To describe the utilisation of antibiotics in children and adolescents across 5 European countries based on the same drug utilisation measures and age groups. Special attention was given to age-group-specific distributions of antibiotic subgroups, since comparison in this regard between countries is lacking so far.Outpatient paediatric prescriptions of systemic antibiotics during the years 2005-2008 were analysed using health care databases from the UK, the Netherlands, Denmark, Italy and Germany. Annual antibiotic prescription rates per 1,000 person years were estimated for each database and stratified by age . Age-group-specific distributions of antibiotic subgroups were calculated for 2008.With 957 prescriptions per 1000 person years, the highest annual prescription rate in the year 2008 was found in the Italian region Emilia Romagna followed by Germany (561), the UK (555), Denmark (481) and the Netherlands (294). Seasonal peaks during winter months were most pronounced in countries with high utilisation. Age-group-specific use varied substantially between countries with regard to total prescribing and distributions of antibiotic subgroups. However, prescription rates were highest among children in the age group ≤4 years in all countries, predominantly due to high use of broad spectrum penicillins.Strong increases of antibiotic prescriptions in winter months in high utilising countries most likely result from frequent antibiotic treatment of mostly viral infections. This and strong variations of overall and age-group-specific distributions of antibiotic subgroups across countries, suggests that antibiotics are inappropriately used to a large extent. Antibiotics are among the most widely prescribed medications in Europe . ResistaSeveral studies have been published in the last decade either assessing antibiotic use in paediatric populations of single European countries -15 or coThe aim of the present study was to compare outpatient prescribing of systemic antibiotics to children and adolescents in the age group 0-18 years between Denmark, Italy, Germany, the Netherlands and the UK for the years 2005-2008, based on a standardised protocol for data extraction and analysis for each database in these countries. Special attention was paid to differences of age-group-specific use of different antibiotic subgroups across countries. Seasonal variations of prescribing rates were described to assess impact of antibiotic treatment of mostly viral respiratory infections during winter months on total use.Data were retrieved from one general practice database , UK), one outpatient pharmacy dispensing database  and three claims databases , Germany; Emilia Romagna regional database, Italy). These electronic healthcare databases cover a total source population of about 23 million persons. All databases are in compliance with European Union guidelines on the usage of medical data for research. The study was given approval by regulatory agencies or by scientific and ethical advisory boards of the databases where applicable. All five databases comprise medical information of a defined population. Detailed descriptions of these databases including specifics regarding approvals for use of data for this study are enclosed as Additional file st 2005 or – if later - the first date a person entered into the respective database. Cohort exit was defined as exit of the person from the database, 18th birthday, death, the first interruption of follow-up in the database or December 31st 2008, whichever came first.The study was conducted in an open (dynamic) cohort design. The study period was from January 2005 to December 2008, since for some databases no more recent data was available at the time of the analysis. The observational period of the Italian Emilia Romagna Database was restricted to the years 2007 and 2008, since data of the years 2005 and 2006 were not available. Cohort start was defined as January 1Over the follow-up period, members of the study population could contribute to more than one age category. Children and adolescents up to the age of 18 years were included and divided into the age groups ≤4, 5-9, 10-14, and 15-18 years. This age group classification was chosen, since it was commonly used in other studies of antibiotic utilisation in the paediatric setting and hence allows comparison of age-group-specific use across studies ,15,19,20Utilisation of systemic antibiotics  code: J01) was measured as the annual prescription rate, i.e. the number of prescriptions divided by 1,000 person years. Person years rather than individuals were used as denominator, given that not all children could be followed for an entire year. Prescription rates were chosen as a main outcome measure instead of Defined Daily Doses (DDDs) per person time, since dosing of antibiotics depends on a patient’s age and body weight. Prescription rates are therefore more appropriate to describe antibiotic use among children and conduct comparison between children in different age groups than DDDs per person time . SeasonaOutpatient prescriptions of systemic antibiotics were divided into the following subgroups (ATC codes in brackets): Tetracyclines (J01AA), broad spectrum penicillins , narrow spectrum penicillins , second generation cephalosporins (J01DC), third generation cephalosporins (J01DD), sulphonamides/trimethoprim , macrolides (J01FA) and nitrofuran derivatives (J01XE). Less frequent antibiotics were pooled in the subgroup ‘others’.To describe differences in the distribution of antibiotic subgroups between countries, age-group-specific proportions of antibiotic subgroups were calculated for each database in the year 2008 based on the respective total number of systemic antibiotic prescriptions per age group.Local data extraction was conducted by using standardised purpose-built Jerboa® software, which was previously developed by the Erasmus University Medical Center and tested against different scripts . MeasureThe average annual total population comprised 334,991 children from Denmark, 773,492 children from the Italian region Emilia Romagna, 1,340,163 children from Germany, 622,450 children from the Netherlands and 798,253 children from the UK.With 957.2 prescriptions per 1,000 person years, the highest annual prescription rate in the year 2008 was found in Emilia Romagna  followed by Germany (560.8), UK (555.2), Denmark (481.0) and the Netherlands (294.2). This ranking did not change over the entire observational period, with the restriction, that data from Italy was only available for the years 2007 and 2008   and SwedHigh antibiotic prescribing in the Italian outpatient setting compared to the other countries in our study might be related to differences with regard to historical backgrounds, cultural and social factors, awareness about antibiotic resistance in the community and among healthcare providers  as well In contrast, several previous studies showed antibiotic utilisation in the Netherlands to be lowest in Europe, overall and in the paediatric setting ,13. The Although antibiotic use was by far the highest among Italian children and adolescents, antibiotic prescription rates in Denmark, Germany and UK still exceeded those in the Netherlands to a great extent. These observed strong variations of total paediatric antibiotic use among the countries of study are unlikely to reflect an actual therapeutic need which would have to be based on marked differences in the burden of infectious diseases between these countries. This assumption is also supported by the observed pronounced increases of prescription rates during winter months which were expectedly highest in Italy, and smallest in the Netherlands. Increases of antibiotic use are most likely related to seasonal rise of predominantly viral respiratory infections and hence should be limited [1].Since our findings could not provide information beyond the 4-year study period, we compared our prescription rates with those of other studies which included other study years or longer time periods. In this respect, our data for the years 2005-2008 in the Netherlands agreed well with the findings by de Jong et al. who reported a variation of the total annual number of antibiotic prescriptions between 282 and 307 per 1,000 Dutch children in the years 1999-2005 . This suWe also detected remarkable differences in the choice of antibiotic subgroups between the countries of our study. Narrow spectrum penicillins formed the majority of systemic antibiotics in Denmark, whereas prescriptions of broad spectrum penicillins were most frequent in the four other countries. In line with that, the highest agent-specific prescription rates were reported for phenoxymethylpenicillin in Denmark, amoxicillin in Germany, the Netherlands and the UK and amoxicillin plus enzyme inhibitor in Italy. Relatively high use of narrow spectrum penicillins in Denmark in comparison to other European countries has also been reported previously . HoweverMacrolides were commonly prescribed in all five countries with the highest use in the age groups 10-14 and 15-18 years. Relative proportions of macrolide use were lowest in Denmark. This finding is in agreement with a Danish practice guideline which recommends restricting the use of macrolides to patients with penicillin allergies in the treatment of common childhood infections . SeveralOur findings regarding paediatric cephalosporin use are in line with previous studies which reported strong variations of cephalosporin prescribing across Europe, with the lowest prescription rates in the Netherlands and Denmark ,18-20,35Our study overcomes limitations of previous studies and facilitates the comparison of paediatric antibiotic prescriptions in five countries based on a common protocol using the same drug utilisation measures. It provides insight into the age-group-specific distributions of antibiotic subgroups in the paediatric setting of the participating countries. Ascertainment of antibiotics prescribed in the outpatient setting was complete in all databases except Denmark, where some antibiotics as e.g. cephalosporins are reimbursable only in particular circumstances and might therefore have been underascertained. Nevertheless, given that the Danish National Health System reimburses antibiotics for the entire spectrum of childhood indications,  the propOur study has some limitations, which have to be taken into consideration. First, for this study only data for the years 2007 and 2008 was available from the Northern Italian region Emilia Romagna. Hence, insight into the development of antibiotic prescribing over time is limited. However, our findings are in good agreement with Gagliotti et al.  In additSince all five databases only provide information on drugs prescribed in the outpatient setting, antibiotics administered to inpatients to treat severe childhood infections could not be studied. Given that indications underlying the issued prescriptions were not available in all databases, the appropriateness of single treatment courses could not be assessed. Additionally, compliance with the antibiotic prescription remains unknown.Comparison of paediatric antibiotic consumption between different European countries revealed a wide variability of antibiotic prescribing patterns. Strong variations of overall and age-group-specific distributions of antibiotic subgroups across countries, suggests that antibiotics are inappropriately used to a large extent. Considerably higher prescription rates along with higher seasonal increases, particularly in Italy, in contrast to the Netherlands suggest frequent utilisation of antibiotics in the treatment of mostly viral respiratory infections. This study showed the benefit of using a common methodological approach to provide comparable and detailed data on paediatric antibiotic prescribing across Europe. Study results allow health care practitioners and policy makers to audit country and age-group-specific patterns of paediatric antibiotic use with regard to both total level of prescribing and the distribution of antibiotic subgroups/substances.ATC: Anatomical therapeutic chemical classification system; DDD: Defined daily dose; GePaRD: German Pharmacoepidemiological Research Database; GP: General practitioner/family physician; GPRD: General Practice Research Database; THIN: The Health Improvement Network; RRE: Remote research environment; SHI: Statutory Health Insurance.JH, TS, GM, FI, AO, EP, AP, SPU and GT declare that they have no competing interest. IB has received grants from several pharmaceutical companies and funding organizations in the previous 3 years. MCS has received grants from Pfizer, grants from Boehringer, grants from Novartis and grants from Eli Lilly in the previous 3 years. MM has received grants from the International Serious Adverse Events Consortium, i SAEC (collaboration of academia and industry) in the previous 3 years. EG is running a department that occasionally performs studies for pharmaceutical industries. These companies include Bayer, Celgene, GlaxoSmithKline, Mundipharma, Novartis, Sanofi-Aventis, Sanofi Pasteur MDS, and STADA. EG has been a consultant to Bayer-Schering, Nycomed, GlaxoSmithKline, Teva and Novartis. EG is a member of the German Standing Vaccination Committee .JH conceived and designed the study, conducted data analysis, drafted the article and had final approval. TS helped acquire the data and interpret the results and revise the article for content, and gave final approval. MM was involved in conception and design of the Study, made revisions to article drafts, and gave final approval for Publication. GM was involved with data acquisition and conception of the study, revised the article, and gave final approval for publication. FI helped design the study, helped with data acquisition and interpretation, and made revisions to article drafts, and gave final approval for publication. AO helped acquire the data and interpret the results and revise the article for content, and gave final approval. IB was involved with data acquisition, helped revise the article, and gave final approval for publication. EP helped acquire the data and interpret the results, and made revisions to article drafts, and gave final approval. AP was involved with data acquisition, helped revise the article, and gave final approval for publication. SPU helped acquire the data and interpret the results, and made revisions to article drafts, and gave final approval. MCS was involved in conception and design of the study, was involved with data acquisition and interpretation, helped revise the article, and gave final approval for publication. GT was involved in conception and design of the study, was involved with data acquisition and interpretation, helped revise the article, and gave final approval for publication. EG helped design the study, and acquire and interpret the data, supervised the publication and made substantial revisions to the article drafts, and gave final approval for publication. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2431/14/174/prepubHealthcare databases. Additional file 1 provides a description of relevant characteristics of included healthcare databases.Click here for file


Journal of Hematology & Oncology is pleased to see the exponential growth in the number of publications from China, especially in hematology and oncology. This editorial calls for putting more weight on the quality of the future scientific output and invites rigorous dialogs among policy makers, granting agencies, academic leaders, physicians, and scientists, followed by concrete actions, to achieve such a goal. The number of scientific publications from China has exploded in the past 10 years. Multiple factors have propelled this exponential growth. Among these factors include but are not limited to a large scientific research base, increases in government funding through Natural Science Foundation of China, acute upholding of the publish-or-perish mantra, monetary reward for publications from local institutions, institutional requirement for publications in quality journals for awarding doctoral degrees, and faculty promotions. Dr. An-mei Deng’s group from Shanghai performed a detailed and focused analysis of hematology research output from China in the past 10 years [China has succeeded in quantitative promotion of scientific research . The nex


In a recent article , cogniti There are distinct, though interconnected, questions surrounding the topic of incest  play a role in moral judgments about incest among the Malagasy, given that there is a widespread belief that committing incest leads to catastrophic consequences  even when incest is unintentional .2In a recent article , cognitiAstuti and Bloch are leading researchers in the area of cognition and culture. In bringing the question of intentionality attributions into the discussion of moral judgments concerning incest, their article speaks to traditional issues in legal and cultural anthropology e.g., , to attrAstuti and Bloch make two sorts of claims. At a more descriptive level, they claim that the Malagasy demonstrate an unusual ambivalence about whether the distinction between intentional and unintentional actions affects their moral judgments concerning incest and its catastrophic consequences. On the one hand, similarly to the way they evaluate other transgressions, the Malagasy consider this distinction to be relevant: for example, a couple is not considered to be “at fault” if they committed the incest unknowingly . On the other hand, when the Malagasy think about and deal with incest’s catastrophic consequences, the distinction is no longer relevant: everyone, including the innocent , is “punished” by these consequences and has a duty of reparation. According to Astuti and Bloch, the irrelevance of intentionality when the Malagasy think about incest’s catastrophic consequences is unusual because it goes against the widely held assumption that considerations of intentionality are a universal component of causal reasoning about wrongdoing.At a more explanatory level, In this article, we critically examine Astuti and Bloch’s claims. Much of our criticism comes from the fact that Astuti and Bloch are not clear enough about the various types of moral judgments invoked in their discussion. Thus, firstly, we provide a characterization of these different types of moral judgments and of our view of the relevance of intentionality considerations in each case. Secondly, we focus on Astuti and Bloch’s claim that the Malagasy demonstrate an unusual ambivalence in their moral judgments concerning incest and its catastrophic consequences, arguing that they do not provide clear evidence for this claim. Thirdly, we focus on Astuti and Bloch’s theoretical explanation, arguing that it lacks coherence and is not supported by beliefs about the incest taboo even in other cultural contexts in Madagascar. Moreover, we argue that their appeal to the notion of dumbfounding is unhelpful, and supports neither their explanation nor its generalization to the US context.Throughout their paper, Astuti and Bloch’s frame their discussion in terms of an inquiry related to moral judgments of wrongdoing, and in the context of what they take to be a widespread assumption that considerations of intentionality are relevant to these judgments. However, there are different moral judgments at stake in their discussion: judgments related to culpability, punishment and duty of reparation, as well as judgments of wrongdoing strictly speaking. Moreover, considerations of intentionality may not play the same role across these different judgments. In order to discuss the import of Astuti and Bloch’s claims about the relevance or irrelevance of intentionality considerations in the case of incest, we provide a brief characterization of how these moral judgments differ from each other and of the extent to which considerations of intentionality are relevant to each of them.3 This judgment is often unrelated to considerations of intentionality, since ordinary norms forbidding an action may simply specify that the action is forbidden rather than specify that the action as intentional action is forbidden. In other words, the irrelevance of whether the forbidden action is intentional or not is already built into the understanding of the norm itself. For example, suppose that destroying someone else’s property is deemed forbidden , and take the case of a person who hits someone else’s vase, breaking it. Given this norm, people will judge that the person did something wrong—breaking the vase is a transgression of the norm, whether the person did it intentionally or unintentionally.A judgment of wrongdoing, strictly speaking, corresponds simply to the judgment that an action is a normative transgression, that is, that an action has been performed when there is a norm forbidding the action  and that one of the variables that calibrates the amount of culpability is related to considerations of intentionality—if, in the vase example, the person hitting the vase was seen as reckless, people would attribute at least some culpability.While a judgment of wrongdoing is the evaluation of an d caused .4 A judga more serious punishment might be dispensed ” .7 for having committed incest because they could not have known that they were siblings. Moreover, they seem to acknowledge this explicitly:Astuti and Bloch seem to accept that when making judgments about the culpability of a couple for committing incest, the Malagasy take intentionality into account. They presented the Malagasy with a story about two adult siblings who had sex, but who, having been completely separated at birth, did not know they were related. They report that the Malagasy judge that the siblings are not “at fault” loza [incest’s catastrophic consequences].  we have also shown that considerations of intentionality are present in the way people access culpability even in the case of incest , p. 5.loza), where they argue that intentionality is not taken into account. Indeed, Astuti and Bloch indicate throughout their article that the principal evidence concerning the irrelevance of intentionality comes from three beliefs (and related behaviors) concerning incest’s catastrophic consequences:As the first passage above also indicates, Astuti and Bloch contrast judgments of culpability with the case of the Malagasy’s moral judgments concerning incest’s catastrophic consequences ((i)the belief that incest leads to catastrophic consequences, whether the incest was intentional or not;(ii)the belief that the catastrophic consequences affect everyone , including the innocent;(iii)the belief that everyone in the community, including the innocent, has a duty to repair the damages that may follow a case of incest.We question whether Astuti and Bloch discussion of these three beliefs and related behaviors provide sufficient evidence that the Malagasy are ambivalent about intentionality in any unusual way.moral judgments concerning incest and its catastrophic consequences, and the claim that intentionality considerations are seen by the Malagasy as irrelevant to more circumscribed causal judgments concerning the relation between an act of incest and catastrophic consequences. The content of the first belief  does confirm the claim that intentionality considerations are seen by the Malagasy as irrelevant to causal judgments concerning the act of incest and catastrophic consequences, but it does not provide any direct evidence for the claim that intentionality considerations are seen by the Malagasy as irrelevant to moral judgments.The content of the first belief does not say anything direct about moral judgments—it just states that an act of incest causes catastrophic consequences regardless of intentionality. Throughout their paper, Astuti and Bloch frame their discussion in terms of an inquiry on “causal reasoning” related to wrongdoing, but appear to be conflating two types of claims—the claim that intentionality considerations  are seen by the Malagasy as irrelevant to We shall leave aside Astuti and Bloch claim that intentionality considerations are irrelevant to causal judgments concerning the act of incest and catastrophic consequences, since this claim is secondary to the topic of this paper, which is the relation between intentionality considerations and moral judgments. Moreover, there is nothing unusual in the fact that the Malagasy do not take into account intentionality in making causal judgments about the relation between the act of incest and catastrophic consequences—what may be unusual here is the belief in a causal link between incest and catastrophic consequences itself, rather than the belief that this causal link exists regardless of intentionality. One can see this point more clearly by noticing that, in general, intentionality considerations are not fundamental to causal judgments concerning the relation between actions and their consequences. Take the case of someone shooting a gun with the aim of killing a person and causing the death of the person, and the case of someone shooting a gun with the aim of breaking a bottle but accidentally causing the death of a person. We imagine people in general would say that in both cases the action of shooting the gun caused the death of a person, regardless of the mental states involved  is still in force and the action  is a transgression of the norm. Although Astuti and Bloch often frame their discussion in terms of judgments of wrongdoing, they do not discuss at all the relation between judgments of wrongdoing qua normative transgression and considerations of intentionality. So, there is nothing clear in their article suggesting that they would like to claim that the Malagasy do not take into account intentionality in making moral judgments of wrongdoing qua normative transgression. More importantly, given that judgments of wrongdoing are often unrelated to considerations of intentionality (as we claimed in the previous section), it would not be in any way unusual for considerations of intentionality to be irrelevant to the Malagasy’s judgments that incest is wrong.punishment, and the content of third belief (“everyone has a duty to repair the damages”) does refer to a moral judgment concerning duty of reparation. We focus on this evidence.Astuti and Bloch describe the content of the second belief above in two different ways: in terms of “catastrophic consequences affecting everyone” or in terms of “punishment befalling everyone.” The first description does not say anything about moral judgments—it just states that the Malagasy think that incest’s catastrophic consequences affect everyone. Therefore, it cannot be evidence for a claim about the irrelevance of intentionality considerations to moral judgments either. The second description  does indicate something about a moral judgment concerning The relevant content of the second belief relates to punishment. Their somewhat implicit argument for claiming that this content would indicate the irrelevance of intentionality considerations to liability judgments is this: if the Malagasy believe that punishment falls on everyone, including the innocent , then intentionality judgments are irrelevant to liability judgments concerning punishment.This argument hinges on the assumption that the Malagasy conceptualize the catastrophic consequences in terms of punishment. However, it is unclear whether Astuti and Bloch’s description of the content of the second belief in terms of punishment (instead of simply in terms of causation) is a faithful translation of the way the Malagasy themselves conceptualize the catastrophic consequences, or whether it is a theory-laden redescription by the anthropologists.The cross-cultural evidence concerning beliefs about a causal relation between incest transgressions and apparently unrelated harmful consequences such as crops failing suggests that this counter-intuitive casual relation may be understood in three distinct ways cf. . The firNow, when Astuti and Bloch mention how the Malagasy discuss incest and its catastrophic consequences, they do not mention anything about the Malagasy discussing the catastrophic consequences in terms of punishment, involving supernatural agency or otherwise. Instead they emphasize an intrinsic mechanistic link between incest and the catastrophic consequences that is present even in the semantics of the verb used to describe an act of incest:loza. The dictionary definition of this term is “calamity” or “disaster”; the verb for committing incest (mandoza) thus literally translates as causing a calamity or disaster  is part of the semantics of the verb counts against any punishment interpretation, and favors the intrinsic-natural-consequence hypothesis. To make a pertinent analogy, to interpret “loza” in terms of punishment would be like saying that the feature death, qua a semantic component of the lexical causative “to kill” (to cause death), can be interpreted as punishment for the behavior that lead to the death. In other words, if the feature catastrophic consequences (“loza”) is seen as an intrinsic component of incest as an action (“mandoza”), it cannot be seen as punishment for a component of the action.8Indeed, the fact that the verb used to describe incest works like a lexical causative in which the feature equally. It may be that, in cases of intentional incest, the Malagasy have intuitions that the catastrophic consequences qua punishment will affect those who committed incest (or those closely associated to them) more than the rest. More importantly, it may be that the Malagasy have intuitions that the catastrophic consequences qua punishment will be more extreme when incest is committed intentionally rather than unintentionally. No evidence concerning these points is provided in Astuti and Bloch’s article.Of course, it is still possible that the Malagasy have different interpretations of the relation between incest and its catastrophic consequences, one of which may be in terms of these consequences being punishment for incest, given that beliefs about a counter-intuitive causal relation between an act of incest and catastrophic consequences constitute a typical reflective belief with a semi-propositional content that is susceptible to various interpretations by the believers themselves , 1997. HLet’s turn to the content of the third belief—everyone in the community has a duty to repair the damages that may follow a case of incest. Astuti and Bloch seem to appeal to a similar argument to demonstrate the irrelevance of intentionality considerations to judgments to liability: if the Malagasy believe that everyone has a duty to make reparation, even the innocent , then intentionality judgments are irrelevant to liability judgments.Again, it is not clear to us whether the description of the content of this belief in terms of a duty of reparation is a faithful translation of the way the Malagasy themselves conceptualize the issue, or is a theory-laden redescription by the anthropologists. Alternatively, and consistent with the alternative hypothesis we put forward above about the Malagasy’s understanding of the catastrophic consequences in terms of intrinsic natural consequences, this belief may indicate simply a duty of mutual help in situations of natural catastrophes that have occurred or are envisaged to occur. Just think about the occurrence of natural disasters like a hurricane in a Western cultural context , where people in the country feel the duty to help those afflicted by the catastrophe; or think about the mutual help involved in preventing the damages of a potential natural disaster.equal duty of reparation. It may be that, in cases of intentional incest, the Malagasy believe those who committed incest (or those closely associated to them) should provide greater reparations than the rest for the disasters that have occurred or may be envisaged to occur. More importantly, it may be that the Malagasy have intuitions that the duty to repair would be more extreme in relation to those who commit incest intentionally than to those who commit incest unintentionally. No evidence concerning these issues is provided by Astuti and Bloch’s article. In fact, Astuti and Bloch do not provide any detailed evidence at all about what is involved in Malagasy’s reparation behavior concerning incest. The most they say is that “… a large number of innocent people are responsible for undertaking the difficult  ritual work that is required to repair the damage and put things right again”  is not without problems. It seems to be inconsistent with Astuti and Bloch’s own suggestion that the Malagasy understand the catastrophic consequences in terms of punishment for incest (see previous section), as it would entail that the Malagasy understand incest’s catastrophic consequences both as punishment, which supports the social order, and as the destruction of the social order. Moreover, as with most symbolist hypotheses a la Durkheim, this hypothesis explains neither why the Malagasy would use such an indirect mode of expression , nor the fact that Malagasy’s behaviors seem compatible with an interpretation of loza simply in terms of intrinsic catastrophic consequences. Finally, the usual move from those who adopt a symbolist hypothesis — claiming that the symbolism is esoteric  or unconscious  — would create more problems than solve them , it is worth pointing out that their explanation is not consistent with other cultural traditions in Madagascar. asy) that can be turned by sacrifice into good or bad. Incest can easily render cattle sterile, cause women to bear ‘creatures’ (biby), harvests to fail, and people to die; but, if handled well, it can make people masine . Its power lies in its essential indeterminacy .A final problem with Astuti and Bloch’s explanation is its incorporation of a notion of dumbfounding, which, in general terms, refers to a person being because the Malagasy are dumbfounded by the attack on the transcendental social order that incest represents, but without providing any clear characterization of the connection between being dumbfounded and neglecting considerations of intentionality. They say:One issue is that Astuti and Bloch claim that considerations of intentionality become irrelevant loza) (“(…) the consequences of incest are indeed understood as catastrophic (…). And yet, when asked why this is so, our Malagasy interlocutors are stumped—or dumbfounded, to use a term used in the psychological literature on moral reasoning (…) they are unable to give a single and sufficient account of the relationship between cause (the breach of the taboo) and effect (loza) , p. 3.However, it is unclear to us why being unable to give an account of the relationship between incest and its catastrophic consequences should impact the relevance of intentionality considerations. It is also unclear to us why Astuti and Bloch need to posit that this dumfounding is the result of the perception of a threat to the transcendental social order. We would argue instead that the Malagasy’s inability to provide a detailed causal account of the relationship between incest and its catastrophic consequences corresponds simply to the well-known fact that people normally do not have plain and systematic ideas about how counter-intuitive causation works.wrongdoing, in the sense of judgments that an action is a normative transgression; the research is not about asking people to provide explanations for any judgments of liability. It is also worth noting that Haidt’s research concerns only cases of intentional incest and is unrelated to the question about the relevance of considerations of intentionality.Another issue is that Astuti and Bloch make an unconvincing analogy between their take on dumbfounding and the notion of moral dumbfounding in the context of Jonathan Haidt’s research (see aforementioned references). “Moral dumbfounding” in the context of Haidt’s research refers to the fact that Americans seem unable to provide an adequate explanation for their judgment that intentional incest is still wrong once the usual Western objections to intentional incest are counteracted . Before explicating Astuti and Bloch’s analogy, it is important to emphasize that Haidt’s research is about people being unable to provide an adequate explanation for their judgments of Turning to the dumbfounding analogy, Astuti and Bloch claim, on the one hand, that, like the American students in Haidt’s research, the Malagasy would evince dumbfounding in response to questions about wrongdoing in the context of Haidt’s scenario of intentional incest. But, as Astuti and Bloch seem to acknowledge in passing, the Malagasy would not have any problem in explaining why intentional incest is wrong if confronted with Haidt’s scenario: incest causes catastrophic consequences even if the aforementioned Western objections were counteracted.post hoc rationalization. However, we find the rationale of their explanation even less persuasive here than in the case of the Malagasy: principles of kinship are much less socially important in individualist societies like the US, and there is no widespread belief in the US context that incest has catastrophic consequences.On the other hand, Astuti and Bloch also suggest that American students’ moral dumfounding can be similarly explained in terms of the perception of an attack on the transcendental social order: “Their dumbfoundedness signals that what they are thinking and care about is the need to align themselves, jointly with others, with what, ultimately and fundamentally, makes people, namely, the transcendental” , p. 6. TAstuti and Bloch are some of the few cognitive anthropologists who combine long-term fieldwork involving participant observation and other qualitative methods with the more controlled tasks deployed by experimental psychologists and cognitive scientists, and we commend them in their perspective and the important contributions they have made. In their recent paper, they claim that their fieldwork has uncovered an unusual pattern of reasoning when the Malagasy think about the catastrophic consequences that are supposed to follow from breaking the incest taboo, and propose an explanatory account that can both explain this finding and potentially provide an account of the moral dumbfounding evinced by American students when they are asked to explain why intentional incest is wrong.We have argued that their article falls short of providing evidence for their descriptive claims and that there are many aspects of their explanation that are unconvincing. We have also argued that many aspects of their central claims are unclear. One of our aims is to push them to address our concerns by developing a more detailed combined methodology and a more rigorous theoretical framework in relation to the current topic, as they have previously pursued in relation to other topics (see, e.g., The primary theoretical arguments of the article were jointly developed by PS and LS. PS took the lead both in terms of theoretical framing and writing, while LS contributed substantially to these aspects.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tumor-infiltrating lymphocytes (TILs) play a major role in anti-tumor immune responses, and their presence is correlated with survival in a variety of tumors. These TILs do not reach the pancreatic cancer (PC) cells in significant numbers due to the presence of stroma and a suppressive microenvironment. Neoadjuvant chemoradiation therapy (CRT) has been advocated as a potential way to improve outcomes of patients with resectable or borderline resectable PC. More importantly, there is recent evidence to suggest that CRT can increase the presence of TILs in the PC microenvironment (PCME), leading to production of interferon-γ (IFN-γ), which could increase the expression of PD-L1 through a negative feedback loop. Accordingly, we hypothesize that blocking the PD-1 receptor will synergize with CRT to increase the density and activation of TILs in the PCME.This is a prospective multicenter randomized trial which will accrue 45 subjects with resectable or borderline resectable pancreatic cancer who had not received prior treatment for PC and have ECOG of 0-1. The primary objectives of the study are: (1) to determine the safety of neoadjuvant CRT in combination with pembrolizumab. (2) To estimate the difference in the number of TILs in pancreatic cancer subjects receiving neoadjuvant CRT in combination with pembrolizumab to the number of TILs in subjects receiving neoadjuvant CRT alone. Eligible subjects will be randomized 2:1 to the investigational treatment (Arm A) to receive pembrolizumab administered IV every 3 weeks on days 1, 22, and 43 during concurrent CRT with capecitabine  through Friday (F), on days of radiation only) and radiation (50.4 Gy in 28 fractions over 28 days) or Arm B to receive only concurrent CRT with capecitabine. In all subjects, restaging CT scan or MRI will be performed at 4-6 weeks after completion of neoadjuvant treatment to determine resectability. Patients without local or distant disease progression will be taken to the operative room for planned surgery (within 2 weeks of imaging). Postoperatively, resected patients will receive off study standard of care adjuvant gemcitabine (1000mg/kg IV weekly for 3 out of 4 weeks for 6 months). Post operatively resected patients will be followed for up for PFS and OS for up to 2 years. Participants are considered evaluable if they satisfy all inclusion and exclusion criteria and have tumor tissue samples sufficient to determine the number of TILs. Clinical Trial Registry Number (NCT02305186).ClinicalTrials.gov identifier NCT02305186.


Synthetic lethality (SL) is a novel strategy for anticancer therapies, whereby mutations of two genes will kill a cell but mutation of a single gene will not. Therefore, a cancer-specific mutation combined with a drug-induced mutation, if they have SL interactions, will selectively kill cancer cells. While numerous SL interactions have been identified in yeast, only a few have beenknown in human. There is a pressing need to systematically discover and understand SL interactions specific to human cancer. In this paper, we present Syn-Lethality, the first integrative knowledge base of SL that is dedicated to human cancer. It integratesexperimentally discovered and verified human SL gene pairs into a network, associated with annotations of gene function, pathway, and molecular mechanisms. It also includes yeast SL genes from high-throughput screenings which are mapped to orthologous human genes. Such an integrative knowledge base, organized as a relational database with user interface for searching and network visualization, will greatly expedite the discovery of novel anticancer drug targets based on synthetic lethality interactions. The database can be downloaded as a stand-alone Java application. Finding effective anticancer therapies is a major goal of biomedical research. As a devastating human disease, cancer kills millions of people each year. In 2008, the World Health Organization (WHO) predicted that, if new anticancer treatments are not discovered, there will be 26.4 million cancer patients around the world and 17 million cancer deaths by 2030 [Recently, synthetic lethality (SL) has emerged as a novel anticancer strategy that is promising to be highly selective. A pair of genes is defined to have synthetic lethal interactions if the mutation to either gene will not kill the cell but the mutations to both genes will lead to cell death  Figure . CompareHowever, the discovery and clinical applications of SL-based anticancer therapies need to overcome several technical obstacles. Most known SL cases are discovered in yeast, and so far only a few SL gene pairs are known in human. A prevalent technique to discover SL genes is high-throughput screening based on chemical or RNAi libraries . Due to  S. cerevisiae has been extensively used to study SL [The technique of SL was originated from yeast genetics . Due to study SL . Computastudy SL . In contstudy SL . Massivestudy SL . It is h Syn-Lethality. From literature, we collected SL gene pairs that have been experimentally discovered and verified and integrated them into a network , Syn-Lethality will search for SL partners of the query genes and display related annotations . The SL network we constructed serves as a roadmap for the whole knowledge base.In this paper, we present an integrative knowledge base dedicated to SL in human cancer, called network , where eTo our best knowledge, Syn-Lethality is the first database dedicated to human synthetic lethality. There are few genome wide screenings for SL interactions with human cancer genes, and they are focused on a few well-known oncogenes . The large-scale screening for human cancer cells is limited by high-cost, false positives, and difficulty to interpret mechanisms, and the information is scattered in the literature. An integrative approach is indispensable for a systematic and mechanistic understanding of human SL. Syn-Lethality database is one of the first attempts to integrate knowledge and data about SL in human cancer. We have also integrated data from yeast and will do so in the future from other model organisms. We believe that it would be a valuable resource and framework that would facilitate novel discovery of potential selective anticancer therapy based on synthetic lethality.The primary aim of our Syn-Lethality database is to collect and maintain a high quality set of SL gene pairs, which serves as a comprehensive, fully classified, and accurately annotated knowledge base for SL-related research. The database also provides extensive cross-references and querying interfaces. The SL pairs in Syn-Lethality database are collected by two alternative methods and we will next introduce them in more detail.The first method for collecting SL pairs is the literature search. We examined the Web of Knowledge and NCBI PubMed databases with the keywords like “synthetic lethality” and then screened with the keyword “human cancer/tumour” from the abstracts. As such, we collected more than one hundred scientific publications. From these articles, we manually extracted more than one hundred SL gene pairs, which have been verified by experiments for cancer treatment. Although the number of SL pairs collected by the literature search is limited, they are highly trustworthy and thus they lay the foundation for our Syn-Lethality database.yi and yj form an SL relationship while two human genes hi and hj are orthologs of yi and yj, respectively. If hi or hj is a gene that is observed to be mutated in a certain type of cancer,  is then a predicted SL pair in the human cancer. In this paper, all the yeast SL interactions are downloaded from BioGrid [http://bioinfo.mbb.yale.edu/genome/yeast/cluster/essential/) and Saccharomyces Genome Deletion Project (http://www-sequence.stanford.edu/group/yeast_deletion_project/) we collected 6,613 SL pairs without any essential genes. In addition, 507 human cancer genes are downloaded from COSMIC: Cancer Gene Census via the link http://cancer.sanger.ac.uk/cancergenome/projects/census/. Finally, we inferred 1,114 SL pairs related with human cancers that are predicted from yeast.The second source of potential SL pairs is the knowledge transfer from the model organism of yeast to human by comparative genomics analysis. Currently, there are quite a few number of SL pairs in yeast which are experimentally detected by various screening techniques. Meanwhile, some human cancer genes  are observed to be highly evolutionarily conserved with yeast cancer genes for inferring human SL pairs of genes based on human-yeast conservation. Therefore, it is possible to infer some SL pairs in human cancers from yeast. We predict a human gene pair to be an SL pair in human cancer based on the following two constraints. First, this human gene pair has a conserved SL interaction in yeast. Second, one of these two genes is a cancer gene. For example, two yeast genes  BioGrid   was identified as a synthetic lethal gene of KRAS . TBK1 isectively . The mechttp://www.ntu.edu.sg/home/zhengjie/software/Syn-Lethality/. This software is a Java executable file and requires the installation of Java. The required version 10 of Java (free) and it can be installed from http://www.java.com/en/download/index.jsp. Once the Java is installed on local machine, just double clicking on the Java executable file will launch the database interface. Since the database is available in the single setup file, the database can be used simultaneously by many end users for performing the query  in each field. Smart Search. Users have flexibility of searching SL gene pairs based on the Boolean logical operators by selecting logical AND and OR operators from the drop down menu. This helps in analyzing various combinations of SL gene pairs.Genetic Alteration Search. The interface of our database provides user flexibility to screen the SL pairs based on various types of the gene alteration which refer to the gene mutated in cancer. The gene alteration types captured in our database includes overexpression, mutation, activation, inactivation, and deficiency.Searching in our database can be divided in the following categories.As of now, it is possible to retrieve complete SL gene pair information based on information such as gene names   and typeTo provide more clear understanding of SL gene pairs, we constructed the network for available SL gene pairs . The diaSyn-Lethality is the first comprehensive database constructed through integrating experimentally validated SL pairs of human cancer with the inferred SL pairs from yeast according to the orthologous relation between human and yeast. It is the first attempt to apply the experimentally verified SL pairs to construct a SL network. In the SL network, each node represents a gene/protein and each edge denotes the SL interactions which can be easily linked to the annotation information including gene/protein alteration type, screening method, pathway, mechanism, and the related literature. It is a valuable resource for better understanding SL mechanism in human cancer and developing useful information for anticancer medicine. Caenorhabditis elegans, Zebrafish, and mouse. With the progress of SL experimental screening technology, it is believed that more SL interactions are expected to be identified. We will continue to collect and curate SL pairs of genes. Additionally, using our SL database, we plan to develop data mining algorithms to quickly extract SL information and mechanistic insights. Moreover, by incorporating the signalling pathways associated with the SL pairs of genes, we will construct a comprehensive and global SL network about human cancer.Considering that our current database only includes the predicted SL pairs from yeast, it is desirable to collect and predict more SL pairs from other model organisms, such as


These findings might contribute to reliable stratification of patients eligible for treatment with biologicals directed against MET.Although the receptor tyrosine kinase (RTK) MET is widely expressed in head and neck squamous cell carcinoma (HNSCC), its prognostic value remains unclear. This might be due to the use of a variety of antibodies and scoring systems. Here, the reliability of five commercial C-terminal MET antibodies  was evaluated before examining the prognostic value of MET immunoreactivity in HNSCC. Using cancer cell lines, it was shown that D1C2 and CVD13 specifically detect MET under reducing, native and formalin-fixed paraffin-embedded (FFPE) conditions. Immunohistochemical staining of routinely FFPE oral SCC with D1C2 and CVD13 demonstrated that D1C2 is most sensitive in the detection of membranous MET. Examination of membranous D1C2 immunoreactivity with 179 FFPE oral and oropharyngeal SCC – represented in a tissue microarray – illustrated that staining is either uniform (negative or positive) across tumors or differs between a tumor's center and periphery. Ultimately, statistical analysis revealed that D1C2 uniform staining is significantly associated with poor 5-year overall and disease free survival of patients lacking vasoinvasive growth (HR = 3.019,  Head and neck cancers (HNCs) are a diverse group of malignant tumors that arise in various anatomical localizations of the upper aerodigestive tract . With anDespite the fact that numerous treatment platforms are available –5, relatMET is synthesized as a partially glycosylated 170 kDa single-chain intracellular precursor, which undergoes cleavage and further glycosylation to yield a mature, cell surface-associated 190 kDa disulphide linked heterodimer consisting of an extracellular 50 kDa a-chain and a transmembrane 145 kDa b-chain . SignaliDeregulated HGF/SF-MET signaling has been implicated in many human solid cancers , which hMET using a siRNA. Next, the antibodies that behaved reliably across all examined conditions  were used to explore MET immunoreactivity across whole tissue sections of a selection of oral SCC.Using the Rimm Lab Algorithm for antibody validation  as guidaFinally, using the antibody that is most sensitive in the detection of membranous MET , it was examined whether MET immunoreactivity is associated with the survival of 179 patients diagnosed with oral and oropharyngeal SCC of whom long-term clinico-pathological follow-up was available.As a guide, the Rimm Lab Algorithm for antibody validation  was usedMET mRNA expression levels were determined in the MET antibody validation cell line panel . It should be mentioned here that we depicted LNCaP as negative for MET mRNA expression in Figure MET fluorescence levels in this cell line are so low that they cannot be observed in the presented bar chart.To establish a baseline for MET expression, s Figure , rangingMET mRNA expression levels will show no or weak immunoreactivity with bands migrating as MET protein products and C-terminal fragments , no immunoreactivity was detected in the MET silenced cell line (DU145#Sh167). When comparing the intensities of the blots generated with D1C2 and CVD13 . The immunoblot generated with C-28 was found too poor to evaluate . In contrast to D1C2 and CVD13, SP44 shows weak immunoreactivity with SK-BR-3 and LNCAP making it the most sensitive antibody under native conditions . Comparing the staining intensities obtained with D1C2 and CVD13 per subcellular localization, reveals that D1C2 has a higher sensitivity for membranous MET and that CVD13 has a higher sensitivity for cytoplasmic MET in FFPE cells . With cores taken from the center and periphery, the design of the TMA allows to explore the behavior of MET immunoreactivity across cancer surfaces. Further details concerning the design of the TMA are described in the Materials and Methods section, paragraph patient material.Before assessing the behavior of MET immunoreactivity across cancer surfaces, the heterogeneity of MET immunoreactivity across cores sampled in the center and periphery was evaluated using the ICC. The ICC for the center cores was 0.930  and the ICC for the periphery cores was 0.894  indicating almost perfect agreement between cores from the same cancer region.Evaluation of membranous MET immunoreactivity in both cancer regions (center and periphery) was possible in 183 (76.3%) cases, of which 4 2.2%) oropharyngeal SCC were HPV-16 positive. Seen this low number, the HPV-16 positive oropharyngeal SCC were excluded from further analysis. The baseline characteristics of the remaining cancers are indicated in Table .2% orophTo establish whether there is a relation between the pattern of MET immunoreactivity  and survival, each data point in the scatter plot was labelled with the 5-year OS or DFS status of the corresponding patient  & S5C. Tp < 0.001) and DFS   in terms of 5-year OS  Figure  & S5E. B) Figure . The sam) Figure .p < 0.001; Figure p < 0.001; Figure n = 136), MET staining pattern has no effect on survival in patients with vasoinvasive growth  and the b-chain (p145MET), D1C2 and CVD13 detected five additional MET C-terminal protein fragments  under reducing conditions. This finding is not unexpected as MET is subject to proteolysis [MET[MET [MET, is subsequently released into the cytosol and degraded by the proteasome [MET and p60MET C-terminal fragments observed in HeLa, HT-29 and PC3 using D1C2 under reducing conditions. More specifically – among the observed shed N-terminal fragments – two fragments were observed of 75 and 85 kDa. These specific N-terminal fragments – theoretically – give rise to MET-CTFs of 70 and 60 kDa.Besides the precursor  in terms of 5-year OS and DFS. The significant association between uniform positive MET staining and poor survival is not unexpected, because MET is a known orchestrator of invasive growth , 45, 46.In addition to MET staining pattern, the prognostic value of clinico-pathological characteristics known to be associated with the survival of patients with HNSCC  was examIn conclusion, this study shows that using a specific and sensitive antibody directed against the C-terminus of MET, both scarce and abundant membranous MET immunoreactivity are significantly associated with poor survival rates of patients with oral and oropharyngeal SCC lacking signs of vasoinvasive growth. These findings might contribute to reliable stratification of patients eligible for treatment with biologicals directed against MET. Yet, whether patients suffering from cancers showing abundant membranous MET immunoreactivity could benefit from treatment with MET inhibitors needs further investigation.®; Leiden, The Netherlands), CVD13 , SP44 , C-12 and C-28 . Additional property information on the antibodies is indicated in Table Western blot analysis, immunocyto- and immunohistochemistry were performed using five commercial antibodies directed against the C-terminus of MET, specifically D1C2 .et al. [et al. [Patients with histologically confirmed oral or oropharyngeal SCC – whose primary treatment was surgery – were included in this study. Patients diagnosed with synchronous primary cancers or previous malignancies in the head and neck region were excluded. Human papillomavirus type 16 (HPV-16) status was determined for all oropharyngeal SCC by means of the algorithm described by Smeets et al.  using th [et al. .To examine MET immunohistochemical reactivity in routinely processed FFPE primary oral SCC, representative tissue blocks of four cancers were randomly collected from the archives of the department of pathology of the Leiden University Medical Center .To investigate the association between MET immunohistochemical reactivity and survival, a tissue microarray (TMA) representing 240 FFPE primary oral or oropharyngeal SCC – surgically removed between 1996 and 2005 in the University Medical Center Utrecht  – was included in the study. Prior to the TMA's construction, hematoxylin and eosin (HE) sections – representing the selected cancers – and their corresponding FFPE tissue blocks were collected from the tissue archive of the department of pathology of the UMCU. Subsequently, a dedicated head and neck pathologist examined all HE slides with special attention to the following pathological characteristics: cancer type, differentiation grade, infiltration depth, growth pattern, perineural invasion, vasoinvasive growth, extranodal growth and bone invasion and selected vital cancer regions that were properly fixated for coring. In total, six tissue cores (0.6 mm diameter) were sampled from each cancer; three from the center and three from the periphery.Using a microtome, 4 μm thick slices were cut from the FFPE cancer tissue and TMA blocks that were processed for immunohistochemistry.MET negative according to the retrieved information), HeLa, HT-29, PC3, DU145 (p145MET positive according to the retrieved information) and DU145#Sh167 (a MET silenced cell line). DU145#Sh167 was derived from DU145 by means of lentiviral infection as previously described [Based on western blot results retrieved from the literature , 54, 55 escribed . Additio2 in DMEM/F12 (Life Technologies™) or RPMI 1640 medium (Life Technologies™), supplemented with varying percentages of Fetal Calf Serum (Life Technologies™) and 1% Penicillin/Streptomycin (Life Technologies™). Only DU145#Sh167 was grown in the presence of Puromycin . More information is indicated in Cells were cultured at 37°C under 5% COFor all experiments  the cell lines were cultured as biological duplicates in view of independent validation of the results.® Reagent (Life Technologies™/Ambion®) for totRNA isolation according to the manufacturer's protocol. In total, 25 μg of totRNA was treated with RNase-free DNaseI (Life Technologies™/Ambion®) for 60 minutes at 37°C. Next, the totRNA was purified using the RNeasy mini kit  and eluted in 30 μL of DEPC treated H2O. Subsequently, cDNA was synthesized using SuperScript® II Reverse Transcriptase (Life Technologies™/Invitrogen™) in accordance with the manufacturer's instructions.Cells were cultured in T25 flasks until 75-90% confluence was reached and harvested in TRIZol® Gene Expression Assays against MET and HPRT1 , TaqMan® Universal Master Mix II, no UNG (Life Technologies™/Applied Biosystems®) and the 7500 Fast Real-Time PCR System (Life Technologies™/Applied Biosystems®) according to the manufacturer's protocol for 25 μL reaction volumes. To determine MET mRNA expression levels, a threshold (0.2) was set that falls in the exponential phase  of both target (MET) and endogenous control (HPRT1) across all samples. Subsequently, within sample normalization of MET expression was performed according to the relative standard curve method .Quantitative real-time PCR (qRT-PCR) reactions were performed in triplicate on two biological replicates (see above) of all cell lines included in the MET antibody validation panel using: TaqMan® BCA Protein Assay Kit . Western blot analysis was performed on the Mini Protean II system . Cell extracts, mixed with 2x Laemmli Sample Buffer (Bio-Rad) including 2-mercaptoethanol, containing 18 μg of protein were separated on a 4-20% gradient polyacrylamide gel (Thermo Scientific). Subsequently, the protein fragments were transferred during 1.5 hour onto a PVDF membrane . After washing, the membrane was blocked for 1 hour with powder milk  dissolved in PBS-Tween 0.1%. Next, the membranes were incubated O/N at 4°C with the primary MET antibodies  was used for detection. β-actin was visualized as loading control for each sample by means of the same protocol, using the primary mouse monoclonal antibody against b-actin  and the secondary Anti- Mouse IgG, HRP-linked whole Ab GPR .Cells were cultured in T75 flasks until 75-90% confluence was reached and harvested in lysis buffer . Protein concentrations of the lysates were measured using the Piercetibodies . After w2O2 solution for 5 minutes, slides were stained with the ABC procedure [® Elite ABC system  according to the manufacturer's protocol. Finally, horseradish peroxidase activity was visualized in 90 seconds with 3, 3′-diaminobenzidine  prepared in accordance with the manufacturer's instructions.Cells were cultured on Nunc™ Lab-Tek™ Chamber slides (Thermo Scientific) until 60-90% confluence was reached. Subsequently, the cells were washed with cold PBS and immersed in fresh, cold Paraformaldehyde (4% v/w) for 20 min. for fixation. After blocking endogenous peroxidase activity with a 0.3% Hrocedure . EndogenCells were cultured in triplicate in T175 flasks until 75-90% confluence was reached. Subsequently, per cell line, all three cultures were harvested in a single volume of PBS (10 mL). After removing the PBS, the cells were fixed O/N at 4°C with 10% formalin (10 mL). After removing the fixative, the cells were resuspended in PBS (500 mL) to be transferred to a flat bottom embedding capsule . Herein, after centrifugation and removal of the supernatant, the cells were dissolved in a PBS-agar  solution (500 mL) of 56°C. Upon solidification on ice, the cell containing agar blocks were removed from their embedding capsules, transferred to cassettes and embedded in paraffin.2O2 for 10 min. Subsequently, antigen retrieval was carried out by heating the sections under high pressure – up to 0.9 bar in case of the FFPE cell lines and up to 1.2 bar in case of the FFPE tissues – in Tris-EGTA buffer .After deparaffinization and rehydration, endogenous peroxidase activity was inactivated by incubating the sections in 3% H®) in 1X PBS for 7 min. after blocking endogenous biotin to reduce non-specific background staining. Instead of 90 seconds, horseradish peroxidase activity was visualized in 2x 5 minutes. The used primary antibody titers are summarized in After antigen retrieval, the tissue sections were stained with the ABC procedure using an almost identical protocol applied for the immunochemical staining of the MET antibody validation cell line panel. However, in contrast to the immunocytochemical procedure, the sections were incubated with 0.22% bovine serum albumin solution  simultaneously assessed the immunoreactivity scores.D1C2. Evaluation of MET staining intensities was restricted to membranous immunoreactivity as defined above. Cancer cells showing no (0) to weak (1) membranous immunoreactivity were assessed as negative for MET, while cancer cells showing moderate (2) to strong (3) membranous immunoreactivity were assessed as positive for MET. Moreover, seemingly suboptimal fixed cores and well-differentiated cancer cells that show no nuclei were omitted during scoring.Two observers (SW and MDH) independently assessed the percentage of MET positive cancer cells for each tissue core . Agreemen = 179) – evaluated for MET immunoreactivity in both the center and periphery – consisted of 114 males (63.7%) and comprised 131 advanced stage (III-IV) cancers (73.2%). Further baseline characteristics are indicated in Table If more than one core was available for evaluation per cancer region , the average percentage of MET positive tumor cells was calculated. Evaluation of MET immunoreactivity in both cancer regions was possible in 183 out of the 240 tumors (76.3%) that are represented on the TMA . Of these 183 tumors, only 4 oropharyngeal SCC were HPV-16 positive. Seen the low number, HPV-16 positive tumors were omitted from further analysis. The remaining patient population  between the three scored cores per tumor region. The ICC is a descriptive statistic which describes how strongly different quantitative measures resemble each other, in this case multiple cores of the same tumor. An ICC < 0 reflects ‘poor’, 0 to 0.20 ‘slight’, 0.21 to 0.4 ‘fair’, 0.41 to 0.60 ‘moderate’, 0.61 to 0.8 ‘substantial’, and above 0.81 ‘almost perfect’ reliability of the measurement. Any measure should have an ICC of at least 0.6 to be useful with regard to reliability of the result. The 95% confidence intervals were indicated between brackets. Calculations were done with SPSS Statistics .http://www.r-project.org). Unless otherwise mentioned, statistical significance was set at p < 0.05.Overall survival (OS) was defined as the time in months from the date of primary surgery to: the date of death due to any cause or the cutoff time (set at 60 months). Disease free survival (DFS) was defined as the time in months from the date of primary surgery to: the date of first evidence of any disease  progression, the date of death due to any cause or the cutoff time (set at 60 months). Individuals who were: lost to follow-up or survived beyond the cutoff time, were considered as censored observations. MET immunoreactivity OS as well as DFS curves were calculated by means of the Kaplan-Meier method and significance of differences in survival times was assessed with the log-rank test. Univariable as well as multivariable Cox proportional hazards regression models were used to evaluate the prognostic value of MET immunoreactivity, demographical, clinical, and histopathological patient characteristics. Variables significantly associated with OS and DFS as well as potential confounders were included in backward selection procedures to select the final models. Calculations were done with SPSS Statistics  and R version 2.15.3 (version


Aims. The purpose was to evaluate the evidence for triple therapy regimen using medicines available in Australia for type 2 diabetes.Methods. A systematic literature review was performed to update the relevant evidence from 2002 to 2014 on triple therapy for type 2 diabetes. A multiple-treatments network meta-analysis was undertaken to summarise the comparative efficacy and harms of different triple therapies.Results. Twenty seven trials were identified, most were six months of duration. The following combinations were included in the network meta-analysis: metformin (MET) + sulfonylureas (SU) (used as reference combination); MET + SU+ dipeptidyl peptidase 4 inhibitors (DPP-4-i); MET + SU+ thiazolidinediones (TZD); MET + SU+ glucagon-like peptide-1 receptor agonists (GLP-1-RA); MET + SU+ insulins; MET + TZD + DPP-4-i; and MET + SU+ sodium/glucose cotransporter 2 inhibitors (SGLT2-i). For HbA1c reduction, all triple therapies were statistically superior to MET+SU dual therapy, except for MET + TZD + DPP-4-i. None of the triple therapy combinations demonstrated differences in HbA1c compared with other triple therapies. MET + SU + SGLT2-i and MET + SU + GLP-1-RA resulted in significantly lower body weight than MET + SU + DPP-4-i, MET+SU+insulin and MET + SU + TZDs; MET + SU + DPP-4-i resulted in significantly lower body weight than MET + SU + insulin and MET + SU + TZD. MET + SU + insulin, MET + SU + TZD and MET + SU + DPP-4-i increased the odds of hypoglycaemia when compared to MET + SU. MET + SU + GLP-1-RA reduced the odds of hypoglycaemia compared to MET + SU + insulin.Conclusion. Care when choosing a triple therapy combination is needed as there is often a risk of increased hypoglycaemia events associated with this regimen and there are very limited data surrounding the long-term effectiveness and safety of combined therapies. Type 2 diabetes is a major health concern worldwide and has an increasing prevalence and impact on health services with an estimated worldwide change in prevalence from 153 million in 1980 to 347 million in 2008  and 1.5 c1 target with regards to each patient , insulins, and sodium glucose co-transporter 2 inhibitors (SGLT2-i). The outcomes of interest were change in glycated haemoglobin, change in body weight and odds of hypoglycaemia events.Our hypotheses are:(1)Triple therapies for type 2 diabetes patients who are insufficiently managed with dual therapies have superior efficacy and inferior safety compared to dual therapy after six months of treatment and(2)Triple therapies for type 2 diabetes have equal glycaemic efficacy and differing safety profiles.The Cochrane Handbook for Systematic Reviews of Interventions  and the The PICO for the systematic review was as follows:Population: All patients with type 2 diabetesIntervention: Any triple therapy combination for treatment of type 2 diabetesComparator: metformin plus sulphonylurea dual therapy, and other triple therapy combinations.Outcome: HbA1c, body weight, hypoglycaemia and adverse eventsThe systematic literature review was performed in three stages: (1) identify the most relevant systematic literature reviews on the efficacy and safety of medications for glycaemic control; (2) update the literature search using the relevant systematic literature reviews as identified in step 1 as a starting point; and (3) identify the relevant randomized controlled trials (RCTs) from steps 1 and 2. Ovid MEDLINE and The Cochrane Library Database were used for searching the literature.Each bibliographic database was systematically searched using search terms for type 2 diabetes medications included in Database searches were carried out to update the three identified systematic reviews in November 2014. Each bibliographic database was systematically searched using different search terms for each search strategy. Supplementary Document—Stage 2 contains full details of each search and the terms used in each database. The flow chart of the selection process is presented in c1, body weight change, frequency of hypoglycaemic events, or frequency of other (serious) adverse events. Publications were excluded if they were not randomised control trials or if they were post hoc analysis of randomised control trials. If two publications used the same data, the most comprehensive version of the paper was included. The flow chart of the selection process is presented in The articles identified through Stage 1 (included in relevant systematic literature reviews) and 2 (updated searches) were title checked by one author MJD and then independently by two authors (MJD and ET) and included in the final analysis if they met all of the following inclusion criteria: randomised controlled trial (RCT), English language publication, patients with type 2 diabetes, at least 100 participants in the trial, duration of treatment at least 24 weeks, published after 2002, trials that include any of the following relevant outcomes: HbAIntention to treat data was extracted, where possible, from the included papers into a template in Excel. Extraction was performed by four different reviewers; for each publication, one reviewer extracted the data and one reviewer checked the extracted data for consistency. Risk of bias assessments were performed by one reviewer, and verified by a second reviewer. Risk of bias for the included RCTs was assessed using the Cochrane Collaboration’s ‘Risk of bias’ tool (Version 5.1.0.) . Where ac1, change in body weight, adverse events, hypoglycaemia , and serious adverse events.The following clinical outcomes were extracted at approximately six months after the start of the RCT: change in HbAc1 at approximately six months. To test the hypothesis that all triple therapies were superior to dual therapy, we considered the minimal clinically important difference (MCID) to be −0.3% (3.3 mmol/mol) as used by the Federal Drug Agency (FDA)   and the cy (FDA)  and alsocy (FDA) . HbA1c adividual . To demodividual .Secondary outcomes were the difference in mean change in body weight (Kg) and difference in the hypoglycaemia odds ratio within six months. For those outcomes, we did not identify any MCIDs. Other outcomes investigated but not reported included adverse events, serious adverse events, severe hypoglycaemia and mortality.Excel was used to consolidate and standardise the outcome measures and measures of variability; standard deviations, standard errors and 95% confidence intervals were imputed where necessary . The datc1, body weight, and hypoglycaemia) using the trial data in the clinical evidence base. Different medications from the same class combinations were pooled, using the assumption that all these medications would have similar efficacy and safety. The network meta-analysis was conducted using STATA network package and mvmeta . The network meta-analysis allowed for heterogeneity between studies during calculation (random effects). An inconsistency model was also applied to test for disagreement between direct and indirect evidence  were examined prior to carrying out the network analysis to ensure similarities in baseline characteristics. The measurements of treatment effect calculated were mean differences and their 95% confidence intervals (CI) for continuous data, and odds ratios and their 95% CI for dichotomous outcomes. Differences between treatments were considered statistically significant if there were no overlap in 95% CI. Some trials only compared treatments from the same combination group . These trials were excluded from the network meta-analyses as they were considered to compare the same treatment and there did not randomise two different treatment regimens.A multiple-treatments network meta-analysis was undertaken to summarise the results of triple therapy for each of the outcomes where common treatment arms existed  or higher. The primary outcome of interest for each study was mean difference in HbAC1 from the control group and the trials were powered to assess this outcome, although Overall, the key features of the triple therapy trials varied see  and S4, c1 varied from 8.1% to 10.3%, duration of diabetes varied from 5 to 10 years and body mass index varied from 27 to 35 kg/m2   (p = 0.996) and body weight (p = 0.431).Two efficacy results were reported in the majority of the trials, change in HbA 9, 144)   . Table Sc1 when compared to MET+ SU dual therapy, with the exception of MET+ TZD+ DPP-4-i triple therapy, which did not provide a significant difference over MET+ SU  reduction in HbA MET+ SU , Fig. 2A DPP-4-i .Only SGLT2-i and GLP-1-RA (added to MET+ SU) showed a significant reduction in body weight compared to MET+ SU dual therapy (mean difference (MD): −1.76 kg; 95% CI: −2.74 to −0.78 kg and MD: −1.55 kg; 95% CI: −2.34 to −0.74 kg respectively) ; SU+ DPPc1 for any of the comparisons   . Data fo 4, 458)  and not All adverse events, serious adverse events and serious hypoglycaemia were not systematically reported in the trials, and therefore no further network analyses are able to be presented.MET+ SU+ TZD, MET+ SU+ DPP-4-i and MET+ SU+ insulin had increased odds ratio of hypoglycaemia , Fig. 2CThere were no statistically significant differences in the odds of hypoglycaemia between most triple therapies; however, MET+ SU+ GLP-1-RA had reduced odds ratio for hypoglycaemia compared to MET+ SU+ insulin  .p = 0.004). The majority of inconsistency can be accounted for by p = 0.144 with The network model was tested for consistency and there were some differences between the direct comparison and the indirect comparison for hypoglycaemia  of each other (MET+ SU+ GLP-1-RA vs MET+ SU+ insulin) as the statistical uncertainties were too great to draw similarity conclusions.The results of the review and network analyses demonstrated that the addition of a third pharmaceutical class to MET+ SU therapy was statistically and clinically more effective  at reducing HbAc1 targets which should be achieved for optimal treatment of type 2 diabetes  or increased weight when compared to MET+ SU .Most clinical guidelines advocate an individualised approach to setting patient HbAdiabetes . For tridiabetes ; howeverdiabetes , with sooutcomes . When asMET+ SU+ insulin had higher hypoglycaemia events than MET+ SU+ GLP-1-RA. Overall, the systematic literature review could identify only limited evidence on the long-term safety of type 2 diabetes medicines when used in triple therapy because the longest trial was 1 year. While there are longer term dual therapy studies for some of the combinations, this is of concern considering that these medicines are intended for long-term use.It is important to note that different drugs of the same therapy class were pooled in the analysis. The assumptions were made that treatments within therapy classes had similar mechanisms of action and hence efficacy. For example; the insulin group contained long-acting basal insulin analogue (glargine)  and fastc1 and had a short duration of 24–30 weeks and the network meta-analyses were done for this time point. The number of participants was generally low and the adverse event rates were also low, for this reason it is difficult to draw conclusions on the long-term effect that these therapies will have on the incidences of adverse events. Also it was not always clear which outcomes occurred but were not reported, or whether they did not occur in the trial period.There were some limitations to the analysis of these trials and these should be taken into consideration when assessing the outcomes of the network analyses. The majority of trials were powered only to assess HbAPerforming a network meta-analysis, which uses indirect comparison analyses, may introduce statistical uncertainty; therefore we applied a random effect model to allow for heterogeneity between studies to be incorporated into the estimates of effect. We also tested the network for consistency to evaluate disagreement between direct and indirect evidence, to ensure that the interpretation of the network model was consistent with the original trials.There are limitations with the available data as there were only three trials that do not include MET + SU as the baseline dual therapy. The network model for hypoglycaemia demonstrated some inconsistency between the direct comparisons and the indirect comparison. There could be a number of reasons for this including differences in the definitions of a hypoglycaemic event and some trials allowing dose titration when an event occurred. In the network, the majority of difference could be accounted for by one trial The network meta-analysis highlights areas where the individualised approach can be used. As examples MET+ SU+ GLP-1-RA or MET+ SU+ SGLT2-i may be a good choice for patients that require weight loss and MET+ SU+ GLP-1-RA for patients at risk for hypoglycaemia, whereas MET+ SU+ insulin may provide good blood glucose control in patients where hypoglycaemia and weight gain is not a concern. Care when choosing a triple therapy combination is needed as there is often a risk of increased hypoglycaemia events associated with this regimen, and there are very limited data surrounding the long-term effectiveness and safety of combined therapies.10.7717/peerj.1461/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.1461/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.1461/supp-3Supplemental Information 3PRISMA ChecklistClick here for additional data file.10.7717/peerj.1461/supp-4Supplemental Information 4Click here for additional data file.