Helogale parvula)—a cooperatively breeding, territorial species—to examine postinteraction responses to the simulated threat of a rival group. Dwarf mongooses invested more in grooming of groupmates, foraged closer together, and more regularly acted as sentinels (a raised guard) after encountering indicators of rival-group presence compared to control conditions. These behavioral changes likely arise from greater anxiety and, in the case of increased vigilance, the need to seek additional information about the threat. The influence of an out-group threat lasted at least 1 h but individuals of different dominance status and sex responded similarly, potentially because all group members suffer costs if a contest with rivals is lost. Our results provide field-based experimental evidence from wild animals that out-group threats can influence within-group behavior and decision making, and suggest the need for greater consideration of the lasting impacts of social conflict.In social species, conspecific outsiders present various threats to groups and their members. These out-group threats are predicted to affect subsequent within-group interactions  and individual behavior . However, experimental investigations of such consequences are rare, especially in natural conditions. We used field-based call playbacks and fecal presentations on habituated wild dwarf mongooses  after encountering indicators of an out-group threat. These behavioral changes likely arise from greater anxiety and, in the case of sentinel behavior, the need to seek additional information about the threat. In many social species, groups and their members face a variety of threats from conspecific outsiders but relatively little is known about the consequences of these so-called out-group threats. From hymenopterans to humans, individuals form stable permanent groups which often defend collective resources . Out-groPhoeniculus purpureus) groupmates was elevated both immediately after intergroup contests and many hours later (Chlorocebus pygerythrus) exhibited greater levels of both within-group affiliation and aggression during extended bouts of intergroup conflict (Neolamprologus pulcher) increased affiliative interactions with groupmates after simulated intrusions by out-group rivals (Cebus apella) increased aggression levels during, but not after, out-group encounters ; in other cases, neighbors are more of a threat than strangers (the nasty-neighbor effect) . These dHelogale parvula), an ideal model species for biological and logistical reasons. Dwarf mongooses are cooperative breeders, living in family groups comprising a dominant breeding pair and nonbreeding subordinates of both sexes  and aggressive within-group interactions are affected by a simulated out-group threat . Second, we considered whether the threat of a rival group influences individual foraging and vigilance decisions, and how those responses are affected by potential variation in the threat level; we played back rival-group close calls to represent a threat from a nearby group and presented rival-group feces to represent a lesser threat (as those who deposited the feces may have moved away). We predicted that, as a consequence of increased anxiety and need for additional information about the threat, individuals would display more within-group grooming and aggression, forage closer together and contribute more to sentinel behavior after the simulated presence of a rival group compared to control conditions. We expected dominant individuals to show stronger responses than subordinates as the former are likely to suffer the greatest costs if rival groups gain access to limited resources . We also expected females to contribute more than males after rival treatments because females are the philopatric sex in dwarf mongooses ; the retData were collected from a habituated wild population of dwarf mongooses as part of the long-term Dwarf Mongoose Research Project (DMRP). This work was conducted on Sorabi Rock Lodge, a private game reserve in the Limpopo Province, South Africa ; full details available in Individuals can be identified from distinctive physical features or from small marks of blonde dye  applied to their fur . The popTwo field-based experiments were conducted to investigate within-group behavioral responses to simulated out-group threats. In Experiment 1, 7 groups each received 2 treatments at their morning sleeping burrow: 1) playback of the close calls of a non-neighboring group  and 2) playback of herbivore grunts and huffs (as a control). The immediate responses to the playback and subsequent within-group affiliative (grooming) and aggressive interactions were recorded. In Experiment 2, 7 groups each received 4 treatments while foraging: 1) playback of the close calls of a non-neighboring group; 2) playback of herbivore grunts and huffs (as a control); 3) presentation of feces from the same non-neighboring group as in (1); and 4) presentation of herbivore feces (as a control). The immediate responses to playback and fecal presentations, as well as subsequent foraging and vigilance decisions, were recorded.Equus quagga), giraffe , blue wildebeest (Connochaetes taurinus), and waterbuck (Kobus ellipsiprymnus), are accustomed to human presence. The microphone was attached to a tree 10 m from an artificial feeding area and left to record for 1 h.Playback stimuli were constructed from original sound recordings. All sound recordings were made with a Marantz PMD660 professional solid-state recorder  and a Sennheiser directional microphone  with a Rycote softie windshield . Recordings were made at a sampling rate of 48 kHz with a 24-bit resolution and stored on a Transcend SD card . Dwarf mongooses are very vocal and rely on acoustic communication to coordinate their cooperative behaviors; they produce close calls  continuously while foraging and moving . Close cFive-minute playback tracks were constructed in Audacity (version 2.1.3). For rival-group tracks, close calls with good signal-to-noise ratio were randomly chosen and extracted from original recordings. Four different call sequences were constructed per group, with each sequence consisting of one close call from each of the 4 recorded individuals. These sequences were selected in a random order and inserted into a 12-s block of ambient sound; ambient-sound recordings were made from the center of the relevant territory with the equipment described above. Five such 12 s blocks were edited together, and this 1 min block was copied 5 times to create a 5-min track. Rival-group tracks had a close-call rate of 75 calls per minute, which is the natural vocalization rate of 4 dwarf mongooses . ControlFecal collection, storage, and usage followed the protocol previously used on this study population by N = 3; Experiment 2: N = 5). Abandoned trials were rerun another day when the above conditions were met. Behavioral responses in all trials were recorded to a Dictaphone ; collecting data through live observation allowed a wider field of view and consideration of more group members than video recording, but precluded blind scoring. The location of each experimental manipulation was recorded using a GPS . Analysis of the GPS data revealed that there was no significant difference between the different treatments in an experiment in the likelihood that they were run in the core or the periphery of the focal group’s territory  and 1 month for Experiment 2 . Treatment order was counterbalanced between groups. Trials were not conducted if there had been an intergroup interaction earlier that day and were abandoned if an alarm call or any other group disturbance  occurred during the experimental manipulation . When the playback track ended, data on within-group behavioral interactions were dictated until 50% of the group had left to start foraging. All aggressive and affiliative (grooming) interactions between adult individuals were recorded, including the identity of those involved and the duration of the interactive bout.For Experiment 1, both sound treatments to the same group  were conducted at the same type of sleeping burrow  when weather conditions were calm. The playback equipment was set up before the first mongoose emerged; the field loudspeaker was hidden from view 5 m from the burrow. Two minutes after all the group members had emerged, the mongooses were attracted to a location 5 m from the loudspeaker and the trial commenced  of the closest group member to all foraging individuals in sight; it was not possible to record individual identities regularly without disrupting foragers. Sentinel scans entailed noting whether a sentinel was present and, if so, its identity and whether it was facing in the direction of the experimental manipulation.For Experiment 2, all 4 treatments to the same group  were conducted when the group was foraging in a similar habitat type during calm weather conditions. Trials were run as per the P < 0.05. Parametric tests were used where the residuals fitted the relevant assumptions of normality and homogeneity of variance. Logarithmic and arcsine transformations were conducted to achieve normality of errors in some cases (details below); otherwise nonparametric tests were used.Mixed models were constructed in RStudio 3.2.2 , while aP-value calculations based on the default asymptotic distribution of the test statistic would be unreliable; we therefore used the Monte Carlo resampling method  to generate P-values. In cases where multiple factors needed to be taken into consideration, repeated-measures analysis of variances (ANOVAs), linear mixed models (LMMs), or generalized linear mixed models (GLMMs)  and a graphical approach was used to confirm normality and homoscedasticity of residuals.For simple paired data, we used Wilcoxon signed-rank tests. As sample sizes were small, To determine if rival-group playbacks induced an increased response relative to control playbacks, and thus simulate an out-group threat as planned, the immediate responses were considered. Sound-treatment differences in the proportion of individuals that looked and orientated towards the loudspeaker and the proportion of individuals that directly approached the loudspeaker were analyzed using Wilcoxon signed-ranks tests.To examine the influence of sound treatment on subsequent within-group affiliative interactions (there were no aggressive interactions observed), grooming bouts of >5 s were analyzed in 2 stages. First, differences in the overall rate  and mean duration of grooming bouts  were considered using Wilcoxon signed-ranks tests. Second, whether the significant difference in grooming bout duration (see Results for details) was driven by individuals of different dominance status or sex was considered. Two LMMs (1 for dominance status and 1 for sex), with identity link functions, were run on the raw data. These included sound treatment  and either dominance status  or sex , as well as their interaction with sound treatment, as fixed effects; individual identity was nested within group identity as the random term.To determine if rival-group playbacks and fecal presentations induced an increased response relative to control playbacks and fecal presentations, and thus simulate an out-group threat as planned, the immediate responses were considered. For the playback trials, sound-treatment differences in the proportion of individuals that looked towards the loudspeaker and the proportion of individuals that directly approached the loudspeaker were analyzed using Wilcoxon signed-ranks tests. For the fecal trials, presentation-treatment differences in the proportion of the group that participated in the latrine and the total time spent sniffing the feces were analyzed using Wilcoxon signed-ranks tests.To examine the influence of experimental treatment on subsequent within-group foraging decisions, log-transformed mean nearest-neighbor distances during the postmanipulation hour were analyzed in a 2×2 repeated-measures ANOVA. Intruder identity , manipulation type , and their interaction were included as predictor variables. To determine if the experimental treatment had a lasting effect, the nearest-neighbor foraging distances from the first and last scans  were compared using a second 2×2 repeated-measures ANOVA. The first analysis revealed no difference in nearest-neighbor distances depending on manipulation type (see Results for details); so, means were calculated from the 2 rival-group treatments and the 2 control treatments for the second ANOVA. Intruder identity , scan period , and their interaction were included as predictor variables in this second ANOVA.To examine the influence of experimental treatment on subsequent within-group sentinel decisions, arc-sine-square-root-transformed proportions of scan samples in which a sentinel was present were analyzed in a 2×2 repeated-measures ANOVA. Intruder identity , manipulation type , and their interaction were included as predictor variables. The likelihood of sentinels facing in the direction of the experimental manipulation was analyzed using a Wilcoxon signed-ranks test for just the period after call playbacks; no individuals acted as a sentinel in the period after a control fecal presentation. Whether the significant difference in sentinel occurrence (see Results for details) was driven by individuals of different dominance status or sex was then considered. Two GLMMs (1 for dominance status and 1 for sex), with binomial error distributions and logit-link functions, were run. Since the first analysis of sentinel behavior revealed no difference depending on manipulation type (see Results for details), values were combined for the 2 rival-group treatments and the 2 control treatments. The models bound the number of scan samples in which an individual was on sentinel duty with the number of scan samples in which the individual was not acting as a sentinel, testing the likelihood of an individual being on sentinel duty over a given period. The fixed effects applied to these models were intruder identity  and either dominance status  or sex , as well as their interaction with intruder identity; individual identity was nested within group identity as the random term.N = 7, Monte Carlo P = 0.014; N = 7, Monte Carlo P = 0.030; The immediate responses to playback at the sleeping burrow were significantly affected by sound treatment. A greater proportion of individuals looked and orientated towards the loudspeaker during rival-group playback compared with control playback  interactions were common (mean ± SD grooming rate: 0.5 ± 0.3 bouts per minute). Although there was no significant sound-treatment difference in the overall rate of grooming interactions . The stronger response to rival-group treatments compared with control treatments lasted for at least 1 h after the simulated intrusion: there was no significant effect of scan period  on nearest-neighbor distances .Foraging decisions in the hour after the manipulation were affected by intruder identity , but not manipulation type . Overall, individuals foraged significantly closer to another group member following rival-group playbacks and fecal presentations compared to control treatments . Moreover, a greater proportion of sentinels were facing in the direction of the manipulation after rival-group playback compared with control playback . There was no significant effect of the interaction between treatment type  and either dominance status  to rival playbacks and fecal presentations, contrary to our prediction that the former might indicate a more imminent threat and so elicit a stronger reaction. One possible explanation is that a playback does not fully replicate the circumstances relating to an approaching rival group. While rival playbacks did simulate an out-group threat, as there were relevant changes in behavior both during and after the manipulation, our playbacks were not followed by visual confirmation of a rival group. Without such visual validation shortly after hearing acoustic cues indicating a rival group presence, dwarf mongooses might not perceive the situation realistic of an imminent contest. Another potential reason for the lack of a difference between experimental treatment types is that encountering relatively fresh rival feces at a latrine might have generated similar anxiety to hearing another group. While acoustic cues might suggest an imminent encounter, uncertainty about the current location of the rival group that deposited the feces might cause equivalent anxiety and thus changes in vigilance and foraging decisions.Conflict is recognized as a powerful selective force, yet relatively few studies have experimentally investigated the consequences of out-group conflict despite its prevalence in the animal kingdom. Both our field manipulations had the predicted effect of simulating an out-group threat—they resulted in clear changes in immediate and subsequent behavior—and therefore represent viable approaches for future work. Combining the ecological validity of studying animals in natural conditions with the power from controlled experimental testing, allows the generation of strong conclusions about the effects of out-group threats on within-group behaviors. Together, the 2 field manipulations provide an insight into 3 neglected avenues of research in this field: studying a broader range of postinteraction behaviors (beyond aggression and affiliation), looking at behavioral changes from an individual level and focusing on behavioral changes beyond the immediate aftermath . Future arz095_suppl_Supplementary-MaterialClick here for additional data file.


Pancreatic cancer remains one of the deadliest cancers. The five-year survival rates have been reported as 3%. Radical surgical tumor resection is critical for improved outcome and the low survival rate for pancreatic cancer is due to lack of other effective treatments and here optical guided surgery could be a solution for better surgical outcome. In the present study, we targeted the urokinase plasminogen activator receptor (uPAR) with a peptide conjugated with the fluophore ICG (ICG-Glu-Glu-AE105) for optical imaging. In the first part of the study we aimed to validate ICG-Glu-Glu-AE105 for resection of the primary tumor and metastases in an orthotopic human xenograft pancreatic cancer model. In the second part of the study we aimed to investigate if fluorescent-guided imaging could locate additional metastases following conventional removal of metastasis under normal white light surgery.Our study showed that ICG-Glu-Glu-AE105 was an excellent probe for intraoperative optical imaging with a mean tumor-to-background ratio (TBR) for the primary tumor of 3.5 and a TBR for the metastases of 3.4. Further, a benefit using intraoperative fluorescent guidance yielded identification of an additional 14% metastases compared to using normal white light surgery. In 4 of 8 mice there were identified additional metastases with uPAR optical imaging compared to white light.In conclusion, the uPAR-targeted optical probe ICG-Glu-Glu-AE105 enables intraoperative optical cancer imaging, including robotic surgery, and may be a benefit during intended radical resection of disseminated pancreas cancer by finding more metastasis than with traditional white light surgery. Pancreatic cancer remains one of the most lethal cancer types and is the fourth leading cause of cancer-related deaths the United States , 2. PatiUrokinase-type plasminogen activator receptor (uPAR) is a membrane-bound protein highly expressed in most cancer types including pancreatic cancer , oral caThe aim of the present study was therefore to investigate if the use of the uPAR-targeted optical probe ICG-Glu-Glu-AE105 in an orthotopic xenograft model of human pancreatic cancer could improve the surgical outcome by detecting additional cancer lesions not found during white light surgery.in vivo. Tumor cells were luciferase (luc) transfected and could thus be detected with not clinically translatable bioluminiscence (BLI) as a reference. Imaging of the primary tumor showed a clear delineation of the tumor lesion located in the pancreas with a very low background signal  were present in the 8 mice. Of these 43 metastases, 29 metastases were found without fluorescent guidance (white light), and an additional 6 metastases were identified only with the Fluobeam®800 camera  there were identified additional metastases with uPAR optical imaging compared to white light. Hence, in 50% of the mice the surgeon operated in white light there would have been left behind metastases that could be found with uPAR optical imaging.The second part of the study aimed to evaluate if optical imaging could identify additional metastases after all metastases visible with white light had been removed . On a le0 camera , 2B. Fin® HD Si surgical robotic system. The mouse was similar to the other mice in the study, with an orthotopic pancreas tumor and the procedure was performed as open surgery. After the abdomen was opened and the region of the pancreas was located, the firefly NIR fluorescence function in the robot was activated  metastases were found using the uPAR optical guidance.In conclusion, our study showed that ICG-Glu-Glu-AE105, targeting uPAR, is a promising imaging agent for fluorescence guided surgery. With uPAR guided surgery additional metastases were found in 50% of the mice suggesting that uPAR optical guidance may lead to better surgical outcome. Our findings support clinical translation of ICG-Glu-Glu-AE105.in vivo injection ICG-Glu-Glu-AE105 was dissolved in (2-hydroxypropyl)-β-cyclodextrin with 2% DSMO. The human pancreas adenocarcinoma cell line BxPC3-luc2 was purchased from PerkinElmer and grown after the recommendations in RPMI-Glutamax with 10% FBS and 1% PenStrep.The peptide AE105  was conj5 BxPC3-luc2 in 25 ml PBS was injected orthotopic in the tail of the pancreas. The incision was closed with 5.0 suture. The primary tumor was followed by luminescence and when a suitable size was reached a similar incision was made and the primary tumor was removed and the incision was closed again as described above. During the study all mice were housed in groups with 12 h light/dark cycle with food and water available ad libitum.All animal experiments were performed under a protocol approved by the Animal Research Committee of the Danish Ministry of Justice (2016-15-0201-00920). The mice were anesthetized with 4% sevoflurane before an incision in the abdomen was made over the spleen and the pancreas was located. 5 × 10® HD Si,  using non-human instruments, in one case for navigation in resection of a primary human xenograft tumor in the pancreas of a mouse. IVIS Lumina XR, a black-box optical camera was used for luminescence imaging for growth monitoring of the tumor and positive control for tumor presence in the resected metastases.The FluobeamR800  used for navigation during resection of the primary tumor and the metastases is a handheld camera with an excitation wavelength at 800 nm where adjustments as sensitivity and zoom are possible. We used the clinically approved surgical robot system da Vinci3, 10 nmol of ICG-Glu-Glu-AE105 was injected i.v. into the mouse 15 h pre-operatively. The primary tumor was then removed and a fluorescent image of the tumor was performed throughout the surgery. Because it is known that removal of the primary tumor does not prevent metastases in the peritoneum from developing, the incision was closed and the mice underwent luminescence imaging to track the development of potential metastases. When potential metastatic burden was assessed in the abdomen, an incision was again made in the abdomen and the fluorescent camera was turned on to locate and image the metastases developed. TBR was later measured using ImageJ software, where a ROI was drawn over the whole tumor and a background ROI was drawn over adjacent tissue.After inoculation of BxPC3-luc2 cells in the pancreas as described above the mice were luminescence imaged to follow the growth of the primary tumor. When the primary tumor reached a suitable size of approximately 50–100 mmex-vivo to validate the presence of tumor cells in the tissue assumed to be a metastasis. The mice were euthanized after the operation.The same work flow as in study I, except that the surgeon was asked to remove all the metastases he could identify with regular white light turned on during surgery. When the surgeon was convinced all metastases were removed the NIR camera was used to identify potentially additional metastases. The mouse was then imaged with bioluminescence to locate further metastases that was missed by both white light and NIR light . All metData are described as mean and 95% confidence interval.


To determine the impact of Sequential Organ Failure Assessment (SOFA) organ sub-scores for hospital mortality risk stratification in a contemporary cardiac intensive care unit (CICU) population.Adult CICU admissions between January 1, 2007 and December 31, 2015 were reviewed. The SOFA score and organ sub-scores were calculated on CICU day 1; patients with missing SOFA sub-score data were excluded. Discrimination for hospital mortality was assessed using area under the receiver-operator characteristic curve (AUROC) values, followed by multivariable logistic regression.We included 1214 patients with complete SOFA sub-score data. The mean age was 67 ± 16 years ; all-cause hospital mortality was 26%. Day 1 SOFA score predicted hospital mortality with an AUROC of 0.72. Each SOFA organ sub-score predicted hospital mortality , with AUROC values of 0.53 to 0.67. On multivariable analysis, only the cardiovascular, central nervous system, renal and respiratory SOFA sub-scores were associated with hospital mortality . A simplified SOFA score containing the cardiovascular, central nervous system and renal sub-scores had an AUROC of 0.72.In CICU patients with complete SOFA sub-score data, risk stratification for hospital mortality is determined primarily by the cardiovascular, central nervous system, renal and respiratory SOFA sub-scores. Risk prediction scores have guided care in the cardiac intensive care unit (CICU) since Killip, et al. reported their classification of patients with acute myocardial infarction. The CICUThe Sequential Organ Failure Assessment (SOFA) score is an illness severity score developed in patients with sepsis, including a 4-point assessment of dysfunction in each of 6 organ systems .–13 The SThe purpose of this study was to determine the relative contribution of each individual SOFA organ sub-score for prediction of mortality in CICU patients without any missing SOFA sub-score data, in order to facilitate potential future modification of the SOFA score to better fit the CICU population. Additionally, we sought to further explore the importance of missing data for mortality risk prediction using the SOFA score in CICU patients, as highlighted in our prior work.This study was approved by the Mayo Clinic Institutional Review Board under an exception from informed consent as posing minimal risk to patients. This is a subset analysis of a historical cohort analysis utilizing an institutional database of patients admitted to the CICU at the Mayo Clinic Hospital, St. Mary’s Campus, as previously described. The MayoAs described previously, demographic and laboratory data and use of invasive ventilation and catecholamine infusions during the first 24 hours of CICU admission were collected. The SOFAhttps://www.r-project.org/).The primary study endpoint was all-cause hospital mortality; secondary endpoints included all-cause CICU mortality and 30-day mortality. Mortality data were extracted from Mayo Clinic electronic databases, the state of Minnesota electronic death certificates and the Rochester Epidemiology Project database, as previously described. CategoriWe screened 12904 adult admissions to the CICU during the study period and excluded 2900 patients, yielding an initial population of 10004 patients .15] Day Day15] Dth percentile for the initial population.[In the final study population, the mean age was 66.7±15.0 years and 459 (37.8%) patients were female . The finShort-term mortality increased progressively with rising Day 1 SOFA score in the final study population . The DayThe distribution of individual SOFA sub-scores in the final study population is shown in On multivariate analysis including all 6 SOFA organ sub-scores as predictors of hospital mortality, only the cardiovascular, central nervous system, respiratory and renal sub-scores were significant predictors of hospital mortality . The AURA simplified SOFA score including only the 4 SOFA organ sub-scores  that were significantly predictive of hospital mortality on multivariate analysis had similar discriminative capacity compared to the Day 1 SOFA score for hospital mortality in the final study population  for hospital mortality in unselected CICU patients. The avaiWe report a novel, U-shaped association between the number of SOFA sub-scores with available data and short-term mortality, with higher mortality in the small number of patients missing data for 3 or more SOFA sub-scores as well as among patients who had available data for the respiratory and liver SOFA sub-scores. This association between laboratory testing patterns and mortality mirrors the prior study by Afessa, et al. reporting that patients with a measured serum bilirubin or albumin level  had higher observed mortality. In the sPrior studies comparing the SOFA score with the APACHE score in general CICU populations have demonstrated similar discrimination for mortality., 15 Our Not all of the individual SOFA organ sub-scores are equally relevant for mortality risk prediction in CICU patients. Because The impact of individual SOFA organ sub-scores on overall mortality prediction has not been previously explored in CICU patients, apart from our prior study. Knox, etThis retrospective single-center cohort study has a number of limitations, including the possibility of unmeasured confounders and potential bias due to local practice patterns. Our patient population may be distinct from other centers, as reflected by a lower hospital death rate and rate of acute coronary syndromes in the initial population than most prior CICU studies., 6, 14 TIn conclusion, CICU patients with the availability of complete data to calculate all 6 SOFA organ sub-scores are a high-risk population, reflecting a bias toward more laboratory testing in sicker patients. Discrimination of the SOFA and OASIS scores for hospital mortality was lower in this cohort than reported in the initial population; the APACHE-III score had the best performance of the scores we examined. The renal and cardiovascular SOFA sub-scores contribute the most to mortality prediction in these CICU patients, and the liver and coagulation SOFA sub-scores contribute little to mortality prediction. These findings emphasize the impact of data availability on performance of the SOFA score, and highlight the potential to refine the SOFA score for mortality risk prediction in CICU patients by replacing the current coagulation and liver SOFA sub-scores with variables more predictive of mortality in CICU populations. We suggest that future prospective studies using the SOFA score as a marker of illness severity use standardized methods for ensuring complete data availability to calculate each of the organ sub-scores, to avoid the limitations of missing data described herein. The suboptimal performance of the SOFA score in this study emphasizes the limitations of using the SOFA score in CICU populations and the need to develop better risk prediction models for CICU patients. Future studies are needed to determine the real-world performance of the SOFA score in CICU patients. This work may ultimately facilitate the future development of a CICU-specific SOFA-derived score or novel CICU-specific risk score that will be more widely applicable to CICU populations.S1 Fig(TIF)Click here for additional data file.S2 FigP<0.001 between groups.(TIF)Click here for additional data file.S3 FigP <0.001 between groups.(TIF)Click here for additional data file.S4 FigP<0.001 between groups.(TIF)Click here for additional data file.S1 TableFor this analysis, all missing data were imputed as normal. All p values <0.001.(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


This research article aims to disclose the features of nanofluidic flow manifested with Cattaneo-Christov model of heat and mass flux over non-linearly stretching surface. An incompressible visco-elastic nanofluid saturates the given porous medium through Darcy-Forchheimer relation. A non-uniformly induced magnetic effect is considered to accentuate the electro-magnetic and thermal conductivity of the base fluid. The model is restricted to small magnetic Reynolds. Boundary layer assumptions are incorporated for the given flow model. Governing equations are remodeled into non-linear ordinary differential equations through transformations. So formulated nonlinear system is solved through homotopy analysis method (HAM) to achieve series solutions for velocity field, concentration of nanoparticles and temperature distribution. It is noticed that the temperature distribution and corresponding thermal boundary layer pattern shows declination for Cattaneo-Christov model of heat and mass flux as compared to classical Fourier’s law of heat flux/conduction. Furthermore, the intensive resistance offered by the addition of porosity factor in the flow model results in rise of temperature profile, however, opposite behavior is noticed in concentration of nanoparticles. The wall-drag intensity, the heat flux and the mass flux are discussed on the premise of numerical information obtained upon simulation of the problem. Fe3O4-H2O Ferro-fluid flow using permeable semi-annulus. The porosity factor resulted in higher resistance offered to the fluid motion following a low velocity field. In another article, Sheikholeslami et al.  is known as embedding factor. z = 0, 1,ith deformations:i = 1 for i > 1, otherwise 0. Finally,Homotopy analysis method (HAM) is an efficient solver for nonlinear systems where small and large physical parameters are involved for analysis of variation in the respective flow profiles. Thus, it is more suitable and economical as compared to that of perturbation method for solving non-linear systems of equations. It is applied in most of non-linear systems developed in engineering, industrial, science and finance problems. Define,The general solutions are,f′), temperature distribution (θ) and concentration of the nanoparticles (ϕ) for the problem under consideration are termed as convergence control parameters. These parameters are critical in choosing appropriate values to speed-up the convergence. The intervals of interest for f, θ and ϕ are presented in th order of approximations are sufficient to achieve convergence in velocity field whereas temperature and concentration requires 10th order approximations.The auxiliary parameters involved in series solutions for the velocity field (Cf), local Nusselt (heat flux) (Nu) and local Sherwood (mass flux) (Sh) is compiled in Tables Cf) and mass flux (Sh) is noted for visco-elastic nanofluid parameter (k1). Furthermore, the Brownian diffusion rate (Nb) and thermophoresis effect (Nt) appeared as reducing factors for heat flux (Nu). Influence of various fluid parameters on the flow profiles i.e. velocity field, temperature distribution and concentration of the nanoparticles in base conventional fluid, is shown in Figs f′(η) and corresponding variations are plotted in Figs Fr. A similar trend is noticed in velocity profile for various values of visco-elastic nano-fluid parameter k1 plotted in k1 result in increasing the fluid viscosity that reduces the flow motion. Figs γ1 reduces the temperature profile and the associated thickness of thermal boundary layer as well. Physically, it confirms that incremental nature of thermal relaxation time parameter requires more time to shift the heat from intensively packed fluid particles to the low energetic fluid particles. One can call it a demonstration of characteristics of non-conducting fluid material. Thus, a decay in temperature profile is noted. The results are plotted in The given non-linear system of Eqs –18) wit wit18) wA locally similar analysis on Darcy Forchheimer visco-elastic nanofluid flow bounded by a non-linearly stretching sheet/surface manifested with Cattaneo-Christov theory of heat—mass flux has been carried out in this analytic research article. The key findings are itemized below:Increasing values of interial coefficient and porosity result in declination of the velocity field and the associated momentum boundary layer.The visco-elastic nanofluid parameter shows reduction in the velocity field however, an increment is noted in the thermal layer for augments in the aforementioned parameter.The intensive resistance offered by the addition of porosity factor in the flow model results in rise of temperature profile, however, opposite behavior is noticed in concentration of nanoparticles.Larger Prandtl number shows reduction in concentration of the nanoparticles.Thermal relaxation time parameter allows more heat to be absorbed by the system. Thus, a decay is noted.Solute relaxation time parameter shows mixed behavior in concentration of nanoparticles.Skin-friction force is intensive for higher thermal relaxation time parameter.Local Nusslt shows increasing behavior for higher values of thermal relaxation time parameter, however, the values are higher for non-linear case as compared with linear case.Local Sherwood is increasing function of concentration relaxation time parameter, however, the values are lower for linear case as compared to the non-linear case.Nomenclature:


Assays that evaluate the functionality of anti-cytokine autoantibodies can supplement such ligand binding assays and add valuable functional information that, when viewed in the context of the clinical phenotype, may guide the use of adjunctive immunomodulatory therapy. This mini review provides an overview of anti-cytokine autoantibodies identified to date and their clinical associations. It also describes the use of flow cytometry for the functional analysis of anti-IFNγ and anti-GM-CSF autoantibodies.Autoantibodies to cytokines are increasingly being detected in association with immunodeficient, autoimmune and immune dysregulated states. Presence of these autoantibodies in an otherwise healthy individual may result in a unique phenotype characterized by predisposition to infection with specific organisms. The ability to detect these autoantibodies is of importance as it may direct treatment toward a combination of anti-microbial agents and immunomodulatory therapies that decrease autoantibody levels, thereby releasing the immune system from autoantibody-mediated inhibition. Ligand binding assays such as ELISA or bead multiplex assays have been used to detect these antibodies. However, not all anti-cytokine autoantibodies have demonstrable function  The association of anti-cytokine autoantibodies (AAbs) with primary or acquired immunodeficiency, immune dysregulation and autoimmunity has been increasingly documented in literature –3. In faAnti-cytokine AAbs can be found in circulation and may mediate diverse infectious and/or immunological manifestations depending on the cytokine that they specifically target. Anticytokine AAbs may decrease bioavailability of cytokines by inhibiting binding to their cognate receptors or by sequestering the cytokine in high molecular weight complexes that subsequently undergo Fc-dependent degradation . AlternaThe ability of anti-cytokine AAbs to mediate disease manifestations may depend on their ability to neutralize or potentiate cytokine function which in turn, may be dependent on the concentration of the AAb in circulation or in tissue, the avidity of the AAb, or the epitopes recognized by the AAb . In an aAnti-cytokine AAbs are generally polyclonal IgG in nature, however, low titer, non-neutralizing IgA AAbs to interleukin-10 (IL-10) have been detected in the serum of patients with inflammatory bowel disease (IBD) . BecauseAnti-cytokine AAbs can also be detected in a majority of healthy individuals and in therapeutic human immunoglobulin preparations . In factUnder physiological conditions, anti-cytokine AAbs may potentially play a role in the regulation of biological activities of cytokines either by neutralizing excessive cytokine production or by prolonging the half-life of cytokines in circulation by forming cytokine-antibody immune complexes . This poThere is growing evidence that suggests certain anti-cytokine AAbs play a direct pathogenic role in increasing susceptibility to infection or development of immune dysregulated states. For example, AAbs to interferon gamma (IFNγ), a cytokine responsible for protective immune responses against intracellular organisms, are associated with chronic, disseminated, treatment refractory infections with intracellular organisms such as mycobacteria  and AAbsIn other clinical conditions, the presence of anti-cytokine AAbs might correlate with disease resolution; for example, an increase in anti-IL-1 AAbs correlates with milder disease course in rheumatoid arthritis , anti-inThe genetic and/or environmental factors that influence the development and potential pathogenicity of these anti-cytokine AAbs in an individual remain poorly defined. Understanding the true prevalence, titer and biological significance of anti-cytokine AAbs in healthy individuals or in specific disease cohorts is confounded by the diversity of techniques used to detect them. Therefore, it is important to measure not only the titer of the AAbs, but to also use functional assays, that may help to determine if the AAbs are functional and therefore more likely to be pathogenic. Appropriate recognition of these AAbs in the context of disease is also important because it may direct treatment toward a combination of adjunctive immunotherapy to modulate the AAb titer while continuing appropriate anti-microbial or other suitable therapy.Although the association of anti-cytokine AAbs with immunodeficiency, dysregulation and autoimmunity is well-recognized, diagnostic testing for these AAbs is limited. Apart from validated diagnostic testing in a few clinical laboratories for the detection anti-GM-CSF AAbs in suspected or confirmed autoimmune PAP, anti-cytokine AAb testing is generally not routinely performed by most clinical laboratories. Analysis of anti-cytokine AAbs can be performed by laboratory-developed ELISA, radioimmunoassay, multiplex bead arrays or other ligand binding assays, and offers the ability to report an antibody titer and/or concentration. Ligand binding assays are, in general, high throughput, cost effective and automatable, and the methods can be fairly easily adapted by most clinical laboratories. Functional assessment of autoantibodies provides further information regarding their biological significance in the context of disease and requires demonstration of their neutralizing or potentiating capacity. These assays, while providing important functional information, are generally performed in laboratories that specialize in high complexity testing. Functional assessment of either the antagonistic or agonistic action of these AAbs has been performed in a variety of ways including demonstration of inhibition of proliferation of cytokine-responsive cell lines, inhibition of the ability of certain cytokines to restrict viral growth in permissive cell lines, inhibition of cytokine-driven differentiation of responsive cell lines, inhibition of cytokine-specific phosphorylation signals or potentiation of cellular function.in vivo. Although currently not widely available, flow cytometry based assays have been established for IFNγ and GM-CSF AAbs by exploiting knowledge of their signaling pathways. These assays and their relevance in diagnosis or monitoring disease states will be discussed below.Flow cytometry is becoming more widely available in clinical laboratories and can be utilized to demonstrate inhibition of cytokine signaling pathways by neutralizing AAbs, and by inference, an indication of their ability to neutralize cytokine function IFNGR1 and IFNGR2, , and STAT1 , that is downstream of the IFNγ receptor, often lead to severe infections with intracellular organisms of low pathogenicity such as the Bacille Calmette Guerin (BCG) vaccine or non-tuberculous mycobacterial (NTM) species  have been reported as well  and to a lesser extent on lymphocytes . BindingGM-CSF mediates its functions through binding to its receptor, a heterodimer that is comprised of a specific a subunit (GMRα) and a dimeric b subunit (βc) that is shared with other cytokines of the βc family. Following binding of GM-CSF to its receptor, the βc subunit associates with Jak2 that then leads to phosphorylation of STAT5, leading to intranuclear translocation and transcription of GM-CSF regulated genes  62). Co. Co62). The IFNγ and GM-CSF signaling pathways have been used to develop flow cytometry based strategies for the functional detection of autoantibodies to these cytokines. The basis of this assay is a short stimulation of isolated peripheral blood mononuclear cells (PBMC) from a healthy donor with either recombinant human IFNγ or GM-CSF to induce phosphorylation of STAT1 or STAT5, respectively. Isolated PBMCs are first stained with fluorophore-conjugated anti-CD14 to identify monocytes. Stained PBMCs are then incubated at 37°C with an optimized concentration of either recombinant IFNγ or GM-CSF for 10–15 min. PBMCs are fixed immediately at the end of the stimulation period with 4% paraformaldehyde to preserve their phosphorylation status and permeabilized using methanol to allow staining for intracellular proteins. Intracellular phosphorylated STAT1 (p-STAT1) or STAT5 (p-STAT5) is detected in monocytes by staining PBMCs with fluorophore conjugated monoclonal antibodies to p-STAT1 (Tyr701) or p-STAT5 (Tyr694) . AdditioAs with most functional assays, flow cytometry analysis of p-STAT1 and p-STAT4 may be affected by factors other than specific AAbs in patient serum that might interfere with these pathways. Functional assays do not directly detect an AAb and only infer its activity through the effect observed on specific biological activity attributed to the cytokine. Thus, it is important to address the specificity of the assay by combining it with a ligand binding assay in order to demonstrate the presence of an AAb, and to utilize a control cytokine that uses the same signaling molecules in order to demonstrate neutralization of a specific cytokine. For instance, the assay for IFNγ AAbs makes use of IFNα as a control for specificity because both cytokines signal through STAT-1. In clinical practice, ligand binding assays may be used as a high throughput screen for anti-cytokine AAbs followed by a functional assay as confirmation.As these assays make use of PBMCs, it is necessary to develop a pool of previously screened donors for the assay. Donor PBMCs for these assays are initially evaluated for their response to IFNγ and GM-CSF and the ability of previous characterized inhibitory serum to effectively neutralize the IFNγ or GM-CSF response to these cytokines. In our laboratory practice, freshly isolated PBMC from acceptable donors are utilized for clinical testing. It may also be possible to utilize cryopreserved PBMC from acceptable donors, however, we have not tested the performance of cryopreserved PBMC for phosphorylation assays extensively. An alternative strategy may be to utilize cell lines that are responsive to these cytokines.Listeria monocytogenes-infected human monocytes to clear infection when activated by IFNγ  and B cell depleting agents such as rituximab have been used to treat autoimmune PAP due to anti-GM-CSF autoantibodies –68. MoniAnti-cytokine AAbs causing immunodeficiency or dysregulation now form a distinct group of PIDs that share phenotypic characteristics with PIDs that occur due to pathogenic variants in genes encoding proteins involved in cytokine signaling pathways. As the management of patients with anti-cytokine AAbs differs from those with genetically disrupted cytokine signaling pathways, laboratory analysis for these AAbs is essential to enable not just improved phenotyping of these patients, but also to provide diagnostic and immune monitoring tools.The neutralizing capability of anti-cytokine AAbs can be assessed by the analysis of the phosphorylation status of signaling molecules downstream of the relevant cytokine receptor. Such assays should be combined with ligand binding assays such as ELISA to demonstrate the presence of an antibody that correlates with neutralizing activity. Since signaling molecules such as STAT1 and STAT5 are phosphorylated by several cytokines, it is necessary to demonstrate specificity of these assays by utilizing alternate recombinant cytokine controls where possible. These assays that demonstrate the neutralizing capability of anti-cytokine AAbs are of utility in immune monitoring, particularly to demonstrate recovery of these cytokine pathways if immune depletion of B cells or plasmapheresis is employed to remove the source of the autoantibody or the autoantibody itself.VK conceptualized and developed content for the manuscript. PM contributed to writing the manuscript. TL performed flow cytometry analysis of the AAb assays.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.


The translation of the navicular bone is thought to be a representative surrogate measure to assess foot pronation and hence foot function; however, it is not known how it is related to multi-segment foot kinematics.Cranio-caudal (NCC) and medio-lateral (NML) navicular translation and multi-segment foot kinematics from the Oxford Foot Model (OFM) were simultaneously assessed during the stance phase of walking in 20 healthy adults. Relationships to forefoot to hindfoot (FFtoHF), hindfoot to tibia (HFtoTBA) and global hindfoot (HFL) motion were explored by cross-correlations at zero phase shift.FFtoHF sagittal, transversal and frontal plane angles showed median cross correlations of -0.95, 0.82 and 0.53 with NCC and of 0.78, -0.81 and -0.90 with NML. HFtoTBA transversal and frontal plane angles had correlations of 0.15 and 0.74 with NCC and of -0.38 and -0.83 with NML. The HFL frontal plane angle showed correlations of 0.41 and -0.44 with NCC and NML, respectively.The strongest relationships were found between FFtoHF sagittal plane angles and NCC and between FFtoHF frontal plane angles and NML. However, cranio-caudal and medio-lateral navicular translation seem to be reasonable surrogates for the triplanar motion between the fore- and hindfoot. The medial longitudinal arch dropped and bulged medially, while the forefoot dorsiflexed, abducted and everted with respect to the hindfoot and vice-versa. The lower cross-correlation coefficients between the rear foot parameters and NCC/NML indicated no distinct relationships between rearfoot frontal plane and midfoot kinematics. The validity of rearfoot parameters, like Achilles tendon or Calcaneal angle, to assess midfoot function must be therefore questioned. The study could also not confirm a systematic relationship between midfoot kinematics and the internal/external rotation between the hindfoot and the tibia. The measurement of navicular translation is suggested as an alternative to more complex multi-segment foot models to assess foot function. The human foot represents a complex biomechanical system  consistiThe cross-sectional study was carried out on active and healthy adults  who were free from any musculoskeletal complaints or acute infections, had had no surgery on the lower extremities during the previous 12 months and did not perform any intensive training session the day before the examination. All participants signed an informed consent and ethics approval for the study was retrieved from the Ethics Committee of the Canton of Bern (KEK-No. 052/15). Twenty-two participants were included and examined but only the data from 20 persons were eligible for further analysis due to data acquisition errors in two cases. One subject was additionally excluded from further statistical analysis in addition because this person presented an atypical and non-representative walking pattern, characterized by making initial contact with the midfoot and a lacking heel rocker phase . Subjets3). Ground reaction forces were measured with a force plate  and served for initial contact and toe off identification (threshold at 25 N) to subsequently extract the kinematics during stance. While the participants were walking at self-selected speed, the kinematics of the right foot were simultaneously captured by a set of four markers to quantify the navicular mobility in terms of cranio-caudal (NCC) and medio-lateral translation (NML) of the navicular tuberosity (www.c3d.org) and the btk-Toolkit [Foot kinematics were measured using a three-dimensional motion capture system with 10 cameras  that provided a measurement volume of (4 x 1.5 x 1.5) mberosity  17] and and3). Gberosity . The foo-Toolkit  were useXc) [xcorr from Matlab’s Signal Processing Toolbox . In accordance with Pohl et al. [Data analysis was carried out in Matlab and included the extraction and normalization of stance phase time-series  and averaging among the trials from each participant. Between 6 and 15 walking trials from each participant were averaged to retain representative stance phase kinematics for further analysis. An analysis was made of how the two navicular mobility measures related to each other and to the forefoot to hindfoot (FFtoHF), the hindfoot to tibia (HFtoTBA) and the hindfoot to laboratory (HFL) angles. Translational  and rotational  displacements from initial contact to toe off were considered in the analysis. The following relationships were explored further qualitatively by displacement-displacement plots and quantitatively by cross-correlation coefficients at zero phase shift % stance phase which corresponded well with the initiation of heel lifting . When interpreting cross-correlation coefficients, which are in fact Pearson correlations in function of a phase shift between the two vectors under consideration, it must be born in mind that they are sensitive to the range of the vector values. Hence, larger cross-correlation coefficients might results from increased ranges of values rather than actually more similarity of curve shapes. In addition, the interpretation of the strength of relationships is dependent from the cross-correlation levels used for classification. Since cross-correlation coefficients are relative estimates on how much of the variance in a dependent variable can be explained by an independent variable, there is no absolute rule from when on a relationship can be said to be strong. However, we set this limit at 0.8 and are more restrictive in this respect than previous examinations . LimitatCranio-caudal and medio-lateral navicular translation seem to be reasonable surrogates for the triplanar motion between the fore- and hindfoot. However, frontal-plane motion seems only to be moderately represented by the cranio-caudal navicular translation. The strengths of cross-correlations indicate that a flattening and rising of the medial longitudinal arch  is primary captured by the cranio-caudal navicular translation and that the forefoot-hindfoot twist  is primary captured by the medio-lateral navicular translation. Cranio-caudal and medio-lateral translation therefore seem to primary measure different aspects of foot function and should be evaluated separately. This statement is also supported by the NCC-NML relationship which was only found to be at the transition from moderate to strong. The relationship between navicular translation and hindfoot motion could not have been confirmed and therefore the Achilles tendon angle and the Calcaneal angle or calcaneal eversion excursion do not seem to be appropriate parameters to assess foot pronation. It is suggested that there is no legitimation to assess midfoot function by measurements at the hindfoot. Similar experiments for other modes of gait like running or stair walking might provide further insights into the relationship between navicular mobility and multi-segment foot kinematics. Taking foot type or symptomatic subgroups into account could strengthen the value of a follow-up study and contribute to a differentiated understanding of foot biomechanics and dysfunction. The approach with a minimal set of four markers provides potential for less complex 3D foot function assessment during dynamic tasks.S1 FigMeans and standard deviations among participants (N = 19) of anterior-posterior (AP) and vertical ground reaction forces  normalized to the body weight force (BWF) and global plantar-/dorsiflexion angle of the foot (FPitch). The vertical lines indicate the transition of the AP ground reaction force from a posterior to an anterior direction (55 ± 2.7% stance phase) which was used to define the power absorption and power generation phases. This transition corresponds to the instance in time where lifting the heel from the ground was initiated.(PDF)Click here for additional data file.S1 DataNCC, NML, FPitch and the GRF  are provided in .mat files (Matlab). NCC and NML are denoted as NEle and NDev, respectively, in the data files. FPitch denotes the global plantar-/dorsiflexion angle Click here for additional data file.S2 DataOxford Foot Model outputs are provided in .mat files (Matlab). One file with all steps under consideration is provided for each participant.(ZIP)Click here for additional data file.


Over the decades the Smiles rearrangement and its variants have become essential synthetic tools in modern synthetic organic chemistry. In this mini-review we summarized some very recent results of the radical version of these rearrangements. The selected examples illustrate the synthetic power of this approach, especially if it is incorporated into a domino process, for the preparation of polyfunctionalized complex molecules. Single-electron reduction of the substrate affords the expected difluoroethyl radical that undergoes a consecutive ipso-addition—SO2 elimination—H atom abstraction process to provide the desired product.In the last two decades increased attention was focused on light-induced chemical transformations, especially in the field of radical chemistry ,19. UV o1 used for the treatment of depression and/or obesity  was proposed by Zard, as illustrated by 2% yield .N-(arylsulfonyl)acrylamides and subsequent radical Smiles rearrangements. In a recent work they demonstrated the synthetic utility of in-situ generated amidyl radical intermediates in additional C-C or C-heteroatom bond forming reactions affording remarkable molecular complexity isoquinolin-6(5H)-ones of potential biological interest acrylamide moiety evidenced the preparative efficiency of this multi-step radical reaction cascade.A systematic substrate scope study concerning the radical sources as well as each part of the ortho-vinyl- or ortho-styryl-substituted N-(arylsulfonyl)arylamide substrates were reacted with carbon or heteroatom centered radicals a multistep radical domino process takes place to afford multifunctionalized indane- and dibenzocycloheptadiene-type products in a stereoselective manner (trans relative configuration between amide and phosphonyl or azide functions) . Th. Thcoll.N-centered radical 19. Addition of this latter to the triple bond and SO2 extrusion led to the phthalazine radical 20. The diarylmethyl anion 21 obtained by reduction of 20 was protonated (or deuterated in CD3OD) to afford the final product 18.Mechanistic investigations  revealedDuring recent years, radical chemistry has played an important role in the development of modern synthetic methodologies, especially for the preparation of highly complex and polycyclic molecular frameworks. This mini-review demonstrates again that the radical Smiles rearrangement is now a well established and powerful addition to the synthetic chemistry toolbox and we are convinced that the selected examples will meet with the interest of researchers who have already been or will be engaged in this research topic.


The10-day EMDR intensive daily treatment (EMDR therapy twice a day for 10 days) produced a similar outcome as to that of the weekly treatment with a 1-year follow-up. Results support the effectiveness of EMDR therapy when offered in both weekly treatment format as well as the intensive 10-day format on an outpatient basis. While recognizing the limitations of this study the results are significant to warrant additional research.The effectiveness of EMDR therapy in treating veterans diagnosed with PTSD was evaluated in this study using two treatment formats: intensive daily EMDR treatment provided twice a day during a 10-day period and a second format of one session each week. The study used archived outcome data previously collected and stored at Soldier Center. Both formats provided 18–20 treatment sessions of EMDR therapy to veterans diagnosed with PTSD that included dissociative exhibitions and moral injury issues. Questions addressed included: (1) does EMDR therapy administered twice daily ameliorate veterans’ PTSD symptoms; (2) does EMDR therapy administered twice daily provide equivalent outcome results as EMDR therapy administered weekly for 18–20 sessions; and (3) does the treatment outcome persist. The effectiveness of the weekly treatment group was also evaluated. Both groups’ results were assessed at pre-treatment, post-treatment and 1-year follow-up. The results indicated that both weekly treatment and intensive daily treatment groups produced statistically significant treatment effects ( With the United States and its allies currently fighting the longest war in its history, a record number of veterans with PTSD diagnoses continues to be reported by the Department of Veterans Affairs . The tolThree decades of research have resulted in 30 randomized controlled trials (RCT) attesting to the efficacy of EMDR therapy in the treatment of PTSD. The Practice Guidelines of the While there has been limited evaluation of EMDR therapy in the treatment of veterans, since 2003 Soldier Center, a community-based outpatient treatment facility, has provided EMDR therapy to veterans. Treatment was initially offered to all clients at Soldier Center on a weekly basis. In 2005, the first intensive treatment, providing EMDR therapy twice a day for 5–10 days, was offered to veterans. This program evaluation study provides a retrospective evaluation of the data regarding both weekly and daily treatment regimes.Research data used in this program evaluation study was regularly collected and archived at Soldier Center as a means of evaluating treatment efficacy. Pre- and post-treatment measures with follow-up were routinely collected from all clients and evaluated to insure the effective treatment of clients. Participants were informed of the nature and purpose of gathering treatment outcome measures noting the use of the collected data would assist in providing effective of other veterans in the future. The importance of the veterans’ consent was stressed as permission to use the collected data in published research studies was verbally requested and received from the veterans. Participants were assured their treatment would not be influenced by whether or not they participated in data collection. The risk associated with treatment were provided as part of the informed consent form. Veterans were assured that their confidentiality would be maintained throughout the archival and evaluation process.Data on 30 veterans’ treatment was collected and divided into two groups of 15 participants. one group was treated with EMDR therapy daily, twice a day over a period of 10 days. The other group was composed of data from veterans treated weekly. Both groups were treated a total of 18–20 sessions. The weekly therapy group consisted of veterans previously treated at veteran centers and local soldiers referred by the military Tricare office for their PTSD symptomatology. Once the data was retrieved, it included in an Excel PTSD spreadsheet containing the names, age, treatment dates, number of sessions and data from the PTSD Checklist (PCL-M/PCL-5), Impact of Events Scale-Revised (IES-R), and the Dissociative Experiences Scale-II (DES-II). The research questions were formulated as: (1) does EMDR therapy administered twice daily ameliorate veterans’ PTSD symptoms; (2) does EMDR therapy administered twice daily provide equivalent outcome results as EMDR therapy administered weekly for 18–20 sessions; and (3) does the treatment outcome persist.Selection criteria limited participants to persons meeting diagnostic criteria for PTSD with no co-occurring disorders such as active addictions, chronic depression or suicidal/ homicidal intent. Persons were stable enough to self-regulate during the early history-taking and preparation phases of therapy. The first clients in the intensive daily group were active duty soldiers referred from Army Warrior Transition Units (WTU)s for an intensive 5-day treatment period. All participants demonstrated symptoms of complex PTSD.According to state legislation and published ethical guidelines, an ethics committee does not need to review the data and approve an exemption for obtaining written informed consent in the use the archival data used in this study. Participants were informed that their treatment would not be influenced by either participation or non-participation in data collection. Accumulated data were gathered based on the clients’ willing participation in providing three-point  data collection. Verbal information was provided regarding how the data would be used in the future to assist Soldier Center in evaluating and improving effective treatment to veterans. The treatment clients received was considered standard care. Fidelity checks, insuring standardized treatment, was conducted on all EMDR therapists by each therapist presenting videos of their psychotherapy sessions to the executive director. The same therapists provided treatment to both the intensive daily treatment and the weekly treatment groups. All active duty soldiers were authorized for PTSD treatment by Tricare. Data were collected by each providing therapist.Supplementary Material section. All participants completed a three-point assessment with pretreatment and post-treatment evaluations completed during their scheduled sessions. Response at follow-up differed with the two treatment groups. The 1-year follow-up evaluation return rate from members of the intensive daily group increased from an initial 28% when the entire packet (listed under measures) was sent and increased to 62% when only the IES-R was sent. In comparison, there was an initial response rate of 36% among the weekly participants at 1- year follow-up. The response increased to 58% when the completed IES-R measure was requested at 1-year follow-up. Dates for the intensive daily group covered the period of 2008–2016. Dates of treatment for the weekly treatment group occurred during an 18 month period, June 2015 through December 2016.A description of the clinical protocol in this study is included under the EMDR treatment was provided to the two groups, the intensive daily group receiving EMDR therapy twice each day for 10 days, and the weekly treatment group. The weekly therapy group consisted of veterans previously treated at veteran centers and local soldiers. Both groups were treated with the EMDR therapy standard protocol. The EMDR intensive successive days therapy model was established in 2005 designed to provide 50 min sessions twice a day for 5 days, with a minimum of 3 h between each session. Fifty-minute sessions were extended to 60 min as needed. Participants in the daily intensive group traveled from out of state. They frequently bore physical wounds from combat as well as psychological distress. Prior to being referred to Soldier Center for EMDR treatment, most of the participants in the daily intensive program had been treated, with varying degrees of success, by means of other models of psychotherapy. Early referrals came from Army Warrior Transitions Units (WTUs) with clients who had completed one and sometimes two combat tours of duty during Operation Iraqi Freedom (OIF) and Operation Enduring Freedom (OEF). As veterans experienced additional deployments the number of traumatic memories increased, producing greater symptom pathology. The daily treatment was expanded to 10 days to treat the expanding number of traumatic memories accompanied by increasing symptom severity. This allowed for the treatment of additional disturbing memories while the veteran was already in the area.The daily intensive treatment allowed for the most distressing memories to be treated during the morning session with the afternoon session used to complete any residual material remaining from the earlier session while insuring the client finished the day with adequate stabilization and calmness. This approach provided for any residual traumatic material to be addressed within a few hours of each previous session.Participants in the intensive daily treatment had been previously treated with other models of psychotherapy. Their personal motivation for treatment was assessed with respect to the impact of previous treatments on their expectations of current treatment outcomes.Although most acknowledged being skeptical that additional treatments would work, all were willing to try another one since they did not want to live with their PTSD symptoms. Response to previous treatments reportedly ranged from compliance to veterans being so upset at the end of sessions they would “go out and get drunk,” which enhanced their alcohol dependency.Other veterans reported prematurely dropping out of therapy to avoid the intensity of treatment. Some soldiers reported having completed treatment with other therapies ranging 2–4 years previously with disappointing results.Treatment for both groups, successive days and weekly, presented a symptom-focused, time- limited approach with the goal of achieving the most effective results in the limited time available. Participants in the weekly treatment group occasionally missed an appointment due to work and pressing personal requirement. Persons traveling from out of state for the 10-day intensive program were expected to proceed with the process with little if any interruption since they were at their location specifically for treatment. No appointments were missed by the successive days intensive group. Both the weekly and daily intensive groups were treated with the standard eight-phase protocol of EMDR therapy. Lodging was provided in military barracks for junior-ranking enlisted soldiers participating in the intensive treatment condition.Officers and non-commissioned officers (NCOs) were authorized by their military units to lodge at local hotels.Veterans in the intensive daily group were referred because previous psychotherapy models had demonstrated limited success in treating them. While all of the participants in the intensive daily group had been previously treated with other models of psychotherapy prior admittance to Soldier Center, this was the case for only four of the members of the weekly treatment group.The weekly group received 50–60 min sessions. In comparison, the successive days sessions during the 10-day intensive format lasted 75 min provided twice daily. The difference in session time between treatment groups was due to the intensive treatment group using an extended closing period to insure the client’s self-regulation and stabilization at the end of each intense session. Weekly group participants lived locally and presumably benefited from their already established support networks.All participants in the intensive daily treatment group had been screened and diagnosed with PTSD by mental health providers prior to their referral to Soldier Center. Four of the 15 participants in the weekly treatment had received a previous diagnosis of PTSD by referring agencies, along with prior treatment, before their referral to Soldier Center. Participants completed the evaluation process with an initial intake appointment. All met the diagnostic criteria for PTSD as outlined by the Diagnostic and Statistical Manual- IV  and the Selection required all participants to have completed treatment and submitted a follow- up evaluation.The PTSD Checklist (PCL-M) was used to validate the participants’ previous PTSD diagnosis made by their referring agencies . At the Change in the Diagnostic and Statistical Manual-IV (DSM-IV) to the Diagnostic and Statistical Manual-V (DSM-5), required a change in the PTSD Checklist with the introduction of the PCL-5. However, as the National Center for PTSD discovered, “PCL-5 scores are not compatible with PCL for DSM-IV scores and cannot be used interchangeably” ,b, p. 1.During pre- and post-treatment, the Beck inventories  measuredDissociative exhibitions, reflecting the participant’s ability to be mentally present and consciously focused, were measured by the Dissociative Experiences Scale (DES). All participants were assessed at three points during treatment at Soldier Center: pre- and post-treatment and 1-year follow-up. The initial intake occurred on arrival at Soldier Center and the second at the completion of the treatment plan. One year later a third assessment was conducted. Some treatment participants maintained occasional contact with the center following treatment while most assimilated into a routine at home with no contact with Soldier Center staff.Previous published research indicate that EMDR therapy can resolve a single-incident trauma memory in three to-six sessions 77–100% of the time , 2004, wBoth treatment formats incorporated the standard EMDR eight-phase approach , 2018. TMany military active duty candidates for treatment in the intensive daily program were referred by a mental health manager at a military installation’s Warrior Transition Unit (WTU). Other participants were veterans discharged from active duty military who had been in treatment programs provided by the Department of Veterans Affairs and who learned of the EMDR therapy intensive treatment program. Initial inquiry was made by the case manager or sometimes by the veteran. Summaries of previous treatments were provided by the referring agency. The veteran was then given an assessment PTSD packet consisting of the PCL- M/PCL-5, IES-R, BAI, BDI, and DES-II. Participation was limited to persons who met the diagnostic criteria for PTSD. When active addiction was noted, the candidate was required to have been clean and sober for 6 months, with no active use at the time of the intensive treatment. While EMDR therapy protocols have been successively used in the treatment of addictions, the intensive daily treatment protocol does not allow time to treat both addictions and PTSD-related issues.Admission into the intensive daily treatment program included a 50-min phone interview between the veteran client and the EMDR therapist. Discussion points included the veteran’s reason for seeking the intensive treatment, their motivation for treatment/change, any secondary gain issues, presenting symptoms, suicidality concerns, any dissociative features such as flashbacks, night terrors/nightmares, number of deployments, other challenging life events (beginning with childhood), relationship with parents and others, social activities and networks, who they can count on, any addiction history, moral injury issues, legal problems, significant losses, anniversary dates with mood changes, social and intra-personal stability, what they do to relax. Key points in each interview included the veteran’s motivation for change and ability to self-regulate when distressed. Persons unable to self-regulate were encouraged to do additional work with their local therapist to enhance self-regulation skills prior to coming for treatment.Upon arrival at Soldier Center, veterans participating in the intensive daily program met with the designated EMDR therapist to begin the initial intake process. The first session was devoted to the History taking (Phase 1) with the client. This phase consists of a broad psychosocial interview in which the therapist learns what life has been like for the client. With a time-limited, symptom-focused approach, identifying the client’s goal(s) in seeking treatment as well as the presenting symptoms were noted. Introduction of the treatment plan addressed how treating specific disturbing memories can result in the resolution of the identified symptoms in accordance with the AIP theory underpinning EMDR therapy. The History- taking session culminated with two lists: a list of positive achievements in the person’s life, known as the “resource list,” and a list of disturbing life events which, when triggered, generated client disturbance. The list of distressing life events formed the basis from which the treatment plan was developed. It identified intrusive memories of past disturbing events that continued to interfere with the participant’s life. Additionally, current triggers and the apprehension of managing future events were included in the plan. The number of sessions required to treat each disturbing memory varied with each person, usually between one and three sessions per memory. The participant’s sense of security was continually addressed as effective treatment of PTSD requires the veteran to feel secure enough to address the vulnerability of recalling memories that are normally avoided.Typically, in the second session of intensive daily treatment each veteran demonstrated the ability to relax or self-regulate by means of various relaxation techniques. The participant was introduced to the basic mechanics of EMDR treatment, which included bilateral stimulation , the introduction of a stop signal (should the client need to terminate the process during a session), and a relaxation exercise with a cue word.With the completion of phases 1 (History taking) and 2 (Preparation), the veteran began treatment of the disturbing memories that were associated with the presenting symptoms. Each targeted disturbing memory in the treatment plan was treated with phases 3–8. For each veteran in the 10-day intensive daily treatment program, 6–10 disturbing memories were treated.Table 1 demonstrates the significance in treatment outcome scores as measured by the IES-R. Descriptive statistics of scores collected at three time-points from each treatment group are reported. As can be seen in the table, the Intensive Daily group mean score at IES-R Pre-test was 53.2, which declined to a mean of 17.4 at the IES Post-test; the Follow-up IES score further reduced to 15.6. The standard deviations were 11.321 at IES Pre-test, 13.092 at IES Post-test and 6.967 at IES follow-up. The Weekly treatment group mean score at IES Pre-test was 51.8, which declined to a mean of 16.1 at the IES Post-test; the follow-up IES score increased to 17.7. The standard deviation was 8.326 at IES Pre-test, 6.923 at IES Post-test and 5.574 at IES follow-up. Variances between the two treatment groups were found to be approximately equal, with a significance level of 0.121. The mean scores between the groups were not significantly different, at 0.703 (Table 2).This study posed the questions: (1) does EMDR therapy administered twice a day have a positive effect on the veteran’s PTSD symptoms as measured by IES-R scores; (2) does EMDR therapy administered twice daily provide equivalent outcome results as EMDR therapy administered weekly for 18–20 sessions, as measured by the IES-R; and (3) does the treatment outcome last. Table 3) with respect to pre-, post- and follow-up IES scores. For the Intensive Daily treatment group, the effectiveness of the treatment was demonstrated by the difference in IES scores among 3 time points, with statistically significant results . Post hoc evaluation of the group differences revealed a significant difference between Pre- and Post-test IES scores, while the Post-test IES and Follow-up IES scores were not significantly different (Tables 4). This outcome was due to the improved scores measured at Post-test remaining only slightly changed at Follow-up.Two repeated measures ANOVAs were employed to compare the two independent groups . Post hoc evaluation of the group differences revealed a significant difference between pre- and post-test IES-R scores, while the post-test IES-R and Follow-up IES-R were not significantly different (Tables 6).The difference in the Weekly groups with regard to the IES-R scores among the 3-time points was found to be significant as well  for PTSD at post-test. These results correlated with the PCL-M/PCL-5 scores of the respective veterans. Additionally, the non-significant difference between post-test and follow-up IES scores indicates that the treatments were also effective in the long-term because the participants were able to retain their treatment gains.To examine whether one treatment had a greater impact on the participants than the other, F statistics and their effect sizes were also compared. In this study, the following formula was used to calculate effect size for the repeated measures ANOVA:where SSw is the sum of squares for the within-subject effect, and SSe is the sum of squares of the total error variance.F-value as well as effect size, and therefore in the current study, the findings suggest that the Weekly treatment had a greater impact on participants with PTSD, while the Intensive Daily treatment demonstrated a slightly greater effect within a limited period of time, an effect that was maintained at 1-year follow- up.The effect size for the Successive Day treatment group is 13493.2/18366.8 = 0.735, and the effect size for the Weekly treatment group is 12200.9/14277.2 = 0.855. Both effect sizes were large. However, the Weekly treatment group evidenced a larger This three-point assessment of two EMDR treatment groups suggest EMDR therapy, when administered twice daily during a 10-day period, produced significant improvement in symptom reduction as measured by the IES-R. The same conclusion was supported when evaluating weekly treatment with 18–20 sessions over a 5–6-month period. Both the intensive daily group and weekly treatment group have significant outcome results at post-test, with results being maintained at 1-year follow-up.These findings indicate that EMDR intensive daily treatment may offer an accelerated treatment model for other therapy programs. As previously noted, all members of the intensive daily group had been treated for PTSD prior to their referral to Soldier Center. Three members of the intensive daily group had previously been treated with EMDR therapy unsuccessfully. Veterans with previous unsuccessful treatment experiences improved significantly in this treatment program. Effective treatment occurred with a change of therapists and treatment centers. The explanation for this observation is unclear. One the one hand, therapists in the current study demonstrated satisfactory treatment fidelity through the monitoring of their videotaped sessions. A meta-analysis of EMDR therapy research  reportedThis study is limited by its sample size, with only two groups of 15 members in each condition evaluated. Future RCT research is needed with a larger sample size. However, the study does evaluate a select group of veterans whose multiple traumas, complex PTSD, and moral injury issues required 18–20 sessions for treatment. This exceeds the number of sessions normally required for the successful treatment of single-incident trauma and adverse life experiences. Future research should more rigorously examine this population.An evaluation of each treatment format found that the Intensive Daily treatment and Weekly treatment conditions were equally effective, with a substantial decrease in symptoms and all of their participants losing their PTSD diagnosis. The intensive daily treatment format provided a rapid resolution of trauma with persons stable enough to self-regulate, while addressing their disturbing memories during a 10-day period. Similar results were found with participants in the weekly treatment group. The intensive daily condition was able to accommodate veterans suffering from extended histories of trauma such as persons serving in critical medical care positions who experienced up to 14 years of military and medical trauma during OIF/OEF assignments. In such cases the 10-day period was not sufficient to resolve all of their traumatic memories, but their major trauma memories were treated as one component of an expanded treatment plan designed to achieve an accelerated healing process of the primary trauma memories before returning home to continue weekly treatment with their local EMDR therapist. However, in all cases they no longer retained the diagnosis of PTSD after the 10 days of treatment. Further, no adverse experiences were precipitated by treatment in either condition. There was a 0% dropout rate in the intensive treatment group and 8% in the weekly group (due to relocation). These findings are consistent with those of a meta-analysis  reportinIt should be noted that all persons in this study completed their treatment plan in 18–20 sessions. Therapists operated from the principle: don’t open psychological wounds you do not have adequate time to resolve. In the beginning of Soldier Center’s intensive treatment program, the goal was to restore the veteran to a level of psychological functioning experienced prior to military service. It was common for a veteran to identify up to 10 combat traumas and one early life adverse event. If there were additional combat deployments with numerous traumatic memories, the goal was to identify and address those targeted disturbing memories that would provide the most psychological healing within the limited period of treatment time. For some veterans this meant that all their traumatic memories could be treated in the designated period. Other veterans with multiple combat deployments identified those disturbing memories that could be treated within the intensive period and then returned to their home region where follow-up treatment was provided by a local EMDR therapist on a weekly basis.F = 105.21, p < 0.001). Post hoc evaluation of the group differences revealed a significant difference between pre- and post-test IES-R scores. The difference in scores between post-test and follow-up scores were not significant indicating the significant improvement if PTSD symptoms as measured by the IES-R at the end of the 10-day treatment. The twice daily treatment was found to be significant in ameliorating veterans’ PTSD symptoms.The research questions addressed in this study were: (1) does EMDR therapy administered twice daily ameliorate veterans’ PTSD symptoms; (2) does EMDR therapy administered twice daily provide equivalent outcome results as EMDR therapy administered weekly for 18–20 sessions; and, (3) does the treatment outcome persist? When comparing the pretreatment, post-treatment, and follow-up IES-R scores, two repeated measures ANOVA noted in the intensive daily group the difference on the three-points were significant (EMDR therapy provided approximately equal outcome results when reviewing treatment results of daily and weekly treatment. At post-test the intensive daily treatment mean changed from 53.20 to 17.40 at post-test and 15.60 at follow-up indicating the treatment change at post-test was maintained at the 1-year evaluation. The weekly treatment changed from the IES-R mean of 51.80 at pre-test to 16.07 at post-test and 17.73 at 1 year follow-up. With a variance of 1.027 between the groups at pre-test, the between group variance at post-test was 1.330. Follow-up 1 year later noted a variance of 2.130.Thte third question, does the treatment persist, was demonstrated at 1 year follow-up. Intensive daily treatment group improved from an IES-R mean of 17.40 at post-test to a mean score of 15.60 at 1 year follow-up. The weekly treatment group IES-R mean increased by 1.66 between post-treatment and 1 year follow-up. It can be stated, based on these findings that both treatments maintained their outcome significance at 1 year follow-up.The intensive daily format and the weekly treatment approach both offer benefits for participating veterans. The intensive program can be formatted for inpatient treatment in VA medical centers. Likewise, active duty personnel can be temporarily transferred to facilities with seasoned EMDR clinicians who can complete treatment within two weeks. The intensive treatment allows any reactivity that might surface to be immediately addressed. Additionally, the intensive program provides momentum in the treatment. Little time is needed for getting caught up with events between sessions since only a few hours elapse between sessions. The time saved due to this momentum can be applied to further treatment work.For some participants the weekly EMDR treatment provides a better fit for their time. They have difficulty getting away for a two-week period due to work or family responsibilities and finances. Weekly appointments allow for the emotional support of family and friends between sessions. The weekly treatment format provides time for psychological triggers to be identified between sessions and added to the treatment plan. This format also allows more time for trust and rapport to develop in the clinical relationship.The intensive daily10-day treatment program offers many possibilities for treatment centers. However, the likelihood of obtaining the same outcomes in other treatment settings is yet to be determined. Based on the findings of this study, additional research using EMDR therapy in an intensive successive-days process is warranted.An additional application of EMDR therapy in military personnel that merits further investigation is the treatment of phantom limb pain. An RCT  and multThe author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.