Baena arenosa, and numerous well-preserved shells of B. arenosa and Chisternon undatum. Baenids from the Uintan NALMA (46.5–40 Ma) are critical in that they provide valuable insight into the morphology and evolution of the diverse and speciose baenid family near the end of its extensive radiation, just prior to the disappearance of this clade from the fossil record. These Uintan specimens greatly increase the known variation in these late-surviving taxa and indicate that several characters thought to define these species should be reassessed. The partial cranium of B. arenosa, including portions of the basicranium, neurocranium, face, and lower jaw, was recently recovered from Uinta B sediments. While its morphology is consistent with known specimens of B. arenosa, we observed several distinct differences: a crescent-shaped condylus occipitalis that is concave dorsally, tuberculum basioccipitale that flare out laterally, and a distinct frontal-nasal suture. The current sample of plastral and carapacial morphology considerably expands the documented variation in the hypodigms of B. arenosa and C. undatum. Novel shell characters observed include sigmoidal extragular-humeral sulci, and small, subtriangular gular scutes. Subadult specimens reveal ontogenetic processes in both taxa, and demonstrate that diagnostic morphological differences between them were present from an early developmental age.We described newly discovered baenid specimens from the Uintan North American Land Mammal Age (NALMA), in the Uinta Formation, Uinta Basin, Utah. These specimens include a partial skull and several previously undescribed postcranial elements of  The Baenidae  were an Baena has been the subject of much debate ..Baena orThe posterior plastron is generally broad in comparison to the narrow anterior lobe. The hypoplastra are dorsoventrally thickened at the midline. The posterior buttresses are robust, although they are flattened in the juvenile UMNH VP 27537 . The posCHISTERNON UNDATUM Leidy, 1873 B. arenosB. arenosa, B. riparia, and B. sima to the newly reinstated “B.” affinis [B. arenosa specimens: two cervical vertebrae of unknown position in UMNH VP 27535, and dorsal vertebrae (D8-10 and S1) in UMNH VP 27191  [C. undatum, while the C2 and C3 of C. undatum are procoelous , and its C4 is amphicoelous (concave anteriorly and posteriorly) [Boremys pulchra [Peckemys brinkman [Thoroughly described and documented postcranial fossils are comparatively rare in the baenid fossil record . In fact affinis . VertebrVP 27191 . The smaeriorly) . This co pulchra  and Peckbrinkman , and likbrinkman .Boremys pulchra [Arvinachelys [Peckemys brinkman [B. arenosa a mechanical advantage in neck mobility. Thus, we infer that B. arenosa, despite having limited neck retraction capabilities, likely possessed powerful neck movement. The extensive bony roofing of the skull is consistent with the inability to pull the head within the shell .Specimen UMNH VP 27535 also appears to have cervical ribs at multiple vertebral levels . Reduced pulchra , but arenachelys  and Peckbrinkman . The funbrinkman  argue thbrinkman , 62. Thibrinkman , thus poB. arenosa and C. undatum by the new Uintan specimens suggested that a new analysis of baenid phylogenetic relationships was warranted. The resulting minimum length cladograms show weak support (52%) for a hypothesis of B. arenosa + C. undatum monophyly . Thus, this relationship should not be considered full resolved, and as more specimens are recovered, the understanding can be refined. Uintan baenid monophyly has not been universally recovered in past studies of baenid phylogeny [Baena” hayi, “Baena” affinis, and “Baena” escavada all fell outside the clade of (Stygiochelys + (B. arenosa + C. undatum)). This finding provides further support for the paraphyletic status of the genus “Baena” and suggest that further taxonomic revision may be required. However, it should also be noted that we did not force a sister taxon relationship between any “Baena” species, and their apparently distant positions on the minimum trees may result from a comparative lack of data in some species , neither of which characterizes the new Uintan Baena specimens. Both groups share only traits that are also found in other eubaenines, including multiple cervical scales, thick neurocranial bone, and vertebrals II-IV that are longer than wide. “Baena” affinis autapomorphies, such as contact between the pectoral and a marginal scale and the presence of three inframarginals, cannot be conclusively assessed in the Uintan specimens due to incompleteness in these regions. If the Uintan specimens described here belong to “B.” affinis, they would represent the youngest “B.” affinis fossils yet described, since this taxon is documented only from the Bridgerian NALMA [Recently, Joyce & Lyson  argued t affinis  should ban NALMA –9, 22.Baena arenosa was found to be polymorphic for six characters, which was higher than any other taxon in the analysis. As additional specimens are discovered, it may become prudent to reassess whether multiple species exist within the B. arenosa hypodigm. However, there are currently few synapomorphies separating Uintan B. arenosa from those of other NALMAS. Therefore, for now, we adopt a conservative approach and retain it as a single species.In our phylogenetic analysis, Baena arenosa and Chisternon undatum in the sample allow an evaluation of the ontogenetic changes that occur during baenid development, as well as permit a more detailed exploration of the individual shell elements, which often become entirely fused in adults. In all subadult baenids described here, a midsagittal ridge courses antero-posteriorly along the dorsal surface of the neural row  generally becomes worn down and obliterated in older adults as the result of years of digging and burrowing in boggy soils [B. arenosa and C. undatum are generally recovered from fluvial and lacustrine-dominated deposits and typically thought to have been predominantly aquatic, they have also been reconstructed as preferring riparian habitats (B. arenosa) and paludal environments (C. undatum) [This may be a simple developmental process; however, it is also possible that this phenomenon is the result of behavior. There is evidence that the mid-dorsal keel of modern bog turtles (genus gy soils –64. Whilundatum) . While tB. arenosa subadult specimens UMNH VP 27540, UCMP 179495, UCMP 179520, and UMNH VP 27537 and C. undatum UMNH VP 27544 permit the comparison of individual shell elements . Smaller turtles, including subadult Baenids, were also likely preyed upon by mammalian predators, including carnivorans , and possibly creodonts , and mesonychians like Harpagolestes [Echmatemys .Predation pressures appear to have been extensive for immature Uintan baenids, and a common cause of death at this developmental stage. For large-bodied adult baenids, however, predation was apparently less frequent. In addition to the generally decreased predation pressures that are typically afforded by a large body size, adult baenids also tend to have extremely thick shell bones due to their sutural fusion , which lgolestes . We haveB. arenosa to 5 C. undatum specimens is almost identical to that described previously. Hutchison [B. arenosa tends to be 2–3 times as abundant in assemblages as C. undatum. Baenids are more abundant in Ui2 strata (20/27 specimens = 74%) compared to Ui3 strata (7/27 specimens = 26%) [C. undatum specimens were recovered, we also found Baena specimens. However, we did observe a difference in the stratigraphic ranges from which the two species were recovered. Baena arenosa was discovered from low in the section (25 m) through the top of the section (366 m), thus ranging from Ui2-Ui3 [C. undatum was recovered only from localities low in the section, within Ui2 strata (25–87 m). However, the distribution of the latter could be an artifact of the relative rarity of this taxon and smaller sample size of C. undatum specimens recovered.The ratio in our sample of 22 utchison  noted ths = 26%) . Interes Ui2-Ui3 , 35. C. B. arenosa and C. undatum specimens, we also found large quantities of the abundant geoemydid, Echmatemys. Uintan baenids are also often found with trionychids, which are a deep channel, highly aquatic family, often found in fluvial and even near-shore brackish environments. In fact, at one locality lower in the section, WU-8 (57-60m), trionychids outnumber baenids. At two localities (WU-34 and WU-223), baenids were also found in association with concentrations of the carettochelyid Anosteira (Pseudanosteira) pulchra. This pattern of co-occurrence with highly aquatic and semi-aquatic turtles reinforces the primarily aquatic habits of B. arenosa and C. undatum. Future studies extending broadly across turtle taxa can elucidate the nature of habitat and resource sharing among sympatric Uintan turtle communities. While collection bias can certainly not be excluded as affecting the higher proportion of baenids recovered from Ui2 strata, it is of note that many other turtle and mammal taxa have been recovered from Ui3 localities [Echmatemys, Anosteira, and trionychids have all been recovered in high concentrations in Ui3, while baenid specimens dwindle in comparison.Perhaps unsurprisingly, many baenids derive from the Ui2 locality WU-22, aptly nicknamed “Terrapin Station”, and thereabouts . In all calities , 35, 66,B. arenosa specimen, UMNH VP 27546, was recovered from the Uinta Formation/Duchesne River Formation contact (WU-123). This is the latest documented specimen of this species. Interestingly, the carapacial and plastral bones in this specimen are substantially thicker than in any of the other Uintan baenid specimens (Baena.One pecimens . Only thThe end of the Uintan NALMA was likely affected by the middle Eocene climatic optimum (MECO) hypothermal event, where a rather long period (500–750 ka) of increased warming has been detected in the marine record and on the North American continent in the Sage Creek Basin of Montana . HoweverBy Ui2 time (~43Ma), within the study area in the eastern Uinta Basin, large river channels and deltaic sands prograding into the westward regressing Lake Uinta comprised the sediments where these turtles were recovered , 70–71. S1 File(TNT)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableB. arenosa and C. undatum based on the new Uintan baenid specimens are highlighted in bold and indicated with an asterisk.Character matrix was taken from Lyson et al., (2016), and codification changes for (DOCX)Click here for additional data file.


This paper demonstrates a framework of processes for identifying potential witnesses of events from evidence they post to social media. The research defines original evidence models for micro-blog content sources, the relative uncertainty of different evidence types, and models for testing evidence by combination. Methods to filter and extract evidence using automated and semi-automated means are demonstrated using a Twitter case study event. Further, an implementation to test extracted evidence using Dempster Shafer Theory of Evidence are presented. The results indicate that the inclusion of evidence from micro-blog text and linked image content can increase the number of micro-bloggers identified at events, in comparison to the number of micro-bloggers identified from geotags alone. Additionally, the number of micro-bloggers that can be tested for evidence corroboration or conflict, is increased by incorporating evidence identified in their posting history. Distinguishing social media posts that originate from witnesses on-the-ground (OTG) in contrast to micro-bloggers merely posting commentary from afar contributes to numerous application domains, including journalism  and emerwitnessing status. The evidence is tested to measure its balance towards a hypothesis, and further evidence is sought about micro-bloggers of interest to improve the certainty of the results. search event.Previous research has used in-depth human analysis to identify and describe characteristics that distinguish individual micro-blogs as Witness Accounts (WA) and inform a model of micro-blog categories , 5. Inittest processes of the framework to be founded on the same concepts. This model supports evidence extracted from a micro-blog’s text, linked image, or geotag content, that can be classified as supporting inferences the micro-blogger is OTG or not OTG (NOTG) , which is interpreted as the lower and upper probability bounds. The belief df to mfdf ≤ 1 is the reliability weight of source i.If evidences are derived from different sources with different reliabilities, it is possible to account for these differences by applying a discount factor mf are aggregated into one mf. Dempsters Rule of Combination was that originally proposed X ⊆ Θ, X ≠ ∅ where K is the degree of conflict between the two mfWhen evidence are derived from multiple sources from the same frame of discernment, these are aggregated by the use of a combination rule. Combination rules state how two and operation) and ignores all conflict through the normalization factor K, which can produce counter-intuitive results notably described by , which may overlap the interval of another subset . To support further analysis, the evidence extracted for Sensor151 and Sensor6 are presented in Figs e15, which has a greater influence than all the preceding evidence. Comparatively, the conflicting geotag e3 for Sensor6 occurs at the beginning of the timeline. This may be an advantage for some real-time applications, an alternative view is that the result does not summarize the majority OTG evidence. It is not possible to interpret directly from the Bel(OTG) value or mf the number of evidence or what content source it was extracted from. Such a metric for summarizing evidence is identified for future work.Interestingly for the ADcomb dataset, the Bel(OTG) values in Bel(OTG) value. The number of ranks increases to 56 for the ADcomb dataset due to the addition of evidence for the same number of micro-bloggers. As predicted in the Theory Section, evidence combinations that cannot be distinguished by mf combination results were identified, and as such are not differentiated in the ranks. For example, the structure of evidence differs for Sensor67 and Sensor15 presented in Eqs Bel(OTG) MIXW categorization in the ADcomb dataset  in the training dataset. These results communicate, that in addition to a reduction in the number of evidence, the impact of supervised classification can be to change a micro-blogs inference categorization.The datasets resulting from supervised classification are labeled OTGB, and five of these micro-bloggers were categorized with a different inference categorization in the training dataset. Unlike the micro-blogs, a single micro-blogger Sensor207 changed categorization to OTGB with corroboration, meaning two evidence where automatically classified for this micro-blogger OTG where no evidence were categorized OTG in the training dataset.The number of micro-bloggers with evidence in the ADon_a dataset is reduced to 240, from 322 in the training datasets, including five who were not previously identified with evidence. Bel(OTG) value from the combined mf result for the example micro-bloggers. The source mf are derived from the predictive rate, computed from the supervised classification results in Bel(OTG) value of 0.73 for two text evidence OTG, a number less than micro-bloggers with evidence extracted from a variety of sources.CB might serve to alert that a conflict is detected in the chain of evidence, which can be further investigated if required. No evidence was discovered for Sensor207 off-hash, to corroborate or conflict this micro-blogger’s status, which results in a recommendation for future research. When conflict is not detected, the status of a micro-blogger might still be questioned if the evidence is limited to a single source/ evidence type.Excluding Sensor129 and Sensor207, it appears the inclusion of the off-hash geotag evidence from the ADcomb_a dataset confirms the micro-bloggers’ status, and is consistent with the training dataset ADcomb. The results are robust to errors introduced from the supervised classification for the example micro-bloggers. The changes in summary inference category Previously, it has been explained that the number of ranks can reflect the number of different combinations of evidence to be found in that dataset. However, the total number of ranks for the ADon_a dataset in mf for geotag evidence, which can be summarized by mf(OTG) curve in OTG or NOTG, and finally manual assignment of mf by experts. Previous analysis of conflict suggests extension of the decision boundary for this case study to approximately 475m from the center of the MCG. The w curve in b = 600 for varying distances d from the center of the event venue. In [w value is adopted to configure mf. The value for b was established though experimentation as the approximate value where w at d = 475m switches from greater certainty OTG to NOTG, that is w = 0.5. Fundamentally, in this geographically weighted approach the categorization of OTG or NOTG is derived by the computed w.This section provides results of experiments exploring a geographically weighted approach to deriving enue. In  the w vaOTG, both to reflect the possibility the location is in error due to limitations of the source sensors (e.g. GPS), and to be able to distinguish results where multiple evidence corroborates an OTG hypothesis. Further questions include what model to adopt for geotag evidence where d > b, and the method for assigning (1 − w) to Θ. The results of this experiment identify the potential to incorporate a geographically weighted approach, but recommend further research is required before implementation can proceed.The differences resulting from the geographically weighted approach are apparent from OTG could be categorized as having multiple evidence with corroboration. And significantly for testing purposes, 45% of micro-bloggers categorized with evidence OTG could be further categorized as having corroboration, confirming the benefit of evidence combination by micro-blogger in comparison to individual micro-blogs. Additionally, the modeling of the counter-evidence category NOTG enabled the distinction between conflicting evidence within a single micro-blog, or between micro-blogs for a micro-blogger. In the training dataset, thirteen of the 136 micro-bloggers with multiple evidence were identified to have conflicting evidence. A detailed analysis of this conflict identified it was caused by two sources of measurement error, geotags categorized NOTG because they were outside the event venue, and incorrectly assigned categories to text content by annotators. The results indicate more restrictive temporal filtering of geotag evidence  type or inference category. However, it was concluded that a number of questions require further research before adoption is recommended. For example, although a geotag may be located within an event venue, an absolute certainty value may not be ideal, both to reflect the possibility the location is in error and to be able to distinguish where multiple evidence corroborates an OTG hypothesis.In the training dataset experiments, the relative uncertainty of evidence was simply distinguished by content source, and represented by a manually configured OTG are typically a small fraction of micro-bloggers posting about an event e.g. [The primary contributions of this research have been to demonstrate a complete framework of processes for identifying potential witnesses of a case study event, with particular focus for this paper on evidence testing. A generalized evidence model has been defined that has supported a DST test implementation. Additionally, it is confirmed that the number of evidence available for this test are increased by searching a micro-blogger’s off-hash posts during the event. It was also confirmed that the proportion of evidence that could be tested was significantly increased by combining evidence for a micro-blogger in comparison to individual micro-blogs. These contributions are significant as witnesses posting from on-the-ground ent e.g. , and conNOTG.The evidence model has defined sets to support the categorization of evidence, micro-blogs, and micro-bloggers, with regards to inference categories, and corroboration and conflict. The model was successfully applied to a case study event, enabling summaries of micro-bloggers’ status to be presented. These summaries confirm the potential benefits of incorporating off-hash datasets, with increased numbers for all categories of evidence detected, the greatest being 84% for geotags not on-the-ground mf in a DST implementation. A range of experiments were completed to explore the development of mf including manual configuration by experts for training datasets, and derivation from a classifiers confusion matrix to demonstrate automatically extracted datasets. The results indicated particular DST behaviors that include conflict might have significant or negligible impact, depending on the order and number of evidence combined, however, the results offered limited transparency as to the contributing evidence.Evidence was defined as being extracted from the text, image, or geotag content of micro-blogs, and characterized by varying uncertainty, which are modeled via mass functions mf, and combination algorithm, adequately responded to individual micro-bloggers’ scenarios for this case study. However, in response to the identified complexity and limited transparency of results, the development of a metric to provide a balance of evidence is proposed for future research. This metric could include a count of evidence for each micro-blogger, with each evidence type weighted so the result provides transparency on that included. Additionally, it could support analysis of whether conflict is significant in comparison to the total count of evidence for the micro-blogger, include factors that distinguish between structural differences in evidence posting, and identify if corroboration is from a variety of evidence types. It is also intended that additional future work can explore alternative formal approaches for evidence combination such as Fuzzy Sets and Possibility theory, supported by the evidence models described in this study.The methods selected to derive For text and image evidence varying uncertainties for different evidence types are envisaged in future implementations. There are currently limited accessible methods to automatically extract the evidence types that have been identified with manual annotation. State of the art machine learning indicates that all evidence will not be extracted with a single method, it is likely that numerous highly specialized methods will be employed for specific evidence types. For example, the needs of this research are methods for extracting spatial and temporal context from text content. In such a scenario, a requirement of the test processes will be handling varying inferential weight for different evidence types, and varying uncertainties that would be associated with different extraction methods for these evidence types. These new evidence extraction methodologies can be tested and utilized by the framework in future work.


Dirofilaria immitis, was isolated from the scrotum of a 14-month-old child. This is the first identification of human dirofilariosis caused by D. immitis in a relatively Northern region (Moscow) of the Russian Federation. The parasite was diagnosed by means of morphological examination of the worm, confirmed by PCR assay. This case raises questions about the range of the D. immitis distribution among humans in Russia. To better understand the geographical distribution of dirofilarioses, detailed clinical and epidemiological information should be collected from human cases with appropriate laboratory confirmation.An immature female worm,  Dirofilaria repens has been documented in dogs and cats, whereas Dirofilaria immitis has been reported in dogs of the South and the Far East of Russia [Dirofilaria ursi has been reported in brown bears (Ursus arctos) of the Primorskiy kray, Sakhalin, Yakutia, and of the Vologda regions [Dirofilaria repens are found more often in the southern regions of the country  [In the Russia Federation, dirofilariosis is commonly identified in domestic and wild carnivores . Dirofilf Russia . Dirofil regions , 21. Humitories) .Dirofilaria spp. in Russia are mosquitos belonging to the Aedes, Anopheles, and Culex genera [In recent years, both animal and human dirofilariosis has spread toward the North of Russia. These zoonotic nematodes are reported more frequently in the Moscow, Tyumen, and Novosibirsk regions, and in the Primorskiy and Khabarovskiy krays, where the environment favors their natural cycles , 22, 25.D. immitis is widespread causing severe cardiac signs in dogs [In Western Europe,  in dogs , 23. In  in dogs , but the in dogs , spermat in dogs , scrotum in dogs , 15, 26, in dogs , ovaries in dogs , liver [ in dogs , or subc in dogs , 7.In this report, we present a case of dirofilariosis in a child.A 14-month-old child residing in a rural settlement of the Solnechnogorskiy district (Moscow region), about 56° North, was brought by the parents to a local health care center on March 2015. Significant edema at the right part of the scrotum, cyanotic tint of the affected area, and tenderness were observed. The urologist suspected torsion of the right testicle and referred the child to the surgery unit.According to the parents, the child, who did not attend a kindergarten, was very often taken outside the home in a baby carriage, lightly dressed or even undressed in the hot days of the 2014 summer. The child was healthy before the 2014 spring but then pain and swelling developed and gradually worsened.The child was operated in March 2015, and an inflammatory infiltrate of solid-elastic texture was removed. A very thin white filamentous worm, about 10 cm in length, was extracted from the biopsy. The worm was surrounded by necrotic tissue, hemorrhage, and it was infiltrated by eosinophils, lymphocytes, and plasma cells. The biopsy was analyzed at the Department of Medical Parasitology and Tropical Medicine of the Clinical and Diagnostic Center, Moscow State Medical University.D. repens.In April 2015, the child was in good health and no abnormal signs were observed, except minor residual edema and induration in the area of the surgical intervention. A case of dirofilariosis was suspected. No microfilariae were detected in the blood. PCR testing of a blood sample tested negative for The morphological examination of the worm showed that it was an immature female (no larvae were present in the uterus) of 130 mm in length and 0.95 mm in width .D. repens, were observed. Fine “cuts” located lengthwise on the cuticle were present. The uteri were formed and large nuclei were clearly visible in the notochord cells. Based on its morphology, the worm was identified as D. immitis.The anterior and posterior ends of the body were slightly narrowed and had a roundish shape. The anus was at 0.8 mm from the tail. The cuticle was white with tender cross striations. No pectiniform longitudinal cross striations, typical for D. repens and D. immitis were tested by PCR using species-specific primers.To confirm the morphological identification of the worm, fragments from the worm isolated from the patient and from reference adult worms belonging to D. repens specific primers were DIR-3 F-5′–CCGGTAGACCATGGCATTAT–3′, and DIR-4 R-5′–CGGTCTTGGACGTTTGGTTA–3′, amplifying a fragment of 246 base pairs. Amplification consisted of 48 cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min [The or 1 min .D. immitis specific primers were CO1 int F-5′–TGATTGGTGGTTTTGGTAA–3′, and CO1 int R-5′–ATAAGTACGAGTATCAATATC–3′, amplifying a fragment of 656 base pairs. Amplification consisted of one cycle at 94 °C for 30 s, then 30 cycles at 94 °C for 1 min, 50 °C for 2 min, and 72 °C for 5 min [D. immitis (amplified fragment of 656 bp), whereas no amplification was observed when the DNA was amplified by the D. repens specific primer pairs  and D. repens in 22 dogs (0.6%) [D. immitis was detected in 19 dogs (36.5%) and 31 dogs (29.2%) with microfilariae of the Voronezh region [D. immitis among dogs in Siberia, this parasite was detected in 61 dogs (43.6%) with microfilaremia in Khabarovsk city [In Russia, data on the prevalence of human dirofilariosis are limited, since the official notification and registration of clinical cases have been introduced very recently. According to Nagornyi and Krivorotova , D. immiRepublic . In the s (0.6%) . D. immih region  and the h region , respectD. immitis in the Russian Federation. The child acquired the infection at 14 months of age. No specific clinical signs and/or symptoms were observed. The parasite was identified by means of morphological examination of the worm and confirmed by molecular analysis. There is a need for more thorough and detailed analysis of each case of human dirofilariosis. We cannot exclude that D. immitis may be more frequent in humans in the Russian Federation than previously reported, since in most cases the worm is not identified at the species level.This is the first documented report of human dirofilariosis due to D. repens is localized most frequently under the conjunctiva, more rarely subcutaneously in areas with areolar cellular tissue [D. repens in humans  have become more frequent [In humans, r tissue , 12, 21.frequent , with thfrequent , 19, 23.frequent .Detailed assessments of epidemiological data would also be very useful and helpful in clarifying the epidemiological situation.


Porphyromonas gingivalis has been invoked in the autoimmune disease rheumatoid arthritis (RA). This association relates to the peptidylarginine deiminase of P. gingivalis (PPAD), an enzyme capable of citrullinating human proteins and potentially contributing to loss of tolerance to citrullinated proteins in RA. PPAD is both retained in the outer membrane (OM) of P. gingivalis cells and secreted into the extracellular milieu, where it is detected in a soluble form and in association with outer membrane vesicles (OMVs). Recent studies showed that certain P. gingivalis proteins are retained in the OM through modification with an A-type lipopolysaccharide (A-LPS). Here, we investigated the possible involvement of A-LPS modification in the association of PPAD to the OM and OMVs. The results indicate that the OM- and OMV-associated PPAD is A-LPS-modified. The modified PPAD species is of low abundance in particular clinical isolates of P. gingivalis, which is not due to defects in the overall synthesis of A-LPS-modified proteins but, rather, to particular traits of the respective PPAD proteins. Lastly, we show that OMV association protects the A-LPS-modified PPAD from proteolytic degradation. Altogether, our observations show that A-LPS modification contributes to OM(V) sorting and ‘protective secretion’ of PPAD.The periodontal pathogen  Porphyromonas gingivalis is an anaerobic Gram-negative bacterium and keystone oral pathogen2 that is mostly known for its role as a causative agent of the inflammatory disease periodontitis3. In recent years, this bacterium has garnered interest for its alleged involvement in the etiopathogenesis of the autoimmune disease rheumatoid arthritis (RA)9. A symptom and possible cause for the disease RA is the loss of tolerance to citrullinated host proteins12. Accordingly, the discovery of a peptidylarginine deiminase in P. gingivalis (PPAD) was a turning point in the study of the etiology of RA, because this enzyme is capable of citrullinating certain host proteins. In turn, this might cause the production of anti-citrullinated protein autoantibodies (ACPAs), which are highly specific for RA14. This notion, coupled with the knowledge that periodontitis is clinically associated with RA, suggests a role for PPAD and therefore P. gingivalis in the etiology of rheumatoid arthritis and highlights the importance of studies on PPAD10. Specifically, understanding the localization of PPAD would ease the search for its targets and cofactors, rendering the sorting a paramount tool to further the knowledge of the molecular role of this virulence factor in the origin of RA.17, a form bound to secreted outer membrane vesicles (OMVs)18 and a secreted form that is present in a soluble state21. This relates to the particular features of the type IX secretion system, which is responsible for the secretion of PPAD and ~30 other OM proteins possessing the same recognition sequence, referred to as ‘C-terminal domain’ (CTD)24. During export these proteins are subject to post-translational modifications, including attachment to the OM via an A-lipopolysaccharide (A-LPS) anchor26. Although this has not yet been shown specifically, a modification with A-LPS would also explain the binding of PPAD to the OM and its association with OMVs. In this respect, it should be noted that OMVs are nanostructures resulting from outer membrane ‘blebbing’. Such OMVs are generally used by Gram-negative bacteria to secrete proteins, or to shuttle their cargo to cells or tissues of the host for infection purposes18. PPAD, in fact, has been reported to be present in OMVs together with several other virulence factors of P. gingivalis20. Of note, our recent studies indicate that two PPAD sorting types can be distinguished amongst clinical isolates of P. gingivalis20. Sorting type I isolates produce a dominant OMV-associated PPAD species and relatively lower amounts of the soluble secreted PPAD. In contrast, sorting type II isolates produce restricted amounts of the OMV-associated form of PPAD and substantially higher amounts of the soluble secreted form of PPAD20. This difference does not relate to differences in the amounts of PPAD or OMVs produced but, instead, it is correlated with the presence of an amino acid substitution at position 373 in the PPAD amino acid sequence. PPAD of sorting type I isolates has a Gln residue at position 373, whereas PPAD of sorting type II isolates has a Lys residue at this position20 P. gingivalis is A-LPS-modified.In view of the likely importance of A-LPS-modification in the subcellular localization of OM- and OMV-associated proteins, and the importance of PPAD as a virulence factor, the present study was aimed at assessing the extent to which PPAD of P. gingivalis isolates were obtained from patients with a periodontal diagnosis . For comparison, a clinical Escherichia coli isolate was included  broth as previously described27; cultures were inoculated with 4 days old colonies on blood agar plates. The E. coli strain was cultured aerobically in Lysogeny Broth (LB) at 37 °C and with shaking (250 rpm), or on LB agar at 37 °C.16 is Table . Additioed Table . P. ging8. This previous study received Institutional Review Board approval from the Medical Ethics Committee of the University Medical Center Groningen (METc UMCG 2011/010). It was performed in accordance with the guidelines of the Declaration of Helsinki and the institutional regulations, and all samples were anonymized.The bacterial samples used in the present analyses were obtained in a previous study upon written informed consentP. gingivalis, cells were separated from the growth medium by centrifugation and proteins in the growth medium fraction were precipitated with 10% trichloroacetic acid (TCA) as previously described27. The collected cells were washed twice with phosphate buffered saline (PBS) to ensure absence of supernatant residues, and frozen at −20 °C. After having been thawed, the cells were resuspended in 300 μL of loading buffer, consisting of lithium dodecyl sulphate (LDS) sample buffer 4× , and sample reducing agent 10× (Life Technologies) in MilliQ water. Subsequently, glass beads  were added to the cells to allow membrane disruption with a Precellys®24 Technology tissue homogenizer  in three cycles of 30 sec at 6500 rpm with 30 sec breaks in between. Afterward, the glass beads and cellular debris were pelleted by centrifugation at 16100 × g, 4 °C for 10 min, and 100 μL of the supernatant were collected and boiled for 10 min at 95 °C. Subsequently, both the proteins from the secreted and the cell fractions were investigated by LDS polyacrylamide gel electrophoresis (PAGE) using NuPAGE gels from Life Technologies27.To collect secreted proteins of 21. To disrupt OMVs mechanically, they were exposed to ultrasound in three cycles using a Misonix sonicator S-4000  prior to precipitation with 10% TCA. To prevent protein degradation, cOmplete Mini protease inhibitors  were added immediately prior to sonication, according to manufacturer’s specifications.The isolation of OMVs by ultracentrifugation of growth medium fractions was performed as previously described20, using Amersham™ Protran® 0.45 μm nitrocellulose membranes  and antibodies specific for either P. gingivalis A-LPS (mAb1B5)28, or PPAD (GP2448)20. Membranes were washed with PBS-Tween20 (PBS-T) after overnight incubation with 5% skim milk in PBS, and incubated with primary anti-PPAD or A-LPS antibodies in a 1:10000 dilution. After one-hour incubation, membranes were washed four times for 5 min with PBS-T prior to 45 min incubation with IRDye® 800CW conjugated goat anti-mouse or goat anti-rabbit secondary antibodies  at a 1:10000 dilution. When it was necessary to probe a blot with the mAb1B5 monoclonal antibody and a rabbit antibody at the same time, IRDye® 680RD Goat anti-Rabbit IgG (LI-COR) was used as a secondary antibody instead of the IRDye® 800CW conjugated goat anti-rabbit antibody. Subsequently, membranes were washed four times in PBS-T and twice in PBS, before analysis with an Odyssey Infrared Imaging System (LI-COR Biosciences).Western blotting analysis was performed as described previously29. The cells present in 2.5 OD600 units of culture were collected via centrifugation at 16100 × g 4 °C for 10 min, and resuspended in 300 μL of TEPI buffer, which is composed of TE buffer (Sigma-Aldrich) supplemented with 1 tablet of the cOmpleteTM Mini Protease Inhibitor Cocktail  per 10 mL. The growth medium fraction was put aside for later use. The cell fraction was vortexed with glass beads four times for one min, with 1-min intervals on ice, before the supernatant fraction containing soluble cellular and OM proteins was collected via centrifugation. The pellets containing glass beads and unbroken cells were washed twice with 300 μL of TEPI buffer and centrifuged. All supernatant fractions with soluble cellular and OM proteins were pooled. Subsequently, 100 μL of Triton X-114 were added to both this pooled fraction with cellular proteins, and to the growth medium fraction. Upon brief vortexing, the Triton X-114-containing samples were incubated for 20 min at 4 °C. Prior to centrifugation at 16100 × g and 4 °C for 5 min bromophenol blue  was added to the samples for a better visualization of phase separation in the subsequent steps. Next, the resulting supernatant was overlaid on a 200-μL cushion of 6% (w/v) sucrose , 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.06% Triton X-114 to provide a better phase separation. The samples were then heated for 20 min at 37 °C for phase separation, and the separated phases were collected by centrifugation at 400 × g for 3 min. To reduce phase contamination, only part of the aqueous phase (800 μL) and part of the detergent phase (90 μL) were collected. The remaining phases were extracted again by adding 90 μL of Triton X-114 to the aqueous phase and 800 μL of TEPI buffer to the detergent phase. Subsequently, proteins in the samples were precipitated with TCA  by incubation on ice for 30 min. Afterward, the precipitated proteins were collected by centrifugation at 16100 × g, 4 °C for 10 min, washed twice with 500 μL of ice-cold acetone, dried at 60 °C for 10 min, separated by LDS-PAGE and analyzed by Western blotting27.To assess modification of the ~75–85-kDa PPAD species with A-LPS, a protein phase separation assay was applied using the non-ionic detergent Triton X-114 P. gingivalis and E. coli was performed essentially as described by Geurtsen et al.30. For each sample, one OD600 unit was collected through centrifugation and resuspended in 50 µL of 2× sample buffer31  before incubation with 50 µL of 1 mg/mL proteinase K for 1 h at 55 °C. Subsequently, the proteinase K was inactivated for 10 min at 95 °C, and samples were diluted ten-fold. 2 µL of the diluted and 2 µL of the undiluted samples were loaded into a Tricine-SDS-polyacrylamide gel. Gels were run at 35 V for the stacking gel part and 105 V for the separation gel part. The separation gel was then fixed overnight in fixing buffer . Afterward, the gel was incubated, shaking at 40 rpm, in oxidizing buffer  for 5 min, prior to being washed thrice, in a clean dish, with demineralized water, shaking at 40 rpm, 15 min per wash. Subsequently, the gel was stained with staining reagent ) for 10 min at 70 rpm. Lastly, the gel was washed thrice with demineralized water in a clean dish for 10 min at 40 rpm prior to incubation at 40 rpm with formaldehyde developer  until browning of LPS bands (2–5 min). Development was terminated using 5% acetic acid.Isolation and analysis of LPS of P. gingivalis cells and the growth medium, we established an approach for two-phase protein separation based on the non-ionic detergent Triton X-114. This approach relies on the principle that mixtures of Triton X-114 and water separate into an aqueous and a detergent-rich phase upon heating above the cloud point of Triton X-114. Accordingly, membrane-associated hydrophobic proteins solubilized in mixtures of Triton X-114 and water will segregate into the detergent phase upon heating, while water-soluble proteins will segregate into the aqueous phase. For validation, this approach was applied to four clinical P. gingivalis isolates showing extreme PPAD sorting type I and II phenotypes. Upon phase separation, a Western blotting analysis was performed using the polyclonal PPAD-specific antibody GP2448. As shown in Fig. 17 represents the OM-bound PPAD, was present as a dominant species in the Triton X-114 phase of the cell fraction of the sorting type I isolates 20664 and 20665. Small traces of the ~75–85-kDa band could also be found in the aqueous phase of the cell fraction, but this was most likely due to phase contamination. This fractionation pattern is consistent with the previously proposed A-LPS modification of the ~75–85-kDa form of PPAD23. Further, we have shown previously that this form of PPAD is also detectable in association with secreted OMVs20. Consistent with our previous finding, the present analysis showed that the ~75–85-kDa OMV-associated form of PPAD of sorting type I isolates fractionated with the Triton X-114 phase of growth medium samples. In contrast, a predominantly cell-associated ~60-kDa form of PPAD as well as a secreted form of ~47-kDa fractionated with the aqueous phase  of P. gingivalis, which might explain the presence of the ~37 kDa PPAD species.Our results show the presence of different PPAD species that are differentially sorted in 32, which was raised specifically against P. gingivalis A-LPS. Upon probing with both mAb1B5 and a PPAD antibody, we discovered that a strong A-LPS signal at around 75-kDa aligns with the PPAD signal at this position in the Western blot. This supports the view that the ~75–85-kDa PPAD species fractionating with the detergent Triton X-114 is A-LPS-modified. Conversely, Western blotting with mAb1B5 indicates that the soluble ~60-kDa form of PPAD does not contain A-LPS, consistent with the periplasmic localization as proposed above. Together, these data allow us to correlate the differentially stained A-LPS and PPAD bands in Figs P. gingivalis isolates, regardless of their PPAD sorting type. This shows that the PPAD sorting types I and II are not distinguished by differences in the overall A-LPS modification of proteins. Further proof of this view was obtained by the analysis of total LPS extracted from cells with different PPAD sorting types, which showed no detectable differences.To verify whether PPAD binding to the OM and OMVs involves an A-LPS anchor, we made use of the mAb1B5 antibodyP. gingivalis, including arginine- and lysine-specific gingipains. Whether this means that the OMV association segregates the A-LPS-modified PPAD physically from the secreted proteases, or whether particular protease cleavage sites are protected by the OMVs remains to be determined. This is a relevant question for future studies, because a previous study has shown that gingipains are also detectable as cargo of OMVs from P. gingivalis strain W5018. Of note, these previous results indicated that the gingipains were predominantly exposed on the OMV surface, but absent from the OMV lumen18.Lastly, by subjecting OMVs of either PPAD sorting type to sonication, the A-LPS-modified PPAD became susceptible to proteolysis. On this basis, we conclude that the A-LPS-modification serves not only to localize PPAD to OMVs but also to place it in a position where it is protected from proteolysis by the highly active secreted proteases of P. gingivalis. Additionally, they show that there are no differences in the overall A-LPS synthesis or overall A-LPS modification of exported proteins in PPAD sorting type I and II isolates. This supports the notion that Gln373 of PPAD is critical for A-LPS modification, and that the Q373K substitution encountered in the PPAD of sorting type II isolates hinders the A-LPS modification of this important virulence factor. It is currently not known whether the difference in A-LPS modification and PPAD sorting observed in the type I and II P. gingivalis isolates may have clinical implications for periodontitis and RA. Both PPAD sorting types I and II have been found in at least one periodontitis patient with RA and at least one periodontitis patient without RA. This leads us to believe that the PPAD sorting type is not a decisive, tipping factor for triggering RA. However, considering the fact that RA is a multifactorial disease and that patient data on the analyzed isolates were in most cases incomplete with respect to the diagnosis of RA, it is presently impossible to draw conclusions on possible implications of the PPAD sorting type in relation to RA.Taken together, the present findings correlate A-LPS-modification of PPAD with the anchoring of this protein to the OM and OMVs of The present research has no particular ethical implications.Supplementary information


Triticum aestivum L., AABBDD genome) by evaluating the expression of pre-pollination  and post-pollination (crossability) barriers in Aegilops tauschii Coss. (the D genome progenitor). In total, 201 Ae. tauschii accessions representing the entire natural habitat range of the species were used for anther length measurement and artificial crosses with a Triticum turgidum L. (the AB genome progenitor) tester. Intraspecific comparisons showed that both barriers were more strongly expressed in the TauL1 lineage than in the TauL2 lineage. The ability of Ae. tauschii to cause reproductive isolation in the hybridisation with T. turgidum might have markedly influenced common wheat’s speciation by inducing lineage-associated patterns of gene flow. The TauL2 accessions with high potential for natural hybridisation with T. turgidum clustered in the southern coastal Caspian region. This provided phenotypic support for the derivation of the D genome of common wheat from southern Caspian populations. The present study underscored the importance of approaches that incorporate the genealogical and geographic structure of the parental species’ reproductive isolation in understanding the mechanism of plant allopolyploid speciation.The ability to cause reproductive isolation often varies among individuals within a plant species. We addressed whether such polymorphism influenced speciation of the allopolyploid common wheat ( How allopolyploid species form is a long-standing question. In contrast to the recent advances in understanding the nuclear and cellular level changes in emerging allopolyploids, much remains uncertain about the genetic and ecological basis of allopolyploid speciation7. Natural allopolyploids may arise through successful species crossing followed by hybrid genome duplication via the union of unreduced gametes, when related but diverged species come into contact due to migration, habitat disturbance, or distribution range shifts. Among other factors, the two mechanisms that act in opposite directions, namely reproductive isolation between the parental species and unreduced gamete production in their hybrids, strongly influence the early stages of allopolyploidy10.Allopolyploidy, defined as the merging of different chromosome sets through hybridisation, is an important driver of plant speciation and diversification14. The mechanism underlying unreduced gamete production in hybrids is largely unknown, but a few studies addressed its genetic basis16. Importantly, the parental plant species are usually polymorphic in their ability for reproductive isolation and for hybrids’ unreduced gamete production. Thus, some parental individuals may be involved in natural allopolyploid formation more often than others may because they frequently hybridise and because their hybrids are likely to produce unreduced gametes. Such polymorphism may have profound impact on the temporal and spatial patterns of allopolyploid speciation particularly when it is genealogically and/or geographically structured. Nevertheless, the influence of such polymorphism on allopolyploid speciation remains understudied.Recent studies increased our knowledge on the genetic mechanisms that underlie reproductive isolation in plants. Types, functions, and causal mutations/variations are now known for several pre-zygotic and post-zygotic barrier genes that reduce the amount of gene flow between species or populations in model and non-model systemsTriticum aestivum L., AABBDD genome). Our goal was to elucidate how the diploid progenitor’s ability to cause early reproductive isolation in hybridisation with the tetraploid progenitor influenced the allopolyploid speciation of common wheat. For this goal, we analysed the patterns of the expression of pre-pollination  and post-pollination (crossability) barriers by measuring the diploid progenitor’s anther lengths and by performing artificial cross experiments. One major finding was that the diploid progenitor’s ability for reproductive isolation might have markedly influenced the allopolyploid speciation of common wheat by causing lineage-associated patterns of gene flow. This and other findings revealed a possible important role of the genealogical and geographic structure of the parental species’ reproductive isolation in the origin of natural allopolyploid plants. The present study underscored the importance of approaches that incorporate information on the parental species’ reproductive isolation structure for studying the genetic and ecological basis of plant allopolyploid speciation.The present study aimed to provide novel insights into the role of reproductive isolation during the allopolyploid speciation of common wheat , is a wild wheat species that is primarily autogamous or geitonogamous. This species has a wide geographic range spanning from central Syria to western China in Eurasia18. In the currently accepted explanation of the origin of common wheat, male Ae. tauschii crossed with female Triticum turgidum L.  under natural conditions. This cross produced triploid F1 hybrids (ABD genome), which, in turn, produced hexaploid F2 hybrids (AABBDD genome), the direct ancestors of the extant allohexaploid common wheat. The hexaploid F2 arose through natural genome duplication via the union of unreduced gametes produced by the triploid F1 hybrids21. The allopolyploid speciation of common wheat was caused by human-mediated migration and/or habitat disturbance. Early in the evolution of common wheat, Ae. tauschii crossed with a cultivated form of T. turgidum, which arose from wild T. turgidum through domestication in the Fertile Crescent . The T. turgidum cultivar came into contact with Ae. tauschii through migration in association with the spread of agriculture across and beyond that region23. The exact place of the critical contact is not known. Molecular evidence suggested that allopolyploidisation was a recurring process during T. aestivum evolution24.The diploid progenitor of common wheat, Ae. tauschii has distinctive genetic lineage structure that may facilitate intraspecific comparisons. This structure comprises two large lineages (TauL1 and TauL2), each including two sublineages , and one small lineage (TauL3)28. While TauL1 is geographically widespread within the species distribution range, TauL2 and TauL3 are restricted to the Transcaucasus-Middle East region and Georgia, respectively. Within the range of TauL1, TauL1a resides mainly in the western part and TauL1b in the eastern part. The ranges of TauL2a and TauL2b widely overlap, but TauL2a tends to occur in the western part of the range of TauL2, whereas the TauL2b mostly occurs in the eastern part. Genetically, TauL2 and TauL3 are much closer to the D genome of common wheat than TauL1 is, suggesting that the ancestors of TauL1 were probably not involved in the allopolyploid speciation of common wheat15. TauL2 seems to be the Ae. tauschii lineage that is closely related to, but not the closest sister of, common wheat’s D genome, because TauL2 and the common wheat D genome diverged long before the origin of common wheat, roughly 0.5 million years ago29. Interestingly, TauL1 and TauL2-TauL3 seem to be reproductively isolated in their natural habitats as, despite their wide overlap in the western part of the species range, the intermediate genotypes are rarely found24. A previous study identified two intraspecific groups of Ae. tauschii based on pollination-related traits: the short anther and long anther groups30. The short anther group consists of strong selfer accessions that produce a reduced amount of pollen . The long anther group consists of the accessions that are capable of facultative outcrossing and produce an increased amount of pollen .Ae. tauschii’s outcrossing potential, the anther length dataset obtained in our previous common garden experiment was used here to examine the outcrossing potential of the accessions30. Anthers were shorter in TauL1 than in TauL2 and TauL3 accessions. The mean length for TauL1 was 1.9 mm , whereas that for TauL2 and TauL3 was 2.6 mm  , indicating that the lineage is a significant predictor of anther length in TauL1 and TauL2 accessions. The marginal R2  for the alternative model was 0.56, whereas the conditional R2  was 0.92. The accession-wise mean length histograms of TauL1 and TauL2 were largely separated, despite a minor overlap, and four TauL2 accessions  had exceptionally short anthers (mean ≤ 2.0 mm)  Fig. . We examly) Fig. . This anmm) Fig. .Figure 1P = 0.23). The marginal R2 for the alternative model was 8.27e-03, whereas the conditional R2 was 0.77. In TauL2, longitude had a positive correlation with anther length (fixed effect coefficient = 0.01). The inclusion of longitude as a fixed effect slightly improved the goodness of prediction of the alternative model (AIC = −472.5) relative to the null model (AIC = −473.7). However, the departure of the alternative model from the null model was not significant . The marginal R2 for the alternative model was 9.29e-03, whereas the conditional R2 was 0.86.Because each TauL1 and TauL2 has a west-east genetic structure, we further examined whether anther length was differentiated across the longitudinal distribution of each lineage. For this purpose, the intralineage relationships of anther length with the longitude of the localities of the accessions were analysed using the LMM-based model selection approach. Longitude was found to be a poor predictor of anther length in both TauL1 and TauL2 (Table P = 6.15e-08), indicating that lineage is a significant predictor of crossability in TauL1 and TauL2 accessions. The marginal R2 for the alternative model was 0.11, whereas the conditional R2 was 0.25 , from 0.00 to 0.42 among the TauL2 accessions , and from 0.19 to 0.32 among the TauL3 accessions  Fig. . We examP = 0.81). The marginal and conditional R2 for the alternative model was 4.96e-04. In contrast, longitude was a significant predictor of crossability in TauL2. Longitude had a positive correlation with crossability (the fixed effect coefficient = 0.24). The inclusion of longitude as a fixed effect in the alternative model improved the goodness of prediction of the alternative model (AIC = 885.8) relative to the null model (AIC = 899.3) (Table P = 8.16e-05). The marginal R2 for the alternative model was 0.09, whereas the conditional R2 was 0.20.We further examined if crossability was differentiated across the longitudinal distribution of each TauL1 and TauL2 lineage using GLMM-based model selection approach. Longitude was a poor predictor of crossability in TauL1 Table ; Fig. 3.3) Table . The depT. turgidum. An index of those traits was produced by principal component analysis (PCA) based on the among-accessions correlation matrix. This index was the first principal component (PC1) in a two-dimensional space defined by the rectangular coordinate axes of mean anther length and crossability. The first and second principal components (PC1 and PC2) of the PCA explained 71.7% and 28.3% of the correlation structure between mean anther length and crossability, respectively (correlation coefficient = 0.43)  Fig. . The PC1ive Fig. . Geograpive Fig. . One Tau30. Moreover, these groups differ in other pollination-related traits, including the number of pollen grains per anther and the number of spikelets per spike30.TauL1 and TauL2 clearly differed in anther length Fig. ; Table 131. Aegilops tauschii has one ovule per flower and anther length is positively correlated with the numbers of pollen grains per anther30. Therefore, outcrossing potential  is expected to be lower in TauL1 than in TauL2 and TauL3. In fact, TauL1 may represent an ecotype that prefers disturbed habitats where strong selfing  is adaptive32. This view is consistent with the fact that TauL1 accessions often show disturbed-habitat-related phenotypes such as high seed production ability, short vegetative growth span, and increased tolerance to abiotic stress33. In addition, Ae. tauschii expanded its distribution range from the Transcaucasus-Middle East region to Central Asia through migration of TauL127. In a scenario where strong autogamy is a derivative state, the low anther length differentiation in TauL1 may suggest that the high-to-low shift in outcrossing potential might have occurred in the western part of the species range prior to the eastward migration on Table .1 hybrids show no or modest reduction in fertility34. Thus, pre-pollination barriers may be the major mechanisms for the intraspecific reproductive isolation of TauL1 from TauL2. The low outcrossing potential of the TauL1 accessions might be a pre-pollination mechanism that reduces the amount of gene flow between TauL1 and TauL2 under natural conditions. Furthermore, the outcrossing potential might be involved in the interspecific reproductive isolation between Ae. tauschii and T. turgidum by limiting the likelihood of the contact between Ae. tauschii pollen and T. turgidum pistils. In this case, the pre-pollination reproductive isolation caused by the low outcrossing potential would be stronger between TauL1 and T. turgidum than between TauL2 and T. turgidum.The strength of post-pollination isolation between TauL1 and TauL2 is very weak, and therefore, their FT. turgidum are genetically diverse. Nevertheless, only one tester was used in the cross experiment. For this reason, care must be taken when interpreting crossability patterns. Crossability with the tester was lower for TauL1 than for TauL2 accessions  for the female progenitor of common wheat35. Accordingly, the present study elucidates on the influence of Ae. tauschii’s ability to cause post-pollination reproductive isolation in the hybridisation with T. turgidum on the evolution of common wheat.The cultivars of ons Fig. ; Table 315. However, the reason why the ancestors of TauL1 were not involved, although the geographic range of this lineage included the probable region of origin of common wheat, is not clear. One hypothesis is that the ancestral TauL1 was reproductively isolated from T. turgidum. This hypothesis is supported by differences between intraspecific lineages of Ae. tauschii regarding the expression of pre- and post-pollination barriers when crossed with T. turgidum: TauL1 expressed the examined barriers more strongly than TauL2 when crossed with the tester. Moreover, hybrid incompatibility phenotypes are more frequent in the tester-TauL1 F1 hybrids than in the tester-TauL2 F1 hybrids36, whereas the extent of unreduced gamete production does not markedly differ between the tester-TauL1 and tester-TauL2 F1 hybrids15.Molecular genetic evidence indicated that the allopolyploid speciation of common wheat involved the ancestors of TauL2 and TauL3T. turgidum. Similarly, post-pollination reproductive isolation caused by crossability reduction and frequent expression of hybrid incompatibility might have been strong between the ancestors of TauL1 and T. turgidum. Accordingly, the ability of Ae. tauschii to cause reproductive isolation in the hybridisation with T. turgidum might have markedly influenced the allopolyploid speciation of common wheat by inducing lineage-associated patterns of gene flow.The ancestors of TauL1 were probably strong selfers, like the current TauL1, and shed a reduced amount of pollen. Thus, the low outcrossing potential of the ancestral TauL1 might have acted as a pre-pollination barrier and limited the likelihood of their hybridisation with Ae. tauschii pollen grains’ arrival on the T. turgidum pistils is not known. In flowering plants, pre-zygotic barriers often contribute more to total reproductive isolation than post-zygotic barriers37. The outcrossing potential, possibly in association with other pre-pollination barriers such as niche differentiation, might have critically contributed to the exclusion of ancestral TauL1 from the allopolyploid speciation of common wheat.Outcrossing potential reduction in TauL1 was 0.24 as compared with TauL2 [1-(TauL1 mean anther length/TauL2 mean anther length)]. Crossability reduction in TauL1 was 0.71 as compared with TauL2 [1-(TauL1 mean crossability/TauL2 mean crossability)]. The reduction in the post-pollination barrier was larger than in the pre-pollination barrier. However, the obtained values do not necessarily indicate that crossability contributed more than outcrossing potential to the exclusion of ancestral TauL1 from common wheat’s speciation. This is because the extent to which the reduction in outcrossing potential lowers the actual frequency of 39. As this might be the case for Ae. tauschii, polymorphism in the reproductive isolation of the parental species may influence the patterns of allopolyploid speciation when this is genealogically and/or geographically structured. Thus, the present study highlighted that the structure of reproductive isolation within the parental species might have an important role in shaping the patterns of plant allopolyploid speciation. Clearly, a better understanding of the genetic and ecological basis of plant allopolyploid speciation requires approaches that incorporate the reproductive isolation structure of the parental species.In plants, reproductive isolation mechanisms are often polymorphic within speciesT. turgidum. The PCA based on mean anther length and crossability data showed that TauL2 and TauL3 have higher potential for the natural hybridisation than TauL1  were pollinated. Usually, pollen of one matured anther was applied to one or two pistils, and crosses used one to 15 tester spikes (median: two spikes) per Ae. tauschii accession . Spikes were immediately rebagged after pollination and ripened. No gibberellic acid solution was applied to the embryos after pollination. The same person performed emasculation and pollination to reduce the effect of possible technical idiosyncrasies. After harvest, the seeds set in each spike were counted and crossability was calculated for each Ae. tauschii accession as the number of well-developed seeds set divided by number of pollinated florets. Thus, both pre-zygotic  and post-zygotic  mechanisms might have influenced crossability values. The crossability data were compiled from published and unpublished observations  (Supplementary Data 42. The lmer function of the lme4 package was used in anther length comparisons to fit a fixed effect (lineage) and random effects (accession and spikelet nested within accession) with a Gaussian error distribution assumption based on a maximum likelihood estimation method. Comparisons were performed for 193 accessions. In the analysis of crossability differences, the glmer function of the lme4 package was used for fitting a fixed effect (lineage) and random effects (accession and spike) with a specific binomial error distribution on a logit link function. The significance of the departure of the alternative model from the null model was examined by a likelihood ratio test using the anova function. The marginal R2 and conditional R2 values of the alternative models were calculated using the r.squaredGLMM function of the MuMIn package for R ver. 3.344.Differences between lineages regarding anther length and crossability were analysed by a model selection approach, based on LMM (anther length comparison) or GLMM (crossability comparison). These analyses were only performed for the TauL1 and TauL2 accessions, because TauL3 contained a small number of accessions. In each anther length and crossability analysis, we compared an alternative model that included the lineage as an explanatory variable to a null model that included no explanatory variable, using the lme4 package for R ver. 3.3P < 0.05.The intralineage relationships of anther length and crossability with the longitude of the accessions’ localities were analysed using LMM-based (for anther length) or GLMM-based (for crossability) model selection approaches. The three accessions with missing anther length data were excluded from the LMM-based analysis. In addition, six TauL1 accessions  from far-east adventive localities were excluded from both LMM-based and GLMM-based analyses. Analytical procedures were identical to those described for the between-lineage comparisons, except that longitude, instead of lineage, was entered as a fixed effect into the alternative model structure. The bobyqa (for TauL1) and Nelder-Mead (for TauL2) optimizer options were invoked in the GLMM-based analysis. In all test procedures, differences were considered significant at 46.A PCA based on the among-accessions correlation matrix was performed using the prcomp function of R ver. 3.3. A map showing the spatial distribution of the PC1 scores was created based on the geographic coordinates of the accessions, using a spatial dataset obtained from DIVA-GIS ver. 7.5.0 and the ggplot2 package for R ver. 3.3All data generated or analysed during this study are included in this published article (and its Supplementary Information files).Supplementary InformationDataset S1Dataset S2Dataset S3


Fibromyalgia syndrome (FMS) is a chronic condition with unknown aetiology. The pathophysiology of the disease is incompletely understood; despite advances in our knowledge with regards to abnormal central and peripheral pain processing, and hypothalamo–pituitary–adrenal dysfunction, there is no clear specific pathophysiological therapeutic target. The management of this complex condition has thus perplexed the medical community for many years, and several national and international guidelines have aimed to address this complexity. The most recent guidelines from European League Against Rheumatism (EULAR) (2016), Canadian Pain Society (2012), and The Association of the Scientific Medical Societies in Germany (AWMF) (2012) highlight the change in attitudes regarding the overall approach to FMS, but offer varying advice with regards to the use of pharmacological agents. Amitriptyline, Pregabalin and Duloxetine are used most commonly in FMS and though modestly effective, are useful adjunctive treatment to non-pharmaceutical measures. Fibromyalgia syndrome (FMS) is a chronic condition characterised by generalized body pain, fatigue, sleep disturbance, impaired cognition, and anxiety with unknown aetiology. Potential causes include genetic, neurologic, psychologic, sleep and immunologic factors . FibromyThe first clinical criteria for the diagnosis of FMS was set in 1990 by the American College of Rheumatologist (ACR) . It was Despite our greater understanding of the disease, there is no definitive treatment for FMS and various guidelines for treatment exist, which are at time contradictory in their advice. The approach to managing FMS has evolved over recent years, as reflected by recently updated guidelines published by European League Against Rheumatism (EULAR) . IncreasIn this paper, we will review the most recent national and international guidelines for the management of fibromyalgia, with a particular focus on the pharmacological agents. We will set out the evidence for the use of each class of pharmacological agent derived from each guideline, and form a synthesis of the evidence with the aim of assisting clinicians in gaining an overview of each intervention based on the agent, rather than divided by guideline. The process and quality of the guideline drafting process will also be assessed.The EULAR 2016 guidelines  were devThe 2012 Canadian guideline ,8 was deThe German guideline  was deveThe comparison of the categorisation of the evidence and recommendations of the three guidelines is listed in Each of the three guidelines was interrogated for its source data, and the levels of evidence for each pharmaceutical agent/class evaluated based on the included articles. Unless stated, the guidelines used systematic review articles and meta-analysis as the basis of their recommendations, although the robustness of these reviews and the quality of the included studies was variable. The summaries of the article are included in Amitriptyline (AMT) is a tricyclic antidepressant known to inhibit both serotonin and noradrenalin reuptake, and has long been used for the management of neuropathic pain and FMS. The AWMF guidelines assessed 12 studies in their meta-analysis, whereas the Canadian guidelines examined only two systematic reviews, whilst EULAR examined five review articles .AMT has received a strong recommendation from AWMF (10–50 mg/daily), while the EUALR guidelines suggest only low dose may be beneficial, although with a high degree of consensus. The Canadian guidelines take a much more a general approach to AMT, and recommend that all categories of antidepressants, including SSRI and NSRI may be used for the treatment of FMS depending on the individual efficacy of the drug, physician’s knowledge, patient’s characteristics, and cost. Nishishinya et al. also concluded that AMT 25 mg/day improved pain, sleep and fatigue at 6–8 weeks with no evidence for 50 mg/day .n = 1481 to 3334) and one clinical trial on gabapentin (n = 150). EULAR recommends use of PGB (weak for), and gabapentin for research purposes only.Pregabalin (PGB) and Gabapentin (GBP) are both structurally similar to the neurotransmitter γ-aminobutyric acid (GABA), although neither drug has activity in GABA’s neuronal system. Their analgesic effect is linked to their ability to bind to voltage-gated calcium channels in the central nervous system (CNS) . An eighUse of PGB and GBP may be limited by their side effect profile such as dizziness, somnolence, weight gain, peripheral oedema, and negative neurocognitive effects .Serotonin (5-HT) and noradrenalin have been implicated in the mediation of the descending pain inhibitory pathways , which hDuloxetine (DLX) has a five-fold stronger effect on serotonin than on noradrenalin . AWMF anMilnacipran (MLN) has three-fold stronger effect on noradrenalin than serotonin. It is recommended by EULAR (seven systematic reviews) and has been shown to be effective ,24,25,26A recent Cochrane review concluded that there was no unbiased evidence with regards to superiority of SSRIs to placebo in treating the key symptoms of fibromyalgia , however they might be considered for treating depression in this group of patients . NationaUse of strong opioids has been discouraged in the treatment of FMS. There is a deficit in opioid mediated descending anti-nociceptive activity in patients with FMS, with increased level of endogenous opioids in the CSF  and decrTramadol is a weak opioid with combined μ-receptor agonist and 5-HT and norepinephrine reuptake inhibition activity . It is tCyclobenzaprine is a centrally acting muscle relaxant which is structurally related to TCA, and which was first developed as an antipsychotic therapy . The EULCannabinoid molecules have been shown to have analgesic properties as well as sleep promoting affects , hence tUse of NSIADs for the management of FMS symptoms are not recommended by EULAR and AWMF. These recommendations come from a small number of studies . A recenDiscordance between the guidelines on recommendations for pharmacological treatments for of FMS is primarily due to lack of high-quality randomized control trials in FMS, hence the guidelines rely on lower-quality evidence and expert consensus. Perhaps most tellingly, the latest guidelines all recommend non-pharmacological therapies, such as exercise, with a greater level of confidence than pharmacological agents, perhaps in part due to low risk of side effects and the generic health benefit of exercise. Despite this, the pharmacological treatment of FMS has an acknowledged role, especially where symptoms of or a concurrent diagnosis of depression is prominent.One potential source of divergence in the guidelines in in the difference between their respective committees. The EULAR committee is formed of experts from 12 European countries with different health care systems, populations needs, and to some degree availability of therapies. EULAR also focuses mainly on systematic reviews , which provide the highest level of evidence, although narrowing the breadth of the guidance. AWMF, however, uses clinical trials as well as the systematic reviews, thus potentially capturing a greater number of admittedly lower quality studies. The Canadian guidelines uses original studies, systematic reviews, and evidence-based guidelines, and was perhaps the broadest approach to evidence gathering, although the number of included studies was actually less than that of the AWMF guidelines.Further to the methodological differences between the guidelines, the divergence of the guidelines is also compounded by the variability with regards to the licensing of drugs in FMS. In the US, the FDA approved medications for FMS include duloxetine, MLN, and PGB, whereas opioids are not approved for treatment of fibromyalgia. Only PGB and duloxetine had Health Canada approval for management of FMS, and the use of other drugs is effectively “off-label” (use the guidance ref on line). In Germany, no drug is specifically licensed for FMS. Each of these factors may influence the prescribing patterns of clinicians managing patients included in the contributory data, and in the future of clinical practice.Additionally, the guidelines do not offer information based on the outcomes for each of the six OMERACT core domains  , which wThe guidelines are however all in agreement that therapy should be tailored to the individual needs of the patient, and that non-pharmacological therapy should be encouraged in the first instance. This is again reiterated in the Canadian guidelines, where a shift to community based care  is emphasized, also highlighting the fact that the ACR diagnosis guidelines (1990 and 2010) were mainly designed for research purposes.EULAR recommends using DLX, PGB, or tramadol (in combination with paracetamol) in severe pain, and that AMT, cyclobenzaprine, or PGB should be considered at night for sleep problems. This is in contrast to the Canadian guidelines, which make only general recommendations on groups of drugs, rather than focusing on a specific agent. Despite the guidelines stating level 1 evidence for antidepressant medications  and anticonvulsants, it does not provide enough information to the prescriber with the regards to the choice of medication, optimum dosage, and the duration of therapy.The AWMF (2012) guidelines recommend AMT as a first-line pharmacological treatment, with duloxetine as a second-line treatment in patients who cannot tolerate AMT and have concurrent depressive disorder. The AWMF guidelines are also more explicit with regards the use and duration of the pharmacological therapy; recommending a trial treatment of at least 6 months, after which, if no effect is observed, the therapy should be stopped. This is based on the maximum duration of RCT study with AMT, Duloxetine and Pregabalin being 6 months .Canadian 2012 guidelines, as well as shifting the diagnosis and management of FMS to the primary care, support the use of combination of non-pharmacological and drug therapy in fibromyalgia. However, their recommendation with regards to the pharmacological therapy is vague and depends heavily on the expertise of the reader.Despite advances in the understanding of the pathophysiology of FMS, the pharmacological management has remained complex and poorly evidenced. What is clear is that an individualized approach to patient care is imperative, and that pharmacological therapy should be considered as an adjunct to non-pharmacological intervention. Where medication is deemed necessary, it should be targeted specifically towards the specific symptoms of the FMS in the individual patient, and if no improvement is observed after a reasonable trial period, should be discontinued. It is also important to note the risks of side effects of these medications, which may add to the complexity of the picture . There i


Training of lay-rescuers is essential to improve survival-rates after cardiac arrest. Multiple campaigns emphasise the importance of basic life support (BLS) training for school children. Trainings require a valid assessment to give feedback to school children and to compare the outcomes of different training formats. Considering these requirements, we developed an assessment of BLS skills using MiniAnne and tested the inter-rater reliability between professionals, medical students and trained school children as assessors.Fifteen professional assessors, 10 medical students and 111-trained school children (peers) assessed 1087 school children at the end of a CPR-training event using the new assessment format. Analyses of inter-rater reliability  were performed.n = 889). The number of comparisons between peer-peer assessors (n = 303), peer-professional assessors (n = 339), and peer-student assessors (n = 191) was adequate to demonstrate high inter-rater reliability between peer- and professional-assessors (ICC: 0.76), peer- and student-assessors (ICC: 0.88) and peer- and other peer-assessors (ICC: 0.91). Systematic variation in rating of specific items was observed for three items between professional- and peer-assessors.Overall inter-rater reliability of the summative assessment was high (ICC = 0.84, 95 %-CI: 0.84 to 0.86, Using this assessment and integrating peers and medical students as assessors gives the opportunity to assess hands-on skills of school children with high reliability. According to the International Liaison Committee on Resuscitation (ILCOR)-guidelines of 2010 the most important determinant of survival from sudden cardiac arrest is the presence of a trained lay rescuer who is ready, willing, and able to act [All over the world initiatives aim to increase the ability and awareness of potential lay rescuers. Initiatives focus on providing adequate training material and recommend to introduce basic life support (BLS) training in schools . DiffereWe designed a study to improve the feasibility to assess BLS skills at schools. By including medical students and trained school children (peers) as assessors we reduced the personnel requirement of professional assessors.The two aims of the study were to develop a context-appropriate assessment of hands-on BLS skills of school children at schools and to test the inter-rater reliability when using professionals, medical students and trained school children as assessors.www.einlebenretten.de”) provided by a physician. Part two consisted of 45 min hands-on training in basic life support (BLS) in groups of 10–14 school children with two instructors using “MiniAnne” . Every group used 4 MiniAnne mannequins and every child practiced all skills evaluated with the formative-assessment in two-rescuer-scenarios. The third part was the “Assessment”.This validation study was associated with a study investigating the effectiveness of peer-led hands-on BLS training for school children compared with professional-led training . The stuFor the practical assessment, we used a standardised three minutes/two-rescuer scenario assessed by a checklist. The examinees were supposed to perform the initial check for responsiveness and breathing and start CPR. They were supposed to tell an assistant rescuer to call the emergency medical service (EMS) and get an automated external defibrillator (AED) and attach it to the mannequin. The assistant rescuer was a child assessed immediately before. We set up numbered assessment-sites (up to eight), each with a MiniAnne and two examiners. The assessors were mixed based on alphabetical lists. We classified professionals and medical students as reference-assessors and tried to combine one reference-assessor with one peer-assessor. At some schools there were more assessment-sites than professional assessors. In this case we used assessment pairs consisting of two peers. If there were more professional assessors we combined one professional and one student or two professional assessors. The school children were randomly assigned to the assessment-sites, read the case vignette, and started the assessment upon an acoustic signal.The assessors judged independently based on a structured assessment checklist consisting of 15 items. All school children received feedback about their performance based on 15 items (formative assessment) from a professional-instructor after their assessment. Eight items of this assessment were relevant to either “pass” or “fail” the assessment. These eight items (summative assessment) were used to compare training outcomes between the peer-led and professional-led training.Trained school children (peers), professional instructors and medical students acted as instructors and assessors at the school event. Peers were school children of the participating schools who were trained beforehand. The training of the peers included two three-hour training sessions provided by two anaesthesiologists who were experienced in instructing medical students in BLS. The first session focused on a knowledge-based access to cardiac arrest and resuscitation as well as hands-on training in BLS. During the second session the peers were taught the 4-step-approach   and pracThe potential peers were recruited by their teachers. Participation was offered to all school children of a class/course by their teachers. The school children participated voluntarily.The medical students were in their final year of study which consists of four months in internal medicine, four months in surgery and four months in a subject of their choice. All students in our study had chosen their elective in the clinic of anaesthesiology of the University Hospital Hamburg-Eppendorf and participated voluntarily in the educational events for the school children.The professional instructors were recruited from the staff of the Clinic of Anaesthesiology at the University Hospital Hamburg-Eppendorf. All of the professionals were experienced in training others in BLS and also volunteered to participate in the training at the schools. All assessors were trained up to 2 weeks in advance to use the formative assessment-checklist. The training was a group-training for 5–20 assessors. BLS skills were rated during three CPR-scenarios by all assessors and a reference rater (ERC-ALS-instructor). The individual results were discussed and aligned with the results of the reference-rater (frame-of-reference training) .BPM Tap by Ignace Maes and free application for i-phone: BPM_counter by Torsten Klaus). Hand-activated stop-watch measurements were used to measure the lengths of time until starting chest-compression and the lengths of time of interruptions of chest-compression.We developed a formative assessment-checklist Table  based onWe developed a summative assessment-checklist to score the outcome of the educational event and to compare the groups. Targeting a binary outcome for every examinee (pass/fail), the trial leader reduced the items stepwise generating pass-relevant items. Quality was ensured with respect to the two most common flaws in examinations, “construct underrepresentation” and “construct irrelevant variance” . ConstruConstruct irrelevant variance was achieved by excluding items with unreliable scoring or representing not mainly task-specific knowledge and skills of the examinee. The final checklist contained 15 Items of the formative assessment and eight of those, Table  were relWe used the intraclass correlation coefficient (ICC) to assess inter-rater reliability of the assessment between the different groups of assessors. Statistical analyses were performed with SPSS Version 21  using a two-way-random-model . ICC is rated moderate for values greater 0.5, strong for values greater 0.7, optimal for values greater 0.8 and excellent for values greater 0.9. To test independency of failure-rates from the assessor, we performed McNemar’s test with GraphPad Prism 5  to compare failure-rates of every item between the different assessor-groups assessing the same school children. With respect to multiplicity, we used the Bonferroni-Method and the two-sided significance level was set at 0.6 % (0.05/number of tests: 0.05/8 = 0.0063) for every hypothesis to keep overall type I error at 5 %. Power-calculation was performed for the associated study and determined the required sample size of 371 school children per group  [One hundred eleven peer-instructors, 10 medical students and 15 professional trainers evaluated 1087 school children with this checklist. Demographic data of the assessors are presented in Table n = 889, 95 % CI: 0.82 to 0.86). The number of comparisons between peer-peer assessors (n = 303), peer-professional assessors (n = 339), and peer-student assessors (n = 191) was high and demonstrated strong inter-rater reliability between peer- and professional assessors , optimal inter-rater reliability between peer- and student-assessors  and excellent inter-rater reliability between peer- and other peer-assessors . The comparison student versus professional demonstrated moderate inter-rater reliability  with a sample size of 44. The sample size for the comparison professional versus professional and student versus student was very low following inter-rater reliability . Agreement between peer- and student assessors was high. Disagreement (assessment judged as “failed” by only one of the two assessors) occurred comparably often in the group of peer- and student-assessors. The scoring of peer- and professional-assessors differed in 65 of 339 ratings. Peers rated the examinations of the school children more often as “failed” (n = 43) compared to professionals (n = 22). Professionals, compared to medical students, rated the examination of school children more often as “failed” .Overall inter-rater reliability Table  accordinp-value of McNemar’s test for “check responsiveness” = 0.004, “correct compression point” = 0.001 and “no-flow-time” < 0.001). The school children failed those items more often, when rated by peers than by professionals. Failure-rates of the other items did not differ between peer- and professional-assessors  and optimal (ICC > 0.8) to excellent (ICC > 0.9) inter-rater reliability between peer- and other peer- or student-assessors, according to intra-class correlation coefficient (ICC) and the 95 %-confidence interval of ICC.School children were more likely to pass the exam when rated by a professional than by a peer because they failed the items “check responsiveness”, “correct compression point” and “no-flow-time” more often, when rated by peers than by professionals. These results are in line with the current literature indicating examinees are more likely to pass the exam, when rated by professional-instructors than by (student-) peer-instructors , 12.Though we can’t explain the differences on item level based on current literature, we assume that the peers rated the items “check responsiveness”, “correct compression point” and “no-flow-time more “text-book” based on definitions and metrics. Whereas professionals may have used their medical experience to judge items “yes” that aren’t necessarily “correct” based on guideline definitions but work out in a real-life.Failure-rates of the school children rated by medical students and professionals did not differ significantly for the whole assessment. There was no systematic scoring difference between these two groups of assessors on single item level. Neither could we demonstrate a significant, systematic scoring-difference between student- and peer-assessors.This study developed a context-appropriate assessment of hand-on BLS-skills of school children. It is reliable using professional instructors, medical students and peers as assessors. Additionally, the new assessment meets other performance-indicators like feasibility, cost effectiveness, specificity and fidelity . Using lThe analysis of inter-rater reliability between the three assessor-groups  demonstrated peers and medical students are capable of reliably assessing BLS skills. We could not show any systematic rating-difference between professional- and student-assessors. But failure-rates of school children rated by peers were higher. The severe examination by peers led to a low-risk of school children passing the exam unjustified when rated by peers.This study adds two important aspects to the current literature:There are several limitations of the study. First of all the dropout rate was high. One hundred ninety-eight assessments could not be included in the analysis because school children forgot their written informed consent or were only evaluated by one assessor. We cannot estimate the consequence of the missing assessments on the results. The development of the assessment-checklist and the assessor-training was performed by one professional-trainer only. Probably a consensus-process like the DELPHI-process  would haThe CPR performance assessed with MiniAnne was not compared to performance assessed with another instrument of assessment e.g. high-quality-feedback mannequin, thus allowing to estimate of dependency of pass-rates and reliability of assessors from the assessment instrument. The “clicking” sound of the MiniAnne did not only offer feedback for the assessors but also for the examinees and probably improved the observed compression quality. The influence of supportive behaviour of the assistant rescuer on performance of the examinee during the two-rescuer scenario could be reduced by using a “standardized” role-play for the assistant rescuer.The summative assessment scores performance of compression-only CPR. According to current literature, we focused on chest-compression as the key determinate to improve survival. We excluded mouth-to-mouth ventilation from the summative assessment, because in the city of Hamburg the emergency medical service arriving time is in 79 % within 8 min and cardiac origin of arrest is very high . In diffUsing this assessment and integrating peers (trained school children) and medical students as assessors gives the opportunity to assess hands-on skills of school children, with high reliability.


Evidence-based public health (EBPH) is defined as public health decision-making based on current best evidence. Debates about the latent tuberculosis infection control program suggested by the Korea Centers for Disease Control and Prevention in 2017 highlight the need to promote EBPH. Three strategies have been proposed, including providing necessary evidence by evaluating policy-evidence gaps; delivering high-quality, relevant, and timely evidence by conducting systematic reviews and adapting public health guidelines; and making value-driven decisions by training and educating public health policymakers. Mycobacterium tuberculosis infections (LTBI) and that prophylactic anti-TB drugs would be provided to those with a positive test result in an attempt to establish a TB-safe country by reducing the incidence of TB to levels comparable to those of developed countries [In commemoration of Tuberculosis (TB) Prevention Day, on March 24, 2017, the Korea Centers for Disease Control and Prevention (KCDC) announced that 18.78 million people, including those in collective facilities, vulnerable populations, and individuals undergoing health examinations would be screened for latent ountries .This is an example of the importance of evidence for scientific validity among the various factors to consider when deciding to conduct public health projects at the national level. Evidence-based public health (EBPH) is based on the use of valid evidence to inform public health-related decision-making . The aboIn 1977, Jenicek  defined Based on these definitions, EBPH and EBM differ in the following three ways: First, EBM focuses on individual patients, whereas EBPH focuses on community and residents . Second,In 2000, Sackett et al.  redefineUnlike the definitions of EBPH that correspond with that of EBM, Brownson et al.  defined The common keywords in the three definitions of EBPH by Jenicek , KohatsuIn order to obtain the relevant evidence required for EBPH compared to EBM, the following differences should be considered. First, there is a difference in the study designs that generate evidence. The results of randomized controlled trials which are mainly used in EBM can be rarely utilized in EBPH; thus, the results of ecologic studies, cross-sectional studies, quasi-experimental studies and time-series analyses that have lower scientific power are often used in EBPH ,13. Furthttps://www.nice.org.uk/) in the UK and the National Evidence-based Healthcare Collaborating Agency  in Korea are representative organizations that pursue EBPH. Second, it is necessary to build a knowledge base to continuously provide information on various risk factors that threaten public health [http://www.cochranelibrary.com/) and Health Research Policy and Systems  are representative knowledge base agencies, while the National Center for Medical Information and Knowledge  under the KCDC in Korea has the same function. Third, it is necessary to actively use systematic reviews (SR) [Because of these differences, it is necessary to obtain data from a number of resources and use special strategies to obtain evidence for EBPH implementation . These sc health . The Cocews (SR) ,22 in EBews (SR) .Because most evidence generated for EBPH is based on the findings of studies conducted in Western countries , evidencTricco et al.  summarizIn summary, good communication between researchers or evidence generators and administrators or decision-makers should be established first in order to promote EBPH. Regarding the various controversies and subsequent changes in the selection of targets for the 2017 LTBI screening program proposed by the KCDC , the KCDMeanwhile, communication between researchers and administrators is more difficult in EBPH than that between patients and medical professionals in EBM  because