The shift from a more didactic to student-centred pedagogical approach has led to the implementation of new information communication technology (ICT) innovations and curricula. Consequently, analysis of the digital competency of both faculty and students is of increasing importance. The aim of this research is to measure and compare the internet skills of medical school faculty and students and to investigate any potential skills gap between the two groups.A survey of medical school faculty and students across three universities in Ireland was carried out using a validated instrument  measuring five internet skills . Three focus groups comprising a total of fifteen students and four semi-structured interviews with faculty across three institutions were carried out to explore further findings and perceptions towards digital literacy, give further insight and add context to the findings.Seventy-eight medical faculty (response rate 45%) and 401 students (response rate 15%) responded to the survey. Mean scores for each internet skill were high (above 4 out of 5) for all skills apart from Creative (mean of 3.08 for students and 3.10 for faculty). There were no large differences between student and faculty scores across the five skills.Qualitative results supported survey findings with a deeper investigation into topics such as online professionalism, use of licencing and mobile application development. Needs based skills training and support were highlighted as areas for faculty development.Both medical educators and students tend to have similar competencies with respect to internet skills. When implementing online and distance learning methodologies however, medical schools need to ensure appropriate skills training and support for faculty as well as providing targeted training to improve the creative skills of both their educators and students.The online version of this article (10.1186/s12909-019-1475-4) contains supplementary material, which is available to authorized users. Medical faculty play a prominent role in educating future clinicians and advancing their skills so that their clinical work and research is informed by evidence-based practice, with the ultimate aim of improving patient care . The levThe transition from more traditional modalities of medical education  towards online and distance learning requires educational resources of a high quality, delivered to a high standard . Furthervis à vis use and creation of online digital content [Although education utilizing online learning has become an increasingly useful and popular part of educational instruction, there remains some hesitance on the part of medical educators  content , 7. Mode content . Student content , and gra content .To the best of our knowledge, none of the previous studies which have employed an internet skills scale has been undertaken to determine medical faculty and students’ digital skills concurrently. This suggests that there is gap in this area, particularly where a concurrent study has yet to be completed. This study therefore seeks to contribute to eliminating this gap by measuring the digital skills of medical faculty and students using a triangulation mixed methodology, employing a combination of a validated instrument , interviWhat levels of digital internet skills do medical faculty and students report?Are there any gaps that might be evident between medical educators and students?Which socio-demographic variables influence internet skills in medical faculty and students?There are a number of questions that guide this study’s focus:This study adopted a concurrent triangulation mixed method design, utilising quantitative and qualitative research methods, namely a large survey followed by focus groups and semi-structured interviews. Data triangulation was employed to improve confidence in the results and conclusions , in addiThe study was approved by the Education and Health Science’s Ethics Committee at the University of Limerick (2016_03_01_EHS).Medical faculty and students from five universities were invited via independent gatekeepers (administrative staff in each university who were not connected to the research study) to participate in an online survey. The survey was distributed to both direct entry (post-secondary education entrants) and graduate entry (who had previously completed an undergraduate degree) medical students across all years of study. Only medical faculty who were responsible for both the development and delivery of the curriculum were to be invited to participate.➢ ‘Operational’ skills scale with 10 items relates to the “skills to operate digital media”. Items on this scale include “I know how to open downloaded files”, “I know how to bookmark a website” and “I know how to adjust privacy settings”.➢ ‘Information Navigation’ skills scale with 8 items relates to the ability to search for information online without navigational issues. These items were all negatively worded. Examples of items on this scale include “I find it hard to find a website I visited before” and “Sometimes I end up on websites without knowing how I got there.”➢ ‘Social’ skills scale with 6 items focuses on skills for engaging in social activities on digital platforms. Items on the ‘Social’ skill scale included “I know which information I should and shouldn’t share online” and “I am careful to make my comments and behaviours appropriate to the situation”.➢ The ‘Creative’ skills scale with 8 items focuses on the ability to create content. Items on this scale included “I know how to create something new from existing online images, music or video”, “I know which different types of licenses apply to online content” and “I know how to design a website”.➢ The ‘Mobile’ skills scale with three items that looks at the skills necessary in using apps on mobile devices. The three items “I know how to install apps on a mobile device”, “I know how to download apps to my mobile device” and “I know how to keep tract of the costs of mobile app use”.The ISS  has beenDemographic information was collected from students on age group, gender, country of origin, university, year of study, mode of entry to medical school (i.e. direct or graduate entry), and type of primary degree (where relevant). Demographic information from faculty included age group, gender, country of origin, university, primary degree, postgraduate qualifications (where relevant), length of teaching experience, and experience creating distance learning content.Cronbach’s alpha (α) was used to measure the internal consistency of the skills scaled with values > 0.8 considered good internal consistency. Mean skill scale and item scores were calculated and compared across groups  using independent samples t tests. A 5% level of significance was used for all tests. Cohen’s d was used to measure effect size with 0.2 considered small, 0.5 medium and 0.8 large. Linear regression was used to predict skill scale scores using demographic data  as predictors for both faculty and students. R squared was used a measure of goodness of fit of the models. Data was analysed using SPSS Statistics version 22 for Windows.Purposive sampling of students who expressed an interest in participating in focus groups after completing the quantitative survey was carried out, and three focus groups comprising a total of fifteen students from three medical schools were undertaken. Four semi-structured interviews also took place with medical school faculty across the three universities. Two interviews were phone interviews and two were face-to-face. In adopting a mixed method approach, results from the survey informed some of the focus group and interview questions  and 78 medical faculty (response rate of 45%) across the three universities. Response rates varied across the three universities for students (7–30%) and faculty (25–57%).Table The demographics of faculty with complete responses are summarised in Table Cronbach’s alpha (α) was calculated for each skill scale  or mean Information Navigation scores   and Social  scores for medical faculty with those aged 40 or more having lower scores (Table p < 0.001) scores for medical students with female students having lower scores (Table p = 0.045).In a multiple linear regression with socio-demographics as predictors for each skill scale, age group was a statistically significant predictor for Mobile  for our online learning materials as a repository and often times you have to click through quite a few folders to get to what you’re getting at.”Faculty discussed issues of usability in relation to navigating information online, and the accessibility of different technology was also highlighted.“Reliability of the source is important.”Many students spoke about the difficulty in navigating information online, specifically relating to the lack of reliable source information for learning purposes, and they also questioned the reliability or trustworthiness of different online platforms. In the face of boundless information that could be sourced online, they noted that it was important to discern what was reputable and what was not.“We are facing an abundance of information but sometimes they are not…well regulated.”“Sometimes I find using the internet there’s nearly information overload... there’s so much stuff…and then you can nearly be overwhelmed.”Some students also expressed interest in being provided with training that would help them differentiate between reliable and reputable online resources. Whilst additional online educational sources were beneficial to students, these supplementary resources often can add to the ‘information overload’ experienced by students.“I would love to but I don’t know how to design a website or update a website.”Follow up interviews and focus groups allowed us to explore students’ and faculty’s experiences of creativity within medical education. The lack of knowledge on designing a website in particular was highlighted:“I can grab an edit video, I can create retro graphs, I can paint pictures.”Whilst most faculty were novice in their creative digital skills, one member of staff was more skilled:“People are reluctant. I think they are also more inclined to possibly infringe in ways that they can see. So people are happier putting up material to which they don’t have rights clearance than they are in putting up something that they feel is homemade looking”Members of faculty also outlined some of the concerns met when putting content online and engaging with different licenses.In a Web 4.0 age, it is of the utmost importance that digital skills are examined for future training opportunities and to ensure higher education institutes (including educators and students) remain competitive and innovative in technologies used in a knowledge-driven environment . This st“I know how to design a website” and “I know which different types of licenses apply to online content”. This raises the question as to the applicability of certain digital skills to medical education – do medical educators / students really need to be able to design a website? The answer is probably ‘no’. However students and faculty both scored low on the item “I know which different types of licenses apply to online content,” a topic which is extremely important in medical education as it is imperative that faculty and students are aware of regulations around the licensing of content online. Training of licensing digital content therefore must be considered when implementing future strategies.Following a deeper analysis of the low scoring Creative skills subscale, two items were identified as having been poorly answered by both groups Qualitative findings highlight the impact of social media in relation to a medical education setting. Social media does have a role to play in a higher education setting; the changing learning environment of higher education is supported through social media’s connectivity , enablinWhilst social media can foster collaborative learning and engagement , qualitaMedical students highlighted difficulty in navigating appropriate routes of information online. The internet allows students and physicians to consume information at high speeds but the quality and reliability of this information is not always guaranteed . The issThe adoption of IT within medical education institutions is dependent on a number of variables including faculty members’ individual and institutional context in addition to their prior IT experience , 28. To To the best of the authors’ knowledge, this study is the first which utilises Van Deursen’s  validateOne significant limitation of this study was the poor response rate amongst faculty and students to the quantitative survey. This may be as a result of survey fatigue, both on behalf of students and faculty. The qualitative study therefore aimed to provide a deeper understanding of medical educator and students’ digital skills via data triangulation .The nature of the survey as an online survey can also be seen as a limitation with delivering it in an online format possibly resulting in an over representation of those with adept internet skills. Furthermore, graduate entry students and younger faculty were over represented in our sample. This younger and more qualified population therefore may have resulted in the higher skills scores even on the Information Navigation and the Mobile subscales. Reassuringly however the findings for the other subscales were similar for the medical and general populations  alike.This study revealed that medical faculty and students appear to have similar competencies with respect to digital skills. However, it is essential that medical schools provide appropriate training and support for faculty in conjunction with specific creative skills, information navigation and social media training for both medical educators and students, in order to address many of the challenges faced in an expanding digital world.Additional file 1:Focus Group Interview Guide. (DOCX 11 kb)Additional file 2:Interview Guide. (DOCX 15 kb)Additional file 3:n = 406). (DOCX 11 kb)Cronbach’s alpha for each skill . (DOCX 12 kb)


MSM) over the last decade. The majority of cases are extragenital and occur at the oropharynx and anorectum. The aim of this narrative review was to review the risk factors and mode of transmission for gonorrhoea and chlamydia at the oropharynx and anorectum among MSM.Gonorrhoea and chlamydia cases have been rising among gay, bisexual and other men who have sex with men (New evidence suggests that oropharyngeal gonorrhoea can be transmitted by kissing in addition to through the established route of condomless oral sex; and anorectal gonorrhoea can be acquired when saliva is used as a lubricant for anal sex and rimming in addition to the established route of condomless penile‐anal sex in MSM. In contrast, condomless penile‐anal sex remains the major route for chlamydia transmission.Substantial transmission of gonorrhoea may occur with practices other than the established routes of condomless oral and/or anal sex and hence condoms may not be effective in preventing gonorrhoea transmission to extragenital sites. In contrast, condoms are effective for chlamydia control because it is mainly transmitted through condomless penile‐anal sex. Novel interventions for gonorrhoea that reduce the risk of transmission at extragenital site are required. Chan and colleagues published a review in 2016 summarising the prevalence of extragenital gonorrhoea and chlamydia from 53 studies  and anorectal chlamydia (9.0% vs. 6.6%) than HIV‐negative MSM; however, the prevalence was similar in both groups for oropharyngeal infection  2.2Oropharyngeal gonorrhoea is relatively short lived and commonly asymptomatic Oropharyngeal gonorrhoea had been thought to be primarily acquired from oro‐genital contact such as condomless fellatio Table  24, 42. Studies of symptoms associated with urethral gonorrhoea have been contradictory. In the 1970s, two studies have reported that about 40% of heterosexual men reporting contact with their female partners with gonorrhoea had asymptomatic urethral gonorrhoea In countries with good access to healthcare, men with symptomatic urethral gonorrhoea usually receive treatment within a few days of the onset of symptoms Neisseria gonorrhoeae in saliva by nucleic acid amplification test (NAAT) and almost half (43%) of men where their saliva samples were detected by culture These same investigators have undertaken a series of studies related to their hypothesis. They undertook a study of 33 MSM with untreated culture positive oropharyngeal gonorrhoea and obtained saliva samples from these men up to 14 days after screening Neisseria gonorrhoeae in the oropharynx. Rather surprisingly, there have been only three epidemiological studies conducted since using NAAT, which is a more sensitive test than culture.Several case reports purposed kissing could be a risk factor for oropharyngeal gonorrhoea in the 1970s et al. was the first longitudinal study examining the association between oropharyngeal gonorrhoea and kissing The Australian Health In Men (HIM) study by Templeton et al. was an age‐matched 1:2 case–control study conducted among 531 MSM in Melbourne in 2015 et al.'s study, Cornelisse et al.'s study also identified that both insertive rimming and receptive fellatio are risk factors for oropharyngeal gonorrhoea in MSM in the univariable analysis The second study by Cornelisse et al.'s et al.'s study et al. addressed this concern et al.'s study defined sex as any oral or anal sexual contacts and the finding showed that both kissing‐only and kissing‐with‐sex partners in the last three months were strongly associated with oropharyngeal gonorrhoea in the adjusted analysis. In addition, the risk of oropharyngeal gonorrhoea increased with an increasing number of kissing‐only and kissing‐with‐sex partners. However, the number of sex‐only partners in the last three months was not associated with oropharyngeal gonorrhoea. This was the first study identified to show kissing in the absence of sex may be an important and neglected risk factor for oropharyngeal gonorrhoea; however, this study did not measure oral sex as a separate sexual act and so could not adjust for it separately.Both Templeton Although studies have shown that kissing may be a risk factor for oropharyngeal gonorrhoea in MSM, the role of saliva in gonorrhoea transmission is still poorly understood. If saliva can carry infectious gonorrhoea, it is hypothesized that men could acquire oropharyngeal gonorrhoea through kissing by contacting infectious saliva, but it is unclear how much saliva is adequate for gonorrhoea transmission. Moreover, the salivary flow and its production vary between individuals. It is estimated that the salivary flow rate is about 0.3‐0.4 ml per min while unstimulated 2.3The majority of the oropharyngeal chlamydia infections are asymptomatic in men and therefore their diagnosis primarily depends on asymptomatic screening Chlamydia trachomatisA number of laboratory studies have been undertaken to examine the role of saliva in oropharyngeal chlamydia transmission. Two studies in the 1990s found that saliva has an inhibitory effect against 2.4Most men infected with gonorrhoea in the anorectum are asymptomatic; Given that saliva can carry infectious gonorrhoea, it is hypothesised anal sex that involves in saliva  may be associated with anorectal gonorrhoea Table . A cross2.5et al. showed that the use of saliva as a lubricant for anal sex is not a risk factor for anorectal chlamydia in MSM Anorectal chlamydia is primarily transmitted through condomless penile‐anal sex in MSM A meta‐analysis published in 2019 has concluded that anal intercourse is associated with anorectal gonorrhoea but not with anorectal chlamydia among women 3MSM can acquire gonorrhoea and/or chlamydia at the oropharynx and anorectum although the epidemiological evidence suggests the modes of transmission differ. For gonorrhoea, infections at extragenital sites are transmitted through non‐genital contacts such as kissing, rimming and use of saliva in addition to condomless oral or anal sex. For chlamydia, condomless anal sex is the main risk factor. However, the uncertainty about the hypotheses of the route of transmission for gonorrhoea and chlamydia via saliva among MSM should be acknowledged EPFC has received consultant fees from Gilead Sciences and speaker's fee from Seqirus Australia and research grants from Merck Sharp & Dohme outside the submitted work. CKF owns shares in CSL limited which markets Gardasil in Australia.EPFC wrote the first draft of the manuscript. All authors contributed to the concept, literature search, writing and revisions of the manuscript. All authors critically revised it for important intellectual content and approved the final version of the manuscript.


Escherichia coli at different antibiotic concentrations for ∼400 generations. Our results reveal fast evolution toward specialization following the first mutational step toward resistance, along with pervasive trade‐offs among different evolution doses. We found clear and regular fitness patterns of specialization, which converged rapidly from different initial starting points. These findings are consistent with a simple fitness peak shift model as described by the classical evolutionary ecology theory of adaptation across environmental gradients. We also found that the fitness costs of resistance tend to be compensated through time at low doses whereas they increase through time at higher doses. This cost evolution follows a linear trend with the log‐dose of antibiotic along the gradient. These results suggest a general explanation for the variability of the fitness costs of resistance and their evolution. Overall, these findings call for more realistic models of resistance management incorporating dose‐specialization.Antibiotic and pesticide resistance of pathogens are major and pressing worldwide issues. Resistance evolution is often considered in simplified ecological contexts: treated versus nontreated environments. In contrast, antibiotic usually present important dose gradients: from ecosystems to hospitals to polluted soils, in treated patients across tissues. However, we do not know whether adaptation to low or high doses involves different phenotypic traits, and whether these traits trade‐off with each other. In this study, we investigated the occurrence of such fitness trade‐offs along a dose gradient by evolving experimentally resistant lines of  The massive increase in use of antibiotics since 1950 has greatly improved standards of living in our societies but has led to an explosion in the number of resistant phenotypes in microorganisms   for ∼400 generations in order to “push” them close to their optimal phenotype. We started with a susceptible reference strain, or “SRef” strain, corresponding to the 10,000th (abbreviated 10 K) generation of Lenski's long‐term adaptation experiment (E. coli strain REL4536). First, we obtained a highly resistant reference strain (“RRef” strain) by screening twice SRef for antibiotic resistance . We then evolved two series of strains at the different Nal doses. In the first main series, six resistant lines per ED  were initiated from independent resistant mutants of SRef obtained after a screen at the same dose than the ED . These lines (hereafter “SRef lines”) provided a diversity of first mutational step and evolved from the screen at a given Nal dose. In the second series, two lines per ED  were initiated from the RRef strain. These lines (hereafter “RRef‐lines”) provided with replicated evolution of the same initial resistant mutant , corresponding to the 10,000th generation of Lenski's long‐term adaptation experiment to Davis minimal broth DM25 (Lenski and Travisano All lines were derived from the −7). They were obtained via an additional fluctuation assay  divided among the five evolution doses and the six control lines  evolving at dose 0. All lines were distributed into four 24‐wells lidded microplates with 1 mL medium per well, with the six Nal concentrations allocated in each plate. Inoculated wells were alternated with noninoculated wells following a checkered pattern, to control for contaminations. The plates were incubated at 37°C, 200 rpm in a water‐saturated atmosphere . Every 24 h, 10 μL of the cultured lines were transferred to new wells containing 1 mL of fresh medium and incubated in the same conditions. The protocol was applied for two months corresponding to ∼400 generations of evolution after what the lines were stored as glycerol stocks.gyrA gene at Tini and Tfin. This sequence covers most of the promoter and 32% of the gyrA gene and its complete quinolone resistance‐determining region . Sequencing was performed using Eurofins mwg operon . Mutations in gyrA gene were identified by comparing the mutant sequences to that of the SRef strain.All lines were sequenced at the ini and Tfin. The “fitness profile” of each line (YFP) across the dose gradient was determined by competitions against the RRef‐CFP strain in DM250 at the five measure doses . Ecotoxicological measures such as MIC (minimum inhibitory concentration) are often used to characterize resistance evolution. However, competitive fitness is better suited in our context, to quantify adaptation and the evolution of specialization. For instance, two lineages with similar MIC can have very different competitive abilities, below their common MIC. The measure dose 150 was preferred to dose 200 because the latter exhibited high growth inhibition of the RRef strain, such that fluorescence measurements were close to the detection limit. The fitness costs of resistance were measured from competitions in absence of Nal (dose 0) against the strain SRef‐CFP.Competitions experiments were performed from glycerol stocks at Tt0). Competitions were initiated by inoculating 2 μL of the competition mix into 200 μL of DM250 . Fluorescence signals were measured again after 24h of growth (t24) in the same conditions as the evolution experiment . Each competition was repeated at least four times at different dates. We assumed that fitness effects were transitive between genotypes competing in the same dose. Deviation from transitivity as measured in Gallet et al. (−3 at most in these experiments).Competition assays were performed in the following procedures. 1:1 volumic ratio competition mixes were prepared from saturated cultures. Fluorescence signals of these mixes were measured on a Tecan Infinite 200  prior to the competition .The selection coefficient per generation associated with each competition was estimated as:lineID, MD (six doses) and time (Tini and Tfin). Random effects included plate and date of the competition assay. The model was used to estimate the fitness values at Tini and Tfin, the fitness changes between Tini and Tfin (pairwise comparisons) of each line in each MD, and their standard errors (performed with mvt adjustments with the lsmeans R package), which are all represented in figures. Polynomial fits of the fitness profiles of evolved lines (from the fitness estimates at Tfin of the previous model) in each MD were performed as functions of the log‐ED of lines (nls function of the R Stats package). The fits were done for the SRef lines alone or for both the RRef and the SRef lines for the comparison of the final fitness profiles of lines evolved in the same ED but initiated from mutants screened at different doses.Fitness measures were analyzed in a linear‐mixed model  with fixed effects on P value = 0.773) nor between replicates   due to adaptation to experimental conditions . The last replicate differed only slightly from the three others due to a fitness jump at generation 400 . The global fitness trajectory of the control0K lines is consistent with that expected from the similar 400 generations evolution in DM25 in the LTEE  Fig. . This reE. coli, the DNA gyrase is the target of Nal and many of the known resistance mutations occur in the gyrA gene coding for the GyrA subunit of this enzyme  and some non‐gyrA mutations (four lines) were initially sampled across the sets of single resistant mutants screened at the doses 3, 8, and 20 .In  20 Fig. . Evoluti 20 Fig. . In all gyrA mutants 87Gly and 119Glu) went extinct early in the evolution experiment. One of the SRef‐lines evolving at ED20 proved to be initially polymorphic for the gyrA resistance mutation. This line was removed from later analysis in order to avoid confusion in the data interpretation. One of the two RRef‐lines evolving at Nal3 was contaminated at one point and removed from the experiment. In all other lines, no additional mutations than the ones detected at Tini were found in the gyrA sequence at Tfin. Among the four lines without gyrA mutations initially (hereafter “non‐gyrA” lines), only one line acquired a gyrA mutation (87Tyr) during the evolution experiment.Two SRef‐lines evolving at ED200 , all fitness profiles show a regular quadratic pattern with log‐ED. Fitted curves are shown on each panel and Table gyrA mutation show a large fitness lag compared to all other lines, at all MD. Given this large and consistent difference, these lines were not included in the fit of the average fitness profiles among ED represented on Figure gyrA mutations after the initial screen, despite that these mutations are frequent and provide a strong fitness benefit. The effect of the first mutational step is also visible on gyrA lines, but is very small. In several cases, lines with the same initial gyrA mutation tend to group together for a given ED and MD . Similarly, RRef lines , tend to group together as well. These effects remain, however, minor compared to the overall trend. Even the RRef lines, which had a quite different starting point than most lines, show a final pattern very consistent with the general trend of SRef lines  and generation 400 of evolution (Tfin) in the MD corresponding to its ED. SRef lines evolved at ED200 are not shown as their fitness were not measured at MD200 (see Methods). For all lines, the fitness change was positive or not significantly different from zero at the dose in which they evolved. Among gyrA lines, the trend shows striking regularity independently of the evolution dose and of the identity of the gyrA mutation carried initially , with a slope close to –1. The trend is also observed among non‐gyrA lines but with a different intercept  and similar slope of about –1. This pattern is expected when evolution corresponds to convergence to a fitness peak, as it indicates that the fitness changes entirely compensate initial maladaptation. This pattern excludes a strong role for “chance” or transient “historical” effects  and show an adaptation (or no change) in all MD, indicating that evolving at low dose increases fitness at high dose. A different pattern is observed for lines evolved at high doses. While they adapted to their ED, they consistently and regularly “deadapted” to low doses.Studying a large sample of mutants screened at different doses in the same system showed that little dose specialization occurred after a single mutational step  but higher for non‐gyrA mutations. They were also variable between single‐mutants and “double”‐mutants of ED100 and ED200, even when they carried the same gyrA mutation. The change in cost between Tini and Tfin is negatively correlated with the ED . The non‐gyrA lines (present only at ED3) have generally higher costs than the gyrA lines . Furthermore, phenotypes evolved at intermediate doses are better adapted to high doses than phenotypes evolved at low doses (consistent with a multiple hit effect). Additionally, long‐term selection at low doses will not result in high fitness  at high doses, and as a result, long‐term selection at high doses will not select for high‐fitness phenotypes at low doses. These conclusions have important implications for understanding and modeling the evolution of resistance in the field, under heterogeneous conditions.To our knowledge, no previous study has clearly established the pattern of dose‐specialization along a gradient so far. First, many studies have investigated the fitness of resistant mutants from field samples . After 400 generations of evolution at ED3, their fitness profiles are still largely different to those of the gyrA lines indicating a strong historical contingency between those “mutational modules” of resistance. Contrary to the specialization pattern, this divergence in fitness patterns between the gyrA and the non‐gyrA lines was already observed immediately after the first resistance screen and interpreted as the occurrence of selective covariances across traits in Harmand et al. gyrA and non‐gyrA resistant lines as good candidates to investigate the effects of historical contingency and selective covariances on long‐term adaptive trajectories.“Historical contingency” or “mutation‐order” effects have been widely discussed in the literature to describe the dependence of adaptive trajectories on initial conditions  often overlook the possibility that dose gradients may represent different phenotypic challenges, in addition to also representing different selection intensities. Our results show that this simplification is not warranted. They show that the evolution of resistance along antibiotic dose gradients is consistent with classic evolutionary models of adaptation on ecological gradients, where each environment corresponds to one fitness peak. The observed patterns of adaptation and maladaptation are also fully consistent with the occurrence of dose‐dependent optima and show pervasive trade‐offs across doses. Hence, our findings call for more realistic models of resistance evolution in heterogeneous dose conditions that include dose‐specialization. Such models are necessary to better evaluate the impact of low doses of antibiotics that are today ecologically widespread.The authors declare no conflict of interest.Associate Editor: K. LythgoeFig. S1. Selection coefficients of control lines evolved in the absence of antibiotic in competition against the non‐evolved wild‐type 10K‐YFP (or 10K‐CFP in the case of 0K‐YFP lines).Click here for additional data file.Fig. S2. Fitness change of the SRef lines during 400 generations of evolution in different evolution doses (colors) versus their fitness just after the screen of resistance in the different measure doses. Symbols indicate the gyrA mutations sequenced after evolution in each line while colors indicate the dose at which they were screened and evolved. Error bars represent standard errors of the mean and the fitness change estimated in the statistical model.Click here for additional data file.Figure S2bClick here for additional data file.Figure S2cClick here for additional data file.Figure S2dClick here for additional data file.Figure S2eClick here for additional data file.Fig. S3. Costs of resistance of the SRel lines evolved for 400 generations at five evolution doses of antibiotic. Symbols indicate the mutation detected in the gyrA sequence or the absence of mutation in the gyrA sequence while colors indicate the evolution dose. The dotted horizontal line at 0 corresponds to an equal fitness with the susceptible ancestor 10K, while negative values correspond to a lower fitness of resistant lines. Error bars represent standard errors among replicates.Click here for additional data file.


The method may offer broad reaching opportunities for the in situ generation organometallic compounds from base metals and their concomitant engagement in synthetic reactions via mechanochemical methods.A form independent activation of zinc, concomitant generation of organozinc species and engagement in a Negishi cross‐coupling reaction via mechanochemical methods is reported. The reported method exhibits a broad substrate scope for both C(sp The controlled and selective synthesis of carbon based molecules is a critical endeavor that is key to the discovery of new medicines, crop protection agents, flavors, and fragrances as well as many more materials with great importance to human quality of life. Whilst synthetic chemistry as a discipline has a good grasp on making molecules succumb to synthesis, focus in the modern era is centered upon achieving sustainable synthesis through reduced numbers of reaction steps, reduced quantities of waste, and milder reaction conditions.ortho‐metallation with organomagnesium or organolithium provides the initial metalation and is followed by transmetallation to a zinc (II) species  after an acidic quench of the jar contents.An alternative tactic involves the metalation of C−X bonds . Thiophenes  and a pyridine (41) derivative were also prepared, highlighting the applicability of this method to synthesize substituted heterocycles.Increasing the quantity of TBAB to 1.5 equivalents further improved the yield to 77 % (GC) corresponding to a 74 % isolated yield –C(sp2) bonds thus rendering it more operationally simplistic than current procedures.In summary, we have developed a novel method for the synthesis and subsequent reaction of organozinc species under mechanochemical conditions, without the need to use inert atmosphere techniques or dry solvents. Organozincs can be generated from an alkyl halide irrespective of the physical form of commercially available zinc metal. A coupling partner could then be added to the reaction mixture along with a palladium catalyst and TBAB to perform the Negishi reaction in a one‐pot, two‐step process. Most excitingly, these conditions were successfully modified to realize the direct generation and consumption of organozinc reagents through a one‐pot Negishi coupling process for the synthesis C should be addressed to the authors.SupplementaryClick here for additional data file.


With an increasingly aging global population, the incidence of neurological diseases such as dementia is set to increase to unmanageable levels, yet there are currently only symptomatic therapies available for treatment. The mechanisms underlying the development of some forms of dementia, such as Alzheimer's disease (AD), are not yet completely elucidated with several competing hypotheses existing. During the closure of the critical period in the brain, significant compositional changes occur to the neural extracellular matrix (ECM). Specifically, condensed mesh‐like structures called perineuronal nets (PNNs) form around subsets of neurons and have a profound effect on axonal growth and limit neuronal plasticity. These PNNs act as a morphological checkpoint and can influence memory and cognition. Manipulating these important ECM structures may provide the key to reactivating plasticity and restoring memory, both of which are severely impaired in AD and other associated neurological diseases. This review explores the current understanding of how PNNs are manipulated and examines potential new methods for PNN modulation.http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.18/issuetocThis article is part of a themed section on Therapeutics for Dementia and Alzheimer's Disease: New Directions for Precision Medicine. To view the other articles in this section visit  ACANaggrecanADAlzheimer's diseaseBCANbrevicanCSchondroitin sulfateCSPGschondroitin sulfate proteoglycansECMextracellular matrixGAGglycosaminoglycanGalNAcN‐acetylgalactosamineGlcAglucuronic acidHAhyaluronanHaplnhyaluronan and proteoglycan link proteinNCANneurocanPNNsperineuronal netsTn‐Rtenascin‐R1Alzheimer's disease (AD) is the most common form of dementia and is clinically characterised by a progressive loss of memory and functional cognition as well as other non‐cognitive disturbances such as anxiety and delusions  proteins  inhibitors , resulting in the restoration of cognition in a mouse model , the most abundant and most crucial component of PNNs, forms a backbone mesh‐like structure which allows for the binding of other important components such as chondroitin sulfate proteoglycans (CSPGs) and ultimately dictates the overall structure of the ECM  units forming long non‐sulfated polysaccharide chains which vary in length, ranging from 25 to 1,000 kDa  and GlcA  staining; however, it is still unclear which component of the nets WFA binds to  simultaneously. All lecticans have an N‐terminal globular G1) domain containing an immunoglobulin‐like loop repeat capable of binding HA chains and certain link proteins (Haplns) and up to two link modules at the C‐terminus  GABAergic interneurons. This creates a lattice structure which blocks the formation of new synapses  specifically binds to the glycosaminoglycan (GAG) chondroitin‐4,6‐sulfate , which is enriched in the PNNs. When bound to PNNs, Sema3A enhances the inhibition of PNNs to neuronal growth and is involved in restricting plasticity , the development of PNNs can be attenuated, leading to the adult mice visual and somatosensory systems plasticity being greatly enhanced to levels that are comparable to juvenile animals  memory in mice. Two mouse models with Tau pathology showing significant impairment in OR memory were injected with chondroitinase ABC (ChABC) at the site of the perirhinal cortex, in order to enzymically digest the PNNs present. One week after treatment, the Tau mice demonstrated similar levels of OR memory and synaptic transmission to control animals, suggesting that ChABC may be effective in restoring memory loss in neurodegenerative disorders such as AD  to chondroitin‐6‐sulfate (C6S) in the PNNs gradually increases  enzyme have been used as an alternative way to interrupt CSPG production. Interestingly, these mice still formed structurally identifiable, albeit abnormal, PNNs  but not chondroitin‐2,6‐sulfate , despite both forms being disulfated chondroitins, suggesting that Sema3A binding has sulfation pattern specificity rather than overall sulfation quantity specificity . This transcription factor also binds to C4,6S in the PNNs of the PV+ interneurons, is then internalised, and modulates gene expression for the maturation of PV+ neurons. It plays an important role in the commencement and termination of the critical period for plasticity  and the leukocyte common antigen‐related phosphatase  was shown to be modestly inhibited by a low MW compound that had been optimised using a high throughput screening and medicinal chemistry (Cheung et al., A promising target for intervention with low MW compounds on PNNs and axonal growth may be the CSPGs. More specifically, preventing the biosynthesis of these structures by targeting the sulfotransferase enzymes that provide the CS chains has been shown to be an effective method to renew axonal growth. In recent studies, the Golgi‐resident There are numerous other components in the PNNs that can potentially be targeted by low MW compounds, including the CNS‐exclusive tenascin, TN‐R, and the HA–CSPG bridging link proteins. Additionally, it can be envisaged that interruption of the binding of the known PNN binding molecules Sema3A and Otx2 may provide success. Further research into the druggability of these proteins is required before an inhibitor can be realised. Other therapeutic avenues may be pursued. However, given the benefits of developing a low MW modulator, as previously mentioned, coupled with the pitfalls frequently encountered with these other modalities, discovery of a low MW drug seems to provide the most promise going forward.6Increasingly, the emerging evidence suggests that PNNs have a vital role to play in controlling plasticity, regulating axonal growth and regeneration and memory storage during development and throughout adulthood. This has obvious implications in several neurological diseases, including the many forms of dementia, in which the underlying mechanisms involved in disease progression are not fully understood. The molecular make‐up and function of the PNNs are increasingly being appreciated, when assessing the scientific literature currently available. This understanding has been used to target and disrupt the PNNs using numerous methods to increase neuronal plasticity. This includes enzymic degradation of the nets, genetic therapy to prevent PNN formation, and blocking of PNN action using low MW compounds and/or biomolecules. The abundance of potential protein targets in the PNNs should inspire the development of novel therapeutic agents with a focus on utilising the ease of discovery and optimisation of low MW compounds to inhibit PNNs, in order to reactivate plasticity and restore cognition in neurological disorders such as AD.6.1http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., Key protein targets and ligands in this article are hyperlinked to corresponding entries in The authors declare no conflicts of interest.


MPP) are important clinical adjuncts in cardiac resynchronization therapy (CRT) pacing aimed at reducing the rate of non‐response to therapy. Mixed results have been achieved using MPP and it is critical to identify which patients require this approach and how to configure their MPP stimulation, in order to achieve optimal electrical resynchronization.Quadripolar lead technology and multi‐point pacing (ECGi), using the CARDIOINSIGHT™ inverse ECG mapping system, could identify alterations in electrical resynchronization during different methods of device optimization. In no patient did a single form of programming optimization provide the best electrical response. The effects of utilizing MPP were idiosyncratic and highly patient specific. ECGi activation maps were clearly able to discern changes in bulk LV activation during differing MPP programming. In two of the five subjects, MPP resulted in more rapid activation of the left ventricle compared to standard CRT; however, in the remaining three patients, the use of MPP did not appear to acutely improve electrical resynchronization. Crucially, this cohort showed evidence of extensive LV scarring which was well visualized using both CMR and ECGi voltage mapping.We sought to investigate whether electrocardiographic imaging (ECGi in the optimization of non‐responders to CRT, as it allows the fusion of activation maps and scar analysis above and beyond interrogation of the 12 lead ECG.Our work suggests a potential role for  Initially devices were programmed according to the default manufacturer settings. The following day after implantation, each patient underwent an iterative CRT optimization procedure, guided by non‐invasive body surface mapping, looking to identify the optimal pacing settings.Patients on optimal medical therapy (OMT) meeting European Society of Cardiology (ESC)2.1Global Right/Left Ventricular Electrical Synchrony (VVsync): the mean activation time in the right ventricle minus the mean activation time in the left ventricle. Previously described as ventricular electrical uncoupling.Global Biventricular Total Activation Time (VVtat): a measurement of the total time required for both ventricles to activate. Previously described as VVTAT.Global Left Ventricular Total Activation Time (LVtat): a measurement of the total time required for all portions of the left ventricle to activate. Previously described as LVTAT.Global Left Ventricular Dispersion of Activation (LVdisp): a measure of the dispersion of the activation times in the left ventricular region of interest.A non‐invasive electrophysiological mapping study was performed using a high resolution electrocardiographic mapping system , as previously described.Given the primary objective of CRT is to restore regional activation synchrony, we defined the optimal activation pattern as that which achieved the most effective degree biventricular resynchronization whilst simultaneously minimizing both biventricular and LV activation times. Electrical synchrony is specifically assessed by VVsync, where a figure of 0 represents identical LV and RV activation time.Epicardial voltage maps were also collected, in order to identify any areas of low voltage which may indicate areas of myocardial scar and fibrosis. Where possible, these were correlated against CMR data, see Figure 2.2The patient's baseline presenting rhythm––either intrinsic sinus rhythm or RV paced rhythm––was first analyzed using directional activation maps.The VV offset was optimized according to the maximal improvement in aortic pulsed‐wave Doppler velocity time integral, as previously described.™  was used to identify the RV‐paced to LV‐sensed timings for each of the poles on the quadripolar lead. MPP was then programmed to pace the pole with the longest delay first and the pole with the shortest delay second. The right ventricular lead was always paced last. We delivered MPP with 5, 10, and 20 ms delays between each stimulus using both a local bipole configuration‐distal (D1) to mid 2 (M2) and proximal (P4), and an extended bipole configuration‐ D1 to RV coil and P4 to RV coil. This resulted in our testing 6 MPP settings per patient; 3 local and 3 extended bipole. In order to test the different MPP vectors for capture threshold and phrenic nerve stimulation, a number of standard biventricular recordings were also performed which served as comparators for individual patients.Once the optimal AV and VV intervals had been established and programmed, differing MPP settings were then acutely programmed. During each configuration, a non‐invasive electro‐anatomical mapping was obtained. The SJM CRT toolkit33.1A total of five patients were enrolled in the study.3.2The effect of changing AV delays, VV delays, the LV pacing vector, and finally the addition of MPP are shown in Table Epicardial voltage mapping showed the presence of scar in all of the ischemic patients which corresponded to LGE on MRI in the 3 cases where MRI was performed, see Figure 3.3Echocardiography demonstrated severe biventricular impairment and CMR demonstrated delayed sub‐endocardial late gadolinium enhancement consistent with prior infarction of the apex, antero/infero‐septum, and inferolateral wall. The epicardial voltage map was consistent with extensive scarring in the same distribution as the LGE visualized during CMR.3.3.1Intrinsic conduction exhibited the activation pattern typically observed in LBBB; ventricular activation is initiated at the distal branching of the right bundle, with activation of the left ventricular endocardium occurring after a significant delay, as a result of slow conduction through the interventricular septum  Extended Bipolar and Local Bipolar MPP resulted in more rapid activation of the left ventricle compared to optimized echo optimized CRT. It is possible accelerating LV activation has the potential to improve the degree of cardiac resynchronization and as such, may explain the greater hemodynamic improvements™ ECGi system represents a non‐invasive technique capable of assessing the acute response to MPP and may be of use in identifying patients likely to gain the most from MPP as well as how best to configure this multi‐polar pacing.The remaining 3 patients showed evidence of extensive LV scarring which was well visualized using both CMR & ECGi voltage mapping. The use of MPP in this cohort did not appear to acutely improve electrical resynchronization. In part, this may be explained by the focal nature of the scar burden,5This is a small study and the results are hypothesis generating rather than conclusive. Our primary objective was to analyze how myocardial activation varied during device programming optimization and whether non‐invasive body surface mapping technology might be capable of identifying these changes in activation. Our hypothesis did not extend to evaluating rates of response to CRT and given the small number of patients, it would be impossible to draw reliable conclusions. Instead, non‐invasive electrical measurements were analyzed acutely and it is unclear whether these results can be extrapolated to the chronic delivery of CRT. Biventricular pacing which improves the degree of biventricular electrical resynchronization has been associated greater response.6Iterative echo guided AV & VV optimization & quadripolar lead technology in conjunction with MPP are important clinical adjuncts to CRT pacing. The range of programming options provide greater cost efficiency by reducing the need for reintervention after implantation for technical issues including high capture thresholds, lead displacement, and phrenic nerve stimulation.Amongst carefully selected patients; however, the use of optimal device programming can achieve a superior degree of electrical resynchronization when compared to conventional CRT with nominal settings. However, these strategies are not without cost. Echo guided device optimization can be expensive and time consumingOur analysis suggests that judicious use of device reprogramming optimization may be a useful strategy; however, the main issue remains identifying which patients may require this approach and then successfully optimizing their programming to achieve optimal electrical resynchronization. ECGi is a non‐invasive technique capable of accurately delineating the electrical effects of CRT pacing as well as the presence and distribution of myocardial scar. Our work suggests a potential a role for this tool in the optimization of non‐responders to CRT, as it allows the fusion of activation maps and scar analysis above and beyond interrogation of the 12 lead ECG.Authors declare no conflict of interests for this article.


Sternula antillarum) and piping plovers (Charadrius melodus) are physiologically adapted to initiate nests concurrent with the cessation of spring river flow rises on two sections of the Platte River, Nebraska. The study by Farnsworth et al. (2017) has several shortcomings which bring into question the authors’ principal assertion that interior least tern and piping plovers are not adapted to occupying and nesting on river sandbars on the Platte River system. We identify these shortcomings and provide information, which, we suggest, would change their conclusions if incorporated.In a recent study, Farnsworth et al. (2017) used distributions of nest initiation dates drawn mostly from human‐created, off‐channel habitats and a model of emergent sandbar habitat to evaluate the hypothesis that least terns (Linked Article: https://doi.org/10.1002/ece3.4097 The authors conclude that (1) these species are not now, nor were they in the past, physiologically adapted to the hydrology of the Platte River, (2) habitats in the Platte River did not, and cannot support reproductive levels sufficient to maintain species subpopulations, (3) the gap in local elevation between peak river stage and typical sandbar height, in combination with the timing of the average spring flood, creates a physical environment which limits opportunities for successful nesting and precludes persistence by either species, and (4) the presence of off‐channel habitats, including human‐created sand and gravel mines, natural lakes, and a playa wetland, allowed the species to expand into the Platte River basin.Historical and contemporary use of large, economically important rivers by threatened and/or endangered species in the United States is a subject of great interest to a wide range of stakeholders. In a recent study of the Platte River in Nebraska, Farnsworth et al.  (hereinaWe suggest the authors (1) overlooked published data on the relationship between formative river stage, sandbar height, and nest heights, (2) used nest initiation dates taken from static off‐channel habitats and overemphasized the importance of mean daily hydrographs to imply that the hydrology of the Platte River system is not suitable for terns and plovers, (3) incorrectly characterized tern and plover biology, population ecology, and metapopulation dynamics, and (4) overlooked portions of the historical record which demonstrate terns and plovers were regularly present and successfully nested along the central Platte River (CPR) and lower Platte River (LPR).2Elevation of sandbars relative to river stage is a foundational component of the authors’ analysis as it determines whether habitat will be available or unavailable  for nesting. The sensitivity analysis presented by Farnsworth et al.  showed tThe authors used mean values for the stage gap, one published  was much larger than the stage gap for sandbars formed by the 2011 flood . Although Farnsworth et al. do not make clear where their value of median stage gap for the LPR was taken from, we believe the value was taken from a “group‐median” value of 2 feet (~0.61 m) reported in the summary of Alexander et al. . This odd stage gap pattern reinforces the need for an explicit description of their sandbar height data collection and analysis methods. Because of the strong control of the assumption of sandbar height on determination of successful nest windows, we suggest that Farnsworth et al. should have accounted for variation in stage gap with discharge rather than using a single value for each reach under all discharges. The larger stage gap for less frequent floods and smaller gap for more frequent floods would have the effect of increasing the number of years with successful nest windows because most years would have a smaller gap than suggested by the constant values used in each reach by Farnsworth et al.Finally, the authors assume parity between median sandbar height and the height of nests on river sandbars, despite the fact that empirical evidence indicates (1) sandbars selected by the species for nesting tend to have mean elevations that are higher than unoccupied sandbars in the same reach and (2) nest sites selected by individual birds tend to occupy the higher regions of a sandbar's topography  for segments of the Platte River with distributions of nest initiation dates for both species  to assert that the annual spring rise typically occurs after the nest initiation date for both species. The authors use this simple overlay to suggest  that the hydrology of the Platte River creates adverse physical conditions for nesting because the typical spring rise would occur after nest establishment and, due to the large stage gaps assumed by the authors, typically inundate established nests.Although the mean daily hydrograph can be useful for understanding basic hydrologic patterns at a location in a river, such hydrographs mask variability, particularly in the timing of the annual instantaneous peak flow, which is the typical emergent sandbar habitat formative event. For example, the mean daily hydrograph illustrated in figure 8 of Farnsworth et al. shows the late spring rise in the historical and contemporary CPR occurs in mid‐ to late June, but the peak flow record at the long‐term stream gage at the downstream end of the CPR  indicates that 60% of annual instantaneous peaks occurred before June 1 (February through May), while 30% occurred sometime in June, and the rest at other times of the year. Although the long‐term gage is not within the Farnsworth et al. study reach , for the overlapping periods of record, more than 80% of peak flows at USGS gages within the AHR  are either earlier, or within 10 days of the peak flows at the long‐term gage (06774000). The peak flow records at these USGS gages on the CPR all indicate that at least 50% of peak flows occurred sometime between February 1 and May 31, and between 36% and 48% of peaks occurred before May 1. On the LPR, the peak flow record  indicates that 26% of instantaneous peak flows occurred before May 1 (February through April), 50% before June 1, and 30% occurred sometime in June. Farnsworth et al. account for variability in flood timing within their sandbar availability model using the daily records, but in Section 3.1 use their figure 8 to suggest a general dissonance between the timing of nest initiation and the timing of annual high flows. A more informative way to visualize and compare the general timing of nest initiation with annual peaks would have been to plot the timing and magnitude of instantaneous peaks for each reach over the nest initiation distributions. Such an overlay would inform the reader of the year‐to‐year variability in flood timing relative to the nest initiation distribution and would be a more accurate portrayal of hydrologic conditions relevant to nesting.n = 1,089) of the nests reported in the CPR during this 13‐year period were found on human‐created off‐channel habitats . Least tern mean nest initiation dates on the Yellowstone River, Montana, where off‐channel habitats are not available, occurred 16 June, 30 June, and 1 July in 1994, 1995, and 1996, respectively, following cessation of spring rises that occurred as late as mid‐ to late June  from the CPR for the years 2001–2013. Although not stated in their paper, nearly all  is influenced by high flows that often extend into June or July  found in a report Lutey,  are neceThe analytical approach used by the authors is too simple to address complex questions about metapopulation dynamics. Metapopulations persist as component populations that appear and disappear over space and time  habitat of the Platte River and suggest human‐created off‐channel habitats were both species’ primary nesting habitat which allowed them to “expand into and persist in a basin where hydrology is not ideally suited to their reproductive ecology .” To support their contentions, the authors refer only to 20th‐century nesting on sandbars and human‐created habitats along the CPR and off‐channel nesting by least terns during 2 years at a single playa wetland in the Rainwater Basin of south‐central Nebraska and along lake shorelines.A more rigorous review of the historical record shows that least terns and piping plovers were found along the Platte and other regional rivers since the earliest recorded ornithological observations. Lewis and Clark observed least terns and piping plovers along the Missouri River in 1803–1804, as did numerous others during the late 1800s and early 1900s  activities directed toward least tern and piping plover recovery away from in‐channel habitat restoration to off‐channel habitat maintenance represents a success of adaptive management that is “unique among riverine restoration programs” . We believe conclusions about threatened and endangered species management and recovery, as well as stewardship of natural resources, must be made considering the full spectrum of tradeoffs and consequences. Loss of habitat due to human alterations of natural systems is the principal reason regional populations of least terns and piping plovers declined, remain small compared to historical levels, and why they were listed under the Endangered Species Act and remain on the federal Endangered Species List USFWS, , 1990. IOn‐channel habitats, such as those used by the birds on the historical CPR and contemporary LPR, existed or presently exist (LPR) only in resilient, dynamic river systems and are maintained by hydrological and geomorphic processes and benefit a diversity of species (Alexander et al., 7We appreciate the authors’ efforts toward modeling sandbar availability in relation to river hydrology; however, their analysis has shortcomings which limit the study's usefulness. These shortcomings, as well as incomplete characterizations of the species’ ecology and the historical record, negate the author's assertions that least tern and piping plovers are not adapted to occupying and nesting on river sandbars on the Platte River system. Decisions relegating imperiled species to conservation reliant status need to be made only after considering the full range of tradeoffs and consequences.None declared.


Chlamydomonas reinhardtii, calcium has been implicated in the regulation of cyclic electron flow—another of those photoprotective mechanisms—via a small calcium sensor protein named CAS  show that increases in the absolute temperature elevate the free calcium concentration in chloroplasts . FurtherThe authors have no conflicts of interest to declare.


However, there are concerns that even short‐term exposure of cervical tissue to dilute proflavine may increase cervical cancer risk. We performed a retrospective analysis of women referred for colposcopy to Barretos Cancer Hospital comparing the risk of cervical disease progression in those whose cervical tissue was (n = 232) or was not exposed (n = 160) to proflavine. Patients in both groups underwent treatment and follow‐up based on histopathologic results and per the local standards of care. Progression of disease was evaluated by comparing histopathology from the initial visit to the worst subsequent histopathology result from all follow‐up visits. Mean duration of follow‐up was 18.7 and 20.1 months for the proflavine‐exposed and controls groups, respectively. There were no significant differences in disease progression from normal/CIN1 to CIN2/3 or from any initial diagnosis to invasive cancer between the proflavine exposed and control groups overall. Risks of cervical dysplasia progression observed in this study are in agreement with those of the natural history of cervical cancer. Our results suggest that cervical exposure to dilute proflavine does not increase the risk of cervical precancer and cancer.Proflavine is an acridine dye used with high‐resolution microendoscopy for  Proflavine is a fluorescent dye that has long been recognized and used as a topical antibacterial agent. Its history of use as an antiseptic in wound care dates back to the early 1900s, and its action as a nucleic acid intercalator has been established as the basis of its antibacterial properties in vivo fiber‐optic microscopy techniques over the past two decades, proflavine and acriflavine (another closely related acridine compound) have been frequently used as topical contrast agents for optical imaging due to their ability to fluorescently label cell nuclei with high contrast. Acriflavine has been reported as a contrast agent for in vivo confocal laser endomicroscopy in the colon in vivo microscopy in the oral cavity in vivo microscopy enables real‐time assessment of the morphology and distribution of cell nuclei, aiding in the accurate identification of precancerous lesions With the development of in vivo imaging. In 1977, the National Cancer Institute published a bioassay of proflavine for possible carcinogenicity, based on administration of proflavine in the diet to groups of rats and mice over a 2‐year period in vitro, their carcinogenic risk to humans could not be classified due to inadequate data in experimental animals and a lack of data in humans. Some view the mutagenicity of these compounds in vitro as sufficient reason for concern. For example, a recent publication recommends the use of methylene blue over acriflavine as an imaging contrast agent, due to concerns about the in vitro mutagenicity of acriflavine Despite their long history of safe clinical use, there remains disagreement on the cancer risk of using proflavine and acriflavine as contrast agents for Yet concerns remain. Therefore, we conducted a retrospective study to evaluate whether proflavine exposure is associated with progression of cervical neoplasia in women with abnormal cervical cytology by comparing the rate of cervical intraepithelial neoplasia progression between two groups, one exposed to proflavine and the other not exposed.This is a historical cohort study. The proflavine group was composed of women who had previously participated in a clinical trial (ID# NCT02335372) at Barretos Cancer Hospital (Brazil) between June 2013 and January 2015. Participants in the trial were recruited from women presenting for colposcopy due to abnormal cytology or previous history of cervical dysplasia at the Prevention Department of Barretos Cancer Hospital. This trial evaluated a new optical imaging device, the high‐resolution microendoscope (HRME), a portable, battery‐powered fluorescence microscope with a flexible fiber‐optic probe Follow‐up data including all histopathology results were collected for patients in both groups from the time of the initial colposcopy until the last appointment available from the medical record. In both groups, the treatment and follow‐up were performed according to the local standard of care based on histopathology results. Those women diagnosed with cervical intraepithelial neoplasia grades 2 (CIN 2) or 3 (CIN 3) underwent treatment with loop electrosurgical excision procedure (LEEP). Those diagnosed with invasive cancer were referred to the Gynecologic Oncology department for care.Data for all patients in the proflavine and control groups were collected from electronic medical records at Barretos Cancer Hospital and compiled into an SPSS database file. The database consisted of a total 499 patient records with 299 records in the proflavine group and 200 in the control group. The following data fields were aggregated for each participant: age, study group, initial cytology result, date of initial colposcopy, initial histopathology result, date of last clinical visit and up to six subsequent histopathology results. For all histopathology results, the date of diagnosis and tissue specimen type , LEEP, or hysterectomy) were documented. The total follow‐up time period was calculated as the time between the first and last colposcopy evaluations on record.in‐situ (AIS) and invasive cancer. NIL category included diagnoses of normal, inflammation, hyperplasia and metaplasia. CIN 2/3 category included diagnoses of CIN 2, CIN 3 and CIN 2/3. Invasive cancer category included diagnoses of invasive squamous cell carcinoma and adenocarcinoma. In cases where follow‐up colposcopy was negative and no biopsies were obtained, the visit was categorized as normal colposcopy/no biopsy performed.Cervical cytology results were grouped into two categories: normal/low grade and high grade. Normal/low grade included the following: negative for intraepithelial lesions or malignancy (NILM), atypical squamous cells of undetermined significance (ASC‐US) and low‐grade squamous intraepithelial lesion (LSIL). High‐grade cytology results included: high‐grade squamous intraepithelial lesion (HSIL), atypical squamous cells cannot exclude high‐grade squamous intraepithelial lesion (ASC‐H), atypical glandular cells (AGC), squamous cell carcinoma and adenocarcinoma. In order to assess cervical dysplasia progression, histopathology results were grouped into five clinically relevant categories by increasing severity of diagnosis: negative for intraepithelial lesion (NIL), cervical intraepithelial neoplasia grade 1 (CIN 1), CIN 2 or CIN 3 (CIN 2/3), adenocarcinoma n = 49; control group, n = 29), and (2) patient had an initial histological diagnosis of invasive carcinoma . After applying these exclusion criteria, 392 patients were included in the retrospective analysis . Using these data, the study population was characterized using descriptive statistics. Categorical variables were compared using Fisher's exact test, and continuous numerical variables were compared using Student's t test. Survival analysis was performed on two clinical endpoints: (1) progression from a baseline histopathologic diagnosis of <CIN2 to a subsequent histopathologic diagnosis of CIN2+, (2) treatment via LEEP. A spreadsheet containing all patients was compiled with the following information: cohort (proflavine or control), time from baseline diagnosis to the event of interest (days), and the status  at the indicated time interval. A survival function was fit using the “survival” package of the R programming language Post hoc statistical power was calculated using PASS .A total of 107 patients were excluded from the analysis for the following reasons: (1) total follow‐up duration was less than 6 months . The mean time of follow‐up was 18.7 months (SD = 6.0 months) and 20.1 months (SD = 5.6 months) for the proflavine and control groups, respectively.There were no statistically significant differences between groups according to their initial diagnosis by cytology and/or histopathology from cervical biopsies Table . The mean = 232) and those not exposed (n = 160). Because removal of proflavine‐exposed tissue via LEEP may mitigate the effect of proflavine exposure, we stratified our analysis within each cohort by those who underwent LEEP and those who did not. A LEEP was performed in 124 of the 232 (53%) patients exposed to proflavine and in 96 of the 160 (60%) control patients (P = 0.21).In order to evaluate cervical dysplasia progression, we compared initial and subsequent worst histopathologic diagnoses for the patients exposed to proflavine (P = 0.73). Additionally, there was one patient in the control group that progressed from an initial diagnosis of CIN 1 to CIN 2/3 . The majority of non‐LEEP patients had no abnormal cervical lesions observed during follow‐up colposcopy, and thus, no additional cervical biopsies were required .Table P = 1.0). There were 28 patients with benign initial histopathology on biopsy who had a worst subsequent diagnosis of CIN 2/3 on the LEEP tissue . In addition, three patients in the control group had benign initial histopathology with a worst subsequent diagnosis of CIN 1 . One invasive cervical cancer was diagnosed both in the proflavine exposed and control groups.Table P = 0.44). Progression from any initial diagnosis to invasive cancer was noted in one of 232 patients exposed to proflavine (0.4%) and one of 160 controls (0.6%) (P = 1.0). Based on the total number of cases where subsequent pathology was worse than initial, there were no statistically significant differences in progression of cervical dysplasia between proflavine‐exposed and nonexposed patients .The risks of cervical dysplasia progression for all groups are summarized in Table n = 125; control group, n = 77) and treatment via LEEP . No statistically significant difference was found for progression of histological diagnosis from <CIN2 to CIN2+ between proflavine‐exposed and control groups . Additionally, no statistically significant difference was found for treatment by LEEP between proflavine‐exposed and control groups .Figure in vivo microscopy during colposcopic examination. Our results showed no significant increases in cervical dysplasia progression in women exposed to proflavine when compared to a control group of nonexposed women. This is important for routine gynecological examinations because proflavine can be used as a diagnostic tool in conjunction with colposcopy, facilitating the recognition of cervical lesions through in vivo microscopy as previously reported by Grant et al. In this study, we assessed whether proflavine might increase the risk of cervical neoplasia progression when used as a fluorescent contrast agent for In a meta‐analysis of 17 studies involving 4504 participants, Östӧr post hoc statistical power was ≥80% (α = 0.05) to detect an absolute increase in risk of upstaging from <CIN2 to CIN2+ of 18% (or more). However, longer‐term follow‐up studies with larger sample sizes will be necessary to provide reassurance for the topical use of proflavine on the cervical epithelium.The total follow‐up duration achieved by this analysis is one limitation of the study. Even with this limitation, the structure of this study provides a meaningful comparison for cervical dysplasia progression in proflavine‐exposed and nonexposed subjects. The In conclusion, the risks of cervical dysplasia progression observed in this study are in agreement with those of the natural history of cervical cancer. Our findings suggest that cervical proflavine exposure is not associated with acute disease progression in women with cervical dysplasia.


Interstitial lung disease (ILD) encompasses a large group of pulmonary conditions sharing common clinical, radiological and histopathological features as a consequence of fibrosis of the lung interstitium. The majority of ILDs are idiopathic in nature with possible genetic predisposition, but is also well recognised as a complication of connective tissue disease or with certain environmental, occupational or drug exposures. In recent years, a concerted international effort has been made to standardise the diagnostic criteria in ILD subtypes, formalise multidisciplinary pathways and standardise treatment recommendations. In this review, we discuss some of the current challenges around ILD diagnostics, the role of serological testing, especially, in light of the new classification of Interstitial Pneumonia with Autoimmune Features (IPAF) and discuss the evidence for therapies targeted at idiopathic and immune‐related pulmonary fibrosis. Interstitial lung diseases (ILD) are a large group of conditions causing morbidity and mortality in all age groups. In addition to highlighting the benefits of a multidisciplinary diagnoses, we review developments in ILD diagnostics, especially around autoimmune disease and current evidence for newer therapeutics. Interstitial lung disease (ILD) is an umbrella term for a large group of over 200 pulmonary disorders characterised by inflammation and/or fibrosis of the pulmonary interstitium with heterogeneous causes, clinical course and treatments. While some ILDs have an identifiable trigger with occupational, environmental or medication exposure and others are strongly associated with systemic disease, in particular the connective tissue diseases (CTD), for many their origin is unknown and the term ‘idiopathic’ is applied.Early work on understanding these diseases was hampered by a lack of shared criteria for diagnosis and treatment trials which were often small and involved mixed groups of patients. More recently, professional societies across the world have published joint position papers on disease classification and approach to treatment which continue to evolve with inclusion of new entities and evolving criteria for diagnosis.For the idiopathic interstitial pneumonias (IIP), there are no identifiable triggers, although genetic predisposition is starting to be documented.As many ILDs are progressive with a poor prognosis, identifying the correct aetiology is clinically important, influencing treatment choice and understanding of prognosis. Additionally, accurate phenotyping informs disease epidemiology, provide insights into pathophysiology of disease and facilitates the design of clinical studies.Treatment options for patients have increased with the availability of anti‐fibrotic therapy, in addition to an increasing range of immunosuppressive agents clinicians can choose from.This review will explore the latest research around making an accurate diagnosis, in the overlapping entities of IIP, CTD‐ILD and IPAF, and will review the implications of recent data on serological testing for connective tissue diseases and the early clinical data on genetics behind ILD. It will explore the implications of having two approaches to therapy, namely with anti‐fibrotic or immunosuppressive therapy and the current evidence behind helping to choose the best for patients.Idiopathic interstitial pneumonias, as shown in Figure Many of the connective tissue disorders  are associated with interstitial disease. The radiological and pathological findings are the same as with the many of the idiopathic forms of interstitial pneumonias with the commonest disease patterns being UIP, fibrotic NSIP and OP with variation across the CTDs being the most prevalent. The interstitial disease may be the most important determinant of prognosis, and as it can precede or be the predominant feature in some of these conditions,For certain patterns of disease, for example, UIP, patients with CTD‐ILD generally have a better prognosis than patients with IPF with a slower progression of disease, with the notable exception of UIP in rheumatoid arthritis‐associated ILD.In addition to those patients who meet the criteria for diagnosis of a CTD, a significant number of patients with ILD have clinical features suggestive of an underlying autoimmune process, but yet do not meet the defined diagnostic criteria for CTD. These patients have been given the designation of interstitial pneumonia with autoimmune features (IPAF). In order to provide a uniform criterion to allow comparative studies of such patients, the European Respiratory Society and American Thoracic society proposed the nomenclature of IPAF for these patients and recommended classification criteria.A multidisciplinary approach to diagnosis with experts in respiratory medicine, radiology, immunology, rheumatology and pathology reviewing findings at a multidisciplinary meeting (MDM) has been shown to increase diagnostic confidence and inter‐observer agreement.An accurate diagnosis starts with a comprehensive clinical assessment. A detailed history should include symptoms, disease course, presence of associated diseases, in particular of connective tissue disorders, history of occupation, hobbies or environmental exposures, medications and family history. Typical signs sought include lung crackles, clubbing and any signs of a connective tissue disease with joint, muscle or skin involvement. Lung function classically shows reduced lung volumes and diffusing capacity with desaturation on walking.Plain x‐rays are often performed during the initial assessment but provide limited information and the key clinical tool is the high‐resolution CT (HRCT) scan. In IPF, the classical HRCT finding of UIP pattern in the setting of a consistent clinical picture equate to a high probability of IPF, and obviates the need for a lung biopsy. The recently published ATS/ERS/JRS/ALAT Clinical Practice Guideline 2018,The challenge in histopathology to make an accurate diagnosis is obtaining a sample of sufficient size. Transbronchial biopsies (TBB) are mostly insufficient, except in a few circumstances , and therefore surgical lung biopsy (SLB) is required. This carries a significant mortality risk of 3.5% , which is lower in the elective setting but higher in a deteriorating patient or one with co‐morbidities.As a consequence of the morbidity and mortality associated with SLBs, there has been growing interest in transbronchial lung cryobiopsy (TBLC) which appears to provide a good yield of tissue with a more acceptable risk (mainly from bleeding), although performance may be less acceptable in non‐expert centres. In a recent study where paired TBLC and SLB were taken in the same patient and read by a blinded expert pathologist, the TBLC and SLB were poorly concordant, with only 38% agreement (95%CI: 18‐62%) and the SLB carrying more weight in making the final diagnosis. It is not quite clear as to where TBLC fits in the investigation of patients with interstitial disease,Early work on the use of genomic classifier or gene expression signatures on multiple TBB samples from a patient can increase the diagnostic yield in differentiating UIP from non‐UIP better than TBB alone.Diagnosis of patients with connective tissue disease ILD (CTD‐ILD) is often challenging, but in the presence of interstitial lung disease, it is important to differentiate CTD‐ILD from idiopathic forms or from IPAF as it has implications for prognosis and treatment, with CTD‐ILD generally having a better prognosis than patients with IIP.Detection of specific autoantibodies in serum plays a key role in the diagnosis of CTD‐ILD, but there remains a paucity of clinical evidence on which of them can guide routine clinical practice. Many guidelines recommended testing for a narrow panel of autoantibodies only, with additional testing performed according to relevant clinical signs and symptoms suggestive of an underlying CTD.Further to the diagnosis of CTD‐ILD, autoantibody screening plays a role in the newer entity IPAF where patients have ILD and features suggestive of an underlying autoimmune process, but yet do not meet the defined diagnostic criteria for CTD. Serology is the key to the definition, along with clinical and morphological findings Table . Given tA significant problem remains the lack of standardisation of laboratory testing. For example, assessment of ANA testing shows significant variability in reported levels for identical samples both within and between laboratories.Exclusion of ANCA antibodies may exclude a large group of potential IPAF patients. In a Japanese study of ILD patients, 26 patients were identified with MPO‐ANCA of whom 16 were positive at diagnosis and 10 seroconverted to positive during follow‐up. Of these patients, only 9 35%) progressed to develop an ANCA‐positive vasculitis over a five‐year observation period .% progresMUC5B, which encodes a member of the mucin family of proteins; TOLLIP, which encodes a toll‐inhibiting protein which inhibits toll receptor signalling; telomere‐related genes, of which TERT encoding telomere reverse transcriptase is the most common; and SP‐C, encoding hydrophobic surfactant protein C essential for lung function and homoeostasis. Genetic studies are an important adjunct to clinical assessment in a number of areas of medicine including in neurodegenerative diseases, where they aid diagnosis, and in oncology, where they may influence the choice of immunotherapy. Genetic studies in interstitial lung diseases have mainly been limited to familial pulmonary fibrosis (FPF) patients and patients with the sporadic form with IPF.The treatment landscape in ILD has changed dramatically in the last decade with available anti‐fibrotics, and whilst a cure remains elusive, meaningful gains in longevity have been achieved.et al. propose an ABCDE approach with Assessment of patients’ needs and values; Backing with education; Co‐morbidities and comfort care; Disease‐modifying treatment and End‐of‐life care . An alternate approach to choosing treatment has been proposed where a patient’s disease is classified by clinical phenotype which captures the rate of progression,Trials of anti‐fibrotics arose from basic research in animal models demonstrating the underlying fibrotic pathogenesis including an appreciation of aberrant wound healing and deposition of collagen in the extracellular matrix as key features. Presently, there are only two licensed drugs in the treatment of IPF: pirfenidone and nintedanib. Pirfenidone (5‐methyl‐1‐phenyl‐2‐[1H]‐pyridone) is a synthetic oral drug shown to inhibit collagen synthesis and fibroblastic proliferation in animal models through regulation of transforming growth factor (TGF) β and tumor necrosis factor (TNF) α.In Australia, patients can access either of these drugs provided they have an MDM diagnosis of IPF, forced expiratory volume in 1 second (FEV1)/forced vital capacity (FVC) ≥ 0.7, FVC ≥ 50% and diffusing capacity of carbon monoxide (DLCO) ≥ 30% and, importantly, must not have interstitial lung disease because of other known causes including domestic and occupational environmental exposures, connective tissue disease or drug toxicity.The CAPACITY trials were two concurrent multinational randomised control trials assessing the efficacy of pirfenidone compared to placebo over 72 weeks in patients aged 40–80 years with an ILD MDT diagnosis of IPF. Inclusion criteria included predicted FVC ≥ 50% (but ≤ 90%), predicted DLCO of ≥ 35% and 6‐min walk test (6MWT) distance of at least 150 m. The primary end point of lower percentage decline in FVC was significantly favorable in the pirfenidone group.P = 0.005), but this was not replicated in INPULSIS‐1. Diarrhoea was the most common adverse event with a high prevalence (rate of 62.4%) but only accounting for complete discontinuation in just under 5% of patients.INPULSIS‐1 and −2 were, again, two concurrent multinational double‐blind randomised controlled trials conducted over 52 weeks comparing nintedanib with placebo. Inclusion criteria were patients aged ≥ 40 years with a diagnosis of IPF (based on radiology expert opinion) established within five years before randomisation, FVC ≥ 50% predicted and DLCO of 30‐79% predicted. Like pirfenidone patients, this group demonstrated a statistically significant adjusted annual rate of change in FVC of −114.7 mL with nintedanib versus −239.9 mL with placebo.Post hoc analysis in both pirfenidone and nintedanib trials demonstrated that the rate of decline regardless of predicted FVC starting point  is similar across all groups prompting health care professionals to think about introducing anti‐fibrotic therapy sooner and perhaps before a patient is symptomatic. This clearly requires further evaluation with appropriate cost‐benefit analysis. To date, there have been no head‐to‐head studies between anti‐fibrotic therapies and so presently choice of anti‐fibrotic lies with the physician and the patient. From trial data, we have derived certain risk criteria – patients with intended high sun exposure would favor nintedanib because of photosensitivity with pirfenidone and patients on anti‐coagulants favor pirfenidone because of increased bleeding risk with nintedanib for example. Other contributing factors include pill burden, history of ischaemic heart disease and gastro‐oesophageal reflux disease to name a few.Prednisolone has been commercially available from the mid‐50s and has been the mainstay for the management of many immune or inflammatory conditions. It is potent, very broad in action and unfortunately associated with many side effects. Historically, it alone or in combination with azathioprine was the treatment given for IPF and many other forms of ILD. It was not until relatively recently definitive evidence became available demonstrating the potential harm of immunosuppression in IPF. The azathioprine, prednisolone and N‐acetylcysteine arm of the PANTHER‐IPF trial was discontinued early because of an increased risk of hospitalisation and death compared to the placebo and N‐acetylcysteine alone arms.In contrast to IPF, immunosuppression is still first‐line non‐licensed therapy in CTD‐ILD. CTD‐ILD patients are a heterogenous group with a wide spectrum of autoimmune conditions including systemic sclerosis (SSc), rheumatoid arthritis (RA), inflammatory myopathies and primary Sjogren’s syndrome. They remain a significant treatment challenge because of their differing (and sometimes multiple) patterns of fibrotic disease, variable rates of progression and limited available clinical data on best time to intervene. Many CTD‐ILD patients can remain stable for years whilst not on therapy but conversely respiratory failure remains a leading cause of death most notably in the SSc cohort and second in RA.Significant variability regarding which immune‐modulating agent, dose and treatment duration exists across the board because of lack of available consensus guidelines. To date, there has been no evidence of superiority amongst the use of azathioprine, mycophenolate, cyclophosphamide or cyclosporine in CTD‐ILD. Choosing immunosuppressant therapy is therefore still merited on an individual basis. In clinical practice, most are used as additional therapy to glucocorticoids to enable the lowest clinically effective dose of steroid .P < 0.05). Concerns regarding side effects and tolerability prompted the SLS II trial to directly compare cyclophosphamide (for 12 months followed by 12 months placebo) with mycophenolate mofetil (for 24 months), with both drugs demonstrating similar improvements in FVC but significantly fewer side effects and lower dropout rates in the mycophenolate group despite the extended duration of use.By far, the most studied CTD‐ILD population is the SSc group with data extrapolated to guide treatment of other CTD‐ILD. In the Scleroderma Lung Study (SLS 1), 158 patients with SSc‐ILD were randomised to receive cyclophosphamide versus placebo.1 in nintedanib versus −93.3 mL year1 in placebo, P = 0.04) was reported in the nintedanib group, there was a high proportion of reported adverse events of which diarrhoea was most common (75.7% in nintedanib group and 31% in placebo respectively). Interestingly, almost 50% of patients were receiving mycophenolate prior to enrolment, challenging discussion as to whether the combination of anti‐fibrotic and immunosuppressant therapy is of additional benefit in this cohort.Announced earlier this year were the results of the much anticipated SENSCIS trial, a randomised placebo‐controlled trial, which assessed nintedanib versus placebo in SSc‐ILD over 52 weeks.With ever evolving understanding of the innate fibrotic processes across the various ILD subtypes, changes in treatment will inevitably follow. Such an approach is the focus of a current placebo‐controlled trial entitled Progressive Fibrosing‐ILD (INBUILD study), comparing nintedanib with placebo. This trial excludes patients with IPF but will include patients with UIP‐like pattern (such as in CTD) or non‐UIP pattern .New agents targeting the various stages in the pathogenesis Figure  of diseaSeveral trials are currently underway exploring combination therapy with anti‐fibrotics and immunosuppression in progressive fibrotic disease of any aetiology, with particular focus on the CTD‐ILD cohort. SLS III is currently recruiting SSc‐ILD patients to trial combination therapy with mycophenolate and pirfenidone. Other forthcoming trials include TRAIL1: Phase ll Study of pirfenidone in Patients with RA‐ILD (NCT02808871) and, as discussed, RECITAL: Rituximab versus Cyclophosphamide in Connective Tissue Disease‐ILD (NCT01862926). A favorable outcome in any or all of these trials has significant potential to change practice relating to anti‐fibrotic use outside the IPF cohort.Comprehensive clinical, morphological and serological assessment by a multidisciplinary expert team in a combined meeting is now the standard of care expected for all patients with ILD requiring monitoring or treatment. The team should be up‐to‐date with evolving criteria for diagnosis and new investigations, but ultimately patients are categorised according to their clinical phenotype, using ratings of strength of evidence to support a diagnosis. These can guide the need for further testing and increase confidence of diagnosis. Genotyping demonstrates great heterogeneity in clinical patterns and at this stage is not helpful other than the finding of short telomeres which indicate greater complications post‐transplantation.Broadly, ILD treatment can be divided into four domains: (1) observation and monitoring; (2) disease‐modifying therapies such as anti‐fibrotics, immunosuppression and lung transplantation; (3) management of co‐morbidity including gastro‐oesophageal reflux, cough and mental health; and (4) non‐pharmacological strategies such as pulmonary rehab, oxygen and education. Palliative care discussion should be encouraged at any stage in the disease focusing on both symptom control and, when needed, end‐of‐life care. The anti‐fibrotic era has been exciting, proving that rate of disease progression can be slowed and exacerbations can be reduced, although their use is currently limited to the IPF cohort.The question remains as to whether we should be characterising disease, and therefore choosing treatment, based on the clinical phenotype or the course of the disease (progressive vs non‐progressive) or the stage of the disease . The question of targeting process or disease pattern instead of specific disease entities is already being challengedIM has received funding to attend an educational meeting from Boerigher Ingelheim, the manufacturers of nintenadib. AMT and FL declare no conflicts of interest.