Among the 40 countries with the highest GDPs, Israel had the highest publications-to-GDP ratio. Among the 20 countries with the most publications, Japan had the highest Attraction Score for induced pluripotent stem (iPS) cells and Italy the highest proportion of review publications. More than 50% of publications in English were from countries in which English is not the primary language. We show an assorted and extensive collection of rankings and charts that will inform scholars and policymakers in studying and improving the research systems both at the national and international level.Studying publication volumes at the country level is key to understanding and improving a country’s research system. PubMed is a public search engine of publications in all life sciences areas. Here, we show how this search engine can be used to assess the outputs of life science-related research by country. We have measured the numbers of publications during different time periods based on the country of affiliation of the first authors. Moreover, we have designed scores, which we have named Attraction Scores, to appraise the relative focus either toward particular types of studies, such as clinical trials or reviews, or toward specific research areas, such as public health and pharmacogenomics, or toward specific topics, for instance embryonic stem cells; we have also investigated a possible use of these Attraction Scores in connection with regulatory policies. We have weighed the statistics against general indicators such as country populations and gross domestic products (GDP). During the 5-year period 2008-2012, the United States was the country with the highest number of publications and Denmark the one with the highest number of publications Some of these efforts have focused specifically on the life science sector by taking advantage of PubMed (http://www.ncbi.nlm.nih.gov/pubmed/)9, a free and public search engine that provides access to over 24 million citations in all fields of life sciences, mostly located in the MEDLINE  bibliographic database. PubMed became accessible to the public at no charge in June 1997 and has been maintained by the United States National Library of Medicine (NLM) at the National Institutes of Health (NIH). Over the years, it has become a very popular database among scientists and nonscientists interested in retrieving research publications in various biological and biomedical fields. One of the most valuable features is users’ ability to restrict searches through different filters and settings. Users can target their searches by restricting the query to publications that meet various requirements—including time of publication, type of publication, type of research, language, authors’ affiliation, journal, and other criteria—and by searching for specific keywords in the full text of the article or in sub-parts, such as title and abstract. The system uses the Boolean operators AND, OR and NOT to facilitate filtering and refining of the searches.Publication output at the country level has been assessed by means of different tools and methodologies. Both academic groups and private companies, by using different and complementary approaches, have offered valuable information to scholars and policymakers11, our study constitutes the most recent, assorted and refined assessment of publication output in the life sciences. Thanks to the ability to attribute a paper to various countries, we present data of publication output by country and supranational regions. Further, we have used various stringency criteria in order to check the method’s robustness. Thus, we have analyzed publication output in different time ranges and calculated publication output in relation to various country-specific statistics  expenditures and presence/absence of English as a primary language). Finally, we have created a new score (Attraction Score) that measures the relative weight of publications for certain kinds of research or certain topics of interest. By analyzing the relationship between the Attraction Scores for human embryonic stem cells and the regulatory policies, we propose an example of how these Attraction Scores could be used to assess the research impact of regulatory policies. We believe that the methodology and the graphic representations of data will provide valuable and easy-to-grasp information that can assist professionals and the general public in understanding and improving biomedical research governance.With the goal of assisting scholars and policymakers in studying and improving research systems at the national and international level, we present a methodology that deploys PubMed to assess “bio” publication output and the sharing of publications for certain types of research and topics of interest. We also present data that can be retrieved using this methodology. Though PubMed search engine and MEDLINE database had been previously used to assess the quantity of research publications of countrieshttp://www.ncbi.nlm.nih.gov/books/NBK3827/#pubmedhelp.Affiliation_AD) . Since this study focuses mainly on publications from 1993 to 2012, we have attributed a paper to a certain country based on the first author’s affiliation. We do not believe that this is a problem: in the vast majority of cases, first author and corresponding author are either the same person or they work in the same research institution/geographical area and therefore have the same country affiliation. To address the problem of countries recorded under multiple names, we have used the different designations separated by the Boolean operator OR. For example, publications of the United Kingdom were searched by inserting “united kingdom OR uk” in the “affiliation” field. Publications of the United States were searched by inserting “us OR usa OR united states” . To identify the year of publication, we used the “Custom range” function . Most of the searches were made for the 5-years period 2008–2012, but we also investigated the 5-years periods 1993–1997, 1998–2002, and 2003–2007 to determine changes in publications volumes over time. Searches were also performed for individual years by using the “Custom range” function or by using the CSV downloads of the automatically retrieved yearly counts. We constrained publication output primarily on papers reporting original research. Therefore, unless otherwise indicated, we excluded reviews from our queries. We did so by using the operator NOT before the word “review” typed in the “Publication Type” field.The number of publications by country was determined by inserting the name/s of the countries in the “affiliation” field in the “advanced” section of the PubMed search engine. In the event of publications with authors based in different countries, we chose to attribute the paper only to the country of the leading author rather than collaborators. This was facilitated by the fact that PubMed only reports affiliation information of the first author of articles published before 2014 . The total numbers of publications for the entire world were calculated by leaving the affiliation fields blank.In addition to the standard criteria discussed so far (criteria A), we also conducted searches using various levels of stringency to criteria A. We thus added the “Journal Article” filter (in “Article types”) and “English” (in “Languages”) to criteria A to generate criteria B; the “Journal Article” filter (in “Article types”) to generate criteria C; the “English” filter (in “Languages”) to generate criteria D. The percentages of publications written in English were estimated by dividing the number of publications obtained using criteria D by the number of publications obtained using criteria A and then by multiplying the result by 100. The countries considered to have English as primary languages are shown in theArea orTopic Attraction Scores”), which were calculated by dividing the number of publications in certain research areas or discussing certain topics, in a certain country, and within a certain period, by the total number of publications in the same country/period and by multiplying it by 10,000 to obtain easy-to-read numbers. The Area/Topic Attraction Scores were calculated by using criteria B because, as the searched areas or topics were English words, we wanted to exclude the (few) publications written in languages other than English. Nonetheless, we run the same query also using criteria A, and we obtained very similar results. Since the results are irrelevant, we decided not to show data of queries with criteria A in this paper. In the case of the “human embryonic stem cells” topic, we also used a “hESC/ESC Score”, defined as the ratio between number of publications obtained by using the search term “human embryonic stem cells” in the “Title/Abstract” field and the number of publications obtained by using the search term “embryonic stem cells” in the “Title/Abstract” field.We chose to retrieve publications in certain specific areas or that discuss specific topics. To this end, we inserted in quotes the chosen research areas or topics of interest  in the “Title/Abstract” field in the “Advanced” section of PubMed. This step allowed as to generate Attraction Scores for areas or topics  and by multiplying the result by 100 (in this case the score is the percentage).http://data.worldbank.org/indicator/SP.POP.TOTL) and GDP data (http://data.worldbank.org/indicator/NY.GDP.MKTP.CD) were retrieved from World Bank databases. We chose 2011 as a reference year. R&D expenditure data were retrieved from Battelle (a private nonprofit science and technology development company) 2014 Global R&D Funding Forecast  and expressed as GERD (Gross Expenditure on Research and Development) of year 2012 with Purchasing Power Parity in US$ billion. Data on population by continent were obtained from estimates published by the US Census Bureau (http://www.census.gov/compendia/statab/2012/tables/12s1330.pdf) for the year 2010. The numbers of total citable documents by country were retrieved from the SCImago Journal & Country Rank (http://www.scimagojr.com/index.php).We studied publication output during the 5-year period 2008–2012 relative to population, gross domestic products (GDP), and R&D expenditure. Population data . This shows that PubMed is rapidly updated and stable across time.http://www.hinxtongroup.org), a consortium fostering international cooperation in stem cell research. Other sources for country-specific policies are referenced in the Results section.The classification of policies that regulate the use of human embryonic stem cells is based on the one previously developed by the Hinxton Group .We determined the number of publications of countries for the 5-year periods 1993–1997, 1998–2002, 2003–2007, and 2008–2012. We divided the sums of publications for all countries by the total numbers of world publications , in the same time periods. We used both criteria A and criteria B for this analysis. We observed that the percentage of publications containing a country name in the affiliation increases with time. In the time period 2008–2012, the proportion of papers with a country name in the affiliation is 87.8% and 97.7% by using criteria A and criteria B, respectively   for the time periods 1993–1997, 1998–2002, 2003–2007, and 2008–2012. Publications increased with a nearly constant rate during the four time periods, with the volume of the time period 2008–2012 corresponding to over 3.8 million publications (reviews excluded according to our above described searching criteria), roughly the double of the volume of the time period 1993–1997  , for the time periods 1993–1997, 1998–2002, 2003–2007, and 2008–2012. Also, this type of publication increased at a nearly constant rate throughout this time span. Interestingly, the share of publications based on clinical trial studies has remained nearly constant  during this time span  . In sheet 3 we show the differences in the total world publications (i.e. no affiliation specification) for the four different criteria. The differences were very small: below 1.5% for all four criteria. We also compared 2013 vs 2014 searches for the publications of countries with the most publications. Even in this case the results were very similar, and the average difference for the 20 countries with the most publications in the time period 2008–2012 was only 0.6%, with a maximum difference for Iran (6.1%) (sheet 4). These data confirm that PubMed is a reliable search engine that accurately retrieves information from databases that are promptly updated.Using the same method described above, we estimated the percentage of publications in English for the 20 countries with the most publications in the time period 2008–2012. With the exceptions of France (88.3%) and China (78.8%), more than 95% of publications of all other countries were written in English  . On average, review publications constituted 9.9% of the publications, ranging from a maximum of 14.7%  to a minimum of 2.9% for South Korea  (sheet 4per capita of countries (sheet 5). We divided the country publications in the time period 2008–2012 by the country population and multiplied by 1,000 in order to obtain the numbers of publication per 1,000 people.per capita. Iran (0.4), Brazil (0.3), China (0.2), and India (less than 0.1) were, in descending order, the countries with lowest publicationsper capita. We also ranked all other countries based on their publicationsper capita.per capita, respectively. The top ranked country was Denmark (which is not part of the group of 20 countries with the most publications) with 4.8 publications per 1,000 people in the time period 2008–2012. Switzerland (4.8), Sweden (4.4), and the Netherlands (4.2) closely followed Denmark in this ranking.We measured the publicationsWe analyzed the publication output of the 40 countries with the highest GDPs for the year 2011 and reported the data for the 20 and 40 countries with the highest GDPs, respectively inMoreover, we calculated the number of publications per GDP for the 20 and 40 countries with the highest GDPs  . We calculated these scores for the 20 countries with the most publications  .http://www.scimagojr.com/). We ran the comparison for a single year (2012).We compared publication output in the life sciences with the overall output of all disciplines. Data on the latter were obtained from the SCImago Journal & Country Rank, which includes journals and country scientific indicators from the information contained in the Scopus® database  in time periods 2003–2007 and 2008–2012. The publications during the time period 2008–2012 were more than one million  sheet 2. The relPubMed provides a function to automatically download CSV files with the numbers of publications year by year. We compared the numbers of publications obtained by using this function with the numbers of publications obtained by using the custom range function to define time periods that we described above. An analysis of the 2012 reveals that the CSV download yielded fewer publications than the custom range approach.We determined the numbers of publications of the 20 countries with the most publications for the years 2013 and 2014. We observed that the patterns of countries' publications were similar in the two years. We also analyzed as before the review publications and noticed that the patterns were similar .Assessing the quantity and quality of a country’s scientific output is key to understanding and improving its research system. In this paper, we present a methodology that focuses on publication in biology and related disciplines that contribute to advancing that field. Based on the search engine PubMed, the method was used to count publications at the country level and at the supranational level (continents and European Union), in different time periods, to trace changes in publication outcome over time and to measure publication ratios relative to country populations, country GDP and R&D expenditure, the share of publications based on clinical trials, whether the publication was a review article, and whether the publication was written in English. We demonstrated how various stringency criteria could be deployed to check data robustness.Box 1. The total number of publications during years 2008–2012  is nearly the double the number of publications during years 1993–1997The proportion of publications based on clinical trials has remained constant around 5% since 1993The proportion of review publications for the 20 countries with the most publications is 9.9%, with a maximum for Italy (14.7%) and a minimum for South Korea (2.9%) (years 2008–2012)The United States, with over one million publications in years 2008–2012 (reviews excluded) is by far the country with the most publications, having almost four times the publications of the second ranking country, Chinaper capita is Denmark , followed by Switzerland  and Sweden  (years 2008–2012)The country with the most publicationsThe country with the highest publications-to-GDP ratio, among the 40 countries with the most publications, is Israel, followed by the United Kingdom, the Netherlands, and South Korea (years 2008–2012)The country with the highest publications-to-R&D expenditure (GERD PPP), among the 20 countries with the highest GDPs is the United Kingdom, followed by Italy, Turkey, and the NetherlandsAmong the 20 countries with the most publications, Iran is the country with the highest relative increase (+220%) in the number of publications from years 2003–2007 to years 2008–2012. Japan is the country with the lowest relative increase (+9.7%)Among the 20 countries with the most publications, Iran is the country with the highest relative increase (+179.6%) in the number of clinical trial publications from years 2003–2007 to years 2008–2012. Israel is the only country with a decrease (-14.6%)The United States is the country with the most publications based on clinical trials , nearly four times the number of publications based on clinical trials in the second ranking country, the United Kingdom (years 2008–2012)Among the 20 countries with the most publications, the Netherlands is the country with the highest “Clinical Trial Attraction Score”, followed by Italy (years 2008–2012)Among the 20 countries with the most publications, the Netherlands is the country with the highest “Pharmacogenomics Attraction Score” (years 2008–2012)Among the 20 countries with the most publications, the United States is the country with the highest “Personalized Medicine Attraction Score” (years 2008–2012)Among the 20 countries with the most publications, Japan is the country with the highest “iPS cells Attraction Score” (years 2008–2012)Among the 20 countries with the most publications, South Korea is the country with the highest “hES cells Attraction Score”, (years 2008–2012)There is a relationship between policies regulating the use of human embryonic stem cells and the “hES cells Attraction Scores”Among the 12 countries with the most publications, Australia, the United Kingdom, and Canada, in descending order, have the highest “Public Health Attraction Scores” (years 2008–2012)The estimated proportion of publications written in English in countries where English is the primary language has decreased from 50.0% (years 2003–2007) to 46.1% (years 2008–2012)per capita  followed by North-Central America  (years 2008–2012)Oceania is the continent with the most publicationsNorth-Central America has the highest proportion of world publications (35.5%); Africa has the lowest (1.4%) (years 2008–2012)The proportion of world publications of the present day 28 countries of the European Union is 30.6% (years 2008–2012)We have also created the Attraction Scores. These assess the focus of publication output on certain types of studies  or areas or topics of interest . Attraction Scores express a proportion of the total publication output rather than the number of papers. For this reason, we believe that they better represent the real attraction of certain types of studies or certain topics of research to scientists.14. The predictable result is that Japan has indeed the highest Attraction Score for iPS cells of all surveyed countries. Variations in Attraction Scores can also be correlated with other factors, including the prevalence of certain diseases, the structure of research workforces , regulatory policies, and cultural and political factors. Attraction Scores can be used not only to assess the effect of putative determining factors but also to predict future trends. For example, the focus toward certain areas (or topics) or types of research can have different consequences such as the development of new avenues of research or new types of interaction between academia and industry.Attraction Scores can be correlated to many different factors. It is no surprise that this attraction for a cutting-edge topic like “pharmacogenomics” is higher in more technologically developed countries . Higher 15. In restrictive policy countries, human embryo research, derivation of hESCs from supernumerary embryos, and somatic cell nuclear transfer (SCNT) are usually prohibited. When permitted, research is possible with imported hESC lines or cell lines that were created before the enactment of any embryo research ban. Predictably, the two countries with the most restrictive policies  report very low Attraction Scores 16. In 2013, “at least 68 centers” with more than 350 researchers were “investigating stem cells and regenerative medicine” )17. The United States, another apparent outlier, is classified as a case of “permissive compromise”. Yet, the reality has been different. Policies were restrictive only at the federal level with theDickey-Wicker Amendment and President Bush’s a ban on federal funding for research on certain stem cells. However, many US states funded and promoted a significant amount of stem cell research18, which resulted in a high Attraction Score for both 2008–2012  for government funding for stem cell research . Moreover, in some cases the name/acronym of a country could be the same as other affiliation specifications. This particular case may lead to overestimating the number of publications for that country. For example, the acronym US could be present in affiliations not related to the United States, and therefore, even if we believe that  this effect is conceivably negligible, there might be the possibility that the searches for the United States are slightly overestimated. Also, there are cases in which a name can be both a country and a region of another country. This is the case of Georgia, whose number of publications are likely overestimated because “georgia” may be listed as a sovereign country or a state that is part of the United States.Third, this method relies on first authors’ affiliation information to attribute publications to a certain country. Given that the first author ordinarily either matches the corresponding author or has the same affiliation country of the corresponding author and that the first author (even when she/he is not the corresponding author) frequently plays a prominent role in the project design or execution, this approach has the advantage of classifying papers based on the effective location of the main ideation and execution of research projects. Furthermore, even if some researchers have multiple affiliations, it is plausible that in many cases the multiple affiliations of a single researcher are from the same country; in addition, the single affiliation listed in PubMed, being the one usually more involved in the execution of the research project that led to that specific publication, is most likely the one that better serves our analytical purposes.http://www.ncbi.nlm.nih.gov/books/NBK3827/#pubmedhelp.Affiliation_AD). Therefore, the method used in this paper cannot be used for papers published in 2014 or after. While PubMed’s decision to include information for all authors of a paper is welcomed, it would be desirable to be able again to select affiliation information only for the first  author. In any case, this recent PubMed change offers the possibility to assess the countries’ biomedical publications by taking in consideration all contributing authors, including authors with minor contributions . Even if assessing the countries’ biomedical publications based on the first author provides slightly different information from an assessment based on all authors, we expect that the patterns presented in this paper will not substantially change even if the new settings of PubMed were to be used. This is also suggested by the very similar patterns of publications in the years 2008–2012, 2013 (the last year with the old settings) and 2014 (the first year with the new settings)  . At any Fourth, PubMed’s records may be incomplete and thus not perfectly accurate. This is the case of information such as the language in which the paper is written and whether the paper is a review.Fifth, even if we excluded reviews from our counts (except where otherwise indicated), we did not exclude all publications that are not based on original research. This is the case of comments and editorials. Our choice was motivated by reasons of simplicity and because we believed that PubMed’s tagging of publications as “letters” or “editorials” could be not fully accurate and possibly unequal among countries. However, we believe this choice does not have an impact on data because it is plausible that these types of publications do not significantly affect the relative differences between countries as suggested also by the fact that, when we chose to count only the publications published as “article journal” (criteria C), we did not observe substantial differences with the standard criteria (criteria A) (sheet 3).20, we argue that any means of measuring the quality of science will always be partial and controversial and for this reason it will always be useful to take in consideration also  the total volumes of publications. Regardless, the methods and results presented in this paper are not to be intended as neither exclusive nor the perfect means of assessing countries’ research output. In fact, they are better seen as complementary to other methods and results.Sixth, the quality  of publications is not taken in account. This methodology quantifies the research output by determining the numbers of publications or ratios between numbers of publications and other variables. Proxies for the quality of the papers, such as numbers of citations, numbers of downloads, and impact factors of the journals, are not taken in consideration. Even if indexes based on the quality of research have been already proposedbona fide representation of the whole literature. Moreover, the fact that the paper is based on a database that is free and easily accessible by everyone in the world, certainly adds value to the results as it provides tools that can be freely used by anyone and it facilitates readers access to the underlying data, reproducibility, and comparison with our approaches.Seventh, though PubMed is a search engine that is based on authoritative and comprehensive databases, it might not retrieve some publications. For this reason, it will be important to confirm these results by analyzing also other authoritative and comprehensive databases like Scopus (Elsevier) and Web of Science (Thomson Reuters) that might contain a few publications not retrieved by PubMed. In any case, given the comprehensiveness and breadth of PubMed databases, we believe that the results presented in this paper are aIf the limitations are taken into account, we believe that the methodology and information presented in this paper can be used, in conjunction with other metrics, to assess research systems in terms of publication output. In particular, we think that the volumes of publications, the relative changes in time, and the Attraction Scores described here provide valuable and unique information about the biomedical and biological research systems of countries. This will assist scholars in studying research systems and policymakers in designing policies to improve scientific production and its benefits to society.The data referenced by this article are under copyright with the following copyright statement: Copyright: © 2015 Xu Q et al.Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).http://dx.doi.org/10.6084/m9.figshare.124689821figshare: Biomedical publication and Attraction Score data based on PubMed searches, My suggestions have been considered and all my questions positively answered.I agree that this article should be indexed.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This paper analyzed the volume of publications in the field of life sciences by country and continent in order to better understand regional differences in life sciences research infrastructure. The authors additionally allocated scores to quantify focal areas of interest and examined the potential use of these scores by exploring the relationship between Attraction Score and research policy on hESCs. This report is interesting, providing a detailed analysis with a graphical representation of data that facilitates a smooth understanding of the study's conclusions. However, as the first reviewer has mentioned, a limitation seems to be present in terms of the data source used for the study. PubMed is a comprehensive and authoritative database but is generally unable to provide an exhaustive data package for scientometric studies. Although the authors have claimed that the study covers the most recent, diverse and refined research output (via PubMed), the number of publications is likely to be only illustrative of the quantitative aspect of the research output. Similarly, the affiliation of the first author provides partial information on the contribution of each country to the publication. Instead, WoS or SCOPUS may be more useful data sources, allowing analysis of the citations and journal impact factors for all authors, as shown in previous studies. In addition, whether the Attraction Scores designated by the authors distinguish between the proportion of studies focused on the topic of interest and the entire number of publications remains open to speculation. Finally, the 'correlation analysis' referred to in this study is not an actual correlation analysis . It is recommendable to change the term in order to avoid confusion for the reader.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. We would like to thank Professor Youngim Jung for taking the time to read the paper and provide their helpful feedback.Here we provide our responses to their comments:bona fide representation of the literature. In any case, we agree that it will be important to crosscheck these data with data obtained by using other databases such as Scopus (Elsevier) and Web of Science (Thomson Reuter). To address this point, we have added the following paragraph to the Discussion section:We agree with the referee (as well as with the other referee) that, although PubMed is a search engine based on comprehensive and authoritative databases, some published papers might not be retrieved by searching in these databases. Having said this, we think that given the comprehensiveness and breadth of the PubMed system, the results presented in this paper are a"Seventh, though PubMed is a search engine that is based on authoritative and comprehensive databases, it might not retrieve some publications. For this reason, it will be important to confirm these results by analyzing also other authoritative and comprehensive databases like Scopus (Elsevier) and Web of Science (Thomson Reuters) that might contain a few publications not retrieved by PubMed. In any case, given the comprehensiveness and breadth of PubMed databases, we believe that the results presented in this paper are a bona fide representation of the whole literature. Moreover, the fact that the paper is based on a database that is free and easily accessible by everyone in the world, certainly adds value to the results as it provides tools that can be freely used by anyone and it facilitates readers access to the underlying data, reproducibility, and comparison with our approaches."           Moreover, in order to stress the point that our method and results are based on PubMed, we have slightly changed the title of the paper.We agree with the referee that the analysis based on the first author has, together with pros, some cons; for this reason we think that it will be useful to complement this analysis with analyses that include all authors. We have therefore added the following paragraph to the Discussion section:"In any case, this recent PubMed change offers the possibility to assess the countries’ biomedical publications by taking in consideration all contributing authors, including authors with minor contributions . Even if assessing the countries’ biomedical publications based on the first author provides slightly different information from an assessment based on all authors, we expect that the patterns presented in this paper will not substantially change even if the new settings of PubMed were to be used. At any rate, repeating this study by taking in consideration all authors could be informative to determine which countries have a propensity for leading (i.e. most of ideation and execution) versus assisting (i.e. least of ideation and execution) roles in biomedical research. However, this can only be done in the future, once that enough literature will be archived under the new settings ."We agree with the referee that analyses of citations and impact factors as well as of other putative quality indicators are not present in this paper. However, this was beyond the purposes of our research and we have already acknowledged this in the first version of the paper with the following paragraph of the Discussion section:1– 6, 10, 11, 20 , we argue that any means of measuring the quality of science will always be partial and controversial and for this reason it will always be useful to take in consideration also  the total volumes of publications.""Sixth, the quality  of publications is not taken in account. This methodology quantifies the research output by determining the numbers of publications or ratios between numbers of publications and other variables. Proxies for the quality of the papers, such as numbers of citations, numbers of downloads, and impact factors of the journals, are not taken in consideration. Even if indexes based on the quality of research have been already proposedMoreover, we have now added the following paragraph:"Regardless, the methods and results presented in this paper are not to be intended as neither exclusive nor the perfect means of assessing countries’ research output. In fact, they are better seen as complementary to other methods and results."Regarding the comment of the referee on the “Attraction Scores”, we do not claim they are a perfect means of measuring the relative focus towards certain topics or areas of research. To avoid any ambiguity on this point we have made a few edits to the text. In particular, the term “assess” has been replaced with “appraise” or “study”. We agree with the referee that the term “correlation analysis’ can create confusion. For this reason, as suggested by the referee, we have changed the term throughout the manuscript. In same cases we have substituted it with the term “connection” while in other cases we have replaced it with the term “relationship”.We would like to thank again Professor Youngim Jung for taking the time to read our paper and provide her helpful feedback. We hope we have addressed all her reservations. We would also like to bring to the attention of our reviewers (Dr Vafa Rahimi Movaghar and Dr Youngim Jung) that we have updated figure S22 and added new figures S23-S25. In these figures we provide an analysis of year 2014. In particular, we observed that the patterns of publications were similar in the years 2008–2012, 2013 (the last year with the old settings regarding the affiliation) and 2014 (the first year with the new settings regarding the affiliation). We have accordingly slightly updated the paragraph added to the Discussion section:“In any case, this recent PubMed change offers the possibility to assess the countries’ biomedical publications by taking in consideration all contributing authors, including authors with minor contributions . Even if assessing the countries’ biomedical publications based on the first author provides slightly different information from an assessment based on all authors, we expect that the patterns presented in this paper will not substantially change even if the new settings of PubMed were to be used. This is also suggested by the very similar patterns of publications in the years 2008–2012, 2013 (the last year with the old settings) and 2014 (the first year with the new settings) . At any rate, repeating this study by taking in consideration all authors could be informative to determine which countries have a propensity for leading (i.e. most of ideation and execution) versus assisting (i.e. least of ideation and execution) roles in biomedical research. However, this can only be done in the future, once that enough literature will be archived under the new settings .”  et al. entitled "Countries’ Biomedical Publications and Attraction Scores" with great interest. Authors evaluated biomedical publications of all countries based on the PubMed and performed great job on different criteria to assess changes during different time periods based on the country of affiliation of the first authors especially during the five-year time period of 2008-2012 versus 2003-2007. The study is an addition to the literature and fruitful. I would recommend reading the paper to all medical scientists in the world. However, there are some concerns. PubMed is not the only search engine to be evaluated. There are other valuable sources such as ISI, Embase and Scopus which may change the results of the study. We would know that there are many journals indexed in the above-mentioned search engines especially written in the developing countries. On the other hand, quantity evaluation does not show the real scientific situation of the countries. I myself prefer the citation without self-citation for the best index of quality assessment. Other indices such as H-index reveal the mixture of quantity and quality assessment. The affiliation of the first author is acceptable, but not ideal to show the comprehensive situation of the countries. All authors’ affiliation or at least first and corresponding authors affiliations are better replacement in the future studies. Authors mentioned that they reported original articles. Therefore, they deleted reviews. However, I would recommend not deleting the review articles unless simultaneously deleting letters and correspondences.I read the paper of Qinyi XuI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We would like to thank Professor Vafa Rahimi Movaghar for reviewing our article. We are glad that the reviewer appreciated our study.The reviewer is right about the fact that PubMed is not the only source of biomedical publications and that some journals are not indexed in PubMed. We will certainly add remarks on this point in the revised version of the manuscript, which we will submit after receiving feedback from all reviewers.The reviewer mentions about the fact that our method measures only the quantity of publications. He is right but we have already addressed this issue in the Discussion section of the paper. In particular, we do not claim that our data can fully measure the efficiency of countries in biomedical research. Our data and methods need to be complemented with other data and methods. Our revised draft will certainly emphasize this point further.The reviewer mentions the limitations of using first authors’ affiliations. His comment has merit and in fact we have already acknowledged the pros and cons of this approach in the Discussion section.The reviewer mentions that it would be better to include reviews publications in the publication count. While we decided not to include reviews in the majority of calculations, we included them in some other calculations. We have observed that reviews amount to a rather small proportion of publications and consequently their inclusion does not substantially affect the rankings (this was done for years 2012 and 2013). We discuss this issue in the Results and Discussion sections. Here are some preliminary answers to his few concerns.We would like to thank again Professor Vafa Rahimi Movaghar for taking the time to read the article and providing his feedbackSincerelyThe authors We would like to confirm our first response to Professor Vafa Rahimi Movaghar. We have waited for a second review before revising the paper based on the reviewers’ suggestion. We now provide a revised version based on the feedback received from both referees.We would like to thank again professor Vafa Rahimi Movaghar for taking the time to read and comment our paper and for this helpful feedback. We would also like to bring to the attention of our reviewers (Dr Vafa Rahimi Movaghar and Dr Youngim Jung) that we have updated figure S22 and added new figures S23-S25. In these figures we provide an analysis of year 2014. In particular, we observed that the patterns of publications were similar in the years 2008–2012, 2013 (the last year with the old settings regarding the affiliation) and 2014 (the first year with the new settings regarding the affiliation). We have accordingly slightly updated the paragraph added to the Discussion section:“In any case, this recent PubMed change offers the possibility to assess the countries’ biomedical publications by taking in consideration all contributing authors, including authors with minor contributions . Even if assessing the countries’ biomedical publications based on the first author provides slightly different information from an assessment based on all authors, we expect that the patterns presented in this paper will not substantially change even if the new settings of PubMed were to be used. This is also suggested by the very similar patterns of publications in the years 2008–2012, 2013 (the last year with the old settings) and 2014 (the first year with the new settings) . At any rate, repeating this study by taking in consideration all authors could be informative to determine which countries have a propensity for leading (i.e. most of ideation and execution) versus assisting (i.e. least of ideation and execution) roles in biomedical research. However, this can only be done in the future, once that enough literature will be archived under the new settings .”


Immunoglobulin type gamma 4 (IgG4)-related disease is a relatively newly described clinical entity characterized by a distinctive histopathological appearance, increased numbers of IgG4 positive plasma cells and often, but not always, elevated serum IgG4 concentrations. The most common renal manifestation of IgG4-related disease is tubulointerstitial nephritis marked with proteinuria, hematuria, decreased kidney function, hypocomplementemia, and radiologic abnormalities. Renal biopsy characteristics include dense lymphoplasmacytic tubulointerstitial nephritis that stains for IgG4, storiform fibrosis, and immune complex deposition in the interstitium and along tubule basement membranes. Treatment traditionally consists of prolonged glucocorticoids but cases refractory to glucocorticoids have been reported.We report a case of a 58-year-old Caucasian man who presented with fatigue, 50 pound weight loss, dyspnea, lymphadenopathy, and nephromegaly. The patient was first misdiagnosed as chronic interstitial nephritis secondary to renal sarcoid and was treated with repeated doses of prednisone. On his third relapse, he underwent a repeat renal biopsy and a diagnosis of IgG4-tubulointerstitial nephritis was confirmed. He was refractory to treatment with prednisone. The patient received Rituximab and had prompt sustained improvement in renal function. At 1 year post Rituximab treatment, his serum creatinine remains at baseline and imaging study revealed reduction in his kidney size.This is the first case report using Rituximab as a steroid sparing option for refractory IgG4-tubulointerstitial nephritis. More information is needed on the long-term effects of using of B-cell depleting agents for glucocorticoid resistant IgG4-tubulointerstitial nephritis. His complete blood count and complete metabolic panels were unremarkable. All further inflammatory and infectious studies were negative, including angiotensin-converting enzyme, antinuclear cytoplasmic antibodies, cryoglobulins, anti-RNP/Smith antibodies, serum and urine protein electrophoresis, hepatitis panel, and testing for human immunodeficiency virus.A 58-year-old Caucasian man presented with fatigue, 50 pound weight loss, and dyspnea. Significant medical history included hypertension, diabetes, bipolar disorder, monoclonal gammopathy of undetermined significance, and remote stage 2 adenocarcinoma of the sigmoid colon treated surgically without chemotherapy or radiation (6 years prior). He initially presented with these symptoms to his oncologist during an annual follow-up. His physical examination was notable for stage 1 hypertension, BMI 30, relative euvolemia with no chest, cardiac, or abdominal abnormalities. His kidneys were not ballotable. He had no palpable lymphadenopathy and no skin lesions. Laboratory work revealed a creatinine of 2.1 mg/dL, up from 0.9 mg/dL 7 months prior, corresponding to a decline in his estimated glomerular filtration rate (eGFR) from 66 mL/min/1.73 m2. Light microscopy . There was persistent heterogeneity of the renal parenchyma and a soft tissue thickening was noted around the proximal renal collecting system bilaterally. His renal ultrasound also showed bilateral nephromegaly  without lesions or masses. He was referred to a nephrologist and kidney biopsy was performed, which showed chronic interstitial nephritis (not shown). Due to his biopsy findings, mediastinal lymphadenopathy and calcified nodules on imaging a diagnosis of sarcoidosis was suspected, and he was started on prednisone 60 mg with a 2-month taper to a maintenance dose of 10 mg. While consuming high-dose prednisone, the patient was hospitalized for an acute manic episode and elevated blood sugar levels resulting in a change in his oral hypoglycemic drug regimen. His creatinine initially fell to 1.0 mg/dL, but a further attempt to taper prednisone resulted in a rebound elevation in his creatinine again treated with high-dose prednisone that was tapered over 2 months. For 7 months he was maintained on prednisone 10 mg/day and despite this he suffered a third relapse in renal function  . Despite this, the patient's creatinine continued to rise to 2.2 mg/dL. Given the patient's glucocorticoid resistance and intolerances, he was treated with Rituximab (4 weekly doses of 375 mg/m2 each equating to 900 mg). One year after treatment with Rituximab, his creatinine was 1.5 mg/dL [and has remained free from all immunosuppressive therapy over 1 year  achieved disease response that was maintained at 6 months.7 Rituximab can be effective in allowing discontinuation of all immunosuppressant's with durable remission in a number of cases of IgG4-RD .8 Other radiographic abnormalities of IgG4-TIN include irregularly shaped hypoattenuated lesions and pseudotumor masses.8 In our case, Rituximab appeared to reduce his bilaterally enlarged kidneys to a relatively normal size . The patients estimated GFR normalized at his baseline GFR of 50 mL/min/1.73 m2.Nephromegaly is not an unusual finding in IgG4-TIN, and potentially caused by cellular infiltration in the renal interstitiumKlebsiella urinary tract infection, and 1 report of cold agglutinin-mediated hemolytic anemia.7 In 1 systematic review, Rituximab was associated with increased leukopenia and cardiovascular morbidity.11 However, evidence from long-term large-scale data is lacking. Our patient tolerated Rituximab well and did not develop any immediate side-effects. More importantly, he was free from glucocorticoids 9 weeks after his initial diagnosis and more than 1 year after his first dose of Rituximab.In general, Rituximab is well tolerated with the most common side effects related to infusion reactions . In a small prospective trial of Rituximab use in IgG4-RD, there was 1 report of 10 A randomized clinical trial is needed to define the optimal use and to determine the appropriate role of B cell depletion in IgG4-RD. More information is needed on the long-term effect of using of B-cell depleting agents for glucocorticoid-resistant IgG4-TIN, and the trajectory of disease in these patients.In summary, we describe the first case report of successful treatment of glucocorticoid refractory IgG4-TIN with Rituximab. Rituximab provided the opportunity for our patient to be free from glucocorticoids 9 weeks after his initial diagnosis. Our patient was a select case, he had an intolerance to glucocorticoids given his mania and hyperglycemia, but he also had lack of clinical effect with glucocorticoids after his third relapse with IgG4-renal disease. This provided the bases for use of an alternative immunosuppressive agent such as Rituximab, in the treatment of IgG4-TIN. Despite the significant degree of tubular atrophy and fibrosis seen on initial biopsy, our patient had preserved renal function and remained free of disease progression and dialysis over 1 year post Rituximab treatment. Rituximab afforded the opportunity to treat IgG4 disease without the toxicities observed by glucocorticoids. Despite the successful use of Rituximab in the treatment of refractory IgG4-TIN in this case, it should highlight glucocorticoids remain the first-line treatment for most patients with IgG4 disease and B-cell depleting agents may be considered in those patients with a major contraindication to glucocorticoid therapy or an incomplete response to glucocorticoids as observed in our patient.


There are errors in the Funding section. The correct funding information is as follows: This project was supported by the President's Emergency Program For AIDS Relief and the NIH Office of AIDS Research, through NIH Grant Number U01 AI100015 to author SR at Boston University, from the National Institute of Allergy and Infectious Diseases (NIAID). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIAID. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


IgG4-related disease is a newly recognized fibroinflammatory disorder that has the ability to affect nearly every organ system. It is characterized by tumefactive lesions and fibrosis and closely mimics neoplasms. Only one case of IgG4-related bladder mass has been reported in the literature, but there are no reports of IgG4-related disease in a urachal mass. Herein, we report a 26-year-old male who initially presented with symptoms of recurrent UTI. Work-up revealed a 6 cm urachal tumor, a 1.4 cm pulmonary lesion, and mediastinal lymphadenopathy; all metabolically active on PET scan and suspicious for urachal adenocarcinoma. Lung lesion fine needle aspiration and TURBT pathology revealed inflammation but no evidence of malignancy. The patient underwent a partial cystectomy and umbilectomy with pathology demonstrating dense plasmacytic cells, a high rate of immunohistochemistry staining positive for IgG4 plasma cells, a storiform pattern of fibrosis, and an obliterative phlebitis. Furthermore, the patient had an elevated serum IgG4 level of 227 mg/dL (range 2.4–121 mg/dL). IgG4-related disease is a newly recognized fibroinflammatory disorder that can mimic neoplastic processes and a high index of suspicion and accurate tissue pathology is necessary for an accurate diagnosis. IgG4-related disease is a newly recognized condition characterized by a fibroinflammatory process that leads to lymphoplasmacytic infiltration containing IgG4-positive plasma cells, fibrosis, and tumefactive lesions than can be locally destructive . ElevateIgG4-related disease can involve the genitourinary system. Reported cases include interstitial nephritis, ureteral obstruction due to ureteral pseudotumor or retroperitoneal fibrosis, prostatitis, and epididymoorchitis . There hThe patient is a 26-year-old male who presented with symptoms of recurrent urinary tract infections despite antibiotic therapy. An abdominal ultrasound revealed a 6-cm mass superior to the bladder consistent with a urachal tumor . ComputeIgG4-related disease is a newly recognized disorder that has been reported in nearly every organ system. IgG4 is the least abundant subclass of IgG, accounting for less than 5% of total IgG. It does not activate the complement pathway and therefore is thought to play little role in immune activation. IgG4 usually only responds to prolonged and repeat antigen exposure and is involved in the anti-inflammatory process. It is theorized that molecular mimicry in genetically predisposed individuals and autoimmunity are responsible for IgG4-related disease. The activation of regulatory T cells is thought to be unique to IgG4-related disease, resulting in overexpression of transforming growth factor beta and promotion of fibrosis. Patients typically have a subacute presentation, either with solitary organ involvement with silent systemic involvement or with multiorgan involvement .In this patient presentation, physical examination and image findings were suspicious for metastatic adenocarcinoma of the urachus. IgG4-positive plasma cells can infiltrate tissue involved by cancer. Therefore, proper tissue sampling and pathologic review must be performed if malignancy is suspected, as the presence of IgG4-positive plasma cells in tissue is not in itself diagnostic of IgG4-related disease. An international symposium was held in Boston in 2011 to better define the histopathology of IgG4-related disease and provide guidelines for pathologic diagnosis, which is based primarily on morphologic appearance . Dense lIgG4-related disease has been described in a wide variety of organs, but to our knowledge, this is the first reported case of IgG4-related disease presenting as a urachal tumor. This case meets the criteria for IgG4-related disease in a new organ, as outlined by the consensus statement on IgG4-related diseases . HistopaTreatment of IgG4-related disease typically consists of glucocorticoid therapy but can vary based more on which organs are involved and the degree of fibrosis present. Progressive fibrosis can obviously impair organ function if left untreated and can be so extensive that it becomes unresponsive to steroid therapy. No clinical trials have been performed to determine the optimal treatment course, but other classes of immunosuppressants have been utilized with some success . In our Clearly, a high index of suspicion by pathologists and clinicians is necessary to appropriately diagnose and treat IgG4-related disease, an increasingly recognized condition. Increased awareness will hopefully improve diagnostic and treatment strategies.


However, the frequency and significance of liver-infiltrating IgG4+ plasma cells in primary sclerosing cholangitis (PSC) applying these cut-offs has not been determined. We sought to determine the incidence of intrahepatic IgG4-positive staining in PSC patients undergoing transplantation, correlating findings with clinical parameters. Immunohistochemical staining was performed on liver explants obtained between 1991 and 2009. Of 122 explants obtained, hilar IgG4+ staining was found to be mild (10–29 IgG4+ cells/HPF) in 23.0%, moderate (30–50/HPF) in 9.0% and marked (>50/HPF) in 15.6%. Marked hilar lymphoplasmacytic infiltration was significantly associated with marked hilar IgG4+ staining (P < 0.001). No patient had marked peripheral IgG4+ staining, although mild and moderate staining was observed in 24.5% and 3.3% respectively. Marked hilar IgG4+ staining was significantly associated with the presence of dominant biliary strictures (P = 0.01) and need for biliary stenting (P = 0.001). There did not, however, exist any significant differences in the age at PSC diagnosis, presence of inflammatory bowel disease or extrahepatic autoimmune disease, frequency of cholangiocarcinoma, interval between diagnosis and transplantation, or post-transplant PSC recurrence or survival. Of 51 control liver sections , none had marked or moderate hilar IgG4+ staining, whereas mild staining was seen in only 10% (P < 0.001). Marked (>50/HPF) hilar IgG4+ lymphoplasmacytic infiltration is frequently observed in PSC and associated with the presence of dominant biliary strictures. However, unlike serum IgG4+, this does not seemingly associate with clinical disease course.Dense tissue infiltrates of IgG4 Primary sclerosing cholangitis (PSC) is a chronic progressive disease of unknown aetiology characterized by fibrosclerotic destruction of the bile ducts resulting in multifocal stricturing of the biliary tree  and at least one histological feature (dense lymphoplasmacytic infiltrate/storiform fibrosis/obliterative phlebitis), this finding would be probable of IgG4-related disease  has been reported to have the highest specificity -associated cholangitis (IAC) often has similar cholangiographic features to PSC , presence of dominant strictures – defined as stenosis of the common bile duct (CBD) with a diameter ≤1.5 mm, or of the left or right hepatic duct with a diameter ≤1 mm; time from PSC diagnosis to transplantation, presence/phenotype of IBD, history of pancreatitis, evidence suggestive of extrahepatic IgG4-related diseases or concomitant extra-hepatopancreatobiliary autoimmune conditions, and/or the presence of clinically unsuspected, incidental cholangiocarcinoma in the explant. Patient survival and evidence of recurrent PSC post-transplant were also evaluated. Recurrent PSC was suspected based on abnormal liver biochemistry post-transplant and confirmed with diagnostic cholangiography. Allograft biopsy was performed in cases of diagnostic uncertainty.Clinical records were reviewed to establish the age at PSC presentation, ductal phenotype  manifestations of IgG4-related disease according to HISORt criteria Chari  was exclFormalin-fixed, paraffin-embedded tissues sampling the liver hilum were retrieved, and haematoxylin/eosin and Masson's trichrome staining performed on all sections. Given that percutaneous liver biopsies routinely sample the peripheral liver parenchyma, paired peripheral sections were also obtained for analysis. All cases were reviewed by a histopathologist with expertise in hepatobiliary disease blinded entirely to clinical characteristics and patient outcome. Hilar lymphoplasmacytic infiltration was characterized into three strata: mild, moderate and severe Figure. Briefly2O2. The detection system used was Biogenex Super-Sensitive Polymer Kit (QD410-YAX) with colour development performed using freshly prepared NovaRed . Finally, sections were counterstained lightly with filtered Mayer's haematoxylin, dehydrated in alcohols, cleared in xylene and mounted with Permount medium .Specimens were immunohistochemically stained with anti-IgG4 antibody  at a dilution of 1:2000 (v/v) and one hour incubation. Briefly, 4-μm sections were dewaxed in five changes of xylene and brought down to water through graded alcohols. Pretreatment with 1% pepsin  in 0.01 NaHCl (pH 2.0) was performed for 15 min (37 °C), and endogenous peroxidase blocked with 3% H+ cells was counted and polychotomized into 4 strata: negative (<10 IgG4+ plasma cells/HPF), mild (10–29 cells/HPF), moderate (30–50 cells/HPF) and marked  had only intrahepatic involvement, whereas 67.2% of patients (n = 82) had coexisting IBD, and 20.5% (n = 25) a history of extra-hepatopancreatobiliary (HPB) autoimmune disease. Of all patients transplanted, 5 (4.1%) were diagnosed as having cholangiocarcinoma in-situ (explant) and 13 (10.7%) developed evidence of recurrent PSC.Over an 18-year period, 157 patients with PSC underwent liver transplantation, of which explant tissue was readily available in 122 cases . The median age of PSC diagnosis was 36 years (range 10–67) with a median interval from diagnosis to transplant of 6 years (8 months–24 years). 27.9% (n = 51) comprised 19 patients transplanted for chronic viral hepatitis type C (HCV) infection, eight with chronic viral hepatitis B (HBV) infection, 18 with primary biliary cirrhosis , and six transplanted for autoimmune hepatitis .The comparator group (n = 58) had positive IgG4 immunohistochemical staining in the hilar tissue, of which 23.0% (n = 28) had mild staining, 9.0% (n = 11) had moderate staining and 15.6% (n = 19) had marked staining. Hilar lymphoplasmacytic infiltration was marked in 52.9% (n = 64), moderate in 28.1% (n = 34) and mild in 19.0% (n = 23) of PSC patients. Neither storiform fibrosis nor significant obliterative phlebitis [which are also characteristic features of IAC , unlike patients with marked hilar lymphoplasmacytic infiltration, who were significantly more likely to have positive immunohistochemical IgG4 staining of any degree compared to those with lesser degrees of hilar lymphoplasmacytic infiltration . This retained significance when restricting the analysis to those with >50 IgG4+ plasma cells/HPF .Only five non-PSC cases  had positive hilar IgG4 immunohistochemical staining, and this was of a mild degree only; no patient in the control group was identified as having marked or moderate staining. The frequency of hilar IgG4n = 30) had mild staining and 3.3% (n = 4) moderate staining. There was no significant association between severities of peripheral IgG4 staining and hilar IgG4 staining, neither did there exist an association between the severity of hilar lymphoplasmacytic infiltration and the severity of peripheral IgG4+ staining (data not shown). Four patients (7.8%) in the comparator group  had evidence of peripheral IgG4+ staining (P < 0.001) although this was of a mild degree only.Thirty-four PSC patients (27.9%) also had positive IgG4 immunohistochemical staining in the peripheral liver parenchyma; 24.6% (+ plasma cells/HPF) was more common in men  and associated with a previous history of acute pancreatitis . On retrospective evaluation, no radiological features indicative of AIP were identified in this subgroup. On comparing with patients who stained negative for IgG4, no statistically significant differences existed with respect to age at PSC diagnosis, presence/phenotype of coexisting IBD or extrahepatic autoimmune disease, ductal phenotype, frequency of cholangiocarcinoma, age at time of transplantation, interval between PSC diagnosis and transplantation , and despite a trend remaining with respect to previous episodes of acute pancreatitis (P = 0.08), this failed to retain statistical significance. Perhaps most strikingly, patients with moderate or marked hilar IgG4+ infiltration were significantly more likely to have a dominant biliary stricture than those with lesser degrees of IgG4+ infiltration  and required biliary stenting more commonly .When restricting comparative analysis using the cut-offs provided by the recent Boston criteria for IgG4-associated systemic disease, no significant gender differences were observed between those with marked hilar IgG4However, there was no significant difference in the frequency of cholangiocarcinoma, median age at PSC diagnosis, median age at transplantation, pretransplant MELD score, interval to transplantation following PSC diagnosis, frequency/phenotype of coexisting IBD, incidence of post-transplant PSC recurrence and all cause mortality after transplantation between groups Table .et al. et al. + staining differentiates IgG4-related HPB disease from other diagnoses such as PSC is heavily debated; however, the recent Boston criteria suggest that >50 IgG4+ plasma cells/HPF is highly specific for the former.PSC is characterized histologically by chronic cholestasis, inflammation and obliterative biliary fibrosis eventually leading to cirrhosis. Presently, it is devoid of effective intervention other than transplantation, and efforts continue to rationalize pathophysiologic understanding with the goal of identifying themes for intervention that account for patient heterogeneity. The repeated observation of detecting elevated serum IgG4 levels in a subgroup of PSC patients has triggered interest in defining IgG4 contributions to disease presentation as well as clinical outcome. In IAC and other conditions falling under the ‘umbrella’ of IgG4-related systemic disease, there exist key histopathological features comprising recognized diagnostic criteria  IgG4+ hilar infiltration, a finding associated with the severity of hilar lymphoplasmacytic inflammation. These results echo those published by Zhang et al. antigen, or to some intrinsic or extrinsic substrate present in bile, and offers a plausible explanation as to the observed association with dominant biliary strictures. Of interest, it is notable that a subset of patients with PSC and dominant biliary strictures may respond to immunosuppressive therapy (Erkelens et al. + status.Zen et al.  recently+ plasma cells, we have shown that there is poor correlation between hilar and peripheral IgG4-positive staining. This implies that peripherally targeted liver biopsies may not be particularly useful in terms of predicting whether a patient may have IgG4-associated cholangitis. Given the infrequency with which hilar biopsies are performed in routine practice, attempting selection of PSC patients for potential therapeutic trials of corticosteroid therapy, based on peripheral-tissue IgG4 positivity alone, should be discouraged.From a practical perspective, it is important to highlight the lack of association between peripheral and hilar samples. Although it is those with marked hilar lymphoplasmacytic inflammation who are most likely to stain positive for IgG4et al. et al. (The main limitations from our report stem from an absence of paired serum IgG4 readings. Given the retrospective nature of the study, the vast majority of included patients were transplanted in an era during which IgG4-related disease had yet to be recognized and whereby routine assessment of IgG4 was neither standard practice nor advocated by international guidelines. Nevertheless, we have recently reported on the frequency and clinical significance of elevated serum IgG4 elsewhere (Alswat . et al. , althoug+ staining, were more likely to have dominant strictures and need for biliary intervention. However, this in itself did not represent a subgroup with worse clinical outcome. It remains the case that understanding the drivers of IgG4 staining is still incomplete, and at present, the routine histological evaluation of peripheral IgG4 staining not clinically indicated.In conclusion, hilar IgG4 staining is apparent on detailed immunohistochemical evaluation of liver grafts of patients with PSC in approximately 50% of cases. Such patients, particularly those with marked hilar IgG4


Met is a tyrosine kinase receptor that is considered to be a proto-oncogene. The hepatocyte growth factor (HGF)-Met signaling system plays an important role in tumor growth, invasion, and metastasis in many types of malignancies. Furthermore, Met expression has been reported to be a useful predictive biomarker for disease progression and patient survival in these malignancies. Many studies have focused on the clinical significance and prognostic role of Met in urological cancers, including prostate cancer (PCa), renal cell carcinoma (RCC), and urothelial cancer. Several preclinical studies and clinical trials are in progress. In this review, the current understanding of the pathological role of Met in cancer cell lines, its clinical significance in cancer tissues, and its predictive value in patients with urological cancers are summarized. In particular, Met-related malignant behavior in castration-resistant PCa and the different pathological roles Met plays in papillary RCC and other histological types of RCC are the subjects of focus. In addition, the pathological significance of phosphorylated Met in these cancers is shown. In recent years, Met has been recognized as a potential therapeutic target in various types of cancer; therapeutic strategies used by Met-targeted agents in urological cancers are summarized in this review. Met is a member of cell surface tyrosine kinase (TK) family that was isolated from a chemically transformed human malignant cell line in the 1980s ,3. ThereSince cell proliferation, migration, and angiogenesis are important steps in malignant cell dissemination, many investigators have paid special attention to the relationship between Met overexpression and malignant aggressiveness, including tumor invasion and metastasis. A recent review indicated that enhanced HGF/Met signaling is found in various types of cancers . In addiMany investigators have suggested that evaluation of the expression and biological activity of Met is important in understanding the pathological characteristics, malignant behavior, and strategies of observation and treatment for patients with malignancies. However, it should be noted that there is a wide range of variability in the quantification and pathological significance of Met in previous reports. In particular, variability has been demonstrated in the immunohistochemical technique most commonly used for evaluation of Met expression in animal and human tissues. Possible reasons that have been suggested for such discrepancies include differences in sample size, antibodies used, methods of evaluation, and condition of the tissue samples . It has MET gene mutation and amplification of the MET gene locus, resulting in receptor overexpression, are also characterized aberrancies. Mutation of the MET gene is thought to be a rare event in cancer, but it is often found in papillary renal cell carcinoma (RCC) and childhood hepatocellular carcinoma [MET gene copy number gain has been reported to be common and is associated with a poor prognosis in patients with lung cancer [The common mechanism of MET signaling activation is transcriptional deregulation, resulting in MET overexpression in malignant cells. In addition, arcinoma ,10. Convg cancer . The facHGF-Met signaling has been reported to interact with various cancer-related molecules that promote tumor growth and metastasis. For example, HGF-Met signaling can promote angiogenesis though interaction with the vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) signaling system ,12. In aThere are three different strategies to inhibit Met signaling: antagonism of receptor-ligand interaction; inhibition of the TK catalytic activity; and inhibition of the interaction between the receptor and intracellular signaling effectors. By understanding the detailed structure, function, and regulative mechanisms of HGF-Met signaling, it has been possible to develop various Met inhibitors . At presin vivo and in vitro. As mentioned above, the Met TK receptor has been shown to play a role in the proliferation and progression of many types of malignancy, including PCa. In this section, the pathological significance, prognostic roles, and possible therapeutic use of targeting Met in PCa are discussed. In addition, the pathological role of Met expression in castration-resistant PCa (CRPC) is shown.PCa is the most common cancer in men. In the United States, it is estimated that 29,000 patients died from PCa in 2013 . NumerouNumerous studies have demonstrated that in human PCa cell lines the MET signaling system plays an important role in cell survival by enhancing cell proliferation and suppressing apoptosis ,19. In ain vivo and in vitro androgen-sensitivity. PCa cells initially depend upon androgen stimulation, through androgen receptor signaling, for cell survival and malignant behavior. In fact, androgen deprivation is the standard treatment for patients with advanced PCa. However, as the cells become androgen-independent, they become more aggressive. Most recurrent and metastatic tumors that occur after androgen-deprivation therapy include castration-resistant PCa cells [In discussing the pathological mechanisms of Met in PCa, it is important to consider Ca cells . There iMET mRNA expression levels are reported to be similar to the protein levels. The androgen receptor-negative PCa cells, PC3 and DU145, have a higher malignant potential, including increased cell proliferation and migration, suppressed apoptosis, and a higher rate of angiogenesis when compared to androgen-receptor-positive cells. Based on these findings, it has been hypothesized that the loss of androgen receptor expression and increased Met expression contribute to the malignant potential of CRPC. Interestingly, continuous androgen-deprivation of the androgen-sensitive PCa cell line, LNCaP, results in decreased androgen receptor expression and increased Met expression [in vitro studies, the pathological activity of Met in CRPC has also been confirmed using CRPC cell lines and animal xenograft models [Among the most widely used human PCa cell lines, MET protein is highly expressed in androgen receptor-negative cell lines (PC-3 and DU145) but only minimally expressed in androgen receptor-positive cell lines  . In thespression . In addit models ,25. Fromt models .Furthermore, the importance of cancer stem cells (CSCs) in prostate cancer should be emphasized. According to the CSC hypothesis, tumor growth is sustained by a subpopulation of cancer stem/progenitor-like cells. Like other malignancies, human prostate cancer tissue contains neoplastic cells with self-renewal and clonogenic potential, which can be enriched and perpetuated in prostaspheres . When MESome investigators have showed the relationships between Met expression and clinicopathological features in patients with prostate cancer ,32,33,34in vitro studies have shown a significant correlation between increased Met expression and androgen-independent growth [in vitro and in vivo findings suggest that the expression of Met is correlated with the emergence of castration-resistant tumor growth, and that Met is a potential therapeutic target in CRPC.For CRPC in particular, though, MET expression has been reported to be upregulated and is thought to play a role in angiogenesis, cell invasion, and metastasis to distant tissues . As mentt growth ,24. Thesversus mitoxantrone plus prednisone on pain response and bone scan response in men with CRPC. However, COMET-1 did not meet demonstrating a statistically significant increase in overall survival, and based on the outcome of COMET-1, enrollment in COMET-2 was halted.Cabozantinib (XL184) is an orally bioavailable TKI with potent activity against MET and VEGFR2. Cabozantinib blocked the progression of osteolytic and osteoblastic lesions in a xenograft model of CRPC in bone . A phaseAnother small molecule inhibitor of Met, tivantinib (formerly ARQ 197), is being studied in PCa. A phase I trial of ARQ 197 was conducted to evaluate the safety of tivantinib. The study showed that tivantinib safely inhibited intratumoral c-MET signaling, but no responses, according to the Response Evaluation Criteria in Solid Tumors, were observed . A phaseSorafenib is a multikinase inhibitor targeting Raf kinase (serine-threonine kinase) and tyrosine kinases, including VEGFR, platelet-derived growth factor receptor (PDGFR), c-kit, c-Met. A phase II trial of sorafenib monotherapy for CRPC reported relative tolerance of this agent. However, although initially 2 of 22 patients treated with sorafenib showed evidence of improved bony metastatic lesions, most patients (21 of 22) had disease progression .Sunitinib is an oral multitargeted inhibitor of VEGFR, PDGFR, c-kit, FLT-3, and c-Met. A phase III trial that evaluated angiogenesis-targeted sunitinib therapy in a randomized, double-blind trial of metastatic CRPC concluded that the addition of sunitinib to prednisone did not improve OS compared with placebo in docetaxel-refractory metastatic CRPC .As mentioned above, various types of MET inhibitors have been studied in clinical trials. However, unfortunately, almost of them showed “negative” results. As reason of such failure, several factors including study design, patient’s selection, and unexpected side effect are speculated. In this review, we emphasize the fact that such molecular-targeting therapies used for patients with CRPC. In addition, in many cases, CRPC patients were previously treated with docetaxel prior to such therapies. From these facts, we believe that more detailed information regarding pathological roles of Met expression in patients with CRPC are essential to improve the anti-tumor effect.VHL) gene should also be considered in this discussion of the pathological characteristics and molecular mechanisms of RCC.Renal cell carcinoma (RCC) is the third most frequent cancer originating from the genitourinary organs. It is estimated that 65,000 patients were diagnosed with RCC in the United States in 2013 . RCC oriMET gene have been described in individuals with papillary RCC [The clinical significance and pathological role of MET has been most widely investigated, and is best understood, in papillary RCC. Several activating missense mutations of the lary RCC . Severallary RCC . Similarlary RCC . Researclary RCC .VHL gene function by mutation or methylation is found in patients with sporadic clear cell RCC, although it is rare in other histological RCC types. Several reports have found that Met protein is detectable in human clear cell RCC tissues [in vitro [The most well-known form of inherited clear cell RCC is that associated with VHL syndrome. Inactivation of  tissues ,47,48,49in vitro . Thus, iet al. have also reported that MET expression in papillary RCC and sarcomatous RCC is significantly higher than in clear cell RCC subtypes [Strong expression of MET is observed in almost all papillary RCC and collecting duct carcinoma, however, it is uncommon in clear cell RCC and chromophobe RCC . Gibney subtypes . Thus, Msubtypes . Furthersubtypes . The cliMET mutations, five (50%) of ten experienced a PR, and the remaining five (50%) patients had stable disease as the best response. A PR was seen in only five (9%) of 57 patients without germline MET mutations. Further investigations are necessary to clarify these results.The drug foretinib, formerly XL880, is a multi-targeted receptor TKI that targets the MET, VEGFR2, platelet derived growth factor receptor (PDGFR) β, Tie-2, recepteur d'origine nantais (RON), KIT, and FLT3 receptors. A phase I study of foretinib has been completed, and two out of four patients with papillary RCC showed a partial response (PR) . A phaseIn vitro studies indicate that tivantinib inhibits colony formation in ACHN and 769P cells [Tivantinib (ARQ197) is an inhibitor of Met receptor TK activity. 9P cells . Followi9P cells , phase IRecently, a phase I study of cabozantinib (XL184), a small molecule inhibitor of TKs including MET and VEGFR2, was performed . The stuUrothelial carcinoma (UC), especially bladder cancer, is the second most common genitourinary malignancy; it accounts for almost 5% of all newly diagnosed cancers. It is estimated that 72,570 patients were newly diagnosed with UC of the bladder in the United States in 2013. More than 90% of cases pathologically originate from urothelial cells. Papillary UCs are typically superficial at first presentation and are often multifocal. These tumors recur with a high frequency >60%), and 10%–15% of them will progress to life-threatening malignancies over a longer period of time ,57. Seve%, and 10in vitro, a detailed understanding of the pathological significance and regulative mechanism of MET in UC cells has not been achieved.In UC cell lines, Met mRNA has been detected by northern blot analysis  and reveIt was initially reported that Met expression could not be detected in the normal urothelium or in cases of UC of the kidney . Howeveret al. [The biological activities of Met are exerted by phosphorylation, and phosphorylation of Met is essential for the stimulation of downstream signaling pathways. For this reason, Miyata et al.  investiget al. .Based on the pathological significance and prognostic role of MET in UC cells, many investigators have suggested that Met is a potential novel therapeutic target for these patients. SU-11274, a selective MET kinase inhibitor, is reported to affect the growth of bladder cancer . In addiin vivo and in vitro. In addition, we have reviewed the preclinical studies and clinical trials of Met-targeted therapies in patients with urological cancers. Numerous investigators have focused on the functions of Met at a genetic, molecular, and clinical level. However, much is still unknown. Based on previous reports and current studies, we believe that Met has the potential to be used as a predictive factor and therapeutic target in patients with urological cancers.In this review, we have summarized the pathological role, clinical significance, and predictive value of Met in urological cancer


IgG4 is the least abundant subclass of IgG in normal human serum, but elevated IgG4 levels are triggered in response to a chronic antigenic stimulus and inflammation. Since the immune system is exposed to tumor-associated antigens over a relatively long period of time, and tumors notoriously promote inflammation, it is unsurprising that IgG4 has been implicated in certain tumor types. Despite differing from other IgG subclasses by only a few amino acids, IgG4 possesses unique structural characteristics that may be responsible for its poor effector function potency and immunomodulatory properties. We describe the unique attributes of IgG4 that may be responsible for these regulatory functions, particularly in the cancer context. We discuss the inflammatory conditions in tumors that support IgG4, the emerging and proposed mechanisms by which IgG4 may contribute to tumor-associated escape from immune surveillance and implications for cancer immunotherapy. H) followed by three constant domains . Light chains  are composed of an N-terminal variable domain (VL) and a constant domain (CL).The four human IgG subclasses were discovered in the 1960s and named according to their time of discovery and order of their relative abundance in human serum  . Fc. Fc12]. H2 and CH3 domains. Residues from these two interfaces belong to a consensus binding site for Fc-binding proteins such as neonatal Fc receptor (FcRn), TRIM21 [IgG4 has been shown to bind other IgGs, in particular IgG1, via Fc-Fc interactions , 64••. T, TRIM21 , rheumat, TRIM21 , and som, TRIM21 . The Fc-, TRIM21 ••. Indee, TRIM21 .A rheumatoid factor-like activity of IgG4 was observed first in rheumatoid arthritis , 70 and H2 interface. Full cleavage to F(ab′)2 fragments is preceded by an intermediate with one intact heavy chain [Tumor microenvironments are rich in proteases such as matrix metalloproteases (MMP), which support extracellular matrix degradation, tissue remodeling, and promotion of cancer cell metastasis. An additional effect of these enzymes may be to partly dissociate or cleave host-produced or therapeutic antibodies . An assovy chain . The samvy chain , suggestvy chain , 74.H2 domain may cause a partial dissociation of the cleaved heavy chain from the rest of the antibody. The partly dissociated IgG1 could facilitate the binding of IgG4-Fc resulting in the blockade, via Fc-Fc interactions, of single-chain cleaved IgG1 on the surface of tumor cells  or unnecessary (payload delivery antibodies) . TherapeA of the same order of magnitude as IgG1 and IgG3, and the inhibitory FcγRIIb receptor with higher affinity than the other subclasses [IgG4 is generally considered an anti-inflammatory antibody due to its functionally monovalent properties, its poor ability to bind C1q and the activating low-affinity FcγRs. Nevertheless, IgG4 binds FcγRI with a Kbclasses , 76. Thubclasses , 78.The overall anti-inflammatory activity of IgG4s, despite their affinity for FcγRI, could be related to their functional monovalency . Therape+ B cells expressing high-affinity tumor antigen-specific IgG4s [Triggering a patient response against cancer may be a desirable feature of active cancer immunotherapy. The emerging field known as AllergoOncology includes the use of IgE antibodies and antibody responses in cancer therapy . IgE claic IgG4s ••, a valic IgG4s –85.The structural and functional attributes of IgG4 described here render it unique among the IgG subclasses. Its ability to undergo FAE may be only partly responsible for its low affinity for activating receptors expressed on immune effector cells and consequently low capacity to engage these cells to trigger ADCP and ADCC and also for its poor ability to bind C1q and fix complement. IgG4 is considered an antibody with low immunoactivatory properties, and it is a suitable subclass for the design of therapeutic antibodies when effector functions are not desirable. However, a better understanding of its properties and functions in vivo is still required to inform the design and optimization of IgG4-based therapeutic antibodies. Reports of IgG4 antibodies and IgG4+ B cells in different cancers suggest the involvement of IgG4 in tumor escape from immune surveillance through a number of potential mechanisms, including IgG4 blockade of IgG1-mediated effector functions. However, IgG4 and its roles in cancer inflammation remain unclear. Dissecting the cross-talk between cancer and humoral immunity and the conditions that can promote IgG4-biased “regulatory” responses can inform the design of novel therapeutic antibodies with improved immune activatory and effector functions and reduced susceptibility to tumor-associated immune blockade.


Our results show that maximal force was not significantly different between KO and WT preparations but length-dependent increase in pCa50 was attenuated in the KO preparations. pCa50 was not significantly different between WT and KO preparations at long SL (5.82 ± 0.02 in WT vs. 5.87 ± 0.02 in KO), whereas pCa50 was significantly different between WT and KO preparations at short SL . The ktr, measured at half-maximal Ca2+-activation, was significantly accelerated at short SL in WT preparations . Furthermore, krel and kdf were accelerated by 32% and 50%, respectively at short SL in WT preparations. In contrast, ktr was not altered by changes in SL in KO preparations (8.03 ± 0.54 s−1 at 1.9 μm vs. 8.90 ± 0.37 s−1 at 2.1 μm). Similarly, KO preparations did not exhibit length-dependent changes in krel and kdf. Collectively, our data implicate cMyBP-C as an important regulator of LDA via its impact on dynamic XB behavior due to changes in SL.Enhanced cardiac contractile function with increased sarcomere length (SL) is, in part, mediated by a decrease in the radial distance between myosin heads and actin. The radial disposition of myosin heads relative to actin is modulated by cardiac myosin binding protein-C (cMyBP-C), suggesting that cMyBP-C contributes to the length-dependent activation (LDA) in the myocardium. However, the precise roles of cMyBP-C in modulating cardiac LDA are unclear. To determine the impact of cMyBP-C on LDA, we measured isometric force, myofilament Ca Our results show that the length-dependent increase in pCa50 was attenuated in the KO preparations compared to wild-type (WT) preparations. Furthermore, length-dependent changes in dynamic contractile parameters ktr, krel, and kdf were blunted in KO preparations compared to WT preparations, indicating that cMyBP-C plays a critical role in the myofilament-mediated response in cardiac LDA.Importantly, recent evidence from low-angle X-ray diffraction experiments showed that cMyBP-C tethers the myosin XBs closer to the thick-filament backbone and that ablation of cMyBP-C results in the radial displacement of XBs closer to the thin-filament , and was conducted according to the guidelines of the Institutional Animal Care and Use Committee at Case Western Reserve University. Mice aged 3–6 months, of both sexes, and belonging to SV/129 strain were used for the experiments. KO mice used in this study were previously generated and well-characterized  also contained 7 EGTA and 7.01 CaCl2; while the relaxing solution (pCa 9.0) contained 7 EGTA and 0.02 CaCl2; and the pre-activating solution contained 0.07 EGTA. The pH of the Ca2+ solutions was set to 7.0 with KOH and the ionic strength was 180 mM. A range of pCa solutions, containing varying amounts of [Ca2+]free, were then prepared by mixing appropriate volumes of pCa 9.0 and 4.5 stock solutions and the experiments were performed at 22°C.Skinned ventricular multicellular preparations were prepared as described previously  i.e., P/Po to construct the force-pCa relationships  of the sarcomeres in the heart muscle , where Hn is the Hill coefficient and k is the pCa required to produce half-maximal activation   and a force transducer  as described previously , and once the muscle preparations attained steady-state isometric force, they were rapidly slackened by 20% of their original muscle length and were held for 10 ms using a high-speed length control device (Aurora Scientific Inc.). The slackening was followed by the brief period of unloaded shortening which resulted in a rapid decline in force due to detachment of the strongly-bound XBs. The muscle preparation was then rapidly restretched back to its original length and the time course of force redevelopment was measured. ktr for each slack-restretch maneuver was estimated by linear transformation of the half-time of force redevelopment, i.e., ktr = 0.693/t1/2, as described previously , with a characteristic rate constant kdf, which is a result of length-induced recruitment of new XBs into the force-bearing state  comparisons.Data were analyzed using Two-Way analysis of variance (ANOVA). One factor in this analysis was cMyBP-C variant (WT or KO), and the second was SL (1.9 or 2.1 μm). Therefore, we used Two-Way ANOVA to test the hypothesis that the effect of the SL on a given contractile parameter depended on the cMyBP-C variant (interaction effect). When the interaction effect was significant, it showed that the effects of SL on various contractile parameters were different in the presence or absence of cMyBP-C. When the interaction effect was not significant, we interpreted the main effect due to cMyBP-C variant or SL. Planned multiple pairwise comparisons were made using Fisher's LSD method  was measured at SL's 1.9 and 2.1 μm in WT and KO muscle preparations  of SL on Ca2+-activated maximal force production. To probe the determining factor for the significant main effect, subsequent post-hoc tests were carried out. These post-hoc tests using multiple planned pairwise comparisons showed that maximal force production was not significantly different between WT and KO groups at either SL (Table max) was significantly decreased by ~34% and ~38% at short SL vs. long SL in WT and KO groups, respectively (Table 2+-independent forces measured at pCa 9.0 (Fmin) in WT and KO groups  and cMyBP-C (P < 0.005) on pCa50. Subsequent post-hoc tests revealed that the main effect of SL was because of the following: pCa50 significantly increased upon increasing the SL from 1.9 to 2.1 μm as indicated by a leftward shift in the force-pCa relationships in both WT and KO groups  was attenuated in KO group when compared to ΔpCa50 of WT group  on nH suggesting that cMyBP-C influenced the effect of SL on nH. Subsequent post-hoc tests revealed that nH significantly increased by ~38% at SL 1.9 μm in WT group, a result that agrees with earlier studies  on ktr suggesting that cMyBP-C influenced the effect of SL on ktr. The cause of the interaction effects was assessed by post-hoc multiple pairwise comparisons which showed that submaximal ktr was accelerated in KO compared to WT group at long SL  + g (rate of XB detachment) according to a two-state XB model  to measure krel and kdf which are measures of the rates of XB detachment and XB recruitment, respectively  on krel suggesting that cMyBP-C influenced the effect of SL on the rate of XB detachment kinetics. The cause of the interaction effect was evident from the post-hoc multiple pairwise comparisons which revealed that krel was significantly accelerated by ~32% at short SL in WT group but such an acceleration of krel at short SL was absent in KO group  on kdf suggesting that cMyBP-C influenced the effect of SL on the rate of XB recruitment into the force-bearing state. The cause of the interaction effect was assessed using post-hoc tests which revealed that kdf was significantly accelerated by ~50% at short SL in WT but such an acceleration of kdf at short SL was absent in KO  was accelerated at short SL compared to long SL in WT group  at short SL compared to long SL in WT group  at long SL in KO compared to WT  upon an increase in SL is a hallmark of LDA  . In this study we found a similar trend, although here the ΔpCa50 was 0.11pCa units in WT skinned myocardium whereas it was 0.07 pCa units in KO skinned myocardium  via changes in the interaction between tropomyosin (Tm) and TnI  and detachment (g)  and force decay (krel), parameters that are analogous to f and g. Our data shows that acceleration of ktr at short SL in WT group was indeed due to a combination of increases in both kdf and krel (Figure kdf and krel did not increase at short SL in KO skinned myocardium such that the values were not significantly different from those at long SL (Figure ktr at short SL in KO group was due to the absence of accelerations in both kdf and krel at short SL (Figure Because l Figure . Our datL Figure . A recenL Figure  showed tL Figure . Thus, okrel, Figure krel (Stelzer et al., krel seen in KO group (Figure To test whether changes in XB detachment (as assessed by p Figure  may havep Figure , becausekdf, Figure nH from the pCa-tension relationships. Our estimates showed that nH values were increased in WT at short SL compared to long SL (Figure nH may be a result of enhanced Ca2+ binding to Tn, near-neighbor interactions among Tn-Tn, XB-Tn, and XB-XB (Razumova et al., nH at short SL was absent in KO (Figure kdf in the KO group (Figure 2+/TnC (Li et al., nH with decrease in SL. Therefore, our data shows that length-dependent changes in cooperative mechanisms are depressed when cMyBP-C is absent in the sarcomere.To test whether changes in XB recruitment, (as assessed by L Figure , a resulL Figure . This suO Figure  which map Figure . In the p Figure , XB-Tn (p Figure  and XB-C2+, XB cycling kinetics, and muscle fiber stiffness. Taken together, our findings suggest that impaired LDA may contribute to depressed myocardial contractile function in human patients harboring mutations in cMyBP-C that ultimately cause a significant decrease in the amount of cMyBP-C expression in the sarcomere.Our study provides evidence to show that cMyBP-C plays a key role in fine-tuning length-dependent cardiac contractile function via its impact on myofilament responsiveness to CaRanganath Mamidi and Julian E. Stelzer contributed to the conception and design of the experiments. Ranganath Mamidi, Kenneth S. Gresham, and Julian E. Stelzer participated in performing the experiments, data acquisition, data analysis, data interpretation, drafting, and revising the manuscript. All authors approved the final version of the 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.