed the plasmids, along with the vector control, into rif2∆ and rif2∆ tlc1-tm cells. After passaging the cells for ~100 generations, we found that expression of wild-type RIF2 partially recovered the telomere profile of rif2∆ tlc1-tm cells , and fully suppressed the growth defect . However, expression of rif2-L44R,V45E,E347R could do neither, indicating that the ability to interact with Rap1 was important for Rif2 protection of tlc1-tm telomeres, but it is also possible that the L44R, V45E, and E347R mutations disrupt the ability of Rif2 to protect tlc1-tm telomeres in a manner independent of the Rap1-Rif2 interaction. This is discussed at the end of the Results section.This is an excellent suggestion. We cloned –You did not investigate Sir3, even though in principle it is an additional player able to bind to Rap1-less telomeres. Could you explain at least what would be the expectation, or better yet, perform experiments with sir3 mutants to check whether its behavior is similar to that of Rif2?SIR3 in wild-type, tlc1-tm, and tlc1-476A strains . We saw no detectable change in cell growth. Telomere length is longer in sir3∆ tlc1-tm than tlc1-tm cells, but there is no detectable difference in telomere profile of tlc1-476A and sir3∆ tlc1-476A. These observations have now been added to the end of the Discussion, which concludes with:We deleted tlc1-tm telomeres serves a different purpose than Rif2. It will be interesting to examine the significance of Sir3 at Rap1-depleted telomeres.”“Thus, the presence of Sir3 at – Figure 6B: cdc13-1 tlc1-tm cells do not exhibit the smear observed in rif2 TLC1-tm but instead show a typical profile of type II-like recombination that suggests that Cdc13 has indeed a role in capping TLC1-tm telomeres. Are cdc13-1 tlc1-tm cells viable in the absence of Rad52?RAD52 in cdc13-1 tlc1-tm strains; the resulting triple mutant is viable but grows very poorly. The type II-like telomere profile of cdc13-1 tlc1-tm cells is also dependent on RAD52. These observations are shown in the new Figure 6—figure supplement 1. We conclude that Rad52 is important for maintaining cdc13-1 tlc1-tm telomeres.We deleted – Figure 7D: How much of the reduction in Rif2 ChIP signal in rad50∆ cells is due to telomere shortening? Adding tel1∆ and tel1∆ tlc1-tm cells as additional controls can address this issue.rad50∆ tlc1-tm cells compared to rad50∆ cells. Since both rad50∆ and rad50∆ tlc1-tm cells have similarly short telomeres , the signal loss is not due to a difference in telomere length. If we compare Rif2 ChIP signal for WT  and rad50∆ , we do see a reduction in signal at the VII-L telomere for rad50∆, but not at Y’ telomeres, so this effect seems specific for the VII-L telomere. At present, we do not have an explanation for the relatively high Rif2 ChIP signal detected for the VII-L telomere, and an experiment with tel1∆ and tel1∆ tlc1-tm is unlikely to solve this mystery. Such an experiment would be further complicated by the fact that Tel1 promotes MRX accumulation at DNA ends (PMID: 26901759), so a tel1∆ mutation may also affect Rif2 ChIP signal independently of telomere length differences.The main conclusion from Figure 7D is that Rif2 ChIP signal is lost in Other comments:– Emphasize what is how your conclusions differ from prior work, considering the large amount of information existing on this topic and the redundancy of Rap1 function with other proteins.We believe that the changes we have made in the revised manuscript have addressed this issue.– Figure 5 is referred to on p. 8 out of order.This has been corrected.– Size markers are missing in Figure 1B.A different blot with size markers is now included.– Figure 1E, Rap1 still binds the mutant telomeric sequences. An estimation of the remaining affinity and its comparison with scramble sequences would help to interpret this result.We have now included a calculation of the percentage of bound DNA for the wild-type and mutant oligos. Scrambled sequences would not be relevant to test because a huge amount of competitor DNA is used in the EMSA, as stated in the Materials and methods:E. coli DNA (~250 bp), salmon sperm DNA, and yeast t-RNA)”.“10 fmol of labelled probe was mixed with 1.5 µg competitor mix  a capping function of Rif2 that acts in parallel with KU and Cdc13.tlc1-tm telomere degradation; tlc1-tm cells lacking Rif2 and the Ku complex are inviable. We have also deleted the sentence in the first paragraph of the Discussion that read:We have amended the sentence as suggested. The new sentence reads: “Rif2 and the Ku complex work in parallel to prevent “Thus, Rap1, Rif2, and the Yku complex perform separate tasks at telomeres that are together essential for telomere capping and cell viability.”– Rif2 interacts with DNA in vitro. Is this relevant for Rif2 recruitment in the absence of Rap1? Can this hypothesis be explored? And is there a remaining telomere specificity in this recruitment?It would indeed be interesting to test whether Rif2’s ability to interact directly with DNA is important for its recruitment to Rap1-depleted telomeres. Unfortunately, a DNA-binding-defective mutant of Rif2 has not been described. Figure 8D of Cassani et al., 2016, examines Rif2 binding to presumably nontelomeric dsDNA while Figure S3 of Hailemariam et al., 2019, examines Rif2 binding to telomeric DNA. Although the exact experimental conditions differ, the results suggest that binding of Rif2 to telomeric and non-telomeric DNA is similar.– A question emerging is whether Rif2 would have a role on DSB repair in general. There are standard NHEJ and HR assays using HO to see whether rif2- cells show DSB repair defects. Wouldn't this be a prediction if Rif2 apparently interacts and work together with non-specific telomeric proteins such as the Yku complex or MRX? Alternatively they can check Rad52 foci accumulation as an indirect measurement. Authors should at least discuss this possibility and future directions in this context.tlc1-tm telomeres by inhibiting the MRX complex”). Specifically, we note that “Rif2 enhancement of Rad50 ATPase activity limits MRX-mediated tethering of DNA ends during double-strand break repair .” In the Discussion, we further note that “the role of Rif2 in DSB repair is more readily detected in the absence of Sae2 .”Rif2 indeed has a role in DSB repair, as has been documented by the Longhese lab. We described what is known in the Results section .We use the term “telomere degradation” because it was previously used by Elizabeth Blackburn’s group to describe such a telomere profile (e.g. PMIDs 10733598 and 14742705). Deletion of – The use of 6Y' in the ChIP experiments makes comparison with TELVII-Lmut difficult. Another X- telomere would have been more appropriate. Explain minimally why it was not used.We chose to examine 6 Y’ telomeres to complement the artificial VII-L telomere because (i) it allowed us to examine several telomeres at once, and (ii) it has been reported by the Petes lab that telomeres lacking subtelomeric elements  are more similar to Y’ telomeres than X telomeres, at least in terms of length regulation (PMID 10430581).– Line 399-400: The reference to Bonetti et al., 2009 is not appropriate. On the contrary, this study was suggesting a role for Sae2 in the formation of the 3'-overhang, a role that was later questioned by Geli's lab .We thank the reviewer for pointing this out to us. We have now changed the reference (PMID 34311383). This reference is a recent review article  that discusses the interplay between Sae2 and Rif2 at DSBs and telomeres.– The vocabulary used to describe the decreased binding of Rap1 to tlc1-tm telomeric repeats should be standardized taking into account that (1) Rap1 binding is not totally abolished (2) telomeres (except TELVII-L mut) retain RAP1 binding sites in their most proximal part as well as on interstitial telomeric repeats in Y' telomeres. It seems therefore more correct to speak of a decreased level of Rap1 rather than loss or lack of Rap1.tlc1-tm telomeres (not lost).In the revised manuscript, we have ensured that we always say that Rap1 levels are decreased or depleted at – The role of recombination in the homeostasis of tlc1-tm telomeres should be discussed because stochastic recombination events or processing of intermediates could be at the origin of variations of the telomere profiles between isogenic tlc1-tm clones. In this line, the figure 5-Figure Supp2 shows a clear difference in the telomere profiles of tlc1-tm and tlc1-tm rad52 cells but this is not commented in the text. Similarly, the large heterogeneity of X-telomeres is not mentioned though it could be due to de novo acquisition of Y' sequences at least in some cases. Finally, Rad52 is required for the growth of rif2 tlc1-tm cells.tlc1-tm telomeres. We had already mentioned the effect of Rad52 on the telomere profile and growth of rif2∆ tlc1-tm cells . We now also note that “rad52∆ tlc1-tm cells also have longer telomeres than tlc1-tm cells , indicating that HR is important for maintaining tlc1-tm telomeres.” The issue of telomere heterogeneity is already discussed in the Results section. In short, the heterogeneity observed in rad50∆ tlc1-tm and rad50∆ rif2∆ tlc1-tm cells is still present in the absence of Rad52, indicating that recombination is not responsible. We speculate that the heterogeneity “may be related to a previous observation that the very tip of the telomere has unique sequence features that affect telomere capping and length regulation .” In contrast, the heterogeneity seen in cdc13-1 tlc1-tm is dependent on Rad52 . We believe that the revised manuscript now more adequately discusses the role of recombination in the maintenance of tlc1-tm telomeres.We agree that Rad52-mediated recombination is involved in the maintenance of


Due to the non-invasiveness and high precision of electroencephalography (EEG), the combination of EEG and artificial intelligence (AI) is often used for emotion recognition. However, the internal differences in EEG data have become an obstacle to classification accuracy. To solve this problem, considering labeled data from similar nature but different domains, domain adaptation usually provides an attractive option. Most of the existing researches aggregate the EEG data from different subjects and sessions as a source domain, which ignores the assumption that the source has a certain marginal distribution. Moreover, existing methods often only align the representation distributions extracted from a single structure, and may only contain partial information. Therefore, we propose the multi-source and multi-representation adaptation (MSMRA) for cross-domain EEG emotion recognition, which divides the EEG data from different subjects and sessions into multiple domains and aligns the distribution of multiple representations extracted from a hybrid structure. Two datasets, i.e., SEED and SEED IV, are used to validate the proposed method in cross-session and cross-subject transfer scenarios, experimental results demonstrate the superior performance of our model to state-of-the-art models in most settings. Emotion is a physiological state of humans, which appears when people are stimulated by external or their own factors. Emotion is the basis of human daily life and work and plays an important role in human psychological development, interpersonal communication, rational decision-making, and cognition. Correct recognition of emotions is of great significance in the fields of education, medical treatment, psychology, cognitive science, and artificial intelligence. For example, in the medical field, emotion recognition can help doctors diagnose and treat patients with expression disorders; in the education field, through emotion recognition, teachers can quickly find and encourage students with poor emotions to create a more positive learning environment.Data sources used for emotion recognition can be roughly divided into two categories: non-physiological signals and physiological signals. Many scholars have carried out researches on emotion recognition using non-physiological signals such as gestures, facial expressions, eye expressions, and voice. With the continuous development of artificial intelligence technology, artificial intelligence has also shown a broader prospect in the field of emotion recognition based on EEG. Employing emerging intelligent technologies like machine learning and computer vision to realize EEG-based quantitative analysis and recognition of emotions has become a research hotspot. Existing EEG-based emotion recognition methods can be roughly divided into two categories: traditional machine learning-based methods and deep learning-based methods. Among the methods based on traditional machine learning, Support Vector Machine (SVM) , BayesiaSince EGG signals are subject-specific, there are large variations between individuals . To solvTo address these problems, we propose a cross-domain EEG emotion recognition method based on multi-source and multi-representation adaptation (MSMRA). In the data processing stage, the EEG signal is divided into multiple source domains to avoid destroying the edge distribution of the source domain data. Since the multi-source will complicate the network structure, we propose a two-partition method to aggregate two EEG data into one source domain. On this basis, the low-level domain-invariant features of different EEG data are extracted by the common feature extraction module. Then for multiple source domains, the multi-domain specific feature extractor (MDSFE) module enhances the representation capability of the source by aligning multiple representation distributions excavated from a hybrid structure. The multiple distributions are converted into a vector and fed into the classifier for final sentiment inference. Our extensive experiments on two public datasets show that MSMRA can achieve remarkable performance compared with state-of-the-art competitors.The contributions of this paper are summarized as follows.(1)We propose the MSMRA network to aggregate the EEG data of different subjects and sessions into multiple source domains, thereby improving the accuracy of emotion classification based on EEG in cross-domain scenarios.(2)A two-partition method is proposed to divide EEG data into multiple source domains, which not only significantly reduces the redundancy of the network structure but also expands the data of a single source to improve the emotion classification accuracy.(3)To obtain more high-level features of EEG signals, we constructed an MRA module to align the distribution of multiple different representations.(4)We conducted extensive experiments on two public datasets to validate the performance of MSMRA compared with state-of-the-art methods.As observed in via 62 EEG electrodes when they were watching 15 Chinese movie clips with negative, positive, and neutral emotions. To avoid subject fatigue, the entire experiment will not last for a long time, and the duration of each segment is about 4 min. Since the video can be understood without explanation and the video only triggers a single target emotion, all EEG signals will be classified into one of three emotional states . The data collection lasts for 3 different periods at approximately 1-week intervals, corresponding to three sessions, so each subject has a total of 45 EEG data trials. In addition, to ensure that the collected EEG data and the movie clips presented to the subjects have the same emotional state, an additional subjective self-assessment will be performed on each subject after the subjects watch the movie. The relevant information about the SEED dataset is shown in To evaluate the proposed methods, we conduct experiments on two public datasets, namely SEED and SEED-IV . In the Similar to SEED, SEED-IV contains a total of 15 subjects  of 62-channel EEG data. These data are the four types of emotions collected by the subjects when they watch emotion-induced movie clips, namely, neutral, sad, fear, and happy. Each movie clip lasts about 2 min. A total of three sessions are collected. Each session includes 24 trials/movies for each subject. Compared to SEED that only uses EEG signals, the database also includes eye movement features recorded by SMI eye-tracking glasses. The relevant information about the SEED-IV dataset is shown in Due to the non-stationarity of the EEG signals between sessions and subjects , the EEGIn the same session, take the EEG data of the new last subject as the target domain, and the EEG data of all the remaining subjects as the source domain. In this article, since the SEED and SEED-IV datasets have a total of 3 sessions and 15 subjects, we will group the EEG data of the subjects in pairs to form a source field, there are 7 in total, and the final subject’s EEG data is used as the target domain.In different session experiments of the same subject, we combine the EEG data of the previous two sessions into a source domain, and the EEG data of the last session is regarded as the target domain (see section “Methods” for details).The collected data is first down-sampled to a sampling rate of 200 Hz, and then a band-pass frequency filter of 0–75 Hz is applied to filter noise and remove artifacts. After the EEG sequence is converted to the frequency domain, the differential entropy features of each frequency band  and each channel (62 channels) are extracted  indicates the probability density of x.where DE represents the differential entropy features, xN, then the calculation of the DE feature can be simplified to Equation 2:We assume that the EEG signal obeys a normal distribution In this work, in both the Cross-Subject Transfer and Cross-Subject Transfer scenarios, we use DE features extracted from EEG raw data as input, and one data from one subject in one session for both databases is in the form of trials  × channel (62) × band (5) × samples, we then merge the channel and frequency band data into trials  × 310 × samples. For the SEED dataset, 15 movie clips contain a total of 3,394 samples corresponding to each session, and for the SEED-IV dataset, 24 movie clips contain a total of 851/832/822 samples corresponding to three sessions.There are two significant concepts in the domain adaptation: the source domain represents a domain different from the test sample but has rich supervision information, and the target domain represents the domain where the test sample is located, without or only have a few labels . The souAs shown in N means Nth source domain. Each source domain consists of two different EEG data, which is expressed as follows:Where S denotes the collection of source domains, STE}. The data of the source domain and the target domain are fed into a common feature extractor module c(⋅) to extract low-level feature of both The target domain can be expressed as T = {Then the low-level features of the source domain and the target domain The multi-domain-specific feature extractor has r different sub-domain-specific feature extractors, and each sub-domain-specific feature extractor can obtain various high-level feature expressions. Compared with a simple single representation, multiple representations can cover more information to better judge emotion classification. After obtaining the multiple representations of the source domain and the target domain t domain , and thecls(⋅) to realize the emotion classification of the EEG signal. Details of these modules are described below.Finally, the multi-representation vector Most existing domain adaptation methods simply aggregate all source domain data into one source domain, which often destroys the edge distribution of the source domain, or increases the difficulty of adaptation. Considering the practicality of the model, if the data of a single participant in a single session is simply divided into a source domain, this will greatly increase the complexity of the model and increase the number of parameters. Therefore, based on the above assumptions, we combine two different EEG data from one subject in one session to form a source domain, which not only satisfies the presumption that domain adaptation has a certain source marginal distribution, but also reduces the complexity of the model.This module is used to map the original data of the source domain and the target domain to a common shared latent space, and then extract the low-level features of the two respectively, which are some low-level domain invariant features that represent similar characteristics of different EEG data.After obtaining low-level features with domain invariance, we build N multi-domain specific feature extractors corresponding to N source domains. Each multi-domain specific feature extractor has r different domain-invariant feature extractors for learning multiple domain-invariant representations. Since diverse neural network structures can extract different representations from low-level features, we apply a multi-domain specific feature extractor to map each pair of source and target domain data to r latent spaces, and then in each latent space extract the high-level features of the two separately to obtain more domain-specific information. To apply DA and make this r pair of high-level features with different representations close to the corresponding latent space, we choose MMD to estimate the distance between the two domains in the latent space. The final MMD loss of the source domain is the MMD of all r latent spaces. Each source domain has r potential spaces, thus the final MMD loss is the sum of the MMD losses of all source domains and target domains, which can be expressed by Equation 11.The multiple high-level feature representations obtained from the multi-domain specific feature extractor are connected into a new vector and sent to the classifier for classification. Each source domain corresponds to a softmax classifier. We use cross-entropy to evaluate classification loss, as shown in Equation 12:In summary, we first synthesize N source domains into two sets of 2N data from one subject in one session and consider the last collected single-session single-subject EEG data as the target domain, and then all source domains and target domain data are fed into the general feature extractor to extract low-level features. Next, we use a multi-domain specific feature extractor to map the low-level features of the source and target domains to multiple different latent spaces and apply MMD loss to measure the high-level feature representations of the source and target domains in various latent spaces. Finally, we connect different expressions into a new vector and send it to the classifier for classification. The final loss function can be expressed as Equation 13:The total loss function of all source and target domain data can be defined as Equation 14:The training is based on Equation 14 and follows the algorithm shown in In our experiment, we use the public SEED dataset and SEED-IV dataset to classify emotions. In addition, we not only compare our proposed method with the baseline method but also conduct a large number of ablation experiments and exploratory experiments.The SEED dataset and SEED-IV dataset are first pre-processed (mentioned in section “EEG Data Pre-processing”) to obtain DE features, and reshaped into trials  × 310 × samples. Then we normalize the dimensions of samples, and the ablation experiment in section “Ablation Study” will confirm the effect. After that, we divide the data into N source domains and one target domain in pairs in the manner mentioned in section “Methods.” As described in section “Methods,” the various modules of MSMRA have many details. For the common feature extraction module, since we do not have as many samples as the original data, so in this layer, we take a simple three fully connected layers, which reduce the dimension from 310 (62 × 5) to 64, each fully connected layer is followed by a LeakyReLU activation function . We alsoWe compare the MSMRA model with the latest various competitors on the SEED dataset and SEED-IV dataset, including Deep domain confusion (DDC) , which tAll the experimental results of the two data sets are shown in •In the SEED data set, our proposed method MSMRA surpasses most of the current state-of-the-art methods in Cross-subject and Cross-session scenarios. It can be seen that our method improves the accuracy of at least close to 2% compared to other methods in the Cross-session scene, and for the first time exceeded the 90% accuracy rate, as far as we know. In the Cross-subject scenario, although our method is 2% lower than MS-MDA, it is better than all other methods, which shows that our method is competitive.•On the SEED-IV dataset, it can be clearly seen that our proposed method significantly exceeds all other competitors, and each has improved by at least 11 and 10% compared to others in Cross-session and Cross-subject scenarios, it is a piece of exciting news. From the results, we can infer that our method can also achieve good classification results on small sample datasets because multiple high-level features containing rich information are extracted from the MDSFE module.To further understand the MSMRA model we proposed, we performed the ablation experiments of removing the initial normalization of the data and removing the MDSFE module on the SEED and SEED-IV datasets respectively, and evaluate the performance of the ablated model. The hyperparameters of all ablation experiments are consistent, and the experimental results are shown in In general, the current emotion classification based on EEG signals is hindered due to the instability of the internal data of Cross-session and Cross-subject when collecting data. Domain adaptation increases the classification accuracy of cross-domain data by finding a mapping relationship to map the source domain and target domain to a latent space, and minimizing the distance between the two. Based on the above, we propose a multi-source and multi-representation domain adaptive network (MSMRA), which regards EEG signals as multiple source domains rather than simply as one source domain, which satisfies the assumption that the source domain data has a certain marginal distribution. It can be seen from https://bcmi.sjtu.edu.cn/~seed/index.html.Publicly available datasets were analyzed in this study. This data can be found here: Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.JC wrote the main manuscript and conducted the experiments. XH and CY participated in the writing of the manuscript and modified the English grammar of the article. SC and ZL made the experiments. ZW analyzed the results. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cassava, a tropical storage-root crop, is a major source of food security for millions in the tropics. Cassava breeding, however, is hindered by the poor development of flowers and a low ratio of female flowers to male flowers. To advance the understanding of the mechanistic factors regulating cassava flowering, combinations of plant growth regulators (PGRs) and pruning treatments were examined for their effectiveness in improving flower production and fruit set in field conditions. Pruning the fork-type branches, which arise at the shoot apex immediately below newly formed inflorescences, stimulated inflorescence and floral development. The anti-ethylene PGR silver thiosulfate (STS) also increased flower abundance. Both pruning and STS increased flower numbers while having minimal influence on sex ratios. In contrast, the cytokinin benzyladenine (BA) feminized flowers without increasing flower abundance. Combining pruning and STS treatments led to an additive increase in flower abundance; with the addition of BA, over 80% of flowers were females. This three-way treatment combination of pruning+STS+BA also led to an increase in fruit number. Transcriptomic analysis of gene expression in tissues of the apical region and developing inflorescence revealed that the enhancement of flower development by STS+BA was accompanied by downregulation of several genes associated with repression of flowering, including homologs of TEMPRANILLO1 (TEM1), GA receptor GID1b, and ABA signaling genes ABI1 and PP2CA. We conclude that flower-enhancing treatments with pruning, STS, and BA create widespread changes in the network of hormone signaling and regulatory factors beyond ethylene and cytokinin. Manihot esculenta) is a perennial tropical plant of the Euphorbiaceae family cultivated as an annual crop for its starchy storage roots  in some genotypes with traits of interest, flowering is late or in some cases there is no flowering at all; (2) premature abortion of inflorescences and flowers before anthesis; (3) disproportionately large number of male flowers and in some cases no female flowers; (4) non-synchronous development of flowers among genotypes; and (5) low probability of fruit development even when flowering and pollination are successful  under field conditions in Nigeria. We also analyzed the transcriptome of young apical tissues in control plants and in plants receiving either pruning or STS + BA, or the combined set of treatments. We specifically studied the expression pattern of differentially expressed genes relevant to hormone signaling and flower development. Our studies indicated that STS or pruning increase flower numbers over the control, but have no effect on the female-to-male ratios. Conversely, BA was able to almost completely feminize developing flowers, while it did not increase the total number of flowers. Combining BA with STS and pruning provided maximal enhancement in the number of female flowers; this methodology has the potential to assist cassava breeding programs. The observed transcriptome changes in response to these treatments provided further insight into the effects on hormone signaling beyond ethylene and cytokinin and provides insight that will assist future investigations in cassava and other plants.Three genotypes, representing three flowering times and flower prolificacy were used for PGR studies. These were IITA-TMS-IBA980002 (early and profuse), IITA-TMS-IBA30572 (intermediate flowering time and quantity), and TMEB419 . In this article, these genotypes will be referred to as 0002, 30572, and 419, respectively. The genotype 0002 was used to optimize the method of PGR application in 2017. This experiment was conducted between June and December of 2017. Experiments examining the effect of PGRs on female flower development were conducted between June and December of 2018 and 2019.All experiments for phenotyping were conducted under field conditions at the International Institute of Tropical Agriculture (IITA), Oyo State, Ibadan . The soil was an Alfisol   dropwise with four parts 0.1 M sodium thiosulfate (Na2S2O3), yielding a 20-mM stock solution. The stock solution was diluted with distilled water to the required concentration as specified in each experiment. BA solution was prepared by diluting a 1.9% (w/v) BA stock  with distilled water to respective concentrations. PGRs were applied either by spray (about 5 ml) to shoot apex, every 7 days or by the “petiole feeding” method every 14 days. In the petiole feeding method, the leaf blade was removed using a surgical scissors and the petiole was inserted into a 15-ml conical-bottom centrifuge tube  containing 5–10-ml of PGR solution. PGR was taken up via the petiole into the xylem from which it was distributed internally to target organs in the apical region (Silver thiosulfate (STS) was prepared by mixing 1 part 0.1 M silver nitrate (AgNOl region . PetioleShoot apexes were inspected weekly using headband magnifier glasses (10 × magnification) to identify forking events. In plants that had forked, young reproductive branches, 2 cm or smaller, were excised carefully without damage to the inflorescence using surgical blades, as previously described  and plants in each plot represented the complete set of treatments in a two-factor design with five BA timing treatments and two STS treatments, creating 10 treatment combinations. At 6 weeks after planting, these 10 PGR treatments were randomly assigned to each plot, with each plant as the experimental unit. BA solution at 0.22 mM was spray-applied at four flower developmental stages; the timing of BA application is summarized in In 2018 and 2019, experiments were conducted using a split–split–split plot design. The experiment was comprised of six plots (blocks), each of which was split into three subplots with one of the three genotypes in each. Each genotype subplot was split into five PGR treatments   as described by Hyde et al.  and expo2; about 0.15 ml of the powder was vigorously mixed for 5 min with 0.4 ml of CTAB extraction buffer [1% [w/v] CTAB detergent, 100 mM Tris-HCl [pH 8.0], 1.4 M NaCl, 20 mM EDTA, and 2% [v/v] 2-mercaptoethanol]; 0.2 ml of chloroform was added and mixed for 1 min, tubes were centrifuged at 14,000 g for 10 min and 200 μL of the top layer was removed to a new tube. To these samples was added 700 μL of Guanidine Buffer  and 500 μL of ethanol (100%). This mixture was applied to silica RNA columns , then washed with 750 μL of each of the following: (1) MOPS-ethanol Buffer [10 mM MOPS-HCl [pH 6.7], 1 mM EDTA, containing 80% [v/v] ethanol], (2) 80% ethanol (twice), and (3) 10 μL RNAase-free water to elute the RNA (twice). The RNA quality was evaluated with a gel system . The three RNA-seq libraries were prepared from ~500 ng total RNA at the Cornell Genomics facility (http://www.biotech.cornell.edu/brc/genomics-facility) using the Lexogen QuantSeq 3′ mRNA-Seq Library Prep Kit FWD .Total RNA was extracted from each sample by a modified CTAB protocol. Samples were ground to a fine powder in a mortar and pestle chilled with liquid N5 demultiplexed reads were excluded from the gene counting analysis. Illumina adapters were removed from the de-multiplexed fastq files using Trimmomatic (version.36)  using the STAR aligner . Samples with fewer than 1.5 × 10M. esculenta genome v7 and the Arabidopsis genome as presented at Phytozome13   and Flowering Interactive Database (FLOR-ID) (http://www.phytosystems.ulg.ac.be/florid/)   . This implies that STS-treated plants were four times more likely to have increased total flower numbers compared with non-STS-treated plants. STS treatment also increased both the number of female flowers , and fruits: . BA treatments alone did not affect the total flower and fruit numbers (P ≤ 0.001) at increasing the fraction of female flowers relative to the no-PGR controls  petiole-fed BA; (2) petiole-fed STS; and (3) a mixture of STS and BA . We also increased the BA concentration to 0.5mM and studied PGR effects with or without pruning . The effect on individual genotypes is shown in From Experiment I, above, we obtained evidence that STS treatment increases flower and fruit numbers and that spraying BA to the shoot apex increased the proportion of flowers that were female. In Experiment II, we tested two additional factors. First was the potential effect on flowering of pruning branch shoots that arise just below the apical meristem where inflorescences initiate . In contrast, BA-only did not affect the total number of flowers relative to the control whether applied by spray (BA) or via the combination of apical spray and petiole uptake methods (BA+BA). STS-inclusive treatments  in the absence of pruning significantly (P ≤ 0.05) increased total flower numbers relative to the control . Pruning plus STS had the largest number of flowers, with an apparent additive effect of the two treatments  more female flowers than BA-only treatments and the no-PGR unpruned control, but was equivalent to BA+BA and control with pruning. STS-inclusive treatments with pruning had the highest number of female flowers  fruit numbers relative to the control in both the unpruned and pruned plants.As with other traits, fruit numbers in the BA-only treatments were not significantly different from control in the absence or presence of pruning . STS-incIn the controls, female flowers represented 35–40% of the flowers in both unpruned and pruned plants, with the remainder male flowers . In contP ≤ 0.05) total and female flower numbers by almost twofold compared with the PGR- or pruning-only treatments. As with field studies, BA-containing treatments increased the number of female flowers and the proportion of flowers that were female  STS+BA without pruning; (b) STS+BA with pruning, (c) pruning without PGR treatment; and (d) control (no PGRs and no pruning). This study was conducted on the model genotype 0002, in a controlled environment greenhouse. The findings in the growth chamber were consistent with those in the field. Similar to the field study, the controls  initiated inflorescences, but did not produce any mature flowers, while pruning or STS+BA as sole treatments produced a modest number of flowers ; in the e female .n = ~pruning), (ii) examining transcriptome changes due to PGR alone (design = ~PGR), and (iii) examining transcriptome changes due to the combination of pruning and PGR (design = ~pruning + PGR). This analysis revealed that a larger number of significant (Padj ≤ 0.05) transcriptome changes occurred in response to PGR or PGR+pruning treatments than pruning alone  (adj ≤ 0.05) in response to pruning and PGR indicated that 2,448 genes were upregulated while 2,952 genes were downregulated. Functional analysis revealed that in the PGR-treated vs. controls, PGR-upregulated genes were enriched in pathways involving cell proliferation, cell maintenance, and biosynthetic processes; while PGR-downregulated genes were enriched in pathways involved in plant hormone signal transduction, photosynthesis, and degradation metabolism, among others  examining transcriptome changes due to pruning alone (desigctively) –5. Princctively) . This grg others .Using the DESeq2 package, we identified genes differentially expressed under the full model (design = ~pruning + PGR) . Among tadj ≤ 0.05) in response to pruning as the sole treatment (2) was used to identify genes from among the genes for which differential expression was found (Padj ≤ 0.05). This yielded a set of 63 genes that were upregulated in both PGR and pruning treatments and 224 genes that were downregulated in both treatments, relative to the control. These genes were subjected to enrichment analysis  and aromatic amino acid biosynthesis . The two categories with the strongest evidence for enrichment among downregulated genes were response to oxygen-containing compound  and response to abiotic stimulus .Twenty-one genes were differentially expressed (Preatment . This sereatment . Also upadj ≤ 0.05) in our samples  in response to our experimental treatments   in response to PGR treatment or pruning (Thirty of the 37 putative MADS-Box MIKC transcription factors (TFs) in cassava  and Arabidopsis, failure to develop fruit in the absence of pollination has been associated with ovary senescence arising from increased ethylene biosynthesis in ovaries , blocking ethylene perception by STS extended ovule lifespan and increased the chance of developing fruit either by pollination or by parthenocarpy in response to gibberellins  affected genes with substantial response to STS+BA treatments were those downregulated relative to their expression in the control. Among the cluster of such genes with the largest fold change  and Magnolia x soulangeana, GID genes are expressed at elevated levels when floral induction takes place  showed that GID and GA2ox2 homologs had higher expression in the control buds than in buds stimulated to form flowers, and this response was interpreted to act in feedback regulation of GA synthesis  and Gibberellin 2-oxidase2 (GA2ox2) were downregulated by PGR treatments , 10. In Jatropha curcas upregulated a PP2C homolog  floral buds, STS decreased expression of PP2C and ethylene-generating treatment increased PP2C  and ABSCISIC ACID HYPERSENSITIVE GERMINATION1  . In contThe expression levels of cassava homologs of INDOLEACETIC ACID-INDUCED PROTEIN16  and TIFY10b   to play a role in carpel development , a repressor of floral development. TEM1 is a member of the RAV family of transcription factors that are involved in suppressing flowering by downregulating FT, and also have roles in abiotic stress responses  and cytokinin (BA) substantially improved cassava female flower and fruit development, and has potential value in promoting reproductive development in cassava breeding programs. It also distinguished treatment effects: BA feminizes floral development and increases the fraction of female flowers, whereas pruning and STS treatment maintain inflorescence development such that more flowers and fruits are produced. Transcript profiling of tissues in the inflorescence region indicated that treatments affected signaling components in multiple hormone and flowering regulatory pathways, including those involving auxin, GA, ethylene, jasmonate, and ABA, with the predominate response in these pathways a downregulation relative to controls. The finding that pruning and BA+STS downregulates TEM1 and other factors that have been found to inhibit flowering advances our understanding of potential regulatory systems that are involved in cassava flowering and are modified by pruning and PGR treatments.https://www.cassavabase.org, a website maintained by the Next Generation (NEXTGEN) Cassava Breeding Project. The data may be accessed at ftp://ftp.cassavabase.org/manuscripts/Oluwasanya_et_al_2021 and are available in accordance with Creative Commons Attribution (CC-BY) license.The datasets presented in this study can be found in online repositories and are available from Cassavabase, DO, PK, and TS obtained funding. PK supervised fieldwork in Nigeria. TS supervised greenhouse and lab work in the United States. DO, OE, and PH conducted the field and greenhouse experiments. DO analyzed the data. DO and TS wrote the manuscript. All authors contributed to the article and approved the submitted version.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.


Homo sapiens. In particular, E2A, a member of the E protein family, plays a major role in the transcriptional regulatory network that promotes the differentiation and development of B and T lymphocytes. E2A-mediated transcriptional regulation usually requires the formation of E2A dimers, which then bind to coregulators. In this review, we summarize the mechanisms by which E2A participates in transcriptional regulation from a structural perspective. More specifically, the C-terminal helix-loop-helix (HLH) region of the basic HLH (bHLH) domain first dimerizes, and then the activation domains of E2A bind to different coactivators or corepressors in different cell contexts, resulting in histone acetylation or deacetylation, respectively. Then, the N-terminal basic region (b) of the bHLH domain binds to or dissociates from a specific DNA motif (E-box sequence). Last, trans-activation or trans-repression occurs. We also summarize the properties of these E2A domains and their interactions with the domains of other proteins. The feasibility of developing drugs based on these domains is discussed.E proteins are transcriptional regulators that regulate many developmental processes in animals and lymphocytosis and leukemia in  Drosophila melanogaster, and HLH-2 expressed in Caenorhabditis elegans . W. W28]. WAlthough AD1 interacts with multiple domains on CBP and ETO, the roles of KIX, NHR1, TAZ2 and AD1 are generally independent and cooperative. The NHR1 binding site on AD1 is consistent with the KIX binding site, and the KIX domain of CBP and the NHR1 domain of ETO competitively bind AD1. Although both the KIX and TAZ2 domains of CBP bind to AD1, the key residues in their identified regions that bind to AD1 are slightly different; Leu20 is the key residue for the KIX interaction, whereas Leu19 and Phe22 are the key residues for the TAZ2 interaction , 28–30.In addition to AD1, E2A contains three other ADs, namely, AD2, AD3, and DES. AD2 is located in the central region of the E2A protein. In the detailed mechanism of E2A activation and transcription reported by Aronheim et al., AD2 was first identified together with AD1 . AD2 is The DES domain (residues 100–239) is adjacent to AD1. It is another conserved ETO-interacting region that was discovered by Guo et al. in 2009 . NHR2 biAD3 is located between the DES domain and AD2. Chen et al. studied the mechanism by which transcription factor II D (TFIID) functions as a coactivator through direct interactions with E proteins. TFIID is a multi-subunit complex composed of TBP and TAFs and was first discovered in AD3 of the E proteins . E proteXenopus proteins showed that this domain consisted of 52 identical amino acid residues and 10 relatively conserved residues. The basic region is almost identical to the E47 bHLH domain of humans, rats, mice, chickens and Xenopus, except for Ser552 in the Xenopus protein, which is replaced with Ala (A) in proteins from other species. Most residues of the HLH region in the VDIR bHLH domain are identical among these species, and a few conserved or variable residues are scattered in several motifs   consists of two helixes and a loop located in the middle of these two helixes. The first helix is composed of 31 amino acid residues from the N-terminus of the bHLH domain. This helix includes a basic region and helix 1 of the HLH region. The basic region is composed of 13 amino acid residues from the N-terminus of the first helix . Helix 1 is formed from 18 residues near the C-terminus of the first helix (Arg560–Leu577). The loop structure consists of seven amino acid residues adjacent to helix 1 (Lys578–Thr584). The remaining residues in the bHLH domain (Lys585–Leu608) constitute the other helix   proteins. ID proteins are also known as the class V helix-loop-helix (HLH) family. Unlike class II bHLH proteins, the HLH domain of ID proteins lacks a key DNA recognition region. When E2A dimerizes with ID proteins, it cannot bind to DNA stably, resulting in the failure of E2A transcriptional regulation [In addition to class II bHLH proteins, E2A proteins can dimerize with gulation , 48. Difgulation .Overall, E2A functions as a transcription regulator, which requires the collaborative participation of ADs and the bHLH domain. First, E2A forms a dimer with bHLH proteins through their HLH regions, and the hydrophobic residues on the HLH region play important roles in the formation of the dimer, followed by AD1 binding to the KIX or TAZ2 domain of CBP through the PCET motif. Thereafter, CBP presents histone acetyl-transferase activity, and the promoter region is exposed , 29, 50.


Exploring and controlling topological textures such as merons and skyrmions has attracted enormous interests from the perspective of fundamental research and spintronic applications. It has been predicted theoretically and proved experimentally that the lattice form of topological meron-skyrmion transformation can be realized with the requirement of external magnetic fields in chiral ferromagnets. However, such topological transition behavior has yet to be verified in other materials. Here, we report real-space observation of magnetic topology transformation between meron pairs and skyrmions in the localized domain wall of ferrimagnetic GdFeCo films without the need of magnetic fields. The topological transformation in the domain wall of ferrimagnet is introduced by temperature-induced spin reorientation transition (SRT) and the underlying mechanism is revealed by micromagnetic simulations. The convenient electric-controlling topology transformation and driving motion along the confined domain wall is further anticipated, which will enable advanced application in magnetic devices. Merons and Skyrmions, two topological spin-textures, have attracted a lot of interests due to their potential use in information storage. Here, the authors demonstrate the transformation of Meron pairs into Skyrmions without an applied magnetic field within domain walls of GdFeCo films. Their nanometer size and electric-current detection/manipulation/generation behavior endow them as competitive candidates for encoding the information bits with dense integration and energy efficiency in the next generation spintronic devices. By exploring and tuning the dedicated interplay among spin–orbit coupling and the competition of magnetic interactions, the materials hosting these topologically nontrivial magnetic textures have been extended from initial chiral magnets with Dzyaloshinskii–Moriya interaction (DMI) to diversified ferromagnets with different stabilization mechanisms17. Currently, the transverse motion of skyrmions in response to external electric current drive in ferromagnets, known as skyrmion Hall effect20 is regarded as a hurdle in promoting skyrmion applications in racetrack memories, where it is preferable for skyrmions to move along the racetrack. Theoretically, compensated antiferromagnets with two equivalent but antiparallel magnetic subsystems can perfectly cancel the transverse motion and allow skyrmions to move along the electric current direction23. However, it is hard to experimentally visualize and study intrinsic antiferromagnetic (AFM) skyrmions by using the standard imaging methods because of their net-zero magnetization. Therefore, it is of great importance to figure out distinctive features and properties of the topological textures in ferrimagnetic materials with partially compensated magnetic moments25.Topological magnetic textures including (anti)meron28. Recently the topologically nontrivial meron29 and skyrmions33 localized in magnetic domain walls have attracted considerable interest. The intrinsic canted spin configuration and the confining potential in the domain wall might advance the specialties of these magnetic textures and restrict them to move only within the domain wall. In this way, the detrimental skyrmion Hall effect can be avoided. The amorphous ferrimagnetic GdFeCo films36 regain the focus in skyrmion generation and manipulation due to the antiferromagnetically coupled sublattices37 and possible DMI35. In this work, we demonstrate the topological transformation between meron pairs and skyrmions in the domain wall via the temperature-induced spin reorientation transition (SRT) and their electric control capability, which does not require any external magnetic field unlike that in chiral lattices38.Due to the compatibility with standard spintronic devices, magnetic thin films or multilayers present good application possibilities, where magnetic bubbles, skyrmions, vortices, and together with magnetic domain walls can be easily tuned by balancing the ferromagnetic exchange, spin anisotropy, and dipolar energy through layer thickness and deposition conditionsx15+(Fe94Co6)x85−  films with thickness about 40 nm are prepared with different RE-to-TM ratios in composition to tune its perpendicular magnetic anisotropy (PMA) x85− and the topological transformation can be adjusted to near room temperature for convenient applications x85− (x = 0.2). It has been known that the topological nature of the magnetic texture is intimately related to the spin anisotropy with (anti)merons3 usually in the magnets with easy plane anisotropy and skyrmions/bubbles in magnets with easy axis anisotropy18. The internal magnetic structure evolution inside the domain wall due to the SRT is unraveled by in-situ L-TEM images  or N = 1/2 (antimeron pair)41. Here, we only demonstrate the spin configuration of the meron pair to present an equal possibility of antimeron pair. When the spin anisotropy is flipped from in-plane to out-of-plane near the SRT, the meron with core polarity antiparallel to the out-of-plane spins remain stable and evolve into skyrmions with topological charge −1, which is well corroborated in the following simulations  software. The in-plane easy axis anisotropy at 243 K can be inferred from the ripple domain and the elongated meron or skyrmion configurations, where the region with spins aligning along the easy axis direction is longer compared to that perpendicular to the easy axis. Also note that domain walls become unstable without the in-plane easy axis anisotropy, because of the in-plane spin rotation symmetry. Across the SRT, an extra out-of-plane easy-axis anisotropy develops and increases with temperature. The above consideration motivates us to introduce the following spin anisotropies all Fig.  is used all Fig.  and 6. Tall Fig. . The sim46. The skyrmion number (Ns) integrated over the x-direction and the top half part along y-direction of the simulation images in Fig. 7n is the direction vector of magnetization. The Ns of a meron is −1/2 despite that the spins away from the meron core are parallel to the domain wall. When the out-of-plane easy axis anisotropy increases across the SRT, the meron with core spin polarization in the same direction (red +mz) as the out-of-plane spin component becomes unstable and shrinks, because there is no energy barrier separating this type of meron and the fully polarized state. At a stronger out-of-plane easy-axis anisotropy, this type of meron disappears completely.These periodic Bloch lines generated in the in-plane Bloch domain walls are different from those in a perpendicular bubble or stripe domainsmz) in the wall, the meron remains stable and evolves into a full skyrmion through continuous growth of the topological charge from −1/2 to −1 x85− (x = 0.2) at different temperatures x85− (x = 0.2)  Fig. . The ove19 is known as the skyrmion Hall effect in a ferromagnet. The ferrimagnetic materials might offer platforms to minimize the skyrmion Hall effect due to the partially compensated magnetization46. Because of the confining effects for domain wall skyrmions, the only movement along the wall is allowed, thereby, avoiding the detrimental skyrmion Hall effect. Theoretically, the skyrmion motion guided by the domain wall is verified in the simulation 6. Here, we leave the underlying mechanism of the current induced SRT and skyrmion motion in nano-fabricated films for future study.The significant transverse deflection of electric-driven skyrmions even within a patterned narrow path33. Our results, therefore, point to a promising direction of controlling the spin topology by simply varying temperatures or electric current at zero field. The guided motion of skyrmions inside the domain wall and the contribution of partially compensated magnetization in the ferrimagnetic film will open great opportunities for a deep understanding of topological Hall effects and their application in electronics, spintronics storage. Given that domain walls are ubiquitous magnetic structures with potential fast DW dynamics, our findings provide critical insights in discovering, yet hitherto unknown, topological spin textures and magnetic phase transition in the domain wall.We have observed the direct topological transformation between meron pair and skyrmion in the domain wall of amorphous ferrimagnetic GdFeCo, which is enabled by spin anisotropy change across the SRT. The critical role of domain walls in generating topological textures and transformation without the requirement of external magnetic fields is revealed through experiments and micromagnetic simulation and agrees with the recently proposed theory about domain wall skyrmionx15+(Fe94Co6)x85−(40 nm)/Pt(3 nm) with different composition ratios of Gd to FeCo were grown by magnetron sputtering on 10-nm-thick Si3N4 membrane windows for direct TEM observation and simultaneously on Si/SiOx wafers for physical property measurements. The higher composition of transition metal (FeCo) presents stronger PMA, therefore, four representative films with different SRT temperatures are selected. The magnetic hysteresis (M-H) loops of the Si/SiOx-based samples were measured at different temperatures using a superconducting quantum interference device magnetometer (SQUID). The amorphous state of GdFeCo thin film is characterized by electron diffraction.The ferrimagnetic thin films Pt (3 nm)/Gdx15+(Fe94Co6)x85−  because the sample cannot be cooled first before heating up by using a high-temperature TEM holder. The magnetic domain wall contrast is imaged under the convergent or divergent electron beam due to the interaction of the electron beam with the in-plane magnetization. The under- and over-focal images were recorded by a charge-coupled device (CCD) camera to work out phase images and then the quantitative in-plane magnetization component on the basis of the TIE equation using commercial software QPt. The colors and arrows depict the magnitude and orientation of the in-plane magnetization according to the color wheel. The skyrmion manipulation behavior by an electric current is conducted using a double-tilt electrical TEM holder with two electrical conducting blocks at two sides of the TEM sample. The dc current was supplied using a source-measure unit instrument (Keithley 2601B).The magnetic domain structures were studied by using a JEOL-dedicated Lorentz TEM (JEOL2100F) equipped with a liquid-nitrogen or high-temperature TEM holder for temperature manipulation. No magnetic field is applied during the L-TEM observation due to the special design of the objective lens. There is about a 10 K temperature difference between L-TEM observation and the magnetic properties measurement in Gd48:The TIE equation is composed of the following two equationsI and phase Ф. λ is the spectrally weighted mean wavelength of illumination. In our experiment, we put the under-focused and over-focused pictures in software QPt to find the value of n is the unit vector along the beam direction, M is the magnetization vector and t is the local sample thickness.The first equation reveals the relationship between intensity 49 to investigate the dependence of magnetic structures on parameters, such as PMA (Ku) and saturated magnetization (Ms) for a fixed exchange stiffness (A = 7 × 10−12 J/m). To aim at the magnetization evolution towards energy minimum for the given conditions, the ferrimagnetic sample is simplified as a ferromagnetic system with reduced saturation magnetization and uniform exchange term. The size of the thin plate GdFeCo film for simulation is about 1400 × 500 × 40 nm3  with periodic boundary conditions in the y-direction and open boundary conditions in the x-direction. According to the simulation, the distance between each Bloch line at steady-state scales around 250 nm based on the experiment parameters  direction is added to stabilize the domain wall during simulation. We then change the out-of-plane anisotropy and study the evolution of the magnetization configuration. We used an exchange stiffness parameter of about 7 pJ/m to make the domain width wider and simultaneously varied Ms, Ku to study the domain evolution. The corresponding L-TEM image simulation is carried by the software called micromagnetic analysis to Lorentz TEM simulation (MALTS)50.Micromagnetic simulations are performed using the OOMMFSupplementary informationSupplementary Video 1Supplementary Video 2Description of Additional Supplementary Files