Korean Journal of Women Health Nursing (KJWHN) received notice from the Scopus Content Selection and Advisory Board (CSAB) on April 13, 2021, that the journal will be included in the Scopus database.I am delighted to announce that the This recognition from Scopus builds on the dedication and tireless work of prior editors-in-chief and editorial board members and signals the continuous work needed for KJWHN to continue its growth while striving for international connectivity.Journal of Korean Women's Health Nursing Academic Society, publishing eight articles in the first issue. Till June 2012 the journal has been published under the current title. Throughout the past 25 years, KJWHN has been “The” scholarly platform for sharing among Korean nurses in maternal and women’s health for nurses in clinical practice, nurse researchers, and students alike. KJWHN was an early pioneer among Korean nursing journals in terms of offering open access since 2008 and being included in CINAHL in 2009, as well as the Directory of Open Access Journals (DOAJ) in 2013.The journal was launched in March 1995 under its previous title, the The following timeline illustrates our path of seeking and achieving Scopus status, which may help other journals: In 2013, the first application to Scopus was submitted. A few months later, the editorial office received notice of rejection with the following comments: “Citedness is below expectations. There is no international diversity among authors. Some part of reference listing is inconsistent with the author instructions.”Acting on these comments, the editorial board was expanded and editors’ efforts to advertise the journal and encourage international authors resulted in a manuscript submission from Turkey, which was eventually published.In August 2015, the second application was submitted, and another letter was received in November 2016, citing the following rejection reasons: “Some part of reference listing is inconsistent with the author instructions. There is no international diversity among editors and low citation activity on editorial board level.”Principles of Transparency and Best Practice in Scholarly Publishing version 3 [https://submit.kjwhn.org). Our new English-based website continues to support full open access (https://kjwhn.org), in addition to translations powered by Google in 80 languages and metrics information. Information on funding sources was made available from 2020.In response, the editorial board worked over the past 4 years to overcome the shortcomings before reapplying in January 2021. It adopted an internal system to screen for inconsistencies with author instructions, including the reference list and conducted regular training of editorial board members. Furthermore, since 2020, KJWHN has invited a professional manuscript editor to ensure consistency with author instructions. Regarding the second limitation, the editorial board and international advisory board were restructured and expanded. While KJWHN had six international advisory board members from three countries in 2016, this was expanded to nine international members from five countries across three continents as of January 2021. These international members are active professionals in their field, and are contributing to the journal via peer-review as well as writing invited papers for the journal. Finally, for citation activity, editorial board members’ academic activity was made available on our website via Scopus Author ID and ORCID links. In addition to addressing the specific comments, editors have worked diligently to improve the journal according to international standards. The journal went through three rounds of revising author guidelines for clarity since 2020, created a statement on After implementing these changes, in January 2021, the third application to Scopus was submitted. The acceptance letter from CSAB, received on April 13, 2021, included the following comments: “The previous and current editorial teams are to be commended on the development which has led to extended citations in international research journals. Many papers have been cited. The development of the ethical standards throughout is very clear and in keeping with both COPE and Scopus guidance. The use of the COPE et al.’s 2018 Principles of Transparency guidance was especially of value and demonstrates an ongoing attention to publishing practice and ethical content of the journal.”While I am delighted the journal is now accepted into the Scopus database, I realize this is a timely opportunity and significant time of reflection for KJWHN to consider how to potentiate its value for authors and reach a wider international readership in the coming future. In this line, I want to add a few notes on how the journal seeks to further improve itself over the next few months.Citations are a constant challenge for all journals. While the adage for academia is ‘publish or perish,’ for journals this seems to have become what I shall term ‘be cited to survive and thrive.’ The total cites in the Scopus database have increased annually as follows: 71 in 2017, 96 in 2018, 123 in 2019, and 125 in 2020. The majority of citations, however, are from Korean journals in Scopus, both nursing and others . Also, mhttps://dataverse.harvard.edu) for authors who wish to share data for transparency and replicability. Also, ascribing to internationally accepted reporting guidelines, e.g., those suggested by the Equator network (https://www.equator-network.org/), is being applied. In addition to these updates to the author guidelines, the following will be applied from the 2021 September issue: Manuscript types for submission have been expanded and requirements for each type have been clarified  and more recently, Child Health Nursing Research, a domestic nursing journal. In the case of APS, since switching to English-only manuscripts in 2012, publication metrics improved and it was accepted for the Scopus database in 2013 and included in the Web of Science Core Collection in 2016 [Readers may notice that the majority of articles are in English in this issue. Some Korean journals have chosen to switch to publishing fully in English, such as the  in 2016 , and the in 2016 , perhaps in 2016 ,8, as a Nevertheless, an analysis of natural science journals from several countries publishing in English or other languages, reported that English publications had a higher number of citations than those published in other languages, even when controlling for the effect of the journal, year of publication, and paper length . It is wThe success of our efforts will largely depend on a solid number of submissions. I invite authors to submit their work, especially systematic review and meta-analysis papers, experimental design studies, exploratory qualitative research, and methodology manuscripts. For descriptive and correlational research design manuscripts, I encourage authors to include a description of the theoretical or conceptual framework used to ground their quantitative research, which has been emphasized as good practice .The present achievements of KJWHN have been possible through the support and cooperation of society members and readers worldwide. I truly appreciate all of them for their contribution. To promote the journal to an internationally top-ranking one, the continuous attention of nursing scholars in Korea and worldwide are essential. The journal will continue to promote maternal and women’s health nursing by following international publishing standards and thoroughly editing and publishing manuscripts.


A long‐standing debate amongst international relations scholars has surrounded the question of whether international institutions with judicial authority enjoy more autonomy and discretion than other global institutions. This is mainly because international courts are established as impartial third‐party actors tasked with performing adjudicative functions for conflicting parties. As such, the delegation contracts of international institutions with judicial authority are expected to minimize control by states, even in cases where the members of a court engage in judicial overreach. This article contributes to that debate by examining the case of the crisis of the WTO Appellate Body. The article analyzes the Trump administration's successful efforts at rendering dysfunctional one of the most powerful courts in the international system. The findings showcase how powerful states are capable and willing to take advantage of the available control mechanisms and the institutional opportunity structures inherent in the design of international courts. The article speaks to the scholarship on the contestation of international institutions. The analysis relies on original data obtained through 22 interviews with WTO officials, state representatives, and experts. Powerful international courts should not be viewed as immortal and shielded from state control. Protectionist, anti‐multilateralist states are willing to take advantage of opportunity structures embedded within the institutional design of such organizations to exert their influence and control on them in pursuit of their interests.Politicization of, and state influence on, international institutions can ultimately lead to a weakening of the authority and autonomy of these institutions. Careful designing of international institutions to ensure that states cannot take advantage of institutional opportunity structures is imperative for avoiding this.The Appellate Body's current dysfunctionality is very costly for the WTO, arguably threatening the future of its multilateral institutional framework for governing global trade. This dysfunctionality should not be understood as unique to the WTO but as a warning to other international legal institutions of the opportunity structures that powerful states may use to disrupt their functionality.1The institutions at the forefront of the global multilateral order are being challenged by a wave of counter‐institutionalism, unilateralism, and bilateralism, the manifestation of which are reflected by the crisis at the World Trade Organization (WTO) , tribunals, and other international organizations (IOs) that have judicial authority, can be referred to as trustees. The logic of delegation to trustees is sometimes argued to be different from that of other international institutions, as the main motivation of states in establishing them is to benefit from their impartial and apolitical third‐party character appointment of all the judges at the Body, effectively disrupting its operations. Denying the re‐appointment of staff is a classic example of a control tactic employed by states against ordinary IOs Alter, . MoreoveDrawing on empirical data obtained through 22 interviews with current and former WTO and state officials, the analysis conducted here demonstrates that overtly protectionist and anti‐multilateralist administrations such as that of President Trump are capable and willing to take advantage of the institutional opportunity structures inherent in the design of international institutions by making use of the control mechanisms available to them post‐delegation. Ultimately, the case explored here highlights the enormous pressures that IOs are facing by deglobalizing actors. This is the case even for IOs with central positions and roles within the international system, such as the WTO.In the following sections, the article delves into the International Relations (IR) literature on the international contestation of international courts, merging this with propositions from the literature on resistance/backlash against international courts in the international law scholarship. The theoretical discussion guides the article's empirical investigation, which focuses on the case of the WTO Appellate Body crisis. The article concludes with an overview of the findings and a discussion of the potential policy and academic implications, as well as the avenues for future research.2This article argues for going beyond a discrete categorization of international organizations/courts for explaining state‐led contestation of these institutions. Even when it comes to international courts, states can take advantage of the opportunity structures inherent in the design of such institutions to block and effectively contest them, as they would with other international organizations. Furthermore, while some forms of contestation may stay limited to specific rulings through rhetorical legal and political critique of the institutions, states may employ opportunity structures to produce favorable institutional outcomes, such as rendering the institution dysfunctional without necessarily disbanding it. Finally, state‐led contestation of international courts does not only arise from divergences from the delegation contracts of such agents, but also from states’ domestic politics and the constellation of factors outside of these institutions that provide inducive conditions for state‐led challenges against them.In other words, contextual factors (domestic politics) and institutional factors  together play a role in the processes behind contestation, as well as the form it manifests itself through. These propositions build on the Principal‐Agent (P‐A) scholarship on delegation contracts of IOs but resonate as well with studies on resistance/backlash against ICs in the international law literature . The resulting theoretical framework therefore fruitfully marries insights from both bodies of research to produce an approach that converges logical conclusions regarding both contextual and institutional factors to contribute to our understanding of contestation of international institutions with judicial authority.ex ante or ex post control mechanisms established within their delegation contract orienting the behavior of institutional actors , as compared to the more serious control and punitive tactics that states may employ against other non‐judicial IOs. Elsig and Pollack  for examex post control mechanisms. More specifically, states may exert hierarchical control over those institutions through the opportunistic use of available ex ante mechanisms, such as appointment of staff and budgeting. Through the use of such opportunity structures, states may obtain outcomes similar to re‐contracting in their contestation of ICs. For example, by denying the (re)appointment of all judges, member states can render a court dysfunctional, effectively shutting down the institution.What is proposed here is that the delegation contract of ICs may create opportunity structures embedded within their institutional design that may allow states to employ these against the institutions despite the lack of viable Moreover, this article argues that it would be unproductive to describe ICs as a discrete type of institution. Courts in the domestic arena are distinct institutions. In contrast, on the international level the distinction between institutions with judicial authority and other types of institutions may be less clear. This is as state principals compromise and tailor the delegation contracts of institutions depending on their specific needs and the particular functions and purposes of the institutions  are contested by anti‐multilateralist governments representing societies in which local grievances against globalization and international institutions take center stage in domestic politics. Examples of this include cases of African regional courts, the International Criminal Court, the European Court of Human Rights, and the WTO Appellate Body  by a powerful member‐state with an anti‐multilateralist agenda to control the court. This allowed the US to effectively achieve an outcome similar to what ex post control mechanisms would have accomplished against an ordinary IO, namely re‐contracting and shutting down the judicial body of the institution. These measures go beyond rhetorical and influence tactics, demonstrating the ability and willingness of powerful states to control and/or disrupt even IOs with judicial authority. Moreover, the findings resonate well with the expectations of the resistance/backlash literature, highlighting the role of domestic contextual factors in the process behind the US contestation of the WTO.The role of contextual factors in IC contestation is also clearly reflected in the case of the US contestation of the WTO Appellate Body during the Trump administration, which is the focus of the empirical investigation in the next section. The case made here is that the crisis at the institution reflects the use of in‐built institutional opportunity structures  (Interviewee #9). The Appellate Body has been accused of engaging in judicial activism, establishing a body of international trade law, and relying on legal precedence .The analysis demonstrates that the opportunity structures available within the institutional design of the WTO allowed for the US, a powerful member with a (at the time) distinctly anti‐multilateralist agenda, to effectively employ The bulk of the analysis relies on the views of relevant actors within and without the organization, obtained though original interview data. In total, 22 interviews were conducted between April 2020 and February 2021. All interviews were based on the condition of full anonymity, hence quotations from interviewees are identified with anonymous labels . The majority of interviews were audio recorded and fully transcribed based on informed consent. For a descriptive list of the interviews please refer to the Appendix.Particular attention was paid on ensuring data triangulation. The interview data draw from the views of former Appellate Body judges, as well as those of current and former WTO Secretariat staff. Interviews were also conducted with current and former state officials and representatives at the WTO, experts on international trade, as well as academics in the field of international trade law. The large number of interviews conducted aimed primarily at tracing the process behind the evolution of the US contestation against the Body in the past decade and throughout various US presidencies. Therefore, interviewees were selected based on the relevant period in which they had professional links with the organization.Interviews were semi‐structured, with a standardized interview questionnaire guiding the process. The questions asked in the interviews were directed at obtaining the views from various actors within the organization, with the specific aim of exploring the various processes that were considered as having contributed to the Appellate Body crisis. Key questions were: ‘What role did the Trump administration play in the crisis?’; ‘How did the previous administrations differ in their approach to the Appellate Body?’; ‘What were the concerns within the Body/WTO regarding the challenge posed by the Trump administration?’; and ‘What incentive and opportunity structures within the organization shaped the Trump administration's strategy against the Body?’.3.1Amicus Curiae briefs, which consist of unsolicited reports and information by non‐governmental organizations (NGOs) appointment process of judges to influence the Body. This tactic became evident during the first reappointment phase of the judges in the late 1990s, which came in tandem with the first accusations of judicial overreach against the Body. The point of contention centered on whether the DSU allowed for panels and judges to receive Discontenting members, particularly the EU, argued that the interpretation provided by the judges regarding the rules set by the DSU went beyond their obligations, and that the Appellate Body was trespassing its mandate by engaging in judicial overreach . Consequently, members resorted to the simplest form of ex ante mechanism available to them for influencing the Appellate Body, namely the process of nominations and (re)appointments of judges appointments of judges in the early 2000s followed a streak of unfavorable rulings for the US Alter, . In the ex ante influence tactics. The US started nominating candidates that held similar views, instead of the previous practice of offering nominees that differed in their background and legal perspectives  resorted to rhetorical attacks against rulings that it claimed reflected the over‐judicialization of the system and unsolicited judicial activism. Nominees that were perceived as favoring that growing aspect of the Appellate Body were vetoed, while those that exhibited a neutral stance were approved appointment of individual judges appointment process came in parallel to early proposals by the US to reform the dispute settlement system by giving members more control over the process. The rhetoric that accompanied the proposals was aggressive, with the USTR declaring that the faulty decisions by the Appellate Body required counterstrategies by the US and the rest of the membership in order to ensure that the organization does not deviate from the trade agreements Alter, . This haex ante control tactics, ultimately the court in its entirety retained its authority and autonomy, as evidenced by the fact that the Appellate Body continued to produce rulings that were unfavorable to the US. As such, the previous paragraphs point to the lack of ability (and willingness) by the US to effectively control and rectify what was deemed as a deviating international court. However, as the next subsection illustrates, the US–WTO relations changed dramatically during the presidency of Donald J. Trump, which reflected not only the contrast in policies by a different administration but also an altered political domestic scene in the US.This period of contestation by the US shows the relational dynamic between international courts and states characterized by more autonomy for the former and less control by the latter. Although the US attempted to influence the individual court's judges through 3.2Even before becoming US President, Donald Trump made it very clear that he intended to bellicosely go after the WTO and its dispute settlement system. The first signs of this approach became evident early on in his presidency. Trump claimed that the WTO and the Appellate Body are a challenge to his ‘America First’ policies, making it impossible for the US to effectively obtain fair trade terms with its international partners and inhibiting its ability to protect its domestic market, workers, and large industries Swanson, . FurtherThe core of the criticism involved the decision by the WTO to allow China to hold the status of a developing country, which the US considered as unfair given that the country's economy is the second largest globally .In response to the claimed inability of the WTO to counter alleged malpractices by China in the area of trade, the US unilaterally imposed tariffs on Chinese products in an effort to protect its domestic producers and gain a political leverage to obtain better trade terms in bilateral negotiations. The Trump administration also applied tariffs against other WTO members, such as the EU, Turkey, Japan, and Brazil, sidestepping the regulatory framework established under the WTO Josephs, . This waThe threat to impose tariffs came in tandem with a full‐on rhetorical assault against the Appellate Body of the WTO. The Trump administration threatened to block the (re)appointment of all of the court's judges. When proposals for reform of the Body were pushed by other members, the US claimed that these did not address its concerns satisfactorily and thus refused to compromise. While the threat of blocking the entire appeals’ process continued, US officials, and in particular the USTR, continuously reaffirmed their belief that the Appellate Body was flawed, had too much autonomy, and its judges had engaged in judicial activism for too long, with the US ambassador to WTO Dennis Shea stating at the WTO General Council meeting in July 2019 that ‘the Appellate Body has felt free to depart from what members agreed to’ . This extraordinary delegation of autonomy was intentional in as much as it reflected the members’ interest in ensuring that the Body and its staff remain insulated from the rest of the organization and guaranteeing their impartiality (Interviewee #6). In fact, Appellate Body reports are adopted through negative consensus, making attempts at authoritative re‐interpretations of Appellate Body reports, and in effect controlling the Body, unfeasible.However, US officials argued that despite its autonomy, the Body was still expected to stick to the rules, and the alleged procedural violations, the judicial overreach, and the unsolicited legal interpretations based on case precedence were unforeseen by the members at the time of the institution's inception, representing a trespassing of authority by the Appellate Body . One interviewee pointed out that the US simply had not predicted that the organization would evolve and become a ‘different animal’ (Interviewee #14).More importantly, the high degree of autonomy of the Appellate Body meant that influencing the nomination and (re)appointment process of judges did not necessarily imply control over the Body. This realization may have been central to the Trump administration's decision to block the entire appeals’ process. Blocking the appointment and reappointment of individual judges had been a practice for a while .Furthermore, the act of blocking the entire Appellate Body represented a disconnect from past US administrations. This tactic reflected the aggressive use of In other words, the Trump administration's actions reflected its strategy for reining in on what it considered as a deviating agent that had become harmful to the domestic interests of the US. The imposition of tariffs on imported goods was a tactic employed by the Trump administration to redress the trade imbalance which was claimed to have been caused by the faulty process of the DSB (Interviewee #3). However, the developments at the Appellate Body were a proper case of full‐on punitive strategies being employed by a disgruntled and powerful state against an international court that was being treated as an ordinary non‐judicial institution.Whereas there may have been a disconnect between the Trump administration's policies and those of its predecessors towards the Body, they were nevertheless in line with broader societal cleavages present within the US. In fact, while the US electorate in the decade preceding the Trump administration overwhelmingly expressed support for IOs such as the WTO, the US public's view has shifted significantly since then . Recently there has also been bipartisan alignment in the US Congress regarding the WTO, reflected by the fact that, as of May 2020, both the House of Representatives and the Senate had resolutions and proposals introduced at their sessions proposing the US withdrawal from the WTO (Levy, The overall domestic support for the Trump administration's policies against the WTO Appellate Body therefore reflected a backlash by the US public against the organization and its institutional framework. Since his inauguration, President Joe Biden and his administration appear to be keeping the same line of policies against the WTO. As of yet no remarks have been made by the Biden administration regarding a change in direction or proposals for reforming the institution. This represents continuity in the country's stance towards the Appellate Body despite the public support for multilateralism expressed by President Biden Howse, .ex ante influence tactics. In this more recent instance, however, it is clear that the US under the Trump administration changed tactics and applied serious punitive measures against the Appellate Body and the WTO as a whole, with effects that were very similar to the ex post control tactic of re‐contracting. Those tactics went beyond the rhetorical and legitimacy politics employed previously by the US and represented the use of opportunity structures within the design of the international institution.In sum, in previous instances of contestation of the WTO by the US, there was a clear degree of discontent exhibited by the US, albeit this was expressed through rhetorical and, in the more extreme cases, vis‐à‐vis international courts that are deemed as harming their interests. This is reflected in a statement by the former chairman of the Appellate Body, James Bacchus, saying that there is very ‘little chance of resolving this [crisis at the Appellate Body] while Donald Trump is still president in a way that will continue to preserve the independence and impartiality of the Appellate Body and the rest of the WTO dispute settlement system’ (Josephs, As the case study illustrates, powerful states with anti‐multilateralist agendas possessing the necessary political clout and means to engage in substantial and direct forms of contestation and institutional control, can and will take such direction Josephs, .vis‐à‐vis the Body is yet to be seen, however the analysis highlights the fact that such change would be conditional on the domestic context within the US.The administration's policies reflected the relevance of the shifting domestic context within the US, and how resistance to international institutions by societal and special interests’ actors within the US was manifested through the case of the demise of the Body. Whether the new administration will change tactics 4ex ante mechanisms to influence the appointment of individual judges at the court. However, the Trump administration employed the same mechanisms to deny the appointment of all the judges at the Appellate Body, therefore rendering the entire court inoperable and resulting in its effective shutdown. This tactic represented the use of existing opportunity structures within the design of the institution with effects that were very similar to the ex post control tactic of re‐contracting.This article has focused on the Trump administration's successful efforts at rendering the WTO Appellate Body inoperable. The findings showcased how powerful states are capable and willing to take advantage of the opportunity structures inherent to the design of international judicial institutions to control them. During the administrations of George W. Bush and Barack Obama, the US expressed its discontent with the Appellate Body through rhetorical attacks and by employing The conclusion reached here is that describing international judicial institutions as being generally shielded from the control of states due to the nature of their delegation contract is not a productive approach for examining the influence of states on such institutions. With a protectionist and anti‐multilateralist administration such as that of President Trump, an international body with judicial authority such as the Appellate Body may receive the same treatment as an ordinary international institution when deemed to be violating the rules of the game. While powerful states may resort to rhetorical attacks and playing the legitimacy politics at first instance, in the long run the sort of tactics available to them for controlling international institutions may become the weapons of choice against international courts as well.The consequences of the previous US administration's policies have already become evident. The current dysfunctionality of the Appellate Body is very costly for the WTO, arguably putting at risk the future of its multilateral institutional framework for governing global trade (Hoekman & Mavroidis, The case explored here highlights the enormous pressures that IOs are facing by deglobalizing actors such as the US and the effects of resistance and backlash against their institutional frameworks. This is the case even for IOs with central positions and roles within the international system. Powerful international courts are not immortal and shielded from state control. Protectionist, anti‐multilateralist states are a threat to the global liberal order and its institutions, and they are willing to take advantage of opportunity structures embedded within the institutional design of such organizations to exert their influence and control.In that regard, institutional reforms at the WTO are critical to the survival of multilateralism and the liberal international trade system Howse, . PoliticThe findings of this article are therefore relevant to the broader IR research agenda on the modes and tactics of contestation of international institutions, as well as the discussion regarding the current challenges to the liberal international order and resistance to international judicial institutions. The IR literature on IOs may benefit from more research on the process of contestation of such institutions, and more focus on the institutional responses of IOs to these challenges could help uncover the role played by institutional actors in ensuring the adaptiveness and resilience of their institutions.


Coronavirus disease 2019 (COVID-19) is the most devastating pandemic of the century, which is still far from over. The remarkable success of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines is the working hope, but the evolving variants are the huge concern that can turn the tide. Potential immune escape mutations (PIEMs) in the past and circulating variants were not studied at large scale . Hence, the conservation of antigenic determinants (epitopes) was analyzed in all available sequences of SARS-CoV-2 according to time (months), proteins, hosts, and variants. Numerous highly conserved B- and T-cell epitopes were identified in 24 proteins of SARS-CoV-2. A decrease in the conservation of epitopes with time was observed in almost all proteins, which was more rapid in neutralizing epitopes. Delta variant still has the highest PIEM in the circulating strains, which pose threat to the effectiveness of current vaccines. The inclusion of identified, highly conserved, and important epitopes in subunit vaccines can increase vaccine effectiveness against evolving variants. Trends in the conservation of epitopes in different proteins, hosts, and variants with time may also help to inspire the counter measure against the current pandemic. The origin and initial cases of coronavirus disease 2019 (COVID-19) were reported from Wuhan, China at the end of the year 2019, which was due to a novel coronavirus later named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The COVID-19 was declared a pandemic on March 11, 2020 by the World Health Organization. The current pandemic has already claimed more than 4.9 million lives from more than 244 million infections globally . The emeFortunately, a large number of epitopes were discovered from the SARS-CoV-2 at a short period of time, which can be utilized for conservation and other studies. Similarly, huge genome sequencing efforts around the world have provided a rich resource of genomic sequences. These resources can be used to calculate the conservation of the different genomic regions, especially the regions that encode epitopes. Earlier conservation analysis in SARS-CoV-2 was conducted on small datasets and not focused on immunity or majorly based on predicted epitopes . Known Bin vivo studies . Therefore, neutralizing epitopes can be considered as more important for inducing protective immune responses. Additionally, epitopes from the region that interact with host protein [angiotensin-converting enzyme-2 (ACE2)] to gain entrance into the host cells were also identified and studied for conservation. The co-crystal structure of spike and human ACE2 protein complex (PDB ID: 6M0J) was used to identify all interacting residue in the spike protein with the help of PDBsum  along with strain IDs and additional information corresponding to all available SARS-CoV-2 genomes from the Global Initiative on Sharing Avian Influenza Data (GISAID)  were extf PDBsum . BCE froEquation 1. Similarly, the monthly conservation of each epitope was calculated every month (from December 2019 to September 2021) according to Equation 2. Variants-wise conservation of epitopes was calculated for all the important variants  listed in GISAID database (Equation 3). Conservation of epitopes was also calculated according to the host in which the sample was isolated (Equation 4).The conservation of each epitope was calculated in the corresponding protein according to the number of strains, variants, months, and hosts. In-house developed PERL scripts were used to organize the data and calculate conservation, and results were saved in CSV files for further analysis. Strain-wise conservation of each epitope was calculated as the ratio of the number of strains in which the epitope was conserved (NSEC) with a number of total strains used in the study [strains in which the epitope was found to be mutated (NSEM) + NSEC] according to M represents the month of sampling (beginning from December 2019).where superscript V represents the variants used in the study.where superscript H represents the hosts from sample were used for sequencing.where superscript Equation 5. In-house developed Perl script was used to organize data and calculate monthly conservation of epitope for every epitope in the corresponding protein. The regression analysis, plot generation, and other statistical analysis were carried out with R using linear regression and plot functions.To study the trends of conservation/mutation of all epitopes with time, the linear regression analysis with a best fit line on month-wise conservation of epitopes was carried out separately for B- and T-cell epitopes in each protein. The linear regression model is depicted in matrix form in Equation 5: linear regression model in matrix form asiα, iβ are arbitrary constants (intersect and slope) for i = 1,2,…, n; n is total number of epitope in the particular protein.where The mutation in the epitope from the spike protein may be the potential immune escape mutation (PIEM), as a mutation in the epitope from (spike protein) most important/only vaccine antigen (in most of the vaccines) may result in reducing immune responses. The variants  with at least 10,000 available genome sequences were studied to calculate the number and percentage of B-cell epitopes with mutation in the spike protein as PIEM. PIEM is calculated every month from the month of origin of the respective variant.To identify common BCE and TCE epitopes with neutralizing response, set analysis was carried out using the list of B-cell, T-cell, and neutralizing epitopes. Common epitopes known to be conserved in 99% strains were selected as common conserved epitopes with neutralizing immune response (CCN) and suggested as subunit vaccine candidates for vaccine design. InteractiVenn was used to perform Venn analysis on sets comprised of BCE, TCE, and neutralizing epitopes .Conservation of each epitope was calculated according to the total number and percentage in all strains, variants, host, and month of sampling. The protein-wise conservation of all epitopes is provided in the excel tables X47. TotThe conservation of both BCE and TCE is found to be decreasing with time according to monthly conservation data of epitopes. Conservation of both BCE and TCE from the spike and N protein is found to be decreased more rapidly among all studied proteins (with more than 50 epitopes) of SARS-CoV-2 according to linear regression S25. TheThe number and percent of PIEM were calculated on important SARS-CoV-2 variants every month until September 2021, since the first reporting month of the variants. In all the variants, the number of escape mutations was found to increase with time to achieve the peak value, and then, a decreasing trend was observed  except for the Delta variant Figure 3A total of 187 epitopes were found to be common in both B- and T-cell epitope lists, i.e., these epitopes were known to provide both B- and T-cell immune responses in the literature Table 2.Vaccination is the main arsenal against the COVID-19, which seems to be highly effective, especially preventing severe disease almost throughout the globe , 26. Howin vivo experiments in animals or humans  in the viruses may be a crucial factor for the reduction in protective immunity conferred through vaccines. The mismatch between vaccine antigen and circulating strain of the virus may result in ineffective vaccination . Consider humans Figure 2r humans . These vr humans , 25. Potr humans . Similarr humans . Furtherr humans , 34. CCNiagnosis . The enzConsidering the fact that more than 244 million cases of infection have already been reported and still there are more than 18 million active cases present worldwide  for the Delta variant in the circulating strains. Highly conserved epitopes from the spike and other proteins were identified in the study, which may be used in vaccine construct to design a more effective vaccine against SARS-CoV-2 and evolving variants.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Conceptualization, VJ and HJL. Methodology, VJ Formal analysis, VJ Investigation, VJ Writing—original draft preparation, VJ. Writing—review and editing, HJL. Visualization, VJ. Supervision, HJL. Funding acquisition, HJL. All authors have read and agreed to the published version of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the “Cooperative Research Program of the Center for Companion Animal Research (Project No. PJ01398402)” of the Rural Development Administration, Republic of Korea.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A contact pressure equivalent to that in a wheel–rail interface was achieved by using a high-pressure torsion test approach. The dynamic change of contact stiffness with slip was measured using ultrasound and the influence of roughness was investigated. The measured changes were validated using a newly developed numerical simulation, and mechanisms to explain the observed behaviour were proposed in terms of fracture and plastic deformation of the asperity bonds. These findings could help in understanding the traction characteristics for different roughness conditions and also assist in understanding damage mechanisms better, such as wear and rolling contact fatigue.Understanding the dynamic condition of the interface between a railway wheel and rail is important to reduce the risks and consider the effectiveness of countermeasures for tribological problems. Traditionally the difficulty in obtaining accurate non-destructive interfacial measurements has hindered systematic experimental investigations. Recently, an ultrasound reflectometry technique has been developed as a direct observation method of a rolling–sliding interface; however, the topography dependence under the high contact pressures in a wheel–rail contact has not been clarified. For this reason, a novel  To achieve these roles, the wheel and the rail roll/slide against each other under extremely high contact pressure conditions. Due to the severe contact condition, the interface is the origin of a number of tribological problems during operation. For example, when contaminants, such as water, oil or fallen leaves, get into the interface, the interaction can lead to wheel spin and brake lock-up. Such significant slipping can cause not only performance problems in terms of delays and safety issues from over-running , but also thermal damage and abnormal deformation of wheel and rail ,2. Also,To consider effective countermeasures for the problems mentioned above and to reduce the risks, it is important to understand the dynamic conditions in the interface. However, the difficulty in obtaining accurate non-destructive interfacial measurements has hindered systematic experimental investigations.The use of pressure-sensitive films is one potential method to evaluate the contact area and pressure ,12. HoweRecently, ultrasonic techniques have been used to observe the contact between wheel and rail –21. ThouThe authors have already investigated the influence of the topography on the friction behaviour between the wheel and rail in dry condition focusing on the mechanisms of flange climb derailments . As a rein situ evaluation of the contact condition, particularly contact stiffness. Transient loading conditions and displacement were also measured during the test. Following these measurements, and the changes of contact stiffness with contact pressure, slip distances were reproduced numerically.The aim of this work was to understand the influence of the roughness on the dynamic friction behaviour between the wheel and rail. To achieve a contact pressure equivalent to that in a wheel–rail interface, a high-pressure torsion (HPT) test approach was used. The HPT testing equipment is capable of applying horizontal relative motion (slip) to two surfaces in contact with each other, while achieving a high contact pressure. The contact area is large enough to evaluate the contact stiffness using ultrasonic waves. Tiny piezoelectric elements which generate the ultrasonic waves were bonded to one of the test specimens. Ultrasonic reflection from the interface was used to conduct . 2 (a)N and the normal stress Nσ is represented as in equation (2.1):r1 and r2, respectively, represent the outer and inner diameters of the hollow circle of the contact area. Equation (2.2) shows the relationship between the torque Tq and the tangential load T [The relationship between the normal load l load T :2.2T=TqT and the tangential stress Tσ can be represented as in equation (2.3):In addition, the relationship between the tangential load  (b)In this study, the contact stiffness was measured in three directions: normal and two tangential directions, slip direction and perpendicular to the slip direction, based on the intensity of the ultrasonic wave reflected from the contact interface. In the following, the principle of the evaluation of contact phenomena by ultrasonic reflectometry will be explained.R, can be represented as in equation (2.4), and it varies depending on the difference in the acoustic impedances of the two materials:z1 and z2 are the acoustic impedances of the materials in contact. The acoustic impedance is determined by the product of the density of the material and the acoustic velocity in the material. Therefore, when the acoustic impedances of the two materials in contact are the same and if the interface is hypothetically perfectly conformal, all the sound waves will transmit at the interface without any loss and no reflection occurs (R = 0). On the other hand, when materials with significantly different acoustic impedances, such as a gas and a solid, are in contact, sound waves are almost completely reflected (R ≈ 1).At the interface between materials with different acoustic impedances, only a part of a sound wave transmits at the interface and the rest of it is reflected back. The reflectivity for an ultrasound at an interface where the materials adhere to each other without any cavities, ω and K, respectively, represent the angular frequency of the ultrasound and the contact stiffness. For the example in this article, the equation can be simplified as follows because it is assumed that the wheel and the rail are made of the same material (z1 = z2 = z):Schoenberg  adopted K if the reflection coefficient R can be determined in an experiment. Generally, the contact stiffness is defined as the normal or tangential stress generated when the relative distance between two surfaces in contact via surface asperities changes by a unit length in each direction as shown in Nδ and Tδ are the normal and tangential stresses, respectively. It is often interpreted that Nδ is the approaching distance between the average height of the roughness asperities distributed on the two contacting surfaces, and Tδ is the relative distance in the tangential direction between the asperities on the two contacting surfaces  is evaluated, as shown in Some of the authors repeatedly measured the contact stiffness distribution in the normal direction in the contact area of the wheel–rail under rolling–sliding conditions using ultrasonic waves and reported that a linear relationship was observed between the contact stiffness in the normal direction and the friction coefficient . It was  (c)The top and the bottom specimens were made from ER8 (EN13262:2009) wheel material and R260 (EN13674-1:2011) rail material, respectively. The hardness values in HV(5) were 267 for the ER8 wheel (top) specimen and 285 for the R260 rail (bottom) specimen. The hardness values were the average for 5 measurements. The measurements were conducted using a Mitutoyo hardness testing machine HV-110 and test force was 5 kgf.The surface type of the top specimens remained constant for all tests and was achieved by grinding to get a repeatable surface finish for all tests, and the surface types of bottom specimens were varied between tests. Three different roughnesses were achieved by grinding, sandblasting and machining (milling), which will be referred to as low roughness, medium roughness and high roughness, respectively. The initial roughness profiles can be seen in §3d. For all the contact tests, specimens were soaked in 2-propanol and washed in an ultrasonic bath before the measurement. Miniature piezoelectric elements were bonded to the top specimen to generate ultrasonic waves that reflected off the contact interface and they also measure the reflected wave. The longitudinal element oscillates a wave in which the displacement of the medium coincides with the direction of motion of the wave, while the transverse element oscillates a wave in which the displacement of the medium is perpendicular to the direction of motion of the wave. The longitudinal and transverse waves reflected on the interface contain information about the contact stiffness in the normal and tangential directions, respectively.When the frictional force acts on the interface, the tangential contact stiffness may behave differently from normal contact stiffness. In particular, the contact stiffness in the tangential direction may also be different between the perpendicular  direction and the slip  direction. Therefore, it is important to evaluate the difference of contact stiffness in different directions so three directions of contact stiffness were measured in this study: normal, radial and circumferential.Off course, it is possible that the friction conditions may be slightly different at each measurement position, but this should provide useful information on whether there are obvious differences between directions. Therefore, measurements were made in all three different directions. Taking into account the direction of polarization of the piezo element, the piezoelectric elements for measurement of the transverse waves were installed in directions parallel  and perpendicular  to the friction direction. The piezoelectric element was made of lead zirconate titanate (PZT) as a base material, with thicknesses of approximately 0.4 mm for longitudinal and approximately 0.2 mm for transverse. The PZT plate was cut into rectangles with a side length of about 1–3 mm. Piezoelectric elements with a central frequency of 5 MHz were used for the measurements of both longitudinal and transverse waves. In this test, a ‘Pitch-Catch’ method was employed in which different piezoelectric elements are used for activation and reception of the ultrasound.Howard scanned the area of the ultrasonic wave emitted by the PZT which was directly attached to the test object as above, using a fine, ball-tipped probe fitted with a PZT for the receiver . The res (d)The measured reflection waveforms were bandpass filtered from 3 MHz to 7 MHz. 1.A normal stress is applied after inserting a pressure-sensitive paper between the specimens to check that the load is uniformly distributed on the contact area.2.After making the specimens directly contact with each other, the normal stress is increased gradually to approximately 600 MPa.3.While keeping the top specimen in the specified position, the tangential stress is applied. (Phase-I)4.The bottom specimen is rotated. (Phase-II)5.The tangential stress is gradually released. (Phase-III)6.After releasing the torque, the specimens are separated.During steps 2 to 6, the contact stiffnesses are measured by using ultrasound. (e)In order to verify whether changes in contact stiffness can occur in the test conditions, numerical contact simulations based on a boundary element method with the half-space approximation were conducted, and the results were compared with the experimental results. There are several methods to perform contact calculations using rough surfaces, such as assuming the asperity distribution to be an ideal distribution (e.g. Gaussian) ,42 or trThe numerical calculations were conducted using three-dimensional surface topography data measured by a non-contact surface profiler  before test cycles. As a consequence of the complexity of the top specimen geometry, the result from the bottom specimen low roughness test was used instead. Both were ground finishes and were judged to have similar roughness profile, as shown in §3d.t) ⊛ h(t) = y(t), then X(f)H(f) = Y(f)) [The boundary element formulation for a normal contact converts the problem of finding displacement at the surface points given a set of pressures on each of the surface points, to a convolution between the surface pressures and a ‘short-form’ influence matrix which depends on the material properties of the surface and the discretization grid. Alternatively, this process can be thought of as a matrix multiplication between a vector of pressures and a `long-form’ influence matrix. In practice, the convolution method is almost always used as this allows the process to be accelerated by completing the convolution in Fourier space (if x( = Y(f)) .For contact between two surfaces, these influence matrices can be summed to give the total deformation of the pair of surfaces due to a set of mutual loads. In general, for a rough surface contact the inverse problem is solved, finding the loads required to produce deformation in the surfaces so that the surfaces do not penetrate each other. The domain of this solution is confined to areas of the surface where this load is positive. This can be solved by a suitable conjugate gradient method.In this work, the domain of the solution is further confined to areas where the normal surface pressure is less than a limiting pressure, based on the material hardness as given by . Areas wL1, the displacement vector caused by these pressures is D1 and the influence matrix is M. The sum of these pressures is the contact stiffness as given by [iG is the gap vector, with superscripts 0 and Δl meaning the original gap and the gap after the application of a small load Δl, iD is the displacement vector caused by this small load and Δd is the approach of distant points in the solids caused by this small load. The overbar signifies the mean value of a vector. As the system is linear, the following can be written:When the normal contact problem has been solved, the contact stiffness must be found. At any point, the response of the system to small perturbations of load can be thought of as linear providing the perturbation is not large enough to change the domain of the solution (the points of the discrete surface profile which are in contact). In practice, we can set the domain of the solution, remove the minimum and maximum load constraints and find the pressures on the currently contacting nodes required to produce a unit deformation of those nodes. In the equation below these pressures are given by . Howevern result ; howeveriD1 = 1: ∀i and this gap stiffness tends to infinity as expected. This method can also be used to calculate the contact stiffness in either the loading or unloading directions; by excluding nodes which are at the maximum pressure the loading stiffness will be found.Clearly, when the entire surface is in contact The normal contact equations were solved for a 1024 by 1024 grid, using the experimentally measured surface profiles shown above. The code used to solve the models has been added to ‘Slippy’ (v. 0.1.4), an open-source contact modelling package. The code used to generate these models is provided in the additional material.. 3 (a)a shows a sharp increase in contact stiffness of the circumferential direction between 300 and 400 MPa in the low roughness condition. Although physical verification is not possible at this moment, a local variation in contact pressure (e.g. sudden increases in contact pressure where it was not previously high) may improve the conformity between the two bodies rapidly. When considering a Hertzian contact, it is known that a smooth surface has a higher maximum pressure and a smaller contact area than a rough surface [Figure 10 surface ,50. Cons (b)T/N), normal stress and rotation position with the time, obtained from steps 3 and 4 of the test procedure. Here, the positive pressure value is defined as the pressure in the compression direction for normal stress. In Phase-I, there was little or no sliding while T/N increased. For this period, the specimens were assumed to be in a static or a quasi-static contact condition except for the possibility of the rig being slightly twisted, and the contact stiffnesses remained at almost constant level or slightly increased. In Phase-II, the rotation position rapidly increased. At the same time, the contact stiffness rapidly decreased at the onset of slip, then slightly increased after that in the case of low and medium roughness. On the other hand, in the case of the highest roughness, the contact stiffness no longer showed the dip and the rotation curve rises gently with T/N. In Phase-III, though the rotation stopped and T/N decreased as the tangential stress was released, the contact stiffness kept a constant level or slightly increased. At the same time, a slight restoration of the rotational position was found. It is considered that a spring-back (relaxation) at the contact interface occurred.T/N decreased to zero when the tangential stress was released after rotation, T/N showed a negative value before Phase-I. It is considered that a slight misalignment in the horizontal direction between the specimen and the testing equipment generated tiny slip even when only normal stress was applied.In all cases of roughness, though the  (c)et al. and Dwyer-Joyce et al. conducted loading–unloading cycles measuring contact stiffness and reported hysteresis trends [figure 13a shows a sharp decrease in contact stiffness of normal direction around 300 MPa in the low roughness condition, this may have been also due to a local variation in contact pressure as in figure 10a.s trends ,38, and  (d)Comparing the roughness profiles, it was found that the shape of the summits of the asperities has been rounded and flattened after the test in the cases of low and medium roughness. The roughness profiles of the top specimen in the case of high roughness show large deformations after the test. It is considered that the asperities of the bottom specimen were stuck and transcribed to the top specimen. (e)The size of this patch greatly influences the result of the calculation; larger patches include more of the edge and give a higher contact stiffness. This is most pronounced for smoother surfaces. Because of this, results from two different sized areas are shown: 2.5 mm square and 1.25 mm square. These sizes represent the projected size of the sensor if the emitter is considered as an area, respectively. Results shown are averaged from 32 measurement regions each offset in the periodic grid direction by 32 grid points (0.16 mm) to account for the random variation in the surfaces. The figure also shows the range of maximum and minimum values of the 32 numerical values as the degree of fluctuation.The contact stiffness tends to be higher for a 2.5 mm square than for a 1.25 mm square. As mentioned above, this is because the greater area is more affected by the locally higher contact stiffness at the edge. In the following sections, the experimental and numerical results of the individual steps will be compared.. 4 (a)W, was calculated by the following equation:L is the accumulation distance and calculated by the following equation:l is the rotation distance, t0 is the time when the tangential stress was applied and t1 is the time when the tangential stress was released.T/N. It should be noted that the contact stiffness was normalized as the initial value by the contact stiffness before the application of tangential stress. It is found that the normalized contact stiffness basically increased with the work for all roughnesses. However, it dropped around 2–5 J in the cases of low and medium roughness before rising again. This period is relevant to the beginning of Phase-II, the dynamic friction. On the other hand, there was no drop in the case of high roughness. Pesaresi et al. [i et al.  also repT/N decreases. This may be due to the accumulation of work energy caused by the slight spring-back and residual tangential stress even in the static friction condition. Fantetti et al. [It should be noted that for all roughness conditions the contact stiffness continues to increase while the apparent increase in slip distance ends and the i et al.  also repi et al.  and ageii et al. , which c (b)The comparison between the experimental and modelled results shows relatively good agreement during the loading step of the experiment. During the sliding step, the deformation and wear of the surface are not captured well by the simple wear model applied, and consequently the modelled results from this and the subsequent unloading steps are generally poor. The range of values which can be produced by the model is relatively large, in the worst case approximately 50% of the maximum value. This high variation is a result of the application of different assumptions of how the ultrasound transducer should be treated and natural variation associated with the random nature of surface geometry. Agreement between the model and the experiment was best for the highest roughness case. In the model, this case also showed the lowest random variation between sampled areas and the least dependence on the size of the area used for the contact stiffness measurement. Further inspection of the results from the highest roughness model showed less pronounced edge effects than seen with either of the lower roughnesses. This is in line with the prediction made in §3a that the larger the roughness, the more gradual the local pressure distribution, as shown in previous literature ,50.The variation between different sampled areas of the same size is a natural result of the semi-random geometry of surfaces, while the variation between different size domains is a result of edge effects which are present both in the model and the real world. The difference between different size areas is large, and unfortunately, there is currently no consensus on what the correct area is. As the ultrasound wave is not uniform on incursion with the contact surface, it is additionally possible that there is no simple size of area which can be used. (c)et al. [et al.  have conc. The slip distance for the high roughness condition was about 0.5 mm (c), which is smaller than the spacing of the asperities. As the asperity pitch is sufficiently long in relation to the slip distance, plastic deformation may continue throughout the entire slip process. Therefore, asperity breakage (wear) and the formation of new contacts, as in the low/medium roughness condition, would be less likely to occur and the increase in contact stiffness could be reduced.On the other hand, in the case of high roughness, macroscale effects (i.e. each large machined peak) dominate as opposed to individual asperities . The summits of asperities also conformed, but the penetration of asperities is greater than the smoother interface because of the higher local pressure . With thfigure 14t 0.5 mm c, which  (d)—Tangential stress is applied.—The asperity deforms until it can withstand the tangential stress and the contact stiffness increases (interface becomes stiffer).—The tangential stress is increased and the contact stiffness is increased again.In other words, this is the initial stage of the running-in process, where the interface becomes harder and more resistant to tangential displacement as the conformity of the interface increases ,27. The  (e)Throughout all the results, there was little difference in the contact stiffness between the directions, normal, radial and circumferential. This can be seen from the fact that there is no difference between the radial and circumferential directions in the roughness profile after the test in the additional data (Dryad). In particular, figures . 5in situ evaluation of the contact condition was carried out. From the results of evaluation, the following conclusions can be drawn:(1)in situ evaluation of the friction interface under extremely high contact pressure and gave the information about continuous change of interfacial topographies and contact stiffnesses with friction. It is thought that this technique can be applied not only to the wheel–rail interface, but also to general high contact pressure interfaces.The application of the ultrasound reflectometry to the HPT test approach enabled (2)The effect of roughness on the change in contact stiffness during the transition from static and quasi-static friction modes to dynamic friction mode could be evaluated. In the low roughness condition, a temporary decrease and subsequent increase in contact stiffness was observed when macroscopic slips occurred, suggesting a breakdown of the interface and the formation of new asperity junctions. In the high roughness conditions, the effect of plastic deformation of the macroscopic roughness asperities was pronounced. The contact stiffness gradually increased with tangential stress loading.(3)Numerical calculations using real surface topography could reproduce the change in contact stiffness under a series of test conditions: loading, slip and unloading. Particularly for the high roughness condition, the experimental and numerical results were in good agreement and the fluctuation due to the different calculation domains was small.(4)There was a positive relationship between the slip distance and the contact stiffnesses. It was considered to indicate that the wear and plastic deformation of the asperities progressed and conformed along with the increase of the slip distance. The dependency of the direction on the contact stiffness was not significant and they were considered to change almost uniformly.Aiming to understand the influence of the roughness on the dynamic friction behaviour between the wheel and rail, ultrasonic reflectometry was applied to the HPT test approach, and


C. elegans using RNAi specifically from the L4 stage and through generation of endogenously tagged 19S RP lid subunit strains. We show functions for the 19S RP in regulation of proliferation and maintenance of integrity of mitotic zone nuclei, in polymerization of the synaptonemal complex (SC) onto meiotic chromosomes and in the timing of SC subunit redistribution to the short arm of the bivalent, and in turnover of XND-1 proteins at late pachytene. Furthermore, we report that certain 19S RP subunits are required for proper germ line localization of WEE-1.3, a major meiotic kinase. Additionally, endogenous fluorescent labeling revealed that the two isoforms of the essential 19S RP proteasome subunit RPN-6.1 are expressed in a tissue-specific manner in the hermaphrodite. Also, we demonstrate that the 19S RP subunits RPN-6.1 and RPN-7 are crucial for the nuclear localization of the lid subunits RPN-8 and RPN-9 in oocytes, further supporting the ability to utilize the C. elegans germ line as a model to study proteasome assembly real-time. Collectively, our data support the premise that certain 19S RP proteasome subunits are playing tissue-specific roles, especially in the germ line. We propose C. elegans as a versatile multicellular model to study the diverse proteolytic and non-proteolytic roles that proteasome subunits play in vivo.The 26S proteasome is a multi-subunit protein complex that is canonically known for its ability to degrade proteins in cells and maintain protein homeostasis. Non-canonical or non-proteolytic roles of proteasomal subunits exist but remain less well studied. We provide characterization of germline-specific functions of different 19S proteasome regulatory particle (RP) subunits in  The 26S proteasome is a ∼2.5 MDa multi-subunit protein complex that maintains cellular homeostasis by degrading old, misfolded, mistranslated, and/or regulatory proteins in cells in both the cytoplasm and the nucleus . Recent via cryo-electron microscopy and atomic modeling has revealed the structure of the eukaryotic proteasome at atomic level  and the resulting edited strains backcrossed with wild type (N2) at least five times and sequenced before being utilized.Strains in this study were generated using CRISPR/Cas9 genome editing technology following the direct delivery method developed by ed edits . SpecifiGFP tags were generated by inserting Superfolder GFP sequence at the N-terminus immediately after the start ATG. Repair templates for the GFP strains were generated by PCR amplifying Superfolder GFP from pDONR221. All the strains generated in this study can be found in rpn-6.1. An ssODN was used as the repair template and contained a minimum of 35 bp homology arms to the genomic region 5′ of the insertion site, the 42 bp OLLAS sequence, and then a minimum of 35 bp homology arms to the genomic region 3′ of the insertion site  in ion site . Approprvia RNAi feeding as previously described  or Open Biosystems ORF-RNAi library  . RNAi clones for rpn-8 and rpn-5 were generated in the lab (see below for details). All RNAi clones were freshly transformed into E. coli strain HT115 cells before usage. Either the L4440 empty vector or smd-1 (RNAi) were used as a control RNAi condition for all RNAi treatments. smd-1 (RNAi) was utilized because it activates the RNAi response yet has no reported reproductive phenotype in a wild-type genetic background. RNAi co-depletions were performed by measuring the optical density at 600 nm wavelength of the RNAi overnight culture for each construct and then mixing the cultures in 1:1 ratio. We performed RNAi knockdown of the genes of interest by feeding the worms for a total of either 24 h at 24°C starting from L4 stage . The following primers were used: for rpn-5, forward oAKA277 5′-aatggctatcgcaaagatgg-3′ and oAKA278 reverse 5′-gtcagtttgtgcacgttgct-3’; and for rpn-8, forward oAKA392 5′-gcgtttctcactgttatgtcg -3′ and reverse oAKA393 5′-ccatgtcgaggaaccatgta-3’. In brief, the vector was linearized with EcoRV, gel-extracted (Bioline Cat. No. BIO-52059), T-tailed, desalted with a DNA Clean Concentrator kit (Zymo Research Cat. No. D4004), and then ligated with either of the previously mentioned PCR product using Quick-Stick ligase (Bioline Cat. No. BIO-27027). Newly generated RNAi clones were transformed into HT115 cells and sequenced using the M13 forward universal primer to confirm successful cloning (Eurofins Genomics).RNAi feeding clones for T-test to find significant differences between the average 24-h brood of control and experimental RNAi conditions. Standard error of the mean (SEM) was calculated by dividing the standard deviation by the square root of the sample size.24-h total brood assays on RNAi-treated worms were performed using the previously published protocol with a minimum of three independent trials . StatistAll fluorescent strains were treated with appropriate RNAi condition at 24°C for 24 h before imaging. 10 µl of anesthetic (0.1% tricane and 0.01% tetramisole in 1X M9 buffer) was added to a 3% agar pad on a slide and 10–15 live worms were transferred to the drop of anesthetic. A glass coverslip was slowly lowered to cover the samples and the coverslip edges were sealed with nail polish and allowed to dry before imaging. Images were obtained on a Nikon Ti-E-PFS inverted spinning-disk confocal microscope using a 60X 1.4NA Plan Apo Lambda objective. The microscope consists of a Yokowaga CSU-X1 spinning disk unit, a self-contained 4-line laser module , and an Andor iXon 897 EMCDD camera. Fluorescence intensities were quantified and image editing done using NIS-elements software.The tube staining method was performed on dissected gonads fixed in 3% paraformaldehyde and methanol . The samrrf-1 or ppw-1 worms exposed to 48 h RNAi by feeding, were dissected in 1X Sperm Salt Buffer , followed by permeabilization with 2% Triton and then fixed in the same buffer containing 2% paraformaldehyde for 5 min. Slides were placed on a frosted metal plate on dry ice before removing the coverslip and then placed in 4°C absolute ethanol for 1 min. Slides were then washed three times for 10 min each in PBST  plus 0.1% BSA and incubated overnight at 4°C with the primary antibodies diluted in PBST. Following three washes of 10 min each in PBST plus 0.1% BSA, slides were incubated in the dark at room temperature for 2 h with secondary antibodies diluted in PBST. Following three 10 min washes with PBST, slides were counterstained with DAPI in the second wash and mounted using Prolong Gold antifade reagent with DAPI (Invitrogen). The primary antibodies used in this study are: Chicken anti-SYP-1  .For the study of synapsis, germ lines from N2, z-Perez) ; Guinea z-Perez) ; Rabbit z-Perez) . XND-1, C. elegans hermaphrodite fertility upon downregulation of individual 19S RP lid and base subunits in comparison to global proteolytic inhibition via the chemical proteasome inhibitor bortezomib. As observed previously, general inhibition of the proteasome with bortezomib resulted in animals with severely reduced fertility, less then 10 progeny in a 24 h period . Whereas the majority of 19S base subunit-knockdown animals had fewer than six offspring (<0.4% of control), rpt-6(RNAi) and rpn-13(RNAi) animals produced substantial numbers of eggs  and rpn-9(RNAi), the hatched embryos developed into larvae but exhibited severe developmental defects, such as L1-L2 developmental arrest and a protruding vulva phenotype, respectively (data not shown). This data, combined with previously published data, suggests that while most of the lid and base subunits of 19S RP of the 26S proteasome play essential roles during C. elegans hermaphrodite reproduction, individual 19S RP subunits may play differential roles in this process.In the RNAi depletion studies, we cannot distinguish whether the embryonic lethality results from maternal deficits in oocyte development, from loss of proteasome function in the developing embryo due to persistence of the dsRNA, or both. Since many of the phenotypes we see in the germ line require prolonged RNAi exposure (>24 h) to be manifest (see below), at least some of the phenotypes likely reflect embryonic requirements, consistent with the zygotic requirement for proteasome function . We noteIn vivo fluorescent reporter systems have been developed to qualitatively assess the proteolytic activity of the 26S proteasome in whole animals and in specific tissues under various conditions (pie-1pro::Ub(G76V)::GFP::H2B::drp-1 3′UTR)  . GFP can1 3′UTR) . If the 1 3′UTR) .rpn-10, rpn-13, dss-1/rpn-15, and rpt-6 resulted in bright, nuclear, germline fluorescence of the Ub(G76V)::GFP reporter compared to control RNAi-treated germ lines  allowed us to ascertain that subunit expression can be effectively inhibited even for those subunits where we observe little to no phenotypic changes , and provide the pool of cells that enter meiosis as they move proximally  and the base subunits rpn-1 and rpn-2, we observed increased numbers of metaphase- or anaphase-like cells  resulted in an extended region of SYP-1 PCs  or the base subunit rpt-6 had no obvious effect on SC assembly or on PC size, number, or persistence  indicate that these phenotypes are not always associated and suggests that multiple regulatory steps feeding into synapsis may be regulated by the proteasome. However, to address whether the defects we see in cell cycle and SC formation are also due to aberrant accumulation of HTP-3, we used immunohistochemistry to determine whether HTP-3 localization is affected by knockdown of the 19S subunits. We previously observed that a subset of HTP-3 can be found in SC polycomplexes upon knockdown of the 20S proteasome  and are retained and enriched on the short arm . The remDespite the congruence between the RNAi studies on distal SC behaviors, the inhibition of the proteasome with bortezomib did not affect SC redistribution in late pachytene. Bortezomib affects the catalytic activity of the proteasome whereas RNAi depletion of a subunit would be expected to disrupt proteasome complex formation. The lack of phenotype with bortezomib may suggest that SC polarization is affected by a non-catalytic role of the proteasome. Alternatively, it may reflect an aspect of timing: the premature polarization is seen after 48 h of RNAi exposure whereas bortezomib kills the animals after only 18 h; the affected nuclei may not move far enough proximally to see the SC polarization phenotype before the animals die from prolonged exposure to the drug.C. elegans strains that are defective in RNAi in different tissues: ppw-1 in germ line tissues; rrf-1 in somatic tissues  on rrf-1, but not ppw-1, led to embryonic lethality (data not shown). These results strongly support the interpretation that specific proteasome targets regulate cell cycle and SC morphogenesis.Since bortezomib ultimately kills the animals, it is clear that proteasome inhibition can have profound effects on organismal health. We wanted to confirm that the phenotypes we observe with RNAi inhibition of the 19S subunits were not simply a consequence of a general decline in proteostasis that is making the animal sick. This is particularly critical for the early SC phenotype which could be considered analogous to age-related aggregation as seen in Alzheimer’s and related disorders. To test this hypothesis, we took advantage of two  tissues . These mf the SC . We alsoC. elegans  profound changes in oocyte transcription and chromatin are occurring to prepare the oocyte for embryonic development and 2) a subset of nuclei is culled by apoptosis. Whether the proteasome plays a pivotal role(s) in promoting these transitions deserves further investigation.In addition to the effects previously described for proteasome inhibition in the meiotic region of the germ line, we also observed that the proteasome is required for the proper down-regulation of XND-1 (X non-disjunction factor 1) protein in late pachytene . XND-1 i elegans . In wildachytene . At thatachytene . The mecoplasmic . Thus, wC. elegans oocytes, like oocytes of most sexually reproducing organisms, undergo meiotic arrest (C. elegans hermaphrodites is maintained by an inhibitory kinase WEE-1.3 phosphorylating the CDK-1 component of maturation promoting factor (MPF) and thus inactivating MPF (C. elegans causes precocious oocyte maturation resulting in infertility (wee-1.3 (RNAi) induced infertility.c arrest . Oocyte ting MPF . Depletiertility . A largeertility . Five ofertility . Therefowee-1.3 (RNAi) are infertile, averaging less than one egg per adult hermaphrodite in a 24 h period (cdk-1 (RNAi) suppresses wee-1.3 (RNAi) infertility and therefore serves as a positive control in these studies (Hermaphrodites fed h period . In the  studies  (Burrows studies . Signifi studies . Depleti studies .gfp::wee-1.3(ana2)] caused aberrant nuclear accumulation of WEE-1.3 (wee-1.3 (RNAi) sterility, RPN-10, RPN-13, DSS-1/RPN-15 and RPT-6, also showed no change in GFP::WEE-1.3 localization (rpn-10 (ana7), a genetic null, results in nuclear accumulation of GFP::WEE-1.3 in oocytes, it is possible that our RNAi depletions of RPN-13, DSS-1 or RPT-6 did not give sufficient knockdown to elicit an alteration in perinuclear WEE-1.3 localization (wee-1.3 (RNAi) infertility nor induced nuclear accumulation of WEE-1.3 ]}. Since an N-terminal fusion of RPN-6.1B would impact expression of RPN-6.1A, we instead attempted to infer its expression from an endogenous GFP tag to the C-terminus of RPN-6.1, which would simultaneously tag both RPN-6.1 isoforms (rpn-6.1[ana12(rpn-6.1::ollas)]}. Lifespan and lifetime brood assays of the gfp::rpn-6.1a and rpn-6.1::ollas strains demonstrated that the N-terminal tag had no effect compared to wild-type control animals, while the C-terminal OLLAS tag results in a slightly reduced lifetime average brood and lifespan compared to wild-type control (data not shown).While the 19S RP subunits  all exist as a single protein isoform, the RPN-6.1 subunit has two protein isoforms, A and B, that differ by an extension of the N-terminus in RPN-6.1A  . Additionally, neither gfp::rpn-6.1a nor rpn-6.1::ollas animals exhibit expression in the pharynx, unlike other tagged proteasomal subunits, for example gfp::rpn-9  or hatching rates  (rpn-9 (RNAi) but not rpn-12 (RNAi) exhibited mitotic zone defects which could explain the brood size defects in the former. Alternatively, there may be regional or cell type-specific differences in the RNAi efficiency for these subunits or different sensitivities of these phenotypic readouts to proteasome impairment. A final possibility, relating specifically to rpn-12, is the previously proposed idea that rpn-10 and rpn-12 are redundant and can compensate for one another during oocyte development  . One poselopment .dss-1, rpn-13, rpn-10, and rpt-6 had mild to no effect on many of the processes examined. While these results may indicate that the RNAi is inefficient at knocking down these subunits, we note that all four knockdowns did have a mild effect on brood size, producing 25%–80% of the number of eggs as wild type, strongly suggesting the RNAi is working. Additionally, although 99% of the embryos hatched upon knockdown of RPN-13, most larvae presented a ruptured vulva phenotype (data not shown). These data strongly suggest that RNAi depletion of these subunits is functional. One possible model for the lack of strong phenotype is that other proteostasis mechanisms may be upregulated when these subunits are inactivated, thereby supporting development and fertility with a partially compromised proteasome. Prior studies have revealed such cross-pathway feedback mechanisms, but whether all tissues respond similarly is not clear -induced sterility, whereas many of the lid subunits did suppress. These data could be explained if the lid has independent, non-proteasomal functions or that it combines with other proteins to make an alternative regulatory particle. In favor of the former model, we previously showed that proteasome inhibition by bortezomib failed to suppress wee-1.3 (RNAi) infertility suggesting that the misregulation of protein turnover is not driving the oocyte maturation defect of wee-1.3 depletion (wee-1.3 (RNAi) infertility occurs is still unknown but future studies may offer new insights into the regulation of this highly conserved WEE-1.3/Myt1 cell cycle kinase.Our studies also point to differences between the behavior of the 19S lid and base. With exception of epletion . The mecC. elegans, RPT-6 interacts with the transcription factor ELT-2 to regulate expression of immune response genes and this role is independent of the proteolytic activity of the proteasome  is expressed throughout the hermaphrodite female germ line but is distinctly absent from both sperm and the pharynx. Since downregulation of RPN-6.1 causes severe dysfunction of the proteolytic activity of the proteasome, we speculate that there is likely to be other RPN-6.1 variant(s) that functions in the pharynx and sperm (Endogenous GFP tagging of a number of the 19S proteasomal subunits indicated strong expression throughout the germ line of nd sperm . Indeed,nd sperm . One criWe currently do not know the mechanism of how either proteasome dysfunction or proteasome complex instability results in many of these varied germline defects. Mining of recent data of ubiquitinated proteins during aging  providesC. elegans proteasome subunits in this study revealed differential roles being played by specific subunits and provides crucial information to fill the knowledge gaps in our understanding of the 26S proteasome and its many functions. Due to the lethality of proteasome knock-down, we relied on RNAi for most of these studies. Since the effectiveness of RNAi can vary gene-to-gene, we cannot rule out that the lack of phenotypes observed with some of the subunit RNAi knockdowns resulted from an inability to fully deplete protein function. Other methods such as auxin-inducible conditional knockdown of the proteasome subunits cannot be used because it relies on the proper function of the proteasome to degrade targeted protein of interest. In the future the application of tissue-specific loss-of-function alleles and/or rescue studies may allow us to overcome this limitation of our study. Generation of the endogenously fluorescently tagged 19S RP subunits reported here, and future tagged subunits, will serve as valuable resources for future proteasome studies. Our current findings in the multicellular model C. elegans and the future ones that stem from this research have tremendous potential to transform the proteasome field and can be translated into better understanding human proteasome function.The spatiotemporal and depletion analyses of the


The second best conduit for coronary artery bypass grafting is uncertain. Theobjective of this study is to determine the second best conduit according tograft patency results from randomized controlled trials using a networkmeta-analysis.A systematic literature search was conducted for randomized controlled trialscomparing the angiographic patency rate of the no-touch saphenous vein(NT-SV), the radial artery (RA), the right internal thoracic artery (RITA),and the gastroepiploic artery (GEA) in reference to the conventionallyharvested saphenous vein (CON-SV). The primary outcome was graft occlusion,and the secondary outcome was all-cause mortality.A total of 859 studies were retrieved, of which 18 were included. A total of6,543 patients and 8,272 grafts were analyzed. The weighted meanangiographic follow-up time was 3.5 years. Compared with CON-SV, RAand NT-SV  demonstrated lower graft occlusion.NT-SV and RA were ranked as the best conduits . There was nosignificant difference in late mortality between different conduittypes.RA and NT-SV are associated with significantly lower graft occlusion ratesand are comparably ranked as the best conduit for patency. A comprehensive networkmeta-analysis (NMA) of graft patency in randomized controlled trials (RCTs) waspreviously completed by our group4][. In the 2005 trial by Gaudino etal.15][. Tian et al.3][. Deb et al.13][. The NT-SV received aClass IIa recommendation in the 2018 European Revascularizationguidelines23][ and was a Best Practice in the 2021 American College ofCardiology/American Heart Association revascularization guidelines22][.Several large RCTs support the long-term patency of RA over CON-SV22][. These results are further substantiated in theRADIAL 10-year extension study’s post-hoc analysis for survival24][. The factorial trial by Angelini et al.involving CON-SV vs. NT-SV and low- vs.high-pressure graft dilation reported that low-pressure distention of CON-SV canachieve wall thickening comparable to NT-SV8][.Limitations of this meta-analysis included a small sample size causing certainpairwise analyses to be underpowered, varying quality of the RCTs included, andno data collected on renal disease, secondary prevention, and antispasmodictherapy, which are additional factors that influence graft patency. It isworthwhile to note that the included studies involving NT-SV grafts used pedicleharvest technique withIn this NMA of 18 angiographic RCTs, the current randomized evidence showssignificantly better patency rates for RA and NT-SV compared with CON-SV, while allconduits were associated with similar rates of late mortality compared with CON-SV.NT-SV and RA were identified as the second best conduits using data from this NMA ofangiographic trials.