Human infection studies (HIS) have generally been used as a tool in the pathway for vaccine development in high income settings. Over the last decade, this model has been implemented in LMICs with the aim of accelerating development of next generation vaccines that would perform better in these settings. However, in most LMICs, the ethics and regulatory framework for the conduct of these studies are not in place. In Zambia, these studies are yet to be conducted and thus we conducted a stakeholder engagement workshop in October 2019. We engaged with bioethicists, regulatory authority officials, and scientists from within Zambia and other African countries to anticipate and address foreseeable ethical and regulatory issues when conducting HIS in Zambia for the first time. The workshop largely focused on sensitizing the stakeholders on the benefits of these studies with the following main points for consideration on the implementation of these studies in Zambia: need for in-country legal framework and guidelines; need for adequate informed consent based on comprehensive understanding of the concept of HIS and study requirements; and requirements for heightened vigilance to assure participant safety including good ethical and clinical practice with regulatory, ethical, data safety, and community oversight. Additionally, the workshop emphasized the need for rigorous health screening prior to enrolment; suitable infrastructure for containment; and personnel to provide appropriate treatment including emergency resuscitation and evacuation if indicated. Specific recommendations included compensation for burden of participation; access to care and provision for study related injury (e.g. no-fault insurance); and withdrawal and exit procedures to preserve individual and community safety. Finally, the meeting concluded that researchers should actively engage key gate keepers including civic leaders such as parliamentarians, universities, researchers, potential participants and laypersons to avoid circulation of misinformation. CIDRZ      Centre for infectious disease research in ZambiaDSMB      Data Safety Monitoring BoardGCP      Good Clinical PracticeGMO      Genetic Modified OrganismGMP      Good Manufacturing PracticeHIS      Human Infection StudiesICF      Informed Consent FormIP        Investigational ProductKEMRI      Kenya Medical Research InstituteLMICs      Low and Middle Income CountriesNBA      National Biosafety AuthorityNHRA      National Health Research AuthorityREGs      Regulatory authoritiesWHO      World Health OrganizationZAMRA      Zambia Medicines Regulatory AuthorityThe views expressed in this article are those of the author(s). Publication in Wellcome Open Research does not imply endorsement by Wellcome. Most of these infectious diseases are preventable. However, children in LMICs, who are the most affected succumb to these diseases1 including children in Zambia4, partly because that life-saving interventions have historically been developed in high-income settings6.It is evident that low- and middle-income countries (LMICs) carry the higher burden of infectious disease in comparison to high income countries7. The lower vaccine efficacy and effectiveness observed in LMICs8 speaks to the need for next generation vaccines developed in these endemic settings to supplement existing vaccines. The urgent need to address this health disparity has led to the introduction of human infection studies (HIS) in LMICs. HIS can accelerate the pace of vaccine discovery and testing by providing an early opportunity to evaluate efficacy, circumventing the difficulties, cost, time and unpredictability of large field trials9. HIS involve deliberate or intentional infection by administration of pathogens to healthy and consenting research participants under controlled conditions with the aim of: (i) evaluating candidate vaccines and therapeutics; (ii) gaining insight into natural infections; and (iii) developing a model of infection10. HIS also facilitate detailed understanding of infectious disease pathophysiology, immunological defence mechanisms, host pathogen interactions11 and provide opportunities to identify useful correlates of vaccine protection12. Historically, the majority of HIS studies have been conducted in high-income, non-endemic settings, which has raised calls to establish HIS models in LMICs so that the findings can be relevant to the population at risk13.Vaccines are among the major cost-effective interventions in global public health with vast social and economic benefitsShigella andSalmonella; with potential to extend to other pathogens includingEnterotoxigenic Escherichia coli (ETEC) andVibrio cholerae. As HIS are new to Zambia, we aimed to create a platform upon which we could initiate the HIS conversation and contribute to strengthening capacity of ethical and regulatory bodies in governing HIS through an ethical and regulatory framework relevant for Zambia and LMICs more widely. Hence, we conducted an engagement workshop in order to: 1) understand views, expectations, and experiences of ethical and regulatory bodies, and other stakeholders involved in HIS in the region; 2) identify core ethical issues for HIS implementation in other LMICs and their implications for HIS in Zambia; and 3) develop modalities to address these issues from lessons learnt. Similar workshops have been conducted in Malawi14, India15, Uganda16, with the main outcomes presented inThe Centre for Infectious Disease Research in Zambia (CIDRZ) is poised to establish enteric disease HIS models targeting Rotavirus,The requirements of ethical and regulatory environment governing the conduct of research and clinical trials in Zambia drove the decision of who to invite for this meeting. First, ethical committees  review study protocols, followed by regulatory authorities , after which the National Heath Research Authority authorises implementation. Thus, representatives from these organizations were critical to achieving the aims of this workshop. Other considerations included gathering experiential knowledge and insights from international  ethics committees and regulators who had reviewed HIS protocols and regulated their implementation as well as collecting the perception and perspectives of local researchers who are knowledgeable about the context and field realities in Zambia. This combination of local and regional knowledge and experience helped highlight issues that maybe unique to Zambia and to HIS implementation respectively.17 helped promote discussions among small specialist and multi-disciplinary groups. Open and debriefing sessions helped collect other views outside the set agenda which might be critical to scientific and ethical conduct of HIS.In order to reduce the burden of participation for attendees while ensuring that they remained engaged and owned the whole process, we used a combination of methods that provided structure to guide proceedings but also the flexibility to allow attendees to share personal stories on key aspects of concern to HIS. Distribution of reading materials ahead of the meeting and didactic sessions aimed to create a common understanding on the current information on HIS implementation and ethical and regulatory issues arising. Participatory methods, empathy and journey mappingThe workshop was set in Siavonga, a Zambian small and quiet town situated 200 kilometres away from the busy city of Lusaka which offered the delegates a serene environment for focused deep conversations.18. The themes and topics covered included: historical perspective and scientific rationale, the benefits and need for HIS in Zambia, methodological issues that can impact implementation and outcome of HIS, an overview of planned HIS in Zambia, defining risks and burdens of participation with application to HIS in Zambia, ethical considerations particularly for compensation supported by experiences shared by Kenyan and Malawian ethical review board members, scientific overview and responsibility of the scientists in the development of a challenge agent, and a funders’ perspective regarding the global agenda and direction for HIS in LMICs. Open questions and discussions concluded these sessions.We used didactic presentations to introduce themes and topics, provide background and rationale for HIS, and to frame the key issues for discussion. Presentations were delivered within 20 minutes in keeping with the expected attention span among adultsWe employed small group discussions to deliberate on specific topics deemed critical. These groups were organised by1.Specialisations  for the discussions around methodological considerations for HIS in Zambia.2.Multidisciplinary for the discussions on: a) risk and burden of participants including through participant empathy and journey mapping; and b) benefits of HIS and participant safety.Plenary discussions were held after each small group session, where group representatives presented their main discussion points to the meeting followed by questions and answers.A total of 29 participants drawn from four different countries namely, Malawi, Kenya, UK, and Zambia. These participants were drawn from specialisations which included ethics, community engagement, social science, funder, regulatory and clinical trial conduct as shown inThe didactic sessions and the group discussions yielded key points to be considered as Zambia prepares to conduct HIS. These key considerations included but were not limited to: (i) legal and regulatory provisions and guidelines; (ii) engagement strategy to introduce HIS; (iii) compensation models to be considered for HIS in Zambia; (iv) identifying population target participants for HIS; (v) risk-benefit ratio coupled with human subject protection; and (vi) balance of adequate information during the consenting process against burden of participation see.Research in Zambia is generally guided by the laws of the land which include the National Health Research Act No. 2 of 2013, Zambia Medicines and Allied Substances Act No. 13 of 2013, the Zambia National Biosafety Act No. 10 of 2007 and the Science and Technology Act No. 26 of 2007.21. They considered the workshop engagement as opportune and timely for their submission of amendments to the current Act to the Ministry of Justice that could propose relevant clauses to cover the conduct of HIS. They discussed another avenue for legal guidance by way of a specific Statutory Instrument which could be proposed by the Ministry of Health specifically covering the conduct of HIS in Zambia.During the meeting, regulatory authorities highlighted the need to further investigate the laws under which the conduct of HIS would be permissible in Zambia and the process of reviewing HIS applications. They undertook to examine whether importation could be regulated using the World Health Organisation (WHO) categorisation of challenge agents as vaccines and HIS as a clinical trial22.A person shall not research on, develop, produce, import, export, transit, carry out any contained use, release or place on the market any genetically modified organism or any product of a genetically modified organism or deal in any manner with any genetically modified organism or a product of a genetically modified organism without the prior approval of the Authority”The National Biosafety Authority (NBA) on the other hand has the mandate to oversee biosafety and regulate importation and use of all products containing any genetically modified organisms (GMOs) and their products. Section 10 of the National Biosafety Act provides that “During the meeting, it was suggested that while the NBA act does not cover for the contained use of challenge agents or strains for HIS, an application can be considered based on scientific merit. It was further noted that, indeed challenge agents for use in HIS will likely meet the category of products overseen by NBA and will require their review and approval before use of the agents in Zambia. Of note, the NBA is required to ensure all such products are notified to the public through open media or press for at least 30 days before their intended importation or use. It was thus suggested that it would be critical to include the need for prior announcement in the media and/or press in the regulatory planning for HIS in Zambia. 23 which is critical to gaining permissions, approvals and legitimacy for any planned study24. Having informed and influential stakeholders both in formal and communal structures are essential to safeguard study volunteers and to stand in defence in any event of issues of public concern25. As one of the methodological benefits, our workshop methods helped identify who were the influential stakeholders in the formal and communal structures.Given that HIS is a new concept in Zambia, the meeting agreed that an engagement strategy must be put in place to sensitise and engage various stakeholders over and above the regular processes used in clinical studies. Correct messaging and delivery through key gatekeepers such as political and civic leadership including the parliamentary committee on health, lecturers, editors for various forms of media houses, advocates, and religious leaders could lead to authentic community and participant engagement in HIS19 were presented and distinguished from each other as inThe four compensation modelsThe meeting stressed the need to separate compensation  from provisions for study related injury . It was concluded that what would work is if Zambia combines the wage and reimbursement compensation models and not the market (demand and supply) or appreciation (token) model. Malawi offered to share their proposed model which could be adapted to the Zambian context. It was also stressed that models that are working for high income settings may not necessarily work in the Zambian context for the same kind of studies. It was suggested that a fair model needed to be put in place that will deal with issues of undue influence and ensure that participants are safe. It was also emphasized that participants needed not to be paid for participating in research but to be compensated for the risk of trial-related injuries. The meeting also emphasized the need for further thought on balancing risk, prevailing economic situations, undue coercion and altruism in HIS.It was noted during the meeting that, there are several reasons which justify HIS in LMIC settings such as Zambia. While capacity to undertake such studies has previously been the main reason to restrict the studies to high income settings, a reassessment was needed to equally justify why such studies may be done in some of high income settings, particularly in the context of vaccine development given that vaccines targeted against infectious diseases are mainly used by LMICs.The meeting pointed out that principles of justice categorically point to LMIC populations to bear some of the risks and burdens of research as they also stand to benefit from it. It was further argued that, this calls to question implementation of HIS in the high income settings where many of the diseases being studied may not be prevalent. It was argued that there are several factors, including environmental and genetic factors, that may affect how people from LMICs respond to vaccines that are developed and tested exclusively in non-endemic countries. Therefore, it was agreed that it is imperative that HIS trials are conducted in environments and individuals for which the vaccine is intended to be used.1 perform poorly in LMICs compared to Western countries. Multiple reports have alluded to context related differences between populations in high income versus low income settings such as repeated exposure to infectious diseases26 resulting in environmental enteric dysfunction, altered gut microbiome, genetic predispositions etc. as some of the reasons why vaccines perform differently27. The meeting further maintained that, these experiences suggest that research studies need to be undertaken in populations with these contextual factors which are similar for the eventual target populations; thus HIS in LMICs are likely to provide more accurate efficacy and eventual effectiveness data especially on oral vaccines.Furthermore, it was argued that findings from vaccine effectiveness reports have overwhelmingly demonstrated that some of the oral vaccines The decision to conduct/approve HIS should be based on considerations of the risk-benefit ratio coupled with the ethical principles that ensure of human subject protection and medical ethic of “do no harm”. The meeting agreed that the ethical considerations and regulatory reviews must be paramount while championing local capacity building to develop and conduct HIS relevant to the Zambian people.The meeting emphasized justice, non-maleficence, beneficence and autonomy as the global ethical principles needed to be applied when considering target population for HIS. The rule of thumb must be to target populations best suited to address the principal research questions FIRST. Once the population is identified then other key considerations should be put in place, particularly if the population has elements of “vulnerability” i.e. children, students, prisoners, low-socio-economic populations etc.It was generally agreed that considerations for target populations must NOT principally avoid vulnerable populations because often those may be the populations affected by problems that require specific research, thus excluding them may only widen the research gap instead of finding specific solutions. Rather, clear justification must be provided why they are chosen and efforts demonstrated on how the vulnerabilities will be mitigated. This discussion included children, if they were to be involved in HIS. The issue of consent versus assent with regard to minors was also discussed and the resolution was that considerations of emancipated minors should be explored further as was done by countries like Kenya. Furthermore, if a child understood processes of a particular research and they refused to participate, researchers are required to grant that particular child their desire, and thus it is not necessary to obtain consent from their guardian.28.The issue of literacy and ability to read and write as a means for better comprehension of the risks involved was discussed at length. It was stressed that while college/university students may be preferred for this reason, there is a need to recognize that there are social-economic vulnerabilities even among this class of society; especially when payment for participation is substantial and may be favourably compared to existing local wages. Examples of students from Western countries were given, in that students usually take part in these studies to make extra money to take care of their everyday needsThe meeting concluded that ensuring adequate provision of information, comprehension of the risks involved, and capacity to make a decision to participate entirely of their own free will is most important for the targeted group of individuals. It further emphasized that HIS must not necessarily avoid the poor or uneducated.The meeting emphasized that the standard principles of informed consent forms (ICF) for trials must also apply to HIS. However, given that typically HIS participants will be healthy and will be deliberately infected, the need to provide very clear information and ensure good participant appreciation of risk cannot be over emphasized.The meeting argued that, more so, the ICF for HIS must be balanced to provide adequate information given the potential and perceived risks but not make the consenting process laborious.For example, for highly infectious pathogens, the ICF must be clear that while subjects are free to leave the study at any point, they may be required to remain as “in-patient” until fully treated as a provision for pathogen containment and thus to protect the community in the spirit of public health. It further stated that there was need to be careful not to be seen to override a participants’ right, but to explain in clear terms that having the participant stay at the study facility was for their own good and that of the community they come from. It was emphasized that ensuring that a participant is cleared of infection was not only ethically imperative but ethically justifiable too. Given the various components and nuances, the meeting suggested group consent where information is shared and to move to individual at the written consent stage. It was also argued that audio-visuals and other materials could also be used to ensure that information is clearly communicated and received. It was argued that a participant needed to understand that signing the consent form meant that if they decided to withdraw from the study, they agree to stay in the study facility until a time when the researchers ascertain that they are free of the infection and safe to mingle with the community so as to contain the pathogen.Nonetheless, after much deliberation, the meeting agreed that “Autonomy is the main principle that justifies HIS”, and the application of informed consent does satisfy the fundamental ethical requirement. The meeting discussed whether proposed challenge agents should be regulated using good manufacturing practice (GMP) or GMP-like regulations. The regulators indicated that there was need for more time to consider what would be permissible in regulatory guidelines for Zambia and that an engagement meeting with stakeholders, particularly the WHO (through the African regulatory and ethics network), would be important to finalise recommendations that uphold the highest standard of quality assurance and support the local conduct and capacity development for HIS implementation including for the manufacture of challenge agents.The workshop approach (i.e. use of participants empathy maps and journey maps) and less didactic techniques provided an opportunity for rich discussion and brought out issues which would ordinarily have been left out. In addition, the ownership of the whole process of considering what needs to be addressed as the country prepares for the conduct of HIS was enhanced with regulators identifying gaps in the legal and policy frameworks and determining how to address HIS applications and conduct. Bearing in mind the local context, the meeting concluded that scientists needed to leverage public engagement with various stakeholders, as a tool to identify key issues that they need to be considered for the successful implementation of HIS in Zambia.When discussing guidelines for importation and development of the challenge agent(s) and whether GMP or GMP-like practices were required, it was clear that there is a need to review both provisions and agree on what would be acceptable for Zambia but that all this should be driven by a balance between ensuring quality assurance and promoting local capacity development in the conduct of research.The workshop also agreed on the need for continuous engagement. Target groups identified included the parliamentary committee on health given their composition and mandate of being law makers; media editors as gatekeepers on information disseminated, published or broadcasted in the press; the use of research champions such as lecturers in tertiary institutions of learning taking advantage of their intellectual independence including their position of high esteem in society; other gate keepers and influential people in target communities as well as the public at large need constant engagement and sensitisation given their hypersensitivity to anything “GMO”.The discussions around what should be considered adequate compensation for participating in HIS brought to light the fact that while the current formula of arriving at compensation for participating in research in Zambia is based on the wage and reimbursement model, there is need to consider a model which can include other aspects such as “Risk” and “Appreciation”. The meeting also agreed to ensure that when talking about compensation, it is important to ensure that both compensation for participation in HIS  and cover for injury due to participation in HIS are considered.Lastly, there was consensus on the need to review the current guidelines and provisions for the conduct of HIS including those under development by the World Health Organisation (WHO) to ensure that gaps in Zambian provisions are covered and updated.The engagement workshop on the conduct of HIS in Zambia successfully introduced these types of studies to regulators, ethicists, scientists and other key stakeholders as well as identified issues to be considered in view of setting up HIS in Zambia. Through the platform that was created: (i) the review of HIS guidelines and legal frameworks to both address local considerations and align with international ones; (ii) review of the GMP and GMP-like requirements and propose what might be acceptable for Zambia; as well as (iii) the development of an engagement strategy for conduct of HIS in Zambia will be ongoing. We summarize the discussions inNo data is associated with this article In the abstract, the authors should add the word "officials" to the sentence, "We engaged bioethicists, regulatory authority officials and scientists in Zambia, other African countries and the funder".et al. Expert voices and equal partnerships: establishing Controlled Human Infection Models (CHIMs) in Vietnam . Wellcome Open Res 2019, 4:143 (https://doi.org/10.12688/wellcomeopenres.15337.1). In this manuscript, the authors define the actual questions that structured the workshop and it is worth including this to also demonstrate how their workshop followed a similar structure particularly in exploring ethical issues around risk, compensation, consent and public engagement.In the last paragraph of the introduction, the authors should also include Vietnam as a country where similar stakeholder engagement workshops have been conducted  - Kestelyn E, Le Phuong C, Ilo Van Nuil J Summary of the considerations for HIS in Zambia to read as follows: Twenty-nine participants were drawn from four different countries: Malawi, Kenya, UK, and Zambia. These participants were drawn from 4 specialisations, including ethics, community engagement, social science, funder, regulatory officials and clinical trial scientists, as shown in Table 2.For clarity, the authors should rephrase the sentence UnderUnder the Legal Provisions and Regulatory Guidelines, were there any suggestions offered on how the process to amend the NHRA Act should be done? If yes, a statement on the process would be valuable to include.On Compensation, did the stakeholders agree on when and how compensation would be provided to participants? Would it be daily or would it be given at the end of the study? How would compensation work if an individual joins the study and decides to leave during the period under quarantine?Were any specific recommendations provided by stakeholders on what key ethical considerations must be considered when researching on children?In the presentation of the report, it would be worthwhile for the authors to compare their findings from other stakeholder workshops that have dealt with the same topics or issues in order to demonstrate how perhaps there s a consensus of views from other engagement workshops on, for example, compensation, the absence of clear regulatory and legal guidance, etc. By weaving finding from across other LMICs that are exploring or have explored the same issues this report could have a section before the conclusion and next steps headed, Summary, that highlights common concerns, challenges in conducting HIS in LMICs while maintaining that the specific findings of this report will inform the implementation of HIS in Zambia leading to conclusions and next steps.The authors have done a good job and presenting the results of the stakeholder engagement workshop in Zambia. The workshop included a variety of individuals who could contribute to the discussion on the establishment and conduct of HIS research in Zambia. Key topical issues covered are the need to do this research in LMICs like Zambia, the review of the legal and regulatory environment, and key ethical issues that need to be considered in the conduct of HIS research. The report covers these aspects well, but there are a few areas that require clarity, amendments and considerations for inclusion.Does the article adequately reference differing views and opinions?PartlyAre all factual statements correct, and are statements and arguments made adequately supported by citations?YesIs the Open Letter written in accessible language?YesWhere applicable, are recommendations and next steps explained clearly for others to follow?YesIs the rationale for the Open Letter provided in sufficient detail?YesReviewer Expertise:Bioethics and Social ScienceI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In “Engagement of ethics and regulatory authorities on human infection studies: Proceedings of an engagement workshop in Zambia,” Kunda-Ng’andu et. al. provide an overview of a workshop held in Siavonga, Zambia on conducting human infection studies (HIS) in low and middle-income countries. In this Open Letter, the authors provide a summary of the central ethical, legal, and regulatory issues raised at similar workshops held in Vietnam, Malawi, Kenya, Uganda, and India, and highlight what “stood out” at the Zambian meeting and stands out in the Zambian context. The findings presented in the aforementioned manuscript are significant and may be used to help inform future engagement workshops on HIS, as well as policy and practice in Zambia, and other geographies and countries.The authors note that one of the main reasons for conducting HIS is due to the health disparity that exists among children in high-income countries and low and middle-income countries. However, other than noting that children may be understood as one vulnerable group among others, the manuscript does not mention any particular regulatory or ethical issues pertaining to conducting HIS with children. Did the participants at the engagement workshop explore potentially conducting HIS involving children?  If the challenges related to conducting HIS involving children were discussed, it is important that these discussions are shared in this paper to help inform an important and timely debate.On page 6, the authors note that “unlike in India, we discussed the need to explore how HIC fits in existing regulatory frameworks” as opposed to developing new regulatory and ethical frameworks in Zambia. Are the authors able to explain why and how participants arrived at this decision? Such insight may prove to be helpful for other stakeholders and policy makers contemplating, and wrestling with this important decision.The proceedings as presented appear to be favorable toward conducting HIS in Zambia. Was there any direct or indirect opposition to this position? It is important that the proceedings reflect dissenting views (if any were present) to provide an accurate, and wholistic portrayal of views presented at the workshop.  When did the engagement workshop take place in Siavonga? The date will help situate the proceedings presented, especially if there was a significant delay between the meeting and publication date.  On page 9, the authors refer to “global ethical principles.” Here, it would be helpful if the authors clarify what particular principles they are referring to in this context.Are the authors able to share the list of pre-read materials that were shared with participants in the workshop? This information may help inform the design of future engagement workshops on HIS as well as help to situate the findings of the workshop.Lastly, while the paper is, generally speaking, clear, and well-written, the paper should be further proof-read as there remain a few small typos throughout  Also, it would be helpful to the reader to use one acronym throughout the paper instead of switching between “CHIM”, “HIC”, and “HIS” to refer to human infection studies.  The following seven queries are intended to draw greater insight from the knowledge exchanged during the workshop, clarify the views presented in this manuscript, and bolster the impact this paper makes on this niche area of research and policy.   et al. provide key insights into stakeholders’ views regarding conducting HIS in Zambia. This Open Letter publication serves as an important guiding piece in the development of future engagement workshops on HIS, as well policy and practice in Zambia, and other geographies and countries. In summary, Kunda-Ng’anduDoes the article adequately reference differing views and opinions?YesAre all factual statements correct, and are statements and arguments made adequately supported by citations?YesIs the Open Letter written in accessible language?YesWhere applicable, are recommendations and next steps explained clearly for others to follow?YesIs the rationale for the Open Letter provided in sufficient detail?YesReviewer Expertise:Global Health Ethics; Ethics of Clinical Trials; Research Ethics; Global Bioethics; and Global Justice.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


As an essential and fundamental process of life, cell development involves large‐scale reorganization of the 3D genome architecture, which forms the basis of gene regulation. Here, a landscape‐switching model is developed to explore the microscopic chromosomal structural origin of embryonic stem cell (ESC) differentiation and somatic cell reprogramming. It is shown that chromosome structure exhibits significant compartment‐switching in the unit of topologically associating domain. It is found that the chromosome during differentiation undergoes monotonic compaction with spatial repositioning of active and inactive chromosomal loci toward the chromosome surface and interior, respectively. In contrast, an overexpanded chromosome, which exhibits universal localization of loci at the chromosomal surface with erasing the structural characteristics formed in the somatic cells, is observed during reprogramming. An early distinct differentiation pathway from the ESC to the terminally differentiated cell, giving rise to early bifurcation on the Waddington landscape for the ESC differentiation is suggested. The theoretical model herein including the non‐equilibrium effects, draws a picture of the highly irreversible cell differentiation and reprogramming processes, in line with the experiments. The predictions provide a physical understanding of cell differentiation and reprogramming from the chromosomal structural and dynamical perspective and can be tested by future experiments. Chromosomes are the structural scaffolds for gene functions, which determine the cell developmental process. A non‐equilibrium landscape model is developed to identify the chromosomal structural and dynamical origins of cell differentiation and reprogramming. The irreversible pathways in differentiation and reprogramming show distinct dynamical rearrangements for the chromosome structure to facilitate the formation of specific gene expression patterns and associated functions. The roles of epigenetic modifications, such as DNA methylation and histone modifications, have been highlighted in modulating the gene expression across different cell types and lineages. As the scaffold for gene expression, genome structure is constantly altered by epigenetic modifications. Therefore, elucidating the microscopic molecular structural origin of cell differentiation at the genomic level is critical for understanding the differentiation mechanism yet still challenging.Cell is the basic unit of life. Cells have two essential functions. One is the division for reproduction, and the other is differentiation for multipurpose functionality. Cell differentiation has been suggested to be determined by the underlying gene regulatory network.1 A fu Derived from the chromosome conformation capture techniques, Hi‐C measures the frequency of genomic loci spatially close to each other, resulting in a high‐resolution chromosome contact map to infer chromosome structure. The prominent findings of Hi‐C show that the chromosome is hierarchically organized through multiple scales from the local self‐interacting domains, termed as topologically associating domains (TADs), to the long‐range segregation patterns, termed as compartments. TADs are characterized as the functional units of the genome in controlling the gene expressions by restricting the promoters and enhancers within one TAD. On the other hand, compartments form two types of mutually excluded segregations, referred to as compartment A and B, corresponding to the gene‐rich, open active euchromatin and the gene‐poor, closed repressive heterochromatin, respectively. TADs and compartments are critical genome structural features that are found to play important roles in many biological processes, including DNA replication, transcription, and gene regulation.Increasing evidence suggests that genome folds into a 3D architecture.6, 7 the cell differentiation process can be metaphorically referred to as an iconic picture, where a ball rolls down on a surface (landscape) from the top to the lowest point. The guidance of the ball in seeking stable positions, which are the differentiated cell states, is controlled by the landscape that is shaped by the epigenetic mechanism. Genome structure, which is in intimate relation to epigenetic modifications, has been found to undergo high‐order structural rearrangement during differentiation, in order to accommodate the required gene expression pattern possibly through modulating the accessibility of the genomic loci to transcription factors. Therefore, cell differentiation should be associated with a landscape at the microscopic molecular level that describes the genome structural dynamics, as a reminiscence of the one widely used in protein folding. The information of this landscape can provide a physical understanding of genome dynamics and microscopic molecular structural origin for cell differentiation, giving insights into the underlying mechanism. Therefore, such structural landscape at the genomic level can serve as the physical and structural basis for understanding and quantifying the Waddington landscape at the gene expression level for cell differentiation and development. However, exploring such structural landscape in practice is very challenging due to the lack of precise determination of the chromosome structure and dynamics, as well as the non‐equilibrium nature of cell differentiation distinctly different from protein folding. A natural initial step toward this ultimate goal is to quantify the chromosomal structural rearrangement during cell differentiation.In the context of Waddington's epigenetic landscape,21 the Cell reprogramming, induced by ectopic expression of the key transcriptional factors, erases the epigenetic modifications established in the differentiated cell, and results in the reorganization of chromosome structure into an ESC‐like form. Understanding how the chromosome undergoes the structural rearrangement during reprogramming has important implications in tissue engineering for developing new approaches to improve the reprogramming efficiency, however, is currently not well addressed. The understanding of cell reprogramming at chromosome structural level can complete our knowledge of the forward and backward cell development processes, however, is still lacking.Embryonic stem cells (ESCs) are pluripotent cells and can differentiate to the somatic cells during cell development. On the other hand, a somatic cell can be reprogrammed back to the induced pluripotent stem cell (iPSC), which genetically behaves like the ESC with high pluripotency.25 CelHerein, we developed a non‐equilibrium landscape‐switching model to investigate chromosome dynamics during cell differentiation and reprogramming. The key element that controls and induces the differentiation and reprogramming was modeled as an energy excitation that triggers the processes for switching of chromosome structural dynamics between the two landscapes, representing the ESC and the terminally differentiated cell, followed by the subsequent conformational relaxation. The model allowed us to accumulate a statistically sufficient number of differentiation and reprogramming trajectories and perform the subsequent chromosome structural analysis. We found that the chromosome shows compartment‐switching in the units of relatively stable TADs during both the differentiation and reprogramming processes. Chromosome in differentiation undergoes gradually monotonic compaction with spatial repositioning of active and inactive genomic loci, while an overexpanded chromosome structure with universal localization of the loci at the chromosome surface appears in reprogramming. Further analysis of the differentiation process exhibited a distinct pathway that bifurcates at the quite early developmental stages to the destined differentiated cell. Our model made extensive predictions that have suggested a chromosomal‐level structural and dynamical picture of cell differentiation and reprogramming.22.1 To simplify the processes, here we used the ESC to replace the iPSC as the destination for reprogramming. This is a reasonable approximation based on the recent evidence that the human ESC and iPSC are equivalent in terms of gene expression patterns. On the other hand, we used the IMR90 as the somatic cell in our study. IMR90 is the terminally differentiated human fetal lung fibroblast cell and has been proved capable of reprogramming to the iPSC by various transcriptional factors.We developed a non‐equilibrium excitation‐relaxation energy landscape‐switching model to investigate the chromosomal structural rearrangement during the differentiation of the ESC to the somatic cell and the reprogramming of the somatic cell to the iPSC. The model is similar to the one used for studying the cell‐cycle chromosome dynamics in our previous study.28 To Figure  This approach provides two effective energy landscapes that generate the experimentally consistent chromosome structural ensembles and kinetically govern the chromosome dynamics at the ESC and IMR90, respectively . It is worth noting that chromosome dynamics at both the ESC and IMR90 are out‐of‐equilibrium. However, an effective energy landscape was demonstrated to be capable of describing the behaviors of non‐equilibrium systems in certain cases not too far from equilibrium. Then we triggered the differentiation/reprogramming process by switching between these two effective landscapes. Finally, the chromosome was relaxed under the new effective landscape. In practice, we termed the switching and relaxation from the ESC to the IMR90 landscape as a differentiation process and from the IMR90 to the ESC landscape as a reprogramming process, respectively. Such switching can be regarded as an energy excitation that activates the system to trigger the escape from the local minima on the energy landscape and achieves the transition to a new one. This excitation requires energy input or cost, which is provided by the underlying molecular processes . The net energy input or cost leads to the detailed balance breaking of the system, resulting in a non‐equilibrium process. Therefore, the model captured the essence of cell differentiation and reprogramming processes and can provide insights for the underlying non‐equilibrium dynamics. Further details of the model can be found in Section 5 and our previous study.We focused on the long arm of chromosome 14 (20.5–106.1 Mb). With a resolution of 100 kb, the chromosome segment was reduced into a coarse‐grained polymer representation with 857 beads. The Hi‐C maps and compartment profiles of the chromosome segment at the ESC and IMR90 show distinct differences Figure 1, indicFigure Pi,jt  between the time frames t = I and t = J, and then obtained a time‐dependent matrix ΔPI, J  versus the genomic distance (s) in the chromosome and we see the slope a in the relation of P(s) ≈ sa gradually changes from –1.5 to –1.0 in differentiation and –1.0 to –1.5 in reprogramming  and is more loosely formed in the ESC .We accumulated a statistically sufficient number of chromosomal structural rearrangement pathways during the ESC differentiation and the IMR90 reprogramming, and then calculated the contact probability map of the chromosome at each time frame, referring to as the evolution of the Hi‐C heat map in differentiation and reprogramming Figure 2. We quJ Figure . These ms Figure , indicats Figure , the chrs Figure . We thenPI, J . Chromosomes at these stages are shown with Hi‐C heat maps and representative structures  is overdecreased under that in the ESC . We see that most of the TAD boundaries are conserved during differentiation and reprogramming  and an anisotropy measure, the aspheric quantity Δ calculated using the inertia tensor. Deviation of Δ from 0, which corresponds to a perfect sphere, measures the extent of anisotropy. As shown in Figure Rg decreases with Δ increasing first and then followed by decreasing due to the anisotropic compaction. Interestingly, the chromosome in reprogramming apparently undergoes distinct pathways, which are not the reversal of those in differentiation. The pathways projected on PA1 and PA3 indicate isotropic expansions during the IMR90 reprogramming to the ESC . Furthermore, we divided Q into different categories varied by genomic distances between the contacting pairs. From Figure QESC and QIMR90 during reprogramming  on the trajectories of the chromosome compartment profiles evolving during differentiation in our simulations , mesendoderm (ME), neural progenitor cell (NP) and mesenchymal stem cell (MS).22 As s Figure . InteresFurther projecting the pathways onto contact probability at various pairwise contact ranges of the chromosome clearly shows that the TB, NP, and MS are neither on the pathways of the ESC differentiating to the IMR90, nor on the pathways of the IMR90 reprogramming to the ESC Figure . A simil53τ to perform the switching and a small number of loci is associated with a slower switching timescale 10τ. Similar phenomena were found in the experiments where the compartment switches universally and gradually to the differentiated cells, while loci may be heterogeneous for participating in chromosomal structural rearrangement during differentiation.Since the compartment profiles of the chromosome change significantly during differentiation, we grouped the chromosomal loci based on the patterns that they adopt for compartment‐switching and then examined the change of their compartmental values along the differentiation times Figure . There antiation.533 Nevertheless, slight structural changes of TADs were observed with a rapid increase and decrease of TAD size during differentiation and reprogramming, as evidence to show that the chromosome structure and dynamics regulate the transcription activity during these two processes. On the other hand, the relatively slow and notable changes in chromosome compartments were observed in terms of switching compartment A/B during differentiation and reprogramming, recapitulating the findings existing in Hi‐C data and the analysis that epigenomes differ significantly for various cell types. Interestingly, we found distinct scenarios for the chromosome to perform the compartment‐switching during differentiation and reprogramming. Compartment‐switching undergoes monotonic change during differentiation, though a small degree of fluctuations may be encountered due to cell stochasticity. In contrast, during reprogramming, compartment profiles show aberrant changes in extensive regions, where compartmental values may be significantly different from the differentiated cell or the destined ESC.The landscape‐switching model developed here and then applied to the cell differentiation and reprogramming processes produced the large‐scale structural rearrangements occurring in a long human chromosome segment. We found that most of the TAD boundaries conserve during these two processes, in concordance with many experimental findings that TADs are conserved between different cell types.54 Nev This implies that the structure of the chromosome in such intermediate state does not lay in between the condensed cylinder in prometaphase or decondensed fractal globule in interphase. Further analysis of the chromatin‐protein binding experiments showed that the chromosome is free of structural maintenance of chromosomes (SMC) complexes at such intermediate state. In our recent study on the cell cycle, we established a correspondence of the overexpanded chromosome observed in the simulations to the intermediate state in the experiments. The overexpanded chromosome topologically increases the accessible surface area for the loading of the SMC complexes, thus it may promote the chromosomal structural rearrangement in favor of an efficient mitotic exit process. Likewise, we suggest a similar biological role of the overexpanded chromosome in facilitating the reprogramming process by opening the structure for protein binding. Different from cell differentiation, cell reprogramming is quite inefficient. From computational efforts, here we can propose a strategy to accelerate the reprogramming by opening the chromosome structure through chromosome remodeling, which has been found to play an important role in reprogramming of the somatic cells to pluripotency.By projecting the trajectories onto several order parameters, we obtained the chromosomal structural rearrangement pathways during differentiation and reprogramming. We attributed the aberrant changes in the compartment profiles during reprogramming to the overexpansion that occurs at both local and non‐local contact ranges in the chromosome, though under different timescales. Such chromosome structure with spatial repositioning of the chromosomal loci universally to the border of chromosome territory Figure , is foll58 Thi Recent experiments interestingly showed that the mouse ESC can differentiate to the same destined state via multiple paths, as evidence of stochasticity. From a physical perspective, these facts underscore the prevalence of non‐equilibrium and stochastic characteristics in cell differentiation and reprogramming, where the cell is constantly under intrinsic and extrinsic fluctuations from the underlying biochemical reactions involving a large number of molecules and the external stimuli in making cell‐fate decision. Therefore, the chromosomal structural rearrangements occur in a highly stochastic manner during differentiation and reprogramming. Still, the averaged irreversible pathways can be determined to some extent, driven by the destined states.The chromosomal structural rearrangement pathways observed in our simulations show high stochasticity and irreversibility for the differentiation and reprogramming processes. This finding is consistent with increasing experimental evidence that reprogramming proceeds in a way that deviates largely from the simple reversal of differentiation.64 Rec The resulting probability landscape provides a quantitative basis for the original pictorial Waddington's epigenetic landscape. This can be used to guide the dynamics of cell development. Furthermore, it was shown that the cell‐fate decision‐making processes are additionally determined by the curl force (flux), which originates from the broken detailed balance in the non‐equilibrium dynamics. The detailed balance breaking leads to the non‐equilibrium thermodynamic cost for maintaining the functional cell states, supplied by the underlying molecular processes . As the epigenetic information is encoded in genome architecture, we can suggest a landscape based on our simulation findings at the chromosomal level  because of the non‐equilibrium effects. Thus, they may bypass the MS, which is a metastable attractor on the landscape. The deviation is controlled by the directions and magnitudes of the flux, which can drive the developmental paths in practice to go through distinct differentiated intermediate states in reaching the destination. As the efficiency of reprogramming from the somatic cells to the iPSCs is generally lower than 1%, the terminally differentiated cells are stable and likely located at the minima of the landscape and reprogramming thus externally requires the introductions of reprogramming factors into the system. In addition, reprogramming undergoes different paths that do not overlap with the differentiation ones, due to the non‐equilibrium effects, in line with the theoretical predictions. Chromosomes on the differentiation and reprogramming paths located at the same level as the same layer on the landscape should be structurally different. This is also observed experimentally, when the mouse B cell was reprogrammed to a state which resembles the epiblast, but is very different at the genomic and genetic level in terms of Hi‐C and RNA‐seq data.By developing a theoretical framework, we previously investigated the landscape and path of cell‐fate decision‐making processes based on the gene regulatory network.68 The the gene regulatory network that determines the cell development process shows multistability, corresponding to different cell fates. Besides, the transitions between these cell fates in the cell development have been found to occur abruptly, similar to a switch between different gene expression states. Recent theoretical studies showed that the simple circuitry of bistable switches between two gene expression states, which correspond to distinct cell fates, is able to capture many characteristics of the cell development in reality. Overall, our landscape‐switching model is in accordance with the switching between the bistable cell fates in cell development. However, the model has two prominent limitations worth noting. First, cell development has been simplified as a bistable system, while the intermediate cell states, often recognized as the multipotent progenitor cells in differentiation, may exist during the process. Second, the model implements the instantaneous switch to trigger the transition between different cell fates, whereas the real biological responses in both cell differentiation and reprogramming should occur with finite reaction rates. The most plausible solution for overcoming these limitations is to add more intermediate cell fates into the model when the experimental data are available, so the model will eventually have the stepwise switches between the multistable cell states during cell differentiation and reprogramming. Besides, our model does not consider the cell cycle dynamics, due to the lack of experimental Hi‐C data and the knowledge of the quantitative relation between the cell cycle and the cell development. In reality, there are usually many cell cycles during cell differentiation and reprogramming. Since we are mainly interested in the development process in this study, for simplicity, we focused on the slow developmental process while treating the fast cell‐cycle dynamics as an averaged background. Therefore, the model provided a prediction of the chromosome structural evolution during cell differentiation and reprogramming at the interphase, which accounts for most of the time in the cell cycle. Further improvements in implementing the cell‐cycle dynamics into cell development should be considered in the future.As evidenced by the experiments,72 the to obtain the time‐evolving Hi‐C maps during differentiation and reprogramming. Cell differentiation and reprogramming depend on both the intrinsic and extrinsic factors, so the processes may vary under different conditions. The switching implementation imposes an instantaneous change of the conditions to the desired ones, so our observations may correspond to the extremes, where the transitions between cell fates are strongly favored. With rapid developments in improving the reprogramming efficiency, we anticipate that the predictions present here can be tested by the experiments in the future.Our computational predictions can be potentially assessed via the targeted experiments. In this regard, it would be highly useful to apply the time‐series Hi‐C experiments, which were recently developed and applied to the cell cycle process,82 to 4In summary, we explicitly considered the non‐equilibrium dynamics and investigated chromosomal structural rearrangement during cell differentiation and reprogramming through a landscape approach. We simplified the non‐equilibrium effects as an instantaneous energy excitation followed by the subsequent relaxation, so the kinetics was driven by the landscape‐switching, which resonates with the cell‐fate switching in multistable cell development. Our approach provides a physical and structural evolution picture of the cell‐fate decision‐making processes for differentiation and reprogramming through the chromosome structural dynamics. The predictions made in this study are in need of assessments by future experiments. Our model can be extended to gain insights into the chromosome pathological disorganization in cancer cells and cell senescence.5 and are publicly available on GEO with accession number GSE35156. Hi‐C data for the four cell lines  during the early ESC development used for comparisons in this study can be downloaded on GEO with accession number GSE52457. Hi‐C data were handled by the Hi‐C Pro software through the standard pipelines. To best minimize the effects of the experimental errors existing in Hi‐C experiments, all the replicas for each cell line were collected and used for generating the Hi‐C contact map at a resolution of 100 kb.Human embryonic stem cell (ESC) and human fetal lung fibroblast cell (IMR90), a terminally differentiated cell, were used as the two start/end points to investigate the differentiation and reprogramming processes. Hi‐C data of these two cell lines were measured by Dixon et al.10 and which is made up of bonded potentials to maintain the chain connection and soft‐core nonbonded potentials to allow chain crossing as an outcome of topoisomerase. Topoisomerases, which are found abundant in cell nucleus, show wide participation in most of the reactions that involve double‐stranded DNA, including transcription and recombination, which are essential for cell differentiation and reprogramming. As noted elsewhere, the underlying kinetic behaviors of the chromosome loci are not affected by the soft‐core potentials when the chromosome is within one cell state. The soft‐core potentials were applied throughout the simulations to keep the effects of the topoisomerase being constantly active.A generic polymer model was used as the basic background,36 whi The potential of the polymer model for the ESC or IMR90 can be written as:VPolymer is the potential of the generic polymer model and the restraint potential from the Hi‐C data (VHi − C) is linearly added with VPolymer as requested by the maximum entropy principle. Hi‐C maps were normalized to be contact probability maps fi, j, where i and j are chromosomal loci, by assuming the adjacent beads always form contacts fi, i ± 1≡ 1. Therefore VHi − C has an expression:Pi, j is the calculated contact probability using step function and αi, j is the parameter that is determined by the maximum entropy principle through multiple rounds of iteration to ultimately match Pi, j to fi, j. Details can be found in previous studies.In order to best reproduce the Hi‐C map for the ESC and IMR90, the experimental restraints were implemented into the generic polymer model by means of maximum entropy principle.32 TheVESC(IMR90), which can generate a chromosome ensemble in matching the Hi‐C map and reproduce the kinetic essence of chromosome dynamics in experiments. The sub‐diffusivity of the genomic loci in the chromosome was observed at both the ESC and IMR90 . The diffusion exponents, which are characterized in the relation of mean square displace (MSD) and time t with MSD ∼ tβ, are found to be β ≈ 0.4, in line with previous experimental observations.A prominent outcome of the maximum entropy principle simulation is the potential  an energy excitation‐relaxation landscape‐switching model was developed to approximately and explicitly include the non‐equilibrium effects in simulations to describe the chromosome conformational transition between the interphase and mitotic phase for the cell‐cycle dynamics. Here, the model herein was extended to the ESC differentiation and the IMR90 reprogramming. In practice, the non‐equilibrium simulations were performed in three steps:In a previous work on the cell cycle by the authors,28 an VESC (preparing for differentiation) or VIMR90 (preparing for reprogramming);i) Chromosome was initially simulated under the potential VESC to VIMR90 (triggered to differentiation) or VIMR90 to VESC (triggered to reprogramming) was implemented;ii) A sudden switch of the potential from VIMR90 (differentiation) or VESC (reprogramming) for a long period of time.iii) Chromosome was relaxed under the new potential  Iterative correction and eigenvector decomposition (ICE) method was then used to normalize the enhanced contact probability map, which was subsequently converted into a Pearson correlation matrix. Finally, the previous suggestion to perform the principal component analysis (PCA) on this matrix was followed, and the first PC (PC1) was assigned as the compartment profiles. The direction of the PC1 values is arbitrary, and the positive and negative PC1 values were set with gene density (positive to gene‐rich and negative to gene‐poor). This is done on the IMR90 data. Then, the direction of the PC1 values during differentiation and reprogramming was determined according to the correlation coefficient with the PC1 values of the IMR90 Hi‐C data.To identify the compartments, the enhanced contact probability map  was first calculated, which is the observed versus expected contact map, at a reduced resolution level (1Mb).93 Ite The same size of sliding square (500×500 kb) within the original application was used to calculate the insulation score. The local minima of the insulation score represent the areas of high local insulation, which may correspond to the boundaries of TADs. A delta vector that measures the slope of the insulation vector to quantitatively determine the boundaries of TADs was then used. Practically, the delta vector with crossing the horizontal 0 and difference between the local left maximum and local right minimum higher than 0.1 signified the boundary of a TAD. To calculate the boundary matching population, 100kb noise error in accounting as an exact matching was allowed. It was found that the average numbers of beads in forming the TADs for the chromosome focused in this study identified from the Hi‐C maps are about 9 and 10 in the ESC and IMR90, respectively. Forming the TADs by such relatively low numbers of beads could be contributed by the thermal fluctuations, which can transiently induce the contacts formed by the beads close in sequence. Therefore, the TAD formation in the model was evaluated for the chromosome ensemble at the ESC and IMR90. This was undertaken by comparing to the polymer ensemble generated by the simulations with applying only the generic polymer potential VPolymer. It was found that the insulation score of the polymer ensemble constantly remains zero, corresponding to no TAD formation . The result indicated that the TADs are robustly formed in the chromosome model under the Hi‐C constraint potentials VESC(IMR90).The boundaries of TADs were identified by the insulation score proposed by Crane et al.44 The0.3%, were chosen. These structures were used as the initial structures for the landscape‐switching simulations. There are 354 and 226 simulation trajectories for differentiation and reprogramming, respectively. All the trajectories were collected and used for calculating the contact probability, compartment profiles, insulation scores, radial density and the averaged pathways. The changes of TAD size and compartmental values for A to B switching with the time were presented as mean ± standard deviation.The hierarchical clustering on the chromosome ensemble generated by the maximum entropy principle simulations was performed on the randomly selected 2400 chromosome structures. The cut‐off criteria were chosen to be the up‐limit of structural difference for chromosome conformational diffusion . By these criteria, chromosomes within one cluster are structurally similar, as shown by the contact maps. Two structures from each cluster, whose population is higher than The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Currently, the Spinal Instability Neoplastic Score system is used in clinics to evaluate the risk of fracture in patients with spinal metastases. This method, however, does not always provide a clear guideline due to the complexity in accounting for the effect of metastatic lesions on vertebral stability. The aim of this study was to use a validated micro Finite Element (microFE) modelling approach to analyse the effect of the size and location of lytic metastases on the mechanical properties of human vertebral bodies. Micro Computed Tomography based microFE models were generated with and without lytic lesions simulated as holes within a human vertebral body. Single and multiple lytic lesions were simulated with four different sizes and in five different locations. Bone was assumed homogenous, isotropic and linear elastic, and each vertebra was loaded in axial compression. It was observed that the size of lytic lesions was linearly related with the reduction in structural properties of the vertebral body (reduction of stiffness between 3% and 30% for lesion volume between 4% and 35%). The location of lytic lesions did not show a clear effect on predicted structural properties. Single or multiple lesions with the same volume provided similar results. Locally, there was a homogeneous distribution of axial principal strains among the models with and without lytic lesions. This study highlights the potential of microFE models to study the effect of lesions on the mechanical properties of the human vertebral body.  •MicroFE models can show the effect of lytic lesions on vertebral properties.•The size of the lesions was more critical than the location of the lesions.•Lesions affecting the cortical shell had a larger effect on the local strains.•Multiple lesions showed a similar effect to single lesions. These l2 = 0.26, n = 45 from T3 to T12 2 = 0.51, n = 10 from T1 to L1 Experimental studies show some controversy about the effect of the size of mechanically induced lytic lesions on the strength of human vertebrae. A few studies performed on human vertebrae tested under eccentric compression found no  or weak Finite element (FE) models based on general geometries or clinical computed tomography (CT) images of human vertebrae, have been used to better understand the importance of the size and location of lytic lesions on the risk of vertebral burst fracture initiation . In thes2 ≥ 0.88 for vertebral strength) (MicroCT based FE models (microFE) can be generated from high resolution images and, by resolving bone microstructure, these models can provide a more detailed understanding about the effect of diseases such as bone metastases on the local and structural properties of bones. These models have been recently used to evaluate the effect of structural changes due to osteoporosis before and after vertebroplasty . The loctrength) .The aim of this study was to develop a computational framework to analyse the effect of the size and location of virtually simulated lytic lesions on the local and structural mechanical behaviour of human vertebrae.22.1One cadaveric spine fixed in formaldehyde was acquired from a 78 years old male donor with no medical history of bone disease  under the approval of the ethics committee of the University of Sheffield (reference number 012716). The vertebral motion segments from T12 to L2 were isolated and the posterior elements were removed. The middle vertebra (i.e. L1) was scanned in saline solution with a microCT scanner  with the following parameters: voltage 70 kVp, intensity 114 mA, integration time 300 ms, and isotropic voxel size 39 μm, similar to . The ima2.2From the reconstructed microCT image a section of the vertebral body was isolated, by removing 20% of its total height from each endplate  . Before 2.3The “control model” was generated by converting every bone voxel of the binary image of the vertebral body section into an 8-noded linear hexahedral element. Bone was modelled as a homogeneous, isotropic, and linear elastic material with elastic tissue modulus equal to 12 GPa  and PoisFrom the control model 20 models were generated with simulated lytic lesions of different sizes and locations . SimulatU) was estimated as the force required to deform 2% of the elements of the models (p3Y = −8000 μɛ) or tension (ɛp1Y = 7200 μɛ) (U) with and without simulated lytic lesions was estimated among the vertebral models. The distribution of the first and third principal strains was obtained from the middle 70% in height of the models with and without simulated lytic lesions. The control model was the largest model, it had 376 million degrees of freedom and took approximately 28 h to solve and post-process  using parallel distributed memory with a maximum of 64 cores .Each microFE model was simulated under the same boundary conditions of the control model. Spring stiffness (K) was computed for each model as the ratio between the sum of the axial reaction forces estimated from the caudal section of each model and the applied displacement. The ultimate force (Fe models  beyond t7200 μɛ) . The per3https://doi.org/10.15131/ shef.data.11958954). The K and FU for the control model and for those with simulated lesions are reported in 2 ≥ 0.99, intercept between −0.004 and −0.049%) (U) caused by simulated single lytic lesions ranged from 3% to 30%. Single lesions occupying 4% and 12% of the VBvol showed similar reductions in structural properties regardless of their location (3%–13% reduction in K and Fu). Single lesions occupying 24% and 35% of the VBvol showed higher reduction in predicted structural properties (19%–30% reduction in K and FU) compared to smaller lesions occupying 4% or 12% of the VBvol  a–b. The he VBvol . For sinobserved .Table 1SSimilar results were obtained between single and multiple lesions with the same total VBvol c–d equalAt the local level, there were generally similar distributions of axial principal strains within the bone tissues of the models with and without simulated lytic lesions  . SimilarLocally, high compressive strains were observed in the cortical shell of the vertebral models with and without simulated lytic lesions, for single and multiple lesions . Lesions4This study aimed to develop a method to evaluate the effect of the size and location of simulated lytic lesions on the local and structural properties of the human vertebral body using a previously validated microFE modelling approach .The results showed that the size of the simulated lytic lesions was linearly related to a decrease in predicted structural properties, with limited contribution of the position of the lesion or of multiple lesions scenario. Nevertheless, it remains to be investigated whether the same linear relationship would hold for larger simulated lytic lesions and for different specimens with different microarchitecture. These results are in line with previous studies, based on low resolution clinical images, which showed that the effect of the size of simulated lytic lesions on the vertebral mechanical properties is higher than that caused by the location of the lesions .At the local level, lytic lesions occupying up to 35% VBvol, had a minor impact on the distribution of axial principal strains, as these were similarly distributed among all the models with and without simulated lytic lesion , Fig. 4.This study has a number of limitations. The simple compressive boundary conditions used in this study does not take into account for potential involvement and deformation of the endplates or of other relevant structures for the physiological loading distribution as the facet joints . The lytNevertheless, the developed computational models have the advantage of considering for the first time the effect of lytic lesions on the trabecular microstructure alone or tangent to the external surface of the cortical shell, lesions that would be impossible to generate with standard experimental approaches. This study aimed to present the methodological approach and preliminary data on one specimen and, therefore, further analyses should be done to evaluate the effect of cortical involvement on the mechanical properties of the vertebral body. Nonetheless, its application to a large cohort of images acquired for different subjects has the potential of revealing the effect of different lesions' parameters on the mechanical properties of the metastatic vertebrae and to improve current scoring systems based on biomechanical assessment of vertebral structural stability.In conclusion, the present study showed how a linear microFE modelling approach can be used to evaluate the effect of lytic lesions on the vertebral mechanical properties, and highlighted that the size of the lesion is the most important parameter to take into account when estimating the effect of lytic lesions on structural and local mechanical properties of the vertebral body. While, the generalization of these results should be supported by a larger dataset and more loading conditions in a future study, these results can be used to improve current scoring systems (e.g. SINS) by including in them a quantitative parameter as the size of the lesion, that could be measured from the medical images.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Deep interactive evolution (DeepIE) combines the capacity of interactive evolutionary computation (IEC) to capture a user’s preference with the domain-specific robustness of a trained generative adversarial network (GAN) generator, allowing the user to control the GAN output through evolutionary exploration of the latent space. However, the traditional GAN latent space presents feature entanglement, which limits the practicability of possible applications of DeepIE. In this paper, we implement DeepIE within a style-based generator from a StyleGAN model trained on the WikiArt dataset and propose StyleIE, a variation of DeepIE that takes advantage of the secondary disentangled latent space in the style-based generator. We performed two AB/BA crossover user tests that compared the performance of DeepIE against StyleIE for art generation. Self-rated evaluations of the performance were collected through a questionnaire. Findings from the tests suggest that StyleIE and DeepIE perform equally in tasks with open-ended goals with relaxed constraints, but StyleIE performs better in close-ended and more constrained tasks. In 1987, Richard Dawkins introduced a system of simulated selective breeding of artificial organisms called Biomorph ,3. EArt This paper explores the intersection of generative adversarial networks (GANs) and interactive evolutionary computation (IEC) within the context of artistic artifact generation and creative expression. GANs are state-of-the-art generative models that have been applied in artifact generation involving video, audio, 3D models, virtual ambiance and videogames ,12,13,14We first began exploring the capabilities of DeepIE for artistic production in , and whiThe rest of this paper is structured as follows. After an overview of the theoretical background in IEC can be defined as an evolutionary computation optimization method whose fitness functions are replaced by the subjective evaluation of a human user. IEC can also be understood as a cooperative endeavor between the human user and the EC system that maps the computational parameters with the psychological space in the user’s mind. Interactive versions of most EC techniques such as genetic algorithms, evolutionary strategy, and genetic programming exist. Genetic operators such as crossover and mutation are common in IEC applications, especially in a subtype of IEC know as simulated breeding or user selection . For somG that learns the data distribution from a noisy sample, denoted by the letter Z, and a discriminative neural network model D that learns to distinguish between the original training data and the fake output of G. The adversarial training corresponds to a minimax game, where the training goal for G is to maximize the probability of D making a mistake. The networks compete until a Nash equilibrium is reached; at this point, the fake samples should be indistinguishable from the real data [GANs were first introduced by Goodfellow et al. , rapidlyeal data . Both theal data . The orieal data , includieal data , convolueal data , and bateal data . Trainineal data  introducW. Ideally, disentangling the latent space means that the dimensions in the latent vectors correspond to no more than one feature. In practice this is still far from being perfectly achieved; however, StyleGAN represents an important step in that direction [Z with a dimensionality of 512. The synthesis network consists of 18 convolutional layers, two for each resolution  encode coarse styles. Middle resolution layers (the next four row-vectors) encode “middle styles.” High resolution layers (the last ten row-vectors) encode “finer styles.” This allows for style transfer within w latent matrices, as seen in The synthesis network works on nine resolutions, from z sampled from a Gaussian distribution fed to the GAN, and the phenotypes are the generated images. DeepIE is a form of simulated breeding that relies on evolutionary operators such as crossover and mutation. The crossover and mutation operators are done at the genotype level, where the components of the latent vector are the alleles. The rationale behind DeepIE is that the trained generator of a GAN can be used as a map between genotype and phenotype within a particular domain. The latent space of the generator becomes the search space of the IGA. The objective of the DeepIE system is to map the search space of the IEC to the psychological search space in the user’s mind. This is the main conceptual appeal of DeepIE compared to other IEC techniques: better artifact representations and a learned search space where every point corresponds to viable artifacts. The generative model produces an initial population of n individuals from random latent vectors. The user selects the images of their preference. The latent vectors of the selected images are included in the mating pool. Pairs of images are selected through random choice and (through uniform crossover and mutation) their respective latent vectors are used to create a new population of latent vectors. The new population can be enriched by the introduction of foreigners. The new latent vectors are used as input for the generative model to create a new set of images. The user is then free to select images from the new population and continue the iterative process. The end condition can be either the user’s decision to stop or a maximum number of generations achieved.DeepIE is a novel approach to the artifact generation problem that combines IEC and GANs to avoid some of the disadvantages of traditional IEC . DeepIE Z) may not translate into a satisfactory combination in the phenotype of the children. A crossover between latent vectors presenting entanglement may lead to non-intuitive results.However, DeepIE still faces some challenges. The principal problem is that traditional GAN generator usually presents feature entanglement in their latent spaces and vectors. In the context of generating images, this means that single visually recognizable features may be entangled within multiple variables of the latent code. Thus, there is no way to modify the latent code in a way that selectively alters a specific feature. This poses a challenge for conducting genetic operators such as crossover in the latent vectors of the model. For example, a crossover in the genotype of the parents , baroque, color field painting, cubism, early renaissance, expressionism, high renaissance, impressionism, magic realism, mannerism (late renaissance), minimalism, naive art (primitivism), neoclassicism, northern renaissance, pop art, postimpressionism, realism, rococo, romanticism, surrealism, symbolism, and Ukiyo-e . Table 1We trained a StyleGAN model using the WikiArt dataset, maintaining the same parameters and training configurations used in . The resz latent vectors.For this study, we adapted the DeepIE algorithm to work with the style-based generator of a trained StyleGAN model. Our implementation did not differ substantially from the one used in  and desca and b be vectors; t is the interpolation amount parameter where In their work , the autEquation :(2)SlerpZ latent space. This way the user could obtain novel options for selection in case the population becomes too homogeneous or if it does not include interesting or useful individuals.We employed the mutation operator with 50% probability and mediated by a strength parameter set by the user. Mutation is applied as a vector of random noise that is added to the latent vectors. The strength parameter is a coefficient between 0 to 1 that controls the magnitude of the noise vector. We also allowed the user to decide the number of foreign individuals to include in the next population. Foreign individuals take the place of children, so the population size remains constant. In our case, the foreign individuals were generated by randomly sampling from the z of 512 m length. If we assume the rest of the model as a black box, the output of the model is a z as an allele and apply genetic operators on them. First, the generative model produces an initial population Z of n individuals from random latent vectors sampled from the normal distribution S of their preference, which adds to the mating pool. The next step is to create a new generation Z of latent vector individuals z. For every z in Z we randomly choose two parents from the mating pool and pair them to generate a new child. This child is generated by slerp crossover between the parents and then receives mutation with a 50% probability, mediated by the strength parameter set by the user. The user selects a number of foreigners F sampled from n remains constant. The new population of latent vectors Z is used to generate a new set of images In our DeepIE implementation, we treat the style-based generator as any other black box GAN generator. StyleGAN accepts latent input vectors Z of individuals in a generation is an n is the number of individuals and m is the length of the latent vectors. In our proposed modification, the population of individuals W is an n is the number of individuals, l is the number of style vectors and m is the length of those vectors. This opens the way to, instead of naively applying the crossover operator on the latent vector z, applying the crossover at the intermediate matrix w. We applied the operator row-wise among the two style matrices of two parents  their success in achieving their goal with the system and how useful they considered the system to be (1 to 5). In the survey we, also asked the participants to express the preference or ambivalence for any of the systems. Results do not seem to strongly favor any specific system, as shown in To gain insight into how far into the evolutionary process the best image occurred, we also measured the ratio between the generation the best image was found in and the total number of generations. When comparing the performance of the same individual when using DeepIE and when using StyleIE, we employed a paired t-test. We found no significant difference between the total number of generations between the two systems. The same was the case with the generation of the best image found. However, the BA group arrived early to the best image and had an overall lower number of generations with both systems. While the t-test indicates a slight statistical difference among both groups, the variances in those metrics were significantly higher in group AB, meaning that this could be just the effect of an outlier See .We found no statistical evidence that the order of the experiments altered the results of the survey or the duration of the process, suggesting that neither user fatigue nor learning effect were major issues. We then proceeded to aggregate the results of DeepIE and SytleIE in both groups See . We founIn the survey, we asked the participants to explain the reason for their preference for one system over the other or the lack thereof. Two participants reported that they chose the second system they tried because of their accumulated practice at that point, suggesting that learning effect had some impact on their decision. More “integration of the images,” precision, and practicality were mentioned as the reasons for preferring StyleIE. A more smooth “flow of the images” and more adaptability were listed as well as a reason for preferring DeepIE.Collect distinct traits: Identifying distinct traits of the target and selecting images that present these traits. The process is continued until the desired traits are merged.Select best likeness: Identifying images that overall resemble more closely the target.Hierarchical trait selection: Similar to the first strategy, but focusing on one single trait at a time.In , the autWe were curious if similar strategies would also naturally arise in our experiment, so we asked in the survey if the participant had used some kind of strategy. Five users reported strategies that resembled collect distinct traits and six users select best likeness. Interestingly, four out of five users that used collect distinct traits preferred StyleIE, while four out of six users that reported select best likeness preferred DeepIE. This could indicate that strategies based on traits can be more suitable for StylyIE, while strategies based on general likeness work better on DeepIE. The rest of the users could not identify any particular strategy.The objective of this experiment was to evaluate and compare the performances and results of DeepIE and StyleIE in a closed-ended problem with strict constraints. For this experiment, we recruited 40 volunteers with an artistic academic background. They were divided into two subgroups AB and BA, of 20 subjects each. The users in each subgroup were asked to approximate the famous expressionist painting “The Scream” by Edvard Munch. As in experiment 1, the AB group first completed the task with DeepIE, and then did it again with StyleIE. The BA group switched the order of the systems. The users persisted in the task until they believed they had found a satisfactory image that fulfilled the requirements but had an upper limit of 20 generations to achieve it. After generating the two images, they answered the questionnaire and chose which of all the selected images from all generations was the most satisfactory. The resulting images can be seen in After completion of experiment 2, both groups AB and BA were asked to answer a survey. In the survey, they self-rated  their success in achieving their goal with the system and how useful they considered the system to be (1 to 5). To measure how both systems compared to each other in the survey results, we compared the rating scores among DeepIE and StyleIE in the same group. StyleIE presented higher mean scores for both self-rated success and perceived usefulness in both AB and BA See . Since wWe proceeded to aggregate the results of both groups and compare the means. The mean ratings of questions for StyleIE were higher than those of DeepIE See . Paired As in experiment 1, we also asked the participants to express the preference or ambivalence for any of the systems. Five participants reported preferring DeepIE, 30 participants preferred StyleIE and five had no preference; 75% of test users expressed preference of StyleIE over DeepIE or no preference see .As in experiment 1, we kept track of the ratio between the generation of the best imaged that was generated and the total number of generations. We compared the total number of generations, the generation of the best image found, and the ratio between them with both DeepIE and StyleIE in the AB group using paired t-tests. We found no statistically significant differences between the means of total number of generations, the generation of best image, and ratio of DeepIE to StyleIE in the AB group. In the BA groups, there was no statistically significant difference in the total number of generations of DeepIE and StyleIE, but we found a statistically significant difference in the means of the best generations found and the ratio. StyleIE found the best image later than DeepIE, but its ratio was closer to 1. The authors in  interpreA participant commented that they preferred StyleIE because it reflected better their intention when trying to combine the image.Another expressed that StyleIE led to more interesting variations and could be useful to explore novel ideas.Another expressed that StyleIE was more predictable.A user noted that while he preferred DeepIE, he often found that an element he wanted to preserve got lost.In the qualitative part of the survey, users expressed their opinion about the systems. We asked them to explain their reasons for a preference of one system over the other. We showcase some of the feedback reported:We also asked them about the strategies that they employed when conducting the evolutionary process. Most users reported using a strategy resembling collect distinct traits or hierarchical trait selection, regardless of their preferred system.We found that users performed equally using DeepIE and StyleIE in an open-ended task with relaxed goal constraints, but StyleIE outperforms DeepIE in a closed-ended task with strong goal constraints. In the first experiment, the totality of users showed no strong preference for either system, suggesting that both were equally effective in aiding them towards the desired goal. In the second experiment, 75% preferred StyleIE over DeepIE, suggesting that StyleIE is more useful the more specific the target. Our self-rated success mean scores in both experiments surpass those reported in  for geneThe scores for the perceived usefulness of the system were generally good in experiment 1 for both systems. In experiment 2, the scores favored StyleIE. We found no correlation between self-rated success and perceived usefulness, meaning that the users judged the usefulness of the system independently of their perceived performance with it. In the qualitative part of the survey, users indicated interest and excitement for the evolutionary system, which in conjunction with the reported usefulness, suggests that deep interactive evolutionary systems have a potential market and applications in the creative industries.According to the best image generation ratios in experiment 1, the best image was usually found in the last generations, which is a positive indicator that the subjective quality of images kept improving for the users. This indicates that the systems are conductive for user-guided search and that the systems have the potential to keep improving the images in subsequent generations. In experiment 2, ratios were closer to 1 when using StyleIE than when using DeepIE. This suggests that StyleIE is more conducice to the search when the goal is more strongly constrained.Survey results in experiment 1 indicate that preference for one or another system may be correlated with the chosen strategy of each user. Most users that preferred StyleIE used strategies based on collecting specific traits in the desired image, reporting a more precise robust combination of the traits. On the other hand, users that preferred DeepIE reported a more steady flow of exploration and employed strategies based on general likeness. This can be interpreted as that DeepIE could be more suitable for best likeness strategies that evolve the generality of the image and StyleIE could favor trait selection that offers the possibility of fine-tuned evolution. However, experiment 2 seems to contradict this idea, since most users preferred trait-based strategies regardless of their preferred system. An explanation for this could be that in experiment 1, the relaxed constraints gave the users more freedom to explore different strategies, while the stronger constraint of experiment 2 prompts the users to employ trait-based strategies. This is an intriguing matter indeed and more research on the optimal strategy for deep interactive evolutionary systems should be worthwhile.IEC can be used to embed the user’s preference as guidance for a search algorithm and deep generative models can learn distributions of large collections of human knowledge and artifacts to later synthesize plausible instances from them. DeepIE was proposed as a new paradigm for combining IEC with deep generative models; however, its suitability for creative and artistic exploration remained to be evaluated under research conditions.This study implemented DeepIE with the style-based generator of a StyleGAN model trained to produce works of visual art. We discovered that the style-based generator allowed us to implement DeepIE in a novel way that takes advantage of the disentangled intermediate space in the mapping network, allowing us to perform genetic operators among specific feature style vectors. We named this modification StyleIE.Finally, we tested the performances of DeepIE and StyleIE in two experiments. The first experiment consisted of a task with relaxed goal constraints. The second experiment had a more strongly constrained goal. DeepIE and StyleIE performed equally under relaxed constraints. StyleIE outperformed DeepIE under stronger constraints. Due to its style-based architecture, StyleIE provides more control over specific features of the generated images. Under the relaxed constraints, the area of the global optima is wider, meaning that more individuals satisfy the constraint requirements and there is no need for fine control over specific features. Therefore, StylIE offers no advantage to DeepIE under those conditions. However, if we constrain the problem, the area of the global optima narrows, meaning that fewer individuals are acceptable solutions. In this case, control over specific features offers a significant advantage to the users.Research on deep interactive evolutionary systems is still in its infancy. However, we have obtained preliminary evidence that deep interactive evolutionary systems have potential applications in creative and artistic tasks, and that our proposed method, StyleIE, offers a significant advantage over DeepIE under certain goal conditions. This could lead to developing interesting applications in diverse fields related to the creative industries such as design, art, and multimedia projects.To our knowledge, this is the first study that explores the potential of deep interactive evolutionary systems for creative exploration using students from a school of architecture, art, and design.This is also the first study to implement DeepIE within the style-based generator, taking advantage of the intermediate space of the mapping network to produce a more complex genotype and offering the possibility of applying crossover over disentangled features.The user interface constitutes a huge limitation, as was also noted in . A more We centered our experiments around a particular artistic style, expressionism. This was done for practical and time limitations. While we did not test the performance of the systems when optimizing for other styles, in principle, there is no reason to believe that the results should not generalize. As long as the target style in question is present in the training data, it should be possible to guide the evolutionary process towards it.Another limitation is that we did not evaluate the usability of the tool. We leave open the possibility of addressing this issue in future work.