Health systems are struggling with unprecedented drug spending and governments have devised various policy options to manage high-priced medicines. Meanwhile, some pricing and reimbursement processes are currently moving under the jurisdiction of international agreements. This study aims to understand trends in international agreements from the perspectives of pricing and reimbursement policies for newly marketed medicines.We proposed the framework to interpret the international agreements as code and applied computational text analysis to understand international agreements as data. In particular, we selected the AUSFTA, KORUS, and TPP to assess the progress and evolution in international agreements and investigate the existing relevant content on the pricing and reimbursement of newly marketed medicines.Similar to the provisions for intellectual property, the scope of international agreements regarding pricing and reimbursement decisions are broadened and strengthened. Over time, the domain of transparency, re-naming procedural fairness, has changed significantly more than the remaining domains. Pharmaceutical companies will have more opportunities to advocate for their positions, to protect their interests in decision processes, to investigate the decisions on listings and setting the amounts of reimbursement, and to challenge these decisions.Recently signed international agreements favor companies over governments with underscoring procedural fairness and timely access. However, access to affordable medicines is the goal towards which international agreements should aim. In a similar vein, substantial fairness and the accountability of companies should be discussed when negotiating agreements or adopting international agreements through domestic legislation. Health systems are struggling with unprecedented drug spending with the marketing of high-priced medicines and sudden price increases among generic drugs –3. IncreNew trade and industry norms are expanding their scopes into low- and middle-income countries through international trade agreements , 11, impMany researchers have been interested in international agreements and their impacts on access to medicines –21. Howe“overall” characteristics of “the TPP or the CPTPP”. In other words, the literature could not address trends in international agreements and their specific impact on pricing and reimbursement policies within the member countries. This study aims to understand trends in international agreements from the perspectives of pricing and reimbursement policies for newly marketed medicines. To this end, we proposed the framework to interpret international agreements as code and applied computational text analysis to understand international agreements as data.Little of the literature has discussed the impact of international agreements on pricing and reimbursement processes within member countries. Researchers have meaningfully analyzed the TPP and the Comprehensive and Progressive Agreement for Trans-Pacific Partnership (CPTPP) and suggested their implications on access to medicines from the perspectives of pricing and reimbursement policies –29. HoweText has become an important data source in social science . With thFew studies have embarked on transforming legal texts into data that can be the subject of computational text analysis. These studies adopting the computational approach put big data at the disposal of scholars and enable scholars to investigate international agreements in unprecedented depth and breadth –36. SchoRecently, the scope of intellectual property in international agreements has been notably broadened and strengthened through bilateral and regional free trade agreements . We seleThe AUSFTA, a bilateral trade agreement between Australia and the United States, was signed in February 2004 and implemented in January 2005 . Annex 2p-value less than 0.05.We formulated the framework to understand international agreements from the perspective of the pricing and reimbursement process for newly marketed medicines. In particular, we undertook a purposive review of the literature on the impacts of international agreements on the pricing and reimbursement schemes in member countries –29 and tThe pricing and reimbursement of pharmaceuticals has been a source of continuous policy debate –41. GoveBuilding upon the discussion of the pricing and reimbursement of pharmaceuticals and the impacts of international agreements on the pricing and reimbursement process, we categorized the framework into principles, process, and outcomes. Principles indicate the guiding values expected for pricing and reimbursement decisions in international agreements. The process indicates how decisions are made and/or the mechanisms through which policies are involved. Outcomes refer to the outputs of coordination between principles and process. Then, we reviewed the related provisions in international agreements to modify the frameworks. The KORUS states 8 provisions in Chapter 5: general provisions, access to innovation, transparency, dissemination of information, ethical business practices, regulatory cooperation, medicines and medical devices committee, and definitions. Similarly, the AUSFTA states 6 provisions in Annex 2-C: agreed principles, transparency, medicines working group, regulatory cooperation, dissemination of information, and definitions. At this stage, we found that the domains of international agreements were consistent with the proposed principles, process, and outcomes framework. Finally, we calibrated the framework into principles, transparency, and value for money.Table Table innovative” pharmaceutical products, while the others include pharmaceutical products and “medical devices”. The KORUS includes not only patented pharmaceuticals but also generics in the agreement. Second, the agreements clarify the importance of research and development in the pharmaceutical industry with appropriate government support, such as intellectual property protection. The KORUS describes incentives for innovation other than intellectual property protection. However, the KORUS does not clarify other incentives beyond intellectual property protection. Third, the agreements underscore “timely and affordable” access. Two themes should be noted to understand access: the subject of the access and the specific methods to achieve “timely and affordable” access. The AUSFTA states that “innovative” pharmaceuticals are the subject of access, and it stresses transparent, accountable and “expeditious” procedures, while the KORUS states that “safe and effective” pharmaceuticals and medical devices are the subject of access, and it emphasizes transparent and accountable procedures. The TPP clarifies that pharmaceuticals and medical devices are the subject, and it stresses transparent, accountable, “expeditious and impartial” procedures. Finally, the agreements discuss how to recognize the value of the medicines. The AUSFTA and TPP provide two methods, either “competitive markets” or “procedures that appropriately value objectively demonstrated therapeutic significance”. However, the KORUS states one method, which is very similar to the “competitive markets” in the AUSFTA and TPP: sound economic incentives and competitive markets. The remaining method, which is “procedures that appropriately value objectively demonstrated therapeutic significance”, is stated in a separate provision of the KORUS, entitled “Access to Innovation”.As presented in Table recognizing the value” in the domain of principles. Furthermore, the agreements cover substantial methods for determining the reimbursement decision for the newly marketed drug and setting the price of the drug in the domain of value for money.Innovation in medicines is closely related to drug development or regulatory processes, but it has little connection with pricing and reimbursement processes. To fill this gap, we defined innovation during the pricing and reimbursement process as higher therapeutic value for a given price, which is linked to value for money. As already explained, the agreements include “procedures that appropriately value the objectively demonstrated therapeutic significance”, as stated in the AUSFTA and TPP. Finally, it offers procedures for applying for the reimbursement process based on additional indications for the medicine.The KORUS provides separate provisions related to innovation. Article 5.2, which is entitled “Access to innovation”, is composed of three parts. First, it clarifies that the procedures, rules, criteria, and guidelines related to pricing and reimbursement decisions should be fair, reasonable, and nondiscriminatory. This provision is similar to provisions on transparency, which applies to the procedures of pricing and reimbursement decisions. However, note that the KORUS includes rules, criteria, and guidelines, which are beyond the procedures, as the subjects of recognizing value. Second, it provides two methods for recognizing the value of medicines, based on either competitive market-derived prices or on nonmarket-derived prices.The domain of transparency applies to the procedures of pricing and reimbursement decisions. Compared to the domain of value for money, transparency is described in detail in the agreements. Interestingly, the heading of the provisions changed from “transparency” to “procedural fairness”, and the content was articulated in the TPP.reasonable” to timely decisions. Second, disclosure rules dictate that rules, methodologies, principles, and guidelines be disclosed to manufacturers and the public. The KORUS also adds “criteria ” to the list of disclosure, and it is closely related to health technology assessment. Third, the agreements guarantee applicants’ participation in providing comments at relevant points during the pricing and reimbursement procedures. The KORUS provides applicants with timely and “meaningful” opportunities to provide comments, which would provide the chance for industry to exert influence on pricing and reimbursement decisions. The TPP states that the public, if appropriate, could provide comments at relevant points. Fourth, the provision of information can be divided into manufacturers and the public. The agreements state that the government should provide “detailed” written information to the manufacturers regarding the basis for decisions. Furthermore, the KORUS requires the government to provide “meaningful” information to manufacturers. In contrast, the AUSFTA and TPP request the government to provide written information to the public. Finally, the agreements state that an independent review process can be invoked at the request of the manufacturer that is directly affected by a decision made by the government. The TPP includes “an internal review process” in its provisions.Table Additionally, the AUSFTA states expedited procedures for processing applications not requiring an economic evaluation and opportunities to apply for an adjustment to the price of pharmaceuticals. Note that the latter is associated with the domain of value for money. The KORUS describes reimbursement decision-making bodies. In particular, the decision-making body is open to all stakeholders and the membership list of all committees is to be publicly available.In this section, we conducted a computational text analysis to supplement the aforementioned content analysis. We selected two domains in the framework, namely, principles and transparency, provided word frequencies, and applied correlation testing to quantify the similarities between the AUSFTA and the KORUS and between the KORUS and the TPP.Table Similarly, we counted word frequencies in the domain of transparency. The TPP comprehensively articulates transparency in the dedicated chapter and its annex. Similarly, the AUSFTA describes transparency in the Annex 2-C Pharmaceuticals and Side letters PBS. We included the related chapter, annex, and side letters for analysis. The subject of the domain of transparency, which is “procedures for listing or setting the amount (or pricing) of pharmaceuticals ”, is frequently observed in the AUSFTA and the KORUS. However, these words are not ranked in the top position in the TPP. Instead, other words presented in Chapter 26, entitled “Transparency and Anticorruption”, are in the top position. Specifically, Chapter 26 states provisions on publication, administrative proceedings, review and appeal, and provision of information. Thus, words related to these provisions, including “administrative”, “proceedings”, “review”, and “information”, are ranked in higher positions.specified period”; and the TPP reads as follows: “(a) ensure that consideration of all formal and duly formulated proposals for such listings of pharmaceutical products or medical devices for reimbursement are completed within a specified period of time”, indicating that the KORUS and TPP place more obligations on member countries than the AUSFTA when a government reviews formal proposals by applicants.Interestingly, we found a word, “reasonable”, only in the KORUS and TPP. For instance, “reasonable time, period, or opportunity” are frequently observed in the KORUS and TPP, while the AUSFTA uses “specified time or timely”. Note that the wording is also stated in the KORUS and TPP. For instance, the KORUS reads thus: “(a) ensure that consideration of all formal requests for the pricing or approval of pharmaceutical products or medical devices for reimbursement is completed within a reasonable, p < 0.0001) is the lowest, the correlation between the AUSFTA and the TPP  is in the middle, and the correlation between the KORUS and the TPP  is the highest. However, we found a different result in the domain of transparency. Interestingly, the correlation between the AUSFTA and the TPP  is the highest, the correlation between the KORUS and the TPP  is in the middle, and the correlation between the KORUS and the AUSFTA  is the lowest. Note that the highest correlation between agreements, which is between the KORUS and the TPP, is marginal when compared to the domain of principles.We quantified similarities in word frequencies among the three agreements by applying a correlation test. Note that a correlation test allows us to quantify how similar and different the sets of word frequencies are . In the Currently, health systems are struggling with the marketing of high-priced medicines and sudden price increases in generics –4. GoverThis study provides several strengths in interpreting international agreements. First, our analysis has a specific interest in provisions related to pricing and reimbursement policy. Furthermore, we suggested three domains, namely, principles, innovation , and transparency, to understand provisions regarding pricing and reimbursement. Second, we introduced a computational text analysis to complement a content analysis and suggested word frequencies and correlations among the subjects to note trends in international agreements.This study has several limitations. First, this study breaks down complex trade agreements to the specific area of provisions related to the domestic pricing and reimbursement policy for new medicines. One may argue that interpretations of the contents of the provisions need to be situated in a whole context and interpreted within the relevant part of the text. For instance, the technical barriers to trade in international agreements, which address regulations, standards, testing and certification procedures, might be relevant in the marketing authorization of new drugs. Similarly, government procurement in international agreements might overlap with the pricing and reimbursement of medicines. However, it is noteworthy that several studies have analyzed “overall” characteristics of the “CPTPP” and their potential impact on pharmaceutical policy –28, indiSimilar to the provisions for intellectual property , 25, theOver time, the domain of transparency has changed significantly more than the remaining domains. A correlation test confirmed that the similarities in word frequencies between the agreements in the domain of transparency continuously scored low. For instance, the values of the correlation between the AUSFTA and the KORUS and between the KORUS and the TPP are 0.4498 and 0.4511, respectively, indicating that the contents of the agreements have not converged over time. Note that the TPP meaningfully stresses transparency in the agreement with detailed form and the headings of the chapter and annex, entitled “Transparency and Anticorruption” and “Transparency and Procedural Fairness for Pharmaceutical Products and Medical Devices”, respectively. According to the provisions, pharmaceutical companies have more opportunities to advocate for their interests and arguments, to influence opinions during the pricing and reimbursement decision processes, to investigate the decisions on listings and setting amounts of reimbursement and to challenge decisions previously rendered. Additionally, interesting words, such as “meaningful” opportunity, “all” procedural rules, and “reasonable” time, have appeared in recently ratified agreements, favoring companies over governments.Conversely, the correlation coefficient of word frequencies in the domain of principles increased over time. The values between the AUSFTA and the KORUS and between the KORUS and the TPP are 0.5984 and 0.8223, respectively, demonstrating that the similarities of word frequencies between the agreements in the domain of principles increased. We also confirmed that the principles of the agreements in the AUSFTA and KORUS are sustained in the TPP. The eligible agreements emphasize “facilitating high quality health care” and “improved public health of their nationals” through public health, research and development, timely and affordable access, and recognizing value. Moreover, interesting words, such as “impartial and expeditious” procedures, appear in recently ratified agreements, also favoring companies over governments.Finally, the domain of innovation  is observed only in the KORUS. The AUSFTA and TPP state the corresponding provisions in the domain of principles or transparency. The AUSFTA and TPP acknowledge “recognizing the value through (1) the operation of competitive markets or (2) by adopting procedures that appropriately value the objectively demonstrated therapeutic significance” in the domain of principle. However, they do not state specific methods to measure the value objectively demonstrating therapeutic significance. The KORUS provides clues for interpreting this provision: health technology assessments. For instance, the KORUS states “permit a manufacturer of the pharmaceutical product or medical device to apply, based on evidence of safety or efficacy, for an increased amount of reimbursement over that provided for comparator products, if any, used to determine the amount of reimbursement;”. Furthermore, the TPP states that companies can participate and voice their interests in the review process of listing applications, as well as in establishing laws, regulations, and procedures in the domain of transparency, indicating that a manufacturer has more chances to promote its interests in introducing laws, regulations, and procedures regarding reimbursement processes.The TPP is a plurilateral agreement among 12 countries, including the United States and low- and middle-income countries, while the AUSFTA and KORUS are bilateral agreements between high-income countries. The United States, which is the main member of the agreements, has been pursuing efforts to ratchet up standards of international agreements, including aspects such as intellectual property and other trading norms .It is interesting to note trends in the investment chapter in the TPP. The investment chapter included in the TPP is not novel compared to similar chapters in other international agreements signed a decade earlier . ApproxiUnderscoring the principles of the agreements is noteworthy for identifying the policy implications of international agreements for access to medicines. The international agreements state that “high quality health care and improved public health” are the basic principles of the agreements. Furthermore, they emphasize that timely and affordable access to new medicines is essential to achieving the aims. The three agreements state the importance of “timely and affordable access to pharmaceutical products” in the domain of principles. For instance, the TPP states “the need to promote timely and affordable access to pharmaceutical products and medical devices, through transparent, impartial, expeditious and accountable procedures”. However, we could not find any provisions regarding the affordability of new medicines in the agreements. Specifically, the word “affordability” is stated only once in all three agreements, indicating that affordability at the individual or payer level should be closely considered when negotiating agreements or adopting international agreements at the country level through domestic legislation.The TPP includes “transparency and procedural fairness” in the agreements. Procedural fairness is concerned with the procedures used in decision making, rather than the actual outcomes reached by the decisions –49. GeneProcedural fairness is desirable in making accountable health policies , 50. HowWe found that the scope of international agreements regarding pricing and reimbursement decisions is increasingly broad and strong. Notably, transparency provisions have changed significantly over time. It is realistic to assume that recently signed international agreements favor companies over governments by underscoring procedural fairness and timely access. Pharmaceutical companies will have more opportunities to advocate for their positions, to protect their interests in pricing and reimbursement decision processes, to investigate the decisions on listings and setting the amounts of reimbursement on new medicines and medical devices previously rendered and to challenge related decisions. However, access to affordable medicines is another goal towards which international agreements aim. Furthermore, substantial fairness and the accountability of companies should be discussed when negotiating agreements or adopting international agreements through domestic legislation.


He was preceded in death by his wife Celia Tabor, a physician scientist and his professional colleague, who died in 2012. He is survived by their four children, Edward, Marilyn, Richard and Stanley, 10 grandchildren and 6 great‐grandchildren  for approximately 50 years, died August 20Herb was born in New York City, was educated in the public schools and graduated from Townsend Harris High School in 1933. He attended City College for two years, then transferred to Harvard College and received his bachelor’s degree and MD there, in 1937 and 1941, respectively. During his last year in medical school, he spent six months working in the laboratory of A. Baird Hastings, who was Chair of the Biological Chemistry Department at Harvard Medical School, and overlapped with such well‐known figures as Birgit Vennesland, Jack Buchanan, Chris Anfinsen and Oliver Lowry. His first paper in the JBC arose from his studies in Hastings’ lab. In 1942, he interned at Yale‐New Haven Hospital and spent some of his spare time in the laboratory of John Peters, who he credits with being an important influence on his thinking. He was also involved in the first major clinical trial of penicillin in the United States and the successful treatment of a case of streptococcal septicemia. After being commissioned in the U.S. Public Health Service in early 1943, he spent his first six months at sea as the sole medical officer on the Coast Guard cutter, U.S.S. Duane. One of us (RB) experienced some of his memories of this service during a birthday festschrift for Bob Hill in the summer of 1995, at the Rosario Resort on Orcas Island in Washington. Several of us were having dinner together in the resort hotel, and as we entered the restaurant Herb stopped abruptly in front of a large group of photographs of various ships and other marine subjects and then announced with a huge grin, “That was my ship.” It was indeed the Duane and we were subsequently entertained through dinner with several anecdotes of a “very young and surgically inexperienced Dr. Tabor avoiding one medical catastrophe after another” (or at least that was how he related it). He did admit he was extremely glad to get to the NIH in the fall of 1943, his next posting, and back to the laboratory.th birthday celebration in 2008, that Herb became so enthusiastic and intent on the seminar presentation on one occasion that he unknowingly ate Arthur’s birthday sandwich that was adjacent to him rather than his own! Over the years, the luncheon meetings expanded to include many other notable figures in biochemical research. The combined efforts of the Tabors heralded the explosive growth of polyamine research, trained many postdoctoral fellows, and laid the foundation of this still active area of research.Herb’s association with the NIH was to be for the rest of his life, totaling some 77 years, the longest term of any NIH employee. When he first arrived at the NIH, he was assigned to the laboratory of Sanford Rosenthal where his initial efforts were on electrolyte changes in burns and shock. When this war time research wound down, Tabor and Rosenthal moved into studies on the metabolism of histamine and histidine that ultimately led to the pioneering work, with his wife Celia, on polyamines, which would characterize the rest of their careers. She was also trained in medicine and joined the NIH in late 1952. They had met several years earlier in Boston and were married in 1946. Three of their children were born prior to her USPHS commission while she was at George Washington University Medical School; the fourth, a couple of years later. Much of their early studies were focused on enzymology, and this emphasis underscores his active interactions with Bernie Horecker, Leon Heppel and Arthur Kornberg, who were colleagues with him at the NIH during his first several years there. He has described with enthusiasm their regular daily luncheon discussions/seminar meetings that started in 1946, and spanned approximately 50 years, and the importance of these interactions to the development of his research career. Arthur Kornberg recalled fondly at Herb’s 90A critically important part of scientific advances is the accurate review of experimental data and entry into the scientific record. It could even be argued that the publishing of science is equally important as the discoveries themselves. Although many accomplished scientists devote significant parts of their career to scientific publishing, few have had the commitment and dedication to these activities that Herb had. Herb was on the editorial board of the JBC from 1961 to 1966 and then became an Associate Editor. Bill Stein, Editor of the JBC and a Rockefeller University professor and Nobel Laureate, contracted Guillain‐Barre Syndrome in the late 1960s, and could not continue in his role as Editor. One of us (RB) remembers events at the meeting in Copenhagen in June of 1969 when Stein was stricken and spending the last night before he became ill at a reception with him at the Carlsberg Laboratory. Herb claimed, in his humble manner, that he was asked to take on the Editor responsibilities because he resided in the immediate vicinity of the American Society of Biological Chemists (ASBC) office and the business side of the Journal. More likely his ability to lead the Journal was well recognized; he assumed the role of Acting Editor in 1969 and Chief Editor in 1971 and remained in that role for the next 41 years.The Journal, which was launched in 1905 by founding editors Christian Herter and John Abel as an independent entity, had enjoyed, in addition to Stein, five earlier editors, four of whom had been managing editors, meaning that they were responsible for both the editorial and financial aspects of journal operations. John Edsall, who took over the editorship in 1958, oversaw the transition of the business side to the “new” offices of the ASBC (which became the American Society for Biochemistry and Molecular Biology (ASBMB) several years later) so that Stein and then Herb were able to concentrate on the editorial side of things. The ASBC had assumed the responsibility for the JBC in 1919. Stein hardly got started as Editor before he was taken ill, but Herb more than made up for any lost time leading a phenomenal 45 year campaign of growth in size and prestige that included such important events as bringing the JBC on‐line, the first biomedical journal to achieve this landmark accomplishment. In his own unassuming but steadfast approach, with a rigid attention to detail, an uncompromising commitment to excellence and a devotion rarely seen, he became a legend in science publishing, and he will long be remembered as such.Most impressively, Herb carried out all of his JBC activities on nights and weekends, while he and Celia worked in their NIH laboratory during the day, scrupulously avoiding any conflict with his government obligations. He oversaw a spectacular growth in the Journal, from 3,100 pages in 1968 to surpassing 55,000 published pages/year at one point. Likewise, the number of Associate Editors increased from 3 in 1968 to over two dozen recently, and editorial board members increased from 54 to hundreds from all over the world, illustrating that the size of his commitment was truly staggering. Herb also resisted pressure to print “hot” or “flashy” science but rather felt that all science that makes a substantial contribution to the biochemical and molecular biological literature should be published in the Journal. This attitude is the basis for the reputation of the JBC for outstanding, high quality publications that advance science and stand the test of time. Herb was also highly protective of the Journal and its assets and would become quite aggressive in shielding it when he thought the Finance Committee or the Council was contemplating using monies he considered were the Journal’s for other activities. This led, on more than one occasion, to some interesting exchanges. One such instance was the development of the online version of the Journal. Although he clearly recognized the value of such a venture, if successful, he also recognized that it could fail and end up financially crippling both the Journal and the Society. One of us (RB) was Treasurer at the time and had many of the same qualms. Witnessing his cautious and careful support of the project while at the same time not allowing it to get out of control was a perfect example of his leadership skills.Herb’s contributions to the ASBC/ASBMB were also substantial and are in many respects underappreciated. The histories of the Society and the Journal are entirely interwoven, even though they started separately, and the combination has been a significant and powerful contributor to the advance of bioscience. Everyone involved in the governance of the Society was aware of his constant presence and thoughtful contributions at meetings of the Council, the Financial Committee, as well as the Publications Committee, which spanned his time as the Editor of the JBC. And he did it all with a fantastic sense of balance and humor. He was nearly unflappable and was a calming influence regardless of how heated discussions became. He was at his best leading JBC Associate Editor meetings, commonly and accurately referred to as “the proverbial herd of cats” and securing consensus on topics that one would swear could not be reconciled.Herb was one of the calmest, most insightful and humblest individuals that either of us has known. He did have his moments though and felt passionately about some subjects. For example, one incident occurred several years ago and came about from a suggestion by the Publications Committee that the journal might be more appealing with a flashier name. On leaving the Publication Committee meeting where this suggestion had been made, Herb joined the ongoing meeting of the Council with the look of a man who had just had to shoot his dog. It was plainly evident that he was thoroughly distressed, and eventually related what the problem was in a private conversation. While this might not seem at first blush to be of such great concern, due to an accident of history, the Publications Committee had been originally created as a separate body within the society and was, at the time, still independent of Council and the elected officers, that is, it could make its own decisions that did not require any ratification or approval from any other part of the society. Thus, if it wanted to change the name of the JBC to something else it had the full authority to do so, and Herb was clearly afraid that it might choose to do just that. The only obvious solution was to change the society’s bylaws and end the autonomy the Publications Committee enjoyed, particularly since there was no longer any apparent reason for it as the circumstances that had engendered its independent authority had long ago been resolved. An appropriate revision was immediately drafted, and a handwritten copy delivered to Chuck Hancock, then the Executive Officer of the ASBMB, with instructions to bring this to Council for a vote as soon as it could be arranged. This was accomplished with dispatch and any crisis that was envisioned was averted.Herb received many honors. He was given the Arthur S. Fleming Award in 1956; the USPHS Meritorious Service Medal in 1970; he was elected to the American Academy of Arts and Sciences in 1971, and to the National Academy of Sciences in 1977; he received the Hillebrand Prize from the American Chemical Society in 1986; he and Celia received the William C. Rose Award from the ASBMB in 1994. The ASBMB created an award in his name, The Herbert Tabor Research Award, presented annually for exceptional established investigators and the JBC created Herbert Tabor Early Career Investigator Awards in his honor. In 2018, the Montgomery County Council in Maryland designated November 28 as “Herb Tabor Day,” and the State of Maryland honored him with a ‘Governor’s citation’ in honor of his many contributions to science.Those of us who have had the good fortune to work with Herb on editing the JBC and/or leading the Society will always remember him as a partner and friend who represented the very best of what the human race has to offer. He leaves a legacy of professional accomplishment in terms of scientific research and publishing that we can only marvel at. To say he will be missed is true but certainly not adequate; but there is also pleasure in knowing he will be fondly remembered, and hopefully emulated, by those who carry on his work Figure .The authors have no conflict of interest.


We report the case of a 13-year-old child who presented to the ophthalmic emergencies for left eye trauma because of a sore eyelid, while fighting with one of his classmates using pairs of scissors. The clinical examination found a wound on the upper left eyelid with loss of substance of the skin without involvement of the orbicularis muscle (A). Examination of the left eye found a visual acuity preserved at 12/10 without optical correction, a calm anterior segment and a flat retina. Faced with the loss of cutaneous substance, it was decided to perform a total skin graft from the abdominal skin (B). The sample was taken using a scalpel and the graft was defatted. The total skin graft was immediately performed, and sutured with fluted edges to prevent healing in the staircase (C). The patient was seen again 10 days after the graft, with good budding of the graft, and removal of the stitches (D). Fat bandage care has been recommended to allow good healing of the graft. Eyelid sores in children represent a multitude of clinical forms. These wounds require early and appropriate management, with a complete initial clinical examination,to look for lesions of the free edge or lachrymal passages, but also a careful examination of the globe in order to rule out a wound of the globe that would be associated. Their management should be early and specialized, in order to avoid the occurrence of skin retraction, or harmful aesthetic sequelae.


Dear Editor,During the Corona virus epidemic, most jobs closed, and according to the policies to reduce crowding, most educational sessions such as clinical rounds, morning reports, and journal clubs barred; therefore, it is necessary to consider ways to deal with these limitations. The use of virtual training used to be limited due to the distance between people, but during the Corona-virus era, there was an opportunity to use this technique more  . One ofNowadays, evidence-based medicine (EBM) is an accepted educational paradigm at all levels of medical education  . EBM reIn this model, unlike journal club session, which starts with an interesting article or a popular journal, an EBM journal club starts with a patient’s problem. In this case, our recommendation is that a mentor and two residents (senior and junior) should identify a patient whose condition is mostly traumatic or emergency due to the closure of elective surgery.Generate a clinical question based on the PICO  model. This question must be certain, well-structured and answerable  . At thaUp to now, only three people have been involved. But at this point, the PICO question and the articles obtained are introduced to other members (other faculty members and residents) in the virtual groups from 3 days before the program, so that the people present at the meeting know about them.On the day of the journal club, the time of which have already been defined, mentors and residents will take control of the meeting. At the beginning, the junior resident explains the clinical question and the how made PICO query and then how to search (about 5 minutes) and the senior resident describes critical appraisal articles (about 10 minutes) which have different items depending on the type of article.Next, after the end of the critiques, the mentor respectfully asks the faculty members and summarizes the excellent results of the articles with the real working environment and the attendance opinions.https://digitalcommons.wayne.edu/crp/.Our recommendation is that the PICO question, the search method, the articles obtained, the critique of the articles, and the comments of the attendees should be written, properly recorded in a special form attached, and be published on certain sites, such asFinally, these days, when there are limitations for the academic community, virtual learning capacities can be properly used in groups.Take home message:In the social constraints of covid-19, it is possible to use virtual communication platforms to make journal club session based on EBM principles in a completely dynamic and productive way, and to continue education at all levels, despite this world-wide crisis.


Stroke has been the leading cause of disability due to the induced spasticity in the upper extremity. The constant flexion of spastic fingers following stroke has not been well described. Accurate measurements for joint stiffness help clinicians have a better access to the level of impairment after stroke. Previously, we conducted a method for quantifying the passive finger joint stiffness based on the pressure-angle relationship between the spastic fingers and the soft-elastic composite actuator (SECA). However, it lacks a ground-truth to demonstrate the compatibility between the SECA-facilitated stiffness estimation and standard joint stiffness quantification procedure. In this study, we compare the passive metacarpophalangeal (MCP) joint stiffness measured using the SECA with the results from our designed standalone mechatronics device, which measures the passive metacarpophalangeal joint torque and angle during passive finger rotation. Results obtained from the fitting model that concludes the stiffness characteristic are further compared with the results obtained from SECA-Finger model, as well as the clinical score of Modified Ashworth Scale (MAS) for grading spasticity. These findings suggest the possibility of passive MCP joint stiffness quantification using the soft robotic actuator during the performance of different tasks in hand rehabilitation. Spasticity, commonly known as a symptom with a broad range of neurological disorders, is frequently found in subjects after stroke. Previous studies indicated that about 40% of subjects with stroke suffer from spasticity , leadingHowever, the only assessments regularly incorporated into neurorehabilitation are through clinical measures , which aThe most common clinical examination of spasticity includes assessment of exaggerated tendon tap reflexes by passive muscle stretch. Several rehabilitation instruments have been built in laboratories to quantitatively examine joint resistance to passive stretch. The evaluation of biomechanical joint properties , such asPreviously, we introduced the first 3D printed soft-elastic composite actuator (SECA) for hand rehabilitation that quantifies stiffness of the impaired fingers . We furtIn this work, we replicated the standalone stiffness measurement device proposed by We have designed the 3D printed SECA that can actively control flexion and extension of a spastic finger. MCP segment on the SECA corresponds to actuation of the MCP joint of the spastic finger, which joint flexion is controlled on pressurization and extension is controlled on depressurization . Flex se0, θ is the bending angle that θm is for the current bending angle of MCP joint and θ0 is the resting angle of the joint and always less than 90°). To demonstrate the pressure-angle relationship, we have obtained the following equation of , wm is the actuator strain energy density function, P is the input pressure, E is the layer Young’s modulus, I is the layer second moment of area, k is the joint stiffness (km for MCP joint), θ is the joint angle  (in which  radian) .0,θ0], the joint stiffness equation isRearranging the analytical model of equation (1) in the region of θ ∈ [0which0) and generating inaccurate results. Therefore, the possible ranges of MCP joint angle and input pressures for stiffness evaluation using equation (2) becomeFurthermore, γ is an empirical coefficient defined for MCP joint angle. The value is set to be 0.7 to avoid reaching singularity  would provide the most stable stiffness results for finger joints and prevent model singularity , and a DC servomotor , as illustrated in Prior to the measurement, the distance between the MCP joint and the tip of the finger splint is measured. Then, the MCP joint is passively moved by the servomotor at specific positions, as instructed by lfin is the distance between the MCP joint and the tip of the finger splint (load cells), Fpassive is the measured tangential force to the circular rotation of index finger, and τpassive is the MCP joint torque. A classic double exponential function-based model developed for finger kinematics is further used to describe the relationship between the passive MCP joint moment and its corresponding joint angle , and A to F are the fitting parameters for the model. They are estimated using the Non-linear least squares (NLS) method in MATLAB based on the minimization of sum of squared differences (SSD) between the experimental values and the fitting model , θFour subjects with chronic stroke who had demonstrated weak hand strength with moderate level of finger flexor spasticity was recruited. Four subjects with no neurological deficit from the laboratory also participated in the study to serve as the control. Both have given their informed consents. The clinical evaluation was registered to the Joint Chinese University of Hong Kong-New Territories East Cluster (CUHK-NTEC) Clinical Research Ethics Committee (Ref. ID: NCT03286309). The subjects are first examined with our stiffness evaluation method. Then, the subjects are further assessed with the replicated stiffness measurement device. Stiffness values obtained from two methods are compared with each other for validation.Index finger is selected as a stiffness indicator of the hand, similar to the approaches from different research groups , 2020. EThe SECA contains MCP segment for the actuation of MCP joint. It is attached to a hand base and secured to the index finger using Velcro Strap, as shown in Index finger is also selected as a stiffness indicator of the hand. The standard stiffness measurement device for measuring passive MCP joint angle and torque in index finger is replicated and used for obtaining the ground-truth stiffness values, as shown in The DIP and PIP joints in the index finger are clamped by the finger splint, allowing solely the MCP joint to be rotated by the servomotor in horizontal plane. Passive extension force at the splint tip is measured by the load cells in the sensor box with the sampling rate of 80 Hz during passive rotation of MCP joint. The force is multiplied by the distance between the MCP joint and the splint tip, thereby obtaining the final values of passive MCP joint stiffness. The posture of upper limb follows the settings in the experiment using SECA, as elaborated previously. A hand holder is placed in the arm rest for holding the other four fingers in vertical plane. Velcro Straps are used to fix the forearm (0° of supination and pronation) and wrist (0° of flexion and extension) at the natural position, as illustrated in A brief physical examination of MAS is first performed on assessing the flexor tone in the spastic finger. The degree of spasticity is assessed by a trained clinical assessor who is blind to the experimental condition. For the subjects with no neurological deficit, the examination of spasticity with MAS is not conducted, and the result can be classified as “no increase in flexor muscle tone” (MAS score = 0) . After cPreparation procedures are required for the subjects prior to both experiments using our SECA and the standard measurement device. To maintain the assessed muscle tone being consistent and stably presented on subjects in two tests, they are instructed to sit vertically for 30 min with the elbow at 90° and supported on the desk to relax the forearm muscles prior to each test. Any passive mobilization to extend the fingers, or any generated movement (either passive or active) on the fingers that will greatly alter the muscle tone is not allowed during the relaxation time. The 30 min resting time before the spasticity measurement was suggested by i_ m and γθm0_(γ = 0.7). Stable stiffness results can be ensured within the defined angle range. Also, only three repetitions are performed to measure the passive MCP joint angle, as it has been suggested that short-term effect of any stretch, e.g., loosening the joints and reducing the muscle tone, may occur if more than three times  and the extra passive MCP joint resistance provided by the joint CLC upon closing to full flexion or extension can be excluded by setting the angle range between θee times .m0_, the MCP joint will slowly (2° per second) return to the extended position θi_ m with the SECA, such that the dynamics of SECA-Finger model can be ignored. This whole process is defined as one complete repetition. Upon completion of all three repetitions, three MCP joint angle values would have been recorded at each specific pressure input, e.g., at 0, 20 kPa, etc., respectively. Average MCP joint angle at each pressure input is taken from the three measured angle values and substituted into the static model in equation (1) – (4) to obtain the measured stiffness value at each specific pressure input. Eventually, the final MCP joint stiffness is determined from the average of all measured stiffness values.During the measurement, the SECA is placed on the index finger in an unpressurized state. Pressure is then increased step by step from zero by 20 kPa one at a time to move the MCP joint to different angles, which the measured angles and the corresponding pressure values are recorded. The SECA holds the MCP joint at each position for 30 s to ensure stable angle values can be collected . As soonOn the other hand, the standard stiffness measurement device is used in the second stiffness test. Previous study used the measurement device to rotate the MCP joint from the flexed position (50°) to the fully extended position (−40°), i.e., ROM of 90°, and obtain the passive MCP stiffness values for this overall range . With sui_ m and γθm0_(γ = 0.7) and substituted into the stiffness equation (7). Eventually, the average of all six calculated joint stiffness is the final MCP joint stiffness measured by the stiffness measurement device. Throughout all the tests, subjects did not indicate any discomfort and pain upon the stretch of their MCP joints.Similar to the stiffness test using SECA, upon completion of all three repetitions, three measured MCP joint torque values would have been recorded at each specific joint position, e.g., at 90°, 80°, etc., respectively. Average MCP joint torque at each joint position is taken from the three measured torque values and fitted into the double exponential function-based model in equation (6) for getting the parameters. The parameters are then substituted into equation (7) for the stiffness equation. Six joint angles are selected by portion, as suggested by Quantification of MCP joint stiffness is conducted. Pressures of 0, 20, 40, and 60 kPa are applied to the SECA. Bending angles of MCP segment, and the corresponding actuation pressures, are recorded for the calculation of stiffness values using equation (2). To avoid singularity, it should be noted again that the maximum MCP joint angle cannot exceed their pre-defined upper limits. The results from two different approaches are similar with each other. Upon the increase of muscle tone (spasticity), the MCP joint stiffness further increases. Only a maximum stiffness difference of 0.021 Nm/rad is observed in subject S2 with chronic stroke, and minimum difference of 0.004 Nm/rad in subject S4 with chronic stroke. From our data, it is also observed that the stiffness from our SECA would be slightly smaller than the actual joint stiffness. Here, we believe that the stiffness evaluation method using our SECA is feasible for finger joint stiffness quantification during any rehabilitation training. It would be possible to extend the quantification to the proximal interphalangeal (PIP) or distal interphalangeal joints (DIP) joints as well using our SECA, which has been hard to be accomplished by the standard stiffness measurement device.Spasticity is a well-known symptom seen in subjects after suffering a stroke. Hand function is severely affected due to the excessive flexor tone in muscles. The purpose of this study was to validate the compatibility of our stiffness quantification method in clinical practices. We quantified the passive MCP joint stiffness in subjects with chronic stroke and no neurological deficit. We also investigated the difference of stiffness results obtained from our SECA and the standard stiffness measurement device. The results between using our approach with the SECA and the standard device were consistent, which only a difference of 0.0044 Nm/rad was observed in the MCP stiffness. The results were also supportive when comparing with MAS.The passive MCP stiffness evaluation was conducted with the replicated standard device to collect the passive joint torque and ROM in each subject. The double exponential function-based model fitted well with the collected finger kinematic data, which the performance was similar to previous research . From thCommon clinical examinations of spasticity include the Ashworth Scale (AS) and Tardieu Scale (TS). To further quantify the MCP stiffness after stroke, there is not a lot of studies conducted the quantify the stiffness of spastic finger joint to the best of our knowledge. We have concluded the results from some existing conclusive studies and presented in As soon as excessive muscle tone further increases in finger flexors, larger MCP stiffness values could eventually be observed. For subjects with chronic stroke having MAS of 2 and 3, their MCP joint stiffness was observed to be much larger than that of subjects with no neurological deficit, e.g., from around 0.22 Nm/rad to 1.12 Nm/rad in This work explored the difference of passive MCP joint stiffness among subjects who experienced a stroke or remain neurologically intact. Experimental results provided some evidence of quantifying the MCP stiffness using our SECA. In the future, our proposed methods could be applied to our rehabilitation robotic hand that reflects the stiffness for individual finger. With the stiffness information, optimal training tasks might be planned for each individual with stroke depending on the finger stiffness condition. Therapeutic progress might also be indicated in detail to motivate patients for achieving better improvement during rehabilitation training.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Joint Chinese University of Hong Kong-New Territories East Cluster (CUHK-NTEC) Clinical Research Ethics Committee (Ref. ID: NCT03286309). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.XS and HH developed the mathematical theories, designed the experimental devices and experimental protocols, implemented the data collection software, performed the experiments, analyzed the experimental results, and participated in manuscript preparation. HH, XS, and ZT performed the experiments, contributed to discussions, analysis, and participated in manuscript revisions. KT and ZL supervised the project, contributed to discussions, analysis, and participated in manuscript revisions. All the authors have approved the submitted manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rational: Increasing evidence indicates that the physical environment is a critical mediator of tumor behavior. Hepatocellular carcinoma (HCC) develops in an altered biomechanical environment, and increased matrix stiffness is a strong predictor of HCC development. C-X-C chemokine receptor type 4 (CXCR4) is known to trigger HCC progression. However, CXCR4 as a mediator of mechanical cues in HCC is not well characterized.Methods: qRT-PCR, Western blot and IHC were used to detect the CXCR4 expression in different matrix stiffness gels. MTT was used to measure the cell proliferation of HCC cells. Immunoblotting was used for detection of epithelial-to-mesenchymal transition (EMT) and stemness on the matrix stiffness. Immunofluorescence (IF) was used to detect the nuclear location in HCC cells. IP was used to show the interaction between YAP, UbcH5c and β-TrCP.Results: We identified CXCR4 as a critical intracellular signal transducer that relays matrix stiffness signals to control mechano-sensitive cellular activities through ubiquitin domain-containing protein 1 (UBTD1)-mediated YAP signaling pathway. We found that CXCR4 expression was remarkably up-regulated in HCC cells with increasing matrix stiffness and mediated proliferation, epithelial to mesenchymal transition, and stemness. Mechanistically, matrix stiffness acts through CXCR4 to decrease the levels of UBTD1, which is involved in the proteasome-dependent degradation of YAP, a major cell mechano-transducer. UBTD1 interacted with components of the YAP degradation complex and promoted the interaction between YAP and its E3 ubiquitin ligase β-TrCP. UBTD1 knockdown decreased YAP ubiquitylation and resulted in the activation of YAP-targeted genes and YAP downstream signaling. Downregulation of UBTD1 in HCC tissues correlated with malignant prognostic features and overall survival. Finally, luteolin, a natural product, suppressed matrix stiffness-induced biological effects and CXCR4-mediated YAP signaling pathway in HCC cells.Conclusion: Our findings reveal CXCR4 as a molecular switch in mechano-transduction, thereby defining a mechano-signaling pathway from matrix stiffness to the nucleus. Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the second most frequent cause of cancer-related mortality worldwide During tumor progression, increased ECM deposition and cross-linking result in increased tumor stiffness. Cells recognize and respond to the mechanical stiffness of the ECM by exerting contractile force and sensing counter-tension through mechano-cellular systems Chemokine receptors are important modulators of cell trafficking and adhesion Ubiquitin domain-containing protein 1 (UBTD1) is highly evolutionarily conserved and has been described to interact with E2 enzymes of the ubiquitin-proteasome system Here, we identified CXCR4 as a mechano-transducer to relay extracellular mechanical signals and induce UBTD1 down-regulation, which decreased YAP ubiquitylation degradation, resulting in the activation of YAP-targeted genes. We showed that UBTD1 was down-regulated in HCC and was associated with poor patient survival. UBTD1 promoted the interaction of YAP with its E3 ubiquitin ligase β-TrCP and decreased YAP ubiquitylation to trigger its activation and downstream signaling. Matrix stiffness acted through CXCR4 and its downstream UBTD1 to modulate a YAP/TAZ-mediated mechano-responsive signaling pathway. This new signaling axis provides novel insights to clarify how mechanical stimuli exerted their effects on cellular bio-response.The mechanically tunable Collagen 1 (COL-1)-coated polyacrylamide gel was established as previously described 2. Luteolin was purchased from Dalian Meilun Biology Technology Co., Ltd.HCC tissues and matched adjacent non-tumor tissues were obtained from our hospital. No patients received preoperative chemo- or radiotherapy before surgery. Written informed consent was obtained from each patient. The human HCC cell lines (Hep3B and Huh7) were obtained from the Chinese Academy of Sciences . The cells were cultured in Dulbecco's Modified Eagle's Medium  supplemented with 10% fetal bovine serum , 100 units/mL ampicillin, and 100 μg/mL streptomycin at 37˚C with 5% COImmunohistochemistry was performed on paraformaldehyde-fixed paraffin sectionsHCC cells were fixed with 4% paraformaldehyde and permeabilized using 0.2% Triton X-100. Then the fixed cells were incubated with the YAP  primary antibody. The secondary antibody was an Alexa Fluor-conjugated IgG . Fluorescence confocal images were captured using an LSM 5 Pascal Laser Scanning Microscope  with a ×40 lens and Laser Scanning Microscope LSM PASCAL software (version 4.2 SP1).Total protein was extracted from HCC cells, and 20 μg of isolated proteins were separated by 10% SDS-PAGE, transferred onto a PVDF membrane , and probed with the respective primary antibodies  for overnight. Subsequently, the membranes were incubated with the HRP-conjugated goat anti-mouse or anti-rabbit IgG antibody . Protein bands were visualized using an enhanced chemiluminescence kit .Cells were seeded in 96-well plates, and the viability of cells was measured by the MTT assay. Briefly, 20 μL of MTT working solution (5 mg/mL) was added into each well and incubated at 37°C for 4 h. Then the supernatants were removed, and the resultant MTT formazan was dissolved in 100 μL of DMSO. The absorbance was measured at the wavelengths of 490 nM.YAP protein stability on matrix stiffness was analyzed by WB in the presence of a protein synthesis inhibitor cycloheximide (CHX). In brief, CHX (50 μg/mL) was added to the cell culture medium for indicated times and cells were harvested for protein extraction. At each time point, cells were washed with cold PBS and lysed in RIPA buffer. Equal amounts of total protein per time point were then loaded onto a PAGE gel for YAP WB.6) were mixed in 150 µL of Matrigel and were inoculated subcutaneously into the flanks of one group of nude mice; the other group received transfected cells (1×106) via tail vein injections for the establishment of the pulmonary metastatic model. The tumor volume for each mouse was determined by the following formula: tumor volume = length × width × width/2. After 3 weeks, the mice were sacrificed by cervical dislocation under anesthesia with ether and the xenograft tumor tissue was explanted for examination. All in vivo protocols were approved by the Institutional Animal Care and Use Committee of Xi'an Jiaotong University.4-6-week-old male BALB/c nude mice  were randomized into two groups (n=5). The transfected cells . After extensive washing, precipitates were analyzed by Western blotting, which was performed using the standard protocol.Data were presented as the mean ± SD from at least three independent replicates. SPSS software, 16.0  was used to conduct the analysis, and a two-tailed Student t-test was employed to analyze the differences between the two groups. Pearson's correlation analysis was used to analyze the correlation between the two indices. Differences were considered statistically significant at P < 0.05.Increasing matrix stiffness constructed with mechanical gels was used to investigate the response of HCC cells. We used the low 1 kPa), medium (6 kPa), and high (12 kPa) matrix stiffness to represent the normal, fibrosis, and cirrhosis HCC tissue background, respectively. We observed the morphological changes of Hep3B and Huh7 cells from small and round to fully spread and outstretched on different stiffness platforms  and inhibit nuclear translocation of YAP and the subsequent transcriptional program To investigate how UBTD1 regulates YAP degradation, we performed Co-IP experiments and demonstrated that UBTD1 was associated with YAP Figure A-B. We uTo determine whether matrix stiffness-induced UBTD1 down-regulation was dependent on CXCR4, we knocked down CXCR4, which abolished the effects of UBTD1 down-regulation by matrix stiffness (Figure We measured UBTD1 levels in tumors and paired adjacent nontumor liver tissues to validate its role in HCC progression. UBTD1 protein was remarkably down-regulated in HCC tissues compared to the corresponding normal liver tissues Figure B. HCC pain vivo. Tumor growth curves revealed that UBTD1 overexpression significantly inhibited the tumor growth of HCC cells in mice (Figure in vivo (We also established a subcutaneous tumor model to measure the effect of UBTD1 on HCC  in vivo . Next, w in vivo F. These in vivo (Figure in vivo (Figure The above data elucidated the molecular mechanisms underlying the matrix stiffness-induced biological behavior in HCC cells. To investigate new agents for the treatment of HCC, we selected luteolin, which is an abundant flavonoid present in many types of vegetables, fruits, and medicinal herbs, and has been used in Chinese traditional medicine to treat various diseases, such as hypertension and cancers o Figure . CollectLiver cancer is a neoplasm with high incidence and mortality that carries a dismal prognosis. Previous reports showed that pathophysiological increases in matrix stiffness, which are encountered in fibrotic and cirrhotic livers, promoted HCC development and progression In the present study, we demonstrated that matrix stiffness profoundly alters the phenotype and behavior of HCC cells, including proliferation, EMT, and stemness characteristics. Chemokine receptors are the major regulators of cell trafficking and adhesion. In particular, CXCR4 has been implicated in both tumor cell dissemination from the primary site and intravasation via trans-endothelial migration. The interaction of CXCR4 and its ligand SDF-1 promotes HCC metastasis, proliferation, and angiogenesis Herein, we confirmed that stiffness induced YAP nuclear targeting and YAP-dependent transcription of target genes. YAP/TAZ is mechano-sensitive transcription coactivators. They reside in the cytoplasm on a soft substrate and translocate into the nucleus by stiffness. In cancer biology, the crucial role of YAP pathway has been widely emphasized at many stages of cancer progression, including proliferation, invasion, and metastasis and, therefore, deregulation of YAP signaling is oncogenic in humans It has been reported that the β-TrCP E3 ubiquitin ligase catalyzed YAP ubiquitination, ultimately leading to YAP degradation. Our data provided converging evidence indicating that UBTD1 was associated with YAP degradation complex and participated in its ubiquitylation by β-TrCP. We also demonstrated that UBTD1 was decreased in human HCC tissues. UBTD1 expression was highly correlated with poor pathological features, including the TNM stage, venous invasion, tumor size, and overall survival. Moreover, UBTD1 overexpression inhibited proliferation, EMT, and stemness. We further confirmed that the natural medicine, luteolin, inhibited the matrix stiffness-induced CXCR4-mediated YAP signaling pathway, proliferation, EMT, and stemness  thus ideIn summary, we have characterized CXCR4 as a new mechano-response linked to the matrix stiffness. The intricate UBTD1 down-regulation controlled YAP signaling and participated in YAP degradation by modulating its ubiquitylation. Significantly, luteolin suppressed matrix stiffness-induced CXCR4/YAP signaling. These results suggest that luteolin is a potential anti-cancer agent for the treatment of HCC.Supplementary figures.Click here for additional data file.


Malaysian Journal of Medical Sciences (MJMS)’ performance as a journal, along with important changes in the administration and achievements of the journal itself. We report here its submission trends, rejection rates, manuscripts accepted based on submitting country/region and impact factor scores for calendar year 2019.Once a year, we report the  Malaysian Journal of Medical Sciences (MJMS) continues to receive increased number of new manuscripts submission every year. In 2019, there were 453 new manuscripts submitted to MJMS (a 26% increase over that of 2018).On average, the New manuscripts submitted to MJMS would initially be assessed to a lesser degree by the Editorial Office to ensure they conform to MJMS requirements and the subject discussed was relevant to MJMS’ main focus. At this level, only a few numbers were desk-rejected and did not make it to the peer-review process. A considerable number of rejections happened to manuscripts which had succeeded to undergo at least the first hurdle of peer-review process and had gone through intense judgement in terms of scientific merit, not technical mistakes. In another word, it depended more on the quality of the manuscripts, not the number of reviewers .™ system  for the second time, after the first award as the Potential Journal received in 2016 from the Ministry of Higher Education, Malaysia.


Enterovirus A71 (EV-A71), Coxsackievirus A16 (CV-A16) and CV-A10 are the major causative agents of hand, foot and mouth disease (HFMD). The conformational epitopes play a vital role in monitoring the antigenic evolution, predicting dominant strains and preparing vaccines. In this study, we employed a Bioinformatics-based algorithm to predict the conformational epitopes of EV-A71 and CV-A16 and compared with that of CV-A10. Prediction results revealed that the distribution patterns of conformational epitopes of EV-A71 and CV-A16 were similar to that of CV-A10 and their epitopes likewise consisted of three sites: site 1 (on the “north rim” of the canyon around the fivefold vertex), site 2 (on the “puff”) and site 3 (one part was in the “knob” and the other was near the threefold vertex). The reported epitopes highly overlapped with our predicted epitopes indicating the predicted results were reliable. These data suggested that three-site distribution pattern may be the basic distribution role of epitopes on the enteroviruses capsids. Our prediction results of EV-A71 and CV-A16 can provide essential information for monitoring the antigenic evolution of enterovirus. Since 1997, numerous great outbreaks of HFMD have occurred in the Asia–Pacific region involving Malaysia, Australia, Taiwan and mainland China8. Thereafter, HFMD was listed under the surveillance by Chinese Center for Disease Control and Prevention. From 2008 to 2015, a total of more than 13 million HFMD cases (probable and laboratory-confirmed) were reported, among which the severe and fatal cases were more than 120,000 and 3000, respectively9. Enterovirus A71 (EV-A71), Coxsackievirus A16 (CV-A16), CV-A10 and CV-A6, members of the enterovirus species A (EV-A), are major causative agents of HFMD. CV-A16-induced HFMD is often benign and mild, while EV-A71-associated infections involve mild and severe cases manifesting neurological complications, and even death13.Hand, foot and mouth disease (HFMD) is a common febrile disease caused by human enteroviruses, which mainly inflicts infants and young children, resulting in the appearance of vesicular rashes on hands, feet and buccal mucosaenterovirus genus within the family Picornaviridae, have similar genome and viral structure15. The genome of EV-A71 is a positive-sense single-strand RNA generating a large polyprotein that is subsequently cleaved into three precursor proteins, termed as P1, P2, and P3. P1 encodes four viral proteins (VPs) including VP1 to VP4, while P2 and P3 encode seven non-structural proteins17. The mature viral capsid is icosahedral containing 60 copies of the protomer composed of four proteins, VP1-4. Of these, VP1-3 together forms the shell of the virion whereas VP4 is linked to viral RNA and entirely internal to the capsid. It is believed that neutralization epitopes resided mainly in VP1-3, but only few in VP4  and Rhinovirus (RV), belonging to the https://www.nmpa.gov.cn) in 2015 and 201622. As the only vaccines against HFMD in the world, three inactivated monovalent vaccines against EV-A71 play an active role in health protection of children. However, it has to be noticed that these vaccines are incapable of inducing sufficient cross-protection against other enteroviruses such as CV-A16 and CV-A1023. This means that they can only provide protection against EV-A71-associated HFMD and be futile to other enteroviruses infections. Moreover, since EV-A71 is constantly mutating and evolving, the protective effect of vaccines will gradually decline. Thus, it is necessary to supervise the antigenic evolution of EV-A71 and update the vaccine in time like influenza viruses. Based on the antigenic epitopes of influenza virus, many models or algorithms had been successfully developed to monitor the evolution of its antigenicity, predict dominant strains and prepare vaccines28.A number of vaccines against EV-A71 in China have made significant achievements in clinical trials and three of them have been successfully licensed by National Medical Products Administration of China  and X-ray crystallography were used to determine the three-dimensional (3D) structure of the virus-Fab complexes and recognize the binding region  of Fab with EV-A7135 or CV-A1636.Antigenic epitopes, also called antigenic determinant, are divided into two categories: linear (or continuous) epitopes and conformational (or discontinuous) epitopes. The linear epitopes are formed by continuous amino acid residues, while the conformational epitopes consist of residues that are distantly separated in the sequence but spatially proximal37. So far, only a few conformational epitopes of enteroviruses have been reported and mainly focused on EV-A71 or PV139. The Bioinformatics prediction algorithm, as a supplement method, can quickly and systematically predict the viral epitopes and screen alternative epitopes for experimental research. The basic principle of the algorithm is to take the 3D structure of a protein antigen as a spherical or ellipsoidal shape and search for the sites extremely exposed or easily binding to antibodies42. For example, DiscoTope integrates the propensity scores into an average surface neighborhood attribute and regards discontinuous amino acid residues with high propensity score as conformational epitopes40. However, presently the majority of predicted algorithms for conformational epitopes are not based on the whole viral capsid proteins but a single protein such as VP1. These algorithms do not take into account the structural features of the viral capsid; therefore, the reliability of the prediction for enterovirus is very low. Borley et al. creatively proposed the multiple chains algorithm to predict the conformational epitopes of foot and mouth disease virus (FMDV)43. The algorithm was proved highly reliable in theory and practice. Both human enteroviruses and FMDV belong to the family Picornaviridae and share a similar viral capsid structure. Hence, the multiple chains algorithm developed by Borley et al. can also be competent in prediction for conformational epitopes of human enteroviruses. Based on the multiple chains algorithm, we developed a conformational epitope prediction algorithm for CV-A10 with high prediction reliability, and found that the conformational epitopes of CV-A10 were clustered into three sites 44.As a powerful approach for epitopes determination, Cryo-EM is expensive, time-consuming and labor-intensiveIn this study, the conformational epitopes of EV-A71 and CV-A16 were systematically predicted with a similar algorithm like before and compared with that of CV-A10. The structural differences of the conformational epitopes  among EV-A71, CV-A16 and CV-A10 were explored.The predicted conformational epitopes were shown in Table The sequence alignments of EV-A71 and CV-A16 were shown in Fig. EV-A71 and CV-A16 had highly consistent secondary structures Fig. . The lenα atoms in a protomer) value of 0.231 Å, revealing the much similar 3D structure shared by those two serotypes .At present, seven conformational epitopes of EV-A71 have been reported, mainly including those combined with mAbs 28-746; SP32 binding to the VP1 BC loop and SP55 to VP1 EF loop constituted site 1a and site 1b, respectively47; VP2-28 lying in the VP2 EF loop formed the site 2b48. Similarly, the linear epitopes of CV-A16 also overlapped with the conformational epitopes. PEP32 was located in the VP1 BC loop and constituted the site 1a of CV-A16; PEP71, sitting in the VP1 GH loop, constituted site 2a49; VP3B was located at the N terminus of VP3 and become a part of site 3a50. Furthermore, it could be seen that each conformational epitope of CV-A16 and EV-A71 was covered by linear epitope excepting the site 3 of EV-A71  with EV-A  51, Heparan sulfate (HS)52, Annexin II (Anx2)53, Sialylated glycans54 and Dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN)55. Among them, SCARB2 was an uncoating receptor of EV-A71, while the other five were attachment receptors that supported viral attachment to the cell surface and failed in triggering uncoating. Luckily, the binding sites of SCARB2, PSGL-1, HS and Anx2 have been clearly identified, while that of Sialylated glycans and DC-SIG remained unknown 57. It interacted with VP1 GH loop and VP2 EF loop which constituted site 2a and site 2b, respectively . In this study, we predicted the conformational epitopes of EV-A71 and CV-A16 and conducted the comparison with that of CV-A10. The final predicted epitopes consisted of core epitopes and surrounding epitopes, with the core epitopes defined by a rigid standard that they were simultaneously predicted via Ellipro, DiscoTope, and Epitopia. Nevertheless, the surrounding epitopes just needed to be predicted as epitopes by any two prediction tools of Ellipro, DiscoTope and Epitopia, and were the core epitopes of other related serotypes of enterovirus. For example, the 94D in VP1 of EV-A71 was predicted by two tools; meanwhile, it was also the core epitope of CV-A16. Thus, it was defined as surrounding epitope. Such algorithm design is based on the hypothesis of “similar structure, similar epitope”, with considering that the resolutions of 3D structures are not completely the same and some random errors occur in determination of structures. This kind of algorithm design will not increase the number of epitopes but enhance the exhibition of epitope distribution pattern.Determination of antigenic epitopes, especially the conformational epitopes, is critical for the development of diagnostic reagents and supervision of the virus mutation. As to influenza viruses, the surveillance and analysis of antigenic epitopes have proceeded for several years, which can help to predict the transformation of virus antigenicity, develop and prepare the vaccines59. Vaccines for various viral diseases can reduce mortality and morbidity worldwide. Friendly, determination of epitopes of enteroviruses can be helpful to monitor their mutations and develop vaccines. Because of the 3D structure of CV-A6 mature virion not identified, our algorithm based on the structure wasn’t permitted. In the present study, we systematically predicted the conformational epitopes of EV-A71 and CV-A16 and compared with our previous study (CV-A10)44. The result revealed that the conformational epitopes of EV-A71, CV-A16 and CV-A10 had the same distribution pattern. Specifically, all epitopes consisted of three clusters: site 1, site 2 and site 3. Site 1 was located on the northern rim of the canyon near the fivefold vertex, and site 2 in the puff. Site 3 was divided into two parts: one was located in the knob, and the other was close to the threefold vertex. As representative enteroviruses, PV1 and RVB14, their antigenic epitopes have been systematically explored and epitope-distribution patterns have been summarized60. The epitopes of PV1 and RVB14 were both clustered into three sites  and these sites corresponded to three sites of EV-A71 and CV-A1660. Overall, these data implied the prediction results were of great reliability and three-site distribution pattern was the fundamental role of the conformational epitope distribution of enterovirus.EV-A71, CV-A16, CV-A10 and CV-A6 are the predominately causative agents of HFMD. Among them, EV-A71 and CV-A16 were the most common pathogens in the early outbreaks of the Asia–Pacific region. However, CV-A10 and CV-A6 have become the dominant agents and responsible for several outbreaks of HFMD in recent years30, 18A7 (CV-A16)36, 1D5 (CV-A6)61 and 11G1 (EV-D68)62 all interacted with this area where could also accommodate the attachment receptors PSGL-151, HS52, and Anx253 of EV-A71. The binding of mAbs or receptors to the virus might be regulated by epitopes or nearby residues. For example, the VP1-145, near the epitope residue VP1-242, could regulate the binding of PSGL-1 to EV-A71. When the 145 mutated from G/Q to E, a negatively charged region was formed. It reduced the prominence of the positively charged 242 K side chain and eliminated the ability of EV-A71 binding to PSGL-151. Analogously, both mAbs 28–7 (EV-A71) and 18A7 (CV-A16) can only bind to 145G rather than 145E36. Puff region, the most protruding structure of capsid second to the fivefold vertex, mainly consisted of VP1 GH loop and VP2 EF loop which accommodated the linear epitopes SP70 and VP2-28, respectively48. As respected, mAbs D5 and NA9D7 combined with this region as well and the location of mAb E18 (EV-A71) likewise overlapped with it36. In addition, the uncoating receptor SCARB2 partially bound to this region. Unluckily, it was still unknown whether the uncoating mechanism of E18 was consistent with SCARB234. Knob region was another protrusion on the capsid surface which interacted with mAbs 10D3, D6, A9, E19 (EV-A71) and 2G8 (CV-A10)37. Also, the binding sites of NA9D7 (CV-A16) involved some residues in the knob. Unlike the binding sites of SCARB2 on EV-A71, the KREMEN1 on CV-A10 covered the entire canyon and contacted with the fivefold vertex, puff and knob region63. Of note, there were some receptor-specific amino acid residues in the EF loop of VP2. For instance, the K140 and P141 of VP2 which the KERMEN1 bound to were conserved in multiple enteroviruses including CV-A10, while the residue on the same position of EV-A71 and CV-A16 was G140 and T/N141, respectively. Conversely, the G137 of VP2 was a conserved residue for SCARB2 binding to in multiple enteroviruses including EV-A71, while the S137 was in the CV-A10. This suggested that the residues in the epitopes were essential for receptor-specific binding.Fivefold vertex (site 1), puff (site 2), and knob (site 3) are important characteristics of the enterovirus capsid and most easy to bind to mAbs and receptors. The VP1 loops of five asymmetric subunits are arranged in a crater-like structure, making the area around fivefold vertex most prominent on the capsid surface. MAbs 28-7 (EV-A71)66. On the other hand, a handful of antibodies/antisera could only cross-react with specific genotype strains, which illustrated that both EV-A71 and CV-A16 had relatively stable antigenicity, but some virus strains might have antigenic variations due to mutations in the epitopes68. Thus, it was important to surveil the variations in epitopes continuously.Intra-serotype genetic diversity analysis revealed that BC and HI loops, and C terminus of VP1, EF and HI loops of VP2, and C terminus of VP3 were the regions with high diversity, which exhibited that these secondary structures involved in epitopes were susceptible to high-frequency mutations. Since only a few residue positions of epitopes had high diversity  and most residues of epitopes were conserved, the genetic divergence such as genotypes/clades and the genetic diversity did not alter the antigenicity significantly till now. The conservation of epitopes was consistent with the results of cross-reactions. On the one hand, EV-A71 vaccine and most antibodies/antisera could provide cross-protection against strains of various genotypesOur Bioinformatics epitope prediction method still has some defects. Firstly, this is a structure-based prediction algorithm, and if the 3D structures of virus particles (such as CV-A6 mature virus particles) are not determined, the algorithm will be denied. Secondly, some missing structures in the 3D structures (such as disordered loops) will affect the prediction. Finally, the object of the algorithm is to find the residues on the surface of viral capsid that are easy to bind to antibodies or receptors. However, the bottom of the canyon, a special area, bound by certain receptors is out of consideration.In summary, our conformational epitope prediction results suggest that EV-A such as EV-A71, CV-A16 and CV-A10 have a similar three-site distribution pattern. And sequence variations lead to noticeable 3D structural differences in the loops, N terminus and C terminus where the epitopes reside. This may yield different serotypes that cannot be cross-protected by vaccines. Our research shows that Bioinformatics prediction results can provide meaningful information for epitope research and identification, linear epitope design and important targets for virus mutation surveillance. In the future, we will use these epitopes to study and predict the antigenic evolution and epidemic trends of the major pathogens of HFMD to help monitor its outbreak.http://www.rcsb.org)69. The deduced protein sequences were downloaded from the National Center for Biotechnology Information (NCBI) Nucleotide database (https://www.ncbi.nlm.nih.gov/nuccore/). Also, the 3D structure (PDB ID: 6AKS) and protein sequence (GenBank accession no: KP009574) of CV-A10 (strain FY01/AH/CHN/2013) were downloaded to conduct the sequence and structure comparison. The protein sequences of EV-A71, CV-A16 and CV-A10 were aligned by MAGE X using MUSCLE70, then uploaded with corresponding PDB files to the ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) for noting secondary structures information71. The sequence alignment was uploaded to BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for calculating the amino acid residue identity. The 3D structures of viral proteins and capsid surfaces were generated and compared by UCSF Chimera72 and PyMOL73. The root mean square deviation (RMSD) of structural difference was calculated by PyMOL. All of software set the default parameters. To perform intra-serotype genetic diversity analysis, all complete genome sequences of EV-A71, CV-A16, CV-A10 and PV1 (used for reliability assessment) were downloaded from NCBI Nucleotide database and the deduced protein sequences for VP1-3 were truncated. Entropy based on amino acid residues was calculated to perform diversity analysis using Augur package of Nextstrain platform74. The whole analysis workflow was presented in Fig. EV71/Fuyang.Anhui.P.R.C/17.08/1 (GenBank accession no: EU703812) and Ningbo.CHN/028-2/2009 (GenBank accession no: JQ354992) strains, whose 3D crystal structures have been determined, were selected as representative strains of EV-A71 and CV-A16, respectively. EV-A71 strain, isolated in Fuyang City, Anhui Province, China in 2008, belongs to subgenotype C4. And CV-A16 strain, isolated in Ningbo City, Zhejiang Province, China in 2009, belongs to subgenotype B1a. The 3D structures of EV-A71 mature virus (PDB ID: 3VBS) and CV-A16 mature virus (PDB ID: 5C4W) were obtained from the RCSB Protein Data Bank (PDB) database  containing 60 chains 41, DiscoTope (http://www.cbs.dtu.dk/services/DiscoTope)40 and Epitopia (http://epitopia.tau.ac.il/index.html)42 were employed to predict the multiple chains and the thresholds were set 0.3, -10.7 and 0.174, respectively43. Residues on VPs were predicted as candidate epitopes for whose prediction values were not less than thresholds.Secondly, predict the epitopes of EV-A71 and CV-A16. Three classic prediction tools ElliPro , were defined as core epitopes. Additionally, the residues were merely predicted by any two tools whilst be predicted as core epitopes by other enteroviruses, can be defined as surrounding epitopes. Ultimately, the predicted epitopes included the core epitopes and surrounding epitopes.75. Predicted epitopes, experimental epitopes and receptor-binding sites were marked on footprints to analyze the relationship among them. PV1 is the representative serotype of EV-C and the antigenic epitopes have been extensively and systematically studied. Therefore, we also predicted the conformational epitopes of PV1 (PDB ID: 1HXS) to help assess the prediction reliability.The binding sites of receptors and antigenic epitopes identified by experiments  were obtained from reported researches. Predicted epitopes and experimental epitopes were compared to evaluate the reliability of algorithm. The viral capsid “footprint” of EV-A71 and CV-A16 were produced by RIVEMSupplementary information.


West Nile Virus (WNV) causes a debilitating and life-threatening neurological disease in humans. Since its emergence in Africa 50 years ago, new strains of WNV and an expanding geographical distribution have increased public health concerns. There are no licensed therapeutics against WNV, limiting effective infection control. Vaccines represent the most efficacious and efficient medical intervention known. Epitope-based vaccines against WNV remain significantly underexploited. Here, we use a selection protocol to identify a set of conserved prevalidated immunogenic T cell epitopes comprising a putative WNV vaccine. Experimentally validated immunogenic WNV epitopes and WNV sequences were retrieved from the IEDB and West Nile Virus Variation Database. Clustering and multiple sequence alignment identified a smaller subset of representative sequences. Protein variability analysis identified evolutionarily conserved sequences, which were used to select a diverse set of immunogenic candidate T cell epitopes. Cross-reactivity and human leukocyte antigen-binding affinities were assessed to eliminate unsuitable epitope candidates. Population protection coverage (PPC) quantified individual epitopes and epitope combinations against the world population. 3 CD8+ T cell epitopes  and 1 CD4+ epitope (VTVNPFVSVATANAKVLI) were selected as a putative WNV vaccine, with an estimated PPC of 97.14%. West Nile Virus (WNV) is a mosquito-borne Flavivirus that causes West Nile Fever (WNF) and West Nile neuroinvasive disease (WNND) in birds, humans, and horses . OriginaWNV infection manifests as one of three disease states: asymptomatic carrier, West Nile Fever (WNF), and West Nile neuroinvasive disease (WNND) . After tWNV is part of the Flavivirus genus, which comprises over 70 viruses and many human pathogens, including numerous mosquito-borne viruses . WNV is The WNV genome comprises a single-stranded nonsegmented positive sense RNA of ~11,000 nucleotides . It is tThere are no current FDA-approved WNV treatments. Reducing exposure to mosquitos is the main strategy, via mosquito nets, protective clothing, and insect repellent and by staying indoors . In the The only trialed WNV inactivated vaccine comprised a minimally pathogenic Kunjin virus incubated with hydrogen peroxide . PrM andγ levels [PrM, E, NS3, and NS4B proteins are commonly targeted by CTLs . Long-laγ levels . Vaccineγ levels .The lack of extant WNV vaccines prompts us to evaluate potential epitope ensemble vaccines as an alternative, exploiting our evolving approach to vaccine design. We have exemplified this by identifying putative vaccines against hepatitis C , influenhttp://www.iedb.org/). Epitopes were restricted to host “Humans,” “T cell Assays,” “Positive Assays,” and “Any MHC restrictions.” CD8+ and CD4+ data were collected separately.Experimentally confirmed West Nile epitopes (ID: 11082) were retrieved from the Immune Database and Analysis Resource (IEDB) (https://www.ncbi.nlm.nih.gov/genome/viruses/variation/WestNile/). Incomplete and duplicated sequences were removed. Sequences were clustered using CD-HIT (URL: http://weizhongli-lab.org/cdhit_suite/cgi-bin/index.cgi?cmd=cd-hit) to form a set of representative sequences. Identified cut-off was 0.99. The clustered sequences were multiply aligned using MUSCLE (URL: http://www.ebi.ac.uk/Tools/msa/muscle/) to form multiple sequence alignment (MSA).Complete West Nile Virus genome sequences for all human variants and lineages were obtained from the National Centre for Biotechnology Information (NCBI) West Nile Virus Variation Database Resource (URL: http://imed.med.ucm.es/PVS/). The first sequence in the alignment was used as a reference. Sequence variability was masked, and only fragments with a length greater or equal to 9 were selected. The Shannon entropy threshold was 0.5. CD8+ and CD4+ epitopes with at least 50% overlap were retained for subsequent analysis.Conserved epitopes were identified by analysing conservation of the MSA, using the Protein Variability Server (PVS)  (URL: hthttp://webclu.bio.wzw.tum.de/icrossr/) after determining class I binding profiles, as below. The number of mismatches was set to “0, 1, 2, and 3” for each binding prediction. Epitopes with a cross-reactivity index (ICR) of 0.01 or greater were removed.CD8+ cross-reactivity was analysed using iCrossR (URL: http://tools.iedb.org/mhci/) and CD4+ (http://tools.iedb.org/mhcii/) T cell epitopes were predicted using IEDB. HLA I reference set was used for MHC I epitopes  and an HLA II reference set was used for MHC II epitopes  selecting all ethnic groups in the US population  and a PPC above 95%. Global PPC values for highly-conserved epitopes were calculated using IEDB (http://tools.iedb.org/tools/population/iedb_input). MHC I and MHC II epitopes were then ranked by PPC. Epitopes were combined within each class to calculate overall PPC values. To create a potential ‘universal' vaccine candidate CD8+ and CD4+ epitopes were combined and PPC calculated using IEDB as above.Binding affinities of conserved CD8+ , AJR27898 , AHB37632 , AMZ00438 , and ALK02494 . AHB37632 was discarded due to sequence anomalies.A multiple sequence alignment (MSA) was performed on the remaining 4 sequences. All four lineages were highly conserved: 3069 positions had identical amino acids (89.37%), and only 64 positions showed variable amino acids (1.87%). The remainder was either partially conserved (3.17%) or highly conserved (5.59%). Analysis with PVS showed that 122 of the 165 epitopes had ≥50% sequence identity to the masked WNV reference sequence: 32 CD8+ 9mers, three 10mers, and one 11mer, with 19 epitopes showing 100% sequence identity; 86 CD4+ epitopes had ≥50% sequence identity, with 19 of the 86 epitopes showing 100% sequence identity.As therapeutic peptides can evoke autoimmune responses, conserved epitopes were screened computationally to detect undesirable cross-reactivity against human tissues. Cross-reactivity (CR) was computed using iCrossR for HLA-I 9mer epitopes only. The output for each mutation was averaged to calculate an overall cross-reactivity index (ICR).Our results show that none of the epitopes have an ICR greater than 0.01; thus, it seems unlikely that self-antigen recognition and toxic effects would occur; when tested at the three mutation levels, many epitopes did exhibit CR. HLA-I binding profiles and PPC were calculated using EPISOPT and IEDB. All CTL 9mer epitopes were entered into EPISOPT to estimate the PCC for the five US ethnic groups see . ITYTDVLUsing three epitopes, the maximum PPC was 97.65%, with 24 distinct class I restrictions: TLARGFPFV, GPIRFVLAL, and ITYTDVLRY. 190 different combinations of four epitopes achieved a PPC > 95%. TLARGFPFV and ITYTDVLRY were present in most highly scoring epitope sets, with many epitopes absent from all candidate ensembles.HLA-I binding profiles were also obtained using IEDB. Peptides in the top 1% rank were retained to ensure strong binding and sufficient immunogenicity. More HLA-A binding alleles (14) were seen than HLAB (9). The IEDB PPC tool predicted that the top EPISOPT ensemble had only an 84.68% PPC. To achieve a PPC of >95%, 6 epitopes are needed: TLARGFPFV, ITYTDVLRY, KSYETEYPK, SYHDRRWCF, MPNGLIAQF, and GPIRFVLAL see .HLA-II binding profiles were estimated using IEDB, retaining epitopes in the top 5% rank. PPC calculation showed that individual epitopes had a relatively high PPC, with 8 having values over 50%. VTVNPFVSVATANAKVLI and GEFLLDLRPATAWSLYAV had the highest value: 70.55%. ILVSLAAVVVNPSVKTVR and VTVNPFVSVATANAKVLI achieved a combined PPC of 81.81%. The addition of further epitopes had no effect. Many CD4+ epitopes had binding profiles that were subsets of other epitopes. These epitopes were removed, leaving a set of HLA-II alleles covering all epitopes see . Many ofBoth CD4+ and CD8+ T cells are important in viral clearance. CD8+ and CD4+ epitopes were combined to calculate a PPC, using the PPC tool on IEDB. By combining the top two epitopes from each HLA subset VTVNPFVSVATANAKVLI and ILVSLAAVVVNPSVKTVR, and ITYTDVLRY and TLARGFPFV, a PPC of 96.36% was achieved. A vaccine ensemble comprising ITYTDVLRY, TLARGFPFV, and SYHDRRWCF and VTVNPFVSVATANAKVLI covered 97.14% of the world's populations. This increased to 99.52% when using 11 epitopes see .West Nile Virus has been recognized as a reemerging global pathogen. Present in Africa for over 50 years, recent geographical transmission has raised its profile as a public health concern. There are no current effective treatments, and the cost-to-benefit ratio of the WNV development pipeline is poor. Vaccination is a key intervention. An efficacious WNV vaccine could significantly benefit the global population. Vaccines are available for closely related Flaviviruses and against equine WNV, resulting in a significant reduction in annual mortalities. Veterinary vaccine Equilis West Nile is an inactivated whole virus vaccine comprising a strain known as Yellow Fever-West Nile. The vaccine is given to horses over six months via 2 intramuscular injections, 3 to 5 weeks apart, with a single booster injection given a year later. In comparative evaluations, correlates of protection were seen in 89-94% of treated animals in different test groups. Most previous candidate WNV vaccines have relied on B cell-mediated immunity. Here, we attempt to identify highly conserved T cell epitopes that might form an epitope ensemble sufficiently immunogenic to protect against geographically diverse WNV strains.Epitopes in adoptive immunotherapies may exhibit undesired side effects , such asNo single epitope provided protection against WNV. A PPC > 95% was only possible when multiple epitopes were combined. A potent immune response needs both CD4+ and CD8+ T cell responses . CombineH < 0.5). This lack of variability is an aid to vaccine design as conserved regions can be exploited as therapeutic targets, as newly emerging strains retain conserved amino acids. CD-HIT was used to remove redundant protein sequences. While remaining sequences represented the most common human WNV lineages , the less common lineages (3 and 4) were excluded. To generate a truly universal vaccine, crossprotection against all WNV lineages and strains should be investigated. Thus, a second-generation WNV vaccine may need inclusion of lineages 3 and 4.Virus replication introduces mutations which can eventually generate new strains or alter existing ones; yet, we found that 90% of the WNV sequence is conserved , NS2A (ITY9), NS3 (SYH9), or NS4B (TLA9). Finding highly conserved epitopes in the E protein, NS3, and NS4B is expected, since previous work has shown these proteins to be highly immunogenic and common targets for CTLs , 28. VarThe C region has the highest proportion of residue alterations (~23%) . The E pBy considering functional cross-reactivity with human proteins, the work described here represents a step forward in our evolving approach to vaccine design. Work based on naive sequence similarity have not previously proved useful –35. Here