This article considers the potential for the COVID-19 pandemic to represent an epochal moment in international politics, one which China can use as a window to maximize its international order-building efforts. It is argued that China’s putative international order-building efforts to date have faltered, in part, due to an inability to create meaningful friendships with prominent international powers. However, the COVID-19 pandemic allows China to play a key humanitarian role in heavily afflicted countries and, through this, perhaps an opportunity to forge more meaningful friendships. It is argued that China is trying to attach its concept of friendship to its humanitarian assistance while also stepping into the clear international leadership void that exists at the moment. This is not without faults or missteps, but the lasting impact could be significant for international politics and the global order. The impact of COVID-19 has been so rapid and globally enveloping that it deserves to be considered as a potential epochal moment for international politics. Since emerging in Wuhan, China, the virus has swept across the globe and has not only caused significant public health costs, especially in Asia, Europe, and North America, but also crippled markets, closed borders, and resulted in tit-for-tat finger-pointing between China and the United States. Although there is much uncertainty to how the COVID-19 pandemic will develop in the coming months and years, it is likely to cause a lasting impact that will be felt in international politics, particularly with regard to international power and order.status quo, resulting in new realities once the dust settles. They are also crucial for order in international politics. While international systems and orders are hardly static and are always evolving, epochal moments make monumental changes suddenly. Think of historical moments like the Thirty Years War or the defeat of Napoleon. These events were critical junctures of great magnitude for European politics at the time. The Thirty Years War ushered in a new system in Europe based on the concept of sovereignty while the defeat of Napoleon led to the Concert of Europe, a system in Europe based on the concept of balance of power . WW2 was arguably the most significant critical juncture experienced in this so-called Westphalian era, leading to significant material and ideational power shifts . The UniIn the years since the United States’ hegemonic moment in 1991, the international order it presides over has come under threat. The cracking of this international order has been, in part, self-inflicted as the United States engaged in disastrous wars in Afghanistan and Iraq as well as numerous other foreign policy blunders . More reblazed a development path that suits China’s actual conditions. This is a path based on China’s realities . . . by drawing on both the wisdom of its civilization and the practices of other countries in both East and West. In exploring this path, China refuses to stay insensitive to the changing times or to blindly follow in others’ footsteps . . . We are not jealous of others’ success; and we will not complain about others who have benefited so much from the great opportunities presented by China’s development. We will open our arms to the people of other countries and welcome them aboard the express train of China’s development.But the cracking of the U.S.-led international order has also been aided by the emergence of challengers to the U.S. hegemonic power position, none more so than China. China’s rise over the past two decades has been remarkable . Its ecoThis article examines whether COVID-19 can be an epochal moment that China can utilize to aid its putative international order building. To do this, first the importance of meaningful friendships to international order building and China’s struggles in cultivating such friendships are examined. Second, the material and ideational costs of the COVID-19 pandemic are evaluated while China’s emerging relative advantage over the rest of the world is gauged. Third, China’s efforts to combine its humanitarian assistance with friendship cultivation is assessed, with a focus on Italy. Last, it is concluded that COVID-19 does represent a useful window that China can use to foster real international momentum with regard to its international friendship and order building.Respublica Christiana, abandonment of Canon Law together with Papal authority, and the spread of the Reformation,” friendship “became one of the few concepts mediating the emergence of the modern political order.” Therefore, given that international politics still exists within something of a Westphalian system, friendships have played an incredibly important role in the rise and fall of different orders in the 400-odd years since.Despite plenty of investment, enthusiasm, and ambition, China’s international order building has yet to produce any tangible results to date. One hurdle it has encountered has been an inability to cultivate meaningful friendships with other countries, especially powerful ones. Friendship has long been crucial to international order building. As It is not for lack of effort as China has long realized that friendships are crucial to order building, and it has attempted to cultivate friendships with many different countries across the globe . One exaTwo problems emerge when canvassing China’s list of “all-weather friends” with regard to order building. First, none of these countries, outside of arguably Brazil, are prominent international agenda setters. China’s desire for parity in the international system requires that more prominent states buy into their international vision; otherwise, their order building will fail to challenge the existing order(s). Second, most of these friendships are unequal and are based not on mutual affinities but rather China’s use of largesse, especially the so-called “no strings attached” loans . States Russia, however, looms large as one prominent international power that China has seemingly become friendly with. Under the leaderships of Xi Jinping and Vladimir Putin, respectively, China and Russia have taken noticeable steps to solidify their relationship. The state of affairs appears so rosy that the term “bromance” is now widely used to characterize this blossoming friendship between the two . And beyHowever, while Sino-Russian cooperation is strong with respect to security matters, it is still relatively weak in terms of trade relations: Russia has significantly higher two-way trade levels with the European Union (EU), while China’s two-way trade with Russia does not even register on its top 10 list. Furthermore, there is a potential for Russia to become nervous about Chinese encroachment into its “sphere of privileged interest,” especially as China’s flagship policy, the BRI targets erstwhile Soviet Central Asia . In addiAs the Russian example shows, China has generated friendships that appeal to strategic or normative affinities, but deeper and lasting friendships usually need stronger ideational glue as well. To this end, cultural diplomacy is one area in which China has invested heavily in recent times to try and cultivate more meaningful friendships. Initiatives under this umbrella included proliferating Confucius Institutes (CI) and Confucius Classrooms abroad and attracting international students to study in China . PiggybaDespite increased efforts to cultivate meaningful friendships, one of the greatest barriers to this involves China’s national identity narratives. Chinese national identity, like other national identities, is predicated on a distinction with “Others.” Looming large among its significant “Others,” are Japan and the United States . AlthougWhen China is misunderstood, it not only reinforces China’s positive distinction with the negative “foreigner,” but can at times provoke anxiety-fueled reactions that lead to declarations from the Chinese state and high-profile figures that the transgressors have “hurt the feeling of the Chinese people” . While fOn the flip side, while China has continued to struggle to create meaningful lasting friendships, the United States, despite all its apparent self-inflicted wounds, still maintains important friendships with numerous key partners. Undeniably, the U.S.-led international order is underpinned and solidified by many key international friendships between the United States and its partners. The most important has been the transatlantic friendship between the United States and the United Kingdom. This is a relationship not built solely on strategic interests but also on a deep mutual normative and cultural understanding as well as being rooted in numerous historical events. This friendship has shaped international politics inexorably over the years, from cooperation in the two world wars of the 20th century to mutual assistance in building the post-1945 liberal international order, to an ongoing partnership in the post-Cold War era .Of course, the relationship has not been without its tensions. Particularly problematic was the changing power relation between the two, especially as the United States rose to become the unquestioned hegemon in international politics post-1945, while the United Kingdom declined to a point of clear subordination . HoweverHowever, COVID-19 is a massive test for the United States and whether it can come through the crisis with its international reputation and its international friendships intact remains to be seen. Given that it was already losing its appetite to underwrite the international order’s provision of public goods—such as international security, international trade, international capital flows, and freedom of navigation—shirking a leadership role during the COVID-19 pandemic could leave the door open for new leaders, and perhaps, new orders .The COVID-19 pandemic has been particularly bad for China domestically. Given that the pandemic first emerged in the Chinese city of Wuhan, much of the initial damage of the pandemic was in China. China was forced to close Hubei province and quarantine the major settlements there, including Wuhan, a city of roughly nine million people. Soon after, much of the country was quarantined and citizens were forced to stay indoors to contain the spread of the virus. The health costs of the pandemic are already significant—including more than 84,000 cases and 4,500 dead at the time of writing . InitialHowever, the Chinese government recovered quickly by co-opting Dr. Li’s “martyrdom” from the outpouring of calls for greater transparency and freedom of speech to enshrining him and other medical staff who died within the national rankings of “martyr” . There hInternationally, the COVID-19 outbreak came on the back of a problematic year for China’s international image in 2019, and by extension, its cultivation of friendships. Further revelations about Xinjiang were printed in major international newspapers, the Hong Kong protests spiraled out of control, the fiasco with the NBA and other Western companies exposed China’s naked use of its market power, and backlashes against its flagship BRI policy occurred in numerous countries, many of which were expected to be key partners for China’s envisaged new world order . If 2020vis-à-vis those countries most severely hit in Europe and North America. Given China has more than 100 cities with at least one million people  in the coming year or two is another question. But the fact that China appears to have the virus under control and has implemented an effective system of surveillance and control that few other countries have the resources or legal wiggle room to copy, it seems China could end up benefiting from being able to get its country back up and running before the rest. Already in early March, less than two months after shutting down, China took steps to kick-start its economy again, working with businesses and employers on plans to increase productivity and children returning to schools in less affected areas .tabula rasa for China’s international image and its efforts to cultivate friendships. Even though the Chinese model for combatting the outbreak of COVID-19 is not easy or realistic for most countries to follow, China is still a source of important information and expertise given it has been first in dealing with the outbreak. As many European countries have struggled to deal with the spread, there have been significant calls from the intelligentsia in these countries to take seriously China’s experience and to find ways to use this to drive policy choices (Zhongguo jia you”]) to that of responsible supporter (“China supports”[“Zhongguo zhiyuan”]).The potential swings are not just material but ideational, because the COVID-19 pandemic is also potentially a  choices . China h choices . The staAnother area of both material but also ideational advantage is China’s increasing position as the world’s largest producer of medical supplies. Although China’s production of high-tech medical equipment is much smaller than countries like Germany and the United States, when it comes to medical supplies it is the undeniable market leader, providing 43 percent of global imports of protective medical equipment . Indeed,Europe offers an interesting case study to evaluate whether the onset of the COVID-19 pandemic has offered China a chance to make some ground there with regard to its friendship initiatives. At a continental level, Europe quickly surpassed Asia in terms of total COVID-19 cases and deaths. And, unlike China, the governments there have been far less effective at stopping the spread of the virus, meaning a much more protracted outbreak has occurred which will likely result in larger societal and economic costs. Italy, for example, reported its first case on January 29, 2020, but did not reach its peak in terms of new cases per day until March 21, 2020, and it was not until mid-May that a significant drop occurred . ComparaOnce it became apparent that Italy was facing a significant outbreak of COVID-19, its leaders initially appealed to the EU, and the member states of the EU individually, for help. However, the EU proved ineffectual in providing an EU-wide solution and also proved powerless to stop Germany and France imposing limits on the export of protective medical equipment . This elThe perceived lack of solidarity and urgency shown by the EU and the United States in Italy created an initial vacuum that the Chinese stepped into. On March 13, 2020, China began by sending nine experts involved in the Wuhan response to Italy to offer advice. This was accompanied by the Chinese Red Cross providing 31 tons of medical equipment—including high-tech equipment like respiratory devices, electrocardiographs, and ventilators . On MarcJD.com  briefings, centered on reciprocity of friendship and in the interconnectedness of existence. The repeated use of the phrase “shared future of mankind” has become commonplace in official press conferences of the Chinese MFA, positioning China as a humanitarian power . Such siThis broad signaling of friendship is also supplemented with more overt references for the countries China has been working closely with since the spread of the virus. For Italy, the support is framed on the interconnectedness of humanity. One example is the use of the quote “we are waves of the same sea” from the Roman stoic philosopher Seneca on the donation boxes from Chinese high-tech firm, Xiaomi. In addition, the Chinese Embassy in Rome has tried to highlight the previous strong ties between the two countries, especially after Italy helped China after the Wenchuan earthquake in May 2008. The Chinese Embassy stated that “Maybe you have forgotten about it, but we will always remember. Now it’s our turn to help you” . As the Right after the epidemic broke out, Pakistan mobilized the whole nation to provide assistance to China, which we will never forget . . . Reciprocating an act of kindness is our nation’s fine tradition. Now the situation in China is getting better and the number of confirmed cases in Pakistan has increased. The Chinese leadership has made it clear that we will do all we can to support our Pakistani friends. Outside Western Europe, China has been even more explicit in signaling to its so-called “all-weather friends.” In response to Pakistan’s initial assistance, an official Chinese spokesperson stated thatRhetorical responses to Africa were similar: “China and Africa are good friends, partners and brothers. We have long been supporting and helping each other” . And in This emphasis on reciprocity casts China as regarding its friends kindly. It also underlines a sharp contrast with the “unfriendly” activities of the United States since the outbreak went global. In early February when the outbreak was mostly only seriously affecting China, the Chinese state condemned the U.S. travel warnings to China, pointing out how that, and associated public comments, lacked the friendly support offered by other parts of the international community. This was highlighted as revealing the lack of friendship emanating from the United States through the evocation of the phrase: “a friend in need is a friend indeed” .However, China’s efforts have not been without issues. One major credibility hit has been problems identified with the quality of supplies purchased from China . And theChina’s noticeable efforts to use the COVID-19 pandemic as a springboard for cultivating deeper friendships to help foster its visions for, and potential impact upon, a new international order has not gone unchallenged, especially from the United States. After President Trump initially praised the Chinese efforts in combatting the outbreak in February 2020 , since MNoticeably absent from the U.S. response to China so far has been a convincing assertion of global leadership . Perhapstruly meaningful international friendships with. At the same time, continued inaction by the United States will likely further erode those friendships crucial to maintaining the international order that country formerly presided over. At the very least, COVID-19 is likely to disrupt the status quo and usher in a new period, if not epoch, where China and the United States compete to etch out international orders more favorable to their grand strategic aims. Cultivating and maintaining international friendships will remain a central feature of this struggle.Clearly, China is not winning hearts and minds everywhere. Many of its efforts to assist other countries in the pandemic have been undermined by either shoddy products, a failure to deliver on promises, or pressing too hard. Furthermore, ongoing revelations as to China’s initial mishandling of the situation and the result this had in enabling the outbreak to become a pandemic will continue to undermine their claims to global  leadership. But for COVID-19 to be something of a window to an epochal moment, China does not need to win everyone over. China’s willingness to help afflicted countries, issues notwithstanding, has reinvigorated its efforts to cultivate friends and, judging by Italy and Spain, it has made significant strides in countries they previously had failed to create


With the development of practical means of human germline genome editing (HGGE) in recent years, there have been calls for stricter regulation and oversight over HGGE interventions with potential for heritable changes in the germline. An international moratorium has been advocated. We examine the practicality of such a proposal, as well as of a regulation through the “traditional” mechanisms of international and municipal laws. We argue that these mechanisms are unlikely to achieve their intended objectives and that the better approach is to engage the international community of stakeholders, researchers, scientists, clinicians, and other workers directly involved in the field in working toward the development of an “informed adaptive consensus”. We offer suggestions as to how this may be achieved and how existing indirect levers of regulation may be harnessed toward this end. Since He Jiankui’s revelations in 2018, there have been calls for an international moratorium on human germline genome editing (HGGE) [We argue that none of these mechanisms alone will achieve their proposed objectives: They themselves are fraught with difficulties relating to enforcement, harmonization, and consistency of regulatory objectives between countries, especially given the need to maintain the appropriate but difficult balance between encouraging and guiding ethical scientific endeavors for the benefit of humankind, while at the same time discouraging or restricting research or clinical applications for which there is a general international consensus that such research or clinical applications are unethical or pose unacceptable and currently unknowable dangers for humanity as a whole. Scientists (and other stakeholders) have a key role to play in the development of these technologies. Instead of focusing on building high walls to contain bad actors, the collateral damage of which is to raise the administrative bar for ethical and beneficial research, and to discourage such research, scientists need to think in terms of how we can devise responsive structures that will support, guide, and encourage ethical and beneficial endeavors in this area while discouraging the dubious. Just as it is their professional and ethical responsibility to speak out if they believe that a particular line of research is not in the public interest, or is unethical (or both), we assert that if scientists believe that particular lines of HGGE can be ethically carried out with potential promise to humankind, they should likewise state their conscience. In other words, these scientists need to be advocates for the potential benefit to humanity of their work and to focus the lay mind and media on the positive and not the negative.During the publication of this work, the World Health Organization (WHO) Expert Advisory Committee on Developing Global Standards for Governance and Oversight of Human Genome Editing released its governance framework  and accoEthical and scientific views evolve with technology over time : Given tWe suggest that the best way forward is to rally the international scientific community toward the formulation of a scientific and ethical consensus on what may be acceptable (or otherwise) at any given time in the development of the technology. We envisage that the stakeholders to such a consensus recognize that it is a consensus that is not a fixed one, but one that develops and changes over time in response to new advances and knowledge, i.e., an “informed adaptive consensus”. The development of such a flexible and responsive scientific and ethical consensus  will also serve the invaluable office of providing assurance and guidance to scientists who are concerned about the ethics of their research in this area. To this end, we explore and suggest avenues for harnessing currently available levers of indirect regulation that can be used to give real teeth to such an international consensus. Finally, we suggest that with this approach, a truly mature and stable ethical and scientific consensus will come about, which can serve the purpose of guiding governments toward informed and harmonized national legislation. Here, we clarify that “informed adaptive consensus” is not to be confused with “broad societal consensus”, which refers to all of society, including lay people. We have critiqued the use of this latter concept in Xafis and colleagues , as it eThe establishment of a moratorium would require a number of features to be addressed explicitly. These include the exact substance of the moratorium, i.e., what exactly is to be prohibited and what may be permitted, particularly in exceptional circumstances. It would also require the exact juridical, ethical, and legal bases and objectives for the moratorium as well as the term of the moratorium. In relation to the latter, by definition, a moratorium is a temporary suspension of an otherwise permitted activity. Moratoriums are therefore articulated for either fixed terms, usually with a clause allowing for extension by agreement, or predicated upon an event/circumstance that would trigger its determination or dissolution. In defining the trigger or determination event (if the moratorium were not to be for a renewable fixed term), justifications would have to be advanced for putting forward the proposed trigger or determination event and how this might serve the bases and objectives of the moratorium. As a result of this key feature of moratoriums, any notion of a permanent or open-ended moratorium cannot hold.The final feature of moratoriums relates to the likely efficacy and impact of the proposed measure. Given the sovereign state principle in international law , a moratorium would need to address issues regarding its ability to have real effect because, if it did not, it would simply result in another ineffectual political expression of intent. Additional considerations regarding its effect relate to a number of critically important considerations. The foremost among these is the “bottom-of-the-barrel” scenario—countries signing up to such a moratorium may find researchers moving off to more receptive climes with more “liberal” regulatory regimes with less oversight and restrictions. Would overly broad regulation or direction have a chilling effect on useful research in allied areas through discouraging researchers, for example, as a result of high administrative burdens, too many added complicated procedures, and having to put forward levels of justifications not required in other areas of research? Finally, most researchers would want to avoid the taint of unfounded “association by guilt” by a public or press that does not understand the vital difference between their work and that sought to be regulated.One approach is to rely on individual states to regulate at the national level through municipal legislation. However, this option does not necessarily promote the cause of international harmonization: In an ideal world, municipal law enacted by countries would be harmonized in the sense that they are at least roughly consistent with each other and have similarly common objectives. A detailed survey of the regulatory mechanisms in 18 countries found that while there is generally a ban on clinical applications of HGGE, some of these bans are not absolute, while other rules are “vague and fail to provide clear guidance as to what is allowed” The greatest drawback of relying on municipal law at this stage is that there is no general clear consensus on precisely what should or should not be permitted in the name of research and in clinical application currently or in the future. The reality is that it is such early days yet in HGGE technology that we cannot know, or be agreed upon, from the ethical, social, as well as the scientific perspective, what kind of restrictive measures we want carved in legislative stone at this stage of development. Ideally, municipal law should reflect an established and mature body of ethical and scientific consensus, but the argument may be that we are currently just opening the debate and far from reaching any mature body of consensus. The act of legislation in most countries is ultimately at heart a political act—ethics and science may inform or be used to drive the legislative debate, but neither have an assured or primary place at the table. Legislators are first and foremost politicians: Science (and sometimes ethics) are often not the controlling premises for their actions. In its most innocent and defensible aspect, this political ranking of priorities may instead prioritize deeply held popular religious perspectives and economic and national interests in preserving the scientific advantages in a given field already established by the scientific or academic community in the country. Once enacted, the regulatory regime tends to ossify and be set in stone—repealing, or amending or “bringing up to date” a law that attracts strong political views on opposing sides of the legislative chamber may prove to be just as difficult as enacting it in the first place, particularly given the nature of HGGE, in which religious sentiments are likely to be a prominent theme in the public debate in some countries. Likewise, the disadvantages of overly precipitate and premature legislation based on an incomplete state of scientific knowledge are obvious.Another approach may be to work toward the promulgation of international norms in an international treaty or instrument regulating the research and clinical use of HGGE. Again, as for muncipal law, the main obstacle to this normative approach is the current lack of consensus on the ethical, legal, and social dimensions of HGGE and how this may be best regulated and directed. A fundamental plank of international law is that all states are sovereign and equal, as enshrined in Article 2 of the United Nations (UN) Charter, which declares that “the Organization is based on the principle of the sovereign equality of all its Members.” This basically means that, with a very few exceptions , no country is entitled to impress its law or will against another (in theory anyway). In short, countries are free to exercise their sovereign right to refuse to play in accordance with rules unpalatable to them.Even when countries accede to a treaty, they are, in most cases, free to withdraw at any time. Even if, in fact, a country signs up to a treaty, it does not mean that the obligations and provisions of the treaty automatically become translated directly into municipal law enforceable by the municipal judicial authorities  or dualism  should be applied to international law in its relation to municipal law date back to the earliest roots of the concept of international law). It suffices to say, for our present purposes, that there is no jurisprudential agreement on this matter, and different countries take different  perspectives. And if a dualist country refuses to implement its international obligations under the treaty, in most cases, there is little that other countries can do—again, this is the sovereignty doctrine at work.But as the United Nations General Assembly (UNGA) experience in the Declaration on Human Cloning amply demonstrates, the biggest obstacle in the way of a clear international treaty or instrument is the simple lack of agreement currently on what should be restricted , as well as the very different competing interests and first premises that different countries may bring to the debate. Many of those countries that refused to sign up to the Declaration on Human Cloning had communities of scientists already engaged in advanced research that would have been affected by the Declaration; for other countries, religious perspectives on human cloning and human embryonic stem cell research were the principal driving forces for their national positions on the debate. Similar divergences are likely to appear for HGGE.Finally, the length of time required to negotiate and ratify an international agreement risks rendering the content of the agreement “obsolete” by the end of this process due to the rapid scientific developments. Governance mechanisms need to be adaptable and responsive, as highlighted in the recent WHO framework . This reIt is often tempting to turn to law  to regulate spheres of human endeavor. But, instead of using the blunt and inflexible instrument of the formal law, working toward the establishment of a body of international norms outside of formal treaties may be an approach that can be used to address the tension between the need for international harmonization and the difficulty in making legally binding international law. This is in line with the trend since the start of the 21st century where international cooperation has moved away from legally binding international laws to nonlegally binding instruments . In recerecommendations issued by the International Committee of Medical Journal Editors (ICJME), which require adherence to standardized ethics guidance to publish in top ICJME journals. Hundreds of non-ICJME journals have also voluntarily adopted these guidelines ;Statement on Benefit Sharing [statements issued by the Human Genome Organization (HUGO), which have impacted on policies developed in the European Union  and on c Sharing , which h Sharing ;the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) consortium initiative, which forms the basis of a harmonized worldwide framework for the conduct of clinical trials and has resulted in consensus guidelines, such as the ICH E6 Good Clinical Practice (GCP) . Most tethe GA4GH Framework for Responsible Sharing of Genomic and Health-Related Data . The fraA key limitation of regulatory initiatives in both the international and municipal legal spheres is that only a very limited number of key stakeholders have a direct hand in the shaping of regulations. In the case of municipal law, legislators not only have a primarily political focus, but are usually also far removed from the actual experience and practice of the science involved, so that both the ethical as well as the scientific implications of developments must be received secondhand from the primary experts. At best, there is a risk of distortion or imperfect translation in such transmission; at worst, there is the possibility of deliberately slanted perspectives by less than scientifically objective experts. The ongoing political struggles over whether lockdowns are justified in the current pandemic is a painful reminder of such a reality. Likewise, the general rule in interstate relations is that only state actors are entitled to a place at the table: All other parties  are permitted a peek in only on sufferance or agreement by the state actors concerned. But, in the initiatives that we have identified above, the fundamental principle is maximum inclusivity of the stakeholders most relevant to the issue at stake: participation of the many who are directly involved, and who have the greatest expertise, instead of a few far removed from the actual practice and impact of the science.To this end, these initiatives have been deliberately fashioned outside of the established mechanisms for the creation of international law, and they demonstrate that state actors are not the only stakeholders who may influence or contribute to shaping the rules. The creation and willing uptake of these and other comparable initiatives illustrate that the views of academia, industry, patient lobbies, public interest groups, non-governmental organizations (NGOs), and other stakeholders exert influence and contribute in substantial ways to developing outputs likely to be palatable to a broader audience because they tend to be a product of consensus. There is, however, always an inherent risk in attempting to reach too widely for a consensus, as the result may be settling for a less meaningful lower common denominator.In the HGGE space, entities with gatekeeping powers capable of considerable influence include biotechnology companies and fertility clinics, which have the power to refrain from offering products or services that are ethically problematic or likely to be used in an ethically problematic way; universities, funding foundations, international consortia and organizations, and WHO itself are other such entities. In addition, the top scientific and medical journals wield enormous normative power and influence in the shaping of norms through requirements they implement, as shown above. Gatekeepers such as professional bodies, scientists, and clinicians who are directly involved in the field are especially well positioned to understand which approaches are likely to result in the best outcomes. We believe that we should do more to explore working through nongovernmental gatekeepers such as those identified here. This approach also appears to be partially explored and supported in the recent NASEM report on HGGE  and is aIn sum, we propose that nonlegally binding instruments are the most promising approach to international governance of HGGE. One of the most important advantages of adopting such an approach that stresses the participation and product of the many, instead of the few, is that the product is one that is constantly being refined and articulated to address and reflect scientific developments as and when they appear: It is a gradual and flexible process in which international  consensus can be gradually built up. In coming to an international consensus with their peers abroad, communities of scientists within a given country are also thereby afforded greater credibility than if they were lobbying alone with their national politicians. Politicians are also thereby quickly made aware of likely policy directions in competing jurisdictions. Non-state actors and participants directly involved in the science can move much more quickly and more expertly to formulate draft guidelines than legislators and state actors relying on secondhand information and bound by the rules of municipal and diplomatic protocols. In this way, scientists and ethicists alike can seize the initiative and provide guidance for the eventual formulation of municipal regulation, rather than the other way around. International and municipal laws have a place when the core consensus has been achieved and when the body of international expert opinion on the way forward is reasonably mature. Until then, all involved in science and ethics should consider what informal levers of power may be available to them and how these may be used to lead responsibly and ethically. A decision to halt any and all involvement in HGGE via a moratorium is not prudent, as it would delay the hard work we will ultimately have to engage in to find prudent solutions, and, importantly, it would bring to a halt an area of science that may have much to offer future generations.


Additive manufacturing (AM) has many advantages and became a valid manufacturing technique for polymers and metals in dentistry. However, its application for dental ceramics is still in process. Among dental ceramics, zirconia is becoming popular and widely used in dentistry mainly due to its outstanding properties. Although subtractive technology or milling is the state of art for manufacturing zirconia restorations but still has shortcomings. Utilizing AM in fabricating ceramics restorations is a new topic for many researchers and companies across the globe and a good understanding of AM of zirconia is essential for dental professional. Therefore, the aim of this narrative review is to illustrate different AM technologies available for processing zirconia and discus their advantages and future potential. A comprehensive literature review was completed to summarize different AM technologies that are available to fabricate zirconia and their clinical application is reported. The results show a promising outcome for utilizing AM of zirconia in restorative, implant and regenerative dentistry. However further improvements and validation is necessary to approve its clinical application. The unique properties of zirconia ceramic as restorative dental material, along with the growing demands from patients for aesthetic and metal-free restorations, have attracted the attention of the dental profession. Currently, zirconia restorations are widely used in dentistry due to their biocompatibility, chemical stability, outstanding mechanical properties, and optical characteristics . Full-coSince its introduction in dentistry in the 1970′s , subtracAdditive manufacturing technology (AM) is an alternative to address the drawbacks of subtractive manufacturing in the CAM step of the dental digital workflow, also known as 3D printing, which is capable of fabricating devices by layering materials using a computer-generated design file; standard tessellation language (STL). Even though AM has yet to be developed, it enables the production of ceramic parts with complex geometries, high precision, and low cost ,14,15.The American Society for Testing and Materials (ASTM) divides AM technologies into seven classifications to construct a product layer by layer: stereolithography (SLA), material jetting (MJ), material extrusion (ME), binder jetting (BJ), powder base fusion (PBF), sheet lamination (SL), and direct energy deposition (DEP), as all can be used to form ceramic components ,17,18.Various AM techniques have been utilized to manufacture zirconia objects including vat photopolymerization (stereo-lithography and direct light processing), selective laser sintering, selective laser melting, ink-jet printing, fused deposition modeling, direct energy deposition, sheet lamination, and binder jetting . AlthougVat photopolymerization technologies are defined as “additive manufacturing processes in which liquid photopolymer in a vat is selectively cured by light-activated polymerization” . This in2 suspension, which provided new possibilities for fabricating high performance ZrO2 objects by SLA.SLA was the first AM technology to be utilized in the medical field and utilized to fabricate surgical models for alloplastic implant surgery in1994 . SLA is Another major issue to address is the significant effect of light scattering caused by the ceramic particles added to the suspension, even if the particles themselves are transparent to irradiation. The light penetration into the suspension is deteriorated by this scattering and it can impact the dimensional accuracy . The cer2 sintering methods differed between groups, with 1450 °C temperature for 0% group, and between 1450 °C and 1225 °C for 20% and 40%. The dimensions of specimens measured by digital caliper and the volume shrinkage percentage of fabrication was computed by comparing the digital design of the bar with dimensions of final AM specimens. Although none of the groups examined were able to fully mimic the specimens’ virtual design, the AM zirconia with a porosity of 40% had the highest fabricating accuracy and the smallest volume change after manufacturing, followed by the 20% porosity and 0% porosity groups. Nakai et al. [Among the different AM technologies, stereolithography (SLA) is the most preferred for dental applications because it provides the highest accuracy, resolution, and a flawless surface finish . There ai et al.  evaluateDigital light processing (DLP) is based on vat-polymerization technology and is similar to SLA and can be classified into same category but it differs in terms of the light source used . An arc DLP can construct the parts in either a bottom-up or top-down layout. In the bottom-up set-up, an object is created upside down on the building platform and dipped in a thin slurry layer deposited on a glass plate rather than entirely immersed in the liquid resin. The advantages of this method are the little quantity of slurry required for producing the parts and less expensive than the top-down printers ,45. DLP Selective laser sintering (SLS) is a powder-based additive manufacturing (AM) technology that uses powder beds containing loose ceramic particles as feedstock to construct three-dimensional objects . As the The SLS process can be classified into two types to manufacture ceramics: direct and indirect. In indirect SLS, ceramic powders are combined with a polymer binder and the scanning laser melts the binder, which bonds the ceramic particles together. The binder is removed and portions are densified during the subsequent debinding and sintering . In direMaterials with a high melting point like zirconia ceramics are particularly difficult for the SLS process as their densification requires high temperatures and a lengthy time to obtain acceptable densities. Another common challenge with these ceramics is that thermal stresses during SLS might trigger crack formation in sintered objects . It was Selective laser melting (SLM) is another powder-based AM technique which works similarly to SLS except that it is a one-step bed fusion by full melting that uses laser sources with substantially higher energy densities and does not require any secondary low-melting binder or post-treatment . SLM is Ceramics, particularly zirconia also have high melting temperatures, extremely low thermal conductivity, and limited ductility. The thermal stress caused by extremely short laser–powder interaction durations and large amounts of energy required for melting, meaning the abrupt heating and cooling rates with each laser scan, is one of the most severe difficulties resulting from the SLM method. As a result of the poor thermal shock resistance of ceramic (zirconia) materials, cracks are more prone to emerge in sintered parts as a result of such thermal stresses combined with the limited ductility of ceramic materials ,57. OnlyDirect Inkjet printing as a material jetting technology is a well-known method which allows for the manufacturing of ceramic objects with full density, high accuracy, complicated shapes, and minimal material usage at a low cost; conversely to other methods which construct porous ceramic structures ,65. A su2 powder layers [The efficacy of Inkjet printing of ceramics is highly dependent on key elements including ceramic powder and ink composition as well as their rheological features like dispersity, viscosity, and surface tension . The perr layers . On solir layers .Binder jetting is a method of additive manufacturing which powder particles are bonded together using a liquid binding agent that is selectively deposited. The bonding of material layers results in the formation of an object. Following that, unbound powder is removed from the green part and appropriate post-processing, such as sintering, is performed ,76. TherMaterial extrusion, also known as fused deposition modeling, in where the ceramic material is heated over its melting temperature and extruding through a nozzle. Then, one layer at a time, it is deposited. The nozzle moves horizontally and the platform travels vertically to allow for the addition to each subsequent layer. The printed part is conducted for debinding and sintering to obtain densification ,79. The 2 is the development of improved ZrO2 inks capable of generating complex 3D structures with micro- and nano-scaled resolution and large span capability [Direct ink writing (DIW), also known as robocasting, was initially designed to process concentrated materials with limited organic component, such as ceramic slurries . The cerpability . The dirpability ,81.Despite its numerous advantages, AM has yet to be certified as a fabrication technology for zirconia ceramic restorations. Although there has been limited research on the 3D printing of dental zirconia ceramics, current studies show a promising future for AM zirconia ceramics in dental applications. So far, some research has concentrated on additive manufacturing approaches for fabricating zirconia dental applications with commercially available systems which in this review paper is classified into three categories: restorative applications, zirconia implants, and bone regeneration. Many recent AM zirconia ceramics research attempts have focused on the use of stereolithography-based technologies, as one of the most promising techniques. Recently, a few AM manufacturers which are using stereolithography-based technologies are available and can print zirconia could be prospective suppliers for zirconia printing for dental applications. Ebert et al.  employedÖzkol et al.  developeLian et al.  investigWang et al.  investigLi et al.  assessedRevilla-León et al.  comparedIoannidis et al.  evaluateIn a recent study, Wang et al.  assessedLi et al.  evaluateAdditive manufacturing has been shown to generate both fully sintered (solid) and partially sintered (more porous) structures by varying manufacturing conditions. As a result, introducing porosities may change mechanical properties, which could be useful in mimicking the mechanical properties of enamel and dentin and enabling the fabrication of functionally graded dental restorations .Cheng et al.  investigMoin et al.  investigOsman et al.  evaluateThe fracture resistance of implant-supported milled zirconia, milled lithium disilicate, and stereolithography (SLA) additively manufactured zirconia crowns were compared by Zandinejad et al. . ZirconiDue to superior mechanical properties and biocompatibility, AM zirconia-based ceramics for bone generation and bone tissue engineering have recently gained much attention. Although, in contrast to the distinguished calcium phosphate-based ceramics for bone-regeneration applications, the biological activities of zirconia-based ceramics for bone-regeneration applications have not been completely investigated and only a few studies have been performed by using extrusion-based, Robocasting/Direct Ink Writing (DIW), DLP and SLS techniques . Li et al.  manufactAdditive manufacturing (AM) is developing in dentistry as a potential approach for fabricating dental restorations and other appliances. However, AM zirconia ceramic techniques are not yet well-developed, and several issues, such as difficulty in preparing raw materials, process control, and ceramic printers development must be addressed. Surface quality, dimensional accuracy, and mechanical properties must all be enhanced in order to make high-quality objects ,32.The feedstock for each AM technology must be given in a form that is compatible with the process. In the case of zirconia ceramics, the raw materials are currently available in the form of slurry, powder, or bulk solid, depending on the kind of AM technique . The proDespite the fact that several commercially available AM systems capable of printing zirconia ceramics have been proven, there are many misalignments between present AM ceramic capabilities and application performance requirements. Surface quality is determined by the AM technology employed, processing conditions, and raw material properties, which impact the thickness of each printed layer . DimensiThe prospects for current AM methods capable of producing zirconia ceramics are thought to be in the following areas. The mechanical properties along with microstructure need to be improved to bridge the gap with milling methods. Also, to obtain the optimal mechanical properties, it is highly needed to determine the best process parameters and optimizing the process in each method. Furthermore, the limits of the AM machinery should be addressed so that zirconia parts of greater sizes, more accurate dimensions, or lower prices can be manufactured. Finally, future study into the application of AM technology should concentrate on producing biomimetic dental restorations that reflect the multi-layered as well as the complex mechanical properties of natural tooth.The preliminary lab studies present different AM technologies for processing zirconia for different clinical applications mainly in restorative and implant dentistry. Each technology has some advantages, however vat polymerization including (DLP and STL) AM technology seems to be the most common in AM of zirconia for dental applications. Although in vitro studies show a comparable mechanical properties and accuracy when compared to milling and the potential of this new technology is very promising however further improvements in various areas including printer development, material development and improving the printing parameters is essential. More information regarding optical properties, biocompatibility, residual resin remnants and bonding to porcelain for layering technique is necessary before validating the technology for clinical applications. However due to its potentials and ability to produce complex geometries with different properties the application of additive manufacturing zirconia in restorative, implant and regenerative dentistry may expand quickly.


Using the RECAST algorithm, taxonomic/functional annotation, and machine learning approaches, the metagenomes from three FMT studies, including healthy volunteers, patients with clostridial colitis, and patients with metabolic syndrome, were analyzed. Using our computational pipeline, the donor-derived and recipient-derived microbes which formed the recipient post-FMT stool metagenomes (successful microbes) were identified. Their presence is well explained by a higher relative abundance in donor/pre-FMT recipient metagenomes or other metagenomes from the human population. In addition, successful microbes are enriched with gene groups potentially related to antibiotic resistance, including antimicrobial peptides. Interestingly, the observed reorganization features are universal and independent of the disease.Fecal microbiota transplantation (FMT) is currently used in medicine to treat recurrent clostridial colitis and other intestinal diseases. However, neither the therapeutic mechanism of FMT nor the mechanism that allows the donor bacteria to colonize the intestine of the recipient has yet been clearly described. From a biological point of view, FMT can be considered a useful model for studying the ecology of host-associated microbial communities. FMT experiments can shed light on the relationship features between the host and its gut microbiota. This creates the need for experimentation with approaches to metagenomic data analysis which may be useful for the interpretation of observed biological phenomena. Here, the IMPORTANCE We assumed that the enrichment of successful gut microbes by lantibiotic/antibiotic resistance genes can be related to gut microbiota colonization resistance by third-party microbe phenomena and resistance to bacterium-derived or host-derived antimicrobial substances. According to this assumption, competition between the donor-derived and recipient-derived microbes as well as host immunity may play a key role in the FMT-related colonization and redistribution of recipient gut microbiota structure.Author Video: An author video summary of this article is available. The gut microbiota is made up of a large community of microorganisms and viruses, which are a key player in the host body metabolism. Metabolic functions of the gut microbial consortia are associated with support of physiological homeostasis, synthesis of vitamins and amino acids, short-chain fatty acids, and other essential functions . Develop2–Clostridioides difficile infection (CDI). Several FMT studies are ongoing in a broad spectrum of disorders. However, neither the therapeutic mechanism of the FMT nor the mechanism that allows the donor bacteria to colonize the intestine of the recipient has been discovered. The changes in the intestinal microbiota under FMT that resulted in colonization with donor bacteria have been described in the case of CDI and metabolic syndrome patients  is currently used to treat recurrent patients –11 and hatients –. Neverthrecipient intestine colonization analysis tool (RECAST). This approach is based on the separation of the donor’s and recipient’s metagenomic reads and allows extraction of read categories by origin: those that came from the donor sample, those that stayed in the recipient intestine, and those with unknown origin. Using the RECAST, we studied the behavior of the donor-derived and recipient-derived microbes after the FMT procedure. Also, we determined which gut microbe features can contribute to the FMT-related restructuring process of human gut microbiota.Here, we present a novel technique that allows the study of recipient gut microbiota reshaping due to FMT—Escherichia coli strain genomes with different nucleotide distances were used. The most probable behavioral scenario that could be observed during FMT has been modeled: donor and recipient strains coexist in the recipient intestine  and recall (B). Also, the different colors denoted the classification quality scores: the precision score is shown in blue and the recall score is shown in green. Download FIG S2, PDF file, 0.2 MB.Evaluation of the RECAST algorithm using simulated data sets with different complexity. The color intensity shows the complexity of the generated data sets. The following numbers of genomes were used for subsets: for “low complexity,” 30 genomes; for “medium complexity,” 100 genomes; for “high complexity,” 300 genomes. The Copyright © 2021 Olekhnovich et al.2021Olekhnovich et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the In summary, the classification quality of the RECAST approach depends on the number of microbes within the post-FMT recipient intestinal microbiota and their nucleotide similarity, as well as the coverage of microbial genomes by metagenomic reads. In addition, the RECAST approach can classify donor and pre-FMT recipient metagenomes (colonizer/noncolonizer and resistant/suppressed categories) with high quality, while the classification accuracy of the post-FMT recipient sample  may be reduced due to high genome similarity. In other words, the proposed method allows limited separation of post-FMT bacteria to donor-derived and recipient-derived bacteria when similar strains are present in both donor and recipient samples. Thus, for additional quality control of the classification, a control group including biologically unrelated metagenomes was used in further analysis of real FMT metagenomic data sets. Also, this strategy can be useful to distinguish the effects of FMT and microbiome natural variation.To determine the behavior of donor-recipient bacteria after FMT and their distribution in RECAST-produced read categories, taxonomic profiles of read categories were obtained. The colonizer category included donor-derived microbes that were found in the post-FMT recipient’s sample. Similarly, the resistant category included recipient-derived microbes that stayed in the recipient’s intestine after the FMT procedure. The noncolonizer and suppressed categories included microbes that were not successful in FMT competition.R2 = 0.09, adjusted P < 0.001, Bray-Curtis dissimilarity metric, 10,000 permutations).Nonmetric multidimensional scaling (NMDS) visualization based on taxonomic profiles and Bray-Curtis dissimilarity shows clear separation of the colonizer and resistant categories from the noncolonizer and suppressed categories . AdditioAnalysis of classified reads separated into different categories also shows a clear difference in microbial composition. The distribution of microbial taxa by read categories is presented in 10.1128/mSystems.00811-21.6TABLE S3Table S3, XLSX file, 0.2 MB.Read counts of microbial species in the basket categories obtained using MetaPhlAn2 . Download Copyright © 2021 Olekhnovich et al.2021Olekhnovich et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00811-21.7TABLE S4Table S4, XLSX file, 0.5 MB.Read counts of microbial species in the basket categories obtained using MetaPhlAn2 (control sample set). Download Copyright © 2021 Olekhnovich et al.2021Olekhnovich et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Clostridioides difficile infection [CDI], or metabolic syndrome [MS]) variable to the analysis.To detect specific features related to microbe separation into the different categories, we used the Random Forest algorithm. To perform this analysis, normalized read quantities of microbial species distributed between read categories were used as a predicted variable  in the models based on the real FMT sample set in comparison to the control set  and strain (C and D) levels due to allogeneic FMT  exceed the range of temporal variation observed in healthy controls without interventions (control). As a comparison, metagenome of the baseline (A and C) or donor (B and D) profiles were used. The horizontal red dotted line shows the maximal dissimilarity value between two taxonomic profiles from one person in the control group. Download Copyright © 2021 Olekhnovich et al.2021Olekhnovich et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the According to the results of the analysis, the donor-derived bacteria and recipient-derived bacteria that can contribute to the post-FMT recipient metagenomes are associated with higher abundance in the human population gut metagenomes. At the same time, the influence of the donor microbiota for control has been reduced. It is consistent with the lack of biological association between control samples. It is also worth noting the similarity of the results obtained for patients with various diseases (the disease variable has the weakest contribution to the prediction). Perhaps, there is a universal feature mediating the human gut microbiota restructuring due to FMT phenomena.For determination of donor-derived and recipient-derived microbes’ contribution to forming gut microbiota after FMT, donor and pre-FMT recipient samples were queried by post-FMT recipient’s metagenomic reads. As a result, new read categories were obtained. These categories included the following categories. The acquired category revealed donor-derived microbes. The common category revealed both donor-derived and recipient-derived microbes. The survived category revealed recipient-derived microbes. The external category revealed microbes (or microbe genome parts) which were not found in the donor and pre-FMT recipient’s metagenomes. Also, sorting of the several post-FMT metagenomic samples from the same patient has shown donor-derived and recipient-derived microbial diversity evolution over time. The obtained results are presented in P < 0.001). At the same time, the recipient’s microbiota is prevalent in the MS group . It is worth noting that the common category is formed by similar microbial reads from the donor and pre-FMT metagenomes, which the sorting algorithm could not clearly distinguish. This category is prevalent in the MS group in comparison with the healthy and CDI groups . Interestingly, the control sample set analysis shows the high microbiota stability over time. According to this analysis, the main part of the microbial diversity comes from the baseline sample. At the same time, the almost complete absence of the acquired category in the control group indicates that the sensitivity and specificity of the RECAST make it possible to achieve a high quality of the classification using real metagenomes.The healthy and CDI groups demonstrate dominance of donor-derived microbes in comparison to MS . It should be noted that in post-FMT samples of the control data set, “donor” microbiota was not found (as expected), whereas the intersection between the “donor” and recipient microbes was ∼25%. The ratio of the categories must be relatively stable. Thus, we can conclude that the gut microbiota has a stable unique structure and overlaps up to ∼25% between two independent persons.In addition, in our analysis, we highlighted the external read category which consists of microbial genomes whose origin could not be determined. These may be metagenomic reads from the uncovered recipient/donor microbes. Also, these may be reads that should have entered the acquired or survived categories but were misclassified. In addition, it could also be explained by transient microflora. There was a decreased external category in the control group in comparison with real FMT . The distance based on species and strain levels from the donor sample decreased over time in the healthy and CDI sample sets, whereas the MS sample set showed a strong decrease 2 days after transplantation, followed by a gradual increase. Thus, the data obtained indicate a strong effect of the donor microbiota on the recipient’s gut microbiota profile after FMT in healthy and CDI sample sets; however, in the MS allogeneic sample set, this effect is reduced.For additional validation of the observed biological effects, the basic analysis using a set of generally accepted metagenomic approaches and original (nonsorting) metagenomes was performed. Taxonomic annotation was performed using MetaPhlAn2, and microbial strain profiling was performed by metaSNV. The obtained results are presented in Thus, results produced using common metagenomic methods such as MetaPhlAn2 or MetaSNV and RECAST are similar. The change in the Bray-Curtis dissimilarity and Manhattan distance to the donor samples corresponds to the increase of the donor fraction in the post-FMT metagenomic samples. However, the RECAST algorithms allow the determination of donor/recipient microbe rate contribution to recipient microbiome assembly after FMT. Moreover, using the RECAST approach, the functional composition of read categories can be studied. These results are presented further.To identify potential functional features associated with gut microbiota restructuring due to FMT, functional profiles of read categories were obtained using the HUMAnN2 pipeline. In total, 5,199 Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology (KO) groups in all categories of reads were identified. Functional differences were determined strictly between dependent read categories, between colonizers/noncolonizers or resistant/suppressed separately. Differences in KO profiles between read categories are presented in 10.1128/mSystems.00811-21.9TABLE S6Table S6, XLSX file, 0.03 MB.KEGG orthology groups are differentially abundant between the colonizer and noncolonizer read categories. Download Copyright © 2021 Olekhnovich et al.2021Olekhnovich et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00811-21.10TABLE S7Table S7, XLSX file, 0.02 MB.KEGG orthology groups are differentially abundant between the resistant and suppressed read categories. Download Copyright © 2021 Olekhnovich et al.2021Olekhnovich et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2 fold change associated with read categories is shown in The top 10 KO by logFurthermore, extended analysis of antibiotic (using the MEGARes 2.0 antibiotic resistance genes [ARGs] database) and nisin (using 5 KOs from the KEGG database described above), resistance gene enrichment in obtained read categories was carried out . AccordiClostridioides difficile infection and other diseases. On the other hand, FMT can be considered a useful model for studying the ecology of host-associated microbial communities. After the FMT procedure, the restructuring processes of the recipient’s gut microbial community are observed. How do donor-derived and recipient-derived microbes contribute to the microbiota reassembly after FMT? Some researchers have tried to answer this question using comparative analysis of gut microbiota taxonomy of donors and recipients obtained by the 16S rRNA gene sequencing approach  is currently used to treat recurrent approach –18. The roach –8–. HoweverUsing the proposed RECAST algorithm, the donor-derived and recipient-derived microbes that formed the recipient post-FMT stool metagenomes were identified. According to our analysis, these microbes have a higher relative abundance in the human population metagenomes compared to noncolonizer donor-derived microbes or recipient-derived microbes which are lost by recipients of FMT. It is worth adding that these results were similar between all samples included in analysis and do not depend on disease.These results are consistent with previous studies. The donor’s metagenome-assembled genomes that colonized all recipients were prevalent, and the ones that colonized neither were rare across the participants of the Human Microbiome Project samples in a FMT study of CDI patients . Other rBlautia obeum is a producer of nisin O, which was isolated from human gut microbiota. This research adds to the evidence that lantibiotic production may be an important trait of gut bacteria  that are not gut microbiota residents. These transient bacteria may be a significant part of gut microbiota diversity. Perhaps, the entire “true” intestinal microbiota of the donor can colonize the recipient’s intestines with various degrees of success. On the other hand, biological sample collection/preparation, sequencing artifacts and/or taxonomic classification and/or other reasons may affect the quality of the analysis and hence the reliability of conclusions. In any case, these observations certainly require additional confirmation.Here, we presented a novel computational approach RECAST to track the restructuring process of the gut microbiota due to FMT. The method is based on sorting post-FMT recipient’s metagenomic reads by origin from the donor or recipient microbiome. The functional analysis of the obtained read categories revealed the enrichment of successful gut microbes by lantibiotic/antibiotic resistance genes. The results obtained with publicly available data sets allowed us to propose the “restructuring hypothesis”: the most prevalent of the donor stool microbiota and likely the most prevalent in the human population stool microbiota can colonize the recipient’s intestine. To summarize, this approach allows researchers to gain novel biological insights via providing the improved resolution of FMT study analysis.recipient intestine colonization analysis tool) algorithm, based on MetaCherchant source code  are extracted from the read and are searched for in the de Bruijn graph. As a result, mean depth coverage of a read by k-mers as well as breadth coverage of a read is obtained. Breadth coverage is defined as a proportion of positions in read, covered by k-mers from de Bruijn graph. Then theoretical estimation of breadth is used to classify each read as found or not found in the de Bruijn graph.We developed the RECAST . Summing up, the read is classified as found if it both satisfies the threshold and falls within the confidence interval. Otherwise, the read is classified as not found. During the processing of paired-end reads, reads from some pairs can be classified into different categories. This indicates the discrepancy between the classification and paired nature that might have been caused by small genome variations or sequencing errors. These read pairs are not credible and are excluded from further analysis. The schematic workflow of the algorithm is presented in A design of FMT experiments to study the behavior of the gut microbiota usually involves the collection and sequencing of stool samples of the donor, the recipient before FMT (pre-FMT sample), and the recipient after FMT (post-FMT sample). The RECAST algorithm takes as input every two out of three metagenomic samples described above and splits reads from each metagenome into different categories based on the origin in the recipient’s post-FMT metagenome. First, donor reads are queried against a post-FMT recipient sample to generate colonizer and noncolonizer categories. Second, pre-FMT recipient reads are queried against the post-FMT recipient sample to generate categories resistant to FMT and suppressed by FMT. Third, post-FMT recipient reads are queried against the donor sample and split into two temporary categories: found and not found. Further, these categories are queried against the pre-FMT sample to split post-FMT reads into four categories: acquired via FMT (reads found only in donor sample), common (reads found in both donor and pre-FMT samples), survived FMT (reads found only in the pre-FMT sample), and external (reads not found in donor or pre-FMT samples). A diagram of the produced read categories is presented in Escherichia coli strain genomes with different nucleotide distances was used for simple reads simulations by the InSilicoSeq tool , as well as associated donor metagenomes. All whole-genome sequencing (WGS) metagenomes containing both donor and recipient samples available at the start of the study were selected. An additional criterion for data selection was the presence of aligned sampling points between recipients.Clostridioides difficile infection . Technical sequences and low-quality bases were trimmed with the Trimmomatic tool (Q > 30. The human sequences from metagenomic samples were removed by bbmap (https://www.ncbi.nlm.nih.gov/assembly/GCF\_000001405.13). Described metagenomics read preprocessing computational steps were implemented in the Assnake metagenomics pipeline (https://github.com/ASSNAKE). The preprocessing results are presented in Raw metagenomic data were downloaded from public repositories using fastq-dump from the SRA Toolkit , qualitytic tool . The thrby bbmap  using GRAfter quality control, samples were sorted using the RECAST algorithm by the categories described above. As a control, sorting was also performed in the control group. Each baseline metagenome was selected as a “donor sample,” while the remaining metagenomes from this subject were not used in the analysis. In total, the 10 sorting series were performed in real FMT data sets and 20 sorting series were used in the control data set. Each sorting series consists of several algorithm runs—one for each post-FMT time point. In total, there were 105 sorting procedures: 33 for real FMT data, 72 for control data.https://ggplot2.tidyverse.org) implemented in GNU/R. PERMANOVA (adonis function from the vegan package for GNU/R) and Bray-Curtis dissimilarity  group levels were calculated. The absolute value (modulus) of the log2 fold change threshold was set at 2. Wilcoxon signed-rank test with false discovery rate (FDR) correction for multiple hypothesis testing was used to determine significant differences in functional profiles (adjusted P < 0.05). Antibiotic resistance genes (ARGs) were identified in the metagenomes by mapping the metagenomic reads to MEGARes 2.0 database  or taxonomic  features, and the relative abundance of microbes in the donor or recipient metagenomic samples as well as in the HMP 2012 data set were added in this analysis. The classification of the pairs of read categories such as colonizers/noncolonizers and resistant/suppressed was performed separately because they derive from different samples: donor and pre-FMT recipient metagenomes, respectively. Microbial species whose MetaPhlAn2 markers were covered with fewer than 100 reads were not included in the analysis. For model building, parameter optimization, and associated data processing, pandas, numpy, and scikit-learn libraries for python 3 and jupyter-lab were used.Nonsorted metagenomic reads of experimental data sets were examined using common metagenomic approaches such as MetaPhlAn2 and metaSNV  profilinPRJNA510036, PRJEB12357, and PRJNA353655, at European Nucleotide Archive (ENA) database ERP009422, and at https://www.hmpdacc.org. The source code for the RECAST algorithm can be found at https://github.com/ivartb/RECAST.The study used data from open sources, which are available at NCBI Sequence Read Archive under the BioProject accession numbers


The corrugated board packaging industry is increasingly using advanced numerical tools to design and estimate the load capacity of its products. This is why numerical analyses are becoming a common standard in this branch of manufacturing. Such trends cause either the use of advanced computational models that take into account the full 3D geometry of the flat and wavy layers of corrugated board, or the use of homogenization techniques to simplify the numerical model. The article presents theoretical considerations that extend the numerical homogenization technique already presented in our previous work. The proposed here homogenization procedure also takes into account the creasing and/or perforation of corrugated board . However, it is not always easy to estimate how exactly these processes affect the bending or torsional stiffness. What is known for sure is that the degradation of stiffness depends, among other things, on the type of cut, its shape, the depth of creasing as well as their position or direction in relation to the corrugation direction. The method proposed here can be successfully applied to model smeared degradation in a finite element or to define degraded interface stiffnesses on a crease line or a perforation line. Colorful boxes and packaging are designed to attract the customers’ attention and, as a consequence, to drive the sales of various goods ranging from bulky products, through food, children’s toys, cosmetics, and many others. A growing awareness of concern for the natural environment has led many companies to opt for packaging that can be easily recycled or disposed of, biodegradable, and space-saving after manufacturing. A corrugated cardboard undoubtedly has all of these qualities. Moreover, it is easy to print on, for example, the brand name. Corrugated cardboard is easy to shape via creasing along the suitable lines and, furthermore, creating openings, ventilation holes, or perforations does not cause much difficulty. The latter is essential with regard to shelf-ready packaging (SRP) or retail-ready packaging (RRP) when the product, after transportation to the site, is placed on the shelves and after tearing off the flap along the appropriately designed perforation, is ready for sale. Thus, a lot of time is saved, which nowadays leads to significant profits for large companies.Of course, one cannot only focus on the aesthetic values because the packaging, in fact, plays a much more important role such as securing the goods during storage or safe transport to the destination place. The load-bearing capacity of the corrugated cardboard boxes and the influence of humidity, openings and perforation arrangement, or the location of flaps is under constant investigation. Therefore, scientific research has become an integral part of a distinct branch of industry . Manufacturers of these packaging types strive for effective, economical, and easy-to-use solutions, which results in the continuous, lasting over many years, development of research on cardboard strength while using various analytical, numerical, and experimental methods.Compressive, tensile, or bursting strength tests are routinely executed to assess the load-bearing capacity of corrugated cardboard boxes. The box compression test (BCT) and the edge crush test (ECT) are the best known. Inextricably related to the mechanical strength of the paperboard or corrugated cardboard boxes are two characteristic in-plane directions of orthotropy  as well as parallel to the fluting—cross direction (CD)).Another option for estimating the compressive strength of the boxes is the application of analytical formulae in which, in general, three groups of parameters such as paper, board, and box parameters are present . Ring crOver recent decades, meshless and meshfree methods  have become popular numerical techniques for solving partial differential equations and have been beneficial while considering corrugated cardboard problems. Wang and Qian  proposedUnquestionably, many determinants affect the compression strength of the corrugated paperboard boxes  includinA very effective, commonly applied in engineering, technique to determine the strength of the boxes is the finite element method (FEM). Thakkar et al.  comparedThe examined models can be facilitated to one single layer described by the effective properties of the composite instead of building layers composed of different materials. Such a method, called homogenization, has been used extensively over the last years by Garbowski et al. ,35,36,37The following article, as the next one in the series, provides theoretical considerations that develop and extend the numerical homogenization technique already presented in the prior works of the authors. The proposed homogenization procedure also takes into consideration the creasing and/or perforation of corrugated board . However, it is not always easy to estimate how exactly these processes affect the bending or torsional stiffness. The fact is that the decrease in stiffness depends, among others, on the type of cut, its shape, and the depth of creasing as well as their position or direction in relation to the corrugation orientation. The method proposed here can be successfully implemented to model smeared degradation in a finite element or to define degraded interface stiffnesses on a crease line or a notch line.Corrugated board, as a fibrous material, is characterized by strong orthotropy. The mechanical properties of its components  depend on the direction of the fibers in the individual layers of the composite. Paper and paperboard are more than twice as stiff in the machine direction (MD) than in the cross direction (CD). This is related to the fibers which, due to the production process, arrange along the MD. In this direction, the material is more resistant to tearing and crushing, although it has lower ductility than in CD see .The linear elastic orthotropic material can be described by the following stress–strain relationships:The material orientation was always the same in all layers see . This isThe paperboard, as already mentioned, was modeled here using classical linear elastic orthotropy (see Equation (1)). The material data were taken from the literature ,44,45. AThe thickness of all flat layers (liners) in both single- and double-walled corrugated boards was assumed to be 0.30 mm; for all corrugated layers (flutes) in both models, the thickness was also taken as 0.30 mm.The main goal of this work was to numerically analyze many cases of perforation with possible creasing and its effect on the stiffness reduction of corrugated board. The variants include not only different types of perforation , but also different orientations of the cuts in the sample (from 0 to 90 deg. every 15 degrees). All cases are compiled in Two hypothetical corrugated boards were analyzed here, namely single-walled (SW) with 8 mm flute period, 4 mm height and double-walled (DW) with 4 mm flute period, 2 mm flute height (for lower layer) and 8 mm flute period, 4 mm flute height (for higher layer). Both the influence of the flute orientation and the cutting orientation on the decrease in the stiffness of the corrugated board were examined. In case C, the cutting orientation changed to 00, 15, 30, 45, 60, 75, 90 degrees see  while thIn case F, the flute orientation were changed to 00, 15, 30, 45, 60, 75, 90 degrees see  while thBoth single-walled and double-walled models with perforations of 4/4 mm, 2/6 mm, and 6/2 mm in the variant 00 deg. of cut and flute rotation were crushed by 10, 20, and 30%. This consideration results from the observation of the serial production of packaging in which crushing is an element built into the entire cutting and perforation process. The additional crushing during cutting is the result of using rubber in the area of perforation knives that additionally crush the cross-section. The crushed geometry of both kinds of samples is shown in All crushed samples were marked with an additional symbol R-xx, where xx means the amount of crush . Therefore, for example, a single-walled specimen with a cut/flute rotated by 0 degrees with a cut version of 44 and crushed by 10% has the symbol SW-44-C-00-R-10.Additionally, what was verified during this research was the influence of the position of the cut in the corrugated boards’ cross-section along the wave on the stiffness reduction. For this purpose, four additional representative volumetric element (RVE) models were created in two variants of the SW and DW samples, in which the flute was shifted by 1/16 of the period (P) from 1/16 P to 4/16 P see .In order to determine the effect of cuts on the stiffness of the corrugated board, the numerical homogenization method was used here. This method, originally proposed by Biancolini  and lateThe displacement based on finite element formulation for a linear analysis can be represented by an equation:Static condensation relies on the removal of unknown degrees of freedom (DOF) and then the formulation of the stiffness matrix for a smaller number of degrees of freedom, called the primary unknown or principal DOF. In the analyzed cases, the eliminated degrees of freedom is the internal RVE nodes and the external nodes are the primary unknowns. The statically condensed FE stiffness matrix is computed from the equation:Static condensation reduces the total elastic strain energy to the work of external forces on the corresponding displacements. The total elastic strain energy can be calculated from the equation:The balance of the total energy for the full 3D shell model and the simplified shell model is ensured by an appropriate definition of displacements in the external RVE nodes and by enabling the membrane and bending behavior. More details can be found in Garbowski and Gajewski . The genWhile using the definition of the elastic strain energy for a discrete model:For a homogenized composite, the stiffness matrix can be easily determined as:The presented homogenization method is based on replacing the full 3D shell model with a simplified shell model and computing the effective stiffness of the RVE. Such a procedure significantly accelerates the computations and maintains a very high accuracy of the results.A, B, D, and R as follows:A represents extensional and shear stiffnesses; B represents extension-bending coupling stiffnesses; and D represents bending and torsional stiffnesses, while R represents transverse shear stiffness.The matrix In general, the stiffness matrix Within all analyses, the 3-node triangular general-purpose shell elements, named S3, were used for the computations. In every examined case, approximate global size equal to 0.5 mm was assumed. Due to the analysis of different orientations of flutings or cuts in the sample, the number of elements changed. For example, in the case of the SW-44-C-00 sample—2002 elements, 1099 nodes, and 6594 degrees of freedom were obtained, and for the DW-44-C-00 sample—3972 elements, 2074 nodes, and 12,444 degrees of freedom were obtained.The proposed numerical method was first verified by direct comparison of the obtained results with the existing solutions from the literature. One example concerns an assembled sandwich structure consisting of a corrugated tooth-shaped core enclosed between two sheets. A reference solution is available from Buannic et al. . AccordiOn the basis of the above validation see  carried This section presents all the results of numerical tests for both single-walled (SW) and double-walled (DW) corrugated board samples. First, Due to the volume limitations of the data that can be presented in all the following tables, only the values from the main diagonals of the A, B, D, and R are non-zero; in particular, matrix B (see Since the DW model is asymmetric, all matrices ix B see , which ction see . Figure tion see .In the process of cutting corrugated board, perforation may occur in various locations relative to the fluting position, therefore the impact of fluting shift on stiffness changes has also been analyzed. Due to noticed increase of As the perforation process is inseparable from the crushing process, this effect on the reduction of stiffness has also been tested. The influence of additional crushing of 10, 20, and 30% of the initial height of the corrugated board on the stiffness degradation of SW and DW samples is presented in On the basis of the conducted analyses and the obtained results, it can be concluded that the perforations to a greater or lesser extent affected the stiffness degradation not only in the For samples with different perforation orientations see , the redFor 2/6 mm perforation in model SW see a, the maodel see d.While considering the 4/4 mm type perforation see b, the obodel see e, a simiThe greatest reductions were observed for the sample with the 6/2 mm perforation type see c,f. Thisodel see f.For samples with different fluting orientations see , the greWhen analyzing the stiffness reductions for models with shifted fluting see , even ind DW see , where aBy also adding to the model the crushing of fluting see  that accThis article presents the comprehensive numerical analyses of the effect of perforation on reducing stiffness while implementing homogenization techniques. The acquired knowledge can be used for numerical modeling, for example, of corrugated cardboard packaging with perforations. Knowing the specific values of the stiffness reduction, it is possible to correctly model the perforation line and thus accurately estimate the load capacity of the packaging. The reduction in individual stiffnesses depends not only on the type of perforation, but also on the orientation of the perforation and the orientation of the fluting, but does not depend on the location of the perforation along the wavelength. Further development of the launched research is planned related to the validation of the proposed model with experimental models while engaging the non-contact displacement measurements .