By application of our modified rules, specificity of ViraChip IgM is significantly increased , and sensitivities of recomBead IgG and IgM are also significantly improved . Further improvement of sensitivity by the rating of strong singular IgG reactions against VlsE as positive can also be shown for recomBead. IgG/IgM result combinations must be interpreted as a function of the assumed disease stage, and the best combinations differ for the various assays. Application of our proposed interpretation criteria significantly improve the discriminatory abilities of two assays; however, this must be confirmed with other data sets. Recommendations from Scientific Societies should be updated as may be necessary.Case-control study for the evaluation of innovative test formats for second-tier testing for the serodiagnosis of Lyme borreliosis (LB). A head-to-head comparison was performed with the test systems ViraStripe, SeraSpot, ViraChip, and recomBead. Serum samples from 62 patients  and 91 controls  were tested. For ViraChip and recomBead, optimised interpretation criteria were developed for both IgG and IgM. The most important modification for the proposed interpretation criteria for ViraChip is the interpretation of strong (> 2.5-fold above cutoff) singular IgG reactions against VlsE as positive. This significantly improves sensitivity (32 to 85%,  Borrelia burgdorferi sensu lato group (B. burgdorferi s.l.) [B. burgdorferi sensu stricto (B. burgdorferi s.s.), B. garinii, B. afzelii, B. spielmanii, and B. bavariensis are considered pathogenic for humans [Mycoplasma pneumoniae as well as autoimmune diseases [Borrelia strains and the successive appearance of antibodies against different antigens in the course of the infection further contribute to complexity [Lyme borreliosis (LB) is the most frequent tick-borne disease in the northern hemisphere and is caused by different species of the ri s.l.) , 2. In Er humans . The disr humans . The diar humans –5. Howevr humans , 3. Unspdiseases , 7. The mplexity , 8–12. Tmplexity .In Europe, as well as in the USA, in most countries a two-step approach is recommended for serological testing , 14, 15.B. afzelii strain PKo and also for immunoblots with recombinant antigens representing this antigen composition  p43, p30, p21, and p14 are important as well [Commercially available assays use a variety of antigen compositions including also antigen homologues derived from different strains. The most important antigens are p100/83, p58, p41 , p39 (BmpA), OspC, DbpA (Osp17), VlsE . For whoIn an attempt to facilitate workflow in high throughput laboratories, innovative test formats like multiplex bead or spot array technologies have been developed to replace strip immunoassays.However, up to now, the introduction of these assays into routine diagnostics is deferred, since only limited evaluation data is available for these assays , 18 and The initial aim of our preliminary study was an evaluation of a bead-based multiplex assay  and two spot array tests  by a head to head comparison with the so far performed line immunoassay  prior to the introduction of one of these test systems into routine diagnostics. Therefore, panels of selected sera from patients with LB meeting European case definitions , as wellBorrelia IgG in CSF, however, no CSF/serum IgG antibody index could be calculated due to the negative Borrelia IgG in serum. All other patients with acute neuroborreliosis showed lymphocytic pleocytosis in CSF, CSF/serum Borrelia IgG antibody indices > = 2.0, and typical clinical symptoms [For the determination of sensitivities, only remnant serum samples from patients with symptoms conform to European case definitions for LB  were tessymptoms . The groThe control group for the determination of specificities comprised 93 samples. Fifty-seven sera were obtained from healthy persons (mostly staff), who negated tick bites, erythemas, neurological symptoms, or joint disorders. A total of 31 sera from patients with diseases potentially leading to cross-reactions  and 5 sera from patients with unspecific symptoms not consistent with LB were also included.After the classification of patients and controls in the respective diagnosis groups, sera were made anonymous by the assignment of new sample identification numbers not connected to patient identity.Borrelia ViraStripe IgG, IgM ; comparison assays: recomBead Borrelia IgG, IgM 2.0 , SeraSpot Anti-Borrelia-10 IgG, IgM , and Borrelia ViraChip IgG, IgM (VIRAMED Biotech AG). Devices and assessment systems were provided by the respective manufacturers. Sera were tested and interpreted according to the manufacturers’ instructions; however, interpretation of the line immunoassay ViraStripe was slightly modified according to in-house criteria including a borderline zone for both IgG and IgM.The following assays were performed: routine assay: Borrelia IgG, IgM 2.0 respective is based on the Luminex system: highly purified, recombinant Borrelia burgdorferi s.l. antigens  are applied separately to different microparticles (beads) with differing fluorescence codings. Antibodies against individual antigens are recorded separately from each other in one solution. The average fluorescence intensities of all individual antigen/antibody reactivities of each sample are compared with the reactivities of an incubation control and a cutoff index (COI) is calculated via a batch-dependent limit value by the software recomQuant. The reactivity against an individual antigen is interpreted as negative for a COI < 0.67, borderline if 0.67 < = COI < 1.00, and positive for COI > = 1.00. The test results are obtained by a point evaluation system for the individual reactions. Therefore, the corresponding point values in the case of borderline and positive results for individual antigens are added. For the development of optimised criteria, only the point evaluation system was modified (see below).The test principle of recomBead Borrelia-10 IgG, IgM respective is a spot immunoassay based on the use of Borrelia antigens printed in an array arrangement (spot array) directly on the bottom of each well of a 96-well microtiter plate. The following recombinant antigens are used for the individual spots: VlsE (B. afzelii), p39 (B. afzelii), p58 (B. garinii), p100 (B. afzelii), OspC (B. afzelii), OspC (B. garinii), OspC (B. burgdorferi s.s.), DbpA (B. afzelii), DbpA (B. garinii), and DbpA (B. burgdorferi s.s.). After test processing, wells with spots consisting of coloured immune complexes are scanned and interpreted automatically by the software Seramun SpotSight scan.SeraSpot Anti-Borrelia ViraChip IgG, IgM respective is an immunoblot in a microarray format carrying highly purified specific native antigens from B. afzelii (strain Pko) and B. burgdorferi s.s. as well as recombinant VlsE. Microarrays are spotted onto nitrocellulose and fixed at the bottom of each well in a standard microtiter plate. The analyte spots of the IgG test serve to detect antibodies against p83, p58, p43, p39, p30, p21, OspC, DbpA/Osp17, p14, and VlsE, whereas the arrays for IgM testing contain p41, p39, OspC, Osp17, and VlsE. Prior to scanning and automatic interpretation, the microtiter plates must be dried. The measured mean intensity of the calibrator controls is multiplied by the lot specific factor for each antigen. The resulting value is used as the cutoff for the assessment of the respective analyte spots (three spots per antigen). Triplets with a mean intensity equal to or higher than the cutoff (i.e. COI > = 100) are considered ‘distinct’ and are incorporated in the interpretation. For our modification of the interpretation criteria calculations with these COIs were performed.Testing of ViraStripe and recomBead was performed in August 2014, ViraChip and SeraSpot were tested with the same sera in July 2017. All tests were performed blindly. Sera were stored continuously at − 20 °C in the meantime. For the determination of the overall sensitivities, all diagnosis groups were included for the IgG assays, whereas for the IgM tests only data from patients with EM or LNB were evaluated. IgM tests were not performed for late LB due to lacking clinical relevance , 5.Sera with discordant results between ViraStripe and recomBead were retested with recomBead for confirmation of the results. All the sera were tested at least twice with different lots of ViraChip. Samples not reacting concordant were tested again with a third lot. Sera with discordant reactivity between ViraChip and SeraSpot were tested also with a different lot of SeraSpot. Reactivity was considered borderline for samples that reacted positive once and negative once in runs with different lots of SeraSpot.Tables with raw data of measured values for each single antigen for all tested sera were analysed for patterns typical for true positive and false positive results. For both recomBead and ViraChip, COIs were taken for this analysis.Sensitivities and specificities for a variety of presumed criteria were calculated and the criteria with the best discriminatory abilities were chosen. Since the optimised criteria for ViraChip focus on the uprate of singular strong IgG reactivity with VlsE, further calculations were carried out also for recomBead and SeraSpot with the subset of sera with singular VlsE-IgG reactivity.The ‘optimised criteria’ for recomBead were developed in 2014 basing on the two-band criterion for a positive IgG blot recommended by the German Society for Hygiene and Medical Microbiology  .n = 91) were not included in the counts. Two sums were calculated for each diagnosis group. The first sum denominated ‘common trunk’ consists of all combinations applicable for all assays. ‘Common trunks’ differ for diagnosis groups but not for assays. For the second sum for ‘best discrimination’ for each assay, the ‘common trunk’ was individually complemented with further combinations adding to sensitivity . Combinations included in ‘best discrimination’ are boxed in the table.Frequencies of all possible IgG/IgM result combinations were compared for patients and controls. The following acronyms were used: GpMp: IgG positive and IgM positive; GpMb: IgG positive and IgM borderline; GpMn: IgG positive and IgM negative; GbMp: IgG borderline and IgM positive; GbMb: IgG borderline and IgM borderline; GbMn: IgG borderline and IgM negative; GnMp: IgG negative and IgM positive; GnMb: IgG negative and IgM borderline; GnMn: IgG negative and IgM negative; and GpMne: IgG positive, IgM not evaluable . For further exploratory analysis, simple arbitrary rules and calculations were applied to describe discriminatory abilities of the different assays. Separate analyses were performed for EM and LNB. The results of the most important combinations for the detection of cases were added. In doing so, specificities equal or greater than 95% were required. Therefore combinations with more than 4 controls -curve) were calculated for comparisons of diagnostic performance since standardisation of specificities was not possible.GraphPad Prism version 6.01 was used for all calculations.Two sera of the potentially cross-reacting group were excluded from the analysis due to their strong reactivity in all IgG and all IgG and IgM tests, respectively. For simplification, the control group is presented without differentiation in healthy persons, cross-reacting sera, or patients with unspecific symptoms.After the determination of sensitivities and specificities by the assessment with the original interpretation criteria, optimised criteria were developed for both ViraChip and recomBead.In Table Table For SeraSpot, no improvement could be demonstrated by a modified assessment of singular VlsE-IgG reactions.Figure p < 0.0001; VC-orig 92 versus 32%: p < 0.0001).For IgG of both ViraStripe and VC-orig, the differences of sensitivities between these two evaluations are significant due to the high proportion of borderline reactions (ViraStripe 90 versus 42%: p < 0.0001) by the use of our in-house criteria and this was linked to a moderate deficit in specificity  only. Sensitivity of recomBead IgG was first improved from 53 to 66% , and then from 66 to 84%  without changes of specificity (100%). Specificity of ViraChip IgM could be raised from 89 to 97% (p < 0.05) with only a minor loss of sensitivity .The evaluation with borderline results included to negatives (shaded bars) showed that the sensitivity of ViraChip IgG was improved from 32 to 85% (p < 0.01) for RB-opt and RB-VlsE, respectively, and this was linked to an insignificant deficit in specificity (100 versus 98%) only. For recomBead IgM, sensitivity could be increased from 57 to 74% (p < 0.01) without deficit of specificity (96%).If borderline results are included to positives (white bars), for recomBead IgG, sensitivity was improved from 69 to 87 or 89%  shows specificities above 95% for both assessments. For all other IgG tests, borderline reactions must be interpreted as negative to achieve a specificity of at least 95%. recomBead has the highest specificity for IgM also . On the other hand, specificities of two other IgM assays are lower than 95% even if borderline reactions are interpreted as negative .The eight sera from patients with late LB reacted positive with all IgG assays.p < 0.01; LNB p < 0.0001), whereas the ‘best discriminations packages’ (summary of the boxed cells in the table) performed in a comparable range  with an exception for SeraSpot for EM with a sensitivity of 48% only . On the other hand, SeraSpot performed very well for LNB (GpMp 25/33).Table For EM, the combinations included for the ‘best discrimination’ differed in respect to the consideration of GpMn and GnMp. GnMp could not be included for SeraSpot and VC-orig due to unspecific reactivity (up to 9% of the controls). For LNB, for VC-ih and for RB-VlsE, the ‘best discrimination’ equals the ‘common trunk’. For the other assays, at least GbMp and GbMb had to be complemented for ‘best discrimination’. The highest AUCs were achieved for VC-ih for EM and with RB-VlsE for LNB.In this study significant improvement of performance of ViraChip as well as recomBead through the application of modified interpretation criteria could be demonstrated Fig. . Most noSince the number of tested sera was rather low in this preliminary study, all modifications were done with caution, e.g. only the grayzone was enlarged for VC-ih IgM and RB-opt IgM. The decision thresholds for the rating of singular VlsE-IgG as positive were also set in favour of high specificity for both VC-ih and RB-VlsE. Setting this threshold at 2.0 instead of 2.5-fold above the cutoff for ViraChip would lead to a gain of sensitivity of 3% (2/62 samples) and a loss of specificity of 1% (1/91 samples) in our data set.The classification ‘borderline’ plays a considerable role for the evaluations in this study. In the case of borderline results, a follow-up sample should be tested if necessary , 5. FurtThe discriminatory abilities of the different assays were analysed further by a combined evaluation of both IgG and IgM results in different stages of LB Table . The patVC-ih and RB-VlsE performed the best, but the application of individually modified ‘best discrimination packages’ showed no significant differences in sensitivities (and specificities) of all tests and AUCs in similar ranges (exception: SeraSpot for EM). This contrasting juxtaposition with arbitrary determinations was intended to demonstrate the ambiguous meaning of certain constellations.However, particularly this data is preliminary since the number of cases and controls is too small for such a differentiated analysis. The descriptive format of this analysis was chosen to reflect the routine situation showing combinations of results in the familiar format (What could this constellation mean?).For EM, GpMn was not included for ‘best discrimination’ for VC-ih. This reflects the higher background IgG reactivity and the higher IgM specificity of our in-house criteria see Fig. .The most critical combination is GnMp, i.e. isolated positive IgM with negative IgG. As demonstrated in the evaluations for both EM and LNB and as is widely discussed in the literature , 5–7, 23For the combined evaluation of tests, Dessau et al. constructed diagnostic scores by logistic regression of data from patients with LNB as well as controls , 24. ThiBorrelia IgG immunoblots, which require at least two bands for a positive result [In our study, the significant improvement of sensitivity of both ViraChip and recomBead IgG was due to the rating of strong isolated reactivities with VlsE as positive. The original interpretation criteria by the manufacturers follow the recommendations by the German Society for Hygiene and Medical Microbiology (DGHM) for the interpretation of e result . Howevere result , 22, 25.e result , 16. Howe result . In the e result . OptimisThe different rating of reactivities as a function of signal strength requires a second (higher) cutoff and sufficient precision in this measuring range. Even if not designed for quantitative analyses, the new test systems might meet this requirement; however, this should be studied further. All of them are processed and analysed automatically, and subjective visual reading is not even possible. In our evaluation, inter assay imprecision of VlsE-IgG was acceptable for the three tested lots of ViraChip IgG  and good for all runs within the same lot of recomBead IgG . This analysis included COIs between 0.5 and 3.0 or 50 and 300, respectively (detailed data not shown). Perhaps, a second calibrator for VlsE could be helpful.A prerequisite for an evaluation of diagnostic assays in a case-control format is the availability of well-defined samples. Our panel from LB patients comprises mainly patients with LNB, as these cases can be unambiguously identified by laboratory findings. Since EM is primarily a clinical diagnosis, most patients are not tested serologically, and the accessibility of sera from typical cases is restricted. However, EM is by far the most frequent manifestation of LB, and therefore should be represented adequately in the test panel. Atypical manifestations of EM constitute a considerable challenge for clinicians. Some authors do not recommend serological testing of these cases ; neverthB. afzelii strain PKo [Borrelia infections existing at least for months or reinfections show a typical broad IgG band pattern when tested with ViraStripe . With VBorrelia burgdorferi s.l. can never be completely ruled out in this group. In some cases, differentiation between unspecific IgG reactivity and past infection is not possible. The potentially cross-reacting sera were collected by laboratory findings only and the history and present or past symptoms of these patients could not be taken into account. Two sera of this group were excluded from the analysis due to a strong reactivity in all IgG and all IgG and IgM tests, respectively. Ten percent of the sera of the total control group were taken from patients with acute EBV or CMV infections. For the evaluation of IgM tests, this means even a higher challenge than in the routine situation.The control group should represent the challenges of the routine situations; consistency is critical and certain single samples can turn the balance. Therefore, high sample numbers are important. For the evaluation of IgG/IgM combinations, we required a specificity of at least 95%. Another approach would have been to intend a discrimination between background reactivity in the local population and active disease, however, this is also problematic to achieve. As far as possible, persons with suspicion of present or passed LB or noticed tick bites were not included in the group of healthy persons or into the group of patients with unspecific symptoms not consistent with LB. However, asymptomatic or passed infections with The strength of our study is the head-to-head comparison of innovative test formats in a case-control design as there is almost no such data in the literature. The main limitation is the low number of samples, especially in the control group.  Another limitation of our study is a certain preselection of sera due to positive or borderline IgG or IgM results with the screening ELISA . Our comparison was meant as an evaluation of the second step of the recommended two-tier testing and thus, included confirmatory assays only. As a matter of course, determined sensitivities in the group of patients with EM are higher as anticipated for unselected cases . This doFurther studies should be performed with larger data sets including also different stages of LB to confirm and refine our proposals. For the interpretation of singular VlsE-IgG as positive, appropriate cutoffs should be determined through ROC analyses. If two cutoffs might be used in the future, reliable inter-lot precision must be assured for both.Probably, fewer antigens would be sufficient to achieve high discriminatory power. Application of regression methods can weigh the role of the different antigens ; howeverThe development of test systems by commercial manufacturers is crucially hampered due to the restricted access to clinically defined sera. In the USA, a collection of well-defined samples is available from the CDC . The estOur preliminary study shows the potential for a significant improvement of innovative commercially available confirmatory tests. For the interpretation of IgG assays, a combination of the established rule ‘reactivity against at least two antigens’ (two-bands criterion) with the rating of isolated strong reactivity against VlsE as positive is favourable. However, further analyses with different and larger data sets are necessary. If our proposals could be verified, the specifications for confirmatory tests from the German Society for Hygiene and Medical Microbiology (DGHM) should be updated.


Furthermore, habitat composition and fallow characteristics were mapped in 2012. We calculated population trends, analyzed habitat associations and revealed the impact of fallow habitat characteristics on territory density. The proportion of fallows in the study site increased from 1.4% (1992) to 8.5% (2012). Population trends of six of 12 censused species increased significantly over the same time, four species showed no trend and trends of two species decreased. Seven species were analyzed in more detail, for five of them fallows were overrepresented around their territory center points compared to arable fields and grassland. The overall territory density of these five species was higher in small fallows which were not placed next to a wood and which held bramble rubus spp., shrubs and the tall‐growing forb goldenrod (Solidago canadensis and S. gigantea). Our study confirms that perennial fallows are a highly suitable option to support different farmland birds in arable landscapes. Yet, we recommend optimizing fallows through careful site selection and management, such that they are not established on shady locations and are structurally diverse by allowing brambles, shrubs, and tall‐growing forbs to occur. We suggest adapting the Swiss AES in this regard. Biodiversity‐related advisory services available for farmers could increase the probability that fallow options are implemented and managed properly for targeted species.Across Europe, patches of un‐cropped land  have been established and managed as part of agri‐environment schemes (AES) to counteract the decrease in farmland biodiversity. Various studies demonstrate a positive impact of such un‐cropped land on different taxa. However, there is potential to further improve the efficiency of fallow options for farmland birds. In a long‐term monitoring, 12 breeding farmland bird species and sizes of perennial fallows were recorded from 1992 to 2015 in a 6.1 km In this project region, perennial fallows were established, providing the possibility to study the relationship between farmland birds and characteristics of the fallows. Our questions were threefold: (a) Do abundances of priority bird species increase over the long‐term study period? We expect the trends to be more positive than European or Swiss bird indices, because of the comparably high percentage of fallows in the study region. (b) Do territories of priority farmland bird species contain more fallow area than expected according to the available habitat in the study site? Studies from other regions and countries revealed a preference for fallow habitat. (c) Which characteristics of fallows are related to territory density of priority farmland birds and is vegetation composition of the fallows related to territory density? Personal observations revealed that small shrubs, brambles, and goldenrod increase the attractiveness of fallows whereas a high amount of grass is less favored by priority bird species of open farmland.22.12) is dominated by intensively cultivated farmland  and some large gravel pits  and holds only a few small settlements and woods , which has a warm and dry climate . The study site  and goldenrod  occurred in different amounts therein.In this study, fallows were either sown wildflower areas/strips, rotational fallows, or naturally vegetated field margins, showing different characteristics of structural and floristical diversity. Wildflower areas/strips and rotational fallows are options of the Swiss AES .Every few years, they were partially mown to reduce the expansion of goldenrod, bramble, and shrubs. Their spatial configuration was quite diverse, most of them being linear strips of 10–25 m width; others were of rectangular (whole parcels) or triangular shape. Size ranged from 0.06 to 3.3 ha (median 0.36 ha). Their age ranged from zero to 22 years (median 13 years). The use of herbicides and pesticides was not allowed. All these fallows stand out from the surrounding arable fields by their high botanical and structural richness. They usually bordered directly on crop fields or meadows with no physical boundary in‐between and only 15 were adjacent to hedgerows. Some of them adjoined roads, gravel‐pits, ruderal areas, or small woods. The proportion of fallows relative to the area of the study site increased significantly from 1.4% in 1992 to 8.5% in 2012 . Eight of the species are listed as “Species of European Conservation Concern” (SPEC). All species are listed in the environmental objectives of the agricultural sector in Switzerland , a habitat mapping was conducted in 2012. Land‐use types of the whole study site were mapped between June 5th and August 10th, assigning each type to one of the habitat categories in Table 2.4All analyses were performed in R 3.3.1  using a Poisson distribution. Territory numbers (response variable) were tested against year and the second and third polynomial of year to account for a possible change in the population trend within the study period. The polynomials of year were removed from the full model if the BIC of the model with the polynomial(s) was less than 2 units lower than the BIC of the reduced model. Overdispersion appeared in the models of Corn Bunting and Common Quail; thus, for these species, we calculated a generalized linear mixed model (GLMM) instead, including observation number (unique number of each row in the data set) as a random factor. Model fit was assessed graphically by plotting the residuals against the fitted values and by plotting the residuals against each explanatory variable.We analyzed the association of birds with the different habitat categories in 2012 Table : Common n = 82) to structural variables as well as vegetation composition of each fallow. This was performed with a generalized linear model  using a Poisson distribution with log link function and the number of territory center points per fallow as dependent variable. Territory center points that did not fall into a fallow were counted as well if they were laying 10 m at most outside a fallow. Explanatory variables were size, age, distance to wood, and amount of shrubs, proportions of bramble, goldenrod, and grass. Because fallows were of different sizes, we included the logarithm of size (in ha) as offset in the model. Thus, we modeled territory densities (number of territories per ha) rather than absolute numbers of territories. The proportions of bramble, goldenrod, and grass were arcsine‐square root‐transformed and distance to wood was square root‐transformed. All explanatory variables were standardized with mean zero and SD = 1. No collinearity was found between the explanatory variables, as variance inflation factors were all <3 . We analyzed the relationship of territory density within each fallow  of the 12 yearly censused species in the study site increased significantly during our study . For four of the seven analyzed bird species  fallows ranked first before all other habitat categories, that is, fallows were strongly overrepresented in the pseudoterritories  of the fallows Table . Territo4Fallows are options of the national and local AES in Switzerland and are designed to promote biodiversity in arable landscapes. In line with former studies, we stress the importance of un‐cropped land like fallows and field margins for breeding farmland birds . In the same period, we measured a large gain in territory numbers of six of 12 bird species. These are species which are usually not increasing in Switzerland or elsewhere in Europe PECBMS , or if sFurthermore, five of the seven studied species  showed a significant association with fallows in the Compositional Analysis. They are typical species of open farmland interspersed with single shrubs , and vegetation composition  had the strongest influence on territory density. The effect of age and the amount of grass were less pronounced.We found a negative effect of fallow size on territory density per fallow. The same relation was found by Zollinger et al.  in westeThe nearer a fallow was established to a wood, the lower was its territory density of studied bird species. Fallows placed 200 m from the nearest wood had six times more territories than areas within 10 m of a wood Figure b. One poDue to sowing or seed pools in the ground, differing soil fertility, vegetation succession and management, fallows show quite a variety of vegetation composition and structural diversity. Our results show that the density of farmland bird territories increases with the proportion of bramble and the occurrence of shrubs in fallows. From experience, we assume that the proportion of bramble in fallows peaks in an optimum with high proportions of bramble being less favorable for most farmland birds. Although we did not find a quadratic effect of bramble, this may arise from the very few samples with high proportions of bramble . The effect of shrubs on territory density was quite variable  and therefore not significant. However, we consider it relevant because it was as strong as the effect of bramble. Its effect may depend on the structure and species composition of the shrubs and would be a topic for further work. It is already known that the Whitethroat favors bramble as breeding habitat, but it seems that some bramble in fallows enhances their attractiveness for several other bird species. Our model estimates an increase of 1.5 territories per ha fallow when the amount of bramble is enhanced from 0% to 10%. Furthermore, predicted territory density increases from 4.3 territories per ha in shrubless fallows to six territories per ha in fallows containing shrubs. Both, bramble and shrubs, are nesting habitat and provide shelter from bad weather and predators, food resources , and song posts or perches. In the Swiss AES directive, bramble and shrubs are permitted in wildflower areas to the amount of 1%. But they are only rarely tolerated by farmers, as these plants can overgrow fallows in a short time, which leads to the withdrawal of AES subsidy payments in Switzerland. We suggest to allow shrubs or bramble on about 10% of the area of fallows and to adapt the Swiss AES in this regard. A similar value of bushes was demanded for pastures in Sweden  are necessary and should be available for all farmers within an agri‐environment scheme. Our results are relevant for arable farmland in simple landscapes  in Switzerland and may also in wider continental Europe. The above‐mentioned management measures provide habitat structures which have become very rare in many European countrysides, and which benefit a variety of farmland bird species.None declared.Jérôme Duplain, Michael Lanz, and Simon Birrer gave substantial contribution to the design of the study and the interpretation of the data. Jérôme Duplain and Bernard Lugrin collected the data. Kim Meichtry‐Stier analyzed the data and drafted the article. Jérôme Duplain, Michael Lanz, Bernard Lugrin, and Simon Birrer revised the article, and all co‐authors gave their approval for the final version to publish.Data on territories numbers and habitat characteristics will be archived on Dryad upon manuscript acceptance. Click here for additional data file.


To describe the identification, adaptation, and testing of an evidence-based pediatric weight management program for a health disparate community.iChoose, was then pilot tested in 3 iterative phases delivered initially by research partners, then co-delivered by research and community partners, then delivered by community partners. The RE-AIM framework was used to plan and evaluate the iChoose intervention across all waves with assessments at baseline, post program (3 months), and follow-up (6 months).A community advisory board (CAB) of decision-makers and staff from local health care, public health, and recreation organizations engaged with academic partners to select an evidence-based program (EBP) for local implementation. Three EBPs were identified  and each EBP was rated on program characteristics, implementation and adaptation, and adoptability. Following selection of the EBP that was rated highest, the POPS-CAB made adaptations based on the program principles described in peer-reviewed publications. The adapted intervention, p < 0.05). Qualitatively, the selection focused on important program characteristics and on matching those characteristics to the potential to fit within the community partner services. The adapted program—iChoose—had 18% reach and with participants that were representative of the target population on age, gender, ethnicity, and race. Effectiveness was demonstrated by modest, but significant reductions in BMI z-scores at post-program compared to baseline . This decrease returned to values similar to baseline 3 months (MΔ = 0.009) after the program was completed. Implementation fidelity was high and implementation fidelity did not differ between community or research delivery agents.Bright Bodies rated highest on program characteristics and adoptability (p’s < 0.05), while Home Environmental Model rated highest on implementation factors (iChoose was successful in initiating changes in BMI z-scores, could be implemented in a low resource community with fidelity, it was insufficient to lead to sustained child BMI z-scores. In response to these data, maintenance of program effects and delivery are the current focus of the CBPR team.The process to help organizations identify and select evidence-based programs appropriate for their community led to consensus on a single EBP. While  A large body of literature suggests that efficacious intervention strategies are currently available for treating childhood obesity . In partPediatrics included the need to have interventions that consisted of a minimum of 26 to 75 contact hours with participants [Unfortunately, there is little evidence that these childhood obesity programs have been systematically translated into regular practice –16. It iicipants . This reicipants . The recAn emerging body of literature suggests that systems-based approaches may be ideal to support the identification, adaptation, and implementation of locally relevant, evidence-based childhood obesity programs –24. IndeiChoose, and the results of pilot testing the iChoose program in cohorts of families. The process of incorporating feedback from families and POPS-CAB members and transitioning from researcher-led implementation to community-led intervention is also described.The purpose of this paper is to describe the steps taken to identify, develop and preliminary feasibility testing of a newly developed, and locally-relevant, childhood obesity treatment program for a medically-underserved community experiencing health disparities. The paper details the systems-based and CBPR processes used to engage local organizations to form a community advisory board (POPS-CAB) that identified and reviewed 3 candidate childhood obesity treatment programs, collectively rated the programs, and selected one for local adaption. This is followed by a description of a newly adapted program, n = 8 community members). Research partners in the POPS-CAB had expertise in childhood obesity treatment, nutrition and exercise, health economics, implementation science, and community capacity development (n = 7 research members). An external consultant facilitated all POPS-CAB meetings to ensure that there was equal input by community and academic partners across the planning process, including the intervention selection process. The RE-AIM framework was used across planning and evaluation activities [The Partnering for Obesity Planning and Sustainability-Community Advisory Board (POPS-CAB) was developed in the Dan River Region (DRR) of south central Virginia and north central North Carolina . Specifitivities . All stuThis intervention development and pilot testing study is part of a larger 3-year, iterative project with two primary goals developed by the POPS-CAB. The first goal was to develop and assess community capacity related to the development, implementation, and sustainability of a local childhood obesity treatment program . The secThe POPS CAB met for a daylong meeting once a month for 3 months to develop a shared vision for the project as well as identify and select an evidence-based program for implementation in the region using the National Cancer Institute’s “Using What Works – Guide to Choosing an Evidence Based Program” process , 41. ThiThe research team used a multistep approach to identify potential paediatric obesity treatment programs, starting with a review of the available literature up to February 2013. Potential programs needed to have been (a) tested in multiple published studies across a range of samples, (b) provided data on efficacy and maintenance results for a period-of-time after the intervention had been completed, and (c) varied in the resources needed for implementation. Four pediatric obesity treatment interventions met these criteria--Traffic Light Diet –44, BrigFor each of the three remaining programs, the following information was presented to the POPS-CAB during their daylong working meetings:  who deliIn a second meeting, the POPS-CAB community partners each presented on the resources that were available to support a childhood obesity program in the region. Resources were identified that included access to the target population of high need families, personnel that could provide program sessions for families, and physical resources available to support a childhood obesity program. Specifically, both the Health District and Children’s Healthcare Center reported seeing a high proportion of low-income families while also documenting height and weight in a high proportion of children who had clinical visits. Parks and Recreation and the Boys and Girls clubs presented several physical  and human resources  that currently engage youth in physical activity and other recreation pursuits.During the third meeting, the POPS-CAB revisited the definition of evidence-based programs and reviewed findings from each of the Traffic Light, Home Environmental Change (Family Connections), and Bright Bodies Interventions using RE-AIM dimensions to characterize the potential reach, effectiveness, adoption, implementation, and maintenance information available. The objective was to evaluate these programs to ensure that the program selected could be adapted for the DRR, was appropriate for families with overweight or obese 8 to 12-year-old children, was likely to help families reduce their BMI status, could reach a large number of families that need the program, and could be adopted, implemented, and sustained in the community once grant funding was complete. The POPS-CAB also discussed modifiable aspects of evidence-based programs  and aspects that were not considered modifiable .After the review of the programs and open discussion, the POPS-CAB members received a comparison chart of the focus and components of each program . Ratings were based on a 5-point Likert-type scale with 1 reflecting strongly disagree and 5 reflecting strongly agree. POPS-CAB members individually rated each intervention. They were then divided into three groups with representation across community organizational and academic partners. The small groups reviewed each of the three programs, worked to develop consensus on the ratings for each program (e.g. groups completed one rating sheet for each program), and met for an additional 30 min after all programs had been reviewed to rank order the top program for each item on the rating sheet and identified the program that they considered the ‘best’ option for local testing.Qualitative data were also collected from each of the small groups during the program rating process described above. First, to facilitate small group discussions, open-ended questions were included on the rating sheet to allow POPS-CAB members to record their perceptions about the program . Second, each small group discussion period was audio-recorded and transcribed verbatim to provide information on the features of the childhood obesity programs that were considered when generating group ratings and rank orders. Finally, once all small groups had completed the rank order and program selection process the full POPS-CAB discussed the findings to decide on the program that would be adapted and tested locally. This discussion was also audio-taped and transcribed verbatim to provide information on areas that were discussed that contributed to the final program selection.iChoose and materials including parent and child workbooks, biweekly family sessions, biweekly telephone support calls to parents, biweekly newsletters for children, and 3 supervised exercise sessions per week.Following the selection process, the POPS-CAB began the process of identifying locally available resources, potential delivery sites and personnel, and local target population characteristics  that should be considered for potential program adaptations. Concurrently, based on the copyright related to the selected evidence-based program materials and a desire not to inappropriately use content without permission, a curriculum subcommittee made up of research and community members of the POPS-CAB used all available research articles on the selected program , 12, 47 The POPS-CAB also identified the need to ensure that the materials were appropriate for the DRR population in regards to cultural and educational factors. As such, all materials were developed using strategies to increase the likelihood that the program would be effective for parents that may have lower health literacy levels . SpecifiiChoose was pilot tested across 3 cohorts with plans to initiate the first cohort with research staff delivery , the second with co-delivery by research staff and community partner staff , and the third with community personnel . To make the transition from research to community delivery we implemented a training structure that followed the consultee-centered protocol and included instruction on session materials, role-playing, self-evaluation, and feedback on program delivery across class, telephone, and exercise sessions [iChoose program. Training for the final cohort included 4, 3-h sessions that followed the same process, but spaced training out over the course of the program due to the experience of the public health nurses and Parks & Recreation staff in delivery from the second cohort.sessions . Public In addition to the training protocol, across the 3 cohorts of pilot testing fidelity checklists were used to increase the likelihood of consistent implementation and to provide instructor feedback. An independent observer completed the fidelity checklists for the family sessions. The observer was unobtrusive, but, in the case that the instructor missed content, provided prompts to increase the likelihood that all content was covered. For the telephone and exercise sessions, fidelity checklists were completed by the delivery staff in real time and embedded within the call scripts or lesson plans to ensure all delivered material was captured. Fidelity checklist data were used formatively to help improve the program for each subsequent cohort and across cohorts as an indication implementation quality.Following the first cohort, the POPS-CAB identified several changes based upon a summative evaluation completed with parents and children and notes included on the fidelity checklists for each session. Specific changes included:  extendinThe POPS-CAB used the RE-AIM framework to provide direction for initial program evaluation and identified the following outcomes for assessment: program reach and representativeness, effectiveness in reducing BMI z-scores, implementation quality for the intervention components, and 3-month, post-program maintenance of changes in BMI z-scores. Program adoption and organizational maintenance were considered in planning, but beyond the scope of assessment for this preliminary intervention development study.Eligible children were between the ages of 8–12 years of age, BMI percentile ranking > 85%, and at least 1 caregiver willing to participate in the program. Potential participants were recruited by the health department or Children’s Healthcare Center using medical chart review to identify potentially eligible families. Nurses involved in the POPS-CAB created initial lists of potentially eligible children. Personalized letters from the child’s paediatrician were mailed to all potential families and a nurse or nurse practitioner followed the letters up with a telephone screening and recruitment call. If, after 3 attempted recruitment calls, the nurses were unable to talk to the family, research assistants made three additional attempts. Eligible and interested families were scheduled for a baseline health screening.Reach was defined as the number, proportion, and representativeness of children who assented to participate when compared to all eligible children. All parents of invited children that were contacted by telephone were asked to complete a brief screener that included gender, age, ethnicity and race, marital status, and socio-economic status related questions .Implementation fidelity of each session or telephone call was assessed based on the proportion of the key objectives that were covered during the intervention session. For physical activity sessions, the key objective was to ensure that approximately 80% of the session time was spent in moderate to vigorous physical activity. Family adherence to the intervention strategies were tracked objectively and reported as the proportion of family and exercise sessions that were attended as well as completion rates for the telephone support calls.iChoose program was operationalized as change in child BMI z-score. Behavioural outcomes related to iChoose program content are reported for both child and parent participants at baseline and post-program and 3-month follow-up. Trained research staff collected anthropometric data and interview-administered surveys as each data point.Effectiveness of the z-scores were computed using CDC standard age for gender algorithms and parent BMI was computed using the established kg/m2 formula and categorical variables created to classify parents as normal weight (BMI = 18–24.9), overweight (BMI =25–29.9) or obese (BMI > 30).Research staff collected height and weight, without shoes and in light clothing using a calibrated digital Tanita scale (Model: 310GS) and research-grade SECA 213 portable stadiometer at baseline, post-program and 3-month follow-up on all children and parents participating in program. For children, BMI Behavioural outcomes were measured using valid and reliable surveys for parents and children. Trained research staff interviewed administered the surveys. Minutes of moderate to vigorous physical (MVPA) activity was measured using the Godin Leisure Time Exercise Questionnaire . Mean miFor the selection process, the POPS-CAB individual and group ratings were summarized and within subjects t-tests were used to determine differences in individual ratings across program characteristics, adoption likelihood, and implementation/adaptability. Though the sample size of stakeholders was small, we also compared community and academic partner ratings to determine if differences emerged in ratings. Group ratings were reported as means and final dispositions of the group rank ordering programs across rating items are presented in tabular format. The transcripts from each small group and large group discussion were reduced to meaning units  and inductively categorized across the areas used to rate each intervention—we provide representative quotes within the results section indicated by quotation marks and italics.For the intervention testing process, all data were entered by trained research assistants with a quality check completed by another research staff member. Data was entered into SPSS 22 and typical measures of central tendency are reported . Chi-square tests of association, Fisher’s exact tests and ANOVA were used to compare reach, implementation and adherence for categorical and continuous variables. Implementation and adherence data were reported by wave and across waves to provide evidence of consistency across cohorts. Due to the small cohort sizes (n = ~ 30 each) all reach, effectiveness, and maintenance data were combined for analysis across waves. Paired t-test were conducted to determine changes in weight status from baseline-post-program and 3-month-follow up- baseline to assess RE-AIM dimensions of effectiveness and maintenance. A bootstrap function was used to control for cohort and M (SE) reported for each change score.p < 0.05) and Traffic Light . In contrast, Family Connections (3.8 ± 0.5) was rated significantly higher on implementation features when compared to Bright Bodies  and Traffic Light . Further, Bright Bodies was rated significantly higher than Traffic Light on implementation features . When rating adoption features the only significant difference in rating was between Bright Bodies  and Traffic Light (3.2 ± 0.4).Following the review of program materials POPS-CAB member ratings across the 3 programs varied across perceptions of program components, adoption, and implementation features. Mean ratings were nearly identical, on average, between community and academic partners and were not statistically or practically significant—as such those data are not presented. When considering program characteristics, ratings for Bright Bodies (4.1 ± 0.4) were significantly higher than Family Connections . Fifth, while the large group discussion led to the selection of Bright Bodies as the intervention to be delivered in the region, during that time and in the small groups, POPS-CAB members indicated that none of the three programs could be implemented without some sort of adaptation. For Bright Bodies, members identified a need to change the number of sessions  and address the health literacy level of the written program materials (“I would only be happy with major adaptations to language”).Data gathered from the transcripts of the small and larger group discussions were used to support the program ranking process and resulted in five consistent themes. First, POPS-CAB members expressed the importance of a program with a good balance of nutrition and physical activity content and opportunities. All groups discussed the benefits of Bright Bodies structured exercise program along with strong nutrition content. Second, there was a strong dislike across community members of the POPS-CAB related to calorie counting. This had the largest impact on Traffic Light, which was the only program that included explicit caloric restriction as part of the program. Third, there was a desire for the program to target both the parent and the child to work towards a family lifestyle change. Fourth, the POPS-CAB members reflected on the practicality and usability of the program in the target setting. Specifically, members noted that there were available resources for nutrition and behavioral counseling as well as physical activity sessions due to the combination of healthcare, public health, and youth serving organizations like Parks and Recreation and Boys and Girls club  were modest but significant . This decrease in BMI z-scores returned to values close to baseline (MΔ = 0.009) at the 3-month follow-up (Table Δ = 52.88 min) but this was not statistically significant. Children significantly decreased calories from SSB (MΔ = − 115.44) and reported significant improvement in quality of life at the completion of the iChoose program (M (SE) = 3.01(1.24)). Unfortunately, these improvements were not maintained after program completion. Minutes of MVPA decreased from post-program but stayed above baseline values (non-significant). Reported kcals from SSB increased at follow-up, nearly matching baseline values. However, children continued to report increases in quality of life at the post intervention follow-up. A similar pattern of outcomes was documented for parents in the study  from across community organizations will result in the selection of an evidence-based intervention that has a strong fit with local values and resources , 63. ThiiChoose program could be delivered with high-fidelity by local partners who had received support and training from the research staff. With support, local health departments and parks and recreation professionals may be an important and underutilized partner for childhood obesity treatment programs in geographic areas where the full range of clinical expertise is not available. Key to this partnership was ongoing meetings and investment by both clinical and community partners using locally available resources and aligning intervention strategies with local community organization missions. In addition to local Parks and recreation partners, the public health department and public health nurses proved key partners in program delivery.We found the adapted iChoose program resulted in modest, but significant reduction in BMI z-score for children. However, these changes were not maintained at three months. There are several possible explanations for why our results differ from the original Bright Bodies studies [The  studies . First, As pragmatic trial, our recruitment strategies were developed to recruit a representative sample of participants in a low income and medically-underserved area. As such, we did not include a run-in period to exclude less motivated families, we rescheduled families who did not show up for initial assessments, and used financial incentives for study assessments that may have encouraged families to enroll in the study simply to receive the incentives which were not tied to participation in the intervention sessions . Thus, tiChoose program was 3-months in duration, which may not be long enough to generate larger effects and an active maintenance phase could extend positive program impacts [iChoose program, participating children and parents could attend twice weekly instructor-led physical activity sessions designed to achieve moderate-to-vigorous intensity physical activity. This increased MPVA for children and parents during the program, but was that increase was not sustained at 3-months. Offering ongoing iChoose specific physical activity programming may provide a much-needed opportunity for children to participate in meaningful physical activity. Importantly, children reported sustained increases in quality of life after the completion of iChoose programming suggesting a sustained positive impact for the children. Parents self-reported quality of life improved during the program but returned to baseline values at 3-months follow-up. Parents may benefit from ongoing support of family sessions to meet challenges of making these family-based changes.For pilot testing purposes, the  impacts . For exaSomewhat related, is the question of adaptations to the program. The process we used focused explicitly on the underlying principles and learning objectives of Bright Bodies and was very flexible in changing the structure in terms of number of in-person sessions, shifting parent support to telephone calls, and shortening the program without maintenance sessions after the program core was delivered. Based on family feedback and the lack of maintained effects, it appears that the shift to a 3-month program without additional booster sessions was problematic. However, POPS-CAB community stakeholders also suggested that longer programs would lead to more difficulty in recruiting and sustaining participation. Recommendations from our stakeholders were to focus on previous, successful program participants as potential role models and support networks for new cohorts of participants and to extend the program using technology approaches that would reduce the ‘class attendance burden’.iChoose program, thus we do not use a control condition for comparison. This does reduce our internal validity. However, we aim to reduce potential biases by using an objective measure (BMI z-score) for our primary outcome. Additionally, the overarching goal of this study was to adapt an effective obesity treatment program for local implementation; thus, we began with programs that have already been shown to be effective. The selected program, Bright Bodies has demonstrated effectiveness in other studies establishing internal validity. We also focus on pragmatic factors related to external validity and the changes in outcomes when delivering an effective program in ‘real-world’ settings. As a feasibility trial, the sample size was modest and with program attrition, our final sample was n = 71. This attrition rate falls within the range (5–46%) reported in a meta-analysis of paediatric obesity treatment programs [z-scores at end of program and during maintenance. Analysing the completers only allows us to more precisely understand the program influences on our primary outcome at the end of program and what turned out to be a lack of sustained effects, both important outcomes to consider for a larger trial. As noted above, using methods to enhance retention of program participants and to extend programming or support to a longer time frame are important considerations for future iterations of the iChoose program.Some overarching limitations to this work should be noted. First, this study was designed as a feasibility pilot trial of the newly adapted programs  and at tprograms . We opteWhile the area of implementation science has gained traction over the past decade, few studies report on the adoption process by which organizations select, adapt, and test an evidence-based program for local delivery. The process and strategies related to initial program identification and selection are distinct from the training and resource deployment that are necessary for initial uptake, adaptation, and implementation—and we provide a process for other community-engaged researchers to follow. Specifically, the process used in our project provides a demonstration of a guided evidence-based program selection process that acknowledges the existing evidence base concurrently with the assessment of available community resources that could be used for on-going implementation—and is likely generalizable to any number of systems attempting to select appropriate evidence-based interventions for implementation. Further, the adaptation and training processes used resulted in high quality implementation across intervention sessions and are likely generalizable to other communities . The fin


We demonstrate the bona fide 2D structure of the IgM pentamer and its association with AIM protein. Soluble immunoglobulin M (IgM) forms a pentamer containing a joining (J) chain polypeptide. While IgM pentamer has various immune functions, it also behaves as a carrier of circulating apoptosis inhibitor of macrophage  protein that facilitates repair during different diseases. AIM binds to the IgM pentamer solely in the presence of the J chain. Here, using a single-particle negative-stain electron microscopy, we found that the IgM pentamer exhibits an asymmetric pentagon containing one large gap, which is markedly different from the textbook symmetric pentagon model. A single AIM molecule specifically fits into the gap, cross-bridging two IgM-Fc that form the edges of the gap through a disulfide bond at one side and a charge-based interaction at the other side. The discovery of the bona fide shape of the IgM pentamer advances our structural understanding of the pentameric IgM and its binding mode with AIM. H-Cμ1-Cμ2-Cμ3-Cμ4/VL-CL) in four polypeptide chains that form a pentameric component (fig. S1). The pentamer also contains an additional polypeptide, the J chain, which assembles the pentamer by firmly bridging the cysteine residues within the C-terminal region of two neighboring IgM monomers (fig. S1)  forms a predominantly pentameric complex that also contains a small polypeptide joining (J) chain (Since portions of the Fc region (Cμ2-Cμ3-Cμ4 chains and the short tail region) and J chain are sufficient to assemble monomers and form pentamers correctly . The rec575), although this cysteine residue makes disulfide bonds to link monomers in other sites , providing a mechanism for stable assembly (414 was replaced by serine (Cys414Ser) could not form the oligomers stably even in the presence of the J chain, forming structures that include tetramers, symmetric pentamers, and asymmetric pentamers with a gap comparable to that in wild-type Fc pentamer  is a circulating protein belonging to the scavenger receptor cysteine-rich (SRCR) superfamily , and other cysteine residues form internal disulfide bonds according to a homology-based structural model of AIM using the crystallographic structure of a conserved SRCR domain present in CD6  and the Cys414Ser Fc oligomers were unable to form the AIM/IgM complex . On 50° gap  is likel complex . The obspentamer , did not294, Lys298, Arg300, Lys301, and Lys340), while the Fc-Cμ4 domain is negatively charged  were used for particle picking. The number of picked particles for each sample were approximately 6500 (mouse IgM-Fc), 4500 (human IgM-Fc), 3400 (native mouse IgM), 8700 (J-negative IgM-Fc), 3700 (Cys414Ser IgM-Fc), 14,000 (mouse AIM-IgM Fc), and 9600 (human AIM-IgM Fc). Particles were extracted with phase-flipping, and subsequently, reference-free 2D class average was performed using RELION-2.1  and removed by filter paper. The grid was immediately stained using 1% uranyl acetate solution. During the staining, we placed a drop of uranyl acetate on the grid, sucked it up immediately using filter paper (without a dry up in the first and second runs), and repeated the procedure in succession. All data were acquired with a JEOL JEM-2010F electron microscope operated at 200 kV using a Tietz 4k by 4k complementary metal-oxide semiconductor camera . Data processing was performed using Scipion software package (Mouse and human IgM-Fc were produced as follows. HEK293T cells were cotransfected with pCAGGS-FLAG-IgM-Fc and pCAGGS–J chain–HA plasmids and then cultured in Dulbecco’s modified Eagle’s medium (DMEM), high glucose, and GlutaMAX medium , supplemented with 10% fetal bovine serum (FBS) for 3 days. Pentameric FLAG-Fc protein associated with the Myc-tagged J chain was purified from culture supernatant using the ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich) and then eluted with glycine-HCl (pH 2.6), followed by neutralization with 1 M tris-HCl (pH 8.5). After the affinity purification using an antibody, the protein was undergone a gel size fractionation to further eliminate undesired protein. Protein was concentrated using Amicon Ultra filter concentrators  and stored at 4°C in phosphate-buffered saline (PBS). Protein concentration was determined by a bicinchoninic acid (BCA) assay according to the manufacturer’s protocol . The protein was then subjected to size-exclusion chromatography on the HiPrep 16/60 Sephacryl S-300 HR column  with Dulbecco’s PBS for negative-stain EM. Mouse and human IgM-Fc (with the J chain) associated with AIM were produced as follows. HEK293T cells transfected with pCAGGS-AIM were cocultured with HEK293T cells transfected with both pCAGGS-FLAG-IgM-Fc and pCAGGS-J chain-Myc in DMEM, high glucose, and a GlutaMAX medium, supplemented with 10% FBS for 3 days. The complex was purified from culture supernatant using the ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich) and then a rabbit anti-AIM polyclonal antibody  conjugated to a HiTrap NHS-activated HP column . The protein was eluted with 0.1 M glycine-HCl (pH 2.3) and neutralized with 1 M tris-HCl (pH 8.5). After the affinity purification using antibodies, the protein was undergone a gel size fractionation to further eliminate undesired protein such as the IgM-free AIM. Protein was concentrated using Amicon Ultra filter concentrators  and stored at 4°C in PBS. Protein concentration was determined by a BCA assay according to the manufacturer’s protocol . Monoclonal IgM proteins were purified using the HiTrap IgM purification column  from the culture supernatant of B cell hybridoma  according to the manufacturer’s protocol.HEK293T cells transfected with pCAGGS-AIM-HA were cocultured with HEK293T cells transfected with both pCAGGS-FLAG-mouse Fc and pCAGGS-mouse J chain–HA in DMEM, supplemented with 10% FBS for 1 day. From the culture supernatant, the Fc-pentamer/J-HA/AIM-HA complex was purified using the ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich), followed by a rabbit anti-AIM polyclonal antibody  conjugated to a HiTrap NHS-activated HP column . The eluate was analyzed for J chain and AIM by Western blotting in a reducing condition using an anti-HA antibody. The intensity of the signal was measured using the NIH ImageJ software.http://swissmodel.expasy.org/). Templates with the highest quality  were selected for model building. Models were built on the basis of target-template alignment and subjected to energy minimization using PRIME . Molecular graphics images were produced using the PyMOL . Colors indicate the following: Blue is positively charged, red is negatively charged, and white is neutrally charged.Homology models of AIM and IgM-Fc were generated using the SWISS-MODEL server (


Insight into the inter- and intra-family relationship of protein families is important, since it can aid understanding of substrate specificity evolution and assign putative functions to proteins with unknown function. To study both these inter- and intra-family relationships, the ability to build phylogenetic trees using the most sensitive sequence similarity search methods (e.g. profile hidden Markov model (pHMM)–pHMM alignments) is required. However, existing solutions require a very long calculation time to obtain the phylogenetic tree. Therefore, a faster protocol is required to make this approach efficient for research. To contribute to this goal, we extended the original Profile Comparer program (PRC) for the construction of large pHMM phylogenetic trees at speeds several orders of magnitude faster compared to pHMM-tree. As an example, PRC Extended (PRCx) was used to study the phylogeny of over 10,000 sequences of lytic polysaccharide monooxygenase (LPMO) from over seven families. Using the newly developed program we were able to reveal previously unknown homologs of LPMOs, namely the PFAM Egh16-like family. Moreover, we show that the substrate specificities have evolved independently several times within the LPMO superfamily. Furthermore, the LPMO phylogenetic tree, does not seem to follow taxonomy-based classification. Renewable feedstocks, such as wheat straw, rice straw and other agricultural waste residues are used by the bioindustry for the production of sugars and value-added products. One of the first steps in this process is the enzymatic breakdown of these raw materials into smaller building blocks. For this, hydrolytic enzyme cocktails are extensively used. However, some biopolymers are resistant to complete enzymatic degradation by available enzyme cocktails. Lytic polysaccharide monooxygenases (LPMOs) are a relatively new class of metalloenzymes that can perform oxidative cleavage and aid breakdown by conventional hydrolytic enzymes (Currently there are seven families of LPMOs defined in the Carbohydrate–Active Enzymes database (CAZy) .After generating the initial dataset, the taxonomic distribution and the presence of accessory domains were analyzed using the HMMER web server . The nonprogram  and pHMMprogram . This reSeveral new features were added to the original PRC program , includiThe original PRC program has the ability to, amongst others, load SAM3, HMMer2 and PSI-Blast profile files . HoweverTo facilitate both pHMM building and fast prefiltering, support for sequence context-specific pseudocounts was added. The idea behind context-specific pseudocounts is that the local environment around an amino acid determines what mutations can occur at that particular amino acid location . For PRCAn additional advantage of implementing support for context-specific libraries is the ability to reduce the amino acid probability vectors of a pHMM to a discretized alphabet. This was achieved by the same method as used by HHblits to translate the amino acid profiles to 219 distinct letters (2) algorithm as implemented in theMUSCLE andClustal Omega alignment programs  and MacOSX (version 10.14.5). The computer system used for testing contained an Intel Core i5 with 8 GB of memory.Jackhmmer program and our previously published LPMO pHMM . The homology between the Egh16-like family and the LPMO family is in part due to the histidine located at the third position of the PFAM HMM, which in the LPMO family is part of the histidine brace. It should be noted that the Egh16-like family HMM is presumably based on an incorrectly predicted signal peptide cleavage site, resulting in the conserved histidine not being the first residue of the PFAM model. When examining several sequences within the Egh16-like family, the latest version of SignalP predicts the signal peptide cleavage site right before the histidine . As expected, the PFAM LPMO_10 (PF03067) and GH61 (PF03443) families were identified as close relatives. Surprisingly, we were also able to identify one distantly related family, namely the PFAM Egh16-like family, formerly known as DUF3129  and sequences contained within where aligned and used to build HMMs. Initially a small tree was constructed, containing a subset of 248 HMMs, using the pHMM-tree program .Extended data;The resulting tree was rooted using the Egh16-like family as an outgroup. A simplified representation is shown inAspergillus niger) . The subfamilies, AA9, AA11, AA13 and AA14 are mostly found in Fungi (>90% of LPMO sequences), the AA16 are found in both Fungi (82%) and Oomycetes (12%), while the AA10 are almost exclusively bacterial (99%) and the AA15 are mainly found predominantly in Metazoa (95%). The recently discovered LPMO16 are mostly found in Fungi (78%), but are also found in Metazoa (4%) and Oomycetes (6%). This observation suggests that LPMOs have found their true functional diversity in the fungal kingdom.After constructing the phylogenetic tree, it is possible to use it in several ways. For example, it is possible to search an unknown sequence against the pHMMs used for the tree building and discover to which LPMO subfamily and specific branch this protein belongs. This might give an indication of substrate specificity and oxidation preference that the newly discovered protein has.It is also possible to extract sequences or pHMMs from the tree that belong to a specific LPMO subfamily or clade. These can subsequently be analyzed for the presence of specific accessory domains or domains of unknown function. This might also give an indication of localization or substrate preference. For example, after extracting all the AA15 pHMMs and searching them against the PFAM database using PRCx, it appears that some of the members have a fasciclin domain. This domain may be involved in cell adhesion, suggesting that some of these proteins are targeted to the cell membrane .Lastly it is possible to take sequences belonging to one or several subtrees and align them using structural alignments. Using this approach, it is possible to get an indication of residues involved in substrate specificities or oxidation preference. This is the first time that a phylogenetic tree showing both the intra- and inter-family relations of LPMOs is constructed. We believe that the new PRCx program will help researchers to determine where their LPMO is located in the phylogenetic tree, what the putative substrate specificities are and identify LPMOs with a yet unknown substrate specificity (e.g. the LPMO16s). Moreover, the PRCx program can also be applied to other large proteins families in which it can aid in discovering long distance evolutionary relations.All data underlying the results are available as part of the article and no additional source data are required.http://doi.org/10.5281/zenodo.3518352 .Table S1 (sequence data used in this study).Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of theSource code for the PRCx program is available from:https://github.com/gerbenvoshol/PRCx.Archived source code at time of publication:http://doi.org/10.5281/zenodo.3518337 , which can be used to give insights into the inter- and intra-family relationships of protein families – based on their structure. The provided tool is, in particular, faster than the conventional tools to assess phylogenetic relationships. A phylogenic tree of over 10000 LPMO sequences was made to illustrate the usefulness of the LPMO-model developed. The authors show that their model indeed shows the expected AA families as listed in the CAZy database. But, what is less clear for me, is how could this LPMO-model be used to make phylogenic trees within one AA family? The authors mention that their model has only a length of 165, starting from the N-terminal histidine. Others  have shown that LPMO-segments important for substrate recognition, all neighboring the active site, are spread over the total sequence. Hence, using the 165 amino acids only would lack information of certain binding-segments. Do I understand correctly that such information cannot be picked up? I would appreciate if the authors can better highlight for which applications their pHMM-approach could be used and for which it would not be suitable. As far as I can judge, still, the tool is an asset to make complex and more complete phylogenetic trees (more sequences) in a relatively short time. The research shown is relevant.Abstract: please specify "speeds several orders of magnitude" to be more specific.Abstract: remove the last sentence, it is not relevant. Or explain.Intro: "aid breakdown by conventional enzymes". Not absolutely clear, please reformulate.Intro: "sequence similarity between LPMO families". The sequence similarities within for example the AA9 family is also rather low even. Add?Results: phylogenetic tree: The authors refer to a paper in which phylogenetic trees per AA family are described, and subsequently write that this approach was not sensitive enough to show the relationship between the different families. Are these two not based on completely different research questions? Instead of being not sensitive enough, could the authors rewrite this section to better highlight the value of trees within and between AA families and which tools/models are best to use?Figure 5: for clarity, the y-axes could be converted to minutes.Figure 6: the model seems to pick up well the large AA families (AA9 and AA10), but in some of the others extra branches appear (i.e. AA16 & LPMO16). Is this a ‘real’ branching or the result of using the short 165 amino acid model? Please discuss and explain the consequences in the manuscript. Overall, the manuscript is clearly written, although several choices made could have been explained better (see itemized points below).I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The authors have developed a UNIX-based program to study the phylogeny of large groups of proteins focusing on LPMOs. The program is novel, performs faster and contains features that no other program currently contains. The manuscript is written well, everything is clearly, accurately and sufficiently explained.  The methods used, prior art and references are addressed appropriately. LPMOs are a very interesting group of enzymes that have emerged as essential in degradation of biomass.  The authors mention this briefly in their Introduction section (first paragraph). Specific comments and questions: How do the authors envisage the utility and impact of their program for people who work with LPMOs in the wet-lab ?The authors mention development of the program for UNIX-based platforms such as Linux and MacOS. Have they considered, or plan to implement an online solution? Without an online solution - the usefulness of the program will be severely limited. In addition, informing readers about how and when such a tool will be implemented, with a provisional online link (even if the implementation is not entirely completed) - will certainly increase the impact of the article. I understand that most Linux code can be run on virtual machines, but from my experience - this is not necessarily accessible to people that are not bioinformaticians. Therefore, in order to make this tool useful for the scientific community - a user-friendly online solution would be highly recommended. This should be reflected upon in the Conclusions section. Could the authors comment, as well as include in the manuscript, their plans regarding such implementation?I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The authors have developed a program to study the phylogeny of large groups of proteins. This new method, named PRC Extended (PRCx), was applied on the lytic polysaccharide monooxygenase (LPMO) from over seven families. Conventional pHMM-pHMM methods failed to perform such a large construction due to the overwhelming calculation requirements. In this study, the authors used both pHMM-sequence searches and pHMM-pHMM alignments, they extended the original Profile Comparer program (PRC) and added several capabilities. It is plausible that the authors updated the popular original PRC program with the ability to load HMMer3.0 pHMM files, this is a very desired feature among the users of the original PRC program. The authors applied PRCx on the large LPMO superfamily to study the inter and intra-family enzyme evolutionary relationship. This study was not feasible without the newly constructed PRCx program due to the large size of the superfamily. The PRCx program was able to build the phylogenetic tree for hundreds of HMMs within a few days, which is a huge improvement comparing to the original pHMM-based method.   During the preparation of the phylogenetic studies, the authors identified the PFAM LPMO_10 (PF03067) and GH61 (PF03443) families to be the in-groups. They also found PFAM Egh16-like family to be a distant relative of the LPMO family. They found the evidence to correct the mis-predicted cleavage site of the Egh16-like family PFAM HMM and used this family as the out-group for the study. The close examination of the resulting tree revealed several interesting features of the LPMO family. Many of these features point to potential experimental targets, for example, altering arginine, lysine and histidine in AA9 LPMOs might give impact on the activity level of the enzyme. Last but not the least, it is interesting to notice that the core LPMO pHMM has a model length of merely 165. Would a much bigger pHMM model compromise the performance of PRCx? Considering the fact that the authors tested the PRCx program on a moderate computer of Intel Core i5 with 8 GB of memory, a high performance computer with more RAM might solve the problem?  As a researcher in the fields of applied Bioinformatics in biotechnology, I would recommend PRCx, together with the phylogenetic analysis of LPMO, to be published. The PRCx program will be a very useful tool to study big enzyme groups beyond the LPMO superfamily. *availability: The authors have made their program available via github, the supporting data is all accessible via Zenodo.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


N = 451; control group: N = 171). We collected data at three time points: pre-, mid- and post-intervention. Using latent growth curve models, we found that the program significantly predicted the change in internalizing symptoms over time. Furthermore, the mediation model showed that only the indirect effect via cybervictimization was significant. In summary, the program reduced internalizing symptoms within the experimental group successfully, through the decrease in cybervictimization more so than through the mediational effect of decreasing victimization.Victims of bullying and cyberbullying present internalizing problems, such as anxiety, psychosomatic and depressive symptoms, and are at higher risk of considering or attempting suicide. Researchers have put great effort into developing interventions able to stop bullying and cyberbullying, and thus buffering possible negative consequences. Despite this, only a few of them have investigated the effects of these programs on the psychological suffering of the victims. The NoTrap! program is an Italian evidence-based intervention able to reduce victimization, bullying, cybervictimization and cyberbullying. The aim of the present study is to analyze whether the NoTrap! program can reduce internalizing symptoms through the decrease in both victimization and cybervictimization. Participants were 622 adolescents, enrolled in the 9th grade of eight high schools in Tuscany (experimental group:  Peer victimization is now recognized as a significant risk factor for an individual’s well-being and adjustment. The symptoms that victims display in response to their bullying experience include internalizing symptoms such as depression, anxiety, suicidality, eating disorders and psychosomatic problems ,3,4,5,6.Looking at the consequences, it seems clear that the prevention of bullying and cyberbullying is crucial. Every intervention in this area aims to reduce the short- or long-term impact of these experiences on physical, psychological, and relational planes, and improve the general well-being ,12,13,14However, until now, few studies have evaluated whether an antibullying prevention program may also have secondary effects on psychological symptoms and reduce the suffering of the victims ,16,17,18In the past three decades, a significant amount of effort has been put forth by researchers analyzing the effects of face-to-face bullying, physically, psychologically, relationally, and in regards to general well-being. As Arseneault and colleagues  emphasizRecent meta-analyses, largely focused on longitudinal studies, help summarize such findings ,22,23,24Only a few studies were able to analyze long-term effects of victimization using longitudinal methods that reassessed participants throughout a large portion of their lives. Using data from a longitudinal study of a birth cohort of 1265 individuals who were monitored until their 30th year  Gibb andIn recent years, the important connection between bullying and well-being was further analyzed considering the possible unique, parallel, additional or synergistic effects played by context: what happens when bullying takes place in a cyber-context? The interactions that occur in the virtual world can affect the everyday reality that students experience elsewhere. As for bullying, the growing literature about cyberbullying often highlights the consequences of this phenomenon with regards to the individual’s well-being. Studies ,8,9,10 hLooking at the results of some metanalyses and systematic reviews, it is clear that cyber victimization is significantly and positively related to depression  and inteResearch appears to show that the negative consequences of cyberbullying often parallel those of traditional bullying, and a main research question arises: are there unique and particularly troubling aspects of the cyber context that could affect the quality and magnitude of the connection between bullying and its negative consequences? We have to keep in mind that, unlike traditional bullying, the cyber context has specific features that could play a role. Scholars have put great effort into analyzing their impact on bullying, and the consequences that follow ,31,32. CAlthough recent research has demonstrated significant connections between involvement in cyberbullying and various psychological health symptoms and difficulties, there is still an open debate as to whether these connections are independent from the involvement in more traditional forms of bullying. The highest risks of poor adjustment were observed in students who were identified as combined bully-victims, in both traditional and cyber contexts . There iIn all the studies cited above, the authors concluded their research by espousing the importance of carrying out effective anti-bullying programs that would have a high benefit/cost ratio in terms of suicide, internalizing symptoms, and so on.Although there is a growing body of research on intervention programs against bullying and cyberbullying ,42,43, uAlthough it is highly recommended in terms of both awareness about the processes involved and consciousness of the overlap between the two phenomena—and the unique effects they have on the victims’ suffering —none of The NoTrap! Program is one of the few interventions that is effectively able to reduce both the act of bullying and its consequent suffering, in both traditional and cyber contexts—i.e., victimization, bullying, cybervictimization and cyberbullying ,42,43,44Participants in the study were part of the quasi-experimental trial of the NoTrap! program, conducted during the 2011/2012 school year . The parThe experimental group was composed of 451 adolescents  = 1.12) attending 22 classes, across 5 high schools. According to the program scheme, 92 students  decided to assume a more involved role in the program by becoming peer educators.N = 171; 69% male; mean age = 15.28; SD = 1.15). Three schools accepted to participate as a control group, comprising of a total of 9 classes.The control group was composed of students who had not received any kind of intervention  released an invitation to participate in the program, which was sent to all of the high schools in Lucca and Florence . No school accepted to participate with a random selection, so we were unable to conduct a randomized control trial (RCT) design. For this reason, we paired schools that requested to participate as project schools with other control schools, specifically with classes with the same type of curriculum . In September, we asked specific schools to participate as control schools. = 1.534; p = 0.464), suggesting that the pairing was appropriate.No differences were found between experimental and control groups regarding the types of school attended  in our sample (χ²(I)Initial evaluation (questionnaire administration in November 2011).(II)Teachers training. Specific course on bullying and cyberbullying focusing on what a school can do against bullying and cyberbullying ; free admission was granted to all teachers of the experimental schools. The goal was to involve schoolteachers and communities, and to start a joint revision (with students) of the school rules and policies on bullying and cyberbullying.(III)Launch of the project and awareness development. Presentation of the project to the participating classes in order to try to raise awareness and communication on issues related to bullying and cyberbullying . We used videos and other materials that were developed in the previous editions of the program. The meeting was followed by another meeting with “a special police unit”  psychologist, and focused on the criminal implications of bullying and cyberbullying.(IV)Selection of peer educators from each participating class through self-nomination.(V)Day training for peer-educators (8 h) focused on communication skills, social skills in real and virtual interactions, victim and bystander emotions, empathy and coping strategies .(VI)Middle evaluation, after the first adult-led part of the project. Questionnaire administration at the end of February 2012. At this stage peer educators have not yet started the activities: they were only trained by the program staff.(VII)Face-to-face peer educators activities in their own class (two meetings—2 h each) on: (1) victim and bystander feelings and emotions, and empathy; (2) how to cope in situations of bullying and cyberbullying, from the point of view of the victim and bystander . They used problem solving strategies in order to decide a variety of possible solutions and they chose whichever they thought was the best. Small groups were led by peer educators. Each student was given a specific role in order to participate in completing the activity. The groups made posters which were later published on the NoTrap! Facebook page. At the end of the activities the students presented their posters to their classmates, and a discussion was held about the solutions they found and how they felt.(VIII)Online peer educators’ activities. We created a rotation schedule whereby all online peer educators worked for two weeks as moderators and publishers and Facebook group  administrators.(IX)Final evaluation. The same questionnaire was re-administered at the end of May/start of June 2012 to evaluate the final situation after the peer-led part of the program.(X)Main conference. Data restitution to the schools and students in October 2012. Before the conference there was a Facebook contest: the class who had gained the most “likes” on the poster they had created during the class activities, won a tablet.The steps of the NoTrap! program were the following:The questionnaires were administered in class by trained researchers during school time (Masters or Ph.D. graduating students). Informed consent procedures consisted of approval by the school, the class council, and the parents: 100% of the families agreed to their children’s participation in the research.Victimization: the Florence Bullying/Victimization Scales were used . Each scCybervictimization: the Florence CyberBullying–cyberVictimization Scales (FCBVSs) were used ,45. ThisInternalizing symptoms: the Youth Self-Report (YSR) ,46 is a 2) statistic, the root mean squared error of approximation (RMSEA), and the comparative fit index (CFI). Recommended cut-off points for these measures are 0.08 [All the analyses were conducted via Mplus 7.0  and PASWare 0.08  or 0.06 are 0.08  for RMSEare 0.08 . Given tThe estimation and prediction of longitudinal development of internalizing symptoms were analyzed through latent growth curve modeling. The latent growth factors (slope and intercept) were regressed on the variable program  in order to test if the program can affect the change over time of internalizing symptoms. In order to interpret the differences between control and experimental group in a meaningful way, multiple-group analyses  were conducted.p = 0.003; cybervictimization: β = 0.216, SE = 0.07, p = 0.002) but did not significantly predict the intercepts, confirming that the control and the experimental groups exhibited no pre-existing differences (intercept) on these variables. Looking at the multiple group latent growth curves, none of the slope means for the control group were significant , while there was a significant decrease over time (significant negative mean of the slope) in victimization and cybervictimization in the experimental group (for further details about these model results see [The same steps were conducted in a previous paper about the efficacy of the program  on victiults see  ). We usAs a subsequent step, we carried out a full mediation process model to test if the change in internalizing symptoms (outcome) is led by the change in victimization and cybervictimization variables (mediators) due to the program (independent variable) . We testWhen the research was planned (in 2011) a formal Ethical Committee was not yet established in our University nor the Italian Association of Psychology was requesting a formal ethical approval. The research was carried out in accordance with the recommendations of the Italian Association of Psychology and we can state that the investigations followed the Declaration of Helsinki principles. The research project was sent and approved by the school and class councils. Parents’ active consent was obtained prior to the questionnaires administration. Parents and students were informed about the confidentiality and anonymity of their responses, that their participation was entirely voluntary, and they were also told that they could withdraw at any time. In Descriptive statistics and correlations among the measures are presented in p < 0.001) while it is not a significant predictor of the intercept , confirming that the control and the experimental groups did not significantly differ initially (pre measure – intercept) in this variable either. The covariance between intercept and slope is significant and negative .Looking at the multiple group latent growth curves, while in the control group the mean of the slope is not significant, in the experimental group there is a significant decrease over time (see p < 0.001) and victimization intercept . Significant covariations both for intercepts  and slopes  between victimization and cybervictimization were found. No effects of the program were found on the three intercepts, confirming the comparability of the experimental and control groups at the pre-measure, in the final model.In p < 0.01) and cybervictimization , confirming the efficacy of this program in reducing both, in the final mediation process model. On the contrary, when introducing the latent growth curves of those variables in the model, the direct effect to internalizing symptoms is no longer significant . The slope of cybervictimization is a significant predictor of the slope of internalizing symptoms  while for the victimization slope, we found a marginally significant effect .The program significantly predicts both slopes of victimization . Upon analyzing the two paths specifically, the only significant indirect effect which led to a decrease in internalizing symptoms appears to be the decrease in cybervictimization led by the program . For victimization the indirect effect is not significant   were not included. However, the indirect paths from the program to the slope of internalizing symptoms were significant both for cybervictimization  and victimization ).We tested for the indirect effect . Overall, the total indirect effect is significant  in victimization.Starting from the increased demand for an evidence-based framework that can inform interventions and policies against bullying and cyberbullying ,54, the On the basis of two recent metanalyses ,43 the NAside from the evidence-based framework ,56, thesAs an important practical issue, we want to underline the evident importance of taking cyber context into consideration, not simply absorbing cyberbullying under the traditional construct of bullying ,59,62. GBefore arriving to the conclusion, it is important that we acknowledge some limitations of this research. First, we lacked an important aspect of evidence-based evaluation: the experimental and control samples were only paired based on the schools’ characteristics . In other words, although we tried, the assignment to one or another condition was not randomized (see the procedure section). Related to this issue, another possible bias can be due to the self-selection inclusion process of experimental schools. For instance, it is possible that a higher risk population missed the opportunity to be involved in the intervention. For this reason, in the analyses, we tested for differences in the two groups and controlled for the possible effects of the sampling . Although the methods we used can be considered at least acceptable, it would be desirable to replicate our findings using RCTs.Second, data have been collected in 2011/2012 and consequently we should consider possible changes in the nature of online risky behavior. It would be desirable to replicate our findings with new experimental trials especially when we consider how fast Information and Communication Technologies (ICTs) and the online context evolve nowadays. This future plan is also relevant in order to verify whether the mechanisms described in the study remain the same across different virtual contexts.Lastly, because of the complexity of the models used to test the effects of the program on internalizing symptoms, no gender analyses were performed. Gender differences are often present when consulting the literature on the effects of interventions on internalizing problems . AlthougDespite its limitations, this study integrates previous knowledge and gives some relevant suggestions to researchers and practitioners currently working on the prevention of bullying and cyberbullying. Using the standards of evidence , we gave