To investigate the impact of Appreciative Inquiry (AI) on learning and developing together for improving the quality of care in a nursing home, and to explore experiences following the four phases of the AI cycle, an AI was performed as part of participative action research. Interviews, group discussions, creative methods of storytelling, and reflections were used in the AI sessions. Staff members were interviewed to evaluate the AI process. All of the interviews were recorded and transcribed. Data were analyzed thematically using Maxqda and were discussed by the interviewers until consensus was reached. The sessions resulted in ten action proposals to improve healthcare. One proposal was worked out in agreements on cooperation. The evaluation revealed that the sessions have given a boost to the team spirit, and involvement at the location leading to more cooperation and communication during the shifts. The evaluation indicated that it is important to convert the action plans of the sessions into actions, which are then evaluated. AI was advised as a way of learning where everyone is involved, and mutual agreements are made. This study has shown that AI can be a valuable way to support learning and development to promote the quality of care in a nursing home. Care in nursing homes is becoming ever more complex, due to the frailty and multimorbidity that many of these residents suffer from. This also makes care in nursing homes time-consuming . KeepingThe provision of person-centered care, which is a dominant indicator of high-quality care, is not easy to achieve . Rutten There is a growing international awareness of the need to perform high-quality care in nursing homes . SkilledThe above elements of learning can also be found in the self-determination theory of Ryan and Desi ; people’While focusing on long-term care for older people, Van Lierop et al.  mapped tAppreciative Inquiry (AI) is a promising approach that is able to bring people together, and for them to connect openly and honestly with each other to achieve change together. AI is defined as a narrative-based process of positive change . AI is aThere are five principles underlying a good AI process: positive, constructionist, simultaneity, poetic, and anticipatory ,17. FirsAI is increasingly being used as a method to conduct research into healthcare and as a way to achieve change in healthcare in general . It is aAI is an upcoming approach in the field of nursing homes in the Netherlands. For example, Sion et al.  advise uTo the best of our knowledge, there have been no studies using the complete 4D-cycle and the principles of AI in Dutch nursing homes to learn about and develop the quality of care. Therefore, the findings from this study contribute to knowledge regarding AI as a method to learn. The goals of this research are: (a) to investigate the impact of a complete AI cycle on learning and improving the quality of care in a nursing home, and (b) to explore the experiences following the entire cycle of AI with a care team in a Dutch nursing home.Within a participative action research design (PAR), AI was chosen as the method for this study. AI, such as PAR, is an action research methodology that involves its subjects as co-researchers , is baseThe study took place from September 2021 until July 2022 in Bovenkerk, a nursing home in the Netherlands that belongs to Zonnehuisgroep Amstelland. This organization was chosen because the second author is affiliated with the organization as a professor of Health and well-being of the frail elderly. Furthermore, the organization encourages conducting scientific research in the care of older people.This nursing home is formed of eight units, which are registered for 48 residents with dementia and employ 90 staff members. This nursing home uses the concept of small-scale living where eight residents live in one unit in a family atmosphere, with a living room and a private bedroom for each resident. The care is provided by a diverse group of caregivers—mainly healthcare assistants, certified nurse assistants, and a few registered nurses. The care is supported by ward assistants and activities coordinators. Management, other disciplines, such as a geriatrician, psychologist, physiotherapist, and also facility staff are also present at the location.The project’s researcher introduced the concept of AI to the workgroup of the project as a promising way of learning and improving. This workgroup included the management of the location, the researcher, the team coach, the quality manager, the practical trainer, the activity supervisor, and one team member (a registered nurse). To prepare and facilitate AI, a trio was composed of the workgroup that included the researcher, the team coach, and a team member. In this way, knowledge about AI and research (researcher), group processes (team coach), and information about the team members and connection with the team (team member) were brought together.All of the employees at the location were invited to participate in three AI (team) sessions. However, because of potential problems with the continuity of care for the residents, not everyone could get out of care at the same time, and therefore two parallel sessions were always organized.Data were collected throughout the AI process by the researcher, the facilitators of the AI process, the participants in the team sessions, and the workgroup of the research project. A variety of  data collection methods has been used as appropriate to the method of AI, including interviews , group discussions , creative methods (dream), and storytelling . Outcomes were written on whiteboards, which have been preserved, and were worked out by the facilitators. Recordings of the explanation of the dream phase were transcribed. The data collection methods of the AI sessions are outlined in To evaluate the AI process, the three facilitators interviewed staff members. To ensure that we got a representative sample from the participants of the AI sessions, some respondents were chosen deliberately, the psychologist, the activity coordinator, and management, for example. The interviews of the care personnel were completed with employees who were working when the facilitators were on site (convenience sample). Then the planning was checked to plan interviews with the care workers who had not yet been interviewed. This was continued until data saturation was reached. Care workers were interviewed individually or, for efficiency reasons, in small groups. See the specification of the interviews in Semi-structured interviews were chosen to enable the participants to ask questions if necessary .An interview protocol was drawn up to ensure the three facilitators conducted the interviews in the same way, which benefits the quality of the data see .The three facilitators themselves also did an evaluation of the AI sessions by interviewing each other based on the topic list that was also presented to the team members.All interviews were conducted orally, audiotaped, and then transcribed. This study is conducted following the Standards for Reporting Qualitative Research (SRQR) .The data analysis involved a participative approach based on Appreciative Inquiry strategies, such as reflection, and deductive and inductive coding . In eachIn addition, the facilitators and the working group reflected on the outcomes and the process of both parallel sessions going through the data, interpreting possible meanings, and identifying important themes. These themes were brought back into the AI sessions to check whether the interpretation of the facilitators and working group matched the interpretation of the team members.The steps of thematic analysis by Braun and Clarke  were useRigor was considered in different ways in the research. In the setup of the research, a specific trio of facilitators was chosen, namely the researcher, the team coach, and a team member of the location. In the process of data collection and data analysis, they were able to inspire, check, and correct each other. Secondly, an experienced external AI facilitator was coaching the three facilitators, ensuring the careful completion of the analysis steps in an AI phase and the proper follow-up of the various AI phases was guaranteed. A third step was to ask the working group to what extent the outcomes of the AI sessions were recognized. They confirmed that the outcomes were consistent with the experiences on the location.During the AI sessions, attention was also paid to rigor by asking the participants in all steps whether they recognized themselves in the (intermediate) outcomes (member check) . Finally, the steps and considerations in data decisions have also been recorded and described for reasons of transparency and replicability of the research. To keep the article readable, those steps and decisions are described in the results.For this study, medical ethics approval was not necessary because particular treatments or interventions were not offered or withheld from respondents. The integrity of the respondents was not encroached upon as a consequence of participating in this study, which is the main criterion in medical-ethical procedures in the Netherlands . PermissIn the workgroup, the researcher explained the 4D-cycle and the principles of AI. The members experienced AI by interviewing each other appreciatively about working together. A positive theme was then established for the AI sessions using an appreciative exercise. The members first filled in a problem tree . The perspective tree was then filled in . The chosen theme was: “Together, we provide the best care for our residents”.The team sessions were scheduled, and invitations were sent out to all of the employees of the location. An employee could sign up for one of the two parallel sessions. Since team members could freely register for a session, the number of participants in a session was variable. The original intention was to schedule the three sessions six weeks apart. However, due to the COVID-19 pandemic, the sessions were held in September 2022, and in May and July 2023. Sessions one and two were held outside the nursing home and lasted five hours (including dinner). Session three was organized in the nursing home and lasted two-and-a-half hours.The facilitators prepared the sessions in which they were coached by an experienced AI facilitator, this was the AI trainer, whereas the researcher followed a basic AI course.In September 2021, two parallel team sessions were planned. The sessions included 25 and 45 participants, respectively. After an explanation about the 4D-AI cycle, we formed groups of two who held an appreciative interview with each other about situations where they had provided good care for the residents and their families and worked well with their coworkers. The pairs were composed of one of the facilitators, the team member, so that as many different perspectives as possible would meet in the interview, taking into account the degree of courage of team members to speak out. Each participant was given an A4 with instructions on it containing the themes to be covered and examples of appreciative questions. Instructions were also given as to what the interviewer should write down from the interview . Each group made an artwork with the following dream question in mind: What is possible for this nursing home if we meet the criteria that we just agreed on in the discovery phase? The subgroups had a variety of creative materials at their disposal. In two sessions, seven artworks were made. Each group told the story of their artwork: What is it about? Why this artwork? The others listened and asked questions. The facilitators encouraged participants to ask questions or asked in-depth questions themselves to the presenting groups to ensure that the meaning of the artwork was well understood. After the first two sessions, the facilitators reflected on the outcomes of the discovery phase and combined the lists of criteria Duplicates were removed. Indicators that were similar but slightly differently formulated were put back in a team meeting to check whether they were indeed the same terms see .In May 2022, two parallel team sessions were planned. The sessions included 25 and 35 participants, respectively. To build upon the previous AI sessions, we started by showing the videotaped recordings of the seven artworks from the dream phase. We formed random teams of five persons and asked them to write down the themes that connected the artwork. The subgroups presented the themes to each other. The facilitators asked the subgroups to make one single list that everyone could agree on. In total, 10 themes were identified see . Due to The participants then chose a theme that they wanted to develop into a change proposal. Each group answered 10 appreciative questions to clarify what needs to be achieved on the chosen theme, what is already going well in the theme, and what needs to be worked on , whether necessary actions to continue the process of AI at the location and if they would recommend AI as a method to learn for the location.The employees generally look back positively on enthusiastic, relaxed sessions, especially the first session in which they used their creativity to dream and craft this dream. They were also asked to think about what is desired instead of what is not possible or not going well:I found it very fun and fascinating. Different from a normal consultation. You did see people in a different way. It was a bit more playful than a normal meeting and you saw colleagues in a different light than usual... It was nice to think creatively and a bit looser and not in ’what is not possible,’ but what would we really like.They enjoyed being with each other. The sessions were often described as instructive because opinions were shared, brainstormed, and discussed together:You can immediately let the whole team know what can be improved or not. Then you immediately get a discussion, brainstorming and that’s good. Yes, exchange of ideas, experiences.(Interview 14 with care worker)The participants have gotten to know each other better and felt more connected to each other:I have seen in those team sessions that people showed more who they were.(Interview 17 with coordinating activity supervisor)I think especially the piece of connection. Not really something very concrete, but more connecting together ...... the feeling. Equal care teams and practitioners: we do this together.(Interview 18 with psychologist)Most team members mentioned openness and honesty in the conversations. In general, team members felt free to express their opinions in the sessions, and felt that they were heard:Even if someone did not agree with it, it was still said.(Interview 6 with care worker)I think everyone misses discussing with the others. And those sessions are very good for that. Everyone listened attentively.(Interview 14 with care worker)The sessions have given a boost to the team spirit. The atmosphere and involvement have been improved at the location:I do think there was a need to change the working atmosphere. I do think that appreciative change has contributed to the team spirit, team building, connection, support for each other’s opinions, and ideas.(Interview 16 with management)If you look at how we were before those sessions, and where we are now as a team, they are two different teams.(Interview 12 with care worker)Most healthcare workers indicated that the sessions motivated them to change their behavior :It has become more helpful, [there is] more cooperation.(Interview 8 with four care workers)Ask sooner, call sooner. Just do it, because we are here for each other. I try to do that more now.(Interview 1 with care worker)Two care employees reported that agreements have now been made with each other and that they can talk to each other about this.The management takes from the sessions that nothing is impossible, that it is important to question the desire behind a story and to align the way of learning with how the team wants to learn:I use the phrase very often: I hear what you say, but what is the wish behind it?(Interview 16 with management)I especially learned how the team wants to learn, especially the how. How do they actually want to learn? Did we offer it the right way?(Interview 16 with management)Suggestions were mentioned in the following four themes: do not just talk but act, keep naming and discussing, come back to agreements made, and finally a clear role for management with a clear division of roles between management and any others.For “not just talk but act”, it is important to follow plan-do-check-act, in which actions are set out and are also evaluated. That is not happening enough now:We have now implemented it and then we have to see how things are going in practice. We can get information from that: how are things going now? Is it well implemented? Is it clear to everyone?(Interview 16 with management)To “keep naming and discussing” it, it is important to create moments where team members see each other and discuss the actions. It is also mentioned to take small steps:That you occasionally have those action points come back: remember, we were working on that and that.(Interview 12 with care worker)If you have eight steps, first try to complete step one. And then the next step.(Interview 6 with care worker)It is also important to come back on joint agreements, take joint responsibility for this, and also hold each other to account:Because we did it together anyway. So, make it your own and if everyone takes their responsibility to make it their own and then we can give each other better feedback. The agreements were made together.(Interview 16 with management)Finally, a clear role is expected from management with a clear division of roles between management and any initiators of certain plans that may be appointed. This is currently insufficiently clear and visible:A clear course. People want clarity.(Interview 4 with care worker)But there isn’t really a division of roles yet.(Interview 16 with management)In the interviews with both care workers and management, it is indicated that AI is a way to learn and develop as a team:It puts me back on my feet every now and then: oh, these are their agreements. If we had not done this, it would not have been their agreements, but more from what we thought from ‘that ivory tower’ would have been good.(Interview 16 with management)Would I advise appreciative change? Always, the part of team building. Despite the fact that you work one-on-one, you hear other people. At least, I hear what other people think.(Interview 6 with care worker)I would advise it. You also have all disciplines and colleagues. you think together, make agreements together. ….. And that everyone gets the opportunity to be involved.(Interview 16 with management)The facilitators also evaluated the AI process themselves using the same interview questions that were also asked of the employees, which resulted in four main points. The first point is the enthusiasm about how the AI brought energy to the team sessions and what AI has subsequently yielded for the location. The second point is the success of facilitating in a trio where knowledge of the method (researcher), group processes (team coach), and the team (team member) came together. The trio also made it possible to discuss during and between sessions to see if the process was going well. The third point is not only the importance of proper preparation and facilitation of the sessions but also the importance of executing the different phases of AI in the right way. It was a good choice to do the sessions outside the nursing home because the choice of location turned out to have had quite an influence on the course of the sessions . Being able to walk in the room, standing by whiteboards, and having space to work in subgroups has been a success factor. Finally, the fourth point is that the action plans were not implemented on their own, the facilitators looked for a more steering role from the management, but this was not sufficiently the case so far.The participants looked back on enthusiastic AI team sessions that resulted in good conversations, a better team atmosphere, and connectedness by thinking together about what good care should look like and how to get there together. These experiences are similar to the results of the review study on AI by Watkins et al.  describiThe client or their representatives were not present during the sessions, which is advised according to the principles of AI . This waAlthough useful action proposals have been made, concrete actions have not been forthcoming so far. Looking back, the destiny phase was insufficiently elaborated because it should have been made clear who should pick up what and when. This has led to delays and to the feeling of the team that nothing is happening regarding their action proposals. It is important here not to make a master plan that cannot be deviated from because the strength of AI is precisely that it moves along with what is considered urgent by the participants: “Therein is a caution against the choreography of Appreciative Inquiry where participant experiences or stories are molded to fit an agenda or a previously drafted master plan”  (p. 2). What has worked powerfully is facilitating with three people: a team member, the team coach, and the researcher; bringing together knowledge of the method (researcher), team processes (team coach), and the team (team member). This gave us the opportunity to discuss the process, and the right substantive steps and/or interventions in the preparation for, and guidance in, the sessions. The team member and team coach can give the team a sense of security because they know them. It also offers the possibility of a smooth transition from the process to the organization or location. Good facilitators are of great importance for the process and outcomes of an AI ,19. The The AI process has been completed in close consultation with the working group and the participants, which guarantees the participatory aspect of the research . HoweverThe AI process was followed at one location with one team, which makes the generalizability of the workable principles of AI small. Therefore, it would be valuable to do follow-up research in more locations and teams to see if the AI process leads to the same experiences and outcomes.Due to the termination of the research project after the third AI session, we were, unfortunately, unable to retrieve data from the evaluation that would have been completed after the implementation of the agreements made about communication and collaboration. With this evaluation, we could have said more about the impact of going through the AI process.Watkins et al.  propose To provide good care to residents in a nursing home, it is necessary to continue learning—both as an individual and as a team. AI is increasingly being used as a method to effect change in healthcare. This study investigated the experiences of a nursing home’s teams while going through the entire AI cycle and asked whether this has resulted in an improvement in care. The participants experienced the AI method as an enthusing method that brought colleagues together and created connections by looking together at where people want to go and how to get there. Going through the AI cycle has resulted in a better atmosphere in the team with increased psychological safety, which is a prerequisite for learning . This st


Schizosaccharomyces pombe (S. pombe), revealing two distinct assembly modes. In the structure of SIN3L, each Sin3 isoform (Pst1 and Pst3) interacts with one histone deacetylase Clr6, and one WD40-containing protein Prw1, forming two lobes. These two lobes are bridged by two vertical coiled-coil domains from Sds3/Dep1 and Rxt2/Png2, respectively. In the structure of SIN3S, there is only one lobe organized by another Sin3 isoform Pst2; each of the Cph1 and Cph2 binds to an Eaf3 molecule, providing two modules for histone recognition and binding. Notably, the Pst1 Lobe in SIN3L and the Pst2 Lobe in SIN3S adopt similar conformation with their deacetylase active sites exposed to the space; however, the Pst3 Lobe in SIN3L is in a compact state with its active center buried inside and blocked. Our work reveals two classical organization mechanisms for the SIN3/HDAC complexes to achieve specific targeting and provides a framework for studying the histone deacetylase complexes.The switch-independent 3 (SIN3)/histone deacetylase (HDAC) complexes play essential roles in regulating chromatin accessibility and gene expression. There are two major types of SIN3/HDAC complexes (named SIN3L and SIN3S) targeting different chromatin regions. Here we present the cryo-electron microscopy structures of the SIN3L and SIN3S complexes from  The SIN3/HDAC complexes, expressed in all eukaryotes, are associated with numerous cellular processes, including embryonic development, cell cycle, cell proliferation, and senescence, or in diseases such as cancer10. The SIN3L complex, consisting of 10–15 protein components with a combined molecular weight of ~1 megadaltons (MDa), is broadly recruited to the promoter of target genes through DNA-binding factors or other corepressors to inhibit transcription14. Moreover, the ~0.6 MDa SIN3S complex contains only five known proteins and targets the transcribed regions to suppress intragenic transcription initiation17.The highly conserved, multidomain-containing protein Sin3 is thought to provide a platform for the assembly of HDACs and non-catalytic subunits, forming two major types of the SIN3/HDAC complexes (SIN3L and SIN3S)28. Recently, dominant loss-of-function mutations in human SIN3 proteins were identified as one of the genetic causes responsible for the intellectual disability and craniofacial dysmorphism characteristic of the rare neurodevelopmental disorder Witteveen-Kolk syndrome (WITKOS)31. In addition, the human SIN3/HDAC complex regulates the expression of several genes important in breast cancer and estrogen signaling, revealing a potential therapeutic strategy33. However, structural information for SIN3/HDAC complex has been slow to emerge, severely limited to only several crystallographic or NMR structures of the isolated domains38. It remains unclear how the other subunits collaborate with Sin3 to furnish the complex organization, as well as how the HDAC associates with Sin3 to enable the deacetylase activity. Addressing these questions necessitates the entire structure of SIN3/HDAC complex.Since the discovery of the SIN3L and the SIN3S complexes we have gained considerable insights into their functional analysis based on extensive genetic and biochemical studiesSchizosaccharomyces pombe (S. pombe). The structure information reveals two distinct representative scaffolds of the SIN3/HDAC complex, and allows the mechanistic understanding of the molecular basis for the underlying SIN3L/SIN3S function.Here we report the atomic cryo-electron microscopy (cryo-EM) structures of the SIN3L and SIN3S complexes from S. pombe by employing a C-terminal Flag-tag on the Sin3 homolog Pst1, Pst2, or Pst3. Cryo-EM analysis finally yielded a reconstruction of SIN3L at an average resolution of 3.2 Å from the Pst3-Flag yeast strain and a map of the SIN3S at 2.9 Å resolution from the Pst2-Flag yeast strain , as well as two copies of the WD40 domain-containing protein Prw1 (Prw1 and Prw1’), and also Dep1, Sds3, Rxt2, Png2, and Rxt3 to form an intricate and asymmetric ‘Large’ scaffold Fig. . The thrNotably, the three isoforms of Sin3  respectively create a lobe  by directly interacting with Clr6 and Prw1. In addition, other subunits of SIN3L and SIN3S play pivotal roles in maintaining structural integrity. This will be discussed in detail in the following sections.42.The SIN3L complex adopts an asymmetric architecture, containing three modules: the Pst3 Lobe , the Pst1 Lobe , and a scaffold  Fig. . The sca43. In the structure of SIN3L, the segment spanning the PAH3, HID, and HCR of Pst1 or Pst3 is well resolved and reveals a different conformation in the organization of the Pst1 Lobe or the Pst3 Lobe , an HDAC interaction domain (HID), and a highly conserved C-terminal region (HCR)obe Fig. . The HIDobe Fig. . In addiobe Fig. , while tobe Fig. . Whereasobe Fig. , the Pstobe Fig. .44 , two histone binding modules , and a scaffold  Fig. . Cph1, CIn the structure of the SIN3S, all five domains  of Pst2 are well resolved and could be modeled Fig. . SimilarS. pombe are homologs of the S. cerevisiae Rco1 and the human PF1, and contain PHD (plant homeodomain) zinc fingers in their N-terminus23, which was proven to serve as the sequence-specific histone recognition element45. Cph2 has two PHDs (PHD1 and PHD2), but Cph1 has one PHD and another basic region (BR) with many positive-charged residues corresponding to the Cph2_PHD2  and the Cph2_NTD  respectively recruit the MRG domains of Eaf3 and Eaf3’ through their SIDs (α2–3), forming two separate modules Fig. . Superpo25. The BR domain, revealing a basic surface, is positioned at one corner of the SIN3S  is occupied by the inositol phosphates, which act as an ‘intermolecular glue’ that cements the HDAC and the SANT/DAD domain together , the fission yeast S. pombe has three . Our work first proved that these three isoforms performed non-redundant functions. They respectively form three distinct lobes   Fig. , providiFurthermore, we realize that the Pst1/2/3 forms different conformations to create different “environments” for the active site. Notably, the overall conformation of the Pst1/2 Lobes are similar and extended with their adjacent active sites highly solvent accessible Fig. , suggestS. pombe, revealing two representative organization mechanisms for the SIN3/HDAC complexes. Combining the structural analysis and previous reports, we find that the distinct assembly modes of SIN3L and SIN3S facilitate the HDACs to target different chromatin regions , which are absent in the SIN3S sample from the Pst2-Flag strain  complex preferentially bound di-nucleosomes18. Therefore, we suppose that the HB1 and HB2 of the SIN3S complex may offer two binding sites for the adjacent nucleosomes. It needs further studies to confirm.The yeast SIN3S complex is reported to recognize the H3K36me3 marker and function in the gene coding regions for suppression of antisense transcription and protection from genotoxic agentsobe Fig. . In the obe Fig. . It was 62, and two HDAC molecules were found to be symmetric in the structures of the human NuRD subcomplex51 and the MiDAC subcomplex52. We show in this study that the SIN3L complex contains two asymmetric deacetylase active sites with one open and the other blocked, and the SIN3S complex only has one. These findings shed new light on the diverse and complex molecular mechanisms for the class I HDAC complexes.Furthermore, two deacetylases were previously suggested to be needed for the class I HDAC complexes 63. The majority of the mutations probably compromise the folding of the structure, and many other mutations are located at the HDAC active site, the hydrophobic pocket of the PAH domains, the Sin3–HDAC interface, and the Sin3–Prw1 interface  at the C-terminus of the Sin3 homolog Pst3 or Pst2. The DNA sequences for the 3× Flag-tag and a HphMX6 marker were amplified by PCR from the plasmid pF6Aa-C3Flag-HphMX6. The PCR product was transformed into a wild-type S. pombe strain (kindly provided by Dr. Rui Bai) by the lithium acetate method66. Transformants were selected on hygromycin containing 2× YES solid medium. Correct integration of the 3× Flag-tag was further confirmed by PCR and western blotting.The 600 of ~6. The collected cell pellets were washed with buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF), and resuspended in lysis buffer containing 50 mM HEPES (pH 7.4), 350 mM NaCl, 15% glycerol (v/v) and protease inhibitors . The cell suspension was dropped into liquid nitrogen to form yeast beads and pulverized to powder in a Retsch ZM200 nitrogen mill. The frozen yeast powder was thawed at room temperature and resuspended in the same lysis buffer. The cell lysate was first centrifuged at 12,000 rpm for 30 min and the supernatant was centrifuged again at 12,000 rpm for another 1 h. The resulting supernatant was loaded into the ANTI-FLAG M2 resin (Sigma) and eluted using eluate buffer containing 0.5 mg/mL FLAG peptide (DYKDDDDK), 20 mM HEPES (pH 7.4), 150 mM NaCl, 5% glycerol (v/v). The eluate was concentrated and then applied for glycerol density gradient centrifugation. The glycerol gradient was prepared with light-buffer containing 10% glycerol (v/v), 20 mM HEPES (pH 7.4), 50 mM NaCl, 2 mM DTT, and heavy-buffer containing 30% glycerol (v/v), 20 mM HEPES (pH 7.4), 50 mM NaCl, 2 mM DTT. After centrifugation at 30,000 rpm for 20 h at 4 °C using a Beckman SW32Ti rotor, the peak fractions containing SIN3L or SIN3S complex were verified by SDS-PAGE  packed with C-18 resin . Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 100% acetonitrile and 0.1% formic acid. The mass spectrometer was operated in the data-dependent acquisition mode using the Xcalibur 4.1 software and there is a single full-scan mass spectrum in the Orbitrap  followed by 20 data-dependent MS/MS scans at 30% normalized collision energy. Each mass spectrum was analyzed using the Thermo Xcalibur Qual Browser and Proteome Discovery for database searching.Uranyl acetate (2% w/v) was used for negative staining. Briefly, 4-μL aliquots of the cross-linked sample at the concentration of 0.02 mg/mL were applied to the glow-discharged copper grid supported by a thin layer of carbon film  for 1 min. The negatively stained samples were imaged on a Thermo Fisher Talos L120C TEM microscope operating at 120 kV. The glow-discharged copper Lacey carbon grids (TED PELLA) were used for cryo-EM specimen preparation. Cryo-EM grids were prepared using Vitrobot Mark IV (FEI Company) operating at 8 °C and 100% humidity. After waiting 1 min, 4-μL sample at a 0.1 mg/mL concentration was blotted and rapidly plunged into liquid ethane cooled by liquid nitrogen.−/Å2, and aligned and summed using MotionCor2 with a binning factor of 2, resulting in a pixel size of 1.087 Å67. Dose weighting was performed concurrently. The defocus value for each image was determined by Gctf68.Cryo-EM specimens were imaged on a 300-kV Titan Krios electron microscope (Thermo Fisher Scientific) with a normal magnification of ×81,000. Movies were collected by a Gatan K3 direct electron detector equipped with a GIF Quantum energy filter (slit width 20 eV) at the super-resolution mode. The micrographs were automatically recorded using EPU (Thermo Fisher Scientific) with a defocus range from −1.8 μm to −2.3 μm. Each stack of 32 frames was exposed for 2.56 s with total dose of ~50 e69 except that is specially mentioned. In total, ~2.1 millions particles were generated from 7294 micrographs using Gautomatch . The generation of initial 3D volume of the SIN3L complex was from preliminary data analysis using cryoSPARC v370. Single-reference and multi-reference guided 3D-classifications were applied to the total particles, and about 562 thousand good partilces were selected. For further processing, local masks were applied to different parts of the initial reconstruction. Finally, about 263 and 389 thousands select partilces yielded the reconstructions at average resolutions of 4.0 Å and 3.2 Å after refinement for the Prw1 and main regions, respectively  0.143 criterionS. pombe SIN3L and SIN3S complexes were built de novo based on our high-resolution EM maps using COOT73 (Supplementary Table 74. Then the individual reliable domains of the predicted structures of each component were fitted into the EM maps using Chimera75, and manually adjusted in COOT. The linker sequences were built de novo based on the clear features of bulk residues.The atomic models of the S. pombe SIN3L and SIN3S complexes were respectively refined according to the 3.2-Å and 2.9-Å EM maps using PHENIX in real space76 and secondary structure restraints that were generated meanwhile. Overfitting of the model was monitored by refining the model in one of the two independent maps from the gold-standard refinement approach, and testing the refined model against the other77 (Supplementary Fig. 78.The final atomic coordinates of the Supplementary materialsSupplementary Table S2Supplementary Table S3


Drosophila melanogaster, the Sin3A gene is alternatively spliced to produce different protein isoforms, two of which include SIN3 220 and SIN3 187. Both SIN3 isoforms are scaffolding proteins that interact with several other factors to regulate the chromatin landscape. The mechanism through which the SIN3 isoforms regulate chromatin is not well understood. Here, we analyze publicly available data sets to allow us to ask specific questions on how SIN3 isoforms regulate chromatin and gene activity. We determined that genes repressed by the SIN3 isoforms exhibited enrichment in histone H3K4me2, H3K4me3, H3K14ac and H3K27ac near the transcription start site. We observed an increase in the amount of paused RNA polymerase II on the promoter of genes repressed by the isoforms as compared to genes that require SIN3 for maximum activation. Furthermore, we analyzed a subset of genes regulated by SIN3 187 that suggest a mechanism in which SIN3 187 might exhibit hard regulation as well as soft regulation. Data presented here expand our knowledge of how the SIN3 isoforms regulate the chromatin environment and RNA polymerase II binding dynamics.The chromatin environment has a significant impact on gene expression. Chromatin structure is highly regulated by histone modifications and RNA polymerase II binding dynamics. The SIN3 histone modifying complex regulates the chromatin environment leading to changes in gene expression. In  Summary: SIN3 cofactors can activate or repress genes. Using bioinformatic analysis, we find that histone modification and RNA polymerase II binding profiles differ at SIN3-regulated genes with distinct transcriptional outcomes. Nucleosomes are composed of DNA strands that wrap around histone proteins. This interaction facilitates DNA compaction into the nucleus of the cell. Histone proteins are subject to post-translational modifications such as acetylation and methylation. The presence or absence of specific histone modifications can impact gene activity . The regSin3A is an essential gene in metazoans. The knockout of Sin3A leads to loss of viability in both Drosophila and mouse models  complex, which regulates the acetylation and methylation of histone tails . The SINe models . Drosophe models . Consistll cycle . Furtherof cells .Sin3A gene encodes multiple isoforms of SIN3 through differential splicing  and DRB sensitivity-inducing factor (DSIF) . HDAC1 iTo further investigate whether SIN3 repressed genes exhibit high RNA Pol II pausing, we asked if the binding of the pausing factor NELF-A is also different between repressed and activated genes. NELF-A is a pausing factor that plays an important role in RNA Pol II promoter-proximal pausing . In lineTin is a homeobox gene that codes for a transcription factor involved in differentiation and development  in gene expression of SIN3 220 active and repressed genes, with a false discovery rate (FDR) of 5%. 242 direct repressed targets of SIN3 220 were identified, of which 99.6% (241/242) of genes demonstrated less than 2 log2FC . We divided the genes into four quadrants  using a We next investigated if SIN3-regulated gene expression is correlated with histone modification and Rbp3 binding profiles. We compared lowly, moderately, and highly expressed genes. The majority of genes repressed by the SIN3 isoforms are moderately expressed (87% of SIN3 220 and SIN3 187 repressed genes), while only a small subset are lowly expressed (∼2% of SIN3 220 and SIN3 187 repressed genes) and highly expressed (1.7% of SIN3 187 repressed genes). Similarly, the majority of genes activated by the SIN3 isoforms are moderately expressed (70% for SIN3 220 and 55% for SIN3 187), while a small subset are lowly expressed (2% for SIN3 220 and 14% for SIN3 187) and highly expressed (∼0.5% of SIN3 187 activated genes). We first analyzed the histone modification profile. Moderately (10-1000 FPKM) and lowly (1-9.99 FPKM) expressed genes showed a similar trend, wherein repressed genes had higher enrichment of H3K4me2 and H3K4me3 on both SIN3 220 and SIN3 187 repressed genes compared to those activated by the isoforms . We alsoNext, we measured the Rpb3 binding profile at lowly, moderately, and highly expressed genes that are regulated by the SIN3 isoforms. Like the trend observed when the analysis was done on the full list of SIN3-regulated genes , moderatC. elegans and mice  using the software package Burrows-Wheeler Aligner (BWA) (Data were downloaded from the Sequence Read Archive (SRA), using NCBI SRA toolkit . To deteer (BWA) . Uniqueler (BWA) . Irreproer (BWA) . Peaks wer (BWA) Using the UCSC table browser , we creaRpb3 enrichment on SIN3-regulated genes was calculated and placed into a matrix using deepTools2: computeMatrix , throughP-value is determined using a null model wherein the P-value is calculated using the null distribution method.MACS2 (version 2.1.1)  was usedRNA-seq data from control cells previously published by our group  was down10.1242/biolopen.060026_sup1Supplementary informationClick here for additional data file.


Periprosthetic joint infection (PJI) is associated with significant morbidity. Management includes surgery and prolonged antibiotic treatment. Few studies have evaluated rates of acquired antimicrobial resistance in bacteria causing PJI. The aim of this study was to describe antimicrobial resistance profiles of bacteria causing PJI and to assess whether there was any apparent association between acquired resistance and clinical outcome.Patients at Mayo Clinic with first episodes of knee or hip PJI whose implants underwent sonication between January 2012 and December 2021 were studied. Sonicate fluid cultures from cement spacers or cultures yielding more than one microorganism were excluded. PJI was defined according to IDSA guidelines. Multidrug resistance was defined as non-susceptibility to at least one agent in three or more antimicrobial classes. Outcomes of PJI recurrence, need for further surgery with negative cultures or cultures yielding a new microorganism and amputation were assessed, within one year. Analysis was performed using SAS software version 9.4 (SAS Inc).S. epidermidis (n=82) and S. aureus (n=50), of which 68% and 30%, respectively, were methicillin resistant. Streptococci, Enterococci, Enterobacterales, and P. aeruginosa were found in 34,16, 8 and 3 cases; none were multidrug-resistant. No differences in PJI recurrence within one year were found in subjects with methicillin-resistant compared to methicillin-susceptible staphylococci , need for further surgery with negative cultures  or with cultures yielding a new microorganism , or amputation .256 PJI episodes were evaluated, 55% from knees and 45% from hips. Median patient age was 66 years, 52% were men. Resistance profiles are shown in the table. The most frequent microorganisms isolated were S. epidermidis with methicillin resistance found in 68% and multidrug resistance in 28%.Acquired antimicrobial resistance in PJI was most common in Matthew P Abdel, MD, OsteoRemedies: Royalties|Springer: Publishing royalties|Stryker: Royalties reltated to hip and knee implants Robin Patel, MD, Abbott Laboratories: Advisor/Consultant|Adaptive Phage Therapeutics: Grant/Research Support|Adaptive Phage Therapeutics: Mayo Clinic has a royalty-bearing know-how agreement and equity in Adaptive Phage Therapeutics.|BIOFIRE: Grant/Research Support|CARB-X: Advisor/Consultant|ContraFect: Grant/Research Support|Day Zero Diagnostics: Advisor/Consultant|HealthTrackRx: Advisor/Consultant|Mammoth Biosciences: Advisor/Consultant|Netflix: Advisor/Consultant|Oxford Nanopore Technologies: Advisor/Consultant|PhAST: Advisor/Consultant|See details: Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic|See details: continued, patent on an anti-biofilm substance issued|TenNor Therapeutics Limited: Grant/Research Support|Torus Biosystems: Advisor/Consultant|Trellis Bioscience, Inc.: Advisor/Consultant


These findings suggest a role for the complement system in the pathogenesis of IPF.Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease characterized by damage to the alveolar epithelium, leading to fibrosis and excessive accumulation of extracellular matrix in the interstitium of the lung. In the present study we performed high-resolution proteomic profiling of bronchoalveolar lavage (BAL) from IPF patients and controls, and found that the complement pathway was highly upregulated in IPF. The proteins C5, C6, C7, C8, and C9, all of which are part of the complement end product, TCC, were all upregulated. We also found that TCC levels were increased in plasma among IPF patients compared to controls, after adjustment for age, sex and BMI [mean (SD) 0.62 (0.24) vs. 0.33 (0.10),  Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease that leads to decline in lung function and early mortality . AlthougThe pathogenesis of IPF is multifactorial and characterized by progressive fibrosis and excessive accumulation of extracellular matrix in the interstitium of the lung, with an imbalance between anti-fibrotic and pro-fibrotic factors leading to collagen deposition .To identify proteins involved in these processes, we performed high-resolution proteomic profiling of bronchoalveolar lavage (BAL) from IPF patients and controls, and identified the complement system to be differentially regulated. The objectives were to (i) categorize the complement factors identified by proteomics, and (ii) assess the clinical significance of circulating soluble terminal C5b-9 complement complex (TCC) in plasma, a key downstream readout of systemic complement activation, and its association with lung function and complement levels in BAL.Twenty-nine patients with IPF , and 10 controls  were included in this cross-sectional study and underwent bronchoscopy with BAL . IPF pat years, 6Bronchoalveolar lavage (BAL) collection and preparation were performed as described previously . In short-test with FDR < 0.05). The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium PRIDE repository with identifier PXD036638  and plasma was snap frozen in liquid nitrogen and stored at −80°C. The fluid-phase terminal complement complex (TCC), consisting of the components C5b, C6, C7, C8, and C9, was measured by ELISA using a monoclonal antibody aE11 as originally described in (Blood (EDTA) was collected <2 h before BAL collection. EDTA plasma was centrifuged  and plasma (TCC) between IPF patients and controls was performed with MANCOVA using complement factors as dependent, diagnosis (IPF or control) as fixed and age, sex and BMI as covariates. Spearman correlation was used to assess associations between proteomic components and BMI as well as with TCC within patients dichotomized at TCC 0.7 CAU/mL.The study was approved by the Regional Committee for Medical Research Ethics (2013/2358) and written informed consent was obtained from all participants.p < 0.05, 0.66 > FoldChange >1.5) in BAL, comparing IPF patients with controls. Pathway analyses of the 567 proteins are listed in In the proteomic dataset, we found 567 differentially regulated proteins (118 upregulated and 449 downregulated) (p < 0.05), except TCC  and C6 . p-values.Focusing on TCC, C1r, complement factor B and C3, C5-9, these factors correlated well with BMI  0.62 (0.24) vs. 0.33 (0.10), = 0.031] . Elevate= 0.031] . We founOur results suggest that complement plays a role in the pathogenesis of IPF. Proteomic analysis of BAL demonstrated that the complement system was highly differentially regulated in IPF patients as compared with controls. Importantly, all five components of TCC were upregulated in the proteomic analyses. TCC in plasma was also elevated in IPF patients as compared to controls, and elevated levels of TCC correlated with C3, C8, and C9 complement factors in BAL.Proteomic analysis of BAL revealed that the complement and coagulation cascade were the top two differentially regulated pathways, with half of the proteins in the complement pathway elevated in IPF patients as compared with controls. Similarly, in a proteomic analysis of peripheral blood, comparing IPF patients to normal controls, O’Dwyer et al. found that complement and coagulation cascade were among the most significantly enriched clusters . Gu et aThe proteomics analyses do not measure activated products of the complement system, but rather native complement components present in the lung. However, elevated levels of plasma TCC in IPF clearly indicate that systemic complement activation occurs in these patients. The proteomics data indicate that there are sufficient amounts of all components needed to activate the terminal pathway to form TCC, and it is reasonable to assume a local activation in the IPF lung. When TCC is formed, the small potent peptide C5a is released. C5a is one of the most important contributors to inflammation, inducing innumerable cytokines and other inflammatory mediators . In accoThe study has limitations. We were not able to match the control group with the IPF-group with respect to age and sex. Since the two groups were not properly matched, we controlled for age and sex in the statistical analyses. In addition, many of the complement factors correlated with BMI, and BMI was therefore also included as an adjustment variable. In a study that involves bronchoscopy and BAL, it is difficult to recruit healthy controls. For practical and ethical reasons, we chose to recruit controls among subjects who should undergo bronchoscopy anyway, as part of annual surveillance after curative resection of carcinoid tumors. They were non-smokers and considered healthy with no previous or current medication that could interfere with the results.In conclusion, IPF was associated with upregulation of terminal complement components in BAL, and increased terminal complement complex in plasma, suggesting a role for the complement system in the pathogenesis of IPF.The mass spectrometry proteomics data to the ProteomeXchange Consortium is available via the PRIDE partner repository with the dataset identifier PXD036638. In the deposited file, there are also mass spectrometry data from patients with sarcoidosis and hypersensitivity pneumonia, which are not included in the current study with only IPF patients and controls. In the supplement of the present paper, the mass spectrometry data is available as a raw data file (excel file) with analysis from Perseus.The studies involving human participants were reviewed and approved by the Regional Committee for Medical Research Ethics (2013/2358). The patients/participants provided their written informed consent to participate in this study.LS and TU participated in data collection and performed the laboratory work. All authors contributed to the design of the study, interpretation of the results, and drafting and revising the manuscript and approved the final version.This study received funding from Norwegian Respiratory Society sponsored by Boehringer Ingelheim. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.MTD has received research funding (to his institution) and speaker/consulting fees from Boehringer Ingelheim and Roche, unrelated to the current study.The remaining 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.


The dynamics of a bilayer of graphene containing one mono-vacancy in the top layer has been investigated in the framework of DFTB in the absence and in the presence of water. Due to the speed of the code, we can describe details of the behavior, which are not directly accessible experimentally and cannot be treated by DFT or classical molecular dynamics. The presence of water enhances the displacement of carbon atoms in the perpendicular direction to the surface. Our results explain very well a variety of experimental findings. In particular, the stabilization of the Jahn–Teller distortion by hydrogenation of one of the carbon atoms at the edge of a mono-vacancy has been elucidated. This work is the first analysis of the behavior of a graphene vacancy at room temperature in contact with water based on a quantum mechanical molecular dynamics method, where both graphene and solvent are treated at the same level. The expectations from the wonder 2D-material had been very high. However, it was soon realized that the magic properties of the idealized, isolated, flat carbon monolayer are strongly affected in real systems.Already in the thirties of the last century, it was demonstrated that even thermal fluctuations should destroy long-range order in two-dimensional lattices at any finite temperature, resulting in their melting. A clear explanation can be found in reference . TherefoThe second issue is that for practical purposes, graphene needs to be supported. According to the strength of the interaction with the substrate, additional perturbations of its properties can be induced. In this case, corrugation can also appear, although of a different nature than the one mentioned above, resulting in Moiré patterns due to the mismatch of the graphene and substrate lattice constants ,6,7. AlsNext, a variety of defects produced during preparation processes are unavoidable. We can distinguish between intrinsic, such as the Stone–Wales defect, created by rotation of one single pair of carbon atoms, single and multiple vacancies, where carbon atoms of the hexagon ring are missing, line defects, caused at grain boundaries of different crystallographic orientations, and extrinsic defects, originated by foreign adatoms or substitutional impurities ,13. In mFinally, a large number of applications, such as water desalination and energy storing, require the presence of a solvent ,17,18. TConcerning the theoretical framework, DFT calculations are widely used to predict structural changes, energetics, and electronic and magnetic properties. These are valuable attempts that have shed light on the understanding of many aspects. However, these results are valid at 0K; therefore, thermal fluctuations effects are missing. A powerful method to investigate the dynamics is classical molecular dynamics (CMD). This is especially efficient to describe the behavior of large ensembles of solvent molecules. However, the lack of reliability of the force field to describe quantum chemically the carbon side of the interface and its interactions with the solvent molecules inhibits the proper description of the whole system.Cicero et al.  investigInterestingly, the development of the self-consistent charge density functional tight-binding method and its combination with molecular dynamics allowed treating both sides of the interface at the same level. This approach has been successfully applied to study the diffusion of vacancies and the annihilation of dislocation pairs in graphene at high temperatures ,22.In the present contribution, we shall apply this approach focusing on the dynamics of a mono-vacancy at the top layer of a graphene bilayer. Also, we shall extend this approach to investigate the interface resulting from the contact of graphene with water, allowing the movement of all atoms, including the carbons of the graphene layers.On one hand, DFT calculations at 0K describe very well the static stable structures. On the other hand, experimental work for structural characterization such as STM and HR-TEM provide images resulting from time periods of the exposures on the order of seconds. Therefore, they represent average configurations. Another limitation of these experimental techniques is the perturbations introduced by the STM-tip and by irradiation with energetic particles, such as electrons or ions. Therefore, our results from QMMD calculations in the framework of DFTB+ can shed some light on details of the dynamics of fast processes occurring in short time scales. This work is the first analysis of the behavior of a graphene vacancy at room temperature in contact with water based on a quantum mechanical molecular dynamics method, where both graphene and solvent are treated at the same level. This also complements a previous publication about desorption of hydrogen from graphene induced by charge injection .We performed our calculations using the package DFTB+ from Bremen ,25, whic12 Hz. In the presence of water, the amplitude of these oscillations is larger and random. It is no more possible to identify any characteristic frequency  in combination with the use of a mono-chromated electron beam  have ideOur simulations confirmed this idea, since we can follow the process in a short timescale not accessible experimentally. The presence of water does not have any significant effect. A better representation is given by the bond distance distribution shown in z-axis; thus, any out-of-plane displacement of the under coordinated carbon atom would lead to an illusory bond compression in the TEM image, due to the corresponding projection of the bond into the 2Dx-y plane. Therefore, we also calculated this distance projected into the plane x-y and obtain effectively a lower mean distance of 1.16 Å.Concerning the stable reconstructed vacancy (r-MV) observed experimentally in , the autIn the supporting information are movies available comparing both systems , with anIn all our simulations, we never detected the diffusion of the vacancy along the surface. This phenomenon has been observed on simulations at higher temperatures (2000–3000 K). In our case, it is not surprising since the activation barrier for this process has been estimated to be larger than 1.7 eV . TherefoAn interesting additional advantage of DFTB+ is that the charge distribution between the atoms can be easily obtained from the orbital occupation analysis. In this section, we shall discuss the atomic charge distribution for the different investigated systems.e−|) is compensated by the total charge on the graphene bilayers . Li et al. [e−|) than ours. Similar to our results for the out of plane coordinates fluctuations, the charge variations in the presence of water are larger than in its absence.i et al.  also foue-|, respectively). The charge distribution in the presence of water is broader than in its absence. This charge is practically compensated by the positive charge (+0.25 |qe−|) on the next neighbor (D + E + F + G + H + I), see It is also interesting to analyze the charge on the neighboring atoms of the vacancy separately. e−|), and the others two forming the weak bond of the distortion are also negatively charged , while the hydrogen bonded to A is positively charged, practically compensating the charge on A .A detailed analysis of the charge distribution on the individual atoms at the border of the vacancy   reveals Finally, two views (into zx and zy planes) of the superposition of the coordinates of all atoms of one typical run and the corresponding charges on the carbon atoms are shown in In the analysis of these results in order to obtain the atomic density, the electrical field and the electrostatic potential across the interfaces are shown in Concerning the electrostatic potential, first it increases negatively into the bulk of the electrolyte until a screening effect of the solvent becomes evident. At the end, since the dipole formed with the bottom layer of graphene is larger, a net dipole potential appears due to the asymmetry introduced by the mono-vacancy in the top layer of graphene.We have performed quantum mechanical molecular dynamics in the framework of DFTB+ for a bilayer of graphene containing one mono-vacancy in the absence and in the presence of water, treating all components of the system at the same level. We have obtained a clear view of the dynamics of the systems on a timescale inaccessible experimentally. We summarize below the most relevant results.12 Hz for the bilayer in vacuum. Therefore, these distortions should be very difficult to be observed experimentally. The presence of water in contact with the bilayer increases the amplitude of the oscillations in the z-direction, and they are not more periodic. There is a polarization between graphene and water, with the graphene bilayer showing a positive charge compensated by the negative charge on water. At the top layer of graphene, the carbon atoms nearest to the mono-vacancy are negatively charged, while the next nearest neighbors are positively charged.The graphene bilayers corrugate at room temperature as a consequence of thermal fluctuations. The twists out of plane show a characteristic high frequency of about 2.5 × 10An interesting result is the dynamics of the Jahn–Teller distortion. In the case of the “clean” vacancy, two of the three carbon atoms at the edge occasionally bond to each other. These switching between the bonds alternate randomly. When one of the carbon atoms with dangling bond is saturated with one hydrogen atom, the Jahn–Teller distortion between the others two carbon atoms is stabilized. These results explain quite well the previous experimental finding .


Multilayer graphene has attracted significant attention because its physical properties can be tuned by stacking its layers in a particular configuration. To apply the intriguing properties of multilayer graphene in various optoelectronic or spintronic devices, it is essential to develop a synthetic method that enables the control of the stacking configuration. This review article presents the recent progress in the synthesis of multilayer graphene by chemical vapor deposition (CVD). First, we discuss the CVD of multilayer graphene, utilizing the precipitation or segregation of carbon atoms from metal catalysts with high carbon solubility. Subsequently, we present novel CVD approaches to yield uniform and thickness-controlled multilayer graphene, which goes beyond the conventional precipitation or segregation methods. Finally, we introduce the latest studies on the control of stacking configurations in bilayer graphene during CVD processes. Graphene is of interest owing to its excellent properties such as its thermal conductivity and electron mobility . Th. Th47]. To further improve the uniformity of multilayer graphene grown on a Cu/Ni alloy, the CVD of graphene on a single-crystal Cu/Ni alloy  was inve−1 K−1, comparable to that of mechanically exfoliated bilayer graphene (~2800 W m−1 K−1), demonstrating the high quality of the synthesized AB-stacked bilayer graphene. In addition, the thickness of the graphene was successfully controlled by tuning the content of Ni in the alloy catalyst.In 2020, Ruoff et al. prepared an extremely large single-crystal Cu/Ni(111) foil as a catalyst for large-area uniform multilayer graphene . First, The graphene growth on the Cu surface is surface mediated, and thus uniform graphene is obtained on the Cu surface. Therefore, uniform multilayer graphene can be obtained once the self-limiting characteristics of the Cu surface are eliminated.4 gases into the inside of Cu pockets is limited, the growth rate of graphene on the inside surface of Cu pockets was considerably lower than that on the outside of the Cu pockets. Therefore, the inside Cu surface can be still open to the adsorption and dehydrogenation of CH4 molecules whereas graphene fully covers the outside Cu surface of the Cu pockets. This leads to asymmetry in the carbon concentration inside the bulk Cu: the carbon concentration is higher near the inner Cu surface. As a result, there is a bulk diffusion of C atoms from the inside surface to the outside surface of Cu pockets, and multilayer graphene can form on the outside even after the monolayer graphene has fully covered the outside Cu surface  have been investigated . The for4 cm2 V−1 s−1 at room temperature . I. I64]. IIn addition to the growth conditions, the crystallographic orientation of the Cu surface is an important factor that determines the stacking orientation of BLG. Cho et al., compared the stacking orientations of BLGs grown on atomically flat (Cu(111)) and stepped Cu surfaces (Cu (311)/Cu(110)) when the other growth conditions were the same for both cases . Bernal-In this review, we covered the CVD method for the synthesis of multilayer graphene, discussing the growth mechanism of graphene on Ni and Cu catalysts and the challenges associated with the provision of uniformity and the control of thickness also see . We highAlthough significant progress has been achieved in the CVD of multilayer graphene, further studies are required to fully understand its growth mechanism and achieve precise control of the thickness and uniformity. An in-depth understanding of the growth mechanism of bilayers is crucial to improve the quality of bilayers, particularly in terms of stacking orientations. Additionally, the production of high-quality graphene at low temperatures is essential for practical applications. Moreover, the development of a catalyst-free CVD method for multilayer graphene synthesis is desirable. Such an approach would eliminate the need for costly catalyst materials and simplify the overall process, including the transfer step, thus reducing production costs.


The harbor porpoise, a small marine mammal related to dolphins and whales, is nowadays very rarely seen in the eastern part of the Baltic Sea, and was not common here in recent centuries. However, the bones of this animal have been found on many Stone Age archaeological sites dating from the period 4000–2000 BC along the coasts of Estonia, Latvia and Lithuania, which shows that it was frequently being hunted by the coastal communities of that time. In this period, the Baltic Sea was saltier than it is today, making conditions more favorable for marine species such as the porpoise. In addition to meat and blubber, the porpoise also provided a tool for decorating pottery vessels made by the Stone Age inhabitants. This instrument was made from the animal’s jaw bone, the teeth being impressed in the clay to produce a distinctive row of marks. Such decoration on pottery may have symbolized the coastal way of life and joint hunting activities. Pottery with porpoise tooth ornamentation has been found on many coastal Stone Age sites in the region and also in smaller amounts at inland sites, providing evidence of contact between coastal and inland communities.Phocoena phocoena) has been quite rare in the eastern Baltic in recent centuries, but according to archaeological finds, its population was quite numerous here ca. 6000–4000 years ago . This paper deals with all known archaeological assemblages of porpoise so far discovered in the eastern Baltic , discusses the hunting strategies and studies the exploitation of this small cetacean by the Neolithic hunter-gatherers. Fauna historical aspects include new archaeological data in addition to those published previously. We consider whether these new data change the temporal and spatial pattern of porpoise hunting and examine how, in addition to the expected use of porpoise meat and blubber, the porpoise’s toothed mandibles were used for patterning ceramics.Compared with the history of seals in the Baltic Sea, the porpoise has received much less research attention. The harbor porpoise ( Phocoena phocoena Linnaeus, 1758) is one of the world’s smallest cetaceans. As the name indicates, it stays close to coastal areas and is, thus, particularly visible to people interested in whale watching. The porpoise can swim up rivers and be found hundreds of kilometers from the sea. However, marine coastal waters constitute its most common environment, and the porpoise is a widespread species in cooler coastal waters of the Northern Hemisphere. In the eastern North Atlantic, the harbor porpoise is distributed along the coast of the Barents Sea, the west coast and fjords of Norway, the coasts of Iceland and the British Isles, in Danish waters and along the West European coast as far as north-western Africa [The harbor porpoise (n Africa . Our main Africa .13C) signature of the porpoises in the Baltic and the Kattegat/Skagerrak, migration between these areas is evident [The Baltic population of porpoise is not large nowadays. The population inhabiting the Baltic Proper has been classified as ‘critically endangered’, on the basis that the current population is likely to be fewer than 250 individuals ,5. Some  evident . DespiteLagenorhynchus albirostris Grey, 1846) were seen as far east as Tallinn Bay in northern Estonia. On 1 June 2020 a couple of dolphins (most probably bottlenose dolphins) were encountered in Kopli Bay (photographed by the captain of the boat Indrek Sülla), and a humpback whale  was seen off the coasts of Poland and Finland. Probably the same individual was finally found dead close to Skulte harbor on the Latvian coast of the Gulf of Riga in July 2006 . Older records provide more evidence of the presence of whales in the eastern Baltic. Besides humpback whales, white whales  are also mentioned [In the coastal waters of Estonia, Latvia and Lithuania, cetaceans have been rare visitors rather than permanent inhabitants. This applies to porpoise as well as other cetacean species . Only recently, in September 2008, white-beaked dolphins  concluded that there are ca. 40 mentions from this period in Estonia, the best known being a picture of “fishermen from Virtsu with a strange fish” ge fish” .merswyn) is mentioned in accounting books among other animal products sold in Tallinn’s markets [In the Middle Ages and Early Modern Era, the porpoise ( markets  (p. 9). Phoca groenlandica Erxleben, 1777), the porpoise has received much less research attention. It has been considered as part of the hunted fauna in zooarchaeological studies, but has not been the subject of any special research in taphonomy. Even though the species has been quite rare in the last centuries, according to archaeological finds, its population was quite numerous ca. 6000–3500 years ago. Thus far, only one paper has summarized the post-glacial spatiotemporal distribution of porpoise in the Baltic Proper [In comparison with the history of seals in the Baltic   up to the brackish water  stage. In cultural terms, the period under consideration corresponds to the New Stone Age or Neolithic. The exploitation of porpoise involved the use not only of hunting products for food but also of bones for practical purposes. Thus, the porpoise mandible with teeth still in place was widely utilized as a tool for decorating pottery.Our study material for clarifying the history of the porpoise comes from archaeological contexts, as no finds of porpoise from geological contexts are known in Estonia, Latvia or Lithuania. Most of the material has been excavated and collected manually from Neolithic dwelling sites situated on islands, in addition to finds from coastal areas of the mainland , and is Porpoise has been identified in the archaeological material of Kunda Lammasmägi by Johannes Lepiksaar ,18, at RThe taxonomic and anatomical determinations were performed using comparative skeletal collections and skeletal atlases. In addition, quantitative methods such as the number of identified specimens (NISP) and the minimum number of individuals (MNI) have been used in most cases . Since m14C (radiocarbon) dates, and both Narva and Comb ceramics have been recorded [From the Lammasmägi site at Kunda, which is considered mainly a Mesolithic site, at least one find of porpoise is recorded ,18 , the hardest part of the skull, which preserves better than other bones of the skeleton. In addition, one unburnt fragment of a vertebral disk may come from a young porpoise, but this assumption is based on the disk’s structure rather than its shape. Namely, the porpoise, like other cetaceans, has a special bone structure, which differs from that of terrestrial mammals. However, as the disk fragment is quite small, some doubt may still remain as to whether it comes from the seal or porpoise (Kõpu XI finds were re-examined by Lõugas in 2022).North of Saaremaa, another island—Hiiumaa—has also yielded a few porpoise bones. Many seal hunters’ campsites are known on Kõpu, the westernmost peninsula of Hiiumaa ,32. The Two Neolithic sites on the coast of the mainland—Tallinn Vabaduse Sq. (Estonia) and Siliņupe (Latvia)—are similar in terms of their geographical location and faunal evidence. Both are situated at the southern shore of a bay close to a river estuary and, besides terrestrial animals, have yielded considerable amounts of harp seal and porpoise bone ,24. It iThe other sites considered here, Riigiküla I, III and IV in north-eastern Estonia and Šventoji 2B and 3B in western Lithuania, are not very rich in porpoise bones ,21,25. O14C dates, it does not provide definite confirmation of the occurrence of porpoise in the mentioned period. Thus, 6A Tatari Street is not far from the Vabaduse Sq. site (ca. 200 m), where the Stone Age finds are partly mixed with the medieval and early modern finds. Although Stone Age layers are absent in Tatari Street, a few finds indicating the Stone Age are known in the vicinity [14C dates and according to the archaeological context, we can relate this porpoise find to the medieval and/or modern period.There is also one lumbar vertebra of porpoise from Tallinn found in mixed layers of medieval/modern age at 6A Tatari Street. In the absence of vicinity . CurrentIn addition to the faunal remains testifying to the porpoise’s importance as a source of meat and blubber for coastal communities in the Neolithic, a recent study has revealed that porpoise hunting also yielded a tool used to create a distinctive kind of pottery decoration. As described by Bērziņš and Dumpe , the eloExperiments using a harbor porpoise mandible  demonstrThe row of tooth imprints may be straight or somewhat curved. In general, the orientation of the long axis of individual tooth imprints tends to follow the overall direction of the row of imprints, although in many cases, individual teeth are somewhat obliquely oriented. In cases where a row of somewhat obliquely oriented porpoise teeth has been used to make a relatively deep imprint in the clay, it may resemble a cord impression; however, in such cases, the tooth imprints will be deeper than imprints left by windings of cord. In cases where the stamp has made a shallow impression, there will be short linear marks left by the tips of the teeth, whereby the overall impression of the stamp resembles a “dashed line” b.Porpoise tooth decoration was first recognized on pottery from the Sārnate site, near the open-sea coast of north western Latvia, and has since been documented in the pottery assemblages from a number of eastern Baltic coastal habitation sites dated to the final part of the Stone Age. These include sites on the western shore of the Gulf of Riga  and at the head of the gulf (Siliņupe and Slocene), all in present-day Latvia, as well as the above-mentioned Estonian sites of Vabaduse Sq. and Naakamäe . The pottery with porpoise tooth impressions from Latvia’s coastal belt is classed as Early Sārnate Ware. Five porpoise . Three dporpoise . One 14C14C dates from these two sites place them in the time interval 3300–2800 BC [The Vabaduse Sq. and Naakamäe sites in Estonia yielded pottery classed as Typical Comb Ware and Late Comb Ware ,39. The –2800 BC ,23,40. AComprehensive study has been undertaken on the pottery from Sārnate, Siliņupe and Riņņukalns. Porpoise tooth decoration is found on the exterior of 28% of the separate vessels identified from rim sherds recovered from dwellings of the Early Sārnate Ware phase at Sārnate, and on 9% of the vessels from area 7 at Siliņupe . The porpoise tooth stamp impressions in the Sārnate assemblage have an overall length of up to 29 mm, with imprints of four to eight individual teeth; at Siliņupe, the overall length reaches 23 mm, with imprints of four to eight teeth. The pottery with decoration now identified as porpoise tooth, classed as belonging to Early Sārnate Ware in Latvia  and to LConsidering that the porpoise could only have been hunted at the coast and along the lower courses of rivers, a very significant discovery is the occurrence of porpoise tooth impressions also on pottery from inland sites; it has been found at Riņņukalns, located at the outlet of the River Salaca from Lake Burtnieks. At this site, 95 km upriver from the coast, porpoise tooth decoration occurs on the exteriors of 6% of the vessels assigned to the phase of midden accumulation , dated tA number of pottery sherds with such decoration are also known from approximately contemporaneous ceramic assemblages in the area around Lake Lubāns , which wAs far as can be ascertained from the highly fragmented material, the designs executed in porpoise tooth stamps on the pottery vessels found at inland habitations resemble those seen on pottery from coastal sites. Various other similarities to the coastal pottery, such as the occasional occurrence of knot, plait and beaver metapodial impressions, indicate that some of the pottery occurring on the inland sites was made by people whose pottery-making techniques corresponded to those of the inhabitants of the coastal belt.14C dating (as in our study). The only exception seems to be a porpoise from the Stora Förvar site on the island of Gotland, Sweden, which has been dated to ca. 4080 cal. BC. This seems to be in accordance with the dates of the first harp seals found on Baltic settlement sites [According to Sommer et al. , the harnt sites . Both sp14C date, is needed. The highest number of bones of this cetacean relates to the time span 3400–3000 cal. BC . This coincides with the period of highest salinity in the Baltic [14C-datings of porpoises are needed from the Baltic region in order to trace the history of the harbor porpoise in this sea. In the eastern Baltic, the Riigiküla site provides the oldest archaeological context with porpoise bone finds, relating to the Narva and Typical Comb Ware cultures, i.e., ca. 4000 cal. BC . The asse Baltic , probable Baltic ,21,27,45In the middle and second half of the 4th millennium BC, porpoise tooth decoration came to be commonly applied on pottery in the eastern Baltic region. One half of the porpoise mandible, with its long, straight row of teeth, could be modified with minimal effort so as to furnish a pottery stamp comparable in length and width to the comb stamps that had already been very widely used to decorate pottery in the region for several centuries before this. Hence, impressed decoration with porpoise teeth could readily be accommodated within the established design grammar of pottery ornamentation, the porpoise tooth stamp taking the place of the comb stamp. However, we may also ask—given the porpoise’s economic importance for certain coastal communities in this period—whether the stamping of pottery with an instrument fashioned from part of this animal also had a special meaning related to the animal’s salience in the consciousness of the pottery-makers. There is practically no other evidence to suggest that the porpoise held a prominent position in the world-view of the Neolithic people in this region. In contrast to the large number of terrestrial mammal and bird representations in the form of figurines and zoomorphically carved artefact parts , so far,It may actually be that decoration on pottery served as the main medium for expressing the value that coastal communities attached to the porpoise. Because of the distinctiveness of this animal’s teeth and the imprints obtainable from them, such a message could be conveyed quite unambiguously. It is also possible to delineate the range of mental associations that the porpoise could have had. In the first place, of course, it would have been connected with the coastal and river-mouth environments that it inhabits. Further, the historical record indicates that a very important method of porpoise exploitation was mass hunting by coordinated drives, involving several boat crews with nets, as on the coast of Denmark, for example . If simiA sense of seasonality must also have featured prominently in the perception of the porpoise among the communities involved in hunting it, given that the animal was present and accessible to hunters in the region only in summer. The warmer part of the year also offered the best conditions for pottery-making (vessels could be dried more quickly for firing), and, thus, the seasonal concurrence of the two tasks may have been a factor promoting their linkage through pottery decoration. It may be significant that mandibles—the bones utilized for this purpose—are absent among the faunal remains, except for a few small fragments. Presumably, they were being curated for this special use, rather than discarded along with other bones.Because of these probable associations—with coastal environments, hunting collectives and the summer season—pottery decoration with the porpoise tooth stamp could have been heavily laden with meaning for the coastal porpoise-hunting groups, potentially conveying a self-identity founded upon the pattern of subsistence activities in these coastal areas. Cerastoderma shells in the Neolithic period in south-western Europe  [No other examples of pottery decoration with marine mammal bone or tooth impressions are known to the authors outside of the Baltic region. However, the widespread practice of impressing pottery with the serrated edges of ottery”)  and the ottery”)  (p. 111)However, how may we interpret the examples of pottery stamped with the teeth of this marine mammal that occur on inland sites? As discussed by Bērziņš and Dumpe , there aThe first possibility to consider is seasonal movement by groups from the inland lake basins to the coast for the purpose of exploiting seasonal resources , whereby pottery made at the coast during this season was being taken to inland sites along with other equipment and maybe used for transporting foodstuffs. However, the evidence from inland faunal assemblages and stable isotope data forces us to reject this hypothesis. The identified faunal remains from Neolithic sites in the Lubāns area do not include any marine species, which is unsurprising, considering the long distance from the seacoast. There is evidence of marine resources from sites in the Lake Burtnieks basin, located much closer to the sea. Thus, the faunal assemblage from Riņņukalns, in addition to mass remains of freshwater and migratory fish, also includes a minor proportion of marine fish , and seaIf we reject seasonal coast–inland mobility as an explanation for the occurrence of porpoise tooth decoration on pottery from inland sites, then we must address the possibility that the pottery vessels were among the items being transferred within the regional exchange network of this period. The exchange of tools used for making the porpoise tooth decoration, although it cannot be ruled out, seems markedly less plausible, since it would not account for the occurrence on inland sites of other characteristic traits of pottery from the coastal sites, as described above. The idea that pottery was being exchanged in the Neolithic does run contrary to the traditional view that early prehistoric pottery was locally made and, thus, serves to characterize the material culture of that particular group. On the other hand, the Neolithic archaeological assemblages from the eastern Baltic and neighboring regions testify to the existence of a highly developed system of exchange, in the frame of which amber ornaments, flint and chopping tools of Karelian metatuff were distributed across a very wide area ,56. PresA third hypothesis to consider is that practices of exogamy contributed significantly to this pattern. If pottery-makers came to live with their spouses in inland lake basin communities, then they may have brought their own tools with them and may have applied their pottery production skills in the new place of residence. The latter two hypotheses are not mutually exclusive: both artefact exchange and exogamy could have functioned concomitantly in the maintenance of links between coastal and inland communities, and so we could potentially be dealing with the movement of potters  as well as the movement of pottery vessels. Future petrographic and chemical analysis to characterize the clays and tempering materials of the region’s Neolithic pottery should permit the isolation of non-locally made ceramics. In addition, we require a better understanding of the social relationships governing the long-distance movement of various materials and finished artefacts in the Neolithic period in the eastern Baltic and neighboring areas. Moreover, this should enable a clearer contextualization of the pottery stamped with porpoise teeth.The evidence from archaeological sites in the eastern Baltic region significantly enhances our understanding of the Holocene history of the harbor porpoise in the Baltic Sea basin. Seldom encountered in eastern Baltic waters in recent centuries, the porpoise was a common seasonal visitor, especially in the period ca. 3400–3000 cal. BC, when it had a major economic importance for the coastal communities of the Stone Age, as reflected by its representation in faunal assemblages. Additionally, the porpoise mandible was very commonly used as an instrument for decorating pottery, producing distinctive imprints that may have served as an expression of the value that coastal communities of that time attached to this animal. The occurrence of this kind of ornamentation also on pottery from inland sites is a very important marker of coast–inland contact, the nature of which remains to be further investigated.


This study proposes a terahertz metamaterial structure composed of a silicon–graphene–silicon sandwich, aiming to achieve quadruple plasmon-induced transparency (PIT). This phenomenon arises from the interaction coupling of bright–dark modes within the structure. The results obtained from the coupled mode theory (CMT) calculations align with the simulations ones using the finite difference time domain (FDTD) method. Based on the electric field distributions at the resonant frequencies of the five bright modes, it is found that the energy localizations of the original five bright modes undergo diffusion and transfer under the influence of the dark mode. Additionally, the impact of the Fermi level of graphene on the transmission spectrum is discussed. The results reveal that the modulation depths (MDs) of 94.0%, 92.48%, 93.54%, 96.54%, 97.51%, 92.86%, 94.82%, and 88.20%, with corresponding insertion losses (ILs) of 0.52 dB, 0.98 dB, 1.37 dB, 0.70 dB, 0.43 dB, 0.63 dB, 0.16 dB, and 0.17 dB at the specific frequencies, are obtained, achieving multiple switching effects. This model holds significant potential for applications in versatile modulators and optical switches in the terahertz range. Surface Plasmon Polaritons (SPPs) , which aGraphene-excited surface plasmons show robust localized field enhancement effects in the terahertz and infrared wavelengths. The plasmon excitations on the surface of graphene can generate plasmon-induced transparency (PIT) , which iIn this study, we present a dual-layer graphene waveguide structure composed of five graphene ribbons  and double vertical graphene ribbons (DVRG) to achieve multiple PIT windows. The proposed structure is a multilayer terahertz metamaterial, in which a silicon substrate is sandwiched between two graphene layers, each connected to its respective electrodes, respectively. Furthermore, the destructive interference between the structure’s four bright modes and one dark mode results in significant PIT. The theoretical results of quadruple PIT are fitted using the coupled mode theory (CMT), which shows high consistency with the outcomes of the finite difference time domain (FDTD) simulations. Importantly, excellent optical modulation characteristics are achieved for our structure, since the maximum and minimum modulation depths (MDs) reach 97.51% and 88.20%, corresponding to the insertion losses (ILs) of 0.43 dB and 0.17 dB, respectively. Therefore, our proposed structure offers significant prospects in areas such as optical switching and modulation.The three-dimensional view depicted in Within the context of our proposed metamaterial structure, Molecular Beam Epitaxy (MBE) presents a promising approach for fabricating the upper Si regions. MBE is capable of growing crystalline thin films with excellent layer thickness and material quality control. This technique involves the precise deposition of atoms or molecules onto a substrate surface under ultra-high vacuum conditions. By finely adjusting growth parameters such as temperature, deposition rate, and silicon precursor flux, the controlled growth of Si layers with nanoscale thickness can be achieved.The Kubo model stands as a well-established theoretical framework in the realm of condensed matter physics. It is harnessed to ascertain the electrical conductivity of graphene, which can be formulated as follows :(1)σ(ω)=μEF/evF2 , the carobtained ,41:(4)σεsThe FDTD simulation process involves numerically solving Maxwell’s equations using a grid-based approach. It discretizes both time and space, allowing us to track the evolution of electromagnetic fields over discrete time steps, enabling analysis of its optical behavior and properties. In the simulation, the graphene layer is discretized into grid cells both in time and space. The electromagnetic field interacts with the graphene layer based on its material properties, and the FDTD algorithm iteratively solves Maxwell’s equations in a timely manner to calculate the propagation of the electromagnetic field and its interaction with the graphene layer. To elucidate the physical mechanism behind the emergence of the quadruple PIT phenomenon, the proposed structure is simulated using the FDTD method, and the transmission spectra are depicted in To further delve into the underlying formation mechanism that governs the creation of the quadruple PIT, the electric field distributions of the bright modes, the dark mode, and the overall structure at the resonant frequencies of the four PIT windows are analyzed, respectively. The SPPs’ excitation of graphene determines the intensity of the electric field, where a higher excitation level results in stronger electric field energy. Based on the analysis process above, it can be understood that in the absence of the dark mode, the energy of the five bright modes remains independently localized. However, with the inclusion of the dark mode, the destructive interferences between the dark mode and the five bright modes give rise to the formation of quadruple PIT. The originally localized bright modes undergo diffusion and transfer under the influence of the dark mode, resulting in the enhanced energy of the dark mode. This mechanism plays a crucial role in the emergence of the quadruple PIT effect.To validate the FDTD simulation results, the CMT is employed to fit the FDTD simulation results. The theoretical coupling diagram derived from the CMT is depicted in s of CMT ,44, the Im(neff) . AbidingIn addition, there is an absence of input wave propagating in the opposing direction in According to Equations (7)–(17), the complete system’s transmission coefficient can be represented as follows:Theoretical transmission characteristics closely associated with the proposed graphene metamaterial structure can be mathematically expressed as As is well known, graphene exhibits satisfactory dynamic tunability. The gate voltage dependency enables dynamic tuning of the transparency window without altering the geometric structure of graphene ,46,47. FThe commonly used metrics for evaluating the performance of optical switch modulators are the MD  and the The transmission spectra curve, acquired via FDTD simulations, is depicted in The MD values and IL values in In essence, our study introduces a dual-layer graphene terahertz metamaterial structure to realize an octa-frequency asynchronous switch, since quadruple PIT transparency windows have been generated. The mechanism of PIT formation is studied by investigating the electric field distribution. The results derived from the FDTD simulation and CMT fitting show high consistency. Through the manipulation of the Fermi level in graphene, dynamic and controllable multi-switch modulation functionality is achieved. For the proposed octa-frequency switch, the MD is generally greater than 88%, and the IL (0.16 dB < IL < 1.37 dB) is also remarkable. In the case of