Despite the importance of behaviour in conservation biology, there have been few studies that address behaviour in areas such as invasion ecology. There is an urgent need to identify specific traits that facilitate the establishment and spread of alien species to prevent biological invasions and their impact on biodiversity. Changes in antipredatory behaviour in captivity have been proposed to explain the higher invasiveness of wild-caught exotic species. We experimentally tested this hypothesis by assessing the response of wild-caught and captive-bred cage birds facing an approaching predator and their ability to escape from human capture, using species available in the Spanish pet market. Results showed the loss of antipredatory responses and escape abilities in captive-bred birds compared with wild-caught ones. An intraspecific comparison between wild-caught and the first generation of captive-bred birds pointed to a rapid behavioural loss in captivity  rather than to differences among species (evolutionary exposure). In the context of current avian invasions, the proportion of individuals showing antipredatory responses within a species was positively related to the likelihood of the species being found escaped and breeding in the wild. These results offer a link between behaviour, fitness, and the invasion syndrome in birds. Exotic species represent one of the major threats to native biodiversity and the correct functioning of ecosystems, with worldwide social and economic implications169101213Contrary to past deliberate introductions, recent (and current) avian invasions mainly arise from the accidental escape of globally traded cage birds15161718Another potential explanation for the higher invasiveness of wild-caught species is the maintenance of antipredatory behaviours, important components of coping in the wild, which could be lost in species bred in captivity14222524Here, we experimentally test the hypothesis that wild-caught and captive-bred individuals differ in their antipredatory responses using a large sample of the exotic cage bird species most commonly traded and maintained in captivity 152 = 215.38, df = 1, p < 0.0001), while controlling for body size  and taxonomic effects.We experimentally assessed the antipredatory behaviour in 422 individuals (157 wild-caught and 265 captive-bred birds) belonging to 93 exotic species (57 species of parrots and 36 species of passerines) available in the pet market. In a first interspecific approach, 94.3% of individuals (n = 157) from wild-caught species (n = 39) showed antipredatory responses, while only 10.9% of individuals (n = 210) from captive-bred species (n = 52) responded to the predator model. Wild-caught species were more prone to eliciting antipredator responses when facing a predator than captive-bred ones , supporting the hypothesis that the loss of this behaviour is related to a bird’s origin but not to interspecific differences.Due to the difficulty of obtaining first generation birds born in captivity from wild-caught parents, sample sizes were reduced to 140 individuals from 12 species of parrots and 4 species of passerines when we evaluated intraspecific changes in antipredatory behaviour (S1). Most of the wild-caught individuals  responded to the predator, contrasting with the low percentage  among the conspecifics born in captivity. It is worth noting that changes in the response to the predator related to captivity were nearly identical when comparing this intraspecific approach  to the i2 = 37.28, p < 0.001, n = 55). This, together with the differential responses of wild-caught and captive-bred conspecifics to the predator model, allowed us to be confident that the above results were indicative of antipredatory behaviour instead of fear of an approaching object.Of the wild-caught individuals introduced into the predation-risk simulator and faced with a plastic object similar in size and colour to the predator , none showed antipredatory responses. This contrasts with the 94.6% of conspecifics of the same origin (wild-caught) that were exposed to the stuffed predator model . As shown in Capture time was measured for 228 individuals belonging to 40 species of passerines (S2). The time taken to capture by hand was shorter for captive-bred birds than for wild-caught birds . These percentages were nearly the inverse to the percentages of captive-bred and wild-caught birds experimentally responding to the predator in both the inter- and intraspecific approaches (see above), and thus inferences from our experimental approach are supported by observations of the outcome of actual escapes from cages.After accidentally escaping from cages, 30 out of 33 captive-bred birds were recaptured  while 21 out of 23 wild-caught birds eluded recapture. The percentage of recaptured captive-bred birds (90.91%) was significantly higher than that for wild-caught ones  of breeding in captivity. On the other hand, all individuals available in the pet market from many other species were imported after being caught in the wild , as shown for Spain in the CITES trade database where the wild-caught or captive-bred origin of traded individuals is reported (www.cites.org). Individuals from these species were sold with accompanying CITES documentation that demonstrated their origin. Finally, there were some predominantly wild-caught species for which some captive-bred individuals were available in the pet market due to the recent efforts of aviculturists to breed them in captivity. We made an effort to sample some of those first-generation (F1) captive-bred individuals for our intraspecific approach. As an example, the monk parakeet (Myiopsitta monachus) was traded worldwide and imported by Spain by the thousands as wild-caught individualswww.cites.org), we were able to get a few individuals bred in captivity by aviculturists. Those captive-bred individuals were easily recognised as they are individually marked as chicks with un-removable closed bands with the year of birth and the code of the aviculturist engraved. Moreover, the price of captive-bred individuals was 2–3 times higher than that of their wild-caught conspecifics, reflecting their rarity in captivity. We classified these species with a mixed origin of individuals as predominantly wild-caught, since wild-caught individuals broadly represented >90 % of individuals available in the market. The origin of all sampled individuals and the main origin of all species sampled are shown in S1 and S2.Wild-caught and captive-bred exotic birds of the Order Psittaciformes and Passeriformes, commonly used as cage birds1515Attending to the individual CITES documentation, wild-caught birds were collected in the field at least one year before initiating our experiments and held in captivity until that time. Both captive-bred and wild-caught birds were at least 1 year old.predation-risk simulator (Buteo buteo) hidden behind an opaque curtain  as study models to record time (in seconds) required to capture by hand focal individual birds in a standard cage of 0.5 × 0.4 × 0.5 m. This size allowed the researcher to easily access all corners of the cage, thus avoiding biases related to handling facilities. Parrots were not included in this experiment since their movements may have been restricted in this reduced space, and variability in aggressiveness and biting defence among species may have affected the willingness and ability of the researcher to capture them. All trials were performed by the same researcher (JLT).During a ten-year period (2005-2015), 47 out of ca. 1,200 exotic parrots and passerines escaped accidentally from the experimental facilities and aviaries, when some cages were accidentally opened or broken due to different causes. Moreover, nine free-living exotic birds were attracted to the aviaries by the captive birds. We tried to catch all escaped birds using a handled net, while some birds were killed by a small-sized dog in the surrounding garden before we had the opportunity to capture them. We used this information, from 18 species of parrots and 18 species of passerines, as additional evidence supporting the hypothesis of a greater ability to elude capture in wild-caught than in captive-bred pet species once escaped from cages.We tested whether the antipredatory behaviour of the experimentally tested exotic species was linked to their invasiveness, using information on invasive exotic birds in Spain4649predation-risk simulator (see before) and included only those belonging to the main origin of each species . For the example of the monk parakeet, only the 11 wild-caught individuals (S1) were used to calculate the proportion of individuals showing antipredatory behaviour.We used Generalized Linear Mixed Models (GLMM) to establish the effect of captivity on the probability of detecting differences in antipredatory responses between wild-caught and captive bred birds . Using sample size as denominator in the binomial GLMMs allowed us to satisfactorily compare prevalence data  when sample sizes are usually small and variable among speciesJLT held the Spanish certificates (B and C) that legally allow the design and conduct of experimental research work using live animals. Work in captivity was done under institutional approval of the competent Spanish wildlife agency  and from the Ethic Committee of CSIC (CEBA-EBD-12-48), in the authorized centre for experimental avian research SE/16/U (REGA ES410910008016). Methods were carried out in accordance with the approved guidelines.How to cite this article: Carrete, M. and Tella, J. L. Rapid loss of antipredatory behaviour in captive-bred birds is linked to current avian invasions. Sci. Rep. 5, 18274; doi: 10.1038/srep18274 (2015).


Most ERP studies using overt speech production tasks have analyzed fixed time-windows of stimulus-aligned ERPs, not exceeding the fastest production latency. These fixed ERP time-windows may cover the whole speech planning process for fast trials or participants, but only part of the planning processes for trials or participants with production latencies exceeding the analyzed period. Two core questions thus emerge when analysing fixed time-windows in overt language production, namely (1) to what extent do ERPs capture “later” encoding processes, especially phonological and phonetic encoding, and (2) how to account for different production latencies across conditions or individuals. Here we review a methodological approach combining waveform and topographic analyses on integrated stimulus- and response-aligned ERPs according to response latencies in each participant and condition. Then we illustrate the approach with a picture naming task. Crucially for the purpose of the methodological illustration, the separate analysis of fixed stimulus- and response-locked ERPs led to a counter-intuitive result . Coherent results with longer lasting periods of topographic stability in the slower condition only appeared when combining stimulus- and response-aligned ERPs in order to cover the actual word planning time-windows. Thus this combined analysis enabled to disentangle the possible interpretations of the neurophysiological processes underlying differences across conditions observed on waveforms and on topographies in the fixed ERP periods. When speakers produce a word, they transform an abstract concept into articulated speech sounds in less than 1 s. This transformation involves a series of mental operations which have been modeled thanks to psycholinguistic experimental chronometric investigations  and moreThe first ERP studies on speech production have used metalinguistic paradigms to avoid overt production because of possible artifacts during motor preparation or execution see . In thesfixed time-window of stimulus- or response-aligned epochs has been analyzed in most studies.Overt “immediate” (not delayed) production paradigms are now widely used in ERP investigations. The most current strategy to avoid artifacts due to motor execution consists in analyzing epochs within a time-period not extending beyond the shortest response latency . Other aA core question when analyzing fixed ERP time-windows in a domain in which processing times vary largely across trials and participants is which underlying processes are being captured in the ERP signal which is distant from the lock point. For instance, when stimulus-aligned epochs of 500 ms , 600 ms Figure 1. For instance, fixed stimulus-aligned ERPs of 500 ms cover the whole speech planning period for the fastest trials or participants (s1), but not for slower trials or participants . The same holds for backward response-aligned ERPs. One way of obtaining ERP data which cover the whole speech planning period in overt speech production tasks is to combine stimulus- and response-aligned ERPs according to the production latency.It is well known that production latencies can vary in typical picture naming tasks and that the slowest trials or participants may be twice as long as the fastest participants or trials. The problem this variability runs into when analysing fixed ERP time-windows has been described in the previous section and is further illustrated in The approach combining stimulus-aligned and response-aligned (backward) ERPs according to each subject’s production latency by removing the overlapping signal has been introduced by Besides studies involving atypical populations, the comparison across participants with variable production latencies is an issue also when typical populations are investigated. In a study by To illustrate the advantages of the procedure of ERP analysis from stimulus to response we recorded EEG during a picture naming task with pictures corresponding to high or low frequency words and to early- and late-acquired words.The participants were 18 undergraduate students  who underwent this picture naming task among other tasks for course credits. They were all right-handed and French speaking with no reported neurological disease.Table 1). Given the difficulty of orthogonalizing these two factors, two different experimental sets were embedded. 56 stimuli corresponded to 28 high and 28 low frequency words, matched on a set of other linguistic and pictorial factors. A second embedded set of 52 stimuli corresponded to 26 early-acquired and 26 late-acquired words, also matched on the other pertinent factors. The properties of the two sub-sets of stimuli are detailed in Table 1.The stimuli were 60 black and white drawings and their corresponding modal names from two French databases . Two psyParticipants sat approximately 70 cm in front of a PC screen in a sound-proof dimly lit room. The presentation of the trials was controlled by the E-Prime software (E-Studio). The experiment started with a familiarization phase, in which all stimuli were presented once on the screen preceded by their modal name. Then, the 60 experimental stimuli were presented twice in pseudo-random order in two blocks with a short break in between. An experimental trial started with a fixation cross presented for 500 ms, then a picture appeared on the screen and remained for 1800 ms. The participants were asked to produce the word corresponding to the picture as fast and accurate as possible. A blank screen lasting 2000 ms was displayed before the next trial. Pictures were presented in constant size (245 × 245) on a gray screen.Word productions were digitized and production latencies (the time separating the onset of the picture and the articulation onset) were systematically checked with a speech analysis software . Offline, ERPs were then band-pass filtered to 0.2–30 Hz and recalculated against the average reference. ERP epochs with amplitudes exceeding ±100 μV were automatically rejected. Stimulus-aligned  epochs of 500 ms and response-aligned epochs of 500 ms  were extracted. Each trial was visually inspected, and epochs contaminated by eye blinks, movements or other noise artifacts were rejected and excluded from averaging. Only trials with correct productions and valid RTs were retained and only trials with both response-aligned and its corresponding stimulus-aligned uncontaminated epochs were retained. Artifact electrodes were interpolated using 3-D splines interpolation  from 128 channels covering the entire scalp. Signals were sampled at 512 Hz with an online band-pass filter set to 0.16–100 Hz. The custom online reference of the system is the common mode sense (CMS active electrode) – driven right leg (CMS-DRL) which drives the average potentials as close as possible to the amplifier zero .Combining stimulus-aligned and response-aligned ERPs. For the topographic analyses (see below), the stimulus- and response-aligned ERPs were combined according to their corresponding production latency. To assure that there was no temporal overlap between stimulus- and response-aligned data , time periods of overlap were removed from response-aligned ERPs. For instance, for an average production latency of 850 ms the 500 ms stimulus-aligned ERP would be combined with response-aligned ERP data from -350 to -100 ms . In other words, the 250 ms overlapping ERP time-window would be removed from the response-aligned signal. This procedure was applied to the group averaged data per condition and on the individual ERs for each condition. As a result, the combination of stimulus- and response-aligned individual and group averaged ERPs covered the exact time interval from picture presentation to 100 ms before vocal onset in each condition. It should be noted that a similar procedure may also be applied to single stimulus- and response-aligned ERPs in case of single trial ERP analysis.The ERPs were first subjected to a sampling point-wise ERP waveform analysis to determine the time periods presenting local differences in ERP amplitudes between conditions. Then, a spatio-temporal segmentation was performed on the group-averaged ERPs to determine topographic differences across conditions and statistically validate them in the responses of single participants as described below.ERP waveform and global field power analyses. Electrode-wise and sampling point-wise paired t-tests served to compare the local ERP amplitudes across conditions separately on the 500 ms stimulus-aligned ERPs and on response-aligned ERPs of 300 ms (from -400 to -100 ms preceding articulation). To correct for multiple comparisons, only ERP differences present in at least five adjacent electrodes and lasting ≥20 ms were retained with a conservative alpha criterion of 0.01.Global topographic erp pattern analysis . These analyses are aimed to capture the global topographic differences across conditions. The global electric field does not change randomly either in EEG or in stimulus-elicited ERP signals; it rather remains stable over periods of tens of milliseconds before changing rapidly into a different stable configuration of the potential map . This procedure is called TANOVA ; it involves a non-parametric randomization test to the global dissimilarity between two electric fields. The global dissimilarity is a quantification of topographic differences between two electric fields independent of their strength ranging from 0 to 2 . The perThen a global topographic ERP (map) pattern analysis called spatio-temporal segmentation was run. This procedure segments ERPs in periods of electrophysiological stability  by compressing the variability of ERPs in a series of template maps which summarize the data and serve to determine which topographic template best explains participants’ ERP responses to each experimental condition . The spaThen the pattern of topographic map templates observed in the group-averaged data was statistically tested by comparing each of these map templates with the moment-by-moment scalp topography (from stimulus onset to 100 ms before articulation) of individual ERPs in each condition. This procedure referred to as “fitting” allows one to establish how well a topographic template map explains single participant responses in each condition. Each data sampling point in each condition was labeled according to the template map with which it best correlated spatially, yielding a measure of map presence in milliseconds and of global explained variance. These measures are then used to statistically test topographic differences across conditions.Mean production accuracy was 95.8% and mean production latency  was 858 ms (range of mean RT per participant per condition: 703–1096).t(17) = -9.8, p < 0.0001], whereas RTs did not vary for high versus low frequency words (see Table 2).Early-acquired words were initialized 63 ms faster than late-acquired words [Figure 2). In the analysis of early- and late-acquired words diverging amplitudes appeared on more than 20 electrodes from about 400–450 ms in the stimulus-aligned ERPs and in two time windows in the response-aligned ERPs: between 380 and 320 ms before articulation onset and around 150 ms before articulation . The global dissimilarity (TANOVA) analysis also revealed significant differences between early- and late-acquired words in the same time-windows.The comparison between high and low frequency items revealed no differences on amplitudes or on global dissimilarity neither on the response-aligned nor on the stimulus-aligned ERPs .The spatio-temporal segmentation applied to the four grand averaged ERPs from picture onset to 100 ms before voice onset revealed eight different topographic patterns accounting for the 97% of the variance. This specific cluster corresponded to a minimum peak in the cross validation criterion and a maximum in the modified Krzanowski–Lai. The distribution of the periods of stable topographic activity are displayed in Figure 2 (around 400–450 ms) different topographic maps appeared between early- and late-acquired words. These differences seem to be due to a shift of a period of global electrophysiological stability (map labeled “E” lasting shorter in early- than in late-acquired words). To verify whether this shift is consistent across participants, maps E and F were fitted in the time-period between 300 and 500 ms in each individual data per condition. The mean duration of map “E” across participants in this fitting period was 94 ms for early-acquired words with 41% of the global explained variance (GEV) and 121 ms with 51% of explained variance for late-acquired words . If we refer to the fixed time-windows of both the stimulus-aligned or the response-aligned ERPs  the following period of stable electrophysiological pattern (map “F”) seems to last longer in the early-acquired than in the late-acquired condition . However, the situation looks differently when considering the combined stimulus- and response-aligned ERPs covering the exact production latencies in each condition . The stable topographic pattern “F” is shifted in the late-acquired condition because of longer duration of the previous pattern “E,” but it has absolute similar duration across conditions. Indeed, the fitting of maps E, F, G, H from 300 ms after picture onset to 100 ms before articulation for each participant and condition revealed no significant difference in the overall duration of map “F” across early- and late-acquired words . Finally, the last period of topographic stability  had longer duration for late-acquired words than for early-acquired words , but had similar GEV .In the first period corresponding to different amplitudes and TANOVA in Here we presented the problems related with the analysis of fixed ERP time-windows in speech production studies with variable initializing latencies. We then proposed a method combining stimulus-locked and response-locked ERPs in order to cover the whole speech planning process according to production speed and we illustrated this approach coupled with topographic analysis by means of a picture naming study.In the illustration, word age of acquisition (AoA) modulated local waveform amplitudes and global topographies after 400 ms, but only the topographic analyses run on the ERPs covering the actual production latencies in each condition led to a coherent interpretation of the underlying neurophysiological difference between early- and late-acquired words.We will only briefly discuss the observed effects of the manipulated factors in relationship with word production models to keep the focus on the purpose of the paper, namely the illustration of the approach.Among the two linguistic factors manipulated in the two subsets of stimuli, only AoA affected production latencies and ERPs. Despite the careful manipulation of both written and spoken frequency values, no lexical frequency effects were observed. At first glance the present results seem in contradiction with two picture naming studies by Word age of acquisition effects on production latencies have been more consistently reported even when stimuli were matched on a large set of other possible confound variables in factorial designs  as well The different waveform amplitudes observed after 400 ms between early- and late-acquired words do not inform us about the nature of the underlying difference. There are indeed several possible interpretations of these differences. Planning early- and late-acquired words may recruit different neuronal networks, suggesting that they are encoded via different brain processes after 400 ms; alternatively, early- and late-acquired words may recruit the same neuronal circuits but with different strengths; finally, the difference between early- and late-acquired words may stem from a shift of identical underlying brain mechanisms, indicating that a given process takes more time in one condition relative to the other. The results of the spatio-temporal segmentation favor the third interpretation, as the same sequences of stable topographic configurations of the potential fields on the scalp appeared in all conditions but with different distributions (different durations).Figure 3) had longer duration in the early-acquired word data than in the late-acquired words, whereas maps “E” and “H” had longer duration for late-acquired words than for early-acquired words. Based on these observations, one may conclude that from about 400 ms post stimulus onset two specific periods of stable electrophysiological activity last longer for late-acquired words, whereas the opposite is observed for the brain process corresponding to map “F,” which lasts about 50 ms longer for early-acquired words. This latter result is counter-intuitive as longer lasting neurophysiological processes when planning early-acquired words would hardly be interpretable in the light of faster production latencies for these words.Crucially for the purpose here, the separate analysis of stimulus- and response-aligned ERPs and the analysis of the combined ERP signal covering the exact production latency led to two very different interpretations of the results. In the separate analysis of fixed stimulus- and response-locked ERPs a stable topographic configuration , but that it had similar overall duration across conditions. The counter-intuitive results observed in the analysis of fixed ERP time-windows were due to the fact that they captured different portions of the planning process across the faster and slower conditions.This situation is disambiguated in the analysis of combined stimulus- and response-locked ERPs according to the actual production latencies, which enabled the observation that the period of stable electrophysiological activity corresponding to map F was shifted in late-acquired words by the previous longer lasting period of topographic stability , the following shifts could only be apprehended with the global topographic analysis.This illustration clearly shows that coherent results on ERP modulations in later time-windows only appeared when combining stimulus and response-aligned ERPs in order to cover the actual RT. It also allows ERP analyses to capture the consequences of earlier modulations on later ERP components: here the shift of later processes due to a longer lasting stable electrophysiological pattern in the stimulus-aligned data. In addition, although the first shift across conditions may be identified by visual inspection of some waveforms (see for instance shift of a P3-like component on Cpz in Here we showed that a coherent interpretation of the results only arose when combining stimulus- and response-aligned ERPs in order to cover the actual word planning time-window in each participant and condition. This combination enabled us to disentangle the possible interpretations of the neurophysiological processes underlying differences across conditions observed on waveform and on topographies in the fixed ERP periods. This approach is very useful in language production studies when comparing conditions or populations presenting different production latencies. Whereas ERPs clearly present sequences of stable periods of global electrophysiological activity enabling the comparison across conditions or groups in terms of global topographic patterns, further research is needed to improve our understanding of the relationship between the observed periods of topographic stability and the mental operations involved in speech planning.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Attention is an important factor that is able to strongly modulate the experience of pain. In order to differentiate cortical mechanisms underlying subject-driven  and stimulus-driven (bottom-up) modes of attentional pain modulation, we recorded electric brain activity in healthy volunteers during painful laser stimulation while spatial attention and stimulus intensity were systematically varied. The subjects’ task was to evaluate the pain intensity at the attended finger, while ignoring laser stimuli delivered to the other finger. Top-down (attention) and bottom up (intensity) influences differed in their effects on oscillatory response components. Attention towards pain induced a decrease in alpha and an increase in gamma band power, localized in the insula. Pain intensity modulated delta, alpha, beta and gamma band power. Source localization revealed stimulus driven modulation in the cingulate gyrus (CG) and somatosensory areas for gamma power changes. Our results indicate that bottom-up and top-down modes of processing exert different effects on pain-induced slow and fast oscillatory activities. Future studies may examine pain-induced oscillations using this paradigm to test for altered attentional pain control in patients with chronic pain. Pain is an aversive experience, which inherently attracts attention, disrupting all ongoing activities and thoughts . In contrast to MEG, which is most sensitive to the tangential orientation of neural dipoles, EEG captures activity from both tangential and radial sources and thus, contains additional information , aged 20–29 years (mean 24.17), participated in this study and received monetary compensation. Basic neurological investigation did not reveal any abnormalities. Subjects were informed that they could terminate the experiment at any time and written informed consent was obtained. The study was conducted in accordance with the Declaration of Helsinki and was approved by the local ethics review board.We delivered brief infrared laser stimuli of 1 ms duration and a beam diameter of 5 mm to the dorsum of the left ring and index finger using a Thulium YAG laser . Prior to the experiment, participants were familiarized with the use of a pain rating scale ranging from zero (no sensation) to 100 . On this rating scale, a value of zero indicates no sensation at all and 30 indicates the threshold for a pain sensation. Sensation higher than zero and below 30 indicates non-painful warm, rarely tactile sensations, whereas sensation at pain threshold indicates the beginning of a painful hot and stinging pain. Individual pain threshold was tested by calculating the average intensity at which subjects reported first a rating value above 30 in three ascending stimulus series and, more- over, first a rating value below 30 in three descending series of laser stimuli using successive intensity increments of 20 mJ. During the experiment subjects were comfortably seated in an electrically shielded and sound-attenuated recording chamber with their eyes closed. The experiment consisted of eight blocks in total, comprising two blocks of 20, 50, 60 and 30 stimuli, respectively. The blocks were presented in counterbalanced order  and BrainVision Recorder software  through four BrainAmp MRplus 32 channel amplifiers with a sampling frequency of 1000 Hz and a band pass filter between 0.1–250 Hz. The electrode impedance was kept below 15 kΩ. The EEG was recorded with nose reference. The data were analyzed offline using EEGLAB  sequences, where T denotes the length of the tapers and W the half bandwidth. These tapers optimally concentrate the spectral energy of the signal over the desired half-bandwidth W. Averaging across trials was finally performed in the frequency domain. A window of 300 ms length was shifted over the data with a step size of 20 ms. Spectral smoothing of 15 Hz was achieved by five slepian tapers. Time-frequency results were expressed as percent signal change relative to baseline, using a baseline interval from −1000 to −500 ms prior to stimulus onset.Frequencies up to 40 Hz were analyzed using a sliding Hanning-window Fourier transformation with a window length of 500 ms and a step-size of 20 ms. For the analysis of frequencies higher than 40 Hz spectral analyses of the EEG data were performed using a sliding window multi-taper analysis  were defined: a frontal ROI consisted of 26 electrodes, a central ROI around Cz of 31 electrodes, an occipital, contra- and ipsilateral ROI of 23 electrodes. Since all time frequency components were most prominent in the central ROI, this ROI was used for visualization and further statistical analysis.p-value was set to P < 0.05.For statistical analysis the statistics toolbox running under Matlab  was used. Because the pattern of activation was most pronounced in the central region, all sensor level analyses were performed using the data of the central ROI averaged across conditions. Visually inspecting the grand average of all subjects and conditions, time-frequency windows for peak detection were chosen from the time-frequency analysis  template brain.t-test was performed on the result for each grid point to estimate the signal change of each condition vs. baseline and for the difference between conditions across subjects. Subsequently t-values were transformed to z-scores. MNI-coordinates of peak voxel were transformed into Talairach-coordinates : subjects classified only 57 percent of high-intensity trials correctly, whereas they responded correctly in 87 percent of low intensity trials. The higher incidence of correct ratings for low intensity laser stimuli was observed regardless of which finger had to be attended. There was no significant difference between the two fingers with respect to the percentage of correct intensity judgments. The percentage of errors in location, resulting from directing the attention to the wrong finger, was low and did not differ between fingers.The classification of stimulus intensity was correct in 70 percent of the trials. This accuracy, however differed significantly between high and low stimuli  using time-frequency peak activation cluster. Hence, the contrasts between high intensity vs. low intensity and attended vs. ignored trials were calculated in source space. Attentional bottom-up modulation . Top-down modulation of laser pain intensity revealed one significant activation located in contralateral sensory motor area and midcingulate gyrus for the gamma band .In order to investigate regional specificity of the above-described gamma modulation we applied a distributed source reconstruction technique termed linear beamforming  with respect to bottom-up and top-down modulation.Recent studies reported consistently that pain stimulation elicits GBR which are linked to cortical perception and integration of pain intensity (Gross et al., In our study, pain induced GBR were also modulated by attention, consistent with an earlier MEG study of our group where we observed that top-down modulation by attention induced stronger gamma band power in bilateral sensorimotor cortex (Hauck et al., Besides pain induced GBR, pain stimulation elicits power changes in other frequency bands as well (Hauck et al., Consistent with other studies on pain processing (Mouraux et al., Recent studies in the somatosensory and visual system (Bauer et al., In conclusion, we were able to show different cortical oscillations and their generators to be involved in bottom-up and top-down modulation of pain processing. Most interestingly, gamma oscillations following laser-induced pain were modulated by both attention and stimulus intensity. Top-down modulation of gamma oscillation were localized in the insula, whereas bottom-up modulation of gamma oscillations were localized in sensorymotor areas and cingulate cortex. This finding suggests a key role of gamma-band oscillations in the routing of pain-related signals and the integration of nociceptive input into the multidimensional experience of pain. Future studies may address the issue of whether oscillatory response patterns of pain patients deviate from those of healthy controls. Enhanced gamma activity could be a sign of a disproportionate integration of noxious input leading to an exaggerated pain experience.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.


Insulin resistance (IR) in humans is related to hypertension and impaired vasodilation. Insulin administration has been shown to lower blood pressure both in insulin-resistant as well as in insulin-sensitive individuals.The aim of the study was to investigate the association between insulin sensitivity and alterations in blood pressure in horses before and after a euglycemic-hyperinsulinemic clamp (EHC).A 3-hour EHC was performed in 13 Warmblood horses . All horses were clinically healthy but had variable degrees of insulin sensitivity, were some horses were defined as insulin resistant. Blood samples for analysis of plasma glucose and insulin were collected before the start of the EHC, every 10-min during the EHC and immediately after the EHC. Mean, systolic and diastolic blood pressure was measured before and after the EHC using an indirect oscillometric device. Insulin and glucose data from the EHC were used to calculate the mean rate of glucose disposal per unit of insulin during steady state (M/I-index).2=0.55), but not diastolic and mean arterial pressure.Insulin administration decreased systolic, diastolic and mean arterial pressure in all horses. The M/I index was positively correlated with the decrease in systolic blood pressure (rThis study indicates that horses with insulin resistance have a blunted response to the cardiovascular effects of insulin.


Prolonged survival in HIV infection is accompanied by an increased frequency of non-HIV-related comorbidities. It is suggested that cardiovascular diseases (CVD) occur earlier among HIV-positive patients compared with HIV-negative patients, and at a higher rate. Several factors have been proposed which can be categorized into traditional and nontraditional risk factors. Immune dysfunction is a nontraditional risk factor that contributes significantly to cardiovascular pathology. Markers of inflammation are elevated in HIV-infected patients, and elevations in markers such as high-sensitivity C-reactive protein, D-dimer, and interleukin-6 (IL-6) have been associated with increased risk for cardiovascular disease. However, the data currently suggest the most practical advice is to start antiretroviral therapy early and to manage traditional risk factors for CVD aggressively. A better understanding of the mechanisms of CVD in this population and further efforts to modify chronic inflammation remain an important research area. The reduction of human immunodeficiency virus- (HIV-) related deaths by introduction of antiretroviral therapy (ART)  has beenGlobally, an estimated 35.3 (32.2–38.8) million people were living with HIV in 2012 . More thIn developed countries, about 9–20% of HIV-positive patients have moderate to high 10-year risk of myocardial infarction (MI) , 12. It In developing countries, about 20% of the daily deaths due to HIV/AIDS are attributed to CVD . This isIn addition to previously mentioned traditional risk factors for CVD, HIV-induced immune dysfunction might partially explain the increased risk of CVD . HIV-ind+ cell count and CVD has been reported by multiple studies. Although in Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) study the CD4+ cell count below 500 cells/μL was associated with increased risk of non-AIDS-related deaths [+ count <500 cells/μL, regardless of the class of ART used [+ cell count is associated with an increased risk of morbidity and mortality of CVD [+ cell count is <200 cells/μL may not achieve a normal CD4+ cell count, even after >10 years of otherwise effective therapy [The association between CD4d deaths , HIV OutART used . In addiy of CVD . Interes therapy . These iElevated C-reactive protein (CRP), interleukin-6 (IL-6), and D-dimer are predictors of CVD events in the general population . However HIV replication below the clinically detectable levels might contribute to persistent immune activation [ Microbial translocation as facilitated by HIV-induced depletion of CD4 T-cells from the gut-associated lymphoid tissue and intestinal barrier dysfunction has been proposed as potential cause for persistent immune activation [ coinfection with subsequent immune activation [The underlying mechanism of immune activation is poorly understood; however, multiple studies investigated the potential causes.tivation . Howevertivation . Microbitivation , 37. Evetivation . Majorittivation . Cytomegtivation .α via downregulation in at least one study [In a study conducted on 49 HIV-infected children with evidence of cardiomyopathy, giving intravenous immunoglobulin (IVIG) was associated with improvement of left ventricular (LV) structure and function as demonstrated by serial echocardiograms . Althougne study .The increasing rates of dyslipidemia and other metabolic changes among HIV-positive patients receiving ART have led to many studies investigating the link between ART use and CVD. In 2003, the D:A:D study demonstrated 26% relative risk increase in rate of MI per year of exposure during the first 4–6 years of use . In a suIn an earlier HOPS study, the investigators found an increased risk of MI in patients receiving a PI compared with those who were not. A multivariate statistical analysis showed that PI use was still strongly, although not significantly, associated with the incidence of MI . In a stThere are inconsistencies in the literature regarding the risk of MI associated with abacavir usage. Although multiple observational studies point towards an increase in MI risk, the evidence is not consistent –51. HoweDespite the available data on immune dysfunction in HIV-infected patients, it is still unclear how patients should be treated on the basis of this information or whether these markers should be used to assess and guide CVD risk management. Currently, early control of the HIV disease activity and managing the risk factors for CVD might partially modulate the immune dysfunction and subsequently reduce cardiovascular complications.Initiation and maintaining HIV viral suppression through ART are crucial for prevention of CVD. In spite of the increased risk of CVD in the population receiving ART, it is known from D:A:D study that the absolute risk is small and the benefits of ART outweigh the risks . Also, SAssessment of CVD risk for ART population is fundamental to guide risk management. However, utilizing conventional tools such as Framingham equation might inaccurately estimate the CVD risk. That is mainly because Framingham equation is used for non-HIV individuals and predicts the risk over a relatively long period. As previously mentioned HIV-infected individuals have significant CVD risk by 6 years' period . RecentlTo date, there is no evidence to suggest that HIV-positive patients need to be offered more aggressive management of dyslipidemia than those used in the general population. In 2013, the American College of Cardiology (ACC)/American Heart Association (AHA) issued updated practice guidelines for the treatment of blood cholesterol to reduce atherosclerotic CVD risk in adults , 64. TheThe prevalence of hypertension in HIV population has not been established with certainty ; howeverSmoking is a classic risk factor for CVD, and in the general population, the risk of coronary heart disease and mortality considerably reduced within the first 2 years of stopping smoking . Data frThere are several small studies conducted to address the impact of smoking cessation programs on HIV population –79. One As HIV-positive patients live longer CVD risk is increasing. The risk of CVD in HIV-positive patients is a complex mix of the traditional cardiovascular risk factors and nontraditional risk factors such as immune dysfunction. The evaluation of an individual's CVD risk should be assessed routinely, and the prevention of CVD should therefore be considered in terms of the patient's overall CVD risk and HIV disease stage. Early initiation of ART, close monitoring of the drugs toxicity, and ensuring high level of adherence are fundamental for CVD risk modification. In addition, control of traditional risk factors such as smoking and dyslipidemia is greatly needed for HIV-positive population.


Interestingly, heterozygous fibroblasts from a Huntington’s disease patient with a 180Q expansion displayed no obvious defects in the early secretory pathway. Thus, our results highlight the requirement for wild-type huntingtin at distinct steps along the secretory pathway.Huntingtin is a large membrane-associated scaffolding protein that associates with endocytic and exocytic vesicles and modulates their trafficking along cytoskeletal tracks. Although the progression of Huntington’s disease is linked to toxic accumulation of mutant huntingtin protein, loss of wild-type huntingtin function might also contribute to neuronal cell death, but its precise function is not well understood. Therefore, we investigated the molecular role of huntingtin in exocytosis and observed that huntingtin knockdown in HeLa cells causes a delay in endoplasmic reticulum (ER)-to-Golgi transport and a reduction in the number of cargo vesicles leaving the trans-Golgi network. In addition, we found that huntingtin is required for secretory vesicle fusion at the plasma membrane. Similar defects in the early exocytic pathway were observed in primary fibroblasts from homozygous  Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder that is caused by a polyglutamine (polyQ) repeat expansion at the N-terminus of the protein huntingtin (HTT) . The ageIn mammals, loss of wild-type huntingtin is embryonic lethal, highlighting the essential cellular role of this protein . HuntingHTT siRNA and homozygous primary embryonic fibroblasts isolated from the 140Q/140QHtt knock-in (KI) mouse as well as primary fibroblasts from an individual with HD who is heterozygous for 180Q at the HTT locus (+/180QHTT). Our results demonstrate that wild-type huntingtin is required in the secretory pathway in HeLa cells for vesicle transport from the endoplasmic reticulum (ER) to the Golgi complex, for the formation and delivery of vesicles from the Golgi to the plasma membrane, and for vesicle fusion with the plasma membrane. Comparing protein secretion in HTT-siRNA-treated cells, homozygous 140Q/140QHtt mouse embryonic fibroblasts (MEFs) and heterozygous +/180QHTT patient fibroblasts suggests that one copy of wild-type huntingtin is sufficient to maintain delivery of secretory cargo between the ER and the Golgi complex in fibroblasts. Interestingly, secretion is not affected by the toxic gain of function of mutant huntingtin protein in heterozygous patient fibroblasts.Transport defects become noticeable during HD progression when reduced levels of brain-derived neurotrophic factor (BDNF) are transported and secreted by cortical neurons and astrocytes. Consequently, less BDNF is taken up by striatal neurons, and this might play an important role in the selective neuronal vulnerability and degeneration associated with this disease . Thus, iTo probe the role(s) of wild-type huntingtin in the secretory pathway, we used a HeLa cell line (termed HeLa C1) stably expressing a constitutively secreted GFP-tagged variant of the human growth hormone (hGH). This reporter construct (GFP-hGH) is attached to multiple aggregation domains, which cause the newly synthesised GFP-hGH protein to aggregate and be retained in the ER. Treating the cells with a small ligand (AP21998) reverses the aggregation, resulting in a synchronous release of GFP-hGH from the ER to the Golgi complex and then on to the plasma membrane for exocytosis . This tiClinical issueHTT gene that lead to an extended polyglutamine repeat at the N-terminus of the huntingtin protein. This mutant protein is prone to aggregation and is thereby cytotoxic for neurons, leading to neuronal loss in the striatum and cortex. The huntingtin protein is ubiquitously expressed in the brain and has multiple roles in cellular membrane trafficking, including anterograde axonal transport of neurotrophic factors [such as brain-derived neurotrophic factor (BDNF)] for release from cortical neurons. During HD progression, transport defects become noticeable, with decreased secretion of BDNF by cortical neurons and astrocytes, and consequently reduced uptake of BDNF by striatal neurons; this might play an important role in the selective neuronal vulnerability and degeneration associated with this disease.Huntington’s disease (HD) is an incurable, autosomal dominant neurodegenerative condition characterised by late-onset motor, cognitive and psychiatric symptoms. HD is linked to mutations in the Results140Q/140QHtt knock-in mice, HTT-knockdown human HeLa cells and primary fibroblasts obtained from a heterozygous individual with HD (+/180QHTT). They use a unique secretory assay that allows dissection of the exact step(s) that require the huntingtin protein during protein secretion. The authors demonstrate that wild-type huntingtin is required for vesicle transport from the endoplasmic reticulum (ER) to the Golgi complex, for the formation and delivery of vesicles from the Golgi to the plasma membrane, and for vesicle fusion with the plasma membrane. The complete loss of wild-type huntingtin in homozygous 140Q/140QHtt knock-in mouse fibroblasts and HTT siRNA knockdown in HeLa cells has a significant impact on protein secretion. In heterozygous HD human fibroblasts (+/180QHTT), one copy of wild-type huntingtin is sufficient to support cargo delivery from the ER to the plasma membrane.To investigate the role of wild-type huntingtin in protein secretion, the authors use a combination of tissue culture cell lines, including primary fibroblasts isolated from homozygous Implications and future directionsIt is well-established that HD progression is linked to the toxic accumulation of mutant huntingtin protein; however, loss of wild-type huntingtin function might also contribute to neuronal cell death. Here, the authors demonstrate the requirement for wild-type huntingtin at several steps along the secretory pathway. Although in heterozygous HD human fibroblasts one copy of wild-type huntingtin is sufficient to maintain protein secretion, in neuronal cells the distances for cargo transport from the ER to the Golgi and then to the plasma membrane are extremely long. Thus, a slight impairment in the efficiency of cargo delivery is likely to result in major defects in protein secretion, which might contribute to the reduced BDNF secretion observed in HD. This study suggests a crucial function for wild-type huntingtin in the secretory pathway that cannot be fulfilled by the mutant protein and, therefore, these results could have important implications for therapies that aim to reduce huntingtin levels.HTT. After confirming huntingtin protein depletion by immunoblotting . Deconvolving of the siRNA SMARTpool against HTT revealed that the individual siRNA oligo 2 leads to efficient knockdown (similar to the SMARTpool) resulting in a significant reduction in ER-to-Golgi transport .First, we investigated the involvement of huntingtin in the delivery of newly synthesised proteins from the ER to the Golgi complex. HeLa C1 cells were either mock-treated or transfected with siRNA targeting blotting , cells wblotting . The ratblotting . Interesblotting , when coHTT siRNA and suggests that huntingtin and dynein might form a functional complex that mediates secretory vesicle transport along microtubules in the early secretory pathway.Dynein, a microtubule-based motor protein and huntingtin-interacting protein, has been shown to mediate cargo transport between the ER and Golgi complex . To deteTo determine whether huntingtin depletion affects the number of GFP-hGH-reporter-containing vesicles leaving the Golgi, a series of 1-minute, confocal spinning-disk time-lapse movies was used to quantify the number of cargo-filled vesicles travelling through defined 6×6-μm regions between the trans-Golgi network and the plasma membrane . siRNA ksupplementary material Movie 2) and an observed accumulation of secretory vesicles underneath the plasma membrane  microscopy, which allows selective imaging of a ~100 nm region beneath the plasma membrane. Due to the high signal-to-noise ratio, single fusion events of fluorescent secretory cargo vesicles with the plasma membrane can be visualised as a short flash in the TIRF field. Loss of wild-type huntingtin resulted in a significant decrease of ~50% in the total number of fusion events as compared with control cells ; GM09197B], and in primary MEFs isolated from wild-type mice  and homozygous 140Q/140QHtt KI mice. The human and mouse fibroblasts were transiently transfected with the GFP-hGH reporter construct and the amount of this reporter molecule in the Golgi area was analysed at 0, 15 and 30 minutes after AP21998 ligand addition. As shown in +/180QHTT patient fibroblasts. However, significantly less cargo accumulated in the Golgi 15 minutes after ligand addition in homozygote 140Q/140QHtt KI MEFs when compared with wild-type MEFs  and an individual with juvenile-onset HD [heterozygous for an expanded polyQ tract of 180Q  and then to the plasma membrane can be followed by live-cell microscopy. The large size and content of fluorescent cargo in stable HeLa C1 cells also enables one to visualise and quantify single secretory cargo fusion events at the plasma membrane . However, in primary MEFs isolated from the 140Q/140QHtt KI mouse or in human patient fibroblasts, single cargo vesicles are smaller and contain much less fluorescent content, making it challenging to analyse the movement of single exocytic vesicles and to image distinct post-Golgi cargo vesicles as well as single fusion events at the plasma membrane in the TIRF field.HeLa C1 cells are an ideal system to visualise cargo transport in the secretory pathway. The movement of large, bright exocytic vesicles between the ER and Golgi complex (see +/180QHTT), which still express one wild-type copy of huntingtin, showed no significant defect in the secretory pathway. The presence of one copy of wild-type huntingtin seems to be sufficient for maintaining secretion, at least in fibroblasts. In addition, the wild-type protein could also have a protective role with respect to the defects caused by expression of the mutant protein, as shown in a Drosophila model of HD , one copy of huntingtin is sufficient to support cargo delivery in the exocytic pathway. The majority of cell types are most likely able to compensate for a slight reduction in secretory efficiency; however, the striatal neurons most affected in HD might be particularly susceptible to alterations in the trafficking of cargo, such as receptors and transporters that are vital for neuronal cell survival. This study suggests a crucial function for wild-type huntingtin that cannot be fulfilled by the mutant protein and, thus, these results could have important implications for therapies that aim to reduce levels of mutant huntingtin.In summary, this novel regulated exocytosis assay has allowed us to demonstrate the requirement for huntingtin at several steps along the secretory pathway. In fibroblasts, the complete absence of wild-type huntingtin in homozygous The following antibodies were used: anti-huntingtin ; anti-optineurin ; anti-GFThe constitutive secretion assay was generated by modifying the RPD Regulated Secretion/Aggregation Kit  as described previously . A stabl2. Human fibroblasts from a healthy control (GM23976) and an individual with juvenile-onset HD with a 180Q expansion (+/180QHTT) were obtained from Coriell Cell Repositories and cultured in Minimum Essential Medium (MEM) with Earl’s salts, non-essential amino acids, and sodium bicarbonate  supplemented with 15% FBS and 2 mM L-glutamine at 37°C and 5% CO2. Mouse embryonic fibroblasts (MEFs) were established from wild-type and 140Q/140QHtt embryos at day E17/18 of development. For preparation of fibroblasts, skin, limbs and muscle tissue was dissected from the embryos, minced and incubated for 20 min at 37°C with trypsin containing 300 U/ml DNase (Invitrogen). After addition of growth medium , the digested tissue was dissociated using a glass Pasteur pipette, centrifuged, resuspended in growth media and plated onto tissue-culture treated dishes.The stable HeLa C1 cell line was grown in Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, penicillin/streptomycin  and puromycin (1.66 μg/ml) at 37°C and 5% COxpansion  (GM09197HTT) were used to knock down huntingtin or dynein intermediate chain. For efficient knockdown, HeLa C1 cells were transfected twice with four siRNA oligos on day 1 and 3 using OligofectAMINE (Invitrogen). On day 5, cells were processed for corresponding assays and the efficiency of protein depletion was assessed by western blotting.All siRNA oligos were obtained from Dharmacon. ON-TARGETplus SMARTpool oligos  using FuGENE 6 (Roche Diagnostics) according to the manufacturer’s instructions.For immunofluorescence, primary MEFs, human fibroblasts or HeLa C1 cells were grown on coverslips, transfected with the corresponding plasmids and, 24 hours later, were fixed with 4% PFA, permeabilised with 0.1% Triton X-100, blocked with 1% BSA in PBS and processed for indirect immunofluorescence using primary antibodies (specified in the figure legends) and secondary antibodies coupled with Alexa Fluor 488 or Alexa Fluor 568 (Molecular Probes). After mounting, cells were imaged using a Zeiss LSM710 confocal microscope.2-independent medium . Images were obtained on a Zeiss microscope equipped with spinning disk and TIRF attachment . Images were acquired with a Hamamatsu Photonics EM-CCD Digital Camera  and Zeiss Zen imaging software . Volocity analysis software  was used to quantify the rate of transfer of the reporter molecule from the ER to the Golgi complex in spinning-disk videos. Imaris vesicle tracking software (Bitplane) was used to quantify the total number of vesicles travelling through 6×6-μm areas between the Golgi complex and the plasma membrane.For live-cell TIRF or spinning-disk microscopy studies, cells were grown on 25-mm round coverslips  and imaged at 37°C in CO


Renal biopsy of the allograft is important to evaluate renal dysfunction . Rare coA 39-year old woman with nephrosclerosis and in hemodialysis for the last 3 years received a KTX from a deceased 20-year old male donor that had a cranio-cerebral trauma. The vascular anastomoses were performed at the right iliac vessels in an end-to-side fashion after 23 hours of cold ischemia.A CNPB, guided by ultrasonography, was indicated due to delayed graft function at postoperative day 10 which revealed acute tubular necrosis. Shortly after the CNPB she experienced tachycardia, hypotension and decreased blood levels requiring 2 units of blood transfusion and remained stable and developed mild hematuria. An allograft ultrasonography performed five days later revealed an AVF and a PA at the middle pole of the allograft and a peri-renal hematoma around the upper pole with 200cc . A super


Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive). These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP) fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking antibody from mice with an affinity of 90 pM.Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG Since Kohler and Milstein first described a method for the generation of monoclonal antibodies (mAbs) via their hybridoma technology in 1975 . As dise−6 efficiency with conventional PEG fusion) due to its reliance on a fusion event . Alt. Alt+ mereening [,43,44.In summary, the generation of large and diverse panels of high quality monoclonal antibodies to therapeutically-relevant target molecules is reliant on the use of a range of discovery technologies that allow for efficient interrogation of the antibody repertoire. The FACS approach described here represents a complementary technology to established antibody discovery methods and provides an additional mechanism by which to identify fit-for-purpose antibodies to an ever expanding array of targets.


The server has a strong emphasis on modeling difficult protein targets for which templates cannot be detected. RBO Aleph's unique features are (i) the use of combined evolutionary and physicochemical information to perform residue–residue contact prediction and (ii) leveraging this contact information effectively in conformational space search. RBO Aleph emerged as one of the leading approaches to ab initio protein structure prediction and contact prediction during the most recent Critical Assessment of Protein Structure Prediction experiment . In addition to RBO Aleph's main focus on ab initio modeling, the server also provides state-of-the-art template-based modeling services. Based on template availability, RBO Aleph switches automatically between template-based modeling and ab initio prediction based on the target protein sequence, facilitating use especially for non-expert users. The RBO Aleph web server offers a range of tools for visualization and data analysis, such as the visualization of predicted models, predicted contacts and the estimated prediction error along the model's backbone. The server is accessible at http://compbio.robotics.tu-berlin.de/rbo_aleph/.RBO Aleph is a novel protein structure prediction web server for template-based modeling, protein contact prediction and  It provides services for template-based modeling, protein contact prediction and ab initio targets for which templates are not available. It therefore complements existing services that mainly focus on template-based modeling . RBO Aleph was one of the top servers in the template-free modeling category.The effectiveness of our To ensure the server's accuracy in the light of ongoing developments, we continuously monitor its performance through the CAMEO project .ab initio modeling), used templates, detected domain boundaries and local prediction error estimates.Our web server provides an intuitive user interface, multiple visualization options and analysis tools to help users interpret the results. These include a visualization of the predicted model structures and contacts, the used modeling methods    guided bch (MBS)  and estich (MBS) . FinallyTo accommodate multi-domain proteins, RBO Aleph attempts to split the query protein into domains. The server infers domain boundaries from retrieved templates. For long sequences without template, RBO Aleph uses a consensus score of two sequence-based domain prediction algorithms, PPRODO  and DomPab initio low-energy structures by evaluating the local physicochemical environment of a contact using a graph-based representation. This novel contact information plays a central role in our pipeline and is used in several parts of RBO Aleph .Residue–residue contact predictions from evolutionary sequence variation are accurate enough to improve three-dimensional structure prediction –24. HoweAb initio structure prediction searches the conformational space of the protein for the native structure. The prediction success highly depends on the employed search strategy because the conformational space is large and the energy landscape is rugged. Model-based search . The contact-based features characterize the ratio of satisfied contacts in each template, while taking into account the contact confidence estimated by EPC-map. High compatibility between predicted contacts and a template improves its ranking.In addition, RBO Aleph uses contact restraints as energy terms to guide conformational space search. These additional energy terms smoothen the energy landscape, thereby effectively guiding search toward relevant regions.Leveraging both evolutionary and physicochemical contact information in protein structure prediction is one of the key features of RBO Aleph.http://www.predictioncenter.org/casp11). The current version of RBO Aleph uses a revised set of parameters for EPC-map, further improving on the performance in CASP11.We provide detailed evaluations of our contact prediction method (EPC-map) and of MBS in separate publications ,8. We evz-score (>0) and third by the sum of z-scores (>−2) of the top prediction based on the assessors formula . Our server is competitive with established automated methods when considering all modeling categories. It ranked seventh by the average z-score (>0) and nineteenth by the sum of z-scores (>−2) of the top prediction.We also evaluated RBO Aleph's tertiary structure modeling capabilities in CASP11. RBO Aleph ranked as one of the top servers in the template-free modeling category for 40 protein domains, demonstrating the effectiveness of MBS as a search strategy. RBO Aleph ranked first by the average http://www.cameo3d.org/).In addition, RBO Aleph continuously participates in CAMEO  as a pubUsers can submit prediction requests on the ‘Submit Job’ page. This requires a job name and the sequence of the query protein. The sequence has to be in the FASTA format, single-chained and shorter than 700 residues. Users may contact the authors to request predictions of larger proteins. Optionally, users can also provide residue–residue contact restraints. The server then linearly combines the input contacts with its own predicted contacts  and leverages the outcome as described in ‘Materials and Methods’ section.ab initio modeling) to be exclusively used in prediction. They can also choose whether to activate the use of domains in modeling. By default, RBO Aleph automatically selects the modeling protocol and the domain boundaries.Users can select a modeling protocol  to guide ab initio structure prediction. RBO Aleph is the first public available server that uses these methods.The main focus of RBO Aleph is RBO Aleph was among the best-performing methods in the template-free protein structure prediction category of CASP11. The performance of RBO Aleph is continuously monitored in CAMEO to ensure high prediction accuracy and reliability.


We present two simple alternative methods to form polymer-derived carbon nanodomains in a controlled fashion and at low cost, using custom-made chemical vapour deposition and selective laser ablation with a commercial CD-DVD platform. Both processes presented shiny and dark residual materials after the polymer combustion and according to micro-Raman spectroscopy of the domains, graphitic nanocrystals and carbon nanotubes have successfully been produced by the combustion of polydimethylsiloxane layers. The fabrication processes and characterization of the byproduct materials are reported. We demonstrate that CVD led to bulk production of graphitic nanocrystals and single-walled carbon nanotubes while direct laser ablation may be employed for the formation of localized fluorescent nanodots. In the latter case, graphitic nanodomains and multi-wall carbon nanotubes are left inside microchannels and preliminary results seem to indicate that laser ablation could offer a tuning control of the nature and optical properties of the nanodomains that are left inside micropatterns with on-demand geometries. These low-cost methods look particularly promising for the formation of carbon nanoresidues with controlled properties and in applications where high integration is desired. Silicon-based polymer-derived ceramics (PDC) are of increasing interest thanks to the relatively controlled production and recent utilization of their unique functional properties. Their great resistance and stability to high temperatures, chemical durability or semiconducting behaviour are indeed permitting new applications as catalysis supports, highly resistant materials for aerospace and automotive industry, microelectronics, optics and even biotechnology . Recent In this work, we report the formation of bulk or localized carbon nanodomains obtained from PDMS, a silicon-based polymer by using two different methods enabled by low-cost custom-made platforms: chemical vapour deposition at 900 °C and selective laser ablation (SLA). In both procedures, it has been found that the combustion of PDMS successfully formed residual nanomaterials of different compositions and ordering depending on the formation conditions. Ease of use at atmospheric pressure and low cost are characteristics of the two alternative techniques employed here for PDC nanodomains production. In CVD, the evaporation of the reagent over the PDMS at high temperatures allowed for the rearrangement of carbon structures in PDMS to produce two layers composed of two different carbon structures. In the case of SLA, a continuous-wave infrared laser beam was focused on the surface of PDMS membranes coated with a thin layer of absorbing carbon nanopowder. This generates superficial laser-induced incandescence and forms highly localized carbon nanodomains inside the microscopic volume of etched material . In thisWhen a commercial CVD reactor is lacking, the low-cost alternative constructed here using conventional laboratory materials showed to be capable of vapour deposition at atmospheric pressure. The setup consists of three essential parts: reagents evaporation stage, deposition chamber and residual gases expulsion stage, as shown in The reagent used in this work for nanodomains production was a solution of ethanol  in water at 43% (v/v). The 3 mm thick PDMS layers on which it was evaporated were prepared from a Sylgard 184 kit (purchased from Dow Corning) mixing the pre-polymer with its curing agent in a 10:1 weight proportion following the conventional procedure. The gel was then deposited on clean glass substrates and cured at 90 °C for 2 h in a convection oven before they were detached and stored in hermetic containers. Vapour deposition of the reagent was performed at 900 °C over one hour and temperature was measured with a type-K thermocouple placed inside the deposition chamber. A post-treatment consisting in heating the sample at the same temperature for 30 min has been implemented to improve deposition and ordering of the structures [−1) and G (ca. 1598 cm−1) bands, characteristics of carbon materials, are clearly present. However, the G′ band (ca. 2700 cm−1) is visible in the dark regions while the 2D band (ca. 2700 cm−1) and the G + D band (ca. 2935 cm−1) are characteristics of the shiny areas [−1) demonstrate that graphitic nanocrystals (GNC) are composing the shiny layers, whereas dark parts consist mainly of single-walled carbon nanotubes (SWCNT) [Two very distinct types of residual layers were always observed after the CVD procedure: dark and shiny layers have been obtained in a highly repeatable manner under the previously described particular conditions . Differeny areas . The difny areas –13, the  (SWCNT) .Selective and localized laser pyrolysis has also been tested to study the formation of nanodomains from PDMS in a controlled fashion, using a low-cost technique that allows laser micropattern formation in PDMS . The setThe SLA process produced nanocarbon residues inside micron-scale laser-etched PDMS channels with on-demand geometry. Dark and shiny domains were also observed by using polarized-light microscopy during which it was evidenced that the laser formation of the nanodomains is extremely localized. This has been clearly identified by the presence of fluorescent compounds inside the channels. The composition of the combustion residues has been characterized first with a NORAN energy dispersive X-ray spectroscopy (EDX) system working at 20 keV in a scanning electron microscope (Jeol JSM-5600LV). The EDX analysis showed that residues are mainly silicon and oxygen, in similar weight proportions, and that carbon is only present in regions etched at the greatest laser intensities. In this case, 47.5% oxygen, 29.3% silicon and 23.2% carbon are found inside the channels demonstrating that greater temperatures reached during incandescence produce carbon nanodomains locally.The composition of the residues was then studied with Raman spectroscopy . This ch


While mRNA stability has been demonstrated to control rates of translation, generating both global and local synonymous codon biases in many unicellular organisms, this explanation cannot adequately explain why codon bias strongly tracks neighboring intergene GC content; suggesting that structural dynamics of DNA might also influence codon choice. Because minor groove width is highly governed by 3-base periodicity in GC, the existence of triplet-based codons might imply a functional role for the optimization of local DNA molecular dynamics via GC content at synonymous sites (≈GC3). We confirm a strong association between GC3-related intrinsic DNA flexibility and codon bias across 24 different prokaryotic multiple whole-genome alignments. We develop a novel test of natural selection targeting synonymous sites and demonstrate that GC3-related DNA backbone dynamics have been subject to moderate selective pressure, perhaps contributing to our observation that many genes possess extreme DNA backbone dynamics for their given protein space. This dual function of codons may impose universal functional constraints affecting the evolution of synonymous and non-synonymous sites. We propose that synonymous sites may have evolved as an ‘accessory’ during an early expansion of a primordial genetic code, allowing for multiplexed protein coding and structural dynamic information within the same molecular context. The nature of codon usage bias and its relation to natural selection acting at synonymous sites is a long-standing problem in molecular evolutionary theory ,2. SinceMethylobacterium extorquens demonstrated that synonymous mutations toward globally favored frequent codons in the genome, which would normally be thought to enhance translational efficiency, actually dramatically decrease Darwinian fitness in this bacterium  Database , a databN-acyl-2-amino-6-ketopimelate aminotransferase (DapC) and N-acyl-L,L-2,6-diaminopimelatedeacylase (DapE) (meso-diaminopimelate is involved in PG cross-linking in most Gram-negative bacteria and lysine serves the same purpose in the PG of Gram-positive bacteria (dapL sequences in Table e (DapE) . dapL sebacteria ,51,52.et al.  proposed by Wan et al. .We used a simple extension of our previous methods for computer simulation of neutral evolution ,56 to decdn) was calculated using both internal P linkages and both external P linkages  were correlated to gene-level codon biases and gene-level deviations from 50% GC content. To further investigate the functionality of different levels of intrinsic DNA flexibility, the average deviations were also correlated with codon bias across the 27 dapL genes. Species and accession numbers are listed in Table To investigate gene-level trends, these local deviations of TRX within protein space were averaged over coding regions to obtain a mean deviation for a given gene. Across all genes in each genome, the average deviations  or transversion (Tv)) and their effect on amino acid composition (i.e. non-synonymous (non-syn) or synonymous (syn)). The average mutational impacts of each class of mutation on a given coding sequence were calculated as followsThe respective dTRX values for each of the four functional combinations of base substitutions  were then calculated for all sequences in all genomes. The rates of substitution on synonymous sites (dS) and non-synonymous sites (dN) were used to calculate omega ratio (dN/dS), a proxy for the level of selection occurring on the protein level for a given gene. The association of dTRX and dN/dS were then examined for evidence of increased dTRX with reduced dN/dS, indicating a potential trade-off between selection at the protein level and mutational events impacting intrinsic flexibility of DNA. Data and graphics files for all genomes are presented in Supplementary File D and summarized in Table For each of the 24 prokaryotic genomes analyzed above, single genome-wide averages for entropy-based codon bias, GC content and TRX score were analyzed for associative trends across species. A linear regression of genome-wide GC contents against the genome-wide TRX averages was produced. Regressions were also conducted for genome-wide codon biases against the genome-wide TRX averages. Because entropy-based codon biases do not discern the direction of bias with respect to GC3 and because codon bias tends to increase with both high and low GC content, two separate linear regressions were produced for species falling above or below the weighted average of the 10 dinucleotides comprising the TRX scale (i.e. TRX = 20.625). Thus, the trending of codon bias in high GC and low GC genomes was analyzed separately.r = −0.729, P = 0.002; r = 0.722, P = 0.018, respectively), demonstrating a multi-genomic trend indicating that genome-wide codon biases are generally associated with unusual shifts away from the value of intrinsic DNA flexibility expected in vitro with equal base composition. The genome-wide intrinsic DNA flexibility was confirmed to be highly dependent on genome-wide GC content  Figure . Codon bThe overall frequencies of natural selection acting on intrinsic DNA flexibility at synonymous sites were estimated by globally averaging the local comparisons of true ancestral → extant dTRX to simulated distributions of local neutral impacts of randomly repositioned silent (i.e. synonymous) mutations on dTRX  demonstrated an even more uncharacteristically high level of DNA flexibility and GC3 across its entire coding region  exhibit less extreme overall values of GC3 and flexibility values . All other graphs for all species analyzed are given in Supplementary File D. For each genome, individual F tests comparing the variance in dTRX for functionally conserved versus adaptively evolving or drifting genes over both synonymous and non-synonymous sites are summarized in Table To investigate the potential trade-offs of evolutionary pressures acting at the level of nucleic acid polymer and protein, we plotted each average gene-level mutational impact on intrinsic DNA flexibility (i.e. dTRX) against the gene's corresponding value of dN/dS Figure . While wThe coding regions of genomes may present a significant informational challenge to the cell because of their need to multiplex the different types of information regarding protein synthesis, genome folding/supercoiling and transcriptional regulation into a singular molecular context of DNA. It has been recently reported that footprints of exonic transcription factors affect codon bias in roughly 15% of the codons in the human genome, supporting that the genetic code itself may have a dual coding feature  We belieThrough our investigations of the evolution of intrinsic DNA flexibility at the level of single genes and whole genomes, we have found evidence for a simple, and yet primary, functional basis for the broad taxonomic variation long observed in codon bias. We have demonstrated a broad general association of DNA polymer biophysics, codon bias and protein evolution in the coding sequences of unicellular genomes. We find an apparently universal constraint connecting the evolution of protein structure and the evolution of DNA polymer biophysics as it may affect genome folding or coiling dynamics through altered local charge and flexibility. We also find a universal relationship across taxa indicating that GC3-related codon bias acts to maintain both local gene-specific, as well as species-specific levels, of DNA flexibility. We utilized a novel method of neutral simulation to identify genes where DNA flexibility governed by synonymous sites is under significant natural selection. We further demonstrate that DNA flexibility is subject to significant and often rather taxon-specific levels of purifying selection, implying that DNA structural dynamics are quite often moderately conserved. However, our observation that bacterial endosymbionts with very extremely low levels in genomic GC and GC3  have ratet al. .What might be the advantage of this more highly-refined genomic architecture enabled via local changes to the structural dynamics of DNA polymer? The packaging of genes into higher order structures obviously has two ultimate effects. First, it allows more genes to be incorporated into a more compact space in the cell, and second, it tends to repress individual gene expression. Both of these genomic characteristics can then allow organisms to better program responses in gene expression to local environments. Botzman and Margalit  have recmeso-diaminopimelate (m-DAP) and lysine. Both m-DAP and lysine are cross linking amino acids in the peptidoglycan cell wall of most bacteria and lysine is one of the 20 common proteogenic amino acids. The dapL gene is well-represented in the three domains of life, yet is not ubiquitous as there are three other anabolic routes toward m-DAP and lysine . There is published evidence to suggest that the genomic packaging and unfolding as it is defined by both DNA backbone dynamics as well as the nucleobase sequence is targeted and shaped by evolution (Scientists in many areas of nucleic acid research are beginning to view the sequence-dependent backbone dynamics of nucleic acids as strong determinants for the fine-tuning of gene regulation ,41–43,69volution –56,71–75volution  are capavolution ,77 and pSupplementary Data are available at NAR Online.


Guidelines recommend to perform head computed tomography (CT) in nontraumatic cardiac arrest only if the patient remains unconscious after return of spontaneous circulation . NeverthWe collected data for all patients with nontraumatic OHCA admitted to the ICU from 1 January 2012 to 31 December 2012. We recorded mean age, male-to-female ratio, cause of cardiac arrest and mortality in the ICU.Thirty-four survive cardiac arrest patients were admitted to the ICU. Mean age was 66 years. The male-to-female ratio was 2:1. Sixteen patients died in the ICU. Head CT scans obtained in the immediate postarrest period found significant abnormalities in four patients (three subarachnoid hemorrhage and one ischemic ictus) with immediate modification of treatment.Head CT abnormalities can be found in cardiac arrest patients. Further investigations are necessary to evaluate the impact of cardiac arrest from neurological causes and to establish the role of neuroimaging in these patients.


Leukocyte-platelet rich fibrin belongs to a second generation of platelet concentrates that does not need biochemical blood manipulation. It is used for tissue healing and regeneration in periodontal and oral-maxillofacial surgery. We report two cases of hyperplastic gingival lesions treated by exeresis and application of leukocyte-platelet rich fibrin membranes in order to improve and accelerate tissue healing.Two patients (a 78-year-old Caucasian woman and a 30-year-old Caucasian man) were treated for hyperplastic gingival lesions. They underwent to exeresis of lesions and application of leukocyte-platelet rich fibrin membranes. Tissue healing was clinically evaluated after 1, 3, 7, 14 and 30 postoperative days. No recurrences were observed after 2 years of semi-annual follow up.We obtained rapid and good healing of soft tissues probably due to the elevated content of leukocytes, platelets and growth factors in the leukocyte-platelet rich fibrin. Based on our results we suggest the use of leukocyte-platelet rich fibrin to cover wounds after exeresis of oral neoformations such as hyperplastic gingival lesions. Hyperplastic gingival lesions consist of fibro-epithelial reactions that are secondary to multiple factors: chronic inflammation, local irritation , hormonal factors , and drugs such as anti-epileptic drugs , immunosuppressive drugs (ciclosporin) and antianginal vasodilators (nifedipine) , 2. A dyWe report two cases of hyperplastic gingival lesions treated by exeresis and application of leukocyte-platelet rich fibrin (L-PRF) membranes in order to improve and accelerate tissue healing. L-PRF belongs to a second generation of platelet concentrates which does not need biochemical blood manipulation , 10. It A 78-year-old Caucasian woman presented with a complaint of a swelling in her right mandibular molar teeth area that was painful at mastication. The swelling had arisen 4 months previously and had grown quickly. On extraoral examination she presented a right facial swelling and the skin of her face was healthy. Her regional lymph nodes were not palpable. On intraoral examination she showed poor oral hygiene. A 45×30×20mm sessile, lobular, soft tissue mass was evident in her right posterior mandibular gingiva Fig. . The mucA 30-year-old Caucasian man presented with a complaint of a swelling in his left hard palate region which had arisen approximately 2 months previously after contact with hot food. The lesion was not painful but was responsible for discomfort on swallowing and phonation. On extraoral examination facial skin lesions or swellings were not highlighted and his regional lymph nodes were not palpable. An intraoral examination showed a sessile and detected lesion of hard palate in correspondence with left maxillary lateral incisor, left maxillary canine and left maxillary first premolar. The lesion was characterized by a diameter of 10mm and the mucosa which covered it looked eutrophic  infiltrative anesthesia (mepivacaine 30mg/mL and epinephrine 0.01mg/mL) was performed. Full thickness incisions were realized by Bard-Parker blades number 15 and they included at least 2 mm of healthy tissue. The lesions strongly adhered to the floor below. They were lifted by Prichard’s periosteal spatulas and surgical tweezers Fig. . At the Although the epulis in its form of giant-cell epulis and fibro-osseous epulis is a benign disease, it is important to find time to make the correct therapy. Both young and old patients must take prompt actions to avoid growth of oral lesions that can begin to become difficult to manage. There is unanimous consensus on the surgical excision of these lesions in toto to prevent recurrence. The target of these case reports is to focus on the management of the L-PRF and its placement on the surgical wounds. The first benefit is given by covering the surgical site, which would otherwise heal by secondary intention, which provides greater comfort to the patient in the postoperative period. The same wound healing appears to be substantially accelerated because of the structural and neoangiogenetical properties of L-PRF . In the According to the encouraging results obtained in these two clinical cases about tissue healing after application of L-PRF membranes, we suggest that L-PRF be used to cover wounds after exeresis of oral neoformations such as hyperplastic gingival lesions.Written informed consent was obtained from the patients for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Vivax malaria with two distinct (short- and long-term) incubation periods has been prevalent in the Republic of Korea since its re-emergence in 1993. Chemoprophylaxis has been conducted among military personnel since 1997. We estimated the time-dependent reproduction number, thereby assessing the protective effect of chemoprophylaxis.A mathematical model has been formulated using a renewal equation, estimating the yearly reproduction number (Ry) from 1993 to 2012 by maximum likelihood estimation method. We also computed Akaike Information Criterion (AIC) to test if there was a detectable change point in the trend in relation to chemoprophylaxis.Three-year average of Ry showed gradual decline through 1993-2012 with a temporary increase from 2003 to 2005, having been under the threshold 1 since 1998. AIC has suggested that the chemoprophylaxis has cut down Ry by 34% from what it would be without chemoprophylaxis.vivax malaria in Korea has been brought under control due mainly to mass-chemoprophylaxis.The epidemic of