diff --git "a/deduped/dedup_0927.jsonl" "b/deduped/dedup_0927.jsonl"
new file mode 100644--- /dev/null
+++ "b/deduped/dedup_0927.jsonl"
@@ -0,0 +1,71 @@
+{"text": "To determine if the search technique that is used to sample randomized controlled trial (RCT) manuscripts from a field of medical science can influence the measurement of the change in quality over time in that field.RCT manuscripts in the field of brain injury were identified using two readily-available search techniques: (1) a PubMed MEDLINE search, and (2) the Cochrane Injuries Group (CIG) trials registry. Seven criteria of quality were assessed in each manuscript and related to the year-of-publication of the RCT manuscripts by regression analysis.No change in the frequency of reporting of any individual quality criterion was found in the sample of RCT manuscripts identified by the PubMed MEDLINE search. In the RCT manuscripts of the CIG trials registry, three of the seven criteria showed significant or near-significant increases over time.We demonstrated that measuring the change in quality over time of a sample of RCT manuscripts from the field of brain injury can be greatly affected by the search technique. This poorly recognized factor may make measurements of the change in RCT quality over time within a given field of medical science unreliable. Considerable effort has been directed toward improving randomized controlled trial (RCT) design, execution, and reporting -6,14. SuAs much as the RCT manuscripts published in a single journal or group of journals would provide a well-defined parent population, the RCT manuscripts from a given field of medical science would be difficult to completely identify. Ultimately no search strategy can claim to identify all manuscripts on a given topic that have been published in every book and journal worldwide. Thus, two search techniques might provide considerably different samples of RCT manuscripts from the same field of medical science depending upon how much and / or what parts of the parent population of RCT manuscripts they can access. The current communication empirically demonstrates this point as a potential pitfall in measuring the change in quality over time of RCT manuscripts sampled from a representative field of medical science.We chose internal validity as a measure of quality according to the definition given by Gehlbach , namely We intended to measure the presence or absence of various criteria of RCT quality as described in the published manuscript. No attempt was made to contact the authors of a manuscript either to clarify the information provided in the manuscript or to gain additional information about a RCT. We acknowledge that relying on the published manuscript in order to assess the quality of a RCT may be biased (1) against well-designed RCTs that were reported in poorly written manuscripts and (2) in favor of poorly-designed RCTs that were reported in well-written manuscripts . Thus, osine qua non of the RCT, additional criteria have been so frequently included in their design and execution that they are now commonly considered as part of quality RCTs. Several sources (themselves located by PubMed MEDLINE and bibliography searches) were used to identify such criteria [Although random allocation and the use of a concurrent control group are the criteria ,9,13-18.We limited our quality scale to measure criteria that have been demonstrated empirically to be associated with the quality of RCTs. This necessarily excluded many items associated with RCT design and execution that are widely thought to affect quality or that are included in commonly-used quality scales, but it provided us with a defensible \"bare minimum\" definition of quality. It should be noted that we did not intend our list of criteria to be encompassing of all aspects of quality; our criteria were intended to serve only as a tool for the comparative analysis of the two sets of RCT manuscripts for the purpose of this study.Each of the seven criteria was scored as being present 1 point) or as absent (0 points) in the RCT manuscript. Definitions of each criterion are shown in Table  point orThe intra-rater reliability for the scoring of the quality criteria was determined by comparing the individual criteria scores given to n = 16 RCT manuscripts by one of the authors of this communication (MKB) on two occasions separated by 3 weeks. The correlation coefficient (Kappa) measured in this manner was 0.94.Inter-rater reliability was determined by comparing the quality criteria scores given to n = 10 RCT manuscripts by two different examiners. One copy of each manuscript was scored by one of the authors of this communication (MKB) while the other copy was scored by an independent examiner  who was provided with a thorough description of the criteria. The correlation coefficient (Kappa) for inter-rater reliability was determined to be 0.74.We chose to evaluate the field of brain injury because two search techniques for sampling the population of these RCT manuscripts were readily available. The first search technique was our own PubMed MEDLINE search. The second search technique was performed by the Cochrane Collaboration Injuries (CIG) Group, and forms the CIG trials registry. Copies of the RCT manuscripts identified by these two search techniques were retrieved through the library holdings and interlibrary loan services of five universities.Next, the manuscripts were read by one of us (CY) to screen-out inappropriately identified manuscripts. Table The design and yield of the two search techniques was as follows:1) the PubMed MEDLINE search: The first search technique we used to identify RCT manuscripts pertaining to brain injury involved the PubMed search engine of the MEDLINE database. It was designed to represent a typical literature search performed by a North American researcher who is fluent only in English. The search term \"brain injuries\" (C10.228.140.199) was used with the limitations of (1) randomized controlled trial, (2) human subjects, and (3) publication in English. The PubMed MEDLINE search included manuscripts indexed from January, 1966, up to February, 2001 (the time at which the search was performed).\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0The PubMed MEDLINE search identified n = 139 manuscripts. During the screening process, n = 41 \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0manuscripts from the original 139 (30%) were discarded leaving n = 98 manuscripts (see Table 2.) the CIG trials registry: The Injuries Group of the Cochrane Collaboration was kind enough to share their list of RCT manuscripts with us for the purpose of conducting this study. The list of manuscripts they provided was compiled by means of the following three steps:Fiona.Renton@lshtm.ac.uk) as \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0well as various hand searches of journals performed during the writing of systemic reviews; it is \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0updated quarterly.\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0step 1) The CIG trials master list was searched using the keywords \"head\" or \"brain\" in conjunction \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0with \"injur*\" or \"trauma*\". The CIG trials master list is a local database maintained at the London \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0School of Hygiene and Tropical Medicine that uses a detailed search strategy to identify RCTs \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0from multiple computerized databases  MEDLINE, EMBASE, and CENTRAL databases were searched using the exploded \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0keyword \"head injuries:ME\" or \"head injuries:TI\". EMBASE includes references from 1974 \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0onward and, while it uses its own database, it is based on an indexing hierarchy which incorporates \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0that used by MEDLINE. Here, MEDLINE was searched with the SilverPlatter search engine, not \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0with the PubMed Search engine. Manuscripts of the MEDLINE database indexed as early as 1966 \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0were accessible to the SilverPlatter search engine. The CENTRAL database is a general list of \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0clinical trials that is maintained by the collaborative efforts of multiple Cochrane specialty groups.\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0step 3) Manuscripts identified by hand searches of relevant journals and from references provided \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0by direct contact with experts in the field of brain injury were also included.\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0The original CIG trials registry was completed in 1998 and was last fully updated in May, 2001; it is that \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0version which was used in our study.\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0The CIG trials registry included n = 312 manuscripts. During the screening process, n = 139 manuscripts \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0from the original 312 (45%) were discarded leaving n = 173 RCT manuscripts (see Table 3.) overlap between the PubMed MEDLINE search and the CIG trials registry: Of the total unscreened samples of manuscripts identified through each search technique, n = 80 manuscripts were present in both samples; this corresponded to 58% of the sample of manuscripts identified by PubMed MEDLINE search and 26% of the sample of manuscripts from the CIG trials registry. After the removal of inappropriate manuscripts during the screening process, and scoring process only n = 56 manuscripts were identified by both the PubMed MEDLINE search and the CIG trials registry. This corresponded to 57% and 32% of the PubMed MEDLINE search and the CIG trials registry samples, respectively.Each of the RCT manuscripts was read by both authors of the current communication (CY and MKB) who, for clarity's sake, will be referred to as \"examiners\". One examiner (\"non-judging examiner\": CY) performed the screening process described previously, then recorded the year-of-publication of each manuscript that survived the screening process in a computerized spreadsheet (Microsoft Excel) and marked them with identification numbers. Then, the non-judging examiner hid the names of the authors of the manuscript, the authors' degrees and departmental affiliations, the journal in which the RCT manuscript was published, and the year-of-publication of the manuscript with black marker. This information was covered wherever it was found in the manuscript so that when the manuscript was scored by the second examiner (\"judging examiner\": MKB) there would be no potential for bias ,19. The As mentioned above, allocation concealment was included in the list of quality criteria after the first evaluation of the manuscripts. Accordingly, the judging examiner re-read all the manuscripts specifically to determine the inclusion of allocation concealment. The manuscripts were still blinded as described above, and the data was entered into a third spreadsheet that was subsequently analyzed independently of the preexisting data.Manuscripts in French and Spanish were read without written translation by the judging examiner, whereas written translations were provided to the judging examiner for manuscripts in Japanese (by CY), German and Italian (by Mrs. Margaret K. Borsody), and Chinese .After completion of the scoring process, statistical analyses were conducted by the judging examiner. The data was considered interval in nature and thus data analysis for discrete variables was used . FurtherSince the samples of manuscripts from the PubMed MEDLINE search and the CIG trials registry are known to be derived from the same parent population of RCTs , it is inappropriate to directly compare them against each other with statistical tests. Rather, it was our goal to analyze the two samples of RCT manuscripts separately, and to make likely conclusions about the parent population from each sample of manuscripts as if there was no other sample of manuscripts available for comparison. Then, knowing that the two samples of RCT manuscripts represent the same parent population, it was our intention to compare the conclusions derived from the separate analyses to determine the impact of the search technique thereupon.Regression analysis of the individual quality criteria against the year-of-publication of the RCT manuscripts was performed to determine if the frequency of reporting of each quality criteria changed over time. For the sample of RCT manuscripts identified by the PubMed MEDLINE search, no significant relationship was found for any individual quality criterion  that themselves can affect the identification of manuscripts from the common database. Whatever may be the cause for the discrepancy between our two samples, it may undermine any claim that a search technique necessarily produces a more representative sample from a field of medical science simply because it identifies a greater number of RCT manuscripts.The two search techniques otherwise differ in several ways. For example, the PubMed MEDLINE search was designed so as to exclude any manuscripts published in a non-English language. This would approximate the typical literature search performed by many researchers in North America, and accordingly all the manuscripts identified by the PubMed MEDLINE search were readily available in local university libraries. Conversely, the CIG trials registry tended to include more references from the non-English language literature (n = 27 manuscripts after the screening process). This inclusiveness of the CIG trials registry seemed to account for the 15 irretrievable manuscripts listed by the CIG trials registry. It is reasonable to state that the non-English language literature is part of medical science and that it should not be discounted solely because of its country-of-origin or the language in which it was written. As another difference, the CIG trials registry involved hand-searches of journals and lists of references provided by authorities in the field of brain injury, which are not features of the PubMed MEDLINE search and which may predispose the CIG trials registry search technique toward recovering more recently-published manuscripts. Recently published manuscripts may be of higher quality, thereby biasing the longitudinal measurement of quality in the RCT manuscript sample provided by the CIG trials registry. Alternatively, such extra efforts would be considered by most to improve on the yield of a search technique by including journals and books that are not indexed by computerized databases.a priori that neither of the search techniques we used necessarily sampled RCTs from the field of brain injury in a representative manner. Furthermore, we do not claim to have accurately measured how the quality of RCT manuscripts is changing over time in the field of brain injury with either one of them. This is because we are not confident that either search technique provided a representative sampling of the field of brain injury . With regards to the field of brain injury, it is clear from our observations that neither the PubMed MEDLINE search nor the CIG Trials Registry can claim to be complete, as each search technique failed to identify a large number of RCT manuscripts that were found by the other search technique. In other fields of medical science there may be specialized databases or registries that claim to identify all relevant manuscripts . The authors of the current communication cannot understand how such a claim can be made or proven, since demonstrating the completeness of a search strategy would require proving that there are no relevant manuscripts that it does not identify. Ultimately, proving that something does not exist is scientifically impossible. Alternatively, it might be claimed that a search strategy identifies the majority of relevant manuscripts. This, of course, depends upon (1) the definition of a majority and (2) the assumption that the finding of even a few relevant manuscripts not identified by the search strategy means there are no other such manuscripts outside of the reach of that search strategy. Again, such a claim would depend upon the assumption that the inability to find further relevant manuscripts indicates that no further relevant manuscripts exist; as described above, this is a scientific impossibility. Rather than claiming perfection or near-perfection, it would seem to us to be more appropriate and accurate to claim that a given search strategy has exhausted all options for identifying relevant manuscripts.Arguments can be made that either of the search techniques provided a more representative sample of RCT manuscripts from the field of brain injury, but which search technique is superior \u2013 if either can be said to be so \u2013 is not a concern of the current study. It was solely our intention to compare the findings provided by two commonly-used search techniques to demonstrate that the search technique can in fact influence the measurement of the change in RCT quality over time. We acknowledge What, then, should be done to avoid a biasing influence related to the search technique during reviews of RCT quality over time? The simplest means of avoiding a such an influence would have apparently been to use multiple search techniques in order to better sample the parent population of RCT manuscripts in a field of medical science. In general, including multiple techniques into a single 'comprehensive' search would be preferable to a simple search involving only a single technique, but even so this does not ensure that the combination is truly comprehensive . Essentially this was the goal of the CIG trials registry, but even it did not completely encompass the sample of manuscripts identified by the PubMed MEDLINE search despite involving a MEDLINE search of its own. Similarly, previous reviews of RCT quality have often involved secondary searches following an initial computerized search, but such efforts certainly cannot match the breadth and thoroughness of that from the Cochrane Collaboration. If such reviews of RCT quality are to judge entire fields of medical science it would seem that the search techniques they employ must be shown to produce a representative sampling of the parent population of RCT manuscripts as well as a high yield from that parent population. We hope that the findings presented here bring more attention to this concern in future reviews of the change in RCT quality over time.We demonstrated that measuring the change in quality over time of a sample of RCT manuscripts from the field of brain injury can be greatly affected by the search technique. This poorly recognized factor may make measurements of the change in RCT quality over time within a given field of medical science unreliable. The search strategy should be accurately reported in any study that attempts to follow trends in the quality of RCT manuscripts over time, and its limitation in sampling the RCT manuscripts from a field of medical science should be acknowledged and evaluated.The author(s) declare that they have no competing interests.Both MKB and CY contributed equally to this study, and the exact nature of their contributions are described in the Methods section.The pre-publication history for this paper can be accessed here:"}
+{"text": "Methodological research to support searching for those doing systematic reviews of epidemiological studies is a relatively neglected area. Our aim was to determine how many databases it is necessary to search to ensure a comprehensive coverage of the literature in diabetes epidemiology, with the aim of examining the efficiency of searching in support of systematic reviews of the epidemiology of diabetesThree approaches were used. First, we defined a set of English language diabetes journals and examined their coverage in bibliographic databases. Second, we searched extensively for diabetes epidemiology articles  to determine which are the most useful databases; and third, we analysed the scattering of these articles to determine the core journals in the area.Diabetes Care contributing 14.3% of the articles, followed by Diabetic Medicine (5.0%); Diabetes Research & Clinical Practice (4.1%); Diabetologia (4.0%); Diabetes & Metabolism (2.4%) and Diabetes (2.0%).The overlap between MEDLINE and Embase for diabetes journals was 59%. A search for diabetes epidemiology articles across both MEDLINE and Embase, showed that MEDLINE alone retrieved about 94% of the total articles. Searching for diabetes epidemiology studies beyond MEDLINE and Embase retrieved no additional English language journal articles. The only diabetes epidemiology studies found by searching beyond MEDLINE and Embase were found in LILACS, and were Spanish or Portuguese language studies from Latin America; no additional English language studies were found. Only 30% of the meeting abstracts were converted to full publication after three years. One third of journal articles were published in just six journals, with Our results show that when searching for articles on diabetes epidemiology, MEDLINE and Embase would suffice for English language papers, with LILACS giving some additional non-English articles from Latin America. Although a MEDLINE-only search will retrieve the vast majority of the relevant literature, Embase and LILACs should also be searched to ensure the search is comprehensive. Searching for meeting abstracts is recommended to alert reviewers to unpublished work. The low rate of full publication of meeting abstracts has the danger of producing bias in reviews. Our findings on scattering show that the core literature in this field is concentrated in just six journals. Review articles have a valuable role in the medical literature, because the volume of journals and articles is such that keeping up to date is very difficult. Reviews are much more valuable if they are systematic reviews done to internationally agreed standards, as non-systematic reviews are known to be subject to bias ,2.Dickersin noted that there was a shortage of systematic reviews in epidemiology, and called for more reviews, and more research into the methodology relating to reviews in epidemiology . A studyOne of the key quality criteria for systematic reviews is the comprehensiveness of the searching, as failure to identify all relevant reports can result in selection bias. The usuUlrich's International Periodicals Directory as a gold standard, found that a MEDLINE search in psychiatry would retrieve only about half the relevant journals [A study, using journals . SimilarThe scattering of the journal literature in a subject area can provide a useful insight into the number and range of journals needed to capture the key literature in a field. Bradford's Law of Scattering states that on any one subject, a small group of 'core' journals (Zone 1) will provide about one third of the articles on that subject, a medium number of less-core journals (Zone 2) will provide another third, and a large number of peripheral journals (Zone 3) will provide the final third of the articles ,10.The aim of this study was to examine the efficiency of searching in support of systematic reviews of the incidence and prevalence of diabetes by providing empirical data to answer the following questions:1. Which databases index diabetes journals (restricted to English language)?2. Which databases outside MEDLINE and Embase index diabetes epidemiology journal articles and grey literature?3. How are diabetes epidemiology articles scattered across the journals, and what are the core journals in this area?Accordingly, this study was divided into three parts..For the purposes of this study, epidemiology articles were defined as studies of incidence or prevalence of diabetes, or of factors affecting those . Basic science studies, e.g. biological mechanisms of disease, were not included.We started from a position that both MEDLINE and Embase should always both be searched, since the overlap between these databases has been estimated to range from 10% to 87% ,11-17. AHence, a three-part approach was used to investigate literature searching to support systematic reviews of diabetes epidemiology, and address each of our aims in turn.Ulrich's Periodical Directory 2003  [Diabetes journals were identified from the 'Medical Sciences \u2013 Endocrinology' section of e world) . This wae world)  for jourUlrich's, the contents pages for the past five years, and where necessary, abstracts or the full journal articles, were examined by a diabetes epidemiologist.The inclusion criteria for the journals were: i) the word stem 'diabet' in the title, ii) contains original scientific studies of an academic or scholarly nature, iii) currently in print, and iv) published in English. If inclusion could not be decided on the basis of the information provided in List of Serials Indexed for Online Users [Journals which fulfilled all the above criteria were then checked against the ne Users  to see iIf any journals were not indexed in either MEDLINE or Embase, searches of BIOSIS, BNI, CINAHL, SCI were done to determine if the journals were indexed in any of these databases.Databases searched were: AMED, ASSIA, BIOSIS (abstracts only), BNI, CINAHL, Conference Papers Index, Dissertation Abstracts US, Health Management Information Consortium (HMIC), Index to Theses UK, ISI Proceedings, PsycINFO, NLM Gateway, LILACS , National Research Register (NRR), SIGLE, SCI (abstracts only), SSCI, and Zetoc.The search strategy used was 'diabetes and ' in the Title (TI) field and restricted to publication years 1998 to 2003.The titles (and abstracts when available) of all records were checked by an expert in diabetes epidemiology, in order to determine their potential usefulness for those doing systematic reviews.MEDLINE and Embase were searched using the search strategy: 'Diabetes Mellitus as the major subject heading, with the sub-heading 'Epidemiology' assigned , and restricted to publication years 1998 to 2003'. All languages were included. Duplicates found in both MEDLINE and Embase were removed. The journal titles in which the articles were found were ranked according to the number of articles contributed by each journal. The cumulated numbers of articles and journals were calculated and plotted. This was used to identify Bradford zones; that is, the number of journals needed to cover about one third, two thirds or all the relevant articles in the field.Ulrich's Periodicals Directory and PubList.com initially identified four English language journals that were of potential interest but not indexed by MEDLINE or Embase. On closer inspection, three of these, Clinical Diabetes, Journal of Diabetes Nursing, and Diabetes and Primary Care were excluded as they did not appear to contain any primary research; the articles were mainly educational, professional news and views, opinions, or narrative reviews. The fourth journal, The Diabetic Foot, contained primary research, but did not appear to contain studies useful for epidemiological reviews of diabetes itself (as opposed to complications). It is indexed by CINAHL only.Searches of As shown in Table The four diabetes journals unique to MEDLINE were all published in the USA. By contrast, only one of the seven journals unique to Embase was published in the USA.The results are summarised below:No English language journals articles that were not also indexed in MEDLINE or Embase were identified.There were 23 Spanish and Portuguese language articles identified in LILACs. On the basis of the English translation of the titles, they all reported studies done in Latin America.We defined grey literature as any literature not published in a peer reviewed journal. After removing duplicates, there were 51 dissertations identified from searches of Dissertations Abstracts US, Index to Theses UK, and SIGLE. The research presented in the vast majority (92%) of the dissertations appeared to have been written up as articles in journals indexed in MEDLINE or Embase. No grey literature studies of any format other than dissertations were retrieved from SIGLE, so there was very little additional information gained by these searches.The National Research Register  gave brief details of 18 projects in progress that had not been otherwise identified. Searching the NRR might be useful if unpublished results could be included in the review, but its main value would be to indicate when the review was likely to need updating.The search of the Conference Proceedings Index retrieved 25 articles, none of which appeared to have been published as journal articles after five years. The Zetoc Conference Search found eight articles, of which 50% had been published as full journal articles in MEDLINE or Embase.The search of Science Citation Index (SCI), restricted to meeting abstracts only, found 171 relevant studies. The time to publication of the SCI abstracts was examined by checking how many had subsequently been published as journal articles indexed in MEDLINE or Embase. It was found that 30% had reached full publication after three years.Diabetes and Metabolism and Diabetes Research and Clinical Practice. Of these, 11 (12%) had been published in journals. The average time delay from the date of publication of the abstract to full publication was 1.4 years.A search of BIOSIS, restricted to meeting abstracts only, retrieved 71 additional relevant abstracts that were not in SCI. Most (65%) of these 71 abstracts came from the supplements of These included AMED, BNI, HMIC, NLM Gateway meeting abstracts, PsychINFO, and SSCI,In summary, the data indicate that when searching for English language journal articles on diabetes epidemiology, searches of MEDLINE and Embase would suffice. The exception would be for studies from Latin America, where LILACS should also be searched. Searching for meeting abstracts may alert reviewers to forthcoming or unpublished work.The searches for diabetes epidemiology articles in MEDLINE and Embase resulted in 2923 articles being found in 696 different journal titles; 39% were found to be in 'diabetes journals' and 14% were non-English language.Figure Applying Bradford's Law of Scattering gives three zones, each providing one-third of the articles.Diabetes Care (contained 14.3% of the articles); Diabetic Medicine (5.0%); Diabetes Research & Clinical Practice (4.1%); Diabetologia (4.0%); Diabetes & Metabolism (2.4%), Diabetes (2.0%) These six journals represent 0.9% (of the 696) total journals, and all are indexed in MEDLINE.The first one-third of articles were from six journals. They are in rank order: Practical Diabetes International; Diabetologia Polska; Diabetes und Stoffwechsel and Journal of the Japan Diabetes Society. Hence, 94% of Zone 2 journals are covered by MEDLINE.The second one-third of articles were from 62 journals, representing 9.1% of the total journals. The four journals in Embase only were: The final one-third of articles were in 628 journals, representing 90.2% of the total journals. MEDLINE indexed 88% of these journals.Overall, for the three zones, the search of MEDLINE and Embase for diabetes epidemiology articles revealed that MEDLINE indexed 89% of the total journals, and these contained 94% of the articles.Our results showed that there was an overlap of only 59% in current English language 'diabetes journals' indexed by both MEDLINE and Embase. Also, a search for diabetes epidemiology articles across both MEDLINE and Embase showed that MEDLINE alone retrieved about 94% of the total articles; therefore, both databases should be searched. Embase appears to index more diabetes journals published outside the USA. Therefore, if searching is limited to MEDLINE only  this could potentially introduce a bias. Also, duplication of searching can be useful, as due to differences in indexing practices, a search of one database may retrieve something missed by the other.We also found that despite a wide range of additional databases searched after MEDLINE and Embase, no additional English language journal articles on diabetes epidemiology were identified. The LILACS database was a useful source of Spanish and Portuguese language articles on the epidemiology of diabetes in Latin American countries.Meeting abstracts appeared to be valuable sources of information on forthcoming studies, but their inclusion in systematic reviews is contentious. Some reviewers exclude abstracts on the grounds that the quality of the study cannot be judged because of the inevitably limited detail. However, others include them on the grounds that abstracts provide the most up-to-date information.Nearly all the dissertations identified had been published as journal articles. However, it was found that only 30% of the meeting abstracts were converted to full publication after three years, which is considerably lower than the figure for RCTs, which is 56% . This haDiabetes Care alone containing about 14% of the articles. A similar concentration effect in journals was also shown in a study of 3400 science journals in the SCI database, where just 100 journals accounted for 22% of the published articles and 100 journals also accounted for 44% of cited articles [Scattering of the diabetes epidemiology articles revealed that the 'core' literature in this field is concentrated in just six journals, with articles .This study has a number of limitations. The search to identify diabetes journals was restricted to English language journals only, as we were unable to assess articles in other languages. We did not compare the quality of the articles identified from databases outside MEDLINE and Embase. Also, when searching for articles, we were necessarily limited to the range of databases available to us.Finally, there may be databases inaccessible or unknown to us that cover foreign language and regional journals not indexed in MEDLINE and Embase. Such journals may carry studies of incidence which may seem of primarily local interest, but which may be useful contributions to the international body of evidence because they may show large variations in incidence, or in its relationship to possible aetiological risk factors.It is often useful to study the epidemiology of a disease where it is rare, as well as where it is common. However it is likely that studies which report high incidence are more likely to be published than those which report low incidence. Similarly with risk factors; a study which finds no link between factor x and disease y may be less likely to be published than one which does show a correlation ,23.There is a need for further research to see whether our findings apply to searching for epidemiological reviews of other diseases, and on measuring the sensitivity and specificity of various search filters to retrieve epidemiological studies in MEDLINE and Embase.We endorse Dickersin's suggestion of an international collaborative effort to establish an 'epidemiological Cochrane-like database' to identify all relevant studies and to begin systematically reviewing available data for important epidemiological questions .Searching MEDLINE and Embase appears to provide comprehensive coverage of the English language journal literature in diabetes epidemiology. LILACs is a useful source of Spanish and Portuguese language articles on diabetes epidemiology done in Latin American countries and published in regional journals not indexed in MEDLINE and Embase. Searching for meeting abstracts is recommended to alert reviewers to unpublished work.The volume of literature on diabetes epidemiology makes it impossible for one person to read everything. However the provision of systematic reviews makes keeping up with research manageable, and more reviews are needed. Our findings on scattering shows that the core literature in diabetes epidemiology is concentrated in a small number of core journals, and that in the absence of reviews, one can follow the field by reading these journals. It may also be reassuring that a good MEDLINE-only search will retrieve the vast majority of the relevant literature.The author(s) declare that they have no competing interests.PR and NW conceived the study and drafted the initial manuscript. PR and LB collected and analysed the data. All authors contributed to and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "General practitioners, mental health practitioners, and researchers wishing to retrieve the best current research evidence in the content area of mental health may have a difficult time when searching large electronic databases such as MEDLINE. When MEDLINE is searched unaided, key articles are often missed while retrieving many articles that are irrelevant to the search. The objectives of this study were to develop optimal search strategies to detect articles with mental health content and to determine the effect of combining mental health content search strategies with methodologic search strategies calibrated to detect the best studies of treatment.An analytic survey was conducted, comparing hand searches of 29 journals with retrievals from MEDLINE for 3,395 candidate search terms and 11,317 combinations. The sensitivity, specificity, precision, and accuracy of the search strategies were calculated.3,277 (26.8%) of the 12,233 articles classified in the 29 journals were considered to be of interest to the discipline area of mental health. Search term combinations reached peak sensitivities of 98.4% with specificity at 50.0%, whereas combinations of search terms to optimize specificity reached peak specificities of 97.1% with sensitivity at 51.7%. Combining content search strategies with methodologic search strategies for treatment led to improved precision: substantive decreases in the number of articles that needed to be sorted through in order to find target articles.Empirically derived search strategies can achieve high sensitivity and specificity for retrieving mental health content from MEDLINE. Combining content search strategies with methodologic search strategies led to more precise searches. Retrieving the best current evidence for a specific medical discipline when searching in large electronic databases such as MEDLINE can be challenging. This challenge is due to the scatter of relevant articles in low concentration across a large number of journals, inherent limits in indexing, and lack of searching skill on the part of the user of the database . For ins as well as the limits screen of Ovid .Researchers have developed search strategies to help retrieve scientifically sound, clinically relevant articles while searching in MEDLINE. To date the majority of the search strategies have been developed when searching for therapy, diagnostic and review articles -13. In aAlthough these search strategies are helpful in identifying scientifically sound, clinically relevant articles for clinical matters , they are not designed to detect content for any particular disorder . When conducting a \"usual\" search in MEDLINE, content terms would be \"ANDed\" to the methodologic search strategies that have been developed . To date, we are unaware of any studies reporting empirically tested search strategies for identifying articles for a particular disease or clinical discipline combined with methodologic search terms.The objectives of this study were to develop optimal search strategies to detect articles of interest to the discipline area of mental health and to determine the effect that content search strategies have on the performance of methodologic search strategies for treatment when the strategies are combined using the Boolean \"AND\".Evidence-Based Mental Health  during the year 2000 .We compared the retrieval performance of mental health content search terms in MEDLINE with a manual review (hand search) of each article for each issue of 29 journal titles for the year 2000. Overall research staff hand searched 170 journal titles. These journals were chosen based on recommendations of clinicians and librarians, Science Citation Index Impact Factors provided by the Institute for Scientific Information, and ongoing assessment of their yield of studies and reviews of scientific merit and clinical relevance for the disciplines of internal medicine, general medical practice, mental health, and general nursing practice . Of these 170 hand searched journals, 161 were indexed in MEDLINE. Search strategies for the study we report here were developed using a 29 journal-subset chosen based on those journals that had the highest number of methodologically sound studies in the area of mental health, that is, those that contributed > 1 article to the journal We compiled a list of 3,395 index terms and textwords (list of terms tested provided by the authors upon request). This list was compiled after surveying 140 mental health specialists from around the world, reviewing the search strategies from 5 mental health focused Cochrane groups, and mapping textwords to MeSH terms. Examples of the search terms tested are '(learn: adj problem)', 'schizoid', 'depression', and 'mania', all as textwords; 'phobic disorders', the index term; and the index term 'aggression', exploded .As part of a larger study , 6 trainPharmacological interventions for persons with mental health problems; cognitive and behavorial approaches to helping any patient ; etiology pertaining to mental health; diagnosis pertaining to mental health; or economic issues pertaining to mental health.The proposed search strategies were treated as \"diagnostic tests\" for sound studies and the manual review (hand search) of the literature was treated as the \"gold standard\". We determined the sensitivity, specificity, precision, and accuracy of each single term and combinations of terms in MEDLINE using an automated process. Sensitivity for a given topic is defined as the proportion of high quality articles for that topic that are retrieved; specificity is the proportion of low quality articles not retrieved; precision is the proportion of retrieved articles that are of high quality; and accuracy is the proportion of all articles that are correctly classified.Individual search terms with sensitivity > 15% and specificity > 80% for articles of interest to mental health were incorporated into the development of search strategies that included 2 or more terms. All combinations of terms used the Boolean OR, for example, \"mania.tw. OR depression.sh.\". For the development of multiple-term search strategies to optimize either sensitivity or specificity, we tested all 2-term search strategies with sensitivity at least 75% and specificity at least 50%. For optimizing accuracy, 2-term search strategies with accuracy > 75% were considered for multiple-term development. 11,317 search strategies were tested in the development of mental health content search filters. To enhance the performance of the most sensitive mental health content search strategy, the single search terms with the highest sensitivity were successively added to the top performing 3-term search strategy until the best sensitivity was achieved while keeping specificity \u226550%.In addition to developing mental heath content search strategies as just described, we also evaluated the performance of the methodologic search filters for treatment articles when \"ANDed\" with the mental health content filters.Indexing information was downloaded from MEDLINE for 12,233 articles from the 29 journals hand searched. Of these 3,277 (26.8%) were considered to be of interest to mental health. Search strategies were developed using all 12,233 articles. Thus, the strategies were tested for their ability to retrieve mental health articles from all other articles.Table Combination of terms with the best results for sensitivity, specificity and optimization of sensitivity and specificity are shown in Tables Each of the top performing strategies for detecting mental health content were \"ANDed\" with the top performing methodologic search strategies for detecting scientifically sound, clinically relevant treatment studies. The results of these combinations are also shown in Tables Our study documents search strategies that can help discriminate the literature with mental health content from articles that do not have mental health content. General practitioners, mental health practitioners, and researchers wanting an overview of the best current evidence in the area of mental health will best be served by the most sensitive search strategy when they have time to sort through articles. This search will have the highest probability of retrieving all relevant articles (in this study one on-target article missed), but will have the lowest precision, retrieving many irrelevant articles. With less time on their hands general practitioners, mental health practitioners, and researchers they may wish to search with the strategy that optimizes the balance between sensitivity and specificity (10 on target articles missed) or the strategy that optimizes specificity (40 on target articles missed).As indicated in our previous papers -21, whenAs reported in this paper, we set out to test whether precision would be enhanced by combining the methodologic search strategies with content specific terms using the Boolean 'AND'. We found a 3- to 17-fold decrease in the absolute number of articles that would need to be sorted through to find articles that are on target. This decrease is substantive and shows that combining empirically derived search strategies for enhancing the retrieval of relevant content with search strategies derived for enhancing the retrieval of scientifically sound, clinically relevant articles can have a profound impact on searching.The example used in this paper is for retrieving high quality treatment papers with mental health content. Treatment was used because the sample size was sufficient to test the performance of combined search strategies (content and methods) in this 29 journal subset (n = 129). Other purpose categories, for example diagnosis, did not lend themselves to this test because the number of scientifically sound diagnostic articles with mental health content in this 29 journal subset was low .Selected combinations of indexing terms and textwords can achieve high sensitivity or specificity in retrieving articles with mental health content in MEDLINE. Combining content search strategies with methodologic search strategies can lead to a substantive decrease in the absolute number of articles that need to be sorted through to find those articles that are on target.The author(s) declare that they have no competing interests.No conflicts of interest. Both authors, Nancy L. Wilczynski and R. Brian Haynes, had full access to all the data in the study and had final responsibility for the decision to submit for publication.RBH and NLW prepared grant submissions in relation to this project. Both authors drafted, commented on and approved the final manuscript. Both authors also supplied intellectual content to the collection and analysis of the data. NLW participated in the data collection and both authors were involved in data analysis and staff supervision."}
+{"text": "Lefebvre and Clarke suggest that reviewers might consider using four revisions of the HSSS. The objective of this study is to validate these four revisions: combining the free text terms We replicated the subject search for 61 Cochrane reviews. The included studies of each review that were indexed in MEDLINE were pooled together by review and then combined with the subject search and each of the four proposed search strategies, the top two phases of the HSSS, and all three phases of the HSSS. These retrievals were used to calculate the sensitivity and precision of each of the six search strategies for each review.versus combined with the top two phases of the HSSS was able to find 3 more included studies than the top two phases of the HSSS alone, or in combination with any of the other proposed search terms, but at the expense of missing 56 relevant articles that would be found if all three phases of the HSSS were used. The estimated time needed to finish a review is 1086 hours for all three phases of the HSSS, 823 hours for the strategy versus, 818 hours for the first two phases of the HSSS or any of the other three proposed strategies.Across the 61 reviews, the search term versus to the top two phases of the HSSS balances the sensitivity and precision in the reviews studied here to some extent but the differences are very small. It is well known that missing relevant studies may result in bias in systematic reviews. Reviewers need to weigh the trade-offs when selecting the search strategies for identifying RCTs in MEDLINE.This study shows that compared to the first two phases of the HSSS, adding the term  The new century has seen a significant proliferation of systematic reviews, and they have become one of the key tools for the evidence-based medicine movement. A quality systematic review involves a comprehensive search for relevant studies on a specific topic, and those identified are then assessed and synthesized according to a predetermined and explicit method . Althougvolunteer, crossover and versus, and the Medical Subject Heading (MeSH) CROSS-OVER STUDIES which was introduced after the search strategy was devised.The MEDLINE database, created and maintained by the United States National Library of Medicine, is the most widely-used database in medicine and other health science fields. It includes 15 million citations dating back to the mid-1960's . The Higvolunteer, crossover, versus, and the MeSH term CROSS-OVER STUDIES, respectively [A few recent studies ,14 have ectively .st Quarter 2003) using the following search strategy:Systematic reviews, which might have used the HSSS to identify RCTs, were selected from the Cochrane Database of Systematic Reviews (CDSR), OVID interface (1(hsss.tw.) or (highly sensitive search.tw.)These systematic reviews were then screened using three eligibility criteria. To be selected, each systematic review had to use at least one phase of the HSSS, report the citations for included and excluded studies, and indicate if primary studies were either RCTs or quasi RCTs.We did a known-item search for the included studies of each systematic review that met the three inclusion criteria in OVID MEDLINE (1966 \u2013 2003) to determine whether they were indexed in MEDLINE or not. The bibliographic records of the included studies that were indexed in MEDLINE were aggregated together by review using the Boolean operator \"or\". Each known-item search strategy was saved in OVID MEDLINE. We recorded the number of included studies that were indexed in MEDLINE for each systematic review. This was used to calculate sensitivity for each review. One sample of the known-item search strategy is listed in volunteer, crossover, versus and MeSH term CROSS-OVER STUDIES was combined with the first two phases of the HSSS to create four test strategies with the Boolean operator \"or\" .Each of the four candidate terms the free-text terms 123), and the top two phases of the HSSS (hereafter abbreviated as SS12) by using Boolean operator \"and\" in OVID MEDLINE (1966-February 2004) to find out how many included studies indexed in MEDLINE were retrieved by each search strategy . We assumed that the subject search presented in each review was a comprehensive search for identifying the subject- related studies. The subject search strategy was combined with each of SSand\" See . If the Precision = We identified 169 systematic reviews from CDSR, which represented about 10% of the reviews published in the database in 2003. Of the 169 systematic reviews, 96 reviews met the three inclusion criteria. Of the 96 reviews, 61 reviews (63.54%) presented detailed subject search to allow us to replicate their subject search See ; 33 . For SS123, 70% of the reviews achieved a perfect sensitivity. A closer examination of the data found that, across the 94 reviews, SSversus was able to find 3 more relevant articles than SScrossover, SSCROSS-OVERSTUDIES, SSvolunteer, or SS12, but SS123 found 56 more relevant articles than SSversus. There is no obvious difference between the sensitivities of the 61 reviews which listed their detailed subject specific search strategies and those of the 33 reviews which did not list their search strategies . The sensitivity of the four test search strategy, SS12, and SS123 are presented in Table We were able to calculate the sensitivity for 94 reviews. The overall sensitivity of the four proposed search strategies, SS12 for each review range from 408\u2013430 studies, and the median of SS123 is 1636 studies. The median of initial retrieval of SSversus is about 1/4 of that of SS123, which means the number of articles needed to read by reviewers would be reduced significantly if SSversus instead of SS123 was used to identify RCTs. When SS123 was used, 36 reviews (59.02%) had a very low precision (less than 1%); when SS12, SScrossover, SSCROSS-OVERSTUDIES, or SSvolunteer was used, 21 reviews (34.43%) had a precision less than 1%; When SSversus was used, 22 reviews (36.07%) had a precision less than 1%. Table 123 (182) is significantly higher than that of SSversus (54), and the ARRs of SS12, SScrossover, SSCROSS-OVERSTUDIES, and SSvolunteer are the same (51). We calculated the estimated time to finish a review for each search strategy based on the regression equation developed by Allen and Olkin [x - 0.0000123x2, where x denotes the number of articles initially retrieved, as shown in Table 123, 823 hours for SSversus, 818 hours for SS12, SScrossover, SSCROSS-OVERSTUDIES, or SSvolunteer.We were able to calculate the precision for the 61 reviews presented a detailed subject search. The precision of the six search strategies can be found in Table iews 36.0% had a pcrossover, SSCROSS-OVERSTUDIES, and SSvolunteer are the same as SS12. SS123 found 56 more relevant articles than SSversus, and SSversus found 3 more relevant articles than SS12, SScrossover, SSCROSS-OVERSTUDIES, or SSvolunteer. The number of articles needed to read per review when SSversus is used is about 1/4 of that when SS123 is used, and the estimated time to finish a review for SS123 is 32% higher than that for SSversus. On the other hand, the number of articles needed to read when SSversus is used is only 5%  more than that when SS12 is used, and the estimated time to finish a review for SSversus is 0.6% (5 hours) more than that for SS12. The result shows that, compared to SS123, SSversus will reduce the number of articles needed to read significantly, thus reducing the reviewers' work in assessing citations for eligibility and the total time to complete a review, while still maintaining a workload comparable to SS12 but a slightly better sensitivity than SS12. Although the other three proposed search strategies also have a lower initial retrieval size than SS123, their sensitivity is the same as SS12.Searching bibliographic databases to identify relevant studies is one of the most important steps of a systematic review . All the123 will miss fewer relevant studies than SS12, and the four variants recommended by Lefebvre and Clarke, including SSversus. Because timelines and financial costs are usually a concern to most reviewers, they must decide whether these benefits justify the extra costs for much broader screening of the initial retrievals and the longer time needed to complete a review.A comprehensive search is considered one of the key factors that distinguish a systematic review from a narrative review, and it is well-known that missing relevant studies will possibly result in bias for systematic reviews. This study confirms that SSversus because it maintains a workload comparable to SS12 but a slightly better sensitivity. If reviewers do not have access to CENTRAL or the specialized registers, we suggest that they still use all three phases of the HSSS to maintain the quality of systematic reviews.The comprehensiveness of systematic review searches not only depends on search filters but also on the varieties of databases searched. The Cochrane Central Register of Controlled Trials (CENTRAL) is the most comprehensive database of controlled trials. It is hoped that CENTRAL can serve as an all-inclusive source of controlled trials. When searching this database, reviewers need only to develop subject search strategies, thus avoiding the problem of selecting search filters. Each Collaborative Review Group (CRG) also develops a subject specialized register of trials to ensure that reviewers within the CRG have access to the maximum number of studies to their topic. All the reviews screened in this study indicated that they searched CENTRAL and/or the specialized CRG registers. The Cochrane Collaboration has gone to great effort to enhance the comprehensiveness of CENTRAL. Therefore, if reviewers do decide to use the first two phases of the HSSS and plan to search CENTRAL and/or the specialized CRG registers as well, they may consider using SSSince the first publication of the Highly Sensitive Search Strategy  few studThe strength of a systematic review over any other kind of review is that it provides readers with an approach to replicate it . The Quaversus would change the outcomes of the 61 reviews. Therefore, we can not judge whether skipping Phase Three of the HSSS would result in bias in systematic reviews. Second, an effective search strategy for systematic reviews usually includes two parts: a subject search and a search filter, both of which are integral to the search strategy. In our study, we replicated the subject search for each systematic review, but the quality of these subject searches is unknown. There is a possibility that some relevant studies could be retrieved if the subject search were more comprehensive. Therefore, the sensitivity of the test search strategies could be lower, and precision higher than those we presented if the subject search were truly comprehensive. More studies should be done on developing high-quality subject searches, which calls for cooperation between medical subject experts and information specialists. In addition, there are limitations in using sensitivity and precision to evaluate the performances of information retrieval, which were discussed by Kagolovsky and Moehr [Our study has some limitations. First, we tested only how many included studies could be retrieved by each of the six search strategies, not whether the 56 relevant studies missed by SSnd Moehr .12 proposed by Lefebvre and Clarke, adding the free text word versus to the first two phases of the HSSS provides a modest balance of the precision and sensitivity in the reviews studied here. We hope that this finding will become part of the evidence used by systematic reviewers and information specialists in making decisions on developing their search strategies for systematic reviews.Since MEDLINE is the most widely used database in searching for evidence for systematic reviews, formulating an optimal search strategy to find RCTs in MEDLINE will greatly increase the efficiency of systematic reviews. Our study demonstrates that of the variants to HSSSThe author(s) declare that they have no competing interests.LZ initially conceived of, designed, and executed the study, and wrote and revised the entire manuscript draft. IA reviewed the study design and results, and participated in the revision of the manuscript. MS participated in the study design, data extraction, drafting and revision of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Highly Sensitive Search Strategy (HSSS) This known item search strategy was to identify included studies in Review 24, as listed in"}
+{"text": "Health technology assessments of surgical interventions frequently require the inclusion of non-randomised evidence. Literature search strategies employed to identify this evidence often exclude a methodological component because of uncertainty surrounding the use of appropriate search terms. This can result in the retrieval of a large number of irrelevant records. Methodological filters would help to minimise this, making literature searching more efficient.An objective approach was employed to develop MEDLINE and EMBASE filters, using a reference standard derived from screening the results of an electronic literature search that contained only subject-related terms. Candidate terms for MEDLINE (N = 37) and EMBASE (N = 35) were derived from examination of the records of the reference standard. The filters were validated on two sets of studies that had been included in previous health technology assessments.The final filters were highly sensitive  with precision ranging between 16.7% \u2013 21.1%, specificity 35.3% \u2013 43.5%, and a reduction in retrievals of over 30%. Against the validation standards, the individual filters retrieved 85.2% \u2013 100% of records. In combination, however, the MEDLINE and EMBASE filters retrieved 100% against both validation standards with a reduction in retrieved records of 28.4% and 30.1%The MEDLINE and EMBASE filters were highly sensitive and substantially reduced the number of records retrieved, indicating that they are useful tools for efficient literature searching. When assessing the safety and efficacy or effectiveness of health technologies it may not be appropriate to restrict the evidence to randomised controlled trials (RCTs) . There aSearching for randomised evidence is relatively straightforward with the introduction of several initiatives to aid retrieval: the CENTRAL database of trials in The Cochrane Library; appropriate indexing terms in MEDLINE and EMBASE; and published highly sensitive filters -6. ThereSearching for non-randomised evidence of safety and efficacy or effectiveness from primary studies, however, is more problematic and there has been little published research to date. Indexing terms are less well established  and, wheThe uncertainty in identifying appropriate search terms for non-randomised evidence has meant that a methodology component is often excluded from search strategies. This can lead to an inefficient use of valuable resources in terms of time involved in screening the titles and abstracts of a large number of irrelevant records. For the purposes of this study, health technology assessments and systematic reviews commissioned by NICE and published on their website at October 2005 were surveyed. 77 health technology assessments from the main NICE program and the seven systematic reviews carried out for the IPP programme were reviewed. 28 (36.4%) of the technology assessments and seven (100%) of the IPP reviews included non-randomised evidence. 31(88.6%) of these reports used no methodology filter in their search strategies while two reviews of diagnostic interventions used diagnostic filters and two strategies used adverse events filters to identify supplementary safety data.Efficient searching for the evidence, for health technology assessments, requires an effective filter. The filter should maintain the sensitivity  of the original subject-only search, retrieving the same relevant studies  with a subject search strategy.In a recent systematic review to assess the effectiveness and safety of laser in-situ keratomileusis (LASIK), the MEDLINE and EMBASE search strategies used to identify the evidence had incorporated terms that pertained only to the intervention and medical conditions of interest to the review and were restricted to the publication years 2000\u20132004 . The MEDThe titles, abstracts, thesaurus controlled subject headings (for MEDLINE and EMBASE) and the publication type field (for MEDLINE) were subjectively assessed by the information specialist for all the MEDLINE and EMBASE records of the reference standard. Terms that explained or gave an indication of methodology employed or systematic assessment of postoperative sequelae were identified and considered as candidate terms for the MEDLINE and EMBASE methodology filters. By incorporating each term individually with the original MEDLINE or EMBASE subject search strategy (using the Boolean operator AND), the sensitivity (proportion of reference standard retrieved); precision ; and specificity (proportion of non-reference standard studies that were not retrieved) of the candidate terms were calculated.Initially all the candidate terms for each database were combined, using the Boolean operator OR, to form the separate MEDLINE and EMBASE filters. These were run in combination with the subject-only search strategies (using the Boolean operator AND). Each candidate term was then tested to establish if its removal from the filter reduced overall sensitivity. If sensitivity was unaffected the term was considered redundant and was excluded from further analysis, while if sensitivity decreased the term was re-instated. To minimise the number of irrelevant records retrieved, two approaches were explored:Precision Terms Filters.1. The terms were tested in order of precision, beginning with the lowest so that preference was given to retaining the terms with higher precision. The resulting MEDLINE and EMBASE filters are referred to as the Specificity Terms Filters.2. The terms were tested in order of specificity, beginning with the lowest so that preference was given to retaining the terms with higher specificity. The resulting MEDLINE and EMBASE filters are referred to as the Precision Terms Filters for both MEDLINE and EMBASE, and Specificity Terms Filters for both MEDLINE and EMBASE.By this process of elimination, redundant terms were removed and the combination of retained terms aimed to minimise the number of retrieved irrelevant records. Four filters were thus developed: Specificity Terms Filters for MEDLINE and EMBASE and the Precision Terms Filters for MEDLINE and EMBASE were also run simultaneously in multi-file MEDLINE and EMBASE searches, the results de-duplicated using the Ovid de-duplication tool, and retrieval parameters calculated.The subject-only MEDLINE and EMBASE search strategies were run with (using Boolean operator AND) and without the resulting filters. The total number of records and the number of reference standard records that were retrieved were used to calculate the retrieval parameters. The The performances of the preferred filters were tested against two validation standards. These comprised the included non-randomised studies from two other reviews: a systematic review of photorefractive keratomileusis (PRK) for myopia  and elecThe validations standards were incomplete in comparison to our reference standard because they did not include all non-randomised studies that met the inclusion criteria in terms of intervention and medical condition. Some non-randomised studies had been excluded at the screening stage because they did not fulfil other criteria, such as sample size or, in the case of the tonsillectomy review, were retrospective. These studies were not readily identifiable for inclusion in this study. The validation standards, therefore, underestimated the total number of non-randomised studies that were identified from the subject-only searches (cells A and C in Table The reference standard comprised 217 articles. Table (compare$ or compara$).mp (65.5%); (postoperat$ or post operat$).mp and (preoperat$ or pre operat$).mp (63.1% and 54.4% respectively); (preoperat$ or pre operat$).tw and (postoperat$ or post operat$).tw (53.4.0% and 51.0% respectively); and the MeSH term Comparative studies (51.5%). Precision varied from 1.3% to 41.7% with highest values for cohort.tw (41.7%), cohort.mp (37.9%) and (non random$ or nonrandom$).tw (37.2%). The ability of the search terms to exclude non-reference standard articles, their specificity, was generally high, with poorest performance found for the terms (postoperat$ or post operat$).mp (54.2%), (postoperat$ or post operat$).tw (68.1%) and evaluat$.tw (70.6%).Table (postoperat$ or post operat$).mp (64.4%), (preoperat$ or pre operat$).mp (55.5%) and the subject term Major clinical study (55.5%) retrieving the highest proportion of the reference standard. Precision varied from 0.3% to 42.1%: the most precise being, as with MEDLINE, cohort.tw (42.1%), (non random$ or nonrandom$).tw (41.3%) and cohort.mp (38.1%). Lowest specificity was again found for (postoperat$ or post operat$).mp (47.1%), (postoperat$ or post operat$).tw (69.7%), (preoperat$ or pre operat$).mp (71.1%) as well as the subject heading Treatment outcome (73.0%).The performances of the 35 candidate terms for the EMBASE records are listed in Table case series, case control$, cohort, observational, non random$ or nonrandom$ and non compara$ or noncompara$, were used infrequently with sensitivity values ranging from 1.5% to 14.1%. Compare$ or compara$, however, occurred in almost half of the titles and/or abstracts ; although this term would not necessarily be used in the context of describing study design. Whether a study was prospective or retrospective was not routinely stated in the titles or abstracts, occurring in only 61.1% of MEDLINE and 63.3% of EMBASE records.The more explicit text words and phrases, used to describe study design, such as Precision Terms search and 5/8 for Specificity Terms search and for EMBASE: 7/10 and 6/10 respectively). The MEDLINE and EMBASE Specificity Terms Filters included the same controlled terms as in the Precision Terms Filters but with the addition of one term each: Time factors for MEDLINE and Clinical trial for EMBASE. There was considerable similarity in retained text words between the strategies with three common to all four search filters  and two text words included in three of the strategies . The term preoperat$ or pre operat$.mp was also included in three strategies.Table The performances of the filters are detailed in Table Specificity Terms Filter. This pattern was also evident for the EMBASE searches ; and for the combined MEDLINE/EMBASE search (from 11.1% increasing to 17.1% for the Specificity Terms Filter). In terms of specificity achieved, the Specificity Terms Filter again performed marginally better. This was a consistent finding for both databases separately as well as in combination. The filters substantially reduced the number of articles retrieved varying from 30.0% to 37.9%. Once again the Specificity Terms Filter showed marginally superior performance, reducing the number retrieved from 1564 to 972 for Medline and from 1521 to 1016 for Embase.The inclusion of each of the filters to the original search strategies improved precision. For MEDLINE, precision increased from 13.2% to 20.9% or 21.1% depending on the filter used, and was highest for the Specificity Terms Filter performed marginally better than the Precision Terms Filter, the former was chosen as the preferred filter and was tested against the validation standards.Given the consistent finding that the Specificity Terms Filters were run against the two validation standards and the results are presented in Table The MEDLINE and EMBASE case control in the abstract. The other MEDLINE records included the text word post-tonsillectomy and were indexed with the term Postoperative complications. For the two EMBASE records, the excluded PRK record contained the textword consecutive while the excluded tonsillectomy record contained the text word retrospective and was inaccurately indexed with the term Retrospective study \u2013 since all the tonsillectomy studies had been assessed as being prospective in design.The six excluded records were examined -18. One Both MEDLINE and EMBASE filters resulted in a substantial reduction in the number of retrievals but was greatest for the MEDLINE filter (PRK: 33.7% vs 21.9% and tonsillectomy: 39.6% vs 29.8%). Using the filters in combination in a multifile search resulted in a reduction of 28.4% (from 792 to 567) in retrievals for the PRK search and 30.1% (from1712 to 1196) for the tonsillectomy search.In a review of methodological search filters, Jenkins describes the identification of a gold or quasi-gold standard as the set of relevant records against which filters are assessed . TypicalIdeally the gold or reference standard should be representative of all indexed records to ensure that the resulting filter has generalizability. This can be a limitation of using a gold standard based on hand searching a particular set of journals . While ocase series, cohort, observational, non-random and non comparative (including variations of these terms) appeared in only a small proportion of records and hence had low sensitivity. The exception was the term compare$ or compara$. Terminology that was used in the abstracts was often non-explicit, giving an indication of general systematic assessment. Those retained in the filters were evaluat$, reviewed, chang$, consecutive$ and preoperat$. The use of structured abstracts, to improve explicit reporting of methodology in the abstract, would facilitate text word searching and could assist in more effective indexing.Reporting of studies in the titles and abstracts infrequently used explicit terms that describe study design. Terms such as Specificity and Precision Terms Filters were similar with the former being consistently marginally better in terms of overall precision and specificity for both databases separately and in combination, while sensitivity was the same. The similar performance is not unexpected given that the majority of the search terms were common to both filters. For MEDLINE 10 out of 12 terms were included in both filters while for EMBASE, 7 were the same out of 8 in the Precision Terms Filter and out of 9 in the Specificity Terms Filter.The search filters were developed using an objective approach. The criterion for inclusion of terms was based on each term's ability to exclude irrelevant articles rather than ability to retrieve relevant ones. This approach aimed to include those terms with highest specificity or precision that in combination produced maximum sensitivity irrespective of the sensitivity of the individual terms. The performances of the post operative and case control for MEDLINE and consecutive and retrospective for EMBASE  but would have reduced the precision of the searches. The one MEDLINE record that did not have an abstract or any methodological related terms would only have been retrieved if no filter had been used.The validation standards were pragmatically derived, comprising the included studies in two other health technology assessments. Although both sets contained a small amount of records, they were independent of the reference standard. The MEDLINE and EMBASE filters retrieved between 85.2% and 100% of the validation standards. These would have been improved with the addition of the terms In combination however, when both MEDLINE and EMBASE were searched, 100% retrieval was achieved, for both validation standards, while reducing the number of retrievals by the same order as found for the reference standard. When comprehensive searching is required, as is usually the case for health technology assessments or systematic reviews, searching both MEDLINE and EMBASE is generally undertaken. The results of the validation would therefore suggest that the deficiencies in sensitivity of the individual filters would be considerably reduced when used in the context of multi-database searching.preoperat$ or pre operat$.mp, the terms used in the filters would be applicable to other interventions and further development could widen its applicability.The resulting filters were developed and tested on sets of studies relating to surgical interventions. With the exception of the terms The preferred MEDLINE and EMBASE filters, in combination with a subject search, maintained the sensitivity of the original subject search while at the same time reducing the number of irrelevant records retrieved by 33.2\u201337.9%. This performance was maintained when assessed against the validations sets. This was an initial attempt using a set of 217 records and small validation sets to develop a suitable filter to identify non-randomised studies for use in research where comprehensive searches are required. Further exploration is desirable to further test this filter using a larger dataset and to adapt it for use to a non-surgical context.The author(s) declare that they have no competing interests.CF developed and validated the filters and drafted the manuscript; AM screened the records for the original review, assessed study design and contributed to the manuscript; JB contributed to the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Information overload, increasing time constraints, and inappropriate search strategies complicate the detection of clinical practice guidelines (CPGs). The aim of this study was to provide clinicians with recommendations for search strategies to efficiently identify relevant CPGs in SUMSearch and Google Scholar.We compared the retrieval efficiency  of search strategies to identify CPGs in SUMSearch and Google Scholar. For this purpose, a two-term GLAD (GuideLine And Disease) strategy was developed, combining a defined CPG term with a specific disease term (MeSH term). We used three different CPG terms and nine MeSH terms for nine selected diseases to identify the most efficient GLAD strategy for each search engine. The retrievals for the nine diseases were pooled. To compare GLAD strategies, we used a manual review of all retrievals as a reference standard. The CPGs detected had to fulfil predefined criteria, e.g., the inclusion of therapeutic recommendations. Retrieval performance was evaluated by calculating so-called diagnostic parameters  for search strategies.The search yielded a total of 2830 retrievals; 987 (34.9%) in Google Scholar and 1843 (65.1%) in SUMSearch. Altogether, we found 119 unique and relevant guidelines for nine diseases (reference standard). Overall, the GLAD strategies showed a better retrieval performance in SUMSearch than in Google Scholar. The performance pattern between search engines was similar: search strategies including the term \"guideline\" yielded the highest sensitivity , and search strategies including the term \"practice guideline\" yielded the highest specificity , and the lowest NNR .SUMSearch is a useful tool to swiftly gain an overview of available CPGs. Its retrieval performance is superior to that of Google Scholar, where a search is more time consuming, as substantially more retrievals have to be reviewed to detect one relevant CPG. In both search engines, the CPG term \"guideline\" should be used to obtain a comprehensive overview of CPGs, and the term \"practice guideline\" should be used if a less time consuming approach for the detection of CPGs is desired. An exploding quantity of information, increasing time constraints, and the inadequacy of traditional sources of information underline the importance for clinicians to search efficiently for evidence-based and up-to-date medical information to support diagnostic, prognostic, and therapeutic decision-making processes . ClinicaThe free-access Internet search engines SUMSearch and Google Scholar are widely used when searching for medical information. SUMSearch was developed by the University of Texas in 1999 . It searGoogle Scholar is a search engine that was launched in November 2004 by Google Inc. It is available in the beta test-version, which is in continuous transition . Google Our analysis was motivated by the fact that we had previously not identified studies that compared search strategies for CPGs in SUMSearch and Google Scholar by means of diagnostic parameters. The model for our study was provided by the analysis methods introduced by the Hedges group to detect different types of studies in different databases ,24-31. TWe compared the retrieval performance (efficient detection of relevant CPGs) of a two-term search strategy in SUMSearch and Google Scholar, using a manual review of retrievals as a reference standard. Our research focussed on nine different diseases currently being evaluated by the German health authorities with regard to their suitability for inclusion in disease management programmes . A \"relevant\" CPG had to fulfil predefined criteria (e.g. the inclusion of diagnostic or therapeutic recommendations). \"Efficient\" meant detecting as many unique and relevant CPGs as possible in a given period of time (which varies depending on the time available to the user).The two primary performance parameters of the evaluation were the completeness of the detected pool of CPGs (measured with the parameter \"sensitivity\"), and the number of retrievals that had to be read  to find one relevant CPG. These two measures of retrieval performance represent the (possibly contradictory) situation of a clinician searching for CPGs: i) he or she has sufficient time for a complete review of retrievals to preferably detect all available relevant CPGs on a topic; ii) he or she would like to detect CPGs on a specific condition at short notice while treating a patient. In the former case, the completeness of the search result is decisive; in the latter, the time invested is. Specificity  was defined as a secondary performance parameter.The individual methodological procedures of the evaluation were continuously developed during the study, according to the experience gained while working with the search engines. These procedures were not specified a priori and were therefore conducted as an iterative process. An overview of the study methodology is shown in Figure Retrievals SUMSearch, Retrievals Google Scholar in Table CPG term + \"back pain\" in Table First, we entered 14 terms commonly used in guideline searches  into SUMThe developers of SUMSearch assessed predictors of successful searches for medical evidence and showed that searches were twice as likely to succeed when no more than two terms were included or MeSH terms were used . On the We adapted the syntax of the GLAD strategy to the individual requirements of each search engine Figure . In SUMSIn Google Scholar, the single or plural CPG terms were used without an operator . We used the advanced interface and selected \"in the title\". We entered the combination of each CPG term  and each MeSH term into the search box \"with all of the words\". We used the \"all in title\" restriction, as prior informal searching in Google Scholar had produced an excessive amount of retrievals without this limit.The retrievals for the nine diseases were pooled. The application of the GLAD search strategy therefore resulted in three retrieval pools (one for each CPG term applied) per search engine.After collecting the raw retrievals in SUMSearch and Google Scholar, we defined a reference standard by manually reviewing all retrievals (links) from the retrieval list of each search engine. Each raw retrieval was regarded as a reading unit that had to be reviewed to identify unique and relevant CPGs. Since the GLAD strategies were to be tested against a reference standard, the retrievals had to be unique .The unique and relevant CPGs of both search engines formed the reference against which each of the three CPG-term search strategies applied in SUMSearch and Google Scholar was tested. As duplicates also involved a relevant input of work and time for the reviewer , they were also considered in the analysis Table .The full-text articles of the retrievals were screened and assessed; if these were not available, abstracts were reviewed. Retrievals were declared \"relevant\" if the detected CPG fulfilled specific criteria with regard to content, language, and publication date Figure . One revWe recorded the number of identified CPGs per GLAD strategy  and per search engine, and documented the corresponding intersections of CPGs Figure .The sensitivity, specificity, and NNR of the three GLAD strategies were calculated per search strategy in each search engine following the terminology of diagnostic tests, using the defined gold standard as a reference Table  26]. It. It26]. Sensitivity for a given strategy is the proportion of identified unique and relevant guidelines among the total number of unique and relevant retrievals identified by the reference search; specificity is the proportion of non-relevant retrievals and duplicates not retrieved by this strategy among the total number of non-relevant retrievals and duplicates not retrieved by the reference search; precision is the proportion of retrieved guidelines that are unique and relevant among the total number of retrievals. The NNR (1/precision) is defined as the number of non-relevant and duplicate retrievals that have to be screened to find one relevant retrieval . For allThe application of the three GLAD search strategies per search engine for each of the nine diseases  yielded a total of 2830 retrievals (for the nine pooled diseases); 987 34.9%) in Google Scholar and 1843 (65.1%) in SUMSearch. The manual review identified a total of 119 unique and relevant CPGs (reference standard) in both search engines , the lowest specificity (78.1%) and a NNR of 16.7. The search strategy including the term \"practice guideline\" yielded the highest specificity (95.7%), a sensitivity of 11.8%, and the lowest NNR (9.3). The search strategy including the term \"recommendation\" yielded the lowest sensitivity (8.4%), a specificity of 92.1%, and the highest NNR (22.4). In the two latter strategies, the low sensitivity seems to be a trade-off for the high specificity.In summary, the GLAD search strategies showed a better retrieval performance in SUMSearch than in Google Scholar. The performance pattern between search engines was similar: in both search engines, the best results for the primary performance parameters were achieved by strategies including the term \"guideline\" (highest sensitivity) and \"practice guideline\" (lowest NNR). This means that the former strategy had the most comprehensive detection of unique and relevant CPGs, i.e. it detected the highest number of unique and relevant retrievals among the reference retrievals. The latter required the lowest time investment in a search, i.e. it required the lowest number of retrievals to identify a relevant retrieval. None of the tested strategies combined the highest sensitivity and lowest NNR. The best results for the secondary performance parameter (highest specificity) were also achieved by the strategy including the term \"practice guideline\", meaning that this strategy disregarded the highest proportion of non-relevant retrievals.With regard to the NNR, about 30% more time was required in Google Scholar than in SUMSearch to detect a relevant CPG (using the strategy including \"practice guideline\") Table . ConcernGoogle Scholar showed better results only for the secondary performance parameter \"specificity\"  strategies in SUMSearch and Google Scholar, using a manual review of retrievals as a reference standard. As the reference standard was solely defined by means of the manual search of the raw retrievals in SUMSearch and Google Scholar and only includes CPGs identified by these engines, it cannot be universally transferred to searches in other search engines or bibliographic databases. It should also be noted that since conducting our search, changes have been made to both SUMSearch and Google Scholar. Therefore, the replicability of results may be affected. A further limitation of this study may be due to the fact that the reviewers were not blinded as to which search engine produced the CPG. They were not blinded because, as stated, the methodological procedures were not specified a priori and were conducted as an iterative process. However, as none of the reviewers previously routinely used either SUMSearch or Google Scholar, we believe it is unlikely that bias caused by a preference for a specific search engine was a relevant factor.The GLAD strategies showed a better retrieval performance when applied in SUMSearch than in Google Scholar. However, one needs to consider that our approach was based on the specifications developed for searches in SUMSearch; therefore, the transfer of a \"SUMSearch-specific\" strategy to Google Scholar might have been unfavourable for its retrieval performance. To our knowledge, there is no 'best search strategy' for CPGs \u2013 or one that is better than our GLAD strategy \u2013 available for Google Scholar.The strength of SUMSearch lies in its expertise in the medical sciences. Since its launch, the search strategies of users have been continuously reviewed. This process has led to the current recommendations for search strategies. SUMSearch's structural characteristic as a meta-search engine in scientific sources supports the specificity of its retrieval performance. The handling of retrievals in SUMSearch is convenient. The links presented in the search results are in reference format and lead directly to the article or abstract of the retrieval.It remains open as to whether the ongoing changes, such as the inclusion of additional filters, in the structure of Google Scholar (which is still in its beta version) will lead to an improvement in retrieval performance. Due to its intuitive approach, Google Scholar has been described as especially quick in locating frequently cited articles and the proverbial \"needle in a haystack\" . DespiteIn SUMSearch as well as in Google Scholar, we found that the term \"practice guideline\" is the CPG-term that should be included in a GLAD strategy by the clinician under time pressure who is looking for quick answers to a clinical problem by means of a CPG. Although this strategy may not identify all relevant CPGs, it has a high probability of detecting a sufficient number to answer the question posed. This is because it has a low NNR and a high specificity, and is therefore likely to detect relevant retrievals quickly without having to review an excessive number of non-relevant ones. If the clinician wants to gain a comprehensive overview of CPGs on a certain topic, a GLAD search strategy including the term \"guideline\" should be used; this was the term with the highest sensitivity. Although this strategy is more time consuming, it considerably increases the chances of detecting a large number of relevant CPGs. However, the dilemma in which the searcher finds him- or herself, time pressure on the one hand and the desire to detect as many relevant CPGs as possible on the other hand, cannot be solved to complete satisfaction.Google Scholar is not a useful tool to search efficiently for CPGs. The advantage of being intuitive does not make up for the laborious and time consuming handling of the retrievals when screening for CPGs. We recommend SUMSearch as a starting point for a search to gain an overview of available CPGs. It specialises in the medical field and has the advantages of a meta-search engine that searches medical databases simultaneously. The retrievals are accessed quickly and reliably by links, enabling swift screening. However, neither SUMSearch nor Google Scholar can replace the commonly used CPG searches in portals/websites of organisations that publish CPGs and bibliographical databases.The developed GLAD strategy is easy to use and to remember. It is applicable in both SUMSearch and Google Scholar. In SUMSearch, however, the GLAD strategy shows a superior retrieval performance.We did not identify the \"ideal\" CPG term for a GLAD strategy. The term \"guideline\" should be used if the aim is to detect a comprehensive pool of CPGs, and \"practice guideline\" should be used if the aim is to rapidly identify CPGs.In future, the introduction of a standardised index term for CPGs, analogous to MeSH terms, may facilitate identification by search engines and help to improve retrieval performance. A further vision for the combination of the advantages of both search principles \u2013 the federated search of Google Scholar and the meta-search of SUMSearch \u2013 could lead to the development of a 'Medical Internet Portal'. In combination with a standardised CPG-index term, relevant CPGs could be identified more quickly and comprehensively.CPG: Clinical Practice GuidelineDARE: Database of Abstracts of Reviews of EffectsGLAD: GuideLine And DiseaseMeSH: Medical Subject HeadingNGC: National Guideline ClearinghouseNNR: Number Needed to ReadThe author(s) declare that they have no competing interests.This study was initiated by HK and MF. All authors provided intellectual content to the literature search and the data analysis. The literature search was conducted by AH. Data analysis was conducted by AH and GS. The draft of the manuscript was prepared by AH. All authors commented on and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "With the rapid growth of medical information and the pervasiveness of the Internet, online search and retrieval systems have become indispensable tools in medicine. The progress of Web technologies can provide expert searching capabilities to non-expert information seekers. The objective of the project is to create an alternative search interface for MEDLINE/PubMed searches using JavaScript slider bars. SLIM, or Slider Interface for MEDLINE/PubMed searches, was developed with PHP and JavaScript. Interactive slider bars in the search form controlled search parameters such as limits, filters and MeSH terminologies. Connections to PubMed were done using the Entrez Programming Utilities (E-Utilities). Custom scripts were created to mimic the automatic term mapping process of Entrez. Page generation times for both local and remote connections were recorded.Alpha testing by developers showed SLIM to be functionally stable. Page generation times to simulate loading times were recorded the first week of alpha and beta testing. Average page generation times for the index page, previews and searches were 2.94 milliseconds, 0.63 seconds and 3.84 seconds, respectively. Eighteen physicians from the US, Australia and the Philippines participated in the beta testing and provided feedback through an online survey. Most users found the search interface user-friendly and easy to use. Information on MeSH terms and the ability to instantly hide and display abstracts were identified as distinctive features.SLIM can be an interactive time-saving tool for online medical literature research that improves user control and capability to instantly refine and refocus search strategies. With continued development and by integrating search limits, methodology filters, MeSH terms and levels of evidence, SLIM may be useful in the practice of evidence-based medicine. There is unprecedented growth of medical information. PubMed, a service of the National Library of Medicine, includes over 15 million citations of biomedical articles \" search tag with the \"[TIAB]\" search tag that redirects the search to keywords in the Title or Abstract. Subsequent levels in the slider involve adding mapped MeSH terms as filters, with options to search MeSH terms as major topics, or exclude MeSH terms below the current term in the MeSH tree. Table MeSH subheadings are topical qualifiers that describe a particular aspect of a subject such as etiology or therapeutic use. Users have the option to require these qualifiers through a checkbox. The MeSH subheadings are grouped and appended as a filter to the search query. This feature is closely linked with the search mapping algorithm. The subheading filter is only added if the search mapping algorithm requires MeSH terms, i.e. second level and above.SLIM was written in PHP and developed on an Apache 2.0.52 server running PHP 4.3.10. The PHP scripts generate a HyperText Markup Language (HTML) and JavaScript search form. JavaScript provides most of the functionality of the search form and search results. Free and open source JavaScript codes were downloaded from the Internet for the slider controls ,18. CustThe application connects to the MEDLINE/PubMed database using tools from Entrez Programming Utilities (E-Utilities) . The ESeAutomatic term mapping is the process where terms entered in the PubMed query box without a search tag are matched against the Medical Subject Headings translation table, the journals translation table, the full author translation table and an author index . To optiDepending on slider bar and search form input, the detailed search query built on the first ESearch pass is modified by appending user-defined limits and study design filters, or by converting search tags. The modified query is processed once again through the ESearch tool to get the final list of PubMed IDs (PMIDs) from the second XML document. The ID list is sent to the EFetch tool to retrieve and display the details of the first 200 citations.To simulate performance testing, benchmarking timer functions were embedded in the PHP scripts to measure page loading and search times. All values generated during the alpha-testing with developers and beta-testing with users were recorded in a MySQL database for data analysis.An online survey form was created to gather preliminary user opinion on stability and usability of the application. All seven questions used Likert scales to record answers. A call for participation in the usability testing was made on a mailing list of an international group of practicing physicians. Users were asked to use the system as a replacement for their regular PubMed search engine for two weeks. Comments and discussion on the application were encouraged.Initial qualitative testing by application developers demonstrated that SLIM is functional and stable. Occasional XML file connection problems occurred, but rarely. These were attributed to technical issues in the E-Utilities server and were quickly resolved by resubmitting the search form. The application was tested for compatibility with multiple browsers and worked in default installations of Internet Explorer 6.0, Mozilla Firefox 1.06, Opera 8.02 and Safari 1.2.During performance testing, the index page was loaded 216 times with a mean page generation time of 2.94 milliseconds and standard deviation of 3.20 milliseconds. Search previews were processed 65 times with a mean page generation time of 0.63 seconds and standard deviation of 0.78 seconds. Complete searches with display of results were done 142 times with a mean page generation time of 3.84 seconds and standard deviation of 6.28 seconds. Table Eighteen physicians from the US, Australia and the Philippines participated in the beta-testing phase of the application and provided performance and usability feedback through an online survey. The mode was used as measure of central tendency as recommended by experts because Likert scales fall within the ordinal level of measurement ,21. TablResearch in data mining, natural language processing and methodology filters move towards developing backend algorithms and protocols for online information search and retrieval systems. Despite all these advances, users continue to use default settings in their PubMed searches. This is a preliminary study of an ongoing project to improve search interface usability by enhancing user interaction with advanced features of PubMed.Limits and filters were chosen based on adaptability to structured series of implementations. Publication date and age groups were obvious choices. The journal subset slider narrowed down the search pool by combining subsets in PubMed with selected filters for availability of text. The hierarchy of levels of evidence provided a structured implementation for publication types and clinical study categories. The search mapping feature increased user involvement with MeSH operations. The project continues to study most of the limits and filters available in PubMed for integration in future versions of the application.The information box was created as an educational tool for PubMed users and researchers by providing another level of feedback. It is nothing more than a short report on the term mapping and translation algorithms of Entrez PubMed. Users are informed of the MeSH term or MeSH subheading equivalents of their search terms. Unmapped terms can prompt users to modify specific keywords in their search.Users who participated in the online survey were practicing physicians interested or involved in Medical Informatics. All were frequent users of PubMed and categorized themselves as intermediate to advanced users. The familiarity with the current Entrez PubMed interface often accounts for the negative responses in the online survey. Although most user comments were positive, one user stated that advanced users of PubMed might feel more comfortable with the consistent interface of Entrez PubMed, whereas slider controls might be more useful for novice or non-expert users. One user suggested that slider labels be more descriptive. In response, some label texts were expanded to include descriptions of the slider settings. A tutorial page was also suggested.The study has several limitations. First, the search interface omits several limits existing in the PubMed limits page like subsets and non-English languages. The developers continue to look into integrating currently available filters in newer versions of the application. Second, only eighteen physicians interested in Medical Informatics projects participated in the beta-testing phase. A broad mix of PubMed researchers is a target for future usability studies. Third, the retrieval performance to measure sensitivity and specificity of the methodology filters was not tested. Review of Clinical Study Category filters not incorporated in the application, e.g. diagnosis and prognosis, is in process. Ongoing efforts to improve the application include evaluating search algorithms for accuracy and precision and adopting validated PubMed filters from previous published studies. The developers continue to monitor advancements in Web technology and add new interactive features in the application.The goal of the project was to create a PubMed search application that allows users of all levels to easily go beyond basic keyword searches and move towards evidence-based principles. This is consistent with the practice of evidence-based medicine which advocates the formulation of effective search strategies to find current evidence. With increasing access to the Internet, online bibliographic databases have become important real-time resources for current evidence at the point-of-care. Although tools to control search parameters are available, these often require advanced familiarity with the search interface elusive to beginners. By exploring progressive Web technologies and creating an interactive search interface, the application may prove valuable in bridging the gap between expert and non-expert users of PubMed.The Web-based application offers an alternative search interface to facilitate MEDLINE/PubMed searches. JavaScript slider bars control search limits, add filters or modify search terms with ease. Textual link controls can hide or display abstracts with a mouse. Initial qualitative testing and user feedback were positive which reinforced the approach of enhancing user interaction to improve online research. SLIM can be an interactive time-saving tool for online medical literature research that improves user control and capability to instantly refocus search strategies. With continued development and by integrating search limits, methodology filters, MeSH terms and levels of evidence, SLIM may be useful in the practice of evidence-based medicine.Project name: SLIM (Slider Interface for MEDLINE/PubMed searches)Project home page: Operating systems: Platform independentProgramming language: PHP, JavaScriptOther requirements: JavaScript-enabled browsers, e.g. Fire Fox 1.0 or higher, IE 5 or higherLicense: Free, anyone may use the serviceAny restrictions to use by non-academics: NoneSLIM: Slider Interface for MEDLINE/PubMed searchesMeSH: Medical Subject HeadingsPHP: PHP: Hypertext PreprocessorHTML: HyperText Markup LanguageXML: Extensible Markup LanguagePMID: PubMed IDThe author(s) declare that they have no competing interests.MM conceived of the study, designed the interface, developed filters and algorithms, wrote the PHP codes, organized the usability testing and drafted the manuscript. PF assisted in the study and interface design, participated in usability testing, developed filters and algorithms and reviewed the initial drafts of the manuscript. FL wrote the custom JavaScript codes and assisted in design of the interface. MA participated in the study design, evaluation study and gave final approval of the version to be published. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Systematic reviewers seek to comprehensively search for relevant studies and summarize these to present the most valid estimate of intervention effectiveness. The more resources searched, the higher the yield, and thus time and costs required to conduct a systematic review. While there is an abundance of evidence to suggest how extensive a search for randomized controlled trials (RCTs) should be, it is neither conclusive nor consistent. This systematic review was conducted in order to assess the value of different resources to identify trials for inclusion in systematic reviews.Cochrane, Simple, Complex, Index), expertise of the searcher , and study design (RCT and CCT).Seven electronic databases, four journals and Cochrane Colloquia were searched. Key authors were contacted and references of relevant articles screened. Included studies compared two or more sources to find RCTs or controlled clinical trials (CCTs). A checklist was developed and applied to assess quality of reporting. Data were extracted by one reviewer and checked by a second. Medians and ranges for precision and recall were calculated; results were grouped by comparison. Meta-analysis was not performed due to large heterogeneity. Subgroup analyses were conducted for: search strategy (Complex and Cochrane search strategies (SS) performed better than Simple SS.Sixty-four studies representing 13 electronic databases met inclusion criteria. The most common comparisons were MEDLINE vs. handsearching (n = 23), MEDLINE vs. MEDLINE+handsearching (n = 13), and MEDLINE vs. reference standard (n = 13). Quality was low, particularly for the reporting of study selection methodology. Overall, recall and precision varied substantially by comparison and ranged from 0 to 100% and 0 to 99%, respectively. The trial registries performed the best with median recall of 89%  and median precision of 96.5% , although these results are based on a small number of studies. Inadequate or inappropriate indexing was the reason most cited for missing studies. Cochrane SS or a Complex SS in consultation with a librarian are recommended. Continued efforts to develop CENTRAL should be supported.Multiple-source comprehensive searches are necessary to identify all RCTs for a systematic review, although indexing needs to be improved. Although trial registries demonstrated the highest recall and precision, the  The aim of systematic reviews is to present the most valid estimate of the effectiveness of the intervention in question. To do so, the identification of relevant studies must be comprehensive and unbiased. Systematic reviews usually include a comprehensive summary of data from both randomized (RCT) and controlled trials (CCT), although other study designs are sometimes incorporated. There is an ongoing debate about the number and type of resources that should be used to identify trials for systematic reviews -3. TheseThe Cochrane Collaboration Reviewers' Handbook notes that MEDLINE, EMBASE, and CENTRAL are the three electronic bibliographic databases generally considered as the richest sources of trials . The CocOur research question was: Does resource-specific searching retrieve RCT/CCTs for systematic reviews with the same recall and precision as those searches which combine two or more distinct resources? Our primary goal was to identify and quantitatively review studies comparing two or more different resources  used to identify RCTs and CCTs for systematic reviews. Specifically, we were interested in determining the value (in terms of identifying unique citations) of searching key resources  in addition to the key resource MEDLINE.Seven electronic databases  were searched from their inception to April 2004. The MEDLINE search strategy was tailored as necessary for each database (Appendix 1). Four journals were handsearched from 1990 to 2004: Health Information & Libraries Journal , Hypothesis, Journal of the Medical Library Association , Medical Reference Services Quarterly. All abstracts presented at Cochrane Colloquia (1993\u20132003) were handsearched. In addition, key authors were contacted via email and references of relevant articles were screened. The searches were not limited by language or date of publication. Searches are available upon request from the corresponding author.Two reviewers independently screened the yield from the searches to identify potentially relevant studies. The full text of these studies was obtained and two reviewers independently applied inclusion/exclusion criteria using a standard form. Any differences were resolved through discussion. Studies were included if they compared two or more sources to find RCTs or CCTs . Inclusion was not limited by the topic/content area in the individual studies.A study was excluded if: it only compared different search strategies within the same database; it only included non-randomized trials; or, if the resource is not currently accessible. If authors searched for all study designs including trials, it was included only if data were reported separately for RCTs or CCTs.RCTs were defined as an experiment in which eligible patients are assigned to two or more study groups using an appropriate method of randomization . CCTs weWe developed a checklist to assess the quality of reporting of the included studies. The quality items were chosen based on threats to the validity of comparative studies that have been empirically supported in the literature . The ite\u2022 Was there an adequate description of what the search was attempting to identify ;\u2022 Was there an adequate description of the methods used to search , words/subject headings used, time period covered, date of search);\u2022 Was there an adequate description of the reference standard  and how it was derived ; and,\u2022 Was bias avoided in the selection of relevant studies .Two reviewers independently applied the checklist to the included studies. Discrepancies were resolved through discussion.Data were extracted independently by one investigator and checked by a second independent investigator. A standard form was used to extract the following information: language of publication, country where study was conducted, year of publication, study design and objective(s), resources being compared, topic being searched, years the search covered, search strategies used, results , and author's conclusions. When data were not available, authors were contacted and asked to supply the missing information.Data were analyzed using Splus 6.2 (Insightful Corporation 2003). Recall and precision were expressed as percentages with 95% confidence intervals which were calculated using exact methods . Recall Cochrane, Simple, Complex, Index), expertise of the searcher , and study design (RCT and CCT).Possible sources of heterogeneity were explored with numerical summaries both by within-study and between-study analyses. Within-study analyses are direct analyses and they occur when two searches are conducted with the same known conditions  and some unknown conditions except for the condition or variable of interest. These are also called direct analyses. Between-study analyses are indirect analyses and are of a lower grade  in that Complex (using a combination of types of search terms); Cochrane (the Cochrane Highly Sensitive Search Strategy (HSSS)); Simple ; and, Index  to check/verify if the study is in a database).Searches were divided into the following four categories : Comple. Approximately half the studies (n = 30) were conducted in the United Kingdom. Three studies were non-English . Thirty studies received funding, some from more than one source. Financial support was received from: 16 government programs, 2 pharmaceutical companies and 35 other sources .Sixty-four studies met the criteria for inclusion in this analysis Figure . Of thesThe included studies searched a variety of topics which fell into four major categories: journal , disease/condition/state , specialty/sub-specialty  and methodology  see . GeneralThe reference standards varied . The specific study design for which authors were searching varied by study: RCTs only (n = 27); RCTs and CCTs (n = 28); and RCTs, CCTs, and other designs (n = 9).There were four major comparisons: MEDLINE vs. handsearch (n = 22), MEDLINE vs. MEDLINE + handsearch n = 12), MEDLINE vs. other reference standard (n = 18), and EMBASE vs. reference standard (n = 13). There were 13 other comparisons with only one to two studies each  stated their inclusion/exclusion criteria. Eighty percent of the studies described (or indicated they were available) reproducible search strategies/methods. Half of the studies stated who developed the search strategies and of these only 2 did not provide reproducible information about their search strategy. Eighty-five percent of articles fully described how the reference standard was compiled. Twenty-five percent reported that at least 2 people independently screened searches and in 35% at least 2 people also independently applied eligibility criteria.Table Forty-nine studies had usable data for the MEDLINE comparisons. Three comparisons were analyzed: MEDLINE versus handsearching , MEDLINE versus MEDLINE plus handsearching , and MEDLINE versus other reference standards . Estimates of both recall and precision for all three comparisons varied substantially, ranging from 7 to 98% and 0.03 to 99%, respectively. The estimates for MEDLINE versus handsearching and MEDLINE versus a reference standard were comparable: median of 53 versus 59% for recall and 35 versus 27% for precision. Median recall and precision for MEDLINE versus MEDLINE plus handsearching were somewhat larger .Eleven studies had usable data for the EMBASE comparisons. Two comparisons were analyzed: EMBASE versus handsearching  and EMBASE versus a reference standard . Individual study estimates ranged from 13 to 100% for recall and 0 to 48% for precision. Summarizing all studies, medians were 65 and 72% for recall and, 13 and 28% for precision.Four studies contained data for the PsycINFO comparisons. Two comparisons were analyzed: PsycINFO versus handsearching , and PsycINFO versus a reference standard . Recall ranged from 0 to 70%. Precision ranged from 8 to 47%. Medians were 69 and 50% for recall and 9 and 39% for precision.Two studies investigated CINAHL and trial registers. Only one study had usable data from other databases . The results from the trial registries versus a reference standard were consistent and high: 89% for median recall and 97% for median precision. The remaining comparisons ranged from 0 to 92% for median recall and 0 to 17% for median precision. Regardless of topic, there were too few included studies in these comparisons for this data to be representative.Simple versus Complex search strategies. All but one study [Complex search strategies. The trade-off which so often occurs between recall and precision did not occur: three out of the four Complex search strategies had larger (better) precision (not including Fergusson [Table ne study  showed gergusson ).Simple search strategy versus the Cochrane HSSS. Again, all but one of the comparisons [Cochrane search strategy. None of these four comparisons reported precision. Fergusson [There were five direct comparisons of a parisons  had largergusson  found neSimple and Complex search strategies in median recall were found: 49 versus 51% for MEDLINE versus handsearching, 48 versus 67% for MEDLINE versus MEDLINE plus handsearching, and 58 versus 40% for MEDLINE versus a reference standard. The precision results were similar: 76 versus 38%, 62 versus 51%, and 23 versus 35%, respectively. And although the median precision estimates for the Cochrane search were much smaller , the median recall estimates  were systematically greater when compared to the Simple and Complex search strategies.Three MEDLINE comparisons were considered for the indirect comparisons Tables  and 4 siComplex search had a recall of 53% and the non-librarian's Complex search had a recall of 34. For the indirect subgroup comparisons, the librarians did not systematically outperform the non-librarians on either median recall or median precision; however the Cochrane HSSS (as author) did outperform the librarians and non-librarians on median recall .Only one study  directlyNo studies directly compared searching for RCTs versus CCTs. Based on indirect comparisons, the three MEDLINE comparisons showed no systematic difference in median recall and precision between design types.A sensitivity analysis excluding duplicate topics was performed due to the concern for non-independence between studies. Studies or comparisons searching on the same topic may include some of the same relevant studies. We picked one comparison randomly from each topic area and eliminated it from the main quantitative results. The results are shown in Table Table Index searches. Six comparisons using the Index searches were found for MEDLINE versus handsearching. The median recall was 93% and the range was 41 to 100%. On average, 7% of studies were not indexed adequately. One study compared MEDLINE to a reference standard, their Index search produced 66% of the included studies. Two further Index searches were performed: EMBASE versus handsearching and PsycINFO versus handsearching; their recalls were 52 and 97%, respectively.In this study, seven studies performed For handsearching, few authors provided information for why trials were missed. Handsearches had high precision and some studies did not miss any references through their handsearches. In the 3 studies where handsearchers missed studies, authors reported the reasons for missing studies were the handsearchers were not trained properly or they had fatigue/boredom. In two studies where trials were missed, authors reported that the journal was not handsearched, yet a database was used to search for this same journal. One of the missed articles was misclassified by a handsearcher as an RCT/CCT and one had a different topic than what handsearchers were meant to identify. Results from the MEDLINE versus MEDLINE plus handsearching comparisons quantify the percentage of trials missed by handsearching. In 13 studies, the median percentage found in MEDLINE but not by handsearching was 6% (range 1 to 15%).For certain topics trial registries may be sufficient , however, the median recall estimates Table  were notOver and above the recalls, the median precisions are quite low and indicate a need for improved indexing in databases. Efforts to improve and standardize the indexing of various databases need to be supported. Guidelines for journals and authors regarding the reporting of key methodological or subject terms when publishing studies would facilitate these efforts . In addiMost of the research has involved MEDLINE and EMBASE, two of the major databases that the systematic review community recommends reviewers search. However, searching multiple databases can be difficult, time consuming and costly. For example, although MEDLINE is available freely on the Internet through PubMed, EMBASE is very costly and many institutions do not subscribe to it. This is of particular concern as studies have demonstrated that there is 17 to 75% overlap between MEDLINE and EMBASE ,23,24 inOptimally, it would be most efficient to search few resources, retrieve a maximum yield of relevant trials, and retrieve a minimum yield of irrelevant trials. The Cochrane Collaboration is trying to achieve this with the Controlled Trials Register (CENTRAL) available through the Cochrane Library. The register now includes over 420,000 RCTs and CCTs. While there are numerous studies that discuss the vast amount of trials that have been added to CENTRAL through handsearching efforts, there are very few studies evaluating whether CENTRAL can be searched exclusively for RCT/CCTs. If one resource  can be searched to identify RCT/CCTs, this would substantially reduce the time and costs associated with searching.There was extensive heterogeneity among topics investigated in the studies included in this review. For the comparisons which had many studies, the values for both recall and precisions covered most of the possible range . Thus, the topic searched may be the strongest determinant of the results. Topics are indexed differentially within and across various sources. Due to the between-study heterogeneity, very little can be concluded about the indirect subgroup results.Over the 20 years that this review covers, it was noted that the older studies were conducted prior to indexing improvements in resources, especially MEDLINE. While there have been numerous changes in search technology in the past two decades, upon conducting post-hoc sub-group analyses, no difference was found. In addition, a sub-group analysis was done of recent studies and no difference was found when compared to the results of older studies. As mentioned above, this may be due to topic heterogeneity, not changing search technology. Thus, including the older studies did not confound our results and did not lead to the conclusion that there is one sufficient resource which identifies RCT/CCTs. Unfortunately, the more recent studies are not showing results which significantly differ from the ones obtained 20 years ago.Complex and Cochrane search strategies performed better in recall than did Simple search strategies without loss in precision. However, the indirect subgroup results for recall showed support for this finding for the Cochrane search strategies, but not for the Complex search strategies. The Cochrane search strategy precisions were poorer in the indirect subgroup results, however data were sparse. Little direct evidence was available comparing searchers with different expertise. No direct evidence was available comparing searches for different design types. Without supporting direct subgroup evidence, conclusions from the indirect subgroup evidence would be too speculative. Other reasons that may explain the heterogeneity include: 1) the time period covered by the search (indexing as well as other search technologies have progressed over time which would affect the accuracy of searches); and 2) the methodological quality of the study. For example the rigor with which handsearching was done, searches were screened, or inclusion criteria applied  would affect the comparability of results across studies.We found that, generally, both The quality of the included studies varied and, in most cases, the poor quality result was due to the lack of rigor in the reporting of the selection methodology. One-third reported that standard inclusion and exclusion criteria were developed and applied to each database/method at the relevance stage. Almost all studies did not indicate that at least 2 people independently screened the searches for potentially relevant studies. In addition, two-thirds of the included studies did not indicate that at least 2 people independently applied eligibility criteria to identify relevant studies. Quality of these studies can be improved by adopting more stringent methodology and reporting its use.Post-hoc, we looked at how the calculated results of recall and precision may have improved over the last two decades considering the changes in search technology . Within our three MEDLINE comparisons, we found no pattern of association between year of publication and results. The correlations ranged from -0.91 to 1. As mentioned above, this quantitative analysis may be too dilute due to topic heterogeneity. We suggest a within topic analysis to robustly test for improvements in search technology.Complex versus Simple search strategies), we also systematically examined the within-study subgroup comparisons which provides more valid information [This paper provides the most current and comprehensive review of the existing evidence comparing any electronic database against any other source or combination of sources. This and previous reviews demonstrate that there is a dearth of evidence regarding the use of different databases to retrieve RCTs, with the notable exception of handsearching and MEDLINE. Therefore, searching multiple resources to retrieve RCTs cannot be ruled out based upon this evidence. There needs to be more research done on major databases such as: EMBASE, CENTRAL, PsycINFO and trial registries in order to gather more information about the value of these databases in identifying RCT/CCTs for various topical areas. This review is more comprehensive than previous work in this area  and reflormation . HoweverThere are several limitations to this study. Foremost, there is a lack of a validated quality score for this type of study . Reference standards are difficult to compare as they are generally different and may not be reported in enough detail to be reproducible. As well, the topic chosen to search can determine the success of the strategy. In addition, there are limitations to using precision and recall which are addressed by Kagolovsky and Moehr .Cochrane search strategy or Complex search strategy in consultation with a librarian are recommended.Since recall is low with single resources, multiple-source comprehensive searches continue to be necessary. The Efforts to enhance and build CENTRAL, a large trial registry, need to be continued. A number of the resources used to find trials for CENTRAL  are not indexed in MEDLINE, therefore CENTRAL has a significant amount of unique information not found in any other source. CENTRAL is also free for researchers in developing countries and available in CD-ROM and on the internet. Based upon the results of why studies were missed, indexing efforts also need to improve. Guidelines should be provided for authors to include MeSH terms and keywords in their abstracts which can then be used by indexers. Other resources that need to be studied include: Trial registries, LILACS, PsycINFO, Science Citation Index, BIOSIS, CABNAR and CINAHL. In addition, those researchers studying searches need to report precision results.CCT \u2013 controlled clinical trialRCT \u2013 randomized controlled trialThe author(s) declare that they have no competing interests.ETC conceived of the study, designed and coordinated it and drafted the manuscript. NW performed the statistical analysis and drafted and read the final manuscript. LH and KC participated in the study and helped to draft the manuscript. TK participated in the study design and read the final manuscript. All authors participated in the design of the study and read and approved the final manuscript.1 medline.mp.2 internet.mp.3 embase.mp.4 (psyclit or psycinfo or psychlit or psychinfo).mp.5 \"web of science\".mp.6 cinahl.mp.7 sigle.mp.8 \"system for information on grey literature in europe\".mp.9 lilacs.mp.10 excerpta medica.mp.11 \"science citation index\".mp.12 \"science citation abstracts\".mp.13 scisearch.mp.14 toxline.mp.15 aidsline.mp.16 cancerline.mp.17 pubmed.mp.18 grateful med.mp.19 cabnar.mp.20 \"health star\".mp.21 healthstar.mp.22 \"current contents\".mp.23 \"cochrane library\".mp.24 .mp.25 \"database of abstracts of reviews of effectiveness\".mp.26 eric.tw.27 \"world wide web\".mp.28 dissertation$.mp.29 thesis.mp.30 \"institute of scientific information\".mp.31 isi.mp.32 \"inside information plus\".mp.33 firstsearch.mp.34 \"international pharmaceutical abstracts\".mp.35 \"biological abstracts\".mp.36 (dare and cochrane).mp.37 pascal.tw.38 or/1\u20133639 search$.mp.40 (handsearch$ or \"hand search$\").mp.41 compar$.mp.42 \"manual search$\".mp.43 or/39\u20134244 .mp.45 clinical trial$.mp.46 .mp.47 (rct or cct).mp.48 or/44\u20134749 and/38,43,48The pre-publication history for this paper can be accessed here:Characteristics of included studies, information about the studies used in this systematic reviewClick here for file"}
+{"text": "Al-Ubaydli shares some useful tips for making the most of search engines. He campaigns for a resource that makes all of humanity's knowledge available to all of humanity.Brewster Kahle, creator of the Internet Archive (www.cochrane.org), for example, posts freely available abstracts of systematic reviews of health interventions (access to the full text of the reviews requires a fee). PubMed (www.ncbi.nlm.nih.gov/entrez/query.fcgi), the United States National Library of Medicine's search service, provides access to abstracts of articles in MEDLINE, PreMEDLINE, and other related databases. PubMed's MyNCBI feature provides useful filters such as \u201cfree full-text,\u201d which shows papers for which the full text is available through the Internet, free of charge. The \u201cHINARI\u201d filter (www.nlm.nih.gov/pubs/techbull/jf05/jf05_myncbi.html#filters) shows papers for which the text is freely available to residents of a small number of developing world countries\u2014those with a Gross National Product per capita below $1,000\u2014who are part of the HINARI agreement . PubMed Central  is the US National Institutes of Health's free digital archive of the full text of biomedical and life sciences journal articles.The Internet certainly provides a number of resources for finding medical evidence. The Cochrane Collaboration , along with other freely available search engines, has made it easier to find information, although the clinical uses of Google have not been as well documented as those of PubMed [The rise of the search engine Google . There are similar search terms that you can use to restrict your search to particular countries, national health systems, or government agencies. For example, \u201csite:nhs.uk\u201d restricts the search to the British National Health Service, while \u201csite:gv.kr\u201d focuses on South Korean government Web sites.Using \u201csite:fr\u201d as a search term will restrict your search to French Web sites, although not all French Web site URLs end with \u201cfr\u201d (for example the French Web site of M\u00e9decins Sans Fronti\u00e8res is www.google.co.in) gives preferential ranking to Indian Web sites in its results and Google Kenya (www.google.co.ke) provides a Kiswahili interface. The full list of country-specific Google sites is available at www.google.com/language_tools.Google also provides country-specific versions of its Web site. For example Google India , which is similar to PubMed in that it is a search engine that focuses on academic papers. In fact, many of the search results it returns are pages from the PubMed site. Google Scholar has a number of useful features that are not shared by PubMed. First, it is more comprehensive, indexing all academic fields, including non-biomedical ones. Second, and more importantly, the ranking mechanism is valuable. As with the rest of Google's technology, the pages are ranked based on the number of links that they receive. In the case of Google Scholar, \u201clinks\u201d are citations from different papers. This means that review papers and seminal papers are most likely to top any list of results from a Google Scholar search.Perhaps the most clinically significant tool is Google Scholar  makes building complex queries easier. Yahoo's Creative Commons search feature (search.yahoo.com/cc) restricts searches to content  that has been published under a Creative Commons license (www.creativecommons.org). These licenses are much less restrictive than the traditional \u201call rights reserved\u201d copyright license. For example, if the content you have found  is licensed under the Creative Commons Attribution License, you are legally entitled to reproduce it, distribute it, and make translations and derivative works, provided you cite the work properly.Google is the most popular search engine, but it is by no means the only one. Other search engines have different approaches with their own advantages. For example, Microsoft Network's query builder (www.teoma.com) clusters search results according to different meanings of the words in the query. This clustering is useful because the medical meaning of some words, such as \u201chip,\u201d is less commonly used than the non-medical meaning. Google lacks this clustering function. Finally, Vivisimo (www.vivisimo.com) can cluster results by subject (www.clustermed.info) tool searches PubMed, while www.biometacluster.com simultaneously searches several relevant sources such as ChemBank and ClinicalTrials.gov. These are useful if you are searching for papers in a narrow specialty.The search engine Teoma ( subject . Its CluAll of these freely available search engines have their limitations, and they rarely give you the perfect answer to your clinical query. But they do at least help to reduce the obstacles to finding medical information online. Kahle would certainly approve."}
+{"text": "It is difficult to systematically search for literature relevant to palliative care in general medical journals. A previously developed search filter for use on OVID Medline validated using a gold standard set of references identified through hand searching, achieved an unacceptably low sensitivity (45.4%). Retrieving relevant literature is integral to support evidence based practice, and understanding the nature of the incorrectly excluded citations  using the filter may lead to improvement in the filter's performance.The objectives were to describe the nature of subjects reflected in the false negative citations and to empirically improve the sensitivity of the search filter. A thematic analysis of MeSH terms by three independent reviewers was used to describe the subject coverage of the missed records. Using a frequency analysis of MeSH terms, those headings which could individually contribute at least 2.5% to sensitivity (occurring 19 or more times) were added to the search filter. All previously run searches were rerun at the same time as the revised filter, and results compared.Thematic analysis of MeSH terms identified thirteen themes reflected in the missing records, none of them intrinsically palliative. The addition of six MeSH terms to the existing search filter  led to an increase in sensitivity from 46.3% to 64.7%, offset by a decrease in precision from 72.6% to 21.9%.The filter's sensitivity was successfully increased using frequency analysis of MeSH terms, offset by a decrease in precision. A thematic analysis of MeSH terms for the false negative citations confirmed the absence of any intrinsically palliative theme or term, suggesting that future improvements to search filters for palliative care literature will first depend on better identifying how clinicians and researchers conceptualise palliative care. It is suggested that a constellation of parameters: stage of disease (advanced or active), prospect of cure (little or none), and treatment goals  may ultimately inform search strategies. This may be similarly true for chronic diseases, which share the inherent passage of time which marks them apart from acute, and therefore more readily identifiable, episodes of care. It is difficult to systematically search for literature relevant to palliative care. It is a diffuse subject, embracing topics from multiple other disciplines, and with relevant papers likely to be published in general medical journals as well as discipline specific journals. To facilitate improved identification of these papers, an earlier study formulated and evaluated palliative care search filters for use in the general medical literature, as part of a larger project, the Evidence Based (CareSearch) Project ,2. This Using a methodological approach often used to evaluate new diagnostic tests in medicine, and previously used to develop validated search filters , four geThis current paper reports a study investigating the nature of this subset of records. The objectives were to improve empirically the sensitivity of the Master Search and to describe the nature of subjects reflected in the incorrectly excluded citations.This research was undertaken May \u2013 November 2006 at the Repatriation General Hospital, Adelaide, South Australia. A set of incorrectly excluded citations was created by comparing records in the gold standard reference set (those identified in the previous study using a hand search) with records retrieved by the Master Search (the best performing MeSH and textword search). Items held in the Gold Set but not retrieved by the Master Search were regarded as false negatives, that is, they were incorrectly excluded when the Master Search was run. A set of 418 records were identified, and was called the Silver Set to distinguish it from the original hand search set of 773 records (the Gold Set). An Excel spreadsheet containing the citation details of the incorrectly excluded items was created.A frequency analysis of all MeSH terms in the Silver Set was undertaken. These were originally derived from four general medical journals; JAMA, The Lancet, BMJ and Annals of Internal Medicine, published from 1999 to 2001. The entire set was downloaded into a Word document, and all data except for MeSH terms were deleted. These terms were reformatted into a list, which was then exported into an Excel spreadsheet, and sorted into alphabetical order. Frequencies were tallied for all terms, including sub headings attached to MeSH terms, but ignoring whether or not terms were tagged as a major heading.th November 2006.A Revised Master Search was devised incorporating the most frequently occurring MeSH terms. It was calculated a priori that an increase of 2.5% sensitivity in the Gold Set n = 773) would equate to 19 additional records being retrieved from the Silver Set. This was a subjective estimate of the percentage increase that might represent a worthwhile improvement when offset against anticipated decline in precision. The Revised Master Search therefore combined the previous Master Search with the additional MeSH terms which demonstrated frequencies of higher than 19 (using the Boolean operator 'or'). Retrieved citations were then compared with those in the Gold Set, using a purposely written computer program called SSV , described elsewhere [73 would drug related therapies (RS) and palliative medications (JT) were grouped with therapeutics for symptoms (DC) using this process. A meeting was then held during which the spreadsheet was discussed, and consensus reached regarding a list of broad themes.As MeSH terms should reflect the major subject foci of an indexed article, the full list of all terms used was provided to RS , JT  and DC . Each separately, and independently, identified the major themes they thought were reflected by the range of these terms. One list (DC's) was tabled in a spreadsheet, and RS and JT then collocated their identified themes against this list according to similarity. For example, th November 2006 compared to 30th June 2005. , prognosis (29), quality of life (26), survival rate (26), treatment outcome (23), and attitude to health (21)  and a total of 10.27 MeSH terms per article (4293 occasions/418 references). When certain terms were disregarded , the most frequently occurring terms were When these six MeSH terms were added to the Master Search to create the Revised Master Search Table  and resuEach researcher identified slightly different individual themes in their individual analyses . Consensus was readily achieved in discussion, resulting in a final list of 13 broad themes, for example, drug therapies for symptoms, non-drug therapies for symptoms, disease modifying therapies, and pain and other symptoms Table .Incorrectly excluded citations from a previously validated palliative search filter were studied using frequency analysis of MeSH terms, resulting in the additional inclusion of headings which empirically improved the sensitivity of the original Master Search. Not unexpectedly, both specificity and precision decreased with this Revised Master Search.Notably, the sensitivity of the Revised Master Search was higher than that achieved by three relevant and published search strategies -7, modifThe overall large number of unique MeSH terms supports the conceptualization of information relevant to palliative care as diffuse, that is, spanning many subject areas . No cleaethics, pain and other symptoms, and drug therapies for symptoms are relevant to many other disciplines, not just palliative care.The thematic analysis of all MeSH terms included in the Silver Set (incorrect exclusions from the first study) was used to investigate this question, and yet did not provide any clear answers. None of the themes, or the individual MeSH terms which we reviewed to identify the themes, are intrinsically 'palliative'. Indeed, a similar analysis in any number of other specialties, such as rheumatology or internal medicine, might reveal a similar range of themes. For example, the themes of arthritis, rheumatoid; cardiovascular diseases; depression; dementia; and asthma. In isolation, none of these diseases are necessarily palliative. Numerous terms relating to cancer were also revealed, for example: adenocarcinoma; breast neoplasms; brain neoplasms; prostatic neoplasms; and neoplasms. Although many cancers are progressively life limiting, the prognosis for many cancers, particularly with early intervention, is good. Thus it cannot be presumed that these are inherently relevant.It is true that summarizing the Silver Set using themes might obscure MeSH terms which are more readily identifiable as palliative, however this was not the case. Using the 'diseases' theme as an example, some of the MeSH terms which led to the identification of this theme included: If the MeSH terms individually do not suggest immediate relevance to palliative care, then it is perhaps the underlying constellation of concepts and terms in individual articles which led to their original identification. For example, perhaps a cluster of MeSH terms and textwords, in unison, reflect something which a clinician recognizes as relevant to palliative care. This would not be surprising, as it would likely reflect the multifaceted nature of what constitutes an episode of palliative care. In developing such a definition for the Australian National Sub-acute and Non-acute Patient (AN-SNAP) Version 1 Casemix Classification, a cluster of parameters identified an episode as palliative, rather than simply diagnosis. These parameters included stage of disease (advanced or active), prospect of cure (little or none), and treatment goals , evidenced by assessment and management of a range of individual needs , and a grief and bereavement process for the individual and their carers/family .prognosis, survival rate and treatment outcome. Yet even for physician-patient relations, quality of life, and attitude to health, all reflect a notion of an inherent timeline.We also speculate that it is the changing nature of a disease over time which can identify it as potentially 'palliative'. All of the six additional MeSH terms incorporated in the Revised Master Search intrinsically reflect the passage of time. This is most identifiable with This study highlights the value of frequency analysis of MeSH terms, a third generation approach considered to be a more rigorous method . Whilst One limitation of this study relates to the online version OVID Medline not being static due to updating over time. Changes were made to the underlying records in between the original study and the current research (approximately 18 months). Whilst this was addressed by re-running all searches at the later date for comparison purposes, it is true that the actual MeSH indexing may be different to the original study from which the Silver Set was created. This means that the underlying frequencies of MeSH terms used to inform the revision of the search may be marginally inaccurate. We do not think the implications are great however, as our original study located 351 records which were in both the Gold Set and the Master Search; when this search was rerun the figure rose to 358 records. In other words, there were an additional seven relevant records retrieved. Given the publication period chosen for the original study was 1999 to 2001, it is sobering to realise that retrospective indexing has so recently been undertaken, that is, in 2006. Such delays in indexing may have implications when identifying the literature for systematic reviews, depending on the topic, and support the need for search strategies that are not solely contingent upon searching bibliographic databases.Generalisability in this study is difficult to assess. Because it builds on the previous study, it shares issues such as being based on a restricted subset of journals, described more fully elsewhere . No valiWhilst this study has described the themes represented by references incorrectly excluded in the use of a validated search strategy, it would be inappropriate to suggest these themes reflect how palliative care overall is conceptualized. A similar analysis of 'correctly included' references, that is, those which were readily identified as relevant to palliative care, would also be required. The most important next step may be an exploration of clusters of MeSH terms and concepts, which together reflect that an article is likely relevant to palliative care.The sensitivity of an existing search filter for identifying literature relevant to palliative care was successfully increased from 46.3% to 64.7% using frequency analysis of MeSH terms. This was notably offset by a decrease in precision from 72.6% to 21.9%. A thematic analysis of MeSH terms for incorrectly excluded references confirmed the absence of any single theme (or term) that was intrinsically palliative, suggesting that future improvements to search filters for literature relevant to palliative care literature will first depend on better identifying how clinicians and researchers conceptualise this discipline. On the basis of an existing definition for a palliative care episode of care, we suggest it may be a constellation of parameters: stage of disease (advanced or active), prospect of cure (little or none), and treatment goals  which may ultimately inform improved searches. This may be similarly true for chronic diseases, which share the inherent passage of time which marks them apart from acute, and therefore more readily identifiable, episodes of care.The author(s) declare that they have no competing interests.RS, JT and DC participated in the conception and design of the study. JT identified and extracted the records referred to as the Silver Set. RS undertook the frequency analysis of MeSH terms. All authors undertook the thematic analysis of MeSH terms. RS, JT and DC discussed the findings and the implications of same, and RS drafted the manuscript to reflect that discussion. All authors were involvedin the revision of paper and authorised the final version for submission.The pre-publication history for this paper can be accessed here:"}
+{"text": "Clinical prediction guides assist clinicians by pointing to specific elements of the patient's clinical presentation that should be considered when forming a diagnosis, prognosis or judgment regarding treatment outcome. The numbers of validated clinical prediction guides are growing in the medical literature, but their retrieval from large biomedical databases remains problematic and this presents a barrier to their uptake in medical practice. We undertook the systematic development of search strategies (\"hedges\") for retrieval of empirically tested clinical prediction guides from EMBASE.An analytic survey was conducted, testing the retrieval performance of search strategies run in EMBASE against the gold standard of hand searching, using a sample of all 27,769 articles identified in 55 journals for the 2000 publishing year. All articles were categorized as original studies, review articles, general papers, or case reports. The original and review articles were then tagged as 'pass' or 'fail' for methodologic rigor in the areas of clinical prediction guides and other clinical topics. Search terms that depicted clinical prediction guides were selected from a pool of index terms and text words gathered in house and through request to clinicians, librarians and professional searchers. A total of 36,232 search strategies composed of single and multiple term phrases were trialed for retrieval of clinical prediction studies. The sensitivity, specificity, precision, and accuracy of search strategies were calculated to identify which were the best.163 clinical prediction studies were identified, of which 69 (42.3%) passed criteria for scientific merit. A 3-term strategy optimized sensitivity at 91.3% and specificity at 90.2%. Higher sensitivity (97.1%) was reached with a different 3-term strategy, but with a 16% drop in specificity. The best measure of specificity (98.8%) was found in a 2-term strategy, but with a considerable fall in sensitivity to 60.9%. All single term strategies performed less well than 2- and 3-term strategies.The retrieval of sound clinical prediction studies from EMBASE is supported by several search strategies. Clinical prediction guides (CPGs), also known as clinical prediction rules or clinical decision rules, are increasingly sought by frontline clinicians to assist in their decision making process. They provide an objective standard by which to gauge which elements in a patient's history, physical examination and laboratory tests are the most important in forming an accurate clinical assessment . CPGs arCPGs can serve as decision aids for determination of causation, diagnosis, prognosis, or patient responsiveness to treatment -3. Some Retrieving CPG studies from the medical literature is problematic for several reasons. First they are relatively few in quantity in comparison to other types of studies and reports posted in major, online, clinical literature databases. For example, EMBASE contains more than 9 million records and is up dated by 6000 \u2013 8000 records per week, spread over more than 4600 journal titles . Second,.Several research groups have identified search strategies  for MEDLINE for topics such as etiology , diagnoset al published CPG search strategies for MEDLINE developed by comparing hand searching 161 journals published in the year 2000 with several search terms and applying comparatively, more strict methodological quality criteria , and these smaller subsets greatly simplify data processing. Hence, 135 journals were further reduced to a 55 journals that were found to contain at least one study that met our criteria for scientific merit.When previously developing search strategies for some categories of articles  for MEDLINE, we split the database into 60% and 40% components to provide a development and validation database. We subsequently found that the comparison between development and validation database results was not statistically significant . For CPGSix research staff were rigorously calibrated for hand searching before reviewing the 2000 literature and inter-rater agreement for application of all criteria exceeded 80% beyond chance . Hand seAn initial list of index terms and textwords relating to studies of different purposes  was compiled in house. The list grew with the addition of terms or phrases suggested by clinicians, librarians and known searchers in the United States and Canada, made upon our request. From here, we compiled a list of 5385 searching terms, of which 4843 were unique and 3524 returned results (terms available on request) for retrieval of studies across all of the purpose categories. Among the 3524 terms were 641 terms that depicted clinical prediction studies such as 'clinical prediction rule', 'derivation set', 'guide', and 'validation cohort', all as textwords; 'validation process', the index term, and the index term 'model', exploded.The search strategies were treated as \"diagnostic tests\" for sound studies and the manual review of the literature was treated as the \"gold standard.\" All CPG study search terms and phrases were run in EMBASE and an automated process determined their sensitivity, specificity, precision, and accuracy. Sensitivity for a given topic is defined as the proportion of high quality articles for that topic that are retrieved; specificity is the proportion of low quality articles not retrieved; precision is the proportion of retrieved articles that are of high quality; and accuracy is the proportion of all articles that are correctly classified.The aim of testing was to identify the best single term, 2-term and multiple-term (greater than two terms) strategies that would optimize sensitivity or specificity or both sensitivity and specificity together. All combinations of terms used the Boolean OR, for example, \"predict.tw. OR guide.tw.\". (The Boolean AND was not used because this strategy invariably compromised sensitivity.) Next, we tested all 2-term search strategies with sensitivity at least 75% and specificity at least 50% to find multiple term strategies that were optimized for sensitivity. For optimizing accuracy in a multiple term strategy, all 2-term search strategies with accuracy >75% were tested. In total, 36,232 search strategies were tested in the development of clinical prediction guide hedges, which represents the second largest number of strategies (next to cost effectiveness studies) tested among EMBASE strategies investigated within our group.A logistic regression approach to developing search strategies for MEDLINE did not improve performance , so it wIndexing information was downloaded from EMBASE for 27,769 articles of various purpose categories, identified from hand-searching the 55 journals. Of these, 163 (0.58%) were classified as clinical prediction guides, of which 69 (42.3%) were methodologically sound.Table Combination of terms with the best results for sensitivity, specificity and optimization of sensitivity and specificity are shown in Table In this study we report search filters found to be effective for the retrieval of clinical predication guide studies from EMBASE. These filters are optimized for sensitivity, specificity or best sensitivity and specificity combined, each lending the searcher unique results that can be geared to his/her needs. The strategy optimized for sensitivity should be applied in cases where retrieval of all relevant articles is key, and substantial weeding of irrelevant content is seen to be acceptable. The most specific search filter is effective when the aim of the search is to retrieve only highly relevant articles, where inclusion of all pertinent matter is less important. Where the intention is to uncover a balance of targeted hits with off topic material then the strategy that maximizes both sensitivity and specificity would be best.When comparing the results of this study to that reported by Wong et al  for CPGsOther parallels occur between the 2 reports with respect to best identified search strategies. For single terms, predict  achieved both highest sensitivity and best optimization for sensitivity and specificity in both EMBASE and MEDLINE. Similarly, validat:.tw. generated best results for specificity in both databases.It is interesting to note that no indexing terms contributed to the optimized search strategies for CPG studies in EMBASE or MEDLINE; textwords were the composite for all winning strategies. This finding is consistent with indexing terminology not keeping pace with research methods, and suggests a means for improving indexing and retrieval in the future.For practical purposes, we restricted our methods filter to just 2 criteria, the use of both a training and test set. Additional criteria that could have been applied include prospective validation, stating the mathematical technique used to develop the rule, clear definition and blinded assessment of predictor variables and outcomes, and prospectively testing the effect of the rule in clinical practice . FurtherFor clinicians to be able to make optimal use of clinical prediction guides in their practice, their accessibility needs to be improved upon. This study highlights search terms that maximize the retrieval of CPG studies, as well as illustrating that there is room for improvement, especially in precision. The application of \"AND\" and \"AND/NOT\" combinations or multivariate statistical techniques may help, but this remains to be determined.The retrieval of higher quality clinical prediction guides from EMBASE is facilitated by the application of search filters that have been optimized for sensitivity or specificity or both.Clinical Prediction Guide: CPGThe author(s) declare that they have no competing interests.NLW and RBH prepared grant submissions in relation to this project. JH, NLW and RBH drafted, commented on and approved the final manuscript. NLW and RBH supplied intellectual content to the collection and analysis of the data. NLW participated in the data collection and all authors were involved in data analysis.The pre-publication history for this paper can be accessed here:"}
+{"text": "It is generally believed that exhaustive searches of bibliographic databases are needed for systematic reviews of health care interventions. The CENTRAL database of controlled trials (RCTs) has been built up by exhaustive searching. The CONSORT statement aims to encourage better reporting, and hence indexing, of RCTs. Our aim was to assess whether developments in the CENTRAL database, and the CONSORT statement, mean that a simplified RCT search strategy for identifying RCTs now suffices for systematic reviews of health care interventions.RCTs used in the Cochrane reviews were identified. A brief RCT search strategy (BRSS), consisting of a search of CENTRAL, and then for variants of the word random across all fields (random$.af.) in MEDLINE and EMBASE, was devised and run. Any trials included in the meta-analyses, but missed by the BRSS, were identified. The meta-analyses were then re-run, with and without the missed RCTs, and the differences quantified. The proportion of trials with variants of the word random in the title or abstract was calculated for each year. The number of RCTs retrieved by searching with \"random$.af.\" was compared to the highly sensitive search strategy (HSSS).The BRSS had a sensitivity of 94%. It found all journal RCTs in 47 of the 57 reviews. The missing RCTs made some significant differences to a small proportion of the total outcomes in only five reviews, but no important differences in conclusions resulted. In the post-CONSORT years, 1997\u20132003, the percentage of RCTs with random in the title or abstract was 85%, a mean increase of 17% compared to the seven years pre-CONSORT . The search using random$.af. reduced the MEDLINE retrieval by 84%, compared to the HSSS, thereby reducing the workload of checking retrievals.A brief RCT search strategy is now sufficient to locate RCTs for systematic reviews in most cases. Exhaustive searching is no longer cost-effective, because in effect it has already been done for CENTRAL. Literature searching for systematic reviews of interventions in health care has been largely based on finding all randomized controlled trials (RCTs), as this study design is considered the gold standard. However RCTs make up only a very small proportion of all the articles included in bibliographic databases, and so the problem for systematic reviewers has been to devise a search strategy which is sensitive enough to find all the RCTs, but specific enough not to bury them in a large number of other unwanted retrievals needing to be manually excluded.Two developments have facilitated searching for RCTs. The first is CENTRAL , the world's most comprehensive database consisting exclusively of controlled clinical trials. It currently contains over 425,000 citations. The majority of the trials in CENTRAL have been identified through systematic searches of MEDLINE and EMBASE. The first two phases of a three phase highly sensitive search strategy (HSSS) have been used to search MEDLINE . CENTRAL\"it may still be worthwhile for reviewers to search MEDLINE using the Cochrane highly sensitive search strategy and to obtain and check the full reports of possibly relevant citations\" [Because identification has relied solely on the titles and, where available, the abstracts, some relevant trials may not have been identified. Therefore, the Cochrane Handbook says tations\" . HoweverThe second development is the CONSORT statement, introduced in 1996 , and sinItem 1 on the CONSORT checklist recommends that the method in which participants were allocated to interventions  is described in the title and abstract. This allows instant identification of RCTs, and should help ensure that a study is appropriately indexed as an RCT in bibliographic databases.As the Cochrane Collaboration has already done exhaustive work to ensure that CENTRAL is as complete as possible, we wanted to examine whether this eliminates the need for individual reviewers to run the HSSS, and the effectiveness of replacing this with a simplified search strategy.1. To determine the effect on the results of Cochrane reviews of using a brief RCT search strategy (BRSS).2. To examine the change in use of variants of the word random in the title or abstract of journal articles, pre- and post-CONSORT.All reviews new to the Cochrane Database of Systematic Reviews in the Cochrane Library 2004 issue 2 were selected. Those that stated that they were considering only RCTs, and which found at least one RCT, were selected.All trials included in each review were identified from the section 'References to studies included in this review'. Each trial was checked to determine whether it was indexed in CENTRAL , and then MEDLINE. If not in either of these databases, EMBASE was checked.The full bibliographic records of all trials that were in either MEDLINE or EMBASE (using the OVID interface) were examined to determine whether random$ was in any field. .The full papers of any trials that were: 1) not in CENTRAL, or 2) did not have random$ in any field in the bibliographic record, were obtained and checked to see whether they were actually RCTs. All non-English articles were translated, apart from those in Japanese and Chinese, as resources were not available.The impact of omitting the RCTs not found with the BRSS was quantified, using Review Manager 4.2.7. The forest plots for the meta-analyses were reproduced, both with and without data from the missing trials, and the results compared to see if omission would make any important difference. The differences could theoretically include;a) There might be less, or no data left, for some outcomes.b) There could be a different result; no benefit over comparator, or vice versa.c) There could be the same result, but with a different effect size.d) There could be the same result and effect size, but with a wider confidence interval, and possibly loss of statistical significance.In summary, the BRSS would involve: 1) searching CENTRAL, and 2) supplementing that with a search of MEDLINE and EMBASE, using a search of 'random$.af.' to pick up trials not in CENTRAL.There were 78 new reviews, of which 57 met the inclusion criteria. They cited a total of 920 trials; 79% (725) were journal articles. The remaining 21% were from the grey literature, 80% of these being conference abstracts).Twenty one reviews were excluded from our study; 14 because they did not find any RCTs that met their inclusion criteria, and seven because they included other study designs in addition to RCTs.It was found that 93.3% (677) of the 725 journal articles included in the systematic reviews were in CENTRAL. It was assumed that these were all RCTs, but this was not checked due to the large numbers involved (and it was not relevant to the aims of the study). This left 48 journal articles not indexed in CENTRAL.The full texts of all 48 of these articles (apart from the four in Japanese and five in Chinese) were translated and checked. It was found that 40 were RCTs, and the remaining eight used non-RCT study designs. (It was assumed that the untranslated articles were RCTs). Eleven of the 40 RCTs were found by searching MEDLINE or EMBASE with 'random$.af'. Therefore, 29 (4%) of RCTs would not have been found with the BRSS.The 29 trials were distributed over 12 reviews. Ten were in MEDLINE, 11 in EMBASE only. Twelve were non-English language.In two reviews ,7, each Therefore, this leaves 27 missing journal articles, in 10 separate reviews that contain at least one trial with data not found by the BRSS. Table Only one review did not do a meta-analysis The nineTwo consequences of missing data were found:1) In three reviews -15, the 2) In three reviews -17, the For the period 1966 to October, 2004, the HSSS search strategy retrieved 2,505,742 records in MEDLINE, compared to 'random$.af.', which retrieved 399,208 records. Therefore, only 16% of the number of records were found using 'random$.af.', compared to HSSS.Using the 725 journal articles in our sample, we compared the frequency of random$ in the title or abstract between pre-CONSORT (published up to 1996) and post-CONSORT trials (published from 1997 onwards). Figure In the post-CONSORT years, 1997\u20132003, the term appeared in 85% of the titles or abstracts, compared to 68% in the seven years before CONSORT (1990\u201396); mean difference was 17% . If a longer pre-CONSORT period is used, 1980 to 1996, the proportion is similar at 62%.Using the BRSS, rather than the much more exhaustive HSSS, to retrieve RCTs in journal articles for Cochrane reviews, affected only a very small percentage of total outcomes of a few reviews, and made no important difference to the conclusions of these reviews. The most affected review had four of the five included trials published in Chinese .The CONSORT statement appears to be associated with a significant increase in the frequency of random in the titles and abstracts of journal articles. Whether this is directly due to CONSORT, or whether CONSORT simply accelerated a pre-existing trend, and raised the general awareness amongst authors and editors for better reporting of RCTs, is uncertain. This improved description of RCTs by authors should mean that all RCTs are indexed with the appropriate publication type in MEDLINE, and also result in more sensitive retrieval of RCTs using the BRSS.The BRSS had a sensitivity of 96% for RCTs in journal articles. Compared to the HSSS, the BRSS reduced the MEDLINE retrieval by 84%. This would represent a major time and cost saving in manual screening.quantified the impact of omitting trials not found with the simplified search.The strengths of this study include firstly that we used Cochrane reviews, as they approximate the 'gold standard' in searching, due to the requirement for exhaustive searches. Hence we can be fairly certain that we were starting with as comprehensive set of included trials as possible. Secondly, we A weakness of this study was that we could not check whether the RCTs would have been in CENTRAL at the time the searchers did their searching, and hence eliminate the possibility that some RCTs were first identified by the reviewers, and then passed to CENTRAL. However, given the extensive searching routinely done for CENTRAL, it is highly likely most RCTs would be identified sooner or later, and therefore be included in a subsequent update of the review.A range of subject areas was included, which helps with generalisability, though it may decrease power in any one subject area. However, a recent study on identifying quality RCTs in pain relief gives general support to our findings . It inveThis study focused only on a simplified search strategy for retrieving RCTs in journal articles, since these made up the vast majority of the references used in Cochrane reviews, and are most important in terms of the quality and quantity of assessable data available. By contrast, most grey literature (the vast majority of which is meeting abstracts) gives very limited data. However, CENTRAL includes many grey literature trials, so these will be identified with the BRSS.There are currently over 2200 Cochrane reviews, and these will need maintaining in the future. Authors may be encouraged to update their reviews, if they can be confident they can identify RCTs comprehensively with a simple search. The simplified search may also be useful for those doing reviews in a tight timescale, or by clinicians who just want a rapid but reliable answer to a question. There is a case for the 'not quite perfect but rapid, easy and almost complete' search'.In practice, some of the few trials missed by the BRSS were small or of poor quality, and as Egger and colleagues have reported, the last few studies found by exhaustive searching could introduce bias, by being of poor quality .The issue is whether the marginal benefits of exhaustive searching justify the extra costs. When the inclusion criteria demand only RCTS, this study suggests that exhaustive searching is now, in the era of CENTRAL and CONSORT, no longer cost-effective.The author(s) declare that they have no competing interests.PR conceived and designed the study, and analysed the data. NW helped with interpretation and with drafting the paper. Both authors approved the final versionThe pre-publication history for this paper can be accessed here:"}
+{"text": "Evaluating the existence and strength of an association between a putative cause and adverse clinical outcome is complex and best done by assessing all available evidence. With the increasing burden of chronic disease, greater time demands on health professionals, and the explosion of information, effective retrieval of best evidence has become both more important and more difficult. Optimal search retrieval can be hampered by a number of obstacles, especially poor search strategies, but using empirically tested methodological search filters can enhance the accuracy of searches for sound evidence concerning etiology. Although such filters have previously been developed for studies of relevance to causation in MEDLINE, no empirically tested search strategy exists for EMBASE.An analytic survey was conducted, comparing hand searches of journals with retrievals from EMBASE for candidate search terms and combinations. 6 research assistants read all issues of 55 journals indexed in EMBASE. All articles were rated using purpose and quality indicators and categorized into clinically relevant original studies, review articles, general papers, or case reports. The original and review articles were then categorized as 'pass' or 'fail' for scientific merit according to explicit criteria in the areas of causation (etiology) and other clinical topics. Candidate search strategies were developed for causation, then run in a subset of 55 EMBASE journals, the retrievals being compared with the hand search data. The sensitivity, specificity, precision, and accuracy of the search strategies were calculated.Of the 1489 studies classified as causation, 14% were methodologically sound. When search terms were combined, sensitivity reached 92%. Compared with the best single-term strategy, the best combination of terms resulted in an absolute increase in sensitivity (19%) and specificity (5.2%). Maximizing specificity for combined terms resulted in an increase of 7.1% compared with the single term but this came at an expense of sensitivity (39% absolute decrease). A search strategy that optimized the trade-off between sensitivity and specificity achieved 81.9% for sensitivity and 81.4% for specificity.We have discovered search strategies that retrieve high quality studies of causation from EMBASE with high sensitivity, high specificity, or an optimal balance of each. Clinical problems encountered by clinicians often involve examining questions about harm that involve genes, treatments, or environmental exposures ,2. KnowlLarge biomedical databases such as MEDLINE and EMBASE provide online access to the medical literature , and conFor clinicians, increasing time demands restrict the practice of evidence-based medicine ,7, despiThe very low concentration of rigorous studies also limits clinicians' awareness and detection of key articles . FurtherMethodologic search filters (which capture relevant articles while eliminating those that are not of interest) are one way of improving the retrieval of scientifically sound and clinically relevant studies from biomedical literature databases . Search In this paper, we report on the evaluation and comparison of the retrieval performance of causation search strategies in EMBASE with a manual review (\"gold standard\") of each article for each issue of 55 journals in 2000. Compared with previous strategies developed for MEDLINE in 1991, the methods we applied for selecting articles for EMBASE were tighter and the calibration database larger . In addition, we tested many more search strategies, which for MEDLINE resulted in the development of search strategies that work better than the ones previously reported. The focus of the strategies is to help clinicians and researchers retrieve methodologically sound study reports on causation, to assist with evidence-based patient care decisions based on the best quality evidence available. To our knowledge, no approach exists that applies such rigorous standards to EMBASE.The study compared the retrieval performance of methodologic search terms and phrases in EMBASE with a manual review of each article for each issue of 55 journal titles for the year 2000. Index terms and text words related to research design features were run as search strategies. The search strategies were treated as \"diagnostic tests\" for sound studies and the manual review of the literature was treated as the \"gold standard.\" The sensitivity, specificity, precision, and accuracy of EMBASE search strategies were determined. Sensitivity for a given topic is defined as the proportion of high quality articles for that topic that are retrieved; specificity is the proportion of low quality articles not retrieved; precision is the proportion of retrieved articles that are of high quality; and accuracy is the proportion of all articles that are correctly classified.Individual search terms with sensitivity > 25% and specificity > 75% for causation studies were incorporated into the development of search strategies that included a combination of 2 or more terms. All combinations of terms used the Boolean OR, for example, \"risk.tw. OR cohort.tw.\". The Boolean AND was not used because this strategy invariably compromised sensitivity. For the development of multiple-term search strategies to either optimize sensitivity or specificity, we tested all 2-term search strategies with sensitivity at least 75% and specificity at least 50%. For optimizing accuracy, 2-term search strategies with accuracy > 75% were considered for multiple-term development. 13,901 search strategies were tested.We did not attempt to use logistic regression to improve search performance in this study because our previous development of regression strategies for retrieving studies of treatment [unpublished observation] and prognosis  showed nSix research assistants hand searched 170 journals titles in total for the year 2000, and applied methodologic criteria to each item in each issue to determine if the article was methodologically sound for 7 purpose categories . All purpose category definitions and corresponding methodologic rigor were outlined in a previous paper . The metThe 170 journal titles reviewed were chosen based on recommendations of clinicians and librarians, Science Citation Index Impact Factors provided by the Institute for Scientific Information, and ongoing assessment of their yield of studies and reviews of scientific merit and clinical relevance for the disciplines of internal medicine, general medical practice, mental health, and general nursing practice . 135 of the 170 journals were indexed in EMBASE. We previously developed search strategies in MEDLINE using the 161 hand-searched journals that were indexed in MEDLINE but found that search strategies developed in much smaller journal subsets are equally robust  and thatTo identify candidate search terms and strategies, we compiled an initial list of index terms and text words by selecting words that related to etiology  and to research methods for establishing causation (see examples below). We then sought input from clinicians and librarians in the United States and Canada through interviews of known searchers, and requests at meetings and conferences. Individuals were asked to identify terms or phrases they used when searching for studies of causation, prognosis, diagnosis, treatment, economics, clinical prediction guides, reviews, costs, and studies of a qualitative nature. We compiled a list of 5385 terms of which 4843 were unique and 3524 returned results in the 55-journal subset in EMBASE (list of terms tested provided by the authors upon request). Examples of the search terms tested are 'adverse drug reaction', 'risk ratio', 'cohort study', and 'harm', all as text words; 'risk', the index term, and the index term 'exposure', exploded.Indexing information was downloaded from EMBASE for 27,769 articles from the 55 hand searched journals. Of these, 1489 were classified as causation, of which 215 (14.4%) were judged methodologically sound. Search strategies were developed using all 27,769 articles. Thus, the strategies were tested for their ability to retrieve articles about higher quality causation studies from all other articles, including both lower quality causation studies and all non-causation studies.The operating characteristics of the best single-term for high-sensitivity, high-specificity, and best optimization of sensitivity and specificity are displayed in Table Combinations of terms with the best results for sensitivity, specificity, and optimization of sensitivity and specificity are shown in Table The two-term strategy, \"cohort.tw. OR relative risk:.tw.\" yielded the best specificity (94.6%) but with an expected trade-off in sensitivity, which was lowered to 53% (38.6% absolute decrease). However, maximizing specificity improved both precision (5.3% absolute increase) and accuracy (33.2% absolute increase). The combination of 3 terms, \"cohort.tw. OR relative risk:.tw. OR adjusted OR.tw\" (where \"adjusted OR\" is not the Boolean OR but rather refers to adjusted odds ratio) achieved a substantive increase in sensitivity (8.4% absolute increase) with a small decrease in specificity (1.7% absolute decrease) (Table Table We developed causation search filters for EMBASE that provide for highly sensitive, highly specific, and highly accurate searches in EMBASE for high quality studies concerning etiology. The utility of these 3 types of search filters will vary according to the needs of end users or the clinical question that is being sought.For example, a person conducting a search to find original articles for constructing a systematic review will have different retrieval needs than the clinician who is looking for quick answers to manage a patient. The best sensitive search would be more beneficial for a systematic review. Although it is time consuming to search through 270 citations that may include some irrelevant articles, key studies that are needed to conduct a robust systematic review would not be missed. In contrast, for quick answers, the narrower yield of a specific search takes less time, and will likely provide a sufficient number of relevant articles to answer the clinical question sought, but with somewhat higher potential for missing key studies. The trade-off between time investment and consequences of missing useful evidence is important to consider .Our results indicate that combination-term strategies generally perform better than single-term strategies. However, in our previous research, \"risk:.mp\", yielded close to best sensitivity in developing causation search filters in MEDLINE . The resA logistic regression approach to developing search strategies was done when deriving search filters for MEDLINE . The anaAnother expected result from our study was that precision was generally low. For a large, multipurpose biomedical database such as EMBASE, it was not surprising to find a low proportion of relevant, high quality causation studies. Although a slight improvement in precision was seen when specificity was maximized, the overall low precision in our study will still require physicians to invest time eliminating irrelevant articles. However, improving precision may be possible by combining search strategies with content-specific terms using the Boolean \"AND\" or \"AND NOT\". Our future research will focus on enhancing precision by developing more sophisticated search filters, and by using the strategies above.We developed several search strategies that can enhance the retrieval of causation articles in EMBASE. The needs of end users play an important role in determining the most beneficial trade-off between sensitivity and specificity.The authors declare that they have no competing interests.The pre-publication history for this paper can be accessed here:"}
+{"text": "Economic evaluations in the medical literature compare competing diagnosis or treatment methods for their use of resources and their expected outcomes. The best evidence currently available from research regarding both cost and economic comparisons will continue to expand as this type of information becomes more important in today's clinical practice. Researchers and clinicians need quick, reliable ways to access this information. A key source of this type of information is large bibliographic databases such as EMBASE. The objective of this study was to develop search strategies that optimize the retrieval of health costs and economics studies from EMBASE.We conducted an analytic survey, comparing hand searches of journals with retrievals from EMBASE for candidate search terms and combinations. 6 research assistants read all issues of 55 journals indexed by EMBASE for the publishing year 2000. We rated all articles using purpose and quality indicators and categorized them into clinically relevant original studies, review articles, general papers, or case reports. The original and review articles were then categorized for purpose  and depending on the purpose as 'pass' or 'fail' for methodologic rigor. Candidate search strategies were developed for economic and cost studies, then run in the 55 EMBASE journals, the retrievals being compared with the hand search data. The sensitivity, specificity, precision, and accuracy of the search strategies were calculated.Combinations of search terms for detecting both cost and economic studies attained levels of 100% sensitivity with specificity levels of 92.9% and 92.3% respectively. When maximizing for both sensitivity and specificity, the combination of terms for detecting cost studies (sensitivity) increased 2.2% over the single term but at a slight decrease in specificity of 0.9%. The maximized combination of terms for economic studies saw no change in sensitivity from the single term and only a 0.1% increase in specificity.Selected terms have excellent performance in the retrieval of studies of health costs and economics from EMBASE. With the rising costs of new technology for the diagnosis and management of disease, data concerning cost effectiveness of health care has become increasingly important, particularly for policy makers and managers of health services when making resource allocation decisions. It is also important that health professionals determine if the benefits will be worthwhile when considering the consumption of health care resources -3.Economic evaluations in the medical literature compare competing diagnosis or treatment methods for their use of resources and their expected outcomes. Several databases provide access to this literature; some are specialty databases such as the U.K. National Health Service Economic Evaluation Database while others are large general purpose biomedical databases. End-users frequently access the medical literature online via the huge biomedical databases such as MEDLINE and EMBASE. Unfortunately, gaining access to this economic and cost literature through these databases can be daunting. The retrieval of relevant information is difficult due to the millions of articles and thousands of journals indexed, the minuscule concentration of articles with economic content, and the inconsistency of indexing within the databases ,4.Researchers have worked to develop search strategies to aid health professionals in the retrieval of relevant information, but this has been mostly for studies of treatment and diagnosis. Few reports exist of empirically validated search strategies for economic analyses and cost studies. Sassi et al  examinedIn this paper, we report on search strategy development and retrieval performance for cost and economic studies indexed in EMBASE.The study compared the retrieval performance of methodologic search terms and phrases in EMBASE with a manual review of each article for each issue of 55 journal titles for the year 2000. Index terms and textwords related to research design features were run as search strategies. The search strategies were treated as \"diagnostic tests\" for sound studies and the manual review of the literature was treated as the \"gold standard.\" The sensitivity, specificity, precision, and accuracy of EMBASE searches were determined. Sensitivity for a given topic is defined as the proportion of high quality articles for that topic that are retrieved; specificity is the proportion of low quality articles not retrieved; precision is the proportion of retrieved articles that are of high quality; and accuracy is the proportion of all articles that are correctly classified.Six research assistants hand searched journals, and applied methodologic criteria to each item in each issue to determine if the article was methodologically sound for 7 purpose categories, including cost and economic evaluations, treatment, diagnosis, prognosis, clinical prediction, etiology, and reviews. Research staff was rigorously calibrated before reviewing the 2000 literature and inter-rater agreement for application of all criteria exceeded 80% beyond chance [The 55 journals were chosen based on having the highest yield of methodologically sound articles across all purpose categories from a larger collection of 170 journal titles chosen based on recommendations of clinicians and librarians, Science Citation Index Impact Factors provided by the Institute for Scientific Information, and ongoing assessment of their yield of studies and reviews of scientific merit and clinical relevance for the disciplines of internal medicine, general medical practice, mental health, and general nursing practice . A total of 135 of the 170 journals were indexed in EMBASE, including the 55 top yielding journals used for this report. We had previously found that the developed search strategies were robust in smaller journal subsets  and thatAn initial list of index terms and textwords was compiled. Input was then sought from clinicians and librarians in the United States and Canada through interviews of known searchers, and requests at meetings and conferences. Individuals were asked to identify which terms or phrases they used when searching for studies of economics, costs, treatment, causation, diagnosis, prognosis, clinical prediction guides, reviews, and studies of a qualitative nature. We compiled a list of 5385 terms of which 4843 were unique and 3524 returned results (list of terms tested provided by the authors on request). Examples of the search terms relevant to costs and economics included 'cost effective', 'incremental costs', 'direct cost', and 'net benefit', all as textwords; 'cost effectiveness analysis', the index term, and the index term 'health economics', exploded.The strategies for economic studies were tested for their ability to retrieve articles about high quality economics studies from all other articles, including both low quality economics studies and all non-economics studies. For costs studies, the strategies were tested for their discrimination between costs studies and all others. Individual terms with sensitivity > 25% and specificity > 75% for a given purpose category were incorporated into the development of search strategies that included a combination of 2 or more terms. All combinations of terms used the Boolean OR, for example, \"effectiveness OR economics\". The Boolean AND was not used because this strategy invariably compromised sensitivity. For the development of multiple-term search strategies to either optimize sensitivity or specificity, we tested all 2-term search strategies with sensitivity at least 75% and specificity at least 50%. For optimizing accuracy, 2-term search strategies with accuracy > 75% were considered for multiple-term development.Indexing information was downloaded from EMBASE for 27,769 articles from the 55 hand-searched journals. Of these 183 were classified as about costs and 148 were classified as economics (a subset of costs studies). Of the economics studies, 31 (20.9%) were rated methodologically sound. A total of 40,116 search strategies were tested in the development of economics hedges and 16,728 for the development of the costs hedges.Table Combination of terms with the best results for sensitivity, specificity and optimization of sensitivity and specificity are shown in Table This investigation shows that selected single terms and combinations of search terms can reach high levels of performance in the retrieval of high quality literature in the area of economic analysis and in the retrieval of literature focusing on the cost of health care services. By assisting in the retrieval of relevant cost literature, clinicians and researchers will be able to find the information they need more dependably and quicker, perhaps improving evidence-based decisions. Single term and combination search strategies have been shown to be highly sensitive and specific in the areas of cost and economics. Although there was little difference when comparing single and combination strategies for cost articles, the economics articles saw much better performance in terms of sensitivity for the single term strategy than the combination strategy when optimizing for specificity. Finally, when optimizing for both specificity and sensitivity, the combination strategies for the cost searches saw a slightly more sensitive return than the single strategies, while there was no real difference when comparing the type of search strategies when applied to the economics searches.It is important to note that several top performing terms are exploded index terms and many are text words. In the event that new index terms relevant to cost and/or economic studies are added to Emtree, it is likely that our reported search strategies will perform similarly in terms of sensitivity and specificity. Text word searching involves only the title and abstract of the article so additions to Emtree will have no effect on the performance of these terms. Additionally, if new index terms are added and if they are closely related to the exploded index term included in the search strategy, the articles indexed with the new term will be retrieved.In all points of comparison, the investigated search strategies performed well in terms of the accuracy of their returns. In fact, all accuracy values were over 92%. Even so, the precision of searches, that is, the proportion of retrieved articles that are on target, is suboptimal. This is simply a reflection of the very low concentration of cost and economics in the huge EMBASE database; for sound economics studies, the concentration was less than 0.1%. Precision is dependent on the concentration of target articles  in the entire database. We tested our search strategies in a subset of EMBASE records. Therefore, the precision figures reported are included only as an illustration of search strategy performance. When searching in the entire EMBASE database, precision will be lower.While two of the single term cost strategies achieved precision levels of 39.4% and 24.0%, none of the economics single term strategies achieved better than 8% precision, meaning that only 8% of the retrieved articles were on target. The overall precision decreased further for the combination search strategies when compared to the single term strategies. Thus the somewhat higher sensitivity of the combination strategies is at the expense of decreased precision and accuracy. In addition to precision being dependent on the concentration of target articles in the entire database, low precision returns could also point to a potential problem of over indexing, that is, index terms that appear to be specific to good quality economic studies are not used solely for those types of articles resulting in the retrieval of many false positive articles .Finally, the methodologic criteria for economic studies are fairly rigorous as noted by the low number of pass economic studies (n = 31) in the database. Since pass and fail economic studies are a subset of cost studies, searchers could use the cost strategies if they fail to find relevant articles when searching using the economic strategies. This is also true for economic studies based on models. Our definition of a pass economic study required that the study be based on data from real patients. Therefore, those that were based on models   would only be retrieved as \"false positives\" when using our economic search strategies but have high likelihood of being retrieved when using the cost strategies.We recently published economics and cost search strategies to use when searching in MEDLINE in the context of retrieving literature relevant to health services research (HSR) . The HSRMultivariate statistical techniques may yield better results than we observed. However, when we tested a logistic regression approach to developing search strategies for MEDLINE, we found no improvement on the same Boolean approach used in the EMBASE study . Even ifMachine learning methodologies may yield better results than we observed. We are currently exploring this possibility through collaborative ventures with two research groups in the United States.By combining specific textwords and terms with  multiple postings (.mp.), one can greatly improve the retrieval of costs and economics literature from EMBASE.The author(s) declare that they have no competing interests.The pre-publication history for this paper can be accessed here:"}
+{"text": "Accurate diagnosis by clinicians is the cornerstone of decision making for recommending clinical interventions. The current best evidence from research concerning diagnostic tests changes unpredictably as science advances. Both clinicians and researchers need dependable access to published evidence concerning diagnostic accuracy. Bibliographic databases such as EMBASE provide the most widely available entr\u00e9e to this literature. The objective of this study was to develop search strategies that optimize the retrieval of methodologically sound diagnostic studies from EMBASE for use by clinicians.An analytic survey was conducted, comparing hand searches of 55 journals with retrievals from EMBASE for 4,843 candidate search terms and 6,574 combinations. All articles were rated using purpose and quality indicators, and clinically relevant diagnostic accuracy articles were categorized as 'pass' or 'fail' according to explicit criteria for scientific merit. Candidate search strategies were run in EMBASE, the retrievals being compared with the hand search data. The proposed search strategies were treated as \"diagnostic tests\" for sound studies and the manual review of the literature was treated as the \"gold standard.\" The sensitivity, specificity, precision and accuracy of the search strategies were calculated.Of the 433 articles about diagnostic tests, 97 (22.4%) met basic criteria for scientific merit. Combinations of search terms reached peak sensitivities of 100% with specificity at 70.4%. Compared with best single terms, best multiple terms increased sensitivity for sound studies by 8.2% (absolute increase), but decreased specificity (absolute decrease 6%) when sensitivity was maximized. When terms were combined to maximize specificity, the single term \"specificity.tw.\" (specificity of 98.2%) outperformed combinations of terms.Empirically derived search strategies combining indexing terms and textwords can achieve high sensitivity and specificity for retrieving sound diagnostic studies from EMBASE. These search filters will enhance the searching efforts of clinicians. Accurate diagnosis is essential for both the clinical care of patients and research about disease conditions. Clinicians increasingly use online access to evidence in the course of clinical care as well as for continuing education and research . For mosResearchers have developed search strategies to assist clinicians with searching, the majority of which have been developed for MEDLINE when searching for therapy and review articles -10. More. Clinicians can easily access and use these search strategies by going to the Clinical Queries page in PubMed. We now report the extension of this research for EMBASE, including the information retrieval properties of single terms and combinations of terms for maximizing the sensitivity and specificity of identifying methodologically sound primary  studies on the diagnosis of health disorders. These search strategies will assist clinicians and researchers when searching for relevant, high-quality articles in EMBASE.In the early 1990s, our group at McMaster University developed search filters for use by clinicians and researchers on a small subset of 10 journals and for 4 types of journal articles  ,16. ThisWe compared the retrieval performance of methodological search terms and phrases in EMBASE with a manual review of each article for each issue of 55 journal titles for the year 2000. Overall, research staff hand-searched 170 journal titles. These journals were chosen based on recommendations of clinicians and librarians, Science Citation Index Impact Factors provided by the Institute for Scientific Information, and the ongoing assessment of their yield of studies and reviews of scientific merit and clinical relevance for the disciplines of internal medicine, general medical practice, mental health, and general nursing practice . Of these 170 hand-searched journals, 135 were indexed in EMBASE. Search strategies were developed using a 55-journal subset chosen based on those journals that had the highest number of methodologically sound studies, that is, studies that clinicians should be using when making patient care decisions. This selection enriches the sample of target articles, improving the precision of estimates of search term performance and simplifying data processing, but is unlikely to bias the estimates of the sensitivity and specificity of search terms.We compiled an initial list of search terms, including index terms and textwords from clinical studies. Input was then sought from clinicians and librarians in the United States and Canada through interviews of known searchers, and requests at meetings and conferences. We compiled a list of 5,385 terms of which 4,843 were unique and 3,524 returned results (list of terms tested provided by the authors upon request). Examples of the search terms tested are 'criterion standard', 'cut point', 'sensitivity', and 'ROC curve', all as textwords; 'diagnosis', the index term, and the index term 'diagnostic test', exploded .As part of a larger study , researcThe proposed search strategies were treated as \"diagnostic tests\" for sound studies and the manual review of the literature was treated as the \"gold standard\". We determined the sensitivity, specificity, precision and accuracy of each single term and combinations of terms in EMBASE using an automated process. Borrowing from the concepts of diagnostic test evaluation and library science, sensitivity for a given topic is defined as the proportion of high quality articles for that topic that are retrieved; specificity is the proportion of low quality articles not retrieved; precision is the proportion of retrieved articles that are of high quality; and accuracy is the proportion of all articles that are correctly classified .Individual search terms with sensitivity > 25% and specificity > 75% for a given purpose category were incorporated into the development of search strategies that included 2 or more terms. All combinations of terms used the Boolean OR, for example, \"predict.tw. OR specificity.tw.\". The Boolean AND was not used because this strategy invariably compromised sensitivity. For the development of multiple-term search strategies to optimize either sensitivity or specificity, we tested all 2-term search strategies with sensitivity at least 75% and specificity at least 50%. For optimizing accuracy, 2-term search strategies with accuracy > 75% were considered for multiple-term development. In the development of diagnosis search filters, 6,574 search strategies were tested.In addition to developing search strategies using the Boolean approach described above, we also evaluated the potential for improving performance using logistic regression. Two approaches were taken. First, we took the top performing Boolean search strategies and ORed additional terms to these base strategies using stepwise logistic regression. The level of significance for entering and removing search terms from the model was 0.05. Adding terms to the model stopped when the increase in the area under the ROC curve was < 1%. Second, we developed search strategies from scratch with stepwise logistic regression using these same cut-off values. Both logistic regression approaches were compared with the Boolean approach to search strategy development when developing strategies for treatment articles and prognostic articles for MEDLINE. Treatment and prognosis were chosen because they represented the best and the worst cases for MEDLINE search strategy performance. For both purpose categories, the logistic regression approaches to developing search strategies did not improve performance compared with search strategies developed using the Boolean approach described above. Thus, for subsequent purpose categories, including diagnosis and databases, including EMBASE, the Boolean approach was used for search strategy development.We also tested search strategies published by other researchers for detecting diagnosis studies.Indexing information was downloaded from EMBASE for 27,769 articles from the 55 hand-searched journals. Of these, 433 were classified as diagnosis, of which 97 (22.4%) were methodologically sound. Search strategies were developed using all 27,769 articles. Thus, the strategies were tested for their ability to retrieve articles about high quality diagnosis studies from all other articles, including both low quality diagnosis studies and all non-diagnosis studies.Table Combinations of terms with the best results for sensitivity, specificity and optimization of sensitivity and specificity are shown in Table Slight modifications to the above-noted most sensitive and most specific search strategies led to some attractive trade-offs in sensitivity and specificity Table . For insOur search strategies were simpler and compared well with two previously published strategies by Bachmann and colleagues for retrieving diagnostic test studies from EMBASE . The mosOur study documents search strategies for use by clinicians and researchers that can help discriminate relevant, high-quality studies from lower quality studies of the diagnosis of health disorders and articles that are not about diagnosis. Those interested in all sound articles on diagnosis, for example researchers conducting systematic reviews of diagnostic tests, will be best served by the most sensitive search. If systematic reviewers wish to include diagnostic test articles that fail the methodological criteria we set, they will still be well served by starting with this strategy: in addition to retrieving all sound studies, the suboptimal specificity (70.4%) of our most sensitive search strategy means the many lower quality diagnostic test studies will also be retrieved. Reviewers may then use additional means to ensure that all pertinent studies are retrieved. Those with little time on their hands who are looking for a few good articles on diagnosis, most likely clinicians, will probably be best served by the most specific strategies. Clinicians could further broaden their search by using the strategies that optimize sensitivity and specificity while minimizing the difference between the two as these strategies provide the best separation of \"hits\" (target citations) from \"false drops\" (undesired citations) but do so without regard for whether sensitivity and specificity are affected.In all cases precision was low. This is the inevitable result of a low proportion of relevant studies for a given purpose in a very large, multipurpose database. This means that clinicians and researchers will continue to need to invest their time in discarding irrelevant retrievals. While low precision in searching can be of concern, the low values here should not be over-interpreted: we did not limit the searches by clinical content terms, as would usually be the case in clinical searches. Precision might be enhanced by combining search strategies in these tables with additional methodological terms using the Boolean 'AND NOT', thereby reducing the possibility of retrieving studies of lower methodological quality; however, this may decrease the sensitivity of the searches. Precision might also be increased by combining search strategies with content specific terms  or journal subsets using the Boolean 'AND' thus reducing the volume of literature searched. The next phases of our project will focus on finding better search strategies through using more sophisticated strategies as outlined above.Comparing the diagnostic search strategies developed for EMBASE with those that we developed for MEDLINE , we founComparing our diagnostic search strategies developed for EMBASE with those previously published , our strSelected combinations of indexing terms and textwords can achieve high sensitivity or specificity in retrieving diagnosis studies cited in EMBASE. The reported search strategies will assist both clinicians and researchers when attempting the retrieve relevant, high-quality diagnostic articles.The author(s) declare that they have no competing interests.NLW and RBH prepared grant submissions in relation to this project. Both authors drafted, commented on and approved the final manuscript. Both authors also supplied intellectual content to the collection and analysis of the data. NLW participated in the data collection and both authors were involved in data analysis and staff supervision.The pre-publication history for this paper can be accessed here:"}
+{"text": "Our objective is to test the ability of an Ultraseek\u00ae search engine to rank MEDLINE\u00ae records of the included studies of Cochrane reviews within the top half of all the records retrieved by the Boolean MEDLINE search used by the reviewers.Most electronic search efforts directed at identifying primary studies for inclusion in systematic reviews rely on the optimal Boolean search features of search interfaces such as DIALOGCollections were created using the MEDLINE bibliographic records of included and excluded studies listed in the review and all records retrieved by the MEDLINE search. Records were converted to individual HTML files. Collections of records were indexed and searched through a statistical search engine, Ultraseek, using review-specific search terms. Our data sources, systematic reviews published in the Cochrane library, were included if they reported using at least one phase of the Cochrane Highly Sensitive Search Strategy (HSSS), provided citations for both included and excluded studies and conducted a meta-analysis using a binary outcome measure. Reviews were selected if they yielded between 1000\u20136000 records when the MEDLINE search strategy was replicated.Nine Cochrane reviews were included. Included studies within the Cochrane reviews were found within the first 500 retrieved studies more often than would be expected by chance. Across all reviews, recall of included studies into the top 500 was 0.70. There was no statistically significant difference in ranking when comparing included studies with just the subset of excluded studies listed as excluded in the published review.The relevance ranking provided by the search engine was better than expected by chance and shows promise for the preliminary evaluation of large results from Boolean searches. A statistical search engine does not appear to be able to make fine discriminations concerning the relevance of bibliographic records that have been pre-screened by systematic reviewers. Systematic reviews in healthcare are designed to summarize and synthesize the existing totality of evidence about a clinical question using rigorous methods that help to safeguard the review from bias. Ideally, the evidence-gathering stage requires preparing as broad a search 'statement' as possible, and then adjusting the electronic search parameters to capture just the right kind of evidence to be useful without overburdening reviewers. More than three quarters of the studies included in systematic reviews are identified through electronic bibliographic databases that are searched using Boolean logic -5. BooleSearch engines can rank search results by relevance based on where in the document the keywords appear, and how often. Using such a search engine to assign relevance to the large result set of a comprehensive Boolean search could reduce the amount of material requiring expert attention, if the documents that meet the strict eligibility criteria of the review are highly ranked.Typically, the manual review of the results of Boolean electronic searches is often done in a two-stage process. In the first stage, reviewers may work from the bibliographic record, or document title, and screen out those that are obviously irrelevant to the review. In the second stage, reviewers usually obtain the full articles associated with remaining records and then decide eligibility based on the complete report, rather than on the more limited information available at the first stage of screening.We evaluated a two-step approach consisting of a comprehensive Boolean search followed by automated relevance ranking for eligible systematic reviews from the Cochrane Library (Cochrane reviews henceforth) to explore the feasibility of such an approach.rd Quarter 2002 was searched through the Ovid interface using the search string (hsss or highly sensitive search).tw. to identify potential studies. Two reviewers assessed each systematic review against the eligibility criteria and resolved any conflicts through consultation. SRS\u2122 was used for all screening and data extraction. SRS is a web-based platform for conducting systematic reviews [We sought Cochrane reviews that used at least one phase of the HSSS  to ident reviews .The size of the MEDLINE retrieval was determined by replicating and running the MEDLINE search strategy. Cochrane reviews with a MEDLINE retrieval of 1000\u20136000 records were selected for testing the performance of the Ultraseek search engine ranking.One librarian extracted descriptive data about the eligible reviews. The following elements were recorded: the number of included studies and the number of excluded studies cited in the review; the number of included studies indexed in MEDLINE; the number of excluded studies indexed in MEDLINE; date of the MEDLINE search reported in the body of the review; level of detail in which the search was reported; phases of the HSSS used; searching techniques employed ; and restrictions such as date, language of publication, age groups or methodological filters. Electronic databases searched as well as other sources used (such as checking reference lists or contacting authors or manufacturers) were also recorded.A known-item search was undertaken in MEDLINE for each included and excluded study listed in the review. A single librarian (MS) completed the searching using the Ovid interface for MEDLINE 1966-April 2003. The indexing status of each study was recorded as indexed or not indexed, and for each review, the set of included studies was aggregated using OR statements, as was a set of excluded studies. Each set was downloaded for subsequent analysis.The Ovid bibliographic records for all studies retrieved from MEDLINE by the replicated search were also downloaded. When the review reported the size of the MEDLINE retrieval, it was compared to ours to validate the replication. Where our search result was smaller than that reported in the review, it was excluded as irreproducible.Produced by Verity, Ultraseek was originally a successful web search engine and is now focused on helping businesses manage their digital information . The UltThe Ultraseek search engine indexes \"collections\". The bibliographic records associated with each systematic review were treated as a collection. Bibliographic records were downloaded from MEDLINE into Reference Manager databases, and tagged according to their inclusion status in the review. A Reference Manager output format was created to write each record with HTML tags. Three sets of fields were written as meta-data \u2013 MeSH headings, title and abstract . A Perl script was used to separate the HTML tagged bibliographic records into individual files. File names encoded the ID number of the review, whether the record was included or excluded from the review, and the reference ID number within that review, in the form http://10included3.html. Thus the collection consisted of the bibliographic records re-written as HTML files tagged with meta-data. The search engine was installed on a laptop computer where the collections resided. The search engine indexed all records in the collection. When a search was run against the collection, the number of items with relevance greater than zero was returned, along with list of up to the first 500 relevant items, sorted by relevance.The Ultraseek search engine was configured to provide weights to the meta-data fields \u2013 index terms, title and description (abstract). When the weights given to the meta-data fields were varied in preliminary testing the relevance scores changed, but not the order of items, which was the variable of interest. Thus, the search engine was configured with all elements equally weighted, and the collections were indexed.For each eligible review, one member of the research team (MS) identified subject terms to be entered into the Ultraseek search. In exploratory work, it became apparent that the number of tied relevance scores depended largely on the number of terms entered. Thus we decided to standardize our Ultraseek searches at 7 terms, the minimum number that seemed to reduce ties to a workable number.We also established that the order in which terms were entered influenced the final relevance score. Terms were entered on the basis of perceived importance  the rankings of included studies within a collection comprising the entire MEDLINE retrieval and ii) the ranking of included studies where only the studies listed as included or excluded in the Cochrane report comprised the collection. As we were concerned that the search engine might be optimized to place highly relevant items in the top few items with less exact ranking further back in the pack, we alsoWhen testing the initial MEDLINE retrieval, recall was determined by considering the proportion of included studies ranking within the top 500. We compared the proportion falling within the top 500, based on their relevance rank, with the proportion expected if ranking was random. When testing listed included and excluded studies, the rankings were analyzed with a Wilcoxon rank sum test using SAS for exact permutations, in order to best handle the small data sets and frequent ties .One hundred and sixty nine Cochrane reviews were retrieved and screened for eligibility. Sixty-four of them were excluded either because they do not use any phase of the HSSS or they did not report the citations of included and excluded trials. The remaining 105 reviews met our inclusion criteria .For three reviews where the Ultraseek ranking performed poorly, we attempted another search, using a different set of terms. Terms and performance on each of the two attempts are reported in Table There was no statistically significant difference between the rankings of studies listed as included versus studies listed as excluded . Further, no obvious pattern between the included and excluded studies could be discerned through visual inspection  a relevance-ranking search engine will have difficulty distinguishing relevant from irrelevant items on the basis of the information contained in the bibliographic record.The approach tested here is interesting as a proof of concept. Truncating the search result at 500 records would have resulted in great efficiencies, reducing the number of records to be reviewed from 26499 to 4500 across the nine reviews, but at the expense of losing 30% of relevant studies \u2013 few reviewers would likely accept such a trade-off.Incorporating additional information into the rankings might facilitate any future exploration of using search engines to reduce the time taken to conduct a systematic review. There are at least four potential sources of additional information. First, giving increased weight to information in the title and index terms is likely to improve performance; we were not able to do this through meta-data weightings. A second source of information to improve accuracy would be the full document. Our results were based on the information contained in a document surrogate \u2013 the bibliographic record. A full text approach will become practical as a larger proportion of articles considered for inclusion in systematic reviews become available electronically.A third source of additional information would be the prior decisions of the reviewers. Novel techniques developed by de Bruijn and Martin  have beevs. excluded lists could not be distinguished points to a role for ranking at the broad screening stage of the review. We did not see a strong enough concentration of relevant records in the top 10 to suggest that some records were so clearly relevant that they should be promoted into the review without manual examination. A useful step would have been to establish a lower cut-off point, below which we could be confident that no relevant studies would fall. This would have permitted us to exclude the lowest ranking (including non-ranked) records without subjecting them to any manual review, thereby gaining efficiency. The search engine selected for the experiment did not enable us to examine any but the first 500 hits.Rankings could be used in several ways. Our finding that included studies receive higher than chance rankings but that the included \u00ae records into HTML format. Ranking holds promise and future research is warranted to explore methods that incorporate more information into the relevance ranking process. A system reliably placing the relevant records within a smaller ranked set derived from the Boolean retrieval can speed the formation of the evidence base for systematic reviews in healthcare.Relevance ranking of Boolean search results is technically feasible using a commercial search engine under academic license to index and search collections built through a simple procedure to render Reference ManagerSample bibliographic record showing HTML markup and meta data tags<html><head><meta name=\"author\" content=\"Fabacher, D., Josephson, K., Pietruszka, F., Linderborn, K., Morley, J. E., and Rubenstein, L. Z.\"><meta name=\"title\" content=\"An in-home preventive assessment program for independent older adults: a randomized controlled trial. -see comments.-\"><meta name=\"subjects\" content=\"Activities of Daily Living;Aged;Comparative Study;Female;Follow-Up Studies;Geriatric Assessment;Health Status;Home Care Services;Human;Male;Non-U.S.Gov't;Patient Compliance;Preventive Health Services;og -Organization & Administration-;Support;United States;Veterans;Voluntary Workers\"><meta name=\"Abstract\" content=\"OBJECTIVE: To evaluate the effectiveness of in-home geriatric assessments as a means of providing preventive health care and improving health and functional status of ... truncated for illustration ... aspects of health and function\"><!-- SR ID = 10--><!-- inclusions status = excluded--><!-- Ph1 or Ph1 = yes--><!-- NBSS = yes--><!-- Ph3 only = no--><title>An in-home preventive assessment program for independent older adults: a randomized controlled trial. -see comments.-</title></head><body><br>Ref ID = 3<br><br>Fabacher, D., Josephson, K., Pietruszka, F., Linderborn, K., Morley, J. E., and Rubenstein, L. Z. An in-home preventive assessment program for independent older adults: a randomized controlled trial. -see comments.-.Journal of the American Geriatrics Society 1994;42(6):630\u2013638.<br><P>MeSH Subject Headings:<br>Activities of Daily Living;Aged;Comparative Study;Female;Follow-Up Studies;Geriatric Assessment;Health Status;Home Care Services;Human;Male;Non-U.S.Gov't;Patient Compliance;Preventive Health Services;og -Organization & Administration-;Support;United States;Veterans;Voluntary Workers<P>Abstract:<br>OBJECTIVE: To evaluate the effectiveness of in-home geriatric assessments as a means of providing preventive health care and improving health and functional status of ... truncated for illustraton ... aspects of health and function<br></body></html>The author(s) declare that they have no competing interests.MS conceptualized the project, screened records for eligibility, undertook data collection and analysis, created the Reference Manager output format, selected search terms, prepared the first draft of the manuscript, and participated in all revisions. NJB obtained funding for the project, designed the statistical analysis and created the Perl scripts, and participated in the drafting and revision of the manuscript. DM obtained funding for the project, advised on the design and conduct of the experiments and participated in all revisions of the manuscript. TJC acted as project leader for the grant, advised on the design and conduct of the experiments and participated in all revisions of the manuscript. RWP obtained funding for the project, advised on the statistical analysis, the design and conduct of the experiments and participated in all revisions of the manuscript. AM screened records for eligibility, undertook data collection, and participated in all revisions of the manuscript. TK obtained funding for the project, the design and conduct of the experiments and participated in all revisions of the manuscript. LZ configured the search engine, replicated the searches, undertook known item searching, built and indexed the collections, created the datasets, and participated in all revisions of the manuscript.All authors reviewed and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "The project aims to create an alternative search interface for MEDLINE/PubMed that may provide assistance to the novice user and added convenience to the advanced user. An earlier version of the project was the 'Slider Interface for MEDLINE/PubMed searches' (SLIM) which provided JavaScript slider bars to control search parameters. In this new version, recent developments in Web-based technologies were implemented. These changes may prove to be even more valuable in enhancing user interactivity through client-side manipulation and management of results.PubMed Interact is a Web-based MEDLINE/PubMed search application built with HTML, JavaScript and PHP. It is implemented on a Windows Server 2003 with Apache 2.0.52, PHP 4.4.1 and MySQL 4.1.18. PHP scripts provide the backend engine that connects with E-Utilities and parses XML files. JavaScript manages client-side functionalities and converts Web pages into interactive platforms using dynamic HTML (DHTML), Document Object Model (DOM) tree manipulation and Ajax methods. With PubMed Interact, users can limit searches with JavaScript slider bars, preview result counts, delete citations from the list, display and add related articles and create relevance lists. Many interactive features occur at client-side, which allow instant feedback without reloading or refreshing the page resulting in a more efficient user experience.PubMed Interact is a highly interactive Web-based search application for MEDLINE/PubMed that explores recent trends in Web technologies like DOM tree manipulation and Ajax. It may become a valuable technical development for online medical search applications. This research continues to investigate innovations in user-computer interface for online storage and retrieval systems in medical research. The goal of the project is to advance the development of a Web-based medical search tool that can enhance user interaction with the MEDLINE/PubMed database and push to the forefront the different strategies and filters in Entrez PubMed that often remain hidden from novice users, such as age groups, clinical study filters and systematic reviews. The long-term objective is to study and implement clean and effective user interfaces for MEDLINE/PubMed that increases utilization and improves search outcomes without overwhelming novice users and limiting the workflow of advanced users. This manuscript reports the development, implementation and technical evaluation of the research application, PubMed Interact.An earlier version of this project is the Slider Interface for MEDLINE/PubMed searches, or SLIM . SLIM isNew approaches in the development of Web-based applications prompted the exploration to look beyond search forms and provide users the ability to further interact with results. Document Object Model (DOM) tree manipulation and Ajax (Asynchronous JavaScript + XML)  have gaiPubMed Interact makes extensive use of DHTML, DOM tree manipulation and Ajax scripting to enhance interactivity and productivity. Although it extracts several features from SLIM, many of its integral features allow interactions with the retrieved set of citations. We hope this project will contribute to ongoing efforts to improve online storage and retrieval systems for medical literature.PubMed Interact introduces an improved version of the SLIM interface Figure . A wide The publication date parameter uses two sliders: the start year slider and the end year slider. With an end year slider, searches are not limited to the current year as the permanent end date. Users can set different date ranges within the past 10 years, e.g. 1998 to 2002, or limit to one specific year, e.g. 2003. No publication date is set by default for the start year slider, i.e. no date limits. The end year slider defaults to the current year.The PubMed database contains several subsets, among them the MEDLINE subset and the Core Clinical Journals. The journal subset slider controls options to search within the whole PubMed database, the MEDLINE database subset, or the Core Clinical Journals. Within each subset, users can further limit the search to articles with abstracts or to those with links to full-text or free full-text. The default setting searches the PubMed database without any abstract or full-text restrictions.The age group slider is a modified version of the age group dropdown menu in the Limits page of Entrez PubMed. It reorders the limits according to age. It starts with 'Newborn' and ends with 'Adult Group'. No limits are set by default.The methodology filter slider can limit searches to case reports, clinical study categories, or systematic reviews. The case report filter uses the publication type search tag of Entrez PubMed to limit the search. The clinical study categories, also called PubMed Clinical Queries, are 10 search methodology filters based on the works of Haynes RB et al . The sysThe MeSH mapping slider, a feature first developed in SLIM as the search mapping slider, is intended for intermediate to advance users of PubMed familiar with search tags and MeSH term operations. A customized PHP function extracts the mapped MeSH terms from the original search and modifies the search tags according to the slider setting. These modified terms are then appended to the current search to refine and redirect the search strategy. The default setting submits the search terms to the ESearch utility as entered in the text box without any modifications.The last slider controls the number of citations to be displayed in the results list. It does not affect the search query. It merely provides users the option to display the number of retrieved citations .To limit the search by subject or language, a dropdown menu below the sliders contains options for human studies, English language or both. To reload the form or reset the sliders to the default settings, users can click on the links found below the dropdown menu.Users can opt to hide the slider bars. A text link at the top right part of the search form beside the search button allows users to hide the search limits. This process is most advantageous when viewing the search results because it maximizes the display area of the browser page Figure  and 3. SAn interactive feature of the search form is the ability to preview the results count without submitting the form or reloading the page Figure . This fuPubMed Interact adapts two features from the results interface of SLIM: the information box and the ability to toggle the display of abstracts. An important distinction of PubMed Interact is the facility by which users can manipulate the search results.The information box is displayed only after the search form is submitted Figure  and 3. PAbstracts can be displayed or hidden from view Figure  and 3. IRemoval of single citations from the search results is seldom found in Web-based medical search applications. In PubMed Interact, users can delete individual citations from the main list by clicking on a link below the citation details Figure . When a The 'Auto-Append Article' feature, also called A3, is linked with citation deletion. If active, the A3 function automatically retrieves the next citation in the results and appends it at the bottom of the list when a citation is deleted. The new citation data is retrieved from the local PMI domain server using Ajax scripting methods, while the action of appending and displaying that citation is done using DOM tree manipulation. All A3 processes are asynchronous and achieved without reloading the page. The appended article acquires the functionality of the original citations on the list. This feature is deactivated by default and can be activated using a checkbox at the top of the list.PubMed Interact implements two relevance lists: high and low. These relevance lists are user-dependent and color-coded. Users can label specific citations according to relevance to the original search. Citations tagged with high relevance will have a light green background, while those with low relevance will have a light yellow background Figure . CitatioAn advanced interactive feature of PubMed Interact is the ability to retrieve the related articles of each citation within the same page. In the current PubMed Interact implementation, only the top 10 related articles are retrieved and displayed where the abstract is positioned Figure . ClickinPubMed Interact was developed on a server running Windows Server 2003 with Apache 2.0.52, PHP 4.4.1 and MySQL 4.1.18. The server-side scripts were coded in PHP and provided the backend engine of PubMed Interact. Client-side display and functionalities were written in HTML and JavaScript. The advanced and interactive features of PubMed Interact rely on DHTML, DOM tree manipulation and Ajax scripting methods, which were all written in JavaScript.A large part of script development adopted the object-oriented programming (OOP) approach. A custom set of PHP classes connect to the Entrez Programming Utilities, specifically the ESearch, EFetch and ELink tools . These PThe retrieved XML files are processed and stored in a local MySQL database to minimize the load on the E-Utilities servers. Instead of several remote queries to E-Utilities, the PHP scripts that retrieve data for the search results send one query and store the top 200 of the citation details regardless of the number of citations to be displayed. Thus, the A3 feature which appends new articles after a citation is deleted retrieves data from the local domain server and not from E-Utilities. The same process is used for the related articles of one citation. The details of all 10 related articles are stored in the local server and retrieved without reconnecting to the E-Utilities server.JavaScript functions were essential in the development of client-side interactivity. DOM tree manipulation captured information within the page and passed the data to the Ajax script engine. The core of Ajax scripting is the XMLHTTPRequest Object of JavaScript which performs HTTP client functions. However, the XMLHTTPRequest Object, by design, can only connect to the local domain server and not to remote servers, e.g. E-Utilities. Thus, custom PHP scripts were written to receive data from the Ajax engine, connect to the E-Utilities server or to the local MySQL database as needed and deliver an output in HTML format. JavaScript then displays the output in the browser page using both the Ajax engine and DOM tree manipulation. This is all done asynchronously without reloading the page and interrupting user activity.PubMed Interact is an experiment in user-computer interface. It is part of an ongoing project to make use of modern Web technologies in the development and improvement of Web-based medical search applications. The growing trend of using the Web as a platform to deliver services opens opportunities for alternative solutions in medical literature research. Web-based applications that function like traditional software, combined with rich user interface and improved user control of data, contribute to the indispensable nature of online information storage and retrieval systems for health resources.Two important components of the trend are DOM tree manipulation and Ajax. By integrating both technologies, PubMed Interact bridges an effective search strategy with a highly-interactive interface. Users not only have the ability to modify searches by setting parameters, they can also label, delete and add from within the existing list of citations. Access to related articles in the same page also provides an additional resource for more relevant citations not found in the original search results.The search interface of PubMed Interact exposes and facilitates the use of several search strategies available in Entrez PubMed. Some options in the Limits page of Entrez PubMed are available in the first four sliders, eliminating the need to go back and forth between pages to set search parameters. Two of the advanced search features of Entrez PubMed \u2013 the clinical study categories, also known as PubMed Clinical Queries, and the systematic reviews subset \u2013 are made available for both novice and seasoned users with the Methodology Filter slider. In Entrez PubMed, the MeSH terms and subheadings of a search are viewed from the Details tab. In PubMed Interact, the MeSH details mapped from the keywords are presented in the information box, which can then be used as guides for the MeSH Mapping slider. Several features for future integration may include adding publication types, language options and subsets and searching in the Journals and MeSH database. These efforts are consistent with the long-term aim of developing a user-computer interface for medical research that empowers novice users with interactive tools for search parameters and provides expert users with easy access to advanced search filters.The application is available online without restrictions. The alpha version and the beta version went live in late November 2005 and February 2006, respectively. The local MySQL database of the beta version contains over 29,900 records of citations in XML format and uses 54 megabytes of disk space. A scheduled maintenance script can be implemented in the future to delete old XML records from the database and keep the storage allocation manageable. This plan is deferred until the implementation is moved out of beta phase to record benchmarks for MySQL usage.Browser compatibility evaluation showed full functionality in Windows versions of Mozilla Firefox 1.5+, Internet Explorer 5.5+ and Opera 8.5+ and in the Linux version of Mozilla Firefox 1.5+. Some formatting inconsistencies were observed in Mac OSX versions of Mozilla Firefox 1.5+ and Safari 2.0+ but no functionality problems were noted.The search form and citation list of the application were tested using the W3C Markup Validation Service . An unsuThis paper is limited to the development, implementation and technical evaluation of PubMed Interact. It does not provide empirical evidence to show increased efficiency in searching or better precision and recall for results. A formal user evaluation of the application is needed to validate the usability and benefits of an alternative PubMed search interface.The technical evaluation of PubMed Interact employed commonly accepted procedures in Web applications, such as functionality, storage space used, markup validations and browser compatibility testing. It was not evaluated against any formal framework or standard criteria for software development.User evaluation is valuable in the continued development of PubMed Interact. The researchers plan to do comparative studies between PubMed Interact and Entrez PubMed. Users with various levels of searching skills will perform structured and unstructured tasks. Through user interviews, online questionnaires and direct observation, the research team will assess the effectiveness of PubMed Interact as compared to Entrez PubMed in usability, performance and search outcomes. The educational impact, speed and stability of the system and the effect on searching attitudes and strategies will also be studied.The projected study will be an opportunity to gather more information on how medical researchers interact with alternative search interfaces and obtain data on usability and functionality. User feedback will determine which features need to be improved or abandoned, and whether new functionalities should be added. As the progress of Web technologies continues, better platforms and methods will be available for further innovations in search interfaces for medical literature search.PubMed Interact is a Web-based MEDLINE/PubMed search application that explores recent trends in Web development technologies like DOM tree manipulation and Ajax scripting methods. Users can control search parameters, refocus search strategies and modify search results easily. Many enhanced and interactive features occur at client-side and allow instant feedback without reloading or refreshing the page. PubMed Interact is a novel approach in the development of online tools for medical information research.Project name: PubMed InteractProject home page: Operating systems: Web-based, platform-independentProgramming language: PHP, JavaScriptOther requirements: JavaScript-enabled browsers, e.g. Fire Fox 1.0 or higher, IE 5.5 or higherLicense: Free, anyone may use the serviceAny restrictions to use by non-academics: NoneSLIM: Slider Interface for MEDLINE/PubMed searchesDHTML: Dynamic HTMLDOM: Document Object ModelAjax: Asynchronous JavaScript + XMLXML: Extensible Markup LanguageHTML: HyperText Markup LanguageMeSH: Medical Subject HeadingsPHP: PHP: Hypertext PreprocessorA3: Auto-Append ArticleOOP: Object-oriented ProgrammingThe author(s) declare that they have no competing interests.MM conceived of the project, designed and developed the application and drafted the manuscript. PF assisted in the design and development of the application and reviewed the initial drafts of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Many questions go unanswered, however, due to lack of skills in formulating questions, crafting effective search strategies, and accessing databases to identify best levels of evidence.Supporting 21This randomized trial was designed as a pilot study to measure the relevancy of search results using three different interfaces for the PubMed search system. Two of the search interfaces utilized a specific framework called PICO, which was designed to focus clinical questions and to prompt for publication type or type of question asked. The third interface was the standard PubMed interface readily available on the Web. Study subjects were recruited from interns and residents on an inpatient general medicine rotation at an academic medical center in the US. Thirty-one subjects were randomized to one of the three interfaces, given 3 clinical questions, and asked to search PubMed for a set of relevant articles that would provide an answer for each question. The success of the search results was determined by a precision score, which compared the number of relevant or gold standard articles retrieved in a result set to the total number of articles retrieved in that set.Participants using the PICO templates (Protocol A or Protocol B) had higher precision scores for each question than the participants who used Protocol C, the standard PubMed Web interface.  95% confidence intervals were calculated for the precision for each question using a lower boundary of zero. However, the 95% confidence limits were overlapping, suggesting no statistical difference between the groups.Due to the small number of searches for each arm, this pilot study could not demonstrate a statistically significant difference between the search protocols. However there was a trend towards higher precision that needs to be investigated in a larger study to determine if PICO can improve the relevancy of search results. Clinicians and educators currently utilize a variety of resources and interfaces to search the biomedical literature to answer clinical questions. PubMed is a service of the U.S. National Library of Medicine (NLM) that provides access to over 16 million citations from MEDLINE and other life science journals dating back to the 1950s . However, the common word \"blacks\" maps to the broader MeSH term \"African continental ancestry group\" and retrieves 48195 citations [searched 4/18/07]. Most of the relevant citations used either the word \"blacks\" or were indexed to the broader MeSH term \"African continental ancestry group.\" The second most common error was related to selecting the wrong study design or Clinical Query for the type of question being searched. This was a common problem for Protocol A, which only listed therapy study designs. The third error affected Protocol C and involved limiting search terms to the title field. While searchers often select their articles based on relevant words in the title, a problem arises when more than one word or phrase is appropriate to the topic. For example, a \"peg\" is also called a \"percutaneous endoscopic gastrostomy tube\" or \"feeding tube.\" Limiting the search to the word \"peg\" in the title eliminates mapping to appropriate subject headings (MeSH) such as intubation, gastrointestinal and therefore may exclude relevant articles on the topic that use alternative terminology. Understanding these errors can help in teaching effective searching and in developing better search systems.In addition to quantitative comparisons, this pilot study provided an opportunity for more open-ended insight into how practitioners search. Some searches within each question  did not retrieve any relevant citations. A qualitative assessment of search strategies that produced no acceptable results revealed three common types of errors: ambiguous mapping of subject headings (MeSH); selecting the wrong publication type; and limiting search queries to just words in the title. The error that accounted for the largest number of non-productive searches was related to the MEDLINE indexing structure or MeSH . In question three, most searchers used the phrase \"Perceptions of ease of use and time spent searching were approximately the same across all three protocols, as were the numbers of terms used in each search, regardless of protocol used. However, of the 30 searches performed using Protocol B , 25 (83%) actually incorporated the Clinical Queries into the strategy, as opposed to only 2 (7%) of the searches using Protocol C (PubMed/Web). Both Protocol A and B facilitated the use of publication types and the Clinical Queries by prompting the searcher to consider these elements in the strategy. While these elements were also available in Protocol C, they are either behind the Limits tab or listed in the PubMed Services menu.Effective searching requires a series of steps to lead the practitioner from the clinical question or bedside to informed decision making. In March of 2005, over 68,000,000 searches were conducted in PubMed, from seven million unique IP addresses.  This stuSearches performed on a PICO-formatted screen retrieved a higher percentage of relevant citations than searches performed on the standard PubMed search interface. However, due to the small number of searches for each arm, this pilot study could not demonstrate a statistically significant difference between the search protocols. There was a suggestion of a trend towards higher precision that needs to be investigated in a larger study to determine if PICO can improve the relevancy of search results.The author(s) declare that they have no competing interests.CS and TO participated in the study design, carried out the study, analyzed the results, and contributed to the writing of the manuscript. MA participated in the study design, carried out the study, and contributed to the writing of the manuscript. SK participated in the study design, analyzed the results, and contributed to the writing of the manuscript. All authors read and approved the final manuscript. FP conceived of the study, participated in the study design, developed the PICO templates, and contributed to the writing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Search filters or hedges play an important role in evidence-based medicine but their development depends on the availability of a \"gold standard\" \u2013 a reference standard against which to establish the performance of the filter. We demonstrate the feasibility of using relative recall of included studies from multiple systematic reviews to validate methodological search filters as an alternative to validation against a gold standard formed through hand searching.We identified 105 Cochrane reviews that used the Highly Sensitive Search Strategy (HSSS), included randomized or quasi-randomized controlled trials, and reported their included studies. We measured the ability of two published and one novel variant of the HSSS to retrieve the MEDLINE-index studies included in these reviews.The systematic reviews were comprehensive in their searches. 72% of included primary studies were indexed in MEDLINE. Relative recall of the three strategies ranged from .98 to .91 across all reviews and more comprehensive strategies showed higher recall.An approach using relative recall instead of a hand searching gold standard proved feasible and produced recall figures that were congruent with previously published figures for the HSSS. This technique would permit validation of a methodological filter using a collection of approximately 100 studies of the chosen design drawn from the included studies of multiple systematic reviews that used comprehensive search strategies. Search filters or hedges play an important role in evidence-based medicine. For example, work of the HEDGES team has enabled focused clinical searches on PubMed  and the Recall and precision are the performance characteristics of search strategies that are most relevant to systematic reviews. Recall has as its numerator the number of relevant records in a database retrieved by a search strategy and as its denominator, the total number of relevant records in the database. It is difficult to measure except in experimental conditions. It is widely believed that valid reviews require as complete as possible an identification of relevant studies, thus recall is the most important search parameter from a scientific perspective. ComprehPrecision has as its numerator the number of relevant records retrieved by a search strategy, and as its denominator, the total number of records retrieved by the search strategy and is thus easily calculated. As precision declines, the burden on reviewers increases as they have more irrelevant items to evaluate for inclusion. Number-needed-to-read is a parameter introduced recently to easily interpret precision figures in the context of systematic reviews \u2013 it is the inverse of precision.The ideal search strategy would show high recall and high precision, but there tends to be a trade off between the two, and the relationship typically takes the form of a sigmoid curve rather tet al. demonstrated that even modest improvements in precision could save a week's effort in a large review[As recall and precision are inversely related, the high recall approach used by systematic reviewers leads to the retrieval of many irrelevant bibliographic records. In practice, restricted by time and cost, reviewers must strive to identify the maximum number of eligible trials, hoping that the studies included in the review will be a representative sample of all eligible studies. The ovege review. MethodoRecall must be calculated to fully evaluate any search strategy, including methodological filters. The real world difficulty in determining the recall of a search strategy is in knowing the denominator. The standard practice amongst those developing search filters for evidence-based medicine has been to establish the gold standard through hand searching of the literature. Jenkins reviewed 20 reports of search filter construction and performance and found that almost all of these filters were validated against a gold standard formed either by hand searching alone, or hand searching and database searching in combination. There aAn approach to filter validation that has not been used widely in the filter development literature is relative recall. Relative recall is the proportion that any specific system retrieves of the total or pooled relevant documents retrieved by all systems considered to be working as a composite. Systemaet al. used the technique, describing it as a pragmatic approach, in developing search filters for diagnostic studies on deep vein thrombosis. They used the included studies of 16 published systematic reviews on the topic as a reference set in order to establish a reference set with a broader range of journals and publication years than could have been practically achieved through hand searching[et al. used relative recall as the basis for comparison of 5 methodological filters for identifying diagnostic studies. The filters were tested against the included studies for 2 systematic reviews in different fields[The relative recall technique that we explore in this paper has been used by a number of authors in the context of information retrieval in evidence-based healthcare, although the term relative recall may not have been used. For example, Vincent searching. Similarnt fields. They suet al. who used the technique in a preliminary study to design a filter for maximally sensitive MEDLINE search for observational studies of a relationship between an exposure and disease. The reference standard was the 58 included studies of a single systematic review[et al. used the technique to calculate recall in a study assessing MEDLINE searches by clinical end-users and librarians, and used the term relative recall to describe it. The basis for recall was the number of relevant citations retrieved from an individual search divided by the total number of relevant citations from all searches on the same topic.Other examples of its use are Wieland ic review. Hersh aic review. McKibboWe explore relative recall as a general technique for forming a reference standard (gold standard) for use in evaluating search filters. In the course of doing a systematic review, reviewers assess items identified from all sources for relevance, and relevant studies are included in the review. The success of any one system used in the review, compared to the pooled relevant documents (included studies) retrieved by all systems working as a composite, is the relative recall of that system.et al. present a case study of such a situation[There are some limitations to relative recall, and these are well described by Fricke. The mossituation. The relsituation. To guar123 and the two-phase search as HSSS12. The HSSS12 was selected for searching all of MEDLINE in the retagging project when a pilot study conducted by the UK Cochrane Centre in 1994 concluded that the terms in the third phase of the HSSS was too broad for use without a subject search[123, McDonald found precision to be 7.8% for the years 1994\u20131997, and reported the precision of individual terms[Since its introduction in early 1990's, the HSSS has been widely used by information specialists and medical practitioners to find reports of randomized controlled trials (RCTs) in MEDLINE. The complete HSSS contains three phases see . We refect search. In an aual terms.To create an additional strategy for comparison, we used the precision of individual terms reported by McDonald to form a more concise search strategy, removing some of the terms with low precision from the HSSS. We call this variant the Narrow Boolean Search Strategy (NBSS) see .rd Quarter 2002 was searched through the Ovid interface to identify systematic reviews that appeared to use elements of the HSSS [see The Cochrane Database of Systematic Review 3To be eligible, a systematic review had to use at least two sections of the Highly Sensitive Search Strategy to find RCTs in MEDLINE. Specifically, we excluded reviews that did not report using the HSSS, that reported using only the first section, or when they reproduced the MEDLINE search strategy, did not reproduce at least 2 sections of the HSSS. We accepted reviews that stated that they used the HSSS, without specifying which sections. In addition to using the HSSS, the systematic review must have reported the citations for included studies. Finally, the review must have had as an inclusion criterion that primary studies were either RCT or quasi RCT. Two reviewers  examined each systematic review for eligibility. Conflicts between the two reviewers were resolved through consultation and consensus.12, the HSSS123, and NBSS to determine the number of trials that were retrieved by each. The number of included trials cited in a systematic review and the number of included trials indexed in MEDLINE was recorded. Additional characteristics of the systematic reviews and the search strategy were extracted by MS.For each eligible systematic review, a known-item search for each included study was undertaken to determine if it was indexed in MEDLINE or not. Searching was completed by a single librarian (LZ) between January 2003 and April 2003, using the Ovid interface for MEDLINE 1966\u20132003. For an example of known item search see We define relative recall as:We use the number of included trials in MEDLINE as the denominator because this represents the composite pool of items available to our filters.169 systematic reviews in Cochrane Database of Systematic Reviews were identified for screening. 64 of these were excluded either because they did not use RCTs or quasi RCTs as the basis for inclusion in the review (n = 7), did not appear to use any part of HSSS (n = 28) or they do not report the citations of included and excluded trials (n = 29). 105 systematic reviews meet all inclusion criteria  Table .Eighty different electronic sources were mentioned in the 105 reviews. Major electronic sources (cited in 10% or more of reviews) and all non-electronic sources are shown in Table 123 would have resulted in the retrieval of 1422 of these , HSSS12 retrieved 1370 of these items , and NBSS retrieved 1322 of these .Among the 2014 included trials, 1456 were found, through known item searching, to be indexed in MEDLINE at the time of our searching (72%). We use this as the denominator in our calculation of relative recall. Using HSSS123 retrieved all MEDLINE included studies in 86 reviews, HSSS12 in 72 reviews, and NBSS in 57. One extreme case was found in which all three search strategies missed a number of included items[Looking at performance of the various filters in individual reviews, of our collection of 105, HSSSded items however,ded items. The dis123, 0.68 for the HSSS12, and 0.656 for the NBSS.Our results support the use of relative recall as a filter validation technique by those without the resources for extensive hand searching. Our figures for recall are somewhat higher than the 0.80 recall of HSSS reported by Hopewell. If stud123 is the broadest. HSSS12 is narrower, as fewer terms are joined with Boolean OR and no new terms are introduced. NBSS is narrower again. As expected, the three phases gave the highest recall, there was a 4 point drop when only phase 1 and 2 were used and a further 3 point drop if the narrower search tested here were used. The HSSS12 provided high relative recall overall and recall as good as the full three section HSSS123 in 72 cases (69%). The NBSS that we introduce here appears to result in a relatively small decline in recall, relative to broader searches. This could be interpreted as providing some support for the technique of picking terms based on performance of individual terms , although the resulting search appears insufficiently sensitive for use in the systematic review context due to the need to maximize recall.We also see our results behave as expected across the three variants tested here. The three strategies are variants on a theme. The HSSSOur findings are based on a large number of reviews from 30 review groups corresponding to different medical specialties. Most of these reviews featured comprehensive searches, using both electronic and non-electronic sources. Thus the composite search result of the individual studies is apt to be quite robust. The pooling of included studies from many reviews helps mitigate against errors or ineffective search performance of individual reviews, and supports the generalisability of these results across subject areas. Thoroughness of the composite search, in terms of number of systems searched and the adequacy of those searches, varied between reviews in our sample. To use this technique of relative recall to validate new filters for other research designs, we suggest restricting the sample to those meeting some minimum criteria for completeness. We also caution that most reviews in this study (87%) benefited from a search of CENTRAL \u2013 a comprehensive register of RCTs. DesignsThe main advantage of using a relative recall approach instead of a gold standard developed through hand searching is efficiency. How many studies of the design sought might be needed for the reference standard created by pooling the included studies of systematic reviews? Sample size calculation are rarely reported in studies of filter development but are The feasibility of the relative recall approach we have described for validating novel methodological filters may depend on the availability of a number of systematic reviews using the methodology of interest. In the example above, 100 included studies of a given design would be needed, from several systematic reviews.The relative recall approach as a gold standard has the added advantage of taking into account user preference, that is, the assessment by expert reviewers that the item is indeed relevant for the review. In addition to providing a gold standard for evaluating searches, relative recall can also be used in testing the contribution of databases,32.This paper has focused on recall and precision as the basis for evaluation of search performance. Numerous criticisms of these measures have been made, and these are well reviewed by Kagolovsky and Moehr Never-thWhile we propose relative recall as an alternative to hand-searching in the formation of a gold standard for search strategy development, our methods are indirect. A useful avenue for further study would be such a direct comparison between a standard based on the included studies of systematic reviews and one derived from hand searching . The most useful comparison would not only examine the information retrieval characteristics of the two approaches, but would also compare the resources required to assemble the collections.Finally, it must be underscored that known item searching of MEDLINE retrieved only 72% of included studies. While the proportion of included studies indexed in MEDLINE is higher than the 51% sensitivity of MEDLINE reported by Dickersin. This reThe relative recall approach of using included studies of a certain design, pooled across a number of reviews appears to be a promising alternative to hand searching for those wishing to develop a reference standard for new methodological filters. It may be possible to validate methodological filters based on approximately 100 included studies of the desired design drawn from a number of systematic reviews with comprehensive searches.The author(s) declare that they have no competing interests.MS conceptualized the project, screened records for eligibility, undertook data collection and analysis, prepared the first draft of the manuscript, and participated in all revisions. LZ undertook the searching, created the datasets, and participated in all revisions of the manuscript. AM screened records for eligibility, undertook data collection, and participated in all revisions of the manuscript. NJB obtained funding for the project, designed the statistical analysis, and participated in the drafting and revision of the manuscript. DM obtained funding for the project, advised on the design and conduct of the research and participated in all revisions of the manuscript. TJC acted as project leader for the grant, advised on the design and conduct of the research and participated in all revisions of the manuscript. RWP obtained funding for the project, advised on the statistical analysis, the design and conduct of the research and participated in all revisions of the manuscript. TK obtained funding for the project, the design and conduct of the research and participated in all revisions of the manuscriptThe pre-publication history for this paper can be accessed here:Appendix 1 Search Strategies Used. Appendix listing search strategies.Click here for fileAppendix 2 \u2013 Included Studies (n = 105). List of studies included in this review.Click here for file"}
+{"text": "Many clinicians and researchers are interested in patients of a specific age . Searching for age-specific publications in large bibliographic databases such as Medline is problematic because of inconsistencies in indexing, overlapping age categories, and the spread of the relevant literature over many journals. To our knowledge, no empirically tested age-specific search strategies exist for Medline. We sought to determine the retrieval characteristics of age-specific terms in Medline for identifying studies relevant for five clinical specialties: adult medicine, geriatric medicine, pediatric medicine, neonatal medicine, and obstetrics. We compared age-specific search terms and phrases for the retrieval of citations in Medline with a manual hand search of the literature for 161 core health care journals. Six experienced research assistants who were trained and intensively calibrated read all issues of 161 journals for the publishing year 2000. In addition to classifying all articles for purpose and quality, study participants' ages were also recorded. Outcome measures were sensitivity, specificity, precision, and accuracy of single and combination search terms. When maximizing sensitivity, the best sensitivity and specificity achieved with combination terms were 98% and 81.2%, respectively, for pediatric medicine, 96.4% and 55.9% for geriatric medicine, 95.3% and 83.6% for neonatal medicine, 94.9% and 64.5% for adult medicine, and 82% and 97.1% for obstetrics. When specificity was maximized, all disciplines had an expected decrease in sensitivity and an increase in precision. Highest values for optimizing best sensitivity and specificity were achieved in neonatal medicine, 92.5% and 92.6%, respectively. Selected single terms and combinations of MeSH terms and textwords can reliably retrieve age-specific studies cited in Medline. Clinicians and researchers seeking research reports for specific age categories, including generalists and those who are engaged in clinical specialties such as adult medicine, geriatric medicine, pediatric medicine, neonatal medicine, or obstetrics, need to target their literature searches so that the information they retrieve is relevant to their patient population. Difficulty in finding pertinent evidence contributes to the challenges\thealth professionals have in keeping up-to-date and practising evidence-based medicine -7.Finding age-specific evidence in Medline is a difficult task for several key reasons. In large bibliographic databases such as Medline, optimal search retrieval for individual topics is hampered by the overwhelming amount of available information that is not pertinent to the question. When users search in Medline they have the potential to retrieve articles from any of the approximately 4800 journals that are currently indexed in the database. The size of this general purpose biomedical database coupled with imperfections in indexing -3 lead tSearching in Medline for a specific patient population by selecting \"age-specific\" journals will not help because studies relevant to any age group are scattered through a wide range of journals, including general journals that cater to no particular age group. Moreover, in Medline, the indexing practices used to identify the ages of those involved in a study are so liberal that they create a very imprecise representation of the age categories of the participants within the study. Medline indexers apply all relevant age-specific index terms to an article regardless of how many participants fall within that category. Thus, if just one patient or participant in the study falls into a particular age category, that age-specific medical subject heading (MeSH) term will be applied. For example, if a researcher was interested in intercultural communication in family medicine around issues of newborn care, the study by Harmsen and colleagues  might beTo assist clinicians searching for studies on age-specific patient populations, we have developed and tested Medline search strategies for detecting studies for specific age categories as well as tested age-specific search terms pertinent to five age-related clinical specialties. In this paper, we report on the evaluation of the retrieval performance of age-specific search strategies in Medline compared with a manual review (the \"gold standard\" search) of each article in every issue of 161 journals in the year 2000.Search strategies are useful tools when searching in large electronic databases. We previously developed search strategies for use in Medline to detect clinically relevant scientifically sound articles in the areas of causation, prognosis, treatment, and diagnosis -15. AfteACP Journal Club, Evidence-Based Medicine, Evidence-Based Nursing, and Evidence-Based Mental Health). The 161 journals include content for the disciplines of internal medicine , general medical practice , mental health , and general nursing practice .The study compared the retrieval performance of age-specific search terms and phrases in Medline (accessed using Ovid) with a manual review of each article in every issue of 161 journal titles for the year 2000. The 161 journals were chosen over several years in an iterative process based on a hand search review of over 400 journals. The journals were recommended by clinicians, librarians, editors, and publishers and were chosen based on Science Citation Index impact factors and ongoing assessment of their yield of studies and reviews of scientific merit and clinical relevance [Six research assistants hand searched the 161 journals for the year 2000 and collected data on age of the study participants according to our hand search categories defined in MeSH terms and textwords related to age  were downloaded from Medline and were treated as \"diagnostic tests\" for detecting studies with an age-specific population as determined by a hand search of the literature from 161 journals (the gold standard). The hand search data were obtained by reading each issue completely. The downloaded Medline data from the 161 journals included the retrieval sets for each of the individual terms. After these two data sources were obtained , a database was created that included the matched merged content from these two sources. These Ovid retrieval sets were then manipulated by our own set of programs to calculate our outcome measures\u2014the operating characteristics of each age-specific searching term  for individual terms and for combinations of terms. When we merged the two data sets (Medline and hand search), we determined the match. If Medline included an item that was not indexed, we went back to the journal and scored it. If we had scored an item that was not in Medline, we removed it from the merged database. Therefore, the final merged database included only items that had hand search scores and Medline indexing. This merged database was used to develop the age-specific search strategies .Borrowing from the concepts of diagnostic test evaluation and library science, we determined the sensitivity, specificity, precision, and accuracy of single- and multiple-term Medline searches. We considered these operating characteristics as the indicators of search term performance. Sensitivity for a given age-specific topic is defined as the proportion of relevant articles  that are retrieved; specificity is the proportion of nonrelevant articles  not retrieved; precision is the proportion of retrieved articles that are relevant ; and accuracy is the proportion of all articles that are correctly classified . Our hand search of the 161 journals indexed in Medline led to the classification of all articles in these journals for age-related content. Search terms were then tested to determine their performance in retrieving age-relevant articles while eliminating those that were nonrelevant. An automated process (which we developed and implemented using a computer program) was used to calculate the operating characteristics (performance) for each single and combination term in Medline. Formulae for calculating the operating characteristics  of searches are shown in Individual search terms with sensitivity > 25% and specificity > 75% for a given age category were incorporated into the development of search strategies that included two or more terms. All combinations of terms used the Boolean \"OR.\" For the development of multiple-term search strategies to either optimize sensitivity or specificity, we tested all two-term search strategies with sensitivity of at least 75% and specificity at least 50%.To construct a comprehensive set of search terms, a list of MeSH terms and textwords was initially generated, and input was sought from clinicians and librarians in the United States and Canada through interviews with known searchers, requests at meetings and conferences, and requests to the National Library of Medicine. These experts were asked which terms or phrases they used when searching for age-specific studies, as well when searching for studies in specific purpose categories. Search terms could be MeSH terms, including publication types and subheadings, or textwords specific to age in titles and abstracts of articles. Various truncations were also applied to the textwords, phrases, and MeSH terms. We compiled a list of 543 age-specific terms (Multimedia Appendix). All terms were tested in Medline using the Ovid Technologies searching system.Age categories for the hand search were modeled from the MeSH terms used to index age content. A comparison of hand search categories and MeSH term definitions is shown in We defined five age-specific specialty areas by collapsing our hand search age categories see  and throTables 3 to 7 show the operating characteristics of top-performing combinations of terms with best sensitivity, best specificity, and best optimization of sensitivity and specificity while minimizing the difference between the two, for detecting studies on geriatric medicine, adult medicine, pediatric medicine, neonatal medicine, and obstetrics in Medline in 2000. Search strategies are reported using Ovid's search engine syntax for Medline .The single term \"exp adult\" yielded the best sensitivity (96.4%) with a specificity of 55.9% for retrieving articles about geriatric medicine. However, by using the next best sensitivity combination, \"aged.sh. OR age:.tw.\", a small sacrifice in sensitivity (1% absolute decrease) resulted in a much better specificity compared with the most sensitive term (absolute increase 14.4%) and improved precision (absolute increase 5.2%) and accuracy (absolute increase 13.3%). As expected, precision improved slightly when specificity was maximized (absolute increase 8.6%). The term that yielded the best optimization of sensitivity and specificity, \"aged.sh.\", resulted in 93.6% sensitivity and 82.7% specificity.The three-term strategy \"adult.mp. OR middle aged.sh. OR age:.tw.\" yielded the best sensitivity (94.9%) and had a specificity of 64.5% for retrieving articles about adult medicine. When specificity was maximized (85.2%) with the single term \"middle aged.sh.\", sensitivity lowered to 72.3%, but precision improved to 62.1% (absolute increase 14.8%) and accuracy improved as well (absolute increase 9.8%). The best optimization of sensitivity and specificity occurred with the combined terms \"middle aged.sh. OR of age.tw.\", with values approaching 79%.The three-term strategy \"child:.mp. OR adolescent.mp. OR infan:.mp.\" yielded the best sensitivity of 98.0% with a specificity of 81.2% for retrieving articles about pediatric medicine. When specificity was maximized (97.1%) with the single term \"children.tw.\", a striking trade-off in sensitivity occurred as it was lowered to 58.2% (absolute decrease 39.8%). Yet, as expected, precision improved (absolute increase 30.9%). The three-term strategy \"adolescent.tw. OR children.tw. OR child, preschool.sh.\" yielded the best optimization of sensitivity and specificity .Best sensitivity (95.3%) was achieved by the three-term strategy \"infan:.mp. OR child:.mp. OR gestation:.tw.\", with a specificity of 83.6% for retrieving articles about neonatal medicine. An expected trade-off occurred in sensitivity (absolute decrease 41.7%) with the most specific term, \"infants.tw.\" (98.7%). However, precision increased to 38.2% (absolute increase 30.8%) and accuracy reached 98.2%. The three-term strategy \"infan:.mp. OR gestation:.tw. OR neonatal.tw.\" yielded the best optimization of sensitivity and specificity, reaching values of 93% .The combination of terms \"gestation:.tw. OR fetal.tw. OR pregnancy.tw.\" produced the best sensitivity of 82.0%, with a very high specificity of 97.1% for retrieving articles about obstetrics. The maximization of specificity  with the single term \"gestation:.tw.\" yielded a 1.8% increase in specificity but with a marked trade-off in sensitivity, which decreased to 52.0% (absolute decrease 30%).Our study shows that selected age-specific search strategies can achieve high retrieval of studies for age-specific populations. Our age-specific search strategies performed differently among the five specialties we investigated. The highest sensitivity and specificity were achieved for pediatric medicine  and neonatal medicine . This finding may be a result of these age groups being more precisely defined and that studies tend to be narrowly focused on them. Search strategies within obstetrics yielded a higher specificity (97.1%) than sensitivity (82%), indicating that this strategy was better at filtering out nonrelevant age-specific articles than retrieving them. The best performing strategy for optimizing sensitivity and specificity was achieved within neonatal medicine . In all cases, precision was low, a consequence of searching in large multi-purpose databases. Future research is focusing on potential ways to improve precision without compromising sensitivity, for example, by searching in journal subsets.A possible limitation to our study is the generalizability of our findings to other publication years as our data was collected in the year 2000. We believe, however, that our search strategies are robust because no major changes have been made to age-specific MeSH terms since the year 2000. Moreover, we have previously shown that search strategies developed in 1990 were robust when searching in 2000 . AnotherThe utility of age-specific filters will vary according to the needs of clinicians and researchers who must weigh the consequences of using a sensitive or specific search. Although a sensitive search will not miss many relevant articles, such searches are less precise and entail time-consuming sorting through irrelevant articles. The narrower yield of a specific search will capture many relevant articles and take less weeding, but it has greater potential for missing key articles.To illustrate the use of age-specific search strategies, if a geriatrician was looking for information about current treatment strategies for Huntington disease, she might begin her search by entering the content term \"Huntington disease\" in Medline, which would yield 5907 articles .However, sifting through such a large number of articles would be time-consuming and many of these articles would not be relevant to treatment studies in geriatric medicine. By combining the content term \"Huntington disease\" with the most sensitive combination of terms for treatment studies , the search can be narrowed to 901 articles. Further, by adding the most sensitive strategy for geriatric medicine (exp adult) to this search string with the Boolean operator AND, the search is refined to 483 articles, which is much more manageable than the original 5907 articles retrieved from searching the content term only. A sensitive search such as this would be an efficient beginning for researchers interested in conducting systematic reviews.A more specific approach may be especially useful for physicians who do not have time to process an exhaustive search. In the above example, by combining the content word \"Huntington disease\" with the most specific search strategy for treatment studies , \"randomSelected age-specific search strategies can enhance the retrieval of studies for clinicians and researchers who need information relevant for a well-defined age-category patient population. The optimal trade-off between sensitivity and specificity should be determined according to the needs of the searcher."}
+{"text": "Plain language search tools for MEDLINE/PubMed are few. We wanted to develop a search tool that would allow anyone using a free-text, natural language query and without knowing specialized vocabularies that an expert searcher might use, to find relevant citations in MEDLINE/PubMed. This tool would translate a question into an efficient search.The accuracy and relevance of retrieved citations were compared to references cited in BMJ POEMs and CATs  questions from the University of Michigan Department of Pediatrics. askMEDLINE correctly matched the cited references 75.8% in POEMs and 89.2 % in CATs questions on first pass. When articles that were deemed to be relevant to the clinical questions were included, the overall efficiency in retrieving journal articles was 96.8% (POEMs) and 96.3% (CATs.)askMEDLINE might be a useful search tool for clinicians, researchers, and other information seekers interested in finding current evidence in MEDLINE/PubMed. The text-only format could be convenient for users with wireless handheld devices and those with low-bandwidth connections in remote locations. Other clinicians may just access resources that evidence-based practitioners might consider less evidence-based,  such as consulting a colleague, or reading a textbook, or perhaps, they may forego searching altogether.askMEDLINE is intended for the clinician, researcher, or the general public who want to simply ask a question and to skip the challenge of learning how to format it in manner that will make the searching MEDLINE/PubMed efficient. It is a tool that allows the user to search MEDLINE/PubMed using free-text, natural language query, just like one would in a clinical setting, or in a conversation. A user enters a clinical question on a Web browser, and then lets the tool retrieve relevant articles in MEDLINE/PubMed. Links are provided to journal abstracts, full-text articles and related items. Moreover, askMEDLINE is formatted for easy viewing on a wireless handheld device so it can be used while mobile, but will work equally well on a desktop computer.askMEDLINE, and an evaluation study on its potential to retrieve references, using published, evidence-based resources.We report our experience in developing askMEDLINE uses a multi-round search strategy. In the first round, the parser ignores punctuation marks and deletes words found on a \"stop-word\" list. The stop-word list includes PubMed stop words, and other words that we found by experience, to be detrimental to the search. The parser, a PHP script, then sends the modified query to PubMed Entrez' E-Utilities. The Extensible Markup Language (XML) file returned by E-Utilities indicates the category of each term in the query. Terms marked as \"All Fields\" denote that they are neither Medical Subject Headings (MeSH) terms nor MeSH Subheadings. These terms are checked to determine if they are found in a \"MeSH Backup vocabulary.\" The backup vocabulary includes words other than MeSH terms, such as MeSH descriptors, that are classified as \"other eligible entries\". If an \"All Fields\" word is in the backup vocabulary, it remains in the query; if it is not, it is deleted. The remaining terms are sent back to PubMed, again through E-Utilities. Human and English language limits are always applied. If the journal retrieval count after the first round is between 1 and 50,000, the first 20 results are displayed in the user's browser and the search process terminates. Further searches are dependent on the user.The search may proceed to Round 2 under two conditions: 1) If no journals are found in the first round, a result that could signify that the search was too narrow , the \"All Fields\" words are deleted from the query, even though they are found in the backup vocabulary. Only MeSH Terms and Subheadings remain (Round 2A.) 2) If the first round retrieval count is larger than 50,000 articles (an indication that the search was too broad) the \"All Fields\" words removed during the first round (words not found in the backup vocabulary) are put back into the query (Round 2B.) Round 2B searches contain all the MeSH terms (or MeSH Subheadings) and \"All Fields\" words in the original question. The updated query from either 2A or 2B is once again sent to Entrez E-Utilities. Retrieved journal articles are sent to the user.Similarly, if the count returned from second round is in the range of 1 to 50000, the search process terminates. If the second round count is still equal to 0 (denoting that the search is still too narrow) another list of \"No-Go Terms\", terms that when removed could result in a successful search is checked. Common MeSH abbreviations, acronyms and words like, \"method,\" \"affect,\" and \"lead\" are examples of terms on the list. New terms are continuously added to this list as they are encountered. The third round modified query is once again sent to E-Utilities and the retrieved journal articles are sent to the user. A result of 1 to 50000 citations terminates the process and displays the first 20 articles.askMEDLINE retrieves only one to four journal articles, a search is automatically done for related articles of the top two articles. All the articles (one to four previous) and the first 25 related articles of the first two are retrieved. As in any of the previous steps, the first 20 are displayed in the browser. In all the search retrieval pages, a link is provided for the user to manually intervene and modify the search process through the PICO interface. Links to related articles, full-text articles and abstracts are shown.If askMEDLINE was evaluated by comparing its accuracy to retrieve an article cited as a reference in a POEM (\"gold standard\".) [askMEDLINE, and for comparison, in Entrez, the integrated, text-based search and retrieval tool for PubMed. New critically appraised topics (CATs) from the University of Michigan, Department of Pediatrics Evidence-Based Pediatrics Web site were also used. [Since November 2002, the British Medical Journal (BMJ) has published a POEM (Patient-Oriented Evidence that Matters) in every issue.  POEMs arndard\".)  Every POso used.  Unlike BThe initial search result was examined to determine if the reference cited in a POEM or CAT was among those retrieved. Subsequent steps were taken if the reference article cited was not: 1) If the initial search retrieved journal citations, but not the specific journals cited in a POEM or CAT, the titles and abstracts were scanned to find out if they were relevant (deemed to answer the question.) If they were, related articles were retrieved, and again evaluated to determine if they matched the cited reference. 2) If no journal articles were retrieved, the question was rephrased, then searched again. Retrievals were again examined for the cited articles and relevancy to the clinical question. Overall efficiency was determined by the accuracy in retrieving a cited article and relevance of citations retrieved for citations that did not match cited references.A simple, handheld-friendly search interface was created where users can enter free-text, natural language searches Figure . The resaskMEDLINE found 62% of the cited articles in POEMs, while Entrez retrieved close to 14% (Table askMEDLINE (8.4% in Entrez.) When three questions were rephrased, askMEDLINE, but none in Entrez retrieved two of the specific cited references, although relevant references were found to one of the questions. For 20 questions, askMEDLINE did not find the specific cited reference, but it found journal citations that were deemed relevant and would be useful in answering the question. Entrez obtained citations for 16 (16.8%) questions that were considered relevant.Clinical questions in 95 POEMs and 28 CATs were searched. After first pass, askMEDLINE retrieved 72/95 exact matches of cited references (gold standard) in POEMs, an accuracy of 75.8%, while Entrez' accuracy was 22% (21/95.) If citations that are not the same as those cited in POEMs, but are relevant and considered satisfactory for answering the clinical question are included, askMEDLINE's total efficiency is 96.8%. Entrez' total efficiency for finding specific and relevant citations for BMJ POEMs is 38.9% (21 specific and 16 relevant citations found.)Overall, askMEDLINE, three searches did not find exact matches or relevant articles (3.1%), while Entrez' results were not relevant (6.3%) for six questions. No citations were found for 52  questions by Entrez.Although citations were retrieved for all POEM questions by askMEDLINE, while it added 7.1% to Entrez. Almost 7% of the searches retrieved relevant citations to rephrased or related articles, but none in Entrez. For CATs' questions, askMEDLINE found 89.2% of cited references, but 14.3% for Entrez. In 21/28 questions, Entrez did not provide a specific or relevant citation, but it was only for one question with askMEDLINE.University of Michigan's CATs yielded a similar total efficiency as POEMs, 96.3%, while it was 14.3% for Entrez Table . First paskMEDLINE is part of a project to develop easy-to-use resources at the point of care that has the functionality of an expert searcher. [earcher.  Special askMEDLINE.MEDLINE now contains more than 13 million citations from over 4,000 journals, with approximately 40,000 added monthly. The Internet also holds millions of Web pages that archive medically related information. The task of the user then is to find the information one needs, sift through the good, bad and dangerous, and after considering other factors  apply them to the management of a patient. This is the practice of evidence-based medicine. Providing decision support for evidence-based practice at the point of care is the goal of askMEDLINE- - Unified Medical Language System (UMLS), International Classification of Diseases: 9th revision (ICD9), but MeSH was selected because it is the controlled vocabulary used for indexing articles for MEDLINE/PubMed. MeSH terminology presents a reliable means of retrieving information that may use different terminologies for the same concept. The multi-round algorithm was developed through testing.Several special vocabularies were considered and tested for The difference in accuracy rates between the POEMs (75.8%) and CATs (89.2%) is most likely explained by the greater number of cited references in CATs. POEMs only had one, but CATs had one or more per question. We considered a single match to a reference is a positive count. The overall efficiency of finding a citation match in POEMs and CATs was consistent, around 96%. We are unable to account for the discordance with between POEMs (38.9%) and CATs (14.3%) questions.Some POEMs' questions did not return exact matches although retrievals may have been relevant, but when the questions were reformulated, positive matches were obtained. One example was the questions, \"Is low dose aspirin safe and effective for the prevention of thrombotic complications in patients with polycythaemia vera?\" which returned 51 relevant citations but no exact match. When it was rephrased to, \"Does low dose aspirin prevent thrombotic complications in polycythaemia vera?\" the second citation matched the cited POEMs reference. The question, \"Is a prolonged period of antithrombotic pretreatment effective for reducing adverse outcomes in patients with unstable coronary syndromes?\" returned close to 40,000 citations, but when rephrased to, \"Is prolonged antithrombotic treatment indicated in unstable coronary syndromes before intervention?\" the first citation was an exact match.There were questions that did not match despite attempts to rephrase them. One, \"Is a one day treatment of Helicobacter pylori as effective as a seven day regimen in patients with dyspepsia?\" The citations retrieved were all relevant, but none was the exact match because there were many treatment regimens variations. Some questions were quite easily modified to obtain exact matches, like, \"Does azithromycin given to patients with acute coronary syndromes prevent recurrent ischaemia?\". But by simply deleting the \"s\" in syndromes, the first of 25 relevant citations was a match. Other examples are shown in Table askMEDLINE also uses MeSH and omits variation that other search engines have that may impact search results as some have found. [Currently, there are not many free-text, natural query search engines that are specifically developed for MEDLINE/PubMed. Requiring clinicians to learn and master search methods for efficient searching is unrealistic.  It is eve found. askMEDLINE is a more modest tool and certainly not comparable to either of the two.Google is a very powerful search engine that searches the entire World Wide Web, including biomedical Web sites. There are ongoing discussions in biomedical lists on the merits of Google and PubMed. They search different databases and comparisons between the two are not valid. askMEDLINE based on two collections of evidence-based resources suggests that it may be a useful resource for clinicians, researchers and the general users interested in finding relevant medical information. Its text-only format will make it convenient for users of wireless handheld devices to use it in the clinical setting where wireless Internet access is available. It will also be helpful for those in with slow connections to the Internet especially those in remote locations in developing countries. Efforts to improve its functionality and clinical usefulness for evidence-based medicine are continuing.Evaluation of Project name: Development of evidence-based medicine toolsProject home page: Operating system: platform independentProgramming language: PHPOther requirements: Apache, MySQL, PHPLicense: Free, anyone may use the serviceAny restrictions to use by non-academics: None, anyone may use the serviceThe author(s) declare that they have no competing interestsPF conceived of the study and design, participated in the development of the search tool, carried out the evaluation studies and drafted the manuscript. FL participated in the design of the study and wrote the search script. MA participated in the study design, evaluation study and gave final approval of the version to be published. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "With the information explosion, the retrieval of the best clinical evidence from large, general purpose, bibliographic databases such as MEDLINE can be difficult. Both researchers conducting systematic reviews and clinicians faced with a patient care question are confronted with the daunting task of searching for the best medical literature in electronic databases. Many have advocated the use of search filters or \"hedges\" to assist with the searching process. The purpose of this report is to describe the design and methods of a study that set out to develop optimal search strategies for retrieving sound clinical studies of health disorders in large electronics databases.To describe the design and methods of a study that set out to develop optimal search strategies for retrieving sound clinical studies of health disorders in large electronic databases.An analytic survey comparing hand searches of 170 journals in the year 2000 with retrievals from MEDLINE, EMBASE, CINAHL, and PsycINFO for candidate search terms and combinations. The sensitivity, specificity, precision, and accuracy of unique search terms and combinations of search terms were calculated.A study design modeled after a diagnostic testing procedure with a gold standard (the hand search of the literature) and a test (the search terms) is an effective way of developing, testing, and validating search strategies for use in large electronic databases. The Clinical Hedges Study was designed with the objective of developing optimal search strategies to improve the retrieval of clinically relevant and scientifically sound study reports from large, general purpose, biomedical research bibliographic databases including MEDLINE, EMBASE, CINAHL, and PsycINFO. The search strategies were developed to 1) assist health care providers to do their own searches; 2) help reviewers of published evidence concerning health care interventions retrieve all relevant citations; 3) provide resources for librarians to help health care providers construct their own searches; and 4) provide input to the database producers about their indexing processes and the organization of their databases.Data for the Clinical Hedges Study was collected throughout the year 2000 and continued into the year 2001. Database construction and analyses are ongoing with some of our pre-study results  appearing in conference proceedings as early as 2001  but the The methods and design of the Clinical Hedges Study are outlined in this paper and are illustrated in the Figure. The study design used to address the above mentioned objective was an analytic survey comparing hand searches of 170 journals in the year 2000 with retrievals from MEDLINE (161 of the 170 were indexed in MEDLINE in the year 2000), EMBASE (135 were indexed in EMBASE), CINAHL (75 were indexed in CINAHL), and PsycINFO (64 were indexed in PsycINFO) through the Ovid web gateway for candidate search terms and combinations. Candidate search terms and combinations were tested in the four electronic databases by treating the search terms as \"diagnostic tests\" for sound studies. Due to the size of these four electronic databases, it is not feasible to determine the total number of relevant citations in each database for a given search. The hand search of the literature for the 170 journals circumvents this problem as it provides a \"gold standard\" for a segment of the literature in the electronic database file, and an approximation for the operating characteristics of search strategies .There are two primary sources of data in the Clinical Hedges Study. The first source of data was generated from a hand search of the literature (the \"gold standard\"). The second source of data was generated from downloads of search terms and citation information from the four electronic databases (the \"test\"). After these two data sources were obtained, we created a database that contained the matched merged content from these sources. This merged database was used for the development and validation of search strategies. In the following sections we outline this process.Annals of Internal Medicine), general medical practice , mental health , and general nursing practice  .To generate the hand search data six research assistants reviewed 170 journals titles for the year 2000. The 170 journal titles reviewed were chosen over several years in an iterative process based on hand search review of over 400 journals recommended by clinicians and librarians, Science Citation Index Impact Factors provided by the Institute for Scientific Information, recommendations by editors and publishers, and ongoing assessment of their yield of studies and reviews of scientific merit and clinical relevance. These journals include content for the disciplines of internal medicine . Research staff were rigorously calibrated before the hand search of the 170 journal titles and inter-rater agreement for identifying the purpose of articles was 81% beyond chance . Inter-rater agreement for which articles met all scientific criteria was 89% (CI 0.78 to 0.99) beyond chance [Each item  in each issue of the 170 journals for the year 2000 was classified for article format Table , age of Hand search data were recorded on a paper based data collection form that was compatible with an optical mark and character recognition system called Teleform . The data collection form was designed using Teleform Designer. On each form data entry fields were available for the journal name, volume, issue and publication date, as well as a tabular data collection area to record the classification of individual items in the journal . Multiple pages of the form could apply to one issue of a journal. Alphanumeric data fields were encoded in Teleform \"comb\" fields, where each hand printed block character had to be drawn within a fixed assigned space. A variety of strategies were used to maximize the reliability of the optical character/mark recognition abilities of Teleform. As many of the data fields as possible were either multiple choice or numeric. For fields containing text, research staff were trained in optimal letter formation and were required to use designated pre-tested pens. A mechanism was also established for correcting data entry errors.All data collection forms were scanned with a Hewlett Packard 610cxi Scanjet scanner using Teleform Reader and the interpretation of the form was verified using Teleform Verifier. Scripts were written in a limited form of Visual Basic for Applications to perform basic validation on the incoming data while being interpreted by Teleform Reader. Data entry staff performed a data verification step by comparing the scanned image of the form alongside the Teleform interpretation. The staff were given the opportunity to accept the interpretation or to correct it. The original data collection sheets and/or the researcher who classified the material may have been consulted to make corrections. Tests to determine data entry error rates were conducted and an overall error rate of 0.01% was found.After data entry and data verification using Teleform, hand search data were exported to a Microsoft (MS) Access database. The hand search data was split into two MS Access tables, one containing journal information and the other containing article information. The two tables were linked on a key field.The hand search database volume was extensive with 11 data fields recorded for a collection of 60,330 articles in 170 publications.The data acquired from the on-line database were matching information , and the results of executing search terms in the on-line database. To generate the second source of data, the on-line data, it was necessary to construct a comprehensive set of search terms. We began a list of index terms and textwords for each of the four electronic databases, MEDLINE, EMBASE, CINAHL, and PsycINFO, and then sought input from clinicians and librarians in the United States and Canada through interviews of known searchers, requests at meetings and conferences, and requests to the National Library of Medicine. Individuals were asked what terms or phrases they used when searching for studies of causation, prognosis, diagnosis, treatment, economics, clinical prediction guides, reviews, costs, and of a qualitative nature when using these databases. For instance, for MEDLINE, terms could be from Medical Subject Headings (MeSH), including publication types (pt), and subheadings (sh), or could be textwords (tw) denoting methodology in titles and abstracts of articles. We compiled a list of 5,345 terms for MEDLINE of which 4,862 were unique and 3,870 returned results (list of terms tested provided by the authors upon request). For EMBASE we compiled a list of 5,385 terms of which 4,843 were unique and 3,524 returned results (list of terms tested provided by the authors upon request). For CINAHL we compiled a list of 5,020 unique search terms of which 3,110 returned results (list of terms tested provided by the authors upon request). For PsycINFO we compiled a list of 4,985 unique search terms of which 2,583 returned results (list of terms tested provided by the authors upon request). Index terms varied by electronic database whereas the same list of textwords were tested in each of the electronic databases.Since the primary goal of the Clinical Hedges Study was to deliver search strategies which could be used by clinicians and researchers to locate the best quality published research specific to their interests it was required that the data be gathered via the same user interface that end users would use. Thus, raw performance data for individual search terms were downloaded via Ovid. Because of the volume of terms and their combinations, we automated the submission of terms using a telnet connection.Ovid provides a simulated Graphical User Interface (GUI) through telnet using a simulated Dec VT-100 terminal interface. To handle a series of exchanges an open loop automation scheme was used. This consisted of a script-reading program written in Visual Basic for Applications, which passed keystroke data that mimicked what a user would enter into a commercially available telnet program. The script reader retrieved specific details from internally derived reference tables such as journal names  and search terms  and inserted this information into the script through a parameter substitution scheme. To retrieve search term data for the Clinical Hedges Study, we \"ANDed\" each search term with a strategy saved on Ovid, which comprised our reading list of 170 journals published in the year 2000.We used MS Outlook to recover the requested data via e-mail. Filters and dedicated \"pst\" files were set up to handle e-mail retrieval from a research project e-mail account. An \"autosave\" program saved the e-mail messages in individual text files for automated processing. A \"downloads\" program read the saved text files and entered the data into the appropriate MS Access tables. These scripts were stored in an MS Access database along with the program, and were organized in tables where keystroke sequences formed individual commands, which could be timed. The sequences of commands were grouped according to their function in the process of connecting to the on-line service (Ovid) or gathering data from it.The approach to matching the hand search data records to the on-line data was undertaken in a two-stage process. First, a minimal set of information was retrieved from the on-line source, organized by journal title, which was used to link hand search data. At this stage, the linking software only attempted to match the hand-coded journal, volume, and issue information from the hand search data to a similarly hand-coded field retrieved from the on-line source. Later, a more complete set of matching information was retrieved from the on-line source, including a unique identifier for the index journals, article titles, authors, abstracts, indexing terms, and publication types. These data were organized by journal and index.The matching algorithm was initially conservative requiring 100% certainty to establish a match. An \"unmatched\" report was subsequently generated which was used to refine the algorithm. For each of the four electronic databases approximately 95% of the records could be matched through an automated process. The remaining unmatched records were processed manually.It should be noted here that there was not a one-to-one relationship between individual items entered in the hand search database and items recovered from the on-line database because an individual article could be determined to serve more than one purpose in the context of the clinical HEDGES study. Thus, an article could be a \"review\" (one format) about \"diagnosis\" and \"therapy\", which were additional purposes.After extensive attempts, a small fraction of the hand-search items failed to be matched to citations in each of the four electronic databases and a small number of citations downloaded from each of the four electronic databases failed to be match to the hand-search data. As a conservative approach, unmatched citations that were detected by a given search strategy were included in cell 'b' of the analysis table Table  leading After the hand search and on-line data were matched the merged data file was prepared for deriving computations. At this point, other than the unique identifiers from both data sources (hand search and on-line data), none of the other matching information  was relevant to the computations. This extraneous data was, therefore, removed from the tables that were used to compute the performance of the search terms, as this information was already stored in a journal table within MS Access.We determined the sensitivity, specificity, precision, and accuracy of each single term and combinations of terms using an automated process. The formulae for calculating these statistics are shown in Table Once the performance parameters of individual search terms were computed, it was possible to select individual terms for the construction of search strategies. In the Clinical Hedges Study, we had a collection of over 4,800 unique search terms for MEDLINE and EMBASE, with up to 1,200 of them returning results for a particular purpose category . A rather simple estimation of computation time required, based on single term calculations and some preliminary two-term calculation runs, indicated that the time to compute search strategies using all of the available terms and an arbitrary limit on the number of terms in combinations, could amount to literally hundreds of years of computing time with equipment available at the time. Many of the combinations of search terms could be predicted to perform poorly as they contained individual terms that had poor performance in terms of returning very few true positives, or returning far too many false positives  there were no statistical differences in performance. Thus, for subsequent purpose categories and databases, the Boolean approach was used for search strategy development and search strategies were developed using all records in the database.Search strategies developed for use in MEDLINE have been translated for use in PubMed by staff of the National Library of Medicine, and compared for performance by the senior author (RBH).A study design modeled after a diagnostic testing procedure with a gold standard (the hand search of the literature) and a test (the search terms) is an effective way of developing, testing, and validating search strategies for use in large electronic databases.Additional research is underway in search strategy development including testing the strategies developed through this research, when combined with disease content terms, and when combined with terms using the Boolean \"AND\" and/or \"NOT\".The author(s) declare that they have no competing interests.NLW and RBH prepared grant submissions in relation to this project and supplied intellectual content to the collection and analysis of the data. NLW participated in the data collection and all authors were involved in data analysis. All authors drafted, commented on and approved the final manuscript.Includes Angela Eady, Brian Haynes, Chris Cotoi, Susan Marks, Ann McKibbon, Doug Morgan, Cindy Walker-Dilks, Stephen Walter, Stephen Werre, Nancy Wilczynski, and Sharon Wong.The pre-publication history for this paper can be accessed here:"}
+{"text": "The past few years have seen a proliferation of search engines for the World Wide Web (WWW), as well as a growing number of specialized subject directories geared to the needs of health care professionals. Yet documentation on scope, coverage, and search features is often uneven at best; and even documented search features may not perform as advertised. This paper will present a group of sample searches to assist users in gauging database size, determining default search operators, and testing for the presence of advanced search features such as case sensitivity, stemming, and concept mapping for medical topics on English-language web sites. Northern Light, HotBot, and AltaVista, undergo constant overhauls. Search engine software changes so frequently that help screens, if they exist at all, are often inadequate. At worst, the help screens may even refer to a previous version of the software. Often, even a system with accurate and up-to-date documentation will present it in a Frequently Asked Question and answer format (FAQ), making it difficult to locate specific facts and features of a search engine.The software used by World Wide Web (WWW) search engines continues to evolve so rapidly that keeping abreast of search features is a never-ending task. Search engines, such as To further add to the confusion, sometimes search engines do not perform as advertised. In other cases, search engines seem to be deliberately vague about the inner workings of their searching software or ranking algorithms because they consider that information to be proprietary.Understanding how an engine combines search terms is essential to effective searching. Many of the search engines, especially in basic or novice mode, employ fuzzy logic, where all search terms are linked with a Boolean OR. This is sometimes referred to as \"Match any of the terms.\" This accounts for the large number of results returned from some search engines.Excite retrieved 129,711 pages. Then add a second word to the search: searching \"ear infections\" on Excite yielded 183,650 pages. Since the second search retrieved more than the first search, the default operator on Excite is OR.It is possible to determine the default operator by performing a few simple searches. First, enter a single search term and record the retrieval: a recent search for the word \"ear\" on If the second search retrieves a smaller number of results than the first one, the default operator may be AND. Searching \"ear\" on Northern Light retrieved 959,152 pages, while \"ear infections\" retrieved 55,560. The smaller results indicates that the default operator may be AND; however, it could also be doing an even narrower search, retrieving only pages with the exact phrase \"ear infections.\" To determine whether the default operator is AND or ADJacency, do a third search with the two terms reversed: \"infections ear.\" If the retrieval is the same as in the second search, as it is for Northern Light, the default operator is almost sure to be AND. If the search result is different, the default is probably an adjacency operator, or an exact phrase search.Stopwords, or noise words, can also be problematic in searching. Some search engines index even the smallest words, including \"a\" and \"the.\" Others have a list of stopwords that are not indexed; these lists are often unpublished. One way to test for the presence of stopwords is to do searches for \"vitamin a deficiency\" and \"\"vitamin k deficiency\" and compare retrieval. In Excite, both of these searches retrieve 96,794 items, indicating that single letters are indeed stopwords. Another test for stopwords is to enter the search term alone; and indeed a search for \"a\" in Excite returns no results.One area where the search engines seem especially prone to hyperbole is in their claims to database size. Here are some claims that have appeared on search engine web pages, either now or in the past:\"Excite Search, the Internet's most comprehensive search tool...\"\"AltaVista gives you access to the largest Web index...\"From Hotbot: \"...the largest and most complete index of Internet documents in the world.\"More than one search engine boasts that its database is the largest and most complete on the WWW. Even when actual numbers on database size are provided, they can be misleading and difficult to compare. One search engine may claim that its database has the most URLs; but this number may be artificially inflated if the database contains many duplicates. And how many of these URLs represent pages which no longer exist? Another search engine may base its claim to be the largest on the size of its database in terabytes . But thiOne word of warning: determining the number of hits retrieved on a web search is not always easily done. In Excite, one must scroll down the page to reveal the number of hits. When searching a highly posted term in HotBot, the number of hits doesn't appear on the first page of results, only on subsequent pages. Sometime in 1998, Lycos removed the number of hits retrieved entirely from their screens, leaving no way to assess relative size using benchmark searches like these. It is also revealing to compare retrieval for medical terms in some of the larger medical directories with search engine results. Medical World Search, with its database of \"nearly 100,000\" pages , is onlyOften the presence of capital letters, or a combination of upper and lower case letters, conveys a specific meaning for a health sciences term. When searching for information on \"AIDS,\" as in Acquired Immunodeficiency Syndrome, a searcher does not want to also retrieve information on hearing \"aids.\" Typically if a search engine recognizes case, it will retrieve both upper and lower case in response to a lower case query (e.g. aids or AIDS), but only upper case if the query is entered that way (e.g. only AIDS). To test for case sensitivity, search for the same word twice: once in upper and once in lower case, and compare the results. If the same number of items is retrieved on both searches, the search engine is not case sensitive.Performing one additional search will test for the ability to search for terms which contain only a special combination of capital and small letters; this is sometimes referred to as \"interesting case.\" An example of interesting case from the medical domain would be MeSH, referring to the Medical Subject Headings published by the National Library of Medicine. In HotBot, a search for \"mesh\" retrieves 175,950 items; \"MESH\" retrieves 7180; but \"MeSH\" retrieves 5480.With most search engines, what you type is what you get; nothing more, and nothing less. The engine does a literal search for exactly what is entered. There are two possible exceptions to this: stemming and concept searching.A search engine which uses stemming will automatically retrieve some words with variant endings. In its simplest form, this operates as automatic right truncation, where a search for \"germ\" also retrieves \"germs,\" \"germinate,\" and even \"Germany.\" Yahoo uses this type of stemming. Other search engines stem more selectively, perhaps where searching a singular word also retrieves the plural form; e.g. searching \"child\" retrieves \"children,\" but not \"childhood.\" To test for the first type of stemming via automatic right truncation, search on a word stem such as \"occlu\" to see if \"occlusion,\" \"occluded,\" etc. are retrieved. The second type of stemming is more difficult to evaluate. Search for a simple plural with and without the \"s,\" then perform the search using both terms linked with OR: first search \"kidney,\" second \"kidneys,\" then \"kidney OR kidneys.\" If all three searches return the same number of hits, simple stemming of singular and plural word forms is in operation. To test for more sophisticated stemming, try an irregular plural: woman vs. women, child vs. children, person vs. people. If results are the same, the stemming is more sophisticated.Some of the search engines, notably Excite and Magellan, claim to be able to conduct concept searches. The user types in a single word, and the search engine purports to search not only that specific word, but also to automatically include synonyms in the search. Unfortunately, this feature is not always optimized for medical terms. One way to tell is by searching on a word such as \"kidney,\" recording the result, and then searching a medical synonym such as \"renal,\" recording that result, and then pooling the two by searching \"kidney OR renal.\" If the last search retrieves many more items than either the first or the second search, one can surmise that concept mapping is weak or perhaps nonexistent in the area of medical vocabulary. Excite searches for documents containing the exact words that you enter into the Search box. But that's not all. Excite takes search technology one step further: Not just words, Excite also searches for ideas closely related to the words in your query.For example, suppose you search on the terms \"elderly people financial concerns.\" In addition to finding sites containing those exact words, the search engine will find sites mentioning the economic status of retired people and the financial concerns of senior citizens .One interesting footnote: Excite and Magellan use almost exactly the same wording and examples when explaining their concept search feature, although the results of these sample search illustrate that the two engines perform quite differently. The only way to account for this, although it doesn't really explain it, is that Excite now owns Magellan, even though the latter is still run as a separate search service with its own look, feel, and capabilities.There is one specialized search engine targeted to a medical audience with relatively sophisticated concept mapping capabilities: Medical World Search (http://www.mwsearch.com). A search of its 100,000 item database of major medical sites retrieves 762 items regardless of whether \"acetaminophen\" or \"tylenol\" is searched, since queries are enhanced with terms from the National Library of Medicine's Unified Medical Language System Metathesaurus . Indeed,Two words of caution apply when applying these benchmark searches. First, they are simply heuristics for determining search engine behavior, and will not provide definitive evidence of the presence or absence of search features in all situations. Second, if these benchmark searches are run during a database update, results may differ by only one or two hits. For example, one evening, when testing AltaVista for case sensitivity, \"aids or AIDS\" retrieved only two more hits than a search for \"aids\" alone had only 5 minutes before. It turned out that these represented two new URLs just added to the database. This was confirmed by re-executing the original search for \"aids\" alone, which then retrieved two more items than it had just minutes before.These same techniques can be used to evaluate the search capabilities of the free MEDLINE sites on the Web . For exaThese benchmark searches evolved partly as an byproduct of the Nothing But 'Net website , an inte"}
+{"text": "Searching the Web for documents using information retrieval systems plays an important part in clinicians\u2019 practice of evidence-based medicine. While much research focuses on the design of methods to retrieve documents, there has been little examination of the way different search engine capabilities influence clinician search behaviors.Previous studies have shown that use of task-based search engines allows for faster searches with no loss of decision accuracy compared with resource-based engines. We hypothesized that changes in search behaviors may explain these differences.In all, 75 clinicians  were randomized to use either a resource-based or a task-based version of a clinical information retrieval system to answer questions about 8 clinical scenarios in a controlled setting in a university computer laboratory. Clinicians using the resource-based system could select 1 of 6 resources, such as PubMed; clinicians using the task-based system could select 1 of 6 clinical tasks, such as diagnosis. Clinicians in both systems could reformulate search queries. System logs unobtrusively capturing clinicians\u2019 interactions with the systems were coded and analyzed for clinicians\u2019 search actions and query reformulation strategies. The most frequent search action of clinicians using the resource-based system was to explore a new resource with the same query, that is, these clinicians exhibited a \u201cbreadth-first\u201d search behaviour. Of 1398 search actions, clinicians using the resource-based system conducted 401  in this way. In contrast, the majority of clinicians using the task-based system exhibited a \u201cdepth-first\u201d search behavior in which they reformulated query keywords while keeping to the same task profiles. Of 585 search actions conducted by clinicians using the task-based system, 379  were conducted in this way.This study provides evidence that different search engine designs are associated with different user search behaviors. Searching for information on the Web to support decision making is now an important part of clinician practice . While mRecent studies of clinical search strategies have concentrated on methods of optimizing queries sent to information retrieval systems that enhance the performance of the retrieval. Hoogendam and colleagues conducted a prospective observational study of how physicians at a hospital used PubMed to search for information during their daily clinical activities . They foFew studies have looked at how clinicians reformulate queries and select sources to retrieve information during a search session to answer clinical questions. In previous studies, we have shown that a task-based search engine design allows for faster clinical decision making  compared with purely resource-based engines at no cost in correctness of answers . SimilarIn all, 75 clinicians  practicing in the state of New South Wales, Australia, were recruited to use an online information retrieval system to answer questions on 8 clinical scenarios within 80 minutes in a controlled setting in a university computer laboratory  9]. Par. Par9]. Participants were randomly allocated to use either a resource-based or a task-based version of an online information retrieval system to answer the 8 questions. All participants were given a brief written orientation tutorial regarding their allocated system. Questions were presented in random order. Each participant was asked to use the allocated system to locate documentary evidence to help answer each question. Participants were asked to work through the questions as they would in a real clinical setting and not spend more than 10 minutes on any one question.The search systems used by participants were essentially identical in that both systems allowed users to first select a profile  to delimit their search and then to enter keywords to specify the focus of their search. The resource-based system first required clinicians to select a profile by specifying one of six online resources. These included PubMed, MIMS , Therapeutic Guidelines , the Merck Manual, Harrison\u2019s Principles of Internal Medicine, and HealthInsite . Of the six resources, five presented evidence in a predigested, summarized form with references available for follow-up.The task-based system first required the clinicians to select a profile by selecting one of six clinical tasks: diagnosis, drug information, etiology, patient education, treatment, and other . Four keSystem logs unobtrusively capturing participants\u2019 interactions with the systems were coded and analyzed for their search actions and query reformulation strategies. For each clinical scenario question, participants were able to reformulate queries and conduct a sequence of searches as they explored information to assist in answering the question. We first coded these query reformulations by the change in profile selection (task or resource) between consecutive searches in a session as \u201cnew profile,\u201d \u201csame profile,\u201d or \u201cpreviously used profile.\u201d We next coded the keyword changes, as indicating a syntactic and/or a semantic reformulation . ExampleChi-square analyses and the test for difference between proportions were conducted to detect statistically significant differences in profile and query search actions between clinicians using the resource-based and task-based systems.Of 75 clinicians, 39 were randomly allocated to use of the resource-based system and 36 to use of the task-based system. Two resource-based scenarios were not completed, giving a total of 310  search sessions, 1708 searches, and 1455 document accesses using the resource-based system. The task-based system generated 288  search sessions, 873 searches, and 1136 document accesses.2                    2 = 103.45, P < .001)    , (2) ref < .001) , and (3) < .001) .P < .001), and 5.9% more likely to select a profile that was previously visited and apply no changes to keywords  (P < .001), 7.5% to keep the same profile and apply both syntactic and semantic changes to the query , and 6.5% to keep the same profile and apply semantic changes to the query   . Also, c < .001) . Further < .001) .P < .001) (P < .001) (P < .001) (P = .006)  , and cli < .001) . At the  < .001) , and cli = .006) .P < .001) (P < .001) (                     < .001) . Among c < .001) .Clinicians using the resource-based system appeared to favor a \u201cbreadth-first\u201d search strategy, exploring different resources with the same keywords in the query before searching in a specific resource with query reformulations. Clinicians using the task-based system were provided with results from multiple resources in each search and so appeared to favor a \u201cdepth-first\u201d search strategy, searching in the same task profile exhaustively with different keyword reformulations in the query before moving to other profiles.We have previously shown that changes in search engine design and interface were associated with changes in clinical decision velocity, number of search actions undertaken, and ultimate decision outcome . To undeFurther study is needed to understand how clinicians assess the results of a search and formulate the next step in their strategy. We have discussed elsewhere that the process of searching can be thought of as a conversation  where inAccording to Grice\u2019s conversational maxims , (which One can hypothesize, when clinicians are faced with a choice of several resources with no clear indication of which is the best, they scan multiple resources to gauge the \"competence\" of each before committing to a detailed conversation with the resource they feel best qualified to help. In contrast, clinicians with a task-based system are simultaneously receiving answers from multiple resources and so should be able to quickly form a view of the overall capabilities of the group of resources being simultaneously searched. Not faced with concerns about the competence of the system they are interacting with, clinicians focus on improving the dialogue with the system. This is done by finding different ways to ask the same question or by changing the question focus if there has been a \u201cmisunderstanding.\u201d As a result, this could explain why users of task-based systems conduct fewer searches and consult fewer documents , that isOverall, given the clear differences in the styles of user-system dialogue demonstrated in this study, and the impact of such behavior on the clinical utility of information retrieval systems, discovering ways of optimizing the dialogue between knowledge sources and users seems a productive line of further enquiry."}
+{"text": "The World Wide Web has increasingly become an important source of information in health care consumer decision making. However, little is known about whether searching online resources actually improves consumers\u2019 understanding of health issues.The aim was to study whether searching on the World Wide Web improves consumers\u2019 accuracy in answering health questions and whether consumers\u2019 understanding of health issues is subject to further change under social feedback.This was a pre/post prospective online study. A convenience sample of 227 undergraduate students was recruited from the population of the University of New South Wales. Subjects used a search engine that retrieved online documents from PubMed, MedlinePlus, and HealthInsite and answered a set of six questions (before and after use of the search engine) designed for health care consumers. They were then presented with feedback consisting of a summary of the post-search answers provided by previous subjects for the same questions and were asked to answer the questions again.P <.001) and after feedback with other subjects\u2019 answers .The proportion of subjects with highly confident correct answers  and the proportion with highly confident incorrect answers significantly increased after searching . Subjects who were not as confident in their post-search answers were 28.5% more likely than those who were confident or very confident to change their answer after feedback with other subjects\u2019 post-search answers .There was an improvement in the percentage of correct answers after searching (pre-search 61.2% vs post-search 82.0%, Searching across quality health information sources on the Web can improve consumers\u2019 accuracy in answering health questions. However, a consumer\u2019s confidence in an answer is not a good indicator of the answer being correct. Consumers who are not confident in their answers after searching are more likely to be influenced to change their views when provided with feedback from other consumers. The World Wide Web is now recognized as an important source of information in supporting the practice of evidence-based medicine  and consMany studies have examined the quality of online health care consumer information , the tooStudies have also shown that people are an important source of influence among consumers with a health-related concern. In a randomized controlled trial conducted by Lorig et al, patients with back pain who had access to an email discussion group demonstrated greater improvement in pain and made less physician visits than those without access . PatientLittle, however, is known about whether consumers are actually able to improve their understanding of health issues after searching the Web. In addition, little is known about the extent to which social feedback affects the way consumers develop their understanding of health issues. This prospective experiment tests the following hypotheses: (1) consumers can improve their accuracy in answering health care questions after searching tested online resources, and (2) consumers\u2019answers to health care questions are influenced by feedback with other consumers\u2019 answers.A convenience sample of 227 undergraduate students was recruited from the University of New South Wales (UNSW). Subjects were asked to use a specific online search engine to answer six consumer health questions. People with Internet access who had previously used an online search engine were recruited by announcements via student email lists, posters, leaflets, weekly student magazines, and a UNSW research news website. Upon completion of the study, subjects were entered into a draw for one of 100 movie tickets. Ethics approval was obtained from the Human Research Ethics Advisory Panel at UNSW.A pre/post protocol was used in this study. Subjects recorded their pre- and post-search answers to each question and their confidence in these answers. After answering each question post-search, subjects were presented with a summary of the post-search answers provided by previous subjects and were asked to answer the question again .                    Each question and the expected correct answer are shown in The search engine retrieved documents from tested resources known to have high relevance in answering health-related questions . These rSubjects\u2019 searches and their selected documents, pre-/post-search answers and confidence, post-feedback responses, time taken from answering the question pre-search to answering post-search, and responses to the pre-search and post-search questionnaire were logged during the experiment. Responses to questions were coded as \u201ccorrect,\u201d \u201cdon\u2019t know,\u201d or \u201cincorrect\u201d according to the predetermined answers for each question. All cases in which subjects did not conduct a search before providing an answer or seeking the social feedback, did not answer the question post-search, or answered \u201cdon\u2019t know\u201d post-search were removed from the data analysis.The test for difference between proportions was used to compare differences between subjects\u2019 pre-search, post-search, and post-feedback answers and to compare changes in confidence in answers pre- and post-search. The chi-square test was used to examine whether there was a statistically significant relationship between subjects\u2019 confidence in their post-search answers and their tendency to change answers after feedback with other subjects\u2019 answers. The McNemar test was used to examine the direction of change in pre- and post-feedback answers.After data exclusion , the stu                    As shown in z= \u22121.21, P <.001). There was also a marginal significant improvement in the percentage of correct answers before and after feedback with other subjects\u2019 answers  in the percentage of correct answers before and after searching, the proportion of subjects with highly confident incorrect answers also increased after searching .More than half of subjects  who did not know the answer pre-search and answered incorrectly post-search (DW) reported that they were confident or very confident with their incorrect post-search answer . In fact\u03c7                    2                    1= 66.65, P <.001; \u03c7                    2                    1= 15.25, P <.001; Those who were not as confident in their post-search answers were 28.5% more likely than those who had higher levels of confidence to change their answer after feedback with other subjects\u2019 post-search answers  [Results of this study for nonclinically trained users are in line with studies that reported search engines can improve the ability of clinically trained users to answer questions ,27,28. TP< .001) .Findings from this research and previous studies have shown that confidence is not always a good indicator of decision accuracy ,29. The Our findings on the impact of social feedback also concur with studies that report people are one of the important sources of information that influence clinicians\u2019 and health care consumers\u2019 actions when confronted with a clinical or health-related concern ,20,33-36"}
+{"text": "Oncorhynchus keta  in Howe Sound, British Columbia. We found a clear gradient in the proportion of terrestrially derived carbon along the tidal gradient ranging from 68% across all invertebrate taxa in the supralittoral to 25% in the high-intertidal, 20% in the mid-intertidal, and 12% in the low-intertidal. Stable isotope values of chum salmon fry indicated carbon contributions from both terrestrial and marine sources, with terrestrially derived carbon ranging from 12.8 to 61.5% in the muscle tissue of chum salmon fry (mean 30%). Our results provide evidence for reciprocal subsidies of marine and terrestrially derived carbon on beaches in the estuary and suggest that the vegetated supralittoral is an important trophic link in supplying terrestrial carbon to nearshore food webs.Stable isotope analysis was used to determine the relative proportions of terrestrial and marine subsidies of carbon to invertebrates along a tidal gradient  and to determine the relative importance of terrestrial carbon in food web pathways leading to chum salmon fry  Subsidies of prey and detritus across ecotones have been shown to affect food webs in both aquatic and terrestrial habitats Supralittoral vegetation in coastal areas may play similar roles in ecosystem functioning as riparian vegetation in freshwater systems 15N signatures of riparian vegetation in 27 watersheds in British Columbia was positively related to total the biomass of spawning chum and pink salmon Marine sources of carbon and nitrogen have also been shown to subsidize terrestrial food webs On coastal beaches, beach wrack is an important food source and habitat that subsidizes both marine and terrestrial food webs. For example, Lewis et al. 15N/14N is positively correlated with trophic level, and the ratio of carbon stable isotopes 13C/12C yields information about the production base of the food web 13C/12C ratio of approximately \u221228\u2030 13C (\u221250\u2030 to \u221210\u2030) relative to terrestrial plants, reflecting site-specific and species-specific factors Stable isotope analysis (SIA) has been used extensively to describe aquatic food webs Oncorhynchus keta , which reside in the estuary from March to June during their transition to the marine environment.In this study we report the results of stable isotope analysis of carbon and nitrogen for a collection of marine, intertidal, and terrestrial organisms collected in the intertidal and supralittoral in Howe Sound, British Columbia, Canada. Our objective was to determine the proportion of terrestrially derived carbon (TC) and marine derived carbon (MC) along the intertidal to supralittoral gradient focusing specifically on the pathways of energy flow to chum salmon fry, Howe Sound is a fjord located on the southeastern shore of the Strait of Georgia, British Columbia, Canada . The Sou13C changed along the terrestrial to marine gradient. Three Orders were sampled in more than one zone: Diptera (primarily Chironomidae) were sampled in the supralittoral (adult), high-intertidal (adult), mid-intertidal (adult), and low-intertidal (larvae) zones; Acariformes were collected from the supralittoral, high- and mid-intertidal zone; Amphipoda  were collected from the high-, mid-, and low-intertidal zones. Gastropods and Mytilus sp. were collected in the mid-intertidal.Ten species of live terrestrial supralittoral plants and six species of live macroalgae were collected by hand at Furry Creek Hyale plumulosa, was identified to species. Invertebrates were washed, frozen, and stored and later combined into composite samples of at least 0.2 mg dry weight . Pooling samples was necessary due to the small size/biomass of most of the invertebrates. When pooled samples were used, variance is reported as the variance across pooled samples.We used a variety of collection methods including epibenthic sleds in the low-intertidal zone and hand vacuums in the supralittoral, high-intertidal, and mid-intertidal zones. Taxa were identified to lowest taxonomic level possible while retaining enough material for stable isotope analysis. One species of amphipod, Chum salmon fry typically migrate downstream to estuaries and nearshore marine habitats where they spend up to three weeks before making the transition to pelagic oceanic conditions Chum salmon fry were kept in plastic bags in a cooler in the field and immediately frozen in the laboratory at \u221220\u00b0C. Fork length and wet weight were measured for 163 individual chum salmon fry and stomachs were removed from 28 fish for gut content analysis. Flank muscle tissue was then removed from 163 fish for stable isotope analysis. Fish samples for stable isotope analysis consisted of 1, 2 or 3 individuals. In total, stable isotope analysis was performed on 44 fish samples composed of 163 individual chum salmon fry. We have previously reported that there is no statistically significant difference in isotope values for fish samples composed of either individual fish or combined samples 2 and N2 were analyzed with a continuous flow-isotope ratio mass spectrometer. Ratios of carbon (13C/12C) and nitrogen (15N/14N) were expressed as the relative per mil (\u2030) difference between the sample and conventional standards (Pee Dee Belemite carbonate and N2 in air) as follows: \u0394X\u200a=\u200a[Rsample/Rstandard\u22121]\u00d71000(\u2030), where X\u200a=\u200a13C or 15N, and R\u200a=\u200a13C:12C or 15N:14N.All samples were oven dried at 60\u00b0C until constant weight. Samples were then sent to the University of New Brunswick Stable Isotope Laboratory or to University of California at Davis Stable Isotope Laboratory where they were ground into powder. Samples of algae, supralittoral vegetation, invertebrates, and fish were oxidized, and the resulting COGut content analysis (GCA) was performed on 28 chum salmon fry. Gut contents were identified to lowest possible taxonomic level and results are shown for fraction of all individuals  and fraction occurrence .13C of supralittoral vegetation and source B was calculated as the average \u03b413C of marine macroalgae. For each taxa we report the \u03b413C and \u03b415N, relative proportion of TC, the standard error (SE) associated with the proportion, and the lower and upper 95%ile confidence intervals when n is\u200a=\u200aor >3. When n\u200a=\u200a1 or 2 we only report \u03b413C and \u03b415N and relative proportion of TC. We were not able to use a three source mixing model using wrack detritus or POM because their isotopic signatures overlapped with either supralittoral vegetation or marine macroalgae  and marine derived carbon (MC) to the assimilated carbon in chum salmon fry were calculated using the procedures and programs outlined in This research was conducted according to relevant national guidelines of the Department of Fisheries and Oceans (Canada).13C and \u03b415N of macroalgae was enriched and isotopically distinct from terrestrial vegetation. The average \u03b413C value for terrestrial vegetation was \u221228.34  in the low-intertidal to \u221218.43 (TC\u200a=\u200a20%) in the mid-intertidal, \u221219.1(TC\u200a=\u200a25%) in the high-intertidal, and \u221224.38 (TC\u200a=\u200a68%) in the supralittoral. TC ranged from 0%  to 87.2% for supralittoral Homoptera  and highest in the mid-intertidal (7.9) with low-intertidal (5.53) and high-intertidal (6.4) displaying intermediate values. \u03b415N for secondary consumers ranged from 0.59 to 9.45  followed by barnacles (9.14) and Collembola (8.85). Supralittoral Homoptera (0.59) and supralittoral Acariformes (0.83) had the lowest \u03b415N. The only taxa to show a trend in \u03b415N along the tidal gradient was Acariformes, with \u03b415N lowest in the supralittoral (0.83) and highest in the mid-intertidal  ranging from \u221223.59 to \u221217.58 . TC ranged from 12.8 to 61.5% (mean 30%) with lower and upper confidence intervals of 12 and 48% (\u00b1 SE 0.07).Chum salmon fry had an average fork length of 37 mm (range 29 to 52 mm) and an average wet weight of 0.48 g (range 0.2 to 1.35 g). \u03b4Corophium sp. , and Harpacticoidea (21%).Twenty-six prey taxa were identified in the gut content analysis of 28 individual chum salmon fry . The fiv15N suggesting that trophic position does not change systematically along the tidal gradient.Our results suggest the importance of reciprocal subsidies in the terrestrial-marine ecotone in the Howe Sound estuary. Not only was marine derived carbon present in consumers present in the supralittoral zone, no supralittoral consumers were characterized by 100% terrestrially derived carbon. Likewise, terrestrially derived carbon was present even in the low-intertidal zone, particularly in amphipods. We found a clear gradient in terrestrially derived carbon down the tidal zone ranging from 68% across all taxa in the supralittoral to 25% in the high-intertidal, 20% in the mid-intertidal, and 12% in the low intertidal. This gradient was particularly clear for Diptera and Acariformes, two of the three taxa that were present in four or three zones respectively. In contrast to our results for carbon, there was no general spatial trend for \u03b4Stable isotope values of chum salmon fry and their prey indicated carbon contributions from both terrestrial and marine sources, with terrestrially derived carbon ranging from 12.8 to 61.5% in the muscle tissue of chum salmon fry (mean 30%). Adult chironomids were the dominant prey item of juvenile chum as has been previously reported at beaches in Howe Sound for juvenile chum salmon 13C averages +0.4\u00b10.12\u2030 (mean \u00b1 SE) from diet to consumer and \u03b415N averages +2.0\u00b10.20\u2030 (mean \u00b1 SE) from diet to consumer. H. plumulosa collected in the high-intertidal are the only groups of prey taxa that fall within potential \u03b413C and \u03b415N ranges for being a primary prey source .15N values may still be an important link Mytilus sp. and Isopoda collected from the mid-intertidal (Mytilus sp. larvae, the only life stage of Mytilus sp. that can be eaten by juvenile salmonids, were found in the gut contents (Taxa that fall outside of the above range of \u03b4tertidal . All of contents .13C fractionation from diet to consumer While the remaining groups fall outside the potential ranges for \u03b4In conclusion, our results show a clear gradient in the proportion of terrestrially derived carbon in invertebrate taxa that decreases down the tidal zone from 68% in the supralittoral to 25% in the high-intertidal, 20% in the mid-intertidal, and 12% in the low intertidal. Stable isotope values and gut content analysis of chum salmon fry indicated carbon contributions from both terrestrial and marine derived sources. Our results suggest that the vegetated supralittoral is an important trophic link in supplying terrestrial carbon to nearshore food webs."}
+{"text": "Randomized trials are essential in assessing the effects of healthcare interventions and are a key component in systematic reviews of effectiveness. Searching for reports of randomized trials in databases is problematic due to the absence of appropriate indexing terms until the 1990s and inconsistent application of these indexing terms thereafter.The Cochrane Library, with the permission of Elsevier, the publishers of EMBASE.The objectives of this study are to devise a search strategy for identifying reports of randomized trials in EMBASE which are not already indexed as trials in MEDLINE and to make these reports easily accessible by including them in the Cochrane Central Register of Controlled Trials (CENTRAL) in A highly sensitive search strategy was designed for EMBASE based on free-text and thesaurus terms which occurred frequently in the titles, abstracts, EMTREE terms (or some combination of these) of reports of trials indexed in EMBASE. This search strategy was run against EMBASE from 1980 to 2005 (1974 to 2005 for four of the terms) and records retrieved by the search, which were not already indexed as randomized trials in MEDLINE, were downloaded from EMBASE, printed and read. An analysis of the language of publication was conducted for the reports of trials published in 2005 (the most recent year completed at the time of this study).The Cochrane Library. Cumulative sensitivity ranged from 0.1% to 60% and cumulative precision ranged from 8% to 61%. The truncated term 'random$' identified 60% of the total number of reports of trials but only 35% of the more than 130,000 records retrieved by this term were reports of trials. The language analysis for the sample year 2005 indicated that of the 18,427 reports indexed as randomized trials in MEDLINE, 959 (5%) were in languages other than English. The EMBASE search identified an additional 658 reports in languages other than English, of which the highest number were in Chinese (320).Twenty-two search terms were used (including nine which were later rejected due to poor cumulative precision). More than a third of a million records were downloaded and scanned and approximately 80,000 reports of trials were identified which were not already indexed as randomized trials in MEDLINE. These are now easily identifiable in CENTRAL, in The results of the search to date have greatly increased access to reports of trials in EMBASE, especially in some languages other than English. The search strategy used was subjectively derived from a small 'gold standard' set of test records and was not validated in an independent test set. We intend to design an objectively-derived validated search strategy using logistic regression based on the frequency of occurrence of terms in the approximately 80,000 reports of randomized trials identified compared with the frequency of these terms across the entire EMBASE database. Randomized trials, involving sufficient numbers of participants are essential to distinguish reliably between the effects of healthcare interventions and the effects of bias or chance. Dissemination and integration of trial results through systematic reviews of the findings provide a basis for informed decision-making about the effects of different interventions. To minimize bias due to the selective availability of data, authors of systematic reviews of healthcare interventions need to identify as many relevant randomized studies as possible to provide reliable evidence on which to base healthcare decisions ,2.Variations in the journals indexed in databases indicate a need to search more than one database to ensure optimal coverage of the published literature both in subject scope and language of report ,4. AlthoSearching for reports of randomized trials presents a challenge in part because this type of study design represents only a small proportion of all the studies included in bibliographic databases. It is important, therefore, to devise a strategy which is sensitive enough to find as high a proportion as possible of all the relevant trials but specific enough not to yield vast quantities of irrelevant material, which is time-consuming, costly to evaluate and can lead to selection error.Trial identification in databases is problematic for a number of reasons. Often the methods are not adequately described by authors in titles or abstracts and not all records in bibliographic databases have abstracts. Sensitive search strategies must, therefore, include both free text terms (used by authors in the titles and abstracts (where available) to describe their studies) and indexing terms (assigned by database indexers to describe studies) for optimal retrieval. Furthermore, suitable methodological indexing terms for randomized trials have only been introduced relatively recently and have not always been consistently applied. For example, in 1991, the United States National Library of Medicine introduced into MEDLINE the Publication Type 'Randomized Controlled Trial' as an indexing term to improve searching for trials. Despite this, a study by one of the authors (CL)  found thIdentifying reports of trials in EMBASE has proved to be similarly problematic. Discussions began between one of the authors (CL) and Elsevier, the producers of EMBASE, in 1992, immediately after the UK Cochrane Centre opened. This led to a representative from Elsevier being invited to a workshop in January 1993, convened by the UK Cochrane Centre. It was confirmed that although the EMBASE thesaurus (EMTREE) contained terms for clinical trials in general, it had no specific term for indexing reports of randomized controlled trials. Elsevier was persuaded of the importance of accurate indexing of clinical trials and of the necessity to differentiate randomized controlled trials from other clinical trials. In September 1993, Elsevier introduced the indexing term 'Randomized Controlled Trial' in EMBASE together with the term 'Multicenter Study' and undertook to index clinical trials \"even more consistently\" in the future .The EMBASE data structure and licensing agreements with third party vendors such as Dialog and Ovid did not, at that time, support record changes in the same way that MEDLINE did and, therefore, 're-tagging' records in EMBASE was not feasible. In addition, because The Cochrane Collaboration did not have its own register of trials at that time no further progress was made with respect to making EMBASE reports of trials available centrally within the Collaboration.In mid-1996, however, as a result of the introduction by Elsevier of a new database platform, it became possible for them to investigate systems for updating their databases in a way that had not previously been possible. Specifically this meant that they could consider upgrading the indexing of EMBASE records by retrospectively adding their new indexing term 'Randomized Controlled Trial' to all those reports identified as such in EMBASE, thus improving retrieval in the future.The Cochrane Library, Update Software, meant that there was now a register within the Collaboration which could provide a vehicle for making these reports accessible.From The Cochrane Collaboration's point of view, the advent of the Cochrane Controlled Trials Register, now known as the Cochrane Central Register of Controlled Trials (CENTRAL), designed and developed by the then publishers of In December 1996, Elsevier requested a further meeting with one of the authors (CL) and the Managing Director of Update Software and agreed to permit the re-publication of EMBASE records in CENTRAL. Until 1996, The Cochrane Collaboration had focussed on the systematic electronic searching of MEDLINE and the systematic handsearching of general and specialized healthcare journals to facilitate access to reports of randomized trials of healthcare interventions. With the developments described above it was possible to extend this searching to include EMBASE.BMJ and the Lancet for the years 1990 and 1994 and that records of reports of randomized trials identified by using this search strategy would be published in CENTRAL.It was decided that a search strategy to identify reports of randomized trials in EMBASE would be devised by two of the authors (CL and SM)  from an The objectives of this study are:1. To devise a search strategy, tested for sensitivity and precision, for identifying reports of randomized trials in EMBASE.2. To identify reports of randomized trials in EMBASE that meet the Cochrane eligibility criteria .3. To identify in EMBASE reports of trials not currently indexed as trials in MEDLINE, as these are already included in CENTRAL.The Cochrane Library, with the permission of Elsevier, the publishers of EMBASE.4. To make these reports easily accessible by including them in CENTRAL in The Medical Subject Headings (MeSH) and Publication Type terms from the Cochrane Highly Sensitive Search Strategy for identifying reports of randomized trials in MEDLINE  were conBMJ and the Lancet, for 1990 and 1994 for all reports of randomized or quasi-randomized trials. These journals had already been handsearched under another project co-ordinated by two of the authors (CL and SM) at the UK Cochrane Centre and funded by the European Union under the BIOMED Programme . Th. Th35]. The systematic search was conducted as a multi-file search across MEDLINE and EMBASE so that duplicate records in EMBASE indexed in MEDLINE with the Publication Type 'Randomized Controlled Trial' or 'Controlled Clinical Trial' could be removed first before downloading records unique to EMBASE for each term sequentially to be checked for eligibility.The search terms were executed sequentially so that the incremental (cumulative) value of each term could be assessed. Cumulative sensitivity is defined as the additional number of reports of randomized trials identified by each term when searched in its position in the search sequence divided by the total number of reports of randomized trials identified, expressed as a percentage. Cumulative precision  is defined as the additional number of reports of randomized trials identified by each term when searched in its position in the search sequence divided by the total number of records retrieved by that term, expressed as a percentage. Terms with low cumulative precision were rejected. Each potentially relevant record was, therefore, only retrieved once, even if it contained more than one of the terms in the search strategy. For example, a record containing the phrase 'randomized placebo controlled trial' would be identified by the first search term 'random$' but would be excluded from the set derived by the search term 'placebo$' Table . The ordSome search terms, as a result of their position in the sequence, had a cumulative precision of less than 10% in sample years  and these terms were not then used to complete the systematic search in all years  offering the ability to search MEDLINE and EMBASE simultaneously were used to identify and download records from EMBASE so that records which were already indexed with the Publication Type terms 'Randomized Controlled Trial' or 'Controlled Clinical Trial' in MEDLINE  could be excluded by the EMBASE search. Initially, we used DataStar as the search interface of choice but this was limited at that time to de-duplicating 3000 records. As there were many more than 3000 records retrieved by our search sets across MEDLINE and EMBASE combined this meant that de-duplication was extremely tedious. We changed to Dialog to increase the limit to 5000 records and eventually changed to Ovid in 1999. Both DataStar and Dialog provided access to EMBASE back to 1974 but Ovid at that time only provided access back to 1980. This change meant that the remainder of the terms could not be searched back to 1974 but only back to 1980.Records were downloaded and printed for the publication years 1974\u20132005 for the first four terms  and 1980\u20132005 for the remainder , placebo$ (12%) and volunteer$ (10%)  of which 35% were found to be reports of controlled trials and contributed the greatest proportion of the total number of reports of trials identified 60%)  approximately 350,000 records have been downloaded from EMBASE and records for all of the approximately 80,000 reports of randomized trials unique to EMBASE at the time of the searches  are included in CENTRAL in The results of the language analysis indicate that for the publication year 2005 searching EMBASE did not identify any more reports of trials in Croatian, Hungarian, Lithuanian, Romanian or Russian than those already found in MEDLINE Figure . SearchiProjects such as this and the systematic electronic search of other bibliographic databases such as MEDLINE tend to under-identify reports of trials as there is often insufficient evidence in the title or abstract of a record to assess adequately whether it is a report of a randomized trial even if it is clearly stated in the methods section of the full journal article. In a recent study, 20 (7%) additional reports of randomized controlled trials were identified only by obtaining the full text of the article . To idenIn addition, further reports of trials could have been identified from EMBASE by using terms with lower cumulative precision. Whilst including these terms was not considered to be feasible in the context of the project that aimed to search the whole of EMBASE, they could be considered by searchers who would be combining their study design search terms with subject- or condition-specific search terms in EMBASE and would thus retrieve considerably fewer records for consideration.Records were de-duplicated within the host system rather than within reference management software. Any use of de-duplication facilities, either within the host system or using reference management software, may lead to over- and/or under-inclusion of records. No systematic quality control of the de-duplication process was undertaken but ad hoc viewing of the duplicate pairs seemed to indicate that the duplicates identified by the host system were valid duplicates. The eligibility criteria for including reports of trials in CENTRAL state that records should be included if they are 'definitely or possibly a report of a randomized or quasi-randomized trial'. Benefit of the doubt is, therefore, exercised where necessary. It should, however, be noted that some reports which claim to be reports of randomized trials in the title or abstract are not in fact randomized trials on the basis of further details given in the Methods section and such reports will have been included erroneously in CENTRAL as a result of this project and other similar projects where records are identified on the basis of the title and abstract only .Many methodological search strategies or 'filters' have been developed in MEDLINE to make it easier to find studies of systematic reviews -44 and rBMJ and the Lancet) and the terms were derived subjectively.Since 1996 when the search strategy reported here was derived, methods of search strategy design have developed from subjectively derived strategies that were not performance tested  to subjeBMJ and Lancet, which were used to derive the search terms in 1996, are general medical journals with medium to high impact factors, published in English.The extent to which the derived search strategy is generalizable depends upon the sample of journals in the 'gold standard'. Boynton and colleagues have warned that the journals used in such 'gold standards' may not be representative of healthcare journals as a whole . FurtherWhether a filter developed and tested in two separate years (in this study 1990 and 1994) will give the same results for other years is likely to be affected by additions and amendments to index terms over time. Although Wilczynski and colleagues established the robustness of search strategies across publication periods 1991 and 2000 in MEDLINE  which leThe validation of a search filter is important in assessing the effectiveness of the filter outside the set used for deriving and testing it. If the same data set is used for both purposes it has been suggested that it can introduce bias resulting in an overestimate of the effectiveness of the filter  because Other initiatives, in particular the CONSORT Statement, aimed at improving the reporting of trials by authors may well have facilitated their retrieval in databases over time. CONSORT was introduced in 1996  and reviIn recent years, the Centre for Reviews and Dissemination and the UK Cochrane Centre have sought to improve the objectivity of the methods used to design search strategies. They have used word frequency analysis and discriminant analysis to derive objectively, using logistic regression, the most efficient search terms and combinations of terms in titles, abstracts and index terms for particular study designs. The Group's most recent research in this area ,61 preseThe Cochrane Library have enhanced access to reports of trials, especially those published in languages other than English. This project has made it easier to identify approximately 80,000 reports of randomized trials by identifying the relevant records in EMBASE and including these, with the permission of Elsevier, in the Cochrane Central Register of Controlled Trials in The Cochrane Library. We have also identified terms that might be useful to people searching EMBASE for randomized trials in the future. However, further work remains to be done to address the limitations in the search strategy reported in this study. We intend to perform an objective analysis, using logistic regression, of the frequency of terms occurring in the approximately 80,000 reports of trials that have been identified to date, compared with their frequency across the entire EMBASE database. The results of this final analysis will be used to generate a highly sensitive search strategy for EMBASE to make reports of trials accessible to authors of reviews and others interested in basing healthcare decisions on the best available evidence.Searching EMBASE for reports of randomized trials and ensuring that they are made available in CENTRAL in EMBASE is a rich source of reports of randomized trials that are either not included in MEDLINE or not indexed as trials in MEDLINE, especially reports in some languages other than English.Due to this project, approximately 80,000 reports of randomized trials are now more accessible through the inclusion of relevant records in the Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library.In addition to searching CENTRAL, people looking for reports of randomized trials should search EMBASE, as well as MEDLINE, for reports published in recent years that have not yet been considered for inclusion in CENTRAL.The abstract of this paper has been translated into the following languages by the following translators (names in brackets):\u2022 Chinese \u2013 simplified characters (Mr. Isaac Chun-Hai Fung and Dr. Yao-Bi Zhang) [see Additional file \u2022 Chinese \u2013 traditional characters (Mr. Isaac Chun-Hai Fung and Dr. Yao-Bi Zhang) [see Additional file \u2022 French (Mr. Philip Harding-Esch) [see Additional file \u2022 Spanish (Ms. Annick B\u00f3rquez) [see Additional file CL and AE work on trial identification at the UK Cochrane Centre and studies such as this may impact on their employment.The Cochrane Library, co-authored reports of each stage of this project which have contributed to this manuscript, created the final manuscript for publication and is guarantor of the manuscript.CL conceived of the study, acquired initial project funding, negotiated publication rights with Elsevier (publishers of EMBASE), conducted the analysis which identified the original search terms, set up the original searches and co-ordinated the identification of the records in 1997, conducted the systematic search in EMBASE on backfiles, processed the resulting data for publication in CENTRAL in The Cochrane Library, co-authored a report of this stage of the project which contributed to this manuscript, wrote the first draft of this manuscript including conducting the literature search and creating the bibliography, updated the analysis of trial reports published in 1997 in languages other than English using 2005 data, and created the final manuscript for publication.AE co-ordinated the identification of the records from 1999 to date, conducted the systematic search in EMBASE on backfiles and prospectively on an annual basis, reviewed and modified the search syntax annually to accommodate changes in database search structure, processed the resulting data for publication in CENTRAL in SM conducted the analysis which identified the original search terms, and co-authored a report of this stage of the project which contributed to this manuscript.The Cochrane Library, conducted an analysis of trial reports published in 1997 in languages other than English and co-authored a report of this stage of the project which contributed to this manuscript.NP co-ordinated the identification of the records from 1997 to 1999, conducted the systematic search in EMBASE on backfiles, processed the resulting data for publication in CENTRAL in All authors approved pre-publication versions of this manuscript and approved the final manuscript for publication, following peer review.The views expressed in this study represent those of the authors and are not necessarily the views or the official policy of The Cochrane Collaboration.Abstract in Chinese (simplified characters).Click here for fileAbstract in Chinese .Click here for fileAbstract in French.Click here for fileAbstract in Spanish.Click here for file"}
+{"text": "Research for developing search strategies to retrieve high-quality clinical journal articles from MEDLINE is expensive and time-consuming. The objective of this study was to determine the minimal number of high-quality articles in a journal subset that would need to be hand-searched to update or create new MEDLINE search strategies for treatment, diagnosis, and prognosis studies.The desired width of the 95% confidence intervals (W) for the lowest sensitivity among existing search strategies was used to calculate the number of high-quality articles needed to reliably update search strategies. New search strategies were derived in journal subsets formed by 2 approaches: random sampling of journals and top journals . The new strategies were tested in both the original large journal database and in a low-yielding journal  subset.For treatment studies, if W was 10% or less for the lowest sensitivity among our existing search strategies, a subset of 15 randomly selected journals or 2 top journals were adequate for updating search strategies, based on each approach having at least 99 high-quality articles. The new strategies derived in 15 randomly selected journals or 2 top journals performed well in the original large journal database. Nevertheless, the new search strategies developed using the random sampling approach performed better than those developed using the top journal approach in a low-yielding journal subset. For studies of diagnosis and prognosis, no journal subset had enough high-quality articles to achieve the expected W (10%).The approach of randomly sampling a small subset of journals that includes sufficient high-quality articles is an efficient way to update or create search strategies for high-quality articles on therapy in MEDLINE. The concentrations of diagnosis and prognosis articles are too low for this approach. For clinicians and clinical researchers, it is important to be able to quickly retrieve articles that are clinically sound and directly relevant without missing key studies or retrieving excessive numbers of preliminary, irrelevant, outdated, or misleading reports. Unfortunately, reliable and precise retrieval of clinical articles from MEDLINE is not easy because of the size of the database  and the and also in Ovid.One possible solution to this problem is to develop methodological search filters or strategies to retrieve original studies and review articles that use the strongest methods to assess clinically important problems ,6. For iThe Clinical Queries search strategies were developed using index terms and text words available in the year 2000. Periodic updating of search strategies is necessary because index terms used in MEDLINE are updated annually as new concepts emerge, some old concepts fall out-of-date, and new journals are added . New seaIn this paper, we set out to determine the least number of high-quality articles in a journal subset that would need to be hand-searched to update the search strategies for retrieving studies of treatment, diagnosis, and prognosis  from MEDLINE.Hand searching of the literature provided a \"gold standard\" classification of article categories. Six research assistants assessed 49,028 articles from 161 journals published in 2000 that were indexed in MEDLINE; all the articles were classified as original studies, review articles, general papers, or case reports; and the original and review articles were then categorized as \"pass\" or \"fail\" studies based on methodological criteria for treatment, diagnosis, prognosis, and other clinical topic areas . ArticleThe sensitivity , specificity , precision (the proportion of retrieved articles that were relevant and sound), and accuracy  for each single term and combinations of terms were determined by using an automated iterative process. All combinations of search terms used the Boolean \"OR\", meaning that articles that included any one of the search terms in the strategy would be retrieved. Search strategies were developed to maximize each of sensitivity and specificity, and to provide the best balance between sensitivity and specificity. These search strategies for retrieving high-quality original treatment, diagnosis, and prognosis studies were used in this study /[(0.05/2)2] = 395.Therefore, n = [1.96A computer program was developed to create journal subsets by randomly selecting journals from the original 161-journal database. Journal subsets that included \u2264 110 journals were gradually and arbitrarily increased by 5 journals  and journal subsets that included > 110 journals were increased arbitrarily by 10 journals. Thus, 26 subsets of journals were randomly created from the 161-journal database. Each journal had the same probability of selection. We presumed that selected journal subsets were independent, and the same journal might appear in more than 1 journal subset. After the creation of the 26 journal subsets, the number of high-quality articles in each subset was counted.The 161 journals were also ordered according to the ascending number of pass articles for the treatment [see Additional file Based on an arbitrarily chosen W of 0.10, we determined the optimal journal subset which included the minimal number of high-quality studies that could be used to develop search strategies. The optimal journal subset was formed by 2 approaches \u2013 random sampling of journals and top journals.To assess whether the 2 optimal journal subsets formed by random sampling and top journal approaches were acceptable for updating the search strategies in MEDLINE, new search strategies were developed in these 2 journal subsets. We used the same method to developing search strategies that was used previously; 3869 unique search terms were tested in each journal subset for their ability to retrieve high-quality articles of a certain category .The new search strategies derived in the small journal subsets  were assessed in the original large journal database, and compared with the existing search strategies.If these new search strategies performed poorly (defined as a sensitivity or specificity < 50%) in the original large database, another journal subset with a total number of pass articles to achieve a smaller W  would be assessed.The chi-squared test (STATA 9.0) was used to compare the performance characteristics between 2 independent journal subsets. A 2-sided significance level of \u03b1 = 0.05 was adopted.Based on the equation noted earlier 1), the sample size requirements for different Ws for the treatment, diagnosis, and prognosis categories are shown in Table , the sam26 subsets of journals were randomly created from the 161-journal database, and the Ws achieved with the corresponding numbers of pass articles for the 3 purpose categories are shown in Table A W of 0.10 was chosen as a starting point to estimate if a small journal subset was good enough to update search strategies for use in MEDLINE in the future. Based on Table The Lancet and Journal of Clinical Oncology) included 158 pass articles  in the treatment category and could also guarantee a W \u2264 0.10. Thus, just 2 top journals could be used when updating treatment search strategies.The 2 top-yielding journals  were developed in 1 subset of 15 randomly sampled journals [see Additional file The new search strategies for the treatment category that were developed in the subset of 15 randomly sampled journals were tested in the original large journal database and the performance was compared with the existing search strategies Table . When coBased on the above analysis, except for the sensitivity of the new high specificity search strategy being lower than that of the existing search strategy, the performance characteristics of the new strategies derived using the 15 randomly sampled journals appeared to be as good as those of the existing search strategies. The new high specificity strategy (\"double-blind.mp.\" OR \"random: assigned.tw.\") and the existing high specificity strategy  retrieved 146,416 and 259,665 eligible articles, respectively, from an Ovid MEDLINE search . Compared with the existing strategy, the new specificity strategy would save about 44% of the time to read the eligible articles. However, this strategy would miss about 57,801 (41%) relevant articles, some of which might be important.In Table The strategies developed using the subset of 15 randomly selected journals and the 2 top journals were compared in the original large journal database  was almost equal to that in the subset of 15 randomly selected journals (191). Similarly, the new strategies from the top journal approach were tested in a low-yielding journal subset that included 97 journals with 158 pass articles that was equal to that in the 2 top journals. Almost all search strategies developed using the top journal approach performed less well in the low-yielding journal subset [see Additional file For both the diagnosis and prognosis categories, even when using the 161-journal database, the smallest W achieved was 0.16, because the numbers of pass articles were very low, 147  for diagnosis and 190 (0.39%) for prognosis, compared with 1587 (3.24%) for treatment category. If we accept a wider W, such as a W = 0.20, we could find a smaller journal subset for diagnosis and prognosis based on the sample size calculation for the number of the pass articles in Table The sample size calculations shown in this study suggest that search strategies developed in small journal subsets will be as good as those developed in larger collections of journals if there are a sufficient number of high-quality articles. In this case, the subsets of 15 randomly sampled journals or 2 top-yielding journals that included \u2265 99 high-quality articles achieved a W of 0.10 for the retrieval of treatment studies. Except for the sensitivities of the high specificity search strategies, the other performance characteristics of the new strategies developed using both the random sampling and top journal approaches were close to those of the existing search strategies Table . The senThis study has some limitations. First, the new search strategies developed using the random sampling approach were done using a subset of 15 randomly sampled journals with \u2265 99 pass articles. As shown earlier, there is a 6% probability [see Additional file The new search strategies developed using the random sampling approach seem to perform better than the new strategies developed using the top journal approach in low-yielding journals. This is not surprising because the subsets of randomly sampled journals included both top-yielding and low-yielding journals.The search strategies that are widely used by clinicians, health researchers, and librarians in the Clinical Queries interface of PubMed and in Ovid were developed in journals published in 2000 and will need to be updated periodically to maintain and improve their performance as well as to address new topic areas. When updating or creating new search strategies for high-quality articles on therapy in MEDLINE in future research, the approach of randomly sampling a subset of journals that includes sufficient high-quality articles provides the most parsimonious way of achieving performance estimates at a specified level of statistical precision. For treatment studies, the number of journals needed is quite small because the concentration of high-quality studies is quite high in clinical journals. For diagnosis and prognosis articles, however, the concentration of high-quality studies is low, and a large number of journals are needed for the development of search strategies. The expense of developing and testing search strategies is high and this research provides a way to estimate how much work will be needed to achieve a robust result.W: width of the 95% confidence intervals.The authors declare that they have no competing interests.XY designed the research, analyzed and interpreted data, and wrote the manuscript. NLW contributed to the analysis of data and revised the manuscript. SDW contributed to the interpretation of data and revised the manuscript. RBH designed the research and revised the manuscript.The pre-publication history for this paper can be accessed here:Click here for fileClick here for fileClick here for fileClick here for fileClick here for fileClick here for file"}
+{"text": "The apparatus,MIASA (methyl iodide automated synthesis apparatus), wasdesigned to operate as part of an automated labelling system in ashielded \u2018hot\u2019 laboratory. The apparatus was designed without thesize constraints of typical instrumentation used in hot cells, althoughit is compact where necessary. Ample use of indicators and sensors,together with compact design of the reaction flasks for small deadspace and efficient evaporation, led to good reliability andperformance. The design of the hardware and software is describedin this paper, together with a preparation of 3-N-[11C]methylspiperoneas a sterile injectable solution in physiological saline.A fully automated apparatus for the routine synthesis andformulation of short-lived"}
+{"text": "Cluster randomized trials (CRTs) present unique methodological and ethical challenges. Researchers conducting systematic reviews of CRTs  require efficient electronic search strategies (filters or hedges) to identify trials in electronic databases such as MEDLINE. According to the CONSORT statement extension to CRTs, the clustered design should be clearly identified in titles or abstracts; however, variability in terminology may make electronic identification challenging. Our objectives were to (a) evaluate sensitivity  and precision of a well-known electronic search strategy  with respect to identifying CRTs, (b) evaluate the feasibility of new search strategies targeted specifically at CRTs, and (c) determine whether CRTs are appropriately identified in titles or abstracts of reports and whether there has been improvement over time.We manually examined a wide range of health journals to identify a gold standard set of CRTs. Search strategies were evaluated against the gold standard set, as well as an independent set of CRTs included in previous systematic reviews.The existing strategy  is sensitive (93.8%) for identifying CRTs, but has relatively low precision ; the number needed to read can be halved to 5 (precision 18.4%) by combining with cluster design-related terms using the Boolean operator AND; combining with the Boolean operator OR maximizes sensitivity (99.4%) but would require 28.6 citations read to identify one CRT. Only about 50% of CRTs are clearly identified as cluster randomized in titles or abstracts; approximately 25% can be identified based on the reported units of randomization but are not amenable to electronic searching; the remaining 25% cannot be identified except through manual inspection of the full-text article. The proportion of trials clearly identified has increased from 28% between the years 2000-2003, to 60% between 2004-2007 .CRTs should include the phrase \"cluster randomized trial\" in titles or abstracts; this will facilitate more accurate indexing of the publication type by reviewers at the National Library of Medicine, and efficient textword retrieval of the subset employing cluster randomization. The randomized controlled trial is widely accepted as the gold standard study design in health research . In someIn the present article, we focus on the problem of identifying CRT reports in the literature for the purpose of conducting systematic reviews. Although systematic reviews often focus on trials of a particular medical treatment or condition, several researchers have conducted systematic reviews of CRTs, to assess the methodological or reporting quality at various points in time -11. MostIn recent years, authors and journal editors may have become more aware of the importance of adequately reporting the design of the study, especially after publication of the Consolidated Standards of Reporting Trials (CONSORT) statement , which iOur objectives in the present article are to (a) determine the sensitivity and precision of a simple, existing electronic search strategy for randomized controlled trials with respect to identification of CRTs, (b) determine the feasibility of alternative electronic search strategies incorporating cluster design-related terms, and (c) determine to what extent authors are appropriately identifying trials as cluster randomized in the titles or abstracts of reports and whether any improvement has occurred over time.We first identified a \"gold standard\" set of CRTs, by manually examining a total of 78 health journals indexed in MEDLINE between January 2000 and November 2007. All issues in a particular year, with the year assigned by computer-generated random numbers, were searched. Journals were purposely selected from a wide range of subject categories in the 2007 Sciences and Social Sciences editions of Journal Citation Reports (JCR), as well as based on our subjective knowledge of their likelihood to publish CRTs , and publication type were exported to a statistical software package for analysis. From the title or abstract, we identified the specific text revealing the trial as cluster randomized  or possibly cluster randomized . We conducted a frequency analysis of the exported text to identify candidate terms for building a search strategy.Unique Identifier (UI) numbers, an 8-digit number assigned by the NLM to uniquely identify a particular record, were obtained for all 162 CRTs in the gold standard set and all 25,545 non-CRTs excluded from the gold standard set. Search strategies were implemented in the MEDLINE database (OVID interface) from 1996 to the third week of January 2009. We calculated the two performance indicators that are most relevant to systematic reviews , namely Because the same CRTs were used to both derive and evaluate the search strategies, we additionally tested search strategies against an independent set of 363 CRTs that had been identified in seven previously published systematic reviews of CRTs. The year of publication for these trials ranged from 1979 to 2005. We obtained UI numbers for these trials and determined the % retrieved by the search strategies .To determine whether the reporting of CRTs has improved over time, we calculated the percentage of trials clearly identified as \"cluster randomized\", \"group randomized\", or \"community randomized\" in the title or abstract, and compared these percentages by year of publication.The number of articles retrieved, as well as sensitivity, 1-specificity, and precision of each search strategy is presented in Table possibly cluster randomized based on the units of randomization   paired on enrolment size, racial composition, urban or rural location, and class structure were randomized\"). Finally, it would be difficult to anticipate in advance all possible units of randomization that could be used in the diverse settings in which CRTs are implemented ). Cluster design related search strategies were therefore developed using primarily the frequency analyses of the MeSH and other text words in the title or abstract.Analysis of the specific text from each trial that identified the trial as cluster randomized or possibly cluster randomized revealed that 78 (48.1%) of the gold standard set had been clearly identified as \"cluster randomized\", \"group randomized\" or \"community randomized\" in the title or abstract; however, 38 23%) could be identified as cluster randomized only by manual inspection of the trial procedures in the full text article. The remaining 46 trials (28%) could be identified as cluster randomized or 3% could The strategy with highest sensitivity Table  line 17,The results of the search strategies evaluated against 363 studies included in previous systematic reviews are presented in Table 2 test for trend = 3.6; p = 0.0003). Because publication year 2006 (unlike the remainder of the years) had not been randomly allocated for searching but represented trials identified from 10 major medical journals, we repeated this analysis excluding the 2006 journals. The results were similar, indicating an improvement over time (p = 0.0012). The lowest percentage of trials clearly identified  was in 2001; this percentage increased to above 50% for the first time in 2003 and remained above that level until 2007. Of the 58 trials published in the first four years (2000-2003), 27.6% were clearly identified; this percentage more than doubled in the second half of the observation period (2004-2007) with 59.6% of 104 trials clearly identified .The results of our analysis of trends in adequate reporting are presented in Table A growing number of studies are using the cluster randomized design to evaluate health care interventions . ClusterOne of the limitations of our study is that the trials which were used to derive the search strategies were not selected by random sampling, representing instead a judgement sample of journals likely to publish CRTs. This was necessary from a practical standpoint as manual searching of electronic journals is a time-consuming process and it was necessary to maximize the yield from these searches. Nevertheless, more than 25,000 articles were examined from a broad range of 78 journals believed to be representative of the disciplines in which these trials are being published. A second limitation is that the cluster design related search strategies were derived using subjective judgement, rather than more objective methods such as logistic regression techniques . This iIt is likely that any search strategy, when evaluated against the same set of studies that was used to derive the search strategy, would provide an over-optimistic view of its sensitivity. It is therefore important to validate a newly derived search strategy against an independent set of studies. We tested three search strategies against an independent set of 363 CRTs identified in previous systematic reviews, by calculating their relative recall. Although these systematic reviews varied somewhat in their focus , they all used the same standard definition of a CRT and are therefore covered by the broader criteria of our search strategy, designed to identify all CRTs regardless of the setting. As expected, the relative recall values of our search strategies were lower when tested against the validation set. It should be noted, however, that the validation set included primarily older trials, ranging from a publication year of 1979 to 2005, whereas our search strategies were derived using trials published in the years 2000-2007 only. Moreover, our results have shown that appropriate reporting of trials as CRTs has improved over time. This is confirmed by the validation set: for example, the results in Table The CONSORT statement extension to CRTs, published in 2004, recommended that authors clearly identify trials as cluster randomized in the title or abstract of reports. Although overall, fewer than half (48.1%) of trials included in the gold standard set had been identified as CRTs in titles or abstracts, there was a significant improvement over time with 60.7% of trials published post-CONSORT (2005-2007) clearly identified. This improvement cannot be solely attributed to CONSORT however, as increases in these proportions were evident even in the pre-CONSORT years; improvements are likely attributable to a combination of factors, including increased awareness of the unique characteristics of the clustered design promoted by several articles and books [e.g., .Although the reporting of CRTs is improving, it is not yet adequate. Simple search strategies will not efficiently retrieve CRTs unless they have been appropriately tagged by MEDLINE indexers as randomized controlled trials. The NLM has, for over a decade now, been concentrating on correctly identifying and indexing randomized controlled trials in MEDLINE . SpecifiThe authors declare that they have no competing interests.MT led the data collection, development of the search strategies, data analysis, and interpretation of results and drafted the manuscript. JM participated in the development of the search strategies, data analysis and interpretation of results. JMG participated in the development of the search strategies and interpretation of results. AM participated in data collection. JCB, MPE and AD participated in interpretation of the results. All authors critically reviewed and edited the manuscript and all authors approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2288/10/15/prepubSubject categories and journals included in manual search to identify gold standard set of cluster randomized trials. Subject categories and journals included in manual search to identify gold standard set of cluster randomized trials.Click here for fileExamples of text in title or abstract suggesting trial as cluster randomized. Examples of text in title or abstract that could be used to identify the trial as cluster randomized or possibly cluster randomized.Click here for file"}
+{"text": "Escherichia coli fit iron transport system consists of 6 genes, fitA, B, C, D, E and fitR. Based on in silico analysis, FitA-E composes a typical bacterial iron transporter, while FitR was deduced to be a regulator. In this paper the regulation of fit expression by FitR was studied using a quantitative RT-PCR technique and a lacZ reporter assay. It was found that fit expression was repressed when FitR was over-expressed and de-repressed when fitR was knocked out by mutation. When the mutation in fitR was complemented in trans- with the wild type fitR gene, repression of fit expression by FitR was restored. Finally, recombinant FitR was found to bind to the fit promoter DNA when employed in an electrophoretic mobility-shift assay. These results demonstrated that fitR encodes an auto-repressor for the E. coli fit system.The  Protein-DNA complexes were separated from unbound DNA on 5% nondenaturing polyacrylamide gels run at 50V for 2 to 3h. The DNA was transferred onto a positively charged Nylon membrane  by electroblotting. The digoxigenin-labeled probes were subsequently detected by an enzyme immunoassay using an antibody  and the chemiluminescent substrate disodium 3-tricyclodecan}-4-yl) phenyl phosphate (CSPD) .The electrophoretic mobility-shift assay was performed as follows. The 347bp intergenic region between lacZ reporter assay was employed to determine if FitR regulated fit expression. The intergenic DNA containing the bidirectional fit promoter, located between fitA and fitB, was cloned in two directions into the lacZ reporter vector, pMP220. The two fit-lacZ reporter plasmids were named pMP-fitA and pMP-fitB, and were used to measure the expression of fitA and fitB, respectively [\u03b2 -galactosidase activities were measured from exponential growth phase cells as described [1), the expression of fitB  in strains containing pMP-fitB was very similar to the strain carrying pMP-fitB and the cloning vector pBAD22. The wild type strain (oy021) containing the plasmid pMP-fitB expressed 139 units of \u03b2-galactosidase .Finally,when the fitR mutation was complemented in trans- by pBAD-fitR1, cells expressed 55 units of \u03b2-galactosidase, which confirmed the observation that fit was repressed by FitR. A similar regulation pattern was obtained for strains containing pMP-fitA . These data demonstrated that FitR does repress fit expression.A escribed . As showitB Fig.  in straifit by FitR was also found even when FitR expression was not induced , which might be the result of leaky expression of fitR from pBAD-fitR1. To test this, we cloned fitR into the plasmid pCR-XL-TOPO, a cloning vector without a ribosome binding site (RBS). The new plasmid was designated pCR64. When this plasmid was introduced into wild type strains oy021 or fitR mutant oy028, strains oy021/pCR-XL-TOPO, oy021/pCR64, oy028/pCR-XL-TOPO and oy028/pCR64 expressed 129, 120, 228 and 217 units of \u03b2-galactosidase, respectively, which demonstrated that fit expression was not repressed without fitR expression . Further, we examined fitR transcription using RT-PCR. As shown in Fig. (1D), the fitR transcript was detected in cells carrying pBAD-fitR1 grown under non-induced condition (lane 2). Thus we concluded that the repression of fit by pBAD-fitR1 when cells were grown in LB containing 0mM arabinose resulted from the leaky expression of FitR from pBAD promoter. The fact that pBAD22 is quite leaky has been found in other studies [fit system by FitR was also examined using a quantitative RT-PCR analysis. Compared to gene expression in wild type strain E. coli i484, fitB was repressed 1.8 fold when FitR was expressed from pBAD-fitR1 and de-repressed 3.2 fold when fitR was mutated. In addition, when the fitR mutation was complemented by pBAD-fitR1, fitB was repressed 6.8 fold compared with gene expression levels in the fitR mutant. Similar results were obtained for fitA expression. These results demonstrated that FitR represses the expression of the fit system.The repression of  studies . The regfit system, it would be expected to bind to the fit promotor. To obtain protein FitR, a fitR over-expression plasmid, pBAD-fitR2, was constructed by cloning fitR into plasmid pBAD22. E. coli DH5\u03b1 cells carrying pBAD-fitR2 were grown in LB and protein expression was induced by 10mM arabinose. As shown in Fig. (2A), the purified recombinant FitR was found to have a molecular weight of 28kDa (lane 1), which is consistent with the predicted molecular weight based on sequence analysis. To determine if FitR bound to the fit promoter, an electrophoretic mobility-shift assay (EMSA) was performed. As shown in Fig. (2B), the purified FitR was found to bind to the fit promoter DNA when used at 20, 50, or 100ng . When a 50 fold excess of non-labeled fit promoter DNA was used as a specific competitor in the reaction, the binding of FitR was found to be inhibited (lane 6). These results suggested that FitR binds specifically to the fit promoter.If FitR directly regulates the E. coli fit system has been found to be repressed by Fur-Fe2+ complex (Ouyang and Isaacson unpublished). Therefore, fit is regulated by two repressors: Fur and FitR. Here we investigated the possibility that these two repressors regulated fit expression independently. Initially, a lacZ reporter assay was performed to determine if FitR needed iron as a cofactor to repress fit expression. As shown in Fig. (3A), the wild type strain containing pMP-fitB (oy021) expressed 168 and 525 units of \u03b2-galactosidase under iron-rich and iron-deficient conditions, respectively. In the fitR mutant (oy028), cells expressed 227 and 777 units of \u03b2-galacto-sidase under iron-rich and iron-deficient conditions, respectively. Similar results were obtained for pMP-fitA . These results indicated that fit expression was still induced by iron deprivation when fitR was mutated, which in turn suggested that iron and FitR separately repressed fit expression. In addition, a quantitative RT-PCR analysis was employed to study if the repression of FitR is independent of expression of Fur. As shown in Fig. (3C), fitA expression was induced 23 fold in the fur mutant, 2.2 fold in the fitR mutant, and 52 fold in the fitR and fur double mutant. These data suggested that FitRandFur repressed fitA independently. Similar results were observed for other fit genes.The E. coli, for those well-studied iron transporters, such as fep, fhu, fec, feo, they are all regulated by the global regulator, Fur (ferric uptake regulator), through binding of Fur-Fe2+ complex to the Fur box in the gene promoters [fec system, most E. coli iron transporters do not have their own regulators [E. coli fit system is regulated by two independent repressors, an auto-regulator FitR and a global regulator Fur. This elegant regulation may indicate the important role of fit in E. coli metabolism. So far we don\u2019t know if FitR is a global regulator or if any other factor is involved in the regulation of the fit system. Further studies, for example, identification of the exact binding site of FitR or Fur on fit promoter, whether there is a synergy in binding by addition of Fur and FitR, and whether other proteins might be involved in the expression of fit, etc, may elucidate the detailed regulation mechanism of this novel iron transporter.In romoters , 11, 12.gulators , 15, 16.E. coli fit iron transport system in this report.In conclusion, an auto-repressor, FitR, was identified for the"}
+{"text": "Other patients\u2019 stories on the Internet can give patients information, support, reassurance, and practical advice.\u00a0We examined which search facility for online stories resulted in patients\u2019 satisfaction and search success.\u00a0This study was a randomized controlled experiment with a 2x2 factorial design conducted online. We facilitated access to 170 stories of breast cancer patients in four ways based on two factors: (1) no versus yes search by story topic, and (2) no versus yes search by writer profile.\u00a0Dutch speaking women with breast cancer were recruited. Women who gave informed consent were randomly assigned to one of four groups. After searching for stories, women were offered a questionnaire relating to satisfaction with the search facility, the stories retrieved, and impact of the stories on coping with breast cancer. Of 353 enrolled women, 182 (51.6%) completed the questionnaire: control group (n = 37), story topics group (n = 49), writer profile group (n = 51), and combination group (n = 45).2                        3 = 3.7, P = .30). Women who had access to the story topics search facility (yes vs no): were more positive about  and more satisfied with the search facility ; were more positive about the number of search options ; were better enabled to find desired information ; were more likely to recommend the search facility to others or intend to use it themselves ; were more positive about how retrieved stories were displayed ; retrieved stories that better covered their information needs ; were more satisfied with the stories retrieved ; and were more likely to report an impact of the stories on coping with breast cancer . Three main effects were associated with use of the writer profile search (yes vs no): being more positive about  and more satisfied with the search facility , and being more positive about how retrieved stories were displayed . For satisfaction with the search facility, an interaction effect was found (P = .03): at least one of the two search facilities was needed for satisfaction.\u00a0Questionnaire completers were evenly distributed over the four groups (\u03c7Having access to the story topics search facility clearly had the most positive effect on patient satisfaction and search success. Patients value having access to stories of other patients as it provides them with emotional support, information, reassurance, and practical advice . The IntStudies of online patient stories have focused on several factors. These include why patients publish their stories online and what this means in a broader sociological context -7. Wise Some qualitative studies have found that patients appreciate the ability to select stories of other patients of a particular age or who have opted for similar treatment ,9. In adIn the present study, we examined which search facilities for patient stories resulted in satisfaction with the search process and the stories retrieved. We also studied the impact of the stories retrieved on coping with illness. Our expectation was that having a search facility would be an improvement compared with not having a search facility. Moreover, we expected that a combination of search facilities would result in higher satisfaction than a single search facility because a combination of search facilities may result in more opportunities to find a relevant story.We focused our study on patients with breast cancer. We contacted the board of The Amazones Foundation, which was founded by a group of young women with breast cancer to provide their peers with information and support. The Amazones Foundation developed a website for young women with breast cancer  that proWe were granted permission by the board of The Amazones Foundation to conduct our study. In January 2007 we downloaded all 170 stories available at that time on their website for use in our study. We facilitated access to the stories in three ways: (1) with a search facility for story topics, (2) with a search facility for writer profiles, and (3) with a combination of these two search facilities. In addition, a control group could access the stories by means of the original alphabetical listing by story writer. We implemented these four ways of facilitating access to the stories on a separate study website. This resulted in four groups based on two independent factors: (1) no versus yes search by story topics, and (2) no versus yes search by writer profile . In eachThe present study is reported in accordance with the CHERRIES checklist, which is a checklist for reporting results of Internet e-surveys . It was Recruitment announcements were disseminated online using banners on the websites of several Dutch patient and health organizations and offline using posters and flyers in waiting rooms of several hospitals. Dutch-speaking women with breast cancer were invited to participate irrespective of other personal characteristics. The offline recruitment announcements gave the URL of the study website, and the online announcements contained a hyperlink to the study website. The study was accessible to each visitor of the site, but only visitors who met the inclusion criteria were further directed to the informed consent page. After finishing the final questionnaire, participants could send a ready-made email message with the URL of the study website to other women who might be interested in participation. The study website was accessible in the period June through November 2007. During this time period, women could choose for themselves on what day and time they wanted to participate. No incentives were offered for participation.The first page of the study website provided the following information about the study: study objective, information about the researchers, study inclusion criteria, details about participation, expected time within which to complete the study, and contact details. When a website visitor chose to participate by clicking the button \u201cI would like to participate\u201d, two questions were presented to check whether the visitor met the inclusion criteria . If this was the case, the visitor was asked to read the informed consent statement. By agreeing with the informed consent statement, the visitor declared that her participation was voluntary, that she understood what participating entailed, and that she was aware of what data would be recorded. To agree with the informed consent statement, the visitor had to check the box \u201cI agree\u201d and then click on the button marked \u201cNext\u201d. After this the visitor was asked whether she was certain that she agreed with the informed consent statement. In this way, we assured that women did not automatically agree to participate. Before the participant was randomly assigned to one of the four groups, she was asked to provide a short description of the information she wanted to search for in the stories. Random assignment of each participant to a study group was carried out by an algorithm that was part of the study website. The chance of being assigned to a group was equal for all four groups, that is, 1 in 4 . Once assigned to a group, a participant could search for and read the stories as long as she liked. When finished with searching and reading she was asked to complete a final questionnaire posted on the study website about satisfaction with the search process and the stories retrieved and the stories\u2019 impact on coping with breast cancer.                                                                                                Participants remained anonymous since no log-in, name, or address were required. In order to minimize traces on each client\u2019s computer, no cookies were used. Recording of log data did not start until participants had agreed to the informed consent statement. Questionnaire responses were not saved until participants confirmed at the end of the final questionnaire that they agreed to submit their responses. Data were saved in a password protected SQL database only accessible to the researchers. Participants could stop at any time without receiving pop-ups or text when leaving the study website.Before the start of the study, our research proposal see  was presTo develop the search facilities, all 170 stories were coded according to a coding scheme for story topics and a coding scheme for writer profile . The topParticipants could search for age using the categories: 20-30 years, 30-40 years, 40-50 years, and over 50 years. Participants could search for time since diagnosis using the categories: less than half a year ago, \u00bd - 1 year ago, 1-3 years ago, 3-5 years ago, and more than 5 years ago. To ensure that participants were aware of all search facilities, the search button was placed at the bottom of the page. In the groups with access to a single search facility, it was possible to search for more than one topic or more than one writer characteristic. In the combination group, participants could choose whether they wanted to search for story topics only, for writer characteristics only, or for both. Searching for more items simultaneously was based on the OR Boolean operator.For every search performed by the participants, a weight between 0 and 1 was assigned to each of the 170 stories in the database. If a story matched exactly with the search objectives, it received a weight of 1. Story weights were calculated with every new search. Therefore, the weight assigned to a story could change with every search.In the story topics group, weights were calculated by dividing the number of topics found in a story by the number of topics that were searched for. For example, when a participant searched for four topics, all stories containing one of these four topics received a weight of \u00bc (0.25).In the writer profile group, a weight was assigned to each of the personal characteristics that a participant searched for. These weights were then multiplied with each other to calculate the weight of a story as a whole.If the age of a writer fell in the age category that the participant was searching for, then \u201cage\u201d received a weight equal to 1. The more the age of the writer deviated from the age category that the participant was searching for, the lower the weight that \u201cage\u201d received. In a similar way, weights for \u201ctime since diagnosis\u201d were assigned.If the partner status of a writer exactly matched the partner status that the participant was searching for, then \u201cpartner\u201d received a weight equal to 1. If the partner status of a writer is unknown, then this characteristic received a weight of 0.5 irrespective of the partner status that the participant was searching for. If the partner status of a writer was the opposite of the partner status that a participant was searching for, then this characteristic received a weight of 0.2. We did not assign a weight of 0 for the latter case because then the weight for the whole story would be 0. In similar ways, weights for the other categorical variables were assigned.In the combination group, weight assignment was similar to that of the previous two groups or a multiplication of these two, depending on whether a participant searched for story topics only, for writer profile only, or for both.It was decided to present participants with at least ten stories after each search. The total number of stories presented after a search depended on the distribution of the weights assigned to the stories. All stories with the same weight as the tenth story were presented because we saw no valid reason for presenting only a portion of the stories with that weight. For example, when five stories matched exactly with the search objectives, that is, weight equal to 1, 20 stories received a weight of 0.80, 45 stories received a weight of 0.60, and 100 stories received a weight of 0.40, then 25 stories (5 + 20) would be presented. Accordingly, if no stories exactly matched the search objectives, still at least ten stories were presented. The list of retrieved stories showed the extent to which the stories matched the search objectives. In this way we tried to present participants with neither too few nor too many stories.When participants did not fill in the search page, no stories were presented to them because we wanted to ensure that participants were aware of the search facility.The retrieved stories were displayed as a list giving for each story the writer\u2019s nickname and the story\u2019s weight. Weights were represented as a number of pink ribbons. In addition, the search criteria that were fulfilled were given in each group, that is, the topics found, the writer\u2019s characteristics, or both see . The lisParticipants were asked to provide demographic information such as age, marital status, children, religion, education, and employment status. They were also asked to report characteristics of their cancer, such as time since diagnosis, type of diagnosis, metastases in axillary lymph nodes or other parts of the body, treatment undergone, and prognosis.The participants were asked to indicate their frequency of Internet use, the type of activities in which they engage on the Internet, and whether they had read stories of other patients on the Internet before. Moreover, they were asked to report how often they had visited the Amazones website before, how familiar they were with this website, and how many of the stories on this website they read before.The constructs listed below were used to measure the three outcomes. Cronbach alphas were calculated using SPSS version 16.0  by conducting reliability analyses. Reverse phrased items were recoded. We found the internal consistency for each construct to be good or satisfactory . The items \u201coverall satisfaction with the search facility\u201d and \u201coverall satisfaction with the stories retrieved\u201d were answered using 10-point Likert scales; all other items were answered using 5-point Likert scales. For an overview of the items belonging to all the below\u00a0mentioned constructs, see the To measure satisfaction with the search process, 13 items were formulated . \u201cOpTo measure satisfaction with the stories retrieved, 18 items were formulated . \u201cOp\u201cThe stories\u2019 impact on coping with breast cancer\u201d was measured with 6 items  which were based on an extensive literature on coping -21. Two We tested the usability and technical functionality of the study website, including the final questionnaire, multiple times, and we solved all appearing errors. During participants\u2019 search processes, log data recorded how long participants surfed on the study website, how many searches they performed, how many stories they accessed, and how long the text of the stories was displayed on the screen. In the control group, clicking on a folder (A to Z) was regarded as performing a search, and subsequently clicking on a name was seen as accessing a story. Also, the time participants needed to fill in the final questionnaire was recorded.Participants who were searching for or reading the stories were reminded to fill in the final questionnaire by a yellow figure on the left side of the screen with the text \u201cDo not forget to complete the questionnaire,\u201d which was highlighted every five minutes. Adaptive questioning was used to reduce the number and complexity of the questions. Questions were not randomized or alternated. The final questionnaire was distributed over five pages in the following sequence:(1) the search process, (2) the stories retrieved, including the stories\u2019 impact on coping with breast cancer, (3) use of the Internet and the Amazones website, (4) disease characteristics, and (5) demographic characteristics. When participants clicked on the \u201cNext\u201d button at the end of a page, JavaScript was used to check for completeness. Unanswered questions were highlighted, and participants were asked to answer these. Yet, answering was not enforced, since by clicking on the \u201cNext\u201d button again, the next page was reached. Participants were not able to review and change their answers in previous parts to prevent a possible influence of questions asked later in the questionnaire.Log data and questionnaire responses were saved automatically in an SQL database. In preparation for data analysis, sessions from the same IP address with a time interval of less than 20 minutes were merged, and those with a time interval of greater than 20 minutes were kept as two separate sessions. We assumed that in the former case the sessions were from the same participant and, in the latter, from different participants. Applying the first rule resulted in 23 merged sessions; the latter rule was applied to 6 pairs of sessions. Merging was possible because in all cases the questionnaire was filled out only once. Participants were only distinguished by IP address. A particular IP address was always assigned to the same intervention group. This was done to prevent women from participating multiple times when trying to get in another study group.The data were imported into SPSS version 16.0. Differences in the log data between questionnaire completers and noncompleters were assessed using Mann-Whitney tests. The noncompleters were excluded from further analyses, since no questionnaire responses for this group were available. For the completers, there was no time frame for filling in the questionnaire. Differences between the four groups in baseline characteristics were assessed using Chi\u00a0square tests, 1-way ANOVA, or Kruskal-Wallis tests (depending on variable type and skewness).Kruskal-Wallis tests were performed to assess differences between the four groups in search behaviour . Significant differences were examined further by performing post hoc tests. We chose to use Mann-Whitney tests with a Bonferroni correction, and as as the critical level of significance we used .05/6 = .008 because with four groups six comparisons were performed.P values above .05 were considered not significant.For each construct of the three outcome measures  a mean total score was calculated. A higher mean indicated a higher satisfaction or impact respectively. The effects of the search facilities on the constructs of the three outcome measures were examined using ANOVA with two independent factors  to assess possible main and interaction effects. This analytical approach was chosen in order to examine the effects of the two search facilities both independently and in combination. 2                    3= 3.7, P = .30). The mean time that participants needed to fill in the final questionnaire was 15.3 minutes . In comparison with questionnaire noncompleters, questionnaire completers spent less time visiting the study website , but completers performed more searches , accessed more stories , and their mean reading time per story was longer .Informed consent was given by 353 people, of whom 182 (51.6%) completed the final questionnaire . No sign                                        P = .008 due to Bonferroni correction) showed that compared with the control group, fewer stories tended to be accessed in both the writer profile group (P = .01) and the combination group (P = .02). In addition, in the control group, the mean reading time per participant per story was shorter than in the writer profile group (P = .007) and tended to be shorter compared with the story topics group (P = .02) and the combination group (P = .009).                    P < .05). Having access to the writer profile search facility compared with not having access to this search facility resulted in a significantly more positive opinion about the search facility (1a), and in a significantly higher overall satisfaction score (1e).                    P = .90). However, when participants could not use the story topics to search the stories, they were more satisfied with having access to the writer profile as a search facility  compared with not having access to any search facility . The effect on satisfaction of having access to the writer profile search facility when not having access to the story topics search facility was significant (P = .009).An interaction effect was found for the overall satisfaction score (1e). When participants could search using the story topics, they were satisfied with this search facility regardless of whether  or not  they could also search with the writer profile. The effect of having access to the writer profile search facility when also having access to the story topics search facility was not significant (Having access to the story topics search facility resulted in a more positive opinion about the list of stories displayed after a search (2c), a greater extent to which the stories retrieved covered one\u2019s information need (2d), and a higher overall satisfaction score with the stories retrieved (2f) compared with not having access to this search facility . Having There were no interaction effects observed in satisfaction with the stories retrieved.                    To our knowledge, this study is the first randomized controlled experiment with a 2x2 factorial design that examined search facilities for accessing online patient stories. We observed that the story topics search factor had a strong impact on patient satisfaction and search success: participants were the most satisfied with this search facility and the stories retrieved. Also, the stories retrieved had a greater impact on coping with breast cancer. The effect of the writer profile search factor was limited. This search facility resulted only in a few effects, predominantly on satisfaction with the search process. The two search factors combined generally had no amplified effect on patient satisfaction or search success as we only had one significant interaction.These findings are contrary to our expectation, which was that the combined search facilities (the interaction) would outperform a single search facility because this combination is more complete and differentiated resulting in greater opportunities to find a relevant story. Apparently, this quantity argument seems to be less important than the type of the search facility . In line with our expectation was that a single search facility was an improvement compared with the alphabetically listed stories in the control group.Participants in the three search facility groups accessed fewer stories and read longer per accessed story compared with the control group. An explanation for this might be that the stories retrieved in the search facility groups were more relevant to the participants. A search facility probably increases the proportion of the documents retrieved relevant to the user's information need .The story topics search facility resulted not only in participants being more satisfied with the search process, but also in participants retrieving stories that better covered their information needs and retrieving stories from which they learned more. Patients might use online stories predominantly for information, and, therefore, the topics described in the stories might be more important for them than the writer\u2019s profile. Patients\u2019 profiles might be more important when seeking face-to-face contact. This difference between seeking information and seeking contact has also been noted by Bennenbroek et al  in theirOur observation that the writer profile search facility compared with not having this search facility resulted in a more positive opinion about the search facility and in a higher overall satisfaction with the search facility is in line with the results of Rozmovits and Ziebland . They foA considerable number of participants performed searches but did not complete the questionnaire. Compared with completers, noncompleters spent more time on the study website while they performed fewer searches, accessed fewer stories, and spent less time reading per story. Noncompleters might not have been sure about how to use the search facilities, or they might not have been as interested. Yet, we could not empirically evaluate these hypotheses nor perform any statistical analyses, since we had no further information about noncompleters. The number of completers and noncompleters was evenly distributed over the four study groups. Therefore, we believe that potential bias equally affected all four groups. In addition, the direction of the bias is probably twofold: dissatisfied participants might have stopped or they might have completed the questionnaire to express their annoyance.More than half of the participants who completed the questionnaire (63.7%) had previously visited the Amazones website. This could introduce bias because participants familiar with the original disclosure of stories might be especially satisfied with the new search facilities. However, frequency of visiting the Amazones site, knowing the site \u201crather well\u201d or \u201cwell,\u201d and the number of Amazones stories read before, were all evenly distributed over the four study groups. Therefore, we do not think this previous experience with the Amazones website affected the results.Since the experiment was conducted completely online , we cannA limitation of the design was a possible confounding between type of search facility  and number of search options . The story topics search might have been more appreciated because it was more extensive than the writer profile search. However, an argument against this reasoning is that the most extensive search facility  was not the most favourite.In addition, one could question the content of the search facilities. Were the most appropriate topics and personal characteristics included in the facilities? Yet, the topics and characteristics we used were chosen based on other websites containing breast cancer stories -13 and oA final limitation is that participants may have been annoyed when stories were presented that did not exactly match their search objectives. However, a search resulting in no stories could also be a cause of annoyance. This is why we chose to present at least ten stories after each search. In order to ease interpretation of the resulting list of stories, weights (as pictured in the form of pink ribbons) were used to indicate to what extent a story matched with the search objective.Earlier studies have shown that patients can benefit from stories of other patients, and that the Internet is an important source of these stories. Our current study suggests that a story topics search facility would be most helpful to patients. With a story topics search facility, participants were better enabled to find the information they were looking for. Also, they retrieved stories that more closely covered their information needs and they learned more from the stories retrieved.Thus, patient organisations or website developers that offer patient stories on their websites can best provide access to them using a story topics search facility. However, constructing such a search facility is very time consuming and labour intensive since stories have to be coded for content. An efficient method might be to use a system analogous to social bookmarking/tagging  in which"}
+{"text": "A case-control, quasi-experimental study was designed (post-test only) to investigate the effect of a performance-based incentive payment scheme on behaviours of public-sector service providers in delivering a basic package of maternal and child-health services in Egyptian primary healthcare units. The results showed significant improvements in the quality of family-planning, antenatal care, and child-care services as reported by women seen in clinics where the incentive payment scheme was in operation as measured by various indicators, including both technical and inter-personal communication content. An analysis of characteristics of the service providers and clients found no significant or meaningful differences between the study groups, and the facilities of both the study groups were essentially the same. Some findings are suggestive of other influences on behaviours of the service providers not captured by the data-collection instruments of the study. Subsequent to this study, the payment scheme has been rolled out to other districts in Egypt. There is an extensive literature on different types of payment methods for healthcare providers and the effects of incentives on organizations and individuals in the healthcare system , 2. The Incentives are known to elicit complex responses from physicians, inducing changes in the number of hours worked for the number of beneficiaries seen per hour, the location of their work, and the type of service provided to a patient , 4. The There are also other risks in the use of incentive payments. For example, performance-based incentive payment schemes increase the level of administrative costs because these require data on the number and type of services provided and have been criticized for these hidden costs , 10. ThuDespite these other well-known difficulties and risks, payments of salary (either in part or whole) that are linked to performance measures are attractive policy options as a means to improving the quality of service and gaining efficiencies.Beginning in the middle of the 1990s, the Government of Egypt began to openly grapple with solutions to pressing problems that the piecemeal approach to reform used previously had failed to resolve. There were significant equity problems in access to services, by both income and geographical groupings, and public spending on health was regressive . The orgThe 1997 Health Sector Reform Strategy Paper responded to these challenges by setting a long-term vision of universal coverage with basic health services for all citizens . The pilAt the close of the HSRP pilot phase, several important initiatives had been introduced (e.g. basic benefit package) while other reforms had not yet been evaluated. Among the later was a set of reforms targeting payments to healthcare providers that were developed in part a response to long overdue increases in salaries in the public sector and also as a means for improving the quality of care. Several types of payment reforms were being explored, including contracting mechanisms (both contracting-in and contracting-out for services) using incentive payments linked to performance measures.The Family Health Fund works through the District Provider Organizations to contract with public and private providers to offer the Basic Benefit Package to the covered populations. Initially, the fund was designed to disburse payments on a per-capita basis system but this was soon put on hold as its implementation required substantial modifications to the existing procedures and policy that could not be achieved during the pilot phase. As a consequence, the Family Health Fund shifted towards the use of salary supplements in the form of incentive payments to encourage facilities to maintain certain operating standards and performance targets.Under this scheme, the incentive payments may reach up to 275% times the total base salaries of all personnel working in the Primary Health Center Unit (PHCU). The payments are metred according to performance measured against a set of standardized indicators and rating criteria . The indThe performance indicators are weighted differentially to encourage service providers to give more attention to indicators of priority programmes, e.g. family planning and immunization. A numeric score which forms the basis for calculating the actual amount of the incentive to be disbursed to each service provider according to a weighting system that differentiates between three categories of staff in each facility: healthcare providers (physicians and nurses), administrative staff, and clerks.The incentive payment scheme was being phased into selected District Provider Organizations and PHCUs at the close of the HSRP pilot phase in 2004. In settings where the incentive payment scheme was not being introduced, all service providers of the Ministry of Health and Population (MoHP) were receiving the same amount of salary supplement but as a top-off of their regular salary, i.e. not based on any performance assessment. This phased-in approach to implementing the incentive payment scheme created a natural experimental setting for studying the effects of the incentive payment compared to a flat-rate salary supplement.This study aimed at testing the hypothesis that providers who receive an incentive payment will provide better-quality services and be more responsive to the healthcare needs of their clients than providers who receive a salary supplement that is not linked to performance.The study used a quasi-experimental post-test only comparison group design that tested the following hypothesis: Providers who receive the incentive payment will provide better-quality reproductive health services and be more responsive to the clients\u2019 needs for reproductive healthcare than providers who received a salary supplement not linked to performance.Results on indicators relating to the performance of service providers and patient outcomes in primary healthcare units where providers received incentive payments were compared with results from primary healthcare units where providers did not receive incentive payments but did receive an equivalent amount as salary top-off. The study included indicators used by the incentive payment scheme and other measures of satisfaction of patients and quality of clinic services.Two (Menoufia and Suhag) of the five Governorates that piloted the incentive payment scheme were purposively selected for this study based upon considerations of location (Lower and Upper Egypt) and length of time that the payment scheme had been in place (e.g. more than 2 years). The two Governorates were selected because they each were working with District Provider Organizations .Within each Governorate, a single district recognized as being the most active and engaged with the implementation of the HSRP was purposively selected for the study sample: El-Maragha health district in Suhag and Quesna health district in Menoufia. This was done to ensure the likelihood of finding the impact of the incentive payment scheme and to hold constant system-wide improvements that could influence the quality of care provided in the study sites.In each of the selected districts, all the PHCUs offer the same basic benefit package of services and have taken part in all other elements of the HSRP to equal measure  but only some have been using the incentive payment scheme. The incentive payment clinics were somewhat better finished than the non-incentive payment PHCUs, e.g. a larger waiting-room and newer benches in the waiting-area. However, all the PHCUs in both the study groups were rated equal by the quality accreditation scheme of the MoHP. Thus, the most salient difference between the two types of PCHU in the study sample is the incentive payment scheme. The former units represent the frame for the intervention units of the study while the latter units are the frame for the comparison group. In total, four PHCUs were selected for the intervention group, and four PHCUs were selected for the comparison group.Within each of the selected PHCUs in each district, all the clinic physicians were interviewed using semi-structured, qualitative discussion guides. The managers of the District Provider Organization were also interviewed. The study sampling frame included all consenting women of reproductive age (15\u201349 years) at the selected PHCUs. These interviews were conducted upon the patient's exit from the clinic using a standardized quantitative questionnaire administered by a trained interviewer.Eighty-one healthcare providers\u201452 in Menoufia Governorate and 29 in Suhag Governorate\u2014were interviewed. Of them, 46 were males and 35 were females. Other than gender, there were no significant differences in the professional or personal characteristics between the physicians in the incentive payment scheme sites and the comparison group, although physicians in the incentive payment sites were somewhat more likely to be younger and have a higher educational degree. Importantly, there were no significant differences in the training programme experiences between the service providers in each study group.In total, 2,414 women were interviewed. Approximately, an equal number (600) was interviewed in each study group by Governorate . Overall, there were no significant differences between the two study groups by age, educational level, working status, or education and working status of their husbands. There were significant differences (p<0.05) between the two study groups by age at first pregnancy, number of living children, number of living sons and girls, and previous history of miscarriage and by the wealth quintiles. For example, women in the incentive group were more likely to have had their first pregnancy at a later age, have fewer children, suffered fewer deaths of children, and be of slightly higher economic status  than women in the comparison group.The volume of services seen in the study clinics during the 11-month period (January-November 2006) before data collection was reviewed to detect any differences in case-load. The average number of reproductive health patients seen per month per PHCU was approximately twice as high in the non-incentive payment scheme units . However, the number of consultations not related to reproductive health was higher in the incentive payment sites, resulting in no significant difference in the total number of consultations for all reasons between the two study groups. Anecdotal information collected during the study suggests that the lower reproductive health case-load in the incentive group can be attributed to the influence of a large general hospital in one Governorate located nearby the incentive payment scheme PHCU. The consultation fee at the hospital is one-third the cost of care at the incentive payment scheme PHCU. There are no other fees charged , and clients are seen by a specialist doctor. A general practitioner sees clients in the incentive scheme units, and the fee includes only 50% of the prescribed medicine cost.The primary reason for visiting the PHCU during the data-collection period of the study is shown in The incentive payment scheme had a clear impact on the performance of family-planning care providers, with significant differences observed with regard to better history-taking, less laboratory investigations, more follow-up visits, and more information about the available family-planning methods . In addiThe incentive payment scheme had several positive effects on the quality of child healthcare services, which was the most common reason for having visited the PHCU during the study. In total, 1,286 women attended the selected units to obtain care for a child during the study , and more likely to ask their patients if they had any questions and encourage them to return for a follow-up.The results of the interviews with the physicians in the PHCU and the district healthcare officers (District Provider Organization) revealed mixed feedback on the design and functioning of the incentive payment scheme. The main highlights of these views were as follows:Too many indicators were used in the calculation of the incentive, and the level of details was overly micro in focus.The indicators were established by national-level decision-makers without consulting local administration. This caused many problems during the implementation as both district-and facility-level care providers needed time to understand the construction of the payment scheme and the rationale for using the selected indicators.Calculations of percentage of the incentive and reasons of its reduction were not clear to most physicians, reflecting an overly-detailed approach.The delays in receiving incentives created an atmosphere of distrust and uncertainty.The phased introduction of the scheme caused some confusion with the overall management of the district\u2014different approaches should be adopted for launching the scheme.The incentive scheme improved those things which scored points in the scheme, such as record-keeping (which was significantly better in the incentivised clinics) and the non-clinical aspects of behaviours of doctors, such as the clarity of their communication and listening to their clients. Although improvements in the quality of care were associated with the introduction of the incentive scheme, the study also showed overall low levels of quality in both the study groups, e.g. women reporting clear communication with the care providers. Sustained attention is required for the continual improvement of quality of service, and greater efforts are needed for consumer education that will empower patients with skills to gather the type of information needed to ensure compliance and the sustained use of contraceptive methods.Contextual differences also emerged in the findings of the study, indicating the limitations of an incentive payment scheme in overcoming external influences on behaviours of the care providers or how management and supervision may also exert an influence on behaviours of the care providers. For example, for many key quality-of-care indicators, the clinics in Suhag Governorate scored higher than those in Menoufia, regardless of whether they were or not in the incentive scheme. These differences between the Governorates are suggestive of other factors influencing behaviours of the care providers beyond the incentive payments. The study did not explore the possibility of how the incentive payment scheme may have changed the overall configuration of services, perhaps leading to a \u2018crowding-out\u2019 of other, non-incentivized services which can occur as the providers focus on services that are linked to the incentive payments and not on other, non-incentivised services.Although the doctors and managers were supportive of the incentive payment scheme, they complained that they were not fully consulted during its design. Consequently, they felt that the scheme was too complicated and that the weights given to different indicators were changed too often.The results from the early introduction of the incentive payment scheme in Egypt are highly suggestive that the care providers do respond to incentives that are carefully integrated into a well-known and established quality of the care-monitoring system. Although the introduction of the new payment scheme was cumbersome for the district officials and caused confusion for the physicians working in the scheme's sites, overall, the experience has been positive. The incentive payment scheme continues to be used in Egypt, expanding into other Governorates as an important element of the national health-sector reform programme."}
+{"text": "Deltavirus genus, which consists of 7 differentiated major clades. In this study, an eighth clade was identified from 3 distinct strains. Deltavirus genetic variability should be considered for diagnostic purposes. Clinical consequences of the diversity have yet to be evaluated.Hepatitis delta virus is the only representative of the  AX741144) to 1,697 nucleotides  (Hepatitis delta virus (HDV) is a subviral agent that can lead to severe acute and chronic forms of liver disease in association with hepatitis B virus. Delta hepatitis is highly endemic to several African countries, the Amazonian region, and the Middle East, while its prevalence is low in industrialized countries, except in the Mediterranean. The HDV genome is a circular, single-stranded RNA virus that ranges from 1,672  is found in Japan  . Phylogenetic analyses were performed with PAUP*4.0\u03b210  from a SOAP sequence alignment that excluded 833 unstable characters. Neighbor-joining (NJ) distance and maximum parsimony (MP) analyses were performed. The robustness of the topologic features was determined by bootstrap methods (103 replicates for NJ and MP). A Bayesian approach  of 15 and a gap-extension penalty (GEP) of 6.66 and 1 with the SOAP program  was identified from 3 complete sequences obtained from strains isolated from patients of African origin. Isolate dFr644, originating from Congo-Brazzaville, was initially described by Radjef et al. and tentatively affiliated with HDV-7 , despite a similarity of only 77.8% with the other HDV-7 sequences (R0 region (defined in ,,Since 1999, a total of 468 HDV isolates collected in France were analyzed in our laboratory for phylogenetic characterization of the Deltavirus genus includes at least 8 major clades, with specific geographic distribution. Future development of molecular assays for diagnosis of delta hepatitis should take into account this high genetic variability. The relationship between HDV diversity and pathogenesis has previously been suggested (,In conclusion, the"}
+{"text": "Childhood systemic vasculitides are a group of rare diseases with multi-organ involvement and potentially devastating consequences. After establishment of new classification criteria (Ankara consensus conference in 2008), it is now time to establish measures for proper definition of activity and damage in childhood primary vasculitis. By comparison to adult vasculitis, there is no consensus for indices of activity and damage assessment in childhood vasculitis. Assessment of disease activity is likely to become a major area of interest in pediatric rheumatology in the near future. After defining the classification criteria for primary systemic childhood vasculitis, the next step was to perform a validation study using the original Birmingham vasculitis activity score as well as the disease extent index to measure disease activity in childhood vasculitis. Presently, there are efforts in place to develop a pediatric vasculitis activity score. This paper reviews the current understanding about the assessment tools  widely used for evaluation of the disease activity and damage status of the children with vasculitis. The primary systemic vasculitides (PSV) in children encompass a group of rare diseases that are characterized by the inflammation of blood vessels . AlthougThe majority of children with HSP experience a full and uneventful recovery. Primary outcome measures for HSP are renal survival and presence of urinary abnormalities or hypertension. For unselected patients with HSP, the risk of end stage renal disease (ESRD) is reportedly very low, between 1.5% and 3% .Prognosis for individuals with PAN varies with a better outcome in children compared to adult onset disease where the mortality rate can be as high as 20-30% despite aggressive therapy . Brogan Despite the new immunosuppressive and immunomodulatory therapies, WG, microscopic polyangiitis (MPA) and Churg-Strauss syndrome (CSS) still carry considerable morbidity and mortality mainly due to progressive renal failure or aggressive respiratory involvement. In a recent report by Cabral et al., significant renal involvement was found in 27 out of reported 117 children with WG (41.5%). In the same report, severe respiratory involvement requiring continuous oxygen therapy or mechanical ventilation was observed in 19% and 11%, respectively . The morAs a result of increased survival and well being in these patients, disease classification and assessment have emerged as important considerations. Established classification criteria are necessary in order to proceed with the development of future epidemiological studies and clinical trials in PSV.http://www.clinicaltrials.gov, NCT01066208). Though children and adults share many features of these rare disorders, ACR classification criteria developed for adults were not appropriate for use in children. The patient groups upon which the ACR had based the vasculitis classification criteria did not include children ..49].In 1994, the original version of BVAS was developed by a group of physicians dealing with vasculitis and validated by the same study. Later on, it was refined as BVAS v2 in 1997 ,51. The This instrument has been developed as a complementary measure to disease activity as measured by the BVAS. It has been validated for use in patients with Wegener's granulomatosis and partially validated in patients with hepatitis C virus-associated cryoglobulinemic vasculitis ,53. DEI Pediatric rheumatologists have recently focused on developing outcome instruments in childhood vasculitis. Although BVAS has been validated in a large number of adult patients and accepted as the gold standard for assessment of disease activity, it may not be suitable for use in children with vasculitis, partly because some items are specific to adults. The items of BVAS could be re-evaluated according to the presence and frequency of clinical features of pediatric vasculitides, with some modification of items, deletion of some items and addition of new pediatric vasculitis specific items, as well as a change to the weighted scores. First and foremost is that the normal reference ranges in children are different from adults such as blood pressure, serum creatinine, amount of proteinuria, glomerular filtration rate or cut-off for significant weight loss. In addition, disease presentations as well as some clinical features and outcome are different in children compared to adults. Arthritis is more common in children with HSP and renal involvement usually increases with age . WG is lAn ideal assessment tool is the one that can differentiate active disease from damage or treatment complications and predict the functional outcome. Presently, there are efforts in place to develop a pediatric version of BVAS that is called pediatric vasculitis activity score (PVAS). The PVAS project, a newly developed tool for the evaluation of systemic vasculitis in children, was built on previous successful international collaboration of the PReS Vasculitis Working Party for childhood systemic vasculitis, in cooperation with members of Childhood Arthritis and Rheumatology Research Alliance (CARRA) from North America. Preliminary work from these investigators has derived a version of PVAS which has been validated in paper cases. Currently, PVAS is in a validation process on real pediatric vasculitis cases that will determine feasibility, reliability, internal consistency and tool responsiveness to change. The PVAS score will be correlated with routine clinical laboratory values such as ESR, CRP and, physician's global assessment of disease activity. The PVAS is meant to help physicians assess an individual patient's response to therapy and also serve as an outcome measures in clinical trials. Thus it might have a positive impact on the ability to research new therapies.PVAS is designed to record new or worsening clinical features of active vasculitis that is attributable to the current state of the disease, after excluding other causes such as infection, hypertension, etc. PVAS includes a set of items that involves nine organ based systems similar to BVAS. The presence or absence of each weighted item gives a maximum score for each organ system. The sum of all nine organ systems determines the total score that shows the disease activity of each patient at the time of evaluation. If the patient is evaluated for the first time by the physician, all features of the disease are considered active/new irrespective of the time period. On subsequent evaluations, time has a more important role in formal distinguishing \"new\" active disease presentation from \"persistent\", low-grade or festering disease. Disease presentation is considered \"active\" if it has been newly present over the 4 weeks prior to the time of evaluation or whether it has worsened since the last evaluation and has been lasting for up to 3 months in total. The paper sheet includes the tick box: \"No new/worse disease\" and should be marked if there is no new/worse abnormality present in any of the systems. On this basis, the calculated score for PVAS will contain only values representing persistent disease. In other words, it represents no new disease and means all of the active features are persistent. If one item among the item list represents either new or worse disease, then the \"No new/worse disease\" box should not be checked.s = 0.80) while correlation with PGA were moderate (rs = 0.49) and poor with CRP and ESR . Responsiveness was good for BVAS (SRM = 1.38) and DEI (SRM = 1.9). BVAS and DEI also seem sufficiently sensitive to detect the change in disease status over time. These two tools have been able to differentiate different vasculitis groups according to their results among childhood vasculitis. The correlation with the physician global assessment of disease activity was moderate in our series, similar to the data reported in the original BVAS paper by Luqmani et al. where the correlation was poor [After the PRINTO and PReS defined the classification criteria for primary systemic childhood vasculitis, they performed the first validation study in this field to use the original BVAS (v.3) and disease extent index (DEI) for the measurement of disease activity in childhood vasculitis . In thiswas poor . With thThe inclusion of PROMs is another standard research practice for rheumatic diseases in adults and children and provides a report of the status of a patient's health condition that comes directly from the patient. There are several examples of disease specific well-defined and reliable instruments in children with JIA such as health-related quality of life (HRQOL), the juvenile arthritis multi-dimensional assessment report (JAMAR), questionnaires for the evaluation of functional ability, and visual analog scales (VAS) for rating of child's overall well-being and intensity of pain. Recently a new tool named simple measure of the impact of lupus erythematosus in youngsters (SMILEY) has been developed for systemic lupus erythematosus (SLE) -61.These instruments can be completed by the patients or parents on their behalf. Despite the development of instruments measuring disease extent, activity, and damage, there are no vasculitis-specific instruments measuring patient-estimated burdens of disease. Herlyn et al. recently published a study of PROMs among patients with vasculitis indicating that there were many manifestations of disease  that were quite important to patients but not measured with the outcome instruments currently included in clinical trials of vasculitis .Patients' subjective experience should be a part of the outcome measurement process since they are the true experts about their disease. Therefore, the importance of the use of PROMs in medical outcome assessment (the OMERACT initiative) is also emphasized by the vasculitis clinical research community. Introduction of patient-based outcome assessments to childhood vasculitis will be one of the future directions.While dealing with disease activity, it is also important not to forget to define the amount of damage present in the patient. In 1997 the Vasculitis Damage Index (VDI) was defined for measuring the amount of damage . It predWhile adult experience provides the backbone of most of the current practice in childhood vasculitis, there are some challenges in treatments and outcome measurement tools. The PReS has established a vasculitis study working group with international collaboration to cope with this problem. On behalf of the PReS and EULAR, PRINTO undertook a multinational, multicenter collaboration to validate BVAS and DEI as the activity measures. Because some of the items in BVAS were not considered appropriate for the pediatric population, the PReS Vasculitis Working Party developed the Pediatric Vasculitis Activity Score \"PVAS\" as a tool to assess disease activity in clinical trials based on the concept of the adult BVAS scoring system. After defining the outcome measures, defining the damage will be an absolute necessity in pediatric rheumatology. Therefore, the PVDI should be developed soon and be an important component of outcome measures to be used in clinical trials of vasculitis treatment in children. Lastly, it is clear that both patient-based outcome assessments and evaluation of the response to treatment during followup are very important as well and better tools for assessment of these parameters need to be developed soon. After the PVAS and PVDI are validated, these tools will be next on the agenda in pediatric rheumatology.PSV: The primary systemic vasculitides; HSP: Henoch-Schonlein purpura; KD: Kawasaki Disease; PAN: Polyarteritis nodosa; WG: Wegener's Granulomatosis; TA: Takayasu Arteritis; ESRD: End stage renal disease; MPA: Microscopic polyangiitis; CSS: Churg-Strauss syndrome; ACR: American College of Rheumatology; EULAR: European League Against Rheumatism; PRES: Pediatric Rheumatology European Society; PRINTO: Paediatric, Rheumatology International Trials Organisation; c-WG: Childhood Wegener's granulomatosis; c-PAN: Childhood polyarteritis nodosa; c-TA: Childhood Takayasu arteritis; JIA: Juvenile idiopathic arthritis; CRP: C-reactive protein; ESR: Erythrocyte sedimentation rate; ANCA: Antineutrophil cytoplasm antibody; CT: Computerized tomography; MRI: Magnetic resonance imaging; PET: Positron emission tomography; EUVAS: European Vaculitis Study Group; VCRC: Vasculitis Clinical Research Consortium; RCTs: Randomized controlled clinical trials; BVAS: Birmingham vasculitis activity score; PVAS: Pediatric vasculitis activity score; CARRA: Childhood Arthritis and Rheumatology Research Alliance; PGA: Physician global assessment; PROMs: Patient-reported outcome measurements; HRQOL: Health-related quality of life; JAMAR: Juvenile arthritis multidimensional assessment report; VAS: Visual analog scales; SMILEY: Simple measure of the impact of lupus erythematosus in youngsters; VDI: Vasculitis damage index; PVDI: Pediatric vasculitis damage index.The authors declare that they have no competing interests.ED is the main author and the corresponding author and has primarily designed and written the article. Coauthors RL, NAA, AK and SO have made substantial contributions to conception and design; have been involved in revising the manuscript and have given final approval of the version to be published. All authors read and approved the final manuscript."}
+{"text": "A/Narita/1/2009 (A/N) was the first H1N1 virus from the 2009 pandemic (H1pdm) to be isolated in Japan. To better understand and predict the possible development of this virus strain, the effect of passaging A/N was investigated in Madin-Darby canine kidney cells, chicken eggs and mice. A/N that had been continuously passaged in cells, eggs, or mice obtained the ability to grow efficiently in each host. Moreover, A/N grown in mice had both a high level of pathogenicity in mice and an increased growth rate in cells and eggs. Changes in growth and pathogenicity were accompanied by amino acid substitutions in viral hemagglutinin (HA) and PB2. In addition, the adapted viruses exhibited a reduced ability to react with ferret antisera against A/N. In conclusion, prolonged passaging allowed influenza A/N to adapt to different hosts, as indicated by a high increase in proliferative capacity that was accompanied by an antigenic alteration leading to amino acid substitutions. In March 2009, a new influenza virus of swine origin, A/H1N1pdm09 (H1pdm), emerged in Mexico and the USA. The virus quickly spread worldwide through human-to-human transmission. In response to the number of countries and communities reporting human cases, the World Health Organization raised the influenza pandemic alert to the highest level (level 6) on June 11, 2009.Genomic analyses were performed on the H1pdm virus as soon as it began to spread rapidly in California and Mexico, and results revealed that it originated from several viruses that had circulated in pigs for a number of years, namely the North American H3N2 triple-reassortant, the classical swine H1N1 lineage and the Eurasian \u2018avian-like\u2019 swine H1N1 virus  or lysinThe prompt production of a vaccine for H1pdm was deemed a global priority in light of the rapid worldwide spread of the pandemic. However, the H1pdm virus was not adapted to growth in eggs. Therefore, in the early stages of the epidemic, classical reassorting processes were used to develop high-growth reassortant vaccine strains X-179A and IVR-153 at the New York Medical College and CSL, Australia. These first vaccine candidates were useful but the yield was only approximately half the amount of antigen typically obtained for a seasonal H1N1 virus . CurrentIn Japan, the first cases of swine flu were confirmed on May 9, 2009 in three people who arrived in Tokyo from Canada . The patFerrets  and mice  were purchaced from Japan SLC  and used in this study. All animal experiments were performed under animal biosafety level 3 conditions. Groups of two ferrets were used to prepare antisera against cell or egg isolates of A/Narita/1/2009 viruses (A/N) in order to evaluate antigenicity. One antiserum for each virus was used because the two ferrets had the same titer. Mice were used to establish mouse-adapted virus and to estimate the lethal dose in mice. All animal experiments were performed in accordance with the Guidelines for Animal Experiments Performed at the National Institute of Infectious Diseases (NIID) and were approved by the Animal Care and Use Committee of the NIID . Virus inoculation and euthanasia were performed under anesthesia with ketamine and xylazine, and all efforts were made to minimize suffering.3-fold with virus isolation medium for subsequent culture in 6-well plates containing 2 mL of virus solution per well. When CPE were observed, all 6 wells of a culture plate were pooled and used in subsequent passages. Egg-isolated A/N was diluted 103-fold with phosphate buffered saline (PBS) and 0.2 mL of virus suspension was inoculated into eggs. After each passage from passages 2\u201315 in eggs, viruses with the highest HA titer were pooled for use in subsequent passages. In addition, viruses passaged 2 or 3 times were propagated in each host to obtain working stocks for this study (A/N-CK or A/N-E). These passaging techniques were described in a manual from World Health Organisation (WHO) Global Influenza Surveillance Network [Egg- or MDCK cell-isolated viruses A/N were used in this study and the accession numbers of each genome segment of A/N isolates are shown in  Network .5 plaque forming units (pfu) of virus under anesthesia. Three days after infection, the mice were sacrificed and their lungs were washed twice by injecting a total of 2 mL PBS containing 0.1% bovine serum albumin (BSA) [Cell-isolated A/N from passage 2 (CK2) was used for subsequent serial passages in mice. Briefly, three 6\u20138-week-old female BALB/c mice  were inoculated intranasally with 20 \u03bcL of culture supernatant containing 7.6 \u00d7 10in (BSA)  as a broTo determine the growth kinetics of the different viral passages in three different hosts, viruses were adjusted to a concentration of 1 \u00d7 HA/mL in PBS. HA titration was performed with 0.5% turkey red blood cells as described by the WHO manual cited earlier . A virusA hemagglutination assay and hemagglutination inhibition assay (HAI) was performed with 0.5% turkey red blood cells (TRBC) using the standard method . Turkey 50 as described previously [50/mL was calculated for each sample using the Reed-Muench method [et al [The virus titer in all viral suspensions was determined with a plaque assay or TCIDeviously . TCID50/h method . The vird [et al . Briefly50), groups of ten BALB/c mice were inoculated intranasally with 20 \u03bcl of 10-fold serial dilutions of A/N in PBS. After a 21-day observation period, the MLD50 values were calculated (as pfu) using the Reed-Muench method [To determine the 50% mouse lethal dose  SWL Viral Genome Segments .p value was less than 0.05.Statistical analysis was performed using the GraphPad Prism statistical software package . Comparisons between more than three groups were performed using non-parametric one-way analysis of variance (ANOVA)  with Dunn\u2019s multiple comparison tests. Data were considered statistically significant if the th, 9th and 15th passages in cell cultures or eggs were assessed  from the first inoculation until the 8th egg passage. Upon the 9th egg passage, however, almost all allantoic fluid samples displayed an HA titer of 256 (28) (data not shown). When A/N was passaged in mice, the virus titer of BAL similarly increased gradually and BAL isolated after the 15th passage displayed a titer approximately 10-fold that of BAL obtained after the 1st passage . Second, the use of cells for production allows vaccines to be used by individuals with egg allergies, and third, cell-based manufacture avoids the selection of egg-adapted variants with altered virus antigenicity [At present, influenza vaccines are largely manufactured using chicken eggs, but a cell culture-based seasonal influenza vaccine is likely to be approved in Japan in the near future. There are several advantages of cell-based manufacturing over current techniques that use embryonated chicken eggs to grow the virus \u201323. Firsgenicity . Howevergenicity . The curgenicity . Earliergenicity ,25. A/Caet al. investigated the adaptation of H1pdm, A/California/04/2009 (A/CA/04/09) and A/Tennessee/1-560/2009 (A/TN/1-560/09), to BALB/c mice and noted amino acid substitutions in the HA protein at positions 155, 183, and 222 for A/CA/04/09 and at positions 119 and 221 for A/TN/1-560/09 [After prolonged passaging in mice, A/N developed a high level of pathogenicity in mice and exhibited faster growth in cells and eggs; however, low HAI titers were detected when using ferret antisera. Ilyushina 1-560/09 . Both st1-560/09 , so ther1-560/09 . The curThis study investigated the effects of passaging A/N multiple times in MDCK cells, chicken eggs, and mice. Results indicated that the virus adapted to these three different hosts and that this adaptation led to an increase in the virus titer. Moreover, viral growth rates increased after viral adaptation to the host in moset instances. The virus that had adapted to eggs or mice also had an increased growth rate in cell culture. The genetic alterations underlying adaptive changes were also ascertained. Taken together, these findings indicate that different virus strains adapt to hosts by similar amino acid substitutions and that viral adaptation occurs in both cell-based and egg-based culture."}
+{"text": "This corrosion behavior was researched using mass-gain, scanning electron microscopy-SEM, laser scanning confocal microscopy-LSCM, X-ray photoelectron spectroscopy-XPS and other techniques. The results showed and revealed that the corrosion was maximal at 60 \u00b0C after 200 h of exposure. The increase of temperature not only affected the solubility of oxygen gas in the thin film, but also promoted the transport of ions (such as Cl\u2212), and the formation of protective AlO(OH), which further affects the corrosion speed.The corrosion behavior of 2A02 Al alloy under 4 mg/cm Atmospheric corrosion results from chemical or physical reactions between a material and the surrounding atmosphere, and is one of the most widely studied topics in the field of corrosion. The corrosion of the Al alloy has been investigated in several studies. However, most of these studies were conducted in a laboratory environment ,11,12,13Al alloys are extensively employed as structural materials in the fields of transportation, electrical engineering, and aerospace applications ,16,17. F\u2212) in the marine atmospheric environmental attack and compromise this passive film, resulting in accelerated corrosion [\u2212 is the most significant factor to accelerate the corrosion rate. That is, chloride plays a critical role in localized corrosion such as pitting corrosion, intergranular corrosion, and stress corrosion cracking [The atmospheric corrosion behavior of Al alloys in various environments has mainly been investigated through field studies ,20,21,22orrosion ,29,30. Aorrosion  studied cracking ,33. \u2212 to cause corrosion of the Al alloy. It is well recognized that atmospheric corrosion takes place only when the metal is wetted [Al alloy corrosion in the marine atmospheric environment is not only affected by the high salt content in the environment, but also by a combination of meteorological and pollution factors . Nishimus wetted . Wettings wetted .2 Cl\u2212 increased with rising temperature. Sharifi-Asla et al. [2 solution containing Cl\u2212. Blucher et al. [Of the key determinants of marine atmospheric corrosion , the effects of different temperatures have been the least systematically investigated. This is despite the fact that the temperature influences the formation of water films on metal surfaces as well as the subsequent electrochemical reactions. High temperatures hinder the formation of water films ,36, in wa et al.  found thr et al.  reportedr et al.  reportedSome recent studies have investigated long-term atmospheric corrosion of Al alloys. Sun et al. ,44,45,46Accordingly, in the present study, laboratory-accelerated modified marine atmospheric corrosion experiments were designed. A special device was built to simulate a marine atmospheric environment identical to the actual service environment of an aircraft engine fan blade. The corrosion behavior of the 2A02 Al alloy was studied at different temperatures in this accelerated modified marine atmospheric environment using mass-gain, scanning electron microscopy (SEM), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDS), laser scanning confocal microscopy (LSCM), electron probe X-ray microanalysis (EPMA), and X-ray photoelectron spectroscopy (XPS) techniques. The effect of temperature on the initial corrosion behavior of 2A02 Al alloy in this marine atmospheric environment is discussed based on these experimental results.The test alloy samples were produced from bare 2A02 Al alloy plates (without Al cladding), with the following chemical composition (in wt %): Si: 0.30, Fe: 0.30, Cu: 2.6, Mg: 2.0, Zn: 0.10, Mn: 0.45, Ti: 0.15, Al: 94.1. The material was cut into coupons with the dimensions 10 mm \u00d7 15 mm \u00d7 2 mm. Before the experiments, the samples were ground with 800 grit SiC paper, degreased and cleaned with acetone and ethanol in an ultrasonic bath, and then dried in flowing cool air. All the samples  were weighed and the surface area (S) was measured before the experiments.2 [The accelerated modified testing involved deposition of a solid NaCl layer on the surface of 2A02 Al alloy samples (not NaCl spray). The deposited NaCl layer was applied to preheat the sample surfaces by repeatedly brushing with a saturated solution of NaCl in water. The surface density of the deposited NaCl was approximately 4 \u00b1 0.2 mg/cm2 ,48. The m), surface area (S) and NaCl solution density  for 5\u201310 min at 80 \u00b0C, after which the samples were rinsed with deionized water [The mass gain measurements were carried out using an analytical balance with an accuracy of 0.01 mg, and the weighing at each temperature included six parallel samples. Taking the weight gain of the exposed samples and uncorroded samples into account, the actual weight gain was obtained. The corrosion products were chemically removed by pickling in the solution  phase consists of Al and Cu, and the AlFeCuMnSi phase consists of Al, Fe, Cu, Mn, and Si. In general, the second phases observed include: the Al-Cu-Mg phase identified as Al2CuMg (S phase), the Al-Cu phase identified as CuAl2 (\u03b8 phase), and the AlFeCuMnSi phase, as illustrated in Longitudinal (L-section), long transverse (T-section), and short transverse (S-section) are the terms conventionally used to label the three directions along the microstructure of 2A02 Al alloy. The surface morphologies of the corrosion scales formed on samples after corrosion at several temperatures for 200 h are shown in In order to get a better understanding of the corrosion behavior of the 2A02 Al alloy, LSCM was used to characterize the substrate surface after removal of the corrosion products. 3, AlCl3, and Al2O3 were present on all samples. For the results at 80 \u00b0C, two diffraction peaks around 2\u03b8 = 14\u00b0 and 2\u03b8 = 49\u00b0 were detected, which are ascribed to AlO(OH). It is noteworthy that AlO(OH) is only detected on the sample at 80 \u00b0C.3, AlCl3, and Al2O3 can be detected on all samples corroded at different temperatures, whereas AlO(OH) was again only detected on samples corroded at 80 \u00b0C.2O3, Al(OH)3, and AlCl3 formed during both time intervals and at all the temperatures, but AlO(OH) was only detected at 80 \u00b0C. By comparing the XRD results for samples obtained after 72 and 200 h at 30, 60, and 80 \u00b0C, it is clear that AlThe corrosion process of 2A02 Al alloy under the thin electrolyte film is subject to the general rule of electrochemical corrosion and also has the characteristics of atmospheric corrosion. The relative humidity was 98 \u00b1 2% in this experiment, and thus a thin water film visible to the naked eye formed on the surface of all samples. The thickness of the thin electrolyte film was very thin, approximately 0.21 mm, which was calculated using Equation (1). In this case, oxygen in the air can diffuse at a high speed through the liquid film to the surface of the metal. Therefore, the overall corrosion process is a couple of reactions: the electrochemical dissolution of the Al alloy and the cathodic reduction of molecular oxygen.2 solid NaCl deposit in 70% humidity at 60 \u00b0C within 200 h. The corrosion occurs along the interface of the second-phase particle, which could be the intergranular corrosion. In this experiment, Mg is exhausted after 200 h of corrosion, as shown by the red circle in Therefore, Al-Cu-Mg and Al-Cu act as the cathode in the electrochemical reaction and the following reaction takes place:The partial anodic reaction occurring at anodic sites (the place around the second phase) during the localized corrosion of Al alloys under the thin electrolyte film is:Thus, as a result of the electrochemical reaction, the concentration of hydroxide ions increases in the localized corrosion sites; therefore, the local pH becomes more alkaline, and the following chemical reaction occurs :Al3+ + 3Meanwhile, it has been generally acknowledged that the presence of chloride ions in an environment leads to pitting corrosion of Al alloys. Several mechanisms have been proposed to illustrate the role of chloride ions in the pitting corrosion of passivating metallic materials such as Al alloys ,53,54.3 identified in this work provides proof that chloride ions react with cations, such as Al3+, to form chloride-containing compounds. Some mechanisms have been proposed to describe the formation of AlCl3 [The AlClof AlCl3 :Al(OH)3 + supports the development of high pH in the cathodic areas, resulting in alkaline dissolution of the alumina passive film and rapid general corrosion.Furthermore, according to Equations (1) and (5), the pH at the corrosion sites will become more alkaline. Therefore, we washed the sample and measured the pH of the cleaning solution by an acidometer. The measured pH was approximately 9.21, which is consistent with the expected alkaline value. NaCl is very corrosive in this environment, because Na3 would further transform into Al2O3 and AlO(OH) [Under some conditions, Al(OH) AlO(OH) :2Al(OH)3AlO(OH) was only found at 80 \u00b0C, not 30 \u00b0C or 60 \u00b0C.It is well known that high humidity can cause liquid films to form on the surface of samples, and that the higher the humidity, the thicker the thin electrolyte film. The thickness of the thin electrolyte film, in turn, affects the diffusion of oxygen and the concentration of salt in the thin electrolyte film. However, in this 98 \u00b1 2% relative humidity environment, salt can be fully dissolved in the liquid film; this is to ensure the samples have the same film thickness and same NaCl salinity under different temperatures. Then, the temperature is the only variable environmental factor. To get a better understanding of the influence of temperature, we discuss the effects of temperature on 2A02 Al alloy corrosion from the following perspectives.\u03b2 is affected not only by temperature but also the composition of the solution. The experimental changeable factor is temperature in the test, and others are stable. Therefore, other factors have not been discussed. According to Henry\u2019s Law, the solubility of oxygen in the thin electrolyte film is related to oxygen partial pressure and the thin electrolyte film temperature at the gas-water interface. The higher the temperature, the higher the partial pressure of water vapor, so that the lower the partial pressure of oxygen results in a lower the content of dissolved oxygen in water. The oxygen diffusivity C in the thin electrolyte film can be estimated by the Arrhenius law. According to the Arrhenius law, the relationship between the oxygen diffusion coefficient and temperature T is C = K Po2exp(\u2212\u03b2/T). When the oxygen diffusion coefficient is calculated, the oxygen diffusivity C can be obtained. It can be seen that the diffusion coefficient increases with increasing temperature.First, the temperature can influence the diffusion of ions in the thin electrolyte film, and especially the oxygen molecules, which directly react via the cathode reaction in local corrosion. Because of the temperature changes, the oxygen solubility and oxygen diffusivity in the liquid film will change, which directly affect the electrochemical reaction of the cathode, and then affect the corrosion. The solubility coefficient The cathodic reaction in Equation (2) is the rate-limiting step of this electrochemical experiment. Therefore, the dissolved oxygen solubility and oxygen diffusivity in the thin electrolyte film can significantly affect the rate of 2A02 Al corrosion. The increase in temperature causes the oxygen diffusion coefficient to increase, and the dissolved oxygen solubility decreases in the thin electrolyte film. Therefore, there is an optimal temperature (in this test it is 60 \u00b0C), at which the dissolved oxygen solubility and oxygen diffusivity are moderate, and in turn maximize the corrosion speed of 2A02 Al alloy. The mass-gain changes with temperature are well associated with the trade-off between decreasing dissolved oxygen solubility and the increasing oxygen diffusivity in the thin electrolyte film.\u2212, or other electric-charged species in the oxide film, which further affects the corrosion speed. Therefore, this is discussed below. In addition to affecting the diffusion of oxygen in the thin film, the temperature can also affect the diffusion behavior of the metal ions, Cl\u2212, and other electric-charged species. High temperatures can also promote the diffusion of the corrosive Cl\u2212 in the thin liquid film to the substrate through the corrosion products, which accelerates the rate of pitting corrosion of Al alloy during the whole corrosion process. \u2212. Rather, Cl\u2212 dispersed throughout the entire corrosion layer. However, an aggregation of Cl\u2212 is then obvious in the inner corrosion layer near the interface between the substrate and corrosion product at 60 and 80 \u00b0C. This aggregation is more significant at 80 \u00b0C than that at 60 \u00b0C. This, once again, proves that Cl\u2212 diffuses faster to the substrate with increasing temperature. Another feature of temperature in the electrochemical corrosion is that the number of the surface reactivity points increases with the increase in temperature for the electrochemical reaction on the surface of 2A02 Al alloy. More active spots thus speed up the reactions (2)\u2013(3) and accelerate the corrosion process. Therefore, the number of pits at 80 \u00b0C is highest in According to the corrosion kinetics curve of 2A02 Al alloy at different temperatures in c values at each temperature, the higher the reaction temperature, the larger Kc, and the easier the reaction to form AlO (OH). Clearly, the more negative values of \u0394G\u00b0 and more positive values of Kc at 80 \u00b0C can make the reaction (6) more easily achievable. From the calculated \u0394G\u00b0 values of reaction (6), it is clear that the reaction will occur when the temperature is more than 30 \u00b0C. The higher \u0394G\u00b0 at 30 and 60 \u00b0C means there will be lower amounts of AlO(OH) at these temperatures, thus accounting for the inability to detect AlO(OH) by XRD at 30 and 60 \u00b0C. At 80 \u00b0C, AlO(OH) is more easily formed, with higher amounts in the inner layer. We have determined that AlO(OH) can penetrate into the pores and cracks in the oxide film on the surface of 2A02 Al alloy through the thin liquid film, and then the colloidal particles close up the pores and cracks, thus slowing down the corrosion rate [ion rate ,59,60. I3. It is well known that Cl\u2212 plays a key role in the chloride-induced pitting model, and that AlCl3 may be formed according to reaction (5). Therefore, the content of AlCl3 contained in corrosion products in this test reflects the degree of the pitting corrosion. Hence, by comparing the relative content of AlCl3 and AlO(OH) in the corrosion products of 2A02 Al alloy formed at different temperatures, we can evaluate the relative rates of the corrosion processes. Reactions (1)\u2013(5) describe in detail the corrosion mechanism of 2A02 Al alloy in the simulated marine atmospheric environment. The relative compositions of the corrosion products directly reflect the reaction conditions during the test. Through the analysis of the corrosion products composition and the electrochemical reactions, we know that a protective product AlO(OH) was produced as well as the destructive product AlCl2O3, Al(OH)3, and AlCl3 formed at all the temperatures, but AlO(OH) only appeared at 70 and 80 \u00b0C. This result is consistent with the XRD analysis. In XPS analysis, all the possible compounds containing Al were deduced from the XPS-peak-differentiation, and the peak area of the possible compound represents its relative content in the corrosion products. However, it should be noted that the relative content of different compounds containing the same element can only be compared in the same sample. In order to give a clearer description about the content of the corrosion products, the intensity curves in the XPS results were processed to be displayed numerically. The compound atomic percentage, which is represented by the XPS-peak-differentiation area percentage, was used to express the content of the corrosion products. The ratio of the AlCl3 peak area to that of the total Al is regarded as the content percentage of AlCl3 in the sample, as shown in 3 in the corrosion products increased with temperature up to 60 \u00b0C. As we know that AlCl3 was produced in the process of electrochemical corrosion (Equation (5)), the involvement of Cl\u2212 as a reactant in Equation (5) is the precondition for pitting corrosion, so the amount of AlCl3 can be used to evaluate the degree of corrosion. The higher the amount of AlCl3, the more reaction (5) may occur, and the more serious the pitting corrosion. Meanwhile, we know the relatively high corrosivity of AlCl3 is explained by the formation of an acidic surface electrolyte and by the high solubility of aluminum hydroxy chloride [3 can accelerate the corrosion of 2A02 Al alloy. The relative content of Al2O3 in corrosion products decreased at first and then increased with increasing temperature, reaching a minimum at 60 \u00b0C. Al2O3 has stable chemical performance, excellent dielectric properties as well as resistance to chemical corrosion. The dense Al2O3 layer thus possesses good corrosion protection properties. The relative content of Al(OH)3 in corrosion products increased at first and then decreased with increasing temperature. The combined XPS results of AlCl3 and Al2O3 clearly show that the corrosion susceptibility of 2A02 Al was highest at 60 \u00b0C. AlO(OH) was mainly detected at 70 and 80 \u00b0C, and has been shown to penetrate and seal up the pit pores, to form a corrosion-resistant layer [The relative content of AlClchloride ,9. Therent layer . Accordi\u2212) and accelerate the formation of Al corrosion products. The formation of AlO(OH) can make the inner layer of the passive film more compact and ultimately hinders further corrosion.In this study, effect of temperature on corrosion behavior of 2A02 Al alloy in a simulated marine atmospheric environment has been systematically investigated. It is found that high temperatures not only decrease the dissolved oxygen solubility and increase the oxygen diffusivity in the liquid film, but also promote the transport of ions  and high content of Al2O3, make the corrosion rate very low. At high temperature (>60 \u00b0C), while the diffusion of Cl is fast, the higher AlO(OH) content causes the inner corrosion layer to be significantly protected, and thus reduces corrosion rate. Therefore, there is a middle temperature, 60 \u00b0C, which has quick Cl diffusion and less AlO(OH) content; hence, the corrosion rate of 2A02 Al alloy at this temperature is maximal.Effect of temperature on the corrosion mechanism is elucidated as follows. At low temperature (<60 \u00b0C), the slower Cl"}
+{"text": "Its corrosion product was Cu2(OH)3Cl, which increased in quantity over time. Cl\u2212 was the major factor responsible for the marine corrosion of copper and copper alloy. Through the nonlinear fitting of corrosion rate and corrosion quantity (corrosion weight loss), degradation data of different corrosion cycles, the quantitative effects of two major factors, i.e., dissolved oxygen (DO) and corrosion medium temperature, on corrosion behavior of copper alloy were analyzed. The corrosion failure prediction models under different ambient conditions were built. One-day corrosion weight loss under oxygenated stirring conditions was equivalent to 1.31-day weight loss under stationary conditions, and the corrosion rate under oxygenated conditions was 1.31 times higher than that under stationary conditions. In addition, corrosion medium temperature had a significant effect on the corrosion of B10 copper sheet.This paper studies the corrosion behavior of B10 copper-nickel alloy in marine environment. Accelerated degradation test under marine environmental conditions was designed and performed based on the accelerated testing principle and the corrosion degradation mechanism. With the prolongation of marine corrosion time, the thickness of Cu Copper-nickel will gradually replace copper to become the mainstream piping material. Many studies have been carried out on copper-nickel alloy, such as corrosion behavior under various environmental conditions, extension of lifecycle, and improvement of corrosion resistance ,3,4,5,6. factors . Kear etnditions . Chen stnditions . Arjmandsolution . In the above-mentioned studies, most scholars studied the marine environmental influencing factors using artificial seawater (3.5% NaCl solution) as the corrosive medium , which iThis paper deals with a laboratory test in natural marine environment based on the accelerated testing principle and the corrosion degradation mechanism in 2O2 was added as the acceleration means, and the corrosion medium temperature was controlled at 30 \u00b0C using a constant temperature water bath. Test cycles included nine cycles: one day, three days, one week, two weeks, three weeks, four weeks, six weeks, eight weeks and 16 weeks. Four parallel samples were used at each cycle, and the test solution was replaced every three days.The test sample used in this study was B10 copper-nickel alloy sheet, and its chemical composition is shown in Samples taken out at various cycles were subjected to surface film observation, micro morphology observation, corrosion product analysis and corrosion quantity/rate determination, respectively. In the surface film observation, the surface microscopic images of samples were observed by scanning electron microscopy (SEM), and the material surface elements of samples were identified by energy-dispersive X-ray spectroscopy (EDS). In the micro morphology observation, surface film was observed and analyzed after removing corrosion products from the sample surface. In the corrosion product analysis, the elemental composition, electronic structure, chemical bonds and elements of material surface substances were analyzed in depth using X-ray photoelectron spectroscopy (XPS). Besides, changes in the structure of surface adsorbed molecules were observed by Raman spectroscopy. Among the four sets of parallel samples, one set was used for surface film and micro morphology observation, while the remaining three sets were used for the measurement of corrosion quantity and corrosion rate.B10 copper-nickel alloy would form a layer of oxide film when immersed in seawater, whose forming quality, density and breakage had significant effects on the marine corrosion resistance of B10 copper-nickel alloy.Macroscopic morphology of B10 was observed during the test. Clean B10 copper sheet surface was silvery white, and after corrosion, it would continuously form an oxide film. etc. that copper alloy substrates originally contain. Nickel content declined slightly, while the oxygen content gradually increased as the corrosion reaction proceeded, indicating the continuation of oxidation reaction on sample surface with the extension of corrosion time.EDS analysis was performed on surfaces immersed for different times, and the results are shown in After full-immersion in indoor simulated seawater and accelerated corrosion of B10 copper sheets for different cycles, the samples were immersed in 10-fold diluted sulfuric acid solution with a relative density of 1.84 for 3 min. Then, the corrosion products were removed with a stiff brush, and the sample surfaces were subjected to micro morphology observation by SEM.The corrosion products of B10 copper-nickel alloy at corrosion different cycles were analyzed by XPS and Raman spectroscopy. 2O series compounds did not have the vibration excitation peak of Cu2p3/2 line, and as can be seen from the Raman spectroscopic analysis on one-week corrosion products, peak positions were at 219 cm\u22121 and 513 cm\u22121 in the spectrogram. Therefore, Cu2O should be contained in the surface layer oxide of the sample after corrosion. As can be from the intensity of peak in the figure, Cu2O film was gradually thickened with the extension of marine corrosion time. Presence of divalent copper could be inferred from the corrosion products where vibration excitation peaks occurred, and generation of Cu2(OH)3Cl could be inferred based on the occurrence of peak positions 219 cm\u22121, 623 cm\u22121 (two weeks), 219 cm\u22121 and 635 cm\u22121 (four weeks) of corrosion products at different times in the Raman spectra. It could be seen from the peak intensity that this product increased continuously over time.As can be seen from +, From the perspective of chemical reaction mechanisms, thermodynamic view indicated that after Cu atom on the B10 copper-nickel alloy surface lost an electron, it left the metal surface into the medium in the form of Cu+ was diffused on the surface of B10 samples, contacted with corrosive factors OH\u2212 and Cl\u2212, and produced complex reaction. The reaction products may include Cu2O, CuCl and CuCl2\u2212, which were Cu\u2212 could more easily react with Cu+ than OH\u2212; moreover, Cl\u2212 concentration was far higher than OH\u2212 concentration in natural seawater, so Cl\u2212 was the main corrosive factor responsible for marine corrosion of copper and copper alloy. As CuCl2\u2212 was easily soluble, yellow-green water film could easily be formed on the sample surface. During the early stage of corrosion reaction, Cu would be oxidized and dissolved in the monovalent form to produce de-copper corrosion of alloy. With the dissolution of copper, Cl\u2212 concentration at the interface of alloy and seawater markedly decreased, making continuation of 2O, 2O generated attached on the sample surface to form a reddish brown oxide film.At the same concentration, Cl2O had P-type semiconductor defect loose structure, when the reaction proceeded to a certain stage, the defect sites of oxide film reacted with the dissolved oxygen and chlorine ions in seawater to produce loose and porous blue-green basic copper chloride, Since CuIn order to study the effect of dissolved oxygen on the corrosion behavior of B10 alloy, a control group where electromagnetic air pump was used for oxygenation and stirring was set up on the basis of the above tests, while the other test conditions remained unchanged.Microscopic corrosion morphologies of B10 copper sheets after corroding for different times under oxygenated stirring and stationary conditions are shown in Under both conditions, the corrosion product films on sample surfaces had cracking and local exfoliation. This may be because the substrates under corrosion product film were located at the grain boundaries, twins or other defects, and due to structural and compositional changes, the growth rate of corrosion product films in these places was slower than the growth rate of inner crystal films. It may also be the greater internal stress of corrosion product film than the film strength that caused cracking of films.In order to study the effect of medium temperature on the corrosion behavior of B10 alloy, the control groups where medium temperatures were 45 \u00b0C and 60 \u00b0C were set up on the basis of the above tests, while all the other test conditions remained unchanged.As can be seen from Corrosion rates under full cycle test conditions were fitted using power function, which has been proven to be effective and accurate for corrosion study . The corFrom the perspective of corrosion weight loss, the corrosion weight loss models under oxygenated stirring and stationary conditions were:Let the corrosion weight losses, w, in the two equations equal, the following conversion relation could then be obtained:Thus, one-day corrosion weight loss under oxygenated stirring conditions was equivalent to the 1.31-day weight loss under stationary conditions, while the corrosion rate under oxygenated conditions was 1.31 times higher than that under stationary conditions, so the corrosion rate acceleration factor was \u03b1 = 1.31 under oxygenated conditions.vice versa.Based on the above results, the rate and quantity of corrosion under oxygenated and stationary conditions could be predicted separately. Corrosion quantity under oxygenated conditions could also be obtained according to the acceleration factor given known corrosion quantity under stationary conditions, and Corrosion weight loss and corrosion rate of B10 copper sheet under different medium temperature conditions were determined by weight loss method, and their changes over time are shown in It can be seen from the trends of changes in the average corrosion rate of B10 copper sheet under different temperature conditions over corrosion time that at the beginning of the reaction, the higher the temperature, the greater the corrosion rate of B10 copper sheet. As shown in Corrosion rates of the entire cycle under different test conditions were fitted using power function , and theR2 was the goodness of fit of the equations.From the perspective of corrosion weight loss, the corrosion weight loss models under corresponding conditions were as follows:Corrosion rate and quantity at different temperatures could be predicted according to the above models to obtain the specific corrosion quantity and rate values at a certain time in the future, as well as the time required for reaching the maximum corrosion quantity.2O2 contributed to the passivation of B10 copper sheet surface while catalyzing and accelerating the cathodic depolarization, thereby thickening the corrosion product film on sample surface, and hindering the corrosion reaction.There were a variety of factors attributable to the decrease in average corrosion rate of B10 copper sheet with increasing temperature. The phenomenon was mainly associated with the dissolved oxygen and the sample surface film. High temperatures could accelerate the decomposition of hydrogen peroxide, on the first day of corrosion, seawater under high temperature conditions contained rich dissolved oxygen, moreover, high temperatures would also accelerate gas diffusion, allowing the corrosion rate of B10 sample at 60 \u00b0C for one day far higher than that under other conditions. As the corrosion reaction proceeded, the dissolved oxygen content in high temperature seawater declined rapidly. Additionally, H2O film thickens gradually with the extension of marine corrosion time. Its corrosion product is Cu2(OH)3Cl, which increases in quantity over time. Cl\u2212 in it is the major corrosive factor responsible for the marine corrosion of copper and copper alloy.Summarily, it could be seen from the presentation above that CuComparative analysis of tests between oxygenated stirring and stationary groups find that the corrosion morphology and products are very similar between two test conditions, except that the corrosion degree of B10 copper sheet is severer under oxygenated stirring conditions than under the stationary conditions. The acceleration factor of oxygenated stirring conditions with respect to the stationary conditions is 1.31. Oxygenated stirring during the accelerated degradation test can achieve better acceleration effect.Based on the experimental results, corrosion prediction models under different environmental conditions were constructed. More specifically, corrosion medium temperature has a significant effect on the corrosion of B10 copper sheet. Corrosion rate is quantitatively measured after observing the stabilization of corrosion from the microscopic morphological perspective. With the increase in corrosion medium temperature, the corrosion rate decreases continuously, which is consistent with other copper-nickel alloy."}
+{"text": "Central nervous system tumours constitute 25% of all childhood cancers; more than half are located in the posterior fossa and surgery is usually part of therapy. One of the most disabling late effects of posterior fossa tumour surgery is the cerebellar mutism syndrome (CMS) which has been reported in up to 39% of the patients but the exact incidence is uncertain since milder cases may be unrecognized. Recovery is usually incomplete. Reported risk factors are tumour type, midline location and brainstem involvement, but the exact aetiology, surgical and other risk factors, the clinical course and strategies for prevention and treatment are yet to be determined.This observational, prospective, multicentre study will include 500 children with posterior fossa tumours. It opened late 2014 with participation from 20 Nordic and Baltic centres. From 2016, five British centres and four Dutch centres will join with a total annual accrual of 130 patients. Three other major European centres are invited to join from 2016/17. Follow-up will run for 12\u00a0months after inclusion of the last patient. All patients are treated according to local practice. Clinical data are collected through standardized online registration at pre-determined time points pre- and postoperatively. Neurological status and speech functions are examined pre-operatively and postoperatively at 1\u20134\u00a0weeks, 2 and 12\u00a0months. Pre- and postoperative speech samples are recorded and analysed. Imaging will be reviewed centrally. Pathology is classified according to the 2007 WHO system. Germline DNA will be collected from all patients for associations between CMS characteristics and host genome variants including pathway profiles.Through prospective and detailed collection of information on 1) differences in incidence and clinical course of CMS for different patient and tumour characteristics, 2) standardized surgical data and their association with CMS, 3) diversities and results of other therapeutic interventions, and 4) the role of host genome variants, we aim to achieve a better understanding of risk factors for and the clinical course of CMS - with the ultimate goal of defining strategies for prevention and treatment of this severely disabling condition.Clinicaltrials.gov: NCT02300766, date of registration: November 21, 2014. Central nervous system (CNS) tumours account for 25% of all cancers in children and over half of these are located in the posterior fossa . For mosThe spectrum of CMS definitions varies greatly , 12\u201314, Cerebellar mutism is thought to be caused by bilateral disturbance of the dentate nuclei and/or their efferents , 20\u201324. Known risk factors are brainstem involvement by the tumour, midline location and tumour type; thus the incidence in children with medulloblastoma is two to three times higher than for astrocytoma or ependymoma but the biological mechanisms behind these associations are uncertain [proposed risk factors include brainstem compression by the tumour, pre-operative language impairment, low socioeconomic level of the families and left-handedness [uncertain risk factors, as previous studies have been inconclusive [unlikely risk factors [ncertain , 32\u201335. ndedness , 36\u201338; nclusive , 40. Gen factors , 42.In traumatic brain injury common host genomic variants are related to the severity of symptoms and degree of recovery \u201345. SimiSupportive speech and rehabilitation therapy is often offered to patients with CMS, but the benefit hereof has not been demonstrated. No publications exist on systematic approaches to pharmacological neuroprotection, and pharmacological interventions are only sporadically reported in the literature \u201350. GlucClinical factors: gender, age, handedness, speech, language and neuropsychological abilities before and after surgery.Tumour factors: histological tumour type and tumour locationSurgical factors: Surgical strategy and surgical trauma including access routes, removal technique, tissue and vascular injury, bleeding and primary surgery vs. re-operationNon-surgical interventions: glucocorticosteroids, other symptomatic medication and chemo- and radiotherapyHost genome variantsThe study focuses on the risk factors for development and severity of CMS including surgery  and host genome variants. The aims of this study are thus to describe differences in incidence, severity and clinical course of CMS related to:Clinicaltrials.gov (file NCT02300766) and EANS . All children younger than 18\u00a0years with a tumour in the posterior fossa requiring surgery or open biopsy at one of the participating centres will be included following informed consent. Patients who have received surgery, chemotherapy and/or radiotherapy previously are also eligible. The study will run for five years with a targeted sample size of 500 patients. It opened late 2014 with participation from 20 Nordic and Baltic centres. From 2016, five British centres and four Dutch centres will join leading to an expected annual accrual of 130 patients. Three other major European centres are invited to join from 2016/17. The target of 500 patients is expected to be reached in 2018. Patients will be followed for 12\u00a0months after inclusion of the last patient, and the study will thus be completed during 2019.The participating centres provide surgery and supportive care according to local practice and register all study information in an online database developed specifically for this study. Consensus concerning the study aims, study design and data registration was achieved at three international planning meetings among the initiating centres during 2013. The annual enrolment from each country will be compared to the number of registered patients in the national cancer registries to document the inclusion rate and representativeness.This open observational study is registered at The primary endpoint is incidence and severity of CMS. Symptoms and severity are scored according to the CMS survey published by Robertsons et al. . Our maiThe secondary endpoint is incidence of \u201creduced speech output\u201d defined as \u201cseverely reduced speech production limited to single words or short sentences which can only be elicited after vigorous stimulation\u201d . The risFurthermore, we want to explore the following:We will analyse the role of host genome variants on development, severity and recovery from CMS by carrying out broad genetic pathway profiling of all study participants using both non-CMS cases from the study cohort and non-CNS tumour patients as controls. Genotyping will use single nucleotide polymorphism (SNP) exome enriched arrays (e.g. Illumina Omni2.5-exome platform). We will apply agnostic genome-wide association studies (GWAS) as well as more complex pathway analyses. Thus, we will interrogate combined effects of multiple SNPs acting in the same pathways or protein-protein interaction complexes using our validated non-linear machine learning algorithm  , 54, whiThe possible effects of chemo- and radiotherapy on recovery from CMS will be investigated. We hypothesize that chemo- and radiotherapy delay recovery from CMS. For descriptive documentation purposes we also ask for information on medications given specifically to treat the symptoms of CMS.We hypothesize that glucocorticosteroids 1) given preoperatively protect against CMS due to reduced oedema; 2) given intraoperatively increase the risk of CMS due to worsening of acute neurological injury by hyperglycaemia; 3) given postoperatively negatively affect the course of CMS as earlier studies have shown a negative effect of glucocorticosteroids on the outcome of traumatic brain injury , 56. It The incidence of the CMS will be correlated to tumour histology using the 2007 WHO classification. We hypothesize that the risk of CMS is highest among patients with medulloblastoma. With increasing focus on subtyping of medulloblastoma  this addTumour location, enhancement pattern, invasiveness and growth velocity may affect the risk and severity of CMS , 18, 58.We will determine whether the risk of the CMS varies according to handedness. We hypothesize that the risk of CMS is increased in left-handed patients, and possibly even more so in patients with medulloblastoma .Our hypothesis that preoperative speech and language impairment increases the risk of postoperative speech and language deficits will be explored by recording pre- and postoperative speech  and language  statuses and relating these to incidence and course of CMS. All speech recordings will be analysed nationally by speech therapists.The following data will be registered at five time points by online standard registration forms:Hospital, country, patient related variables such as date of birth, handedness, comorbidities, bilingualism, gender and date of diagnosis, medical history and preoperative neurological status. A speech and language test will be performed and recorded. If the patient is younger than two years a bedside assessment of speech will be performed instead of a formalized test. A two millilitre blood sample for genetic analysis will be collected.Surgery related variables such as date, patient position during surgery, surgical approach, tumour removal method , duration and course of operation, damage to non-tumour tissue, complications, technology employed , surgeon\u2019s estimate of tumour resection extent and presence of preoperative hydrocephalus.Approximately one to two weeks post-operatively: neurological examination, postoperative speech and language status including speech and language recording or bedside assessment and medications used for treatment of CMS.Approximately four weeks post-operatively: Development and treatment of postoperative intracranial haematoma and hydrocephalus, leakage of cerebrospinal fluid and need for ventilator. These complications are usually seen earlier but we wait until the fourth post-operative week to register these in order to ensure no complications are missed.Neurological examination, CMS-survey, speech and language recording or bedside assessment and any medications given to treat CMS since last registration.Neurological examination, speech and language status including speech and language recording or bedside assessment, medications given since last registration to treat CMS, chemo- and/or radiotherapy, neuropsychological assessment(s) if performed, final neuropathological classification of tumour, and additional neuroimaging performed since the first follow-up. Copies of the neuroimaging and descriptions performed pre- and postoperatively will be collected for central review.In case of emergency surgery (e.g. due to risk of incarceration or coma) information about the study and invitation to participate can be given within seven days postoperatively. These patients will be included in all parts of the study except for the recording and analysis of preoperative speech and language status.In cases with repeat tumour surgery during the twelve months follow-up, the patient can re-enter the study and start a new follow-up programme  and the remaining 65% of patients are operated using other approaches (assumed carrying a 20% risk), a total of 450 patients have to be included to identify a 5% significance level and 80% power. Based on a projected overall risk of CMS of 20%, an estimated frequency of a specific SNP of 30%, and a projected doubled risk of CMS with this particular SNP, we will need to include a total of 343 patients to identify such a genetic predisposition at a 5% significance level with 90% power.The study will be the largest prospective international study on CMS to date, and the first one to 1) systematically register surgery, use of steroids, standardized speech samples and 2) to investigate the influence of host genome. Detailed information on neuroradiological features, tumour and patient characteristics (incl. Handedness and pre-language impairment) will also be gathered, and may help further elucidate the incidence and clinical course of the syndrome for various patient and tumour types.On-line registration compliance rates to Nordic/Baltic multicentre trials are in general above 95% . FurtherCurrently, a randomized intervention study is unrealistic due to limited data supporting any specific neurosurgical approach and given the diversity of tumour subtypes, localisation and invasiveness. However, such a randomisation may be realistic if the present study does not clearly identify surgical approaches with statistically significant reduced risks of CMS."}
+{"text": "A peripheral blood interferon (IFN) signature  has been described in a subset of patients with rheumatoid arthritis (RA). In the present study, we systematically assessed the association between this IRG expression and clinical parameters.Expression of 19 IRGs was determined in peripheral blood from 182 consecutive patients with RA and averaged into an IFN score per individual. Correlation and unpaired analyses were performed on the complete patient group. The analyses were internally validated by using an algorithm to randomize the patient group 1000 times into two equally sized sets, and then analyses were performed on both sets.n\u2009=\u200995) did not reveal any significant associations either.Associations were assessed between IFN score and disease duration, 28-joint Disease Activity Score and its components, the occurrence of erosions and nodules, autoantibody positivity, and immunosuppressive treatment. This analysis revealed lower IFN scores in patients using hydroxychloroquine, prednisone, and/or sulfasalazine, but it did not show significant associations between the other parameters and the IFN score. Selecting patients who were not treated with hydroxychloroquine, prednisone, and/or sulfasalazine  contains supplementary material, which is available to authorized users. Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic joint inflammation. It manifests as a heterogeneous disease that is partly reflected at the level of gene expression. Genome-wide gene expression analysis revealed evidence for molecular differences between patients with RA, in particular in the type I interferon (IFN) system. Approximately 50% of patients with RA display a peripheral blood IFN signature  .Type I IFNs were initially known for their antiviral effects, but increasing insight into their activities revealed their role as pleiotropic cytokines with a critical role in modulating immune responses, such as cellular activation, major histocompatibility complex upregulation, induction of apoptosis, and inhibition of angiogenesis . It is tn\u2009=\u2009182) in combination with a random sampling algorithm to systematically investigate whether the peripheral blood IFN signature in RA could be associated with parameters such as disease activity, laboratory parameters, and the use of immunosuppressive treatment.Earlier studies have addressed whether the IFN signature in RA could be associated with clinical parameters, which inconclusively revealed a potential relationship of the IFN signature with anticitrullinated protein antibody (ACPA) titers , 8. Howen\u2009=\u2009182) were consecutively recruited from the Jan van Breemen Research Institute, Reade center, Amsterdam, The Netherlands. All patients were Caucasian and were diagnosed with RA according to the American College of Rheumatology 1987 criteria [Patients with RA n\u2009=\u200982 were c2-transformed.Total RNA was isolated from the PAXgene tubes according to the manufacturer\u2019s instructions. The quantity and purity of the RNA samples were checked using a NanoDrop spectrophotometer . We reverse-transcribed 0.25\u00a0\u03bcg of RNA into complementary DNA (cDNA) using a RevertAid H Minus cDNA Synthesis Kit . A single aliquot of each cDNA sample was first subjected to 14\u00a0cycles of specific target amplification using a 0.2\u00d7 mixture of all TaqMan gene expression assays in combination with the TaqMan PreAmp Master Mix . Following preamplification, the samples were diluted 1:5 (vol/vol) in Tris-ethylenediaminetetraacetic acid buffer, pH\u00a08.0. Multiplex real-time quantitative polymerase chain reaction was performed using the 96.96 Biomark Dynamic Array systems  at ServiceXS  according to the manufacturer\u2019s instructions. Quantities were calculated relative to GAPDH using the standard curve method. Expression levels were log2-transformed expression levels of the IRGs were highly correlated ; therefore, an IFN score was calculated by averaging these values of all genes for each sample.Nineteen IRGs described to be components of the IFN signature in RA  were meaU analysis for dichotomous variables on each set [p Values <0.05 were considered to be significant. Correction for multiple testing was performed using the method of Benjamini and Hochberg.Data were analyzed using IBM SPSS Statistics version 22 , R version 3.1.3 , and Graeach set . p Valuep value <0.05 was detected in both sets in 521 of the 1000 iterations, in 1 of the 2 sets for 479 of 1000 iterations, and never in none of the sets . A slight trend was also observed for prednisone use and dose and SSZ use , although significance was never found in both sets for these variables. Each IRG was also analyzed individually, which revealed similar results (data not shown).We studied the association between the peripheral blood IFN score and the following parameters: disease duration, DAS28 and its individual components, the occurrence of erosions and nodules, autoantibody positivity, and immunosuppressive treatment. As demonstrated in Table\u00a0p\u2009=\u20090.0080 with Benjamini-Hochberg correction, median cross-validation p\u2009\u2264\u20090.012)  at the moment of blood collection. This did not result in any significant associations between the IFN score and other variables  , we cannot fully exclude the possibility that significant associations could have been found if the cohort had included more patients with low disease activity. Inclusion of patients with inactive and/or early disease could provide a more complete view of the IFN response in different states of the disease.Our cohort consisted mainly of established patients with RA with a DAS28\u2009\u2265\u20093.2 despite treatment with at least two DMARDs. Although our data do not suggest any association between IFN score and DAS28 (Remarkably, the IFN scores were decreased in HCQ-, prednisone-, and/or SSZ-treated patients, even though the beneficial effects of these treatments were supposedly diminished. Moreover, cotreatment with these agents appeared to have an additive suppressive effect. Interference of both prednisone and HCQ with type I IFN signaling has been described before , 15, butAs previously described, suppression of the IFN score by certain treatment could affect the applicability of the IFN signature as a biomarker for therapy response, particularly to rituximab , 19. ThaOur data indicate that there are no evident associations between the peripheral blood IFN signature in established RA and clinical parameters. This suggests that the IFN signature is not an indication of disease activity per se, but its presence could indicate a potential difference in pathology or immune pathway activation compared with patients without this signature. Consequently, this could influence the response to therapy, particularly to biologics because these are specific modulators of these immune pathways."}
+{"text": "Polycystic ovary syndrome (PCOS) affects 6\u201310% of women in reproductive age and is characterized by hyperandrogenism, insulin resistance, and chronic anovulation . On thatIn this scenario, we are honored to introduce this special issue, which contains five articles that may shed new light on the topic. In particular, three articles are focused on metabolic disturbances in PCOS women: the first one (\u201cFree Testosterone Reflects Metabolic as well as Ovarian Disturbances in Subfertile Oligomenorrheic Women\u201d) found that sex hormone-binding globulin and calculated free testosterone are associated with both ovarian ultrasound and metabolic parameters, such as the body mass index (BMI) and insulin resistance, suggesting a pivotal role for androgen excess in PCOS-related subfertility and ovulatory dysfunction; the second article  found that pericardial fat measured using 1H-magnetic resonance spectroscopy and imaging is positively related to atherogenic lipid profiles, BMI, waist circumference, and liver fat in women with PCOS, suggesting it as a potential noninvasive tool to predict metabolic prognosis in this population; the third article  highlights that a low-dose combination of insulin sensitizers and an antiandrogen is able to normalize fetuin-A levels in adolescent girls with PCOS. Considering that high levels of fetuin-A have been associated with greater risks for type 2 diabetes and with features of metabolic syndrome, this treatment may significantly reduce metabolic consequences and prevent acute events.Besides these three articles related to metabolic disturbances and their treatment, another paper (\u201cThe Place of In Vitro Maturation in PCO/PCOS\u201d) depicted a clear and accurate summary of available evidence regarding the optimization of culture media, laboratory protocols, pregnancy rates, and neonatal outcomes following in vitro maturation (IVM) of human oocytes in PCOS women, which are known to have a variable incidence of infertility and worse outcomes following assisted reproductive technology.Finally, the last paper  investigated differences in the uterine artery pulsatility index (UtAPI) between pregnant women with PCOS and healthy controls and explored the possible effects of metformin on this parameter. Interestingly, the authors found that there was no difference in the UtAPI between women with PCOS and healthy controls in the first and second trimesters of pregnancy; in addition, metformin was not found to have an immediate effect on the UtAPI.Overall, the manuscripts published in this special issue add significant and novel elements for the understanding of the etiology, pathophysiology, diagnosis, and treatment of this complex and multifaceted syndrome. We offer these new insights to the readers, hoping that they will stimulate further debate and address new fields of investigation in the next future."}
+{"text": "The second author's name is spelled incorrectly. The correct name is: Qianchuan He."}
+{"text": "Review of our experience in treating thymic carcinoma patients using a combination of surgery, chemotherapy and radiation therapy.An institutional review of thymic carcinoma patients treated between 2007 and 2014 was performed analyzing clinical characteristics, treatment intent, surgical margin status, and radiation treatment dose. Survival curves were generated using the Kaplan-Meier method.Nine individuals were treated for newly diagnosed thymic carcinoma. Three patients had unresectable disease at presentation; two of these were treated with definitive chemoradiation therapy while another received neoadjuvant chemotherapy. Seven subjects underwent surgical resection (one after neoadjuvant chemotherapy) with pathological staging ranging from IIa \u2013 IVb disease. Patients were planned for adjuvant radiotherapy followed by chemotherapy; however, one developed liver metastases prior to initiating radiotherapy and was therefore treated with palliative chemotherapy alone. A second patient was non-compliant with radiation treatments and was considered as treated with palliative chemotherapy alone. Of the seven patients who completed definitive treatment, median time to progression and overall survival has yet to be reached. Only one of these patients developed progressive disease 10 months after completing treatment and eventually succumbed to disease 41 months after completing definitive therapy. With a median follow up of 30 months, two year overall survival is 67% for all patients.Resection with an emphasis on best possible oncologic margins, followed by radiation and chemotherapy remains an effective treatment strategy for advanced stage thymic carcinoma. In patients who present with unresectable tumors, neoadjuvant chemotherapy or definitive chemoradiation therapy may also be considered as viable treatment strategies. Thymic epithelial tumors collectively represent a rare set of anterior mediastinal tumors comprised of both thymomas and thymic carcinomas. Thymic carcinomas comprise 15% of all thymic tumors . This beThe central importance of maximal surgical resection with an emphasis on best possible oncologic margins in the management of thymic carcinoma is well-appreciated . Thymic Unfortunately, advanced stage disease (\u2265 Masaoka stage III disease) at the time of diagnosis is common and often prevents negative surgical margins . ConsequThe rarity of thymic carcinomas precludes its prospective evaluation in a large series. Most thymic carcinoma reports are multi-decade retrospective series with many reports including both thymomas and thymic carcinomas. The extended period over which these cases occurred may also confound conclusions due to the the significant evolution in treatment strategies over the period of observation . ConsequPatients with a pathological diagnosis of thymic carcinoma treated atour institution with planned defintiive radiation therapy between January 2007 and December 2014 were identified and their records were reviewed. Thymic carcinoma cases were characterized using Masaoka-Koga staging and International Thymic Malignancy Interest Group (ITMIG) conventions. Cases were analyzed in terms of clinical presentation, diagnostic workup, immunohistochemistry markers , surgical resection margin status, pathology subtype, and overall clinical course. This study was approved by our Institutional Review Board.All patients underwent CT-based radiation treatment planning. Patient immobiliziation was obtained using either an arm board or custom-made positioning cushion. Patients received a contrast Computed Tomographic (CT) scan of 2 mm slice thickness. The Clinical Target Volume (CTV) included the thymic tumor bed and areas of concern for residual microscopic or residual disease based on discussion with the thoracic surgeons. The Planning Target Volume (PTV) included an additional 5 mm margin around the CTV. All patients received a 3D-conformal or IMRT based radiation. A curative intent radiation dose ranged from 45 Gy to 60 Gy, depending upon disease status at time of treatment planning, and was delivered using daily conventional fractionation Monday through Friday. Prior to radiation treatment delivery, an orthovoltage conebeam CT scan was acquired for localization.2 intravenous (IV) day 1, doxorubicin 40 mg/m2 IV day 1, vincristine 0.6 mg/m2 IV day 3, and cyclophosphamide 500 mg/m2 IV day 4) followed by surgical resection. In the adjuvant setting, patients received either carboplatin-paclitaxel (carboplatin AUC 5 and paclitaxel 200 mg/m2 IV day 1 of every 3 weeks) or ADOC chemotherapy following completion of radiation therapy. In the two cases of definitive concurrent radiation and chemotherapy in the setting of unresectable disease, one patient received concurrent carboplatin-paclitaxel and the second who had synchronous multiple myeloma received bortezomib and dexamethasone. Two patients receiving palliative chemotherapy received either carboplatin-paclitaxel (carboplatin AUC 5 and paclitaxel 200 g mg/m2 IV day 1 of every 3 weeks) or etoposide-cisplatin (etoposide 100 mg/m2 and cisplatin 60 mg/m2 day 1 of every 3 weeks).Chemotherapy treatments included neoadjuvant, adjuvant, and definitive treatment regimens. In the neoadjuvant setting, one patient received 3 cycles of ADOC . Time to progression was defined as the number of months elapsed from obtaining tumor histology to tumor recurrence based on CT-based imagingas defined by the absence of progression at the treatment site, per RECIST 1.1 criteria or development of metastatic disease. Overall survival was defined as the number of months elapsed from obtaining tumor histology to death from any cause. Survival curves were generated by the Kaplan-Meier method.Between January 01, 2007 and December 31, 2014, nine individuals were identified who met the study inclusion criteria. The patient characteristics are listed in Postresection, patients were planned to receive conformal adjuvant radiotherapy to the thymic tumor bed to a dose of 45 Gy to 54 Gy in conventional 1.8 Gy to 2.0 Gy fractions). Patients with residual disease or those with unresectable disease received further radiation treatments up to a total dose up to 66.6 Gy utilizing a shrinking volume/cone-down approach. However, one patient with R2-surgical margins had poor compliance with treatment sessions and pre-maturely stopped therapy after receiving 30 Gy. He went on to receive palliative chemotherapy with carboplatin-paclitaxol. Another patient with R2-surgical margins rapidly developed liver metastases prior to starting radiation therapy and was subsequently treated with palliative chemotherapy with cisplatin and etoposide. In general, radiation therapy was followed by 4\u20136 weeks of chemotherapy with either ADOC or carboplatin-paclitaxel regimens.Overall, patients tolerated adjuvant radiation therapy well with development of only mild (grade 1) erythema in four patients which shortly resolved after completion of radiation therapy. There were no reported or observed long term radiation-related sequelae.In the context of clinical outcomes, the patient who received neoadjuvant ADOC chemotherapy followed by an R0 resection had disease progression 10 months after therapy. This individual demonstrated a 41 month overall survival, and ultimately succumbed to metastatic brain disease . CurrentThymic carcinomas are rare tumors of the mediastinum that comprise 15% of all thymic tumors . CompareThe mainstay of treatment for thymic carcinoma patients without evidence of extra thoracic metastases is surgical resection with an emphasis on optimal oncologic margins \u201312. All Though patterns of care analyses utilizing national cancer databases suggest that the utilization of radiation treatment in the management of thymic carcinoma continues to increase, there are conflicting reports as to whether adjuvant radiotherapy confers any survival benefit ,16. WherSurvival rates vary depending on stage, resectability and completeness of resection. Patients with Masaoka stage II, III, IV a disease have a 5-year overall survival of 81%, 51% and 24% respectively . In our Our results support the importance of up-front maximal resection for optimal outcome , with diIf the likelihood of residual disease is high after surgery, or if disease is unresectable, neoadjuvant chemotherapy should be considered as 1 of our patients who presented with initially unresectable disease received neoadjuvant chemotherapy and achieved a R0 resection. In our experience, treatment failure consistently occurred outside of radiation fields, which highlights the importance of adjuvant RT. In this framework, a vital role for adjuvant radiation and chemotherapy is demonstrated with excellent and sustained time to progression and overall survival of patients who presented with locally advanced thymic caricinoma.Maximal safe surgical resection with an emphasis on the best possible oncologic margins in those deemed surgically resectable, followed by adjuvant radiotherapy and chemotherapy, and remains an effective treatment strategy for locally advanced stage thymic carcinoma. In patients who present with initially unresectable disease, neoadjuvant chemotherapy may be a viable approach for local control. Alternatively, definitive radiotherapy and chemotherapy with dose escalation to \u2265 60 Gy may also be considered as an effective treatment option for those unable to have resection."}
+{"text": "Zika virus (ZIKV) is a mosquito-borne positive sense RNA virus. Recently, ZIKV emerged into the Western hemisphere as a human health threat, with severe disease associated with developmental and neurological complications. The structural envelope protein of ZIKV and other neurotropic flaviviruses contains an extended CD-loop relative to non-neurotropic flaviviruses, and has been shown to augment ZIKV stability and pathogenesis. Here we show that shortening the CD-loop in ZIKV attenuates the virus in mice, by reducing the ability to invade and replicate in the central nervous system. The CD-loop mutation was genetically stable following infection in mice, though secondary site mutations arise adjacent to the CD-loop. Importantly, while shortening of the CD-loop attenuates the virus, the CD-loop mutant maintains antigenicity in immunocompetent mice, eliciting an antibody response that similarly neutralizes both the mutant and wildtype ZIKV. These findings suggest that the extended CD-loop in ZIKV is a determinant of neurotropism and may be a target in live-attenuated vaccine design, for not only ZIKV, but for other neurotropic flaviviruses. Zika virus (ZIKV) is a mosquito-transmitted virus that was recently introduced in Brazil and subsequently spread throughout the Americas. ZIKV is highly similar to the related dengue virus but causes unique disease outcomes including neurological disease in adults and fetal developmental complications. The ZIKV envelope protein coats the surface of the virus and allows entry into host cells. Here we investigate a portion of the ZIKV envelope protein, the CD-loop, which extends further than in dengue virus, and its role in ZIKV neurological disease. Our study finds that shortening the CD-loop reduces the ability of ZIKV to replicate in neuronal cells, that the longer CD-loop is a key factor for invasion of the central nervous system in mice, and that a deletion in the CD-loop is genetically stable with passage. Additionally, we show that infection with the CD-loop mutant induces a potent antibody response that can neutralize wildtype ZIKV, suggesting it may offer protection in mice. Shortening of the ZIKV CD-loop, and the CD-loop of other neurotropic flaviviruses, could contribute to development of rapid, effective, and safe vaccines. Zika virus (ZIKV) is a positive-sense single-stranded RNA flavivirus. Though first isolated in 1947 and after years of relatively benign infections throughout Southeast Asia, ZIKV was identified in large human disease outbreaks in Yap Island, French Polynesia, and then the Americas . ZIKV inThe ability of ZIKV to cause neurological disease is not unique among flaviviruses . West NiMultiple groups have shown that ZIKV is more structurally stable than DENV, hypothesizing that it can persist longer in body compartments and fluids, potentially leading to an increased chance of neuroinvasion \u201325. One in vivo infection, secondary site mutations emerge in the envelope glycoprotein. A successful ZIKV vaccine should have no risk of neurological complications in immunocompetent populations, and as our shortened CD-loop virus results in decreased neuropathogenicity, we characterized the antibody response elicited by the \u0394346 virus in mice. Mice infected with the \u0394346 mutant mount an antibody response that similarly neutralizes both \u0394346 and WT ZIKV in cell culture. Together, this data suggests that shortening the CD-loop of ZIKV results in decreased neurovirulence while maintaining wildtype ZIKV antigenicity.In this study we further investigate the role of the ZIKV CD-loop in neurotropism and antigenicity. We find that the CD-loop mutant is delayed in disseminating to the brain, but once present can replicate and cause lethal disease. Importantly, when delivered via intracranial infection, \u0394346 ZIKV is still less pathogenic, indicating that the attenuated phenotype is not solely due to a defect in neuroinvasion. While the \u0394346 deletion is genetically stable after long-term in vitro neurological model of ZIKV replication , observing severe attenuation with no development of disease  was approved by the UNC\u2019s IACUC (Permit Number A-3410-01).-/- mice on C57BL/6 background with 103 or 104 FFU of WT or \u0394346 ZIKV in 10ul diluted in PBS into the left hind footpad (n = 13 for each group). Infected mice were monitored daily for weight loss and clinical signs of disease. On day six post infection, a subset of mice were euthanized and perfused with PBS for tissue analysis. The remainder of mice were euthanized via isoflurane overdose and cervical dislocation upon losing 20% of their starting body weight or showing signs of severe disease and tissues were collected for titer analysis. No mice died prior to humane euthanasia. Tissues were homogenized in 1ml PBS, centrifuged to pellet debris, immediately frozen at -80\u00b0C, then clarified supernatant was used for virus titering as described above. Survival curves were analyzed by log-rank test. Log-transformed titer data was analyzed by Mann-Whitney test.Footpad infections were performed in 8- to 10-week-old male and female IFNAGR-/- mice with 103 FFU of WT or \u0394346 ZIKV in 20ul diluted in PBS into the left cerebral hemisphere via insulin syringe while under brief isoflurane anesthesia and monitored as described above . Mice were euthanized on days two, six, or upon losing 20% of their starting weight, and tissue was collected and titered as described above. No mice died prior to humane euthanasia. Survival curves were analyzed by log-rank test. Log-transformed titer data was analyzed by two-factor ANOVA followed by Sidak\u2019s multiple comparisons.Intracranial infections were performed in 5- to 7-week-old male and female C57BL/6J or IFNAGRAntibody response experiments were performed in five-week-old female C57BL/6J mice, which were given 1mg of anti-IFNAR1 antibody  via intraperitoneal injection one day prior to footpad infection and monitored as described above (n = 5 mock infected and n = 8 for each ZIKV infected group). Blood was collected on day three post infection via submandibular bleed, allowed to clot for at least 10 minutes and clarified by centrifugation prior to storage at -80\u00b0C. Mice were euthanized on day 28 post infection for terminal blood collection. Log-transformed titer data was analyzed by one-factor ANOVA followed by Tukey\u2019s multiple comparisons. All mouse experiments were performed once.Virus stock RNA was isolated via Viral RNA Mini Kit (Qiagen). Total brain RNA was isolated from brain homogenates and TRIzol LS (Invitrogen) and Direct-zol RNA MiniPrep kit (Zymo Research). cDNA was reverse transcribed using SuperScript III (Invitrogen) using random primers. The ZIKV envelope sequence was PCR amplified, and PCR amplicon was sequenced via Sanger sequencing and analyzed in Geneious (Version 11.0.04).96-well high bind plates were coated with human MAb EDE1 C10, previously shown to bind WT and \u0394346 ZIKV equally . Plates Plates were seeded with C6/36 cells one day prior to neutralization assay. Mouse immune sera were serially diluted four-fold beginning at 1:20, then mixed with WT or \u0394346 ZIKV viruses diluted to ~45 FFU/well. Virus:Ab mixture was incubated for one hour at 32\u00b0C, added to C6/36 cells and incubated for an additional hour at 32\u00b0C. Overlay was added and cells were incubated for 4\u20136 days, then fixed and immunostained as described above. FRNT data was analyzed by two-factor ANOVA followed by Tukey\u2019s multiple comparisons.S1 DataMicrosoft Excel file containing data used to generate figures in Prism (version 8.0.1). Individual sheets correspond to each figure panel. ZIKV titer data provided as the log transform of foci forming units per ml or gram of tissue (FFU/ml or FFU/g). Limits of detection are also provided. Mouse weight data provided in grams and as percent of starting weight. Includes full ZIKV envelope protein amino acid sequence for each sample.(XLSX)Click here for additional data file."}
+{"text": "Ifnar1\u2212/\u2212 mice. ZIKV-IG successfully protected mice from lethal ZIKV challenge. In particular, ZIKV-IG treatment at 24\u2009hours after lethal ZIKV infection improved survival by reducing weight loss and tissue viral burden and improving clinical score. Additionally, ZIKV-IG eliminated ZIKV-induced tissue damage and inflammation in the brain and liver. These results indicate that ZIKV-IG is efficacious against ZIKV, suggesting this human polyclonal antibody is a viable candidate for further development as a treatment against human ZIKV infection.Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that represents a major threat to global health. ZIKV infections in adults are generally asymptomatic or present with mild symptoms. However, recent outbreaks of ZIKV have revealed that it can cause Congenital Zika Syndrome in neonates and Guillain-Barr\u00e9 syndrome in adults. Currently, no ZIKV-specific vaccines or antiviral treatments are available. In this study, we tested the efficacy of convalescent plasma IgG hyperimmune product (ZIKV-IG) isolated from individuals with high neutralizing anti-ZIKV titers as a therapeutic candidate against ZIKV infection using a model of ZIKV infection in  Flaviviridae, similar to dengue, West Nile, Japanese encephalitis, and yellow fever viruses1. ZIKV was first identified in a sentinel rhesus monkey in the Zika Forest of Uganda in 1947 and was isolated from mosquitoes (Aedes africanus) in 19482. ZIKV is transmitted through the bite of infected female Ae. aegypti mosquitoes3 and potentially Ae. albopictus mosquitoes4, as well as alternative non-vector routes which have been identified, including vertical 8, transfusion11, and sexual transmission12. From the 1950\u2019s to 1990\u2019s, serological evidence of ZIKV was reported in multiple Asian16 and African22 countries, but no outbreaks and only 14 cases of human ZIKV disease were described25. The first ZIKV outbreak was observed in 2007 on Yap Island in the Federated States of Micronesia26, followed by a second outbreak in French Polynesia in 201327. The most recent reported outbreak was on a larger scale that occurred from 2014 to 2016 in Latin America30. Interest in this virus increased after these outbreaks in part due to the emergence of ZIKV outside its previously known geographic range, showing the potential of the virus to spread wherever the mosquito vector is present. In addition, prior to the French Polynesia outbreak, ZIKV was known to be asymptomatic or cause only mild symptoms . However, since 2007, severe complications of ZIKV infection, in particular Guillain\u2013Barr\u00e9 Syndrome in adults32 and Congenital Zika Syndrome in babies born to ZIKV-infected women36 have been observed. These findings led the WHO to declare ZIKV a public health emergency of international concern in 2016 and expanded efforts for the development of vaccines and therapeutics to combat the disease.Zika virus (ZIKV) is an arthropod-borne virus belonging to the family 37. The role of convalescent serum therapy expanded to many infections beyond influenza during the first half of the 20th century with clinical benefit demonstrated for other viral diseases like measles38 and polio39, and for invasive bacterial pathogens, including pneumococcus, Haemophilus influenzae B, and meningococcus41. Passive immunization with antibody-based therapies has emerged as a promising strategy for treating emerging infectious diseases, and include both monoclonal (mAb) and polyclonal antibodies (pAb), each of which has its advantages and disadvantages. For example, mAbs can be easily manufactured in large quantities and have a greater inherent biological consistency due to their epitope specificity as compared to pAbs. However, mAbs have limitations, including development of escape mutants and high production costs. In comparison with mAbs, pAbs can have more robust activities, neutralizing several virus strains even after viral mutations43. Although several studies have demonstrated that mAbs can provide therapeutic protection against ZIKV in various human and mouse models44, only a single study to date has shown therapeutic potential of human pAbs produced from transchromosomal cows against ZIKV infection in mice. As no study has as yet assessed the efficacy of pAbs isolated from humans as a potential therapeutic against ZIKV, herein, we evaluate the therapeutic potential of a pAb preparation from human plasma containing high anti-ZIKV titers (ZIKV-IG).Antibodies (Abs) have been shown to play a critical role in the protective immune response against infectious diseases and have been used for passive immunization, in the prevention and treatment of both bacterial and viral infections, for more than a century. Immune animal sera were first used in the late 1800\u2019s for treatment of disease, followed by an era of immune human serum therapy for both viral and bacterial diseases. Notably, during the 1918 influenza pandemic, serum from recovered patients was used successfully to treat acutely ill patientsIfnar1\u2212/\u2212 mouse model as a stringent challenge system to evaluate the therapeutic potential of ZIKV-IG. Prior to the recent ZIKV epidemics, only a few studies had been performed in mice and these required many serial passages of ZIKV in mice to produce consistent disease phenotypes47. Within the last three years, substantial efforts have been focused on generating new mouse models. ZIKV evades the anti-viral type I interferon (IFN) response, in part through inhibition of the STAT2 and STING pathways in human but not mouse cells50. This antagonism of the type I IFN receptor (Ifnar) signaling in a species-specific manner by ZIKV explains the more severe pathogenesis of ZIKV infection in mice with immature or compromised immune systems compared to adult immunocompetent mice, and why disruption of the Ifnar1 signaling increases susceptibility of mice to lethal ZIKV infection. Accordingly, wild type mice treated with blocking anti-Ifnar1 mAb54, and mice gene-deficient for Ifnar158 or for both Ifnar1 and type II IFN receptors61 have been widely used as models of ZIKV infection. We measured the effectiveness of ZIKV-IG therapy on survival, viral burden and tissue pathology in key organs, including spleen, kidneys, liver, sciatic nerves and brain, of Ifnar1\u2212/\u2212 mice following lethal ZIKV challenge. ZIKV-IG treatment at 24 hrs post-infection increased survival by reducing viral burden and ZIKV-induced tissue damage and inflammation in several key organs. These findings demonstrated that a single dose of ZIKV-IG is efficacious against lethal Zika disease in a highly stringent mouse challenge model.Specifically, we used the Ifnar1\u2212/\u2212 mice were infected with ZIKV  route) and then treated with ZIKV-IG  24 hrs post-infection (p.i.). ZIKV-IG used for this study exhibits high neutralization activity against multiple ZIKV strains, including strain FSS13025  followed by treatment at 24 hrs p.i. with ZIKV-IG . Sera and organs were harvested on days 3 and 7 p.i., and levels of viral RNA and infectious virus to BHK cells were determined by qRT-PCR and FFA, respectively. The 0.1\u2009mg/kg dose was not used in this experiment due to the observed similarities of mortality and morbidity between this group and the 0.5\u2009mg/kg group. In the serum, only 50\u2009mg/kg ZIKV-IG-treated mice had significant reductions in both viral RNA  and infectious virus levels  at day 3 p.i. relative to vehicle-treated control mice . ice Fig.\u00a0. No signice Fig.\u00a0. In the ice Fig.\u00a0. Spleensice Fig.\u00a0. In the ice Fig.\u00a0. Kidneysice Fig.\u00a0. In the ice Fig.\u00a0. At day ice Fig.\u00a0; howeverice Fig.\u00a0. The incvs. control mice. At day 3 p.i., similar levels of ZIKV RNA and infectious particles were present in the sciatic nerves of control and ZIKV-IG-treated mice, but at day 7 p.i., both viral RNA  and BHK cell-infectious ZIKV  levels were reduced in the sciatic nerves of animals treated with 50\u2009mg/kg ZIKV-IG relative to control mice    and late (day 7) time points after infection. In comparison to 50\u2009mg/kg ZIKV-IG, lower doses of ZIKV-IG are less effective against ZIKV infection, reducing viral burden in select tissues.To confirm the robust efficacy of 50\u2009mg/kg ZIKV-IG treatment against ZIKV infection in this mouse model, we next localized ZIKV in tissues by performing immunohistochemistry (IHC) for expression of ZIKV nonstructural protein 2B (NS2B), which is absent in virions and thus serves as a marker of viral replication. In the livers of control mice Fig.\u00a0, NS2B wa06) Fig.\u00a0. In the 06) Fig.\u00a0, whereas06) Fig.\u00a0. Quantit06) Fig.\u00a0. Thus, 5Ifnar1\u2212/\u2212 mice. Therefore, to evaluate the extent of brain tissue pathology and other signs of injury in ZIKV-IG-treated and control mice following ZIKV infection, hematoxylin & eosin-stained brain slide sections were examined and scored by a blinded board-certified pathologist. Scores for these sections are shown in Table\u00a0ZIKV\u2019s neurotropism and associated brain pathology has been extensively documented in humans and In this study, we evaluated the therapeutic potential of human anti-ZIKV pAb (ZIKV-IG) against lethal ZIKV infection in a highly stringent mouse model. ZIKV-IG treatment was effective in protecting mice against ZIKV-induced mortality and morbidity by decreasing viral replication and dissemination into key target organs and ZIKV-induced pathology in the brain. These findings support further development of ZIKV-IG as a candidate for prophylaxis or treatment of Zika disease.64 and human polyclonal antibody produced in transchromosomal bovines65 were shown to have therapeutic potential against ZIKV infection in mice. Similarly, transfer of convalescent sera from a human to pregnant mice prevented ZIKV infection and associated fetal birth defects66, and convalescent human plasma or sera obtained from ZIKV-infected individuals were able to neutralize both African and Asian ZIKV strains in vitro67. Here, we report that a single administration of 50\u2009mg/kg human polyclonal Ab (ZIKV-IG) given at 24 hrs post-infection in Ifnar1\u2212/\u2212 mouse model of lethal ZIKV infection prevented both severe disease development and mortality. In addition, ZIKV-IG reduced ZIKV burden and ZIKV-induced tissue damage in target organs, confirming therapeutic potential against ZIKV infection. Recent outbreaks of several emerging and re-emerging viral diseases for which no approved treatment or vaccine exists have rekindled interest in the development of plasma-derived immunoglobulin therapeutic products. In our current study, we used human polyclonal IgG antibodies purified from convalescent donor plasma containing high titers of anti-ZIKV Abs. Plasma collection was based on criteria set by FDA and Emergent\u2019s standards for virus screening and donor qualification. The immunoglobulin fraction was purified using a validated hyperimmune platform manufacturing process68.A variety of Abs, including mAbs isolated from ZIKV-immune individuals67. This premise is supported by the report that primary infection with ZIKV African strain in macaques protected the animals from secondary heterologous re-challenge with ZIKV Asian strain69. Thus, an effective ZIKV therapeutic candidate could potentially neutralize infection with any of the ZIKV virus lineages. Our in vitro potency based on anti-ZIKV neutralization titer and in vivo mouse results provide evidence that ZIKV-IG can effectively neutralize ZIKV infection.Although, ZIKV strains have been phylogenetically characterized into African and Asian/American lineages, the virus has very little genome variability and is classified as a single serotypeFlaviviridae75. During the ADE process, pre-existing non- or sub-neutralizing Abs that recognize DENV enhance subsequent DENV infection and pathogenesis78. ZIKV is antigenically and genetically similar to DENV with ~56% genome sequence homology79, with in vitro and in vivo mouse studies demonstrating that Ab response to DENV and ZIKV can cross-react and cross-enhance infection and pathogenesis of each virus83. Although recent macaque and mouse studies have provided further support for pre-existing ZIKV Ab-mediated enhancement of subsequent DENV infection and disease severity86, passive transfer of vaccine-induced Abs before ZIKV challenge did not result in ZIKV infection enhancement or disease in non-pregnant mice and monkeys88. Consistent with these studies88, treatment with various sub-protective doses of ZIKV-IG showed no evidence for ADE of ZIKV infection in our mouse model as suggested by both survival and viral RNA results. No increase in mortality or viral burden were observed even using low ZIKV-IG concentrations which are potentially sub-neutralizing. However, viral load data obtained through focus forming assays using BHK cells may not be appropriate for drawing conclusions around the presence or absence of ADE as BHK cells lack expression of Fc\u03b3 receptors that support the ADE mode of infection.Development of Ab products intended for use as a therapy against ZIKV infection should consider the risk of antibody dependent enhancement (ADE) of infection, which has previously been described for dengue virus (DENV), another member of the family Whether ADE is clinically relevant to human Zika disease is currently unknown, and thus, the possibility of ZIKV-IG-mediated ADE for Zika clinical disease remains a theoretical question for the development of Ab therapies against ZIKV. Further studies should be performed to assess whether ZIKV-IG has the potential to enhance DENV infection when given under pre-exposure setting.In summary, we report that a single administration of 50\u2009mg/kg of ZIKV-IG 24 hrs after infection protected mice against lethal ZIKV infection. Non-protective doses of ZIKV-IG did not induce ADE of ZIKV infection. These results provide the evidence that, at appropriate doses, ZIKV-IG treatment could be effective at preventing the deleterious effects of ZIKV in humans. Therefore, further testing in relevant pregnancy models to determine the impact of treatment on maternal and fetal infection is warranted.Key reagents, Abs, primers, and probes used in this study are outlined in Supplementary Table\u00a089. PRVABC59 is an Asian lineage strain isolated in 2015 from the blood of a human in Puerto Rico90. MR766 is an African lineage strain isolated in 1947 from a sentinel rhesus monkey (766) in Uganda46. Viruses were cultured using C6/36 Aedes albopictus mosquito cells, as described previously92. Virus was titrated using a baby hamster kidney (BHK)-21 cell-based focus-forming assay (FFA) as described previously52.ZIKV strains MR766, FSS13025, and PRVABC59 were obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA). FSS13025 is an Asian lineage strain isolated in 2010 from a pediatric case2 for 1\u2009hr with either 1\u2009\u00d7\u2009105 FFU of ZIKV strain FSS13025, 2\u2009\u00d7\u2009104 FFU of ZIKV strain MR766, or 1\u2009\u00d7\u2009104 FFU of ZIKV strain PRVABC59. Standard flow cytometry-based neutralization assays using U937-DC-SIGN cells were then performed as described previously93.Na\u00efve-ZIKV-IG and ZIKV-IG were initially diluted to 0.1\u2009mg/mL and then serially diluted 1:3 for 11 dilutions in RPMI 1640 medium supplemented with 1% Hepes and 1% penicillin/streptomycin. Antibody dilutions were incubated at 37\u2009\u00b0C with 5% COIfnar1\u2212/\u2212 mice (C57BL/6 mice deficient in type I interferon receptor) were originally obtained from Dr. W. Yokoyama  and subsequently bred under specific pathogen-free conditions at the animal facility of La Jolla Institute for Immunology. All experiments were approved by the Institutional Animal Care and Use Committee under protocol number AP028-SS1-0615/AP00001029. All experiments included age- and sex-matched mice. At 5\u20136 weeks of age, mice were randomized at first by gender and secondly per weight. Mice were inoculated intravenously via the retro-orbital (r.o.) route with 1.0\u2009\u00d7\u2009103 FFU of ZIKV FSS13025.68 were employed for manufacturing of ZIKV-IG (lot PD_740_ZKP_16_001_003_ER_v1) used in this study. This lot contained a total protein concentration of 54\u2009mg/mL (>98.9% human IgG). Potency was determined using a microneutralization assay that measured the cytopathic effect of ZIKV strain PRVABC59 on Vero E6 cells using an xCELLigence\u00ae real-time cell analyzer . Briefly, Vero E6 cells were added to an xCELLigence 96-well electronic microtiter plate (E-plate) then pre-incubated overnight at 37\u2009\u00b0C in a humidified 5% CO2 incubator. The E-plate contains electrodes in each well; adherent cells in the wells impede the electric current passing through the electrodes. An equal volume of ZIKV PRVABC59, diluted to 100 TCID50, was incubated for one hour at 37\u2009\u00b0C with serial dilutions of ZIKV-IG, then added to the E-plate. The E-plate was then incubated at 37\u2009\u00b0C in an xCELLigence unit contained in a humidified 5% CO2 incubator. Cells were monitored in real-time by measuring ZIKV-induced changes in cell impedance at 30-minute intervals. Sample dilution data from the defined end point was analyzed by fitting the impedance measure (cell index) to the log dilution using a 4-parameter logistic curve fit to determine the 50% neutralizing titer (NT50). The initial potency value for ZIKV-IG product was 18,480, indicating a high degree of Zika virus neutralization.ZIKV-IG was a purified human IgG product manufactured using plasma collected from US FDA licensed plasma centers screened for Ab reactive to ZIKV. Established processesNa\u00efve-ZIKV-IG was manufactured using the same processes as ZIKV-IG, with the exception that the source plasma used did not contain Ab reactive to ZIKV. Mice were injected via r.o. route with 50, 10, 2.0, 0.5 or 0.1\u2009mg/kg ZIKV-IG (100 \u03bcL/mouse) at 24 hrs following lethal ZIKV infection.Following infection, mice were weighed and observed for clinical signs and scored daily. Clinical scores were based on mouse appearance, mobility, and attitude on a 7-point scale Table\u00a0. AnimalsViral quantification was conducted by qRT-PCR and by Focus Forming Assay (FFA) on serum, spleen, kidney, liver, sciatic nerve, and brain. Sera were collected after centrifugation  of blood harvested by cardiac puncture into collection tubes . Following mouse perfusion with PBS, tissues were harvested and stored either in tubes containing RNA Later (Invitrogen) for qRT-PCR or in pre-weighed tubes containing MEM\u03b1 medium and steel beads  for FFA. Tissues were then homogenized in RTL buffer (Qiagen)\u2009+\u20091% beta mercaptoethanol for qRT-PCR and MEM\u03b1 medium for FFA followed by clarification .91 and expressed as genome equivalent per 18S (GE/18S), while serum RNA levels were expressed as GE/mL.RNA from serum and homogenized tissues were isolated using the QIAmp Viral RNA Mini Kit (Qiagen) and the RNeasy Mini Kit (Qiagen), respectively. ZIKV RNA levels in sera and tissues were quantified by qRT-PCR. Specific primers and probes used are listed in Table\u00a052. Tissue samples were homogenized and clarified by centrifugation. Tissue supernatants and sera were diluted serially before infection on BHK cells. BHK cells used in this assay do not express Fc\u03b3 receptors. Cells were plated (2.0\u2009\u00d7\u2009105 cells/well in a 24-well plate) and incubated overnight at 37\u2009\u00b0C, 5% CO2. Confluent monolayers were inoculated with undiluted or 10-fold serially diluted sera or clarified tissue supernatant, and were incubated for 1\u2009hr at 37\u2009\u00b0C. After incubation, the inoculum was removed, and each cell monolayer was overlaid with CMC-media and incubated at 37\u2009\u00b0C, 5% CO2 for 1.5\u20132 days. Cells were then fixed, permeabilized, and incubated with pan Flavivirus anti-envelope (E) Ab clone 4G2 (BioXCell), followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch) and staining with True-Blue peroxidase substrate (Sera Care). Foci were counted, virus levels in the serum were expressed as Focus Forming Units (FFU) per mL, and for most tissues as FFU/g. As it was not technically feasible to weigh sciatic nerves, viral levels in these tissues were expressed as FFU/tissue.FFA procedures were performed as previously describedhttp://www.mbl.org), resulting in 3 brain sections . Tissues were processed routinely for paraffinization and cut at 4 \u03bcm thickness for H&E staining. Slides were blinded before review with an Olympus BX40 brightfield microscope at 2-60X magnification. Lesions were graded on a 4-point scale (0 to +++). All categories of lesions were tabulated  for histopathologic evaluation. Tissues were fixed for 48 hrs at room temperature. Brains were cut transversely at points corresponding to Bregma +2\u2009mm and Bregma \u22123 mm of Mouse Brain Atlas celloidin case #170 .Paraffinized liver was cut onto slides (4 \u03bcm) and routinely deparaffinized. Slides were microwaved  on high setting in Antigen Unmasking Solution (Vector Laboratories) and cooled at room temperature. Endogenous enzyme activity was blocked with BLOXALL (Vector Laboratories) and nonspecific protein binding was blocked with 10% Normal Goat Serum (Thermo Fisher). Slides were incubated with anti-ZIKV NS2B Ab (Genetex) at 4\u2009\u00b0C for 14 hrs. Slides were incubated with ImmPRESS HRP (Vector Laboratories) secondary Ab followed by incubation with ImmPACT NovaRED (Vector Laboratories) substrate. Slides were counterstained with Modified Mayer\u2019s Hematoxylin (Thermo Fisher) and routinely processed for mounting. Positive, negative, and rabbit IgG (Vector Laboratories) controls were included with each batch. Morphology of immuno-reactive cells were confirmed by a board-certified pathologist, and immunoreactivity was quantified by ImageDxhttp:/www.mbl.org). Tissues were transferred to cryomolds, embedded in OCT (Electron Microscopy Services), and frozen on dry ice followed by \u221280\u2009\u00b0C freezer. Tissues were cut at 10\u2009\u03bcm thickness for immunofluorescence. Slides were microwaved  on high setting in Antigen Unmasking Solution (Vector Laboratories) and cooled at room temperature. Slides were incubated in Tris-Urea buffer and permeabilized with 0.1% Triton X-100 (Acros Organics). Nonspecific protein binding was blocked with 10% Horse Serum (Thermo Fisher). Slides were incubated with anti-ZIKV NS2B Ab (Genetex) at 4\u2009\u00b0C for 14 hrs, followed by incubation with anti-rabbit AlexaFluor 488 (Invitrogen) secondary Ab and DAPI (Invitrogen) counterstain. Cover slips were mounted with Prolong Gold (Invitrogen). Positive, negative, and rabbit IgG (Vector Laboratories) controls were included with each batch. Morphology of cells in the brains was confirmed by a board-certified pathologist. Brain immunoreactivity was quantified by ImageDxTM .At the time of necropsy, brains were collected in 4% paraformaldehyde (Alfa Aesar) at 4\u2009\u00b0C and allowed to fix for 24 hrs. Tissues were then cryoprotected by serial immersion in 15% sucrose (Affymetrix) followed by 20% sucrose until the brains floated. Brains were cut transversely at points corresponding to Bregma +2\u2009mm and Bregma \u22123 mm of Mouse Brain Atlas celloidin case #170 . Survival rates and median time to death (MTD) were estimated using the Kaplan-Meier method. Data were analyzed either with Fisher\u2019s exact test , Log-rank test (MTD) or exact Wilcoxon rank-sum test . All these tests were followed by a Bonferroni correction. Immunohistochemistry and immunofluorescence data for both brain and liver were analyzed with Kruskal-Wallis rank-sum test with Dwass, Steel, Critchlow-Fligner correction for multiple comparisons. Results were considered significant when p\u2009<\u20090.05.Supplementary file"}
+{"text": "Puerarin exerts therapeutic effect on osteoporosis due to its inhibitory effect on the formation of osteoclasts. Puerarin is also widely established as an autophagy inhibitor. The study aimed to investigate the significance of autophagy in Puerarin-treated osteoclast formation.Osteoclast precursors (OCPs) derived from bone marrow-derived macrophages (BMMs) were treated with Puerarin along with RANKL or without RANKL, and then the autophagic parameters of OCPs  were observed through Western Blotting, Transmission Electron Microscopy and Immunofluorescence assays. Next, after using overexpression vectors of autophagic genes  to alter autophagy activity, OCP proliferation was measured by Ethynyl deoxyuridine (EdU) assays and Cell Counting Kit-8 (CCK-8) kit, and osteoclast differentiation was assessed by Tartrate-resistant acid phosphatase (TRAP) staining.The results showed that Puerarin could directly inhibit the autophagy and proliferation of OCPs. Importantly, overexpression of autophagic genes Atg5, Atg7 and BECN1 reversed Puerarin-inhibited OCP autophagy and proliferation. What\u2019s more, RANKL could promote the autography of OCPs, which was recovered by Puerarin treatment. Interestingly, different from single-Puerarin treatment, we found that in the presence of RANKL, only BECN1 overexpression significantly reversed Puerarin-inhibited osteoclast differentiation and OCP autophagy.In conclusion, Puerarin could inhibit the OCP autophagy in the presence or absence of RANKL, which blocked the OCP proliferation and osteoclast differentiation respectively. Moreover, BECN1 plays an essential role in Puerarin-inhibited osteoclastogenesis. Our study provides potential clue to further complete the intrinsic mechanism of Puerarin in treating osteoporosis. Pueraria lobata is a leguminous plant in China, which is widely used in the treatment of cardiovascular diseases, diabetes, osteonecrosis and neurodegeneration [Pueraria lobate, Puerarin is a phytoestrogen with significant bone-protective effect. Its therapeutic effect has been broadly reported in the treatment of osteoporosis. Cho et al. [neration . As an eo et al.  found tho et al. . In addio et al. . The inho et al. . Puerario et al. . In addio et al. . HoweverPrevious studies disclosed that the protective autophagy exerts an indispensable effect on the osteoclast formation as well as bone absorption activity of osteoclast \u201310. In aOsteoclastogenesis consists of the proliferation and differentiation of OCPs. RANKL is a key inducing factor in osteoclast differentiation. Autophagy not only plays an important role in OCP proliferation , but alsThis study showed a role of Puerarin in inhibiting the OCP autophagy in the absence or presence of RANKL, which contributed to the reduction in OCP proliferation or OCP differentiation, respectively. Therefore, by clarifying the significance of Puerarin in the OCP autophagy, the present study revealed an autophagic mechanism underlying Puerarin-treated osteoclastogenesis for the first time.Recombinant M-CSF and RANKL were purchased from Peprotech . Puerarin, E64d, Pepstatin A (PEPS A) and TRAP staining kit were obtained from Sigma-Aldrich . Rabbit LC3B, Atg5, Atg7, Beclin1, and \u03b2-actin antibodies were purchased from Cell Signaling Technology . Cell Counting Kit-8 (CCK-8) kit was obtained from Dojindo . The EdU kit was purchased from Roche . The SYBR Premix Ex TaqTM kit was from TakaRa . After dissolving in 1% BSA, different working concentration of Puerarin  were prepared by complete \u03b1-minimum Eagle\u2019s medium (\u03b1-MEM).2. The experimental protocols were approved by the Institutional Animal Care and Use Committee of Southern Medical University (No.44002100017774). To further elucidate the direct effect of Puerarin on the expression of autophagic proteins, cells were treated with Puerarin at different concentrations for 6\u2009h without RANKL.4\u20138\u2009week old littermate C57BL/6\u2009J female mice were purchased from Slaccas Experimental Animal Centre . The mice were housed in a common environment in which the room temperature was 20~30\u2009\u00b0C and the humidity 60~80% and fed a general laboratory diet. The mice were sacrificed by cervical dislocation, and the tibia from mice were flushed using \u03b1-MEM without serum. Bone marrow cells were incubated with a-MEM containing 10% FBS, penicillin (100\u2009U/ml) and streptomycin (100\u2009mg/ml) for 24\u2009h. Non-adherent cells were collected as Bone marrow-derived macrophages (BMMs). BMMs were induced to OCPs (adherent cells) after treatment with M-CSF (20\u2009ng/ml) for 3\u2009days as previously described , 22. Cel5/well) were incubated in 24-well plate in \u03b1-MEM involving M-CSF (20\u2009ng/ml) plus RANKL (100\u2009ng/ml) supplemented with other relevant treatment for 5\u2009days to induce mature osteoclasts. TRAP staining-positive multinucleate cells (having more than three nuclei) were regarded as the differentiated osteoclasts.OCPs (1\u2009\u00d7\u2009105/well) were cultured in 6-well plate, and treated with indicated treatment. Then, EdU assays were performed by using the EdU kit in accordance with the instruments. The results were collected and quantified under the Zeiss Photomicroscope  on the basis of at least ten random fields. For CCK-8 analysis, OCPs were incubate in 96-well plate at a density of 1\u2009\u00d7\u2009104/well, and then treated with different interventions. Next, cells were treated with the CCK-8 reagents for 1\u2009h. Ultimately, the optical density at 450\u2009nm (OD450) was observed by using Varioskan Flash reader .Cell proliferation was evaluated using EdU assays and CCK-8 kit. In EdU assays, cell proliferation was quantified by observing EdU-positive cells, and in CCK-8 analysis, cell proliferation was measured by observing relative cell number. For EdU assays, OCPs  were constructed by homologous recombination between expression vector (EX-Puro-Lv105) and cDNA in 293 cells using the construction kit  as previously described . After 26/well) with indicated interventions in 6-well plates were prepared. Lysates were packed into 10% SDS-PAGE gels and polyvinylidene fluoride (PVDF) membranes were incubated with the antibodies against Atg5, Atg7, Beclin1, and LC3B, and \u03b2-actin after trarsmembrane. Horseradish peroxidase-linked secondary antibodies were used as secondary antibodies. Bands were visualized using a chemiluminescence system .The whole-cell lysate protein from cells  measurements were performed as described previously . The preCathepsin K (CTSK): 5\u2032-GGAAGAAGACTCACCAGAAGC-3\u2032 (forward) and 5\u2032-GTC-ATATAGCCGCCTCCACAG-3\u2032 (reverse); Matrix metalloproteinase-9 (MMP-9):5\u2032-CC-TGTGTGTTCCCGTTCATCT-3\u2032 (forward) and 5\u2032-ACCCGAATCTAGTAAGGTCGC-3\u2032 (reverse); TRAP: 5\u2032-GCTGGAAACCATGATCACCT-3\u2032 (forward) and 5\u2032-TTGAGCCAGG-ACAGCTGAGT-3\u2032 (reverse); Atg7, 5\u2032-GTTCGCCCCCTTTAATAGTGC-3\u2032 (forward) and 5\u2032-TGAACTCCAACGTCAAGCGG-3\u2032 (reverse); Atg5, 5\u2032-ATGCGGTTGAGG-CTCACTTTA-3\u2032 (forward) and 5\u2032-GGTTGATGGCCCAAAACTGG-3\u2032 (reverse); BECN1: 5\u2032-CTAAGGCAGGCAGGAGGATG-3\u2032 (forward) and 5\u2032-GCTGGCCTCAA-GAGATCCAT \u2212\u20093\u2032 (reverse); Cyclophillin A: 5\u2032-CGAGCTCTGAGCACTGGAGA-3\u2032 (forward) and 5\u2032-TGG-CGTGTAAAGTCACCACC-3\u2032 (reverse).qRT-PCR analysis was carried out by SYBR Premix Ex TaqTM kit and using ABI7500 analyzer .5) were incubated on 6-cm dishes as described above, followed by indicated treatments. The preparation of cell slices and subsequent staining were performed according to the protocol  as previously described [Cells  were enriched in flow tubes, and then fixed by using 4% paraformaldehyde (PFA). Following perforation, cells were blocked using 1% bovine serum albumin (BSA), and incubated with the antibody targeting LC3B at 4\u2009\u00b0C for 12\u2009h. Then, cells were stained with Cy3-labeled Goat Anti-Rabbit IgG for 1\u2009h. Next, cell suspensions were transferred to the adhesive slide. Thirty\u2009minutes later, the suspensions were removed, and the cells were counterstained using DAPI. Ultimately, the cells were observed and recorded under the fluorescence microscope . The cells with more than five LC3-punctas were regarded as LC3-puncta positive cells [Cells were incubated on 6-cm dishes, and stimulated with different reagents. The treated cells (1\u2009\u00d7\u200910ve cells , 25.P\u00a0<\u20090.05. The statistical analyses were carried out using SPSS 19.0 software.The data are presented as mean\u2009+\u2009SEM. Statistical differences among groups were evaluated with one-way ANOVA analysis. Bonferroni test was used for Post Hoc Multiple Comparisons. Statistical significance was set at We first observed the direct effect of Puerarin on OCP autophagy without RANKL. The results showed that directly intervened by Puerarin, the expression of autophagic proteins Atg5, Atg7 and Beclin1 in OCPs decreased in a concentration-dependent manner (Fig.\u00a0Next, we assessed the effect of autophagic regulation on the proliferation of OCPs treated with Puerarin. EdU and CCK-8 assay showed that after 24\u2009h intervention, Puerarin at 50\u2009\u03bcm significantly inhibited the relative number of OCPs, which was recovered with the overexpression of Atg5, Atg7 or BECN 1 (Fig.\u00a0The direct effect of Puerarin on regulating the OCP autophagy and subsequent OCP proliferation was documented. It is known that RANKL could promote the autophagy of OCPs , 27. TheNext, we observed the effects of the autophagic activity on RANKL-regulated OCP autophagy and osteoclast differentiation in the presence of Puerarin treatment. Under the intervention of RANKL, the LC3 transformation of OCPs inhibited by Puerarin was reversed by BECN1 overexpression, but the overexpression of the other two autophagic genes Atg5 or Atg7 could not affect Puerarin-treated OCP autophagy Fig.\u00a0a, b. In As a phytoestrogen, Puerarin is known to inhibit the formation of osteoclasts, yet the detailed mechanism remains unclear, which raises an interesting scientific question for investigation. The effect of Puerarin on autophagic response has been verified. Here, we provide the first evidence demonstrating the effect of Puerarin on regulating the autophagic activity of OCPs. Combining up-to-date molecular manipulations and classic pharmacological inventions, our findings revealed an important autophagic mechanism underlying Puerarin-treated osteoclastogenesis.Osteoclast formation is known to consist of the proliferation, fusion and differentiation of OCPs . FirstlyPuerarin is well accepted as an autophagy regulator and osteoclastogenesis inhibitor. However, the role of autophagy in Puerarin-regulated osteoclastogenesis is still unclear. The present study clarified the potential mechanism regarding osteoclastogenesis treated by Puerarin from the angle of autophagic response. It proved the effect of Puerarin on inhibiting the autophagic activity of OCPs in the absence or presence of RANKL, which is responsible for Puerarin-inhibited proliferation and differentiation of OCPs respectively. This study not only explored the molecular mechanisms of Puerarin-inhibited osteoclastogenesis, which laid the foundation for further investigation, but also presented novel clues for improving the therapeutic strategies of Puerarin in treating osteoporosis. In addition, the role of autophagy in the resorption activity of the osteoclast treated by Puerarin has not been studied, which suggests research gaps to be bridged in future studies."}
+{"text": "Foot-and-mouth disease (FMD) is a highly infectious viral disease, recognised to affect animals in the order Artiodactyla. The disease is rarely fatal in adult animals, however high mortality is associated with neonatal and juvenile infection.Five puppies died after being fed lamb carcases, the lambs having died during an outbreak of FMD in Iran. Following a post-mortem examination, cardiac\u00a0tissue from one of the dead puppies was subjected to virus isolation, antigen ELISA, real-time RT-PCR, sequencing and confocal microscopy to assess the presence and characteristics of any FMD virus. The virological and microscopic examination of the cardiac tissue provided evidence of FMD virus replication in the canine heart.The data generated in this study demonstrate for the first time that FMD virus can internalise and replicate in dogs and may represent an epidemiologically significant event in FMD transmission, highlighting the dangers of feeding diseased animal carcases to other species. The reporting of this finding may also focus attention on similar disease presentations in dogs in FMD endemic countries allowing a better understanding of the prevalence of such events. Syncerus caffer caffer). Infection of non-artiodactyla species has been reported in natural and/or experimental settings such as in hedgehogs, rats, cats, kangaroos and dogs [Foot-and-mouth disease (FMD) is an important transboundary animal disease, the aetiological agent of which is a picornavirus FMD virus (FMDV). The most commonly recognised signs of infection are vesicles on the feet and in the mouth, often associated with pyrexia. Acute myocarditis and death may also occur during infection of young susceptible animals. FMD directly impacts the livelihoods of farming communities in sub-Saharan Africa and large parts of Asia via reduced productivity and loss of draft-power, as well as imposing barriers on access to lucrative export markets for animals and animal products . In FMD-and dogs , 4, althand dogs , 6, howeand dogs , Germanyand dogs  demonstrand dogs . Due to and dogs .Canis lupus familiaris isolate CAN001). RNA was extracted from the submitted tissues, and FMDV RNA was detected by real-time RT-PCR targeting the 5\u2032 untranslated and 3D polymerase regions [In February-March 2016, cases of FMD due to serotype O were reported in Iranian provinces to the south and east of Tehran resulting in widespread disease in livestock, including reports of FMDV-associated mortality among young animals. In February, the Iran Veterinary Organisation (IVO) were informed that 2 puppies in Esfahan Province, and 3 in Kurdistan Province had become lethargic and weak 2 days after being fed carcases of lambs which themselves had died during an outbreak of FMD. During the following 1\u20132 days all\u00a05 puppies died. Subsequent post mortem examination of these puppies by the IVO revealed lesions in heart tissue suggestive of FMD infection, and cardiac tissue from one case was collected and found to be positive for FMDV RNA via RT-PCR. This tissue was included in a batch of clinical samples collected from the region and submitted to the FAO World Reference Laboratory for Foot-and-Mouth Disease (WRLFMD), UK, for FMDV confirmation and characterisation. The canine origin of this sample was confirmed by using PCR amplification and sequencing of a fragment of the cytochrome c oxidase subunit 1 (COX1) gene  which de regions . The Ct  regions  and the QOM\u2009\u2212\u200915 lineage closely related to FMDVs circulating concurrently in the region . The 3A region of the FMDV genome has previously been associated with host specificity, with deletions present in the 3A region of isolates of swine origin found to be attenuated in cattle. Interestingly, a reverse mutation in the same position (I248T) has previously been observed in an FMDV isolate experimentally adapted to the guinea pig which may indicate importance in this site for species specific tropism [Complete FMDV genomes (8189 nucleotides) obtained from\u00a0both the canine heart sample and isolate IRN/9/2016 (GenBank accession numbers MT944980 and MT944981 respectively) were determined using next-generation sequencing . PhylogeThe presence of FMDV proteins and microscopic cardiac damage was imaged using confocal microscopic examination. The cardiac tissue, which had been stored in a 50:50 v/v PBS:glycerol solution at -20\u00a0\u00b0C, was placed in PBS at 4\u00a0\u00b0C overnight to remove the glycerol and the following day was frozen in a cryomould containing optimal cutting temperature (OCT) compound. Tissue sections were immunolabelled using a mouse IgG2a monoclonal antibody (mAb), 2C2, targeting the FMDV non-structural protein 3A and a mouse IgG1 mAb, IB11, previously described as targeting the FMDV capsid . PrimaryIn summary, the data presented here support the conclusion that canine fatalities occurred as a result of FMDV infection in puppies which had fed upon infected carcasses in an outbreak of serotype O FMDV in Iran. This represents the first description of natural infection of dogs by FMDV. Specifically, evidence is provided to show that FMDV can infect and damage the cardiac myocytes and was the cause of death in at least one of these animals. The wider epidemiological importance of this finding is unknown. However, wider awareness of this case report should ensure that a provenance of FMDV\u00a0infection is given greater consideration in scenarios entailing canine deaths associated with FMD outbreaks in endemic countries. Importantly there is a clear risk of feeding young dogs the carcases of animals which have been infected with FMDV, and therefore this practice should be avoided."}
+{"text": "In the past two decades, there has been a significant improvement in the understanding of the molecular pathogenesis of Renal Cell Carcinoma (RCC). These insights in the biological pathways have resulted in the development of multiple agents targeting vascular endothelial growth factor (VEGF), as well as inhibitors of the mammalian target of the rapamycin (mTOR) pathway. Most recently, checkpoint inhibitors were shown to have excellent clinical efficacy. Although the patients are living longer, durable complete responses are rarely seen. Historically, high dose interleukin 2 (IL2) therapy has produced durable complete responses in 5% to 8% highly selected patients\u2014albeit with significant toxicity. A durable complete response is a surrogate for a long-term response in the modern era of targeted therapy and checkpoint immunotherapy. Numerous clinical trials are currently exploring the combination of immunotherapy with various targeted therapeutic agents to develop therapies with a higher complete response rate with acceptable toxicity. in this study, we provide a comprehensive review of multiple reported and ongoing clinical trials evaluating the combination of PD-1/PD-L1 inhibitors with either ipilimumab  or with anti-VEGF targeted therapy. Kidney cancer is the second most common malignancy arising from the urinary system. In 2019, about 73,820 new cases and 14,770 deaths were estimated to occur in the United States [In the past two decades, there has been significant progress in the understanding of the molecular pathogenesis of ccRCC. These insights in the biological pathways have resulted in the development of multiple agents targeting vascular endothelial growth factors (VEGF) such as sunitinib, pazopanib, cabozantinib, axitinib, and lenvatinib, as well as inhibitors of the mammalian target of rapamycin pathway (mTOR) such as temsirolimus and everolimus. These agents have improved the objective response rates (ORR) and overall survival (OS); however, they are short-lived. The ability of the tumor cells to evade the host immune surveillance by PD-1-PD-L1 interaction, lead to the development of checkpoint inhibitors that target either PD-1 or PD-L1, and restore immune competence. Nivolumab, a fully human IgG4 programmed death 1 (PD-1) checkpoint inhibitor selectively blocks the interaction between PD-1 (expressed on activated T cells) and PD ligands (expressed on immune cells and tumor cells). In a Phase III trial of patients previously treated with one or two VEGF inhibitors, nivolumab, when compared to everolimus, showed significantly longer overall survival along with fewer adverse events . DespiteHigh or Angiolow), anti-tumor immune response (TeffHigh or TeffLow), and myeloid inflammation (MyeloidHigh or MyeloidLow) [low subgroup, the combination therapy had better PFS when compared to sunitinib. Additionally, the high Teff gene signature expression, a maker of a high preexisting anti-tumor immune response, was associated with improved PFS with atezolizumab\u2009 + \u2009bevacizumab when compared to sunitinib monotherapy. A higher level of PD-L1 expression by immunohistochemistry was also predictive of a better response to the combination therapy. However, tumor mutation and neoantigen burden did not correlate with PFS [Vascular endothelial growth factors (VEGF) play a crucial role in tumor angiogenesis by binding to VEGF receptors. In addition to a proangiogenic effect, they also exert immunosuppression in the tumor microenvironment by not only inducing the accumulation of myeloid-derived suppressor cells and regulatory T cells but also by impeding the migration of T lymphocytes towards the tumor microenvironment. VEFG inhibition can restore antitumor immunity by normalizing the vasculature and endothelial cell activation . AdditioloidLow) ,8,9,10. Numerous of these combinational studies are ongoing, and some have been recently published. In this review, we summarize the clinical trials evaluating the combination of PD1/PDL1 inhibitors with either ipilimumab  or angiogenesis inhibitors.p < 0.001). Progression-free survival (PFS) and overall response rates (ORR) also favored the checkpoint inhibitors when compared to sunitinib and were 11.6 months vs. 8.4 months and 42% vs. 27% (p < 0.001), respectively. The complete response (CR) rate was 9% in the combination immunotherapy arm. However, the PFS and ORR were better with sunitinib monotherapy in patients with IMDC favorable risk cancer. Additionally, PDL-L1 status was not predictive of response to the combination therapy. Treatment-related grade 3 or 4 adverse events (AE) occurred in 250 (46%) and 335 (63%) patients in nivolumab + ipilimumab and sunitinib groups, respectively. The most common grade 3 or 4 AEs in the combination group were elevated lipase levels, fatigue, and diarrhea. While in the sunitinib group, the most common grade 3 or 4 AEs were hypertension, fatigue, palmar-plantar erythrodysesthesia, and elevated lipase levels. About 35% of patients in the combination immunotherapy group required high-dose steroids for the management of immune-mediated adverse events. There were eight treatment-related deaths in the combination group and four in the sunitinib group. Based on the study results, the US Food and Drug Administration (FDA) approved the combination immunotherapy for intermediate and poor-risk patients in the first-line setting for metastatic ccRCC and also received a category 1 recommendation by the National Comprehensive Cancer Network (NCCN). Additionally, Grunwald and colleagues studied the depth of response as an indicator for long term survival among the 1096 patients in Checkmate 214 with previously untreated ccRCC [In Checkmate 214, a Phase III randomized open-label multicenter trial, nivolumab (3 mg/kg) + ipilimumab (1 mg/kg) was compared with sunitinib monotherapy (50 mg daily for 4 weeks on and 2 weeks off every cycle) in patients with treatment na\u00efve metastatic ccRCC  summari. Of the ed ccRCC . They foed ccRCC .p < 0.001) irrespective of IMDC risk groups and PDL1 status. The median OS was not reached, but the risk of death was 47% lower with combination therapy when compared to sunitinib. This OS benefit was also evidenced in all subgroups irrespective of age, metastatic sites, and IMDC risk groups. The ORRs were better with the combination therapy when compared to sunitinib and were 59.3% vs. 35.7%, respectively. The most common grade 3 and 4 treatment-related adverse events in both groups were diarrhea and hypertension. The incidence of hepatic toxicity was higher in the pembrolizumab-axitinib group; however, there were no deaths related to hepatoxicity. Based on the significant efficacy and acceptable toxicity profile, this combination therapy was approved by the FDA for treatment na\u00efve metastatic ccRCC, irrespective of PDL1 status or IMDC risk stratification. Brian Rini and colleagues presented a subgroup analysis of KEYNOTE-426 during the American Society of Clinical Oncology (ASCO) annual meeting in June 2019, examining intermediate and poor-risk groups in addition to the sarcomatoid group [In Phase III, randomized KEYNOTE-426 clinical trial of the efficacy of checkpoint PD-1 inhibitor, pembrolizumab (200 mg IV every 3 weeks) in combination with axitinib  was compared to sunitinib monotherapy in previously untreated patients with metastatic ccRCC . In thisid group . Analysiid group . This stTK Choueiri and colleagues evaluated the combination of avelumab, a PD-L1 antibody with axitinib in a Phase 1b clinical trial, where it was not only shown to be safe, but also had 58% objective response rate in patients with metastatic ccRCC . These en = 101), atezolizumab alone (n = 103) and sunitinib (n = 101), respectively, and was not statistically significantly. In PD-L1 positive patients (defined as \u22651% PD-L1 expression on IC by IHC), the atezolizumab plus bevacizumab arm had a PFS of 14.7 months as opposed to 7.8 months with sunitinib. Treatment-related grade 3 or 4 adverse events were seen in 40% vs. 17% vs. 57% in atezolizumab plus bevacizumab, atezolizumab, alone, and sunitinib arms, respectively. In the atezolizumab plus bevacizumab group, proteinuria was the most common adverse event leading to treatment discontinuation. While in the atezolizumab monotherapy group, it was nephritis, pancreatitis, and demyelination. In the sunitinib group, there was increased blood creatinine and palmar-plantar erythrodysesthesia syndrome. Of note, there were two treatment-related AEs leading to death in the sunitinib group secondary to sudden death and intestinal hemorrhage, and one in the atezolizumab plus bevacizumab group secondary to intracranial hemorrhage [In a Phase II randomized study (IMmotion150) reported by McDermott et al., atezolizumab monotherapy or in combination with bevacizumab was compared with sunitinib in 305 patients with treatment na\u00efve metastatic renal cell carcinoma. In the intention to treat population, the median PFS was 11.7 months vs. 6.1 months vs. 8.4 months  with atezolizumab plus bevacizumab  and fatigue (n = 1). Also, 70% (14/20) patients experienced grade 3 and 4 treatment-related AEs. This study arm was subsequently closed due to significant toxicities. The objective response rate was 45%, and all were partial responses. The median PFS and OS were 7.2 months and 27.9 months, respectively.Twenty patients were enrolled in the nivolumab plus pazopanib group. However, 4 DLTs were seen with this combination, including elevated liver enzymes  patients, and the most common were hypertension, hepatotoxicity, diarrhea, and fatigue. The objective response rate was 54.5%, with two complete responses and 16 partial responses. The median PFS was 12.7 months, and median OS was not reached. Though there were signals for antitumor efficacy, due to significant toxicities, this combination was also not considered for further evaluation .In the Big Ten Cancer Research Consortium sponsored Phase Ib/II clinical trial, the safety and antitumor activity of pembrolizumab in combination with bevacizumab was evaluated in 61 patients with metastatic ccRCC, who at received at least one prior systemic therapy . No doseRET and KIT [Lenvatinib is an oral multi kinase inhibitor that targets VEGFR1\u20133, FGFR1\u20134, PDGFR\u03b1, and the oncogenes  and KIT . A Phase and KIT . Patient and KIT . Median  and KIT .Preliminary safety and antitumor effects of the Phase I part of the KEYNOTE-018 study were presented at the ASCO annual meeting 2017 . Twenty COSMIC-021 clinical trial is a Phase Ib, multicenter trial that evaluated the safety and efficacy of the combination of cabozantinib (VEGFR/MET/AXL inhibitor) at 40 mg or 60 mg daily dose along with atezolizumab 1200 mg every three weeks in seven genitourinary cohorts, including a cohort of advanced RCC. There were no DLTs but the 40 mg cabozantinib dose was chosen for the expansion cohort. Of the 10 evaluable patients, the ORR was 50% with 1 CR, 4 PR. The most common grade 3 AEs were hypertension, diarrhea and hypophosphatemia .Several other studies investigating other combinations of antiangiogenic agents and immunotherapies in mRCC are ongoing. Ongoing trials include VEGF/PD-1 blockade combination, including lenvatinib with everolimus and pembrolizumab, and nivolumab with cabozantinib as first-line treatment in patients with mRCC are summarized in The therapeutic landscape of renal cell carcinoma has rapidly advanced in the past decade. The combination strategies of checkpoint inhibitors along with ipilimumab or antiangiogenic agents have demonstrated good synergy. Most notably, for the IMDC intermediate and poor-risk patients, the dual checkpoint inhibitor combination of nivolumab with ipilimumab and the combination of pembrolizumab with axitinib have shown improved response and overall survival leading to FDA approval in the first-line setting. Similarly, for the IMDC favorable-risk, the combination of pembrolizumab with axitinib or single-agent VEGF inhibitors such as sunitinib and pazopanib are preferred first-line options . The comIt is imperative to understand that not all patients benefit from combination therapy, and some have a higher incidence of serious toxicities. Biomarkers and genomic studies that can predict the efficacy of either monotherapy or combination therapy should be explored to individualize the treatment. A few combinations appeared to be beneficial in limited cases with specific histology and immunologic biomarkers and may be considered in particular situations, as seen in atezolizumab and bevacizumab in comparison to sunitinib monotherapy, where PFS was more significant in sarcomatoid variant mRCC and PD-L1 expressing tumors . Not allCombination therapy can be associated with severe side effects, with a significant overlap in the toxicity profile of the drugs. For example, diarrhea and hepatoxicity are common side effects that can be associated with either axitinib or checkpoint inhibitors. The management varies based on the etiology and also the severity, i.e., treatment discontinuation/dose reduction and use of loperamide for diarrhea in case of axitinib and timely initiation of high dose corticosteroids for autoimmune colitis and hepatitis secondary to checkpoint inhibitors. Checkpoint inhibitor and antiangiogenic combination therapy requires intense monitoring on treatment that entails frequent office visits, physician and nurse assessments, investment of health-care resources, and higher financial burden on the patient. A multidisciplinary approach and an in-depth knowledge of complications are crucial before embarking on the combination therapy.In summary, the combination of checkpoint inhibitors and anti-angiogenic agents is a valuable addition in the therapeutic armamentarium for mRCC. The choice of combination therapy or monotherapy should be individualized after a thorough discussion between the physician and patients based on cost, efficacy, and toxicity profile."}
+{"text": "The introduction of nanoparticles made of polymers, protein, and lipids as drug delivery systems has led to significant progress in modern medicine. Since the application of nanoparticles in medicine involves the use of biodegradable, nanosized materials to deliver a certain amount of chemotherapeutic agents into a tumor site, this leads to the accumulation of these nanoencapsulated agents in the right region. This strategy minimizes the stress and toxicity generated by chemotherapeutic agents on healthy cells. Therefore, encapsulating chemotherapeutic agents have less cytotoxicity than non-encapsulation ones. The purpose of this review is to address how nanoparticles made of polymers and lipids can successfully be delivered into lung cancer tumors. Lung cancer types and their anatomies are first introduced to provide an overview of the general lung cancer structure. Then, the rationale and strategy applied for the use of nanoparticle biotechnology in cancer therapies are discussed, focusing on pulmonary drug delivery systems made from liposomes, lipid nanoparticles, and polymeric nanoparticles. Many nanoparticles fabricated in the shape of liposomes, lipid nanoparticles, and polymeric nanoparticles are summarized in our review, with a focus on the encapsulated chemotherapeutic molecules, ligand\u2013receptor attachments, and their targets. Afterwards, we highlight the nanoparticles that have demonstrated promising results and have been delivered into clinical trials. Recent clinical trials that were done for successful nanoparticles are summarized in our review. Lung cancer is the most frequently diagnosed cancer in the world and a common reason for cancer-related deaths . For patAdenocarcinoma is a popular type, representing around 40% of all total diagnostic cases. It usually occurs in smokers and nonsmokers . It arisLarge cell carcinoma comprises 5\u201310% of lung cancer patients. It arises from the central region of the lungs, the area nearest to the lymph nodes, and the wall of the chest .SCLC represents 25% of all invasive cancer types worldwide, and it is found exclusively in smokers . It origThe last type of lung cancer is lung carcinoid tumor. It originates from neuroendocrine cells, which is are special cells located in the lungs. The growth of this type of cancer is typically very slow and it rarely spreads see  17]..17].Here, we try to highlight the development and application of nanoparticles in lung cancer treatment, mainly those made of liposomes, lipids, and polymer materials. Many nanoparticle-based therapies have been developed for the treatment of metastatic NSCLC, such as liposomes, polymeric nanoparticles (NPs), albumin NPs, Lipid NPs, inorganic NPs, and metal NPs. However, few have been translated successfully into clinical trials. In our current study, liposomes, solid lipid nanoparticles (SLNPs), polymeric nanoparticles (PNPs), and hybrid polymeric materials were studied, and they are summarized in The possible solution for lung cancer treatment involves chemotherapy, surgery, radiotherapy, and the development of targeted nano-therapies . Indeed,First, the term nanotechnology can be used to describe the possible control of matter in the size range between 1 and 100 nm. This size range is strongly recommended for biomedical applications . The appEPR can increase the distribution of encapsulated cargo molecules, whereas nano-sized particles show good accumulation in tumor tissues because they can rapidly reach a specific location with a higher concentration than other formulations . MeanwhiPulmonary drug delivery systems provide a good opportunity to delivery therapeutic molecules directly into the locations of lung cancer cells, through both systemic and localized treatment forms . The anaFor this reason, targeted nano-carriers are used extensively to localize chemotherapeutic agents inside the lungs. This decreases the effect of systemic dilution by increasing the chance of their accumulation within the tumor site as well as reducing the cytotoxicity of chemotherapeutic agent . This haIt has been reported that micro and macro particles are deposited at the oropharynx and upper respiratory tract via impaction, while nano-sized particles can reach the site of action easily. Therefore, the particle size range must be carefully controlled. In addition, many pulmonary clearance mechanisms have been observed, such as alveolar macrophages and the mucociliary escalator system, which prevents the adsorption and stabilization of nanoparticles. The mucociliary system can remove most of the deposited insoluble particles with sizes > 6 mm, and most of them can be phagocytized . VariousLiposomes are phospholipid bilayer spherical systems composed of a core surrounded by a shell . They arThe PEGylated process can cause changes in the physicochemical properties of nanoparticles, resulting in changes to their hydrophilicity, diameter, conformation, and intermolecular interactions . Both PEKoshkina and her coworkers used 9-nitrocamptothecin (9NC)-loaded liposomes as an inhalation treatment against lung cancer, leading to reduced lung weight and minimizing the number of tumor foci in two different lung metastasis models . In the Additionally, a dual target therapy was developed by Wang and his team using the specific ligand peptide HAIYPRH (T7) and the cationic cell-penetrating peptide TAT that were connected with phospholipids via a polyethylene glycol (PEG) spacer to prepare the dual-ligand liposomes (T7/TAT-LP-PTX). This structure was evaluated on the tumor spheroids, which revealed that T7/TAT-LP was more efficaciously internalized in tumor cells than TAT-LP, T7-LP, and LP, respectively . Also, AZhu and his coworkers  investigTagami and his team incorporated Poloxamer 188 (P188) into liposome-DOX membranes. The results suggested that DOX-loaded DPPC/P188 liposomes may be useful for treating lung cancer .Zhang and his team developed poly-conjugated octadecylamine, and then the mixture was attached to liposomal pixantrone. The structure represents a promising approach for lung cancer treatment . Many chSLNs are formed by the replacement of the liquid lipid core with a solid one. They are generally solid at 37 \u00b0C with diameters of less than 80 nm after filtration . SLNs haVideirae and his team evaluated the use of paclitaxel inserted into lipid nanocarriers to determine their therapeutic efficiency in lung cancer. It was reported that these nanocarriers reduced the number and size of lung metastases more in comparison to intravenous injection of the same non-encapsulating drugs . AdditioDrugs that have been encapsulated inside SLNPs are summarized in Kabary et al. designed layer-by-layer alternate adsorption involving the use of lactoferrin (LF) and hyaluronic acid (HA) on the surface of lipid nanoparticles (NPs) for dual delivery of berberine (BER) and rapamycin (RAP) to lung cancer. The obtained results showed a high recommendation for the use of LbL for enhanced functionalization of NPs and cellular uptake .Khatri et al. optimized Artemether-loaded solid lipid nanoparticles to treat lung cancer. The results exhibited an anti-lipolytic effect against lung cancer .Rosi\u00e8re, et al. designed solid lipid nanoparticles coated with folate-grafted polyethylene glycol (PEG) and chitosan. Then, paclitaxel was loaded into the SLN. The results showed a positive impact of the coated SLN on the delivery of paclitaxel by inhalation .Soni, et al. developed solid mannosylated lipid nanoparticles loaded with gemcitabine using emulsification and solvent evaporation process. The result was improved cellular uptake and drug efficacy of gemcitabine inside lung cancer .Shao, et al. developed transferrin (Tf)-decorated nanostructured lipid carriers as a multifunctional nanomedicine for the co-delivery of paclitaxel (PTX) and enhanced green fluorescence protein plasmid .Wang, et al. developed a method involving combined delivery of PTX and DOX using a melt-emulsification technique with nanostructured lipid carriers. The results showed a highly cytotoxic effect for all formulations in vitro, as compared to single drug delivery .PNPs are produced in the final colloidal nanoparticle suspension, and they can be synthesized by polymer self-assembly . Good reYordanov and his coworkers fabricated epirubicin-loaded poly butyl cyanoacrylate nanoparticles in an aqueous dispersion. They were assembled with Pluronic F68 and Polysorbate 80, as non-ionic surfactants. Fluorescent imaging done in the adenocarcinoma cells showed that the free cargo molecules were highly internalized into the cell nucleus, whereas the drug loaded nanoparticles were accumulated in the cytoplasm. This may explain why cellular internalization was done by endocytosis. The final result suggested that nanoparticle-based anthracycline should be developed as a therapeutic tool for lung adenocarcinoma .Polymeric nanoparticles can be formed by self-assembly or they can be conjugated with proteins like albumin or gelatin as hybrid polymeric protein nanocarriers (HPPNCs). In this special case, the platform takes advantage of both proteins and polymers for optimizing drug carriers. These hybrid materials are used to encapsulate genetic materials and naturally derived vectors, such as viral vectors . Using t2O. For this reason, a thickness reduction of the shell from 300 to 100 nm can cause rapid drug release from 79% to 85% in 24 h. In other assemblies, the shell of PHLNPs can be formed by polymers and lipids included as a core. This assembly can minimize the clearance rate from the lung  (DSPE-PEG2000). The structure obtained particle sizes of about 170 nm, Polydispersity index(PDI) of <0.2, and a drug entrapment efficiency of about 66% with good serum and storage stability .Xiong and his coworkers designed a combined therapy using cisplatin and metformin. Cisplatin was conjugated to polyglutamic acid to form an anionic mixture, and then the mixture was reacted with cationic polymeric metformin. The payload was then inserted into a liposome composed of DOTAP-trimethylammonium/Cholesterol/DSPE-PEG-anisamide aminoethyl.Cisplatin-loaded nanoparticles exhibited significantly increased tumor accumulation compared to free ones without causing any nephrotoxicity .Mottaghitalab and her team optimized SP5-52 peptide conjugated silk fibroin nanoparticles loaded with gemcitabine to treat induced lung tumors in a mice model. The results showed a significant reduction in lung tumor size compared with the untreated groups .As a comparison between liposomes, solid lipid nanoparticles and hybrid polymeric lipid nanoparticles, it can be summarized that liposomes are mostly coformulated by the presence of a phospholipid and cholesterol core and shell in their structure . Their dIn this way, liposomes have great advantages as biodegradable and biocompatible materials with the potential for large-scale production during fabrication, allowing them to be used widely in biomedical applications. Besides that, entrapped drugs can be modulated inside the core and shell.The disadvantage of liposomes is mostly attributed to the possible oxidation of the liposomal phospholipid layer, leading to hydrolysis of the membrane and degradation of the liposomal structure. This drawback leads to the leakage of drugs out of the liposome. Furthermore, lipid peroxidation leads to damage to the properties of the lipid structure, particularly cellular permeability. Additionally, temperature, pH, and light may induce instability into the physical and chemical properties of liposomes, leading to a reduction in the shelf life of liposomes during long-term storage. The other disadvantages could be related to the use of the freeze-drying technique during liposomal fabrication , since tSolid lipid nanoparticles are just a solid core of lipids formed by one of the following techniques at 37 \u00b0C: high-pressure homogenization, double emulsion, high-shear homogenization, or emulsifier evaporation . The lipThe disadvantage of this structure is mostly associated with the type of lipid used, as this can produce cytotoxicity related to free fatty acids and can cause a growth in the diameter during fabrication . AdditioPolymeric nanoparticles can be identified as an interaction of two opposite polymeric materials that form a cross-linked network and condensed core . This coRecently, advanced nanotechnology has undergone significant progression by using materials for the production and design of an optimal drug delivery system, since the physicochemical properties of polymers are used as an advantage to develop both liposomes and solid lipid NPs and to overcome their drawbacks during fabrication. These new structures are called polymeric hybrid lipid nanoparticles. Polymeric hybrid lipid nanoparticles contain three main structures, as follows: (1) a hydrophobic/hydrophilic polymeric core inserted inside liposomes or coated by a lipid monolayer; (2) a solid lipid core or liposomal phospholipid/cholesterol layer surrounded by a polymeric shell; (3) polymer materials incorporated inside solid lipid or liposomal phospholipid/cholesterol layers and then and an outer component consisting of a PEGylation.Clinical trials represent strategy-based scientific research that attempts to evaluate the influence of a new therapeutic molecule on human health outcomes. It includes several steps, as follows: Phase I\u2014the study of new therapeutic molecules on a small group of volunteers; Phase II\u2014monitoring and evaluation of safe drugs on a large group of volunteers; Phase III\u2014the study of safe drugs in different groups of volunteers located in different regions and countries; and Phase IV\u2014monitoring of safe drugs after obtaining approval for their use in a wide population over long time frame . During Nanotechnology has been used recently as an interesting approach to develop cancer therapies ,123,124."}
+{"text": "As the COVID-19 pandemic escalates worldwide, it is apparent that many patients with more severe illness will also experience delirium. These patients pose a particular challenge in the application of optimal care due to issues with infectious risk, respiratory compromise and potential interactions between medications that can be used to manage delirium with antiviral and other treatments used for COVID-19. We describe a guidance resource adapted from existing guidelines for delirium management that has been tailored to the specific challenge of managing delirium in patients with COVID-19 infection. Issues around the assessment and treatment of these patients are examined and distilled into a simple (one-paged guidance resource that can assist clinicians in managing suspected delirium. It is supported by at least 11 validation studies (involving >2500 patients) that indicate high sensitivity (83\u2013100%) with moderate to high specificity (70\u201399%) for delirium  but also commonly occurs in response to disturbances that are primarily located systemically, such as peripheral infection, organ failure or metabolic disruptions. The precise mechanisms by which COVID-19 may cause neurological manifestations are still unclear but may include direct CNS infection, access due to reduced blood\u2013brain barrier integrity, retrograde neuronal transport, hypoxic damage, vascular mechanisms and neuroinflammatory responses, along with the many other causes that have been associated with increased delirium propensity. In addition, patients in isolation, requiring mechanical ventilation, with reduced sensory input and mobilisation are all more prone to developing delirium . The PINCH-ME algorithm see Fig.\u00a0 is frequper se , while haloperidol remains a useful option due to the range of routes by which it can be administered . Moreover, use of antipsychotics brings with it a risk of a variety of other potential adverse effects, with a significantly increased risk of cerebrovascular incidents especially in patients with pre-existing cognitive issues, such as dementia (Rao www.covid19-druginteractions.org). This information indicates a favourable profile for olanzapine in terms of interactions with antiviral agents, while haloperidol, risperidone and quetiapine increase the exposure to potential adverse effects of many antiviral agents and haloperidol warrants particular caution in respect of potential for effects on cardiac conduction.A further consideration relates to potential interactions between psychotropic agents and other treatments used in these patients, with antiviral agents a particular focus of concern. The Liverpool Drug Interaction Group , in collaboration with the University Hospital of Basel (Switzerland) and Radboud UMC (Netherlands), has collated information regarding interactions between over 400 medications (including psychotropics) and experimental COVID-19 therapies  and practical, addressing four steps in decision-making: (1) assessment for delirium using the 4AT, (2) assessing potential aetiological factors using the PINCH-ME algorithm, (3) guidance on non-pharmacological management and finally, (4) guidance on use of pharmacological interventions. While in many cases, clinicians will be comfortable in detecting and managing delirium, this guidance can assist where a more structured approach is needed. It can also serve as a useful support to guide efforts to assess and manage delirium in consultation with psychiatry services. This guidance provides a rapid response to the need to focus our efforts to manage delirium during the pandemic that has been disseminated to support everyday practice in local services and beyond. It can also provide a document that can be further developed in a more systematic way (e.g. consensus guidelines) over time as further evidence  emerges."}
+{"text": "Individual difference analyses further show that participants who benefit from flavanols intake during hypercapnia are also those who do so in the cognitive challenge. These data support the hypothesis that similar vascular mechanisms underlie both the peripheral and cerebral effects of flavanols. They further show the importance of studies combining physiological and graded cognitive challenges in young adults to investigate the actions of dietary flavanols on brain function.Cocoa flavanols protect humans against vascular disease, as evidenced by improvements in peripheral endothelial function, likely through nitric oxide signalling. Emerging evidence also suggests that flavanol-rich diets protect against cognitive aging, but mechanisms remain elusive. In a randomized double-blind within-subject acute study in healthy young adults, we link these two lines of research by showing, for the first time, that flavanol intake leads to  In particular, cocoa flavanols, a sub-group of flavonoids  have been shown to improve endothelial function in humans quite rapidly (within 1\u20132\u00a0h) by enhancing vasodilatory properties of peripheral arteries6. Acute benefits translate effectively into short-term (2\u20138\u00a0weeks) clinically relevant improvements in blood pressure and endothelial function 8, comparable to those of drugs, such as statins10. Mechanistically, the beneficial effects of cocoa flavanols on endothelial function have been linked to increases in bioavailability of nitric oxide (NO)6, which is known to be affected in the earliest stages of vascular disease11.Whilst the acute effects of flavanols have been mainly attributed to phase I/II-derived (\u2212)-epicatechin metabolites, the short to long term benefits may be also driven by gut-derived metabolites13, although this remains to be established. Another emerging line of research further suggests that this class of plant-derived compounds may protect against cognitive decline in aging16 and cognitive resilience to neuropsychiatric disorders and stress18. Yet, the extent to which increases in circulatory levels of NO by flavanols can translate into benefits in the brain vasculature, and effectively influence cognitive performance in humans, is poorly understood.Lifespan wear and tear of the vascular system due to poor nutrition and lack of fitness, among other factors, can accelerate cognitive aging and lead to dementia. There is epidemiological evidence suggesting that flavonoids, a group of small molecules present in fruits and vegetables, can protect against vascular disease and cardiovascular-related mortality2)19. Relevant to our hypothesis is the fact that NO is known to contribute to CO2-dependent increases in cerebral blood flow in humans (hypercapnia)20. Furthermore, cerebrovascular reactivity to CO2 is widely accepted as a key biomarker of cerebrovascular health, and has been closely associated with cognitive function in health and disease states25. Hence, hypercapnia represents a robust model to test whether flavanol-mediated increases in endothelial function (as assessed by gold-standard FMD) mediate benefits in cerebrovascular and cognitive function.Cerebral blood flow is controlled by neuronal activity but also by levels of arterial blood gases, in particular carbon dioxide . Further, modulation of cerebral physiological outcomes by flavanols in the context of neuronal/cognitive challenges frequently and surprisingly fail to translate into cognitive benefits32. A possibility is that the benefits of flavanols may only be visible at high levels of task difficulty. This highlights that, whilst some of these studies could provide ecological validity (as they target aging adults with cognitive and/or vascular problems), they were not designed in a manner that allows for an evaluation of the underlying physiological effects of these compounds in the human brain. This leaves some uncertainty about whether flavanols\u2019 benefits in peripheral vascular function are reflected by similar effects on cerebrovascular reactivity, and whether the cognitive and vascular benefits are related.Only a handful of studies have previously reported effects of flavanols on the human cerebral vasculature, both in a resting stateThere is a need to determine whether the peripheral vascular benefits of flavanols extend to the cerebral vasculature. This requires well-controlled experiments to demonstrate that flavanols (a) can modulate the brain\u2019s vasculature; (b) that these effects, similarly to those found in the periphery, are revealed during physiological challenges likely involving the NO pathway; (c) that they affect cognitive performance, at least in challenging conditions; and (d) that cerebrovascular and cognitive benefits are linked.2-breathing challenge (hypercapnia) before and after intake of either a high- or low-flavanol intervention. During hypercapnia, we measured cortical haemoglobin concentration using functional near-infrared spectroscopy (fNIRS)33, which allowed us to finely quantify the dynamics of cerebrovascular reactivity, providing information not only about the amplitude of this response, but also about its time course. Similar to peripheral measures, cerebral CO2-reactivity has been shown to be mediated by the NO pathway20, which is the hypothesized mechanism underlying the beneficial effects of flavanols on peripheral endothelial function6. To measure the cognitive effects of flavanols, we employed tasks with escalating levels of difficulty34, which could inform us of the level at which the cognitive benefits of flavanols may emerge.In the current study, we employed separate physiological and cognitive challenges in a double-blind, within-subject, placebo-controlled acute (2\u00a0h) study to assess the underlying physiological actions of cocoa flavanols on cerebral and peripheral vascular and cognitive function. To measure cerebrovascular reactivity, we employed a CO2-breathing challenge \u2009=\u20096.08, p\u2009=\u20090.025 , but a significantly greater oxy-haemoglobin level after the high-flavanol in comparison to the low-flavanol (t(16)\u2009=\u2009\u2212\u20092.37, p\u2009=\u20090.030) (1c). No significant differences between interventions were detected for deoxygenated haemoglobin  at different times during the CO2-breathing challenge , with darker grey background indicating the recording region. The maps show that increases in blood oxygenation were most evident in lateral frontal regions and that high-flavanol intake led to earlier and larger responses than the other three conditions, which were similar to each other \u2009=\u200913.61,\u00a0p\u2009=\u20090.002\u00a0, but latency of oxygenation was significantly shorter (by approximately 1\u00a0min) after the high-flavanol intervention (t(16)\u2009=\u2009 \u2212\u20095.68, p\u2009<\u20090.001).Figure\u00a0p\u2009=\u2009\u2009<\u20090.001), as previously described in this population8.Efficacy of the high-flavanol intervention on endothelial function, as measured by brachial FMD Fig. , was con38. A lower IES value (expressed in seconds) indicates more efficient cognitive processing. As shown in Fig.\u00a0Figure\u00a0F\u2009=\u20093.267,\u00a0p\u2009=\u20090.029. A planned contrast between the difference in the Double-Stroop and the average of the other three conditions yielded a significant cognitive advantage in the high flavanol versus low-flavanol condition,\u00a0t(16)\u2009=\u20092.142,\u00a0p\u2009=\u20090.042 (3b). The contrast of the Stroop task with the other two tasks was not significant,\u00a0t(16)\u2009=\u20091.050,\u00a0p\u2009=\u20090.309, confirming that flavanols only generated an advantage in the most difficult condition (Double-Stroop), but not in the other conditions.Results indicate that only the Double-Stroop task differentiated between the high- and low-flavanols conditions. The interaction in the two-way repeated-measure ANOVA  was significant,\u00a02-breathing challenge after the intake of high-flavanol in comparison to the low-flavanol intervention. However, some individuals , and reached a maximum lagged cross-correlation (r\u2009=\u20090.897) when the high-flavanols waveform was anticipated by 18\u00a0s relative to the low-flavanols waveform. In contrast, in most participants (N\u2009=\u200913), the fNIRS waveforms were not as strongly correlated at lag 0 (r\u2009=\u20090.567), but reached a much higher cross-correlation  when the high-flavanols waveform was anticipated by 117\u00a0s relative to the low-flavanol waveform (i), the phenomenon reflected by the full group analyses  showed a clear and significant improvement in performance in the Double Stroop (t(12)\u2009=\u2009\u2212\u20093.784,\u00a0p\u2009=\u20090.003, N\u2009=\u200913), whereas group ii  did not (t(3)\u2009=\u20090.86,\u00a0p\u2009=\u20090.453, N\u2009=\u20094). This highlights the coupling of these two effects: those individuals showing improved cerebrovascular reactivity after high-flavanol intake also showed benefits during cognitive performance, whilst those that did not improve cognitive performance had no increase in cerebrovascular reactivity following high-flavanol intake.Crucially, cerebrovascular reactivity to flavanols for these two sub-groups was predictive of the responsiveness to flavanols under the cognitive challenge. Figure\u00a02-challenge in healthy young individuals. This suggests that, similarly to peripheral vascular benefits, flavanols result in clinically relevant improvements in cerebrovascular reactivity in a healthy brain. Such benefits further translate into improvements on prefrontal-dependent cognitive performance, but only for the highest level of cognitive demand. More importantly, only individuals who benefited from flavanol intake during hypercapnia experienced cognitive benefits, suggesting that these effects are likely linked. Finally, we confirmed that flavanol\u2019s benefits on peripheral endothelial function  are in line with those reported in previous studies39 in this population within\u00a0an acute timeframe (2\u00a0h), where maximal levels of flavanol metabolites reach circulation in humans13.The present study shows, for the first time, that cocoa flavanols lead to more efficient tissue oxygenation responses in frontal areas of the brain during a CO8 and replicated in this study . Flavanols\u2019 ability to increase bioavailability of circulating NO in humans is believed to underlie its benefits on peripheral endothelial function40 and we suggest that this mechanism is also contributing to the benefits observed in cerebrovascular reactivity. Although a direct causal link between NO and hypercapnia-induced vasodilation in humans remains to be established42, previous studies have shown that inhibition of NO results in decreased reactivity to hypercapnia in cerebral arteries44. More importantly, pharmacological interventions that directly increase NO production and improve endothelial function, such as NO-donor sodium nitroprusside45 and L-Arginine 46 also result in improvements in CO2 reactivity of cerebral vessels in humans. This is consistent with the hypothesis that flavanols enhance hypercapnia-induced release of NO from endothelial cells locally within the cerebral vasculature.The use of fNIRS allowed us for the first time to observe how flavanol intake can modify the dynamics of cerebral tissue oxygenation: our data suggest that improved efficiency of the coupling between hypercapnia and vasodilation is driven by not only larger but also faster tissue oxygenation Figs.\u00a0, 2. This48, which normally stimulates the production of NO by the endothelium49. This suggests a similar mechanism to brachial FMD, in which increases in shear-rate following hyperaemia result in NO-dependent arterial dilation51. As such, more efficient tissue oxygenation after flavanol intake might also be the result of NO-mediated increases in dilation of upstream cerebral conduit arteries  in response to hypercapnia.Recent evidence further suggests that hypercapnia induces vasodilation in the internal carotid artery  by inducing increases in shear-rate28 or during cognitive manipulations31, producing contradictory results (increases and declines in blood flow/velocity). However, neither of these contexts engages directly with NO within the vasculature, which might contribute to the discrepancy in outcomes.Previous studies that assessed the impact of flavanols on the cerebrovasculature employed other imaging methods  either at rest254. Cerebrovascular reactivity to CO2 is also depressed in populations at higher risk for cardiovascular disease and cerebral small-vessel disease, as well as in aging56. Importantly, lower cerebrovascular reactivity has been consistently associated with cognitive impairments57, and shown to be predictive of future cognitive decline24. Therefore, if the acute benefits demonstrated in the present study were to be sustained by continued intake of flavanol-rich foods , this may be particularly beneficial for populations at higher risk.In physiological, real-world conditions, flavanol-induced faster vascular reactivity may help recovery from injuries such as mild brain injury or stroke, conditions that are also associated with impaired cerebrovascular reactivity to CO31, a finding we replicate in our study. It has been previously suggested that failure to find cognitive effects in healthy young participants might be related to a high level of cognitive ability in this population, which is unlikely to be improved upon26. We suggest that the level of cognitive and physiological demands associated with the task are key factors, and we clearly demonstrate this by varying the level of conflict in the cognitive task used. Most interestingly, this indicates that flavanols may help cognition only when there is a substantial demand for information processing at the neuronal level, which, in turn, requires appropriate levels of blood oxygenation. Given the benefits in efficiency of cortical oxygenation we observed in the present study, we propose that flavanols may facilitate increases in blood oxygenation during complex tasks as a direct consequence of increased information processing demands in frontal cortical areas of the brain. Diets rich in dietary flavanols might be particularly beneficial when executive function becomes more limited . Future work should focus on systematically employing difficulty-graded cognitive challenges when assessing the efficacy of dietary flavonoids on human cognitive function.Our behavioural findings indicate that flavanols can improve cognitive performance acutely in healthy young adults, but only when the level of cognitive demand is high  were individuals with particularly large and rapid responses to hypercapnia at baseline (or after low-flavanol intake). This indicates that lack of flavanol efficacy may be due to a ceiling effect, with high baseline cerebrovascular reactivity leaving little room for improving it further. Most remarkable is the observation that these same individuals did not benefit cognitively from flavanol intake. In contrast, after flavanol intake, individuals with lower baseline cerebrovascular reactivity to hypercapnia were brought up to levels similar to those of the \u2018high performers\u2019, effectively equalizing individuals.58. However, previous research also suggests that these factors alone cannot explain the entirety of the variability observed in health benefits of these compounds59. Our data illustrate that there might be other factors at play: for example, there is evidence indicating that individuals with high cardiorespiratory fitness tend to have higher CO2 cerebrovascular reactivity61. While we did not assess the fitness of the participants in the present study, one possible explanation for the differential responses observed between sub-groups was that flavanols may have improved the cerebrovascular reactivity of less fit individuals to levels similar to those of potentially highly fit individuals. Future work should formally address this hypothesis, which may have significant translational implications.Variability of the effects of flavanols on human vascular function have been reported previously and partially attributed to variations in flavanol metabolism and/or absorption: previous research reported up to 39% variability in flavanol plasma concentration after acute intake of flavanols in young adultsWe have demonstrated that acute flavanol intake can improve efficiency in blood oxygenation (amplitude and speed) during hypercapnia in frontal cortical areas of young healthy subjects and that it is likely to contribute to improvements in cognitive function, but only when cognitive demands are high. We also show that only individuals with lower baseline cerebrovascular reactivity benefit from flavanol intake, with acute improvements in cerebral vascular function and cognitive performance. We suggest that the underlying mechanisms at play centrally may be similar to the ones detected in the peripheral vasculature, through hypercapnia-induced increases in NO release from the endothelium in cerebral arteries. Future work should confirm this by conducting studies in animal models where direct assessments of NO levels in the cerebral vasculature are possible in real time during hypercapnic challenges.The findings reported here can have important future implications for using dietary strategies containing plant-derived flavanols for enhancement of blood oxygenation and cognitive performance in healthy populations, as well as for populations at higher risk  or to help recover and treat brain injuries and disease. Most importantly, our data can potentially open new avenues for precision-medicine research with regard to understanding individual responses to flavanol intake and helping to identify populations that might benefit the most from these\u00a0interventions.fNIRS measures have high temporal sampling, which allow for studying the dynamics of oxygenation effects, but only provide information about superficial areas of the brain, which is a common limitation to all diffuse optical imaging measures. We also only focused on frontal cortical areas and observed that, within this region, the effects of flavanols in response to hypercapnia seemed to be relatively homogeneous. However, we are unable to establish whether there are regional differences across the brain. Whilst previous studies suggest that flavanols can increase NO in humans in an acute manner, in the present study we have not assessed NO species and cannot conclude that flavanol-induced responsiveness to hypercapnia is specifically linked to this mechanism. Other important limitations of this study are the low number of participants, particularly to address cognitive outcomes, and the exclusion of females. Additionally, the evaluation of efficacy of flavanol intake in an at-risk population (e.g. older adults) would have more ecological validity and this should be addressed in future work.Eighteen healthy male volunteers (18\u201345\u00a0years old) were recruited from the University of Birmingham and surrounding areas according to the following inclusion criteria: (i) non-smokers; (ii) normotensive; (iii) no history of cerebrovascular, cardiovascular or respiratory disease; (iv) no allergies or intolerances to ingredients present in cocoa powders; (v) not taking long-term medication ; (vi) not suffering from blood-clotting disorders; (vii) no known infections at the time of the study; (viii) not on a weight-reducing regimen\u00a0(Table 2). Following baseline measurements volunteers consumed either the high-flavanol intervention or control low-flavanol intervention (within 10\u00a0min). Two hours post intake, BP, FMD and fNIRS-based optical reactivity to the hypercapnic challenge were assessed, as well as cognitive function using a Modified Stroop Task34. Cognitive function was assessed only at the 2-h point to minimize practice effects. During the time between ingestion of the beverage and post measurements, optical sources and detector locations were digitized (for each visit) using a Polhemus FASTRAK 3D Digitizer . The design of the cognitive tasks was not compatible with accurate fNIRS analysis, mainly due to the variable duration of the task between subjects and the small number of trials. Methods and results regarding the standard FMD and BP measures are included in supplementary materials.The study was based on an acute, randomised, placebo-controlled, double-blind, cross-over design. After ascertaining eligibility, participants attended two visits, separated by a minimum of two weeks, in which they consumed either a high-flavanol cocoa drink (HF) or a low-flavanol (LF) cocoa (control) drink, to which they, as well as the experimenters, remained blinded throughout the study (for composition of the two cocoa interventions see Table 39 and plasma NO levels40. A similar dose of (\u2212)-epicatechin could be achieved through diet by consuming foods rich in flavanols, particularly apples, black grapes, blackberries, cherries, raspberries, pears, pulses, green tea and red wine62. Total levels of polyphenols in the powders were assessed by a Folin-Ciocalteu reagent calorimetric assay as described previously63. Individual monomer levels and procyanidin levels as well as levels of methylxanthines, were confirmed by HPLC as described previously65. Maximal levels of flavanol monomers metabolites are reached in the human circulation at approximately 2\u00a0h post-intake13. Individual doses of cocoa powder were kept at \u2212\u200920\u00a0\u00b0C and identified by a three-digit code to ensure double-blindness. Intervention beverages were identical in texture, consistency and taste, and were delivered to participants in an opaque container with an opaque straw. Participants and researchers involved in data collection and analysis were blind to the intervention conditions until all data analysis was completed.Intervention beverages were prepared immediately prior to consumption by dissolving 8.3\u00a0g of cocoa powders in 300\u00a0ml of room temperature bottled water containing low levels of nitroso species (Buxton). The cocoa powders used are commercially available : the low-flavanol control is a fat-reduced cocoa powder alkalized  and the high-flavanol cocoa powder a non-alkalized fat reduced powder . Both are matched for macronutrient and micronutrient content (including caffeine and theobromine). The high-flavanol cocoa delivered 150\u00a0mg of (\u2212)-epicatechin and 35.5\u00a0mg of catechin (flavanol monomers), whilst the low-flavanol intervention delivered\u2009<\u20094\u00a0mg of both monomers , based on 32 sources  and 14 detectors . Source and detector fibres were held in place using custom-built helmets matched to a range of head circumferences. Hair was combed away from the points of contact between detectors and scalp, whilst the smaller source fibres allowed them to be placed directly through the hair onto the scalp. Source fibres were paired such that each location was illuminated by two fibres connected to one 830\u00a0nm and one 690\u00a0nm laser, but the two fibres were never on at the same time. Each detector received light from 16 time-multiplexed sources, which stayed on for a period of 1.398\u00a0ms and off for 20.971\u00a0ms, yielding an effective sampling rate of 44.704\u00a0Hz. To avoid crosstalk between channels , the optical montage ensured that a detector could not receive light from more than one source contacting the head at less than 70\u00a0mm distance at any one time.3 resolution. All MRI scanning was carried out either for the purpose of this study or acquired from previous studies with the written consent of the participant and original researcher, with the following parameters: flip angle\u2009=\u20098\u25e6, TE\u2009=\u20093.8\u00a0ms, TR\u2009=\u20098.39\u00a0ms, inversion time\u2009=\u20091150\u00a0ms, 175 sagittal slices, voxel size 1.0\u2009\u00d7\u20091.0\u2009\u00d7\u20091.0\u00a0mm with a field of view of 288\u2009\u00d7\u2009232\u2009\u00d7\u2009175 mm (FH x AP x RL). The structural MRI was used for co-registration of optical data onto each individual\u2019s anatomy66 using nasion and pre-auricular points as references, the spatial locations of sources and detectors were digitized. Digitization points were then co-registered with T1 structural MRI scans, following procedures described by66. This approach has been shown to have errors of less than 4 mm67. See supplementary Figure All participants underwent a high-resolution structural MRI scan in a 3-T Philips Achieva MRI scanner . A whole-head T1-weighted anatomical image was produced using an MPRAGE sequence with 1 mm2 (in air) via the open-circuit Douglas bag method. Once participants were instrumented with the fNIRS device, baseline data were collected while breathing room air for 4\u00a0min, followed by 4\u00a0min breathing the 5% CO2 gas mixture (hypercapnia), then a 2-min recovery period breathing room air68. Respiratory gases were analysed for changes in end-tidal CO2 using a continuous gas analyser . Throughout this hypercapnic cerebrovascular reactivity procedure, participants remained seated whilst optical data were recorded.Hypercapnia was induced with 5% CO69  or right (\u2018/\u2019) key, corresponding to response options displayed on the screen  with an average AC count\u2009<\u2009100 digitizing units for either wavelength were discarded. For the remaining channels, raw fNIRS signals were normalised by dividing each value by the mean value across the time points for each block and channel. To determine the overall haemodynamic response across CO2 manipulations, data were corrected for vascular pulsation71. Pulse-corrected data were then motion corrected72 and low-pass (zero-phase shift) filtered at 0.05\u00a0Hz. The data were then down-sampled to one value every 9\u00a0s, and the changes in light intensities for each sample and channel pair were transformed into oxy- and deoxy-haemoglobin concentration changes using the Beer\u2013Lambert law73. Finally, to eliminate the effects of non-brain phenomena, the time course of the response for channels with the shortest source-detector distance  was regressed out from each channel data74. In-house software Opt-3D75 was used to merge channels whose diffusion paths intersected within a given brain volume (modelled as a curved ellipsoid path76). Only source-detector distances within a 20\u201350\u00a0mm range were used for analysis, in order to focus on signal extraction from deeper brain regions and exclude superficial tissue effects, dominant at shorter source-detector distances. An 8-mm spatial filter was applied to spatially reconstructed source-detector data, which were projected onto the axial surface of a model brain image in Talairach space. The data from 1 volunteer were excluded from fNIRS analysis due to excessive noise.Optical data were obtained for 10\u00a0min epochs starting 4\u00a0min before the beginning of the 5% COp\u2009<\u20090.05 (95% CI) for all outcome measures. Sample size was estimated based on previous data from our laboratory on flavanol acute changes in brachial FMD in this population : the selected sample size (N\u2009=\u200918) was expected to afford us a 95% probability of detecting a difference  of 0.6% FMD (the expected effect size) due to flavanol intake, using an alpha value of \u03b1\u2009=\u20090.05.fNIRS and FMD and cognitive data were analysed using two-way repeated measures ANOVA with intervention (low- or high-flavanol) and time  as within-subject factors. Cognitive data were analysed using a two-way repeated measures ANOVA with intervention (low- or high-flavanol) and task levels  as within-subject factors. fNIRS data were averaged across the whole cortical area recorded for each participant. Planned a priori comparisons between high and low-flavanol were tested by 2-tailed t-tests. Significance was defined as Supplementary Figures."}
+{"text": "Short Birth Interval negatively affects the health of both mothers and children in developing nations, like, Ethiopia. However, studies conducted to date in Ethiopia upon short birth interval were inconclusive and they did not show the extent and determinants of short birth interval in developing  regions of the country. Thus, this study was intended to assess the short birth interval and its determinants in the four developing regions of the country.http://dhsprogram.com). A sample of 2683 women of childbearing age group (15\u201349) who had at least two alive consecutive children in the four developing regions of Ethiopia was included in this study. A multilevel multivariable logistic regression model was fitted to identify the independent predictors of short birth interval and Akaike\u2019s Information Criterion (AIC) was used during the model selection procedure.Data were retrieved from the Demographic and Health Survey program official database website (In this study, the prevalence of short birth interval was 46% . The multilevel multivariable logistic regression model showed women living in rural area , women attended secondary education and above level , have no media exposure , female sex of the index child , breastfeeding duration , having six and more ideal number of children  and having preferred waiting time to birth two years and above  were the predictors of short birth interval.The prevalence of short birth intervals in the developing regions of Ethiopia is still high. Therefore, the government of Ethiopia should work on the access of family planning and education in rural parts of the developing regions where more than 90% of the population in these regions is pastoral. The World Health Organization (WHO) and Ethiopian Demographic and Health Survey (EDHS) reports on birth spacing recommended a birth to conception interval of at least 24 months in two consecutive births , 2.Demographic and Health Survey (DHS) data from 18 developing countries  and an International comparison study of 77 countries using DHS data revealed that a birth interval of three or more years interval improves the survival status of mothers, under-five children and infants , 4.Ethiopia is the second-most populous country in Africa, with a population size of more than 100 million and a fertility rate of 4.6 children per woman , 5. LikeIn developing nations, more than 200 million women either want to space or limit pregnancies and yet they lack access to modern family planning options , 9\u201312. DGlobally, a birth interval of fewer than 18 months is associated with increased risk for neonatal mortality, infant mortality, under-five mortality, and maternal mortality , 15\u201321. Studies conducted across the globe have identified various factors associated with SBI. These include; maternal age, maternal education level, husband education level, death of the index child, sex preference of the parents, no use of contraceptives, the ideal number of children, socio-cultural factors, religion, short breastfeeding duration (less than 24 months), and poor wealth index , 22\u201327.The Sustainable Development Goals (SDGs) of 2030, which combine multisystem strategies at global, regional, and national levels, have three focuses to ensure healthy lives and promote wellbeing for all at all ages. Of these goals, one main objective is to reduce the neonatal mortality rate to lower than 12 per 1,000 live births \u201330 whichDespite the implementation of various strategies and interventions at global and national levels to decline burden of under-five children, infant and neonatal mortality rates , 33\u201336, The results of this study will offer crucial information for policymakers, program planners, and other stakeholders to plan and implement proper interventions to prevent short birth interval in developing regions of the country.Therefore, this study was aimed to address both the individual and community-level determinants of short birth interval among women resided in the four developing regions  of Ethiopia.The study was conducted in developing regions of Ethiopia which are found mainly in lowland parts of the country. These regions are; Afar, Somali, Gambella, and Benishangul-Gumuz regions. These four regions are not well achieving most of the indicators related to health, human development and Millennium Development Goals compared to other developed regions of Ethiopia . The maihttp://dhsprogram.com), that was conducted in nine regions , Gambella, and Harari), and two city administrations (Addis Ababa and Dire-Dawa) of Ethiopia from January 18, 2016, to June 27, 2016.The data were retrieved from the Demographic and Health Survey (DHS) program official database website , a two-stage stratified cluster sampling technique has been employed. Enumeration areas were selected in the first stage. In the second stage, 28 households per enumeration area were selected with an equal probability of systematic selection per Enumeration Area (EA). Nationally, a total of 645 EAs were selected with probability proportional to EA size, and nationally a total sample size of 16,515 women aged 15\u201349 years was collected.The study populations for this study were 2683  women who had at least two consecutive live births in the 5 years preceding the survey, nested within four developing regions of the country Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: NoReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: No**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Adequate birth spacing fetches great health benefits to both Mother and Child. The topic of the present study is of great interest for improving the Maternal and Child health in pastoral region of Ethiopia. The authors have used data from Demographic and Health Survey, 2016 on women in reproductive age-group 15-49 years. The present study aimed to address both the individual and community-related factors for short birth interval on four pastoral regions of Ethiopia using multilevel logistic regression technique. The manuscript does not contain line or page numbers which makes it difficult to point out observations. Therefore, I am mentioning the observations by sections in the manuscript.TitleAs the study is focused only on women in Child-bearing ages 15-49 years and not all women, the present title, \"A Multilevel Analysis of Short Birth Interval and Its Determinants among Women in Pastoral Regions of Ethiopia\" needs to be modified.Abstract\" Avoid use of abbreviations in the abstract section.\" Please mention the four pastoral regions where the study was based.\" What is the sample size included in the study?\" Replace \"independent predictor\" with \"predictor\".\" The statement \"The statistical significance level was declared at a 95% confidence interval\" can be dropped from the abstract.\" The conclusion section needs to be strengthened, as the present one looks vague.\" The statement, \"government should encourage local communication channels to promote the health of women and children\" is ambiguous in interpretation. What measures are suggested by the authors to promote the health of women and children.Introduction\" Paragraph 3 gives data related to Nigeria. Please provide similar data for Ethiopia.\" Also, total fertility rate is given per 1000 women. Please mention the units.\" In view of the statement \"Like many other African countries, Ethiopia has shown so far little change in fertility reduction because of socio-cultural and religious factors.\" Can author give instances or examples of the cultural practices and religious factors responsible for higher fertility rates.\" Please mention the full form of the abbreviation SBI when first mentioned in the Introduction section.\" In view of the statement, \"The Sustainable Development Goals (SDGs) of 2030, which combine multisystem strategies at global, regional and national levels, have three focuses to ensure healthy lives and promote wellbeing for all at all ages. Of these goals, one main objective is to reduce the neonatal mortality rate to lower than 12 per 1,000 live births [32-34] which is at a steady stage in developing nations.\" Can authors mention the present level of Infant and under five mortality in Ethiopia and Country specific goals to reduce IMR and U5MR (set by government or SDG).\" In view of the statement \"Moreover, studies conducted in Ethiopia were limited and inconclusive to show the determinants of short birth interval at the community level . Therefore, this study aimed to address both the individual and community-related factors for short birth interval on four pastoral regions of Ethiopia using multilevel logistic regression analysis which is the appropriate model to handle community-level factors of short birth interval\" it would be better to re-write the need for the study by adding more literature stating the importance of studying SBI in pastoral regions and use of multi-level analysis.\" It is not mentioned anywhere what is the meaning of pastoral region and why is it important to study it. It is difficult to understand the use of the present study for international audience.\" Dedicate the last paragraph of the Introduction section to mention only the need for the study and study objectivesMethodsData Source\" What was the total sample collected by DHS in Ethiopia?\" What are these nine regions and two city administrations included by the survey?\" The statements \"All women of reproductive-age group were included in the first stage\" and \"The information was collected from a nationally representative sample of 16,515 women aged 15-49 years\" can be combined.\" Is child birth outside of wed-lock is common in Ethiopia? If not, unmarried women sample needs to be removed from the analysis. Were any measures taken to do the same?\" The author has used the term Pastoral region throughout the manuscript. There are a few questions which arise in my mind: 1) What is a pastoral region? , 2) What four pastoral regions have been selected in the study and what are the reasons for specifically selecting these regions? 3) What are the sample sizes from each of the regions? It would be better if the authors can provide an elaborate description on the sample selection.\" How have the authors adjusted for \"twins\" birth?Ethics Statement: Although the present study rests on a publically available dataset, still as the study included human subjects, it is advised to add a section of ethical consideration in the methods section after data source.Study Variables-Dependent Variable\" Use either of the two terms: Short Birth Interval or Optimal Birth Interval.https://dhsprogram.com/pubs/pdf/CR28/CR28.pdf (section 1.2).\" The authors have calculated the birth interval as the time that elapsed between the birth date of the first child and the birth date of the second child which I think is the standard procedure. It is however advisable to include a reference of the same. You can find a reference here: \" I would recommend using \"Croft, T. N., Marshall, A. M., Allen, C. K., Arnold, F., Assaf, S., & Balian, S. (2018). Guide to DHS statistics. Rockville: ICF\" to check and revise the computation procedure of the variables included in the study.Independent Variables\" I am concerned regarding the choice of predictor variables. The predictor variables can be clubbed into following sections, namely reproductive, behavioral, and child status. However, it is not clear that which conceptual framework has been utilized to select these predictor variables. Please mention the same.\" Also, the abstract suggests that a multilevel multivariable analysis has been used, it is not clear what are the levels included for the analysis and which variable was introduced at which level? Please provide a detailed description of the same.\" The explanatory variables included in the study can be correlated with each other. Are any measures taken to check for the same? If yes, include a section explaining the same in the methods section after variable description.Analysis Plan\" Data Analysis section needs to be restructured as there are a lot of repetition.\" The section \"In data with a nested structure\u2026\u2026. mixed- effect logistic regression analysis was used in this study.\" needs to be re-written as the meaning is not clear.\" The statement \"Data were weighted before analysis and merge and re-categorize to make suitable for analysis\" looks ambiguous. Please elaborate.\" A section on Bivariate analysis needs to be added before proceeding to the multivariate analysis. It is evident that Table 4 provides a 2x2 contingency table for all the predictor variables, however, it would be great if you can include chi-square/unadjusted odds ratio, and p-values in the table.\" Authors state that they have utilized software R, it is not clear which part of the analysis or data visualization was done in R.Results\" Table 1 and 2 provides the descriptive account of the variables included in the study. These two tables can be combined to form a single table using sub-headings in the table.\" In both Table 1 and 2 the authors have mentioned Weighted frequency and percentage (unweighted) which needs to be substituted with unweighted frequency and weighted percentages.\" Include a row \"Total\" for the Table 1 and 2 or mention total sample size (N) in the table.\" The categorization of the explanatory variables needs an urgent attention, for instance variables like Religion, Respondent's educational Status, Husband's Educational Status, Respondent Occupation, Husbands Education have very small frequencies for certain categories which needs to be corrected.\" Additionally, are there any specific reason for categorizing number of births, number of living children, and Survival of index child as they are presently. Can these variables be used as continuous?\" For Media Exposure the variable is not directly available in the DHS dataset. What is computation procedure of the variable \"Media Exposure\". Also, what is the meaning of the categories \"no\" and \"yes\". Does \"Yes\" includes partial exposure to media? Please add a section describing the computation and categorization procedure in methods.\" Variables like ethnicity and social segregation play a vital role in affecting the behaviors and decision related to child birth and spacing. Are these variables present in the dataset? If yes, why are these not included in the analysis.\" Prevalence is generally not reported in Percentage, it is therefore recommended that the authors report prevalence per 100 individual (denominator). Also, it is advised to mention the exact prevalence (count) in the figure.\" Figure 1 seems unnecessary. It can be deleted.\" Add the data source and year to the headings of all the Tables and Figures.\" The titles of all the Tables and Figures needs to be modified more meaningfully.\" In the section \"Prevalence of Short Birth Interval\" the statement, \"From a total of 2111(weighted) women who had at least two consecutive live births in four pastoral regions of Ethiopia\", the authors have mentioned 2111 as weighted women, which needs to be substituted for unweighted number of women.\" As the existing literature points out that birth spacing can vary across the reproductive age-group. It is therefore advised to use the age-adjusted prevalence rates in the analysis.Discussion\" The statement \"This finding is higher than \u2026\u2026and United States (35%)\". As the population size, socio-economic and developmental levels of Ethiopia and United States is quite different, it is better not to compare the two in discussion section.\" Also, the word \"This finding\" is an unclear reference. Please modify as required.\" The statement \"Thus, the prevalence of SBI is \u2026..and non- pastoral regions of the country\" looks repetitive.\" In reference to the statement, \"This could be\u2026\u2026.In addition, this variation could be explained by a difference in cut-off values used to determine SBI\". What were the definitions of SBI previously used (cut-offs).Limitations of the StudyPlease add a section on the limitations of the study.Conclusions\" The statement, \"government should encourage local communication channels to promote the health of women and children\" is ambiguous in interpretation. What measures are suggested by the authors to promote the health of women and children.\" What is Xaagu system? How will it improve the present scenario?Style of tabulation and presentation: The manuscript will benefit with major changes in the style of presentation. I would recommend the following paper published in PloS One , to help the authors improve the style of tabulation and presentation:1. Goli, S., Moradhvaj, A. R., & Shruti, J. P. (2016). High spending on maternity care in India: What are the factors explaining it?. PloS one, 11(6).2. McNay, K., Arokiasamy, P., & Cassen, R. (2003). Why are uneducated women in India using contraception? A multilevel analysis. Population studies, 57(1), 21-40.Language IssuesIt is quite understandable that the authors of the manuscript are not native English speakers. There are grammatical and English language errors throughout the manuscript. The manuscript can be benefitted from an extensive grammar and language check. It is advised to take help from a native English speaker in order to achieve the English standard of the article published in PloS One.PlagiarismThirteen percent of the text matches 15 sources or archives of academic publications. It is, therefore, advised to change the wording of Introduction and Methods sections. In majority of the instances the references are provided, however, the wordings of the entire paragraph are similar in a couple of occasions, which needs to be revisited and changed. I am unable to mention the exact paragraph which needs revision as no line numbers are provided in the manuscript. Majority of the text is similar to the following articles and report:1. Birhanu, B. E., Kebede, D. L., Kahsay, A. B., & Belachew, A. B. (2019). Predictors of teenage pregnancy in Ethiopia: a multilevel analysis. BMC public health, 19(1), 601.https://dhsprogram.com/data/Guide-to-DHS-Statistics/Place_of_Delivery.htm2. 3. Kawo, K. N., Asfaw, Z. G., & Yohannes, N. (2018). Multilevel analysis of determinants of anemia prevalence among children aged 6-59 Months in Ethiopia: classical and bayesian approaches. Anemia, 2018.4. Woday, A., & Ayesheshim Muluneh, C. S. D. (2019). Birth asphyxia and its associated factors among newborns in public hospital, northeast Amhara, Ethiopia. PloS one, 14(12).Reviewer #2: Short Birth Interval is a critical determinant of both maternal and child health and is an issue of concern in the developing world. The topic of the paper is an interesting one. However, here are some review points that might help improve the present work.1. Authors can leave out the data collection method of DHS from the abstract. More importance should be given in explaining the tools and techniques used in the present research paper.2. Introduction lacks continuity and flow. First authors should address why SBI is an important issue, the global scenario and then discuss its pertinence to African countries and the study region. Discussion of explanatory variables either should be better placed or put in the methods part where explanatory variables are listed out.3. The need for a community-level study in the pastoral regions should be highlighted.4. Why have the authors given weighted frequencies? It is difficult to understand the actual sample number that was collected. Only weighted percentage estimates should be enough.5. Table 1 and 2 are both of explanatory variables. They can be merged with some partition within the table.6. Rather than elaborating the explanatory variables more emphasis should be given on prevalence of SBI.7. Figure 1, 2 are not of publishable quality.8. Results on multilevel regression needs to be rewritten narrowing it down to only the results the authors find pertinent to the objectives of the study.9. Usually wealth index of the household, women\u2019s education play a significant role in determining spacing and limiting decisions, why are these variables not significant in Table 4? Authors might want to add it in the discussion.10. No ante-natal care variables are taken in the study. During ante-natal care, women are exposed to various materials on how to practice spacing and limiting for the next birth. The multilevel analysis has not controlled for this variable.11. Sex of preceding birth might be a better explanatory variable for SBI, as many communities around the globe has a preference for male child. If the preceding birth was female, there might be a shorter birth interval for the next child.12. Authors should revisit the variables they have taken for multilevel analysis based on multicollinearity. Eg: No. of live births and No. of living children capture similar aspects of reproductive choices. It will be advisable to generate a cumulative score that captures all these measures together or choose the more critical one for the analysis.13. Breastfeeding duration variable needs further explanation to understand whether this is for the preceding birth or all births. Breastfeeding duration for the index birth might not explain SBI for the last 2 births.14. Authors highlight all previous studies were individual level and their study considers community-level factors. It will be more effective if a few community level variables are taken in the multilevel analysis, such as health infrastructural support, sanitation and hygiene practices in the neighbourhood, etc.15. In discussion, authors merely summarise their results and point them to be similar to other studies. Discussion should have more content on implications of their results and suggest some policy revisions based on the findings.16. Tense of the manuscript needs critical revision. Grammatical errors need to be reviewed. English editing might be beneficial.All the best!**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,\u00a0 25 Jul 2020Dear Editors and reviewers, Thank you for giving us the opportunity to revise our manuscript entitled \u201cA Multilevel Analysis of Short Birth Interval and Its Determinants among Reproductive age Women in Developing Regions of Ethiopia\u201d before decisions. The authors have intensively discussed and addressed the raised concerns of the editor, and reviewers using point-by-point response as stated below. The amendments made on the manuscript have been presented using track change in the second attachment titled \u201cRevised Manuscript with Track Changes\u201dPoint-by-point responses for the questions and suggestions raised by Reviewer #1Adequate birth spacing fetches great health benefits to both Mother and Child. The topic of the present study is of great interest for improving the Maternal and Child health in pastoral region of Ethiopia. The authors have used data from Demographic and Health Survey, 2016 on women in reproductive age-group 15-49 years. The present study aimed to address both the individual and community-related factors for short birth interval on four pastoral regions of Ethiopia using multilevel logistic regression technique. The manuscript does not contain line or page numbers which makes it difficult to point out observations. Therefore, I am mentioning the observations by sections in the manuscript.TitleQ1. As the study is focused only on women in Child-bearing ages 15-49 years and not all women, the present title, \"A Multilevel Analysis of Short Birth Interval and Its Determinants among Women in Pastoral Regions of Ethiopia\" needs to be modified.Response: We have revised it per raised constructive concerns of the reviewer.AbstractQ2. Avoid use of abbreviations in the abstract section.Response: We have corrected it in the revised manuscript.Q3.\" Please mention the four pastoral regions where the study was based.Response: We have mentioned the four regions in the revised manuscript Q4\" What is the sample size included in the study?Response: 2683 total sample size Q5 \" Replace \"independent predictor\" with \"predictor\".Response: We have replaced it.Q6 \" The statement \"The statistical significance level was declared at a 95% confidence interval\" can be dropped from the abstract.Response: We have dropped as suggested by the reviewer.Q7\" The conclusion section needs to be strengthened, as the present one looks vague.Response: We have revised it based on your important suggestion. Q8\" The statement, \"government should encourage local communication channels to promote the health of women and children\" is ambiguous in interpretation. What measures are suggested by the authors to promote the health of women and children.Response: We have revised and incorporated in the revised manuscript.IntroductionQ9\" Paragraph 3 gives data related to Nigeria. Please provide similar data for Ethiopia.Response: We have revised and incorporated data related to Ethiopia in the revised manuscript.Q10\" Also, total fertility rate is given per 1000 women. Please mention the units.Response: We have revised and mentioned the units Q11\" In view of the statement \"Like many other African countries, Ethiopia has shown so far little change in fertility reduction because of socio-cultural and religious factors.\" Can author give instances or examples of the cultural practices and religious factors responsible for higher fertility rates.Response: We have addressed it in the revised manuscript.Q12\" Please mention the full form of the abbreviation SBI when first mentioned in the Introduction section.Response: We have corrected it.Q13\" In view of the statement, \"The Sustainable Development Goals (SDGs) of 2030, which combine multisystem strategies at global, regional and national levels, have three focuses to ensure healthy lives and promote wellbeing for all at all ages. Of these goals, one main objective is to reduce the neonatal mortality rate to lower than 12 per 1,000 live births [32-34] which is at a steady stage in developing nations.\" Can authors mention the present level of Infant and under five mortality in Ethiopia and Country specific goals to reduce IMR and U5MR (set by government or SDG).Response: We have incorporated the required information based on your important suggestion.Q14\" In view of the statement \"Moreover, studies conducted in Ethiopia were limited and inconclusive to show the determinants of short birth interval at the community level . Therefore, this study aimed to address both the individual and community-related factors for short birth interval on four pastoral regions of Ethiopia using multilevel logistic regression analysis which is the appropriate model to handle community-level factors of short birth interval\" it would be better to re-write the need for the study by adding more literature stating the importance of studying SBI in pastoral regions and use of multi-level analysis.Response: We have revised it in the revised manuscript per raised constructive concerns of the reviewer.Q15\" It is not mentioned anywhere what is the meaning of pastoral region and why is it important to study it. It is difficult to understand the use of the present study for international audience.Response: We have revised this section in revised manuscript Q16\" Dedicate the last paragraph of the Introduction section to mention only the need for the study and study objectivesResponse: We have revised this part as per the recommendation of the reviewer.MethodsData SourceQ17\" What was the total sample collected by DHS in Ethiopia?Response: a total sample size of 16,515 women aged 15\u201349 years was collected. Q18\" What are these nine regions and two city administrations included by the survey?Response: We have corrected it in the revised manuscript. The nine regions are Tigray, Afar, Amhara, Oromia, Somali, Benishangul-Gumuz, Southern Nations Nationalities and Peoples Region (SNNPR), Gambella, and Harari), and the two city administrations are (Addis Ababa and Dire-Dawa). Q19\" The statements \"All women of reproductive-age group were included in the first stage\" and the information was collected from a nationally representative sample of 16,515 women aged 15-49 years\" can be combined.Response: We have corrected it as per suggested by the reviewer. Q20\" Is child birth outside of wed-lock is common in Ethiopia? If not, unmarried women sample needs to be removed from the analysis. Were any measures taken to do the same?Response: We have revised it in the main document. Women who had never been married and those who have multiple births out of wedlock were excluded from the analysis.\" The author has used the term Pastoral region throughout the manuscript. There are a few questions which arise in my mind: Q21 1) What is a pastoral region? Response: we have revised this term and substituted pastoral region by developing region to make it clear for the audiences and we have briefly elaborated in the study area and data source section of the revised manuscript. Q22 2) What four pastoral regions have been selected in the study and what are the reasons for specifically selecting these regions? Response: We have revised this concern and incorporated all the required information in the revised manuscript.Q23 3) What are the sample sizes from each of the regions? It would be better if the authors can provide an elaborate description on the sample selection.Response: the authors corrected and incorporated the required data in the revised version of the manuscript. The sample sizes from each of the regions were 388 from Afar, 1706 from Somali, 489 from Benishangul-Gumuz, and 100 from Gambella Q24 \" How have the authors adjusted for \"twins\" birth?Response: We have described in the revised version of the manuscript under study area and data source section. Q25 Ethics Statement: Although the present study rests on a publically available dataset, still as the study included human subjects, it is advised to add a section of ethical consideration in the methods section after data source.Response: We have revised this concern and included ethics statement in the main revised manuscript. Study Variables-Dependent VariableQ26\" Use either of the two terms: Short Birth Interval or Optimal Birth Interval.Response: We have corrected it in the revised manuscripthttps://dhsprogram.com/pubs/pdf/CR28/CR28.pdf(section 1.2).Q27\" The authors have calculated the birth interval as the time that elapsed between the birth date of the first child and the birth date of the second child which I think is the standard procedure. It is however advisable to include a reference of the same. You can find a reference here: Response: we have cited the recommended reference in the revised version of the manuscriptQ28\" I would recommend using \"Croft, T. N., Marshall, A. M., Allen, C. K., Arnold, F., Assaf, S., & Balian, S. (2018). Guide to DHS statistics. Rockville: ICF\" to check and revise the computation procedure of the variables included in the study.Response: Thank you very much for your recommendation, we have considered this guideline for computation of this DHS dataset.Independent VariablesQ29\" I am concerned regarding the choice of predictor variables. The predictor variables can be clubbed into following sections, namely reproductive, behavioral, and child status. However, it is not clear that which conceptual framework has been utilized to select these predictor variables. Please mention the same.Response: this concern is incorporated in the revised version of the manuscript under study variable section. we have developed the conceptual framework by reviewed literature published before. All the independent variables were selected based on those literature.Q30\" Also, the abstract suggests that a multilevel multivariable analysis has been used, it is not clear what are the levels included for the analysis and which variable was introduced at which level? Please provide a detailed description of the same.Response: we have revised this issue and incorporated in the revised version of the manuscript under the independent variable section. Here in our study, independent variables were classified in to individual level variables and community level variables.Q31\" The explanatory variables included in the study can be correlated with each other. Are any measures taken to check for the same? If yes, include a section explaining the same in the methods section after variable description.Response: we have corrected and included a section which describes this concern in the revised version of the manuscript.Analysis PlanQ32\" Data Analysis section needs to be restructured as there are a lot of repetition.Response: we have corrected this concern in the revised version of the manuscript Q33\" The section \"In data with a nested structure\u2026\u2026. mixed- effect logistic regression analysis was used in this study.\" needs to be re-written as the meaning is not clear.Response: we have corrected this concern in the revised version of the manuscript Q34\" The statement \"Data were weighted before analysis and merge and re-categorize to make suitable for analysis\" looks ambiguous. Please elaborate.Response: this concern is revised and incorporated in the revised version of the manuscript.Q35\" A section on Bivariate analysis needs to be added before proceeding to the multivariate analysis. It is evident that Table 4 provides a 2x2 contingency table for all the predictor variables, however, it would be great if you can include chi square/unadjusted odds ratio, and p-values in the table.Response: we have addressed in the revised version of the manuscript. In this contingency table the frequency of each categories of predictors is putted to compute crude/unadjusted odds ratio, thus, including the crude/unadjusted odds ratio in this table will be redundancy. As we know all, to know statistically significant predictors in multivariable analysis, we can use either p value  or confidence interval of adjusted odds ratio . Here in our case, we putted the AOR with its corresponding confidence interval to know statistically significant predictors. So, we believed that including the p value will not add any new information if the AOR with its confidence interval is given.Q36\" Authors state that they have utilized software R, it is not clear which part of the analysis or data visualization was done in R.Response: We have used R for graphical presentation of data , we have corrected this concern in the revised version of the manuscript.ResultsQ37\" Table 1 and 2 provides the descriptive account of the variables included in the study. These two tables can be combined to form a single table using sub-headings in the table.Response: we have revised it as suggested.Q38\" In both Table 1 and 2 the authors have mentioned Weighted frequency and percentage (unweighted) which needs to be substituted with unweighted frequency and weighted percentages.Response: we have revised it as suggested. Based on the recommendation of the EDHS 2016 report, we have used sample weight for the frequency as well as for the percentage. Q39\" Include a row \"Total\" for the Table 1 and 2 or mention total sample size (N) in the table.Response: we have corrected as suggested and we include total sample size(N) in the table.Q40\" The categorization of the explanatory variables needs an urgent attention, for instance variables like Religion, Respondent's educational Status, Husband's Educational Status, Respondent Occupation, Husbands Education have very small frequencies for certain categories which needs to be corrected.Response: we have revised and recategorized those independent variables as suggested.Q41\" Additionally, are there any specific reason for categorizing number of births, number of living children, and Survival of index child as they are presently. Can these variables be used as continuous?Response: we have revised this concern in the revised manuscript. We have used their continuous form for predictors number of births and number of living children based on your important suggestion but the predictor survival of index child is categorical by its nature ( i.e survived or not) in the EDHS data set, So we have used as it is.Q42\" For Media Exposure the variable is not directly available in the DHS dataset. What is computation procedure of the variable \"Media Exposure \". Also, what is the meaning of the categories \"no\" and \"yes\". Does \"Yes\" includes partial exposure to media? Please add a section describing the computation and categorization procedure in methods.Response: this concern is revised and incorporated in the revised version of the manuscript.Q43\" Variables like ethnicity and social segregation play a vital role in affecting the behaviors and decision related to child birth and spacing. Are these variables present in the dataset? If yes, why are these not included in the analysis.Response: Social segregation was not present in the dataset; ethnicity is available in the dataset however there were no variability in respondents to the predictor ethnicity. Due to this we did not included it in the analysis.Q44\" Prevalence is generally not reported in Percentage, it is therefore recommended that the authors report prevalence per 100 individual (denominator). Also, it is advised to mention the exact prevalence (count) in the figure.Response: The authors addressed this concern in the revised manuscript.Q45\" Figure 1 seems unnecessary. It can be deleted.Response: we have deleted it as suggested Q46\" Add the data source and year to the headings of all the Tables and Figures.Response: we have included the data source (i.e EDHS) and year (i.e 2016). Q47\" The titles of all the Tables and Figures needs to be modified more meaningfully.Response: we have revised it as suggested. Q48\" In the section \"Prevalence of Short Birth Interval\" the statement, \"From a total of 2111(weighted) women who had at least two consecutive live births in four pastoral regions of Ethiopia\", the authors have mentioned 2111 as weighted women, which needs to be substituted for unweighted number of women.Response: As we know, the allocation of the sample in the EDHS data to different regions as well as urban and rural areas were non-proportional, therefore based on the recommendation of EDHS 2016 report, all proportions and frequencies were estimated after applying sample weights to the data to adjust for disproportionate sampling and non-responses. We have explained the detail on data analysis section of the revised manuscript. Based on your suggestion we have included the unweighted number of women in the commented statement under \u201cPrevalence of Short Birth Interval section\u201d of the revised manuscript.Q49\" As the existing literature points out that birth spacing can vary across the reproductive age-group. It is therefore advised to use the age-adjusted prevalence rates in the analysis.Response: we have revised it as suggested by the reviewer. DiscussionQ50\" The statement \"This finding is higher than \u2026\u2026and United States (35%)\". As the population size, socio-economic and developmental levels of Ethiopia and United States is quite different, it is better not to compare the two in discussion section.Response: we have corrected it as suggested.Q51\" The statement \"Thus, the prevalence of SBI is \u2026..and non- pastoral regions of the country\" looks repetitive.Response: we have removed the repetition as suggested.Q52\" In reference to the statement, \"This could be\u2026\u2026.In addition, this variation could be explained by a difference in cut-off values used to determine SBI\". What were the definitions of SBI previously used (cut-offs).Response: Those previous studies considered SBI, if birth interval less than 36 months, whereas our study defined it as less than 24 months.Q53 Limitations of the StudyResponse: we have included limitation of study in the revised manuscript.ConclusionsQ54\" The statement, \"government should encourage local communication channels to promote the health of women and children\" is ambiguous in interpretation. What measures are suggested by the authors to promote the health of women and children.Response: we have revised this section in the revised manuscript. The government should mobilize the community using health extension workers, women\u2019s group like Women\u2019s development Army (WDA). Q55\" What is Xaagu system? How will it improve the present scenario?Response: we have revised this section in the revised manuscript. Xaagu or dagu system is a local means for news exchange. It is a social institution with particular purposes in the daily life of the Afar communities. It functions within a defined set of regulations and expectations, though the rules are necessarily unwritten. The law of dagu means that whenever you meet someone on the road who has travelled reasonably far, say from a nearby village, you are required to pause andengage in a news exchange session.Q56 Style of tabulation and presentation: The manuscript will benefit with major changes in the style of presentation. I would recommend the following paper published in PloS One , to help the authors improve the style of tabulation and presentation:1. Goli, S., Moradhvaj, A. R., & Shruti, J. P. (2016). High spending on maternity care in India: What are the factors explaining it?. PloS one, 11(6).2. McNay, K., Arokiasamy, P., & Cassen, R. (2003). Why are uneducated women in India using contraception? A multilevel analysis. Population studies, 57(1), 21-40.Response: Thank you very much for your recommendation, we have seen those recommended articles and revised the tabulation and presentation style of our manuscript in the revised version of the manuscript.Language IssuesQ57 It is quite understandable that the authors of the manuscript are not native English speakers. There are grammatical and English language errors throughout the manuscript. The manuscript can be benefitted from an extensive grammar and language check. It is advised to take help from a native English speaker in order to achieve the English standard of the article published in PloS One.Response: Thank you for your important suggestion. We have addressed in the revised version of the manuscript.PlagiarismQ58 Thirteen percent of the text matches 15 sources or archives of academic publications. It is, therefore, advised to change the wording of Introduction and Methods sections. In majority of the instances the references are provided, however, the wordings of the entire paragraph are similar in a couple of occasions, which needs to be revisited and changed. I am unable to mention the exact paragraph which needs revision as no line numbers are provided in the manuscript. Majority of the text is similar to the following articles and report:1. Birhanu, B. E., Kebede, D. L., Kahsay, A. B., & Belachew, A. B. (2019). Predictors of teenage pregnancy in Ethiopia: a multilevel analysis. BMC public health, 19(1), 601.https://dhsprogram.com/data/Guide-to-DHS-Statistics/Place_of_Delivery.htm2. 3. Kawo, K. N., Asfaw, Z. G., & Yohannes, N. (2018). Multilevel analysis of determinants of anemia prevalence among children aged 6-59 Months in Ethiopia: classical and bayesian approaches. Anemia, 2018.4. Woday, A., & Ayesheshim Muluneh, C. S. D. (2019). Birth asphyxia and its associated factors among newborns in public hospital, northeast Amhara, Ethiopia. PloS one, 14(12).Response: Thank you for your crucial advice. The authors addressed this issue in the revised version of the manuscript.Point-by-point responses for the questions and suggestions raised by Reviewer #2Short Birth Interval is a critical determinant of both maternal and child health and is an issue of concern in the developing world. The topic of the paper is an interesting one. However, here are some review points that might help improve the present work.1. Authors can leave out the data collection method of DHS from the abstract. More importance should be given in explaining the tools and techniques used in the present research paper.Response: we have revised it as suggested 2. Introduction lacks continuity and flow. First authors should address why SBI is an important issue, the global scenario and then discuss its pertinence to African countries and the study region. Discussion of explanatory variables either should be better placed or put in the methods part where explanatory variables are listed out.Response: we have revised it as suggested3. The need for a community-level study in the pastoral regions should be highlighted.Response: we have addressed this issue in the revised manuscript.4. Why have the authors given weighted frequencies? It is difficult to understand the actual sample number that was collected. Only weighted percentage estimates should be enough.Response: Since, the allocation of the sample in the EDHS to different regions as well as urban and rural areas were non-proportional, therefore based on the recommendation of EDHS 2016 report, all proportions and frequencies were estimated after applying sample weights to the data to adjust for disproportionate sampling and non-responses. We have explained the detail on data analysis section of the revised manuscript.5. Table 1 and 2 are both of explanatory variables. They can be merged with some partition within the table.Response: we have merged it as suggested 6. Rather than elaborating the explanatory variables more emphasis should be given on prevalence of SBI.Response: the authors have addressed this concern in the revised manuscript 7. Figure 1, 2 are not of publishable quality.Response: we have corrected it based on the suggestions of you and reviewer #1 in the revised manuscript8. Results on multilevel regression needs to be rewritten narrowing it down to only the results the authors find pertinent to the objectives of the study.Response: we have tried to address it based on our objectives9. Usually wealth index of the household, women\u2019s education plays a significant role in determining spacing and limiting decisions, why are these variables not significant in Table 4? Authors might want to add it in the discussion.Response: the authors addressed this concern. As you said that wealth index of the household, women\u2019s education predictors of birth interval, in our study women\u2019s education (category secondary education and above level) is significantly associated with short birth interval. But wealth index of the household not statistically significant. 10. No ante-natal care variables are taken in the study. During ante-natal care, women are exposed to various materials on how to practice spacing and limiting for the next birth. The multilevel analysis has not controlled for this variable.Response: You are wright but this variable is not available in this data set.11. Sex of preceding birth might be a better explanatory variable for SBI, as many communities around the globe has a preference for male child. If the preceding birth was female, there might be a shorter birth interval for the next child.Response: Thank you very much for your important concern. In our case sex of preceding birth is one of the important predictors of short birth interval that is women having female sex of the index child had 1.13 times greater risk of short birth interval compared to those women having male index children.12. Authors should revisit the variables they have taken for multilevel analysis based on multicollinearity. Eg: No. of live births and No. of living children capture similar aspects of reproductive choices. It will be advisable to generate a cumulative score that captures all these measures together or choose the more critical one for the analysis.Response: we have considered the issue of multicollinearity in multivariable multilevel analysis and we have included a section described about this concern in the revised version of the manuscript.13. Breastfeeding duration variable needs further explanation to understand whether this is for the preceding birth or all births. Breastfeeding duration for the index birth might not explain SBI for the last 2 births.Response: Breastfeeding duration is asked for the preceding index child and it is known that women who breastfeed their children for longer duration have a longer period of amenorrhea which results in postpartum infertility. So that it determines the birth interval of the next births of a woman.14. Authors highlight all previous studies were individual level and their study considers community-level factors. It will be more effective if a few community level variables are taken in the multilevel analysis, such as health infrastructural support, sanitation and hygiene practices in the neighbourhood, etc.Response: Thank you for your important suggestion, Variables like health infrastructural support, sanitation and hygiene practices in the neighbourhood not available in the EDHS dataset. However, variables like residence site, region, and cluster are available in the dataset. The authors included those available variables in the multilevel analysis. The detail is explained in the variables section of the revised manuscript.15. In discussion, authors merely summarise their results and point them to be similar to other studies. Discussion should have more content on implications of their results and suggest some policy revisions based on the findings.Response: The authors tried to review this concern in the revised version of the manuscript 16. Tense of the manuscript needs critical revision. Grammatical errors need to be reviewed. English editing might be beneficial.Response: Thank you for your important advice. The authors addressed this issue in the revised version of the manuscript.AttachmentResponse to Reviwers.docxSubmitted filename: Click here for additional data file. 30 Jul 2020A Multilevel Analysis of Short Birth Interval and Its Determinants among Reproductive age Women in Developing Regions of EthiopiaPONE-D-20-07800R1Dear Dr. Birara,We\u2019re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you\u2019ll receive an e-mail detailing the required amendments. When these have been addressed, you\u2019ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they\u2019ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Srinivas Goli, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :The revisions are satisfactory.\u00a0Reviewers' comments: 14 Aug 2020PONE-D-20-07800R1 A Multilevel Analysis of Short Birth Interval and Its Determinants among Reproductive age Women in Developing Regions of Ethiopia Dear Dr. Aychiluhm:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Srinivas Goli Academic EditorPLOS ONE"}
+{"text": "Short birth interval is a universal public health problem resulting in adverse fetal, neonatal, child and maternal outcomes. In Ethiopia, more than 50% of the overall inter birth spacing is short. However, prior scientific evidence on its determinants is limited and even then findings are inconsistent.A community -based unmatched case-control study was employed on 218 cases and 436 controls. Cases were ever married reproductive age women whose last delivery has been in the past five years with birth interval of less than 3 years between the latest two successive live births whereas those women with birth interval of 3\u20135 years were taken as controls. A multistage sampling technique was employed on 30% of the kebeles in Dessie city administration. A pre-tested interviewer based questionnaire was used to collect data by 16 trained diploma nurses and 8 health extension workers supervised by 4 BSc nurses. The collected data were cleaned, coded and double entered into Epi-data version 4.2 and exported to SPSS version 22. Binary logistic regression model was considered and those variables with P<0.25 in the bivariable analysis were entered in to final model after which statistical significance was declared at P< 0.05 using adjusted odds ratio at 95% CI.In this study, contraceptive use , optimal breast feeding for at least 2 years , age at first birth <25 years , having male preceding child  and knowing the duration of optimum birth interval correctly  were significant determinants of short birth interval.Contraceptive use, duration of breast feeding, age at first birth, preceding child sex and correct understanding of the duration of birth interval were significant determinants of short birth interval. Fortunately, all these significant factors are likely modifiable. Thus, the existing efforts of optimizing birth interval should be enhanced through proper designation and implementation of different strategies on safe breastfeeding practice, modern contraceptive use and maternal awareness about the health merits of optimum birth interval. Birthinterval refers to the time gap between two consecutive live births . In 2005Short birth interval is a universal public health problem having association with adverse maternal, fetal, neonataland child outcomes such as low birth weight and perinatal death , 5, pretEthiopia had high population size as it was projected to reach more than 100 million and 4.0 total fertility rates in 2015. The country had also higher estimated pregnancy-related mortality ratio (PRM) of 412 deaths per 100,000 live births. Moreover, 1 in every 35 children dies within the first month; 1 in every 21 children dies before celebrating the first birthday; and 1 of every 15 children dies before reaching the fifth birthday (16). Therefore, the Ethiopian Federal Ministry of Health (FOMH) recommends spacing of childbirth at intervals of three to five years to reduce maternal, perinatal and infant mortality by optimizing the fertility rate in the country. However, in Ethiopia, more than 50% of the pregnancies occur within 3 years of their prior birth  which isSince short birth interval is a potentially modifiable problem, a better knowledge and understanding of its determinants is imperative and essential to improve maternal health by designing and applying specifically targeted interventions thereby decreasing catastrophic pregnancy outcomes , 16.HoweDessie city administration is located in northern part of Ethiopia at a distance of 401 km from Addis Ababa, capital of the country. It has an altitude of 2470 meters above sea level, situated between Tosa and Azewa mountains at11\u00b0 05\u00b4 North latitude and 39\u00b0 40\u00b4East longitude. The city administration has 5 sub cities. Besides, for administrative sake, the city is categorized into 18 urban and 8 rural kebeles (the lowest administrative levels in the study area). Based on the 2014 Ethiopian population projection, Dessie district had a total population of 212,436 of whom 83.6%  lived in urban areas . The stuA community based unmatched case-control study was conducted on a sample of eligible cases and controls. All the ever married reproductive age women who had at least two consecutive live births and whose last delivery within the past five years before the survey were eligible for the study.The eligible women who had history of less than 3 years birth interval between their two successive live births were considered as cases. Besides, controls were considered to be those eligible women with birth interval of 3\u20135 years (including 3 and 5) between their two successive live births.Taking several exposure variables into account, we calculated the respective sample size just by considering the assumption of case to control ratio of 1: 2; CI: 95%; Power: 80%; minimum detectable AOR = 2; design effect of 1.5 and 5% non-respondent rate. Among the given factors, we selected \u2018contraceptive use\u2019 because it yielded the maximum sample size as given in the following table . Therefokebeles\u2019 , were selected by simple random sampling technique. For those rural kebeles, the authors first checked family folder from health extension workers. We reviewed the family folder of permanently residing women in each kebele that fulfilled the inclusion criteria ) by registering the birth date of the last two successive children in a family with their corresponding household identification number. However, for urban \u2018kebeles\u2019, house to house visit (census) was conducted to identify permanently residing women that fulfilled the inclusion criteria (cases and controls) by registering the birth date of the last two successive children in a family with their corresponding household identification number. Using the respective household identification number, a sampling frame of the households containing cases and controls was prepared for each kebele. Then, proportional allocation of sample size was employed to determine the study participants from each kebele. Finally, cases and controls were selected by simple random sampling technique from the existing sampling frame. Whenever more than one eligible woman was found in same selected household, only one woman was chosen by lottery method. Thus, a sample of 678 women (226 cases and 452 controls) was recruited from the sampling frame for the study , 24 eligible women (8 cases and 16 controls) weren\u2019t accessed even after 2 different return visits. Therefore, these 24 absentees were replaced by other 24 randomly selected eligible mothers. The replaced mothers weren\u2019t systematically different from the original mothers because the replaced mothers were randomly selected from the already prepared sampling frame of eligible mothers (i.e. volunteers weren\u2019t included).Then, all the selected cases and controls were approached to be interviewed about factors related to their socio-demography, obstetrics, breastfeeding practice and modern contraception. Besides, the respondents were asked about their knowledge and attitude of birth interval. To determine children\u2019s birth dates, birth certificate or immunization cards were used. For those who were not immunized, health extension workers or mother\u2019s memory was consulted.A structured English version interviewer based questionnaire  was firsData were coded and double entered into Epi-Data software version 4.2 and then exported to SPSS version 22 for further processing and analysis. Descriptive statistics of different variables was done by cross tabulation. Binary logistic regression model using bivariable  and multivariable analyses  with 95% Confidence interval [CI] was employed. During bivariable analysis, variables whose p<0.25 were reserved for inclusion into the multivariable analysis in the final model after which statistical significance was declared at P< 0.05 using adjusted odds ratio. Both Hosmer-Lemeshow\u2019s test (p = 0.753) and Omnibus Tests (p = .000) were used to check model fitness. Multi-collinearity was checked to see the linear correlation among the independent variables by using variance inflation factor and standard error. It was tried to minimize bias from intra-cluster correlation effect (dependencies) by considering only one of the eligible women in a selected household. Besides, standard error was used during multivariate regressions and there was no any factor whose standard error greater than two indicating no dependency between mothers regarding the considered factors.Wealth index of the studied households were given scores based on the number and kinds of consumer goods they own including chairs, tables, chicken, transport (vehicles) and household characteristics like source of drinking water, toilet facilities, wall, roof and flooring materials. Among the nine characteristics, eight of them were extracted.SPSS version 22 software was used to perform principal component analysis (PCA). Finally, wealth status was categorized into five groups and ranked from poorest to wealthiest quintile. Kaiser-Meyer-Olkin Measure of Sampling Adequacy was 0.751 and Bartlett\u2019s Test of Sphericity was significant.Ethical approval and consent to participate. Ethical approval with ethics approval number of HU-CHMS-001 was obtained from Haramaya University, College of Health and Medical Sciences, Institutional Health Research Ethics Review Committee (IHRERC). An informed and voluntarily signed written consent (thumb print for those unable to write) was obtained from all the eligible mothers. Parental consent wasn\u2019t required because all the respondent mothers were above 16 years of old.From the overall sample of 678 mothers, 654 women (218 cases and 436 controls) agreed to be interviewed, thus making a response rate of 96.5%. Median age of the respondents at last delivery was 32 years. Twenty four (11%) of the cases and 82 (18.8%) of the controls were married at their age of 18 or less years. One hundred thirty five (61.9%) of the cases and 287 (65.8%) of the controls were within the age of25\u201334 years. Regarding their residence, 130 (59.6%) of the cases and 273 (62.6%) of the controls were urban residents. Nearly one fourth of the cases 58 (26.6%) and controls 110 (25.2.0%) had college and above level of education. One hundred twenty five (57.3%) of the cases and 232 (53.2%) of the controls were house wives. Moreover, 37(17.0%) of the cases and 107(24.5%) of the controls had the richest wealth index .One hundred sixty five (75.7%) of the cases and 352 (80.7%) of the controls had ever heard about optimal birth interval. One hundred thirty four (61.5%) of the cases and 291 (66.7%) of the controls agreed that a minimum of 3 years birth spacing is essential between two successive births. Regarding husbands\u2019 perception of birth spacing, 120 (55%) of the cases and 246(56.4%) of the controls had encouraging perception to birth spacing. One hundred forty four (66.1%) of the cases and 298(68.3%) of the controls had nobody to influence them to give birth with short interval. Two hundred and four (93.6%) of the cases and 404(92.7%) of the controls perceived that short birth interval have disadvantages on both maternal and child health. Regarding respondents\u2019 knowledge of the optimum birth interval, 130(78.8%) of the cases and 280(79.5%) of the controls knew the appropriate cut point correctly. The source of information for majority of the cases 112(67.9%) and controls 289(82.1%) were health workers .The mean maternal age at first birth was 23(\u00b13.47) years. The median length of time from marriage to first birth was 24 months. Equal proportion (12.4%) of the cases and controls had bad fetal outcome at first delivery. Among the respondents, 5% of the cases and 2.5% of the controls experienced neonatal mortality. Besides, 3.7% of the cases and 1.8% of the controls had experienced stillbirth in their life time. Twenty five (5.7%) of the cases and 9(4.1%) of the controls had high birth order of their preceding child. From the overall respondents, 38(17.4%) of the cases and 34 (7.8%) of the controls reported that their previous pregnancy was unplanned.Forty six (21.1%) of the cases and 49(11.2%) of the controls had not ANC follow up for their previous pregnancy. Twenty five (11.5%) of the cases and 39(8.9%) of the controls had home delivery of their previous and last children. Majority of the cases 197(90.4%) and controls 397(89.9%) had spontaneous vaginal delivery of their previous child. Twenty six (11.9%) of the cases and 61(13.9%) of the controls ever had history of postpartum complications during their previous to last deliveries. From these complications, bleeding was reported among 6 (23.1%) of the cases and 30(49.2%) of the controls. The median duration of resuming postpartum sexual activity was 45 days. From the total respondents, 16 (7.3%) of the cases and 44(10.1%) of the controls ever had chronic diseases like hypertension and diabetic mellitus before their last childbirth. The median ages of last and preceding child were 17and 60 months respectively .Most of the cases 198(90.9%) breast fed their children for less than 24 months whereas 178(40.8%) of the controls breastfed for at least 24 months. Moreover, more than half of the cases 80 (52.6%) and three fourth of the controls 295(73.8%) practiced exclusive breastfeeding to their preceding child. Ninety eight (44.9%) of the cases and 411 (94.3%) of the controls have utilized modern contraceptive methods after delivering their preceding child. Nearly all of the cases 213 (97.7%) and 434(99.5%) of the controls knew at least one type of modern contraceptive. One hundred eighty three (83.9%) of the cases and 428(98.2%) of the controls agreed that family planning method is necessary for birth spacing. Regarding decision making about family planning in the house hold, ninety seven (44.5%) of the cases and 227(52.1%) of the controls decided based on couple agreement .Concerning the practice of modern contraceptive methods, forty three (43.9%) of the cases and 183(44.5%) of the controls utilized injectable type after delivering their preceding child .From the total fourteen variables that were entered to the multivariable logistic regression analysis, only five of them namely contraceptive use , optimal breast feeding for at least 2 years , age at first birth<25 years , having male preceding child  and knowing the duration of optimum birth interval correctly  had significant odds of association with short birth interval. We used backward stepwise method to identify variables which had the largest contribution to the regression model. The result in forward or a stepwise variable selection method was similar on significance of the variables, but little change in adjusted odds ratio, p value and confidence interval were observed.The odds of short birth interval among mothers who breastfed their prior child for at least 24 months were 90.2% lower  as compared to those having less than 12 months of breastfeeding duration. The odds of short birth interval among mothers having male preceding child was 54.0% lower than those whose child was female . Besides, the odds of short birth interval among those who didn\u2019t use modern contraceptives was11.2 times higher as compared to the users . Concerning maternal knowledge about the duration of birth interval, those mothers who knew the duration correctly had 55% lower odds of association with short birth interval  as compared to those who didn\u2019t know the duration correctly. Lastly, it was found that mothers who gave their first birth at the age of less than 28 years had 64% lower odds of association with short birth interval when compared to their counterparts  .Despite no statistical significance in the adjusted analysis, the crude odds of short birth interval was lower among mothers who had planned preceding pregnancy  than those whose pregnancy wasn\u2019t planned. Besides, mothers who abstained in the post partum period had lower crude odds of short birth interval  than those who didn\u2019t abstain. Similarly, mothers who had ANC follow up Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors have conducted an interesting descriptive analysis of factors that predict birth spacing of < 3 years as opposed to between 3 and 5 years in Ethiopia. They find that contraceptive use, breastfeeding, age at first birth, preceding child sex and underlying knowledge of existing advice regarding birth spacing all influenced the likelihood of a short birth interval. I have several questions for clarification.1) You need to put the Ethiopian guidelines regarding birth spacing in a wider context in the introduction. Most high-income countries have a much shorter birth spacing. Why does Ethiopia (and a lot of other low and middle income countries) recommend a minimum of 3 years? It would benefit the reader a lot if you described the reasoning.2) Please describe and justify the sampling frame in more detail. You should also provide the response rates for cases and controls. There should also be a figure 1 showing exactly how many cases and controls were recruited as opposed to the number included in the analysis.3) For your power calculations, you have not described how prevalent you estimated the relevant predictors of short interpregnancy interval to be? Some of the predictors you considered are very rare and you are not adequately powered to evaluate them. You should specify the minimum prevalence of the predictors you were powered to detect in relation the estimated minimum effect size.4) How did you decide what background factors to explore? What informed the questions that you initially decided to ask the study participants?5) Was there really no missing information for any of the covariates? If there was any missing information in any of the covariates, how was this dealt this? I can\u00b4t see that this is described in the methods. If you have any missing data, this should be dealt with using multiple imputation.6) Were any of the women included in the study related? I was wondering whether you have any dependencies in the data that should be dealt with in the regression analysis. For example by using robust standard errors.7) You should clearly show the p-values from the bivariate analyses in all tables (1-4). As far as I can tell, these bivariate analyses provided the basis for your decision for to carry some covariates forward to the regression analysis.8) You used a backwards approach to your variable selection, if I am interpreting the methods correctly. Were the results similar if you used a forward or a stepwise variable selection procedure?9) I would recommend that you have a native English speaker help you look through the manuscript.Reviewer #2: This was an interesting manuscript and I enjoyed reading your manuscript. However, the authors should consider addressing the following:1. The entire manuscript needs to be revised for grammatical errors and punctuation issues. For example, on page 2, the conclusion section of the abstract, the authors started a sentence with a lower case alphabet. Also, on page 3 (introduction) the first sentence needs revising as we express maternal mortality per 100,000 live births and not \"live birth\".Similarly, on page 3, the last sentence in paragraph 1 needs revising particularly the sentence ....\"the problem is still major public concern.\"2. The authors stated on page 3 that ....\"national guideline for family planning services according to the Ethiopian FMOH\u2019s recommendation\" I will recommend that the authors should provide a sentence or two from this guidelines that are applicable/relevant to their study.3. The authors need to state the aim in the last paragraph of their introduction. At present, this is not really clear.4. On page 8, Table 1, the authors need to correct the word 'college' which is wrongly written as 'collage'. Additionally the word 'widowed' is also wrongly written as 'windowed'5. On page 14, Table 5, the authors need to double-check the p-values as there as selected p-values <0.05 that were not highlighted.6. On page 15, paragraph 2, the authors should correct the word 'consistency' which was misspelt as 'consistence'.7. The limitations of the study (page 17) needs revision and should be reported before the conclusion. Specifically, there was no mention of how the recall and social desirability bias reported in the manuscript were dealt with.8. The authors also need to provide a few sentences on the key strength of their study.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  12 Oct 2020Response letterDear editorAfter going through the entire manuscript, you forwarded your constructive editorial comments which we missed to touch. Therefore, we are glad enough to express our sincerest thanks for your in-depth review and comments that could help improve the tone and readability of our paper.https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf. Editor comment 1: Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at  Authors\u2019 response: Very important comment it is! Thus, we have ensured that our manuscript meets PLOS ONE's style requirements, including those for file naming by finding the aforementioned link for PLOS ONE style templates.Editor comment 2: Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information. In addition, please refrain from stating p values as .000, either state the exact value or use the format p<0.001.Authors\u2019 response: Undoubtedly! There is a need for including the survey or questionnaire in the study. Besides, we have ensured that we have provided sufficient details that others could replicate the analyses as provided in the additional information file. We didn\u2019t develop questionnaire as part of this study and hence no worry about copyright. In addition, we have refrained from stating p values as .000, rather we stated the exact value as it can be noticed from the yellow highlight in the revised version manuscript.The questionnaire is newly added as additional information as it can be seen from the yellow highlight on pages 7 and 24 of the revised version manuscript.https://www.youtube.com/watch?v=_xcclfuvtxQEditor comment 3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to \u2018Update my Information\u2019 (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account:  Authors\u2019 response: the corresponding author didn\u2019t have an ORCID iD before. Thus, based on your recommendation, the corresponding author created a new ORCID iD (0000-0002-5972-4818) in the PLOS Editorial Manager.Editor comment 4. Your ethics statement must appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please also ensure that your ethics statement is included in your manuscript, as the ethics section of your online submission will not be published alongside your manuscript. Authors\u2019 response: Absolutely! It is a comment of technical relevance as per the journal requirement. Thus, our ethics statement appears in the methods section of our manuscript. Besides, our ethics statement is deleted from the declaration section and moved to the methods section as shown by the yellow highlighted text on page 8 in the ethical approval and consent to participate subsection, methods section of the revised version manuscript.Additional Editor Comments (if provided):Reviewers have returned some substantive comments to improve your manuscript. Please carefully consider each comment and respond appropriately. Please prepare a table indicating how you have responded to each comment and please follow the advice to secure expertise in the correction of English grammar within the script. Authors\u2019 response: No doubt! We have tried our best to carefully consider each reviewer\u2019s comment and respond appropriately. We have also followed the advice to secure expertise in the correction of English grammar within the script. Besides, we have included a point by point response letter as detailed below. Dear reviewer 1 After going through the entire manuscript, you forwarded your constructive comments which we missed to touch. Therefore, we are glad enough to express our sincerest thanks for your in-depth review and comments that could help improve the tone of ourpaper.Reviewer suggestion: The authors have conducted an interesting descriptive analysis of factors that predict birth spacing of < 3 years as opposed to between 3 and 5 years in Ethiopia. They find that contraceptive use, breastfeeding, age at first birth, preceding child sex and underlying knowledge of existing advice regarding birth spacing all influenced the likelihood of a short birth interval. I have several questions for clarification.Authors\u2019 response: We are really grateful for your appreciation of our efforts. Besides, we have tried our best to address all your comments point by point as detailed below. Reviewer comment 1: You need to put the Ethiopian guidelines regarding birth spacing in a wider context in the introduction. Most high-income countries have much shorter birth spacing. Why does Ethiopia (and a lot of other low and middle income countries) recommend a minimum of 3 years? It would benefit the reader a lot if you described the reasoning.Authors\u2019 response: Definitely! Description of why does Ethiopia recommend a minimum of 3 years of birth spacing in a wider context in the introduction would benefit the reader a lot for easy understanding. Therefore, the following quoted text is added to the introduction section of the revised version manuscript as it can be seen by the yellow highlighted text on pages 3 and 4, under background section. \u201cEthiopia had high population size as it was projected to reach more than 100 million and 4.0 total fertility rates in 2015. The country had also higher estimated pregnancy-related mortality ratio (PRM) of 412 deaths per 100,000 live births. Moreover, 1 in every 35 children dies within the first month; 1 in every 21 children dies before celebrating the first birthday; and 1 of every 15 children dies before reaching the fifth birthday (16). Therefore, the Ethiopian Federal Ministry of Health (FOMH) recommends spacing of childbirth at intervals of three to five years to reduce maternal, perinatal and infant mortality by optimizing the fertility rate in the country.Reviewer comment 2) Please describe and justify the sampling frame in more detail. You should also provide the response rates for cases and controls. There should also be a figure 1 showing exactly how many cases and controls were recruited as opposed to the number included in the analysis.Authors\u2019 response: What a comment of relevance! It would help increase understandability of the sampling procedure! Thus, the following detail is given. \u201cMulti stage sampling technique was employed to select the cases and controls. At first, 30% of the overall \u2018kebeles\u2019 , were selected by simple random sampling technique. Then, for those rural kebeles, the authors first checked family folder from health extension workers. The family folder is an extension of the Ethiopian Community Health Information System (CHIS) at the most basic level of rural health system. Health extension workers (HEWs) make individualized household family member and assessment of each household\u2019s health behavior and assign a set of health cards for individuals in each household. We reviewed the family folder of permanently residing women in each kebele that fulfilled the inclusion criteria  by registering the birth date of the last two successive children in a family with their corresponding household identification number. However, for urban \u2018kebeles\u2019, house to house visit (census) was conducted to identify permanently residing women that fulfilled the inclusion criteria (cases and controls) by registering the birth date of the last two successive children in a family with their corresponding household identification number. Using the respective household identification number, a sampling frame of the households containing cases and controls was prepared for each kebele. Then, proportional allocation of sample size was employed to determine the study participants from each kebele. Finally, cases and controls were selected by simple random sampling technique from the existing sampling frame. Whenever more than one eligible woman was found in same selected household, only one woman was chosen by lottery method. Thus, a sample of 678 women (226 cases and 452 controls) was recruited from the sampling frame for the study. But, from these recruited 678 women, 654 women (218 cases and 436 controls) agreed to be interviewed, thereby making a response rate of 96.5% \u201d.Amendment is located on pages 5 and 6 , methods section, subsection of sample size determination and sampling procedure in the revised version manuscript as shown by the yellow highlighted text. Reviewer comment:3) For your power calculations, you have not described how prevalent you estimated the relevant predictors of short inter-pregnancy interval to be? Some of the predictors you considered are very rare and you are not adequately powered to evaluate them. You should specify the minimum prevalence of the predictors you were powered to detect in relation the estimated minimum effect size.Authors\u2019 response: Yes indeed! For our power calculations, we have not described how prevalent we estimated the relevant predictors of short inter-pregnancy interval to be. Besides, as you said, some of the predictors we considered are rare and we are not adequately powered to evaluate them. Therefore, taking this comment and several exposure variables into account, we calculated the respective sample size just by considering the assumption of case to control ratio of 1: 2; CI: 95%; Power: 80%; minimum detectable AOR =2; design effect of 1.5 and 5% non-respondent rate. We selected the factor \u2018contraceptive use\u2019 because it yielded the maximum sample size as given in the following table (Table 1). Therefore, the sample size for this study was 678 (226 cases and 452 controls). Table 1: Sample size determination using different factors in the literature and the respective assumptions using Open EPI INFO version 7 software.Factors Assumption Total sample size References Contraceptive user P of exposure in controls =66.7% 678 Residence/urban P of exposure in controls =52.1% 540 Husbands\u2019 occupation /Employee P of exposure in controls =51.7% 537 Mothers\u2019 education /Has formal education P of exposure in controls =48.3% 524 Parity />=5 children P of exposure in controls = 49.2% 524 Sex of the index child /male P of exposure in controls = 64.2% 638 Age of the mother/ 25-29 P of exposure in controls = 24.9% 576 Status of index child /Alive P of exposure in controls = 41.3% 509 Wealth index/ Richest P of exposure in controls = 25.2% 509 Amendment is located on pages 5 and 6 in the revised version manuscript as shown by the yellow highlighted text. Reviewer comment 4) How did you decide what background factors to explore? What informed the questions that you initially decided to ask the study participants?Authors\u2019 response: After reviewing different literature  [Ethiopia] and ICF, 2016; Hailu and Gulte, 2016; Tsegaye Dereje et al., 2017; Yohannes et al., 2011) that addressed proximate, intermediate, socio- demographic and economic determinants of birth interval, we decided the background factors to be explored in this study. Reviewer comment 5) Was there really no missing information for any of the covariates? If there was any missing information in any of the covariates, how was this dealt this? I can\u00b4t see that this is described in the methods. If you have any missing data, this should be dealt with using multiple imputations.Authors\u2019 response: There is no missing information for any of the covariates in this study. This was because incomplete questionnaires were returned to the data collectors for completion by referring to the respective household identification number on a daily basis of checking all the questionnaires.Amendment is located on page 7, methods section, subsection of data quality control, in the revised version manuscript as shown by the yellow highlighted text. Reviewer comment 6) Were any of the women included in the study related? I was wondering whether you have any dependencies in the data that should be dealt with in the regression analysis. For example by using robust standard errors.Authors\u2019 response: What a comment of paramount importance! Answering this comment helps assure data quality and management of bias. As mentioned in the sampling procedure, it was tried to minimize bias from intra-cluster correlation effect (dependencies) by selecting only one of the eligible women in a selected household. Besides, standard error was used during multivariate regressions and there was no any factor whose standard error greater than two indicating no dependency between mothers regarding the considered factors. Amendment is located on page 8, methods section, subsection of data processing and analysis, in the revised version manuscript as shown by the yellow highlighted text. Reviewer comment 7) You should clearly show the p-values from the bivariate analyses in all tables (1-4). As far as I can tell, these bivariate analyses provided the basis for your decision for to carry some covariates forward to the regression analysis.Authors\u2019 response: Undoubtedly! Considering your constructive comment, we have now clearly shown the p-values from the bivariate analyses in all tables . As you said, these bivariate analyses provided the basis for our decision to carry some covariates forward to the regression analysis. The detailed response is given below and also shown by the yellow highlighted column of the P-value for tables 2-5.Table 2: Socio-demographic characteristics on short birth interval among ever married mothers  in Dessie city administration, Dessie , Ethiopia 2019.Amendment is located on page , line of the revised version manuscript as shown by the yellow highlighted text. Factors Category Case (%) Control(%) P valueRsidence Urban 130(59.7%) 273(62.65) 0.460 Rural 88(40.3%) 163(37.4%) Marital status Married 186(85.3%) 364(83.5%) 0.759 Divorced 21(9.6%) 44(10.1%)  Widowed 11(5.1%) 28(6.4%) Religion Orthodox 92(42.2%) 173(39.7%) 0.287 Muslim 124(56.9%) 249(57.1%)  Protestant 2(0.9%) 14(3.2%) Ethinicity Amhara 200(91.7%) 399(91.5%) 0.926 Tgrai 7(3.2%) 11(2.5%)  Oromo 6(2.7%) 14(3.2%)  Others1 5(2.3%) 12(2.8%) Mother\u2019s education No formal education 45(20.6%) 70(16.1%) 0.546 read and write 42(19.3%) 86(19.7%)  Elementary 34(15.6%) 81(18.6%)  Secondary 39(17.9%) 89(20.4%)  Collage and above 58(26.6%) 110(25.2%) Husband education No formal education 50(22.9%) 69(15.8%) 0.104 read and write 32(14.7%) 69(15.8%)  Elementary 13(5.9%) 42(9.6%)  Secondary 41(18.8%) 72(16.5%)  College and above 82(37.6%) 184(42.2%) Mothers\u2019 occupation employee(GO/NGO) 43(19.7%) 91(20.9%) 0.730 house wife 125(57.3%) 232(53.2%)  Merchant 28(12.8%) 53(12.2%)  Student 9(4.1%) 29(6.7%)  Farmer 10(4.6%) 19(4.4%)  daily workers 3(1.4%) 11(2.5%)  Others2 0(0%) 1(0.2%) Husband occupation employee(GO/NGO) 84(38.5%) 164(37.6%) 0.086 Merchant 66(30.3%) 129(29.6%)  Student 0(0%) 2(0.5%)  Farmer 63(28.9%) 107(24.5%)  daily workers 4(1.8%) 23(5.3%)  Others3 1(0.5%) 11(2.5%)  Number of wiveswealth index One 216(99.1%) 434(99.5%) 0.478 More than one 2(0.9%) 2(0.5%)  Poorest 57(26.1%) 84(19.3%) 0.096 Second 35(16.1%) 80(18.3%)  Middle 47(26.6%) 83(19.0%)  Fourth 42(19.3%) 82(18.8%)  Richest 37(17.0%) 107(24.5%) 1Afar, Gurage2 House servant,3Religious leaderTable 3: Knowledge and attitude of birth interval among ever married reproductive age mothers  in Dessie city administration, Dessie, Ethiopia 2019.Factors Category Case (%) Control (%) P valueHeard about optimal birth interval Yes 165(75.7%) 352(80.7%) 0.336 No 53(24.3%) 84(19.3%) Optimum number of years between two successive births Below three years 19(11.5) 46(13.1%) 0.701 Three to five years 130(78.8%) 280(79.5%)  Above five years 13(7.8%) 23(6.5%)  I am not sure 3(1.8%) 3(0.8%) 0.562A minimum of 3 years of birth interval is essential between two successive births Strongly agree 81(37.2%) 139(31.9%)  Agree 134(61.5%) 291(66.7%)  no idea 2(0.9%) 3(0.7%)  Disagree 1(0.5%) 3(0.7%) Husband's perception regarding birth spacing Disagree strongly 28(12.8%) 27(6.2%) 0.001 don't mind 57(26.1%) 152(34.9%)  Encouraging 120(55.04%) 246(56.4%)  Unknown 13(5.96%) 11(2.5%) External influences to give birth in short interval My family 37(16.97%) 61(13.99%) 0.258 Mother in law 21(9.63%) 60(13.76%)  Father in law 7(3.2%) 12(2.75%)  Societies norm 9(4.1%) 5(1.1%)  None 144(66.1%) 298(68.4%) Perceived advantages of optimum birth spacing Yes 205(94.04%) 406(93.1%) 0.655 No 13(5.96%) 30(6.9%) Perceived disadvantages of short birth interval Yes 204(93.6%) 404(92.7%) 0.665 No 14(6.4%) 32(7.3%) Table 4: Obstetrics related factors of short birth interval among ever married reproductive age mothers inDessie city administration, Dessie, Ethiopia 2019.Factors Category Case (%) Control (%) P valueFetal outcome of first delivery Live birth 191(87.6%) 382(87.62%) 0.352 still birth 11(5.04%) 13(2.98%)  Abortion 3(1.4%) 13(2.98%)  Neonatal mortality 13(5.96%) 28(6.42%) Prior history of infertility Yes 4(1.83%) 3(0.69%) 0.279 No 214(98.17%) 433(99.31%) Ever given birth to any child who died Yes 31(14.2%) 58(13.3%) 0.723 No 187(85.8%) 378(86.7%) Male to female ratio of living children More than one 71(32.6%) 160(36.7%) 0.355 One 63(28.89%) 135(30.96%)  Less than one 49(22.48%) 74(16.97%)  Males only 15(6.9%) 36(8.26%)  Females only 20(9.17%) 31(7.11%) Previous to last pregnancy is planned Yes 180(82.6%) 402(92.2%) 0.001 No 38(17.4%) 34(7.8%) Practice postpartum abstinence before the last child Yes 161(73.85%) 359(82.3%) 0.011 No 57(26.15%) 77(17.7%) Mode of delivery of previous to last birth Vaginal delivery 197(90.4%) 392(89.9%) 0.981 Cesarean section 14(6.4%) 29(6.7%)  Instrumental delivery 7(3.2%) 15(3.4%) ANC follow up in preceding pregnancy Yes 172(78.9%) 387(88.8%) 0.009 No 46(21.1%) 49(11.2%) Place of delivery of previous to last birth Home 25(11.5%) 39(8.9%) 0.308 Health institution 193(88.5%) 397(91.1%) Pattern of menstruation in previous to last deliveries Regular 185(84.9%) 362(83.02%) 0.550 Irregular 33(15.1%) 74(16.97%) Ever had chronic diseases  before the last child Yes 16(7.3%) 44(10.1%) 0.255 No 202(92.7%) 392(89.9%) Ever had history of postpartum complications in previous to last deliveries Yes 26(11.9%) 61(13.99%) 0.464 No 192(88.1%) 375(86.01%) Last child sex Male 121(55.5%) 238(54.6%) 0.824 Female 97(44.5%) 198(45.4%) Is last child alive Yes 217(99.5%) 434(99.5%) 0.741 No 1(0.5%) 2(0.5%) previous to last child sex Male 72(33%) 235(53.9%) 0.001 Female 116(53.2%) 201(46.1%) Is previous to last child alive Yes 215(98.6%) 434(99.5%) 0.254 No 3(1.4%) 2(0.5%) Parity <5 180 (82.5%) 370(84.8%) 0.450 >=5 38(17.5%) 66(15.2%) Table 5: Breast feeding duration and contraceptive use among ever married reproductive age mothers in Dessie city administration, Dessie, Ethiopia 2019 Factors Category Case (%) Control(%) P valueDid you breast feed previous to last child Yes 152(69.7%) 400(91.7%) 0.001 No 66(30.3%) 36(8.3%) Did you exclusively breastfeed previous to last child Yes 80(52.6%) 295(73.8%) 0.001 No 72(47.4%) 105(26.2)  Breast feeding duration 0-11 134(61.5%) 61(13.99%) 0.001 12-23 64(29.4%) 197(45.18%)  >=24 20(9.2%) 178(40.83%) Using any of the modern methods before the conception of your last child Yes 98(44.95%) 411(94.3%) 0.001 No 120(55.05%) 25(5.7%) Decision maker about Family planning Self 104(47.7%) 190(43.58)  Both husband and wife 97(44.5%) 227(52.06%) 0.261 Husband only 3(1.4%) 13(2.98%)  No one 14(6.4%) 6(1.38%) Perception of family planning method Agree 183(83.9%) 428(98.2%) 0.001 Disagree 34(15.6%) 4(0.9%)  Neutral 1(0.5%) 4(0.9%) Distance from health institution Less than 30 minutes 93(42.7%) 197(45.2%) 0.799 30-1hrs 123(56.4%) 236(54.1%)  Greater than 1 hr 2(0.9%) 3(0.7%) Reviewer comment 8) You used a backwards approach to your variable selection, if I am interpreting the methods correctly. Were the results similar if you used a forward or a stepwise variable selection procedure?Authors\u2019 response: We used backward stepwise LR to identify variables which had the largest contribution to the model. The result in forward or a stepwise variable selection method was similar on significance of the variables, but little change in adjusted odds ratio, p value and confidence interval were observed. Amendment is located on page 14, results section, subsection of determinants of short birth interval as shown by the yellow highlighted text in the revised version manuscript. Reviewer comment 9) I would recommend that you have a native English speaker help you look through the manuscript.Authors\u2019 response: what a similar comment with reviewer two. Therefore, similar response is given as mentioned below in the quoted text.\u201cFrom repeated proof-reading of the manuscript, we found several grammatical errors, interlinings, police titles, punctuation errors, wordings and spelling errors. Therefore, finding our colleague who has Master of Arts in English, we have tried our best to thoroughly copyedit the manuscript for English language usage. These changes are found throughout the revised version manuscript.\u201dDear reviewer #2After going through the entire manuscript, you forwarded your constructive comments which we missed to touch. Therefore, we are glad enough to express our sincerest thanks for your in-depth review and comments that could help improve the tone of our paper.Reviewer suggestion: This was an interesting manuscript and I enjoyed reading your manuscript. However, the authors should consider addressing the followingAuthors\u2019 response: We are really grateful for your appreciation of our efforts. Besides, we have tried our best to address all your comments point by point as detailed below. Reviewer comment 1: The entire manuscript needs to be revised for grammatical errors and punctuation issues. For example, on page 2, the conclusion section of the abstract, the authors started a sentence with a lower case alphabet. Also, on page 3 (introduction) the first sentences need revisiting as we express maternal mortality per 100,000 live births and not \"live birth\". Similarly, on page 3, the last sentence in paragraph 1 needs revising particularly the sentence ....\"the problem is still major public concern.\"Authors\u2019 response: Sure! From repeated proof-reading of the manuscript, we found several grammatical errors, interlinings, police titles, punctuation errors, wordings and spelling errors. Therefore, finding our colleague who has Master of Arts in English, we have tried our best to thoroughly copyedit the manuscript for English language usage. These changes are found throughout the revised version manuscript. Moreover, the aforementioned reviewer\u2019s specific concerns are addressed as listed below.Conclusion: Contraceptive use, breast feeding duration, age at first birth, preceding child sex and knowing the duration of birth interval correctly were independent determinants of short birthinterval.Inter birth interval refers to the time interval from one child\u2019s birth date until the next child\u2019s birth date between two consecutive live births.Amendment is located on pages 2 and 3 of the revised version manuscript as shown by the yellow highlighted text. Reviewer comment 2. The authors stated on page 3 that ....\"national guideline for family planning services according to the Ethiopian FMOH\u2019s recommendation\" I will recommend that the authors should provide a sentence or two from this guidelines that are applicable/relevant to their study.Authors\u2019 response: Considering the given comment, the following sentence was taken from the Ethiopian national guideline of family planning services. \u201cThe Ethiopian Federal Ministry of Health (FOMH) recommends spacing of childbirth at intervals of three to five years to reduce maternal, perinatal and infant mortality by optimizing the fertility rate in the country.\u201dAmendment is located on page 3, paragraph 3 of the revised version manuscript as shown by the yellow highlighted text. Reviewer comment 3. The authors need to state the aim in the last paragraph of their introduction. At present, this is not really clear.Authors\u2019 response: Absolutely! At present, the aim of this study is not really clear. Therefore, it is now stated in the last paragraph of the introduction concisely as shown by the yellow highlighted text on page 4 of the revised version manuscript. The aim is also given below. \u201cTherefore, this study was aimed at identifying factors that have significant odds of association with short inter-birth interval among a community-based sample of Ethiopian women in Dessie city administration, 2019.\u201dReviewer comment 4. On page 8, Table 1, the authors need to correct the word 'college' which is wrongly written as 'collage'. Additionally the word 'widowed' is also wrongly written as 'windowed'Authors\u2019 response: Certainly! The misspelt words are corrected accordingly as shown by the yellow highlighted text in table 2 of the revised version manuscript which is also given below.Table 2: Socio-demographic characteristics on short birth interval among ever married mothers  in Dessie city administration, Dessie , Ethiopia 2019Factors Category Case (%) Control(%)Rsidence Urban 130(59.7%) 273(62.65) Rural 88(40.3%) 163(37.4%)Marital status Married 186(85.3%) 364(83.5%) Divorced 21(9.6%) 44(10.1%) Widowed 11(5.1%) 28(6.4%)Religion Orthodox 92(42.2%) 173(39.7%) Muslim 124(56.9%) 249(57.1%) Protestant 2(0.9%) 14(3.2%)Ethinicity Amhara 200(91.7%) 399(91.5%) Tgrai 7(3.2%) 11(2.5%) Oromo 6(2.7%) 14(3.2%) Others1 5(2.3%) 12(2.8%)Mother\u2019s education No formal education 45(20.6%) 70(16.1%) read and write 42(19.3%) 86(19.7%) Elementary 34(15.6%) 81(18.6%) Secondary 39(17.9%) 89(20.4%) Collage and above 58(26.6%) 110(25.2%)Husband education No formal education 50(22.9%) 69(15.8%) read and write 32(14.7%) 69(15.8%) Elementary 13(5.9%) 42(9.6%) Secondary 41(18.8%) 72(16.5%) College and above 82(37.6%) 184(42.2%)Mothers\u2019 occupation employee(GO/NGO) 43(19.7%) 91(20.9%) house wife 125(57.3%) 232(53.2%) Merchant 28(12.8%) 53(12.2%) Student 9(4.1%) 29(6.7%) Farmer 10(4.6%) 19(4.4%) daily workers 3(1.4%) 11(2.5%) Others2 0(0%) 1(0.2%)Husband occupation employee(GO/NGO) 84(38.5%) 164(37.6%) Merchant 66(30.3%) 129(29.6%) Student 0(0%) 2(0.5%) Farmer 63(28.9%) 107(24.5%) daily workers 4(1.8%) 23(5.3%) Others3 1(0.5%) 11(2.5%) Number of wiveswealth index One 216(99.1%) 434(99.5%) More than one 2(0.9%) 2(0.5%) Poorest 57(26.1%) 84(19.3%) Second 35(16.1%) 80(18.3%) Middle 47(26.6%) 83(19.0%) Fourth 42(19.3%) 82(18.8%) Richest 37(17.0%) 107(24.5%)1Afar, Gurage2 House servant,3Religious leaderAmendment is located on page 9, in table 2 , results section, sociodemographic characteristics subsection of the revised version manuscript as shown by the yellow highlighted text. Reviewer comment 5. On page 14, Table 5, the authors need to double-check the p-values as there as selected p-values <0.05 that were not highlighted.Authors\u2019 response: Based on the given comment, Table 5  has been double-checked if there is any P-value <0.05 that were not highlighted. Thus, the double-checked table is given below with yellow highlight of P-values that were not highlighted.Table 6: Multivariable analysis on the determinants of short birth interval among ever married reproductive age mothers in Dessie city administration, Dessie, Ethiopia 2019. Factors Case Controls Crude OR(95% CI) p-value AOR(95%CI) p-valuePreceding pregnancy was plannedyesno 18038 40234 0.401(0.244-0.657)1 .001 0.800 (.348-1.839)1 .599practice of postpartum abstinence in preceding childyesno 16157 35977 0.606(0.410-0.8941)1 .0120.875(0.482-1.587)1 .659ANC follow up in preceding pregnancyYesNo 17741 38749 0.547(0.348-0.858)1 .009 0.895(0.400-2.003)1 0.787breast fed duration from previous to last child0-1112-23>=24 1346420 61197178 10.148(0.098-0.224)0.051(0.029-0.089) .001.001 10.291(0.154-0.550)0.098(0.047-0.208) .001*.001*previous to Last child sexmalefemale 72146 235201 0.422(0.300-0.592)1 0.01 0.463(0.282-0.761)1 .002*using any of the modern methods before the conception of your last childyesno 98120 41125 120.1(12.407-32.662) .001 111.221(5.953-21.151) .001*knowledge to appropriate duration of birth interval correctly know not correctly know 13088 280156 0.823(.589-1.149)1 0.2530.446(0.245-0.811)1 .008*Husband educationNo formal educationHad formal education 82136 138298 1.302(0.926-1.830)1 0.129 1.236 (0.633-2.416)1 .535age at first marriageless than 1818-25Greater than 25 2415440 8232826 11.604(0.979-2.628)5.256(2.68-10.286) 0.061.001 1 1.148(0.550-2.398)0.478(0.113-2.024) .713.316age at first birth (years) less than 28>=28 16058 41323 0.154(0.092-0.257)1 0.0010.363(0.166-0.793)1 0.011*no of living children 0-23-4>=5 5512538 9028066 10.731(0.492-1.086)0.942(0.559-1.587) 0.1200.8231 .617(0.338-1.124)1.109(0.489-2.514) .115.696Husband perception to birth spacingDisagree stronglyDont mindEncouragingUnknown 285712013 2715224611 10.362(0.196-0.666)0.470(0.266-0.833)1.140(0.436-2.980) 0.0010.0100.790 10.376(0.136-1.036)0.557(0.221-1.401)0.873(0.195-3.908) .059.214.859Wealth indexPoorest SecondMiddleFourth 57354742 84808382 1.962(1.187-3.245)1.265(0.733-2.183)1.638(0.976-2.747)1.481(0.874-2.510) .009.0.398.062.144 2.012(0.872-4.645)1.486(0.606-3.647)2.378(1.086-5.2101.823(0.780-4.262).101.387.0300.166Richest 37 107 1*for Significant association at p<0.05)Amendment is located in table 6, on pages 15 and 16 of the revised version manuscript as shown by the yellow highlighted text. Reviewer comment 6: On page 15, paragraph 2, the authors should correct the word 'consistency' which was misspelt as 'consistence'.Authors\u2019 response: Yes indeed! On page 15, paragraph 2 of the original document, the word 'consistency' was misspelt as 'consistence'. Thus, it has been corrected as listed below.\u201cThe consistency could be due to the fact that contraceptive use contributes to birth spacing thereby reducing the total fertility rate by different mechanisms on normal reproductive process [25].\u201dAmendment is located on page 17, first paragraph of the revised version manuscript as shown by the yellow highlighted text. Reviewer comment 7. The limitations of the study (page 17) needs revision and should be reported before the conclusion. Specifically, there was no mention of how the recall and social desirability bias reported in the manuscript were dealt with.Authors\u2019 response: Quite sure! The limitations of the study , is currently revised and reported before the conclusion. Specifically, a mention of how the recall and social desirability bias reported in the manuscript were dealt with is stated clearly as given below.  \u201cThe recall bias was dealt with enabling mothers attach their children\u2019s birth dates to unforgettable Ethiopian holidays and calendar days. Besides, it was tried to minimize social desirability bias by conducting probed maternal interviews of the events (factors) by the trained data collectors.\u201dAmendment is located on 18, strength and limitation section of the revised version manuscript as shown by the yellow highlighted text. Reviewer comment 8. The authors also need to provide a few sentences on the key strength of their study.Authors\u2019 response: Certainly! The key strength of our study was \u201cUsing community based unmatched case control study design, high response rate and inclusion of both urban and rural communities.\u201dAmendment is located on page 18, strength and limitation section of the revised version manuscript as shown by the yellow highlighted text. We look forward to receiving your immediate and kind response!Habtamu Shimelis Hailemeskel Attachmentwubet 1Response letter for plos (2).docxSubmitted filename: Click here for additional data file. 9 Nov 2020PONE-D-19-32845R1Determinants of short birth interval among ever married reproductive age women: A Community based unmatched case control study at Dessie city administration, Northern EthiopiaPLOS ONEDear Dr. Habtamu Shimelis,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE\u2019s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by 10 December. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsIf applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see:\u00a0We look forward to receiving your revised manuscript.Kind regards,Sharon Mary BrownieAcademic EditorPLOS ONEReviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the \u201cComments to the Author\u201d section, enter your conflict of interest statement in the \u201cConfidential to Editor\u201d section, and submit your \"Accept\" recommendation.Reviewer #1:\u00a0(No Response)Reviewer #2:\u00a0All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1:\u00a0PartlyReviewer #2:\u00a0Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified.The Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0The authors have adressed most of my comments. I only have a few minor points.1) Delete this text from the conclusion:\"We want to emphasize that our study involved neither experimental nor observational design andhence our recommendations are not based on causal mechanisms. Our recommendations arerather based on the assumption that short birth interval is a potentially modifiable risk factor ofadverse pregnancy outcomes. Hence, intervening on its identified independent predictors(significant factors) helps optimize inter-birth interval.\"Your study is observational and this suggested deleted text does not add any useful information.2) Move the last paragraph away from the conclusion section to the discisttion:\"Based on our findings, local health care providers , the city health department and policy makers should focus on differentstrategies for creating parental awareness about the importance of modern contraceptive use,being primiparous before 28 years old and maternal knowledge of birth spacing. Moreover, westrongly recommend that mothers should prolong their breastfeeding practice for at least twoyears because its effect for optimizing birth interval has been witnessed by many other studies,WHO and UNICEF. However, encouraging breast feeding up to two years may not warrant areduction of birth interval because increasing breast feeding duration merely does not increaseperiod of amenorrhea. This could in turn be due to differences among maternal breastfeedingpractices, maternal age and parity. Women who are partially breast-feeding are at higher risk ofconceiving than women who are fully breast-feeding. The period of lactational amenorrhoeatends to be longer for older and multiparous than for younger and primiparous women. Besides,regardless of their breastfeeding practices, the other possible independent factor that may affectlactational infertility is maternal nutritional status. Therefore, despite the aforementionedconfounders, maternal practice of optimal breastfeeding helps them optimize not only theirhealth but also feto-neonatal and childhood survival.\"3) You can consider shortening the section on the sample size determination and selection procedure, and the section on data quality control.Reviewer #2:\u00a0The authors have meticulously addressed my comments and the manuscript has been further strengthened.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,\u00a0 13 Nov 2020Dear academic Editor (Sharon Mary Brownie)After going through the entire revised version manuscript, you forwarded your constructive editorial comment. Therefore, we are glad enough to express our sincerest thanks for your helpful comment that could help improve the tone and readability of our paper.Editor comment: Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE\u2019s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Authors\u2019 response: We are really delighted with your constructive editorial comments. Hence, we have addressed the points raised during the review process and the corrections are incorporated within the second round revised version manuscript. All the improved changes are shown by tracked insertions and deletions.Dear reviewer 1 After going through the entire manuscript, you forwarded your constructive comments which we missed to touch. Therefore, we are glad enough to express our sincerest thanks for your in-depth review and comments that could help improve the tone of our paper.Reviewer comment: The authors have addressed most of my comments. I only have a few minor points.Authors\u2019 response: We are most grateful for your acknowledgment of our efforts in addressing most of your comments. Besides, we have tried to address your current concerns point by point as detailed below. Reviewer comment: 1) Delete this text from the conclusion:\"We want to emphasize that our study involved neither experimental nor observational design and hence our recommendations are not based on causal mechanisms. Our recommendations arerather based on the assumption that short birth interval is a potentially modifiable risk factor ofadverse pregnancy outcomes. Hence, intervening on its identified independent predictors (significant factors) helps optimize inter-birth interval.\"Your study is observational and this suggested deleted text does not add any useful information.Authors\u2019 response: We strongly agree with the reviewer\u2019s comment and hence the aforementioned text has been deleted from the conclusion section as it can be appreciated from the tracked deletion, paragraph 1 of the conclusion, on page 19 of the revised version manuscript.Reviewer comment: 2) Move the last paragraph away from the conclusion section to the discussion:\"Based on our findings, local health care providers , the city health department and policy makers should focus on differentstrategies for creating parental awareness about the importance of modern contraceptive use,being primiparous before 28 years old and maternal knowledge of birth spacing. Moreover, westrongly recommend that mothers should prolong their breastfeeding practice for at least twoyears because its effect for optimizing birth interval has been witnessed by many other studies,WHO and UNICEF. However, encouraging breast feeding up to two years may not warrant areduction of birth interval because increasing breast feeding duration merely does not increaseperiod of amenorrhea. This could in turn be due to differences among maternal breastfeedingpractices, maternal age and parity. Women who are partially breast-feeding are at higher risk ofconceiving than women who are fully breast-feeding. The period of lactational amenorrhoeatends to be longer for older and multiparous than for younger and primiparous women. Besides,regardless of their breastfeeding practices, the other possible independent factor that may affectlactational infertility is maternal nutritional status. Therefore, despite the aforementionedconfounders, maternal practice of optimal breastfeeding helps them optimize not only theirhealth but also feto-neonatal and childhood survival.\"Authors\u2019 response: Well! We are grateful for your comment of importance. Thus, the last paragraph has been moved away from the conclusion section to the discussion as shown by the tracked insertions and deletions in the discussion and conclusion sections of the revised version manuscript on pages 18 and 19. Reviewer comment: 3) You can consider shortening the section on the sample size determination and selection procedure, and the section on data quality control.Authors\u2019 response: Great thanks! Based on the given comment, the section on the sample size determination and selection procedure, and the section on data quality control have been shortened to a reasonable extent. The amendment can be appreciated from the tracked deletion on pages 5, 6 and 7, in the methods section, subsection of sample size determination and selection procedure and data quality control.AttachmentResponse letter (Revised 2 version).docxSubmitted filename: Click here for additional data file. 16 Nov 2020Determinants of short birth interval among ever married reproductive age women: A Community based unmatched case control study at Dessie city administration, Northern EthiopiaPONE-D-19-32845R2Dear Dr. Habtamu Shimelis,We\u2019re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you\u2019ll receive an e-mail detailing the required amendments. When these have been addressed, you\u2019ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they\u2019ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Sharon Mary BrownieAcademic EditorPLOS ONEReviewer comments have been satisfactorily addressed 20 Nov 2020PONE-D-19-32845R2 Determinants of short birth interval among ever married reproductive age women: A Community based unmatched case control study at Dessie city administration, Northern Ethiopia Dear Dr. Shimels Hailemeskel:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofProfessor Sharon Mary Brownie Academic EditorPLOS ONE"}
+{"text": "Arachnoiditis ossificans is a rare disease, characterized by intradural ossifications, representing the end stage of chronic adhesive arachnoiditis. We describe the case of a 55-year-old patient who developed symptoms of a cauda equina syndrome after an open microdiscectomy at the L5 to S1 segment. A subsequent exploratory surgery revealed an intradural concentric bony structure with partly incorporated and partly adherent nerve roots. A partial removal of the intradural calcifications was performed. Postoperatively, the patient showed neurological improvement. The removed intradural calcifications were submitted for histological analysis and proved to be normal bone tissue, notably containing yellow bone marrow. To our knowledge, the presence of yellow bone marrow within bony cavities of arachnoiditis ossificans has not previously been reported. A 55-year-old man presented with a 1-month history of low back pain and a left-sided weak plantarflexion. The Las\u00e8gue's sign was positive on the left side and a hyperactive patellar reflex on the left side was detected. There was no history of trauma, previous spine operations, or intrathecal injections. His clinical history revealed an episode of low back pain and sphincter disturbances 16 years ago.The initial spinal lumbar magnetic resonance imaging (MRI) showed a left-sided herniated disc at the L5 to S1 segment .Due to the failure of nonsurgical management, the patient was submitted to an open microdiscectomy. The surgical intervention was uneventful; however, postoperatively the patient developed symptoms of a cauda equina syndrome with saddle anesthesia and bladder dysfunction (urinary retention).A spinal MRI was performed immediately and demonstrated a cystic enlargement of the dural sac from L5 to S2 .An exploratory spinal surgery was performed and the dural sac at L5 to S1 was exposed. An extremely hard dural sac was detected, leading to the decision of a durotomy. A concentric bony structure was discovered in the dural sac. Nerve roots were partly incorporated by calcifications and partly adherent to the wall of this concentric bony structure . A partThe removed intradural calcifications were submitted for histologic examination and revealed normal osseous tissue. Yellow bone marrow was found in the bony cavities .Postoperatively, the patient showed neurological improvement; the saddle anesthesia regressed, the bladder function turned normal, and the strength of the left plantarflexion improved.A computed tomography (CT) was subsequently performed and demonstrated the extent of the intraoperatively detected intradural ossification .The findings were consistent with spinal arachnoiditis ossificans.Arachnoiditis ossificans has to be distinguished from common benign meningeal ossifications.Arachnoiditis ossificans is most frequently located in the thoracic spine (66%); this is explained by the highest concentration of arachnoid cells in these segments.The disease has shown to be associated with infective, traumatic, iatrogenic, or vascular events.The mean age at diagnosis is 53 years and there is no gender predilection.Given that arachnoiditis ossificans mainly occurs at the thoracic and lumbar level, symptoms typically consist of longstanding backpain and radiculopathy proceeding to progressive myelopathy.In the absence of CT scanners, only severe forms of arachnoiditis ossificans have been detected by plain X-rays of the spine.Nowadays, arachnoiditis ossificans can be reliably diagnosed by MRI or CT. On MRI, clumped nerve roots refer typically to a spinal arachnoiditis. Intradural ossifications appear hyperintense in T1-weighted images and may be hypo- or hyperintense in T2-weighted imaging.The pathogenesis of arachnoiditis ossificans is not yet sufficiently resolved. It is suggested that inflammatory processes occur, similar to chronic arachnoid inflammations of other causes.Arachnoiditis ossificans is affecting nerval structures  by inflammatory processes or direct mechanical impairment, leading subsequently to neurological deficits. Moreover, the circulation of the cerebrospinal fluid may be affected by space occupying intradural osseous structures that can result in the formation of syringomyelia and arachnoid cysts.Spinal vascular abnormalities, such as vascular malformations, have been found in patients presenting with arachnoiditis ossificans. It is hypothesized that recurred bleeding generates inflammatory processes.Pathohistological specimens of these intradural ossifications reveal normal bone tissue, trabecular or lamellar in structure.Due to the rarity of the disease, no general treatment recommendations exist.The therapeutic strategy should be based on the clinical presentation, the ossification pattern, and associated conditions like syringomyelia. Surgical treatment should be reserved for patients in whom rapid neurological deterioration is noted. Goals of surgery include decompression of the affected neural structures  and reestablishment of a physiologic cerebrospinal fluid circulation. Therefore, decompression of the spinal canal over the entire length of the ossification, for example via a laminectomy, is recommended as the treatment of choice.Removing calcified plaques adhering the spinal cord or the nerve roots should be avoided. Particularly in type III arachnoiditis ossificans, an aggressive removal of adherent osseous plaques from neural tissue can cause significant deterioration of neurological deficits.A few authors propose the placement of syringopleural or ventriculoperitoneal shunts to normalize altered cerebrospinal fluid dynamics.In our case, arachnoiditis ossificans was detected accidentally when performing an exploratory spinal surgery in a patient presenting with secondary deterioration after an uneventful lumbosacral microdiscectomy.It is noticeable that the clinical history revealed no predisposing events disregarding a temporary episode of low back pain and sphincter disturbances more than a decade ago. These findings had not been further explored by imaging and disappeared spontaneously.Retrospectively, the cystic intradural formation was clearly apparent on the spinal MRI on admission. Neither the radiological findings nor the surgeon performing the initial procedure paid attention to this alteration. No suspicious findings particularly with regard to nerval structures or the dural sac were described in the surgical report.It can be speculated that the postoperative development of the cauda equina syndrome in our patient is most likely attributable to intraoperative manipulations of the dural sac and the osseous incorporated nerve roots while performing microdiscectomy.However, in the knowledge of an arachnoiditis ossificans, a (hemi)laminectomy of L5 followed by the removal of the herniated lumbosacral disc may be regarded as an optimal surgical approach in this case. It has to be debated whether this approach would have sufficiently reduced surgical manipulations during microdiscectomy to prevent postoperative neurological deterioration.Histological analysis revealed normal bone tissue with yellow bone marrow. This is the first report describing this finding in arachnoiditis ossificans. We suggest that the formation of yellow bone tissue is caused by an adipose metaplasia of the arachnoid.In conclusion, arachnoiditis ossificans is rare, but should be kept in mind, particularly in patients with a previous history of spinal interventions or trauma. In these patients, clumped nerve roots and signal alterations, suspicious of intradural ossifications on MRI, should be further evaluated by CT scan. If arachnoiditis ossificans diagnosed, surgery should be performed only in patients with neurological deterioration. The recommended procedure is a bony decompression of the affected nerval structures; durotomy and resection of osseous plaques should be avoided."}
+{"text": "Pseudotolithus senegalensis) nuggets during frozen storage were investigated. The moisture, protein, fat, ash, and carbohydrate contents varied among the cooking methods and frozen storage times. The deep-fried nugget had a higher fat content, which resulted in a higher energy value (p < 0.05). The free fatty acid content and peroxide value (PV) of the oven-baked nuggets were higher than the deep-fried ones (p < 0.05). The PV tended to increase with increasing storage time, but it was still within the recommended range for consumption. The deep-fried nugget showed a vivid orange\u2013yellow color, with higher L*, a*, and b* values, while oven-baked nuggets showed a pale-yellow color. The baked nuggets had relatively lower total expressible fluid than the deep-fried nuggets at all time points (p < 0.05). The hardness, springiness, and chewiness of deep-fried nuggets were higher than baked nuggets throughout the storage period (p < 0.05). The total plate count and yeast and mold counts produced by the two cooking methods were within the acceptable range throughout the storage.The effects of deep-frying and oven-baking on chemical, physical, and microbiological, properties of cassava croaker ( Consumprombosis . Additiorombosis . Cassavat Africa . It belot Africa . Thus, tAlthough the fish have high nutritional value, the time for their preparation and limited shelf life may discourage some consumers to purchase, leading to a preference for convenience products . Fish nuFish-based food products can be cooked by several methods to retain and/or improve the quality of the final product, and nutritional value and cooking also positively associated . SeveralAll of the ingredients for the fish nuggets such as garlic, ginger, seasoning, black pepper, parsley, mustard, lemon, and salt were purchased from a local supermarket in James Town, Accra, Ghana. All analytical chemicals were purchased from Sigma\u2013Aldrich .P. senegalensis) weighing 1\u20132 kg were purchased from a local market in James Town, Greater Accra, Ghana, West Africa. The fishes were placed in ice with a fish/ice proportion of 1:2 (w/w) and transported to the Laboratory of Food Research Institute, Accra, Ghana, within 50 min. Then, the fish were immediately washed with cold tap water (4 \u00b0C), gutted, clean, and filleted. The fish fillets were cut into 17\u201318 g square shapes .Ten cassava croaker , 7.5 g of black pepper, 3.2 g of parsley, 37.5 g of mustard paste, 15.5 g of lemon juice, and 16.1 g of salt. The seasoned CFC were later dipped in 274.8 g of wheat flour. The floured CFC were dipped into 286.6 g of egg white, allowing any excess to drip off, and then rolled in 357.1 g of dried breadcrumbs. The nuggets were kept on the tray for 30 min to allow the coating to set. According to AOAC [The nuggets were produced using 3.2 kg of fish fillets for each treatment (deep-frying and oven-baking). For each treatment, approximately 182 of cut fillet cubes (CFC) were used. CFC were mixed with 70.6 g of garlic, 96.7 g of ginger, 27.8 g of ready-made seasoning powder (Knorrw/v). The second portion of fish nuggets (26 nuggets) was oven-baked at a temperature of 160 \u00b1 5 \u00b0C for 45 min using a JY-OE60T5 electric convection oven  [One part of the fish nuggets (26 nuggets) was deep-fried in 5-L King Rice Bran Oil   at a temperature of 160 \u00b1 5 \u00b0C [, China) . The deeThe standard methods of AOAC  were useLipid was extracted from processed fish nuggets by the method of Bligh and Dyer  and usedL*a*b* color parameters of processed fish nugget samples were examined with a Hunterlab Miniscan/EX instrument .The g/25 \u00b0C/3 min) using an RC-5B plus centrifuge . The pelleted samples were removed and weighed (Wp). Then, the TEF was calculated using Equation (2) [For the TEF, two cooked nuggets (25 g each) (Ws) were placed in a preweighed centrifuge tube and then centrifuged (2960\u00d7 tion (2) .(2)TEF\u00a0TPA of nuggets was examined using a TA-XT plus texture analyzer  fixed to texture expert software following the methods described by Bonfim et al. . TextureTotal plate count (TPC) and yeast and mold counts of deep-fried and baked fish nuggets were enumerated as per Panpipat and Chaijan , over 90t-test was used in the case of pairwise comparison.A completely randomized design was used, and the experiment was triplicated. Data were subjected to one-way analysis of variance. Mean comparison was performed by Duncan\u2019s Multiple Range Test using the SPSS program . The p < 0.05). The results obtained were comparable to those published by Marimuthu et al. [p > 0.05). During storage, the moisture and carbohydrate content decreased with associated increase in protein, fat, and ash content in both cooking methods. Moisture loss, due to dehydration, during frozen storage may explain the decrease in moisture content of the cooked nuggets. At the same time, the loss of breadcrumbs from the coating during frozen storage and cooking may cause the decrease in carbohydrate content of the cooked nuggets. Loss of coating can occur at any stage during the preparation or frozen storage [p > 0.05) among the two cooking methods at all time points. However, an increase in ash content was noticed with the increasing storage period, while the opposite was observed in the carbohydrate content, as shown in The effects of deep-frying and oven-baking on the proximate compositions and energy values of cassava fish nuggets are shown in u et al. . During u et al. ,25,26,27 storage . The proThe energy values ranged from 243.3 to 277.4 and 207.2 to 233.3 kcal/100 g for deep-fried and oven-baked fish nuggets , respectFFAs are the products of lipid hydrolysis. There was a general increase in the FFAs among the two cooking methods during storage a. The FFp < 0.05). Results revealed that the PV of cooked samples increased with storage time, reaching 4.30 meq/kg fat in oven-baked nuggets and 4.03 meq/kg fat in deep-fried nuggets after 90 days. Findings from Moosavi-Nasab et al. [b et al.  showed tb et al. ,32,33,34b et al. , may havb et al. . In addib et al. , could ab et al. . The lowb et al. .p < 0.05) were observed for L*, a*, and b* values during the storage among the treatments, indicating that the different cooking methods and storage periods affected the color parameters. The findings from this study showed that deep-fried nuggets tended to have higher L*, a*, and b* values throughout the storage period. The L* values of the nuggets cooked using both methods were positively correlated with their final moisture contents (R2 = 0.9294 for deep-fried nuggets and R2 = 0.9521 for oven-baked nuggets). Throughout the storage period, the a* value of deep-fried nuggets was significantly higher than that of oven-baked nuggets (p < 0.05). The b* values of deep-fried nuggets were likewise greater than those of oven-baked nuggets. As a result, the deep-fried nuggets were a vivid orange-yellow color, but the oven-baked nuggets were a pale yellow tone. Benjakul et al. [The color values of deep-fried and oven-baked cassava fish nuggets are illustrated in l et al.  reportedl et al. , the mosl et al. ,42,43. TWith increasing storage time, all the color parameters of both treatments tended to decrease . This map < 0.05). In addition, the TEF of both samples increased with storage time (p < 0.05). The baked fish nuggets had relatively lower TEF than the deep-fried nuggets at all time points (p < 0.05), which is expected because the wet process (frying) will intuitively retain and express more fluid than the dry process (baking). The low fat content of oven-baked nuggets (The TEF of fish nuggets from the two cooking methods are shown in  nuggets  is respo nuggets . Becausep < 0.05). According to Bechtel et al. [2 = 0.6203\u20130.7786). Moisture content was negatively correlated with hardness, whereas protein and fat levels were positively correlated with hardness. As a result, the final textural quality of the fish nuggets could be influenced by compositional variation. Springiness and cohesiveness of both samples showed similar degrees of change at different time points. Springiness represents the deformation when the compressive force is taken away [p < 0.05) whereas the cohesiveness values of both samples were not different (p > 0.05). Based on the findings, oven-baked fish nuggets were softer compared to the deep-fried nuggets. The chewiness had the same trend with the hardness and springiness. Chewiness is the derived textural parameter, and its behavior is governed by the primary parameters it is dependent on [p < 0.05). It has been reported that the existence of crust impacts the food\u2019s mechanical qualities, as well as its texture and acceptability [TPA results for deep-fried and oven-baked cassava fish nuggets were determined as hardness, springiness, cohesiveness, and chewiness . There wl et al. , baked bken away . Cohesivken away . Springindent on . Chewinendent on . The chetability . As a retability  and gluttability . With intability ,54. Thostability  and, henp < 0.05). Then, no significant differences were seen among the two cooking methods from Day 30 until the end (p > 0.05). However, there was a decrease in TPC in both treatments as the storage period increased (The TPC and yeast and mold counts are shown in ncreased a. The yencreased b. HoweveThe cooking methods (deep-frying and oven-baking) and the frozen storage influenced the overall quality of the cassava fish nuggets. Proximate composition, FFA, PV, color, texture, and microbial quality of the nuggets varied among the two cooking methods and frozen storage time. In general, deep frying produced fish nuggets with a better quality, as evidenced by lower PV, FFA, TPC, and yeast and mold count compared to oven-baked nuggets during frozen storage. However, the oven-baked nuggets had the lower fat content and energy value and relatively higher moisture content. Fish nuggets can be kept at \u221218 \u00b0C for up to 90 days without marked deterioration. Microbial indices of fish nuggets evaluated during 90 days of frozen storage were also found to be within acceptable quality standards for cooked fish products. This study has demonstrated that deep-frying and oven-baking produced nuggets of high eating quality during frozen storage. However, for food safety concerns, it is suggested that GMP and GHP be used in the production of fish nuggets."}
+{"text": "We study systemscontaining oppositely charged colloidal particlesunder applied alternating current electric fields (AC fields) usingoverdamped Langevin dynamics simulations in three dimensions. We obtainjammed bands perpendicular to the field direction under intermediatefrequencies and lanes parallel with the field under low frequencies.These structures also depend upon the particle charges. The pathwayfor generating jammed bands follows a stepwise mechanism, and intermediatebands are observed during lane formation in some systems. We investigatethe component of the pressure tensors in the direction parallel tothe field and observe that the jammed to lane transition occurs ata critical value for this pressure. We also find that the stable steadystates appear to satisfy the principle of maximum entropy production.Our results may help to improve the understand of the underlying mechanismsfor these types of dynamic phase transitions and the subsequent cooperativeassemblies of colloidal particles under such non-equilibrium conditions. Such systems, in the presence ofa constant driving field, have been well-studied theoretically.23 It has been found that a lattice-gas model for the two-colored particlesystem has a propensity to form so-called \u201cjammed\u201d structures,wherein interfaces between layers of similarly colored particles formperpendicular to the driving field.22Colloidal dispersions, in particular, occur in many areasof interest, 26 When the field is oscillatory, it appears that bothjammed and so-called \u201claned\u201d structures may form, dependingupon factors such as the magnitude of the driving field and its oscillatoryfrequency. For charged particles, the magnitude of the surface chargealso plays a role. Laning generally occurs for a high enough fieldstrength and a low frequency of oscillation.24 Here, the colloidal mixture forms long rows (lanes) of similarlycharged particles moving parallel to the field. Some evidence pointsto this being a second-order transition from the uniform randomlymixed state, with the correlation length of the lanes growing exponentiallywith the field strength.27 However, morerecent studies suggest that this observation may be an artifact ofthe slow dynamics of lane formation from an initially random configuration.A correspondence between density-dependent dynamics and attractivefluids at equilibrium suggests that laning is instead a first-order(discontinuous) transition.28 Related theoreticalstudies have considered driven colloidal particles in channels andin spatially periodic fields.31L\u00f6wen and co-workers have simulated mixtures of chargedcolloidal particles driven by external fields in a continuum two-dimensional(2D) space. They also obtained ordered steady-state structures forsuch systems.32 Of particular relevance to the work presented here areexperiments by Vissers et al. on charged colloids,where both jammed33 and laned34 steady states were observed in the presenceof an applied AC field in three-dimensional (3D) systems. On the otherhand, comparisons with such experimental work have usually involvedsimulations performed with 2D planar systems. In this work, we revisitthe problem of charged colloidal particles in the presence of an electrolyteand an AC electric field. However, unlike most previous work, we willcarry out a simulation study of the dynamic behavior of this systemin 3D.Experimental studies ofthe behavior of colloidal particles inexternal fields have also been quite prevalent.It is well-known that for equilibrium systems the phasebehaviorcan be crucially dependent upon the dimensionality of the allowedfluctuations. For example, fluctuations of interfaces in 1D systemspreclude phase separation. Furthermore, (infinite) 2D interfaces canalso be subjected to unconfined capillary waves, leading to divergencesin the interfacial width. Here, we investigate the propensity of thedriven colloidal system in 3D to form steady-state jammed and lanedstructures seen in 2D studies and to determine the putative phaseboundaries as a function of particle charge, as well as the strengthand frequency of the driving fields.q, varying from \u00b110e to \u00b150e. The interactions between the colloids are assumed tohave a screened-Coulomb (Yukawa) form. An AC electric field, E(t) = E0 sin(2\u03c0\u03c9t), is applied to the dispersion with frequencies \u03c9ranging from 10 Hz to 1 kHz. The colloidal particles are assumed tobe dispersed in a highly viscous solvent, which is mimicked usingoverdamped Langevin dynamics; see Supporting Information (SI). Hydrodynamic and electrofriction effects are ignored in thisstudy. Details of the simulation setup are given in SI.We modeled binary mixtures of oppositelycharged colloidal particles with a range of charge magnitudes, q = \u00b140e in the presence of AC fields with the same maximum fieldstrength, E0 = 0.115 V/\u03bcm, but withdifferent \u03c9 values. For the largest frequency (500 Hz), onlyhomogeneous steady-state structures could be obtained in the system, respectively. The \u03c8i values are then averaged over all particles to obtain \u03a8. Forthe completely disordered (homogeneous) state, we have \u03a8 = 0,whereas for structures with regions where particles have the samecharge, 0 < \u03a8 < 1. Larger \u03a8 corresponds to moreordered structure.In order to quantify the structure of the system, we calculatedan order parameter, \u03a8, as follows. The quantity \u03c8E0 = 0.115 V/\u03bcmand vary the values of q and \u03c9. The stablephase regions were established by noting the steady states which evolvedin the simulations over a transient period, starting from the uniformmixture. After this transient period, \u03a8 values were calculatedat the end of each oscillating period and averaged over a simulatedtime of t = 2 s. During this time, the observed structureand total energy do not vary significantly over different periodsof oscillation, which indicates the systems reach a steady state.The putative dynamic phase diagram, describingthe stable steadystates, will ostensibly depend upon several variables, including fieldstrength and frequency as well as particle charge and density. Here,we consider two \u201cslices\u201d through this multidimensionalphase diagram. First  are ableto inhibit the formation of ordered domains. For intermediate charges,the field is able to create bands of like-charged colloids correspondingto jammed steady-state structures. This re-entrant behavior is clearlyreflected in the \u03a8 values, which show a nonmonotonic behaviorwith respect to the particle charge. At lower field frequencies (between\u03c9 = 100 and 200 Hz), jammed bands with greater \u03a8 valuesare obtained, with these values increasing with particle charge. Thisis due to a stronger coupling with the field, promoting phase separation.However, if instead \u03c9 < 100 Hz, laned structures  are found in systems with a high particle charge. Atthese lower frequencies, the parallel interface of the jammed structuresis disrupted by the strong field coupling, and instead, lanes (whichextend over the length of the box) form over the longer field period.F0 = E0q. Thus, we also considered a \u201cconstantforce\u201d slice through the full phase diagram, where we varyboth \u03c9 and q as before, but the absolute maximumforce is kept constant, i.e., F0 = 0.552 pN (E0 and q vary inversely). The corresponding results are given in q forthe same \u03c9 and force, the system tends to become disordered,due to the repulsion within regions of similar charge. This effectdominates earlier at high field frequencies, due to the shorter periodover which the force is able to drive the separation of particles.That is, for larger charges the homogeneous phase has a greater cohesiveenergy, which means the driving field needs to be applied for a longerperiod in order to drive the phase separation. Thus, at low frequencies(\u03c9 = 100 and 50 Hz), these jammed bands are still observed,even at an absolute charge of 50e. On the other hand,at very high frequencies, \u03c9 = 1 kHz, the charged particles onlyoscillate locally within a relatively small region and the jammedband phase is not exhibited, except at the lowest charge. It is noteworthythat, for q = \u00b110e, the jammedband phase is seen under all frequencies investigated. However, theorder parameters behave nonmonotonically. For example, the order parameterfor the q = \u00b110e system at\u03c9 = 10 Hz is lower than that at \u03c9 = 50 Hz, perhaps becauseof some incipient laning.As the external forces on colloidal particles depend upon theircharge at a fixed field strength, the phase diagram z direction, with a field strength Ec = E0/\u221a2.In this case, only lane structures  are obtained.Representative snapshots are shown in Figure S2.Laned structures parallel with ACfield direction are observedin the more highly charged system for a field frequency of \u03c9= 10 Hz. We also investigated the system in a constant electric field(DC field) along the 37riz(t) is the coordinatealong the field direction of particle i of speciesA at time t. The results are given in In order to characterizethe kinetic mechanisms by which the observed steady states evolved,we considered the time evolution of \u03a8, as well as the mean squareddisplacement (MSD) along the field direction. This gave us some indicationof the correlations between the structural evolution and dynamicalproperties of the colloidal particles. The MSD is determined by theequatione charged particlesat a field strength E0 = 0.1725 V/\u03bcmand a frequency \u03c9 = 100 Hz. The system was started in the homogeneousmixed phase. Both the MSD and the \u03a8 initially increase rapidly,suggesting that the particles initially diffuse quickly to form like-chargedclusters. The order parameter \u03a8 initially peaks at t = 0.15 s, and the corresponding snapshot shows that a jammed structurewith relatively thin bands is initially formed. This is a relativelyunstable jammed state, and the MSD curve is lowered only marginallyafter it is formed. In fact, diffusion in the period between 0.15and 0.28 s remains somewhat high, and during this period, \u03a8decreases until a local minimum occurs at 0.28 s. This can be attributedto the penetration of one band into another, momentarily creatinga larger total interface between unlike charges and reducing \u03a8.Between 0.28 and 0.47 s, regions of opposite charge move through eachother and then merge to create thicker jammed bands, increasing \u03a8. This is another (metastable) jammed statecorresponding to a transient period whereby the MSD (immediately after0.47 s) is rather flat. However, this is followed quickly by an increasein the MSD as two bands (in the middle of the system) repeat the processof merging and reordering. The \u03a8 values oscillate again duringthis period. The \u03a8 value reaches a plateau after 1 s and doesnot increase any further. The MSD curve increases more slowly after1 s, again because of the jamming effect. The penetration and mergingappear to cease, as the period for the oscillation is insufficientlylong to bring this about. Thus, jammed structures appear to emergefrom the homogeneous phase via a series of metastablestates with progressively thicker bands. The order parameter \u03a8will oscillate as the system evolves, progressing to sequentiallyhigher values until it reaches a steady state.e, in a field withsame maximum strength as above (E0 = 0.1725V/\u03bcm) but with a frequency of \u03c9 = 10 Hz. Although thefinal phase is a laned structure, oriented parallel with the AC fielddirection, an intermediate jammed structure (perpendicular to thefield) is observed to form initially corresponding to a maximum \u03a8value at 0.177 s. This is also characterized by the initially flatterregion in the MSD curve. This jammed structure starts to break upat around 0.23 s. This is initiated by a tilting of the interfaceleading to oppositely charged bands moving parallel with the fieldand temporarily opening of a void, which is subsequently filled ata later time. This process appears to form clusters of like chargedparticles which eventually percolate to form a laned steady state(after approximately 0.5 s). The MSD shows that the colloidal particlesdiffuse relatively quickly once the jammed structure is dismantled.Movies of the full trajectories for these two systems , along the field direction (z-axis)using the definition due to Kirkwood and Buff:40x\u2013y plane with Vs being the volume of each slice. For everyparticle i in a given slice centered at z (with thickness \u03b4z), we calculated the componentof the virial in the z direction, Fijzrijz, by summing the pairwise force with every other particle, j. This, in turn, is summed over every particle in the slice.The bracket \u27e8\u27e9 represents a specific average over thetrajectory (as described below). An average over all the slices inthe simulation box gives the total virial pressure tensor of the system.The ideal gas contribution to the pressure is much smaller than thevirial contribution, and we neglect it.We also calculated the local pressuretensor (due to particle interactions), F0 = 0.552 pN (as described in pzz) is plottedagainst the field frequency, \u03c9. The relative magnitudes of therepulsive and Yukawa contributions to the virial can be appreciatedby choosing a pair of particles at contact (with separation r = \u03c3 = 150 nm). Detailed information on these potentialsare provided in the SI. In e. At this separation, thesteric term is much greater than the Yukawa contribution. The averagesteric and Yukuwa contributions to the virial pzz are given in 42 As expected, the short-rangedsteric repulsions dominate the virial pressure over the range of \u03c9considered, at least for these colloidal charges.In pzz generally decreases with large \u03c9. At smaller\u03c9, where the time period over which particles in the jammedphases are forced against each other is longer, the pressure in thecompressed bands will increase. This increase in pzz with decreasing \u03c9 reaches athreshold value (located at \u03c9 \u2248 50 Hz). Below this threshold,the system is unable to maintain the perpendicular bands of the jammedstructure and the steady state adopts a different form, with a significantdecrease in pzz. At \u03c9= 10 Hz, particles with charge \u00b120e and \u00b130e form laned structures. For particles with \u00b110e, lanes do not appear explicitly, and the order parameter,\u03a8, is reduced but not zero, indicating mixing of particles withopposite charges and perhaps some incipient laning. The maximum valueof pzz, at which phasechange occurs, is similar for all of the systems investigated . This indicates the possible occurrenceof an instability in the jammed phase, wherein parallel bands of like-chargedparticles are unable to remain perpendicular to each other beyonda threshold value of the perpendicular pressure. Instead, at or beyondthis threshold value, the surfaces of the bands undergo significantbuckling via concerted motions that cause the bandsto penetrate one another. This ultimately gives rise to laned steadystates, at least at the higher charges considered .Over most of the frequency range (50\u2013600Hz), the systemadopts the jammed steady-state form where pzz(t), resolved as a function of time, for thefrequencies \u03c9 = 100 and 10 Hz. These are the same systems displayedin pzz oscillates inresponse to the applied field with a magnitude that appears to becommensurate with the order parameter \u03a8. For example, in pzz(t) increases during phase separation process andthen decreases until about 0.3 s. This is the same behavior seen forthe order parameter, \u03a8, in this system  together with the AC field , at specific times in a single period, as shownin p(z) profiles are small in magnitude andrelatively flat at the same time when the total pzz is at its minimum (p(z), has larger distinct peaks at thecollision interfaces when pzz is at its maximum (p(z) are much smaller than the steric repulsion,especially when pzz reachesthe maximum values. In addition, there are small minima in the Yukawacontribution to p(z) at the collisioninterfaces, due to the electrostatic attraction between the bandswith oppositely charged particles.stem see 2a. The aield see 5b. The o minimum 6a,c. On  maximum 6b,d. Thepzz values increase rapidly to about 160Pawhen the jammed structure forms at 0.177 s  profiles at the maximum and minimumvalues of pzz under 10Hz in an oscillating period are shown in p(z) profiles showpeaks and wells at the maxima of pzz in an oscillating period, and they are flat at the minimaof pzz.In the system with \u03c9 = 10 Hz, the  0.177 s 2b, such via the overdamped Langevin dynamics. The dynamics, being nonreversible,also produce entropy in the system, which at steady state is matchedby the entropy dissipated via the heat loss to thereservoir. From the first law of thermodynamics, the heat dissipatedis also equal to the work performed by the external force under steady-stateconditions. The heat dissipation rate is given by 46 where \u1e59i is the velocity of particle i, \u03b3 isthe friction coefficient, \u03bei isthe random force in the overdamped Langevin equation and \u201c\u00b7\u201dis a Stratonovich product. The averaged dissipation rate for the systemover an oscillating period \u03c40 isSI. The first term on the right-handside corresponds to the contribution from the applied force, whichhas an amplitude F0. It is the work performedon ideal particles by the applied force. The secondterm accounts for contributions from particle interactions and isgiven by47U(t) is the nonbonded interaction between particles(steric repulsionand Yukawa). The quantity \u27e8\u1e87\u27e9can be thought of as the work done by the restoring forces generatedby the colloidal particles, as a consequence of them responding tothe applied field.The systems investigated here, drivenby AC fields, dissipate heat to the implicit reservoir \u1e87\u27e9 at different oscillating periods for the \u00b120e charged particle systems are displayed in Figure S7, in order to provide more comprehensive view forthe time evolution of the rate of work). At 1 kHz  initial configuration to the jammed steady state,suggesting that the latter corresponds to a condition of least workbeing performed by the restoring forces of the colloidal particlesper oscillation period. This is equivalent to a maximum rate of dissipationof heat to the surrounds or maximal entropy production rate.48 A similar analysis for the system where the applied force oscillatesat \u03c9 = 10 Hz  scaled by the factor \u03bb(=2). The density profiles of the system are shown in Figure S5d,f, which displays again a centrallane of one type of particle surrounded by the opposite type of particles,similar to the original simulation (\u03bb = 1). As the main contributionto the work comes from the interfaces between the lanes , \u27e8\u1e87\u27e9,which is a per particle quantity , we expect, and see, a decrease in \u27e8\u1e87\u27e9 compared with the smaller simulation . For both the geometry ofthe uniform and jammed steady states, we do not expect to see sucha significant size dependence. Compared to the other results, we nowsee a monotonic behavior with respect to frequency.As \u27e8dy state 7a\u2013c show\u1e87\u27e9 for various laned andjammed structures under those conditions where they do not appear to be the preferred steady state. For instance, the lanedstructure formed under 10 Hz was used as the initial configurationfor the simulation at 100 Hz in order to determine if the laned structuretransitions to the apparently preferred jammed structure  or viceversa. The rates of work \u27e8\u1e87\u27e9 for the simulations with varying frequencies as well assome representative snapshots are shown in \u1e87\u27e9, i.e., to maximize the rate of entropyproduction. This can be understood as a tendency of the system torespond in a way to minimize the restoring forces in the fluid. Inthe initial laned structure, the sum of collisional forces betweenthe particles is generally smaller than those in the random disorderedstate, hence, \u27e8\u1e87\u27e9 for the lanedsteady states at the beginning of the simulations is lower than thatwith a random state. However, both kinds of initial states find theirway to the jammed states . In addition, the steady-state value of\u27e8\u1e87\u27e9 at 400 Hz, initiated fromthe laned structure, is lower than that emerging from the random disorderedstate. This is because of the presence of fewer bands in the lattercase  did transition to the laned structure appropriateto \u03c9 = 10 Hz  = E0 sin(2\u03c0\u03c9t), where E0 is the maximumelectric field strength. The electric field is chosen to be uniformand directed along a particular axis of the simulation box. The fieldinfluences the dynamics by the subsequent force on the colloidal particles,which was assumed to have the magnitude, Fext(t) = E(t)q (q is the particle charge), and actsin the field direction. The field frequency \u03c9 is the reciprocalof the oscillation period \u03c40, ranging between 10Hz and 1 kHz.We implement an effective AC electric field of the form SI. The effective particlediameter \u03c3 is set to 150 nm in real units. We use a 3D simulationbox with Lx = Ly = 3 \u03bcm and Lz = 9 \u03bcm, and the externalfield acts along z direction. Periodic boundary conditionsare applied in all three dimensions. Altogether, we use 19200 colloidalparticles in the system. In any one system, we use equal numbers ofpositive and negative particles, with identical absolute charges,and the particle charges vary from \u00b110e to \u00b150e. The Bjerrum length \u03bbB is set to 10 nmin order to mimic an organic solvent, and the Debye length \u03bbD = 20 nm reflects the screening of smaller ions. The totalsimulation time is about 2.5 s in real units.The charged colloidal particle is representedby a single chargedsphere model. Details of the model in our simulations are providedin 52 The particles are assumed tobe dispersed in a highly viscous solvent, so that their dynamics areoverdamped. The overdamped Langevin dynamics method has been implementedin LAMMPS, as described in the SI. We keepthe solvent viscosity to be approximately 1.17 cP in the systems westudy.All of the simulationsare performed with the software packageLAMMPS."}
+{"text": "However, missing information on factors that stimulate or inhibit these hetero\u2010transglucosylation reactions limits our insight into their biological functions. To explore factors that influence hetero\u2010transglucosylation, we studied Equisetum fluviatile hetero\u2010trans\u2010\u03b2\u2010glucanase (EfHTG), which exhibits both CXE and MXE activity, exceeding its xyloglucan:xyloglucan homo\u2010transglucosylation (XET) activity. Enzyme assays employed radiolabelled and fluorescently labelled oligomeric acceptor substrates, and were conducted in vitro and in cell walls (in situ). With whole denatured Equisetum cell walls as donor substrate, exogenous EfHTG (extracted from Equisetum or produced in Pichia) exhibited all three activities  in competition with each other. Acting on pure cellulose as donor substrate, the CXE action of Pichia\u2010produced EfHTG was up to approximately 300% increased by addition of methanol\u2010boiled Equisetum extracts; there was no similar effect when the same enzyme acted on soluble donors (MLG or xyloglucan). The methanol\u2010stable factor is proposed to be expansin\u2010like, a suggestion supported by observations of pH dependence. Screening numerous low\u2010molecular\u2010weight compounds for hetero\u2010transglucanase inhibition showed that cellobiose was highly effective, inhibiting the abundant endogenous CXE and MXE (but not XET) action in Equisetum internodes. Furthermore, cellobiose retarded Equisetum stem elongation, potentially owing to its effect on hetero\u2010transglucosylation reactions. This work provides insight and tools to further study the role of cellulose hetero\u2010transglucosylation in planta by identifying factors that govern this reaction.Certain transglucanases can covalently graft cellulose and mixed\u2010linkage \u03b2\u2010glucan (MLG) as donor substrates onto xyloglucan as acceptor substrate and thus exhibit cellulose:xyloglucan endotransglucosylase (CXE) and MLG:xyloglucan endotransglucosylase (MXE) activities  Equisetum stem elongation, suggesting a role for HTG in growth.The enzyme HTG can graft segments of cellulose molecules onto xyloglucan (a hemicellulose), thereby re\u2010structuring the cell wall via hetero\u2010transglycosylation. In native walls, two endogenous hemicelluloses competed with cellulose (and with each other) as substrate. HTG more readily selected cellulose as substrate if an expansin\u2010enriched preparation was added. Hetero\u2010transglycosylation was inhibited by cellobiose \u2013 a potential tool for exploring HTG\u2019s biological functions. Interestingly, cellobiose retarded  This can serve as a solid foundation for future \u2018chemical genetics\u2019 studies and will inform the biotechnological use of cellulose hetero\u2010transglucosylation  have recently been documented in native cell walls  impregnated with XyG, MLG or konjac glucomannan (KGM)  plus [3H]XXXGol (XGO acceptor substrate) for transglucanase assays. While the water holding capacity of untreated or alkali\u2010pre\u2010treated filter paper was similar (approximately twice their dry weights), water washing removed 59\u00a0\u00b1\u00a05% of XyG and 46\u00a0\u00b1\u00a05% of MLG from untreated papers but only 39\u00a0\u00b1\u00a09% of XyG and 28\u00a0\u00b1\u00a06% of MLG from alkali\u2010pre\u2010treated filter papers . The removed XyG and MLG most likely represent loosely bound donor substrates.In the cell wall ) Figure\u00a0. The hemEfHTG\u2019s detectable CXE activity  the enzyme\u2019s ability to catalyse XyG\u2010to\u2010[3H]XGO transglycosylation (XET activity) in competition with cellulose\u2010to\u2010[3H]XGO transglycosylation (CXE activity). To distinguish (a) and (b) as factors affecting detectable CXE activity, we can consider the detectable XET reaction rates occurring in the same assays: as expected, negligible \u2018XET\u2019 activity was detected on non\u2010impregnated paper ; compared with this rate, infiltrating a higher concentration of XyG (1% w/v) diminished the detectable XET rate by only approximately 40%. Of factors (a) and (b) above, only (a) is relevant in the case of measured XET activity \u2013 viz. undetectable polymer\u2010to\u2010XyG transglycosylation competing with detectable polymer\u2010to\u2010[3H]XGO transglycosylation. The difference between the effect of 1% XyG impregnation on CXE and that on XET (78% versus 40% inhibition) suggests that the main competitive factor operating in the CXE assays was (b) \u2013 the ability of XyG to compete with cellulose as donor substrate.Infiltrating a 1% (w/v) solution of XyG into filter\u2010paper cellulose (followed by washing away loosely bound XyG) strongly decreased EfXTH\u2010H does not exhibit appreciable CXE activity  transglycosylation in XyG\u2010impregnated paper  would not apply in the case of MLG impregnation because EfHTG cannot use MLG as an acceptor substrate , which is only a poor transglucanase substrate  had little if any effect on measured CXE activity. This may be due to a diminished ability of hemicelluloses to firmly bind to the surfaces of cellulose I as we found for our washing experiments, as previously demonstrated by an approximately 2.5\u00d7\u00a0lower ability of XyG to bind to cotton fibre cellulose than to alkali\u2010washed pea cellulose . For comparison, we also investigated the native form of cotton cellulose . As expected (Simmons EfHTG can act on both XyG and MLG that are tightly attached (resistant to water\u2010washing) to cellulose. At the same time, EfHTG can utilise cellulose as donor substrate even if the cellulose fibres are coated by hemicelluloses.In conclusion, EfHTG transglucanase activities operate at high rates on pure cellulose and on cellulose impregnated with competing hemicellulosic donor substrates, we employed a transglucanase assay that mimics in\u2010planta conditions. The donor substrates were ethanol\u2010denatured cell walls ; Figure\u00a0Equisetum shoots at different developmental stages  and CXE:XET ratios  in naturally relevant concentrations and architecture. Total Equisetum extracts and Pichia\u2010produced EfHTG often gave MXE:XET ratios of roughly 1 rather than, as above, MXE predominating. Indeed, when acting on AIR from the youngest shoots  regardless of the AIR selected. The main conclusion is that both the hetero\u2010transglucanase activities (MXE and CXE) can operate simultaneously on intact Equisetum cell walls.XET:MXE:CXE ratios were remarkably different when transglucanases acted on Equisetum cell wall material being acted on by protein extracts from differently aged Equisetum shoots produced different amounts and ratios of XET, MXE and CXE products. This suggests that the cell wall composition (e.g. polysaccharide ratios) and architecture (e.g. accessibility to enzymes), and/or the presence of co\u2010extracted substances  had negligible effect on the XET and MXE activities of Pichia\u2010produced EfHTG  at pH 4  yet still considerable at pH 5.5 and pH 6.8  stimulation of CXE activity by extracts occurred at pH 6.8 ) than those from blackish stem tissue ). This and the observation that stimulation is strongest on cellulose I and at low pH agrees with recent results showing that bacterial expansin strongly augments cellulose hetero\u2010transglucosylation  without affecting XET activity. Such an inhibitor would allow us to block EfHTG\u2010catalysed hetero\u2010transglucosylation and thus study its potential functions in planta. A set of sugar mimics from the EDI collection  inhibited MXE and CXE activity and inhibited XET activity of EfHTG by 70\u201390% . Cellobiose at 80\u00a0mm almost completely inhibited all three activities  activity and action in vitro and in situ, while having little effect on XET activity or action  relative to the control  and 0.31\u00a0\u00b1\u00a00.12\u00a0cm/day (no cellobiose); Figure\u00a0m) did not affect elongation  can graft both cellulose and mixed\u2010linkage glucan onto XyG oligosaccharides, resulting in the formation of very stable hetero\u2010polymers. We show here that the rate of hetero\u2010transglucosylation is strongly influenced by many factors, including the presence of stimulating or inhibiting substances, pH, temperature and the nature of the donor substrate .Hetero\u2010trans\u2010\u03b2\u2010glucanase , y Figure\u00a0b. This set al., et al., et al., et al., Equisetum extracts to CXE assays produced a similar effect on the CXE activity of Pichia\u2010produced EfHTG, and the highest stimulation occurred at the lowest pH tested. Since plant expansins have their pH optimum in the acidic range  rather than with the transglucanase itself.Expansin augments the activity of cellulose\u2010active enzymes, an effect which has been intensively studied owing to its potential in enhancing cellulose utilisation during saccharification and other industrial processes . The latter additive would allow recovery of most of the enzyme after use, helping to establish highly efficient production cycles.Cellulose hetero\u2010transglucosylation has great biotechnological potential, allowing covalent incorporation of a commercially valuable \u2018cargo\u2019 (attached to an XGO) into cellulosic materials by non\u2010polluting procedures  are efficient inhibitors of Equisetum HTG, while glucose has no inhibitory effects. MXE and CXE activity \u2013 using unbranched donor substrates  \u2013 were affected strongest, while the XET activity of HTG required higher oligosaccharide concentrations for inhibition. However, a comparison of the XET activities of HTG and a standard Equisetum homo\u2010transglucanase (EfXTH\u2010H), which exhibits negligible MXE and CXE activity, showed that the XET activity of HTG is more susceptible to inhibition than that of XTH\u2010H. These different inhibitory effects of cellobiose might be explained by 3D modelling, which showed that (i) XyG exhibits three more interactions with HTG\u2019s active site than does cellulose or MLG, but (ii) other standard XTHs still exhibit two more interactions with XyG than does EfHTG \u2010hydrolase, which breaks down supplied cellobiose and thus increases the availability of glucose as a carbon source . In contrast to, for example, cellotriose , we propose that plant expansins are involved. This provides a solid foundation for further studies, exploring potential roles of synergy between transglucanases and expansins for cell wall formation and remodelling, which govern plant cell expansion and organ strengthening. Furthermore, efforts to introduce cellulose hetero\u2010transglucanases genetically into other plants for, for example, tissue strengthening could benefit from a simultaneous upregulation of expansin action. Our inhibitor studies show that (hetero\u2010)transglucanase activities/actions can be inhibited, opening the potential for \u2018chemical genetics\u2019 studies. This would be particularly valuable for inhibiting functions of transglucanases, which are difficult to knock out or knock down, for example in non\u2010model organisms such as Equisetum or if excessive genetic redundancy precludes deactivation of all relevant genes. Finally, we suggest that future studies aiming at drawing conclusions from in\u2010vitro experiments \u2013 with commercial donor substrates and/or heterologously produced enzyme \u2013 on the actual biological roles of transglucanases should consider that numerous factors exist that heavily influence the rate and ratios of transglucosylation activities.In conclusion, this study showed that Equisetum fluviatile was grown in a pond at the Institute of Molecular Plant Sciences of the University of Edinburgh  or collected from the Pentland Hills (Edinburgh). Tamarindus indica seed XyG was from Dainippon Pharmaceutical Co. ; barley (Hordeum vulgare) MLG  and lichenase (from Bacillus subtilis) were from Megazyme Inc. . XXXGol\u2013sulphorhodamine (XXXGol\u2010SR) was prepared as previously described  (the nomenclature of XGOs (e.g. XXXGol) follows Fry et al., (3H]XyG) was prepared according to Herburger et al. . Radiolr et al. . Other cEfHTG and EfXTH\u2010H in Pichia pastoris strain SMD1168H was done as described before , 3% (w/v) polyvinylpolypyrrolidone; 5\u00a0ml\u00a0g\u22121 FW) at 0\u00b0C, and the supernatant was either stored at \u221280\u00b0C until processed or immediately used for assaying XET, MXE and CXE activity.Production of y et al. . In brie3H\u2010labelled compounds were quantified by scintillation counting in ScintiSafe 3 scintillation cocktail , 3H bound to cellulosic substrates was assayed by scintillation counting in GoldStar \u2018O\u2019 scintillation cocktail .Soluble m of the potential inhibitor in a reaction mixture  containing 5\u00a0\u00b5l of filtrate from Pichia cultures expressing EfHTG or EfXTH\u2010H or Equisetum protein extracts, 0.1\u00a0m succinate , 0.1% (w/v) BSA, 0.4\u20131.0\u00a0kBq acceptor substrate ([3H]XXXGol or [3H]XyG) and 0.5% (w/v) donor substrate . For CXE activity, 20\u00a0mg of cellulosic substrate  or pre\u2010treated with 6\u00a0m NaOH (thus cellulose II)) was soaked with 20\u00a0\u00b5l reaction mixture lacking a soluble donor substrate. Controls contained heat\u2010inactivated enzymes and the values obtained from these samples were subtracted from non\u2010mock groups, thus correcting values for unspecific [3H]XXXGol or [3H]XyG binding. Mixtures were incubated for 24\u00a0h at 20\u00b0C or at 0\u201360\u00b0C in a temperature chamber. XET and MXE products were dried on Whatman No. 3 paper, washed in running tap water overnight, re\u2010dried and quantified by scintillation counting. After the reaction had been stopped with 6\u00a0\u00b5l of 90% formic acid, cellulosic substrates were washed sequentially in 6\u00a0m NaOH for 12\u00a0h at 20\u00b0C, 6\u00a0m NaOH for 0.5\u00a0h at 100\u00b0C and running tap water overnight, and assayed for bound 3H. Control groups contained heat\u2010inactivated enzyme preparations and the signal obtained was subtracted as \u2018background 3H\u2019 from experimental groups if not otherwise stated.Practical methodology for transglycanase assays is presented by Frankov\u00e1 and Fry . To testet al., m NaOH (CXE on cellulose II). Dry papers were then impregnated with approximately 5\u00a0\u00b5m XXXGol\u2010SR, dried again, loaded with 5\u00a0\u00b5l of reaction mixture  and incubated in darkness at 20\u00b0C between acetate stationery sheets to maintain humidity for approximately 18\u00a0h. Papers were then washed in ethanol/formic acid/water  for 1.5\u00a0h, rinsed twice with water and dried. Fluorescence emitted by bound XGO\u2010SR was visualised under 254\u00a0nm UV.In complementary experiments, a fluorescent dot\u2010blot assay testing for XET, MXE and CXE activities was used  or citrate (pH 2.1\u20137.6) buffers (counter\u2010ion Na+).The pH dependency of transglucanase activities was measured in 0.1\u00a03H]XXXGol , 0.9% MLG in 0.225\u00a0m citrate  and 0.45% chlorobutanol) for between 0.2 and 31\u00a0days at 6, 23 or 37\u00b0C. Reactions were terminated with formic acid and [3H]polysaccharide products were assayed by paper binding as above.To test the longevity of HTG activity, we incubated MXE assay reaction mixtures  BSA) was pipetted onto 20\u00a0mg cellulose , incubated for 16\u00a0h and dried at 20\u00b0C, and then the cellulose was washed in 0.05\u00a0m succinate buffer  under gentle shaking at 20\u00b0C for 6\u00a0h. Washing solutions were collected, freeze\u2010dried and re\u2010dissolved in water. Control samples received the same treatment except that they were not applied to cellulose. The protein recovered from cellulose by washing  and controls that had not been in contact with cellulose were then assayed for MXE activity in a reaction mixture  containing 10\u00a0\u00b5l of cellulose\u2010recovered or control EfHTG, 1\u00a0kBq [3H]XXXGol, 1% (w/v) BSA and 0.5% (w/v) MLG.Recovery of et al., Equisetum stems (approximately 250\u00a0mg) were sectioned with a razor blade and incubated with 50\u00a0kBq [3H]XXXGol (in 750\u00a0\u00b5l 25\u00a0mm succinate (Na+) + 0.1% (w/v) chlorobutanol with or without 10\u00a0mm cellobiose) for 24\u00a0h, and after the reaction had been stopped with 0.5% formic acid in 96% ethanol, specimens were washed thoroughly with ethanol (90\u201330%). Hemicelluloses were extracted with 6\u00a0m NaOH (4\u00a0\u00d7\u00a024\u00a0h at 37\u00b0C) and digested with lichenase (releasing MXE\u2010diagnostic Glc2\u2022[3H]XXXGol) and then with XyG endoglucanase (releasing XET\u2010diagnostic [3H]XXXGol). 3H in both the MXE\u2010 and XET\u2010diagnostic products was quantified by scintillation counting. The cellulosic pellet obtained after hemicellulose extraction was boiled in 6\u00a0m NaOH (1\u00a0h), re\u2010digested with lichenase  and then treated with a series of cellulose digesting enzymes  and any additional released 3H was quantified. CXE action was recorded as the total radioactivity released from the cellulosic pellet by cellulose\u2010digesting enzymes and TFA treatment.An assay for quantifying XET, MXE and CXE action in native plant cell walls was described in detail previously  connected to a segment of horizontal rhizome, were selected. The rhizome was cut under water giving a length of approximately 20\u00a0cm and the explants were transferred into 250\u2010ml beakers. Water from the pond in which the plants had been growing was filtered through a nylon mesh. Five randomly selected explants were grown indoors  in 200\u00a0ml filtered pond water  supplemented with 10\u00a0mm cellobiose. Five control plants were not exposed to cellobiose. The water was changed every 2\u00bd days to avoid algal and fungal growth. After 5\u00a0days, the water was changed to sugar\u2010free pond water and elongation was recorded for another 10\u00a0days. After 15\u00a0days, cellobiose (10\u00a0mm) was added again and shoot growth was recorded for a further 15\u00a0days. In an additional experiment, five plants were exposed to 10\u00a0mm glucose instead of cellobiose and their growth was compared with control plants lacking any treatment over 10\u00a0days. Shoots and rhizomes were sectioned, and transglucanase action was visualised by incubation of approximately 200\u2010\u00b5m sections in 150\u00a0\u00b5l 25\u00a0mm succinate  containing approximately 5\u00a0\u00b5m XXXGol\u2010SR and 10 or 0\u00a0mm (control) cellobiose for 2\u20134\u00a0h. Sections where then washed in ethanol/formic acid/water  for 10\u00a0min and in aqueous 5% (v/v) formic acid overnight to remove non\u2010incorporated XXXGol\u2010SR. After rinsing in water, sections were examined with a Leica DM2000 LED microscope equipped with a Leica DFC7000 T camera and a Leica EL6000 external light source. Incorporated SR was visualised with a GFP filter cube (excitation band pass (BP) 470/40\u00a0nm , emission BP 525/50\u00a0nm). Images were taken with LAS X software and assembled in Adobe Photoshop CC. Minimal contrast adjustments were applied equally across entire image plates. Fluorescence intensity on images (n\u00a0=\u00a04) was quantified with ImageJ. To test solute transport from explants\u2019 rhizome segments into their vertical shoots, we added 0.05% safranin O to pond water  in a separate experiment, and blotted the stem cross\u2010sectional area onto filter paper. Safranin O in stem cross sections (approximately 200\u00a0\u00b5m thickness) was visualised by brightfield and fluorescence microscopy. Controls lacked safranin O.Fifteen m NaOH, thoroughly washed in water and dried) were dipped into 0\u20131% (w/v) XyG, MLG or other plant polysaccharides, dried and washed in water for 4\u00a0h, removing loosely bound polysaccharides. The amount of hemicelluloses removed by washing in water (4\u00a0h) was assessed from paper weights, which were dipped in 0.5% (w/v) XyG or MLG, before and after washing and drying. Reaction mixture  BSA, total volume 20\u00a0\u00b5l) was added to the dried, impregnated, approximately 20\u2010mg pieces of paper, and after 24\u00a0h incubation, the papers were washed in running tap water overnight and re\u2010dried, and the bound 3H was quantified by scintillation counting. Papers were recovered, the scintillant was removed with acetone and the papers were washed for 24\u00a0h in 6\u00a0m NaOH, which removes bound hemicelluloses; after washing in water and re\u2010drying, papers were assayed for firmly bound 3H.Pieces (20\u00a0mg) of Whatman No. 1 filter paper  were tested for XET, MXE and CXE activity on their respective pure donor substrates as described above. The Equisetum tissue remaining after protein extraction was washed in 75% (v/v) ethanol (thus denaturing any remaining enzymes) until the supernatant was colourless, yielding AIR. The AIR (30\u00a0mg) was soaked with 30\u00a0\u00b5l reaction mixture  BSA) and incubated at 20\u00b0C for 24\u00a0h; then, after the reaction had been stopped with formic acid, hemicelluloses were extracted with 2\u00a0ml 6\u00a0m NaOH (4\u00a0\u00d7\u00a01\u00a0day at 37\u00b0C under constant shaking). NaOH extracts were slightly acidified with acetic acid, dialysed against tap and distilled water (3\u00a0\u00d7\u00a01\u00a0day), freeze\u2010dried and digested with 250\u00a0\u00b5l of lichenase , for 6\u00a0h at 20\u00b0C). Digestion products were dried, dissolved in aqueous 72% ethanol and centrifuged (2500\u00a0rpm for 10\u00a0min). The lichenase\u2010resistant pellet  and supernatant (containing Glc2\u2022[3H]XXXGol) were assayed for 3H. The remaining NaOH\u2010insoluble cellulosic material containing CXE products was digested by Saeman hydrolysis in H2SO4 , which were assayed as described above, we mixed Pichia\u2010produced EfHTG with Equisetum extracts prepared from young emerging shoots, green or black internodes or roots. These extracts had either been boiled for 5\u00a0sec, inactivating extracted enzymes, or not boiled. A reaction mixture (20\u00a0\u00b5l) contained: 5\u00a0\u00b5l enzyme solution ), 0.1\u00a0m succinate , 0.05% (w/v) BSA, 1\u00a0kBq [3H]XXXGol and donor substrate (0.5% (w/v) XyG or MLG or 20\u00a0mg of cellulosic substrate (untreated or pre\u2010treated with 6\u00a0m NaOH at 20\u00b0C)). In an additional experiment, freeze\u2010dried Equisetum extracts were boiled (approximately 65\u00b0C) in 99.8% methanol (5\u00a0min), dialysed against water , dried and added (at 0.1\u20131% (w/v)) to assays testing for CXE activity of Pichia\u2010produced EfHTG on plain or 6\u00a0m NaOH\u2010pre\u2010treated Whatman No. 1 paper.To test the effect of t\u2010test or one\u2010way analysis of variance (anova) followed by Tukey\u2019s post hoc test (Origin 8.5).Experiments were usually carried out with three to six independent replicates. Data are represented as the mean and standard deviation. Statistically significant differences between groups were determined by standard A patent application (WO2015044209) has been filed by BASF Agricultural Solutions Belgium NV and University of Edinburgh for the use of hetero\u2010transglycosylase. LF, FM, AH and SCF are inventors.3H]xyloglucan, AX performed some of the inhibitor screenings, KH, SCF and LF analysed the data, KH prepared the figures and drafted the manuscript and SCF and LF edited the manuscript. All authors approved the manuscript.SCF, KH, LF, AH and FM planned and designed the study, KH performed most of the experiments, MP synthesised and assayed the [Figure S1. Extractable transglucanase activities from different Equisetum parts.Figure S2. Effect of BSA on EfHTG activities .Figure S3. Statistical evaluation of stimulatory effect of non\u2010enzymatic Equisetum polymers.Figure S4. Safranin O uptake by hydroponically grown Equisetum fluviatile shoots.Click here for additional data file.Table S1. pH and temperature optima of the three transglucanase activities of HTG.Click here for additional data file."}
+{"text": "This c\u22a5 orientation, unprecedented for PLLA films, can be achieved by the crystallization of amorphous films as induced by low-temperature sorption of molecules being suitable as guests of PLLA co-crystalline forms, such as N,N-dimethylformamide, cyclopentanone or 1,3-dioxolane. This kind of orientation is shown and quantified by two-dimensional wide-angle X-ray diffraction (2D-WAXD) patterns, as taken with the X-ray beam parallel to the film plane (EDGE patterns), which present all the hk0 arcs centered on the meridian. PLLA \u03b1-form films, as obtained by low-temperature guest-induced crystallization, also exhibit high transparency, being not far from those of the starting amorphous films.Poly(\u029f-lactide) (PLLA) films, even of high thickness, exhibiting co-crystalline and crystalline \u03b1 phases with their chain axes preferentially perpendicular to the film plane (c Co-crystallization of polymers with suitable guest molecules can lead, without any stretching procedure, to films (even of high thickness) with high degrees of crystal phase orientation. In fact, planar and uniplanar orientations, i.e., the preferential orientations of a crystal axis or a crystal plane with respect to the film plane, can be easily achieved ,9,10,11.These planar and uniplanar orientations of co-crystalline and nanoporous-crystalline films can lead to the control of relevant properties  in amorphous films can lead to co-crystalline phases with a high degree of orientation. In particular, a uniplanar orientation with ac-planes of crystallites being preferentially parallel to the film plane  has been recently observed [Furthermore, for PLLA, a relevant commercial biodegradable polymer, crystallization as induced by sorption of several guest molecules  [\u22a5 oriented films also lead to high transparency, being not far from those of starting amorphous films.In the present paper, we show that suitable procedures for guest-induced crystallization on amorphous PLLA films can lead to an unprecedented co-crystalline and crystalline phase orientation with chain axes being preferentially perpendicular to the film plane (c-perpendicular orientation or more shortly cntation) ,3,4,5. Ww ~152,000 and Mn ~99,000), N,N-dimethylformamide (DMF), cyclopentanone, 1,3-dioxolane, chloroform, hexane, methanol, and CaCl2 were supplied by Aldrich, Milan, Italy, and used as received.PLLA polymer  of amorphous PLLA films.Two-dimensional wide angle X-ray diffraction (2D-WAXD) patterns were collected by a D8 QUEST Bruker diffractometer, Karlsruhe, Germany . EDGE or THROUGH patterns were collected by sending the X-ray beam parallel or perpendicular to the film surface, respectively.cf, was calculated by using Herman\u2019s orientation function [2 \u03b3 was calculated by the azimuthal distribution of the main reflection , for the EDGE patterns. Based on these assumptions, when cf is equal to 0, a random crystallite orientation occurs, while when cf is equal to \u20130.5 the c axes of all crystallites are perfectly perpendicular to the film plane.The degree of orientation of the crystalline phase, function ,18:(1)fcOptical transmittance of PLLA films, with thicknesses of 25 \u03bcm and 100 \u03bcm, was measured by using a Shimadzu, Duisburg, Germany UV\u2013Vis spectrophotometer (UV-2600). Films, after guest removal, were placed between two quartz plates, and the transmittance was measured as a function of wavelength in the 200\u2013800 nm range.2, at room temperature. The degree of crystallinity of the films (\u03c7d) was evaluated by using the density of \u03b1 crystalline phase [Density of PLLA films was measured, after guest removal, by the flotation method, in aqueous solutions of CaClne phase ,20,21. EDifferential scanning calorimetry (DSC) TA Q2000 equipment, New Castle, DE, USA, was used to measure the melting behavior of the PLLA films. All the measurements were made under the control heating and cooling rates (at a rate of 10 \u00b0C/min) see .Scanning electron microscopy  was used to study the PLLA films\u2019 surface morphology. Before imaging, all films were coated with thin gold layer  at 30 mA for 5 min.N,N-dimethylformamide (DMF), [It is well known that PLLA is able to form host\u2013guest co-crystalline forms with suitable guest molecules like cyclopentanone, tetrahydrofuran, 1,3-dioxolane, \u03b3-butyrolactone and e (DMF), ,23,24,25e (DMF), ,22.For an amorphous PLLA film crystallized by DMF sorption at \u221218 \u00b0C, 2D-WAXD patterns as taken with the X-ray beam perpendicular (THROUGH pattern) and parallel (EDGE pattern) to the film plane, are shown in CuK\u03b1 = 16.7\u00b0, corresponding to Miller indexes 110/200 [As for the THROUGH pattern, a diffraction radial profile is shown in the upper part of  110/200 ,21) indi\u22a5 orientation). This kind of orientation has been described for co-crystalline phases of s-PS [\u22a5 orientation of co-crystalline and \u03b1 form phases, as evaluated from the azimuthal scans of their 020 and 110/200 reflections, is not far from cf = \u22120.3 for both phases.As for the EDGE pattern, a scheme of the diffraction arc is shown in the lower part of  of s-PS ,3 as wel of s-PS  while it\u22a5 orientation of their co-crystalline phases, although with a lower degree of orientation (cf \u2248 \u2212 0.1).It is worth adding that guest-induced crystallization of amorphous PLLA films, by sorption at \u221218 \u00b0C of cyclopentanone or 1,3-dioxolane also leads to c\u22a5 orientation. The degree of the c\u22a5 orientations as evaluated by EDGE patterns of \u22a5 orientation.The 2D-WAXD patterns of the film including a PLLA/DMF co-crystalline phase a,a\u2032, aft\u22a5 orientation of d = 35%  and is fully unoriented .The unoriented \u03b1 form films as obtained by cold crystallization c is partThis transparency information was quantified by UV\u2013Visible spectra in the range 200\u2013800 nm, for two sets of PLLA films with a thickness close to 100 \u00b5m and 25 \u00b5m, which are shown in The great differences in transparency of the PLLA \u03b1-form films as crystallized by DMF sorption at \u221218 \u00b0C and room temperature  can be easily rationalized by their SEM images a obtaineFilm transparency, of course, is an important factor for many PLLA applications, mostly for packaging and coatings ,41,42.\u22a5 orientation), are reported for the first time.PLLA macroscopic films, with the orientation of the chain axes of their co-crystalline and crystalline phases being preferentially perpendicular to the film plane .These c\u22a5 orientation both for co-crystalline and derived \u03b1 form PLLA films is not far from fc = \u22120.3 .The kind and degree of orientation was established by 2D-WAXD patterns, as taken with X-ray beam parallel to the film plane (EDGE patterns). For instance, for an amorphous PLLA film crystallized by DMF sorption at \u221218 \u00b0C, the degree of c\u22a5 orientation exhibit high transparency, being not far from those of the original amorphous films. The observed behavior is rationalized by formation at low crystallization temperatures of very small polymer crystallites. High transparency of PLLA films is an important factor for packaging and coatings applications.This paper also shows that \u03b1 form PLLA films with c"}
+{"text": "Whenever I think about Michael\u2019s passing, a sad feeling still strikes me. Two months before his eighty-ninth birthday, Michael left us and his beloved scientific research. His legendary achievements have been reviewed in several memorial articles ,2,3. ThuBefore I met Michael, I read his seminal publication on the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) structure. I used that structure as a template to model Rice GAPDH, which I manually sequenced in China. Therefore, I felt so lucky when I joined Michael\u2019s group in 1998. I still remember the very first thing Michael taught me: \u201cdo not call me Professor Rossmann; call me Michael.\u201d He described the historical association with the title \u201cProfessor\u201d in the UK, stating that he would rather earn respect from his knowledge not his title. In the introductory lecture to my large undergraduate biochemistry classes, I always relay this story and tell my students that they can call me by my first name, River. This is just one example of how Michael influenced the culture at Purdue, where it is common for students to address professors by their first names, which is not the normal practice at many other places.Once, on my way to a regional tourist site after a seminar talk at El Paso, I was stopped by the Border Patrol Police. Since I was traveling inside the US, I did not carry my passport. I was almost deported back to China. After hearing this story, Michael was livid and insisted on writing to his US Senator. In his letter, he described his childhood in Nazi Germany where all Jewish people were mandated to wear the Star of David. He believed that the US was becoming a police state by requiring immigrants to carry passports at all times. Obviously, Michael had a very difficult childhood in Nazi Germany . For exaEach of us have our own memory of Michael. Thus, before writing this article, I asked these questions: how can I best describe Michael\u2019s personality? And which characteristics were his most important ones? My answers: Michael had a childlike heart that shined through his personality, even during his last days. It was so pure and innocent, full of curiosity and energy. It was manifested in his exceptional wisdom and sometimes intimidating seriousness when discussing science.Michael loved skiing, a sport he learned from his postdoctoral mentor Max Perutz . I was lThis childlike eagerness and impatience to adventure is reflected in his beloved science and research. We all have stories of Michael asking for experiments and calculations to carry out, not tomorrow but today, and maybe even yesterday! Perhaps this explains why Michael pursued protein crystallography when the field could only solve organic compounds, or his courage to pioneer virus structure determination when most considered it a crazy endeavor. The enthusiasm behind Michael\u2019s young heart never diminished, which has influenced many of us to dive into \u201cdangerous\u201d slopes with him .Another of Michael\u2019s long-time hobbies was sailing, a sport about which I knew nothing until I became his crew for almost ten years. When sailing, his childlike desire to win was always apparent, especially at the finish line. However, unlike a child, he understood that winning required planning and hard work. We spent hours cleaning his sailboat, checking the lines and the knots. Ironically, in our eagerness to win, our boat had capsized. I felt I was drowning and climbed onto a rescue boat. When I looked back, Michael was still in water, dragging the boat to the shore. When Michael set his mind on something, he did not give up, and strived to insure everything was perfect, both in sailing and in science. I remember generating a figure for my first publication, Michael pulled out a ruler and found that there was a small error, something was off by less than 1 mm. He said, \u201credo the figure.\u201d Computer software was not as sophisticated as it is today. Thus, remaking the figure was a time consuming and laborious endeavor. Just as a sailing race: as we approached the finish line, Michael would not tolerate any mistakes, no matter how tiny, to the line and knot settings. His strong desire to win, requisite and persistent hard work, and perfectionism also showed in his research, which led to many breakthroughs.After Michael\u2019s passing, I began to collect pictures from mentees, colleagues, and friends. Michael always has a very attractive and genuine smile . HoweverMichael did not want to leave us or his beloved science. In his last days, he was still planning \u201cpost-recovery\u201d research. Perhaps, he is still doing research in another dimension. When I teach about the Rossmann fold, I hear him saying, \u201ccall it the nucleotide binding motif. The name should inform its function not who solved the structure.\u201d In closing, I would like to quote one of my students, \u201cIt is our responsibility and honor to carry on Michael\u2019s legacy and keep him alive in our memory.\u201d I think this special issue well serves this purpose."}
+{"text": "H. polygyrus much earlier in life than previously anticipated. This includes the more extensive induction of IFN-\u03b3 competent, nematode-specific Th2/1 hybrid cells in BALB/c mice older than three months compared to younger animals. In C57BL/6 mice, Th1 cells accumulate more rapidly at steady state, translating to elevated Th2/1 differentiation and poor control of parasite fitness in primary infections experienced at a young age. Blocking of early IFN-\u03b3 and IL-12 signals during the first week of nematode infection leads to sharply decreased Th2/1 differentiation and promotes resistance in both mouse lines. Together, these data suggest that IFN-\u03b3 competent, type 1 like effector cells spontaneously accumulating in the vertebrate host progressively curtail the effectiveness of anti-nematode type 2 responses with rising host age.The efficient induction of type 2 immune responses is central to the control of helminth infections. Previous studies demonstrated that strong Th1 responses driven by intracellular pathogens as well as a bias for type 1 activity in senescent mice impedes the generation of Th2 responses and the control of intestinal nematode infections. Here, we show that the spontaneous differentiation of Th1 cells and their expansion with age restrains type 2 immunity to infection with the small intestinal nematode  However, type 2 responses develop slowly and inefficient protection against re-infection often results in lifelong chronic infections with GI nematodes in human populations, livestock and wild animals4. The small intestinal nematode Heligmosomoides polygyrus is a natural murine parasite that establishes chronic primary infections in several mouse lines and thus provides a suitable model for investigating long-lasting infections with GI nematodes in humans5. The infection results in the differentiation of Th2 cells releasing interleukin-4 and IL-13 along with strong B cells responses primarily characterized by IgG1 production. IL-4/-13 promote mucus production and the release of effector molecules by goblet cells as well as the alternative activation of macrophages. In concert, these responses eventually permit the expulsion of adult worms and provide protection against challenge infections5. Parasite clearance is sharply impaired in mice lacking CD4+ T cells or IL-4R\u03b17. Furthermore, inbred mouse lines such as C57BL/6 and BALB/c differ in resistance, i.e., the time required to control the infection and the quantity of eggs released by adult worms7. This was shown to correlate with the extent of both innate and adaptive type 2 responses, and likely also involves discrete differences in the activity of regulatory T cells8.Experimental work in rodent models and epidemiological surveys of human populations clearly showed the importance of Th2 responses for the control of gastrointestinal (GI) nematode infections10. Similarly, our group showed that Th2 differentiation in response to H. polygyrus infection is prevented in mice harboring high frequencies of IFN-\u03b3 competent Th1 and CD8+ T cells as a result of preceding exposure to Toxoplasma gondii infection11. Furthermore, we showed that, irrespective of the pre-exposure to other infections, a high proportion of na\u00efve T cells activated in primary H. polygyrus infection differentiates into GATA-3+ T-bet+ Th2/1 hybrid cells12 and that IFN-\u03b3 signals integrated along with IL-4R\u03b1 signaling are critical for the commitment to the Th2/1 hybrid phenotype13. Th2/1 hybrid cells stably express T-bet along with intermediate GATA-3 expression and produce IFN-\u03b3 together with modest amounts of Th2 cytokines13. In line with the modest contribution of Th2/1 cells to IL-4 and IL-13 production and their identification as the main source of parasite-specific IFN-\u03b3 production, expanded Th2/1 hybrid cells lead to a further delay in the control of infection in highly susceptible C57BL/6 mice12.In accordance with the concept defining Th2 and Th1 generation as the result of opposing differentiation programs, high doses of type 1 cytokines applied during infections with intestinal nematodes were shown to block Th2 development and to severely impair the control of primary as well as challenge infections with GI nematodes15, the current study addressed if the phenotypical composition of GATA-3+ T effector cells generated in nematode-infected mice differed depending on host age. Investigating BALB/c mice displaying high genetic resistance to H. polygyrus infection, we show that IFN-\u03b3 competence of small intestinal CD4+ T cells increased with age and correlated positively with parasite fitness. Resistance was further increased after blocking of type 1 cytokines early during infection, whereas IFN-\u03b3 supplementation during priming of the CD4+ T cell response selectively promoted the outgrowth of Th2/1 hybrid cells, resulting in the impaired control of nematode fitness. The IFN-\u03b3-Th2/1 hybrid-susceptibility axis was also evident in comparing partially resistant BALB/c to fully susceptible C57BL/6 mice, the latter exhibiting stronger accumulation of Th1 at steady state, more robust Th2/1 differentiation upon infection and a significant rise in resistance upon blocking of early type 1 cytokine signals.Based on the importance of IFN-\u03b3 signaling for Th2/1 hybrid cell commitment and because IFN-\u03b3 competent memory-phenotype CD4+ and CD8+ T cells expand independent of pathogen exposure in an age-dependent mannerH. polygyrus13. Here, we asked whether the rise in IFN-\u03b3 competence resulting from the spontaneous generation and expansion of memory-phenotype (MP) Th1 cells and CD8+ T cells after birth16 might predispose the host for more extensive Th2/1 hybrid formation upon first encounter with an enteric nematode infection. BALB/c mice displaying high genetically controlled resistance to H. polygyrus infection were used to estimate the IFN-\u03b3 competence of lymphocytes isolated at steady state covering an age range of 1.5 up to 18 months according to PMA/ionomycin induced IFN-\u03b3 production  with rIFN-\u03b3 twice daily (2.5 \u03bcg/dose) until day 4 post infection Fig.\u00a0. IFN-\u03b3 s.5 monthsH. polygyrus at the age of 1.5, 3 or 9 months . Parasite-specific CD4+ T cells responding to TCR activation by the upregulation of CD40-ligand (CD40-L) were surveyed for the expression of GATA-3/IL-13 and T-bet/IFN-\u03b3. The vast majority of parasite-specific CD40-L+ cells expressed GATA-3 in all age groups  harbored significantly higher percentages of T-bet+ Th1 cells in spleen, mLN and siLP compared to na\u00efve BALB/c mice with a similar age range (mean age: 2+/\u2212 0.6 months)  Fig.\u00a0. As expehs) Fig.\u00a0. This wahs) Fig.\u00a0. Poor rehs) Fig.\u00a0. At day hs) Fig.\u00a0. The earhs) Fig.\u00a0. SJL michs) Fig.\u00a0, poor smhs) Fig.\u00a0 and strohs) Fig.\u00a0.Fig. 6HiNext, we compared classical Th2 and Th2/1 hybrid responses in both mouse lines infected at the age of three or six months and determined the ratio of Th2 to Th2/1 cells in lymphatic organs and small intestines at day 14 post infection Fig.\u00a0. In bothH. polygyrus infection was sufficient for converting the immunological phenotype of C57BL/6 mice to the more effective response seen in BALB/c mice. To that end, C57BL/6 mice were infected at the age of three months, treated with blocking antibodies against IFN-\u03b3 and IL-12 as described above and dissected at day 14 post infection. As seen in BALB/c mice  Th1 cells expand after birth, reaching a plateau at the age of six months. MP Th1 induction is not contingent to the presence of foreign antigens, but depends on IL-12 constitutively produced by CD8\u03b1+ dendritic cells21. MP Th1 were reported to rapidly produce IFN-\u03b3 in an innate-like manner in the context of Toxoplasma gondii infection, and thereby support the development of adaptive Th1 responses16. Hence, while MP Th1 cells were shown to be beneficial in the context of Toxoplasma infection, our data suggest that the expansion of MP Th1 cells along age exerts detrimental effects on the development of anti-nematode Th2 responses.Focusing on BALB/c mice displaying a genetic bias for the induction of robust Th2 responses to nematode as well as 25. IL-12 and IL-18 synergize in driving IFN-\u03b3 production by Th1 and NK cells26 and the inflammasome dependent release of IL-18 was previously shown to promote IFN-\u03b3 production by CD4+ T cells in whipworm infected mice24. Furthermore, several recent studies reported a type-1 signature, including the production of IFN-\u03b3, in small intestinal tissue surrounding the larval stage of H. polygyrus29. It hence seems likely that higher numbers of Th1 cells present in mature mice drive the more vigorous Th2/1 hybrid responses in nematode-infected mature compared to younger individuals, resulting in the less efficient control of parasite reproduction. Compliant with the differential resistance of the two mouse lines investigated here, we further show that the accumulation of Th1 MP-like cells is delayed in BALB/c compared to C57BL/6 mice. This may relate to the poor maintenance of the IL-12R\u03b22 chain and the more limited IL-12 production by antigen presenting cells reported for the BALB/c compared to other mouse lines31.Importantly, several studies reported the expression of type 1 factors that may support IFN-\u03b3 release by MP Th1 as well as CD8+ and NK cells in the context of helminth infections. This comprises the constitutive expression of IL-12 by CD103+ migratory dendritic cells and the release of IL-1\u03b2 and IL-18 by macrophages responding to nematode products or helminth driven tissue damage34. Similarly, high doses of IFN-\u03b3 or IL-12 applied early during nematode infection result in poor Th2 differentiation and delayed worm expulsion35. Furthermore, the IL-4 driven inability of BALB/c mice to control Leishmania infections was shown to be confined to young animals, whereas old mice controlled the infection, partially depending on elevated IL-12 production by macrophages in aged mice36. Similarly, defective Th2 differentiation and strongly Th1-biased responses were reported for nematode-infected senescent mice (age\u2009\u2265\u20091.5 years)40. Our study expands on these findings, suggesting that the age-dependent IFN-\u03b3 availability impacts the phenotype of the developing T cell response in nematode-infected mice and thereby impedes the type-2 dependent control of nematode fitness.Previous studies showed that type 2 responses are impaired in mice simultaneously infected with helminths and Th1-inducing pathogensWe further show that the divergent patterns seen in young adult BALB/c compared to C57BL/6 mice upon nematode infection tend to level out with progressing age of the BALB/c line, associated with the rise in Th1 cells at steady state. Of note, our demonstration of age- and IFN-\u03b3 dependent shifts of the immune response and resistance to a GI nematode infection is based on mice exposed to a single high dose infection under highly controlled environmental conditions. This does not necessarily imply that age-related differences have a similarly clear effect on the anti-helminth responses in natural systems. Our data may rather suggest that differential IFN-\u03b3 availability related to host age and genotype may take part in the modification of anti-helminth immune responses in e.g., wildlife and human populations. However, given that GI nematodes are typically acquired at a young age, IFN-\u03b3 availability depending on the coinfection status as well as the structure of the host microbiome may be more important and result in similar consequences for anti-helminth immunity in natural systems, which is as addressed further below.th stage larvae from small intestinal tissue17. Under the given culture conditions, larvae migrate towards the abluminal side17, whereas in situ migration back to the small intestinal lumen is accomplished around day eight post infection5. Although not reflecting the natural behavior, our finding of accelerated egress of the 4th larval stage from host tissue infiltrated by more Th2/1 cells indicates that larval fitness is affected by the early local T cell responses, which translates to the differential reproductive fitness of the adult stage shortly later.Worm expulsion was largely intact in mature BALB/c mice exhibiting prominent mucosal Th2/1 hybrid accumulation and strong parasite-specific IFN-\u03b3 production. Nevertheless, egg release by adult worms proved a reliable correlate with the extent of intestinal Th2/1 hybrid accumulation depending on host age Figs.\u00a0, genotyp41, the age-dependent rise in IFN-\u03b3 availability primarily promoted the number of IFN-\u03b3 competent Th2/1 cells within the parasite-specific T cell pool in our model .Female BALB/c and C57BL/6 mice were purchased from Janvier, Saint-Berthevin, France. Mice were house in specific-pathogen-free conditions in individual ventilated cages. Mice were infected by oral gavage of 200 3\u03b1IL-12p40  and \u03b1IFN-\u03b3  antibodies were administered at a dose of 0.5\u2009mg intraperitoneally at days 0, 3 and 6 post infection. Recombinant IFN-\u03b3 (Peprotech) was given at a concentration of 2.5\u2009\u03bcg in 200 \u03bcl DPBS intraperitoneally twice a day from day 0 to 4 post infection. Where possible, treated and control mice were housed together to reduce bias.2 in 200\u2009\u00b5l of RPMI  on 96 well plates, followed by counting of the deposited eggs. To assess larval fitness by means of egress of the L4 stage from the infected tissue, a segment of 2\u20133\u2009cm in length was cut from the proximal third of the freshly retrieved small intestine, followed by individual culture with both ends tied on 6 well plates in in serum free RPMI (PAN Biotech containing L-glutamine and 1% P/S). Larval egress was monitored for a total duration of 4\u2009h and expressed as % of total L4 larvae present in each gut segment.To determined fecal egg counts, weighed pellets collected from individual mice were briefly soaked in water (1\u2009ml), meshed uniformly using a glass rod and mixed with saturated NaCl solution (6\u2009ml) and counted using McMaster slides (FiBL). For the quantification of parasite fecundity, where possible, eight adult female worms were collected per mouse and cultured for 24\u2009h at 37\u2009\u00b0C and 5% CO11. 1\u20132 drops of blood were collected in FACS buffer  and treated with FACS Lysing solution (BD Biosciences) to remove red blood cells before staining.Single cells were prepared from spleen, mLN and small intestinal lamina propria (siLP) as previously describedH. polygyrus adult worms  for 5\u20136\u2009h. Splenocytes were added to the pre-loaded dendritic cells and co-cultured overnight in the presence of Brefeldin A  and subsequently stained for surface and intracellular markers.Bone marrow derived cells were harvested from tibia and femur of na\u00efve BALB/c mice and cultured for 6 days with 20\u2009ng/ml GM-CSF  to allow the differentiation of dendritic cells. Dendritic cells were pulsed with excretory/secretory products of 2 for a total of 4\u2009h, brefeldin A (3\u2009\u00b5g/ml) was added after 30\u2009min. Cells were either fixed using IC fixation buffer or fix/perm buffer when targeting transcription factors (both from Thermofisher) and stained using the following reagents: CD4 , FoxP3 , T-bet , GATA-3 , IFN-\u03b3 , IL-4 , IL-13 , CD154 , CD44 , CD62-L , RELM-\u03b1 (Biotin), F4/80 , Siglec-F . Streptavidin coupled to PE, PE-Cy7 or APC was used as secondary conjugates. Non-specific binding of antibodies was prevented by adding Fc\u03b3RII/III blocking antibody (clone 93). Dead cells were excluded using Fixable Viability Dye eFluor 780 or eFluor 506. All antibodies and other reagents were from BioLegend, Thermofisher, or BD Biosciences. Cells were acquired on FACSCantoTM II (BD Biosciences) or FACSAriaTM III (BD Biosciences) and data analyzed using FlowJo .For detection of intracellular cytokines, cells were stimulated with 1 \u03bcg/ml phorbol 12-myristate 13-acetate (Sigma-Aldrich) and 1\u2009\u03bcg/ml ionomycin (Sigma-Aldrich) at 37\u2009\u00b0C and 5% COt test or Mann\u2013Whitney test.All statistical analysis were performed using GraphPad Prism Software . Normality was tested with the Shapiro\u2013Wilk test, followed by ordinary one-way-ANOVA or Kruskal\u2013Wallis test and Tukey\u2019s or Dunn\u2019s multiple comparison test. Comparisons of two groups were performed with an unpaired Supplementary Materials"}
+{"text": "Inhaled antimicrobials enable high local concentrations where needed and, compared to orally administration, greatly reduce the potential for systemic side effects. In SARS-CoV-2 infections, hydroxychloroquine sulphate (HCQ) administered as dry powder via inhalation could be safer than oral HCQ allowing higher and therefore more effective pulmonary concentrations without dose limiting toxic effects.To assess the local tolerability, safety and pharmacokinetic parameters of HCQ inhalations in single ascending doses of 5, 10 and 20 mg using the Cyclops dry powder inhaler.Twelve healthy volunteers were included in the study. Local tolerability and safety were assessed by pulmonary function tests, electrocardiogram and recording adverse events. To estimate systemic exposure, serum samples were collected before and 0.5, 2 and 3.5 h after inhalation.1 post inhalation. The serum HCQ concentration remained below 10 \u03bcg/L in all samples.Dry powder HCQ inhalations were well tolerated by the participants, except for transient bitter taste in all participants and minor coughing irritation. There was no significant change in QTc-interval or drop in FEVSingle doses of inhaled dry powder HCQ up to 20 mg are safe and well tolerated. Our data support that further studies with inhaled HCQ dry powder to evaluate pulmonary pharmacokinetics and efficacy are warranted. In late December 2019 an outbreak of the novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), started in Wuhan, China, and caused the spread of corona virus disease 2019 (COVID-19) .Since the emergence of SARS-CoV-2 many treatment options have been studied. Current treatment for patients needing hospitalization with oxygen therapy consist of dexamethasone with addition of IL-6 inhibitors in patients with high inflammation and respiratory deterioration necessitating high oxygen supply . Monocloin vitro Vero cell systems infected with SARS-CoV-2 in two separate Chinese studies , but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the \u2018author contributions\u2019 section.\u201dIf your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.\u00a0http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and\u00a0 there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: \"This does not alter our adherence to\u00a0 PLOS ONE policies on sharing data and materials.\u201d , along with any other relevant declarations relating to employment, consultancy, patents, products in development or modified products etc. Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, as detailed online in our guide for authors This information should be included in your cover letter; we will change the online submission form on your behalf.http://journals.plos.org/plosone/s/competing-interestsPlease know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article\u2019s retracted status in the References list and also include a citation and full reference for the retraction notice.Additional Editor Comments (if provided):[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to Questions <font color=\"black\"> Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions? </font> The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1:\u00a0YesReviewer #2:\u00a0YesReviewer #3:\u00a0YesReviewer #4:\u00a0PartlyReviewer #5:\u00a0Yes********** <font color=\"black\"> 2. Has the statistical analysis been performed appropriately and rigorously?  </font> Reviewer #1:\u00a0N/AReviewer #2:\u00a0YesReviewer #3:\u00a0YesReviewer #4:\u00a0N/AReviewer #5:\u00a0I Don't Know********** <font color=\"black\"> 3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified. </font> The Reviewer #1:\u00a0YesReviewer #2:\u00a0NoReviewer #3:\u00a0YesReviewer #4:\u00a0NoReviewer #5:\u00a0Yes********** <font color=\"black\"> 4. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0YesReviewer #2:\u00a0YesReviewer #3:\u00a0YesReviewer #4:\u00a0YesReviewer #5:\u00a0Yes********** <font color=\"black\"> 5. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0This is a well written concise manuscript describing a small safety study in healthy volunteers to explore the use of inhaled dry powder hydroxychloroquine to treat COVID 19 in the future. The authors correctly point out that this study does not provide proof of principle that the dose and administration used here produces the drug levels in alveolar tissue that would definitelyinhibit viral invasion. The authors could add some sentences to discuss whether a separate study to attempt to measure alveolar levels in healthy subjects should be done, I think the authors should comment on whether this inhalerwould produce alveolar deposition or whether it is limited to larger airways.A comment on ANY DEPOSITION STUDIES would helpReviewer #2:\u00a0The major problem with the study is the sample size which is 12. However, due to the fact that the authors use only a t-test, this is potentially acceptable.The authors should add a sample size estimation based on reasonable assumptions for the effect size to demonstrate that for the studied problem this small sample size is tolerable.In addition, please emphasize in the discussion this issue.Reviewer #3:\u00a0This manuscript is a well written paper with clear study design and conclusions. However, there are some minor points to be addressed.1. Please describe more details for the study design including dosing sequence, wash-out period, food (fasting or fed), restrictions.2. Please describe or discuss the rationale for the time points of FEV1 assessments and ECG assessment.3. It seems to be better to present the adverse events according to dose in Table 3.4. This study aimed to evaluate the tolerability and PK after a single dose. However, in clinical setting, multiple doses will be needed for the treatment of COVID-19. Please add the limitation of this single dose study in Discussion.Reviewer #4:\u00a0This paper describes a simple but useful trial to test, in healthy volunteers, the pulmonary safety and tolerability of an inhaled, dry-powder formulation of hydroxychloroquine. It deserves rapid publication, because it's of topical interest as regards the treatment of COVID-19.The authors should add comments or discussion about the following weaknesses of the trial:1) The trial is open-label and has no placebo control, so no statistical hypothesis testing is justified. Also, the lack of placebo greatly weakens the assessment of tolerability and safety, including the main pulmonary outcome measure (FEV1).2) the trial did not include any measures of pulmonary inflammation  or gas permeability (eg CO transfer factor)3) In line 235, the authors refer to the rapid \"elimination\" of hydroxychloroquine: the word \"distribution\" would be more accurate.4) There are a few typographical errors to be corrected. Also, the phrase \"allow for\" has been used throughout: the \"for\" is incorrect in this context, and \"allow\" should be used on its own.5) Complete data are not availableReviewer #5:\u00a0The article is well written and can have an impact in clinical practice. The major disadvantage is related to the time when the blood samples were taken, too late to measure the plasma concentration of the drug.Table 3 mention \"respiratory infection\", but this term does not appear in the manuscript. How did you evaluate this side effect?In my opinion, \"inhaled dry powder HCQ is safe\" is too enthusiastic statement, because you have followed a small number of patients for a short time after single increasing doses of 5, 10 and 20 mg. I think you need to mention this within the limits of the study.********** <font color=\"black\"> 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose \u201cno\u201d, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. </font> Yes:\u00a0Diana BiltonReviewer #1:\u00a0Reviewer #2:\u00a0NoReviewer #3:\u00a0NoYes:\u00a0Steve WarringtonReviewer #4:\u00a0Reviewer #5:\u00a0Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at\u00a0figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,\u00a0 26 Apr 2021We would like to thank the editor and reviewers for their time, critical view and suggestions to improve our manuscript. The following additional requirements were implemented:1. We applied the PLOS ONE style requirements2. We provided the full name of the Institutional Review board [line 116].3. A and C: Additional information about the participant recruitment method, recruitment date range [line 109-112] and table with demographic details of the participants [table 2] are added. B: We made a statement in the discussion section whether our sample can be considered representative of a larger population [ see comment on reviewer 2]4. We included the tables with separate captions in the manuscript and removed them as separate files. 5. The figure was given a separate caption in the manuscript. 6. We did not add new supporting information files with this resubmission7. We updated the Competing Interests Statement and the Funding Statement as mentioned in cover letter and in the manuscript. 8. We declared the use of a patented inhaler called the Cyclops with number WO2015/187025 in the cover letter. 9. We reviewed and completed the reference list. The references to the unpublished data of Dayton et al were replaced , since their results were not directly available to us and only published after personal communication in the article of Kavanagh et al [reference 34]. The references belonging to the retracted article of Mehra et al [56] and the retraction notice by Watson et al [57] are removed. New references are added as mentioned in the response to the reviewers.Below is our response to each point raised by the reviewers. Line numbers correspond with the lines in the \u2018revised mancuscript track and changes\u2019Reviewer 1: This is a well written concise manuscript describing a small safety study in healthy volunteers to explore the use of inhaled dry powder hydroxychloroquine to treat COVID 19 in the future. The authors correctly point out that this study does not provide proof of principle that the dose and administration used here produces the drug levels in alveolar tissue that would definitely inhibit viral invasion. The authors could add some sentences to discuss whether a separate study to attempt to measure alveolar levels in healthy subjects should be done.I think the authors should comment on whether this inhaler would produce alveolar deposition or whether it is limited to larger airways. A comment on ANY DEPOSITION STUDIES would helpThank you for these suggestions. It is correct that this study does not provide proof of principle that the dose and administration used here produces alveolar tissue concentrations that inhibit viral invasion or replication. The limitation of the study is that we only measured hydroxychloroquine systemically \u2013 in blood samples \u2013 but ideally this should be measured in lung tissue, BAL, and epithelial lining fluid as well. It would be of great interest to know what concentration can be achieved after inhalation and compare this with oral dosing. We added this limitation to the discussion section [line 290-294]. We made the expectation that there will be alveolar deposition with use of the Cyclops more clear in the introduction section by adding information about the particle size produced by the inhaler [line 92-97].Reviewer 2: The major problem with the study is the sample size which is 12. However, due to the fact that the authors use only a t-test, this is potentially acceptable. The authors should add a sample size estimation based on reasonable assumptions for the effect size to demonstrate that for the studied problem this small sample size is tolerable. In addition, please emphasize in the discussion this issue.We agree on the comment of the small sample size. It is difficult to make a specific calculation of the number of subjects needed in this pilot study. Low numbers have provided good results in other dry powder inhalation studies of antibiotics [41-44], so we think that 12 participants are sufficient to make a good impression of safety and tolerability. This is supported specifically for HCQ by the phase 1 study of aerosolized HCQ in 31 healthy individuals and the phase 2 with aerosolized HCQ in patients with asthma [34]. We added a note of this in the discussion section and provided new supporting references accordingly [line 271-287]. Reviewer 3: This manuscript is a well written paper with clear study design and conclusions. However, there are some minor points to be addressed.1. Please describe more details for the study design including dosing sequence, wash-out period, food (fasting or fed), restrictions.We provided extra information in the manuscript regarding the wash-out period of at least 4 days in the methods section. As mentioned in the manuscript the dosing sequence consisted of single doses, starting with a dose of 5 mg, ascending tot 10 and finally 20mg [line 112-114]. Feeding state and restrictions are important point to address in drug research, but since the drug in this study is deposited and resorbed from the lungs it holds less relevance. Therefore we did not document feeding state or other restrictions in the manuscript. 2. Please describe or discuss the rationale for the time points of FEV1 assessments and ECG assessment.The timepoints for spirometry were chosen shortly after inhalation, because bronchoconstriction as a side effect is expected to occur shortly after inhalation due to direct irritating effects. This has been shown in other studies with inhaled antimicrobials, for example in the article of Hoppentocht et al where drop in FEV1 > 10% is observed within 95 minutes after inhalation of dry powder tobramycin [41][line 135-138]. The risk of QTc prolongation increases with higher systemic HCQ concentrations. We added ECG assessment as a safety parameter, although (high) systemic concentrations were not to expected after local administration in a dose that is just a fraction of the oral dose [line 260-268]. Since there was no data available about the absorption rate of HCQ from the lungs we based our time point on the time that HCQ reaches a maximum concentration after oral dosing, which is within 2-4.5 hours. For logistical reasons we planned it at the end of the study day which was 3,5 hours after inhalation [line 295-296]. 3. It seems to be better to present the adverse events according to dose in Table 3.Thank you for this suggestion. We updated the table en presented adverse events according to dose and in absolute numbers instead of percentages in table 3. 4. This study aimed to evaluate the tolerability and PK after a single dose. However, in clinical setting, multiple doses will be needed for the treatment of COVID-19. Please add the limitation of this single dose study in Discussion.We agree that the single dose is a limitation in our study since in COVID-19 treatment multiple doses will be needed. We added this to the limitations in the discussion section [lines 275-277].Reviewer 4:This paper describes a simple but useful trial to test, in healthy volunteers, the pulmonary safety and tolerability of an inhaled, dry-powder formulation of hydroxychloroquine. It deserves rapid publication, because it's of topical interest as regards the treatment of COVID-19. The authors should add comments or discussion about the following weaknesses of the trial:1. The trial is open-label and has no placebo control, so no statistical hypothesis testing is justified. The lack of placebo weakens the assessment of tolerability and safety, including the main pulmonary outcome measure. (FEV1).The study indeed is open label without placebo control. We believe this limitation is more important when one does find safety or tolerability problems and perspective is needed, but not so much when problems found are minor anyway.2. The trial did not include any measures of pulmonary inflammation  or gas permeability (eg CO transfer factor)We agree that pulmonary inflammation as such was not measured, nor potential proxies such as FeNO or KCO. This will be interesting to consider in a next, phase 1b study in which we will aim for measuring direct local concentrations in the airways and BAL.3. In line 235, the authors refer to the rapid \"elimination\" of hydroxychloroquine: the word \"distribution\" would be more accurate.Indeed the word distribution is more accurate, we replaced the word elimination. 4. There are a few typographical errors to be corrected. Also, the phrase \"allow for\" has been used throughout: the \"for\" is incorrect in this context, and \"allow\" should be used on its own.We deleted the word \u2018for\u2019 in this context and checked the manuscript again for typographical errors. 5. Complete data are not availableAll relevant data are within the paper. The complete data set can be requested from the authors.Reviewer 5: The article is well written and can have an impact in clinical practice. The major disadvantage is related to the time when the blood samples were taken, too late to measure the plasma concentration of the drug.We agree this is an disadvantage and added this as a limitation of the study in the discussion section [line 293-299]. Timepoints for drawing the blood samples were based on the rate of absorption after oral administration of HCQ, since data after pulmonary absorption were lacking at time of writing of the study protocol. Based on results and current knowledge blood samples should be taken at earlier time points as well in future studies. Table 3 mention \"respiratory infection\", but this term does not appear in the manuscript. How did you evaluate this side effect?In table 3 we changed \u2018respiratory infection/sore throat\u2019 to \u2018sore throat\u2019. Participants complaining of sore threat or other upper respiratory tract infection symptoms were provided a swab for respiratory viruses of which one tested positive for SARS-CoV-2, as mentioned in the manuscript [line 191-194]. In my opinion, \"inhaled dry powder HCQ is safe\" is too enthusiastic statement, because you have followed a small number of patients for a short time after single increasing doses of 5, 10 and 20 mg. I think you need to mention this within the limits of the study.We agree on this comment and added this as a limitation in the discussion section. See comments made by reviewer 2 and reviewer 3. In addition we nuanced the conclusion [line 45-47 and line 334-335].AttachmentRespons to reviewer.docxSubmitted filename: Click here for additional data file. 22 Dec 2021 <div> PONE-D-20-37157R1 </div>  <div> Tolerability and Pharmacokinetic Evaluation of Inhaled Dry Powder Hydroxychloroquine in Healthy Volunteers </div>  <div> PLOS ONEDear Dr. Akkerman,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE\u2019s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Feb 05 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at\u00a0 </div> A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. <div> If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.Please include the following items when submitting your revised manuscript:https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Kaisar RazaAcademic EditorPLOS ONEJournal Requirements:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article\u2019s retracted status in the References list and also include a citation and full reference for the retraction notice.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to Questions <font color=\"black\"> Comments to the Author </font> 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the \u201cComments to the Author\u201d section, enter your conflict of interest statement in the \u201cConfidential to Editor\u201d section, and submit your \"Accept\" recommendation.Reviewer #3:\u00a0All comments have been addressedReviewer #6:\u00a0(No Response)********** <font color=\"black\"> 2. Is the manuscript technically sound, and do the data support the conclusions? </font> The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #3:\u00a0YesReviewer #6:\u00a0Yes********** <font color=\"black\"> 3. Has the statistical analysis been performed appropriately and rigorously?  </font> Reviewer #3:\u00a0YesReviewer #6:\u00a0Yes********** <font color=\"black\"> 4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified. </font> The Reviewer #3:\u00a0YesReviewer #6:\u00a0Yes********** <font color=\"black\"> 5. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #3:\u00a0YesReviewer #6:\u00a0Yes********** <font color=\"black\"> 6. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #3:\u00a0All comments and suggestions are properly reflected and addressed. So, it is acceptable for a new publication.Reviewer #6:\u00a0The study design was clear, and the manuscript was well written in general. I would recommend authors to briefly mention the method of development of HCQ dry powder formulation and it\u2019s aerosolization behavior in vitro as this study couldn\u2019t test the lung concentrations of drug before acceptance for publication.********** <font color=\"black\"> 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose \u201cno\u201d, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. </font> Reviewer #3:\u00a0NoReviewer #6:\u00a0Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at\u00a0figures@plos.org. Please note that Supporting Information files do not need this step. </div> While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,\u00a0 4 Jan 2022We would like to thank the editor and reviewers for their time, critical view and suggestions to improve our manuscript. The following additional requirements were implemented based on the suggestion from reviewer 6. The study design was clear, and the manuscript was well written in general. I would recommend authors to briefly mention the method of development of HCQ dry powder formulation and it\u2019s aerosolization behavior in vitro as this study couldn\u2019t test the lung concentrations of drug before acceptance for publicationIn response to reviewer 6 we added additional information to the methods section, under 'study drug', about the development of the HCQ dry powder formulation and it\u2019s behavior in vitro. [line 107-115].The HCQ Cyclops was produced by PureIMS B.V. . For this, hydroxychloroquine sulphate was comicronized by air jet milling with 4% L-leucine to improve formulation dispersion. Each Cyclops contained a nominal dose of 5 mg or 10 mg HCQ; the 20 mg dose was administered as 2 successive inhalations with 10 mg of HCQ. The doses were comparable with known doses of inhaled nebulized HCQ and thus expected to be safe . Furthermore, to enhance dose emission from the inhaler, 5 mg of coarse lactose was added to the dose compartment. At 4 kPa, 85% of the nominal dose was emitted from Cyclops with a fine particle fraction < 5 \u00b5m of 74% , as determined by laser diffraction analysis.AttachmentRespons to reviewer 02-01-2022.docxSubmitted filename: Click here for additional data file. 18 May 2022 <div> PONE-D-20-37157R2 </div>  <div> Tolerability and Pharmacokinetic Evaluation of Inhaled Dry Powder Hydroxychloroquine in Healthy Volunteers </div>  <div> PLOS ONEDear Dr. Akkerman,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE\u2019s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. </div>  <div> Please revise on the following grounds too: </div>  <div> \u00a0This is a dose-escalation Phase I repeated-measures trial. The authors studied the safety and tolerability of dry HCQ through inhalation with dose escalation among the healthy volunteers. The tolerability was assessed by the measures on PFT and ECG and the safety through the adverse events. The design of the study seems appropriate and well-written manuscript. However, the authors need to make more changes to shape the manuscript nicely. Below are some comments: 1. The topic is misleading on 'Pharmacokinetics Evaluation. I have not seen any in the manuscript related to that. 2. The statement in the first two paragraphs of the introduction section is obsolete. The authors need to update at least as of December 31, 2021. 3. The authors must mention many available treatment modalities, including the recent EUA COVID pill. 4. Why was the oral HCQ not advised by FDA and EMA? The authors need to refer to the RECOVERY Trial and the prospective European DISCOVERY and WHO SOLIDARITY trials. Those details would enhance the manuscript. 5. The statistical analytic plans are critically important in ensuring appropriate reporting of clinical trials. The significance reported using the methods seems irrelevant due to the small sample size and the authors' need to consider using the exact tests. The main focus is the safety and tolerability, not the statistical significance of the measures collected. 6. There is no uniformity in using the terminology. In some places in the manuscript, the authors use 'volunteer' and state 'participant' in many areas. 7. The second paragraph in the results section (lines 165- 184) should be converted into a table by listing all event names with a number of participants. Then, delete the entire text from that section. 8. Not clear, the authors screened 12 participants, and all are eligible and enrolled in the study? If it is more than that, it has to be reported. 9. The statements in the discussion section (Lines 217-227) should highlight the importance of the study and focus on elaborating the contents based on the data with supporting evidence (any prior cited research). The statistical values reported in that paragraph in the lines 229-240 should be deleted. 10. The authors described why the timing was chosen (Lines 268-272) for blood collection. These statements should move to the methods section. 11. There is not enough in the discussion to claim this study is warranted. A strong justification is still missing: why is the clinical research warranted? How would the proposed method help in the clinical arena? 12. Line 198: Table 1: Check the spelling for 'Cough.' It says 'Couch.' 13. Line 203: Table 3: Last column 'Percentage of patients' is meaningless- should be excluded. Add the percentage under each dose column N (%). 14. Line 205: The change in value for 10 mg HCQ from baseline to 95 minutes post inhalation is reported as 0.21. All the other values are less than zero. The authors need to double-check the calculation of this declared value.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Jul 02 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at\u00a0 </div> A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. <div> Please include the following items when submitting your revised manuscript:If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Kaisar RazaAcademic EditorPLOS ONEJournal Requirements:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article\u2019s retracted status in the References list and also include a citation and full reference for the retraction notice.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to Questions <font color=\"black\"> Comments to the Author </font> 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the \u201cComments to the Author\u201d section, enter your conflict of interest statement in the \u201cConfidential to Editor\u201d section, and submit your \"Accept\" recommendation.Reviewer #6:\u00a0All comments have been addressed********** <font color=\"black\"> 2. Is the manuscript technically sound, and do the data support the conclusions? </font> The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #6:\u00a0Yes********** <font color=\"black\"> 3. Has the statistical analysis been performed appropriately and rigorously?  </font> Reviewer #6:\u00a0Yes********** <font color=\"black\"> 4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified. </font> The Reviewer #6:\u00a0Yes********** <font color=\"black\"> 5. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #6:\u00a0Yes********** <font color=\"black\"> 6. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #6:\u00a0I would appreciate authors for addressing all the comments. Now, I would recommend accepting this manuscript for publication.********** <font color=\"black\"> 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose \u201cno\u201d, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. </font> Reviewer #6:\u00a0Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at\u00a0figures@plos.org. Please note that Supporting Information files do not need this step. </div> While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,\u00a0 1 Jul 2022We would like to thank the editor and reviewer for their time, critical view and suggestions to improve our manuscript. 1. The topic is misleading on 'Pharmacokinetics Evaluation. I have not seen any in the manuscript related to that. The pharmacokinetic evaluation consisted of measuring serum HCQ concentrations predose, 30 minutes, 2 and 3,5 hours after inhalation. Serum HCQ concentrations are reported in the manuscript. Please refer to line 202-206\u201cIn all participants, the serum HCQ concentrations sampled predose, 30 minutes, 2 and 3.5 hours, were below the detection level of 10 \u00b5g/L, irrespective of dose. The mean delivered dose was 3.16 mg (63%) after administration of 5 mg HCQ, 6.95 mg (70%) after administration of 10 mg HCQ and 14.86 mg (75%) after administration of 20 mg HCQ. Based on the recorded inspiratory flow parameters, all participants correctly performed the inhalation maneuvers.\u201d2. The statement in the first two paragraphs of the introduction section is obsolete. The authors need to update at least as of December 31, 2021.Thank you, we agree. Unfortunately, the manuscript has been under review for two very long periods at the journal. The information is now updated. 3. The authors must mention many available treatment modalities, including the recent EUA COVID pill. Thank you for this suggestion. We added a paragraph with a short summary of current treatment advice according to the WHO living guideline accessed June 26th, 2022. 4. Why was the oral HCQ not advised by FDA and EMA? The authors need to refer to the RECOVERY Trial and the prospective European DISCOVERY and WHO SOLIDARITY trials. Those details would enhance the manuscript. Yes, it is important to know that oral HCQ was not adviced because of a lack of effect on mortality. In line 78-81 we now refer to this decision and the mentioned trials. 5. The statistical analytic plans are critically important in ensuring appropriate reporting of clinical trials. The significance reported using the methods seems irrelevant due to the small sample size and the authors' need to consider using the exact tests. The main focus is the safety and tolerability, not the statistical significance of the measures collected.We would like to thank the reviewer for this advice. Since the main focus is indeed the safety and tolerability and the sample size is small we decided not to mention the statistical analysis anymore and only use descriptive statistics. Therefore we changed the text in the data management and statistics section and removed the p-values from the tables. 6. There is no uniformity in using the terminology. In some places in the manuscript, the authors use 'volunteer' and state 'participant' in many areas. Thank you for this suggestion to make the manuscript more uniform. As volunteers participate in the study they can also be referred to as participants. The term healthy volunteer is now only used in the methods sections, in the rest of the manuscript we use the term participant. 7. The second paragraph in the results section (lines 165- 184) should be converted into a table by listing all event names with a number of participants. Then, delete the entire text from that section. Thank you, we agree that a table gives the best overview of the events, they are mentioned in table 3. To our opinion, the text adds more detailed information to this. Therefore we decided not to remove the text. 8. Not clear, the authors screened 12 participants, and all are eligible and enrolled in the study? If it is more than that, it has to be reported. All participants screened were eligible and were enrolled in the study. This has now been made more clear in the text.9. The statements in the discussion section (Lines 217-227) should highlight the importance of the study and focus on elaborating the contents based on the data with supporting evidence (any prior cited research). The statistical values reported in that paragraph in the lines 229-240 should be deleted. Thank you, we tried to make the importance of the study more clear in the first paragraph of the discussion and added citations to previous mentioned research in the first and second paragraph as well. 10. The authors described why the timing was chosen (Lines 268-272) for blood collection. These statements should move to the methods section. We moved the statement to the methods section as suggested. 11. There is not enough in the discussion to claim this study is warranted. A strong justification is still missing: why is the clinical research warranted? How would the proposed method help in the clinical arena? There is not a good explanation why hydroxychloroquine lacks a clinical effect in the treatment of COVID-19 despite the promising results of in vitro studies and the assumed mechanism of action. We tried to state this more clear in the first section of the discussion section, addressing that we hypothesize that high enough concentrations are not reached in the lung where needed when dosed systemically, because of limitations of systemic toxicity, but might be reached by inhaled dosing. 12. Line 198: Table 1: Check the spelling for 'Cough.' It says 'Couch.' We corrected the spelling12. Line 203: Table 3: Last column 'Percentage of patients' is meaningless- should be excluded. Add the percentage under each dose column N (%). Thank you. We excluded the last column and added the percentages under the remaining column as suggested. 14. Line 205: The change in value for 10 mg HCQ from baseline to 95 minutes post inhalation is reported as 0.21. All the other values are less than zero. The authors need to double-check the calculation of this declared value.We checked this value and the value of +0.21 % is correctly documented.AttachmentRespons to Reviewer iHCQ.docxSubmitted filename: Click here for additional data file. 13 Jul 2022Tolerability and Pharmacokinetic Evaluation of Inhaled Dry Powder Hydroxychloroquine in Healthy VolunteersPONE-D-20-37157R3Dear Dr. Akkerman,We\u2019re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you\u2019ll receive an e-mail detailing the required amendments. When these have been addressed, you\u2019ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they\u2019ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Kaisar RazaAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 26 Jul 2022PONE-D-20-37157R3 Tolerability and Pharmacokinetic Evaluation of Inhaled Dry Powder Hydroxychloroquine in Healthy Volunteers Dear Dr. Akkerman:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Kaisar Raza Academic EditorPLOS ONE"}
+{"text": "The present screening data provide a useful resource for GPCR targets and biomarkers for improving the efficacy of cisplatin treatment.The screening of siRNAs targeting 390 human G protein-coupled receptors (GPCRs) was multiplexed in combination with cisplatin against lung cancer cells. While the cell viability measure hardly captured the anticancer effect of siGPCRs, the direct cell count revealed the anticancer potential of diverse GPCRs . In combined treatment with cisplatin, siRNAs against five genes  enhanced the anti-proliferation efficacy on cancer cells and reduced the self-recovery ability of surviving cells after the removal of the combined treatment. Further on-target validation confirmed that the knockdown of TACR3 expression exhibited anticancer efficacy under both single and combined treatment with cisplatin. Q-omics ( Their roles and functions have been recently reported in the cancer context, providing new strategies for cancer drug development. Several drugs or drug candidates targeting GPCRs are already in clinical use or in trials. For example, there are a few FDA-approved cancer drugs available, such as cabergoline, lanreotide, degarelix, plerixafor, vismodegib and raloxifene, that target DRD1 (dopamine receptor D1), SSTR (somatostatin receptor), GnRH (gonadotropin-releasing hormone receptor), CXCR4 (C-X-C chemokine receptor type 4), SMO (smoothened homolog) and ES (estrogen receptor)3. More compounds targeting GPCRs, such as CCR2/CCR4 (C\u2013C chemokine receptor type 2/4), FZD4 (frizzled class receptor 4), and PE2/PE4 (prostaglandin receptors), are under clinical trials6. However, GPCRs have not been widely investigated as cancer targets, partly because of their low inhibitory outcome from conventional in vitro cytotoxicity  assays6.G protein-coupled receptors (GPCRs) are major targets of therapeutic drugs against diverse diseases9. In our previous study, we observed negative correlations between the total cell count and the viability of surviving single cells after treatment with anti-proliferative short interfering RNAs (siRNAs)9. However, the image-based direct cell count provided greater resolution than conventional viability measures for the quantification of the anticancer efficacy of siRNAs.Cell viability measures based on mitochondrial activity have been widely used as surrogates for the mass change of cancer cells in screens. However, recent studies showed that cell viability measures from surviving cells also represent the resistant phenotype overcoming the effect of anticancer treatments; thus, the measure is not directly correlated with the cell number, particularly in the screen with the treatment of stress-inducing apoptotic anticancer agents11. Data from well-based reverse screening using a siRNA library have limited availability from public domains but have advantages in compatibility with drug screen data and in employing diverse (or multiplexed) phenotypic assays13.Here, we carried out multiplexed screening of siGPCRs by using simultaneous measures of cell viability  and image-based cell counts Fig.\u00a0. This mu15. Although many combination targets for cisplatin have been reported, GPCRs have not been systematically investigated as potential targets for cisplatin combination therapy or for overcoming cisplatin resistance in cancers. Together with a single treatment of siGPCRs, we comparatively screened the combined treatment of siGPCRs with cisplatin against lung cancer cells. We expect that the present multiplexed screening provides sufficient resolution to capture the additive or synergistic effect of siGPCRs on cisplatin treatment.Cisplatin is a first-line chemotherapeutic against lung cancers and many other cancer types16  hits from cell count assays  Fig.\u00a0E. The reAmong 46 siGPCR hits, we selected seven siGPCRs  for further validation  of the treatment was used for the second validation  using Q-omics software (http://qomics.io)18, TACR3 expression was not associated with patient survival in any sample filtering conditions  , H1793, and H1437. The cell lines were maintained in RPMI 1640 medium containing 10% fetal bovine serum  and 1% penicillin. For H1437, RPMI 1640, HEPES  was used. The cells were cultured in a humidified atmosphere of 5% COThe siRNA screen was performed by using four pooled On-Target Plus siRNAs to target each of the 390 genes in the human GPCR siRNA library. Screening was performed in triplicate for four days in a 384-well plate format (400 cells per well). Each plate was supplied with a negative control siRNA  and positive control siRNA  to distinguish sequence-specific silencing from nonspecific efficiency and the effects of knockdown. Reverse transfection was performed using a MultiFlo microplate dispenser (BioTek Instruments) with siRNAs  and Lipofectamine RNAiMAX  diluted Opti-MEM  in a black 384-well plate . After 24\u00a0h, each set was treated with 0.1% dimethylformamide (DMF) and cisplatin . Cisplatin was obtained from Abmole Bioscience and dissolved in 100% DMF at a final concentration of 50\u00a0\u00b5M. Aliquots were stored at -20\u00a0\u00b0C and thawed and diluted (1\u00a0\u00b5M) immediately before use. After 72\u00a0h, the cells were assayed using the CellTiter-Blue Cell Viability Assay Kit  according to the manufacturer\u2019s protocol, and the fluorescence produced was proportional to the number of viable cells. Plates were read on a SYNERGY H1 microplate reader (BioTek Instruments). After the cell viability assay, the cells were stained with Hoechst 33342 , and the plates were imaged on a CYTATION3 imaging reader (BioTek Instruments) in 2\u2009\u00d7\u20092 montage mode.16:For the quality control metric in siRNA screening, Z\u2019 factor is used which is defined as followswhere SD is for standard deviation and AVG is for average.The range of z\u2019-factor is negative infinity to one, with\u2009>\u20090.5 indicating an excellent assay and\u2009>\u20090 a borderline assay.Raw data were converted into log2 scale to calculate the inhibitory effect of each siRNA: cell count and cell viability compared to the negative controls per plate. After normalization, negative values indicate that treating the target gene has a lower number of cells than the negative controls. All data were on a log2 scale, unless stated otherwise. Statistical significance was calculated by the independent 2-sample Student\u2019s t test. Inhibitory siRNA hits were selected by combination of cisplatin and siGPCR transfection. The cisplatin-sensitive hits were selected with a p-value\u2009<\u20090.01 in combined treatment with cisplatin. The common hits were selected by p-value\u2009<\u20090.01 in both single and combined treatment with cisplatin. We calculated the cell counting value as a percentage for siNC-DMF. All data analysis including t test is done using Excel.Validation was performed to identify false positives. siRNAs from On-Target Plus siRNAs  were rescreened with A549 using the same transfection protocol of 10\u00a0nM by reverse transfection using Lipofectamine RNAi Max and Opti-MEM with DMF and a concentration of 1\u00a0\u00b5M cisplatin according to the manufacturer\u2019s protocols. Repeated experiments were performed twice. Another validation experiment was conducted on five siRNAs  from siTOOLs Biotech with A549, EKVX, H1437, H1793. siRNAs were transfected  by reverse transfection using Lipofectamine RNAi Max and Opti-MEM and a concentration of 1\u00a0\u00b5M cisplatin with DMF. The experiment was repeated four times. For the anticancer effect, % Growth inhibition was defined as follows:A self-renewal experiment was conducted to identify how the pretreated chemical can affect the regeneration and growth of cancer cell lines even though it was replaced with fresh medium. We seeded the A549 cell line in a 96-well plate (Thermo Fisher) (2500 cells per well) for pretreatment. Transfection of siNC and siGPCRs was performed. After 24\u00a0h, the cells were treated with 1\u00a0\u00b5M cisplatin and DMF to identify four conditions . After a total of 96\u00a0h, the cells were harvested and seeded in 384-well plates by seeding number  in fresh medium. After 96\u00a0h, the cells were assayed using Hoechst 33,342 (Sigma\u2013Aldrich) to count the cells.For transfection of cisplatin-pretreated A549, we performed the same protocol with the A549 cell line prepared in advance. Cisplatin (1\u00a0\u00b5M) in the culture media was added to prepare cisplatin-pretreated A549 cells. We normalized the cell counting value as a percentage for siNC. The experiment was conducted with three repeats.23. The RNA sequencing data in FPKM (Fragments Per Kilobase Million) were converted to log2 (TPM\u2009+\u20091) (Transcripts Per Kilobase Million). Both FPKM and TPM indicate expression levels of RNA transcript. TPM was introduced as a measurement to correct the inconsistences among independent samples24. For calculation of survival analysis, the following patient details were obtained: vital status, gender, days to death, days to last follow-up and days to new tumor event after initial treatment from clinical data.RNA sequencing data, mutation data and clinical data were obtained from The Cancer Genome Atlas (TCGA) GDC data portal25 and the log-rank test26 via R. Gender  and stage  were divided by TCGA clinical data. The patients were divided into high expression and low expression groups based on the median and quartile  gene expression among all patients for each gene. Overall survival (OS) was defined as the length of time from either the date of diagnosis or the start of treatment for a disease, such as cancer, to the date of death, and patients diagnosed with the disease who were still alive were censored. Disease-free survival (DFS) was defined from randomization to the first evidence of recurrence, second primary malignancy or death, whichever occurred first. To obtain a p-value indicating the significance of this analysis, the log-rank test26 was performed.The genes were analyzed using the Kaplan\u2013Meier method27. Favorable prognostic gene expression was associated with a lower survival rate in the group with low gene expression, and unfavorable prognostic gene expression was associated with a lower survival rate in the group with high gene expression.Survival analysis was calculated for 48 different measures using the TCGA dataset. The patients were divided according to the clinical data, including gender  and stage . Gene expression groups were divided into medians or quartiles and calculated according to OS and DFS. Among the 48 survival p-values calculated in this way, the best p-value was used. We analyzed favorable and unfavorable prognostic gene expressionhttps://www.gsea-msigdb.org/gsea/msigdb/).We used GO (Gene Ontology) term to find out with which function each group of genes is associated. Among them, we focused on the biological process term. GO database were obtained from msigdb (Oncoprint was used to display mutation information for LUAD (Lung Adenocarcinoma) patients whose mutation information were obtained from TCGA. Data processing was done via R.Supplementary Information."}
+{"text": "ObjectivesThe current study aimed to evaluate the effectiveness of plasma skin regeneration (PSR) in the treatment of cleft lip scars in cleft lip patients.\u00a0Materials and methodsTwenty patients, 10 females\u00a0and 10 males, with a mean age of 19 years and who had a cleft lip scar, were included in the current study. All patients were treated with a plasma skin regeneration pen device in one treatment session. The thickness, relief, and pliability of the scars were assessed by external observers using a 10-point numeric rating scale (NRS).ResultsThe thickness, relief, and pliability of the scar were significantly improved according to the observers\u2019 opinions .ConclusionsWithin the limits of this study, the PSR appeared to be safe and effective for treating cleft lip scars with minimal complications. A cleft lip is a congenital development defect during the embryonic development period, resulting from the lack of connection of the upper lip tissues . The clePlasma skin regeneration (PSR) is the fourth state of matter, and it is a new technique of resurfacing that creates a thermal effect on the skin using plasma energy . The plaThe current study aimed to evaluate the effectiveness of this novel method in the field of maxillofacial surgery, especially in patients who have a cleft lip, due to its advantages and the few reported side effects.Trial designThis was a cohort study. The current trial was achieved at the Department of Maxillofacial Surgery at the Faculty of Dentistry, Damascus University, Syria, between September 2019 and January 2022. Ethical approval was obtained from the Local Ethics Research Committee at the University of Damascus Dental School (Reference Number: 297325082019-DEN).Participants and eligibility criteriaTwenty adult Syrian patients aged 17-23 years  were enrolled in the current study.Inclusion criteria: Patients who had a chronic cleft lip scar of type III or IV according to Fitzpatrick skin classification . The ageExclusion criteria: Patients with pregnancy and lactation, a history of keloid formation, previous laser treatment, a history of collagen vascular disease, a history of dermal fillers, and an inability to avoid significant sun exposure during the follow-up period were excluded.Informed consent was obtained from all patients before any procedure was applied.Sample size calculationA sample size calculation was done using Minitab\u00ae 18.1  according to the following assumptions: The minimal clinical difference to be detected was 2 degrees on the Numeric Rating Scale (NRS); the standard deviation was determined to be two as well; the level of significance  was set at 0.05 with a power of 85%. According to the calculation, the required sample size was 20 patients.The plasma skin regeneration procedure (PSR)The patients were treated with plasma skin regeneration in one stage, using the PSR device  Figure . The appFirst, the area was disinfected with a povidone-iodine solution. Before the PSR treatment, patients were given cephalexin 500 mg twice daily (BID) and acyclovir 400 mg three times a day (TID) for bacterial and viral prophylaxis, respectively. A topical 4% lidocaine gel  was applied to the treatment site, followed by an injection of 2% lidocaine .After anesthesia, the skin was disinfected with hexamidine , followed by saline irrigation.A single pass of high-energy plasma was delivered to the scar and the surrounding normal skin for 15 minutes. The tip of the pen did not touch the surface of the treated skin. All patients were asked to use a moisturizing cream and not itch the treatment site for a week. Figure Postoperative care consists of an open dressing with petrolatum-based ointment, which is applied three to four times daily, along with gentle cleansing with a non-detergent-based cleanser. Sunscreens and makeup are generally safe to use following desquamation by postoperative day seven following treatment.Outcomes assessmentThe thickness, relief, and pliability of the cleft scar were assessed before treatment and six months after the treatment by three external observers from the Oral and Maxillofacial Surgery Residents . The observers had no conflict of interest either. They used a 10-point numeric rating scale (NRS), with one referring to an appearance very similar to normal skin and 10 referring to the worst scar shape Figure . The meaStatistical analysisAll statistical analyses were performed using Statistical Package for Social Sciences (SPSS) software . The Shapiro-Wilk test was used to assess the shape of the data distribution and its normality. A Wilcoxon signed-rank test was used to detect significant changes in the scar properties after PSR treatment.The percentage of improvement for the thickness, relief, and pliability of the scar was assessed according to the opinions of the observers using the following equation:The Improvement Percentage = (the amount of improvement after treatment / pre-operative rate) * 100.The thickness, relief, and pliability of the scar were improved according to the observers\u2019 opinions   Table .The current study assessed the PSR as a modality for enhancing the scar of the cleft lip. Carbon dioxide lasers are more frequently used in the field of facial scar treatment. Unfortunately, carbon dioxide lasers may cause damage to the skin, and side effects include transient or prolonged erythema, temporary and permanent hypopigmentation, hyperpigmentation infection, and scarring , 18.Plasma skin regeneration technology has been evaluated in several medical fields, including complete regeneration of the facial skin at low energy . It was During the first ten days of PSR, which includes the superficial skin layers and uses a high energy rate (3-4 joules), the regeneration reaches the upper papillary layer. Continuous collagen production starts from the beginning of the treatment and lasts for a year around the area of thermal damage resulting from the treatment. It differs from the carbon dioxide laser in two main points: the tissue surface contraction, as a result of dermal collagen thermal denaturation, and the thermal disruption of solar elastosis; and the activation of fibroblasts stimulate a wound healing cascade necessary for neocollagenesis and the reduction of solar elastosis , 9, 11.In the current study, high energy (3-4 joules) was used to treat the scars. This energy can produce a high-frequency thermal effect on the surface of the skin .The results of the current report showed that PSR improved the thickness, relief, and pliability of the scar. These results can be explained by the polished surface after treatment as a result of the denaturation of collagen fibers due to carbonizing the spot, the reorganization of collagen and elastin fibers into tissue on the surface of the dermal-epidermal junction, and regeneration processes that include the epidermal layers and the upper layers of the dermis in cases of high energy treatment .This thermal effect caused by plasma energy helps to improve the characteristics of the scar and the skin adjacent to it in terms of pliability, thickness, and relief. These results agreed with the study of Kono et al., who evaluated the traumatic scars and found an improvement in the superficial and medium-depth scars of up to 50%, while deep scars showed resistance to treatment .The results of the current study can be linked to the treatment of wrinkles. The same findings were observed in the study of Foster et al., who assessed the wrinkles around the mouth after the PSR treatment and reported a high improvement rate . These fThe main limitation of the current study is that the complications of the PSR procedure were not assessed. Another limitation is that the current study did not evaluate symmetry during dynamic aspects of lip movements. Finally, the current study is limited in that it did not study the effect of repeating the application of the procedure (PSR) on its effectiveness.Within the limits of the current study, a 15-minute single pass of high-energy plasma can make an average improvement in chronic cleft lip scar thickness, relief, and pliability, according to the opinions of external observers. The PSR can be considered an effective modality of treatment for enhancing the scarring properties of cleft lips in adult Syrian cleft lip patients."}
+{"text": "Myocardial bridging is a rare anatomical variant that can lead to detrimental cardiac consequences when undiagnosed and untreated. This rare variant can induce anginal-type symptoms due to disrupted blood flow to the myocardium during systole. The patients presented in this report of two cases had previously undiagnosed myocardial bridging of the left anterior descending artery, however clinically, they presented quite differently. Here we present two cases discussing the course of diagnosis and treatment of myocardial bridging of these two patients. The goal of this case report is to highlight the significant cardiovascular injuries that can be a result of undiagnosed myocardial bridging. Myocardial bridging is a rare anatomical variant in the coronary artery potentially leading to myocardial ischemia, left ventricular dysfunction, cardiac arrhythmia, and even sudden cardiac deaths\u00a0-4. SurprPatients become symptomatic when the coronary vessel tunneling through the myocardium gets compressed during systole. Patients are medically managed with beta-blockers and calcium-channel blockers and further managed with surgical interventions with myotomy, and/or\u00a0coronary artery bypass grafting (CABG) and stenting of tunneled arteries ,9. We reA 40-year-old male with a past medical history of hypertension and obesity came to the emergency department (ED) with complaints of progressively worsening shortness of breath and intermittent exertional central chest pain for the past six months. These symptoms had progressively worsened over the last few days resulting in severe impairment of his functional capacity and routine daily activities. These symptoms were additionally associated with swelling of the legs bilaterally, orthopnea, paroxysmal nocturnal dyspnea, and occasional nonproductive cough. He denied palpitation and any episodes of syncope. Review of systems was otherwise negative. He has a smoking history of 8 pack-years, drinks socially, and occasionally smokes marijuana. Family history was significant for heart disease in the mother and brother; however, the nature of the disease was unknown but he\u00a0denied any sudden cardiac death in the family.On physical examination, he was obese with a BMI of 36.5. Triage vitals showed blood pressure of 138/97 mmHg, heart rate (HR) of 114 beats/min, respiratory rate of 23, and oxygen saturation of 97% on room air. Jugular venous pressure was elevated. A cardiovascular exam revealed sinus tachycardia without any murmur, rub, or gallop. Bilateral vesicular breath sounds were heard with mild wheezing and bilateral basal crepitation. He had bilateral pitting edema. On admission, brain natriuretic peptide (BNP) was 1865 pg/mL, high-sensitivity troponin was 49.5 ng/L, D-dimer 709, high-density lipoprotein (HDL) was 22 mg/dL, low-density lipoprotein (LDL) was 115 mg/dL, triglycerides (TG) were 89 mg/dL, and total cholesterol was 155 mg/dL. Urine toxicology was positive for cannabinoids. All other lab parameters, including complete blood count (CBC), comprehensive metabolic panel (CMP), and thyroid-stimulating hormone (TSH), were within normal limits. EKG showed: sinus tachycardia, QTc 454 as described in Figure A chest x-ray showed borderline vasculature with cardiomegaly. CT pulmonary angiogram (CTPA) was negative for pulmonary embolism and aortic dissection. Echocardiogram showed severely reduced systolic function ejection fraction (EF): 16%, diffuse hypokinesis, decreased left ventricular diastolic compliance, increased left atrial pressure, and a mild mitral and tricuspid regurgitation. Furosemide\u00a040 mg\u00a0every 12 hours, lisinopril 20 mg daily, aspirin 81 mg daily,\u00a0and\u00a0atorvastatin 20 mg\u00a0daily were subsequently started and he was admitted to telemetry for continuous cardiac monitoring. A beta-blocker was not started initially in view of acute exacerbation of heart failure. Troponin and EKG were trended and were not suggestive of acute ischemic changes at the time of admission. On day 2 of admission, he complained of constant severe central chest pain.\u00a0High-sensitivity troponin was 681.2 ng/L, and EKG showed new T wave inversion in leads II, III, and aVF as described in Figure He was given a loading dose of aspirin 325 mg, nitrates , morphine 2 mg,\u00a0and a therapeutic dose of Lovenox 120 mg\u00a0and was transferred for urgent cardiac catheterization. Cardiac catheterization revealed severely depressed global left ventricular function without significant coronary artery disease (CAD). Severe myocardial bridging was present in the distal left anterior descending artery (LAD), as shown in Figure Medications were subsequently optimized to metoprolol-XL 50 mg daily, spironolactone 25 mg\u00a0daily, Entresto 24-26 two times daily, dapagliflozin 10 mg daily, aspirin 81 mg daily, and atorvastatin 20 mg\u00a0daily. He was discharged with a life vest and was instructed to follow up with the cardiology clinic for a device check. On follow-up, he improved clinically with an increase in his functional capacity. The follow-up device check did not reveal any significant arrhythmia.Case 2A 25-year-old male with a past medical history of uncontrolled type 2 diabetes, hypertension, childhood bronchial asthma, dyslipidemia, and obesity presented to the ED with complaints of acute pressure-like central chest pain for 15-20 minutes with an onset of 1 hour before the presentation. This was associated with diaphoresis and one episode of non-projectile, non-bilious vomiting. He denied shortness of breath, cough, palpitation, and syncopal episode. All other reviews of systems were unremarkable. He has a smoking history of 10-15 cigarettes/day for 3-4 years with a pack-year of two. He is a social drinker and takes marijuana occasionally. Family history is not significant for any cardiac disease or sudden cardiac death.On physical examination, he was obese with BMI of 38. Triage vitals included: blood pressure of 148/104\u00a0mmHg, HR of 70 beats/min, respiratory rate of 16,\u00a0and oxygen saturation of 98% on room air. On cardiovascular exam: normal S1/S2, with a regular rate and rhythm with no murmurs, rubs, or gallops were observed. Chest exam: bilateral vesicular breath sounds with no added sounds. All other physical findings were normal. Admission labs were significant for elevated high-sensitivity troponin of 1833 ng/L, D-dimer of 587, random blood glucose of 254 mg/dL, HDL 26 mg/dL, LDL 185 mg/dL, TG: 276 mg/dL, total cholesterol 266 mg/dL, and urine toxicology were positive for cannabinoids. All other labs, including CBC, CMP, BNP, and lipase, were within normal limits. EKG showed HR of 79, QTc: 438 but no ST-T wave changes as in Figure The patient received a loading dose of aspirin 325 mg, clopidogrel 225 mg, atorvastatin 40 mg, and one therapeutic dose of Lovenox 100 mg. A bedside echo was done, which was normal with EF: 55-60%. CTPA was negative for pulmonary embolism and aortic dissection. High-sensitivity troponin trended up to 2749 ng/L, however, repeat EKG did not show any new changes. He was immediately transferred for cardiac catheterization. Cardiac catheterization was performed and revealed no significant CAD with normal left ventricular ejection fraction (LVEF): 55% but revealed bridging of the mid LAD segment as described in Figure He was discharged with oral diabetic metformin 500 mg every 12 hours, lisinopril 10 mg daily (for hypertension), and atorvastatin 10 mg daily for diabetes mellitus. On the second day of admission, approximately 8-9 hours post-cardiac catheterization, he left against medical advice even after extensive counseling.Myocardial bridging commonly involves the LAD artery as in our cases,\u00a0though rarely the right coronary artery is involved.  PatientsIn an earlier study, myocardial fibrosis and interstitial edema were significantly associated among patients with hemodynamically significant myocardial bridges in sudden cardiac death . MyocardPatients with myocardial bridging presenting with angina symptoms can have normal resting EKG like in our second case. Even on exercise stress tests, the absence of EKG changes is common in\u00a0myocardial bridging, however, patients can present with angina . AkdemirThe degree of vessel narrowing, depth, and number of tunneled arteries determine the manifestations and subsequent complications of the myocardial bridging ,18.Coronary angiography is the gold standard for diagnosing myocardial bridging. In coronary angiography, the systolic compression of the tunneled artery is classically depicted as a \u201cmilking effect\u201d, with retrograde blood flow on systole and subsequent antegrade flow during diastole. Intravascular US and intracoronary Doppler sonogram are other diagnostic modalities of choice. Our patients underwent coronary angiography which showed myocardial bridging without significant CAD.Recent advances in cardiac imaging with cardiac CT, cardiac MRI, can also be used in making the diagnosis ,20. CardManagement of myocardial bridging involves reducing intramyocardial artery compression by shortening the systolic phase and lengthening the diastolic phase, which can be achieved with the administration of beta-blockers and calcium-channel blockers. Sublingually administered nitroglycerine could even exacerbate the angina symptoms as reported in previous studies ,22. Our Other common differentials including MINOCA , hypercoagulability, vascular spasm, or coronary dissection should be considered when evaluating the cause of chest pain syndromes in young patients with low pretest probability for atherosclerosis. Although coronary angiograms were negative for obstructive CAD in both of our cases, full imaging studies should be considered to diagnose coronary anomalies in patients with a low pretest probability of atherosclerosis who come with angina equivalents, as these defects can have serious consequences. Our cases of two young patients are unique with similar cardiovascular risk factor profiles\u00a0and with the overlapping presentation of angina symptoms but yet different left ventricular systolic function leading to severe clinical heart failure as demonstrated in our first patient.Myocardial bridging is one of the infrequent benign congenital anomalies featuring coronary arteries where an epicardial coronary artery follows an intramyocardial course, commonly involving LAD. Changes in flow dynamics from this condition can result in accelerated atherosclerosis in the portion proximal to the bridged segment sparing the bridging segment itself. Though several detrimental cardiac conditions including sudden cardiac death have been considered as a consequence of myocardial bridging, making it attributable as the only cause of the events is still debatable. Cardiac computed tomography angiography, intravascular ultrasound, and CT-based noninvasive fractional flow reserve could be proved helpful in clinical practice in terms of the study of this clinical attribute but yet need more evidence. Medical therapy is preferred as first-line therapy whereas interventional procedures have also been attempted for refractory symptoms. Further correlation and research should be encountered to define prevalent patient communities to provide most benefit from intervention as well as to categorize best treatment strategy."}
+{"text": "The combination of flexible-printed substrates and conventional electronics leads to flexible hybrid electronics. When fabrics are used as flexible substrates, two kinds of problems arise. The first type is related to the printing of the tracks of the corresponding circuit. The second one concerns the incorporation of conventional electronic devices, such as integrated circuits, on the textile substrate. Regarding the printing of tracks, this work studies the optimal design parameters of screen-printed silver tracks on textiles focused on printing an electronic circuit on a textile substrate. Several patterns of different widths and gaps between tracks were tested in order to find the best design parameters for some footprint configurations. With respect to the incorporation of devices on textile substrates, the paper analyzes the soldering of surface mount devices on fabric substrates. Due to the substrate\u2019s nature, low soldering temperatures must be used to avoid deformations or damage to the substrate caused by the higher temperatures used in conventional soldering. Several solder pastes used for low-temperature soldering are analyzed in terms of joint resistance and shear force application. The results obtained are satisfactory, demonstrating the viability of using flexible hybrid electronics with fabrics. As a practical result, a simple single-layer circuit was implemented to check the results of the research. Printed electronics is a recent technology that permits the manufacture of electronic circuits on a wide variety of substrates. Among the materials used as substrates, the electronic circuits developed with flexible substrates, such as textile substrates ,2, have Printing technologies have evolved, providing a gradual improvement of the resulting printed circuits. The technologies for printing, such as screen printing ,10, inkjRegarding the employed materials, great progress has also been made in the development of functional inks. They incorporate new materials ensuring, at the same time, adequate viscosities, and suitability. This allows them to be printed on a wide variety of printing equipment and substrates. Nanoparticle-based inks of many conductive materials have been developed, such as Ag ,19, Au ,21, Cu ,22,23, NThe conductive tracks printed with the mentioned materials can be combined with surface mount electronic devices (SMD) giving rise to the so-called flexible hybrid electronics. Flexible hybrid circuits have had a great development on plastic substrates, but this kind of electronics using textile substrates presents some issues to be addressed. On the one hand, those relative to the printing of the conductive tracks on the textile substrate and, on the other hand, those concerning the incorporation of the SMD to these conductive tracks by soldering. Moreover, there exist problems that are intrinsic to the textile itself, such as washability, behavior under environmental changes (temperature and humidity), or bending that must be considered as well ,29,30,31Regarding the printing of conductive tracks on textile substrates, the first issue found is related to the lack of specific inks for this kind of substrate. This means that it is necessary to check the most compatible ink with the textile substrate used. Moreover, not all textiles are likely to be used as substrates, since parameters such as thread material, thread diameter, roughness, or type of weave can make printing impossible . In connThe number of layers to be printed must also be considered, since usually, and due to the complexity of the circuits, the designs are multilayer. In this case, it is necessary to print conductor and dielectric layers, alternately, until the multilayer design is achieved. With textile substrates, short circuits between conductive layers are highly probable due to the difficulty of ensuring a good printing of the dielectric layers . Only whRegarding SMD, printing issues are found when using textile substrates, related to fit pad size limits when printing . HoweverThe contribution of this paper is double. On the one hand, the paper determines the optimal design parameters (pattern classes) of screen-printed silver tracks on textiles, focused on printing an electronic circuit on a textile substrate. Even though the printing of the tracks has been successfully achieved in several works ,41, it iThe mechanical behavior of fabric can be characterized by parameters such as resistance or deformation. In electronic soldering, it is crucial to study the deformation of the fabric when applying temperature. For instance, a shrinkage of the fabric will reduce the size of the solder pads and may resist soldering or produce conductivity failures in it.Natural fibers such as cotton, linen, silk, or wool have a burning point ranging from 148 \u00b0C for silk and up to 570 \u00b0C for wool . ManufacIn addition, below the Ts, mechanical deformations may occur. In polyester PET, with a Ts of 238 \u00b0C, mechanical deformations begin to occur from 140 \u00b0C , in cottThe preeminent solder in the area of electronics has been based on Pb. Sn-Pb eutectic solder has been used for decades due to its excellent properties, good wettability, high reliabilities, and low cost . The RoHLow-temperature solders permit less thermal deformation of the components, increasing the quality of the solder joint due to a lower deformation of the components. In addition, they provide a reduction in costs due to the materials used and the energy-cost saving .In general, low-temperature solders are based on Sn or In alloys. Depending on these two elements, three groups can be established: Sn-Bi based, Sn-Zn based, and In (In-X) based.Sn-Bi solder has good properties due to its low melting point. 42Sn-58Bi eutectic has a melting point of 138 \u00b0C. Other advantages are its low cost, due to the lower cost of Bi , the redIn-based solder has a very low melting point, good conductivity, good thermal conductivity as well as, fatigue resistance, ductility, and wettability. 52In-48Sn eutectic has a melting point of 118 \u00b0C. However, its cost is appreciably higher, due to the high cost of In ,50.Sn-Zn lead-free solder has a low melting point. 91.2Sn-8.8Zn eutectic has a melting point of 199 \u00b0C. However, this alloy has some problems such as poor wettability, coarse structure, and low oxidation resistance .Sn-Bi-based solder alloys have a low melting point solder and reliable properties. Sn-58Bi with a melting point of 138 \u00b0C is a representative solder with low cost, good solderability, and environmentally friendly characteristics .To improve the properties of the different alloys, additional alloying elements can be incorporated. Jiang, N et al.  reviewedThe fabric used for the study is a 100% waterproof cotton substrate with the characteristics shown in A shrinkage study was carried out to determine the percentage of shrinkage of this fabric and provide a treatment that allows a 0% shrinkage for the thermal treatment of curing the silver ink and soldering. In the first process, the fabric was subjected to washing with neutral soap using six resistant cotton programs at temperatures of 90, 60, 50, 40, 30 \u00b0C, and a cold wash, with a spin cycle at 1200 rpm in all programs. The washing machine used was the model WM12E467EE . In the second process, the fabric was subjected to steam ironing at 215 \u00b0C using the G9222F0 model . The third process was to introduce it into an oven FED-115  at 130 \u00b0C for 30 min, using the same curing cycle as conductive silver inks. Finally, it was subjected to an oven temperature of 200 \u00b0C for 3 min, higher than 191 \u00b0C which is the melting point temperature of the Sn-Pb eutectic, using the same temperature cycle as the one used in a typical solder paste.When an electronic circuit needs to be printed, it needs to be as dimensionally stable as possible. Therefore, it is not necessary to use shrinkable inks, these can be used to join different circuits in a textile . The mosIn printed electronic circuits, it is necessary to determine the minimum sizes for the track (conductor) and the gap (insulation) to make electronic circuits on any substrate. These two parameters determine the so-called pattern classes. The track width (TW) and the gap between conductors, namely track-to-track (TT), track-to-pad (TP), and pad-to-pad (PP) gaps need to be defined.To carry out a study of these parameters using the aforementioned three inks, a specific pattern  with a sThe designed pattern consists of tracks of different sizes and orientations, as well as a circle and a serpentine that permitted the investigation of the printing that resulted. On the other hand, a series of SMD land patterns were included to assess the effect of printing on its dimensionality. The dimensions of the land patterns were based on the recommendations of the standard IPC-SM-782. For these land patterns, the minimum track width (TW) was 10 mils (0.254 mm), and the minimum pad-to-pad gap (PP) was 8 mils (0.203 mm).Groups of four tracks were drawn, in the direction of the weft (0\u00b0), warp (90\u00b0), and at 45\u00b0 and 315\u00b0. The widths of these tracks (TW) were 12, 15, 20, 25, 30, 40, and 50 mils . The gaps between tracks (TT) in each group were 12, 15, 20, 25, 30, 40, and 50 mils , respectively. In addition, four tracks of 80 mils (2.032 mm) with a length of 3937 mils (100 mm) were drawn.In some of the SMD land patterns, tracks were drawn between pads, in this case, the width used was 10 mils (0.254 mm). Finally, two serpentines at 0\u00b0 and 90\u00b0, and a ring with a width of 30 mils (0.762 mm) were also drawn.In order to print the pattern, the manufacturing technology used was serigraphic technology. To check the effect of the thickness and resolution of the conductor layer, three types of mesh were used for the screens, namely PET 1500 90/230-48 PW, PET 1500 130/330-34 PW, and PET 1500 150/380-34 PW  with 90, 130, and 150 threads/cm, respectively. The main features of the mesh are shown in Printing was carried out using an E2XL from an EKRA screen printer  with a shore 75\u00b0 hardness squeegee, 60\u00b0 squeegee angle, 1 mm snapoff, 3.5 bar force, and 100 mm/s. After the deposition of the inks, these were cured in an air oven FED-115 from BINDER at 130 \u00b0C for 15 min to use the same curing characteristics for all the inks.One parameter that affects the thickness and width of the track is the number of printed layers. Normally a single print is made, but it is usually insufficient in textiles since, in this case, the tracks do not conduct due to the rough surface of the textile itself that makes printing correctly difficult. J. Ferri et al.  demonstr\u00ae software .The dimensional characterization was carried out with a magnifying glass AM9915MZT from Dino-Lite . Resistance measurements and electrical continuity were made with a Fluke 8845A multimeter . The resistance was measured using the four-probe method. Data were sent to a PC via RS232 connection, they were stored and analyzed with FlukeViewThe solder pastes used in this study are commercially available. Indium NC-SMQ80 Indalloy #1E of Sn-In alloy and Indium5-LT Indalloy#281 of Sn-Bi alloy , TS391lT50  of Sn-Bi-Ag alloy, and SSLTNC  of Sn-Bi-Ag alloy were used .In order to check the soldering of the four solder pastes, different tests with a second pattern  were desOn the 0805 land pattern, 10 k\u03a9 resistors (CRGP0805F10K) were soldered; on 1206, LED diodes (LTST-C150CKT), on SOT23, double diodes (TPS7A0533PDBZR), and finally, on SOIC8, operational amplifiers (NCV4949CDR2G) were used.Solder paste printing was carried out using stencil printing. The screen printer was an E2XL from the EKRA  with a stainless steel stencil of 150 \u00b5m, 90\u00b0 squeegee angle, 0 mm snap-off, 2.5 bar force, and 50 mm/s. Then, components were placed using a pick and place ProtoPlace S .The continuous five-zone reflow oven AE-R330  was used for soldering the samples.The profiles supplied by the ink manufacturers were based on their use with PCBs, so the profile had to be modified to suit textiles. For the experiment, the soldering profiles were based on datasheets by recommendation of solder paste manufacturers and were adjusted until a correct soldering was obtained. The measurements of the temperature of the different profiles were obtained using a thermocouple data logger USB TC-08 .\u00ae software .The joint electrical resistance among the pad of the component, the soldering, and the land pattern, can give an idea of a good soldering. Resistance measurements were made with a Fluke 8845A multimeter . The soldering joint resistance was measured using the four-probe method. Data were sent to a PC via RS232 connection and they were stored and analyzed with FlukeViewThe shear test method is designed to test and evaluate the endurance of the solder joint between component terminals and lands on a substrate, using a shear-type mechanical stress (standard IEC 62137-1-2:2007). \u00ae voice coil linear actuator with force sensor from Physik Instrumente (PI) GmbH & Co. KG  was used.To measure the shear strength of welds, a V-275.431 PIMagAll optical characterization was carried out with a magnifying glass AM9915MZT from Dino-Lite .Washing at 90 \u00b0C, ironing, and curing at 130 \u00b0C will be used as a previous treatment applied to the textile to obtain a substrate as stable as possible for the soldering stage.The most relevant data obtained from the study on pattern one are presented since the whole study exceeds the scope of this publication. The used nomenclature is the following: M3, M4, and M5 corresponding to the silver inks SCAG-003, SCAG-004, and SCAG-005, respectively; T12 and T15 for the tracks of 12 mils (0.305 mm) and 15 mils (0.381 mm), respectively.To determine the correct printing of the pattern, an electrical continuity test was carried out using a Fluke 8845A multimeter. As a result, it can be noted that a high percentage of the results present open circuits a or shorThe percentage of shorts was low, with the worst case being lower than 30%, due to the excessive ink deposition because of the increase in the number of printings. Most of the shorts were found with four printing layers, with meshes of 90 and 130 threads/cm, and, particularly with M4.Thus, from As observed in Once the best printing configurations were determined to ensure the physical integrity of the track, the influence of the printings on the track resistance was studied. Based on this unique printing configuration, a study of the value of the resistance as a function of the orientation was conducted. The individual value of resistance can be seen in A fundamental parameter in the development of electronic circuits is the resolution of the track width (TW) obtained in the printing. Several factors can influence this parameter: First, the type of ink due to the particle size it contains. Mateprincs does not give exact information about the particle size but indicates that it is less than 10 \u00b5m; Second, the mesh of the screen, since the greater number of the mesh, the higher the resolution obtained; Third, the number of printed layers, since the greater number of layers, the higher the width obtained.From the results, the average variation of the track width depending on the orientation and for tracks between 15 and 80 mils (0.381 and 2.032 mm) is 6.14% for 0\u00b0, 1.09% for 45\u00b0, 6.47% for 90\u00b0, and 5.26% for 315\u00b0. These data coincide with the average resistance of the set of tracks from 15 to 80 mils (0.381 to 2.032 mm), shown in As mentioned previously, the number of prints influences the resolution. The results obtained show that the track width of 15 mils (0.381 mm) printed at 0\u00b0 with M5 ink and a mesh of 90 threads/cm, ranges between 17.74 \u00b1 1.90 mils (0.45 \u00b1 0.05 mm) with two printings, and 21.95 \u00b1 1.78 mils (0.56 \u00b1 0.05 mm) with four printings. The number of the mesh also influences the result, the track width of 15 mils (0.381 mm) printed at 0\u00b0 with M5 ink and a mesh of 150 threads/cm, ranges between 15.64 \u00b1 1.27 mils (80.39 \u00b1 0.03 mm) with two printings, and 15.75 \u00b1 2.76 mils (0.40 \u00b1 0.07 mm) with four printings.The variation in the width of the tracks implies changes in the land patterns, but this variation is not so significant that welding failures occur. The reflow solder oven used only allows for configuring the temperature in each of the five stages of the curing process. The temperature of each stage was adjusted until the temperature curve was as close as possible to the profile indicated by the solder paste instructions. During the adjustments, it was noted that the reflow point changed depending on the substrate. Hence, the reflow soldering profiles were tuned adapting the temperature values to the substrate used. The final profiles obtained are shown in The soldering joint resistance of the packages presented in X axis for three types of land pattern. In general, the solder joint cannot be considered to be very good compared to similar solder joints on PCBs, but based on the results of shear-force tests, they can be considered acceptable. The observation of the optical magnifications  shows anA simple oscillator circuit based on the IC555 was designed  and implThe direct realization of electronic circuits in textiles is not feasible in the case of complex circuits, with multilayer layouts and small track widths or minimum separation between tracks. In these cases, it is better to implement the circuit on a flexible PCB and incorporate it into the textile. However, there are basic circuits that may be interesting to implement directly onto textile fabrics, for instance, an amplifier as close to an electrode as possible. In this case, the layout can be a single layer, with less-demanding track sizes and widths. For those cases, it would be interesting to carry out a study to determine the best way to implement the circuit on a textile substrate.The choice of the textile substrate is critical, and tests must be carried out to obtain a good printing of the conductors. Once the best textile substrate has been selected, previous treatments, such as washing, ironing, and drying, must be applied to avoid size modifications of the textile during the track printing and SMD soldering stages. Next, the minimum track width and separation between tracks must be established to be able to implement the circuit. The influence of the tracks on the resistance of each net must also be analyzed. This will be determined by the type of ink, the number of prints, and the type of mesh used. Lastly, the best low-temperature solder paste, compatible with the component and conductive trace, must be determined.In this work, a 100% waterproof cotton substrate with the most compatible silver ink was used. The best printing option was achieved for three layers of conductive ink, printed with a mesh of 130 threads/cm. Under these conditions, the minimum track width obtained, as well as the separation between tracks, was 15 mils (0.381 mm). The best results were obtained with SRA SSLTNC solder paste, possibly due to its composition of In and Ag. The verification of this process was assessed with a simple single-layer circuit."}
+{"text": "An effective System-on-Chip (SoC) for smart Quality-of-Service (QoS) management over a virtual local area network (LAN) is presented in this study. The SoC is implemented by field programmable gate array (FPGA) for accelerating the delivery quality prediction for a service. The quality prediction is carried out by the general regression neural network (GRNN) algorithm based on a time-varying profile consisting of the past delivery records of the service. A novel record replacement algorithm is presented to update the profile, so that the bandwidth usage of the service can be effectively tracked by GRNN. Experimental results show that the SoC provides self-aware QoS management with low computation costs for applications over virtual LAN. Basic Internet services are usually delivered on a\u00a0best effort basis, without taking any quality requirements into consideration. To\u00a0satisfy the\u00a0demands of applications and users in\u00a0the\u00a0network, Quality-of-Service (QoS) management ,2 is usuSoftware-defined networking (SDN)  is a\u00a0tecThe study in  builds aAn approach for solving the\u00a0latency issues for QoS management is the\u00a0employment of a hardware accelerator for NFV. A field programmable gate array (FPGA) ,12 impleThe objective of this paper is to present a\u00a0novel system-on-chip (SoC) with record replacement for self-aware QoS management over a\u00a0virtual LAN. An\u00a0FPGA architecture of the\u00a0GRNN algorithm is proposed as\u00a0a\u00a0hardware VNF for the\u00a0prediction of delivery quality for a\u00a0service. Based on\u00a0the\u00a0prediction results, optimal bandwidth allocation is performed to the\u00a0service so that the\u00a0service can be delivered with desired quality. The\u00a0VNFs for traffic control operating in\u00a0conjunction with the\u00a0proposed FPGA-based QoS management VNFs are also implemented for the\u00a0virtual\u00a0LAN.In addition to providing fast computation for producing prediction results for delivery quality, the\u00a0proposed FPGA architecture contains a\u00a0dedicated circuit for profile updating. Apart from basic record appending and removal operations, the\u00a0dedicated circuit support online record replacement based on a\u00a0novel record replacement algorithm. In\u00a0this way, the\u00a0profile for GRNN prediction can be effectively updated even for a\u00a0small profile buffer. In\u00a0the algorithm, the\u00a0past records are separated into two groups: positive response group and negative response group. A\u00a0record with positive response implies the\u00a0delivery is achieved with satisfactory quality. Conversely, a\u00a0negative response record reveals that the\u00a0quality of the\u00a0delivery is below expected level. One of the\u00a0groups will be dynamically identified as the\u00a0insignificant group based on\u00a0the\u00a0most recently received record. In\u00a0the insignificant group, the\u00a0oldest record is\u00a0removed.Both analytical and numerical evaluations are provided for the\u00a0algorithms and systems presented in\u00a0this study. Analytical results show that the\u00a0GRNN-based prediction with the\u00a0proposed record replacement algorithm achieves self-aware prediction. Furthermore, numerical evaluations reveal that the\u00a0proposed smart SoC system offers accurate quality prediction for services for the\u00a0efficient bandwidth allocation of the\u00a0virtual LAN. All\u00a0the results reveal that the\u00a0proposed smart FPGA-based SoC is effective for the\u00a0exploitation of network resources for dynamic and self-aware QoS\u00a0management.The remaining parts of this paper are organized as follows. A number of neural networks, such as multilayer perceptron (MLP) and recurrent neural network (RNN) ,15,16, cThe auto-regressive integrated moving average (ARIMA)  and GRNNA drawback for the\u00a0GRNN-based prediction is the\u00a0high computational complexities because of the\u00a0employment of Gaussian kernels. One approach to accelerate the\u00a0computation is the\u00a0employment of FPGA techniques. Because\u00a0of high flexibility and high computation speed, FPGA has been found to be effective for the\u00a0hardware VNF implementations . ExampleThe GRNN prediction in  is impleThis section covers the\u00a0infrastructure for the QoS server, QoS level definition, GRNN-based quality prediction, and\u00a0the proposed QoS management algorithm in\u00a0detail. To\u00a0facilitate an\u00a0understanding of the\u00a0proposed algorithms, For the\u00a0virtual LAN considered in\u00a0this study, there are two or more domains. A\u00a0multi-link core network is responsible for the\u00a0communication among different domains. Only the\u00a0bridges in\u00a0each domain are connected to the\u00a0core network. There is a\u00a0QoS server in\u00a0the\u00a0LAN for QoS management. The\u00a0bridges carry out data forwarding operations subject to the\u00a0constraint of the\u00a0bandwidth allocated by a\u00a0QoS server. The\u00a0block diagrams of a\u00a0bridge and a\u00a0QoS server in\u00a0the\u00a0virtual LAN are shown in\u00a0We can see from The bridges in\u00a0the\u00a0LAN can operate in\u00a0a\u00a0general-purpose computing platform. It\u00a0contains a\u00a0virtual switch supporting link aggregation and traffic shaping based on\u00a0the\u00a0commands from QoS server by the Openflow protocol. In\u00a0addition, each bridge supports the\u00a0delivery of data packets to/from the\u00a0other bridges in\u00a0the\u00a0LAN by user datagram protocol (UDP). In\u00a0j reserved for the\u00a0service, and\u00a0n is the\u00a0number of links in\u00a0the\u00a0core network. LetR be the\u00a0actual source data rate of the\u00a0service. Note that R and In this study, we\u00a0define a\u00a0service as\u00a0a\u00a0dataflow between two appliances from different domains. The\u00a0service is delivered subject to a\u00a0QoS level, which is dependent on\u00a0the\u00a0redundant bandwidth reserved for a\u00a0service. Let L be the\u00a0number of quantization levels. Based on L, let Clearly, when ERAB in\u00a0 is positIn the\u00a0proposed algorithm, the\u00a0quantization result ls. From\u00a0, the\u00a0ERAT of expected service qualities for the\u00a0data delivery, where T values imply better service qualities. Given the\u00a0quantizer in\u00a0\u20039: \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Wait till the\u00a0end of the current time slot.\u200310: \u00a0\u00a0\u00a0\u00a0end if\u200311: end looprom P by .\u20034: \u00a0\u00a0\u00a0\u00a0Algorithm 1 summarizes the\u00a0operations of the\u00a0proposed algorithm. As\u00a0shown in\u00a0Algorithm 1, each service based on\u00a0the\u00a0 bandwidth allocation T, a\u00a0response T. Firstly, after\u00a0a negative response is acquired, the\u00a0algorithm will increase the\u00a0total bandwidth allocated to the\u00a0service. In\u00a0addition, the\u00a0algorithm will maintain or reduce the\u00a0total allocated bandwidth after a\u00a0positive response is obtained. In\u00a0the remaining parts of this section, we\u00a0show that the\u00a0proposed profile updating algorithm has the\u00a0advantage of being self-aware.To facilitate the\u00a0presentation of the\u00a0profile updating algorithm, we\u00a0first define positive responses, negative responses, and\u00a0self-aware QoS management. Given a\u00a0QoS level T, we\u00a0can rewrite (Given a\u00a0service with QoS level  rewrite  for the\u00a0te (O by  as(14)Op from ,,y\u2032 is deWe then rewrite  as(16)OFrom , we\u00a0see ows from  that S1\u2265We next consider the\u00a0scenario where the\u00a0new record Two cases are then studied separately: a\u00a0new positive response 21), it\u00a0f|x*(p+1)|For the\u00a0case of ive from  thatS1>0, S2>Therefore,Both  and 25)25) conclp grows as new records are acquired during the\u00a0service. Therefore, for\u00a0a service with long transmission, a\u00a0large profile may be produced. This\u00a0would increase the\u00a0computation overhead for QoS management. One way to solving the\u00a0issue is to maintain\u00a0the\u00a0profile size p as it\u00a0reaches a\u00a0predefined upper limit C. That is, when The profile size p reaches C, let Because the\u00a0replacement only occurs after the\u00a0profile size We can rewrite  and 27)27) asy\u2265T), it\u00a0Let Consequently, when the\u00a0new response is positive, and\u00a0the selected old record Define a\u00a0set We can then see thatAs a\u00a0result,Therefore, for\u00a0the cases of new negative responses, when the\u00a0old record to be replaced satisfies Although the\u00a0self-awareness can be achieved by the\u00a0proposed algorithm by record replacement, high computation complexities may be required for the\u00a0search of old record tisfying  or 36).. g y\u2265T and y yq\u2265T by  and 38)y\u2265T and y yq\u2265T by  and 36)y\u2265T and yOnly simple comparisons are necessary for  and 40)40) for tGiven a\u00a0new record tisfying  or 40)..{x*,y}, Algorithm 2 The Profile Updating\u00a0AlgorithmprocedureProfile_Update\u20032: \u00a0\u00a0\u00a0\u00a0if\u00a0then\u20033: \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u20034: \u00a0\u00a0\u00a0\u00a0else\u20035: \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Determine set \u20036: \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0if\u00a0then\u20037: \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u2003\u20038: \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0else\u20039: \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u2003\u200310: \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0end if\u200311: \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u200312: \u00a0\u00a0\u00a0\u00a0end if\u200313: \u00a0\u00a0\u00a0\u00a0\u200314: \u00a0\u00a0\u00a0\u00a0return\u00a0p.\u200315: end procedure\u20031: set Q by .\u20036: \u00a0\u00a0\u00a0\u00a0The target record to be replaced It is usually desired to employ a SoC for the QoS management in a LAN because of low power consumption and low deployment costs. However, because the processor of the SoC may have only limited computation capacities, the computation time of the software implementation of the algorithm in the SoC is high. By adopting the dedicated hardware circuits as the accelerator for the processor, we are then able to achieve realtime QoS management for the GRNN-based delivery quality prediction with dynamic profile updating.As shown in T provided by the interface unit, the goal of GRNN unit is to search for The GRNN prediction unit is a hardware implementation of operations in . Based oGiven a candidate x,xi) in , respectn format . In the he \u03c32 in  is choseThe ACC module is responsible for the computations of both In the ACC module, the computation of There is only a single FP divider for the computation of y\u2032 in  in the Dlt y^ in . As showK be the latency for updating K is given byp. They are determined by the latency of FP adders, multipliers, comparators, exponent operators, and/or dividers. Therefore, the latency K only grows linearly with p.Given a bandwidth allocation J be the number of search candidates. The number of candidates is dependent on the search step size The pipelined operations can also be extended for different search candidates lgorithm . FurtherWhen operations for different search candidates are not overlapping, is case,  decreaseThe profile updating unit contains the profile fined in  plays anAs shown in C-stage shift register supporting serial-in parallel-out (SIPO) operations, where C is the upperbound of the profile size. Therefore, each buffer accepts at most one response record at a time. All the registers in the buffers are connected to the output multiplexer shown in Both the positive response buffer and negative response buffer have the same architecture, as revealed in u and v be the value of the counter associated with positive response buffer and negative response buffer, respectively. Therefore, u valid records and v valid records are located in the first u stages and the first v stages of the shift registers in positive response buffer and negative buffer, respectively. Only the stages with valid records in the shift registers are accessed by the GRNN prediction unit.Because the actual profile size Given a newly received record p and u are incremented by 1, and v remains the same. When p and v are incremented by 1, and u remains the same.In this case, only record appending is necessary. Dependent on the value of The record replacement is required in this case because the size of the profile has already attained its upperbound. To carry out the replacement operations, the set v-th stage in the shift register of negative response buffer contains the oldest record. It is then removed. The record p remains the same. However, the value of v is decreased by 1 because of the removal operation for the negative response buffer. Furthermore, since the new record is appended to the positive response record, we increase the u by 1.From , we obseuffer by . The v-tu-th stage of the shift register of the positive response buffer is the oldest record, and is removed. The record u and v are incremented by 1 and decremented by 1, respectively. The profile size p remains the same.By contrast, when An example of record replacement for This section presents some experimental results of the proposed smart SoC, which have been deployed in the real LAN for QoS management. The setup for the experiments is first provided in detail. This is followed by the evaluations of hardware costs and computation speed of the SoC. The performance metrics of the SoC for QoS management in terms of DLR and RAB for different services are subsequently included with comparisons.As shown in Raspberry Pi 4 computers are adopted for the implementation of Bridge 1 and Bridge 2. The QoS server is built on Terasic DE-10 Nano board. The FPGA device for the DE-10 Nano board is Intel Cyclone V 5CSEBA6. The HPS associated with the DE-10 board is based on ARM Cortex A9 processor with 800 MHz clock rate The proposed FPGA accelerator has been simulated, implemented and mapped in FPGA using Cyclone V 5CSEBA6. The ModelSim is the simulator for the RTL level verification. Furthermore, the Qsys is used for building the SoC for the evaluation of the proposed algorithms and architectures for QoS management. The FPGA accelerator operates at maximum clock rate of 50 MHz.j-th link of the core network. In the proposed link aggregation scheme, R is the total source data rate. The flow tables for packet matching operations in the virtual switch is used for traffic shaping. The matching rules for the flow tables are updated by the SDN controller in the QoS server. An Openflow controller operating in accordance with the bandwidth allocation results from the proposed algorithm is adopted as the SDN controller. The OpenFlow protocol [The virtual switch in each bridge is implemented by the Open vSwitch (OVS) . In our protocol  is used C, the number of links n, and the number of service quality levels L for the GRNN prediction unit and profile updating unit. The analysis is based on the big-O function.In the proposed FPGA accelerator, both the arithmetic operators and memory buffers are the major contributing factors to the utilization of hardware resources. The arithmetic operators are the FP adders, FP multipliers, FP accumulators, exponent operators, FP dividers, and FP comparators. The memory buffers are the shift registers. C, n and L. This is because only 2 FP accumulators, 1 exponent operator, and 1 FP divider are used for GRNN prediction, as revealed in It can be observed from n because of the squared distance computation in SDC unit. We also conclude from L for the quantization operations in QUAN unit shown in C and n. This is because the shift registers are used for the implementation of positive response buffer and negative response buffer, as shown in n and/or high number of quality levels L. However, it is usually desired to have a high upper bound of profile size C so that robust GRNN prediction could be achieved.We see from C to profile sizes. The area costs considered in the table are Adaptive Logic Modules (ALMs), dedicated registers, embedded memory bits and DSP blocks. It can be observed from C. This is because the DSP blocks are mainly used for the implementation of arithmetic operators. Both ALMs and dedicated registers are used for the implementation of buffers for the profile updating unit. Therefore, their utilizations grow with C. In fact, when J in   are alsoWe can observe from T. In addition to QoS level Because the proposed algorithm is self-aware, it has low RAB and DLR for tracking source data rates, as revealed in C, C has lower average RAB and average DLR values. This is because larger number of past response records are available for more accurate quality prediction. Furthermore, given C, lower average DLR values can be attained by adopting QoS level with higher T at the expense of larger average RAB values. While attaining accurate tracking for input source data rates, the proposed algorithm is able to provide high flexibilities for QoS management by specifying different upper bound for profile sizes and QoS levels for data delivery.To evaluate the impact of the performance of the proposed algorithm on the upper bound of profile size A smart SoC has been successfully deployed for QoS management in a virtual LAN. An FPGA accelerator has been implemented for the GRNN-based service quality prediction so that the bandwidth allocated for a service can be optimized with low computation latency. Both analytical and numerical studies have been provided for demonstrating the self-awareness of the proposed algorithm for QoS management. Subject to the constraint on the profile size, the analytical study shows that the proposed profile updating algorithm is still able to maintain self-awareness. Numerical results reveal that the proposed FPGA accelerator utilizes only limited hardware resources, even for large profile size upper bounds. When applied for QoS management, the SoC based on the FPGA as an accelerator has low latency for finding the optimal bandwidth allocation and profile updating. The proposed SoC therefore is beneficial as a hardware VNF for effective QoS management over virtual LAN with low implementation costs."}
+{"text": "Spectrum sensing in Cognitive radio (CR) is a way to improve spectrum utilization by detecting spectral holes to achieve a dynamic allocation of spectrum resources. As it is often difficult to obtain accurate wireless environment information in real-world scenarios, the detection performance is limited. Signal-to-noise ratio (SNR), noise variance, and channel prior occupancy rate are critical parameters in wireless spectrum sensing. However, obtaining these parameter values in advance is challenging in practical scenarios. A lifting wavelet-assisted Expectation-Maximization (EM) joint estimation and detection method is proposed to estimate multiple parameters and achieve full-blind detection, which uses lifting wavelet in noise variance estimation to improve detection probability and convergence speed. Moreover, a stream learning strategy is used in estimating SNR and channel prior occupancy rate to fit the scenario where the SU has mobility. The simulation results demonstrate that the proposed method can achieve comparable detection performance to the semi-blind EM method. With the rapid development of mobile intelligent terminals, wearable devices, and industrial monitoring in 5G applications, the amount of mobile data traffic is growing exponentially . To ensuGenerally speaking, different detecting techniques require different amounts of wireless environment information. According to the amount of prior information required, spectrum sensing methods can be divided into un-blind detection, semi-blind detection, and full-blind detection. The likelihood ratio test (LRT) based on the Neyman-Pearson criterion is currently known as the optimal detection method, which requests a known distribution of the received signal that is generally related to the PU information, the noise variance, and channel statistic characteristics. Therefore, the LRT is an un-blind detection method . ConversOwing to the intricacy of the full-blind approach, several studies employed a semi-blind approach. The semi-blind detection method expects knowledge of certain parameter values such as noise variance, PU information, or channel occupancy. Energy detection (ED) is widely used in spectrum sensing due to its easy implementation. However, the detection accuracy is susceptible to noise uncertainty, as the noise variance should be known to calculate its threshold value . In addiCompared with full-blind detection, semi-blind detection has the advantage of being easy to implement and relies on the assumption of known noise variance. In practical scenarios, it is difficult for semi-blind detection methods to obtain real-time noise variance values, which makes semi-blind detection face challenges in practice where noise uncertainty exists. The semi-blind detection method with known noise variance can availably serve as a full-blind detection method in spectrum sensing when noise variance estimation is conducted. In noise variance estimation, some special channels for noise variance estimation can effectively avoid interference from the PU signal, but this estimation method sacrifices spectrum utilization ,20. MoreDue to the limitations of path loss and multipath fading, local spectrum sensing performance may be affected. When reusing the licensed spectrum, it is usually hoped to minimize the impact on the PU, which relies on highly reliable spectrum sensing techniques for global sensing of the wireless channel. Cooperative spectrum sensing (CSS) using spatial diversity among SUs is a good solution to achieve highly reliable spectrum sensing . In CSS,In this paper, we investigate full-blind cooperative spectrum sensing based on the classical optimal LRT. In order to eliminate the coupling between the unknown parameter estimates, we independently discuss the noise variance estimation problem and combine it with the EM estimation algorithm to achieve fully-blind detection. The main contributions are: (1) in un-blind optimal likelihood ratio detection, many parameters need to be known in advance. Traditional full-blind detection approaches are hard to use in practice as they have high computational complexity. When the parameters are unknown, the detection problem becomes a composite hypothesis. In order to achieve full-blind spectrum sensing with multiple unknown parameters, the EM algorithm is used to estimate the noise variance, SNR of the received signal, and channel prior occupancy rate. (2) Besides, a simple noise variance estimation method based on the lifting wavelet transform is used in the EM algorithm to further shorten the detection time and improve the detection performance and fitness for actual scenarios with noise uncertainty. (3) Finally, the stream learning strategy based on historical information is used in estimating SNR and the channel prior occupancy rate, making the proposed method adaptable to the scenario where the SU has mobility.The remainder of this paper is outlined as follows: The corresponding system model and the optimal detection problem are described in J SUs, which is shown in It is assumed that the cognitive radio network consists of one PU, one FC, and j is defined as [j is denoted by m-th sampling point of the PU signal, which follows a Gaussian distribution with a zero mean. j with variance According to Neyman-Pearson criteria, the spectrum sensing problem can be modeled as a binary hypothesis test, and the received signal sample at the SUfined as (1){yj, the probability density function (pdf) of The detection statistic, SNR, and noise variance for all SUs in the entire network are denoted as According to the likelihood ratio detection principle, the decision expression based on the log-likelihood ratio function for the whole work can be written as (9)L\u210b1(TBy substituting (5) and (7) into (9), the decision expression can be simply described asAccording to (10), the decision can be made if the SNR and the noise variance for each SU are known. However, these parameters usually vary in a dynamic environment. In order to determine the state of the channel, it is necessary to estimate the SNR and the noise variance of each SU. The unknown parameter vector is defined as Since the channel occupancy is unknown when SUs acquire the detection statistic of ariables . In the n-th detection, the n-th detection statistic for the j-th SU. According to Bayes\u2019 rules, In the The conditional expectation of (8) given the posterior probability of the channel occupancy state of n-th round of detection are obtained by maximizing the In the M-step, the corresponding estimation parameters of From (15), the conditional expectation By substituting (18) into (15) and let the Finally, we compute the The detailed solution to the optimization problem is given in According to the above derivation, this multi-parameter estimating method using the EM principle is denoted as a fully Blind-EM estimation and detection method.Because the estimated values of noise variance and SNR are coupled with each other, the performance of multi-parameter estimation will be affected in fully Blind-EM estimation and detection. Consequently, we consider decoupling the two estimates. Since the noise variance in the received signal is mainly concentrated in the high-frequency part and the wavelet transform can realize the variable resolution decomposition of the signal, the noise variance value can be estimated by using the high-frequency coefficient decomposed by the wavelet transform.The dynamic environment caused by SU mobility puts forward a higher demand for detection delay; a shorter detection delay will help the fusion center estimate and track the changes in various parameters promptly. The traditional wavelet transform relies on the Fourier transform and has complex operations, so it is not suitable for dynamic scenarios. The lifting wavelet can realize the specific wavelet function by designing different prediction operators and update operators, which has the advantages of fast decomposition speed and small memory consumption and is completely equivalent to the traditional Mallat algorithm . A low-cp-th layer is shown in k represents the number of prediction times.The decomposition process of the Similarly, p = 1 is considered, the db2 wavelet transform can be achieved by performing two predictions and one update, and the corresponding operator can be written asIf the case of The specific calculation process is as follows:In the end, by multiplying the normalization factors by the final wavelet factorization coefficient, the following can be obtained:The noise variance can be estimated using the high-frequency coefficients obtained from the current period\u2019s decomposition .(27)\u03c3^jThe noise variance estimation conducted in the Blind-EM algorithm is replaced by the estimation through the lifting wavelet, which is called the lifting wavelet-assisted EM iteration joint estimation method (WEMJD), which performs estimation of other unknown parameters by setting Compared with batch learning, which needs to wait until all the observed data are obtained, online learning is better suited for scenarios where data arrives continuously . The parBy replacing (19) and (21) with (29), the final estimating expression of SNR and channel prior occupancy rate can be expressed, respectively, asAs the noise samples are independent of each other and do not have a correlation, it should be noted that historical learning is not suitable for predicting and tracking the irregular changes in the noise variance. Hence, only the sliding window is used to smooth the noise variance estimation results.Due to the sensitivity of the EM algorithm to the initial values, reliable initial values can help the EM algorithm converge to the global maximum and greatly reduce the iteration time of the algorithm. Since the PU activity status is independent of the noise variance and SNR, it is assumed that the probability of the channel being occupied and unoccupied is equal at the initial time, therefore To evaluate the performance of a detection method, its ability to correctly detect the presence of a target signal can be determined by using the false alarm probability and detection probability.The ability of the detection method to correctly detect the presence of a target signal can be described by using the false alarm probability and detection probability. The detection probability Based on the Neyman-Pearson criterion, the threshold value should maximize the detection probability with constraint d degrees of freedom forms like In general, the exact distribution of the weighted sum is difficult to obtain, So we approximate R=\u03b1\u03c7d2+\u03b2 ,41,42. TLet the first three cumulative moments of The approximate distribution of The value of Refer to J SUs, M sampling points, and N detection times. At FC, for WEMJD, Semi-blind EM and GLRT have the same computational complexity is M is the same size as N, while J is usually smaller than M and N, so the proposed method has a lower degree of complexity. At SUs, we assume that the length of the prediction operator is p, the complexity of the noise estimation part is The algorithm complexity is evaluated under the case of In this section, the simulation results under different settings are shown. To make the results more realistic and reliable, we performed j.In the network, due to the random distribution of SUs, differences in geographic location also bring about differences in received SNR. Therefore, we define the global average SNR asn-th detection is defined asIn order to evaluate the estimation performance of the proposed algorithm, the mean squared error (MSE) is used as the evaluation metric. The MSE for the To discuss the convergence of the method, it is first assumed that the secondary users are stationary and have the same nominal noise variance of The setting of the false alarm probability is related to the threshold value. From By taking account of the scenario of changing channel conditions, it is assumed that the SUs exhibit mobility, which means that the received SNR at the SUs changes over time. In the presence of To observe the sustained performance of the method, the simulation parameters are set as To observe the performance of different estimation and detection methods with noise uncertainty. It is assumed that the noise variance follows a uniform distribution within a certain range, It may be noted that the false alarm probability of the proposed method fluctuates around the set value as the global average SNR is varied, which is shown in As it is often difficult to obtain accurate wireless environment information in real-world scenarios, the estimation and detection methods of cooperative spectrum sensing in dynamic environments are studied in this paper. Based on the LRT theory, it is required to estimate the noise variance, the signal-to-noise ratio, and the channel prior occupancy in the blind scenario with varied channel conditions. A lifting wavelet-assisted EM joint estimation and detection algorithm is proposed to estimate multiple parameters and achieve full-blind detection. Moreover, the lifting wavelet technique is applied in noise variance estimation to improve detection probability and convergence speed. At the same time, a stream learning method is used in estimating SNR and channel prior occupancy rate to fit the dynamic environment. The simulation results demonstrate that the proposed full-blind method can achieve comparable detection performance to the semi-blind EM method. It can adapt to mobile scenarios where the SNR varies over time and exhibit a certain robustness against noise uncertainty."}
+{"text": "Hematoloechus sp., H) and their impact on systemic and individual capillary variables measured in pithed Rana pipiens, a long-standing model for studies of capillary physiology. Three groups were identified based on Hematoloechus attachment: no Hematoloechus (No H), Hematoloechus not attached (H Not Att), and Hematoloechus attached (H Att). Among 38 descriptive, cardiovascular, and immunological variables, 18 changed significantly with H. Symptoms of H included weight loss, elevated immune cells, heart rate variability, faster coagulation, lower hematocrit, and fluid accumulation. Important capillary function discoveries included median baselines for hydraulic conductivity (Lp) of 7.0 (No H), 12.4 (H Not Att), and 4.2 (H Att) \u00d7 10\u22127 cm\u00b7s\u22121\u00b7cmH2O\u22121 (P < 0.0001) plus seasonal adaptation of sigma delta pi . Pro- and anti-inflammatory phases were revealed for Lp and plasma nitrite/nitrate concentration ([NOx]) in both H Not Att and H Att, whereas capillary wall tensile strength increased in the H Att. H attachment was advantageous for the host due to lower edema and for the parasite via a sustained food source illustrating an excellent example of natural symbiosis. However, H attachment also resulted in host weight loss: in time, a conundrum for the highly dependent parasite. The study increases overall knowledge of Rana pipiens by revealing intriguing effects of H and previously unknown, naturally occurring seasonal changes in many variables. The data improve Rana pipiens as a general scientific and capillary physiology model. Diseases of inflammation and stroke are among the clinical applications.This prospective, descriptive study focused on lung flukes ( Rana pipiens (North American leopard frog) has been a reliable animal model used for a wide variety of scientific investigations into organ and tissue physiology as well as for student laboratory demonstrations. In the United States, Rana pipiens are caught in the wild and sold by companies located primarily in the eastern half of the country. In our laboratory, Rana pipiens was selected to study capillary physiology as it is a lower vertebrate that spawned and matured in a natural environment.For over a century, Hematoloechus sp. (H) was reported first in the early 19th century. H have a complicated taxonomy and at least 89 species of the genus are known to exist . Within the snail, the eggs hatch into sporocysts that produce cercaria, the larval form of trematodes. Cercaria are then shed from the snail and enter dragonflies or damselflies (odonata). When a frog (or toad) eats a dragonfly or damselfly and if that odonate has cercaria, the parasite makes its way through the esophagus and lodges in the lungs within 5 days. Once the lung fluke is located in its definitive host, the life cycle of H is complete , Rana pipiens has long been considered a gold standard model. Data from frog capillaries have been published for decades in primary and review articles  and surface area (S) of each capillary are measured directly. Blood perfusion of each capillary is maintained up to the moment of cannulation, and neural input and lymph flow remain intact. The approach was first used by Landis in 1927 ; weight = 34.8 (SD 6.0) g; length = 19.7 (SD 0.8) cm; # H (median (\u00b1 25%/75%) = 2 (\u00b1 1/4), range = 1\u201322); 22 samples over 7 yr; Charles D. Sullivan Co., Nashville, TN] were determined to be healthy by visual inspection. Upon arriving at the animal care facility, each frog was examined and placed in a gentamicin (1.3 mg\u00b7mL\u22121) bath for the first 24 h. The frogs were then moved to containers with dry areas and fresh, dechlorinated water that was filtered continuously. The frogs were cage sedentary and housed six per container. Containers were placed on racks in a room where the environment was maintained at 15\u00b0C, and light was controlled on a 12-h:12-h light:dark cycle. The frogs were fed weekly. Once every 2 weeks they were fed individually using a 1-mL syringe filled with vegetable beef baby food (Gerber), and on the alternate week, crickets were released into each container. Data were collected from 2010 to 2017. Protocols were approved and monitored by the Animal Care and Use Committee at Montana State University, Bozeman, MT.Wild-caught \u22121) from a reservoir and siphoned away with a vacuum line. The tissue and its microvascular bed remained intact, attached to the frog, and blood-perfused during the entire protocol.Each frog was pithed cerebrally, and one drop of blood was placed on a glass slide for measurement of clotting time. Cotton was placed in the cranial cavity to separate cerebral and spinal column nerve tissue. Body weight, length, and displaced volume were measured, and the frog was placed supine on a pad and heart rate was counted. An incision was made through the skin and muscle wall to expose the contents of the abdomen. A loop of small intestine was exteriorized and draped carefully over a quartz pillar, which allowed the mesenteric tissue to lay flat atop the pillar. The entire tissue preparation was kept cool  and moist with frog Ringer solution superfused continuously (\u2248 3 mL\u00b7minx]). Next, a small incision was made in the abdominal wall, and lymph fluid was collected for later analysis of abdominal cavity fluid protein concentration ([abpr]). Surgical and body fluid sampling procedures have been published previously . After the area between the skin and abdominal wall was carefully swabbed dry, blood was drawn into three heparinized microhematocrit tubes  from a peripheral vein located along the interior surface of the skin near the right axilla and exterior to the abdominal cavity. The first sample was used for measurement of systemic hematocrit (sysHct) and plasma protein concentration ([plpr]), the second for systemic red blood cell concentration ([RBC]), white blood concentration ([WBC]), activated white blood concentration (a[WBC]), and hemoglobin concentration ([Hb]), and the third was prepared for measurement of plasma nitrite/nitrate concentration  was dialyzed  and dissolved in frog Ringer . BSA was made fresh daily, mixed with human red blood cells , and kept on ice until use. The same BSA lot number and human donor were used for all experiments to minimize variability.Frog Ringer solution was prepared fresh daily from 5\u00d7 concentrated stock (pH 7.4 at 15\u00b0C) to the following concentrations (mM): 111.0 NaCl, 2.4 KCl, 1.0 MgSOTime 0 for the clotting time measurement was designated as the time when the blood contacted the glass slide. The drop was observed continuously until a clot formed. Time to clot formation was recorded.Coagulation of a single drop of blood was determined visually. g for 3 min (IEC Micro-MB Microcentrifuge fitted with microhematocrit rotor). sysHct was measured using a microcapillary reader  placed consistently at the same height and angle for each measurement with the indicator line placed at the interface of the red blood cell (RBC) column and white blood cell (WBC) layer. Intrasample reliability was within 1%.Hematocrit tubes were double sealed with critoseal (Thermo Fisher Scientific) and spun within minutes of blood collection at 13,460 7.A 5-\u00b5L sample of whole blood was diluted 1:200 in filtered  frog Ringer solution. Diluted blood (10 \u00b5L) was loaded into a Reichert bright line hemacytometer . Red blood cells (RBCs) were counted (Fisher Laboratory counter) using a Zeiss light microscope . RBCs located completely within the ruling lines of the hemacytometer were included in the count. If a portion of a cell was located outside the line it was not counted. Mean corpuscular volume (MCV) was calculated as MCV = (sysHct/[RBC]) \u00d7 10The [RBC] procedure was followed for [WBC] with the only change being a 1:20 dilution of a 12.5 \u00b5L whole blood sample. The diluted sample was loaded onto the Neubauer slide, and WBCs were differentiated by size  and counted. To measure concentration of activated white blood cells ([aWBC]), a solution of nitroblue tetrazolium dye was added to 12.5 \u00b5L of whole blood and incubated at 37\u00b0C for 20 min and then at room temperature for 20 min. The dye/whole blood sample was then diluted 1:20 with frog Ringer, loaded onto the Neubauer slide, and inspected for [aWBC], defined as those that had burst after taking up the dye . The sam\u22121, Stanbio Diagnostics Co., Boerne, TX). One milliliter of the blood/Drabkin\u2019s mixture or standards solution was pipetted into microcuvettes and read on a spectrophotometer  at a wavelength of 540 nm. Absorbance was recorded, and [Hb] calculated from the standard curve. Mean corpuscular hemoglobin concentration ([MCHb]) calculation was [MCHb] = ([Hb]/sysHct) \u00d7 100.[Hb] was determined from a 20-\u00b5L sample of whole blood mixed with 480 \u00b5L of Drabkin\u2019s Reagent  in a glass test tube. A standard curve was prepared with each assay using cyanomethemoglobin (range 20\u201380 mg\u00b7dLg for 15 min (4\u2013 5\u00b0C). Plasma was removed, injected into siliconized vacuum tubes, and stored at \u221270\u00b0C.Samples of whole blood were placed immediately into EDTA tubes, put on wet ice, and centrifuged  within 1 h at 3,200 x] was measured using chemiluminescence  as reported previously (x to nitric oxide at 85\u00b0C (3 standards (range 0.5\u20135.0 nM). Water was collected from each housing container, and [NOx] measured to assure consistent housing conditions.Within 2 to 3 mo of sample collection, . \u03c3(\u03c0c\u2013\u03c0i) is the x-axis intercept of the Pc\u2013Jv/S relationship. Capillary Lp was calculated for each of the nine measures of Jv/S at two pressures (see above) and averaged.The Starling equation assumes a linear relationship between Jv/S measurements. Capillary pressure was then uniformly raised by 1 cmH2O every 10 s until one or more hRBCs burst through the capillary wall. The capillary was observed closely on the video monitor and through the microscope to ensure accurate assessment. If a hRBC escaped at the occlusion site (an infrequent occurrence), that measurement was excluded.The first occlusion was maintained after a sufficient recording of the hRBC marker cells for Frogs were fasted for 4 days before each experiment. Fresh water was available ad libitum to prevent dehydration. Frogs with one or more [aWBC] were excluded from the study.2O (7.4 mmHg), and the balance pressure was measured (see above). The pressure was raised by 1 cmH2O to establish low flow during a 2 min equilibration period (steady state 1). At the end of equilibration, the capillary was stimulated mechanically with a square wave change in shear stress (\u0394\u03c4) via an abrupt change in pressure to 30 cmH2O (22.1 mmHg). The higher \u03c4 was maintained for 2 min (steady state 2). Jv/S was measured followed by measurement of burst pressure. At the end of the protocol, the animal was euthanized and a necropsy performed.After blood collection and surgical procedures, one capillary per frog was cannulated at 10 cmHsteady states 1 and 2. \u0394\u03c4 for a given capillary was calculated from plateau values as \u0394\u03c4 = \u03c4steady state 2\u2013\u03c4steady state 1. A physiological range for \u0394\u03c4 occurred in accordance with the downstream resistance of the microcirculation, which varied in situ from animal to animal. In general, it was assumed that filtration during occlusion reflected filtration at steady state 2. An important advantage of this technique was that the downstream end of each capillary remained undisturbed, and the \u0394\u03c4 stimulus was applied uniformly to each intact capillary  or median (\u00b1 25 and 75% quartiles) as appropriate. Values identified using box and whiskers plots were designated as outliers and excluded. A secondary analysis of Lp outliers and associated variables was reported. Time series data included two or more data points obtained in 2 or more years. Single time course data points were included in the figures but not in the analyses. One-way analysis of variance with parasite or month as the main effect was used to determine differences between means followed by Tukey\u2019s post hoc tests when the main effect was significant. Differences between medians were determined using van der Waerden test. In the case of unequal variance (Bartlett test), a Welch analysis of variance was employed. Significance was set a priori at P \u2264 0.05.JMP software  was used for statistical analyses. The dataset was control data pooled from experiments performed over the course of 7 years. Averages and time series data are presented below for the three parasite conditions. Results are organized according to systemic and capillary variables.H Att compared with No H and both groups displayed seasonal variation. No H body weights were highest in four winter months and lowest in July/August. H Att displayed low body weights in summer, similar to No H, with highs in late fall and early winter. No H body weight decreased at slower rate than its increase, 1.2 versus 3.6 g/month, respectively. Similarly, H Att\u2019s body weight rate of change was slower (0.9) compared with the increase (3.6 g/month). Average displaced volume /month, respectively].Average eosinophils B were hiH Not Att and H Att compared with No H. Seasonal variation occurred in No H, with lows in January and March and a high in September .Averages for lymphocytes C were hiH Att compared with No H. Similar to lymphocytes, No H was the only group that demonstrated seasonal variation in total [WBC], which peaked twice during the year, once in April/May and once in September. Three low months occurred through the winter in January, March, and November, and a fourth low occurred in June (change rates = \u2212459.3 and 302.7 (cells\u00b7cm\u22123)/month).Similar to eosinophils, average total [WBC] D were hix] /month] with a sustained peak in March, April, and May that was threefold higher compared with No H during the same timeframe. For H Att, [NOx] did not peak until August [rate = 10.5 (nmoles\u00b7mL\u22121)/month], a peak that was 100% lower (58.1 nmoles\u00b7mL\u22121) than the highest monthly average for H Not Att (101.1 nmoles\u00b7mL\u22121). Troughs occurred in July and November for H Not Att [rate = \u221238.0 (nmoles\u00b7mL\u22121)/mo] and March and May for H Att [rate = \u22124.4 (nmoles\u00b7mL\u22121)/month], revealing opposite cycles for the two parasite groups. Variability within each month also differed between the three groups. H Not Att showed high monthly variability compared with No H and was much reduced for H Att.Averages for plasma [NOx]  differedH Not Att versus No H and both groups demonstrated steady heart rates throughout the year. In contrast, the average heart rate for H Att did not differ from the other two groups; however, seasonal changes in H Att were identified, with peaks in April and July and trough in November/December (rate of change between significant monthly averages = 4.5 beats/min/month increase and \u22123.8 beats/min/month decrease).H clotting time averaged 5 s longer than H Att and demonstrated distinct oscillations across the year with higher month-to-month variability in spring months that diminished as the year progressed. Average clotting time for H Not Att did not differ from the other two groups; however, a significant seasonal decline of 17 s from February to December was notable (rates of change = \u22121.9 and 5.7 s/mo).H was higher than H Not Att and H Att. No H sysHct demonstrated seasonal changes that were highest in late winter and spring and lowest in summer into fall /month], a pattern that was similar to sysHct. H Att also showed seasonal changes in [RBC]  with both peaks shifted earlier in the year compared with No H. H Att [RBC] declined steadily from a high of 367.3 \u00d7 1,000 cells\u00b7cm\u22123 in February to 231.1 \u00d7 1,000 cells\u00b7cm\u22123 in July, a low that was \u223c50 \u00d7 1,000 cells\u00b7cm\u22123 below that of No H in the same month .Average [RBC] B did notNeither parasite condition nor season influenced [Hb] C, MCHb D, or MCVH [skpr] rose by 1.5 mg\u00b7mL\u22121 from April to June and fell in August by a similar amount (change rates = \u22120.7 and 0.8 (mg\u00b7mL\u22121)/month, respectively). For H Att, [skpr] had two peaks (February and October) that were similar in magnitude compared with No H. Importantly, H Att [skpr] dropped by almost 50% from 4.9 in February to 2.9 mg\u00b7mL\u22121 in May, a low that was well below the lowest No H trough average of 3.8 mg\u00b7mL\u22121 (rates = \u22120.7 and 0.5 (mg\u00b7mL\u22121)/month, respectively).Average protein concentrations, [abpr] /month], and displayed normal distributions in April, May, August, and September. The \u0394\u03c4 mechanical stimulus for each capillary averaged 29.0 (SD 10.5) dynes\u00b7cm\u22122 and did not differ for parasite conditions or season.H and Lp. The data displayed a dose/response curve at the limit with a negative slope that extended from a high value of 38.3 to a low value of 3.9 \u00d7 10\u22127 cm\u00b7s\u22121\u00b7cmH2O\u22121. H Not Att accounted for the highest values of Lp shown under the curve and approached the lower, H Att values of Lp, as the number of H increased.H balance pressure, which oscillated around an average of 10.0 cmH2O. In contrast, absence of regular oscillations was the most notable feature of the balance pressure time series data for H Not Att and H Att.The three pressure variables for individual capillaries are presented in c\u2013\u03c0i), which is the x-axis intercept of the pressure/Jv/S plots used to assess Lp and the pressure at which filtration flow through the capillary wall is zero. Average \u03c3(\u03c0c\u2013\u03c0i) was lowest for H Not Att compared with both No H and H Att. A distinct seasonal pattern was revealed for No H where \u03c3(\u03c0c\u2013\u03c0i) was lowest in March, increased steadily to a peak in December at a rate of 0.5 cmH20/month, and declined at a faster rate (\u22121.5 cmH2O/month) back to March. For the two parasite groups, the annual time course of \u03c3(\u03c0c\u2013\u03c0i) was more variable and no seasonal changes were detected.c\u2013\u03c0i) pressure are shown in H Not Att was less than H Att. H Not Att displayed seasonal variation with average difference between the two pressures rising above zero in May, June, and September, an increase that would favor flow through the tissue during those months if precapillary resistance remained constant. In contrast, average pressure difference for No H and H Att remained negative throughout the year, which would favor filtration into tissues if resistance upstream was constant.Average differences between balance pressure and \u03c3 versus 33.0  \u00d7 10\u22127 cm\u00b7s\u22121\u00b7cmH2O\u22121 for Lp outliers in the No H group. Of the 38 variables tested, five systemic and four capillary variables distinguished the animals with Lp outliers from control. Basophils, eosinophils, lymphocytes, total [WBC], clotting time, capillary \u03c3(\u03c0c\u2013\u03c0i), and capillary burst pressure were lower, and balance pressure minus \u03c3(\u03c0c\u2013\u03c0i) was significantly higher for the Lp outliers versus control (Median (\u00b1 25/75%) control  control .Lp was 12.4  versus 68.0  \u00d7 10\u22127 cm\u00b7s\u22121\u00b7cmH2O\u22121 for the Lp outliers in the H Not Att group. In contrast to No H, only capillary density and [plpr] distinguished control from Lp outliers for H Not Att (Median (\u00b1 25/75%) control  Not Att .Lp was 4.2  versus 20.0  \u00d7 10\u22127 cm\u00b7s\u22121\u00b7cmH2O\u22121 for Lp outliers. The variables that were unique to H Att were spleen weight and capillary balance pressure. Both were lower for H Att Lp outliers compared with No H (Median (\u00b1 25/75%) control ith No H .1) to determine the impact of Hematoloechus sp. (H) on Rana pipiens and 2) to establish baselines across the year for a broad range of systemic and individual capillary variables. The first of the two study objectives revealed that 47% of the 38 descriptive, cardiovascular, and immunological variables showed one or more significant changes in Rana pipiens with H. In time, we recognized that H attachment status was an important effector and, ultimately, three groups were identified: no lung Hematoloechus (No H), Hematoloechus not attached in the lungs (H Not Att), and Hematoloechus attached to the inner lung surface (H Att). The second objective indicated previously unknown, naturally occurring seasonal changes for 10 variables in No H, 3 in H Not Att, and 8 in H Att. Chronic diseases of inflammation, parasitic diseases, and stroke are among the significant clinical applications of the study. Past, present, and future studies of capillary physiology using Rana pipiens are among the basic sciences expected to benefit from the data.The present prospective, descriptive study had two primary objectives: H had on Rana pipiens. Ten combinations of differences between average values were discovered among the variables across the three parasite conditions. Of note among those listed is a cluster of eight pro-inflammatory indicators in H Not Att\u20141) elevated lung weight, 2) elevated abdominal cavity fluid volume, and 3) decreased heart rate, all of which were secondary to 4) higher capillary Jv/S, 5) higher capillary Lp, and 6) lower capillary \u03c3(\u03c0c\u2013\u03c0i), and, in turn, secondary to 7) elevated lymphocytes and 8) elevated plasma [NOx]. Of interest, lymphocytes were the only leukocyte subtype that increased and remained elevated in both parasite groups. Basophils had a statistically significant parasite effect; however, differences between groups could not be distinguished, and eosinophils were elevated only in the H Att group. Thus, it appears that the host response in H Not Att was specific enough to blunt or inhibit parasite-induced leukocyte responders (basophils and eosinophils), but unable to prevent lymphocytes and/or plasma [NOx] from triggering the array of inflammation indicators listed above.H Att. The average coagulation rate was fastest, and eosinophils and total [WBC] were elevated compared with No H. These observations plus the fact that pro-inflammatory indicators  were elevated in H Not Att, but did not resolve with H attachment suggest that the presence of unattached H induced symptoms of inflammation that were sustained upon H attachment and that eosinophils, total [WBC], and coagulation responded subsequently to H attachment.Blood clotting is also associated with inflammation. Here, eosinophils, total [WBC], and coagulation rate differed in the H Att reversed the increases in plasma [NOx] and capillary Lp that were observed in H Not Att. H Att had the lowest average plasma [NOx], which not only fell below H Not Att, but also was significantly lower than No H. Although mechanism/s for the dramatic changes in [NOx] are not known, it appears that the ubiquitous nitric oxide molecule was involved with both proinflammatory and anti-inflammatory processes, all associated with the H parasite and its attachment status. Sources of plasma [NOx] include endothelial, immune, and nervous system cells. An important aim for future investigations would be to discover how H increases and decreases nitric oxide in Rana pipiens and if these outcomes are achieved dose dependently.A very important distinction between the two parasite groups was that inflammation in Lp, H Att capillaries were \u201ctighter\u201d (lower Lp) than H Not Att and, surprisingly, also tighter than No H, which would be considered an anti-inflammatory phenotype profile for capillaries. It appears that H attachment resulted in parasite-host biochemical communication that not only bypassed or counteracted inflammatory processes at the capillary level of the cardiovascular system, but actually lowered median Lp by more than 100% below that for No H. The number of H/Lp dose/response curve further supported a correlation between H and capillary Lp. Higher capillary burst pressure, an index of wall tensile strength, also occurred with H attachment and did not change in parallel with changes in capillary Lp. Capillary wall strength for No H and H Not Att remained stable in the face of dynamic changes in Lp reflecting two separate sets of molecular machinery acting in parallel\u2014one that maintains wall strength and is not sensitive to H and a second that induces a wide range of changes in Lp and is sensitive to H. Mechanisms for these results are unknown at this time. Investigative attention to this important distinction is warranted.Regarding H on the host. Fluid accumulated in the lungs, abdominal cavity, and cardiovascular space (lower hematocrit) of H Not Att and the excess fluid (organ weight) did not resolve with H attachment. Higher levels of organ fluid for both parasite groups in the face of opposing barrier functions suggested two different mechanisms leading to increased organ fluid/weight based on H attachment status: 1) excess fluid filtered into tissues and compartments via leaky capillaries (H Not Att) and 2) fluid trapped in tissues and compartments via tight capillaries (H Att). Of the two mechanisms that accounted for similar fluid accumulation in organs, cavities, and spaces, only the \u201ctight\u201d capillaries in H Att would trap fluid and also restrict nutrients. In actual fact, average body weight was lower only for H Att, a consequence that likely was due to diminished nutrition secondary to low permeability.Overall, organ and body weights provided useful indicators of the additive effects of Rana pipiens that was created from measurements performed across the calendar year for all 38 variables is discussed below and organized according to the three parasite conditions. In many cases, waveforms were identified in the times series data and these are summarized in The catalogue of baseline information on H dataset. Best examples were the time series traces for sysHct (H) and [RBC] (H). Both curves were similar with peaks in winter/spring and troughs in summer, suggesting that changes in RBC, not plasma volume, were causal of sysHct changes. Capillary \u03b3 , total [WBC] (H), and plasma [NOx] (H). The spleen is the main erythropoietic organ in Rana pipiens , capillary tHct (H), and capillary Lp (H), suggesting possible interconnections. The curves for Lp synchronized with tHct starting in March and with clotting time starting in May. Of note was the observation that Lp and tHct cycles correlated positively, and the relationship strengthened coincident with a unique feature of the Lp trace that showed oscillations increasing in amplitude as the year progressed. Positive coupling of capillary tHct with capillary Lp in situ represents a unique outcome from this study that has not been documented in the past.Three oscillations across the year were displayed by clotting time , No H, cary tHct G, No H, llary Lp B, No H, H). The steady nature of the waveform across the entire year suggested internal controls with remarkable timing and precision. Although no statistical differences between months were observed, the time trace pattern was noteworthy for its rhythmic activity that most likely reflected the well-known \u201chunting\u201d mechanism displayed by vascular smooth muscle, in this case, on the venous, downstream side of the capillary bed.A striking example of a sine wave with five oscillation cycles was the trace for capillary balance pressure A, No H. H) approached a square wave curve. The trace was distinguished by an abrupt drop from winter to summer, sustained plateau in summer, and then a steep rise from fall into winter. We are not aware of any reports of a seasonal, >200% drop, and similar recovery in liver weight in Rana pipiens. This new information is fundamental to understanding annual processes of digestion, metabolism, and protein synthesis in this model.Liver weight C, No H aH) and capillary \u03c3(\u03c0c\u2013\u03c0i) (H). The trough time points in March were similar between the two curves; however, each had a different rate of rise to peak. Lymphocytes displayed the steeper slope compared with the slower rise of capillary \u03c3(\u03c0c\u2013\u03c0i) with time. Speculation of a connection between these two variables was tempting. The initial increase in lymphocytes could signal an increase in \u03c3(\u03c0c\u2013\u03c0i) via changes in Jv, S, and/or capillary pressure, which would increase resistance to water movement into tissues and, thus, preserve fluid in the vascular space during winter. In spring, lower lymphocytes could signal lower \u03c3(\u03c0c\u2013\u03c0i), which would reduce the resistance threshold required to move fluid out of the vascular space and, thus, restore tissue hydration. Experiments to test these ideas are required.A single saw tooth waveform was demonstrated by lymphocytes C, No H a\u03c3(\u03c0c\u2013\u03c0i) B, No H. H confirmed stability in the animal model. Steady heart rate (H) across the year was an index of the successful, consistent, low-stress housing environment that was provided for Rana pipiens. Capillary burst pressure (H) also displayed little to no variation, indicating that capillary wall strength was maintained while dynamic changes in capillary Lp and \u03c3(\u03c0c\u2013\u03c0i) occurred. Heart rate and burst pressure plus the additional flat line curves listed in Rana pipiens.Flat line time series data for No art rate , No H acpressure D, No H aH were replicated in H Not Att. Instead, two variables associated with inflammation and four inflammation-responding variables were identified. First, plasma [NOx] , which displayed a sawtooth waveform that was inverse and shifted right relative to the time trace for eosinophils , suggesting possible stimulus-responses between these two variables. The second, third, and fourth inflammation-responding variables were capillary Jv/S (H Not Att), capillary Lp (H Not Att), and capillary balance pressure minus \u03c3(\u03c0c\u2013\u03c0i) (H Not Att). Each displayed accentuated oscillations between March and September, indicating vigorous responses of capillaries during the same timeframe as the increases in plasma [NOx] and eosinophils. The accentuated intramonth variability of Jv/S, Lp, balance pressure minus \u03c3(\u03c0c\u2013\u03c0i) compared with No H and H Att was additional compelling evidence of disrupted/chaotic barrier control due to inflammation in H Not Att.The first of the inflammation-responding variables was clotting time , it was clear that H Att were stressed at different time points during the year, a result that differed from the flat heart rate traces for No H and H Not Att. All animals in the study lived in the same controlled environment and received the same care, as such, an external source of stress was not apparent. Investigators were blind to presence or absence of H until necropsy. We conclude that the presence of attached H was likely an internal source of stress that was sufficient enough to influence heart rate. Seasonal changes in heart rate for H Att may be one of the best indicators of H activity/potency within Rana pipiens.Heart rate, plasma  (H Att) also had a significant annual time series in H Att that was not present in No H and, when compared with H Not Att, was most instructive. The first prominent peak in spring that was the signature for plasma [NOx] in H Not Att was noticeably absent in H Att, and the second peak in August was muted. These traces suggested that H, when attached, introduced a nitric oxide-targeted inhibitor into the host. The high levels of plasma [NOx] in H Not Att during March, April, and May plus the absence of that peak during the same timeframe in H Att indicated the greatest proinflammatory (H Not Att) and anti-inflammatory (H Att) impact of H, an observation that could be useful for designing future experiments focused on nitric oxide, H, and Rana pipiens.Plasma [NOx] , H Att aH Att time series data revealed a seasonal change in capillary Lp (H Att). Once recognized, it was obvious that H attachment silenced an internal clock that was generating the Lp oscillations in No H as well as the accentuated excursions in Lp observed in H Not Att. The end result was a parasite-induced flat baseline for Lp with one internal clock still ticking, a seasonal change in Lp that was highest in January and lowest in November and that had gone undiscovered previously  had the lowest \u201cn\u201d and the lowest median Lp of the three outlier groups further emphasizing the rarity of the data reported here and consistent with capillaries being less reactive to stimuli when H are attached. Finally, average total [WBC] were lower, not higher, for Lp outliers in No H, suggesting a minimum [WBC] that protects and maintains the integrity of the capillary barrier in Rana pipiens.The three unique outcomes for variables associated with Rana pipiens has been valued for studies of capillary physiology , which adjusted naturally over a period of 9 mo and Lp that required 11 mo to demonstrate adaptive change to H attachment. Each of these clues expand our collective knowledge about systems physiology and must be accounted for when translating this research into creative new ways to improve health and minimize the impact of disease.The annual time series data provide more specific details of naturally occurring versus Schistosoma mansoni is a water-borne trematode (blood fluke) with snail as its intermediate host and human is its definitive host. S. mansoni is the most prevalent parasite in humans causing the tropical disease, schistosomiasis. The fluke penetrates through the skin, lives in blood vessels, and comes into contact with endothelial cells during its migration through the body. In 1997, Coulson and Wilson  disappearance of oscillations in spleen weight, balance pressure, tHct, and Lp in H Not Att and H Att, 2) disappearance of seasonal changes in \u03c3(\u03c0c\u2013\u03c0i) in H Not Att and H Att, 3) highest Lp in H Not Att, 4) highest [NOx] in H Not Att, 5) lowest Lp in H Att, 6) lowest [NOx] in H Att, and 7) increased capillary wall tensile strength in H Att compared with No H. These results with the lung fluke (H) support the conclusion that H shift the in vivo endothelium from a pro- to anti-inflammation profile in Rana pipiens and are consistent with reports from blood flukes (S. mansoni). S. mansoni is well-studied, yet, how the host cells respond to the various proteins, lipids, glycans, and nucleic acids released by the blood fluke remains unknown  when referencing previous studies, and reporting H status in future studies will facilitate a new and higher \u201cgold\u201d standard for studies of single capillaries in situ and improve Rana pipiens as a long-standing, classic model.Our awareness expanded when we recognized that c\u2013\u03c0i) are obtained when flow through  and out of [\u03c3(\u03c0c\u2013\u03c0i)] the capillary are both zero. Calculating the difference between balance pressure and \u03c3(\u03c0c\u2013\u03c0i) pressure  have reported low and narrow baseline ranges for their systems. Some examples are the following: 1.65 to 4.94 \u00d7 10\u22127 ; 0.7 to 43.9 \u00d7 10\u22127 (H Not Att); and 0.7 to 9.3 \u00d7 10\u22127 cm\u00b7s\u22121\u00b7cmH2O\u22121 (H Att) with low values that were not attained in BAEC and high values well above BAEC. In addition, Sill et al.  and 2.0 \u00d7 10\u22127 cm\u00b7s\u22121\u00b7cmH2O\u22121 (H and more than two times lower than H Att (4.2 \u00d7 10\u22127 cm\u00b7s\u22121\u00b7cmH2O\u22121). We performed a comprehensive review of past capillary physiology studies based on whether results were conducted with or without the presence and effects of the Hematoloechus and found that none reported H status. We conclude that, in the past, low values of Lp likely were obtained from a mixture of Lp assessments performed in No H, H Not Att, and H Att plus Lp values > 10.0 \u00d7 10\u22127 cm\u00b7s\u22121\u00b7cmH2O\u22121 were excluded. The net result produced a left-skewed distribution of Lp and a false, low, target value of 2.0 \u00d7 10\u22127 cm\u00b7s\u22121\u00b7cmH2O\u22121 for baseline Lp.Although using one number for \u00b7cmH2O\u22121  is close\u00b7cmH2O\u22121  is more Lp for BAEC experiments and for testing mathematical models. Specifically, low Lp in H Att was associated with body weight loss in vivo  and the Dean\u2019s office, College of Nursing, Montana State University.No conflicts of interest, financial or otherwise, are declared by the authors.D.A.W. conceived and designed research; D.A.W. and M.H.F. performed experiments; D.A.W. analyzed data; D.A.W. and M.H.F. interpreted results of experiments; D.A.W. prepared figures; D.A.W. drafted manuscript; D.A.W. edited and revised manuscript; D.A.W. approved final version of manuscript."}
+{"text": "CXC chemokine ligand 3 (CXCL3) is a member of CXC-type chemokine family that is identified as a major regulator in immune and inflammation responses. Recently, numerous evidence indicated that CXCL3 is broadly expressed in various human tumor types, and it is also known to play a critical role in mediating tumor development and progression. However, the expression profile of CXCL3 and the exact molecular mechanism behind the role of CXCL3 in colon adenocarcinoma (COAD) has not been fully elucidated.The expression and clinical significance of CXCL3 mRNA and protein in the tissues from COAD patients were estimated using bioinformatics and immunohistochemistry assays. The expression and roles of exogenous administration or overexpression of CXCL3 in HT-29 and SW480 COAD cells were determined using enzyme-linked immunosorbent assay(ELISA), Cell Counting Kit-8 (CCK-8) and Transwell assays. Mechanically, CXCL3-induced malignant behaviors were elucidated using western blotting assay and extracellular signal-regulated protein kinase 1/2 (ERk1/2) inhibitor PD98059.(TCGA)-COAD data analysis revealed that CXCL3 mRNA is highly expressed and has high clinical diagnostic accuracy in COAD. Increased expression of CXCL3 mRNA was associated with patient\u2019s clinical stage, race, gender, age, histological subtype, nodal mestastasis and tumor protein 53 (TP53) mutation status. Similarly, immunohistochemistry assay also exhibited that CXCL3 protein in COAD tissues was significantly up-regulated. Gene expression associated assay implied that CXC chemokine ligand 1 (CXCL1) and CXC chemokine ligand 2 (CXCL2) were markedly correlated with CXCL3 in COAD. Protein-protein interaction (PPI) analysis revealed that cyclin B1 (CCNB1), mitotic arrest deficient 2 like 1 (MAD2L1), H2A family member Z (H2AFZ) and CXCL2 may be the important protein molecules involved in CXCL3-related tumor biology. Gene set enrichment analysis (GSEA) analysis revealed that CXCL3 was mainly enriched in the cell cycle, DNA replication, NOD-like receptors, NOTCH and transforming growth factor-\u03b2 (TGF-\u03b2) Signal pathways. In vitro, exogenous administration or overexpression of CXCL3 resulted in increased malignant behaviors of HT-29 and SW480 cells, and down-regulation of CXCL3 expression inhibited the malignant behaviors of these tumor cells. In addition, overexpression of CXCL3 affected the expression of genes related to extracellular signal regulated kinase (ERK) pathway, including ERK1/2, p-ERK, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax) and Cyclin D1. Finally, CXCL3-induced malignant behaviors in HT-29 and SW480 cells were obviously attenuated following treatment with ERK inhibitor PD98059.The cancer genome atlas CXCL3 is upregulated in COAD and plays a crucial role in the control of malignant behaviors of tumor cells, which indicated its involvement in the pathogenesis of COAD.The online version contains supplementary material available at 10.1186/s12885-023-11655-y. Colon adenocarcinoma (COAD) is one of the leading causes of cancer deaths worldwide, which is the second most common prominent cancer among women and the third in men, respectively . It was Chemokines, also defined as chemotactic cytokines, are a class of secretory proteins with a low-molecular weight of 8 to 12 KD, which are initially chartered to play pivotal roles in inducing chemotaxis of various kinds of cells . ChemokiCXCL3 belongs to the CXC family of chemokines and can be further known as a ELR\u2009+\u2009CXC chemokine due to the presence of a conserved Glu-Leu-Arg sequence. CXCL3 exerts its biological functions mainly related to its interaction with CXC-chemokine receptor 2 (CXCR2) . Our prehttps://portal.gdc.cancer.gov/), and the expression levels of CXCL3 mRNA in tissues were analyzed using the university of alabama at birmingham cancer data analysis portal (UALCAN) database . Receiver operating characteristic (ROC) curve was used to calculate the area under the curve (AUC). The genes closely related to CXCL3 in COAD were obtained from UALCAN database, and the top 25 genes positively correlated with CXCL3 were displayed in the form of heatmap. The protein interaction network (PPI) was constructed through String database (https://cn.string-db.org/) and Cytoscape Software, and the tumor immune estimation resource (TIMER) database (https://cistrome.shinyapps.io/timer/) was used to determine the correlation between CXCL3 expression and CXCL3-related genes expression (or immunocyte infiltration). The TCGA-COAD data set was used for GSEA analysis, both p value and false discovery rate (FDR) q value less than 0.05 was considered to be significantly rich.The original CXCL3 mRNA-seq data including 284 COAD and 41 normal colon tissues were obtained from the Cancer Genome Atlas (TCGA) database . Briefly, the microarray was incubated with an antibody targeted to CXCL3  at 4\u02daC overnight, followed by treatment with a SABC Kit (Boster) according to the supplier\u2019s instructions. After color development with 3,3\u2032-diaminobenzidine (DAB), Hematoxylin was applied for nuclear counterstain. CXCL3 staining was scored using the sum of the following two indicators: (1) the intensity of staining: negative-weak (1), medium (2), strong (3) and super strong (4); (2) the percentage of staining cells: \u226425% (1), >\u200925\u2013\u226450% (2), >\u200950\u2013\u226475% (3) and >\u200975% (4). The final score = (1)+(2). Low expression was defined as a final score\u2009\u2264\u20094 and high expression with a final score\u2009\u2265\u20095.Human CC HT-29 and SW480 cells were provided by ATCC. Cells were grown in RPMI-1640 medium (for HT-29) or L-15 medium (for SW480) containing 10% fetal bovine serum  and 100 U/ml penicillin/streptomycin, and the medium containing FBS with penicillin/streptomycin was referred to as complete medium.Following construction of empty lentiviral vector and lentiviral expression vector harboring CXCL3 gene sequence or interfering sequence targeted CXCL3, the pseudovirus particles containing these vectors were produced by GeneCopoeia Inc. . CXCL3-overexpressing cells, CXCL3-deficiency cells and their mock control cells were established by infection of the pseudovirus particles according to the supplier\u2019s recommendations.5 cells were cultured in each well of six-well plates containing 1 ml serum-free medium. After 24\u00a0h of inoculation, the supernatant was obtained by centrifuging the medium. The concentration of CXCL3 in supernatant was measured and calculated using an ELISA Kit (Mskbio) in strict accordance with the manufacturer\u2019s protocol. The experiments were repeated in triplicate.A total of 4\u2009\u00d7\u2009103 HT-29 or SW480 cells were seeded in each well of a 96-well plate containing different concentrations of recombinant CXCL3 (peprotech). (b) Endogenous experiment: 4\u2009\u00d7\u2009103 CXCL3-overexpressing or CXCL3-deficiency HT-29 or SW480 cells, as well as their mock control cells were seeded in each well of a 96-well plate containing 100\u00a0\u00b5l of complete medium. In addition, the complete medium supplemented with DMSO (control) or the ERK1/2 blocker PD98059 was utilized to assess whether the role of CXCL3 is mediated by ERK1/2 pathway. After 48-hour culture in the experiments, 10\u00a0\u00b5l of CCK8 reagent was added to each well, and the OD value of each well was measured after 40\u00a0min of reaction. The experiments were repeated in triplicate.Cell counting kit-8 (CCK-8) reagent (Med Chem Express) was employed for cell proliferation experiments: (a) Exogenous experiment: 4\u2009\u00d7\u2009104 HT-29 or SW480 cells in serum-free medium were seeded in each upper chamber of Transwell, while the bottom chamber was filled with 20% FBS medium containing different concentrations of recombinant CXCL3. (b) Endogenous experiment: 3\u2009\u00d7\u2009104 CXCL3-overexpressing or CXCL3-deficiency HT-29 or SW480 cells, as well as their mock control cells in serum-free medium were seeded in each upper chamber of Transwell, while the bottom chamber was filled with 20% FBS medium. Similarly, 20% FBS medium supplemented with dimethyl sulfoxide (DMSO) (control) or the ERK1/2 blocker PD98059 was also utilized in the endogenous experiment. After 48-hour culture in the experiments, the migratory cells that had invaded through the filter were fixed and stained with crystal violet solution in ethanol. The experiments were repeated in triplicate.Cell migration analys1s was performed using 24-well Transwell systems (Biofil). (a) Exogenous experiment: 3\u2009\u00d7\u2009102 cells were grown in each well of a 24-well plate containing 500\u00a0\u00b5l of complete medium. After 2 weeks of routine culture, the medium was discarded, and the plate was soaked twice with PBS followed by staining with 0.1% crystal violet for 40\u00a0min. The number of colonies was counted under an eletron microscope. The experiments were repeated in triplicate.2\u2009\u00d7\u200910A total of 30\u00a0\u00b5g protein extracts was resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently electroblotted onto polyvinylidene difluoride (PVDF) membranes. Following blocking with 5% skimmed milk, the membranes were treated with various specific antibodies, including a rabbit antibody against ERK1/2 , a rabbit antibody against p-ERK1/2 , a rabbit antibody against Bcl-2 , a rabbit antibody against Bax , a rabbit antibody against Cyclin D1  and a mouse antibody against \u03b2-actin . The relative intensity of ERK1/2, Bcl-2, Bax and Cyclin D1 was normalized to \u03b2-actin, whereas p-ERK1/2 was normalized to total ERK1/2. Immunoreactive protein bands were detected using 5200 multifunctional imaging system , and the intensity of each band was quantified using image analysis software . The experiments were repeated in triplicate.P values\u2009<\u20090.05 were considered significant.Statistical analyses were executed with SPSS statistics 22.0 software. Mann-Whitney U test was used for comparing differences in the expression of CXCL3 mRNA-seq data. Univariate and multivariate Cox regression models were used to analyze the prognostic significance of CXCL3 in patients with COAD. The correlation between two-group was analyzed with Pearson correlation analysis, and comparison of the rates was conducted using the Pearson Chi-square test. Comparisons between the two-group in the cell experiment were made using T test or Rank sum test, whereas multiple-group comparisons were assessed by one-way ANOVA and LSD test. Differences with P\u2009<\u20090.0001) was calculated by ROC curve, indicating that the expression level of CXCL3 mRNA has very high accuracy in the clinical diagnosis of COAD  of the COAD tissues, but in 7.41% (4/54) of the normal colon tissues  and their mock control cells (mock) were identified by ELISA Fig.\u00a0A and B. Similarly, CXCL3-deficiency cells were established by transfection with interference sequence targeting CXCL3 Fig.\u00a0A and B, The underlying mechanism mediating CXCL3-related malignant behaviors was evaluated by detecting gene expression at protein level using western blotting assays. As shown in Fig.\u00a0The biological behaviors of COAD cells in response to ERK1/2 blocker PD98059 were assessed by CCK-8 and Transwell assays. The results demonstrated proliferation , GRO\u03b3 or Macrophage inflammatory protein-2-\u03b2 (MIP-2\u03b2), is a single-chain protein which is encoded by human growth-related oncogene (GRO) located in the chromosomal region of 4q21 . CXCL3 iIn this study, the results from bioinformatics and immunohistochemistry assays exhibited that CXCL3 mRNA and protein are surprisingly upregulated in COAD tissues, and its high-level expression is closely correlated with several clinicopathological parameters, including patient\u2019s clinical stage, race, gender, age, histological subtype, nodal mestastasis and TP53 mutation status. Our results also presented that CXCL3 expression has a strong positive association with CXCL1 and CXCL2 expression, as well as neutrophil attraction, but has a negative association with macrophage attraction. Chemokines are best known for their roles in mediating the attraction of distinct populations of immune cells that are responsible for the balance between pro- and anti-tumorigenic immune responses. The important immune cells implicated in the elimination of tumor are natural killer (NK) cells, T helper 1 (Th1) cells, monocytes and CD8\u2009+\u2009T cells, whereas several immune cell subsets including neutrophils, myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) have the ability to facilitate tumor development . CXCL1, In addition, our results showed that CXCL3 overexpression also exerts its role to regulate Bcl-2, Bcl-2/Bax and Cyclin D1 expression that are responsible for apoptosis and cell cycle, the processes upon which tumor cells depend for proliferation, survival, and metastasis. Apoptosis is an energy-dependent cell suicide event that is strictly regulated by several apoptosis-related gene families, in which Bcl-2 family plays a key role. Bcl-2 family includes both pro- and anti-apoptotic genes, and the relative ratio of pro-/anti-apoptotic genes, especially Bcl-2/Bax, is considered to be a major determinant for the balance between cell survival and death . NumerouIn summary, our experiment showed that chemokine CXCL3 is highly expressed in COAD, and its high-level expression is closely associated with several tumor-associated clinicopathological parameters, chemokines, genes, signaling pathways and immunocyte recruitment. In vitro, CXCL3 plays a crucial role in promoting the carcinogenic potential of COAD cells through endogenous and exogenous pathways. Mechanically, ERK\u3001Bcl-2 and Cyclin D1, the MAPK/ERK pathway related genes that are most notably related to the apoptosis and cell cycle, are intimately involved in CXCL3-mediated malignant behaviors. Our findings indicated that activation of MAPK/ERK signal pathway controlled by upregulation of CXCL3 might be one of the key determinants in tumor biology of COAD.Below is the link to the electronic supplementary material.Supplementary Material 1"}
+{"text": "Phosphate toxicity, the accumulation of toxic levels of phosphate in the body, is associated with tumor growth and bone mineral abnormalities. Based on shared associations with phosphate toxicity, the hypothesis of the present study proposes that breast cancer is associated with bone mineral abnormalities in middle-aged women from the U.S. Study of Women\u2019s Health Across the Nation. The results of the present mixed methods\u2013grounded theory study confirmed that women self-reporting breast cancer had a greater magnitude of changes in bone mineral density over time compared with women who remained cancer-free. These findings have implications for phosphate toxicity as a potential cause of bone metastasis in metastatic breast cancer, and future studies should investigate a low-phosphate diet to reduce tumorigenesis and bone mineral abnormalities in breast cancer patients.Breast cancer is associated with phosphate toxicity, the toxic effect from dysregulated phosphate metabolism that can stimulate tumorigenesis. Phosphate toxicity and dysregulated phosphate metabolism are also associated with bone mineral abnormalities, including excessive bone mineral loss and deposition. Based on shared associations with dysregulated phosphate metabolism and phosphate toxicity, a hypothesis proposed in the present mixed methods\u2013grounded theory study posits that middle-aged women with incidence of breast cancer had a greater magnitude of changes in bone mineral density over time compared with women who remained cancer-free. To test this hypothesis, a mixed-effects model was used to analyze the associations of breast cancer incidence with spinal bone mineral density changes in the U.S. Study of Women\u2019s Health Across the Nation. Compared with women in the cohort who remained cancer-free, women who self-reported breast cancer had higher bone mineral density at baseline, but had more rapid losses in bone mineral density during follow-up visits. These findings agree with the hypothesis that a greater magnitude of changes in bone mineral density over time is associated with breast cancer in a cohort of middle-aged women. The findings also have implications for studies investigating dysregulated phosphate metabolism and phosphate toxicity as causative factors of bone metastasis in metastatic breast cancer. Additionally, the authors previously found increased breast cancer risk associated with high dietary phosphate intake in the same cohort of middle-aged women, and more studies should investigate a low-phosphorus diet to reduce bone mineral abnormalities and tumorigenesis in breast cancer patients. An association of breast cancer with high bone mineral density (BMD) has been reported in the literature , but theFurther contributing to the research literature on bone mineral density and breast cancer is the opposite finding of increased osteoporosis risk associated with breast cancer in postmenopausal women, suggesting that breast cancer may share common \u201cbiochemical links\u201d with low bone mineral density . HoweverAdding to the controversy, other studies have failed to find an association between breast cancer and BMD ,9,10,11.2D3, also known as calcitriol. The kidneys reabsorb Pi to maintain normal serum Pi levels and excrete excess Pi in the urine. Fibroblast growth factor 23 (FGF23), released from bones, and parathyroid hormone (PTH) released from the parathyroid glands, help to regulate Pi levels by inhibiting kidney reabsorption of excessive Pi and increasing urinary phosphate excretion.The metabolism of serum inorganic phosphate (Pi) is regulated through endocrine hormones secreted by the bone\u2013kidney\u2013intestine\u2013parathyroid axis . IntestiPhosphate toxicity from excessive accumulation of phosphate in the tissues of the body can accelerate aging, cause bone deformities, and reduce longevity . Importap = 0.07) [Using a mixed-methods approach to analyze both quantitative and qualitative data , the pre = 0.07) . The pre = 0.07) . This st = 0.07) : a quali = 0.07) , and a hIn the qualitative analysis of the present MM-GT study, research findings on phosphate toxicity, breast cancer, and bone mineral density were selected from all relevant sources for comparative analysis of concepts consisting of pathophysiological relationships and mechanisms. Concepts are the building blocks of qualitative analysis in the present MM-GT study.\u201cConcept formation in qualitative research is a systematic process whereby the researcher sets definitions for important concepts that emerge during the research. These definitions help to provide the parameters for the qualitative study\u201d.Concepts were organized into themes and eventually synthesized into a novel qualitative theory explaining the metabolic mechanisms by which phosphate toxicity and dysregulated phosphate metabolism are associated with changes in bone mineral density and tumorigenesis, i.e., the \u201csensitizing concepts\u201d that guided the development of the MM-GT study . Science as far back as 1946 noted the detection of breast tumors through increased uptake of the radioactive isotope phosphorus-32, compared with lower uptake of the phosphorus isotope by normal breast tissue [The following includes a brief summary of the findings cited in a review of phosphate toxicity and tumorigenesis . Elevatet tissue . A compat tissue .Hyperphosphatemia in patients was associated with chromosome instability and increased proliferation of parathyroid cells , and eleNormal bone metabolism renews bone tissue through a balance of mechanisms that break down and remove worn bone tissue, and replace the discarded tissue with deposits of new bone: \u201cBone remodeling is the process by which bone is renewed to maintain bone strength and mineral homeostasis. Remodeling involves continuous removal of discrete packets of old bone, replacement of these packets with newly synthesized proteinaceous matrix, and subsequent mineralization of the matrix to form new bone. The remodeling process resorbs old bone and forms new bone to prevent accumulation of bone microdamage\u201d.If bone remodeling mechanisms that normally build up and break down bone become unbalanced, metabolic bone disorders may occur, such as osteoporosis, in which \u201cbone resorption outpaces bone formation\u201d . Of rele\u201cPTH can produce catabolic or anabolic effect(s) on bone metabolism depending on the level of the hormone, periodicity, and duration of exposure\u201d.Loss of healthy bone in cancer is found in combination with increases in abnormal bone deposits, or osteoblastic skeletal lesions ,50. AbnoLow vitamin D levels associated with dysregulated phosphate metabolism are common in CKD , and breMetastatic breast cancer, stage IV breast cancer that has spread to other organs, is the most advanced form of breast cancer affecting approximately 30% of women with the disease, and is \u201cgenerally incurable\u201d . Bone is\u201cMetastases leading to overall bone loss are classified as osteolytic. Those leading to excess bone deposition are considered osteoblastic. However, both bone degradation and deposition likely occur early in the metastatic process. The majority of breast cancer metastases ultimately cause bone loss\u201d.Although breast cancer bone metastases are predominantly osteolytic, 15\u201320% of breast cancer bone metastases cases \u201chave a predominant osteoblastic component\u201d . ExcessiA synthesis of concepts from the previously reviewed literature explains how abnormal bone mineralization and tumorigenesis share associations with dysregulated phosphate metabolism and phosphate toxicity. The rationale used to inform the hypothesis of the present study is based on transitive inference\u2014\u201cthe process of inferring the relation between two items based on their shared relation with a third item\u201d . For exaThe quantitative analysis in the present MM-GT study uses frequent repeated measures to investigate the association of self-reported breast cancer with longitudinal changes in bone mineral density based on a secondary analysis of follow-up data from the SWAN study . The SWA2 for absolute bone mineral density with cross-calibration applied at each visit number.Between 1996 and 1997, 3302 women aged 42\u201353 years who were free of breast cancer were enrolled in the SWAN cohort . Partici2 are listed in the data set as variable SPBMDT. Analysis was performed by fitting a linear mixed-effects regression model to the data using the PROC MIXED statistical analysis procedure in SAS, release 9.04.01M3P06242015. Fixed effects in a mixed-effects model are the constant or fixed relationships assumed between independent and dependent variables, so that \u201conly the dependent variable changes in response to the levels of independent variables\u201d [2  in women self-reporting breast cancer incidence vs. women remaining cancer-free (the main independent variable of interest). In addition to fixed-effect responses in groups, the model\u2019s random effects include the analysis of BMD values from individual participants, which adds more detailed response information to the model. Importantly, random effects are specifically related to some unknown or latent variable in individuals, and \u201cby including random-effects in the model, it is possible for researchers to account for multiple sources of variation\u201d [The present study examined longitudinal data from the SWAN cohort totaling 151 self-reports of cancer incidence in at least one breast and over 17,000 DEXA scans of the lumbar spine . The BMDriables\u201d . The fixriation\u201d .ij denotes the ith individual\u2019s continuous BMD values (the dependent variable) at the jth repeated measurement;The general formula for the linear regression mixed-effects model used in the present study is based on Hedeker and Gibbons :yij = \u03b20\u03b20 is the y-intercept between individuals;\u03b21ijt is the time or trend effect between individuals denoted by the jth individual annual visit = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9;\u03b22jx is the ith individual\u2019s self-reported breast cancer status (the independent variable) = 1 if yes for breast cancer, 0 otherwise;\u03b23ij \u00d7 xj), meaning that the factors represented by the missing data were unlikely to have contributed to the cause of the data\u2019s absence . FurtherThe hypothesis in the present MM-GT study was tested by analyzing longitudinal data from the SWAN cohort of middle-aged women. A mixed-effects linear regression model was used to examine bone spinal mineral density changes in women who self-reported breast cancer compared with women who remained cancer-free. A stepwise recursive procedure was used to fit the mixed-effects model to the SWAN data . Table 1The notated formula for the final selected mixed-effects model is:p < 0.05. Of note, the stepwise recursive procedure  of breast cancer through nutritional interventions that lower patients\u2019 intake of dietary phosphate. Furthermore, the restoration and maintenance of normal serum phosphate levels in patients can be assisted by pharmacotherapies, such as phosphate binders, that reduce the intestinal absorption of dietary phosphorus . The limIn the present MM-GT study, a grounded theory\u2013literature review method was used to synthesize findings in the research literature, leading to a hypothesis positing that a greater magnitude of changes in BMD over time are associated with breast cancer in middle-aged women. A mixed-effects linear regression model based on the SWAN cohort confirmed that longitudinal BMD changes were higher in women self-reporting breast cancer, but declined at a faster pace than BMD changes in women without breast cancer. Future clinical studies are needed to further investigate the causative role of dysregulated phosphate and phosphate toxicity in BMD abnormalities and bone metastasis in metastatic breast cancer. Furthermore, the authors previously found that high dietary phosphate intake was associated with increased breast cancer risk in the SWAN cohort, and a low-phosphate dietary intervention should be tested to decrease abnormal bone mineral density changes and tumorigenesis in breast cancer patients. Future studies should also monitor the endocrine hormonal levels that regulate phosphate metabolism in breast cancer patients, as well as bio-indicators of decreasing renal function."}
+{"text": "The proliferation of IoT devices has led to an unprecedented integration of machine learning techniques, raising concerns about data privacy. To address these concerns, federated learning has been introduced. However, practical implementations face challenges, including communication costs, data and device heterogeneity, and privacy security. This paper proposes an innovative approach within the context of federated learning, introducing a personalized joint learning algorithm for Non-IID IoT data. This algorithm incorporates multi-task learning principles and leverages neural network model characteristics. To overcome data heterogeneity, we present a novel clustering algorithm designed specifically for federated learning. Unlike conventional methods that require a predetermined number of clusters, our approach utilizes automatic clustering, eliminating the need for fixed cluster specifications. Extensive experimentation demonstrates the exceptional performance of the proposed algorithm, particularly in scenarios with specific client distributions. By significantly improving the accuracy of trained models, our approach not only addresses data heterogeneity but also strengthens privacy preservation in federated learning. In conclusion, we offer a robust solution to the practical challenges of federated learning in IoT environments. By combining personalized joint learning, automatic clustering, and neural network model characteristics, we facilitate more effective and privacy-conscious machine learning in Non-IID IoT data settings. In recent years, federated learning has emerged as a prominent research domain at the crossroads of artificial intelligence and decentralized data processing. The ever-evolving landscape of AI and machine learning necessitates a critical reassessment of the state-of-the-art in federated learning to ascertain its significance and potential applications . HoweverCommunication Cost: As federated learning inherently involves distributed learning, the transmission of training data, such as model parameters, to participating clients remains a substantial cost factor . Even thEquipment Heterogeneity: The performance capabilities of devices participating in federated learning can vary significantly, leading to issues like stragglers or dropout effects. Stragglers, devices with limited storage, computational power, or communication capabilities, can substantially prolong the duration of a federated learning round . While dPrivacy Issues: Federated learning aims to address privacy concerns inherent in traditional distributed learning. It achieves heightened privacy by transmitting only the data necessary for training, avoiding the exchange of personally sensitive information . HoweverNon-IID Data: Federated learning faces the challenge of non-independent and non-identically distributed (Non-IID) data, in contrast to traditional machine learning\u2019s assumptions of IID data ,16. In fConcerning the issue of complex, dynamic data sources, hybridizations of distributed policy and heuristic augmentation represent pivotal components that can augment the federated learning approach by amalgamating the strengths of policy-based strategies with heuristic-based techniques . Policy-Clustering Approach: We propose a clustering approach integrated into the federated learning framework to address the Non-IID data challenge.Experimental Design: We establish multiple Non-IID environments for comparative evaluations, offering insights into the strengths and weaknesses of our approach.Future Directions: We identify potential areas for further improvement in our method, facilitating future research in this domain.In this paper, we introduce an innovative clustering approach designed to address the challenges associated with Non-IID data within the federated learning framework. By grouping clients with similar data distributions, our approach aims to enhance the efficiency of federated learning without imposing additional burdens on the existing methodology. Our method significantly improves the accuracy of the final model in Non-IID scenarios, as demonstrated through comparative analyses with FedAvg and other clustering methods across various Non-IID settings. Our contributions in this paper encompass:In summary, our research endeavors aim to enhance the applicability and effectiveness of federated learning in addressing contemporary challenges. We aspire to advance AI development while safeguarding privacy and data integrity.In the rapidly evolving landscape of distributed learning, numerous research efforts have made substantial contributions to the optimization of algorithms and the enhancement of communication efficiency. These studies have addressed a wide range of challenges associated with scaling machine learning algorithms to distributed environments, ensuring convergence guarantees, and enabling effective communication. Notable among these contributions is the work of Boyd et al., which delved into the realm of distributed convex optimization, pushing its boundaries to extend applicability into non-convex settings, thereby unlocking new possibilities for versatile optimization techniques .Communication Cost Reduction:In a parallel vein, Dekel et al. introduced a distributed mini-batch algorithm designed to expedite the training speed of distributed learning models, resulting in significant linear acceleration benefits . In the Efficiently reducing communication costs represents a critical aspect of optimizing federated learning systems. Researchers have explored two primary directions to achieve this: data compression and minimizing the frequency of communication.2.Data Compression:An effective approach for reducing communication overhead involves data compression techniques. Model compression has proven to be particularly efficient in this regard. One practical method is to operate within a low-dimensional space by applying techniques such as low-rank approximation or random masking to model gradients . Alterna3.Communication Methods:In federated learning, communication methods play a vital role in determining system efficiency. There are two main communication methods: synchronous and asynchronous. In synchronous communication, all participating devices must transmit their data before the aggregation process can begin. While this approach ensures data consistency, it is vulnerable to the impact of stragglers, significantly prolonging training time. One commonly used algorithm in this context is Federated Averaging (FedAvg) . FedAvg In the field of federated learning, researchers have undertaken extensive efforts to tackle the challenges posed by data heterogeneity, particularly in scenarios involving non-identically and independently distributed (Non-IID) data. These endeavors encompass global and personalized strategies, each tailored to mitigate the impact of data heterogeneity and enhance the efficiency and efficacy of federated learning algorithms.Global Strategies:Global strategies are designed to ensure model consistency across all participating clients, thereby preventing divergence and promoting convergence. FedProx introduces a proximal term in local client updates to maintain model parameters in proximity to the global model. While it succeeds in preserving model consistency, it may exhibit slower convergence rates in cases where the data are independent and identically distributed (IID). In response to weight divergence issues, SCAFFOLD incorporates control variates in local client updates to adjust gradient deviations from the global gradient. However, this method comes at the cost of increased communication overhead and client resource utilization. Qu demonstrated that self-attention-based architectures  enhance federated learning over heterogeneous data, addressing challenges like convergence and catastrophic forgetting, through a rigorous empirical investigation across various federated algorithms, real-world benchmarks, and heterogeneous data splits. . In resp2.Personalization Strategies:Personalization strategies acknowledge the diversity of client data and aim to create individualized models for each client, ultimately aligning the models more closely with the respective data distributions. Deng et al. advance TailorFL that is a dual-personalized federated learning framework that tailors sub-models to individual devices based on their resource capabilities and local data distribution, resulting in improved inference accuracy and reduced resource usage compared to existing methods . Tan delThese research efforts collectively contribute to the advancement of federated learning, particularly in scenarios involving data heterogeneity. By offering a spectrum of strategies, both global and personalized, these approaches help address challenges related to data distribution diversity, ultimately enhancing the efficiency and effectiveness of federated learning algorithms.Selection and Broadcast:Federated learning is an innovative approach that enables collaborative model training across multiple client devices while ensuring data privacy. The architecture of federated learning comprises two main components: the server-side and the client-side. On the server-side, there exists a central server that manages the global neural network model\u2019s parameters. Importantly, the server does not have direct access to the training data. The primary responsibilities of the server include selecting clients to participate in federated learning rounds, transmitting model parameters and necessary hyperparameters to the selected clients, and aggregating and updating the global model based on the contributions from the clients. On the client-side, each participating client possesses its own training data. Clients perform two key tasks. First, they wait for the server to send the parameters of the neural network model, and during this waiting period, clients collect and store data locally. Second, after receiving the model parameters from the server, clients use their local data to train the model. Once the training is completed, clients send back the trained model parameters or gradients to the server. A typical federated learning round consists of four steps:The server selects clients to participate in the federated learning round. It encrypts and transmits the model parameters and other necessary training-related parameters to the selected clients.2.Local Training:Clients receive the model parameters and perform local gradient descent on the neural network model using their respective local data.3.Upload Parameters:After local training, clients send the trained model parameters back to the server. This step also involves encryption for privacy.4.Aggregation and Update:The server aggregates the model parameters or gradients received from the clients based on a predefined aggregation strategy. Subsequently, it updates the global neural network model with the aggregated parameters.These four steps are repeated multiple times throughout the federated learning process, either until the model converges or for a specified number of training sessions.Federated learning aims to minimize a global objective function that aggregates local objective functions across clients. The mathematical formulation of federated learning can be described as follows:The global objective function, denoted as x \u2208 X and y \u2208 Y. We combine them to be a pair of labeled data  \u2208 X \u00d7X \u00d7D. kth client. These coefficients, Commonly, loss functions such as cross-entropy loss are utilized for classification problems, When the client finishes local training, it sends the result of the training to the server. The server aggregates all of the result returned by clients using aggregate weight.In the previous section, we discussed the limitations of existing federated learning methods, particularly in handling Non-IID  data. These methods primarily rely on weight aggregation to align local and global objectives for accelerated convergence. However, when data distributions on client devices are highly skewed, these skewed gradient updates receive very low weights in the aggregation process. This inefficiency can lead to wasted computational resources and hinder the federated learning system\u2019s overall efficiency.To address this challenge, we introduce a novel approach, federated cluster, which utilizes clustering to group clients with similar data distributions. The goal is to achieve accelerated convergence while ensuring efficient resource utilization. Before delving into the formal introduction of federated clusters, we provide an overview of the FedAvg algorithm to highlight the differences and showcase how our approach can enhance model accuracy with minimal additional complexity.Algorithm 1 Federated Averaging (FedAvg)Server-sideInput:N, T, C\u00a0\u00a01: Initialize \u00a0\u00a02: for each round t = 0, 1, ...T do\u00a0\u00a03: \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a04: \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a05: \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0for each client do in parallel\u00a0\u00a06: \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0ClientUpdate describes the pseudocode of FedAvg in Algorithm 1, a commonly used federated learning algorithm. It involves initializing the neural network model and selecting a group of clients for training in each round. The global model parameters are transmitted to the selected clients, who then perform local gradient updates. After training, clients send back their updated model parameters, and the server aggregates these parameters based on the amount of data owned by each client.Algorithm 2 Federated Adaptive Cluster (FedAC)Server-sideInput:N, TC\u03b1\u03b2\u00a0\u00a01: Initialize \u00a0\u00a02: for each round t = 0, 1, ...T do\u00a0\u00a03: \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a04: \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a05: \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0for each client do in parallel\u00a0\u00a06: \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0ClientUpdate\u00a0\u00a01: \u2206\u00a0\u00a02: for each client k \u2208 C do\u00a0\u00a03: \u00a0\u00a0if cosine_similarity as part of our proposed personalized federated learning approach in Algorithm 2. The primary objective of FedAC is to address the issue of clients with smaller contributions to Federated Learning. These clients, which may have highly skewed or less representative data, can hinder the overall efficiency of the federated learning system.Our clustering approach allows us to harness their potential effectively. The key aspect of our clustering algorithm is the strategy for separating clients. We acknowledge that the initial model parameters and distance threshold settings can significantly influence the accuracy of client clustering. To mitigate this, we have opted to delay the clustering process until after a certain amount of training has been completed. This strategic decision ensures that the model parameters have reached a suitable state for accurate clustering, as illustrated in Input Parameters: FedAC introduces additional parameters compared to FedAvg. The input parameters include N (proportion of clients participating), T , and C (the set of clients in a cluster).Client Selection: Clients to be trained in each round are sampled from the cluster based on the defined proportion of participating clients (N).Local Training: Selected clients receive the global model from the server and perform local updates using gradient descent. This step ensures that the algorithm does not increase the computational burden on client devices, aligning with the federated learning principle of minimizing client resource consumption.Aggregation with Evaluation: After clients complete local training and send back their models, the server aggregates these models. Importantly, FedAC introduces an evaluation step. The server assesses whether the current global model is close to convergence or has already converged.Clustering Decision: The decision to perform clustering relies on two conditions. Firstly, we assess the degree of loss reduction to determine the proximity of the global model to convergence. Secondly, we employ cosine similarity to evaluate the correlation between client gradient updates and the gradient update of the global model. Cosine similarity measures the angle and direction of these updates, helping us identify clients whose gradient updates significantly deviate from the direction of the global model. Such deviations may indicate variations in data distributions among clients.Clustering: If clustering is warranted, the server selects a subset of clients for clustering based on their gradient updates. The selected clients are divided into two clusters using a dichotomy approach. This process is designed to efficiently group clients with distinct data distributions.The clustering process shown in We delve into the experimental results, presenting our findings based on various parameter settings, datasets, and model structures. We also provide insights into the effectiveness of our proposed algorithm.Number of Classes: 10;Total Dataset Size: 60,000 images (6000 images per class);Training Dataset Size: 50,000 images;Test Dataset Size: 10,000 images.We utilized the CIFAR-10 dataset for training and classification tasks in our federated learning experiments . CIFAR-1IID Situation: Clients have the same probability of having data from all classes.Non-IID Situation (two clusters): Clients are divided into two clusters, each with five classes of data.Non-IID Situation (three clusters): Clients are divided into three clusters, with two clusters containing three classes of data and one cluster with four classes of data.Non-IID Situation (four clusters): Clients are divided into four clusters, with two clusters containing two classes of data and two clusters containing three classes of data.Despite CIFAR-10\u2032s inherently IID  nature, we introduced Non-IID scenarios for federated learning. Our Non-IID scenarios aimed to simulate more realistic data distributions across clients, considering factors such as geographical location, age, and social environment. We categorized the data distributions into four distinct cases:This classification allowed us to examine how our algorithm performs across varying degrees of data distribution heterogeneity among clients.Number of Clients: 100;Total Number of Rounds: 200;Proportion of Participating Clients: 0.1;Local Epochs: 5;Learning Rate: 0.001;Momentum: 0.9;Batch Size: 32;Clustering Thresholds (\u03b1 and \u03b2): \u03b1 is set to 5 for assessing convergence, while \u03b2 employs a 90-degree threshold for detecting inconsistent data distributions within clusters.We outline the hyperparameters used in our experiments, implemented using the Flower framework. Key hyperparameters include:In the IID scenario, we observe that the performance of FedAC, FedACP, and FedAvg is nearly identical in In the Non-IID scenarios, we observe distinct differences in performance. FedAvg exhibits significant oscillations in both accuracy and loss due to the inherent Non-IID nature of the data distributions among clients. In contrast, FedAC stabilizes quickly after clustering, as evidenced by Our algorithm demonstrates robustness and effectiveness in transforming Non-IID data environments into IID-like scenarios, making it particularly valuable in privacy-sensitive applications. In healthcare scenarios, for instance, where patient habits may significantly impact the data distribution, our FedC algorithm allows us to create separate training groups without revealing the sensitive habits of individual patients. However, while discussing the capabilities of our algorithm, it is important to highlight the inherent privacy and security aspects of Federated Learning, which have been an integral part of our work. We outline key areas for future research and development to further enhance the capabilities and robustness of our algorithm. Our current clustering initiation method relies on straightforward threshold-based criteria. Future research should focus on devising more sophisticated and adaptable detection methods. These methods should be capable of accurately identifying the optimal timing for clustering in scenarios with oscillating loss variations, thus improving the clustering process\u2019s efficiency. While clustering generally leads to improved model performance, there are instances where clustering does not yield optimal results, resulting in suboptimal or inefficient clusters. Future work should concentrate on enhancing the efficiency and effectiveness of the clustering process. This may involve refining the clustering algorithms or introducing adaptive mechanisms to adjust to changing data distributions. Scanning all clients within a cluster during each clustering round can be computationally demanding. Future efforts should aim to optimize the computational efficiency of the clustering process. This includes reducing computation time and addressing potential issues caused by straggling clients. While our algorithm currently utilizes Federated Averaging (FedAvg) for aggregation and training, it is worthwhile to explore alternative global strategies such as Federated Proximal (FedProx). Investigating these alternative strategies can help determine if they offer performance improvements in specific scenarios. Conducting additional experiments is essential to evaluate the benefits of alternative global strategies. The use of model parameters for clustering may raise privacy concerns, particularly in scenarios involving sensitive data. Future research should focus on developing techniques that ensure effective clustering while preserving privacy. Differential privacy and other privacy-preserving methods should be explored to mitigate privacy risks associated with clustering. Lossy compression techniques used for communication may impact efficiency. Future work should investigate approaches to minimize the communication overhead caused by lossy compression. This involves optimizing compression algorithms and strategies to improve communication efficiency without compromising data integrity. By addressing these recommendations, researchers and practitioners can further refine and expand the capabilities of our personalized federated learning algorithm with adaptive clustering. This will make the algorithm more adaptable and robust, particularly in the context of Non-IID IoT data scenarios."
\ No newline at end of file