Background: With a recurrence rate of over 30%, techniques that offer stronger acromioclavicular (AC) joint reconstruction through increased graft strength may provide longevity. The purpose of our study was to determine the biomechanical strength of a novel tendon graft sutured throughout compared to a native tendon graft in Grade 3 anatomical AC joint reconstruction.Methods: For this in vitro experiment, nine paired (n = 18) embalmed cadaveric AC joints of three males and six females  were harvested. Anatomic repair with fresh bovine Achilles tendon grafts without bone block was simulated. Specimens were divided into two groups; with group 1 using grafts with ultra-high molecular-weight polyethylene (UHMWPE) suture ran throughout the entire length. In group 2, reconstruction with only native allografts was performed. The distal scapula and humerus were casted in epoxy compound and mounted on the mechanical testing machine. Tensile tests were performed using a mechanical testing machine at the rate of 50 mm/min. Maximum load and displacement to failure were collected.Results: The average load to failure was significantly higher for group 1 compared to group 2, with mean values of 437.5 N ± 160.7 N and 94.4 N ± 43.6 N, (p = 0.001). The average displacement to failure was not significantly different, with 29.7 mm ± 10.6 mm in group 1 and 25 mm ± 9.1 mm in group 2 (p = 0.25).Conclusion: We conclude that a UHMWPE suture reinforced graft can provide a 3.6 times stronger AC joint reconstruction compared to a native graft. Acromioclavicular (AC) joint injuries are common in the active population –3. The iMore than 60 surgical procedures to treat AC joint separation have been reported. These can be categorized into four different types which are (1) primary AC and coracoclavicular (CC) fixation, (2) the Weaver-Dunn procedure, (3) anatomic reconstruction, and (4) arthroscopic reconstructions [Primary AC and CC fixation accomplished with Kirschner wires, sutures, and Bosworth screws are one of the first fixation methods. Though good results have been reported, these methods have fallen into disfavor due to hardware migration, of which some have been devastating –9. The pSubsequently, this method has been more commonly augmented with sutures, wire cerclage, or tendon grafts for reinforcement. In order to replicate biomechanical behavior of the native ligament, anatomic reconstructions, wherein the conoid and trapezoid ligaments are recreated, have become more popular . HoweverSo far, the orthopedic literature does not contain recommendations for a single operative technique as the optimal reconstruction method for AC joint separations. Postsurgical recurrence rates of AC joint separation after reconstruction can range between 20 and 30% or higher and occur frequently within one year of the initial surgery –18. ReceWhile tendon graft repair techniques augmented with cerclage cables have been described, to our knowledge, no study has evaluated the effect of an intra-tendinous suture that functions similarly to a coracoid cerclage construction, on the strength of the AC joint repair .We hypothesized that running a suture throughout the tendon graft itself would increase load to failure and the longevity of reconstruction techniques using tendon grafts. Therefore, the purpose of our study was to determine the difference in tensile strength and displacement to failure, between a novel tendon graft with ultra-high molecular-weight polyethylene (UHMWPE) suture incorporated throughout and a standard nonaugmented tendon graft in an anatomic AC joint reconstruction.n = 18) cadaveric AC joints  were harvested. After thorough inspection, none of the 18 specimens showed signs of prior injury to the shoulder or prior operative intervention and thus were all included for biomechanical testing. The mean age of the embalmed cadaveric donors at the time of death was 86 years . Surrounding muscles and other soft tissues were removed. The AC and coracoclavicular (CC) ligaments were severed in order to simulate a Type III dislocation.For this biomechanical laboratory study evaluating tensile load to failure and displacement to failure of a novel graft augmentation technique, nine paired  across the entire length . The contralateral AC joint received the standard native bovine Achilles tendon graft and represented group 2 (n = 9). Prior to joint reconstruction all grafts were tensioned to 10 N to remove any creep.The matched pairs of AC joints were separated. Randomly, either the left or the right shoulder was assigned a graft sutured throughout with UHMWPE suture and became group 1 . The epoxy end was mounted in a shop vise that was bolted to the mechanical testing machine  with calibrated load cells. Custom-made fixture and grips, made from a bronze alloy ASTM B150, were used to grab the clavicle . For statistical analysis a paired Student’s t test was performed using SPSS 22.0 .p = 0.001). The difference in displacement to failure (p = 0.25).All failures except two were graft failures at the mid-substance. Each group had one clavicular fracture just distal to the grip. These specimens were not included in the statistical analyses. Load to failure was significantly higher in group 1 compared to group 2 , with me failure , though There was a 363% increase in average load to failure in UHMWPE augmented tendon grafts compared to the nonaugmented group. An increase in average displacement to failure was noted to be 19% in the augmented group.The AC ligament, the CC ligaments (trapezoid and conoid), and to some degree the coracoacromial (CA) ligament, are the primary static stabilizers of the AC joints . TherefoRecently the focus has been on repair using tendon grafts. In this study, we determined whether a difference in strength of AC joint repair exists between the use of a standard tendon graft and a tendon graft augmented throughout with UHMWPE suture.As expected, our data supports the proposed novel technique to run UHMWPE suture throughout the entire length of the tendon graft for reinforcement. One explanation for why constructs using such augmented tendon grafts are stronger than nonaugmented is the added material that leads to greater time zero strength. Results of this study show that the proposed tendon graft augmentation technique significantly increases load to failure (356%).Grutter and Petersen  comparedIn this current study a substantial improvement in graft performance when augmented with a suture within the graft was observed. The maximum load to failure recorded in the augmented group was 438 N. The tremendous increase compared to the nonaugmented group (94 N) suggests that our method may be a viable option to improve the biomechanical strength of tendon grafts and provide longevity to AC joint repairs.Charlick and Caborn  developeConcerns have been raised regarding clavicular fractures. Costic et al.  reportedIn this biomechanical study, none of our samples fractured at the bone bridge between the tunnels. Each group experienced one clavicular fracture just distal to the part where the custom-made grips grabbed the clavicle. This may be due to the fact that our cadaver specimens had a higher average age (86 years) than the patients who generally require this procedure. To compensate for advanced age and possible osteoporosis the fractured specimens were excluded from statistical analysis.Current graft augmentations, such as metal or suture cerclage techniques, have been linked to erosion of the coracoid and clavicle –35. The Our method may offer greater biologic fixation compared to all synthetic grafts and may also restore normal arthrokinematics because it is strong but of nonrigid nature, which is shown by the lack of statistically significant displacement to failure when compared to the nonaugmented tendon graft.TM has been added to the tendon graft, displacement to failure did not change significantly. This might be partially explained by the relatively circular shape of the stitch loops that changed to an oval shape with increasing tension. Cyclic loading may provide greater insight and should be the focus of future investigations.Almost half of all shoulder injuries in athletes involved in contact sports are AC joint injuries –38. AlsoAnother limitation was the use of calf tendon for reconstruction, which may be slightly different from human-derived tendon grafts. Ideally, tendon grafts similar to those used in actual patient surgeries, for example semitendinosus allografts should have been used. However, since the basic microarchitecture of collagen fibrils is shared among mammals, there is no difference between the strength of individual bovine tendon fibers compared to human fibers. Therefore the grafts should be of similar strength, if the dimensions of the grafts are similar . Also thThe results of our tendon graft augmentation method are very promising in terms of reconstructive strength in both maximum load and displacement to failure.This method may be used with different AC joint reconstruction techniques that use tendon grafts for repair. However, further biomechanical and clinical studies using human allografts are warranted to explore the feasibility of our novel method.All authors certify that they have no financial conflict of interest  in connection with this article. No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.


Bidding farewell and breaking up from a good company is never easy, yet every table setting has its end. For me, after serving an 11-years term as joint Editor-in-Chief (2005–2015), it is now time to look back at a remarkable journey before I say ‘adieu’. Indeed, a journey that turned out more fabulous than anyone would have believed or maybe even dreamed of at the time of its onset. My inaugural article in the Journal . I guess this only testifies to the Journal eventually having gained the love and acclaim of the community – j’taime SJTREM!The name change  and transition was not easy, and among the several options, we landed on a name that is not easily spoken in one breath: the The evolution of the Journal has been documented in the past –29, withThus, it is with much pride and few concerns that I shall demit office after 11 exciting years. The Journal appears to be in sound shape, with good prospects and with a steady course towards the future. I am proud of having been part of its inception, evolution, development and eventual establishment as a Journal with a strong foothold in Scandinavia and beyond.Knowing continuity will be assured with professor Hans Morten Lossius still involved, I welcome professor David Lockey from London HEMS to succeed as joint EiC of the Journal. The past managing editor Kristi G. Bache PhD now takes over as EiC and will continue to maintain the day-to-day contact with authors, referees and publishers alike. New faces and new places will be introduced only to the better advancement for the Journal. Still, I hope a Scandinavian focus will remain visible, although the world is now the playground for us all.Last but not least, I sincerely need to thank the most important contributors to the success of the Journal. For one, the many named and unnamed referees who, relentlessly and with dedication, have put time, knowledge and expertise into reviewing the numerous papers submitted to the Journal over the years. Without your efforts and dedication, the Journal could not exist. Second, to the number of authors who have contributed original and review papers of very high quality at a time when the Journal was trying to get a foot in the door to the academic community of people interested in trauma, resuscitation and emergency medicine. Many high-ranked and high profiled authors devoted of their time and expertise to contribute timely pieces on their pet topics. For sure, this has largely contributed to the growth and development of the journal. Finally, my sincere gratitude and expressed thanks goes to those who peruse the Journal material, be it as readers of the content, presenters at conferences and as citations in further publications. The steady and increasing use of SJTREM material in other media and in meetings testifies to its having a role and being a vehicle for transmission of knowledge across Europe and beyond. Lastly, the ultimate goal of the Journal and its content is to advance the medical care for the benefit of critically ill patients around the world.takk for meg’ but also include an ‘adieu’, forward a sincere wish of ‘godspeed’, and, definitely state an “auf wiedersehn” to the global supporters of the Journal.So, it is only appropriate that I shall say not only ‘


In this letter from the editors, we wanted to inform you that our journal has been selected for coverage in Thomson Reuter’s products and services. Beginning with volume 8, issue 1, 2012, Advances in Cognitive Psychology (ACP) will be indexed and abstracted in:•Social Sciences Citation Index•Journal Citation Reports/Social Sciences Edition•Current Contents/Social and Behavioral Sciences This is of course very good news, and implies that we soon will receive an ImpactFactor. As our journal is both an Open Access journal and charges up to now nopublishing fees this may imply that the number of submissions increases.We especially wanted to thank all the people who helped us in making this possible.First, and most important of all, we want to thank our good friend and colleague,Prof. dr. hab. Piotr Jakowksi. Piotr was the great inspirer of our journal. Manyyears ago, he came up with the idea of a pure electronic version of a new openaccess journal in the field of cognitive psychology, and after gathering peoplewilling to be part of the editorial board, ACP was launched and the first volume gotout in 2005. Piotr led the journal until his very unfortunate death in January 2011.Shortly thereafter, the decision was made by the editorial board members that thejournal should get a chance to persist and it was decided that we (Rob and Ulrich)would try to do this by serving as the co-editor and the editor in chief,respectively. This, however, would not have been possible without support from theUniversity of Finance and Management, in particular Krzysztof Godlewski, thefinancial director of the university, and also the help of Marcin Staniewski.Krzysztof continued to believe that it might be possible to successfully expand theimpact of the journal, and due to this support we were finally able to get access tothe ISI (Institute for Scientific Information) Master list of scientific journals.Obviously, this also would not have been possible without help from people from theIT department of Vizja that helped us with maintaining and renewing the website.Furthermore, several people were involved with improving the graphical design of thewebsite, and also in developing flyers to promote ACP at national and internationalconferences.A scientific journal cannot exist without an editorial manager. Several personssupported the journal in this role. We want to thank Anita Biauska, MagdalenaBerkowska, and Jacek Grunt-Mejer for all their past work for ACP. During the lastyears, Jacek was of great help, especially when this involved solving more technicalproblems, and without him the transition to the new ACP website in 2014 would nothave been possible. Other crucial technical support comes from Marlena Bartczak, whotook care of the technical editorial work, and who is responsible for the nicelayout of all that appeared in our journal. Due to maternity and parental leave herduties have temporarily been taken over by Dobrosia Jakowska, who was alreadyinvolved in the implementation of hyperlinks. Thanks for all your work!Special thanks go to all the members of our advisory and editorial board whocontinued to support us: Christian Buechel, Markus Kiefer, Isabelle Peretz, ErnstPoeppel, Bruno Repp, Ingrid Scharlau, Vincent Walsh, Elger Abrahamse, GisaAschersleben, Arndt Broeder, Claus-Christian Carbon, Simone Dalla Bella, MagosiaFajkowska, Anna Grabowska, Peter Keller, Iring Koch, Grzegorz Króliczak, MichaKuniecki, Markus Lappe, Bernd Leplow, Severine Samson, Elisabet Service, MichaelSpivey, Kate Stevens, Rolf Verleger, Martin Voracek, and Dirk Vorberg. As weindicated in our previous letters, we are also grateful for all the efforts that thereviewers put in evaluating submissions to our journal (see the lists on ourwebsite), and especially to the authors that submitted their papers to ourjournal.Thanks a lot. Without all your work this would not have been possible.We decided that our letter concerning the year 2014 would come out in the first issueof 2015. In the last year, there were no major changes concerning our editorialboard, but Iza Szumska was appointed as a new web-editor in December 2014. Shereplaces Jacek Grunt-Mejer.Out of all of ACP’s submissions in the year 2014, one was withdrawn at a laterstage, 8% were rejected without external review, 45% were rejected after externalreview, 12% were accepted, and the remaining manuscripts are currently being revisedor are under review.We would like to draw your attention to the Neuronus conference(http://neuronusforum.pl/). This conference will be held in Kraków from the17th to the 19th of April 2015, and with this conference we have a special bound,which already resulted in a special issue devoted to Neuronus conferences in 2013.For the coming year, there is again the plan to organize a new special issue thatmay come out in 2016. More information will be available on the Neuronus conferencein Kraków.In the last year, there were no major changes in the journal’s budget. We arevery grateful to the University of Finance and Management in Warsaw that continuedto financially support ACP in the last year. Nevertheless, there is a possibilitythat we will have to ask authors for (voluntary) contributions to cover ACP’spublication costs, which include maintenance of the website, costs ofcross-referencing, and costs for proof-editing and language corrections.In conclusion of this letter, we kindly wanted to remind you that we continue towelcome your submissions to our journal. Also, should you have any questions orcomments regarding this letter, or ACP, please feel always free to contact us.Rob van der LubbeSincerely yours,Ulrich Ansorge


This is a comment on “New insights into the Steen solution properties: breakthrough in antioxidant effects via NOX2 downregulation” .The addressed paper investigated reactive oxygen species (ROS) production and NADPH oxidase (NOX2) activity in platelets, polymorphonuclear leukocytes, and lymphocytes-monocytes, while being incubated in buffer solution and in Steen solution. The results showed significant role of Steen solution to reduce ROS production and to downregulate NADPH oxidase activity . HoweverDuring ex vivo lung perfusion (EVLP), antagonizing ROS production would be important for the attenuation of cytokine production, which may constitute the stimulus for subsequent graft dysfunction and rejection after transplantation  .ATP channels [During graft ischemia, the absence of flow would be translated into membrane depolarization and increased ROS production, with associated increased NOX2 activity and inhibition of KTo antagonize ROS during EVLP, antioxidants or NOX2 inhibitors might be used. However, caution should be taken with that because EVLP might be considered as an ischemic preconditioning model. This hypothesis is supported by the observation that lung grafts are tolerant to both cold and normothermic post-EVLP ischemia . In suchAccordingly, the use of Steen solution with its antioxidant potential and its inhibitory effect on NOX2 during the graft cold static preservation phase would be expected to result in a better outcome than that of the currently applied protocols.+ channel agonists would have a dual benefit through decreasing ROS production during initial ischemia and graft conditioning and protection during the subsequent ischemic-reperfusion injury [In addition, the presence of albumin and dextran in Steen solution would provide further protection for the graft during cold preservation. Furthermore, supplementation of Steen solution with Kn injury , 5.+ channel agonists) during cold static graft preservation as well as EVLP [Formerly this year, a new EVLP protocol (Shehata protocol) was introduced by the author which recommended the use of Steen solution (supplemented with antioxidants and K as EVLP . The res


Plasmodium falciparum cyclic amine resistance locus (PfCARL) are associated with parasite resistance to the imidazolopiperazines, a potent class of novel antimalarial compounds that display both prophylactic and transmission-blocking activity, in addition to activity against blood-stage parasites. Here, we show that pfcarl encodes a protein, with a predicted molecular weight of 153 kDa, that localizes to the cis-Golgi apparatus of the parasite in both asexual and sexual blood stages. Utilizing clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene introduction of 5 variants , we demonstrate that mutations in pfcarl are sufficient to generate resistance against the imidazolopiperazines in both asexual and sexual blood-stage parasites. We further determined that the mutant PfCARL protein confers resistance to several structurally unrelated compounds. These data suggest that PfCARL modulates the levels of small-molecule inhibitors that affect Golgi-related processes, such as protein sorting or membrane trafficking, and is therefore an important mechanism of resistance in malaria parasites.Mutations in the  in vitro evolution studies have implicated the Plasmodium falciparum cyclic amine resistance locus (PfCARL) as a potential target of imidazolopiperazines, potent antimalarial compounds with broad activity against different parasite life cycle stages. Given that the imidazolopiperazines are currently being tested in clinical trials, understanding their mechanism of resistance and the cellular processes involved will allow more effective clinical usage.Several previous  Plasmodium, remains a devastating disease worldwide, yielding approximately 200 million symptomatic infections and 438,000 deaths in 2014  and liver-stage (IC50 = 4.5 nM) parasites and also prevent transmission (0 oocysts with 5 nM KAF156) in standard membrane feeding assays  detection methods (whole-genome sequencing and high-density oligonucleotide arrays)  (PF3D7_0321900), a previously uncharacterized protein-coding gene (Saccharomyces cerevisiae homolog EMP65 (endoplasmic reticulum [ER] membrane protein of 65 kDa) suggests that this protein serves as a chaperone in the ER  domain  speculated role as a transporter of rather than as a direct target of chloroquine   have bee arrays) , 7. Whiln the ER , 11. Thefunction . The moufunction . Finallyfficking ) and posoroquine .pfcarl convey resistance against a variety of antimalarial compounds and the degree to which different pfcarl mutations confer differing levels of drug resistance. These findings lead us to conclude that mutations in pfcarl most likely induce a generalized drug resistance mechanism and that the PfCARL protein is not the direct target of the IZPs. These studies of pfcarl will expand our understanding of the mechanism of action of the imidazolopiperazines and also demonstrate a new multidrug resistance mechanism in P. falciparum.This issue formed the basis of this study. On the basis of PfCARL’s localization to the parasite Golgi apparatus and its predicted structural domains and amino acid conservation, we hypothesize that the PfCARL protein plays a role in protein export and localization within the parasite. This demonstrates both the degree to which mutations in P. falciparum laboratory strains (Dd2 and 3D7) treated for several months with sublethal concentrations of different IZPs, including GNF179 and KAF156, showed that parasites acquired multiple mutations in pfcarl  (pfcarl SNVs. The variants in pfcarl that most frequently conferred resistance corresponded to amino acid changes at codons 830 and 1076 (50 of 2.7 µM) in drug-selected parasite clones (All except 1 of 13 reported n 747”]) . The onland 1076 . Four prin vitro-isolated drug-resistant SNVs (V1218F) was found in the set of 60 naturally occurring pfcarl SNVs which have been identified within 203 P. falciparum sequences available on PlasmoDB    into two independent laboratory strains (Dd2 and NF54) using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system  and are associated with large predicted IZP IC50 shifts (I1139K in particular) .–21.pfcar system –. These sticular) . Enginee50 shift of greater than 5-fold to represent drug resistance (pfcarl clone containing the I1139K amino acid change gave the largest increase in GNF179 IC50 (1.4 µM), which is similar to the 2.6 µM value reported previously for a drug-selected line  were likely due to double and triple pfcarl mutant combinations  (pfcarl might convey resistance against the antigametocyte activity of GNF179. We generated synchronized stage V gametocytes in an NF54 pfcarl L830V parasite clone and then assessed GNF179 activity using a MitoTracker viability assay (pfcarl L830V mutation had an EC50 of 2.55 µM (50 = 9 nM). This indicated that pfcarl mutations play an even greater role in sexual stages and could impact the transmission-blocking activity of the IZPs. Importantly, no increase in resistance conveyed by the pfcarl L830V mutation was seen in our control compound, puromycin, which was previously demonstrated to have potent anti-stage V gametocidal activity (GNF179 and KAF156 both have potent transmission-blocking activity in standard membrane feeding assays  and poss = 9 nM) . This thty assay . The NF5 2.55 µM , a 274-factivity  and therpfcarl do not give rise to resistance to other clinically relevant antimalarial compounds, we tested the engineered pfcarl L830V and I1139K clones against artemisinin, chloroquine, mefloquine, pyrimethamine, and atovaquone in asexual blood-stage parasites , represent a common pathway leading to enhanced drug resistance in P. falciparum, such as the chloroquine resistance conveyed by the K76T mutation in pfcrt , which reduces disulfide bonds of protein, leading to a dramatic rearrangement of global protein structure and therefore a strong induction of the unfolded protein response (UPR)  but no sensitization to artemisinin with DTT . This enhanced sensitivity to DTT not only confirms the importance of pfcarl for GNF179 activity but also suggests that both GNF179 and PfCARL may play a role in parasite protein trafficking, particularly when combined with PfCARL’s localization to the Golgi apparatus.In se (UPR) . Given tse (UPR) , the senwith DTT . This sepfcarl was the actual target of the IZPs or whether mutations could convey resistance via an indirect mechanism. Our first approach was to examine whether pfcarl was essential, as the antimalarial potency of the IZPs suggests that they target an essential parasite process. In order to investigate the essentiality of pfcarl, we attempted to disrupt the pfcarl gene. We used the CRISPR/Cas9 system described above to attempt a variety of disruption strategies. We attempted to insert a stop codon, via point mutation, at codon 81 because it had been previously mutated in a drug-selected strain as a pfcarl double mutant  compared to wild-type Dd2 parasites .On the basis of this slight growth defect, we speculated that mutations in  of time , demonstpfcarl did not convey resistance to a series of currently used antimalarial compounds , the cytosol (pyrimethamine), or mitochondria (atovaquone) were not affected. In order to determine whether mutations in pfcarl might provide resistance against other antimalarial compounds, we tested the P. falciparum Dd2 wild-type strain and two representative resistant pfcarl lines  against asexual blood-stage P. falciparum , but the pfcarl S1076I mutant was not  . The asymmetric indolylmaleimide (pfcarl P822L mutant than against either the wild-type strain or the pfcarl S1076I mutant (pfcarl P822L IC50 = 348 nM versus pfcarl S1076I IC50 = 1.2 µM and Dd2 WT IC50 = 979 nM), further highlighting differences in pfcarl compound specificity based upon the mutated region of the protein. Interestingly, this pattern of pfcarl mutations conveying sensitivity to certain antimalarial compounds extended to KDU691, a preclinical antimalarial shown to target PI4K (pfcarl rendered parasites 2-fold more sensitive to KDU691 than either the wild-type strain or the P822L parasite clone (pfcarl S1076I IC50 = 44.3 nM versus pfcarl P822L IC50 = 79.6 nM and Dd2 WT IC50 = 88.6 nM).The  108 nM) . The pfcaleimide , in contget PI4K   . Additionally, the alteration of the benzene ring to which R1, R2, and R4 were attached to any other cyclic structure also dramatically reduced compound activity. Finally, substitutions at the R3 position seemed to have minimal effect upon compound potency, consistent with previous lead optimization studies in which even removal of the associated ketone group had a minimal effect upon compound activity  . Consistctively) , 5, struactivity .pfcarl drug-selected clones exhibited (at least some degree of) resistance against all 38 active IZP analogs  also had increased P822L resistance, but compounds with an R2 nitrile group  did not   . These cpfcarl have been implicated in resistance to the IZPs, these mutations had never been shown to be sufficient to directly cause resistance to the IZPs. In addition, many of the drug-resistant lines had mutations in genes other than pfcarl, suggesting that other genes may have been responsible for the IZP resistance of those IZP-selected clones. In this study, we definitively demonstrated that mutations in pfcarl, introduced via the CRISPR/Cas9 system into a clean genetic background, are sufficient to give resistance to the IZPs. The CRISPR/Cas9 experiments also demonstrated that resistance to the IZPs extends to sexual blood stages as well.While mutations in P. falciparum  have already been identified in lciparum , 33–37, parum 33–. Here, weridines , furtherpfcarl suggests that the IZPs have a distinct mechanism of action relative to other antimalarial compounds. Since mutations in pfcarl are clearly sufficient to introduce resistance to the IZPs, the presence of the PfCARL protein at the cis-Golgi apparatus strongly suggests that the IZPs exert their parasite-killing activity within the Golgi apparatus as well. The Golgi apparatus plays the primary cellular role in protein trafficking and export within the malaria parasite, and the likely translocation of the IZPs to the Golgi apparatus suggests that the IZPs alter parasite protein export and remodeling of the host erythrocyte. This is doubly true given the enhanced sensitivity of P. falciparum parasites to GNF179 when the unfolded protein response is induced via exposure to DTT. Therefore, the IZPs are likely to inhibit parasite protein export, and mutations in pfcarl are therefore likely to induce resistance to the subset of compounds which also inhibit these processes.However, the combination of different patterns of localization and the lack of cross-resistance to known antimalarials conveyed by mutations in pfcarl as a Golgi-localized drug resistance gene has significant implications not only for the IZPs but also as a means by which the malaria parasite could develop resistance to other antimalarials as well. The identification of new resistance pathways will significantly enhance the development of new antimalarial drug compound combinations, as has been done for several of the previously mentioned resistance loci  whole blood was obtained from the Blood Bank of The Scripps Research Institute (TSRI) . Leukocyte-free erythrocytes are stored at 50% hematocrit in RPMI 1640 washing media at 4°C for 1 to 3 weeks before experimental use. Cultures were monitored via Giemsa-stained thin smears.The nditions . Human Opfcarl  to a parasite population of 109 parasites. Excessive debris resulting from the death of drug-sensitive parasites was removed using a 63% Percoll purification. Subsequently, viable parasites were cloned into 96-well plates for continuous culturing in which each well contained 200 µl of complete media at 2.5% hematocrit. Parasites were grown under constant drug pressure with replacement of complete media, blood, and 38.4 nM GNF179 every 48 H. After 25 days in culture, mutants were selected microscopically using Giemsa-stained slides.Three additional Dd2-resistant lines with mutations in 50 determinations, and puromycin was used as a positive control. EC50s were then calculated using a nonlinear regression curve fit in GraphPad Prism 5.0.Gametocytes were induced in NF54 or derived clones as previously described . AsexualP. falciparum parasites (Dd2 and NF54 strains) were cultured under standard conditions  and pelleted again as described above. The PBS was removed, and lysis buffer (0.15% saponin–PBS) was added on ice in 10 pellet volumes. Upon lysis of red blood cells, indicated by a clear red supernatant, the lysed cultures were centrifuged at 3,200 × g for 12 min at 4°C. The supernatant was removed by aspiration, and the cells were washed twice in microcentrifuge tubes using chilled PBS. The cell pellets were then stored at −80°C until genomic DNA isolation was performed with a Blood and Cell Culture DNA mini-extraction kit .Cultures were grown to 4% to 5% parasitemia in 50 ml of RPMI media at 5.0% hematocrit. Cultures were then transferred to 50-ml conical tubes, pelleted via centrifugation at 800 × P. falciparum 3D7 reference genome (PlasmoDB v. 11.0) as previously described, using the previously reported Platypus pipeline  were prepped for sequencing using a Nextera XT kit , following standard dual-index protocols. Libraries were clustered and run on an Illumina HiSeq 2500 instrument using the RapidRun mode and sequencing of 100 bp in depth on either end. Paired-end reads were aligned to the pipeline . Geneticin vitro selection were confirmed by introducing those mutations into the parental Dd2 line using the CRISPR/Cas9 system . Primers for the donor templates were as follows: pfcarl Editing Domain F and pfcarl Editing Domain R.The gRNA sequence (listed as gRNA plus a protospacer-adjacent motif [PAM]) used for P. falciparum Dd2 WT culture was mixed separately with the pfcarl S1076I mutant line in mutant/WT ratios of 1:10, 1:1, and 10:1. The cultures of mixed mutant and WT were adjusted to have an initial parasitemia level of 1% in a 12-ml culture at 4% hematocrit. Parasites were then passaged at a 1:6 ratio every other day for 20 days (10 generations). Genomic DNA was also harvested every second day using a QIAamp DNA Blood Minikit . Genomic DNA was subsequently used for droplet digital PCR (ddPCR) to amplify an amplicon between 75 and 150 bp within the target of interest containing the mutant allele.The P. falciparum genome. Primers and probes, synthesized by Integrated DNA Technologies, were added at final concentrations of 900 nM and 250 nM, respectively. Wild-type probes were 5′-hexachloro-fluorescein (HEX) labeled, while mutant probes were 5′ 6-carboxyfluorescein (FAM) labeled. Both probes were also 3′ labeled with Iowa Black quencher. Emulsions were generated in the droplet digital PCR droplet generator and then transferred to an Eppendorf TwinTec PCR plate and sealed with a foil seal. Emulsions underwent 40 cycles of amplification in a Bio-Rad C1000 thermal cycler under the following conditions: 95°C for 10 min, followed by two-step amplification at 94°C for 30 s and 57.6°C  for 60 s with a 2.5°C/s ramp rate, then 98°C for 10 min, and finally a 12°C hold. Droplets were read on a QX150 reader and manually gated into double-negative, single-positive, and double-positive populations using QuantaSoft software, as indicated in pfcarl were ddPCR pfcarl F and ddPCR pfcarl R for a 154-bp amplicon. Probe sequences were ddPCR pfcarl WT and ddPCR pfcarl Mut (S1076I).Droplet digital PCRs were performed using 2 ng of genomic DNA template as input and the 2× droplet PCR supermix for probes (without dUTP), supplemented with additional 50 µM dATP/dTTP due to the AT richness of the P. falciparum Dd2 wild-type (WT) strain, the drug-selected pfcarl P822L clone (mutant no. 2), and one pfcarl S1076I drug-selected clone (mutant no. 1) were tested for cross-resistance against 60 imidazolopiperazine chemical analogs, as well as against a library of 10 preclinical antimalarials and 42 unrelated compounds with antimalarial activity, using the SYBR green I growth assay described above. In addition to performing a 0.1% dimethyl sulfoxide (DMSO) assay and the 10 µM artemisinin assays described above, both artemisinin and GNF179 were included as controls for dose-response analyses in all plates. For the chemogenomics analysis, the median IC50 is used for mutants that share the same SNP. The biological activity data were converted to pIC50 values , and fold shifts were calculated for the mutants relative to the wild type. The data were clustered using Software R 3.2.0 using the following method: hclust. The complete linkage method finds similar clusters with Euclidean distances between activity profiles.The pfcarl mutants containing drug-selected P822L (mutant no. 2) or drug-selected S1076I (mutant no. 1) or CRISPR-engineered I1139K mutations were cultured without drug pressure for up to 6 months. Phenotypic reversion was defined by a change in IC50s, and morphology. IC50 assays, using the SYBR green I parasite growth assay, were carried out at the indicated time points to evaluate changes in IC50s.pfcarl (PF3D7_1113300) was amplified from genomic DNA using primers pfcarl LocF/pfcarl LocR and subcloned into a pDC2-based expression vector downstream of the P. falciparum calmodulin promoter, yielding the expression plasmid pDC2-PfCAM-pfcarl-FLAG-attP-BSD. This expression plasmid was transfected into the NF54-attB parasite line together with the pINT plasmid for use in integrase-mediated recombination performed with attP × attB  and pFLAG R  and primers pfcarl LocF  and pfcarl LocR .For pfcarl mutation introduction , the following primers were used: L830V (TATGAAATTATATGTGGTTGTAAATATGTTAGAAATTTTAG), S1076N (GTTTTTAATCCTAGTGATGAACAATTTTACAGAGATAAAATC), S1076I (GTTTTTAATCCTAGTGATGATCAATTTTACAGAGATAAAATC), V1103L (GTAGCGTCTGATGCACTTGAAAGATTTTATTTGTTCATAG), I1139K (CCAGTTGGCTGATTATTAAATTATTACTTGAGGTGGGTGTAG), and CRISPR mut (GTTAGAAATTTTAGAAAGATTGTTGAGATCTTTAGGAAAAGATTTGATCG). For generation of the pfcarl editing domain (before mutation), the following primers were used: pfcarl Editing Domain F primer (KasI+Primer) CTTCTTGGCGGCCATGAACGTGCAGGTAAAAAAAATAG and pfcarl Editing Domain R primer (AAtII+Primer) CTATCAGACGTCCCCATTTTTAGTTAAAAGCTTTTATAAGG. The primers for pfcarl droplet digital PCR (for comparative growth assays) were ddPCR pfcarl F (GATATTAATATGTTGCACCTGTG) and ddPCR pfcarl R (GTAAATTTTTTTAAAAACTGTGGATTTTTATC) for a 154-bp amplicon. Probe sequences were ddPCR pfcarl WT (CACCATGTTTTTAATCCTAGTGATGAGCTTTTAC) and ddPCR pfcarl Mut (S1076I) (CACCATGTTTTTAATCCTAGTGATGATCTTTTAC). The underlined sequences represent mutated codon.For Figure S1 pfcarl probes. Download Digital drop PCR for growth competition assay schematic for experiment performed as described for Figure S1, EPS file, 1.4 MBTable S1 SNVs and indels identified in GNF179-selected parasite lines. All SNVs and indels within three new (sample set no. 1) and four previously reported (from reference Table S1, PDF file, 0.1 MBTable S2 pfcarl within 203 lines whose sequences are publically available (SNPs present in vailable . MutatioTable S2, PDF file, 0.1 MBTable S3 50s for each indicated parasite line and the structure of each compound listed.IZP analogs tested as described for Table S3, PDF file, 0.1 MB


In many applications, a family of nucleotide or protein sequences classified into several subfamilies has to be modeled. Profile Hidden Markov Models (pHMMs) are widely used for this task, modeling each subfamily separately by one pHMM. However, a major drawback of this approach is the difficulty of dealing with subfamilies composed of very few sequences. One of the most crucial bioinformatical tasks affected by the problem of small-size subfamilies is the subtyping of human immunodeficiency virus type 1 (HIV-1) sequences, i.e., HIV-1 subtypes for which only a small number of sequences is known.To deal with small samples for particular subfamilies of HIV-1, we introduce a novel model-based information sharing protocol. It estimates the emission probabilities of the pHMM modeling a particular subfamily not only based on the nucleotide frequencies of the respective subfamily but also incorporating the nucleotide frequencies of all available subfamilies. To this end, the underlying probabilistic model mimics the pattern of commonality and variation between the subtypes with regards to the biological characteristics of HI viruses. In order to implement the proposed protocol, we make use of an existing HMM architecture and its associated inference engine.We apply the modified algorithm to classify HIV-1 sequence data in the form of partial HIV-1 sequences and semi-artificial recombinants. Thereby, we demonstrate that the performance of pHMMs can be significantly improved by the proposed technique. Moreover, we show that our algorithm performs significantly better than Simplot and Bootscanning. Profile Hidden Markov Models (pHMMs) are widely employed to model nucleotide or protein sequence families, in particular for database search ,2. In nuTo overcome this drawback, one can use an information sharing protocol which makes use of the information available about other subfamilies to model subfamilies composed of only a few sequences. Although they are usually named differently, information sharing protocols are a widely used concept in bioinformatics and other fields. For example, deducing pseudocounts  or DiricS is based on the idea to add the nucleotide frequencies of other subfamilies T (in weighted form) to the ones of S as done with pseudocounts. Hereby, the added frequencies of T are weighted by the probability of the amino acid frequencies of T under a model whose emission probabilities are constructed based only on the frequencies of S.Sjölander et al.  suggesteHIV-1 is classified into the four main phylogenetic groups M, N, O, and P. All four were introduced into humans by separate zoonotic events, whereby the sources were different simian immunodeficiency viruses from chimpanzees  and goriThe subtypes of HIV-1 Group M can be divided into two groups: On the one hand, the ones for which at least 30 full-length sequences are available (subtypes A–G), on the other hand, subtypes H, J, and K, for which up to now only four (subtypes H and J) or two (Subtype K), respectively, complete-length sequences have been sampled. Thus, we refer to the first group – subtypes A–G – as “large-size” subtypes and to the second one – subtypes H–K – as “small-size” subtypes in what follows. Moreover, 11 full-length and about 1000 partial sequences have been labeled as “unknown”. That is, these sequences do not form an independent subtype or CRF. One can expect that by further sampling activities new small-size subtypes will be found, by finding sequences which are closely related to sequences of an unknown subtype already known, as well as by discovering sets of epidemiological independent sequences which are unrelated to the sequences now available [Up to now, roughly 50 tools have been developed for the purpose of recognition of recombinants and breakpoint detection in HIV-1 sequences. Examples are Bootscanning , SimplotThe aim of this paper is to develop an information sharing protocol suitable for the application to HIV-1 Group M sequences. The introduced protocol is incorporated into the HIV recombination detection tool jumping profile Hidden Markov Model ,20,21, sThe effect of modeling subfamilies with very few sequences separately is illustrated in Table Since a vast majority of columns in the HIV-1 MSA are strongly conserved over subtypes, it is very probable that in fact the most realistic way to model the emission probabilities in this example consists of using a joint emission probability for all subtypes. Hence, the modeled emission probabilities of Subtype K should be much closer to the ones of Subtype A and C. They should still be smaller (but to a lesser extent) since one has to account for the following (rather improbable) scenario: The nucleotide frequencies of Subtype K only coincidentally suggests that all subtypes should be modeled jointly, but the real emission probabilities are, in fact, considerably different.A workaround for this kind of situation consists of using the same emission probabilities for all subtypes in case a column is completely conserved (notice that in our example this does not apply to the column under consideration). But given that there are about 2000 non-recombinant full-length HIV-1 Group M genomes reported by now, almost all columns in an alignment composed of all these sequences are not 100% conserved.In a preparatory step, Sjölander’s approach performs a sequence weighting based on the total numbers of independent counts. Unfortunately, this procedure results in sequence weights that are far too low if applied to HIV nucleotide sequence data instead of protein amino acid data. Trying to circumvent this problem by leaving this step out causes the influence of other subtypes to become too strong. Since Sjölander’s approach is, in addition, an ad hoc one and aims at protein sequence data which are considerably more variable than HIV-1 sequences, we developed an alternative algorithm. This new algorithm is suitable for HIV-1 nucleotide sequence data and is derived from a probabilistic model of the interdependence of different HIV-1 subtypes.Our model of the emission probabilities is based on the observation that for almost all sites in an HIV-1 MSA at least some of the subtypes share the same emission probabilities. In fact, for the majority of sites, it would be most plausible to assign equal emission probabilities to all subtypes. Neglecting the trivial case of all subtypes having the same emission probability assigned to them, the phenomenon that some but not all of the subtypes exhibit equal emission probabilities could be explained biologically as follows: Assume a site allows for more than one nucleotide to be present . Then, there are only very few characteristics of the virus which determine which degree of fitness the virus has for the different nucleotides possible at the respective site. Moreover, these characteristics take only very few values. As the characteristics and their values at a particular site are small in number, the number of different nucleotide distributions observed at the respective site is also small.i in the MSA the dependence of the virus fitness on the nucleotide at site i is determined by a binary characteristics  of the virus. For example, it could be the case that some part of the virus can assume two distinct shapes, influencing which nucleotides can be present at site i such that the virus is able to survive. Then, the following might hold for the nucleotide distributions of the subtypes: To clarify this reasoning, let us assume that for a site i. This leads to a nucleotide distribution where adenine has a probability very near to one.• If the characteristics takes the value 0, it might be that the virus can only survive if adenine is present at site • In case it takes the value 1 the virus might also be able to survive if cytosine is present, but with a significant disadvantage with respect to its fitness. This might lead to a nucleotide distribution where adenine has a probability of, say, about 90% and the one of cytosine is about 10%.i: One part of the subtypes will show one nucleotide distribution and the other subtypes will show another. In the following, we will call the different nucleotide distributions (resp. emission probabilities) at the site “source”. That is, in the example just given there are two sources.In this case we would observe two different nucleotide distributions at site A,C,G,H}→1, {B,K}→2, {D,F,J}→3 constitutes the source combination of the respective site. In a more general context, the source combinations are called the set of partitions . Since a[In order to estimate the emission probabilities of the subtypes, we iterate through all possible source combinations for each position. For each source combination we estimate the emission probabilities of each source based on the nucleotide frequencies of the subtypes assigned to it using a Dirichlet priora. We obtaIn HIV, the distribution of gaps in the MSA provides little help in distinguishing different subtypes. Most parts of the genome are highly conserved, possessing nearly no insertions and deletions. In contrast, small parts are highly variable (possessing lots of gaps), but they vary so strongly that they provide little information for distinguishing different subtypes. For this reason, we estimate the transition probabilities (using a Dirichlet distribution as prior) for all subtypes jointly from the complete MSA. The same approach was applied by  for subfThe recombination and breakpoint detection tool jpHMM requires a pre-calculated MSA of the HIV-1 subtypes as input  indices. The individual sources in a source combination are indexed by 1,…,R. The space of all source combinations is denoted by S, the source of subtype i by si. For each source j of a source combination j by F=4 and the subtypes 1, 2, and 4 are assigned to Source 1, and the Subtype 3 to Source 2, we have m1=3, m2=1, R and the N, is equal to 4.Let 1,…,j by P(R = j) = ρj. These probabilities are estimated as described in [R is estimated by the relative frequency of for how many alignment columns R was found to be the most likely number of sources. Since the frequencies for R>6 are zero or near to zero, we set the maximum number of sources Rmax to 6. The estimated values are (ρj)j=1,…, 6=.We denote the probability of a given number of sources ribed in : For eacS to (Sr)r≤3, where Sr is the space of source combinations composed of r sources. Since |Sr|=S (with S the Stirling numbers of the second kind) and |S|=B(F) (with B the Bell numbersb) a brute force search in the entire space S would imply a considerable computational burden: Increasing Rmax from 3 to 6 would result in an increase of the computational effort by about a factor of 6. Hence, we restrict the search in S by the following procedure, illustrated in Figure Sr)r≤6 successively, starting with S1, which only contains one source combination. Before searching Sr, r≥2, we determine the most likely element of Sr−1 , with pmated in ). The paν∈{1,…,N} of Subtype 1 for the sake of simplicity. We denote the probability to be estimated by i at the position under consideration by For the general case of more than one subtype, we here restrict ourselves to the estimation of the emission probability of nucleotide with That is, we estimate the emission probability of Subtype 1 for each fixed source combination m: We now consider the two factors in (1). In this, we assume that 1. In  , (4), (5), and (6) into (1), we obtain with With these two formulas, Trec and Tpure , the middle part stems from one of the small-size subtypes . The choice to group the subtypes this way into normal- and small-size ones, is canonical since the subtypes H, J, and K contain only 2 resp. 3 sequences, whereas the other subtypes contain 30+ sequences. For each pair composed of a normal- and a small-size subtype, Trec contains 10 semi-artificial recombinants composed of sequences from the respective pair of subtypes. That is, we have 180 sequences in total. The first position of the 750 bps long genome part from which a semi-artificial recombinant is constructed is chosen randomly between position 1007 and 8501 with respect to the HIV-1 reference sequence HXB2 (see [The sequences in XB2 (see ).Tpure is composed of parts of sequences from regular-size pure subtypes, i.e. partial subtype sequences. The purpose of Tpure is to test the specificity of our algorithm with respect to the detection of segments originating from small-size and regular-size subtypes. Each sequence in Tpure has a length of 2000 bps. For each subtype, Tpure contains 20 partial sequences, i.e. 180 sequences in total. The starting position of the sequence part taken is chosen randomly between position 1007 and 8501 with respect to HXB2.The set spos of sequence positions correctly classified, ii) the conformance ssegm of the predicted and the correct subtype pattern. Hereby, ssegm is computed like follows  the fraction spos and ssegm consists in each concentrating on a different type of classification error: the primary purpose of spos is to identify smaller shifts of the breakpoints, under the assumption that the predicted recombination patterns are correct. On the contrary, ssegm does not aim for these errors, but is designed for detecting different recombination patterns. Furthermore, these scores are suitable for being used in an automatic evaluation. In contrast to that, using the distances between the correct and the predicted breakpoints  would require to deal with differences in the recombination patterns (like the recombination patterns AGA and AGAGA) manually. We use the arithmetic mean of spos and ssegm as an overall score for a single test sequence, denoting it stot.The motivation for using the two measures Trec we also consider misclassification in the first and last segment and for Tpure we do not distinguish between wrongful inserts of segments of normal- and small-size subtypes. One can rather view Trec and Tpure as boundaries between which all reasonable application scenarios lie: No standard user will want to emphasize sensitivity more than measured by one of the introduced scores on Trec and, analogously, no one specificity more than measured on Tpure.Notice that applying the introduced measures on the two test sets does not exactly measure what we stated as purpose of the test sets : For prob: the probabilistic model utilizing an information sharing protocol introduced in this paper, ii) jpHMM ml: the regularized linear discriminant learning presented in [scal: a heuristic approach scaling the pseudocounts depending on the number of sequences in a subtype already evaluated in [semi: the original algorithm introduced in [We compare four different versions of jpHMM, as summarized in Table ented in , iii) jpuated in , and iv)duced in .semi, jpHMM scal, jpHMM ml, and jpHMM prob on the test sequence set Tpure and all of them but jpHMM scal on Trec. The results of this evaluation are illustrated in Figures scal is not tested on Trec since the purpose of introducing jpHMM scal was to demonstrate that it is not possible to reach the performance of jpHMM ml or jpHMM prob by employing an arbitrary heuristic approach to estimate the emission probabilities. For this purpose, testing only on Tpure is sufficient.We compare the four jpHMM versions jpHMM Tpure, jpHMM semi classifies 82.2% of the sequences correctly, jpHMM scal 85.6%, jpHMM ml 97.2%, and jpHMM prob 98.9%. Applying the sign test yields that jpHMM prob is better than jpHMM semi with p-value p=9·10−10, than jpHMM scal with p=6·10−8, and than jpHMM ml with p=0.125.On Trec, jpHMM semi achieves an average stot of 0.74, jpHMM ml of 0.83, and jpHMM prob of 0.90. Based on the Wilcoxon signed rank test jpHMM prob performs better than jpHMM semi with p<2·10−16 and outperforms jpHMM ml with p=3·10−6.On Simplot employs a distance-based method, whereas Bootscanning is based on a phylogenetic approach. Both can be applied to both single sequences and groups of sequences, such as HIV-1 subtypes. Simplot as well as Bootscanning require a certain number of sequences  as input .Tpure and 36 sequences of Trec. Since it turned out during our evaluation that correctly classifying segments from large subtypes is not a challenge for any algorithm, we compared the methods i) for each large-size subtype on four randomly selected sequences from Tpure and ii) on all sequences of Tpure from small-size subtypes. For the comparison on semi-artificial recombinants, we randomly picked two sequences for each combination of a large- and a small-size subtype from Trec.As neither Simplot nor Bootscanning allow for automatic execution, we restrict our comparison to 84 sequences of prob misclassifies 2, jpHMM ml 5, Simplot 11, and Bootscanning 6. Applying the sign test yields that jpHMM prob performs better than Simplot with p=0.006 and better than Bootscanning with p=0.11.All four methods classify the 24 pure segments from large-size subtypes correctly. Out of the 60 segments from the small-size subtypes, jpHMM Trec, jpHMM prob achieves an average spos of 0.926, jpHMM ml of 0.863, Simplot of 0.833, and Bootscanning of 0.839. Hereby, jpHMM prob achieves a higher score than Simplot for 28 sequences with Simplot better performing for the remaining 8 sequences. Comparing jpHMM prob with Bootscannnig, jpHMM prob performs better in 32 out of 36 cases, with Bootscanning being better for the rest. Applying the Wilcoxon signed-rank test yields that jpHMM prob performs better than Simplot with p=2·10−4 and better than Bootscanning with p=2·10−6.On the 36 semi-artificial recombinants taken from prob, we evaluate the performance of jpHMM prob on semi-artificial recombinants. Since the results from pure sequences are less conclusive, we restrict our evaluation to recombinants. As Trec is too small to achieve significant results in this regards, we employ an extended version of Trec. Instead of 10 sequences per subtype pair we use 50. We measure the degree of conservation by the pairwise percentage identity of all sequences from our input MSA in the genome region from which the respective semi-artificial recombinant was taken. The results are shown in Figure In order to measure the influence of the variability of the genome part from which a sequence stems on the performance of jpHMM prob. The score achieved by jpHMM prob for sequences with identity lower than 70% is significantly lower than the one for sequences with identity higher than 70%. Above the threshold of 70%, the degree of conservation does not seem to have a significant influence on the prediction performance.The degree of conservation has an influence on the performance of jpHMM prob is capable of handling full-length sequences which exhibit a recombination pattern observed in a real recombinant, we apply jpHMM semi, jpHMM prob, and jpHMM ml to the sequence AF119819 classified as recombinant of type CRF04 [pjump=10−8 for jpHMM semi, pjump=10−6 for jpHMM prob and pjump=10−7 for jpHMM ml. We here have to use a considerably smaller jump probability for jpHMM semi than for jpHMM prob since we have to smooth out the misclassifications jpHMM semi does due to its less performant estimator of the emission probabilities. The results and the classification given on the LANL website are given in Figure In order to verify that jpHMM pe CRF04 ,31 in thNotice that subtypes F and K as well as subtypes A, G, and J, respectively, are closely related . Hence, prob change with different parameter values. Increasing pjump to 10−5 does not change the prediction at all, while for pjump=10−4 the number of segments is increased by four. Decreasing pjump to 10−7 or 10−8 yields the same result. The fragment from Subtype G, predicted in the vif gene, vanishes and instead Subtype A is predicted in this location.We also explored how the results of jpHMM prob to a sequence from Trec (length 750 bps) takes about 16 minutes. The lion’s share of the computational time is due to the calculation of the emission probabilities. It could be decreased considerably by implementing further heuristics to neglect source combinations with very low likelihood.On an Intel Core 2 Duo E8400 with 3 GHz and 4 GB of RAM, the application of jpHMM In this paper, we presented a novel information sharing protocol, realized in the form of a probabilistic model, for estimating the emission probabilities of pHMMs. We incorporated this protocol into the recombination detection program jpHMM and applied it to semi-artificial and real-world HIV-1 Group M sequences. As we were able to confirm an improvement of the prediction accuracy of jpHMM by our protocol, we demonstrated the value of the protocol for a crucial biological and medical application employing pHMMs.The work of biological and medical researchers can be supported in various ways by improving the precision of recombination and breakpoint detection for HIV-1. For example, discovering recombination-free genome regions allows for the identification of barriers to genetic crossing between different viral strains. This kind of barriers then may help identifying vulnerable aspects of HIV biology in form of combinations of mutations strongly decreasing the survivability of an HI virus. This can facilitate finding promising targets for antiviral strategies .Furthermore, recombination analysis and breakpoint detection do not only play a role for numerous viruses, e.g., flaviviruses, coronaviruses, alphaviruses, avian oncoviruses, rotaviruses, and influenza viruses ,33-39. IWe have applied i) the novel version of jpHMM incorporating our information sharing protocol, ii) former versions of jpHMM, and iii) the established recombination breakpoint detection tools Simplot and Bootscanning to i) sequences designated (in the LANL HIV database) as originating from a pure subtype, ii) semi-artificial recombinants of two real-world subtype sequences, iii) a full-length sequence of CRF04. In total, we compared the novel version of jpHMM with three formerly introduced ones, differing with regards to how they estimate the emission probabilities: i) a machine learning approach using linear discriminant learning, ii) a heuristic approach of scaling the pseudocounts, iii) the original approach without adaptions for small-size subtypes.The results were compared using appropriate performance measures. On all test sequence sets, the novel version of jpHMM either significantly outperformed the other algorithms or achieved better results, but without reaching statistical significance. The latter is mostly a result of the fact that for most algorithms the task of correctly classifying segments of large-size subtypes is not a challenge, thus yielding very similar results for the corresponding type of test.ml) introduced in [prob) seems to be considerably better performing on very small subtypes: On the one hand, jpHMM ml achieves an average score (stot) of 0.87 on the semi-artificial recombinants constructed from Subtype H or J, respectively, whereas the average score on recombinants constructed from Subtype K is 0.76. That is, there is a difference in regard to the average score of 0.11 between recombinants built from Subtype H and J, respectively, and Subtype K. On the other hand, for jpHMM prob the average score is 0.92 for recombinants built from Subtype H and J, respectively, and 0.87 for Subtype K. That is, a difference of 0.05 can be observed. Thus, our model-driven information sharing approach seems in particular more suitable to model very small subtypes than a machine learning approach based on regularized linear discriminant learning and a transformation of the resulting weights into valid probabilities via a softmax function.Compared with the machine-learning approach , we can conclude that the low performance for recombinants built from Subtype J and A is probably simply coincidental and that the influence of the phylogenetic distance is at most weak for recombinants between HIV-1 Group M subtypes.Astonishingly, for the tests on semi-artificial recombinants, the phylogenetic distance between the respective small- and large-size subtypes does not seem to have a notable influence on the performance of jpHMM prob on partial sequences stemming from weakly conserved genome parts can easily be explained by the fact that jpHMM is not well suited for this kind of sequences: pHMMs assume that the group of sequences to be modeled can be well aligned. If not, the resulting pHMM consists mostly of insert states which do not constitute a sophisticated model for unalignable sequences.The relatively weak performance of jpHMM One might wonder whether our test setting, with very short segments, is appropriate if the normal application requires considerably lower jumping probabilities. That is, one could suppose that our test setting does not prove that the algorithm performs well in correctly classifying a very long fragment  stemming entirely from one subtype. But if the algorithm tended to assign such a fragment to more than one subtype, it would also misclassify shorter subfragments. Hence, by testing it on partial length sequences from one subtype we have in fact also covered these long fragments from one subtype.prob is capable of handling real world data, we applied it to a CRF04 sequence. This application shows – to some extent – why we stuck to semi-artificial recombinants instead of evaluating jpHMM prob on CRFs. For CRFs, it is unclear which classification is the “correct” one. This results from the scientific community not coming to any consensus on which subtype classifications are the correct ones. Beyond that, it is even discussed whether the entire classification system exhibits flaws: For example, it has been debated whether Subtype G is in fact a recombinant and CRF02 a pure subtype [prob on semi-artificial recombinants, despite one could perceive testing on CRFs as more “realistic”.In order to verify that jpHMM  subtype -48. More subtype ,46,49,50We recently have developed a probabilistic algorithm called the “Unknown Subtype Finder (USF)”  for the Beyond this, one might think of several extensions to the probabilistic model presented in this paper: • The probability of whether the emission probabilities of a given set of subtypes are modeled jointly might depend on the subtypes in the set. Like this, it could be accounted for the fact that phylogenetic close subtypes should tend more to be modeled jointly than phylogenetic distant ones. As the phylogeny of the subtypes, which subtypes tend to be jointly modeled might change along the genome.• The information sharing protocol could be based on a hierarchical classification system  instead• The probability of jumping between subtypes could be modeled as varying along the genome, motivated by the fact that the distribution of recombination breakpoints is not uniform among the genome .http://jphmm.gobics.de.The C++ source code of jpHMM and Matlab code for evaluating the different versions of jpHMM is available at a The explanation here emphasizes understandability. Due to computational efficiency, the actual computations are carried out in a quite different way.b The first ten Bell numbers B(1) through B(10) are 1, 2, 5, 15, 52, 203, 877, 4140, 21147, 115975.The authors declare that they have no competing interests.IB conceived the approach and developed, implemented, and tested the algorithm. AKS carried out modifications on jpHMM. CC provided statistical expertise. TM and FS assisted in the data handling. All authors read and approved the final manuscript.Supplements.Click here for file


Rat models are widely used in preclinical studies investigating fracture healing. The interfragmentary movement at a fracture site is critical to the course of healing and therefore demands definition in order to aptly interpret the experimental results. Estimation of this movement requires knowledge of the fixation stiffness and loading. The characteristic loading for the rat femur has been estimated, but the stiffness of fixation used in rat studies has yet to be fully described. This study aimed to determine the 6 degree of freedom stiffness of four commonly used implants, two external fixators (RatExFix and UlmExFix), a locking plate, and a locking intramedullary nail, in all degrees of freedom and estimate the interfragmentary movement under specific physiological loads. The external fixator systems allow the greatest movement. Mounted 45° anterolateral on the femur, the RatExFix allows an average of 0.88 mm of motion in each anatomic direction while the stiffer UlmExFix allows about 0.6 mm of motion. The nail is far stiffer than the other implants investigated while the plate allows movement of an intermediate magnitude. Both the nail and plate demonstrate higher axial than shear stiffness. The relatively large standard deviations in external fixator shear motion imply strong dependence on bone axis alignment across the gap and the precise orientation of the specimen relative to the loading. The smaller standard deviation associated with the nail and plate results from improved alignment and minimization of the influence of rotational positioning of the specimen due to the reduced implant eccentricity relative to the specimen axis. These results show that the interfragmentary movement is complex and varies significantly between fixation devices but establishes a baseline for the evaluation of the results of different studies. These s studies , 2. A di studies –7.Although the fixation stiffness is frequently explicitly considered and tested in large animal models, to date, little data is published for devices used in small animal models and most studies do not characterize the devices at all –14. Thisin vivo IFM. Although such simplified characterization may hint at the mechanical environment, it also ignores the complicated and potentially highly influential effects of coupled response between the primary loading mode and motion in all other secondary degrees of freedom. For example, the relationship between a force within the anteroposterior plane and the shearing motion response which develops in the medial lateral plane may be significant but would be unquantified and unconsidered without rigorously specifying the 3-dimensional stiffness.The few studies which address the mechanical environment using rat models place emphasis on the axial compression within the fracture gap and disregard the potential shearing motion. For this reason, it is common for researchers to characterize the axial stiffness of their chosen implant system through some sort of uniaxial compression test –17. SomeThe IFM is a function of the load applied through weight bearing and muscle tension as well as the stiffness of the fracture fixation , 21, 22.et al. have estimated the physiological loading occurring in the hind limb of the rat throughout gait [The aims of this study were therefore to develop, for the first time, the 6DOF stiffness matrices for the most important fixation devices used in rat fracture healing studies as well as present a method by which other researchers may characterize their chosen fracture fixation systems. Since Wehner out gait  we have Four different fixator designs commonly used in fracture healing studies employing the rat femur , 23–27 wThe remaining fixator designs are standardized and commercially available research implants . These implants include an external fixator  GmbH & Co. KG, Karlsruhe, Germany) using custom made mounts. The upper aluminum fixator mount was clamped into the immobile frame surrounding the Hexapod. The lower aluminum fixator mount is radially clamped within a fixture directly attached to a 6 DOF FT-Nano 43 load cell  . For eacguration , 29 whicij is a representative element of a stiffness matrix in the ith row and jth column, Uj is the jth element of a column displacement vector acting on the system relative to a chosen coordinate system, and Ri is the ith element of the column vector of the reaction forces and moments. Note that the Einstein summation convention is applied to dummy indices.The following analysis assumes each fixator behaves in a linear elastic manner within the applied loading regime in accordance with previous studies characterizing the 6DOF stiffness of fixation , 29. Thexv would correlate the force which develops along the x axis due to a rotation applied about the y axis. The units of each element of the stiffness matrix are determined by the constitutive coupling it represents. Let T represent any subscript x, y, or z and R represent any subscript u, v, or w. Elements of the form KTT will have units of N/mm. Elements of the form KTR will have units of N/degree. Elements of the form KRT will have units of Nmm/mm. Elements of the form KRR will have units of Nmm/degree. Taking our previous example, element Kxv will have the units of N/degree since it maps a reaction force to a rotational displacement.A typical load case will be represented by a six element vector of displacement and rotation values, only one of which will be non-zero. A single load case in which all but a single displacement or rotation is non-zero will therefore provide a system of six algebraic equations which can be solved directly to determine six of the unknown stiffness values. In order to solve for all 36 of the stiffness values, we must therefore apply six independent load cases.kj references an independent load case and each column j of Rij refers to the reaction forces and moments which develop in response to load case j of Ukj. The matrix representation thus appears asij references the specific load case where 1–6 indicate the columns of matrix Ukj. This system maps each of the 36 unknown stiffness values to a specific force or moment as the result of an applied translational or rotational displacement. This system of equations was solved by post-multiplying Rij with the inverse of Ukj using MATLAB software .The resulting matrices give the forward relationship mapping an applied IFM to the virtual reaction forces and moments within the fracture gap. In order to make this data useful for the prediction of IFM, the calculated stiffness matrices must be inverted to give flexibility matrices which map a vector of physiological loads to an expected IFM at the fracture gap. This is accomplished by matrix inversion and was calculated using the inversion function of MATLAB.et al. The first coordinate transformation simply reoriented the coordinate system of Wehner et al. to the coordinate system established for the testing of the implants  were applied to this basis physiological load vector for calculation of the IFM with the fixator mounted at each 30, 45, and 60°. The transformed vectors correspond to the loading that would be acting on the fixator relative to the coordinate system used to calculate the stiffness and flexibility matrices . These aIn calculating the stiffness matrices from the collected data, it was found that certain components of the mathematical sign of certain components of the stiffness matrices varied between specimens of the same fixation. This can be explained by the variability of implantation and the resulting angular deviation and offset displacement between the proximal and distal ends of the specimen. Therefore, the IFM is reported as an average of the absolute value of the motion within a specific anatomic plane for each specimen. This is a valid approach as it is the magnitude and mode  which dictates the healing response independent of the exact direction of motion .The maximum translational and rotational physiological IFM Figs  and 7 weA comparison of the A-P translation for each fixator indicates that the RatExFix is significantly different from both the RatNail (p < 0.001) and the RatFix (p = 0.020) while the UlmExFix allows significantly greater motion than the RatNail (p = 0.020).The RatExFix is the most compliant implant tested allowing an average initial axial motion of 0.84 ± 0.07, 0.88 ± 0.08, and 0.92 ± 0.08 mm of motion for 30, 45, and 60° anterolateral implantation, respectively. When mounted at 45° the magnitude of IFM is nearly equal in all anatomical directions, allowing an average of 0.88 mm of mediolateral (M-L), anteroposterior (A-P), and axial interfragmentary translation. When mounted at 30°, the peak A-P translation exceeds the axial IFM while the M-L translation is reduced. When mounted at 60°, a similar trend is observed with the M-L translation exceeding the axial motion and the A-P translation decreasing. The axial IFM is much more consistent than either the M-L or A-P translation across all mounting planes.For all mounting positions, the A-P bending  and axial rotation are nearly equal with approximately 7° of motion. The M-L bending  is markedly lower in all cases Figs  and 7.When the fixator frame is mounted 12 mm from the surface of the bone, the UlmExFix is stiffer than the RatExFix but demonstrates similar trends across the mounting angles. The axial IFM is more consistent than for the RatExFix, measuring 0.61 ± 0.03 mm in every mounting angle, and when mounted at 30°, the A-P translation is equal to the axial motion. When mounted at 60°, the M-L translation exceeds the axial IFM. The axial rotational stiffness and the A-P bending stiffness are much higher for the UlmExFix than for the RatExFix at every mounting angle. Notably, the axial rotational motion is nearly twice that of the A-P or M-L bending motion for this fixation system Figs .The translational stiffness of the RatFix is greater than either of the external fixator systems, but it is more compliant in axial rotation and A-P bending than the UlmExFix. Of interest, when mounted in the 30° position, the A-P translation is approximately twice the M-L translation and axial IFM. The M-L bending motion tends to be equal to or greater than the M-L bending of the UlmExFix, however, the standard deviation is relatively large in this direction, averaging ±1°. Furthermore, the pattern of rotational motion is more similar to RatExFix than the UlmExFix is to the RatExFix Figs  and 7.The RatNail is, by far, the stiffest implant system investigated allowing an average of only 0.03 ± 0.01mm of axial IFM. For each calculated mounting angle, the average M-L translation was greatest, followed closely by A-P translation. However, the M-L and A-P translation demonstrated larger standard deviations than the axial IFM. In contrast, the average amount of axial rotation was much greater than either A-P or M-L bending but with a standard deviation larger than ±1.3°, nearly an order of magnitude greater than the deviation in the bending modes Figs  and 7.This study presents a method for the calculation of a 6DOF stiffness matrix for the most important fracture fixation devices used in rat models. Using the musculoskeletal model of Wehner et al., the IFM in the fracture gap could be estimated for use as a basis of comparison of fixation behavior. The results demonstrate that the IFM is always a combined and complex movement that also depends on the anatomical location of the fixation device.in vitro boundary conditions do not correspond to the in vivo boundary conditions. Because the complex relationships between displacements and reaction forces and moments are not defined, one cannot mathematically compensate for the variation in boundary conditions. Although this study applied confined torsion, the reactions are recorded in all 6DOF and the coupling of displacements with the 6 DOF forces and moments in the other five load cases provide a comprehensive model freeing it from the boundary conditions applied during testing. This information is vital; literature indicates that shearing motion may inhibit bone healing [Typical confined torsion testing of implant constructs yields little useful information because the  healing , so largThe larger standard deviations associated with A-P and M-L shear in the external fixators and variation in the sign of respective stiffness matrix components of different samples of the same fixator indicate strong dependence on a few variables including alignment of the femoral axis across the fracture gap and the precise angular position of the fixation relative to the directions of applied load during testing. The latter is analogous to varying rotational positioning about the femoral axis and is the likely cause of sign discrepancy. If the direction of loading varies relative to fixation plane, the reaction forces and moments will vary accordingly due to the “anisotropic” structural stiffness. Furthermore, for the RatExFix, measurements of our specimens indicate that the actual mounting distance from the bone varies between 3.8 and 4.5 mm using the recommended mounting technique. These factors also help explain the much smaller standard deviations associated with the RatNail and RatFix systems in which the implant is coincident with and minorly eccentric to the specimen axis, respectively. This improves the reproducibility of alignment across the fracture gap compared to the large eccentric positioning of the external fixators and reduces the variability in structural stiffness resulting from small angular deviations relative to the loading direction.The two external fixators allow very large translational motion in every plane relative to the typical gap size used in fracture healing experiments employing the rat. A 1 mm osteotomy stabilized with this implant will lead to an average initial axial strain of 84 ± 7, 88 ± 8, and 92 ± 8%. Strains of this magnitude may delay healing relative to optimal fixation stiffness. Furthermore, it may illicit a biochemical response that would not correspond well to studies using other, more stable fixation techniques. Importantly, it is known that certain biological factors have a biphasic dose response , 31. TheIn the present study, the UlmExFix is visibly stiffer than the RatExFix Figs  and 7, hThe RatFix is intermediate in its stiffness characteristics and has relatively small standard deviations across all translational DOFs. However it is more flexible, in torsion and the bending modes than the UlmExFix and the RatNail, likely due to the PEEK material from which it is made in contrast to the metal construction of the two other implants.in vivo. Additionally, the loss of functionality of the proximal anchoring thread would likely contribute to a larger lateral toggle than that present in vivo.The very stiff RatNail fixation is due to the combination of its unusually large diameter in comparison to traditional Kirschner wire intramedullary fixation as well as its locking feature provided by the four locking screws which pass through the bone and nail. These features are in stark contrast to the typical K-wire internal fixation used in many rat fracture studies –37, whicIn vivo conditions will vary greatly between individual specimens based on exact bone morphology, pain tolerance, weight, and implantation variables. For this reason, the resulting IFM magnitudes should not be taken as precise values. Furthermore, the predicted motion does not consider the potential difference between fixation stiffness of screws in aluminum tubes rather than bones and the stabilization resulting from resistance due to surrounding soft tissue and the callus, or the decreased post-surgical weight bearing.The method for measuring implant flexibility and the resulting IFM trends under loading are independent of the exact physiological load. However, the physiological loading vector is based on values determined by inverse dynamic analysis for a single specific rat. in vivo IFM values due to the system nonlinearities and variations as well as the large discrepancy in physiological loading across individuals resulting from body weight variation, morphological variation, post-surgical pain tolerance, and implantation variations.This study assumes that a linear elastic coupling exists between the load in any given degree of freedom and the resulting kinematic response in any other. We have checked the validity of this assumption by plotting the incremental motion steps against each of the six reaction force and moment values corresponding to the respective position steps to assess the linearity of every force and moment response with respect to motion in each degree of freedom. An example of this is given in In order to compare and understand the results of different fracture healing studies, the experimental conditions must also be understood. The results of this study show that the IFM associated with physiological loads is complex and varies significantly between commonly used rat fixation devices and their application relative to anatomical planes. Studies which characterized the stiffness of the fracture fixation in only one loading direction missed information regarding the IFM in the other loading planes and may have led to the misinterpretation of results. In addition, this study establishes a baseline for the evaluation of the results of different studies. This study adds to the body of data and understanding of research implant behavior allowing researchers using any of these particular systems to better understand their experimental set-up. Furthermore, it provides a general method and encouragement for researchers to characterize and publish complete implant stiffness data for whichever fixation system and technique they employ.By including a full mathematical description of the behavior of fixation systems, researchers will be able to mine the work of others more efficiently and potentially draw important conclusion that are currently obscured by undefined mechanics thereby increasing the utility of experimental results. This characterization can be completed at any time, and may potentially help uncover new information useful for the improvement of experimental design for future studies.S1 Dataset(ZIP)Click here for additional data file.


Dear Friends and Colleagues,Rambam Maimonides Medical Journal was once a new and unknown publication. Today we have more than 17,000 subscribers from 146 nations and territories. We published 39 scientific medical papers in 2017 out of 61 submitted manuscripts.We are now indexed by PubMed and Thompson Reuters Emerging Sources Citation Index, to name a few. Next year, the Journal is scheduled to receive an official impact factor from Thompson Reuters.We are not so unknown anymore.Rambam Maimonides Medical Journal established in 2017 the Maimonides Best Published Original Research Prize. This annual prize of $1,000 is to be awarded to the first author of the best original research paper published in the journal over the previous year.As a new Journal, most of the papers submitted were naturally reviews. However, the most important aspect for the promotion and advancement of medicine is publication of original research. To promote such efforts the editors of no advertising—perhaps the most unusual feature of this publication. Rambam Maimonides Medical Journal is donor-supported by those who share in our vision to ensure that anyone anywhere in the world can submit their scholarly work with no fear of commercial bias and no fees to impede publication.This prize is an added benefit to authors. Since asking for fees from authors who have few or no research funds or grants may preclude publication of their papers, our Journal has no subscription fees, no submission fees, and no publication fees. Equally noteworthy is that although published by Rambam Health Care Campus, the Journal is unaffiliated with any organization or society. A decision was also made when the Journal was established to seek Rambam Maimonides Medical Journal has brought to the world 273 original scientific medical papers on a variety of topics.This editorial effort has been fruitful and has led to excellent results. As we enter our ninth year of publication, it is rewarding to note that With the announcement of the Maimonides Best Published Original Research Prize, 25% of our published papers in 2017 were original research articles. Determining the best published paper from so many well written articles presenting many important research findings was no small task. Nevertheless, after careful deliberation, the RMMJ Original Research Prize Committee selected a winner.I am pleased to announce that the first recipient of this prize is Dr. Anatoli Stav for his paper titled “Femoral versus Multiple Nerve Blocks for Analgesia after Total Knee Arthroplasty,”Rambam Maimonides Medical Journal has the freedom to present topics of varied and specific interest. To that end, we not only ensure publication of papers with a Jewish flavor,e.g.e.g.e.g.As a general medicine publication, Rambam Maimonides Medical Journal includes a special supplement: Abstracts from the 14th Annual Rambam Research Day. We would like to encourage other medical organizations to contact us regarding publication of supplements representing the research from their esteemed institutes.The January 2018 issue of Rambam Maimonides Medical Journal will serve the international scientific medical community with a broad range of scholarly papers that might not have been published via any other venue.Our hope is that


European Journal of General Practice should like to express their gratitude to all reviewers who have advised us during the year 2016. To acknowledge their indispensable contribution to the European Journal of General Practice, we have listed the names of all referees who completed at least one review report between 16 December 2015 and 16 December 2016.The Editors of the Heinz-Harald Abholz, Erik Abilsnes, Jeffery Adams, Gisele Apter, Enric Aragonés, Chris Arden, Davut Baltaci, Tadej Battelino, Carol Bayona, Jaume Benavent, Ria Benko, Anette Berendsen, Christophe Berkhout, Sonja Bidmon, Peter Birner, Bjørn Bjorvatn, Marco Blanker, Jan Bosmans, Colin Bradley, Carlos Brotonos, Mateja Bulc, L.H. Burridge, Manuel Campiñez, Meltem Ciceklioglu, Peter Clarys, David Cohen, Gloria Cordoba, Lizzie Cottrell, Anne-Marie Cox, An de Sutter, Jan Degryse, Dorner, Thomas, Chris Dowrick, Sandra Fahrenkrog, Gabriele Fischer, Thomas Frese, Zbigniew Gaciong, Luis Galvez, Jochen Gensichen, Sherly George, Juan Gervas, Christoph Gisinger, Liam Glynn, Maciek Godycki-Cwirko, Alexandre Gouveia, Tomasz Grodzicki, Dilek Güldal, Ronny Gunnarsson, Peter Hjertholm, Kathryn Hoffmann, Eva Hummers-Pradier, Thomas Kahan, Laszlo Kalabay, Ruth Kalda, Gustav Kamenski, Bridget Kane, Brian Kelly, Martina Kelly, Marko Kolšek, Evan Kontopantelis, Dionne S. Kringos, Ron Kusters, Ilias-Ioannis Kyriopoulos, Toine Lagro, Amnon Lahad, A. J. Larner, Asam Latif, Klaus Linde, Christos Lionis, Carl Llor, José Ramon Loayssa, Nicole Lowres, Heidi Lyshol, Roar Maagaard, Aldo Pietro Maggioni, Christian Mallen, Niina Markkula, Inaki Martin-Lesende, Mercè Marzo-Castillejo, Marion McAllister, Saskia Mol, Jose M. Molero, Ana Moragas-Moreno, Maria Luisa Morato, Tania Morris, Luke Mounce, Anders Munck, Miguel Muñoz, Peter Murchie, Markus Nielen, Caitlin Notley, Peter Nygaard, Abimbola Obimakinde, Zeliha Ocek, Eric Olsman, Graziano Onder, Ennio Ongini, John Paget, Davorina Petek, Soeren Prins, Enriqueta Pujol-Ribera, Jesus Pujol, Maja Racic, S. Ramlall, Sofia B. Ravara, Shmuel Reis, Claire Rondet, Jim Ross, Danica Rotar Pavlič, Marie-Eve Rougé Bugat, Guy Rutten, Antonio Sarría Santamera, Willemijn Schäfer, Nynke Scherpbier, Henrik Schroll, Michaela Schunk, Polona Selic, Monika Seltenhammer, Helmut Sinzinger, Jef Sisler, Antoni Sisó Almirall, Marleen Smits, Hilde Solberg, Sal Stapley, Igor Ŝvab, Dorota Szczesniak, Angela Taft, Clare Taylor, Gabriela Heiden Telo, Berend Terluin, Bratt Thombs, Tomasz Tomasik, Salvador Tranche, Michael J. Twigg, Anne-Marie Uijen, Steven Uittenbogaart, Sabina Ulbricht, Aysegul Uludag, Juan Valdes-Stauber, Joan Antoni Valles, Wil van den Bosch, Marc van der Wel, Tineke van Geel, Evelien van Riet, Piet Vanden Bussche, Juha Varis, José Vázquez Villegas, Theo Verheij, Ernest Vinyoles, Johannes Wancata, Jane Wilcock, Oriol Yuguero Torres.European Journal of General Practice accepts around 30% of all submitted manuscripts.We want to thank you all for your appreciated assistance! Your review reports were very valuable feedback to the authors and helped us to make our editorial decisions. Currently, the http://mc.manuscript-central.com/ejgp and log in to modify your profile. In addition, we invite all readers of the European Journal of General Practice who would like to review manuscripts for this journal, to visit http://mc.manuscriptcentral.com/ejgp and to register as ‘new user’, i.e. to complete the details of their expertise. We provide a format to facilitate the review.Although our database of authors and reviewers steadily increases, the Editors do not always find it easy to find referees within the journal’s deadlines. Therefore, we sincerely hope that all reviewers want to continue their review work in the future. If you would like to update your reviewer account, e.g. to specify your expertise, please visit ejgp-agermeraad@maastrichtuniversity.nl).If you have comments regarding the journal or its peer review process, we invite you to contact the editorial office (Ms. Anneke Germeraad-Uriot, Editorial Assistant: