Oncolytic virotherapy has shown impressive results in preclinical studies and first promising therapeutic outcomes in clinical trials as well. Since viruses are known for a long time as excellent vaccination agents, oncolytic viruses are now designed as novel anticancer agents combining the aspect of lysis-dependent cytoreductive activity with concomitant induction of antitumoral immune responses. Antitumoral immune activation by oncolytic virus infection of tumor tissue comprises both, immediate effects of innate immunity and also adaptive responses for long lasting antitumoral activity, which is regarded as the most prominent challenge in clinical oncology. To date, the complex effects of a viral tumor infection on the tumor microenvironment and the consequences for the tumor-infiltrating immune cell compartment are poorly understood. However, there is more and more evidence that a tumor infection by an oncolytic virus opens up a number of options for further immunomodulating interventions such as systemic chemotherapy, generic immunostimulating strategies, dendritic cell-based vaccines, and antigenic libraries to further support clinical efficacy of oncolytic virotherapy. Oncolytic viruses are novel antitumor agents with the ability to selectively replicate and lyse tumor cells while sparing healthy tissue. This intriguing characteristic is either an inherent feature of certain virus species or a result of targeted genetic engineering, which harnesses tumor-specific molecular alterations for virus replication and tumor cell lysis . The idein situ vaccinative and immunostimulatory effect of virus infection. The GM-CSF-expressing oncolytic vaccinia virus JX-594 has shown promising results in phase I/II clinical studies in hepatocellular carcinoma . Since signs of autophagy also occur as a reversible process in the context of nutrient starvation, it is not completely clear whether autophagy is causative for cell death or is an epiphenomenon of other cell death triggers. However, autophagy plays a definitive role in triggering immune responses. Autophagic mechanisms are involved in the clearance of intracellular microbial or viral pathogens not only by intracellular digestion but also by improved processing of microbial/viral antigens for antigen presentation on MHC I as known for herpes simplex virus infections . AutophaAntigen-presenting cells such as dendritic cells (DC) fulfill a central role in triggering effective T cell responses in case of a pathogenic threat. Antigen-presenting cells are activated when encountering pathogen-derived structures, called PAMPs (pathogen-associated molecular patterns), which reflect conserved components of microbes and viruses. Classical PAMPs are microbial DNA with unmethylated CpG, defective viral genomes that occur during viral lysis, double stranded RNA, single stranded RNA, 5′-triphospate RNA, lipoproteins, surface glycoproteins, and bacterial membrane components such as LPS. PAMPs are recognized by pattern recognition receptors (PRRs) present on innate immune cells, antigen-presenting cells, and also on epithelial cells. PRR include toll-like receptors, retinoid acid inducible gene I (RIG-I)-like receptors (RLRs), AIM like receptors (ALRs), and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) . In 1994When cells succumb to necrosis, they also externalize and release the high mobility group box 1 (HMGB1) protein into the cellular environment, which is known for its proinflammatory properties . The relFurther, important DAMPs are released heat shock proteins, such as HSP70 and HSP90, and uric acid. Heat shock protein release has been demonstrated to play a role in induction of tumor-specific immune responses by the oncolytic parvovirus H1 . Uric acA further important mechanism that provides dying cells with an immunogenic signature is ER-stress. The ER is a central production site for proteins and membrane components involved in the secretory pathway. The ER is also an important sensor for ER-stress, a physiological reaction to dysbalanced protein synthesis, e.g., in the context of viral infections. Under homeostatic conditions, the luminal ER-stress sensors IRE1α, ATF6, and PERK are bound and silenced by the molecular chaperone Grp78/BiP. Once unfolded proteins accumulate in the ER due to an unphysiologic increase in protein synthesis, Grp78/BiP is competitively displaced from the ER-stress sensors leading to their subsequent activation for downstream induction of an unfolded protein response (UPR) . WhereasImmunogenic cell death is basically the first aspect in innate and adaptive immune effects that have been recognized as a central mode of action in virotherapy . The tumAside of neutrophils, macrophages and monocytes belong to the initial defense response by the innate immunity against pathogens. These populations are highly activated after viral infections, are capable of phagocytosis, support the professional antigen-presenting cells, and contribute to adaptive immunity. Within an intact tumor, secretion of immunosuppressive cytokines determines the phenotypic differentiation of these innate immune cells to adopt an immunosuppressive status to promote tumor progression and metastases . ConsequIn recent years, myeloid-derived suppressor cells (MDSC) population has been described as one of the most important immunosuppressive within the tumor microenvironment. These cells have been observed in primary tumors as well as in metastases of patients , 84. MyeAmong the cells of the innate immune system, NK cells play a crucial role in clearing viral infection and in fighting malignant cells. Trying to escape from adaptive immune responses by downregulation of MHC, virus-infected cells, and tumor cells become a natural target of NK cells. In line with a role of NK cells in immunoediting of tumors, tumor-infiltrating NK cells correlate with a favorable prognosis in humans . NK cellOn the other hand, several studies showed an antitumoral effect of NK cells after oncolytic viral treatment. Depletion studies with VSV in the B16 melanoma model revealed an NK cell and T cell dependent tumor regression . FurtherRegulatory CD4 T cells (Tregs) are an immunosuppressive cell population that has frequently been discussed as a critical contributor to the tumor microenvironment. It has been shown that the ratio of intratumoral cytotoxic T cells and Tregs is a prognostic factor for the patient’s outcome and studies using antibodies blocking CTLA-4 (which is expressed on Tregs) for increased immune activation have shown that Tregs can be interesting targets for immunotherapeutic approaches , 102. ThTo elicit enhanced immune stimulation, Treg depletion has therefore been considered a supportive measure during oncolytic virotherapy. Studies have shown that tumor preconditioning with IL-2 and Treg depletion using a depleting antibody or low-dose cyclophosphamide led to increased intratumoral uptake of systemically delivered reovirus or vesicular stomatitis virus. IL-2 in combination with Treg depletion generated “hyperactivated” NK cells with enhanced antitumoral activity and secreting factors that facilitated oncolytic virus spread throughout the tumor by disrupting the tumor architecture , 106. SuHowever, the consequences of Treg depletion on long-term antitumoral T cell responses that can be induced by oncolytic virotherapy are not clear. Observations in classical infection models have shown that migratory activity of Tregs plays a central role in eliciting a protective immunity to viral infection . Consist−/− mice ] cured established tumors after repetitive intravenous injections. The use of ASEL conferred significantly better protection against TC2 cells than a self-antigen library from normal mouse prostate tissue. Upon application of ASEL, a TNK cells . A subseNK cells . Virus-eTo date, identification of novel tumor-antigens that can be addressed by targeted therapeutics appears to be a crucial step toward the establishment of clinically effective immunotherapies and toward induction of sustained adaptive T cell responses. In the past, antitumoral vaccine research has focused on finding non-mutated, tumor-associated antigens such as telomerase or MAGE, which can be found either in a relevant numbers of patients and/or across several entities to promise broad applicability. Disappointingly, corresponding vaccination approaches have so far delivered insufficient effects in the clinic . A limitIn a pioneering study targeting the mutanome by vaccinations, NGS was used for immunoepitope identification in B16F10 melanoma cells. Selected from 563 non-synonymous SNV candidates, the immunogenicity of 50 validated mutations was determined using corresponding peptide immunizations in mice. The authors showed that immune responses could be raised against 60% of these epitopes and the vaccinations against these predicted and validated epitopes successfully raised antitumoral adaptive immune responses and significantly slowed tumor-growth . This il+ cells, natural killer cells, and type I IFNs. Overcoming systemic resistance to immune checkpoint blockade by oncolytic virotherapy moreover led to protection from tumor rechallenge in poorly immunogenic tumors, even in a cell line refractory to NDV-mediated lysis. An alternative to checkpoint blockade is the direct activation of costimulation using oncolytic viruses expressing the costimulatory CD40L (The recent clinical success of immune checkpoint blockade  has confry CD40L . Furtherry CD40L . Strong ry CD40L . An intery CD40L . PD-1/PDThere is increasing evidence that oncolytic virotherapy shows antitumoral efficacy in clinical application even as monotherapy. However, most preclinical data suggest that virotherapy can be ideally combined with other treatment options to raise significant therapeutic synergies on several levels (see also an overview in Figure 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.


We hypothesized that disruptions in neuroendocrine function in cocaine-dependent individuals would explain their impairments in fear recognition by modulating the relationship with the underlying gray matter networks. We found that cocaine-dependent individuals not only exhibited significant impairments in the recognition of fear, but also for facial expressions of anger. Although recognition accuracy of threatening expressions co-varied in all participants with distinctive gray matter networks implicated in fear and anger processing, in cocaine users it was less well predicted by these networks than in controls. The weaker brain-behavior relationships for threat processing were also mediated by distinctly different factors. Fear recognition impairments were influenced by variations in intelligence levels, whereas anger recognition impairments were associated with comorbid opiate dependence and related reduction in testosterone levels. We also observed an inverse relationship between testosterone levels and the duration of crack and opiate use. Our data provide novel insight into the neurobiological basis of abnormal threat processing in cocaine dependence, which may shed light on new opportunities facilitating the psychosocial integration of these patients.The ability to recognize facial expressions of emotion in others is a cornerstone of human interaction. Selective impairments in the recognition of facial expressions of fear have frequently been reported in chronic cocaine users, but the nature of these impairments remains poorly understood. We used the multivariate method of partial least squares and structural magnetic resonance imaging to identify gray matter brain networks that underlie facial affect processing in both cocaine-dependent ( It is conceivable that these impairments may predate stimulant dependence as adolescents with conduct disorder—who have an increased risk for developing drug addiction in adulthood7—exhibit impairments in recognizing negative facial affect, including fear.9 An alternative, but not mutually exclusive, possibility is that prolonged drug abuse impairs emotion processing abilities. However, prior studies failed to find a direct relationship between the duration of stimulant use and fear recognition impairments,10 suggesting that these relationships are more likely to be indirect.Facial expressions of emotion are important social signals of information, reflecting the emotional state of others and helping us to predict their behavior and adjust our responses accordingly.11 Cortisol directly influences autonomic neural systems associated with the processing of threatening information, such as those conveyed by fearful or angry facial expressions,14 while testosterone is thought to modulate the behavioral response to threat.16 Both hormones have mutually antagonistic actions—that is, cortisol attenuates the production of testosterone by suppressing HPG-activity, and testosterone reduces cortisol by inhibiting HPA-activity at the level of the hypothalamus.17 An imbalance between the HPA and HPG axes, as measured by peripheral levels of cortisol and testosterone, has been shown to mediate facial affect processing through their endocrine actions on neural activity in the amygdala and orbitofrontal cortex.23 These areas, along with the inferior occipital and fusiform gyri and the prefrontal cortex, make up the distributed network of cortical and subcortical brain structures that process facial affect.24 Thus, as both hormones can affect brain structure26 and functional connectivity during facial emotion recognition,28 it is conceivable that hormonal changes mediate threat processing through changes in these underlying structural brain networks. Indeed, decreased functional connectivity or damage to structures within this network have repeatedly been linked with impairments in emotion perception, including fear recognition.31Imbalance in the levels of steroidal hormones, such as cortisol and testosterone, may have a mediating role. Cortisol and testosterone are the end products of the hypothalamus–pituitary–adrenal (HPA) axis and the hypothalamus–pituitary–gonadal (HPG) axis, respectively, and together they regulate an individual's response to threat.32 as reflected by increased cortisol and decreased testosterone levels.35 However, studies of hormonal changes in human long-term cocaine users have been inconsistent.37 Using data-driven multivariate analysis methods, we sought to first determine the neural correlates associated with facial threat recognition, and subsequently investigate the influence of steroidal hormones on this brain-behavior relationship. We hypothesized that imbalances between HPA and HPG axes activity in cocaine-dependent individuals (CDIs) would explain impairments in recognizing fear via associated neuroanatomical networks.Preclinical evidence suggests that repeated use of cocaine enhances HPA axis activity and suppresses the HPG axis,38 criteria for cocaine dependence, whereas the remaining 30 individuals were healthy and without a personal and family history of drug abuse. Exclusion criteria for all volunteers included a lifetime history of a psychotic disorder, neurological illness or traumatic head injury, an autoimmune or metabolic disorder, or HIV-infection. All volunteers consented in writing before being screened for current psychiatric disorders using the Mini-International Neuropsychiatric Inventory.39 Psychopathology in drug users was further evaluated using the Structured Clinical Interview for DSM-IV.40 All participants completed the National Adult Reading Test41 to provide an estimate of verbal IQ, the State–Trait Anxiety Inventory42 to record temporary and stable feelings of anxiety, and the Beck Depression Inventory (BDI-II)43 to measure depressive mood. Before behavioral tasks, a blood sample was drawn in all participants to measure serum levels of cortisol and testosterone. Urine samples were tested for undeclared drugs; all samples provided by the healthy volunteers were negative and all except four urine samples provided by CDIs tested positive for stimulants. The protocol was approved by the National Research Ethics Committee ; data from both groups have been published previously.45Sixty-five male volunteers, aged between 20 and 60 years, were recruited from the local community. Thirty-five individuals had a history of chronic cocaine abuse, satisfying the DSM-IV-TRn=12) or injected (n=9) crack-cocaine, while the other half used powdered cocaine intranasally (n=14). Besides meeting the DSM-IV-TR criteria for cocaine dependence, several CDIs also met criteria for dependence on another substance  and used other drugs sporadically . Eleven CDIs were prescribed methadone (mean dose: 55 mg±16.2 s.d.), three were prescribed buprenorphine (mean dose: 3 mg±2.6 s.d.) and four received narcotic-like pain relief medication on prescription. Three CDIs were prescribed antidepressants, three were prescribed benzodiazepines and one received d-amphetamines on prescription. None of the healthy volunteers satisfied criteria for alcohol abuse or dependence, nor were they taking prescribed or illicit drugs on a regular basis. Seventy percent of healthy volunteers were either past or current smokers of tobacco, and 57% reported having social experiences with cannabis.All CDIs were non-treatment seeking and had been actively using cocaine/crack-cocaine for an average of 17 years (±8.0 s.d.). Half of the CDIs smoked . Faces were shown for a maximum of 5 s, and participants were asked to judge by button-press which of the six emotions each face was expressing. Participants had no time limit for their judgments and received no feedback about the accuracy of their responses. Task performance was reflected by the percentage of correct responses for each emotion.All participants first completed the Benton Facial Recognition Test,www.cuh.org.uk), where serum levels of cortisol and testosterone were determined using competitive chemiluminescent immunoassays on the Siemens ADVIA Centaur Immunoassay System. Participants then underwent a whole-brain T1-weighted magnetic resonance (MR) scan at the Wolfson Brain Imaging Centre, University of Cambridge, UK. The MR images were acquired using a Siemens TIM-Trio 3T system with a magnetization-prepared rapid acquisition gradient-echo sequence . Out of the final sample of 58 participants, three MR imaging scans  were of poor quality and therefore not included in the analysis.After completion of the behavioral tasks (between 1200 and 1300 hours GMT), blood samples were drawn from each participant and sent to the Department of Pathology at Addenbrooke's Hospital (n=4) were also excluded to avoid potentially confounding effects of drug abstinence, leaving a final sample of 58 participants . Data were analyzed using the Statistical Package for the Social Sciences . Group differences regarding demographic, behavioral and endocrine measures were analyzed using independent samples t-tests. Performance on the Hexagon task was analyzed using multivariate analysis of variance to take into account the relationships between the six emotions. We initially included verbal IQ, depressive mood and trait-anxiety as covariates in the analysis to statistically control for group differences in these variables, but all variables interacted with group status, suggesting that they are defining features of these groups. Consequently, co-varying for them would not be appropriate.48 To determine potentially confounding effects of the aforementioned demographic variables and the influence that comorbidity had on the results, we performed a two-step hierarchical regression model. In step 1, we included dependence status as three separate dichotomous variables  and then added the continuous variables verbal IQ, depressive mood and trait-anxiety to the model in step 2. Significant results of the regression model were tested for mediator effects using the process software tool (version 2.13) by Andrew F. Hayes (www.afhayes.com), which was implemented in SPSS. We also calculated Pearson's correlation coefficients to evaluate putative relationships between outcome variables and drug-taking measures. All statistical tests were two-tailed and significance levels were set at 0.05.Three participants' behavioral data were lost due to technical failure . CDIs without a stimulant-positive urine screen . Non-brain tissues were removed using the brain extraction tool of FSL and tissue-type segmentation was performed using FAST. The resulting gray matter partial-volume images were aligned to MNI-152 standard space using the affine registration tool FLIRT, followed by nonlinear registration using FNIRT, which uses a b-spline representation of the registration warp field. A study-specific gray matter template was made to which the native gray matter images were nonlinearly re-registered. To correct for local expansion or contraction, the registered partial-volume images were modulated by division with the Jacobian warp field. The modulated segmented images were then smoothed with an isotropic Gaussian kernel with full width half maximum=2.3 mm. Group comparisons of the gray matter maps were performed using CamBA software for permutation testing,49 v.2.3.0 (http://www.bmu.psychiatry.cam.ac.uk/software/) and thresholded at cluster-level statistics of η=1 error clusters per image.Gray matter density maps were analyzed using FSL-VBM (50 to determine covariance between brain regions (voxels) across the entire brain and successful task performance (recognition accuracy). For the calculation of the correlation maps, we only included brain regions where the mean gray matter density over all participants was >0.01. We determined the overall strength of each correlation map by calculating the root-mean-squared correlation coefficient over the entire brain.51 The significance of the correlation pattern was then tested using permutation methods—that is, the input images were randomly re-labeled and the root-mean-squared correlation was re-calculated to generate a null distribution for the hypothesis .We used partial least squares methods51 and multiple stepwise regression was used to determine the influence of testosterone and cortisol levels on the brain scores for fear and anger recognition. We then conducted bootstrap-mediation analysis restricted to the significant results of the regression analysis using the process software tool  implemented in SPSS.In partial least squares method, each voxel in the correlation map is given a numerical weight  reflecting how strongly that voxel is related to the behavioral variable of interest. Salience can be either positive or negative depending on whether the voxel shows a positive or negative relationship with the pattern identified. Partial least squares method also computes a brain score that reflects the strength of each participant's brain-behavior relationship. As CDIs exhibited recognition impairments for both fearful and angry facial expressions, we investigated both expressions separately. Brain scores were imported into SPSS for statistical group comparisons,t56=1.12, P=0.247), systolic  and diastolic blood pressure , indicating that the CDIs were not acutely intoxicated.Descriptive data is shown in P>0.1). However, significantly lower levels of testosterone were found in CDIs with comorbid opiate dependence compared with their non-opiate-dependent counterparts , suggesting that the low levels of testosterone observed in the cocaine group may have been driven, at least in part, by the low testosterone levels in CDIs with comorbid opiate dependence.No differences were observed between subgroups of CDIs with comorbid opiate or alcohol dependence regarding demographics, intelligence, anxiety, mood, physiological measures and duration or compulsive pattern of stimulant use , confirming no significant group differences in facial discrimination ability. However, one participant in each group (<4% of the entire sample) scored outside the normal range; this is in keeping with predictions that ~8% of normal individuals without a known pathology underperform on this test.52 To ensure that our findings were not confounded by deficits in facial perception, all analyses were repeated after removal of these two individuals.Compared with their healthy peers, CDIs performed equally well in matching the unfamiliar faces  but also angry  facial expressions. On closer inspection, we noticed that CDIs misclassified expressions of fear significantly more often than control volunteers as expressions of either sadness (Fisher's exact P=0.032) or surprise (Fisher's exact P=0.070). No systematic misclassifications were observed for angry faces. The two groups did not differ with respect to time needed for classifications  accounted for a quarter of the variance of fear recognition , and the inclusion of demographic variables to the model  explained an additional 13% of the variance . As can be seen from β=−0.37; P=0.023), but this effect did not survive when verbal IQ  was included in the model at step 2, suggesting that the significant direct effect of cocaine dependence was mediated by verbal IQ. Drug-dependency also had a significant effect on the recognition of anger , explaining 38% of the variance . When demographic variables were added to the model , they explained an additional 11% of the variance . As shown in β=−0.60; P<0.001) and this effect survived when verbal IQ  was included in the model, suggesting that opiate dependence had a critical role in the observed impairments in anger recognition. Indeed, when opiate-dependent patients were excluded from the sample, the groups no longer differed in terms of anger recognition  but remained significantly different for fear recognition .Hierarchical regression revealed that drug dependency (P=0.035) and anger (P<0.001). The networks for fear and anger recognition both encompassed distributed clusters across both hemispheres in the cingulum, inferior orbitofrontal gyrus, middle frontal gyrus, middle and superior temporal cortices, and fusiform gyrus, as well as parietal regions and subcortical structures including the amygdala and the hippocampus  and anger  compared with control volunteers , explaining a quarter of the variance . Inclusion of demographic variables to the model  accounted for a further 12% of the variance . Although cocaine dependence significantly predicted the brain score of fear , this effect did not survive when verbal IQ  was added to the model in step 2  became nonsignificant  when IQ  was included in the model , which accounted for 30% of the variance . As shown in β=−0.516, P=0.001), and the inclusion of demographic variables  did not explain any further variance .With regard to the brain score of anger, there was also a significant effect of dependence ; cortisol had no explanatory value. We subsequently discovered significant group-by-testosterone interactions on variations in the anger brain score . A mediation analysis confirmed that the direct effect of cocaine on testosterone was significant , as were the direct effects of both testosterone  and cocaine dependence  on the anger brain score, respectively. However, when testosterone was added to the model, the direct effect of cocaine dependence was no longer significant , but not with duration of powdered cocaine or opiate use (both P>0.5). Testosterone levels were also inversely correlated with the duration of crack  and opiate use , but not with the use of powdered cocaine . No relationships were identified with regard to fearful face recognition brain score or cortisol levels.Brain scores related to the recognition of angry faces in CDIs were significantly inversely correlated with duration of crack use  intellectual ability,62 revealing a potential relationship between IQ and fear recognition in CDIs. Given that stimulant drugs have been shown to enhance facial emotion recognition,63 it is tempting to speculate whether fear recognition impairments might impact the risk of cocaine abuse in individuals with low IQ. The relationship between intelligence and cocaine use is generally an interesting one: high IQ has been associated with an increased likelihood of experimenting with illicit drugs, including cocaine,64 while the risk of developing dependence in individuals with low IQ is thought to be mediated by conduct problems during childhood.65 One may speculate whether the impairments in fear recognition frequently reported in adolescents with conduct disorder might influence their increased risk of developing drug dependence.Out of the six basic emotions, fear is consistently the most difficult facial expression to recognize,67 it is unsurprising that disruptions in brain networks during emotion recognition have been reported not only in psychiatric patients, but also in their unaffected first-degree relatives.69 Shared variance between family members in brain-behavior networks implicated in fear processing is particularly interesting in light of the social context in which fear is learned,70 as childhood adversity has been shown to either improve72 or impair fear recognition performance.73 Comparisons between CDIs and their siblings who share adverse childhood experiences74 may thus provide insight into compensatory mechanisms in fear processing in those siblings who do not abuse drugs, possibly elucidating sources of resilience.In light of the strong genetic contribution to both intelligence and brain morphology,76 but in our study fearful faces were shown overtly for an extended period of time, and as such these observations may not be comparable. Second, although the HPA axis is involved in fear processing, cortisol levels and fear recognition performance were unrelated in both groups. Indeed, although acute changes in cortisol have been linked with processing threat-related stimuli,77 basal levels seem to be unrelated to fearful recognition accuracy, except in individuals with increased risk for recurrent depression.78Finally, attention should be drawn to two observations that may, at first glance, appear counter-intuitive. First, variations in trait-anxiety did not affect fear recognition performance in either group. Prior research suggests that anxious individuals exhibit hypervigilance to facial signals of covert fear,81 However, although striatal dopamine function is diminished in cocaine dependence,82 impairments in anger recognition have rarely been reported previously in CDIs, except in those who are co-dependent on a variety of other drugs.83 Additional factors are thus likely to mediate the processing of angry faces. Testosterone levels have been suggested to interact with striatal dopamine function,84 possibly by modulating neural reactivity to facial threat,19 thereby mediating individuals' attention and behavioral reactions to angry expressions.85 Indeed, mediation analysis confirmed that low testosterone levels in CDIs largely explained their poor recognition of angry faces and the weak relationship between recognition performance and underlying gray matter networks (In the current study, emotion recognition impairments in CDIs also extended to expressions of anger. Converging lines of evidence from clinical and experimental research indicate that striatal dopaminergic transmission modulates recognition accuracy of angry faces.networks .86 Regression analysis further confirmed the significant effect of comorbid opiate dependence on anger recognition, suggesting that the low testosterone levels associated with opiate dependence drive the impairments in anger recognition. This raises the question as to why impairments in angry facial expressions have previously not been reported in opiate-dependent individuals without concomitant crack-cocaine use.87 Possibly, the observed impairments in angry face perception reflect a combined effect of chronic cocaine and opiate use on HPA and HPG axes hormones,88 potentially mediated by altered vasopressin secretion in the hypothalamus89 and thereby influencing social behavior.91 Given that neuropeptides like oxytocin and vasopressin are being discussed as potential targets for treatment of drug addiction,93 facial affect recognition tasks could be useful for predicting social implications associated with this treatment in this highly prevalent but difficult-to-treat subgroup of crack-cocaine and heroin-dependent individuals.94It is of note that testosterone levels were significantly reduced in CDIs with comorbid opiate dependence, possibly due to the inhibitory effects of opiates on gonadal function.95 and may, therefore, be perceived as threatening,96 triggering avoidance responses in anxious individuals or aggression in dominant, status-seeking individuals.15 Failure to recognize angry faces thus has dramatic implications for social behavior,97 with a potential subsequent impact on rehabilitation in CDIs.98 The ratio between testosterone and cortisol levels seems to determine individuals' responses to confrontation,99 with an increased testosterone–cortisol ratio associated with reactive aggression.100 As stated above, in our sample, CDIs' testosterone levels were significantly reduced relative to cortisol, suggesting a socially avoidant response tendency,101 an effect previously reported in animal models of cocaine dependence.102Angry faces are an important social sign of disapprovalAlthough our findings shed new light on disruptions associated with emotion processing in cocaine dependence, they also raise a number of questions that warrant further investigation. The group difference in National Adult Reading Test scores and the restriction of threat cues to facial expressions should be considered as a limitation to the study; specifically, the latter precludes inferences as to whether the impairments seen extend to other threat signals. The role of gender also warrants clarification as the current study only included male volunteers. Further, our study does not address the question about the sequelae of threat recognition impairments in CDIs during addiction recovery. Future studies may, therefore, consider determining whether the observed impairments are reversed following prolonged drug abstinence.24 Functional pharmacological and endocrine challenge studies are now needed to identify the mechanisms underpinning the described disruptions in threat processing. Relationships between recognition performance and brain structure, as we have shown here, may open up new avenues for elucidating the nature of abnormal functional brain activity previously reported in individuals at risk for addition69 and with particularly high recognition accuracy.103The multivariate nature of partial least squares provides the closest structural analog to functional connectivity studies, which has allowed us to relate the complex neuropathology of addiction to recognition performance in the same manner as the associated functional networks of threat processing that have previously been identified.


Phoebastria nigripes, and P. immutabilis, respectively). Extensive field studies have shown that paired (but not unpaired) males of both of these albatross species use rape as a supplemental reproductive strategy. Between species differences in size, timing of laying, and aggressiveness suggest that Black-footed Albatrosses should be more successful than Laysan Albatrosses in heteropspecific rape attempts, and male Black-footed Albatrosses have been observed attempting to force copulations on female Laysan Albatrosses. Nuclear markers showed that the six hybrids we studied were F1s and mitochondrial markers showed that male Black-footed Albatrosses sired all six hybrids. Long-term gene exchange between these species has been from Black-footed Albatrosses into Laysan Albatrosses, suggesting that the siring asymmetry found in our hybrids has long persisted. If hybrids are sired in heterospecific rapes, they presumably would be raised and sexually imprinted on Laysan Albatrosses, and two unmated hybrids in a previous study courted only Laysan Albatrosses.Conspecific rape often increases male reproductive success. However, the haste and aggression of forced copulations suggests that males may sometimes rape heterospecific females, thus making rape a likely, but undocumented, source of hybrids between broadly sympatric species. We present evidence that heterospecific rape may be the source of hybrids between Black-footed and Laysan Albatrosses ( Unidirectional hybridization is common in nature. A recent review showed that 50 of 80 cases involving at least five hybrids were predominantly unidirectional . From a Here we suggest that predicting siring asymmetries offers a promising way to evaluate the importance of heterospecific rape as a source of hybrids between broadly sympatric species. In general, rape supplements male reproductive success when directed toward conspecifics  but the Phoebastria immutabilis and P. nigripes, respectively) because we had genetic samples for both parental species and for hybrids that could be used to test for a siring bias in F1 hybrids and to evaluate long-term gene exchange between the parental species. Paired males of both of these albatrosses are known to force copulations on conspecific females. If hybrids are sired through heterospecific rape, differences between these albatrosses in behavior and the timing of egg laying (detailed below) suggest that Black-footed Albatrosses should sire most F1 hybrids. It is important that only F1 hybrids are used to evaluate siring biases predicted for heterospecific rape because siring asymmetries will be lost if backcross hybrids are generated through random mating with either parental species. Siring bias in F1 hybrids is easily assessed using mitochondrial DNA (mtDNA) to identify the maternal species.We illustrate our predictions using hybrids between Laysan and Black-footed Albatrosses . Although interbreeding between Black-footed and Laysan Albatrosses is rare, putative hybrids have been noted for decades  and up tMature Black-footed Albatrosses are primarily dark brown, whereas Laysan Albatrosses are largely white on the body and dark grey to black on the wings and back. Presumed hybrids are intermediate between the parental species in plumage and soft part coloration, ranging in plumage from very pale grey to fairly dark, with pale under wings . The ligConspecific rapes are observed in both Laysan and Black-footed Albatrosses , and BlaImportantly, differences in the timing of breeding, body size, and aggressiveness all suggest that F1 hybrids are sired when the larger and more aggressive male Black-footed Albatrosses force copulations on female Laysan Albatrosses. Particularly important is that Black-footed Albatrosses arrive at the breeding colonies and lay earlier than do Laysan Albatrosses . BecauseWe evaluated the F1 status of hybrids using fixed and near-fixed differences in their nuclear genome, and we assessed siring bias using mtDNA from the hybrids. We also used an isolation-migration (IM) model to test the hypothesis of asymmetric gene flow between these species following their divergence approximately 1.03 million years ago .′N, 177°22′W). Genomic DNA was extracted from blood samples either by a standard phenol:chloroform procedure  at Midway Atoll National Wildlife Refuge (28°13rocedure  or usingcytochrome-b (cyt-b) locus. Anonymous loci were derived from a fosmid library for Black-footed Albatross  gene , and thelbatross . “FWD” aKF475302–KF475698).We assigned quality scores to base calls in sequence trace files using Phred  and aligPutative hybrids were sexed using primers 2550F and 2718R ; sex wasWe computed two hybrid indices, both varying from 0 (pure Laysan Albatrosses) to 1 (pure Black-footed Albatrosses). The first is most intuitive and includes only loci with fixed or near-fixed sequence differences between Black-footed and Laysan Albatrosses . Using tSiring asymmetries for the hybrids were assessed using a binomial test on mtDNA data.To assess the rate and direction of gene flow between Black-footed and Laysan Albatrosses, we used the IM model implemented in IMa2 . We appl−5 substitutions per locus per year for our fragment of cyt-b; . Three of the hybrids were male and three were female, suggesting no inviability of the heterogametic sex . The mean rate of gene flow (2 Nm) was 0.09 gene copies per generation from Black-footed Albatross into Laysan Albatross (95% highest probability density (HPD) 0.024–0.23), whereas this rate was zero in the reverse direction (95% HPD 0–0.10).Under the IM model, the rate of gene flow was significantly higher from Black-footed Albatrosses into Laysan Albatrosses (k = 3 vs. 4) and the maximized log-likelihood value of model #5 is similar to that of the best model. This suggests that the larger model #5 is not competitive with the best model and instead is “close” only because it adds one parameter, even though the fit is not improved .Asymmetrical gene flow from Laysan to Black-footed Albatross was constrained to zero in the top four models, which, together, account for 55% of the variation in the weighted AIC . A commoUsing diagnostic nuclear loci, we show that all six presumed hybrids between Laysan and Black-footed Albatross were F1 hybrids. All six carried Laysan mtDNA haplotypes, indicating that male Black-footed Albatrosses were their sires. This contradicts the hypothesis that a scarcity of mates for females of the rare species results in hybrid pairings  because Black-footed Albatrosses lay eggs 10 days to two weeks earlier than Laysan Albatrosses , so mostThe asymmetry in gene exchange revealed by the isolation-migration model suggests a long history of unidirectional gene flow from Black-footed Albatrosses into Laysan Albatrosses. Although modern hybrids appear to have no success in attracting mates , two carWe can think of two alternatives to our hypothesis of heterospecific rape as the cause of the observed asymmetry in gene flow between Black-footed and Laysan Albatrosses. First, is the possibility that F1 backcrosses into the Black-footed Albatross population have not been viable. Definitively addressing this alternative would require breeding experiments, but Fisher’s  observatSecond, if hybrids were intermediate in their breeding schedule relative to the parental species, then hybrids may have had greater opportunity to mate with Laysan Albatrosses, which return later to the breeding colonies than Black-footed Albatrosses. However, this explanation untenably assumes that hybrids form life-long pair-bonds and breed the first year that they return to their breeding islands. Instead, pre-breeding Laysan Albatrosses typically spend one or two years choosing mates , making It seems most likely to us that the gene flow revealed by the IM analysis reflects gene exchange that took place as the species were diverging in coloration. This is supported by the fact that courting birds focus their attention on the breasts of their dance partners, where the two species differ most in color , and by Notiomystis cincta) (The contrast between species in which conspecific extra-pair copulations (EPC) are forced, as opposed to species in which females accept or solicit such copulations, is critical to our thesis that hybrids between broadly sympatric species will be more common in groups where forced copulations are frequent. Although EPC are common in many passerines, they are mostly unforced and apparently controlled by females to increase the genetic quality of offspring . Unfortu cincta) ). ObviouAnas clypeata) and both Mallards (Anas platyrynchos) and Northern Pintails (Anas acuta) are reported from North America and Eurasia   . Becausectively) , F1 hybrHirundo rustica) and House Martins (Delichon urbica) in Europe and between Barn Swallows and Cliff Swallows (Petrochelidon pyrrhonota) in North America. Barn Swallows are characterized by many EPCs, but females choose whether or not to accept these EPCs, which are almost never forced (P. fulva) were found, in all cases but one, in Barn Swallow nests, attended by two Barn Swallow parents  are facultative brood parasites of Canvasbacks , whereas Canvasbacks do not parasitize Redhead nests  and Common (B. clangula) Goldeneyes parasitize each other, so males of both species are expected to be sires of hybrids. Although rare, hybrids between both the Bucephala and the Aythya species pairs are regularly reported , census population sizes are too small for certain shared alleles to have persisted for more than 2 and 2.6 million years. But these alleles, which are shared with Mallards, are estimated to have persisted for 6.2 and 7.9 million years, respectively, suggesting a long history of horizontal gene exchange with Mallards, which have a much larger effective population size  informed us of a hybrid albatross  at Midwa


Leishmania immunobiologicals in dogs naturally exposed to parasite transmission. Most of the samples (1716) were from 387 dogs enrolled for anti-Leishmania vaccine studies. The safety of the method was particularly assessed on these dogs that as per study protocol were submitted to repeated bone-marrow aspirations (2–4 per year) in follow-up examinations.Bone-marrow aspirate material is commonly considered as one of the most sensitive tissues for a reliable diagnosis of leishmaniasis. The procedure herein described may permit less experienced veterinarians to be familiar with a quick and safe assessment method for leishmaniasis diagnosis in their patients. Animals are positioned in right lateral recumbency, and the area corresponding to the second, third, or fourth sternebra is identified and aseptically prepared. A 18-gauge needle connected to a 10 mL syringe is driven through the skin, up to the bone wall, and firmly pushed forward while rotating. Entry into the sternebra's cavity is clearly perceived by the fall of resistance offered by the cortex. Some 2,500 sternal bone-marrow samplings were safely and efficiently performed on 887 dogs of different breeds and aging from 6 months to 14 years, during eight years of clinical activity for routine diagnosis of canine leishmaniasis in pets or for the efficacy evaluation of anti- Leishmania detection and/or at the evaluation the host's immune responses [Leishmania amastigotes in macrophages of affected tissues. In dogs without clinical signs involving organs or tissues but yet suspected as having leishmaniasis because of exposure to infection risk or clinical recovery following drug treatment, a diagnostic sample should be obtained from tissues where parasites are more likely to be detected, such as spleen, bone marrow, lymph nodes, or buffy-coat from peripheral blood, in descending order of diagnostic sensitivity, respectively, [Leishmania in smears or culture from these tissues [Canine leishmaniasis (CanL) has emerged as a major veterinary and public health problem in endemic areas but also in nonendemic ones where individual clinical cases or outbreaks of disease are reported, such as in northern Europe, the USA, and Canada –3. Diagnesponses . Parasitctively, , 6. Part tissues . However tissues . The aimLeishmania immunobiologicals in dogs naturally exposed to parasite transmission. The dogs were aged 6 months-14 years and were of both sexes and of different breeds. Most of the samples (1716) were obtained from 387 dogs enrolled in five clinical trials approved by the Italian Ministry of Health and designed for testing immunobiologicals against CanL. Of them, 237 dogs were Beagles, whereas 150 belonged to 30 different breeds. The studies consisted of long-term follow-up evaluation (from 2 to 4 years) starting from a naive condition followed by natural exposure to infection transmission in endemic foci of CanL. Due to the longitudinal nature of these studies, dogs were submitted to frequent bone-marrow aspiration, for an average of 3 aspirations per year.2,500 sternal bone-marrow samplings were performed on 887 dogs during eight years of clinical activity for routine diagnosis of canine leishmaniasis in pets or for the efficacy evaluation of anti-Owner dogs coming for clinical suspicion of CanL were usually submitted to a single bone-marrow aspiration, except for those that required a reevaluation of parasitological load during or after therapy. Equipment includes sterile gloves, a 18-gauge needle, a 10 mL syringe, and sterile gauze. No pharmacological sedation was usually required because of the quick procedure that may last 1-2 minutes. Slight sedation by medetomidine  was only necessary in aggressive or agitated dogs. Pain control was obtained with butorphanol  administered by intramuscular route (i.m.) 10–15 minutes before the sampling.Briefly, the animals were positioned in right lateral recumbency with the right foreleg kept forward and the left foreleg kept backward, both parallel with the body axis. The area corresponding to the second, third, or fourth sternebra  was idenLeishmania amastigotes within macrophages or free in the extracellular environment, after the macrophages membrane destruction. Parasites count was done using the technique described by Chulay and Bryceson [A conventional Giemsa blood staining was then applied; in rare occasion a Diff-Quick staining was performed as alternative, with no difference of smears quality. Smears were first observed with low-power objective to gain an appreciation of the overall cellularity and determine the adequacy of megakaryocyte number. For each dog 3 appropriated slides were considered, and all of them were scanned using a high microscopic magnification (100x) to determine a complete 500 cell differential counts, the myeloid to erythroid ratio calculation, and the final interpretation of the cytologic findings. Parasitological examination was performed with the same magnification objective to show the presence of Bryceson . To avoiμL) were obtained regardless of the clinical condition. Samples were used for Leishmania nested (n)-PCR examination as previously described [In dogs enrolled in clinical trials of immunobiologicals against CanL, bone-marrow samples , which included also several clinically healthy dogs in the early stages of infection. Except for a dog fraction that showed intermittent bone-marrow positivity during repeated investigations, n-PCR was constantly positive when a well-established infection was confirmed by a standard serological assay  at titers higher than 1 : 160 . Our PCRLeishmania amastigotes were detected in 344/748 bone-marrow aspirate smears (46.0%), such rate being higher among dogs showing advanced stages of CanL and/or high IFAT titres. Also in these dogs concordance between IFAT titers higher than 1 : 160 and cytological parasite demonstration was equal to 100%, when anti-Leishmania antibodies were detected at first clinical examination in sick dogs.On the remaining 502 dogs (corresponding to 748 bone marrow samples) consisting of sick animals, No significant accidents due to the technical procedure were recorded, including those dogs submitted to more than 4 samplings per year. in vivo or in vitro evaluation of cellular immune responses) [Leishmania amastigotes. Lymph node tissue is similarly rich in parasites especially in sick dogs [Leishmania/Ehrlichia coinfections commonly seen in endemic areas [ Leishmania parasite may be applied for both during clinical practice and for long-term studies that required repeated bone marrow samplings.Diagnostic methods for diagnosis of CanL infection are grouped into 2 main categories: direct methods  and indirect methods  . Regardiick dogs ; howeverick dogs , thus, cic areas . It has ic areas , 15. It ic areas , 17. Theic areas . Althougic areas . Furtheric areas . In conc


Ensemble forecasting is advocated as a way of reducing uncertainty in species distribution modeling (SDM). This is because it is expected to balance accuracy and robustness of SDM models. However, there are little available data regarding the spatial similarity of the combined distribution maps generated by different consensus approaches. Here, using eight niche-based models, nine split-sample calibration bouts (or nine random model-training subsets), and nine climate change scenarios, the distributions of 32 forest tree species in China were simulated under current and future climate conditions. The forecasting ensembles were combined to determine final consensual prediction maps for target species using three simple consensus approaches . Species’ geographic ranges changed (area change and shifting distance) in response to climate change, but the three consensual projections did not differ significantly with respect to how much or in which direction, but they did differ with respect to the spatial similarity of the three consensual predictions. Incongruent areas were observed primarily at the edges of species’ ranges. Multiple stepwise regression models showed the three factors  to be related to the observed variations in consensual prediction maps among consensus approaches. Spatial correspondence among prediction maps was the highest when niche model accuracy was high and marginality and specialization were low. The difference in spatial predictions suggested that more attention should be paid to the range of spatial uncertainty before any decisions regarding specialist species can be made based on map outputs. The niche properties and single-model predictive performance provide promising insights that may further understanding of uncertainties in SDM. Species distribution modeling (SDM) techniques, which attempt to provide detailed predictions of distributions by statistically relating present-day species distribution to environmental predictors, have been widely used to model and project the changes of species’ geographic distributions in response to climate change. The mosIn practice, any modeling exercise into an unknown future involves uncertainty. Four sources of uncertainty in niche-based SDM have been identified, including initial dataset conditions (IC), model classes (MC), model parameters (MP), and boundary conditions (BC) –12. IC rUncertainty in distribution projections can skew policy making and planning intended to address and respond to climate change, especially where the conservation of threatened and endangered species is concerned . One recMost available studies have demonstrated that species distribution traits could substantially influence the accuracy of species distribution predictions ,20,21. TIn this study, using eight niche models, nine random data-splitting bouts and nine different climate change scenarios, the distributions of 32 forest tree species in China were simulated under current and projected future climate conditions. Forecasting ensembles were combined by means of three widely-used consensus approaches, i.e., on the basis of median (PCA), average, and frequency of species occurrence under given climatic conditions. The primary objectives were 1) to determine whether there is substantial variation in consensual prediction maps among different consensus approaches and 2) to determine whether these variations could be best explained by species traits and niche mode predictive performance.http://westdc.westgis.ac.cn).The study area encompassed all of China. Thirty-two common forest tree species, which collectively account for more than 50% of forest cover in China, were selected for comparison of various consensus approaches in projecting the species distribution under current and future climate and potential range shifts. See Seven climatically derived variables are considered critical to plant physiological function and survival: mean annual temperature , mean warmest month temperature , mean coldest month temperature , difference  between MWMT and MCMT, mean annual precipitation (mm), mean annual summer precipitation , and degree-days above 5°C . Baseline climate data were averaged for the period 1961–1990. These seven climatic variables were calculated using ClimateChina (Ver 4.4) , which wTo assess the uncertainties related to future projections of climate change, three SRES emissions scenarios  and three GCMs  were used in this study. Climate change scenarios were averaged for three 30-year periods: 2010–2039 (2020s), 2040–2069 (2050s), and 2070–2099 (2080s). For the future climatic projections, the same set of seven climate variables were calculated using ClimateChina software for all 8 km×8 km grids.The distribution of the 32 tree species was predicted using a BIOMOD framework  programmAll eight niche-based models require species presence and absence records. One solution was to generate pseudo-absences when no reliable absence data were available ,32,33. RThen 70% true absences were selected for model development. This may prevent bias attributable to inclusion of an extremely high number of absences and reduce the computation burden ,35. The The evaluation dataset generated by split-sample was used to assess the accuracy of the model. Model accuracy was determined using three measures: The Kappa, true skill statistic (TSS), and area under the curve values (AUC) of receiver operator characteristic (ROC) curves. These three measures attribute different weights to the various types of prediction errors . AUC is an effective, threshold-independent model evaluation indicator and is also independent of prevalence (i.e. the frequency of occurrence) of target species . AUC valA total of 648 projections (72 models × 3 GCMs × 3 SERSs) were generated for each tree species. To reduce uncertainty in species distribution projections, the following three most-widely used consensus approaches were used to combine ensemble model projections after deleting models with AUC < 0.70, Kappa, and TSS < 0.4.A two-step method was used: (1) An individual projection was selected among the eight niche-based model projections for each of the nine split-sample bouts and each of the nine climate scenarios by using principal components analysis (PCA) e.g. ,24). The. The24])The simple average of all models outputs (predictions or projections) was calculated.The frequency of predictions or projections that indicated a given species was present in each grid was calculated after transforming each probability map into binary values (presence/absence) at threshold 0.5.Repeated-measures ANOVA was used to analyze the variations in model performance (AUC) between model classes and species. Model classes and species types served as fixed factors and nine split-sample bouts served as random factors. The analysis was performed using the linear mixed effect model of R statistical package (“lme” function) .http://www.esri.com/). The coordinates of the centroid were used to calculate distance and direction of habitat shift. A two-way ANOVA was performed to investigate variations in the changes in species range among three consensus approaches for each time period using species changes in range (i.e. area change percentage or shifting distance) as a response variable and consensus approach and tree species as factors. Because the levels in the factor (tree species), are a sample of possibilities (i.e. other trees), we could think the factors as random effects.Because all consensus approaches produced predictive probabilities, the comparisons of range area changes among different projections require a threshold to classify predicted presences and absences. A species is considered present at a given grid if the probability of occurrence is above 0.5. To track changes in latitudinal distributions, we compared geographic centers (or centroids) of current and future species range. The geographic center for probability value of each species was calculated by using the mean center function in ArcGIS9.3  and six species ecological and biogeographical properties  were defined as explanatory variables. Pairwise map correlations (Pearson’s correlation and Kappa) were averaged among all consensus approaches for each species for baseline and future time to use as the dependent variables and related differences in map correlation to explanatory variables using multiple stepwise regression models.The latitudinal and elevation ranges were described as the differences between the average values of the 10% most extreme sites (maximum and minimum) where each species was found. To define the species thermal range, a PCA was performed on the five thermal variables . The first two axes of this PCA, which account for more than 95% of the total variability, were kept as a synthetic variable describing thermal gradients. The thermal range was calculated as the difference between the average positions of the 10% highest and lowest values along this synthetic variable where each species was found. Prevalence is here defined as the proportion of species’ presence in the model-training data. The two measures of environmental niche, specialization and marginality, were calculated using ecological niche factor analyses (ENFA in R package “adehabitatHS” ). SpeciaLarix principis-rupprechtii, niche models, like RF, GAM, GBM, and GLM, showed higher predictive accuracy  and were less sensitive to the procedure than other models (The split-sample procedure (or data-splitting process) influenced the model performance . For Larr models .The predictive accuracies of the models also varied among modeling techniques when data were pooled for all species and split-sample bouts, with MDA showing the worst average performance and largest deviation. It was followed by ANN, CTA, and MARS. RF, GLM, GAM, and GBM showed the better average performance .Variations in predictive accuracy were clearly demonstrated among the 32 species when data were pooled for all niche models and split-sample bouts . These oA simple regression model was used to evaluate the relationship between average model performance  and species traits . Results from this analysis showed that AUC and TSS were negatively related to latitudinal and elevation ranges . PrevaleChanges in the potential distribution area of tree species and changes in distance and direction of mean centers of suitable habitat were predicted for the periods of 2020s, 2050s and 2080s using three different consensus forecasting methods. The period 1961–1990 served as a baseline. Results are presented in Pinus yunnanensis, but it increased with time for Pinus tabulaeformis  among pairs for each time period.Mean correlation among consensual prediction maps varied according to the consensus approaches used to produce the maps, and there was substantial variability in the correlations among species . For botWhen averaging the Pearson’s correlation coefficients across all trees species, the correlation between probabilistic maps between average and frequency was higher than that between median (PCA) and average or frequency . PairwisWhen all of the explanatory variables  were included in multiple stepwise regression models, only Kappa, marginality, and specialization remained significant predictors, of map correlation . Under bIdeally, SDM models would be verified on an entirely independent dataset. Model evaluations should be performed in at least two different time periods undergoing climate change . AlternaFor each species, predictive accuracy varied from one model to another when using the same mode-training and testing data sets. Some models (e.g. MDA with AUC < 0.5) even failed to be calibrated, and others, such as RF, GBM, GAM, and GLM frequently produced models with better predictive accuracy . This isSpecies traits might affect the model performance due to a large range of variations in climatic and ecological requirements for these species, which makes it difficult to find a consistent relationship between species distribution and environmental conditions ,21. TherAlthough studies using ensemble forecasting to predict habitat suitability have identified areas of spatial uncertainty by comparing maps of projections ,12,15,21Species traits can substantially influence the vulnerability of range changes to environmental changes ,41,42. TProjections vary among models. One response to this is to build a set of models across more than one set of IC, MC, MP, and BC combinations for analysis of the range of predictions and achievement of consensus among different predictions. The reason to use consensus approach is based on the central limit theorem in statistics . ConsensIn the current study, none of the three consensus approaches performed equally well in projecting species distribution range onto a future scenario. A non-significant difference in species’ relative range changes among the three consensus approaches did not reflect the spatial correspondence among the distribution maps derived from the three consensus approaches. Both in terms of probabilistic and binary maps, there was a substantial difference between three consensus approaches. Although a few studies have compared the predictive performances of different consensus approaches e.g. ,24), non, non24])Ensemble forecasting assists the recent efforts to capitalize on the growing availability of species occurrence records, modeling techniques, and future climate scenarios. Although a wide spectrum of modeling approaches and GCM predictions based on alternative emissions scenarios exist, it does not necessarily mean that they should be incorporated in ensemble forecasting. Araújo et al. (2005) argued tThe present study provides conceptual insights regarding the uncertainty of modeling the response of species to climate change. Results support previous findings that specialist species have more accurate results under modeling than generalist species and that model robustness is related to model complexity. These findings, along with those showing that different modeling techniques show various degrees of susceptibility to model-training data, could have important implications for preselecting models in SDM. Spatial uncertainty in ensemble forecasting of species distributions was here found to be related to the accuracy of single models and the positions of species in ecological space. The finding that generalist species yield distribution maps with lower spatial uncertainty than specialist species in projecting their distributions under current and future climate conditions could have important implications for consensus forecasting of species distributions. The positive relationship between spatial correspondence among consensual predictions and model performance suggests that additional efforts should be made to select or develop a new niche model with high spatial-temporal transferability. We conclude that species niche properties and model performance should be taken into account more critically in ensemble forecasting of species distributions, and particularly in the assessments of climate change impacts.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S1 Fig(DOC)Click here for additional data file.S2 Fig(DOC)Click here for additional data file.S1 Appendix(DOC)Click here for additional data file.


Chylothorax is a rare complication of surgical neck dissection. This is the first reported pediatric case of bilateral chylothorax following cervical surgery and the first to occur after tracheoplasty. Chylothorax can lead to significant complications, including hypoxemia and shock, and requires timely treatment. This case report discusses the clinical presentation, diagnosis, and treatment of our patient and reviews possible pathophysiologic mechanisms to explain the development of postoperative bilateral chylous effusions.An 18-month-old white baby girl with a complex past medical history including choanal atresia, atrioventricular septal defect, failure to thrive, developmental delay, and tracheostomy dependence developed significant hypoxemia and shock following a routine tracehostomy revision. She was subsequently found to have developed massive bilateral chylothorax, requiring escalation of mechanical ventilation, thoracostomy tube drainage, vasoactive support, and eventual surgical ligation of her thoracic duct.Massive bilateral chylothorax is a rare but potentially life-threatening complication following tracheoplasty. Clinicians caring for this patient population postoperatively should be aware of this potential complication and its management. Chylothorax is the accumulation of chyle in the pleural space due to disruption or obstruction of the thoracic duct. Chyle is lymphatic fluid absorbed from the intestinal tract; it is rich in triglycerides, lymphocytes, immunoglobulins, and other small-molecular-weight proteins. Chyle is transported from the intestinal tract to the upper central venous system by the thoracic duct. The thoracic duct begins at the cisterna chyli located at the level of the first or second lumbar vertebra. It enters into the thoracic cavity via the aortic hiatus and continues to ascend in the posterior mediastinum between the aorta and the azygous vein. At the fifth thoracic vertebral level, the duct crosses over from the right side to the left and continues its course until it reaches and empties into venous circulation. The termination point is highly variable but most commonly occurs at the junction of the left internal jugular and subclavian veins.The etiology of chylothorax can be divided into four categories: malignancy, trauma, idiopathic, and miscellaneous. Traumatic chylothorax most commonly occurs as a complication of thoracic surgery . In the Diagnosis of chylothorax usually begins with radiographic imaging revealing a pleural effusion on chest X-ray after development of respiratory distress. The diagnosis of chylothorax requires laboratory evaluation of pleural fluid in order to differentiate it from other types of pleural effusion. The presence of chylomicrons is diagnostic. Table The management approach for chylothorax is variable. Management initially involves dietary modification to reduce chyle flow as well as somatostatin analogs to reduce lymphatic flow. Drainage of pleural fluid may be necessary if it causes respiratory distress. The patient must be monitored for sequelae from chyle loss and abnormalities corrected. Conservative management is often successful but surgical management is occasionally necessary. A systematic trial of conservative treatment for 7 to 10 days followed by tiered surgical intervention can minimize the length of Intensive Care Unit (ICU) stay, hospital stay, and length of medical device use .Here we present the first pediatric case of bilateral chylothorax after cervical surgery and the first case of chylothorax following tracheostoma revision.An 18-month-old white baby girl who is tracheostomy and ventilator dependent underwent cartilage graft tracheoplasty and developed the postoperative complication of bilateral chylothorax. Her past medical history is complex and includes choanal atresia, atrioventricular septal defect, complete heart block, congestive heart failure, failure to thrive, tracheobronchomalacia, developmental delay, and hypothyroidism. Her past surgical history is significant for bilateral choanal atresia repair, patent ductus arteriosus (PDA) ligation at age 1 month, partial atrial septal defect/ventricular septal defect (ASD/VSD) closure at age 2 months, subsequent cardiac pacemaker implantation at age 2 months complicated by implant infection requiring explantation at age 3 months. Due to heart failure and tracheobronchomalacia, she underwent tracheostomy at age 3 months with an intraoperative note of continuity of the cervical field with the mediastinal surgical fields. Her pacemaker was replaced at age 4 months and was complicated by left chylothorax, which resolved with chest tube drainage and NPO diet with parenteral nutrition. Other significant procedures include gastrojejunostomy (GJ) tube placement at age 5 months complicated by small bowel perforation requiring open repair.She was first discharged home after birth at age 9 months with a cuffed tracheostomy tube, ventilator dependent, and gastrostomy tube dependent. During out-patient follow-up, evaluation of her tracheostomy site revealed significant inferior tracheostoma breakdown with difficulty maintaining a seal for adequate airway pressures as well as imminent innominate artery exposure. She was also noted to have significant hearing loss due to chronic otitis media with effusion. Pediatric surgery separately evaluated her gastrostomy tube with leakage noted around the site. Therefore tracheoplasty, bilateral myringotomy and tympanostomy tube insertion, and gastrostomy tube revision were scheduled.She underwent combined procedures with otolaryngology and pediatric surgery under a single anesthetic. Bilateral myringotomy and tympanostomy, direct laryngoscopy, bronchoscopy, and anterior cartilage graft tracheoplasty were performed by otolaryngology. Pediatric surgery performed gastrocutaneous fistula closure and Broviac catheter placement. During the skin incision for tracheoplasty, an obvious chyle leak was encountered just deep to the skin and 2mm left of the tracheostoma. This was controlled using a figure-of-eight stitch with a 2–0 silk suture. No further chyle leak was detected during the remainder of the case.2O, positive end-expiratory pressure (PEEP) 8cmH2O, and fraction of inspired oxygen (FiO2) 21% to PIP 30cmH2O, PEEP 12cmH2O, and FiO2 45%. A chest X-ray revealed a large right pleural effusion and a smaller left pleural effusion  24cmHThe patient proceeded to have significant chest tube output particularly from the right side (>130mL/kg/day). Output was significant enough to cause hemodynamic instability. Four 10ml/kg fluid boluses with either 0.9% normal saline or 5% albumin were administered for hypotension during the first 24 hours post-chest tube placement. Due to continued significant chest tube drainage and subsequent fluid refractory hypotension, vasoactive support was provided with an epinephrine infusion for 3 days. In order to prevent further hemodynamic instability from chest tube losses, ml per ml volume replacement of chest tube output with 5% albumin was started.She met our institution’s criteria for high-output chylothorax (>20mL/kg/day), and treatment was initiated according to protocol [This is the first known report of bilateral chylothorax after neck surgery in a pediatric patient as well as the first known report of chylothorax following tracheostoma revision.Given the anatomical structure of the thoracic duct, postsurgical chylothorax tends to occur unilaterally. Thoracic duct injury below the fifth thoracic vertebral body usually results in a right-sided pleural effusion while injury above usually results in a left-sided effusion . AlthougTreatment recommendations for chylothorax are variable –7 but prAside from direct management of chyle leak, patients require close monitoring for complications of chyle loss. Chyle contains triglycerides, small-molecular weight proteins , and lymphocytes. Therefore, patients are at risk for malnutrition, hypovolemia, metabolic and electrolyte abnormalities, and immunodeficiency. If enteral nutrition is possible, a fat-free diet can be instituted using a medium-chain triglyceride formula. Medium-chain triglycerides are saturated fatty acids that are of 8 to 12 carbon lengths and their use is indicated in chylothorax because they are directly taken up by portal circulation, bypassing lymphatic circulation. Response to a medium-chain triglyceride diet should be seen within the first week with full resolution of the chylothorax by the end of the second week  [Hypovolemia can result if a chyle leak is significant enough from both general volume loss and albumin loss . HypoalbWhile our patient did have a cervical chyle leak identified and repaired intraoperatively, the development of massive bilateral chylothorax following cervical surgery remains puzzling. There was no intrathoracic injury to the thoracic duct nor was there injury to the pleura evidenced by the absence of pneumothorax. Two pathophysiologic mechanisms have been suggested as the etiology of chylothorax after cervical surgery. The first is direct leakage of chyle into the mediastinum from the thoracic duct. Leakage of chyle results in an inflammatory reaction that allows penetration of chylous fluid from the mediastinum into the pleural space through the inflamed pleura. The second is ligation of the distal thoracic duct, which leads to increased hydrostatic pressure within the fragile lymphatic vessels. The build-up of pressure within the thoracic duct, combined with negative intrathoracic pressure, leads to either rupture or non-traumatic leakage of chyle into the mediastinum and pleural spaces , 10. EitIn summary, chylothorax is a rare complication of neck surgery. This is the first reported pediatric case of bilateral chylothorax following cervical surgery and the first to occur after tracheoplasty. Clinicians caring for patients after neck dissection should be aware of this rare but significant complication and the various strategies for effective treatment.Written informed consent was obtained from the patient’s mother for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Thalassarche chrysostoma tracked from two major populations, South Georgia (Atlantic Ocean) and the Prince Edward Islands (Indian Ocean). Spatial segregation was greater than expected, reflecting distinct seasonal differences in habitat selection and accessibility, and avoidance of intra-specific competition with local breeders. Previously failed birds segregated spatially from successful birds during summer, when they used less productive waters, suggesting a link between breeding outcome and subsequent habitat selection. In contrast, we found weak evidence of sexual segregation, which did not reflect a difference in habitat use. Our results indicate that the large-scale spatial structuring of albatross distributions results from interactions between extrinsic and intrinsic factors, with important implications for population dynamics. As habitat preferences differed substantially between colonies, populations should be considered independently when identifying critical areas for protection.Many animals partition resources to avoid competition, and in colonially-breeding species this often leads to divergent space or habitat use. During the non-breeding season, foraging constraints are relaxed, yet the patterns and drivers of segregation both between and within populations are poorly understood. We modelled habitat preference to examine how extrinsic (habitat availability and intra-specific competition) and intrinsic factors  influence the distributions of non-breeding grey-headed albatrosses  Partitioning of resources between ecologically-similar animals promotes their coexistence, and often involves the use of different areas or habitats1346Colonial species such as seabirds frequently target seasonally productive areas, which can lead to intense competition among breeding birds constrained to return to land to incubate eggs or feed dependent young. Thus, seabirds are useful tools for studying the relationship between extrinsic factors such as resource availability and competition as well as intrinsic habitat preferences69Outside the breeding period, seabirds have fewer constraints and disperse over wider areas e.g., and so 11121471218Thalassarche chrysostoma (hereafter GHA) forage in highly seasonal environments and often associate with frontal systems between Antarctic and sub-Antarctic waters11Grey-headed albatrosses In this study we modelled the distributions of non-breeding GHA from different populations within a use-availability frameworkWe tracked the non-breeding migrations of 66 grey-headed albatrosses from Bird Island, South Georgia (hereafter SG) and Marion Island, Prince Edward Islands (hereafter PEI), comprising 46 previously successful and 20 failed birds. The successful breeders were tracked for 490 ± 78 days (range 92–815), covering the whole non-breeding period (winter-summer-winter) for all but three birds with logger batteries that failed before retrieval. For both SG and PEI, bootstrapping analysis confirmed that our sample sizes were sufficient to make population-level inferences . We tracP < 0.001; P < 0.001; P < 0.001; P = 0.58), although birds in 2002 were more likely to use the southeast Pacific , but did not differ in return dates . There was no evidence that males from either population travelled further than females, or varied more in their migration characteristics , probably due to their shorter non-breeding period (8 vs. 16 months), as they covered a similar mean distance per day  . The incDistributions of both populations were best explained by models that included habitat preferences and constraint variables associated with competition and accessibility see .Birds from both sites used markedly cooler waters during summer than winter; birds from SG preferred lower sea surface temperature (SST) in both seasons . GHA alsIn slight contrast to the observed distributions, the models including only habitat variables predicted occurrences of GHA over large swathes of the Southern Ocean , indicatWe found no significant differences in model performance scores with the inclusion of sex-specific preferences, providing no evidence of sexual segregation by habitat . During In this study, we model the non-breeding distributions of a highly mobile marine predator as a function of multiple extrinsic (habitat availability and competition) and intrinsic drivers . We find that patterns of spatial segregation are explained well by season-specific habitat preferences and competition at both meta-population and population scales.It is challenging to model the distributions of pelagic seabirds as they are highly vagile and often respond to dynamic environmental cues e.g.. In part23Thalassarche melanophris and Campbell albatrosses T. impavida are more specialized in targeting shelf-edges than GHA, in areas such as the Patagonian Shelf and New Zealand sub-Antarctic islands, prey depletion around colonies may be enhanced by the presence of these closely-related speciesWhilst there is substantial evidence that spatial segregation reduces competition in breeding seabirds e.g., few stuDist. own explained the greatest variance. Thus, despite their extreme vagility, albatrosses have preferences for areas around their own colony that are unrelated to habitat. These findings are counterintuitive, suggesting that albatrosses are willing to tolerate birds from their own population rather than exploit distant profitable patches. The costs of migration may therefore be greater than the negative interactions that might arise from local competition, or perhaps in the case of SG birds, the southwest Atlantic is productive enough to support a very large population, year-roundWe incorporated the movement constraints of albatrosses in our null models, yet for all models 13C provide limited resolution of spatial overlap, particularly along longitudinal gradients. Segregation of habitat therefore appears to be the main partitioning mechanism in non-breeding albatrosses, which feed opportunistically at the surface and tend to have relatively wide dietary niches18Whilst niche partitioning can lead to spatial structuring among seabirds347Non-breeding GHA have season-specific habitat preferences related to dynamic features , and birds from the two populations exploit different water masses and productivity regimes. During summer, resident SG non-breeders use similar foraging areas to breeding birds101514Within a population, there is growing evidence that processes operating in one season may influence subsequent performance, known as carry-over effects19The reasons why breeding outcome affected habitat use in summer are unclear, but could arise from variation in individual condition over short or long timescales. Failed breeders may be in better condition due to their reduced parental effort and so may withstand foraging in poorer quality habitats that are closer to other colonies than their own . Alterna19Thalassarche spp.35In our study the link with subsequent breeding outcome is unknown, yet failed GHA return to the colony slightly later than successful birds. Late arrival at the breeding grounds can reduce reproductive success in some birds e.g., but not13C, but not δ15N, is higher in feathers of female than male GHA, suggesting the two sexes feed at similar trophic levelsIn large, dimorphic species such as albatrosses, sex-differences in distributions have been attributed to competition, or the effects of wing-loading on flight performance910Finally, although we did not test explicitly for individual differences, in assessing model performance it was apparent that habitat preferences of some individuals differed substantially from the population mean. Similarly, stable isotope analyses indicate that GHA from Bird Island moult in a wide range of habitats, from Antarctic to subtropical latitudes, although the timing of moult is uncertainDissostichus eleginoides fisheries in the south Indian than Atlantic OceanAlthough predictive models are increasingly used to identify suitable habitat for poorly-known populations, there is increasing evidence that for pelagic seabirds, they have poor transferability  and Marion Island, PEI in the southwest Indian Ocean . Together these archipelagos represent >60% of the global annual breeding population . The expWe processed light data in Multitrace, providing two positions per day with a mean error of 186 km ± 114 km26Distributions of birds were compared in summer  and winter (mid-May to mid-September). To determine if samples were sufficient to represent each population, a bootstrapping approach was used to randomly sample home-range area with an increasing number of individualsP-values were determined as the proportion of randomized overlaps that were smaller than the observedTo test whether albatrosses segregate by population, sex, breeding outcome and season, we calculated the overlap between home ranges using the utilization distribution overlap index (UDOI), which is considered the most appropriate measure of overlapping space useWe used a habitat preference modelling approach to quantify space use as a function of available habitat, by comparing where an animal was observed (tracking location) with where it could have gone (pseudo-absence), if it had no particular preferenceSST) and 2) gradient as measured by the standard deviation of SST (SST std), indicative of water mass and frontal regions; 3) chlorophyll α concentration (Chl) as a proxy of ocean productivity; 4) ocean floor depth (Depth) and 5) slope, represented by the standard deviation of Depth (Depth std), to indicate productive bathymetric areas such as shelf-breaks, seamounts and upwelling; 6) sea-level anomaly (SLA) and 7) eddy kinetic energy (EKE) as indicators of mesoscale oceanography associated with currents and eddies; and 8) wind speed (Wind) as albatrosses are known to be constrained by high flight costsDist. own) and 10) minimum distance to the closest other colony (Dist. closest) to determine the importance of habitat accessibility and intra-specific competition, which limit distributions of breeding albatrossesAs animals rarely respond to the environment in a linear way2031We constructed separate models for different classes of birds because of computational demands and difficulties of interpreting high order interactions. Initial models testing for interactions between population and season were significant, so we split the model into four components, by population (SG vs. PEI) and season (summer vs. winter). For each model, we ran all possible combinations of predictors and calculated AIC values using the dredge function in the R package MuMInTo identify the main extrinsic drivers, we constructed models representing only habitat (habitat variables), the constraints associated with competition and accessibility (distance variables), and the full model . We used area under the receiver operator characteristic curve (AUC) to evaluate performance of models on a weekly basis, in the PresenceAbsence package in RTo test for intrinsic influences of sex and breeding outcome (failed or successful) on space use, we ran models with and without these variables as smoothers. To assess sex differences, we used the original four population models (above), and to assess breeding outcome differences, used the pooled dataset of failed and successful birds from South Georgia during summer and winter. We tested model performance on each individual, as above, comparing the resulting AUC scores of models with and without sex- and breeding outcome-specific smoothers. All analyses were conducted in the software R v. 3.1.1How to cite this article: Clay, T. A. et al. Proximate drivers of spatial segregation in non-breeding albatrosses. Sci. Rep.6, 29932; doi: 10.1038/srep29932 (2016).