On length scales from nanometres to metres, partial adhesion of thin films with substrates generates a fascinating variety of patterns, such as ‘telephone cord’ buckles, wrinkles, and labyrinth domains. Although these patterns are part of everyday experience and are important in industry, they are not completely understood. Here, we report simulation studies of a previously-overlooked phenomenon in which pairs of wrinkles form avoiding pairs, focusing on the case of graphene over patterned substrates. By nucleating and growing wrinkles in a controlled way, we characterize how their morphology is determined by stress fields in the sheet and friction with the substrate. Our simulations uncover the generic behaviour of avoiding wrinkle pairs that should be valid at all scales. The phenomena we describe are not graphene-specific, but we make a case study of graphene because it is an exemplary thin sheet, with much greater stiffness against tension than bending, as well as being fascinating in its own right22232526We simulate controlled growth of wrinkles in mono- and multilayer graphene sheets. These simulations reveal the role played by interactions in determining whether wrinkles merge or form avoiding pairs. We quantify how the initial geometry of a pair of growing wrinkles determines its final shape and examine how substrate friction suppresses wrinkle formation. By varying sheet thickness, we demonstrate how the phenomenon of avoiding pair formation is length scale-independent. Our results open a new perspective on how thin films delaminate and point to approaches for controlling film morphology.We are primarily interested in universal phenomena, rather than graphene-specific behavior. We therefore use a linearized model that captures the essential physics of a sheet interacting with a substrate, in which the sheet is a triangular lattice of springs that provide a stretching cost, with pairs of plaquettes joined by hinges that have a harmonic energy favouring a flat sheet, which provides a bending cost. Both stretching and bending energies must be accounted for in order to model accurate wrinkle profiles8μm2 regions of monolayer and 5-layer graphene, with particles of radius 8 nm placed at positions determined by digitizing the atomic force micrograph of a system with nanoparticle density of 90 μm−2We verify our model through comparison with experimental studies of graphene sheets deposited on substrates decorated with nanoparticles of diameter ~10 nm, as published recentlyet al., we find increasing the thickness of the graphene sheet hinders its conformation to the substrate . We attribute this to lower noise levels in simulation data allowing more wrinkles to be detected.Beyond visual comparisons, we compare simulation and experiment statistically, by measuring the distribution of the number of wrinkles emanating from each particle for the graphene monolayers, as plotted in Features reminiscent of avoiding pairs of wrinkles can be observed also on macroscopic lengthscales, as demonstrated in 2 is subject to upward pulling in “wrinkle nuclei” regions. The tips of the nuclei are separated by distances X and Y, as defined in We first study the growth and interactions of wrinkles, with a focus on avoiding pairs. In order to characterize these pairs, we require a controlled method for creating and growing them. Inspired by studies of interacting cracks grown from a pair of notchesX/Y and distance X/Y > 1, the joining condition is simply a distance threshold of ~30 nm. However, for X/Y < 1, wrinkle merging becomes more favourable and the minimum distance required for the formation of an avoiding pair increases rapidly.Whereas an isolated wrinkle propagates directly forward, parallel to its nucleus, nearby wrinkles interact and either join or form an avoiding pair. To uncover the role of interactions, we fix driving conditions and make a systematic study of the effect of wrinkle nucleus separation, which we characterize by aspect ratio X/Y = 1. For small R, wrinkles are directed toward each other, but for larger R they are initially repelled before being attracted. To quantitatively describe the wrinkle shapes, we focus on two characteristics which are indicated in μ/ν. For fixed X/Y, ξ depends on R and is maximized at a distance ~230 nm, as shown in X/Y, and avoiding pairs of wrinkles formed by nearly head-on nuclei are substantially longer than wrinkles formed from nuclei that are separated in the X direction. Remarkably, for nucleus separations X/Y has little effect on the aspect ratio μ/ν of wrinkles, which decreases approximately linearly with R.In addition to the conditions for avoiding pair formation, we also characterize how the morphology of such pairs depends on wrinkle nuclei separation. Typical wrinkle shapes are shown in σ) = xxσ + yyσ + zzσ. Unlike, e.g, tensile fracture, where hoop stress θθσ determines crack propagationσ) < 0). The wrinkle grows via buckling of the sheet, and deformation occurs primarily as bending rather than stretching which is highly unfavourable for graphene.To understand how wrinkle interactions affect their shape, we characterize the stresses induced by wrinkles, using the trace of the stress tensor Tr2 where The graphene sheet is modeled as a triangular lattice of particles connected by linear springs with rest length r from the substrate isk = 1.0 eV/Å2 is a spring constant and cr = 1 Å. We have also tested a radial Lennard-Jones potential with parameters to match experiments  for r < 1.01 Å, with A drawn from a uniform random distribution over [−5, 5) eV and B = 10 Å−2. The centres of these lie on a plane at distance z0 below the minimum of a Lennard-Jones potential. The LJ potential is the dominant contribution so that in the absence of external forces the graphene sheet sits near the minimum of the LJ potential. Varying z0 tunes the strength of the random contributions to the sheet's energy.Our simulations of substrates with random pinning use a Lennard-Jones potential for the substrate, with parameters to match experiments (S.Z. designed the research. Zoe B. and A.L.S. performed simulations. Zoe B., A.L.S., Zsolt B. and S.Z. analyzed data. Zoe B. wrote the paper with input from all authors.four_particles_spacing160nm_1layerfour_particles_spacing160nm_5layerswrinkle_pair_const-forcewrinkle_pair_ramp-upwrinkle_pair_StressXYZ_ramp-upSupplementary Information for 'Wrinkle motifs in thin films'


Significant change was observed in heart rate including a downtrend of heart rate within 15 min prior to I-HCA (p < 0.05). There was a significant difference in heart rate and QRS duration during the last hour prior to I-HCA compared to the previous hours (p < 0.05). Inferior ECG leads showed the most significant changes in QRS morphology and ST segments prior to I-HCA (p < 0.05). Subjects with an initial rhythm of asystole demonstrated significantly greater ECG changes including QRS morphology and ST segment changes compared to the subjects with initial rhythms of PEA (p < 0.05). Diagnostic ECG trends can be identified prior to I-HCA due to PEA and asystole and can be further utilized for training a predictive machine learning model for I-HCA.Survival after in-hospital cardiac arrest (I-HCA) remains < 30 %. There is very limited literature exploring the electrocardiogram changes prior to I-HCA. The purpose of the study was to determine demographics and electrocardiographic predictors prior to I-HCA. A retrospective study was conducted among 39 cardiovascular subjects who had cardiopulmonary resuscitation from I-HCA with initial rhythms of pulseless electrical activity (PEA) and asystole. Demographics including medical history, ejection fraction, laboratory values, and medications were examined. Electrocardiogram (ECG) parameters from telemetry were studied to identify changes in heart rate, QRS duration and morphology, and time of occurrence and location of ST segment changes prior to I-HCA. Increased age was significantly associated with failure to survive to discharge ( In-hospital cardiac arrest (I-HCA) accounts for approximately 200,000 cardiac arrests in the United States each year  and survApproximately 70 % of initial rhythms in patients with I-HCA are pulseless electrical activity (PEA) and asystole, and these rhythms are associated with higher mortality rates as compared to initial rhythms of ventricular tachycardia and fibrillation –6. TelemA retrospective study was conducted among subjects who experienced I-HCA with an initial rhythm of PEA or asystole in the medical and surgical intensive care units (ICU) and step-down units of three acute care hospitals. All I-HCA subjects were on continuous ECG monitoring  prior to I-HCA, per standard of care required by each hospital, for the entire duration of stay of these patients in the ICU or step down units.Subjects who experienced I-HCA and underwent cardiopulmonary resuscitation (CPR) were screened from February 2010 to June 2012 for eligibility in the study. Subjects were included if they were ≥21 years of age and had a history of structural heart diseases (see definitions below). If a subject had more than one I-HCA, only the first episode of I-HCA was included in the analysis. Subjects were excluded from the study if resuscitation was initiated out-of-hospital, in the emergency room, in a procedure or operating room, or for trauma, drug overdose, hypothermia, drowning, or if they had an active cancer  or were comatose. Clinical characteristics of subjects were obtained from the medical record. Laboratory data including potassium, calcium, hemoglobin, blood urea nitrogen and creatinine were obtained within 24–48 h of I-HCA, and troponin and B-type natriuretic peptide (BNP) levels were recorded if obtained during the same hospitalization in which I-HCA occurred. A list of cardiac medications, sedatives, and narcotics administrated within 72 h of I-HCA was obtained. Cardiac risk factors including a history of diabetes, hypertension, and hyperlipidemia were noted. The ejection fraction (EF) from any echocardiogram obtained within 6 months prior to I-HCA was recorded. All available 12-lead ECGs prior to I-HCA were examined for evidence of bundle branch blocks.1, V6). Since lead V6 was not routinely recorded in the majority of subjects, only seven leads were used for analysis. Each full disclosure ECG was 10 s long. Full disclosure ECGs were analyzed for 2 min at the time of I-HCA and for any significant rhythm changes within 1 h of documented I-HCA. Additionally, full disclosure ECG papers were analyzed for 2 min in each of the 8 h prior to I-HCA. All rhythms prior to I-HCA were analyzed except for paced rhythms.The full disclosure ECG prior to I-HCA from continuous monitoring systems was obtained. The full disclosure ECG was recorded at a paper speed of 25 mm/s, at 10 mm/mV gain, and typically consisted of 7–8 ECG leads  to the end of the S wave in each lead of the full disclosure ECG and the mean QRS duration for each lead was calculated over three beats in each full disclosure ECG paper. QRS prolongation was defined as an increase in QRS duration ≥20 ms (ms) observed within 7 h prior to I-HCA. QRS duration prior to I-HCA was noted in two ways: (1) a single longest QRS duration in any available lead for each hour and (2) the longest mean QRS duration in any available lead for each hour.ST segment elevation or depression of ≥1 mm and QRS morphology including notching, fractionated QRS (fQRS), rSR′ and rSr′ in any available ECG lead were identified visually by experts including a physician (cardiac electrophysiologist) and the primary author. We reported only those changes in QRS morphology and ST segment elevation and depression that were not present initially at 8 h prior to arrest. The time between I-HCA and changes in QRS morphology, ST segment elevation and depression were obtained by recording the earliest change that was present in any ECG lead. The ECG locations of the earliest changes and subsequent changes were also recorded. Subjects with left bundle branch block (LBBB) confirmed through 12 lead ECG were not included in the sample set for detecting ST segment changes.Research assistants were trained to review and evaluate ECG changes, and the correlation of measurements between research assistants and primary investigators was >90 %. A cardiac electrophysiologist was available as a consultant to review the ECG data. Additionally, we digitized ECG paper recordings for 39 subjects and performed automated measurements including heart rate . The agrFor this study, cardiac arrest was defined as a sudden cessation of cardiac function, precipitated by pulseless electrical activity or asystole (ventricular tachycardia and ventricular fibrillation were excluded) . The preDescriptive statistics were reported as means, standard deviation, and standard errors for continuous variables. Categorical variables were expressed in numbers and percentages. The Chi-squared test was used to determine the statistical significance of differences among categorical/continuous variables .p value <0.05 was considered statistically significant.Logistic regression was used to assess association between immediate outcomes of cardiac arrest, survival to discharge, and ECG changes (e.g. changes in heart rate and QRS duration) and demographic variables. The change in heart rate within 15 min prior to I-HCA for all subjects was calculated. ANOVA and Kruskal–Wallis tests were used to detect significant differences in heart rate and QRS duration within 8 h of cardiac arrest and their comparisons with the last hour of cardiac arrest for all subjects. Statistical analyses were performed using SAS software , and Python. A During the study, 39 of 80 subjects with I-HCA from intensive care and step-down units met inclusion criteria. Clinical characteristics are summarized in Table Troponin I (μg/mL) values were available in 26 subjects (67 %), of whom 16 (62 %) had elevated levels (ranging from 0.23 to 46.77). Brain natriuretic peptide levels were available in 18 subjects (46 %) with a median of 801 pg/mL (absolute median deviation = 418.5).p < 0.05) and only older age was significantly associated with poor survival to discharge (p < 0.05). Covariate analysis found no significant association between demographic values, heart rate changes or QRS prolongation.Among covariates analyzed for all subjects, only older age and high creatinine were negatively associated with resuscitation from I-HCA (p < 0.05) as supported by parametric (ANOVA) and non-parametric  multiple comparison tests. Pairwise hourly comparisons showed that the hour prior to I-HCA contributed to the significance of the tests. Mean heart rate decreased 3.0 beats per min (bpm) within the last hour prior to I-HCA (p < 0.01), 4.6 ± 2.5 bpm within 15–10 min (p = 0.24), 5 ± 0.5 bpm within 10–5 min (p < 0.01) and 7.6 ± 1.3 bpm within 5–0 min (p < 0.01). There was no difference with respect to the change in heart rate within the last hour or 15 min prior to I-HCA in patients with an initial rhythms of PEA or asystole. Different cardiac arrhythmias during the period of decreased heart rate were observed including sinus bradycardia, with and without first degree atrioventricular (AV) block (47 %), junctional rhythm (28 %), atrial fibrillation (19 %), and third degree AV block (6 %).There were 32 subjects (82 %) who had decreased heart rate prior to cardiac arrest. Figure Nine subjects (28 %) had a sudden decrease in heart rate over 10–25 s during the 15 min prior to I-HCA. This sudden decrease in heart rate was either followed by a continuous downtrend of heart rate or an increase in heart rate to the previous rate followed by a downtrend of heart rate leading up to I-HCA. The maximum and minimum heart rates were 100 and 38 bpm respectively, and the range of difference in heart rate was 21–60 bpm with a median of 34 in subjects with asystole and PEA. Junctional rhythms, progression of first degree to third degree atrioventricular block, and sinus bradycardia with premature atrial contractions were observed during periods with a sudden decrease in heart rate.p < 0.01) was observed within the last hour prior to I-HCA compared to previous hours among all subjects. QRS prolongation was significant for both the longest mean QRS and the single longest QRS measured in full disclosure ECG leads. There was no significant difference between the longest mean QRS and the single longest QRS in any ECG lead.QRS prolongation was observed in 20 (51 %) subjects, including 19 in the group with decreased heart rate prior to I-HCA. QRS prolongation  and ST depression (1–4 mm) were observed in seven subjects (18 %). The median time for the earliest transient changes in QRS morphology, ST depression and ST elevation was 14, 4, and 5 min prior to I-HCA, respectively.If there was more than one change in QRS morphology (e.g. notched R wave progressing to fQRS) in one lead for a subject, then we only counted the change once. Figure Among cardiovascular subjects, we found significant ECG changes prior to PEA and asystole. Gradually decrease in heart rate, QRS prolongation, changes in QRS morphology and ST segment depression and elevation were the main findings prior to I-HCA with initial rhythms of PEA and asystole.We found evidence that continuous ECG monitoring systems/telemetry are currently incapable of detecting important ECG changes prior to cardiac arrest and alerting clinicians of these changes. For example, the ECG system sounds an alarm when the heart rate is high or low . However, it does not alert the clinician when the heart rate decreases gradually from 130 to 60 beats per min. ECG changes such as a gradual decrease in heart rate within 15 min might have an immediate prognostic impact on the timely treatments of some subjects with PEA and asystole, which may be particularly important given the fact that the survival after asystole is approximately only five percent. Our study confirms previous findings that bradycardia and/or a decreased heart rate occur in adults experiencing I-HCA . HoweverThe findings suggest that the mechanism of the decrease in heart rate prior to I-HCA might be due to an imbalance between the sympathetic and parasympathetic nervous system –19. It hTo the best of our knowledge, this study is the first to report abnormalities in ventricular conduction including both duration and morphology of the QRS prior to I-HCA due to PEA and asystole among cardiovascular subjects. QRS prolongation has been considered a prognostic marker for mortality among patients with a variety of cardiovascular diseases –23. MoreThe demographic findings of our study are consistent with previous findings that age is associated with mortality after I-HCA  and the An additional issue inherent with PEA is that the time of arrest may not be determined precisely because patients who experience PEA have no palpable pulse but the monitoring system may show an ECG that is compatible with life. We have identified diagnostic ECG clues that warrant patient evaluation by clinicians prior to PEA and asystole as an initial rhythm of the I-HCA. This may help shorten the response time to cardiac arrest especially with PEA. Future studies will be required to determine whether these diagnostic clues can be used as a guide for appropriate interventions.An important clinical implication of this pilot study is increased awareness of the need to use full disclosure ECGs while monitoring two ECG leads continuously. Analysis of the full disclosure ECG is not a standard feature for all ECG monitoring systems. Typically only one or two ECG leads (e.g. leads II and V1) are displayed continuously. Because the full disclosure ECG is not displayed automatically, it is not clear whether clinicians are aware they can use this feature if it is part of their telemetry system. The presence of QRS morphology and ST segment changes prior to I-HCA in leads other than II and V1 in our study supports the importance of using available full disclosure ECGs. Identification of ECG morphology changes by analyzing full disclosure ECGs may preempt or decrease response time to I-HCA due to PEA and asystole.In conclusion, this study has identified ECG changes using full-disclosure ECGs from continuous monitoring systems that precede I-HCA due to PEA and asystole. The identification of these indicators may be a first step in better understanding the mechanism of I-HCA due to PEA and asystole. More importantly, if these changes are more widely known by medical professionals, they may be used, in concert with better telemetry and continuous monitoring systems, to prevent adverse outcomes among hospital patients from I-HCA, including death.Telemetry is one of the most common monitoring systems being utilized at hospitals and ECG changes from telemetry can be potentially incorporated in the Early Warning Scores (EWS). Different formats of EWS  predict the deteriorating condition of patients by analyzing continuously recorded data including a patient’s vital signs and neurological status. ECG changes from telemetry prior to asystole and PEA can also be used to assess patients at the risk for adverse events. Establishing the ECG predictors prior to these lethal arrhythmias through multi-center studies and creating a standard of care for using monitoring systems including access to digital data at hospitals may improve our ability to predict onset of I-HCA in high risk patients, over existing clinical risk prediction methods .Only cardiovascular disease subjects were included in this study resulting in a small sample size. However, this more homogenous population may have served as a better indicator for the potential underlying mechanisms, as opposed to a mixed population with different underlying diseases such as trauma and cancer. Nevertheless, I-HCA is a complex public health problem, and a larger, multi-center study should be conducted to verify these results and shed more light on the mechanisms of I-HCA. The present findings should be viewed with caution before making population-based conclusions because of small sample size studied. Our study indicate the potential role for telemetry in identifying patients at the risk of I-HCA and ECG changes might add meaningful clinical data to existing predictors .ECG data of hospitalized patients from telemetry is not typically saved as part of the patient’s electronic health records in the majority of US hospitals . Therefo


Neuropsychological and functional MRI data suggest that two functionally and anatomically dissociable streams of visual processing exist: a ventral perception-related stream and a dorsal action-related stream. However, relatively little is known about how the two streams interact in the intact brain during the production of adaptive behavior. Using functional MRI and a virtual three-dimensional paradigm, we aimed at examining whether the parieto-occipital junction (POJ) acts as an interface for the integration and processing of information between the dorsal and ventral streams in the near and far space processing. Virtual reality three-dimensional near and far space was defined by manipulating binocular disparity, with -68.76 arcmin crossed disparity for near space and +68.76 arcmin uncrossed disparity for near space. Our results showed that the POJ and bilateral superior occipital gyrus (SOG) showed relative increased activity when responded to targets presented in the near space than in the far space, which was independent of the retinotopic and perceived sizes of target. Furthermore, the POJ showed the enhanced functional connectivity with both the dorsal and ventral streams during the far space processing irrespective of target sizes, supporting that the POJ acts as an interface between the dorsal and ventral streams in disparity-defined near and far space processing. In contrast, the bilateral SOG showed the enhanced functional connectivity only with the ventral stream if retinotopic sizes of targets in the near and far spaces were matched, which suggested there was a functional dissociation between the POJ and bilateral SOG. Visual information processing draws upon two anatomically distinct ‘streams’ of projections from the primary visual cortex V1): A ventral stream projecting to the inferior temporal cortex and a dorsal stream projecting to the posterior parietal cortex : A ventr–5. FolloElectrophysiological and tract tracking studies in monkeys suggested role of visual area 6 (V6) complex as a potential candidate region in which information from the ventral and dorsal streams is integrated. The V6 complex is well-suited for this integrative function as it contains both visual and somatomotor cells . MoreoveThe primary aim of the present study was to directly examine whether the POJ acts as an interface for the integration and processing of information between the dorsal and ventral streams. By adopting the virtual reality technique, we attempted to provide a three-dimensional space as the setting for a visual-manual task. We hypothesized that, in a visual-manual task, there were higher demands for the integration of information from two streams when a visual-manual task demands the processing of a visual target presented in the far space than in a visual-manual task that requires the processing of a visual target presented in the near space. Previous neuropsychological and imaging studies revealed that the dorsal and ventral streams differentially supported the near and far space processing –24. SincMoreover, in three-dimensional space of the present study, stimuli in the near and far spaces could be either retinotopic size matched (same size in retina) or perceived size matched (same size in perception). If the retinotopic size were matched, bottom-up visual inputs between the near and far space processing could be equated, but participants would subjectively perceive larger objects in the far space. In contrast, if the perceived size were matched, the subjective perception could be equated, but the retinal inputs differed between the near and face spaces processing. However, previous studies mostly focused on the former and ignored the latter, which might confound the effect of near/far space with the stimulus size –29. In oIn the present functional MRI study, we directly addressed whether the interaction between the ventral and dorsal streams took place in the POJ . By employing virtual reality technique, we asked participants to perform tasks with a three-dimensional object presented either in the near or far space via stereo goggles, under both the ‘match’ and ‘natural’ conditions. Near and far space was defined by manipulating different binocular disparities, i.e. -68.76 arcmin crossed disparity for near space and +68.76 arcmin uncrossed disparity for near space. Since we were most interested in the differential processing between the near and far spaces, we contrasted the near and far space processing  and then conjoined the ‘Near > Far’ contrasts for the ‘match’ and ‘natural’ conditions, thus creating a near or far space preference region that was independent of object size. Then, we performed psychophysiological interaction (PPI) analysis to investigate the functional connectivity between the resulting region (POJ) and the dorsal and ventral streams. With the neural activity in the POJ as the physiological factor, and the ‘Near > Far’ contrast as the psychological factor, we predicted that the POJ would show enhanced functional connectivity with both the dorsal and ventral streams during the far space processing.Before the experiments, all participants gave their informed written consent in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki), and the study protocol was approved by the Ethics Committee of the South China Normal University.Nineteen participants  took part in the present study. They were all right handed, and had normal or corrected to normal visual acuity. None of them had a history of neurological or psychiatric disorders.http://www.blender.org), exported as Direct X files, and presented on a grey background by custom made Presentation scripts .A goggle-based MR-compatible system  provided two separate VGA with digital dual video inputs for stereoscopic display, each with a resolution of 800  × 600  pixels at 60 Hz refresh rate. The horizontal extent of the field of view was 30 degree. The default viewing distance was 75 cm. The dual-display stereoscopic video, with 0.5 mega pixel resolution in a 0.25 square area, yielded 3D images by delivering slightly disparate images to each eye (binocular disparity). The 3D objects were generated by Blender , the diameter of the plate was 45° of visual angle in the far space (150 cm), see Thus, the experimental design was a two  by two  by three  blocked design. For each of ‘match’ and ‘natural’ runs, this design resulted in six experimental conditions: egocentric task in near space (‘Near_EGO’), allocentric task in near space (‘Near_ALLO’), high-level baseline task in near space (‘Near_HLB’), egocentric task in far space (‘Far_EGO’), allocentric task in far space (‘Far_ALLO’), and high-level baseline task in far space (‘Far_HLB’). Accordingly, for each run, participants alternated between six types of experimental blocks. Each block began with a 3 second visual instruction, informing participants about the type of task in the following block. The target presentation time during each trial was 150 ms, such a short stimulus-on time was chosen to minimize eye movements. Participants were required to keep their gaze straightforward and converge towards the frontal plane in which the near space or far space stimuli appeared during the near space blocks or far space blocks, respectively. The importance of not moving their eyes was repeatedly emphasized. The duration of each trial was 1650 ms. There were 10 trials per block, resulting in a block duration of 16.5 seconds. There were 8 repetitions for each of the 6 block types.3. Thirty-six transversal slices of 3 mm thickness that covered the whole brain were acquired sequentially with a 0.3 mm gap . There were two runs of functional scanning, each lasted for 16.5 minutes (450 EPI volumes). The first five volumes were discarded to allow for T1 equilibration effects. Additional high-resolution anatomical images were acquired using a standard T1-weighted 3D MP-RAGE sequence, the voxel size was 1 × 1 × 1 mm3.A 3T Siemens Trio system with a standard head coil was used to obtain T2*-weighted echo-planar images (EPI) with blood oxygenation level-dependent (BOLD) contrast. The matrix size was 64 × 64, and the voxel size: 3.1 × 3.1 × 3.0 mmhttp://www.fil.ion.ucl.ac.uk). Images were realigned to the first volume to correct for interscan head movements. Then, the mean EPI image of each participant was computed and spatially normalized to the MNI single participant template using the ‘unified segmentation’ function in SPM8. This algorithm is based on a probabilistic framework that enables image registration, tissue classification, and bias correction to be combined within the same generative model. The resulting parameters of a discrete cosine transform, which define the deformation field necessary to move individual data into the space of the MNI tissue probability maps, were then combined with the deformation field transforming between the latter and the MNI single participant template. The ensuing deformation was subsequently applied to individual EPI volumes. All images were thus transformed into standard MNI space and re-sampled to 2 × 2 × 2 mm3 voxel size. The data were then smoothed with a Gaussian kernel of 8 mm full-width half-maximum to accommodate inter-participant anatomical variability.Data were preprocessed with Statistical Parametric Mapping software SPM8  as implemented in SPM8. At the first level, ‘match’ and ‘natural’ runs were modeled separately. In each run, the GLM was used to construct a multiple regression design matrix that included two conditions: ‘Near’ and ‘Far’. Each condition was modeled by a boxcar reference vector (16.5 s) convolved with a canonical synthetic hemodynamic response function (HRF). Additionally, all the instructions and the six head movement parameters derived from the realignment procedure were included as covariates of no interest. Task blocks in which participants did not adhere to the task instructions (error rates within such blocks were above 60%), were separately modeled as another regressor of no interest. This occurred in two of the nineteen participants who each misperformed in one of a total of 48 task blocks. Parameter estimates were subsequently calculated for each voxel using weighted least squares to provide maximum likelihood estimators based on the temporal autocorrelation of the data. No global scaling was applied. For each participant, simple main effects for both of the two conditions were computed. The two first-level individual contrast images in both the ‘match’ and ‘natural’ runs were then fed into a within-subjects ANOVA at the second group level employing a random-effects model . In the modeling of variance components, we allowed for violations of sphericity by modeling non-independence across parameter estimates from the same participant and allowing unequal variances both between conditions and participants using the standard implementation in SPM8. We were especially interested in the differences between the two conditions (‘Near > Far’ and ‘Far > Near’). Areas of activation were identified as significant only if they passed a conservative threshold of orrected . For theorrected , 32.p < 0.001, FWE correction for multiple comparisons at cluster level with an underlying voxel threshold at p < 0.005, uncorrected).We used the POJ and bilateral SOG  as a source region to estimate the context-specific functional modulation of neural activity across the whole brain caused by neural activity in the POJ and bilateral SOG using psychophysiological interaction (PPI) analysis. PPI analysis allows for detecting regionally specific responses in one brain area in terms of the interaction between input from another brain region and a cognitive/sensory process . We usedWe first identified brain regions that were associated with differential processing of spatial domains (both of the two conditions) in the ‘match’ run. We found that an extended activation cluster, including the bilateral SOG and the POJ, showed significantly higher neural activity in the near space processing than in the far space processing  . No signWe also identified brain regions that were associated with differential processing of spatial domains in the ‘natural’ run. We found an extended activation cluster, including the right lingual gyrus, the left calcarine sulcus, the bilateral SOG, and the POJ, showed significantly higher neural activity in near space processing than in far space processing  . For theMore importantly, we isolated the brain regions, which showed near space preference independent of object size. A conjunction analysis between the main effect of spatial domain (‘Near > Far’) in the ‘natural’ and ‘match’ runs was performed. The conjunction null hypothesis, instead of the global null hypothesis, was tested as implemented in SPM8 , 32. ThePPI analysis was performed with the main effect of spatial domain  as the psychological factor and with the neural activity in the POJ as the physiological factor. The POJ showed increased neural coupling with both the dorsal and ventral visual streams in the far space processing than in the near space processing. The ‘match’ and ‘natural’ runs revealed consistent results see . No signPPI analysis was performed with the main effect of spatial domain  as the psychological factor and with the neural activity in the bilateral SOG as the physiological factor. For the ‘natural’ run, the bilateral SOG showed increased neural coupling with both the dorsal and ventral visual streams for far space processing than near space processing. The left and right SOG showed the same pattern, but connectivity was observed only between the left SOG and the dorsal and ventral visual streams see . HoweverThe present study directly examined whether the POJ acts as an interface for the integration and processing of information between the dorsal and ventral streams in disparity-defined near and far space processing. Although there was a variety of depth cue to implement depth perception, the present study defined near and far space by manipulating binocular disparities. Thus the observed effects of spatial domain were results of different binocular disparities. We first identified the brain regions that were associated with differential processing of spatial domains (near vs. far space). The POJ, along with the bilateral SOG, showed significantly higher neural activity in the near space processing than in the far space processing, independent of object size . AnatomiSecond, and most pertinent to the aim of the present study, our PPI results found that the POJ showed enhanced functional connectivity with both the dorsal and ventral streams during the far space processing, independent of the retinotopic or perceived size of the targets, which lent support to the proposal that the POJ acted as an interface for the integration and processing of information between the dorsal and ventral visual streams. The areas showing enhanced functional connectivity in the PPI analysis overlapped with the areas of the dorsal and ventral visual streams identified in previous studies , 25, 26.Three-dimensional spatial processing of targets in the near reachable space depended on sensorimotor representations in the dorsal stream; in contrast, processing of targets that placed in the far space outside the distance of an outreached hand depended on perceptual representations in the ventral stream , 25, 26.Moreover, the PPI results in the present study demonstrated functional dissociation between the POJ and SOG. Previous studies usually defined the combination of the POJ and SOG regions as the superior parieto-occipital cortex (sPOC), which included the regions on either the posterior or anterior side of the superior parieto-occipital sulcus , 45–49. In the present study, the different target distances of near and far spaces were simulated by adjusting the binocular disparity. Therefore, the differential effects of near and far spatial domains on the neural activity level and neural coupling were resulted from binocular disparity depth cue. Please note, the perception of depth or distance arises from a variety of depth cues: monocular cues, such as perspective, and binocular cues, such as disparity. Among all depth cues, disparity is the most frequently used to define spatial domain  in human , 56, 57 Taken together, by manipulating binocular disparity as depth cue, the present study found that, in addition to its role in the near space processing, the POJ showed enhanced functional connectivity with both the dorsal and ventral streams during the far space processing irrespective of the target size, which directly supports that the POJ acts as a neural interface between the dorsal and ventral streams in disparity-defined near space and far space processing.


Protozoa are eukaryotic cells distributed worldwide in nature and are receiving increasing attention as reservoirs and potential vectors for the transmission of pathogenic bacteria. In the environment, on the other hand, many genera of the protozoa are human and animal pathogens. Only limited information is available on these organisms in developing countries and so far no information on their presence is available from Sudan. It is necessary to establish a molecular identification of species of the protozoa from drinking and environmental water. 600 water samples were collected from five states  in Sudan and analysed by polymerase chain reaction (PCR) and sequencing. 57 out of 600 water samples were PCR positive for protozoa. 38 out of the 57 positive samples were identified by sequencing to contain 66 protozoa species including 19 (28.8%) amoebae, 17 (25.7%) Apicomplexa, 25 (37.9%) ciliates, and 5 (7.6%) flagellates. This study utilized molecular methods identified species belonging to all phyla of protozoa and presented a fast and accurate molecular detection and identification of pathogenic as well as free-living protozoa in water uncovering hazards facing public health. Protozoa are eukaryotic cells distributed worldwide in nature and are receiving increasing attention as human and animal pathogens and potential vehicles for the transmission of bacteria in the environment. Unfortunately, one of two persons in the world is affected by waterborne or foodborne parasites . The proThe protozoa are subdivided into four phyla depending on their methods of locomotion. Mastigophora (flagellates) move by using one or more flagella. Sarcodina (amoebae) have extensions of the cytoplasm called pseudopodia assisting phagocytosis and motion in the organisms. Ciliophora (ciliates) move by means of cilia. Sporozoa (Apicomplexa) have no locomotion .Over the past few decades, pathogenic enteric protozoa have been increasingly implicated in compromising the health of millions of people, mostly in developing countries. These protozoa contribute significantly to the staggering caseload of diarrheal disease morbidity encountered in these regions and are also significant concerns in industrialized countries despite improved sanitation .Acanthamoeba is a genus of free-living amoebae (FLA), which are environmental eukaryotic cells distributed worldwide in nature [ Acanthamoeba includes two stages, a feeding trophozoite and a dormant cyst stage. Acanthamoeba supports bacterial growth and survival and saves the bacteria from chlorination [ Acanthamoeba. Acanthamoeba species are showing an increased role as human pathogens causing encephalitis, keratitis, pneumonitis, and dermatitis [n nature , 6. Liferination –9, incrermatitis , 11 overCryptosporidium species and Giardia intestinalis are major pathogens in the waterborne transmission of infections and they are able to persist in the environment due to the robustness of the oocysts and cysts. The occurrence of these microorganisms in different types of water has been confirmed, and a considerable number of waterborne outbreaks have been reported worldwide [orldwide –14. Cryptosporidium species is an important cause of diarrhea in children, and it is suggested that intrafamilial spread occurs [ Cryptosporidium. The aims of this study were to detect protozoa from water samples by polymerase chain reaction (PCR) using primer (P-SSU-342f and Medlin B) and identify species of the protozoa by sequencing.In Sudan,d occurs . The higd occurs . However600 samples were collected from water sources  that belonged to different areas  in Sudan for a one-year period in 2008 .The hafir   is one oThe samples were centrifuged for 10 min at 4000 rpm. The supernatant was removed and the sediment was used for DNA extraction. The DNA was extracted using routine proteinase K procedures using Qiagen DNA mini kit .μM of each primer. The PCR amplification step was carried out and the final PCR reaction mixture was divided into two parts. One part was used for 2% agarose gel electrophoresis with ethidium bromide staining and the other part was used for DNA sequencing.A protozoa-specific forward primer (P-SSU-342f) and Eukarya-specific reverse primer (Medlin B) targeting 18S rRNA gene were used. The forward primer was P-SSU-342f (5′ CTTTCGATGGTAGTGTATTGGACTAC-3′) and the reverse primer was Medlin B (5′-TGATCCTTCTGCAGGTTCACCTAC-3′),  in PCR tA. castellanii was used as a positive control. Detection sensitivity was performed by serial dilution of A. castellanii (10 cells/mL to 106 cells/mL).After samples collection, DNA extraction, and PCR analysis, direct sequencing of the protozoa 18S rRNA gene was performed using purified nested PCR products. The sequencing was carried out by MWG operon . The nucleotide sequences were then edited and aligned using Blast program; the sequencing confirmed protozoal species according to the GenBank accession numbers for their nucleotide sequences.χ2 test was used to verify differences in the existence of protozoa in water samples collected from different sources. A P value of ≤0.05 was considered statistically significant. Acanthamoeba according to the serial dilutions , Apicomplexa species (25.7%), ciliates (37.9%), and flagellates (7.6%). A. castellanii, A. polyphaga, and Hartmannella vermiformis, were identified from tap water [ Cryptosporidium, Giardia, and Acanthamoeba were isolated from stations of recreational lakes in Malaysia [In this context, studies by Leiva et al.  and Tsveap water . In addiMalaysia . Cryptosporidium, and Giardia only in water samples compared to our study that identified 49 microorganisms comprising 16 species belonging to four different phyla of protozoa, which gives an alert about the risk of water contamination since most of these protozoa are human pathogens or zoonotic parasites.However, these studies detected a limited number of amoebae, Cryptosporidium was an important cause of diarrhea in children in Sudan. Most of the international studies in the field detected limited numbers of protozoal species such as Cryptosporidium and Giardia [To our knowledge, no proper studies were performed in Sudan searching for protozoa in water sources. However, Adam et al.  reported Giardia , 26. Acanthamoeba, Balamuthia, Naegleria, and Sappinia. It has been shown that Acanthamoebae benefits from E. coli and Klebsiella aerogenes as food. In contrast, the role of Acanthamoebae as hosts and vectors for bacteria has been proposed for many pathogenic bacteria [It is well known that a complex interaction between organisms is found in aquatic environment, especially amoebae and bacteria. Free-living amoebae are eukaryotic cells found in nature and including several genera such asbacteria . P. aeruginosa kill the amoebae. However, the extracellular bacterium E. coli does not possess TTSS and therefore it is ingested as food by the amoebae.The interaction between bacteria and amoebae is very complex. Output of that interaction is depending on whether the interacted bacterium is extracellular or intracellular and if it possesses a type three secretion system (TTSS) or not since TTSS effector proteins are observed to affect strongly output of the interaction . Extrace F. tularensis multiplies symbiotically inside the amoebae, while TTSS possessing intracellular bacterium E. coli K1 and Shigella flexneri lyse the amoebae according to activation of TTSS [An intracellular bacterium multiplies inside amoeba cell. The intracellular bacterium that does not possess TTSS of TTSS . Vibrio cholerae are able to multiply in water and inside the environmental phagocytic eukaryotes such as free-living amoebae. Therefore, presence of the amoebae in water will enhance presence of bacteria such as V. cholerae [ Acanthamoeba species and V. cholerae were detected from the same water samples collected from different water sources in Sudan [ Legionella species [ V. cholerae and Legionella pneumophila are well known as causative agents for the waterborne diseases cholera and legionellosis.Facultative intracellular bacteria such ascholerae , 28, 29.in Sudan , 30 and  species , 32. V. The protozoa have a double infectious role in public health. They can cause infections by themselves such as amoebiasis, cryptosporidiosis, giardiasis, amoebic encephalitis, and amoebic keratitis and they can act as a vector to their intracellular bacteria to cause multiple infections of protozoa and bacteria . G. intestinalis, Cryptosporidium spp., and Entamoeba spp. were the most commonly reported protozoa associated with enteric infections and were associated mainly with food- and waterborne outbreaks. These enteric protozoa are isolated frequently from diarrheal patients in developing regions such as Asia and Sub-Saharan Africa [ Acanthamoeba from 172 wastewater samples [ A. castellanii, T. gondii, C. owczarzaki, B. hominis, C. muris, and T. vaginitis from zeers found in Gadarif, Juba, and Khartoum.In addition to our findings about the identified protozoa in this paper, Fletcher et al.  reviewedn Africa . However samples . Our stuχ2 test (P > 0.05). Moreover, the prevalence of identified protozoa in Wad Madani compared to those of Gadarif, Juba, and Kordofan was highly statistically significant . But the prevalence of identified protozoa in Wad Madani and Khartoum was not statistically significant; P value was 0.1210.Overall prevalence of detected protozoa by PCR was 9.5% and the prevalence in Gadarif, Juba, Khartoum, Kordofan, and Wad Madani was 14.3%, 14.0%, 9.1%, 14.0%, and 2.8%, respectively. The prevalence of identified protozoa in Gadarif, Juba, Khartoum, and Kordofan was not statistically significant by This result uncovers the danger coming from water to spread infections between populations and our study presented a fast and accurate molecular detection and diagnosis of protozoa species in water.


Excessive alcohol use is assumed to affect maturation of cognitive functioning in adolescence. However, most existing studies that have tested this hypothesis are seriously flawed due to the use of selective groups and/or cross-sectional designs, which limits the ability to draw firm conclusions. This longitudinal study investigated whether patterns of alcohol use predicted differences in maturation of executive functioning in adolescence. Additionally, gender was tested as a possible moderator.We used data from the Tracking Adolescents’ Individual Lives Survey (TRAILS), which comprises a cohort of 2,230 Dutch adolescents. Maturation of executive functioning was measured by assessing the standardized improvement on each of four basic executive functions  between ages 11 and 19. Participants were assigned to one of six (heavy) drinking groups . We conducted linear regression analyses, and adjusted for relevant confounders.B = -0.14, 95% CI [-0.41 to 0.14], working memory: B = -0.03, 95% CI [-0.26 to 0.21], shift attention: B = 0.13, 95% CI [-0.17 to 0.41], sustained attention: B = 0.12, 95% CI [-0.60 to 0.36]. Furthermore, gender was not found to be a significant moderator.The six drinking groups did not reveal significant differences in maturation between drinking groups. E.g., maturation executive functioning of chronic heavy drinkers in comparison to non-drinkers; inhibition: Four years of weekly heavy drinking  did not result in measurable impairments in four basic executive functions. Thus, regular heavy drinking in adolescence does not seem to affect these basic behavioural measures of executive functioning. In adolescence, significant increases in alcohol consumption are usually found, with prevalence rates of last month alcohol use rising from 16.1% at age 12 to 84.8% at age 16 in Dutch adolescents ,4 are asMost studies that compared alcohol abusing adolescents with non-abusing adolescents on a broad range of neurocognitive functions, such as language and general intelligence , attentin = 75 and 40 respectively) have analysed the effects of alcohol use on neurocognitive maturation in adolescence using a longitudinal design with pre- and post-measurements of neurocognitive functioning in a general population [To the best of our knowledge, only three small scale studies , since this set of cognitive control functions mediates the ability to guide and direct behaviour and is therefore essential for success in school and at work . Furthern = 2,230, mean age 11.1 years, SD = 0.56, 49.2% male) , the response rate was 81.4%. At the fourth (T4) wave , the response rate was 70%.The present study used data from the first, second (for descriptive statistics only), third, and fourth wave of the TRacking Adolescents’ Individual Lives Survey (TRAILS). This is a prospective cohort study of Dutch pre-adolescents at age 11. The target sample involved children living in the North of the Netherlands, covering urban and rural areas Demographic information from five municipalities for all inhabitants born between October 1, 1989, and September 30, 1990, in two of the municipalities, and October 1, 1990, and September 30, 1991, in the other three was obtained. These children attended 135 schools that were approached for participation, of which 13 refused to participate (excluding 338 children). Next, parents of eligible children were informed about the study and then invited to participate. After excluding 210 children who were unable to participate because of serious health or language problems, we invited 2,935 eligible children and their parents to enter the study. Seventy-six percent of eligible adolescents and their parents agreed to participate and were enrolled in the study at baseline (T1) ?” up until that time point, on a 5-point scale ranging from 0 to 7 times or more. We dichotomised these answers into ‘never or once’ and ‘twice or more’. At T2-T4, adolescents were asked to report their average drinking habits between the previous data collection wave and the present. They were asked four questions: “On how many week(end) days do you normally drink alcohol” and “On an average week(end) day on which you drink alcohol, how much alcohol  do you drink?” By multiplying and adding the answers, average weekly quantity of alcohol use can be computed. A Dutch standard drink contains 10 grams of alcohol. Furthermore, at T2-T4 adolescents were asked how many times they had been drunk in the last 12 months. We dichotomised these answers into never and once or more. These measures of alcohol use were used as descriptive statistics.Drinking groups: Since the legal age for buying alcohol in the Netherlands was 16 years at time of the data collection, we used data from T3 and T4 to determine group assignment. As to be expected, prevalence rates of frequent heavy drinking (see below) at T2 were indeed very small (<3%). For constructing drinking groups we used measures of both quantity and frequency of alcohol use. The average amount of glasses on a weekend day (since previous measurement wave) was used since weekend quantity of alcohol use has been shown to be a useful and specific measure of alcohol use at this age [this age . We furtnot drinking; those who indicated that they did not consume alcohol on a regular weekend day. Next, for the respondents who consumed alcohol, we set a cut off score at 6 glasses on a weekend day for boys and 5 glasses for girls [drinking, not heavy drinking. For participants scoring above the cut off , we looked at frequency of alcohol consumption. Those scoring above the cut off but drinking on an irregular bases  were considered to be infrequent heavy drinking. Finally, respondents falling above the cut off and drinking regularly  were classified as heavy drinking.In step one, per measurement wave, we started with identifying participants that were or girls . Particinot drinking at T3 and heavy drinking at T4 was longitudinally classified as increasing heavy drinker. For all possible combinations and descriptive statistics, see In step two, these categorizations per measurement wave were combined into longitudinal drinking groups. Participants were divided into six groups: non-drinker, light drinker, infrequent heavy drinker, increasing heavy drinker, decreasing heavy drinker, and chronic heavy drinker. For example, a participant Baseline executive functioning was examined at T1, using three computerised reaction time tasks from the Amsterdam Neuropsychological Tasks (ANT) . The ANTThe use of computerised tasks guarantees standardised assessment while working with reaction times allows detection of subtle improvements in performance. Working memory, inhibition, and shift attention were calculated by subtracting the performance time of a baseline version from a more difficult version of the task. Performance results reflecting more than 50% errors were coded as missing and imputed because they were assumed to reflect either misunderstood instructions or false computer settings, undermining the validity of the testing. In accordance with previous research on neurocognitive maturation , we calcp < .05) he drinks per week on average and how much his or her partner drinks. Delinquent behaviour was measured at T1 Youth Self Report (YSR) [Smoking was also measured at both T3 and T4 by asking: “How many times have you smoked cigarettes during the last month?”. Cannabis use was assessed at T3 and T4 by asking: “How many times have you used weed (marihuana) or hash during the last year?”.In accordance with previous studies , age at upations ). These upations . Paternart (YSR) , consistMultiple data sets in SPSS 20 were imputed, using fully conditional specification (MCMC) with Predictive Mean Matching because there were missing values due to attrition on both predictors and outcome measures . This ap2 with standardized residuals was used to identify observed counts that were significantly different (p < .05) from expected counts. For continuous variables, MANOVA’s with post-hoc tests were used. For these two specific analyses, the five datasets were analysed separately, as pooled analyses are unavailable for these tests.The six drinking groups were validated by comparing them on alcohol related behaviour, such as prevalence of last year drunkenness and drinking in early adolescence. For variables measuring prevalence, Pearson χTo examine the influence of drinking groups on maturation of executive functioning, we conducted four bivariate and multivariate linear regression analyses. Standardised change scores of executive functioning were entered as the dependent variable. Dummy variables of drinking groups were constructed and all dummy variables were entered in the model simultaneously as predictors (non-drinkers served as the reference group). First, we conducted four separate linear regression analyses without confounders. In the second model, we conducted four multivariate linear regression analyses adjusting for control variables. Main effects of drinking groups and drinking patters*gender interaction were entered in separate blocks and interpreted accordingly. To reduce Type 1 error, we set α at < .01. For the regression analyses, data from the pooled dataset were used for interpretation.2 yielded significant differences between groups for prevalence rates of other substance use, drinking in early adolescence, and drunkenness. Non-drinkers and light drinkers scored lower than expected on all variables while the chronic heavy drinkers scored higher on all variables.Descriptive statistics are depicted in p < .001). Parental alcohol use and delinquency scores in pre-adolescence were largest in the frequent heavy drinking groups . Also, those who later would be classified as frequent heavy drinkers consumed significantly more alcohol at age 13 than the future non-drinkers, light drinkers, and infrequent heavy drinkers. Chronic drinkers, as to be expected, drank most at ages 16 and 19 with an average weekly consumption finally exceeding 14 glasses. Taken together, the results indicate significant differences between the drinking groups on all measures of alcohol related behaviour, validating our identification of the six drinking patterns. The patterns become more differentiated as the adolescents get older.A MANOVA indicated significant differences between groups on all continuous measures (Raw scores of baseline (T1) and follow-up (T4) executive functioning and standardized change scores are depicted in The results of the multivariate linear regression analyses are depicted in All analyses were also conducted with observed cases only see  Tables. p>.01). Second, we tested whether the findings changed when drinking groups were formed according to the definition of heavy drinking by the National Institute on Alcohol Abuse and Alcoholism . The analysis did not yield significant results (p>.01). Third, non-drinkers may consist of a non-normative group of adolescents; therefore, we also examined whether the findings changed when we used light drinkers or infrequent heavy drinkers as reference group instead of non-drinkers. The results were not significant (p>.01). Finally, since there are differences in maturation in executive functioning between males and females, we also undertook separate analyses for boys and girls, instead of using gender as a moderator. The results were not significant (p>.01). .Furthermore, we conducted four additional analyses. First, we were interested if the use of a continuous measure of alcohol consumption resulted in different outcomes. Therefore, we conducted linear regression analyses where the average number of glasses per week at T3, at T4, and the sum of these two measures as predictors. This did not result in significant findings , it may seem to be flexible enough to cope with these effects of alcohol at least at the level of behavioural development and neuropsychological maturation. That is not to say that the damaging effects of alcohol can be neglected s Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Solenopsis geminata  and S. invicta . Colonies of these species may produce two different monogyne queen types—claustral queens with heavy abdomens that found colonies independently, and parasitic queens with small abdomens that enter conspecific nests. Claustral and parasitic queens were similarly sized, but the abdomens of claustral queens weighed twice as much as those of their parasitic counterparts. Their heavier abdomens adversely impacted morphological predictors of flight ability, resulting in 32–38% lower flight muscle ratios, 55–63% higher wing loading, and 32–33% higher abdomen drag. In lab experiments maximum flight durations in claustral S. invicta queens decreased by about 18 minutes for every milligram of abdomen mass. Combining our results into a simple fitness tradeoff model, we calculated that an average parasitic S. invicta queen could produce only 1/3 as many worker offspring as a claustral queen, but could fly 4 times as long and have a 17- to 36-fold larger potential colonization area. Investigations of dispersal polymorphisms and their associated tradeoffs promises to shed light on range expansions in invasive species, the evolution of alternative reproductive strategies, and the selective forces driving the recurrent evolution of parasitism in ants.In the Found or Fly (FoF) hypothesis ant queens experience reproduction-dispersal tradeoffs such that queens with heavier abdomens are better at founding colonies but are worse flyers. We tested predictions of FoF in two globally invasive fire ants,  Wing mass density—a measure of stiffness and durability—is total wing mass divided by total wing area (mg/mm2).After processing the specimens we calculated flight morphology metrics for each queen. Flight muscle ratio (FMR)—the ratio of flight muscle mass to body mass—is probably the most important predictor of insect flight performance [ficiency , 50,57.To control for body size differences when comparing flight morphology we first checked whether parasitic and claustral queens differed in head width. We then tested whether parasitic queens had lighter abdomens than claustral queens (A1). To examine how abdomen mass impacts flight morphology (A2) we regressed flight muscle ratio, wing loading and drag against abdomen mass separately for each queen type. We then tested flight morphology predictions (P1 to P5) by comparing queen types .S. invicta queens. We used claustral S. invicta queens because they were reliably available in sufficient numbers, perform well in lab conditions, and are routinely used as models in ant biology [We followed up flight morphology comparisons with a live flight experiment to link morphology to dispersal ability. The experiment was performed from June to July using claustral –r2 = 0.98) and the results would be similar for either measure. After the flights we preserved queens in ethanol and processed them as above to compare their performance to their flight morphology. We removed as an outlier one queen with an anomalously light abdomen who had likely just eclosed and was not prepared for her mating flight, leaving 32 queens for analysis.To examine how abdomen mass impacts flight endurance (P6) we observed 33 queens from three colonies during tethered flight [S. invicta, as its rate of spread in the United States  has exc48 km/yr , 65), du48 km/yr , 66. Desr2 = 0.13), likely because of the unrealistic limits of the flight chamber and method of flight initiation (dropping from a height versus taking off from a surface), and we excluded it from our results as uninformative.As an additional measure of flight performance we attempted to measure queen flight distances when dropped. Fifty-six queens were dropped from a height of 170 cm above the center of a 2 x 2 meter chamber, and their resultant flight distances measured. This experiment detected a possible unimodal relationship between abdomen mass and flight distance. The explanatory power, however, was low . For the tethered flight experiment, flight muscle ratio was log transformed to meet normality assumptions. Quantile regressions were performed with the quantreg R package [p-values of regressions of flight morphology versus abdomen mass within queen types, and to regressions of flight duration versus morphology.All statistics were performed in R [S. geminata, 38 parasitic S. geminata, 58 claustral S. invicta, and 33 parasitic S. invicta, S. geminata  or S. invicta . Despite having similar body sizes, the abdomens of claustral S. geminata queens (A1) were 2.3 times heavier than those of parasitic queens . Likewise, abdomens of claustral S. invicta averaged nearly double the weight of their parasitic counterparts . We obtained similar results when comparing averages among colonies  or S. invicta , and abdomens of claustral queens were 2.5 times heavier than parasitic queens in S. geminata  and 2.1 times greater in S. invicta . Within each queen type heavier abdomens adversely impacted flight morphology (A2) by decreasing flight muscle ratio by 11 to 53%, increasing wing loading by 35 to 122%, and increasing drag by 23 to 95% over their respective ranges of abdomen mass , and those of claustral S. invicta queens 38% lower , than their parasitic counterparts  and 55% higher in claustral S. invicta  than in parasitic queens of the same species  and 33% higher drag in claustral S. invicta  than in parasitic queens  and 42% lower in claustral S. invicta  than in their parasitic counterparts  and 63% higher in claustral S. invicta  than in parasitic queens  and 43% higher in claustral S. invicta  than in their parasitic counterparts  of claustral terparts . Likewis species . Larger c queens . The samterparts . Colony c queens . Finallyterparts . These fS. geminata wing areas (P4) were 16% larger  and their forewings 8% longer  than in parasitic queens  and 6% longer  than in parasitic queens (S. invicta queens (P5), in contrast, had slightly smaller  and shorter wings  than parasitic queens  and 1% difference in forewing length (0.09 mm)—and disappeared when comparing colony averages . Aspect ratios did not differ among queen types in either species  and neither did wing mass density in S. geminata . These similarities held when comparing colony averages. Colony average aspect ratios did not differ among queen types in either species , and neither did wing mass density in S. geminata . Individual claustral S. invicta queens, on the other hand, had lighter wings than parasitic queens , but again the difference is slight (0.0012 mg/mm2) and disappeared when comparing colony averages .Wing morphology also differed among queen types as predicted. Claustral c queens . Similarc queens . Claustrc queens . The difp = 0.45), log-transformed flight muscle ratio (ANOVA p = 0.18), abdomen mass , or wing loading , nor was colony identity a significant factor in any of the three quantile regressions , and we therefore pooled the data. Among individual queens, the range of queen flight durations (P6) decreased with abdomen mass, so that the heaviest queens flew only for short time periods but light queens could have long or short flights , resulting in a 6-fold increase in flight range (3420 m versus 540 m). These predicted flight ranges agree with independent estimates that most claustral fire ant queens fly only a few hundred meters and for less than half an hour .Claustral and parasitic oduction . All elsSolenopsis geminata and S. invicta. The heavier abdomens of claustral queens . Claustral queens, in contrast, can potentially found a colony in any vacant patch of soil. If parasitic queens experience greater dispersal ability, it would grant them larger search areas and more search time to locate potential hosts. Assuming a purely horizontal flight and a constant maximum flight speed of 1.5 ms-1 [2. If the same flight relationships hold in both queen types, the average parasitic queen could fly for 3,800 seconds and have a potential colonization area of 102 km2—a 17-fold difference. Using the lower and perhaps more realistic flight speeds of 0.9 and 0.6 ms-1 for parasitic and claustral queens, the difference more than doubles to 36-fold (36 versus 1 km2). Models of the reproductive success of the two queen types [,Parasitic fire ant queens require specific nest sites—orphaned host colonies—that occur at low densities across the landscape Click here for additional data file.


Thylacinus cynocephalus) was Australia’s largest marsupial carnivore until its extinction within the last century. There remains considerable interest and debate regarding the biology of this species. Studies of thylacine biology are now limited to preserved specimens, and parts thereof, as well as written historical accounts of its biology. This study describes the development of the immune tissues of a pouch young thylacine, one of only eleven in existence, and the only specimen to be histologically sectioned. The appearance of the immune tissue of the developing pouch young thylacine is compared to the immune tissues of extant marsupials, providing insights into the immunity, biology and ecology of the extinct thylacine.The thylacine ( Thylacinus cynocephalus) is extinct [th September, 1936 at Beaumaris Zoo in Hobart, Tasmania [The thylacine  [Bettongia penicillata) [Sarcophilus harrisii) . The rate at which Tasmanian devil pouch young head lengths increase is 0.4mm/day for at least the first 50 days postpartum . If it is assumed that the thylacine also had an unchanging incremental daily increase in head length, and given that the largest adult Tasmanian devils weighed on average around 12.5kg [The age of the pouch young specimens was estimated based on the following assumptions. Marsupials have an unchanging incremental daily increase in head length for the first 50 days of their pouch life, for example brushtail possums (lpecula) , brush-tcillata) , and Tasd 12.5kg , and thyd 12.5kg , double Although we are unable to measure the head length of the sectioned thylacine pouch young, the head lengths were provided in Sleightholme et al.  for the One male pouch young (C5754), having been preserved in pure alcohol  was decaThe histological slides from this specimen  were examined using an Olympus CX31 microscope  and photomicrographs taken using an Olympus DP71 camera. Specifically, the thymus, bone marrow, spleen, gastrointestinal tract, liver, lungs and kidneys were examined. The thymus and bone marrow were examined as they are primary lymphoid tissues, whilst the spleen is a secondary lymphoid tissue along with the gastrointestinal tract and lungs. The gastrointestinal tract and lungs are potential sites for mucosal-associated lymphoid tissues, specifically, gut-associated and bronchus-associated lymphoid tissues, respectively. The liver was investigated because it is a primary site of haematopoiesis shortly after birth in marsupials . OnThe thylacine pouch young was processed for histology, sectioned and stained in 1994, having been stored in pure alcohol for nearly a century. The decades of long term storage in preservative, time since sectioning and staining had impacted the quality of the slides. It was difficult to discern specific cell types when viewed using high power magnification, with some sections showing evidence of other processing artifacts and damage, presumably also due to long term storage and age of the tissue generally. Despite some damage occurring to the sections and slides, most tissues could be described in some detail.Although it was not possible to estimate the head length of this thylacine pouch young specimen , the mean head length of its three siblings was 36mm . The ageOnly a thoracic thymus was identified in the thorax section of the pouch young thylacine, with no cervical thymus located. The lobes and lobules of the thymus were separated by connective tissue septa . These sThe early and mature lymphocytes of the thymus were uniform in appearance and size. Rare Hassall’s corpuscles were observed in the medullary regions of the thymus tissue sections .Bone marrow was present in the long bones, the ribs and bones of the axial skeleton. Islands of haematopoietic areas were observed in the marrow spaces. These appeared to be mainly erythroblastic, although it was difficult to discern many of the cells.Some sections of bone marrow were beginning to involute, with adipocytes present, despite many areas of haematopoiesis still evident . Other sThe bones had some calcification occurring and some compact bone was visible. Some sections showed active growth of the bones and developing trabeculae were present. At a higher magnification, maturing cartilage cells were observed towards the bone epiphysis . ChondroThe bone collar was composed of compact bone, and the trabeculae were visible. The periosteum had shrunk from the bone collar presumably due to the age and length of time the tissue had been in preservative solution, and tissue processing artifacts.The liver had a characteristically mature appearance. There were hepatocytes, central and portal veins, and hepatic arteries visible , but no Due to the pouch young being sectioned transversely, only very thin sections of spleen could be viewed. The spleen was relatively uniform in appearance with some areas of white pulp beginning to appear . These aNo mucosal associated lymphoid tissue was evident. Specifically, neither bronchus-associated lymphoid tissue in the lungs, nor gut-associated lymphoid tissues in the gastrointestinal tract were evident.The lungs were composed of large sacs, with some smaller saccules present at the extremities of the tissue . Small aThe gastrointestinal tract itself was essentially a thin-walled sac. There were no villi, or intestinal glands and the mucosal and submucosal layers had not yet formed .Glomeruli or renal corpuscles were visible with areas of white capillary loops and podocysts . Each glAs one of only a few pouch young thylacines in existence, and the only one to have been serially sectioned, this specimen provided invaluable histological descriptions of the immune tissues of an extinct marsupial. It allowed a comparison of these tissues to those of some extant marsupials and provided insights into the biology of this extinct marsupial, albeit relying on a limited sample size.Dasyurus maculatus) [Myrmecobius fasciatus), and generally a paucity of available literature on many of these key milestones for most marsupials. Only one study, for example, has described the developmental appearance of Tasmanian devil pouch young in captive bred animals of known age, but it was limited to one litter [The age of the pouch young in this study is unknown. Le Souef & Burrell  suggest culatus) . Howevere litter .Phascogale calura), has claws present on all toes by day 30, fine fur appearing by around 34 days postpartum, and the young first detaching from the teat at day 42 postpartum [Boardman  providedstpartum , whilst stpartum . Therefostpartum , the pouIn contrast to the specimen examined in this study, Johnstone  describeIn this study of the pouch young thylacine only a thoracic thymus was observed. The lack of a second cervical thymus in the pouch young thylacine is similar to the findings in all other polyprotodont marsupials studied to date  and furtIn marsupials the thymus is the first lymphoid tissue to mature . PrIn marsupials like other vertebrates, lymphocytes differentiate in the cortical regions of the thymus and then move to the medullary regions. In marsupials this has been demonstrated with the use of a cross-species reactive antibody to the CD3ε chain developed by Jones et al. , and useOnly mature cells in the bone marrow could be identified with confidence. Ideally, fresh bone marrow samples and cross-species reactive antibodies would allow for less mature cells to be identified; however these options are currently not possible  [Comparatively little is known about bone marrow in marsupials, with only four species being investigated to date , 32, 33.Macropus eugenii) [Isodoon macrourus) [Given the relative maturity of the bone marrow in the pouch young thylacine specimen, it was not unlikely to see a lack of haematopoiesis occurring in the liver. Marsupials are born with a haematopoietic liver rich in erythroblasts and to a lesser extent some granulocytopoiesis occurring , 34, 35.eugenii) , day 60 crourus) .Didelphis virginiana) spleen, for example, lacked sinusoids, and trabeculae arteries were rare when described by Hayes [Although not fully developed the spleen was starting to mature, as there were large areas of red pulp, and some white pulp observed. Hartwig & Hartwig  suggesteby Hayes .The mature spleen is both an important site of immune defence, and storage site for the rapid release of erythrocytes. Hartwig & Hartwig  describeAlthough no lymph nodes were observed in this study, it is highly likely that they were present in some form. When Johnstone  describeLymph nodes have previously been difficult to locate in a range of marsupials , 42, 43.Although no lymphoid tissues were evident in the lungs or gut of the pouch young thylacine, this was not unexpected given the developmental stage of the specimen. In all marsupials to date the mucosal-associated lymphoid tissues are the last immune tissues to develop, and this also appears to be the case in the thylacine.Whereas in peripheral lymphoid tissues such as the spleen and lymph nodes antigens are required for the tissues to fully mature, this is not always the case for bronchus-associated lymphoid tissues (BALT), with the appearance and frequency of BALT differing between and among species, and may or may not require antigen stimulation . BienensTrichosurus vulpecula) or northern quolls  [The thylacine pouch young lungs were more developed than those of newborn brushtail possums (lucatus) . The newlucatus) .Isoodon macrourus) lung was similar to the adult in appearance at 61 days after birth and corresponded to the time the young possum left the pouch. In addition, although Runciman et al. [The Virginian opossum does not have well developed alveoli until after 85 days postpartum when the pouch young starts to the leave the pouch intermittently . Cooke &n et al.  describen et al. . The thyn et al. . Based on et al. , 55, 56.Gemmell & Nelson  also sugin utero in eutherians, hence could be considered as an ‘external gestation period’. During the second lactation or switch phase (80–120 days) the milk becomes more similar in composition to that of eutherian mammals. The third or late lactation phase occurs from 120–200 days postpartum in the possum . The swiewed in . In the phase 80–0 days thKidneys were included in the description as they may provide some insights into the water conservation capacity of the thylacine, even though they are not immune tissues in mammals. Many Australian marsupials living in arid zones have thicker renal medullas  capable of producing urine of a higher concentration than those living in moister environments . A thickDespite the need for an adult kidney to accurately determine the relative renal thickness, and hence water conservation ability of the thylacine kidneys, the recorded distribution of the thylacine in Tasmania and New Guinea (currently moist habitats), suggests that thinner renal medullas are likely. However, thylacines were also previously distributed throughout mainland Australia  includinUnfortunately, the extinction of the thylacine precludes the collection and examination of additional samples for comparative purposes from the species. Nevertheless the description of the immune tissues in this one thylacine pouch young provides insights into the development of the pouch young thylacine immune system, and thylacine biology.


Proliferating trophoblast stem cells (TSCs) can differentiate into nonproliferating but viable trophoblast giant cells (TGCs) that are resistant to DNA damage induced apoptosis. Differentiation is associated with selective up-regulation of the Cip/Kip cyclin-dependent kinase inhibitors p57 and p21; expression of p27 remains constant. Previous studies showed that p57 localizes to the nucleus in TGCs where it is essential for endoreplication. Here we show that p27 also remains localized to the nucleus during TSC differentiation where it complements the role of p57. Unexpectedly, p21 localized to the cytoplasm where it was maintained throughout both the G- and S-phases of endocycles, and where it prevented DNA damage induced apoptosis. This unusual status for a Cip/Kip protein was dependent on site-specific phosphorylation of p21 by the Akt1 kinase that is also up-regulated in TGCs. Although cytoplasmic p21 is widespread among cancer cells, among normal cells it has been observed only in monocytes. The fact that it also occurs in TGCs reveals that p57 and p21 serve nonredundant functions, and suggests that the role of p21 in suppressing apoptosis is restricted to terminally differentiated cells. Genes that regulate cell proliferation and differentiation during mammalian development are often deregulated in human cancer, thereby permitting either unrestrained cell proliferation or increased survivability. One example is the phosphatidylinositol-3 kinase (PI3K) signal transduction pathway that influences cell proliferation, survival, metabolism, and metastasis Remarkably, during normal mammalian development, cytoplasmic localization of p21 has been reported only in monocytes where it promotes resistance to apoptosis in vitro by TSCs.Here we show that Akt-dependent cytoplasmic localization of p21 and its ability to suppress DNA damage induced apoptosis also occurs when trophoblast stem cells (TSCs) differentiate into trophoblast giant cells (TGCs). The rapidly proliferating TSCs exit their mitotic cell cycle in response to environmental signals and initiate multiple rounds of endoreplication (termed ‘endocycles’) to produce the nonproliferating, polyploid, viable TGCs that are essential for embryo implantation and placental development TSCs are derived from the trophectoderm of the blastocyst and give rise exclusively to all of the trophoblast lineages in the placenta The p27 and p57 proteins are essential for normal mouse development only after midgestation Based on RT-PCR and Western immuno-blotting analyses, previous studies have shown that all three Cip/Kip genes are transcribed in both TSCs and TGCs, but the p21 and p57 proteins are expressed primarily, if not exclusively, in TGCs In contrast with p27, only trace amounts of p57 protein were detected in TSC populations , and thiIn some TGCs, EdU, anti-p27 and anti-p57 either did not stain the nucleus or stained the nucleus only lightly . This rep21−/− TGCs, whereas p57 protein was readily detected in the nuclei of p21−/− TGCs. A second distinction between p21 and p57 was that p21 protein was present throughout the TGC population, whereas p57 protein was present only in the nuclei of G-phase cells. Immuno-staining of wild-type TGCs, in which replicating DNA had been pulse-labeled with BrdU, confirmed that p21 protein was present in the cytoplasm of S-phase as well as G-phase cells  increased the sensitivity of TGCs to etoposide , thereby stabilizing the protein and localizing it to the cytoplasm in a variety of human cancer cells CIP/KIP genes are transcribed in trophoblast cells, but in TSCs the Chk1 kinase selectively phosphorylates p57 and p21 proteins, thereby targeting them for ubiquitin-dependent degradation TGCs express low levels of M-phase genes and high levels of genes required for placenta function, including p57 The fact that p27 is expressed at comparable levels in both TSCs and TGCs  suggestsThe p27 protein is expressed ubiquitously throughout mouse development, whereas expression of the p57 protein is confined primarily to maintaining either terminal cell differentiation As for the p21 protein, the results presented here reveal that p21 plays the same role in TGCs as it does in human cancer cells. In human cancer cells, Akt1 phosphorylates p21 at T145 and S146, thereby preventing its binding to PCNA (the processivity cofactor for DNA polymerases δ and ε), decreasing its inhibitory effect on cyclin CDK complexes, increasing p21 protein stability, localizing it to the cytoplasm, and protecting cells from apoptosis The results presented here also reveal that TSC differentiation is governed by three protein kinases: Cdk1, Chk1 and Akt1. Cdk1 activity is essential for mitotic cell cycles; selective inhibition of Cdk1 in TSCs triggers endoreplication and differentiation into TGCs Remarkably, both Chk1 and Akt1 phosphorylate the same amino acids in p21, but the fate of the phosphorylated protein is cell-type specific. Phosphorylated p21 in TSCs is targeted for ubiquitin dependent degradation p21−/− and p57−/− TSC lines were previously derived and characterized Mus musculus p57 , p21 , and p27 .Wild-type, Cdkn1A/p21/Cip1 cDNA was amplified by PCR  to produce recombinant p21 proteins tagged with cMyc and His6 at their C-termini. NIH3T3 cells were co-transfected with pcDNA/6TR (containing the tetracycline repressor gene) and pcDNA/TO/p21-myc-His plasmids (ratio 4∶1), as indicated in the figure legends. Twenty-four hours after transfection, tetracycline (1 µg/ml) was added to the medium for 18 hours to induce p21 expression. Alternatively pcDNA/TO-p21-myc-His plasmids were transfected alone into NIH3T3. Twenty-four hours post-transfection, the cells were fixed and stained for p21 protein. For bacterial expression, wild-type and mutant p21 genes were cloned into pET-15b (Novagen) to produce N-terminal His6-p21 protein in Escherichia coli. Recombinant proteins were purified as described previously Mouse d by PCR  and clonWestern immuno-blotting and immuno-fluorescence were performed as previously described in vitro kinase assay previously described Recombinant mouse p21 proteins were purified from bacteria and tested as Akt1 (Sigma SRP-0353-10UG) substrates using an Akt1 and p27 shRNA oligonucleotides were designed against three different sequences within their respective target RNA  and clon