In utero exposure to environmental factors can modify the development of allergies later in life whereby the mechanisms of the feto-maternal crosstalk still remain largely unknown. Murine studies revealed that inflammatory maternal signals elicited by chronic helminth infection within the placenta imprint a distinct gene expression profile related to the Vitamin-D-receptor (VDR)-inflammation-axis. We thus investigated whether pro- or anti- inflammatory immune responses as well as VDR and related gene expression within the placenta differ between women from helminth-endemic and non-endemic areas. A prospective pilot study was conducted in Munich, Germany (helminth non-endemic) and Lambaréné, Gabon (helminth-endemic). At delivery, clinical information alongside placenta tissue samples and maternal and cord blood were obtained for further laboratory analysis. Schistosoma haematobium infection was detected in 13/54 (23%) Gabonese women. RT PCR revealed significantly lower gene expression of VDR, Cyp27b1, Foxp3 and IL10 in Gabonese compared to German placentae as well as significantly lower levels of plasma IgG4 in newborns resulting in a significantly higher IgE/IgG4 ratio. These findings demonstrate that exposure in utero to different environments alters placental gene expression and thus possibly plays a role in the development and modulation of the immune system of the offspring. Approximately 220 million people are infected with schistosomes5 and thus, approximately 40 million women of childbearing age. Yet little is known about the specific morbidities inflicted on pregnant women and their offspring6. Complications such as anaemia and low-birth-weight7, as well as skewed immune responses, which can alter susceptibility for other diseases8, are just some examples of the potential impact of a pregnancy modulated by helminth infection. Such infections are known to modify host immune responses through a variety of mechanisms9, minimizing host-damage to enable survival of the parasite10. Stable, chronic helminth infections can endure for long periods (up to 20 years) in an individual host, facilitated through helminth-induced recalibration of host immunity towards a state of immune hyporesponsiveness that can be considered a form of immunologic tolerance13.Chronic inflammatory disorders, such as allergic airway inflammation, are negatively associated with high levels of helminth infection such as schistosomes, particularly in low-income countriesin utero and leads to enhanced immunological maturity at birth14. Indeed, it was recently demonstrated, that experimental chronic infection with the helminth Schistosoma mansoni during pregnancy influences the outcome of allergic asthma in offspring15. This was further associated with downregulation of genes associated with either Vitamin-D-metabolism and –pathways such as the transcription factor Vitamin-D-receptor (VDR)16, the enzyme 1α-hydroxylase (Cyp27b1), responsible for vitamin D activation, as well as hydroxy-delta-5-steroid dehydrogenase (Hsd3b1)15, which is crucial for roles in the biosynthesis of all hormonal steroids17.Recent research indicates that early exposure to such a modified environment during gestation already primes the fetal immune system 2D, whereas Cyp24a1 is responsible for the inactivation. VDR and Cyp27b1 are expressed in almost all immune cells as well as in both decidua18 and trophoblast19 suggesting that the placenta itself converts 25(OH)D to the active form and may thereby function in an autocrine or paracrine fashion20. Indeed, the loss of 1,25 (OH)2D production in the fetal compartment of the placenta has been shown to cause generalized dysregulation of placental inflammation after immune challenge20, which are known to be induced during helminth infection22. IL-10 is produced by Forkhead-Box-Protein P3 (Foxp3) expressing regulatory T cells alongside other cell types such as the villous cytotrophoblasts within the placenta, where it appears to work as a key facilitator of successful pregnancy20.Therefore, defective VDR signalling in placental tissue might result in increased risk of placental inflammation and expression of inflammatory cytokines or dampening of anti-inflammatory and tolerogenic cytokines, respectively. Placental VDR expression, as well as Cyp27b1, has also been linked to regulation of key cytokines involved in inflammatory responses, namely interleukin 10 (IL-10) or interferon gamma (IFN-γ). Cyp27b1 hydroxylases’ 25(OH)D to the active form 1,25 (OH)23 to avoid rejection of the fetus as well to protect it from maternal infections. The placental leukocyte population is compromised by around 70% uterine natural killer cells (uNKs), about 20% macrophages and 10% T-lymphocytes (with 10–15% regulatory T-cells), but also dendritic cells and mast cells can be found in the early placental bed25. The fetal side is composed of the chorionic plate. The feto-maternal zone in-between consists in the intervillous space with maternal blood and the villous trees providing the fetal blood.The placenta plays a decisive role in pregnancy maintenance and the development and protection of the fetus. Besides the production of hormones, the placenta is a barrier between mother and fetus and maintains immunological tolerance. The mature, disc-shaped placenta can be divided into three zones. First the basal plate or decidua which is predominantly the maternal side of the placenta and consists of up to 30–40% leukocytes26. In multivariate models adjusted for geohelminths, maternal schistosomiasis was associated with increased levels of inflammatory cytokines in maternal peripheral blood, placental, and cord blood, as well as acute subchorionitis27. Granulomatous inflammation in the placenta28 and the cervix29 in the context of female genital schistosomiasis (FGS) can occur if immature worms or eggs directly become lodged in the placenta6. In general, placental inflammation is associated with substantially lower feto-maternal immunoglobin G (IgG) antibody transfer efficiency30 which is tightly regulated and mediated by neonatal Fc Receptor (FcRn)31. IgG is the only antibody subclass which is able to cross the placental barrier. IgG, especially its subclasse like IgG4, and IgE, which acts in a competitive way with IgG4, are strongly induced during helminth infections32. Strong anti-parasite IgE responses are associated with resistance to infection33, whereas high levels of IgG4 have been associated with susceptibility34. However, in contrast to IgG4, IgE is not able to cross the placental barrier. Thus, any detection of fetal IgE can be considered as evidence for in utero priming of the fetal immune system. Indeed, schistosome specific IgE have been detected in cord blood from S. haematobium infected mothers35.Environmental triggers and maternal stress can lead to significant changes within to the placenta, with important outcomes for fetal health and developmentConsidering that the placental anti- as well as proinflammatory gene expression can be skewed by the environment including helminth infections, we compared placental gene expression as well as inflammation markers in maternal and cord blood of Germans and Gabonese cohorts in a cross-sectional study.For the study, 54 pregnant women in Gabon, and 47 in Germany fulfilled all inclusion criteria and were successfully included into the study Table . At birtS. haematobium infection, none of the mentioned outcome parameters differed significantly  or immunological  genes as well as vitamin D plasma levels. Vitamin D has recently been shown to induce the antimicrobial peptide LL-37 which belongs to the cathelicidin family44 and has a potent antimicrobial activity against a wide assortment of bacterial, mycobacterial, protozoal, fungal and viral pathogens. Yet, until now, nothing is known about its role in helminthic diseases such as schistosomiasis. Within this study however, the schistosomiasis-infected group was too small to allow assessment of this parameter but will be included in future studies. In conclusion, environmental and perhaps genetic factors might suppress anti-inflammatory signals and alter the regulation and activation of vitamin D and the regulation of tolerogenic and anti-inflammatory markers.48. The main cause is proinflammatory cytokines such as TNF-α or IL-66. The women from Gabon, which is a highly endemic area for schistosomiasis49, indeed suffered from anaemia in contrast to the European women even though lower haemoglobin concentrations during pregnancy are physiologic (cut-off for pregnant women: haemoglobin <11 g/dL). This confirmed findings from another study with 998 pregnant women in Gabon, where 49.9% of the participants were anaemic7. Anaemia however is most likely to have multifactorial causes in this geographical region which besides schistosomiasis and pregnancy itself includes malaria, soil transmitted helminths and nutritional deficiencies such as iron deficiency.Helminths provoke a subtle inflammation in their hosts and cause anaemia, also known as anaemia of infection or of chronic disease45 and adverse birth outcomes, such as low birth weight7. Indeed, neonates in Gabon were significantly smaller (minus 3 cm) and lighter (minus 255 g) than those born in Germany. In addition, young maternal age has been shown to be associated to low birth weight, too50 and mothers in Gabon are younger than the Germans, but this might be an additional factor beside geographical difference.Placental inflammation and pro-inflammatory cytokines might also contribute to fetal hypoxiaPlasmodium falciparum in her thick blood smear and was thus excluded from this study. Malaria could contribute to disturbed placental transfer taking into consideration that infections may be asymptomatic in high endemic areas for malaria and infections therefore obscure49.Another consequence of placental inflammation is disorder of the placental barrier with a disruption of the active Fc receptor mediated antibody transfer. Cord blood IgG4 compared to maternal levels was significantly higher in Germany, whereas Gabonese cord and maternal IgG4 levels did not differ from each other. As this isotype represents a maternally-derived antibody, these results indicate disturbances to the processes involved in the transfer from maternal circulation in Gabon. Because of the small number of schistosome infected mothers  in our study, the noted differences are more likely to be influenced by other factors than acute schistosomiasis. Six Gabonese women (11%) stated to have had malaria during pregnancy whereas at time of delivery only one Gabonese mother had 32. IgG4 predominantly supports an immune-tolerant environment for helminths by counteracting helminth-specific IgE responses. High IgE levels are associated with resistance to infection33, whereas high levels of IgG4 have been associated with susceptibility34. In our study population total IgE levels were significantly higher in the Gabonese cohort and within this cohort also higher in the maternal plasma samples of the infected group. The high levels in some maternal German plasma samples were correlated to their history of allergy since severe autoimmune diseases were part of the exclusion criteria and therefore excluded. Regarding interactions between IgE and IgG4, a high IgE/IgG4 ratio correlates with protection against helminth infections. Indeed, in Gabon the maternal total IgE/IgG4 ratio is significantly higher than in Germany and lifelong exposures might induce protection against reinfection.Overall levels of IgG4 were significantly lower in Gabonese plasma, although the infected sub-group showed a trend towards higher IgG4. AWA-specific IgG4 levels were significantly higher in cord and maternal blood of the schistosome-infected group as expected in a population endemic for helminth infections since there exists substantial cross reactivity between helminth antigensin utero. Unlike the German cohort, about 98% Gabonese cord blood samples had detectable levels of IgE antibodies. In a study conducted in Coast Province, Kenya, cord blood lymphocytes were stimulated in vitro and about 36% of healthy newborns spontaneously produced polyclonal IgE and IgG51. Polyclonal and parasite-specific IgE were equally present in Kenyan cord and maternal sera whilst it remained undetectable in cord blood sera from North American infants52. Likewise, in Gabonese cord blood Schistosoma-specific IgE antibodies were detected previously35. However, in this study, even though levels of AWA-specific IgE were high in maternal blood, none could be detected in the cord blood. An explanation could be adult worm antigens may not cross the placenta in the same quantity as egg-related antigens35. The immune reaction to AWA is less strong than to the eggs and SEA54. Worms are able to survive for many years in the human host due to their ability to mask their surface tegument with host antigens55 and the excretion of an only small amount of antigenic molecules.In contrast to IgG4, IgE cannot cross the placental barrier thus the IgE antibodies detected in cord blood are most likely already produced by the fetus in utero exposure to (helminthic) antigens. Furthermore, as one of the first studies to compare inflammatory genes between geographically distinct populations, including subgroups based on helminth infection, as well as localized gene expression to placental and fetal placental subregions, we provide an initial setup for further investigation into how infection status can modify the complex crosstalk between placental inflammatory responses and healthy fetal development.In summary, our results indicate that pregnant women in Gabon might have a higher risk for placental inflammation, with a lower anti-inflammatory milieu when compared to German women. This is associated with reduced transplacental IgG4 transfer whereby the detection of IgE in offspring could be a hint for early priming events such as the de novo antibody class-switching due to The neonates in Gabon and Germany were examined by different midwives. Except for Vitamin D, Ca++ and CRP levels, maternal hemogram was done in two different laboratories for each cohort and helminth diagnostic only for the Gabonese cohort, whereby we attempted to reduce the probability of infection to a maximum by using a questionnaire in the German group. Only 30 Gabonese women provided stool samples to investigate for geo-helminth infections.The placenta samples were not washed, and gene expression analysis might be influenced by the presence of blood cells.The study was approved by the Institutional ethic committee of CERMEL in BP 242 Lambaréné, Gabon, (CEI-007/2017) and the Ethikkommission der Fakultät für Medizin der Technischen Universität München, Ismaninger Straße 22, 81675 München, Munich, Germany, . It was financed by Deutsche Forschungsgemeinschaft DFG (CO 1469/14–1) and Deutsches Zentrum für Infektionsforschung (DZIF) e. V. (TI 07.003). The methods were carried out in accordance with the relevant guidelines and regulations. The study took place at two sites. First between June and August 2015 and Mai and August 2017 at the Klinikum rechts der Isar, Munich, Germany, where 57 pregnant women were screened and samples of 47 participants as European non-endemic control group could be collected and be included for analysis. All mothers were Caucasians. As the samples were all collected in Germany, we call them “German samples”. The second population consisted of 59 women living in the province of Moyen-Ogooue, Gabon, in central Africa and delivering their newborns between August and November 2017 either at the Albert Schweitzer Hospital or the Centre Hospitalier Rérgional Georges Rawiri (CHRGR) in Lambaréné. 54 women and their newborn fulfilled inclusion criteria. We call these samples “Gabonese samples”. The purpose of the study and the procedures involved were explained and only those mothers granting written informed consent were enrolled as participants. To participate, all adolescents were at least 18 years old, had ≥ 37 weeks of gestation, and were giving spontaneous vaginal birth. Caesarean section, as well as acute severe infections such as malaria or knowing of other chronic diseases such as hepatitis and HIV at time of delivery were exclusion criteria.4-Heparin (BD Vacutainer S-Monovette®) tubes for further peripheral blood mononuclear cell- (PBMC) and cord blood mononuclear cell- (CBMC) processing and for plasma separation. Plasma  was separated by centrifugation and frozen at – 20 °C. Moreover, for the Gabonese cohort, EDTA blood for parasitology diagnostic and for clinical analyses was taken at the same time point and immediately analysed for the presence of protozoan blood parasites.Paired umbilical cord and maternal peripheral venous blood samples were collected at delivery. To avoid admixture of maternal and cord blood, cord blood was obtained not by squeezing the umbilical cord but instead by direct needle aspiration, taking care to clean the cord of maternal blood beforehand. All blood samples were collected in 9 ml NH3 directly from a macroscopic not calcified region in the middle of the placenta. For the fetal samples, we prepared the chorionic plate away to take a 0,5 cm3 big sample from the intervillous space. The tissue was put into labelled cryotubes containing 700 μl RNAlater® solution to stabilize placental RNA. The samples were stored at 4 °C for 24 to 48 hours and then transferred to −20° for long time storage and shipment on dry ice to Munich, Germany.Placenta tissue from both, maternal and fetal side, was collected. For the maternal side we took about 0,5 cmS. haematobium egg-count by microscopy; where possible the samples were frozen at −20 °C for shipment and further analysis. Furthermore, levels of schistosome circulating antigen (circulating anodic antigen [CAA]) were measured in cord and maternal peripheral plasma as well as in urine utilizing an immunochromatography based assay56.This lateral flow (LF) test applies luminescent upconverting particles (UCP) to quantitatively measure CAA levels with a 10 pg/mL lower limit threshold, analysing respectively 20 or 10 μL plasma or urine57. A total number of 13 Gabonese women were positive for S. haematobium by egg count and/or CAA in plasma . Sample collection for parasitological examination was only performed at the time of delivery.The concentrations of 25(OH)D using the Diasorin assay , as well as CRP and Calcium levels in all plasma samples were measured at the Institute for Clinical Chemistry and Pathobiochemistry at the Klinikum rechts der Isar of the Technical University of Munich according to the manufacturer’s specifications.™ Mammalian Total RNA Miniprep Kit, according to manufacturer’s instructions. RNA yield and purity were measured using a NanoDrop® 1000 Spectrophotometer. The resulting RNA was stored at −80 °C until further processing. cDNA was prepared following the Promega usage information for first-strand cDNA synthesis with M-MLV RT (H-) Point Mutant and the resulting cDNA was stored at −20 °C until further processing. qRT-PCR was performed to measure the relative concentrations of certain genes, using Roche Universal Probe Library and HPRT as house-keeping gene. The LightCycler® Probes Master (Roche) is a ready-to-use reaction mix containing the Taq Polymerase and appropriate buffers. Gene sequences and probes are shown in Supplemental Table Thawed fetal and maternal placenta samples were homogenized (using Precellys®24 tissue homogenizer) and RNA was isolated from using GenEluteS. haematobium adult worm antigen (AWA) specific IgG4 and IgE were measured by ELISA modified from previous protocols58. Briefly, maxisorp plates  were coated overnight with 5 mg/ml AWA diluted in carbonate buffer pH 9.6. For IgG4, the plates were blocked with PBS-5%BSA while for IgE with PBS-2%BSA. The plasma was diluted in PBS-0.05%Tween-5% FCS  or Tris-HCl–0.05% Tween for IgG4 and IgE ELISA, respectively. The presence of IgG4 was shown by using HRP-labelled anti human IgG4 (1:3000) . For IgE assay, the detection antibodies used were biotinylated goat anti human IgE  followed by streptavidin HRP conjugate . The assays were developed with tetramethylbenzidine (TMB) and stopped with 10% H2SO4. Absorbances were measured at 450 nm. The levels of antibody present in a given sample are expressed in arbitrary units (AU/ml) according to the standard curve of pooled positive S. haematobium plasma.Plasma samples were transported on dry ice to Munich, Germany, for quantification of total IgE and IgG4 and to Leiden, the Netherlands, for AWA specific IgE and IgG4. Levels of total IgE and IgG4 were measured in all European and African maternal and cord plasma samples using the Invitrogen IgE, or IgG4 Human ELISA Kit, respectively, according to manufacturer’s instructions (Thermo Fischer). The plasma was 1:10 diluted for IgE measurement and 1:1000 for IgG4. Acquisition of the multiplex immunoassay was performed by using the LSRII flow cytometer (Becton Dickinson). Statistical analysis was performed by using SPSS for Windows version 23.0  and PRISM® 5.01 . D’Agostino and Pearson omnibus normality tests were performed, and parametrically distributed data was analysed with unpaired T test (2 groups) and two-tailed Mann-Whitney-Test was used for non-parametric data. Statistical dependence was analysed by Spearman’s rank correlation coefficient. Categorical data were analysed by Pearson chi-squared test. Data are presented as median with interquartile range (IQR) or mean with standard deviation (SD).In all cases, statistical significance was assumed with a p value < 0.05 and where significance level was reached, p values are indicated in individual graphs.Supplementary figuresSupplementary tables


The drift and deformation of sea ice floating on the polar oceans is caused by the applied wind and ocean currents. Over ocean basin length scales the internal stresses and boundary conditions of the sea ice pack result in observable deformation patterns. Cracks and leads can be observed in satellite images and within the velocity fields generated from floe tracking. In a climate sea ice model the deformation of sea ice over ocean basin length scales is modelled using a rheology that represents the relationship between stresses and deformation within the sea ice cover. Here we investigate the link between emergent deformation characteristics and the underlying internal sea ice stresses using the Los Alamos numerical sea ice climate model. We have developed an idealized square domain, focusing on the role of sea ice rheologies in producing deformation at spatial resolutions of up to 500 m. We use the elastic anisotropic plastic (EAP) and elastic viscous plastic (EVP) rheologies, comparing their stability, with the EAP rheology producing sharper deformation features than EVP at all space and time resolutions. Sea ice within the domain is forced by idealized winds, allowing for the emergence of five distinct deformation types. Two for a low confinement ratio: convergent and expansive stresses. Two about a critical confinement ratio: isotropic and anisotropic conditions. One for a high confinement ratio and isotropic sea ice. Using the EAP rheology and through the modification of initial conditions and forcing, we show the emergence of the power law of strain rate, in accordance with observations.This article is part of the theme issue ‘Modelling of sea-ice phenomena’. These floes are continually breaking apart, sliding against one another and thermodynamically healing. These interactions result in discontinuous drift and deformation fields. Sea ice is continually under the influence of external wind and ocean forcing that, along with the internal ice stresses resulting from floe interaction and deformation and Coriolis acceleration, make up the sea ice force balance ,2.Accurately representing the sea ice force balance is essential for modelling the drift and thickness distribution of sea ice over seasonal and climate time scales. The resulting sea ice drift is important in the momentum transfer from the atmosphere, through sea ice, to the ocean below, driving basin-scale currents in the Arctic Ocean.The relation between stress and deformation of sea ice is known as sea ice rheology. When modelling the drift and deformation of sea ice within a numerical climate model, a numerical implementation of the rheology of sea ice over ocean dynamical length scales is needed. In recent years, new anisotropic rheologies have been developed that take inspiration from observable oriented features in satellite observations, for example, the elastic anisotropic plastic (EAP) rheology used in this paper  and the Rint (as described in §2a(i)), is found to control the mechanical failure of the ice. For Rint below a critical confinement ratio Rcrit, ice fails with Coulombic lines of shear deformation, lines that are inclined to the principal components of the stress tensor. For Rint > Rcrit there is a different failure mechanism, with either a single unoriented feature or the disintegration of the sample [The deformation and force balance of 10 cm samples of sea ice have been investigated in laboratory settings . Typicale sample . These de sample ,8, whichrc < 0. This distribution has been used to investigate how well climate sea ice models represent the deformation and thus the rheology of sea ice over climate length and time scales [The drift and deformation of sea ice have been observed both from floating arrays of remote buoys , and froe scales , with hiIn this paper, we document our study into the link between observable deformation features and the underlying internal stress characteristics. We use a state of the art sea ice climate model, the Los Alamos sea ice model (CICE), with the EAP and EVP rheologies on an idealized domain, documented in §2.(a)u the sea ice drift velocity and m the mass of sea ice per unit area, balanced by, in order: the Coriolis acceleration −mfck × u, with fc the Coriolis parameter and k the unit vector normal to the sea surface; the ice concentration C weighted applied stress from the atmosphere τa and ocean τo; gravitational acceleration from the ocean tilt S set to zero for this study; and the divergence of the internal ice stress tensor ∇ · σ.The numerical sea ice model CICE calculates the drift and deformation of sea ice using the vertically integrated, horizontal momentum balance :2.1The Ua with τa = ρaCa|Ua|Ua, where ρa is the air density and Ca is a drag parameter. Ocean stresses are typically calculated from the difference between ice and ocean velocities along with a turning angle. In this study, we have a still ocean and no turning angle with τo = − ρoCo|u|u|, where ρo is the ocean density and Co an ocean drag coefficient.The atmospheric drag is calculated from the 10 m wind speed (i)et al. [et al. [σfs, and ridging, σfr, stresses are obtained for the floe orientations (given by unit vector r) and their relative motion (h) are the ridging and sliding ice strengths for ice of thickness h. The individual floe stresses are expanded over an arbitrary area ψ(r) = ψ( − r)  biaxial tension causing longitudinal splitting; (ii) uniaxial compression/tension causing axial splitting; (iii) biaxial compression with a low confinement ratio causing in-plane shear rupture; and (iv) biaxial compression with a large confinement ratio causing out-of-plane shear rupture. Modes (i), (iv) cause no realignment of floes and modes (ii), (iii) align floes with r parallel to the direction of greatest compressive stress (σ2). The formulation for Ffrac isS is a tensor that results in the major principal axis of A aligning with the principal direction of σ associated with σ1; kf reflects the rate of fracture formation in the sea ice cover and, as with [kf = 10−3 s−1 which gives 90% anisotropic alignment within 6 h of case (ii) or (iii) occurring. The value of Rc = 0.3 used is in accordance with the laboratory-scale observations of Golding et al. [Fiso = 0) to allow us to focus on floe reorientation due to fracture Ffrac. Feltham , we use  as with , we choo(ii)η and ζ are the shear and bulk viscosities, e defines the elliptical plastic yield curve aspect ratio, p is the ice strength as discussed below.The EVP rheology  is a num(b)fc = 1.46 × 10−4 s−1 to make the model applicable to the polar regions. The size of the domain is d = 2000 km square. To simplify the model dynamics, we use a single thickness category and the ice strength parametrization of Hibler [p* = 2700 N m−2 and c = 20 are constants. Tests showed that using the alternative strength parametrization of Rothrock [et al. [We use a square grid with constant Coriolis acceleration of f Hibler  with  whRothrock  and five [et al. , though et al. [dbc ≈ 50 km from the ice edge  by balancing the wind forcing and a linear ocean drag withUog = 0 represents the still ocean. As experienced by Hutchings et al. [dtrans = 300 km from the domain edge, the uniform strain rate changes into observable features. The inner region beyond dtrans is the area analysed within the results sections , givings et al.  this bousections .Figure et al. [The model is initialized using data from an Arctic-wide CICE run using the restart configuration. There are many variables required to restart a model run. Most we take from a single point in the Arctic-wide run with 2% noise, with idealized: sea ice velocity near zero, sea ice concentration of 0.999, a constant thin snow cover of 0.1 m and constant idealized sea ice structure tensor and thickness, which we discuss in §et al.  who solv(c)et al. [et al. [Uog is set to zero. We construct wind forcing by imposing the gradient of the wind stress, with τayy/τaxx = Rwind a constant and the maximum wind speed Uamax. The wind velocity field can be generated from ua, va) = |y=d/2x=0 and  = |y=d/2x=d, measuring  from the southwest corner of the model domain withRwind < 0  = 0 that increases linearly to the idealized wind forcing set over 6 h of model time.In order to gain insight into the role of sea ice rheology in producing sea ice deformation features, we perform simulations with idealized atmospheric and oceanic forcing. To induce near constant internal stress conditions over the idealized domain, we impose a wind forcing similar to that used both by Wilchinsky et al. ,21 for d [et al. . In all (d)(i)The model resolution has been tested at 10 km, typical of modern high-resolution global climate models, 2 km, typical of very high-resolution regional models, and 500 m. The time resolution is decreased for finer spatial resolution to allow the equations of motion to be solved, with a time step of 600 s at 10 km, 30 s at 2 km and 5 s at 500 m. For all cases, we use 200 elastic sub-cycles within the CICE numerical solver. These time steps give model convergence at each resolution with the model results not changing by more than 1% for shorter time steps or increased sub-cycles. The model responses have been widely tested to confirm a correct response. For example, for the 2 km EVP runs presented in Rwind = 0.4 and the appearance of comb cracks at 2 km and 500 m resolution for Rwind =  − 0.4. When using the EVP rheology Rwind =  − 0.8  from after 10 h of model time. From 12 h to 10 days, the features have little variation. All differences between these two time points are due to thickening sea ice, primarily in areas of ridging.For the EAP rheology oriented lines of deformation are observable from 3 h of model time, and the sea ice is strongly aligned A1 = 0.5): the case of Rwind =  − 0.8 and divergent shear deformation  is aligned to the principal component of the wind stress gradient, not the wind direction. This is in agreement with Heorton et al. [In n et al.  who founRwind = 0.8 shown in For the convergent wind field with (b)Rint and the deformation characteristics of sea ice displayed in figures Rint is an emergent property we cannot directly impose it and so we perform a set of simulations with different values of imposed Rwind, which directly determines Rint. We calculate Rint = σ1/σ2 from the principal components or eigenvectors of the internal ice stress tensor σ for each grid cell point, with the values plotted in dtrans from the domain edge.Here we investigate the relationship between the internal stress confinement ratio (i)Uamax = 15 m s−1 over the first 6 h of the model run. The magnitude of internal stress reaches its maximum from 3 h of model run whereas the strain rate increases with the wind speed to its maximum at 6 h of model run , due to the symmetry of equations  of shear strain rate  approaches a linear relationship and thus the power law of sea ice deformation suggested by Girard et al. [et al. [The experiments with ain rate , indicatd et al.  and Ramp [et al. .(ii)Rwind reveals two failure modes of sea ice. The runs with a large positive Rwind in the range 0.1 < Rwind < 0.8 produce shear strain rate concentrated into near parallel lines that diverge towards the edge of the domain; see For an initial isotropic cover of sea ice, varying Rwind < 0.1 and features diagonally intersecting lines of high shear strain rate. The number of lines and incident angle between them both appear to increase for decreasing Rwind; for example the run with Rwind =  − 0.8 has the greatest number of intersecting lines of shear that intersect at an angle of ≈ 90°. Hutchings et al. [Rwind. We attribute this failure mode to in-plane shear rupture with the sea ice pack, the inspiration for mode (iii) in equation  displays both peaks, as the domain is split into isotropic regions with parallel deformation features and anisotropic regions with Coulombic deformation features, as seen in et al. [Rcrit when performing laboratory experiments with imposed stress confinement. A laboratory experiment where the internal stresses could be observed in comparison to imposed external stresses, if possible, would produce a distribution of stress confinement ratio that can be used to contrast with those we show for the EAP and EVP rheologies in this paper.The wind stress confinement n et al. , for exa(c)Rint < Rcrit, the sea ice structure tensor becomes aligned . To investigate these relationships we perform the runs described above in §When the sea ice internal stress confinement is less than the critical confinement ratio (i)A1 aligned with the x axis), the parallel slip line failure mode for Rint > Rcrit shown in Rint < Rcrit but the sea ice internal stress confinement ratio Rint is much more closely concentrated around the critical confinement ratio Rcrit for all the runs to produce Rwind ≤ 0.2 for this pre-alignment. For Rint > Rcrit (for winds with Rwind≥0.2 in this case) the increased shear stress of aligned sea ice and the converging sea ice combine, resulting in little shear deformation and no identifiable features. For the case of Rint ≈ Rcrit a new deformation pattern is observed as shown in the last figure in this paper. This failure mode has bands of shear failure that are similarly oriented to the Coulombic slip lines but are much wider and have a blurry appearance. In some cases the bands may be formed of closely packed comb cracks perpendicular to the overall band of shear, though more investigation is needed to confirm this. This failure mode is also observed for the Rwind = 0.0 run in A1 ≈ 1 and conditions are very similar to the pre-aligned case. Apart from the visual features this aligned failure mode has similar characteristics to the Coulombic failure mode: high shear stress, Rint ≈ Rcrit and a similar pdf of strain rate magnitude.For anisotropically aligned sea ice, pre-aligned to the expected alignment for the given wind field , then there are two failure modes encountered. For Rwind > 0.0 the sea ice remains anisotropic as in the initial conditions and the increased shear stresses compared to the isotropic case result in no obvious deformation characteristics. For these cases (‘warm’ coloured lines in figure 7c), Rint remains closer to Rcrit than in the isotropic case, with increasing Rwind resulting in decreased Rint. For Rwind ≤ 0.0, the sea ice is able to realign with Rint going lower than Rcrit within the run time. These runs all go through an ≈ 6 h period of realignment where the structure tensor A and internal stress confinement change rapidly. During this period and afterwards, Coulombic slip lines occur though they move orientation and location much more than in the runs illustrated in Rint. Those that realign appear to have strictly Rint < Rcrit and likewise those that do not realign have Rint > Rcrit for all cases.When the model is initiated with sea ice aligned perpendicular to the expected alignment for the applied wind field ua, va = 0) conditions at t = 0 to a wind field with Uamax = 15 m s−1 and Rwind = 0.4 at t = 6 h with the wind field then held constant until t = 12 h. From t = 12 to 18 h, the wind pattern is smoothly varied to Rwind =  − 0.4, with the wind speed held constant. From t = 24 to 30 h, the wind field is interpolated back to have Rwind = 0.4 and then held constant until t = 36 h, as shown in the line plot in Rwind = 0.4 run in Rint > Rcrit Uamax = 15 m s−1 and Rwind =  − 0.4 rotates by 45° anticlockwise about the centre of the grid during t = 12–18, 24–30, 36–42 and 48–54 h to return to the wind conditions at 6 h, as this wind field has horizontal and vertical symmetry  with minimal adaptation so it runs with idealized initial conditions on a square grid. We run the model at 10 km, 2 km and 500 m spatial resolutions using both the anisotropic EAP and isotropic EVP rheologies. This set-up enables us to illustrate the sea ice dynamical phenomena that can be expected to occur within state of the art high-resolution sea ice climate models and is suitable for testing and comparing all sea ice model rheologies.Rwind, see §Rwind and the internal stress confinement Rint. This result links the EAP rheology and laboratory experiments such as Golding et al. [Rwind and initial alignment have revealed five characteristic failure modes as illustrated in Rint > Rcrit, there are two failure modes. When the sea ice is isotropic, parallel shear slip lines can form. However, when the sea ice is anisotropic, its increased shear strength resists the formation of shear slip lines and the sea ice deforms compressively over large length scales. For Rint < Rcrit, the sea ice becomes anisotropically aligned due to the mechanics of the rheology in equation (Rwind = 0.4 with the wind on a bearing from the centre to the edge of the domain. In this case, the sea ice fails in divergence, Rint < 0 and the sea ice becomes anisotropically aligned.We have successfully imposed internal sea ice stress states using winds fields with a constant confinement of the wind stress gradient (g et al.  where a g et al. . With thequation  and therPrevious attempts at characterizing the rheology of sea ice over basin length scales have focused on the distribution of strain rate , observaIn this paper, we have shown clear contrasting features of the EAP and EVP rheologies when using them as part of the currently available CICE model set-up. The EAP rheology consistently gives observable deformation features for all the model resolutions studied, but only for specific situations when using the EVP rheology §d. The EA


Pasteurella multocida from a case of BRD, designated ICEMh1PM22 for its structure and host genome insertion site, and to identify consequences for host fitness and antimicrobial therapy. The modular structure of ICEMh1-like elements found in several related livestock pathogens was compared to ICEMh1PM22, and the repertoire of cargo genes in variable ICE modules was functionally categorized. AMR genes were identified as frequent additions to the variable modules of ICEMh1-like elements. Random PCR-based mapping of ICEMh1PM22-genome junctions in transconjugants provided evidence that ICEMh1PM22 integrates into the tRNA-leu for the UUG codon, and not into tRNA-leu for other codons. This was separately confirmed in the genomes of ICEMh1-like-harboring livestock pathogens. Bacterial genera harboring receptive tRNA-leuUUG were identified to establish the potential host range of ICEMh1-like elements. ICEMh1PM22-carrying transconjugants in P. multocida and Mannheimia haemolytica were less fit than isogenic strains without the ICE when grown without antimicrobial selection. This fitness cost was abrogated in the presence of subinhibitory concentrations of antimicrobials. Despite this cost, ICEMh1PM22 was retained in transconjugants in extended culture. To identify possible therapeutic efficiencies, antimicrobial combinations were screened for synergistic interactions against AMR ICEMh1PM22-carrying transconjugants. No antimicrobial combination tested exhibited synergistic interactions against AMR P. multocida or M. haemolytica harboring ICEMh1PM22. In conclusion, this study provided information on the structural variation of ICEMh1-like elements, refined the ICE insertion site and potential host range, and demonstrated the risk and consequences for AMR following horizontal transfer of ICE into BRD pathogens.Horizontal gene transfer of integrative and conjugative elements (ICE) in bacterial pathogens of the bovine respiratory disease (BRD) complex has emerged as a significant cause of antimicrobial resistance (AMR) and therapeutic failure and mortalities in cattle. The aim of this study was to assess an AMR ICE occurring in  Pmu1 and ICEMh1, in Pasteurella multocida strain 36950 . Furthermore, resistance genes found in these ICE are often highly related to AMR genes from other human and veterinary bacteria (Integrative and conjugative elements (ICEs) are mobile genetic elements that can transfer autonomously by conjugation in bacteria. In general, ICEs can excise from the host chromosome to form a circular extrachromosomal intermediate which can be transferred and integrated into a receptive host . These win 36950 , b and Mectively . Recentlectively . Of partectively . In addius somni , as wellbacteria .Pmu1, the first BRD ICE characterized in P. multocida, was shown to integrate into a copy of the leucine tRNA (tRNA-leu) (Pmu1 was flanked by 13 bp direct repeats (5′-GATTTTGAATCAA-3′), which corresponded to the attB attachment site for site-specific recombination. In the circular form of ICEPmu1, the direct repeat sequence is flanked by left and right ‘attachment’ sites, attL and attR, which are thought to be the recombination sites for a XerCD-like pair of tyrosine recombinases   are syntenous (Pmu1 has 12 resistance genes: strA and strB (conferring resistance to streptomycin), aphA1 (gentamicin), sul2 (sulfonamides), tet(H) (tetracyclines), floR (phenicols), erm(42) (macrolides and lincosamides), aadB (gentamicin), aadA15 (streptomycin and spectinomycin), blaOXA–2 (β-lactams), and msr(E) and mph(E) (macrolides)   . The chrmbinases . In the AATC-3′) . Thus, Iyntenous . Both ICrolides) , b. ICEM, tet(H) . Both ICcipients .Pmu1 and ICEMh1 can easily move between genera and species highlights the extreme risk for rapid acquisition of AMR in BRD bacteria via horizontal gene transfer. The acquisition of these AMR genes poses an obvious threat to effective antimicrobial therapy, and could exacerbate economic losses associated with cattle mortalities. Greater knowledge of basic ICE biology is necessary to understand their potential host range, including acquisition by human pathogens, and the threat they pose to the efficacy of antimicrobial therapy. We explored these attributes using isogenic transconjugants of an ICEMh1-like element (ICEMh1PM22) previously identified in a bovine isolate (P. multocida PM22) from a BRD mortality  predict host range based on the insertion site, (III) explore the effect of ICEMh1PM22 acquisition on host fitness, and (IV) identify any synergistic antimicrobial combinations effective against bacteria harboring ICEMh1PM22. We tested for synergy using antimicrobials against which PM22 was both non-susceptible and susceptible to identify any combination capable of restoring susceptibility. We hypothesized that this information could be used to ensure prudent antimicrobial usage, inform future BRD mitigation strategies, and identify the potential risk in other areas of human and veterinary medicine.The observation that ICEortality . The objP. multocida PM22 was initially isolated following lung tissue necropsy of a beef cattle BRD mortality (acute fibrinous pneumonia) in a Texas feedlot (E. coli were maintained with Luria-Bertani (LB) or Mueller-Hinton II  agar/broth (BD Difco) supplemented with antibiotics where appropriate. All susceptibility testing was performed in MH broth. Spontaneous rifampin-resistant (RifR) mutants of P. multocida CCUG 17976 and M. haemolytica ATCC 33396 were obtained by plating OD600 2.0 on BHI supplemented with 50 mg/L rifampin , followed by incubation for ∼48 h at 37°C. To generate luciferase-expressing E. coli DH5α, the pAKlux2 plasmid (E. coli DH5α and transformants were selected on and routinely maintained on LB agar supplemented with 100 mg/L of ampicillin (MilliporeSigma). feedlot . Pasteur plasmid  was elec600 1.0 equivalents of P. multocida PM22 and RifRP. multocida CCUG 17976 or M. haemolytica ATCC 33396 were mated in 100 μL of BHI broth spotted on TSA blood agar, incubated aerobically at 37°C for 4 h, harvested and then plated on BHI agar supplemented with rifampin (50 mg/L), tetracycline , and spectinomycin . Six colonies were selected from each mating  and verified for ICEMh1PM22 acquisition by susceptibility testing and PCR screening . To map the ICEMh1PM22 insertion site, transconjugant DNA was purified  and amplified using the CEKG set of nested and random  oligonucleotides in combination with nested ICEMh1PM22-specific oligonucleotides: PM22-A1: 5′-CACCTTTAGTTGAAGCAATAG-3′; PM22-A2, 5′-TGGTAA AAGGTTTGTTTGTAC-3′; PM22-B1, 5′-ATCGTAGTAAGTG TGTATTTG-3′; and PM22-B2 5′-ATTATTTGAACAGTTC TACGC-3′  in tandem rounds of PCR as described by Bacterial conjugation assays were performed as previously described , with micreening . To furtRP. multocida CCUG 17976 and M. haemolytica ATCC 33396 and their isogenic ICEMh1PM22 transconjugants were performed in 200 μL volumes of MH broth inoculated at an OD600 of 0.005 in 96-well plates (Nunc) sealed with gas-permeable film . Bacterial growth, with shaking at 37°C, was monitored continuously for 12 h in a plate-reader (Biotek HT Synergy). For 2-strain co-culture experiments, luciferase-expressing E. coli DH5α pAKlux2 and test strains (including DH5α without pAKlux2 as a control) were each inoculated at an OD600 of 0.0025  into black clear bottom 96-well plates . Bacterial growth was monitored as above, with the addition of luminometry. The maximum light produced  in each competition was recorded, and used to generate a competitive index . Thus, values <1 indicated that test E. coli DH5α pAKlux2 was outperformed compared to an E. coli-only control. For long-term repeated passage experiments to assess if ICEMh1PM22 could be lost from the host strain, ICEMh1PM22 transconjugants were inoculated into 2 mL of MH broth or MH broth supplemented with 0.5 MIC (subinhibitory for the susceptible WT) oxytetracycline (0.125 mg/L), spectinomycin (32 mg/L), or tylosin (16 mg/L) in a sterile 96-well block , and sub-cultured (1/100) every ∼3 d into fresh media supplemented with the same antimicrobials for 150 d. To assess for loss of ICEMh1PM22, CFU from OD600 0.1-equivalents from each culture were enumerated in parallel on MH agar  and MH agar supplemented with each antimicrobial at concentrations selective for ICEMh1PM22: oxytetracycline (10 mg/L); spectinomycin (512 mg/L); or tylosin (64 mg/L). Likewise, the effect of subinhibitory concentrations on co-culture competitions with luciferase-expressing E. coli was assessed with 0.5 MICs (for WT strains) of oxytetracycline (0.125 mg/L) and spectinomycin (32 mg/L) as above for co-cultures. Tylosin was not tested because E. coli is intrinsically resistant to macrolides.Growth curves of RifR and isogenic ICEMh1PM22P. multocida CCUG 17976 and M. haemolytica ATCC 33396 transconjugants according to the CLSI approved standard M07 - Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically . Plates were grown aerobically at 37°C for 20 h and MIC was registered as the lowest concentration inhibiting visually detectable growth. For checkerboard synergy assays, two-dimensional arrays of serial concentrations  of antimicrobials were created for two experiments: (I) all combinations of 5 antimicrobials  representing major antimicrobial classes to which transconjugants were non-susceptible, and (II) selected combinations of ‘non-susceptible’ antimicrobials  with antimicrobials to which ICEMh1PM22 transconjugants were susceptible . Growth and sterility controls were included in each checkerboard assay, which were performed in triplicate. MICs were interpreted by eye and OD600 was measured in a plate reader to facilitate calculation of a fractional inhibitory concentration index (FICI). For discrepancies between replicates, the highest antimicrobial concentration determined the MIC. The FIC for each antimicrobial (A or B) was determined by the MIC of the antimicrobial in combination and divided by the MIC of the drug alone, according to:Minimum inhibitory concentrations (MICs) were determined for the Rifobically . Escheriassigned  in accorassigned . AntimicJournal of Antimicrobial Chemotherapy (Interactions were conservatively interpreted as ‘synergistic’ (FICI ≤ 0.5) or ‘no interaction’ (FICI > 0.5–4.0) in line with synergy testing guidelines in the otherapy .Mh1-like-containing Pasteurellaceae genomes were identified by default BLAST alignment to ICEMh1PM22, using only results with >80% sequence identity and >50% query coverage and eliminating isogenic or partial sequences. Sequence manipulations and alignments were performed in Geneious 8.1.9 using default MUSCLE parameters or progressive Mauve alignment  sequence from M. haemolytica M42458 with parameters: Program, blastn; Word size, 11; Expect value, 10; Hitlist size, 20,000; Match/Mismatch scores, 2/-3; Gapcosts, 5/2. This resulted in 9604 hits/alignments, which were filtered for those containing the ICEMh1-associated direct repeat  and palindrome (with 100% identity). From this, a taxonomic report was generated to show bacterial genera.Statistical tests and graphing were performed with Sigmaplot 13.0 (Systat Software Inc.), with error bars indicating standard error of the mean (SEM). ICElignment  for idenh PROKKA  and manuh PROKKA . Sequench Circos . Predicth Circos . To idenMh1PM22 is a 77,786 bp element in P. multocida PM22 (accession # CP045724) bound by two copies of a tRNA for leucine (tRNA-leu), one of which is disrupted and potentially non-functional  region—resistance region 1, encodes aminoglycoside (aph(3′)-Ia, aph(6)-Id, aph(3″)-Ib), sulfonamide (sul2), and macrolide (erm42) resistance genes, and the second—resistance region 2—encodes genes for tetracycline , aminoglycoside (ant(2″)-Ia, aadA25), macrolide (mphE) resistance, and a potential extended-spectrum β-lactamase (blaOXA–2) previously shown to be phenotypically non-functional in transconjugants , sometimes annotated as XerC and XerD, members of which are thought to work in tandem by mediating site-specific recombination  using two recognition sites which separately constitute the attL and attR sites in integrated ICE, and which are found on either side of a central attachment site involved in crossover . XerC and XerD are likely part of the core or conserved backbone of ICEMh1-like elements, as are genes annotated as part of the conjugation machinery, such as the tra genes  were identified as a direct repeat (5′-GATTCAAAATC-3′)  PCR oligonucleotides  were present  ribosome recognition site  copy of tRNA-leuUUG (not shown). In the 41 ICEMh1-like-containing genomes, at least 1 functional copy of each tRNA was present, although sometimes full-length tRNA-leuUUG was present twice  of the ICE was absent in some ICEMh1-like-containing genomes, potentially indicating ICEs incapable of mobilization (not shown).It was previously shown that ICEmolytica . The terAATC-3′) . Given tleotides  with oli. Enterobacterales were also frequently represented, and included many well-known food-borne and cattle-associated pathogens such as Escherichia coli and Salmonella spp. For E. coli, only some strains harbored the receptive tRNA-leu sequence, suggesting that only some members of any genus might be capable of transconjugation. Burkholderiales (β-proteobacteria) also harbored similar tRNA-leu, suggesting the potential for ICEMh1-like horizontal gene transfer to Burkholderia spp. and Bordetella spp. Likewise, bacteria from α-, δ-, and ε-Proteobacteria were also represented and may be receptive to ICEMh1, such that cattle-associated food-borne bacteria like Campylobacter could conjugate with ICE-bearing Pasteurellaceae. Some Gram-positive Firmicutes, specifically Clostridia, were also host to ICE-receptive tRNA-leu. Thus, if the tRNA-leuUUG sequence was the only requirement for mobilization and integration, ICEMh1 might be capable of crossing into these Gram-positive organisms. At present, it has only been experimentally confirmed that ICEMh1PM22 can transfer into RifRP. multocida and M. haemolytica (E. coli strains K-12 and DH5α (not shown).Given the conservation of the tRNA-leustructed . As expemolytica , and E. M. haemolytica ATCC 33396 transconjugants harboring ICEMh1PM22 were noticeably slower-growing than the isogenic WT on agar media supplemented with selective concentrations of antimicrobials. To assess the effect of ICE acquisition on growth parameters, we performed OD600-based growth curves for RifRP. multocida CCUG 17976 and M. haemolytica ATCC 33396, and their respective ICEMh1PM22 transconjugants. The transconjugant of RifRP. multocida CCUG 17976 exhibited both an extended lag phase and decreased growth rate compared to the ICE-minus strain . To test this, the ICEMh1PM22 transconjugants for RifRP. multocida CCUG 17976 and M. haemolytica ATCC 33396 were repeatedly passaged over 150 days in media without antimicrobials or supplemented with sub-inhibitory concentrations of oxytetracycline, spectinomycin, or tylosin. The chosen concentrations were sub-inhibitory (0.5 MIC) to the parental WT strains. Every 30 days, CFU from each repeated passage were enumerated on MH agar  and MH agar supplemented with each antimicrobial at a selective concentration . The presence of ICEMh1PM22 was also confirmed in selected isolated colonies by PCR (not shown). This showed that repeated passage of ICEMh1PM22-carrying strains did not result in any measurable loss of the ICE  and spectinomycin (32 mg/L) were selected as bacteriostatic antimicrobials and used at 0.5 MIC for both P. multocida and M. haemolytica RifR WT. At these concentrations, oxytetracycline was sub-inhibitory to E. coli DH5α pAKlux2 (MIC: ∼1 mg/L), but spectinomycin was inhibitory (MIC: ∼16 mg/L). Co-cultures of the transconjugants or RifR WT with E. coli DH5α pAKlux2 were monitored for OD600 and luciferase production over 12 h. For oxytetracycline, increased luciferase production was observed for both P. multocida and M. haemolytica ICEMh1PM22 co-cultures compared to the ICE-minus RifR WT . These findings suggest that the presence of ICEMh1PM22 negatively affected the efficacy of these antimicrobials against E. coli in co-culture, without horizontal gene transfer.Given that previous work had shown that ICE RifR WT , left. Fonjugant , right. Mh1PM22 harbors an extensive repertoire of antimicrobial resistance genes for drugs in frequent and common use for BRD, we investigated whether or not combination antimicrobial therapies were effective against P. multocida or M. haemolytica ICEMh1PM22 transconjugants. Checkerboard antimicrobial synergy assays  and those that it does not . Following calculation and strict interpretation of the FICI scores for each combination, we found that no pairwise combination of oxytetracycline, spectinomycin, tilmicosin, tylosin, and sulfamethazine demonstrated synergism  cannot be identified. We speculate that these represent on-going recombination post-integration, and may be mechanisms through which ICEs become non-mobile, or acquire new genes from the host chromosome.Many ICEs site-specifically insert into tRNAs and, in general, are not able to change their location within the same host cell . PrevioutRNA-leu . Here, wMh1-like elements and tRNA-leuUUG, the 11 bp that constitute the ICE’s direct repeat and the tRNA’s anticodon loop were conserved with 100% identity in all ICEMh1-like-containing genomes. Exploiting the anticodon loop in this manner also likely enables a broader host range compared to more variable features of the tRNA. Likewise, in the case of tRNA-leuUUG, we speculate that other conserved bp in the tRNA provide for the attL and attR recognition and/or recombination sites of the putative XerCD tyrosine recombinases. Although we did not explore that possibility here, this would be consistent with known XerCD mechanisms . The host range of other conjugative elements in nature has been shown to be narrower than that observed in in vitro experiments (Mh1-like element transmission to bacteria, despite the presence of a receptive tRNA-leu. These may include phage (Mh1-like elements identified here includes many pathogens capable of causing significant human and veterinary disease. The transfer of AMR determinants into such bacteria could significantly compromise antimicrobial therapy.It is difficult to accurately assess the potential host range of ICEeriments . There ade phage , certainde phage , and CRIde phage . Furtherde phage . The putMh1PM22 transconjugants do not lose the ICE after long-term passage. This is likely due to ‘addiction’ via the action of toxin-antitoxin genes common to most ICE (Mh1-like elements may have a fitness cost to the host cell in the absence of antimicrobials, elimination of antimicrobial usage could help non-carriers to outcompete hosts. However, ICEMh1-like elements frequently contain metal-resistance and other genes conferring benefits under dynamic selective pressures in vivo, so elimination of antimicrobial pressure may not be effective at dampening ICE-carrying populations. We also used ICEMh1PM22 transconjugants to test for synergistic combinations of antimicrobials, hoping to identify combinations that might be effective against AMR BRD pathogens, potentially reducing antimicrobial usage and restoring drug effectiveness. Ultimately, we did not detect any synergistic interactions between the drugs tested using these organisms. This was not unexpected, and illustrates the relatively limited therapeutic options available to veterinarians for BRD treatment. The presence of multiple AMR determinants conferring resistance to most drug classes in these elements also underscores the dwindling effectiveness of antimicrobials. In conclusion, our results suggest that there is no easy solution to the emerging problem of AMR ICE in BRD pathogens using current antimicrobials and usage practices.Despite a fitness cost in the absence of antimicrobial selection, we also found that ICEmost ICE . For praCP045724.The PM22 sequence was deposited in GenBank under accession number AC and TM: conceptualization. AC and RZ: methodology, software, validation, formal analysis, and data curation. AC: investigation, visualization, writing, and original draft preparation. TM: resources, supervision, project administration, and funding acquisition. AC, RZ, and TM: writing, review, and editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rhizoctonia solani, Zymoseptoria tritici, and Botrytis cinerea) and three human pathogens , showing that 48 combinations inhibited strongly the growth of the pathogens; the growth inhibition effect was significantly greater with the agents combined than by a simple product of their individual effects at the same doses. Of these, 23 combinations were effective against more than one pathogen, including combinations comprising food-and-drug approved compounds, e.g., quinine with bicarbonate, and quinine with hygromycin. These combinations [fractional inhibitory combination (FIC) index ≤0.5] gave up to 100% reduction of fungal growth yield at concentrations of agents which, individually, had negligible effect. No synergy was evident against bacterial, plant or mammalian cells, indicating specificity for fungi. Mode-of-action analyses for quinine + hygromycin indicated that synergistic mistranslation was the antifungal mechanism. That mechanism was not universal as bicarbonate exacerbated quinine action by increasing drug uptake. The study unveils chemical combinations and a target process with potential for control of diverse fungal pathogens, and suggests repurposing possibilities for several current therapeutics.A range of fungicides or antifungals are currently deployed to control fungi in agriculture or medicine, but resistance to current agents is growing so new approaches and molecular targets are urgently needed. Recently, different aminoglycoside antibiotics combined with particular transport inhibitors were found to produce strong, synergistic growth-inhibition of fungi, by synergistically increasing the error rate of mRNA translation. Here, focusing on translation fidelity as a novel target for combinatorial antifungal treatment, we tested the hypothesis that alternative combinations of agents known to affect the availability of functional amino acids would synergistically inhibit growth of major fungal pathogens. We screened 172 novel combinations against three phytopathogens ( Candida albicans and Aspergillus fumigatus, and the basidiomycete Cryptococcus neoformans, cause life-threatening infections particularly for individuals with compromised immunity. Invasive fungal infections have mortality rates between 20 and 95% and are estimated to cause ∼1.5 million deaths annually, comparable to the mortality associated with tuberculosis and malaria . Mode of action studies were performed with S. cerevisiae BY4743 (MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met15Δ0/MET15 ura3Δ0/ura3Δ0). The yeasts were maintained and grown in YPD medium , or YNB medium . OD600 was then measured with a BioTek EL800 microplate spectrophotometer. Fractional inhibitory concentration (FIC) as an indicator of synergy was calculated as described (S. cerevisiae) treated as above were transferred to YPD agar and incubated for 24 h at 30°C to determine colony forming ability.All culturing and preparation for checkerboard assays adhered to EUCAST guidelines , with thescribed . To test-1 overnight. Cultures were diluted to OD600∼0.01 in RPMI 1640 medium and 100 μl aliquots transferred to 96-well microtiter plates  before static incubation for 2 h. Non-adherent cells were removed by three gentle washes with PBS, then 100 μl of fresh RPMI 1640 were added to each well. Plates were incubated at 37°C with orbital shaking at 100 rev. min-1 for 24 h. After biofilm formation, biofilms were washed and drugs were added as specified. Cultures were incubated for a further 24 h. The wells were washed three times with PBS and the XTT reaction was performed in YNB by adding XTT to 210 μg.ml-1 and menadione to 4.2 μM. After 3 h, 100 μl of the reaction solution was transferred to a new 96-well plate and the absorbance at 490 nm was measured using a BioTek EL800 microplate spectrophotometer.Biofilm metabolic activity was measured by the XTT  (Sigma) reduction assay . Single 600∼0.5 in fresh YPD medium and cultured for a further 4 h with shaking. Quinine hydrochloride was added in combination with sodium bicarbonate, hygromycin B or no drug and cells incubated for 20 min at 30°C, 120 rev. min-1. Cells were harvested, washed, resuspended in 10% (w/v) perchloric acid – 2 M sodium methanesulfonate, and then lysed using acid-washed beads. Supernatant was collected after centrifugation  and A350 measured with an Ultrospec 2000 UV/visible spectrophotometer .Quinine uptake was assayed by measuring quinine absorbance at 350 nm as described previously . BrieflyS. cerevisiae was transformed with a dual luciferase reporter plasmid encoding firefly and Renilla luciferases separated by a UAA stop codon  could offer novel antifungal combinations against this target. As fungal demand for particular amino acids can be satisfied by exogenous supply , which could undermine targeting of amino acid availability, we reasoned that methionine and tryptophan availability could be good candidates in human pathogens; serum levels of these amino acids are low .Following recent work suggesting translation fidelity to be a new target for combinatorial treatment of undesirable fungi , we hypo are low . Further are low , we exam are low ], amino  are low ], non-prno acids ], and agno acids  and coppno acids ]. These B. cinerea and Z. tritici, and the basidiomycete R. solani) and three human pathogens . Screen hits were determined as compounds giving a percentage inhibition in combination that was greater by 20% than the sum of inhibitory effects of the compounds when supplied individually, adapted from Figure 1) produced synergistic-type inhibition when combined with at least one other agent . Twenty-three of these combinations showed significant inhibition against more than one pathogen . Among these 23, five combinations comprised a sulfate transport inhibitor and an aminoglycoside, with the chromate + hygromycin combination exhibiting synergistic-type inhibition of four of the test fungi. These included A. fumigatus, previously shown to resist synergy from chromate with the alternative aminoglycoside paromomycin . These combinations did not work against Z. tritici and only six of the 172 combinations gave inhibition of this phytopathogen, whereas ≥12 combinations were effective against each of the other tested species. No combination appeared to act synergistically against all three plant pathogens . On the other hand, ziram + hygromycin, like chromate + hygromycin, inhibited all three human pathogens. Generally, combinations involving a dithiocarbamate (ziram or thiram) and an aminoglycoside seemed to target specifically fungal pathogens of humans, and encompassed 3 of the 23 combinations . Of those 23, nine did not include an aminoglycoside antibiotic: these were quinine combined with three different sulfate transport inhibitors, orthovanadate + cyprodinil, selenate + norvaline, and a dithiocarbamate with either copper, molybdate, or norvaline . The dithiocarbamate + copper combination is used in agriculture. Synergistic-type inhibition by bicarbonate + quinine was observed in four of the six test fungi and so had one of the broadest spectra. This combination conferred very strong growth inhibition, with ∼100% reduction of C. albicans growth yield at quinine and bicarbonate concentrations which individually had little effect .Combination screens were performed against three plant pathogens . Most of the combinations of interest detected from the screen were shown to be synergistic (only 2 exceptions out of 17), with the lowest FIC index values being 0.06 for chromate + hygromycin against C. albicans and 0.19 for paromomycin + eugenol (B. cinerea) as well as quinine + hygromycin . Whereas there were 2 exceptions among hits from the screen, 4 of the checkerboard outcomes indicated synergies that were not detected in the screen, suggesting the screen could be conservative. Three of the outcomes from checkerboards that did not match screen results were for C. neoformans, which grew poorly in the static cultures used for the checkerboard assay.Checkerboard analysis was performed against all six pathogens to corroborate synergies for some of the key combinations observed from the screen . We also tested key combinations against the yeast S. cerevisiae, to enable this model’s use for insights to mode of action (below). With the exception of orthovanadate + cyprodinil, the main combinations that produced synergistic-type inhibition against more than two fungal pathogens also did so against S. cerevisiae, albeit less markedly in the case of ziram + hygromycin . Regarding combinations of an aromatic amino acid transport inhibitor with an aminoglycoside, the data suggested a stronger synergistic effect of quinine + hygromycin than eugenol + paromomycin on S. cerevisiae.The results revealed a number of combinations that produced synergistic growth inhibition of the fungi. Some of the most effective combinations were tested in the bacterium Figure 2A). The antimalarial quinine is a food and drug approved agent, like hygromycin and bicarbonate, so these novel combinations provided a good focus for further investigation. Resistance erodes the efficacy of current antifungal drugs, but resistance is typically less common against fungicidal than fungistatic drugs .Several of the most effective combinations in the above screens involved quinine, with quinine + hygromycin and quinine + bicarbonate appearing especially effective cross-species . Therefore, the synergy was effective against C. albicans within biofilms.patients . We testB. cinerea and C. albicans). In both organisms, quinacrine gave strong synergistic-type inhibition in combination with bicarbonate, but not hygromycin. Primaquine and mefloquine exhibited synergy with both compounds, particularly bicarbonate . Quinoline-derived compounds are used in medicine, but not yet in agriculture and plant toxicity might be an issue. Therefore, we tested for effects of the agents on leaf material, from lettuce. The compounds were sprayed at different concentrations onto leaves and the average surface area of any arising lesions determined. Treating the leaves with quinine or quinacrine, at the concentrations used above against the plant pathogen , produced negligible damage (lesions) . The subtoxicity concentrations in planta of bicarbonate and hygromycin were also determined (not shown), according to doses that produced <10% surface-area lesions; these concentrations were respectively, 5 and 50 times higher than the subinhibitory concentrations used against B. cinerea. We tested whether quinacrine + bicarbonate (the most effective combination against B. cinerea) had a synergistic effect on plant material, at the same concentrations used against B. cinerea. No leaf lesions were observed . In conjunction with the data for bacterial and human cells , the data indicated that synergistic inhibition by the combinations involving quinoline-derived compounds was selective for fungi.Quinine is one of several quinoline-derived antimalarial drugs. We hypothesized that antifungal synergy with bicarbonate or hygromycin may not be restricted to quinine, so tested different examples of these agents for synergistic interactions against representative plant and human pathogens  . However, trp1Δ cells were no more sensitive to the quinine + bicarbonate combination, relative to quinine alone, than the wild type . In contrast, the trp1Δ mutant was hyper-sensitive to the quinine + hygromycin combination . This suggested that tryptophan depletion in conjunction with quinine exposure exacerbates the action of hygromycin specifically.The hypothesized mode of action of the combinations was perturbation of mRNA translation fidelity, via amino acid depletion. The principal mode(s) of quinine drug action is unresolved, but one effect detected first in the yeast model is that quinine competes with tryptophan to cause tryptophan starvation . To testeviously , the auxFigure 8A). Bicarbonate additions can make media more alkaline (the media used here were unbuffered) and we observed in a previous study that NaOH had a stimulatory effect on quinine uptake , which is sufficient to explain some exacerbation of growth inhibition by quinine: an increase of ∼0.36 pH units increased quinine-dependent growth inhibition from ∼25% to >55% relative to control growth at the same pH values . The results show that stimulation of growth-inhibitory quinine action by bicarbonate is correlated with increased quinine uptake, and may be related to medium alkalinization by bicarbonate.Cellular quinine levels were assayed to determine whether bicarbonate or hygromycin may alter drug uptake, an alternative potential explanation for the synergies. Hygromycin did not have a significant effect on quinine uptake. In contrast, cells incubated with bicarbonate showed ∼3-fold greater quinine uptake . However, readthrough was increased ∼4-fold by a combination of quinine + hygromycin, where neither agent alone had a strong effect at the doses used .To test whether synergistic growth inhibition was reflected by synergistic mistranslation, we used a quantitative mistranslation assay. The assay tests for inappropriate readthrough of a UAA stop codon, positioned between ORFs encoding Figure 9C). The absence of rescue by anaerobicity leaves open the starting hypothesis: that amino acid limitation by quinine (rather than ROS generation) could be the relevant mechanism exacerbating mistranslation caused by hygromycin. In contrast, we showed that bicarbonate enhanced quinine uptake (and resultant growth inhibition), without stimulating stop codon readthrough. The data indicate that the different combinations involving quinine could exert different modes of synergistic antifungal action.Quinoline-containing compounds are known to provoke reactive oxygen species (ROS) production . As ROS Previously, it was reported that combination of a sulfate transport inhibitor with an aminoglycoside synergistically inhibits growth of several fungi . The prein vivo. Furthermore, chemicals targeting amino acid biosynthetic enzymes can exert good antifungal activity in vitro . Furthermore, we showed that medium pH can markedly influence quinine toxicity. The high-bicarbonate, high-pH nature of DMEM used for human cell culture could explain the relative quinine-susceptibility apparent in TE671 cells, for example (Table 1). Finally, as discussed further below, the present data provide proof of principle for novel synergistic interactions which provide a basis for rational formulation of more effective combinations, if needed, centered around similar chemistries.Our experimental design, to supply drugs at concentrations which individually were just subinhibitory, demanded concentrations of some agents which were higher than could be realistic for therapeutic use in humans or for large scale crop treatments. However, it should be borne in mind that the design was intended to give the most sensitive detection for discovering synergistic interactions, rather than replicate mponents  or, partmponents . We havetrp1Δ yeast mutant supported this, as did synergistic stimulation of mistranslation by this drug combination. In contrast, our observation that bicarbonate increased quinine uptake could alone account for that synergy as increased intracellular quinine should increase quinine toxicity. The alkalinizing effect of bicarbonate could impact quinine uptake though altered membrane energetics, for example. Moreover, collectively the data indicate that some but not all of the synergies observed here may be underpinned by translation-error, the starting hypothesis which originally informed our choice of agent-classes to test. Certainly several of the test agents will, like bicarbonate, have effects on cells additional to perturbation of translation fidelity. Given that, it will be interesting to resolve mechanism for a broader range of the new synergistic combinations discovered here.We had hypothesized that tryptophan starvation arising from competition between quinine and tryptophan for uptake  would stAspergillus niger and C. albicans  could be used as a basis for computational chemistry and other rational approaches to further optimize properties and synergies. In addition, while we focused primarily on agents targeting sulfur-containing amino acids and aromatic amino acids, it is likely that agents affecting availability of other amino acids may also produce synergies. For example, inhibitors of the NADP- and NAD-glutamate dehydrogenases are known to be active against fungi such as albicans  and coulR. solani and C. neoformans). Also, with certain exceptions, there was no consistent relationship within the human or plant pathogens. Drug resistance phenotypes can arise from a resistant (or absent) molecular target, reduced drug uptake, increased efflux, or drug inactivation, modification or sequestration. In addition, resistance of certain fungi to certain drugs was so high  that just-subinhibitory concentrations were not attained for some synergy assays. It is possible that synergy may become detectable at higher concentrations in such cases. The different test-fungi are cultivated on different media, where contents of amino acids, sulfate and other relevant substrates differ, potentially impacting the phenotypes reported here. Previously, it was reported that B. cinerea was not susceptible to synergistic inhibition by chromate + paromomycin (B. cinerea when combined with aminoglycosides. This emphasizes the need, where possible, to assess a range of agents associated with a similar mode of action.None of the combinations were synergistic against all six of the fungal pathogens tested. The overall pattern of susceptibility to the different combinations did not map clearly to phylogenetic relatedness of the fungi. For example, whereas 23 combinations strongly inhibited more than one fungus, none were effective against both of the basidiomycetes (omomycin . HoweverIn conclusion this work unveils a panel of novel chemical combinations, of agents affecting amino acid availability or translation, which synergistically inhibit fungal pathogens. Besides the therapeutic potential for exploitation of these new insights, some of these activities could provide valuable tools for understanding the control of translation fidelity in fungi, and how it may unravel.Supplementary Files. Any additional raw data supporting the conclusions will be made available by the authors on request, without undue reservation, to any qualified researcher.Most of the relevant data are included within the manuscript and SA and CV devised the study and drafted the manuscript. CV and RR performed the experiments. SA and MD provided resources and intellectual input which supported the study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Multiplayer games have emerged as a promising approach to increase the motivation of patients involved in rehabilitation therapy. In this systematic review, we evaluated recent publications in health-related multiplayer games that involved patients with cognitive and/or motor impairments. The aim was to investigate the effect of multiplayer gaming on game experience and game performance in healthy and non-healthy populations in comparison to individual game play. We further discuss the publications within the context of the theory of flow and the challenge point framework.A systematic search was conducted through EMBASE, Medline, PubMed, Cochrane, CINAHL and PsycINFO. The search was complemented by recent publications in robot-assisted multiplayer neurorehabilitation. The search was restricted to robot-assisted or virtual reality-based training.Thirteen articles met the inclusion criteria. Multiplayer modes used in health-related multiplayer games were: competitive, collaborative and co-active multiplayer modes. Multiplayer modes positively affected game experience in nine studies and game performance in six studies. Two articles reported increased game performance in single-player mode when compared to multiplayer mode.The multiplayer modes of training reviewed improved game experience and game performance compared to single-player modes. However, the methods reviewed were quite heterogeneous and not exhaustive. One important take-away is that adaptation of the game conditions can individualize the difficulty of a game to a player’s skill level in competitive multiplayer games. Robotic assistance and virtual reality can enhance individualization by, for example, adapting the haptic conditions, e.g. by increasing haptic support or by providing haptic resistance. The flow theory and the challenge point framework support these results and are used in this review to frame the idea of adapting players’ game conditions.The online version of this article (10.1186/s12984-018-0449-9) contains supplementary material, which is available to authorized users. Neurological deficits can result in impaired motor function that affect a person’s quality of life. Researchers have been working to restore the nervous system and reduce the neurological deficits of people suffering from stroke, spinal cord injury, or traumatic brain injury . For peoRobot- and virtual reality-assisted single-player games are well integrated in neurorehabilitation schedules. Recently, multiplayer games have been tested to complement neuromuscular therapy. Multiplayer games are expected to motivate the patients and increase the potential of robot- and virtual reality-assisted neuromuscular therapy.Multiplayer games incorporate social interaction to promote the enjoyment of the involved players. The additional player adds new possibilities to the game environment, generally missed in single-player gaming against preprogrammed challenges or artificially controlled opponents. The multiplayer environment and related game mechanics can facilitate social interaction, ranging from conversation to haptic interaction. Due to the this added social interaction, the game experience is thought to be better in multiplayer compared to single-player gaming .The mode of the game specifies whether the players compete or cooperate with one another . In lineIn positive game experience players increase their game performance , 10. IncAccounting for the individual’s skill level is not only important for game experience, but also for motor learning. According to the challenge point framework , motor lVarious solutions have been offered to account for the differences in skill level in multiplayer gaming in neurorehabilitation. These can be differentiated into adaptation of game mechanics (e.g. frequency of actions) and adaptation of the interface’s mechanics (e.g. robotic support). In multiplayer games, adaptations of the game mechanics affect the game condition for all players. In contrast, adaptation at the interface level allows for individual adjustment of the task difficulty. Thus, in settings with differently skilled players, individual interface mechanics can ensure that all players are challenged according to their skill level and can therefore increase game experience.In health-related gaming, task difficulty adaptation in multiplayer games, i.e. accounting for the skill level of the opponents, bears a challenge for game developers: Adaptations of game mechanics–as commonly done for single-player games–affect the game conditions for all players. In health-related gaming environments – particularly in patients with neuromuscular deficits – this challenge is even more prominent due to large variability in information processing abilities and motor skills. This large variability leads to differences across active range of motion, muscular strength, interlimb coordination or spasticity, among others. In multiplayer games, these deficits can make it so that two players cannot play against one another. It is therefore of interest to understand how to manipulate game conditions to balance the skill levels of patients and enable multiplayer gaming.In this review, we investigated whether multiplayer environments have improved game experience or game performance in serious games for health-related disciplines and neuromuscular therapy. We further compared different multiplayer game modes regarding game experience and game performance. To facilitate the transfer and consolidation of multiplayer gaming from health-related gaming to robot-assisted neuromuscular therapy, we systematically reviewed the literature regarding multiplayer gaming in health-related games. In this article, we describe the available literature in the context of the flow model and the challenge point framework. To further facilitate a transfer to neuromuscular rehabilitation, this review discusses measures regarding perceived game experience and physical performance, which have been applied in studies using multiplayer modes offering social interaction. Furthermore, this review complements definitions of task difficulty with the term “conditional task difficulty” to facilitate the discussion of haptic difficulty adaptation in robot-assisted rehabilitation. All discussions refer to our research question: Do multiplayer games enhance experience and performance in robot-assisted and virtual-reality assisted neurorehabilitation?The review protocol was based on an initial search review of the promising aspects of social interaction in virtual environments for rehabilitation interventions.We developed a database adjusted electronic search strategy for EMBASE, Medline, PubMed, Cochrane, CINAHL and PsycINFO in collaboration with a librarian from the Medicinal Library of the University of Zurich. The search was restricted to English and German language literature. There was no limitation in publication date or restriction by study design. The first search was performed in April 2016 and was complemented by papers published since (until January 2018).We used medical sub-headings as search terms, including the following main terms for the population: motor impairments, cognitive impairments, stroke, elderly people; for interventions: competition, collaboration, cooperation, coopetition, competitive behavior, collaborative behavior, exergame, multi-player-exergame, serious game, rehabilitative game, rehabilitative exergame, education game, computerized training, robot-assisted rehabilitation, robot-aided rehabilitation, virtual reality, virtual reality therapy, virtual world, social community, community integration, virtual community; for outcome: persuasion, compliance, motivation, engagement, effort, adherence, therapy progress.The search strategy  determined which articles should be included within this systematic review by scanning the titles, abstracts and keywords while applying the inclusion and exclusion criteria Table . An artiThis review includes a transfer to neurorehabilitation training gaming from related disciplines, such as health related serious gaming. For this purpose, we considered typical types of exercise interfaces as described by Tanaka . FeasibiThe full-text article was read if the title, abstract, or keywords provided insufficient information to decide on inclusion Fig. .Fig. 1SThe following data were extracted from the studies: (1) characteristics of the studied population: number of participants, disease and age, 2) characteristics of the interventions: the design, frequency and duration of the intervention, co-interventions and control intervention; 3) characteristics of the outcomes: outcome measures and results (Tables  characte charactePerceived game experience is discussed as the expressed opinions gathered from the activity. The opinion was assessed either during or after the task and either by answering a questionnaire or by assessing performance attributes (e.g. player’s decision on duration of task performance). Physical fitness is considered as a set of attributes that people have or achieve to perform a physical activity as defined by Caspersen . PhysicaBecause we expected the interventions and reported outcome measures to be markedly varied, we focused on a description of the studies and their results, and on thematic synthesis as defined by Thomas and Barnett-Page , 20.Our critical appraisal of the studies was based on a checklist designed for assessing the methodological quality of both randomised and non-randomised studies of healthcare interventions . The cheβ<70%=0points) to sufficient (β≥70%=1point). To assess the level of agreement between the investigators a Cohen’s kappa analysis was performed on all items on the checklist. In accordance with Landis and Koch’s benchmarks for assessing the agreement between raters a kappa-score of 0.81...1.0 was considered almost perfect, 0.61...0.8 was substantial, 0.41...0.6 was moderate, 0.21...0.4 was fair, 0.0...0.2 were slight and scores <0 were poor [The remaining 21 items were applied by two reviewers  to assess the methodological quality of the selected full text studies   was usedAssessment of goal commitment , 42 was The Koehler effect , assessePsychosocial factors such as self-efficacy , efficacIntervention specific questions for perception assessment of the external forces and fatigue were used in one study .Five out of thirteen articles assessed physical fitness by calculating energy expenditure Table : weight Five out of thirteen studies, –30, 36, In three studies –32, asseThe majority of the reviewed studies state that social interaction through multiplayer game settings improve both game experience and game performance Fig. . All stuα = 0.05). The percentage of agreement between the two reviewers was 99.3%. The mean quality score was 12.1 points , the median value was 12 points and the mode was 11 points. The mean score for reporting was 6.2 points , for external validity 0.8 points , for internal validity (bias) 3.9 points , for internal validity (confounding) 1.2 points  and for power 0.0 points , see Additional file The agreement on study quality between the two reviewers was almost perfect. The estimated Kappa value was 0.99 with a confidence interval ranging between 0.96 and 1.00  facilitIn general, measures of game experience can be acquired using physiological measures and questionnaires; only the latter were present in the included papers. Physiological measures, e.g. sensors measuring cardiac activity, significantly improve the assessment of game experience . HoweverInventories regarding psychosocial attractiveness of game design in the dimensions of social interaction, collaboration, individual feedback and team feedback, can extend motivation inventories when different multiplayer modes are compared . The perThe benefit of social interaction could be increased by integrating visual, auditory/verbal, and haptic elements. In the cognitive task study of Yu et al.  converseEnergy expenditure is a common measure for physical fitness regarding physical performance . VariousPhysical performance corresponds to training intensity and, therefore, predicts improvement in motor function, too . HoweverIn games where the end effector of the input device controls the avatar, the end effector position affects the success or failure within the game task. Assessing the end effector position and its impact on task success does not demand functional movement patterns in arm joints. However, functional movements are important in upper limb training. In exoskeleton devices used for rehabilitation training it is possible to measure the movements of non-game controlling joints . One solWe used a structured protocol to guide our search strategy, study selection, extraction of data and statistical analysis. However, limitations of this review should be noted: a publication bias may be present, as well as a language bias, given that we considered only interventions described in published studies and restricted our search to English and German language publications. A bias regarding research fields was generated since we mainly focused on neuromuscular therapy and health-related training.Multiplayer modes can enhance the players’ perceived game experience and positively influence the players’ performance. Based on the small number of relevant studies published so far, a conclusion cannot yet be drawn about which multiplayer mode is best during neurorehabilitation training. A meta-analysis of game experience and game performance outcomes may be suggested as soon as more multiplayer studies with homogeneous outcome measures will be published. Nevertheless, this review demonstrated that the players’ individual skill levels and personalities, as well as their role in the game, must be taken into account when selecting and designing multiplayer modes.Based on the model of flow and the challenge point framework, we suggest an individual adaptation of game conditions, i.e. conditional task difficulty, to assimilate differently skilled players for an optimal game experience. Furthermore, player specific selection of multiplayer modes may result in more robust interventions regarding game experience and requires less assimilation of differently skilled players.We suggest breaking the limited variety in multiplayer modes and fully exploring multiplayer modes and co-player’s characteristics such as the co-players presence, skill level, personality and relation to the player. We further suggest that future studies use a more stringent research design in which participants are allocated to either single play or multiplayer modes of exercise through randomised assignment.Additional file 1Search strategy. (DOC 31 kb)Additional file 2Assessment of methodological quality. (DOC 78 kb)