Perioperative antibiotic prophylaxis in non-infected orthopedic surgery is evident, in contrast to prophylaxis during surgery for infection. Epidemiological data are lacking for this particular situation.nd debridement. However, we failed to define an ideal hypothetic prophylaxis during antibiotic therapy to prevent further SSIs.It is a single-center cohort on iterative surgical site infections (SSIs) in infected orthopedic patients. We included 2480 first episodes of orthopedic infections (median age 56 years and 833 immune-suppressed): implant-related infections (n = 648), osteoarticular infections (1153), and 1327 soft tissue infections. The median number of debridement was 1 . Overall, 1617 infections (65%) were debrided once compared to 862 cases that were operated multiple times (35%). Upon iterative intraoperative tissue sampling, we detected pathogens in 507 cases , of which 241  corresponded to the initial species at the first debridement. We witnessed 265 new SSIs (11% of the cohort) that were resistant to current antibiotic therapy in 174 cases (7% of the cohort). In multivariate analysis, iterative surgical debridements that were performed under current antibiotic administration were associated with new SSIs ; mostly occurring after the 2nd debridement. The new pathogens are however unpredictable. The prevention, if feasible, probably relies on surgical performance and wise indications for re-debridement instead of new maximal prophylactic antibiotic coverage in addition to current therapeutic regimens.Selection of new pathogens resistant to ongoing antibiotic therapy occurs frequently during iterative debridement in orthopedic infections, especially after the 2 The ideal regimen for perioperative antibiotic prophylaxis for prevention of surgical site infections (SSI) is evidence-based for the majority of clean, non-infected orthopedic procedures –4. HowevPractically, when performing a second look during ongoing antibiotic therapy, surgeons continue with the current therapeutic antibiotics or, if clinical evolution is unsatisfactory, empirically broaden the spectrum after obtaining new intraoperative tissue samples. Alternatively, few colleagues administer the standard perioperative prophylaxis, independently of the pathogens, simply because they lack specific protocols. New intraoperative cultures during re-operation may remain negative because of the influence of systemic antibiotics , but theThese new pathogens indicate a dilemma. If clinical evolution is satisfactory, physicians might interpret them as a selection or contamination, and usually continue with the antibiotic treatment in place. However, besides a pre-planned re-intervention , mostly the evolution has been unsatisfactory; hence the indication for re-debridement. Consequently, these new pathogens are interpreted as new SSIs, with broadening of the spectrum and prolongation of total antimicrobial therapy .In this study, we aimed to evaluate the missing epidemiology and specifically link the occurrence of new SSIs to the numbers of iterative re-debridement that we performed under current therapeutic antibiotic agents and. We wonder if these patients would profit from extended prophylaxis during re-debridement; and if the nature of possible secondary SSIs would be predictable.), including all adult patients hospitalized for clinically moderate and severe orthopedic infections, including the diabetic foot 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—e.g. participant privacy or use of data from a third party—those 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 article titlted: “Changing perioperative prophylaxis during antibiotic therapy and iterative debridement for orthopedic infections?” Is a description of the microbiological findings in patients with orthopedic infections that required a new re-operation for failure of the infection during antibiotic treatment. This is a very original work, well written and with a large number of patients included in the analysis from a retrospective database. I have some comments:1.- In the abstract and in your conclusions, you highlight that (abstract) “Selection of new pathogens resistant to ongoing antibiotic therapy occurs infrequently” (conclusion) “According to our cohort of 2480 adult patients with orthopedic infections, new SSIs occur at ten percent’s risk during iterative debridement and concomitant antibiotic therapy.” From a clinical perspective, I think that the impresive data is that from 862 patients that required a re-debridement 507 (59%) had a positive culture and in 265 (52%) the isolated microorganism is different from the first one. This means that from all that required a re-debridement 862, 30.7% (265) had a different pathogen and indeed this is not a minor problem particularly taken into account that the microorganisms were often more resistant.2.- According to material and methods section: “We excluded cases that were amputated [11], cases with antibiotic-free windows before re- debridement…” so all patients included in the study were under antibiotic treatment. Then I dont understand what you say in results section: “Table 2 shows clinical variables related to new SSIs. Current antibiotic administration and…” All patients were under antibiotic treatment, so you mean a specific antibiotic? Indeed, in table 2 you show different antibiotics  but no one was significantly associated with new G+ or G-.3.- In methods section for the multivariate analysis you mention: “An unmatched multivariate logistic regression analysis determined associations with the outcome “SSI resistant to antibiotic therapy”.” So until now the endpoint of the study (table 2) was new SSI and for the multivariate adjustment you change to antibiotic-resistant (to the current antibiotic treament) SSI. It is reasonable to expect that the majority of new pathogens were resistant to current antibiotics but this was indeed the case? Could you confirm that all new pathogens were resistant to current antibiotic treament. In addition, in multivariate adjustment you mention: “We confirmed that current antibiotic use was associated with new antibiotic-resistant SSIs ,…” but in table 5 there is no reference to current antibiotic variable??? Indeed, all the patients were under antibiotic treatment so I dont understand this as in point 2.4.- The reason for a new infection (isolatation of a different pathogen from the first one) could be the consequence of 1.- miss identification during the first surgery for infection  and 3.- superinfection of the wound after surgery. Considering only 2 and 3 as possible, the third is not modifiable by antibiotic prophylaxis but the number 2 could be significanlty reduced. In the text you talk about prophylaxis for the new debridement but this is not correct. New pathogens are indeed new infections so you have to talk about additional empirical treatment when a patient requires a new debridement, at least 30% had a new infection so in my opinion we have to recommend to broad the spectrum until deffinitive results are available . A different point is whether the previous debridement to the current one with no pathogens, the same pathogen or a new pathogen, required a different prophylaxis to avoid contamination by a different pathogen . Could you evaluate the antibiotics  received during the prior debridement to the one with or without a new pathogen? This would be really interesting.Reviewer #2: Dear Authors,I read with interest the paper. It is a comprehensive analysis of complex set of data of a large cohort of patients with orthopedic infection with a clear aim. It needs a huge effort to organize this type of data. And it is nearly impossible to study the study hypothesis in any other prospective and more controlled way. The aim of the study is clearly defined and important. The issue of antibiotic prophylaxis and treatment in unsuccessful orthopedic infections is a very important and still unsolved topic touching every specialist involved in treatment of this complex pathology.Despite the complexity of the cohort data the authors were able to organize it in a systematic way. The presenting cohort is thus clearly outlined in the text and trough the tables.Despite the study did not give a clear answer to the original question it still revealed the complexity of the clinical field and gave thought lines for the readers to integrate in their clinical decisions.Some comments:Page 3 last paragraph: Why were there revisions in satisfactory clinical evolution?Page 4 last paragraph: Why episodes with new organisms that did not change the antibiotics were interpreted as contamination, just because they were sensitive to original antibiotic therapy? According to the definition of the infection it should thus be mentioned that only high grade infections were included.Page 10 second paragraph: bundle of measures [1-3]. "Thus, avoiding unnecessary re-debridement, evidence-based surgical indications and techniques, and experienced surgical skills are certainly as important as mere addition of a new molecule." This sentence is very brave and to me lacking evidence besides common thinking.Page 10 second paragraph:"At the same time, numerous reported transient kidney injuries by aminoglycosides [27] or combined vancomycin prophylaxis [28] in orthopedic surgery." The sentence has no verb.**********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,  27 Nov 2019Revision of PONE-D-19-23445 manuscript entitled “Changing perioperative prophylaxis during antibiotic therapy and iterative debridement for orthopedic infections?” by Wuarin et al. Dear Dr Daniel Pérez-PrietoWe thank you for your email of 1 November 2019 in which you invite us to submit a revised version of our above-mentioned manuscript. We also want to thank both Reviewers for the careful reading and suggestions provided. Of note, we incorporated all their suggestions into the manuscript. All changes are tracked. All authors agreed to the final version of the paper.Thanking you in advance for your consideration of our revised manuscript and we look forward to the final decision of the Editorial Committee.Yours most sincerely,Ilker Uçkay, MD Editors CommentsA marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.Answer: Done.An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.Answer: Done.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 athttp://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfAnswer: We now adapted the manuscript according to this editorial guidance.2. Please include your tables as part of your main manuscript and remove the individual files.Answer: Done as advised. However, we have five large Tables. By fitting them into the manuscript, the Tables become difficult to read. Therefore, we take the liberty to resumes all five Tables on a separate file, too. http://journals.plos.org/plosone/s/supporting-information3. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: Answer: We have no Supporting Information.Reviewers' comments:1. Is the manuscript technically sound, and do the data support the conclusions?Reviewer #1: YesReviewer #2: YesAnswer: We thank both Reviewers.________________________________________2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesAnswer: We thank both Reviewers.________________________________________3. Have the authors made all data underlying the findings in their manuscript fully available?Reviewer #1: YesReviewer #2: YesAnswer: We thank both Reviewers.________________________________________4. Is the manuscript presented in an intelligible fashion and written in standard English?Reviewer #1: YesReviewer #2: YesAnswer: We thank both Reviewers.________________________________________Reviewer #1: The article titled: “Changing perioperative prophylaxis during antibiotic therapy and iterative debridement for orthopedic infections?” Is a description of the microbiological findings in patients with orthopedic infections that required a new re-operation for failure of the infection during antibiotic treatment. Answer: Yes. Exactly. Thank you.This is a very original work, well written and with a large number of patients included in the analysis from a retrospective database. Answer: Thank you very much. We introduce this sentence now on page 16, lines 272-273.I have some comments:1.- In the abstract and in your conclusions, you highlight that (abstract) “Selection of new pathogens resistant to ongoing antibiotic therapy occurs infrequently” (conclusion) “According to our cohort of 2480 adult patients with orthopedic infections, new SSIs occur at ten percent’s risk during iterative debridement and concomitant antibiotic therapy.” From a clinical perspective, I think that the impressive data is that from 862 patients that required a re-debridement 507 (59%) had a positive culture and in 265 (52%) the isolated microorganism is different from the first one. This means that from all that required a re-debridement 862, 30.7% (265) had a different pathogen and indeed this is not a minor problem particularly considered that the microorganisms were often more resistant.Answer: Exactly. We like this  way of resuming by Reviewer 1. We now introduce his/her phrasing into the Discussion .2.- According to material and methods section: “We excluded cases that were amputated [11], cases with antibiotic-free windows before re- debridement…” so all patients included in the study were under antibiotic treatment. Answer: Yes; besides the very first debridement for the orthopedic infection . We explicitly say this in the Methods  and repeat it at various parts of the manuscript.Then I don't understand what you say in results section: “Table 2 shows clinical variables related to new SSIs. Current antibiotic administration and…” All patients were under antibiotic treatment? Answer: Yes. The entire analyzed population was already under systemic antibiotic therapy . In Table 2 you show different antibiotics  but no one was significantly associated with new G+ or G-.Answer: Indeed. No antibiotic class  was associated with the Gram-staining of the new pathogens. The analyzed population were entirely under some systemic antibiotic therapy . 3.- In the Methods section for the multivariate analysis you mention: “An unmatched multivariate logistic regression analysis determined associations with the outcome “SSI resistant to antibiotic therapy”.” So, until now the endpoint of the study (Table 2) was new SSI and for the multivariate adjustment you change to antibiotic-resistant (to the current treatment) SSI. Answer: No. In the multivariate analyses, the outcome variables were always “resistant microorganisms” . We now reword better on page 10, line 193 and on page 14, lines 246-253.It is reasonable to expect that the majority of new pathogens were resistant to current antibiotics but this was indeed the case? Could you confirm that all new pathogens were resistant to current antibiotic treatment. Answer: No, not every new pathogen was resistant to current (prior) antibiotics. We already say it on page 8, line 180: "resistant to current antibiotics in 174 cases ". We also add now the number of new susceptible pathogens and display some key, non-resistant, pathogens in Figure 1, too. In addition, in multivariate adjustment you mention: “We confirmed that current antibiotic use was associated with new antibiotic-resistant SSIs , …” but in Table 5 there is no reference to current antibiotic variable? Indeed, all the patients were under antibiotic treatment?Answer: Yes. Every patient in the corresponding analysis was under systemic antibiotic therapy. Therefore, there is no sense to introduce a variable “antibiotic” into the final model, if every patient already is under antibiotics. We now explain this aspect better in Results , and repeat it as Limitation .We equally modify the former sentence "We confirmed that current antibiotic use was associated with"… to a new phrasing .4.- The reason for a new infection (isolation of a different pathogen from the first one) could be the consequence of 1.- miss identification during the first surgery for infection  and 3.- superinfection of the wound after surgery. Considering only 2 and 3 as possible, the third is not modifiable by antibiotic prophylaxis but the number 2 could be significantly reduced. Answer: Thank you very much. This is a probably a better résumé than we initially wrote. We thank Reviewer 1 for this short résumé and introduce it into the Discussion .In the text you talk about prophylaxis for the new debridement but this is not correct. New pathogens are indeed new infections so you have to talk about additional empirical treatment when a patient requires a new debridement.Answer: We beg to differ. The Reviewer 1 may think that we mention replacing a current antibiotic treatment by a new one; basing on presumed changes in microbiology and continuing with this new regimen during several days in a therapeutic manner. No. Wherever we talk about prophylaxis, we truly mean additional prophylaxis under current therapeutic antibiotic therapy. Just one dose of a perioperative antibiotic prophylaxis, which prevents new infections, and is not continued after surgery. We now make this distinction clearer in the Methods .A different point is whether the previous debridement to the current one with no pathogens, the same pathogen or a new pathogen, required a different prophylaxis to avoid contamination by a different pathogen . Answer: In our study population, we had 83 different antibiotic regimens prior to the occurrence of 273 new pathogens . A given episode could witness different antibiotic regimens throughout the therapeutic course. To these microbiological differences, we also add a variety of 867 new antibiotic regimens . Such resulting two-by-two tables become too much detailed and individualized to be displayed in this manuscript. We have to group the different subpopulations, which we did. However, Reviewer 1 is right in a conceptual way. We acknowledge it now in the "Limitation" section .Could you evaluate the antibiotics received during the prior debridement to the one with or without a new pathogen? This would be really interesting.Answer: The same question and answer as above. Of note, all patients during iterative surgeries were already under systemic antibiotic therapy . Therefore, we cannot analyze if a prior antibiotic therapy per se was associated with an enhanced risk of new infections . We can only analyze of a particular antibiotic class was associated with a particular group of new pathogens, which we already displayed on Table 2 and Figure 1.Reviewer #2: I read with interest the paper. It is a comprehensive analysis of complex set of data of a large cohort of patients with orthopedic infection with a clear aim. It needs a huge effort to organize this type of data. And it is nearly impossible to study the hypothesis in any other prospective and more controlled way. The aim of the study is clearly defined and important. The issue of antibiotic prophylaxis and treatment in unsuccessful orthopedic infections is a very important and still unsolved topic touching every specialist involved in treatment of this complex pathology.Answer: Thank you very much. We now use the last sentence in our revised manuscript .Despite the complexity of the cohort data the authors were able to organize it in a systematic way. The presenting cohort is thus clearly outlined in the text and trough the tables. Despite the study did not give a clear answer to the original question it still revealed the complexity of the clinical field and gave thought lines for the readers to integrate in their clinical decisions.Answer: We thank you very much.Page 3 last paragraph: Why were there revisions in satisfactory clinical evolution?Answer: Planned second or third looks in order to decrease the bacterial inoculum, for which the indication has been decided already during the first debridement. Unfortunately, it is very common in surgical settings to re-operate the patient in a planned way in order to (wrongly) accelerate the healing, especially when the patient was instable or has other parameters to consider. We now underline this possibility of “planned reinterventions” on page 3, lines 64-65.Page 4 last paragraph: Why episodes with new organisms that did not change the antibiotics were interpreted as contamination, just because they were sensitive to original antibiotic therapy? Answer: There are three answers, which we all explain better in this revised version of our paper:a) The interpretation of a “contamination” clinically based on a microbiological finding during a good evolution, meaning that the newly detected bacteria had no new clinical (worse) impact .b) In contrast, pathogens sensitive to original antibiotic therapy can truly cause a clinical worsening, but are identified as a new infectious constellation .c) Theoretically, new bacteria can also be a new infection that was not severe enough to worsen the clinical evolution (yet). In that sense, when there is good clinical evolution, it is impossible to distinguish between colonization and clinical new infection, which we now acknowledge it the “Limitation section” .According to the definition of the infection it should thus be mentioned that only high-grade infections were included.Answer: Yes. “Clinically moderate and severe” infections. We now add this prerequisite to the inclusion criteria .Page 10 second paragraph: bundle of measures [1-3]. "Thus, avoiding unnecessary re-debridement, evidence-based surgical indications and techniques, and experienced surgical skills are certainly as important as mere addition of a new molecule." This sentence is very brave and to me lacking evidence besides common thinking.Answer: Yes, this is common thinking. And we have now deleted this sentence entirely.Page 10 second paragraph: "At the same time, numerous reported transient kidney injuries by aminoglycosides [27] or combined vancomycin prophylaxis [28] in orthopedic surgery." The sentence has no verb.Answer: Yes, thank you. The missing verb was “documented” .________________________________________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 “no”, your identity will remain anonymous but your review may still be made public.Answer: It is up to you. I do not mind to have my personal identity to be 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.Reviewer #1: NoReviewer #2: No________________________________________https://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 us at figures@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, Answer: Yes, we have uploaded them.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 5 Dec 2019Changing perioperative prophylaxis during antibiotic therapy and iterative debridement for orthopedic infections?PONE-D-19-23445R1Dear Dr. Uçkay,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Daniel Pérez-Prieto, PhDAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 10 Dec 2019PONE-D-19-23445R1 Changing perioperative prophylaxis during antibiotic therapy and iterative debridement for orthopedic infections? Dear Dr. Uçkay:I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. 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Adhesion-mediated activation of FAK/ERK signalling pathway, enabled by the formation of filopodial protrusions (FLP), has been shown to be an important event for triggering of dormancy-to-proliferation switch and metastatic outgrowth of breast cancer cells (BCC). We studied the role of actin-binding protein profilin1 (Pfn1) in these processes.Quantitative immunohistochemistry (IHC) of BC tissue microarray (TMA) and survival analyses of curated transcriptome datasets of BC patients were performed to examine Pfn1’s association with certain clinicopathological features. FLP formation and single cell outgrowth of BCC were assessed using a 3D matrigel culture that accurately predicts dormant vs metastatic outgrowth phenotypes of BCC in certain microenvironment. Gene expression studies were performed to identify potential biological pathways that are perturbed under Pfn1-depleted condition.Lower Pfn1 expression is correlated with lower nuclear grade of breast tumours and longer relapse-free survival of BC patients. Pfn1 depletion leads to defects in FLP and outgrowth of BCC but without impairing either FAK or ERK activation. Guided by transcriptome analyses, we further showed that Pfn1 depletion is associated with prominent SMAD3 upregulation. Although knockdown and overexpression experiments revealed that SMAD3 has an inhibitory effect on the outgrowth of breast cancer cells, SMAD3 knockdown alone was not sufficient to enhance the outgrowth potential of Pfn1-depleted BCC suggesting that other proliferation-regulatory pathways in conjunction with SMAD3 upregulation may underlie the outgrowth-deficient phenotype of BCC cells upon depletion of Pfn1.Overall, these data suggest that Pfn1 may be a novel biomarker for BC recurrence and a possible target to reduce metastatic outgrowth of BCC. Disseminated BCC that fail to initiate FLPs become incompetent in metastatic outgrowth and therefore remain dormant. Furthermore, FLPs are also abundant in the sub-population of BCC that are endowed with stem-cell like features and have higher tumour-initiating potential (whether at the primary or metastatic sites) than the non-stem-like pool of BCC. Therefore, FLP abundance has been regarded a general deterministic indicator for tumourigenic ability of BCC cells. Formation of FLPs requires the actions of Rho-GTPases  and actin nucleation and/or elongating factors such as Arp 2/3 complex, formin family protein mDia2  and Ena/VASP (enabled/vasodilator activated phosphoprotein) proteins. Loss of function (LOF) of these actin cytoskeletal regulators reduces the incidence of primary tumours from experimentally implanted BCC as well as causes impairment in metastatic outgrowth of extravasated BCC.7 These data further suggest the general importance of actin assembly factors or their upstream regulators in outgrowth and tumour-initiating abilities (whether at the primary or the metastatic sites) of BCC.Tumour metastasis is a multistep process that requires cancer cells to escape from the primary tumour, survive in the circulation, invade the target organs and finally reinitiate secondary tumour outgrowth at the metastatic sites. Oncogenic transformation causes alterations of the actin cytoskeleton through deregulating expression and/or activity of a wide range of actin-regulatory proteins. These changes are often correlated with acquisition of a motile phenotype of cancer cells. Recent studies have also underscored the importance of actin cytoskeleton in governing tumour initiation and metastatic outgrowth of cancer cells. For example, metastatic outgrowth of extravasated breast cancer (BC) cells (BCC) is promoted by signalling triggered by the extracellular matrix (ECM) component of the local microenvironment, and this involves changes in actin cytoskeletal architecture. Specifically, ECM-induced activation of integrin receptors potentiates actin stress-fibre assembly through a FAK -Src-ERK  signalling axis. Preventing actin stress-fibre assembly or blocking integrin signalling dramatically inhibits FAK-Src-ERK signalling and subsequent metastatic outgrowth of disseminated BCC cells.8 Although these two Pfn isoforms are structurally similar and generally bind to the similar types of ligands, they differ in their affinities for various ligands. Pfn1 expression is partially downregulated in the primary tumour in human BC, more prominently in those associated with distant metastasis.10 In contrast to the pro-migratory role of Pfn in most physiological contexts, there is evidence that downregulation of Pfn isoforms can induce hypermigratory phenotype in certain BCC lines.13 At least in MDA-MB-231  cell line) xenograft model, we showed in vivo evidence of increased proficiency of BCC to escape from experimentally established primary tumours upon Pfn1 depletion. Collectively, these studies implied that downregulation of Pfn1 can promote the early steps of BC metastasis . Contrasting these pro-metastatic features associated with LOF of Pfn, we showed that depletion of Pfn1 can severely impair the development of pulmonary metastases from extravasated MDA-231 cells suggesting there may be Pfn1-dependency for metastatic colonisation of TNBC cells.10 However, whether (a) it is the inability to survive or reduced metastatic outgrowth in the lung parenchyma that causes the colonisation defect of BCC upon Pfn1 depletion and (b) there is any potential association between Pfn1 expression and growth-related clinical features in human BC, remain unclear.Profilin (Pfn) family of actin-binding proteins serve as a common denominator in various cellular pathways of actin assembly including those driven by formin and Ena/VASP proteins (these proteins are capable of binding to Pfn and recruiting polymerisation-competent actin monomers via Pfn). There are two main cellular isoforms of Pfn: Pfn1 (the major isoform) and Pfn2 .matrigel-on-top) culture: it closely approximates the compliant mechanical microenvironment of lung and mammary gland) that accurately predicts the metastatic outgrowth competency of BCC in the lungs,15 we show that Pfn1 deficiency causes defects in FLP abundance (a feature of outgrowth-competent cells) and reduces single cell outgrowth ability of TNBC cells. Our studies further suggest that Pfn1 depletion induces outgrowth defect in TNBC cells by affecting an ECM-stiffness sensitive signalling pathway that is important for BCC outgrowth other than the FAK/ERK signalling.In this study, we demonstrate that lower Pfn1 expression correlates with lower nuclear grade (NG: an indicator of growth-aggressiveness of tumour cells) of BC cells and longer relapse-free survival (RFS) of BC patients. Using a three-dimensional (3D) basement membrane matrix (BME or matrigel) culture  outgrowth of BCC was assessed using MoT assay in either 384- or 96-well format as previously described.15 Essentially, cells were seeded at different densities on a layer of 100% growth factor-reduced matrigel  and then overlaid with culture medium containing 2% matrigel. In experiments involving collagen-I, matrigel was mixed with collagen-I (BD Biosciences) with final concentrations of matrigel and collagen-I equal to 7.9 mg/mL and 2 mg/mL, respectively. After 7–10 days of culture, cells were fixed overnight with 4% paraformaldehyde and 5% sucrose in PBS, permeabilised for 5 min with 4% paraformaldehyde, 5% sucrose, and 0.25% Triton X-100 in PBS before staining for 25 min with a mixture of 4% paraformaldehyde, 5% sucrose, and 10 μg/mL Hoechst 33342 (Invitrogen) in PBS followed by washing for 10 min with PBS twice. Cells were imaged on an ImageXpress Ultra confocal high content reader using a ×4 objective. A total of twenty 50 µm thick planes was acquired and collapsed using a maximum projection algorithm. Objects were enumerated using the MetaXpress multiwavelength cell scoring algorithm. For FLP assay, cells were cultured in 8 chamber glass slide system  coated with 50 μL growth factor-reduced matrigel (without or with collagen-I) for 3 days. Following fixation and permeabilisation as described above, cells were incubated overnight with either Alexa Fluor 488- or rhodamine-phalloidin , and washed with PBS for 15 min for three times. The slides were then mounted overnight with gelvatol mounting medium in dark. The images were taken using Nikon A1 Spectral Confocal Microscope.Generation and culture of sublines of MDA-231 cells stably expressing control- and Pfn1-shRNAs have been described previously.17 Doxycycline (Dox)-inducible TRIPZ Pfn1 Lentiviral shRNA vector  was purchased from Dharmacon. Adenovirus encoding EGFP (Ad-GFP) and SMAD3 (Ad-SMAD3) were obtained from University of Pittsburgh Viral core and Vector Biolabs , respectively. For SMAD3 knockdown, two different sets of siRNAs with no targeting sequence overlap were used: siRNA#1 ON-TARGETplus Human Smad3 siRNA – SMARTpool  and siRNA #2 Human Smad3 Stealth siRNA . All siRNA transfection was performed using transfection reagent 2 from Dharmacon. The cells were transfected with the siRNAs for 24 h before performing outgrowth assays. Adenoviral infection was performed overnight at 500 MOI before performing outgrowth assays. The efficiency of transduction was assessed by % GFP-positive cells 48–72 h after infection. Lentivirus was produced by the Magee-Women’s Hospital of University of Pittsburgh lentiviral core facility. Lentiviral infection of MDA-231 was performed at MOI of 500 in the presence of 8 μg/ml of Polybrene. Cells with the strongest RFP signal after dox induction (2 μg/ml) were selected and the best Pfn1 knockdown clone was selected for further experiments. For outgrowth experiments, cells were treated initially with dox (2 μg/ml) for 2 days before seeding on matrigel and dox was replenished every third day.The sequences of single target and smart-pool siRNAs targeting Pfn1 isoforms (source: GE Dharmacon) and smart-pool of Pfn2 (source: Santa Cruz) were reported in our previous studies.Cells cultured on coverslips were fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were permeabilised using 0.25% Triton X-100 in PBS, blocked with 10% goat serum in PBS, and incubated with anti-Collagen-I alpha1 antibody  in Ab-buffer (10% goat serum/0.25% Tween 20/1X PBS) for 1 h. After primary antibody incubation, cells were washed three times with 1X PBS and incubated with Alexa 488-conjugated secondary antibody for 1 h. Cells were then washed three times with PBS before mounting on glass slides with mounting media containing DAPI for fluorescence imaging using an Olympus IX71 microscope. Fluorescence images were background subtracted, and the fluorescence intensity of collagen-I immunoreactivity was analysed on a cell-by-cell basis for computing the average fluorescence intensity based on the analyses of several randomly selected fields containing 150–200 cells pooled from two independent experiments.Total cell lysate from 2D monolayer culture was prepared by extracting cells with modified RIPA buffer  supplemented with 50 mM NaF, 1 mM sodium pervanadate, and protease inhibitors. Total cell lysate from 3D culture was prepared by incubating cells at 4 °C in the modified RIPA buffer supplemented with 0.5% SDS and protease/phosphatase inhibitor cocktail (Pierce). The extracts were clarified by centrifugation at 13,000 rpm for 30 min and the supernatant was used for immunoblotting. Sources of different antibodies were: anti-Pfn1 (Abcam), anti-phospho-FAK (Y397) (Invitrogen), anti-Smad3 (Biorad), anti-ERK1/2, anti-phospho-ERK1/2 and anti-phospho-Smad3 (S423/S425) , anti-Pfn2 and anti-FAK (Santa Cruz), and anti-GAPDH and anti-Tubulin (Sigma-Aldrich) Immunoblotting concentrations for different antibodies were: 1:3000 for anti-Pfn1, anti-GAPDH and anti-tubulin; 1:500 for anti-Pfn2, anti-ERK1/2, anti-pERK1/2 and anti-FAK; and 1:1000 for anti-Smad3, anti-pSmad3 and anti-pFAK.19 Fluorescence staining of TMA with anti-Pfn1, anti-pan-cytokeratin and DAPI, and quantification of Pfn1 immunostaining in cytokeratin-positive areas (representing tumour cells) were performed accordingly to a previous protocol.20We used the YTMA49–12 breast cancer TMA  constructed from archived invasive breast cancer patient samples from 1961 to 1983. This TMA represented ER (oestrogen receptor)-positive and ER-negative tumours approximately at a 1:1 ratio and has been used in many previous studies for cancer biomarker identification and validation.2 ratios. A total of 13,008 probes showing consistent direction of fold-changes in all experiments were retained for further analysis. Pathway enrichment analysis was conducted via 2 different approaches. First, gene-set enrichment analysis (GSEA21) was conducted on a list of 13,008 probes, ranked by the magnitude of their median fold-change across the 3 studies. Specifically, we used the ‘pre-ranked GSEA’ option with enrichment statistic set to ‘classic’, and queried the canonical pathway (C2:CP) and oncogenic signature databases (C6:oncogenic signatures), available from the Molecular Signature Database, MSigDb.22 False discovery rate (FDR) were controlled by the q-value and pathways with FDR ≤10% were considered to be significantly enriched. Significant pathways were further clustered based on shared gene members through the EnrichmentMap application in Cytoscape.23 In addition to GSEA, we employed the Ingenuity Pathway Analysis, IPA (Qiagen) tool to identify potential upstream regulators that influence gene expression upon Pfn1 knockdown. We pre-selected 349 genes with an absolute fold-change ≥1.5-fold as input for IPA. Overlap of regulator target genes with the experimental input gene list was assessed by the Fisher’s exact test; whereas, the consistency of the overlapping gene expression profiles with activation or inhibition of the regulator (predicted from prior data in the Ingenuity Knowledge Base) was quantified by the z-score method, as described . Regulators with an overlap p-value ≤0.01 and absolute z-score ≥2 were considered significantly affected by Pfn1 KD treatment.Gene expression signals from each hybridised microarray (Affymetrix platform) were derived by the MAS5 algorithm. Fold-change estimates (Pfn1 KD vs control) were calculated from each of the two independent Pfn KD experiments  and converted to log24 Curated genomic and clinical data for each of these cancers were obtained from Gene Expression Omnibus (GEO), European Genome-Phenome Archive (EGA) and The Cancer Gene Atlas (TCGA). Overall survival(OS) or relapse-free survival(RFS) was evaluated on all available patients using the Kaplan–Meier approach, after dividing the patient populations based on the median gene expression across all samples tested. Differences in survival between two groups were compared using log-rank tests, with statistical significance ascribed at P < 0.01. The relative rates of time to death in the two groups (above and below median gene expression) were expressed via hazard ratios.The effects of Pfn gene expression on survival outcomes in cancer patients was conducted via informatic analysis of transcriptome datasets for breast cancer patient tumour samples available from kmplot.com.T-test or one-way ANOVA, and a p-value of <0.05 was considered significant. Data in bar graphs represent mean ± standard deviation(SD) values.Statistical differences were assessed by Student’s P = 8E−08)  for luminal B  BC. The only exception was HER2-enriched BC  where no significant difference in Pfn1 expression was noted between the low and the high NG tumours. Whether this deviation is due to any biological effect of HER2 signalling on Pfn1 expression or lower sample number in luminal B and HER2-enriched BC compared to the other two subtypes is not clear. Overall, these clinical correlation data support the notion of Pfn1 being an important determinant for growth aggressiveness of BCC, and suggest better patient outcome corresponding to lower Pfn1 gene expression at least in terms of BC recurrence.We first asked whether there is any association between Pfn1 expression and growth-related clinical features in human BC. Since BC recurrence involves either metastatic or locoregional outgrowth of therapy-resistant tumour cells, we interrogated the effects of Pfn1 and Pfn2 transcript levels on the RFS and the OS of BC patients (3951 samples) using pre-assembled transcriptome datasets available from kmplot.com. Univariate Kaplan–Meier and log-rank analyses demonstrated that although there was no significant association between the gene expression of any of the two Pfn isoforms and the OS of BC patients  Fig. . Nuclear08) Fig. . For bot10 Immunoblot in Fig. To assess the impact of LOF of Pfn1 on the outgrowth competency of BCC, we first analysed the single cell outgrowth of sparsely seeded MDA-231 TNBC cells stably expressing either Pfn1 shRNA or a non-targeting control shRNA, as a function of seeding density in BME-MoT assay. We used MDA-231 as a model system as this cell line exhibited post-extravasation lung colonisation defect upon depletion of Pfn1 in our previous study.7 Consistent with our outgrowth data, stable KD of Pfn1 significantly inhibited the FLP-forming ability of MDA-231 cells in MoT culture, as judged by the relative % of FLP-positive cells in control (~55%) vs Pfn1-KD (~10%) groups  of FAK and ERK in MDA-231 cells, at least at a global level, upon transient KD of either Pfn1 or Pfn2 Fig. . SimilarIn order to gain further insights into the biological mechanisms that might underlie outgrowth defects of MDA-231 cells upon LOF of Pfn1, we initially performed differential gene expression analyses of control vs Pfn1 KD MDA-231 cells  in 2D culture settings and further carried out pathway enrichment and over-representation analyses from the microarray data. GSEA identified several canonical pathways that were significantly enriched for differentially expressed genes in Pfn1 KD samples (46 upregulated and 25 downregulated pathways at FDR ≤ 10%)  whose target genes showed significant overlap with Pfn KD responsive genes  . Figure 27 Given our findings that depletion of Pfn1 leads to outgrowth defect of BCC in BME matrix and this can be rescued by stiffening of ECM through addition of collagen-I, we asked whether these phenotypes are correlated with alterations in SMAD3 activation. Therefore, we performed immunoblot analyses of C-terminal phosphorylation of S423/S425 (pSMAD3C: this phosphorylation confers growth-inhibitory ability of SMAD3) as well as the total protein level of SMAD3 in 3D BME-MoT cultures of control vs Pfn1 KD MDA-231 cells. We found that consistent with the upregulation of gene expression signature of SMAD3, loss of Pfn1 in both transient and stable KD settings was associated with elevated levels of both total and phosphorylated SMAD3  in MDA-231 cells in BME-MoT culture (note that Pfn2 KD did not elicit this effect). Addition of collagen-I to the MoT culture dramatically reduced the total and the phosphorylated levels of SMAD3 in Pfn1 KD cells normalizing their levels to those in control cells  and dox-treated (represents Pfn1-depleted condition) MDA-231 cells without or with SMAD3 KD using with a second siRNA (SMAD3 siRNA #2). Similar to our results shown in Fig. To further explore the candidate gene regulatory networks active in Pfn KD samples, we analysed Pfn KD responsive genes (absolute fold-change >1.5-fold) via the upstream regulator analysis (URA) function in Ingenuity. We identified 2 transcription factors, SMAD3  and SPDEF (an ETS family transcription factor that is required for tumourigenesis of certain subtypes of BCly) Fig. . The dirlls Fig. . The invlls Fig. . Since tlls Fig. . The avells Fig. . It is alls Fig. . To furtlls Fig. . As dox 7 and may even indirectly suggest possible role of Pfn1’s interaction with those actin-binding proteins, in this context, there is an important distinction between the previous and our findings. Although impaired FAK/ERK activation resulting from FLP defect has been causally linked to outgrowth deficiency of BCC upon LOF of various actin-cytoskeleton regulatory proteins, surprisingly, this does not appear to be true in the case of Pfn1. This might suggest that FAK/ERK activation may be promoted by but is not absolutely dependent on FLP induction in BCC. Furthermore, physiological levels of FAK/ERK signalling may be necessary but not sufficient to drive efficient outgrowth of BCC as it has been postulated in the literature based on the experimental findings involving overexpression of constitutively active FAK construct .In this study, we demonstrated that Pfn1 deficiency significantly impairs FLP forming ability and single cell outgrowth competency of BCC. In accord with these data, clinical correlation findings further revealed lower Pfn1 expression associated with lower nuclear grade tumours and longer RFS of BC patients. While reduced FLP abundance and outgrowth deficiency of BCC upon Pfn1 KD is consistent with previously reported LOF phenotypes of other Pfn1-binding actin-cytoskeleton regulatory proteins (such as mDia2 and Ena/VASP),14 In our case, we do not believe that loss of Pfn1 is promoting SMAD3 transcription indirectly through affecting Pfn2 activity (possibly through relieving a competition between two isoforms for a common binding factor) for two reasons. First, we did not see any appreciable change in either SMAD3 and phospho-SMAD3 level in MDA-231 cells upon Pfn2 KD. Second, our microarray data did not reveal any change in the SMAD3 mRNA level upon Pfn1 KD, it is possible that SMAD3 is not transcriptionally regulated . It has been shown that SMAD3 level can be post-translationally regulated through protein-stability control via phosphorylation of certain residues  in its linker region . Therefore, it will be interesting to further explore whether loss of Pfn1 somehow impacts protein stability of SMAD3 through modulation of its linker phosphorylation, and there is any dependence on substrate stiffness for this event. MAP kinases  have been shown to promote linker phosphorylation of SMAD3.32 As we did not find any evidence of enhanced ERK activation in response to Pfn1 KD, it is unlikely that SMAD3 upregulation in Pfn1 KD cells is ERK signalling dependent; however, potential role of other members of MAP kinase family cannot be ruled out.In this study, we showed that loss of Pfn1 leads to SMAD3 upregulation in BCC when cultured in soft ECM environment (such as BME matrix) but this effect is abrogated when cells are cultured in a stiffer environment (i.e. either rigid tissue-culture plate or collagen-I-stiffened BME matrix). Interestingly, a recent study has shown that Pfn2 can promote SMAD3 transcription epigenetically in lung cancer cells through suppressing the recruitment of HDAC1 to the SMAD3 promoter.34 our MoT-outgrowth experiments in SMAD3 overexpression and knockdown settings demonstrated an inhibitory effect of SMAD3 on single cell 3D outgrowth of BCC. While these data are generally consistent with the tumour-suppressive functionality of SMAD3, the relatively modest effect of SMAD3 overexpression on single cell 3D outgrowth can be potentially explained by the possibility of the FLP-promoting effect (an outgrowth-promoting feature in single-cell outgrowth setting) partly negating the anti-proliferative action of SMAD3. In fact, our preliminary observations of reduced abundance of FLP in MDA-231 cells in MoT assay upon SMAD3 KD  and AKT signalling in MDA-231 cells.36 A recent study has shown that p53 activation in BCC is substrate stiffness-sensitive and that p53 elevation can suppress FLP.37 Since it was shown that softer substrate actually represses p53 activation, it is somewhat unlikely that modulation of p53 pathway accounts for collagen-I-induced reversal of outgrowth-deficient phenotype of Pfn1-depleted cells. Therefore, unbiased proteomic analyses of cellular extracts prepared in BME vs collagen-I-supplemented BME matrix culture environments will be necessary to identify Pfn1- and stiffness-sensitive proliferation-regulatory pathways for further mechanistic studies in the future.Our studies clearly suggest that there are other proliferation-restraining signals which can dominate over proliferation-inducing effect of FAK/ERK signalling. Specifically, in the context of Pfn1, based on collagen-I’s ability to concomitantly relieve Pfn1-deficient cells from SMAD3 hyperactivation (likely a direct result of SMAD3 upregulation) and outgrowth-arrested phenotype, probing a possible causal relationship between hyperactive SMAD3 signalling and outgrowth-deficient phenotype upon Pfn1 depletion was clearly a logical step in our study. Although previous studies have shown that SMAD3 can promote FLP in breast (including MDA-231) and gastric cancer cells ,10 The dichotomous effect of Pfn1 on early vs late steps of BC metastasis could be a possible reason why the OS (affected by both dissemination and growth of BCC) of BC patients has no significant association with Pfn1 expression at least at the gene expression level. Outgrowth-deficient phenotype of MDA-231 cells in BME matrix  upon Pfn1 KD suggest that metastatic colonisation defect of these cells in the lungs as shown previously our group can be a result of reduced proliferation and outgrowth. Pfn1 dependency for efficient outgrowth of BCC also seems to be consistent with lower Pfn1 expression correlated with longer RFS (dictated primarily by outgrowth) of BC patients. Therefore Pfn1 may be a novel biomarker for BC recurrence and a possible target to reduce metastatic outgrowth of BCC. However, a more comprehensive study with different molecular subtypes of BCC supported by in vivo studies is needed to fully justify this conclusion.Mortality of BC patients primarily results from overt metastatic growth from disseminated cancer cells in the distant organs. Therefore, molecular strategies that could induce dormancy-like phenotype in BCC may lead to a conceptual framework of new lines of targeted therapy against metastatic BC. We previously showed that loss-of-Pfn1 expression promotes dissemination but impairs metastatic colonisation ability of MDA-231 BCC in the lungs.Finally, our studies show that outgrowth-deficiency of BCC upon Pfn1 depletion is cell-density dependent and rescuable by collagen-I. Therefore, it is possible that Pfn1 depletion may become an ineffective strategy to halt metastatic outgrowth of BCC in certain microenvironment (such as a stiffer organ or possibly under fibrotic condition in otherwise a soft organ) or at an advanced stage of the disease when too many micro-metastases have already developed. Future in vivo experiments are needed to address these outstanding questions.Supplementary materialFig S1Fig S2Fig S3Fig S4Fig S5Fig S6Fig S7Fig S8Fig S9Fig S10Supplementary table S1Supplementary table S2


This study aimed to compare the effects of diets with and without supplements of methionine/lysine (met/lys) and methionine/α-tocopherol (met/α-tocopherol) on the performance, carcass traits and meat quality of Hanwoo steers.A total of 21 Hanwoo steers were divided into three different groups. Each group consisted of 7 animals that received different dietary treatments for 120 days as follows: a control (C) diet composed of a basal diet with 74% total digestible nutrient and 12% crude protein; treatment 1 (T1) composed of a basal diet enriched with methionine 69%+lysine 31%; and treatment (T2) composed of a basal diet enriched with methionine 84.65%+α-tocopherol 15.35%. Daily supplementation was given using the top-dressing method .longissimus lumborum with increased water holding capacity (p<0.01) and lower shear force value (p<0.05). Dietary met/lys did not have an adverse effect on the animal performance and carcass traits. Instead, these supplements contributed significantly to the higher protein content compared to the control diet (p<0.05). Myristic acid (C14:0) was the only fatty acid affected by the supplementation. While the met/α-tocopherol group led to significantly higher protein and oxymyoglobin contents during storage (p<0.05). It also contributed significantly to the lower shear force value and 2-thiobarbituric acid reactive substances score during storage (p<0.05) compared to the control and treatment 1 since the initial storage day. The met/α-tocopherol diet also yielded meat with a redder color (p<0.05) after 3 days of storage. However, it did not significantly contribute to the fatty acid profiles of Hanwoo steers.The met/lys groups yielded a Met/lys supplementation resulted in higher protein scores, water holding capacity and lower shear force scores. While met/α-tocopherol supplementation contributes to the production of redder meat, reduces lipid oxidation, production of more tender meat and increases the content of protein and oxymyoglobin percentage. Efforts have been applied to improve the production and meat quality of cattle. One of these efforts is through supplementation with limiting amino acids, alone or in combination with other nutrition, to meet the metabolizable protein requirements. This is not a new technological approach applied in livestock industries to achieve the optimum performance of the animal . MoreoveIn addition, it is well recognized that lipid oxidation is one of the major factors leading to meat deterioration, lowering the quality of meat by the production of off-flavors, reduction of polyunsaturated fatty acids (PUFAs), and consequently lower consumer preferability due to the unfresh color of the meat and possibly the formation of toxic compounds such as peroxides and aldehydes . TherefoThe protocol for animal handling in this study was approved by the Institutional Animal Care and Use Committee (IACUC) at the Gangwon Province Livestock Research Institute.This study utilized twenty-one Hanwoo steers with an initial weight of 712.57±17.26 kg. These animals were then randomly assigned into three different pens. Each pen consisted of 7 animals. The formulation of the diet given was based on the animal’s body weight to meet their nutritional requirements. It was formulated based on the guidelines given by NRC  for the Individual body weight was recorded at the beginning and after 120 days of feeding. Total weight gain and average daily gain (ADG) were calculated.longissimus lumborum) of each animal was cut and packed in polyethylene zipper bags at 24 h postmortem and subsequently transported to the laboratory. Samples were stored in a chilling room at 2°C±2°C overnight. The strip loin from one animal was sliced into six steaks (2 cm thick). To observe the storage effect on color, pH, lipid oxidation and freshness, three steaks from each animal were put on styrofoam, wrapped with oxygen-permeable film and stored at 5°C±0.5°C in the dark for 3, 6, and 9 days. The other two were used for cooking loss and shear force analyses, while the remainder was used for the analyses of color, pH, lipid oxidation and freshness at day 0 of storage. The analysis of proximate composition, water-holding capacity (WHC) and fatty acid composition were also performed using day 0 samples. Analysis of day 0 samples was performed on day 2 postmortem. Samples used for fatty acid analysis were stored at −24°C under vacuum and thawed overnight prior to analysis.After 120 days of the finishing period, animals were slaughtered to measure the effects of the treatment diets on carcass traits. Carcass weight, yield grade  and quality grade  were evaluated based on the Korean carcass grading system. The strip loin  with medium speed for 10 s. Proximate composition was determined according to Association of Official Analytical Chemists (AOAC) method . The conThe meat color was measured right after blooming phase was completely achieved on its surface (1 h after slicing). Colorimeter  was used to record sample’s Lightness (L*), redness (a*), and yellowness (b*). The source of illuminant light C (2° observer) 8 mm aperture was calibrated using a white plate . Measurement was performed 5 times for each sample.g for 10 min at the temperature of 24°C . The supernatant was then decanted and measured. Moisture content of both raw and the supernatant was determined according to AOAC method [The WHC was measured based on method by NRC  with sliC method .Cooking loss is defined as weight loss after subjected to heating until medium degree of doneness was achieved . Briefly, samples were weighed to get initial weight (W1) of 70±5 g. Samples in duplicate were then placed in the polyethylene zipper bags and cooked by immersing in water bath with temperature of 80°C. Cooked samples were subsequently discarded from the bags, cooled down at 2°C±2°C for 30 min and weighed (W2). The percentage of cooking loss was obtained by calculating weight loss (W1W2) against W1.i Plus . The method was according to [Samples from cooking loss was subsequently used to measure tenderness (toughness) of the meat by performing Warner-Bratzler Shear Force test using TA-XT2rding to  where thMethod to obtain the pH score was according to method by Utama et al  where 5 The measurement of lipid oxidation was performed using the 2-thiobarbituric acid reactive substances (TBARS). Sample in amount of 0.5 grams in 25-mL TBARS test tube was prepared in three repetitions, 0.1 g of antioxidant mixture  was transfered into the tube, subsequently 3 mL of 1% thiobarbituric acid in 0.3% NaOH was added into the mixture. Right after vortexing, 17 mL of 2.5% trichloroacetic acid in 36 mM HCl was added and the tube was closed. The sample was subjected for heating in a water bath  with temperature of 100°C for 30 min. The tube was then immersed in cold water for another 15 min. Aqueous sample in amount of 5 mL was transfered into a new 15 mL centrifuge tube and mixed with 3 mL of chloroform. The mixture was then subjected for centrifugation with the condition of 2,400×g for 30 min at 4°C  to separate from the dirt. The absorbance was measured at 532 nm by using uv-spectrophotometer  against blank (distilled water was used to replace sample). Each sample was repeated three times. Data was expressed in the form of mg malondialdehyde/kg sample.One gram of beef sample was homogenized with 8 mL sodium phosphate solution . It was subsequently centrifuged at 10,000 g for 15 minutes. Supernatant formed was filtered through a Whatman filter paper. Extract absorbance was read at 572, 565, 545, and 525 nm against a blank using a spectrophotometer . The proportion of the DMb, OMb, and MMb was calculated by using a formula.The determination of fatty acid composition was performed using an Agilent gas chromatography system  with an auto sampler . Meat fat was extracted according to  with a cThe determination of significances for treatment groups  during storage days was by using linear mixed model of two-way analysis of variance (ANOVA). While to determine the significant different for animal performance, carcass traits, proximate content, WHC, cooking loss, shear force, and fatty acid composition were compared with those of control sample by performing one-way ANOVA. Analyses were conducted using R version 3.5.1 with the “Agricolae” library .The effects of dietary supplements of met/lys or met/α-tocopherol are shown in Feeding cattle with diets supplemented with either met/lys or met/α-tocopherol yielded meat with higher protein content (p<0.01), as shown in Cattle from both treatment groups responded positively to the diet by producing meat with a significantly lower shear force value (p<0.05), which indicates that both treatments produced meat with more tender characteristics. However, there were no significant differences between treatment diets. For the WHC score shown in The storage experiment was carried out for 9 days in a chilling room at 2°C±2°C. The rate of meat lipid oxidation was recorded through TBARS analysis which shown in The effect of met/lys and met/α-tocopherol supplementation on meat pH during storage is reported in The Commission Internationale de l’Eclairage (CIE) value changes were recorded in The formation of oxymyoglobin, metmyoglobin and deoxymyoglobin is shown in Diet supplemented with either met/lys or met/α-tocopherol had a limited effect on the intramuscular fatty acid of Hanwoo beef which can be seen in Hanwoo steers were assessed for their performance after 120 days of feeding with either met/lys or met/α-tocopherol supplementation. Significant differences were not observed for final weight, weight gain or ADG of cattle either treated with diets supplemented with met/lys and met/α-tocopherol. This is in agreement with explained by Park et al and Prado et al ,14, wherIt was expected that treatment could enhance carcass traits, as occurs in nonruminant animals. Theoretically, methionine and lysine are two limiting amino acids that cannot be sufficiently provided by animals with a low protein diet , and thelongissimus lumborum muscles of Hanwoo steers fed either treatment compared to the control diet. Dietary supplementation with either met/lys or met/α-tocopherol seemed to promote more protein synthesis. The mechanism relates to its role in the initiation of translation of protein synthesis [Differences were obtained for the protein content of ynthesis . Supportynthesis , supplemynthesis . HoweverThe water content of the meat is an essential quality parameter to be considered by the meat processor. Due to its relation to the final yield of the processed meat, it has economic implications. However, it is also essential in terms of eating . Meat teAll samples showed an initial pH score below 5.6, which indicates that animals were not in a stressful condition when slaughtered. It also indicates that the meat was not pale soft exudative meat . TreatmeMyoglobin reflects the freshness of the meat and is an important marker for meat discoloration, which correlates with the rate of lipid oxidation . The myoNine fatty acids were identified within the beef samples. Dietary supplementation with both met/lys and met/α-tocopherol had a limited effect on the intramuscular fatty acid profile of Hanwoo beef. The content of PUFAs did not differ across samples. This finding agrees with that of the previous study by Parr et al  that altIn conclusion, supplementation of both met/lys and met/α-tocopherol during fattening to Hanwoo steers had no significant effect on growth performance and carcass weight and yield grade. Supplementation of met/lys resulted in a higher score of protein content, WHC and a tendency of lower shear force value and amount of cooking loss compared to control diet, which is economically profitable due to more augmentation of protein, and less product shrinkage. Therefore, supplementation with met/lys could be beneficial for Hanwoo steers during the fattening program. While met/α-tocopherol supplementation contributes to the production of redder meat, reduces lipid oxidation, production of more tender meat and enhances the protein and oxymyoglobin content, which is preferred by the consumer. However, further study to determine the optimum ratio of supplementation is needed to achieve the best effects on the desirable traits of Hanwoo meat.


This paper investigates the possibility of applying novel microwave sensors for crack detection in reinforced concrete structures. Initially, a microstrip patch antenna with a split ring resonator (SRR) structure was designed, simulated and fabricated. To evaluate the sensor’s performance, a series of structural tests were carried out and the sensor responses were monitored. Four reinforced concrete (RC) beam specimens, designed according to the European Standards, were tested under three-point bending. The load was applied incrementally to the beams and the static responses were monitored via the use of a load cell, displacement transducers and crack width gauges (Demec studs). In parallel, signal readings from the microwave sensors, which were employed prior to the casting of the concrete and located along the neutral axis at the mid-span of the beam, were recorded at various load increments. The microwave measurements were analysed and compared with those from crack width gauges. A strong linear relationship between the crack propagation and the electromagnetic signal across the full captured spectrum was found, demonstrating the technique’s capability and its potential for further research, offering a reliable, low-cost option for structural health monitoring (SHM).  The maintenance of structures is essential in various industries, including civil, offshore, marine and transportation engineering. The inspection of structures in order to detect structural damage and to assess the condition of materials is an important part of the maintenance process. Crack formation and propagation due to fatigue or earthquake loading, corrosion in metals and delamination in composites can be addressed via effective maintenance . EffectiStructural health monitoring (SHM) provides continuous feedback regarding the “state” of the constituent materials and structural members. Continuous monitoring over a period of time offers a full history database for a structure, which can be used to provide a prognosis  . DeterioIn recent years, different SHM technologies have been investigated aiming to minimise the costs related to damage assessment and maintenance works, while securing the reliability of the structural performance. Traditional sensor technologies  can be utilized, along with traditional visual inspection and damage detection, for structures subjected to various load conditions, such as earthquakes, wind and fatigue loading. In addition to traditional sensors, new measuring approaches  have been applied for structural health monitoring purposes . Non-desAiming to complement existing sensing technologies for SHM, the present study investigates the potential of using microwave sensors for crack detection in concrete structures. Recently, electromagnetic sensors have received interest as a method to monitor and predict the excess moisture content in concrete structures due to exposure ,8,9. TheAn experimental programme was designed and carried out in order to investigate the accuracy of the microwave sensing technology in predicting the crack formation in reinforced concrete beams. The current section provides details regarding the design of the RC beams  and of tFour RC beams were designed in accordance with Eurocode 2  to be usIn this study, a microstrip patch antenna with a split ring resonator (SRR) structure was designed, simulated and fabricated as a potential microwave sensing technique. SRR structures have been utilised to realise microwave resonators, filters and microwave antennas , and hav11, reflection coefficient) were realised in the frequency range of 1 to 10 GHz and with 4000 sweep points. As the sensor was aimed to be embedded into the concrete, a concrete sample was modelled and simulated using HFSS. According to Piladaeng , the con11). The full simulated frequency spectrum  was scanned across using a bespoke LabVIEW (v2013) program  aiming to identify a linear relationship between the sensor response (S11) and the crack width (mm). 11 and the crack width (mm) changed at 6.2 GHz, with R2 = 0.91. The figure demonstrates that S11 increased as the crack width grew, i.e., less energy/power was absorbed by the concrete sample as there was an opening air gap, which changed the penetration of the electromagnetic (EM) signal, as well as changing the reflection of the signal. Based on these simulation results and previous work [The simulation results are shown in ous work , the invThe proposed antennas were fabricated on a double-sided 1.6 mm FR4 PCB (Printed Circuit Board) sheet using an LPKF ProtoMat D104 unit see . Then thIn order to evaluate the capability of the microwave sensors to predict the crack formation, four reinforced concrete beams, in line with the design described in Initially, the formwork for the concrete beams was constructed and the longitudinal steel reinforcement bars and the shear links were fabricated. Upon careful treatment of the surfaces, six strain gauges  were attached at the tensile reinforcement bars of the specimens. In particular, the strain gauges were adhered to the bottom and the top of a tensile reinforcement bars and at the mid and quarter span. A microwave sensor was located at the neutral axis (NA) of the cross-section mid-span of each specimen, where negligible longitudinal stresses and strains were expected. 2. Crushed limestone aggregates  (20 mm down), sand (Mersey Grit) , OPC Type II  and a water-to-cement ratio equal to 0.45 were used. A slump test was also performed to ensure that the mix was in accordance with the design guidelines. The beams were cast and the concrete was vibrated using a poker vibrator according to the design guidelines [Following this, the concrete mix was prepared in accordance with the BRE (Building Research Establishment) concrete mix design , which gidelines . Note thConcrete cubes with dimensions of 150 × 150 × 150 mm were also prepared in order to check the compressive strength of the concrete at 7 and 28 days. The specimens’ preparation, including the details of the attached sensors and gauges along with the concrete casting, are shown in Upon demoulding, the beams were painted on both lateral sides in order to make the crack formation and propagation detectable. Demec studs were applied at the central tension region of the beams in order to monitor the crack opening within the most heavily stressed areas. Four stud sets were bonded on the concrete’s surface at a set horizontal distance of 50 mm on either side of the centreline and at vertical distances equal to 80 mm, 60 mm, 40 mm and 20 mm from the beam’s neutral axis (NA).Having attached the required instrumentation , the specimens were cured for 7 days and were then located at the test rig for the experimental investigation. The beams spanned 1200 mm with an overhang of 100 mm from both sides of the supports. Simply-supported conditions were achieved with the use of roller supports. A calibrated hydraulic 100 kN load actuator was used for the load application. A constant slow loading rate, excluding any dynamic effects, was implemented, while a load cell was used for the recording of the load increments. Two strain gauge type displacement transducers (DTs) were placed at the mid-span in order to measure the vertical deflections of the specimens during testing. Synchronised data acquisition systems were used for the structural response measurements. The experimental set-up, along with the employed instrumentation, are shown in The structural response for all specimens is shown in 2 and 57.15 N/mm2 for the 7- and 28-day tests, respectively. In For each of the specimens, a concrete cube compressive strength test was performed. The average compressive strength was 49.92 N/mm11, reflection coefficient), namely complex data  and magnitude from the Vector Network Analyser (VNA) . The data was captured via a bespoke LabVIEW program on a laptop connected to the VNA. The data acquisition took place at each load increment. The recorded measurements were in the 1 to 10 GHz frequency range using the maximum number of sweep points available (4000). The captured results were analysed against the crack width of the samples in order to determine a relationship between the changes in the EM response and the crack width. The microwave measurements were provided by the S-parameter , a novel microwave sensor, specifically designed for this application, was inserted into each beam’s reinforcement cage before the concrete’s casting. In order to allow for a reduced size of the sensor and to shift the resonant frequencies to lower frequency ranges, hence increasing the antenna’s penetration capability, a microstrip patch antenna with an SRR structure was designed and subsequently fabricated. 11 reflection coefficient, and were within the 1–10 GHz frequency range. For the largest anticipated tensile crack at the mid-span of the beam, the readings for the crack width gauges and the sensors were processed and compared. A strong linear relationship with R2 values very close to unity were observed. The variance on the sensor response was related to the different crack patterns observed in each specimen. The present study considered crack widths below 1 mm and thus only small changes in the concrete structure. It is noteworthy that the sensor was able to detect these small changes in the concrete’s structure, demonstrating that the proposed technique could be further studied in order to detect cracking in RC structures. Further investigation is required to obtain more data for various crack patterns and widths, thus enhancing the measurements’ repeatability and reliability and allowing for the technique’s validation.During the load application, both the structural performance and the signal measurements were incrementally recorded. As anticipated, a linear load–displacement structural response was found to be very similar for all specimens. The microwave data sets, which were captured via a bespoke LabVIEW program, were provided by the SFuture studies could also investigate the possibility of insulating/sealing the antennas to avoid direct contact with the concrete, which might change the properties of the antenna owing to the physical and chemical properties of the concrete mixture. The extra layer could protect antennas from possible corrosion/oxidation of the copper part of the antenna. Finally, additional sensors  could be added to provide additional variables for the sensor algorithm, which could further improve crack detection.


X-ray computed tomography (XCT) is used to trace and contrast the corrosion profiles of the concrete reinforcement bar with and without the microcapsule. The results show that the OH−-regulated microcapsule exhibits effective corrosion protection by delaying corrosion initiation and cracking. An SEM study revealed that the microcapsule could be broken as Cl− invades the concrete. However, intelligent OH− regulation was realized by releasing CaO.Corrosion is a challenging problem for marine concrete infrastructure projects. In this study, an intelligent OH Reinforced concrete has been widely applied in civil engineering projects due to its complementary advantages . HoweverTraditionally, corrosion inhibitors, such as nitrites or sodium monofluorophosphate, have been frequently used to improve resistance to chloride invasion ,9,10,11.−/OH− ratio /[OH−] value and then alleviated the corrosion development. In contrast, without the help of OH−-regulated microcapsules, corrosion initiation and propagation of the control samples occurred more easily. The adverse trend of icorr was shown in Additionally, − penetrated through the mortar matrix, as Cl element was found in the energy-dispersive X-ray spectroscopy (EDS) results (2), provided hydroxide ions and directly regulated OH− in the pore solution. The XCT image in During cement hydration, the pore solution was highly alkalized pH > 12.5) and was principally comprised of sodium and potassium hydroxides 2.5 and w. It has  results c,d. Thus3 and 0.823 mm3 of steel volumetric loss, respectively, but still no cracks could be found on them, while Sample A cracked with a volumetric loss of only 0.626 mm3 , indicating that an outflow of OH− may cause an inconspicuous effect on anti-corrosion of the reinforcing bar.When more CaO/EC microcapsules were added, better corrosion protection performance was theoretically expected, since more CaO/EC microcapsules contribute to [OHsolution . However− regulation as a corrosion inhibitor or pH buffer in combination with pH-sensitive microcapsules was successfully designed to achieve and prevent chloride-induced corrosion in concrete reinforcing bars.Intelligent OHThe X-ray computed tomography (XCT) method was used to trace and contrast the corrosion profiles of the concrete reinforcement bars with and without the microcapsules. The efficiency of the microcapsules was verified by XCT testing, which allowed visualization of the corrosion process  and provided quantitative information.− regulation in the concrete material, which delayed corrosion initiation and cracking of the cover concrete.The microcapsules showed highly effective corrosion protection due to intelligent OH− regulation.An excessive addition of microcapsules may compromise the inhibitive efficiency of OHFrom the above analysis and discussion, the following conclusions can be made:


To evaluate the duration of prescriptions for antibiotic treatment for common infections in English primary care and to compare this with guideline recommendations.Cross sectional study.General practices contributing to The Health Improvement Network database, 2013-15.931 015 consultations that resulted in an antibiotic prescription for one of several indications: acute sinusitis, acute sore throat, acute cough and bronchitis, pneumonia, acute exacerbation of chronic obstructive pulmonary disease (COPD), acute otitis media, acute cystitis, acute prostatitis, pyelonephritis, cellulitis, impetigo, scarlet fever, and gastroenteritis.The main outcomes were the percentage of antibiotic prescriptions with a duration exceeding the guideline recommendation and the total number of days beyond the recommended duration for each indication.The most common reasons for antibiotics being prescribed were acute cough and bronchitis , acute sore throat , acute otitis media , and acute sinusitis . Antibiotic treatments for upper respiratory tract indications and acute cough and bronchitis accounted for more than two thirds of the total prescriptions considered, and 80% or more of these treatment courses exceeded guideline recommendations. Notable exceptions were acute sinusitis, where only 9.6%  of prescriptions exceeded seven days and acute sore throat where only 2.1% (2.0% to 2.1%) exceeded 10 days (recent guidance recommends five days). More than half of the antibiotic prescriptions were for longer than guidelines recommend for acute cystitis among females . The percentage of antibiotic prescriptions exceeding the recommended duration was lower for most non-respiratory infections. For the 931 015 included consultations resulting in antibiotic prescriptions, about 1.3 million days were beyond the durations recommended by guidelines.For most common infections treated in primary care, a substantial proportion of antibiotic prescriptions have durations exceeding those recommended in guidelines. Substantial reductions in antibiotic exposure can be accomplished by aligning antibiotic prescription durations with guidelines. Streptococcus pneumoniae.Clostridium difficile infection, highlighting the need to reduce the duration of treatment where possible.Many countries, including the United Kingdom, are trying to tackle antibiotic resistance by reducing unnecessary or inappropriate prescribing.Evidence about the contribution of excessive treatment duration to antibiotic overuse in primary care is limited. A recent study from the US showed that more than two thirds of antibiotic courses for acute sinusitis in adults were 10 days or longer, whereas the Infectious Disease Society of America recommends treatment of uncomplicated cases for five to seven days.An up-to-date picture on prescribed antibiotic durations for common infections in English primary care is lacking. Such an overview is especially relevant given the increasing evidence from randomised controlled trials and meta-analyses that shorter antibiotic courses clear infection comparable to longer courses, while minimising selection, proliferation, and spread of antibiotic resistant bacteria and the likelihood of side effects from antibiotic use.We therefore assessed the extent to which durations of antibiotic courses prescribed for common infections in English primary care are in line with relevant guidelines. If substantial proportions of antibiotic prescriptions are longer than recommended, this would indicate that there is potential to safely reduce total antibiotic use simply by better application of guidelines in clinical practice.Data were obtained from The Health Improvement Network (THIN), a primary care electronic database that contains anonymised data on patients, practices, and consultations and is representative of the general UK population, with consultation and prescribing rates similar to national data.For each indication we calculated the proportion of prescriptions longer than the recommended duration separately for children (<16 years) and for people aged 16 years or older, and by antibiotic where guidelines recommended different durations for particular drugs. Because guidelines recommend longer antibiotic courses to treat acute cystitis in males than females,We used multiple imputations through chained equations using sequential regression trees to impute duration data that were missing in 10-20% of the consultations (see supplementary file 2 for more details).In addition, we calculated the total number of excess antibiotic days, defined as the total number of days beyond the recommended duration in the guidelines.All analyses were performed using R version 3.4.3 .No patients were involved in setting the research question or the outcome measures, nor were they involved in developing plans for design or implementation of the study. No patients were asked to advise on interpretation or writing up of results. Results will be disseminated to relevant patient communities through news media.Between 2013 and 2015, 931 015 consultations for the 13 included indications led to antibiotic prescriptions. This subset—which focused on common conditions, but excluded chronic and recurrent cases, repeat prescriptions, and antibiotic prophylaxis—covered about 20% of total antibiotics being prescribed (for any condition) during the study period. The most common indications were acute cough and bronchitis , acute sore throat , acute otitis media , acute sinusitis , cellulitis , and acute cystitis . Durations of antibiotic treatment for the included indications showed poor guideline adherence for several indications  for acute cough and bronchitis, 2 (2-8) days for acute otitis media, and 3 (1-7) days for acute sinusitis. This was 7 (3-8) days for cellulitis (using seven days as threshold), and for acute cystitis 4 (2-4) days for females and 7 (3-32) days for males. In general, the tendency was to write prescriptions with a duration of five or seven days or multiples thereof  comprised a substantial proportion of the total number of days of antibiotics prescribed for these indications—for example, 29.5% for acute cough and bronchitis .Fewer prescriptions exceeded recommended durations for non-respiratory tract indications, but still more than half of the antibiotic prescriptions were for longer than guidelines recommend for acute cystitis among females  . AlthougThe percentage of antibiotic prescriptions for which the actual duration was beyond the recommendation was much lower for pyelonephritis , acute cystitis among males , impetigo , and cellulitis , but this still translated into 155 238 excess antibiotic days .For acute prostatitis and acute cystitis among males, there appeared to be substantial under-treatment, with a substantial number of courses shorter than the guideline recommendations, with 52.3% below 28 days and 26.0% below 14 days for acute prostatitis and 31.8% below seven days for acute cystitis among males . In addiv 84.7%), acute otitis media (89.9% v 78.6%), impetigo (12.1% v 6.4%), pyelonephritis (27.2% v 17.7%), and gastroenteritis (70.7% v 51.0%).There was no clear tendency to prescribe longer courses for children (<16 years) compared with adults (≥16 years) (supplementary table S2). Nevertheless, the percentage of prescriptions that were longer than recommended was higher among younger patients for acute cough and bronchitis . In addition, for sore throat, pyelonephritis, and scarlet fever, longer prescriptions are recommended for specific antibiotics. In these cases, observed durations less frequently exceeded durations recommended in the guidelines compared with other antibiotics prescribed for the same condition (supplementary table S3). For most conditions, 75% or more of the prescribed antibiotics were mentioned in the guidelines for treatment of that condition (supplementary table S3). A notable exception was gastroenteritis, where only 12.8% of the patients were treated with the recommended antibiotic.When restricting the analyses to antibiotics only mentioned in the guidelines, results were generally similar to the analyses including all antibiotics (supplementary table S3). A notable exception was gastroenteritis. The percentage of antibiotic prescriptions longer than guidelines recommended became lower for gastroenteritis when only considering antibiotics mentioned in the guidelines .For most common infections treated in primary care, a substantial proportion of antibiotic prescriptions have durations exceeding those recommended in guidelines. This is most noticeable for most respiratory indications and acute cystitis among females. If all patients receiving antibiotics for the included indications received treatment for durations recommended by guidelines over the study period , this would equate to 1.1 million fewer days of antibiotics for respiratory tract indications and 100 000 fewer days for acute cystitis among females. Our findings indicate substantial scope for reducing antibiotic prescribing through better adherence to recommended durations of antibiotic treatment. In contrast, the duration of antibiotic prescriptions for pyelonephritis, acute cystitis among men, impetigo, and cellulitis generally did not exceed guideline recommendations, with only 4-18% of prescriptions exceeding them.This study assessed the extent to which the duration of antibiotic prescriptions, for a wide range of common infections, follow guidelines in English primary care. In contrast with a recent US study only focusing on acute sinusitis,3) colony forming unit (CFU)/mL has been found in only 25-65% of patients aged less than 65 years given antibiotic treatment. In older adults, asymptomatic bacteriuria is common and does not require antibiotic treatment.We only assessed whether the durations of antibiotic prescriptions followed guidelines and not whether antibiotics should have been completely avoided. Previous work showed that a substantial proportion of prescriptions for acute cough and bronchitis, acute otitis media, acute sinusitis, acute sore throat, and impetigo in primary care are unnecessary.For two of the antibiotic indications we studied, treatment was frequently prescribed for shorter durations than recommended—both urinary tract infections in men: acute cystitis and acute prostatitis. This could arise because prescribers appreciate that recommendations for these conditions are based on expert opinion and historical precedent rather than evidence, and that treatment durations for urinary tract infections in women have declined considerably in recent years. It needs to be established whether shorter treatment durations are effective for men with urinary tract infections, as this is a small but important patient population with a risk of harm from under-treatment. Notably, the conditions for which course durations tended to be less than recommended in the guidelines were those for which evidence supporting duration of treatment is weak (supplementary figs S5-S7).In general, the preference seemed to be for antibiotic prescriptions with durations of five or seven days or multiples thereof, without a clear evidence base for this preference. The preference for such durations has been observed previously in other settings and depended more on local practice and subspecialty than on clinical features such as fever, comorbidities, and severity.One important limitation is that some antibiotic prescriptions may have been falsely linked to certain conditions in our study, because there is no automatic link between prescriptions and diagnoses in THIN.We were unable to account for the possibility that treatment durations for some patients are influenced by pack sizes, which may include more than the prescriber would like dispensed, or by protocolised durations set in electronic prescribing decisions. Pack size could result in unintended antibiotic overuse, whereas electronic based decisions might be expected to guide prescribers to recommended durations. Both, however, are system factors that could be addressed to help prescribers make more patient tailored decisions about minimum effective duration of treatment.The evidence base for optimal durations of treatment for respiratory tract infections in primary care is relatively smallAlthough previous work has shown that antibiotic use can potentially be substantially reduced by not prescribing antibiotics when they are unnecessary,Overall, substantial reductions in antibiotic exposure can be accomplished by aligning duration of antibiotic prescriptions with guidelines.A clear causal link exists between antibiotic exposure and antibiotic resistanceStrategies to reduce antibiotic use in primary care focus on decisions to start treatmentIt is not known to what extent excessive treatment duration contributes to antibiotic overuse in primary careFor many common infections treated in primary care, a substantial proportion of antibiotic prescriptions have durations that exceed those recommended in guidelinesRates of treatment duration beyond guidelines are highest for respiratory tract infections and are similar among patients with and without comorbiditiesSubstantial reductions in antibiotic use in primary care could be achieved by closer compliance with recommended treatment durations