Recently, fluorescence imaging following the preoperative intravenous injection of indocyanine green has been used in clinical settings to identify hepatic malignancies during surgery. The aim of this study was to evaluate the ability of photoacoustic tomography using indocyanine green as a contrast agent to produce representative fluorescence images of hepatic tumors by visualizing the spatial distribution of indocyanine green on ultrasonographic images. Indocyanine green  was preoperatively administered to 9 patients undergoing hepatectomy. Intraoperatively, photoacoustic tomography was performed on the surface of the resected hepatic specimens (n = 10) under excitation with an 800 nm pulse laser. In 4 hepatocellular carcinoma nodules, photoacoustic imaging identified indocyanine green accumulation in the cancerous tissue. In contrast, in one hepatocellular carcinoma nodule and five adenocarcinoma foci , photoacoustic imaging delineated indocyanine green accumulation not in the cancerous tissue but rather in the peri-cancerous hepatic parenchyma. Although photoacoustic tomography enabled to visualize spatial distribution of ICG on ultrasonographic images, which was consistent with fluorescence images on cut surfaces of the resected specimens, photoacoustic signals of ICG-containing tissues decreased approximately by 40% even at 4 mm depth from liver surfaces. Photoacoustic tomography using indocyanine green also failed to identify any hepatocellular carcinoma nodules from the body surface of model mice with non-alcoholic steatohepatitis. In conclusion, photoacoustic tomography has a potential to enhance cancer detectability and differential diagnosis by ultrasonographic examinations and intraoperative fluorescence imaging through visualization of stasis of bile-excreting imaging agents in and/or around hepatic tumors. However, further technical advances are needed to improve the visibility of photoacoustic signals emitted from deeply-located lesions. In vivo fluorescence imaging using indocyanine green (ICG) has been clinically applied as an intraoperative navigation tool that enables the real-time identification of biological structures, such as the lymphatic system In the last decade, photoacoustic (PA) tomography has been actively developed as a novel optical imaging technology that enables the real-time visualization of deeply-located biologic structures on ultrasonographic images through the “photoacoustic effect” This study was conducted with the approval of the Institutional Ethics Review Board of the University of Tokyo Hospital. Written informed consent was obtained from all patients. The animal study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health. The protocol for the animal study was approved by the University of Tokyo's Committee on the Ethics of Animal Experiments. All surgery was performed under isoflurane anesthesia, and all possible efforts were made to minimize suffering.First, the ability of PA tomography to detect ICG-containing biological material was evaluated using a human liver tissue-mimicking phantom  In 9 patients who underwent hepatic resection for malignancies , ICG  was preoperatively injected  as part of routine liver function testing performed for surgical planning Because direct use of the Vevo LAZR imaging system in patients had not yet been approved, PA tomography's ability to visualize hepatic tumors from the body surface was evaluated in a mouse model. Three male NASH-HCC model mice  were created by administering low-dose streptozotocin after birth (first hit) followed by a subsequent high-fat diet (second hit) For the plasma encapsulated in the tissue-mimicking phantom, at ICG concentrations of 0.001, 0.01, 0.1, and 1.0 mg/mL, the average PA signal intensity under excitation with 800 nm of light was 0.218, 0.239, 0.202, and 0.612, respectively. In contrast, the fluorescence intensity of the ICG-containing plasma increased with increases in ICG concentration up to 0.01 mg/mL, but then started to decrease; this was most likely due to absorption of near-infrared light by ICG .Photoacoustic tomography's ability to identify hepatic malignancies was evaluated in the mice that had undergone subcutaneous implantation of a well-differentiated human hepatoma cell line (HuH-7). Fluorescence imaging at 48 hours after the intravenous injection of ICG demonstrated ICG accumulation in the implanted tumors. Photoacoustic tomography also enabled identification of tumor-specific ICG signals and their visualization on US images under conditions of 800-nm excitation light and 54-dB PA gain .Photoacoustic signal amplitude according to ROI  is demonstrated in P = 0.01 [Wilcoxon's rank-sum test]). In contrast, the ratio of PA signal amplitudes in Peri tissue relative to that in NC tissue was lower in the former group.The ratio of PA signal amplitude in CA tissue relative to that in NC tissue was higher in the 4 tumors showing cancerous-type ICG fluorescence compared with the 6 tumors with rim-type fluorescence  The major limitation of the current technique used for PA tomography with ICG lies in the small region observable from the liver surface. Although PA signals can be detected from depths of up to 7 cm in theory In conclusion, PA tomography using ICG is a promising technique that can support US examination of the liver by providing information on biliary excretion disorders in cancerous tissues and peri-cancerous hepatic parenchyma. However, further technical improvement is needed to enable the visualization of deeply-located lesions.Video S1Three-dimensional reconstruction of PA tomography from a human hepatic specimen containing well-differentiated HCC.(WMV)Click here for additional data file.Video S2Three-dimensional reconstruction of PA tomography from a human hepatic specimen containing CRLM.(WMV)Click here for additional data file.Data S1Raw data of the present study.(ZIP)Click here for additional data file.


To trigger innate behavior, sensory neural networks are pre-tuned to extract biologically relevant stimuli. Many male-female or insect-plant interactions depend on this phenomenon. Especially communication among individuals within social groups depends on innate behaviors. One example is the efficient recruitment of nest mates by successful bumblebee foragers. Returning foragers release a recruitment pheromone in the nest while they perform a ‘dance’ behavior to activate unemployed nest mates. A major component of this pheromone is the sesquiterpenoid farnesol. How farnesol is processed and perceived by the olfactory system, has not yet been identified. It is much likely that processing farnesol involves an innate mechanism for the extraction of relevant information to trigger a fast and reliable behavioral response. To test this hypothesis, we used population response analyses of 100 antennal lobe (AL) neurons recorded in alive bumblebee workers under repeated stimulation with four behaviorally different, but chemically related odorants . The analysis identified a unique neural representation of the recruitment pheromone component compared to the other odorants that are predominantly emitted by flowers. The farnesol induced population activity in the AL allowed a reliable separation of farnesol from all other chemically related odor stimuli we tested. We conclude that the farnesol induced population activity may reflect a predetermined representation within the AL-neural network allowing efficient and fast extraction of a behaviorally relevant stimulus. Furthermore, the results show that population response analyses of multiple single AL-units may provide a powerful tool to identify distinct representations of behaviorally relevant odors. Heliothis virescens it is shown that plant odorants can interfere with the signaling of a major component of a sexpheromone already at the receptor level [Agrotis ipsilon) computational interactions between pheromone and general odors are demonstrated to occurs at the receptor level [Innate behaviors should be hardwired, meaning that animals respond with a more or less stereotypic behavior when confronted with a specific stimulus or combination of stimuli . In flieor level . In anotor level  and at tor level ,14. But or level . Furtheror level .Bombus terrestris.In the present study we introduce a system that allows to investigate stimulus separation of chemically related, but behaviorally distinct odor components at the AL level and characterized their neural representation in the AL-network of the bumblebee, Honeybees and bumblebees are well established model organisms to investigate olfactory learning and memory formation –21. MoreThe first-order olfactory neuropil of the insect brain, the AL, shows analogous organization compared to the vertebrate olfactory bulb . TheBombus terrestris) colonies were obtained from a commercial breeder  and kept in a climate chamber under controlled laboratory conditions . In the morning before the experiment started, workers were caught and harnessed as described in Sommerlandt et al. [No specific permissions were required. Bumblebee  were soaked with 6 μl of odor solution and placed in the syringes. During the three-second odor stimulation, only half (2.5 ml) of the syringe air volume was injected into the constant air stream. The olfactometer was controlled via TTL pulses which were generated and synchronized to the recording channels using the trial control software from Neuralynx . We tested four different odor stimuli, geraniol (CAS#: 106-24-1), citronellol (CAS#: 106-22-9), citronellal (CAS#: 106-23-0) and farnesol (CAS#: 4602-84-0) all purchased from . Each stimulus application was repeated ten times in a pseudo-randomized order.A 12-channel olfactometer similar to the one described earlier ,47 was uWe used a 16-channel digital data acquisition system from Neuralynx. The extracellular electrode consisted of three micro wires . Detailed instructions of electrode building and visualization of the recording position are described in detail elsewhere . A silveWe applied a semi-automatic spike sorting technique (template-matching) provided with the Spike2 software . For details see ,47,49. Ihttp://find.bccn.uni-freiburg.de/). From the ten repetitions per stimulus time-resolved (milliseconds) mean firing rates were calculated by means of kernel convolution [τ. We applied a baseline correction by calculating the mean activity of 1000 ms before stimulus onset and subtracted that baseline from the units’ firing rate. The resulting mean rates of all units were used to construct stimulus dependent population vectors in the following way. For a given stimulus, a, and an ensemble of n neurons we constructed the n-dimensional rate vector va at each point in time. To calculate the population mean activity the rate vectors were averaged. All vectors together were used for principal component analysis (PCA). The factor loadings or weights of each single unit were used afterwards to order the units with respect to their contribution to the first principal component.Data analysis was carried out with MatLab and the FIND open source toolbox  were calculated for each pair of rate vectors (va- vb) using the following equation d(t) = (Σ(via(t)–vib(t))2)1/2.Time-resolved Euclidean distance  using the Friedman-test for dependent data. We tested post-hoc the pair-wise differences (six pairs) using the Wilcoxon rank sum test with an adjusted alpha-level of p<0.0083 (0.05/6) to account for multiple comparisons. To test for statistical differences between all six Euclidean distances a two-sided Wilcoxon rank sum test was performed with an adjusted alpha-level of p<0.0033 (0.05/15).We recorded extracellular neural activity close to the dorsal rim of the AL in worker bumble bees . In contDuring the experimental procedure each of the different odorants and the background (paraffin oil) was presented ten times in pseudorandomized order. To be able to analyze the 100 AL-units as a neural ensemble or population including the whole spectrum of neural response patterns , we used principal component analysis (PCA). PCA was performed taking into account time as the source of sample points, and number of neurons as the dimensions of the original component space. Comparison of the time courses of the ensemble responses of the first three principal components (PC1-3) revealed a clear separation of all stimuli by the AL-unit population . PromineInterestingly, farnesol was separated most clearly from all other stimuli. To visualize which neurons were responsible for that separation, we extracted AL-neurons dominating the PCA. We used the factor loadings to rank the recorded single units with respect to their contribution to the variation in PC1 (loading) and color coded their individual response profiles over time . PC1 conThe analysis of the subset of 32 AL-units supports that the farnesol induced activity evolves over time. In the next step we tested if this delayed temporal evolution was also reflected if we treated the 100 recorded AL-neurons as one ensemble. We calculated the Euclidean distances ) between the pairwise population vectors of the different odor stimuli . Using EOur results show that farnesol evoked a distinct and prolonged population activity of the AL-neuron network ensemble which was significantly increased compared to all other tested chemically related odor molecules. This most likely reflects a unique computation of farnesol within the AL-network that may enhance extraction of the biological relevance of this pheromone component in bumblebee workers.Drosophila melanogaster [Bombyx mori [Agrotis ipsilon [Schistocerca americana [Danio rerio [Bombus terrestris we also found these characteristic firing patterns of AL-neurons  in OSNs innervating only one glomerulus (DC3) facilitates the farnesol response [The neural activity of the AL consists of a complex network of temporal excitation and inhibition, resulting in a spatio-temporal pattern coding for an odor stimulus –35. The r tuning  or an inr tuning ,68 and or tuning . Both tyr tuning . As a coresponse . HoweverBumblebees can be easily trained to differentiate odors under controlled laboratory conditions . CombineDrosophila [5 mm Hg) is 1000 times lower compared to the other odor compounds tested . The odor concentration of farnesol in the headspace was therefore much lower compared with the monoterpenoids. The concentration of odor molecules in the headspace of the antenna usually correlates with the number of activated OSNs, and thus farnesol activation should be lower compared to the activation by geraniol, citronellol and citronellal. However, this was not the case, supporting our hypothesis of a distinct neuronal representation of farnesol in the AL-network.To investigate insect-plant or insect-insect interactions it is important to test how biologically relevant stimuli are detected and processed. A common way is to record antennal activity  during odorant stimulation ,71–73. Hosophila . As the osophila , it is iosophila , and accThe prominent perception of farnesol might be conserved in different insect species since farnesol can be found as a compound in floral volatiles in many plant families ,77, and Bombus terrestris AL or if it is related to its pheromonal nature, we plan additional experiments comparing different bumble bee casts, in particular workers and drones. Our results further show that the electrophysiological population analyses are a useful tool to investigate the perceptual space of behaviorally relevant odor stimuli in insects.Here we report, for the first time, multi-unit extracellular recordings and analyses of individual AL-neuron activities in bumblebees. Using population response analyses we show that farnesol, a component of the recruitment pheromone in bumblebees inducing an innate behavioral response, evokes a unique activity pattern at the AL network level compared to other odor stimuli used for localizing floral resources. To confirm, if this effect is a general feature of the


However, the potential of Brillouin microscopy is currently limited to transparent samples, because Brillouin spectrometers do not have sufficient spectral extinction to reject the predominant non-Brillouin scattered light of turbid media. To overcome this issue, we combined a multi-pass Fabry-Perot interferometer with a two-stage virtually imaged phased array spectrometer. The Fabry-Perot etalon acts as an ultra-narrow band-pass filter for Brillouin light with high spectral extinction and low loss. We report background-free Brillouin spectra from Intralipid solutions and up to 100  To address these challenges, historically, Brillouin spectroscopy has relied on multi-pass scanning Fabry-Perot (FP) interferometers.Brillouin light scattering spectroscopy has been a powerful technique in applied physics and material science for several decades by enabling the noninvasive characterization of material properties through the measurement of acoustic phonons.in vivo.In the past few years, a new approach to Brillouin spectroscopy has emerged, which dramatically enhanced measurement throughput. Using virtually imaged phased array (VIPA) etalons,Here, we report a spectrometer configuration featuring a tunable, high-throughput and narrow-bandpass filter based on a low-finesse Fabry Perot etalon. Thanks to this innovation, we increased the overall spectral extinction by more than 10 fold with respect to state-of-the-art spectrometers with less than ∼2 dB insertion loss. This enabled us to perform rapid Brillouin spectral characterization deep into non-transparent biological tissue without any limitation due to elastic scattering background.The spectrometer consists of a triple-pass Fabry-Perot (3PFP) bandpass filter and a two-stage VIPA spectrometer Fig. . The 3PFBy changing the angle between the etalon and the incoming beam, the filter bandpass can be tuned with high precision to isolate the desired Brillouin scattering signal (Stokes peak from a water sample in this case), as shown in Figure g), exposure time (t), and using a neutral density filter of throughput (f). The intensities (I) for each acquisition were then scaled according to the relation The spectral extinction of the spectrometer is characterized in Figure To quantify the performances of our setup in the measurements of turbid media, we connected the Brillouin spectrometer to an inverted confocal microscope and measured the Brillouin spectra of intralipid solutions. Light from a 532 nm single mode laser was focused into an intralipid solution sample, placed in a transparent plastic dish. Figure Figure 2, placed on the bottom of glass dish plate. The laser light was focused into the tissue through the glass cover-slip at a power of 6 mW and exposure time of 300 ms. Fig. μm. We observed an exponential decay of SNR yielding a mean free path of ∼150 μm, consistent with the intensity loss due to elastic scattering in chicken breast tissueNext, we demonstrated the ability to detect background-free Brillouin signal from biological tissue. The tissue sample was a thin slice of chicken breast of area of about 1 cmImportantly, other methods of rejecting elastically scattered light that use absorption lines of gas cells require experimentalists to perform Brillouin measurements with specific laser wavelengths and are prone to degradations due to laser frequency drift. In contrast, our spectrometer maintains its performance over a spectral band only limited by the coatings of the optical elements . Therefore, the spectrometer can accommodate Brillouin experiments with a wide variety of excitation wavelengths; moreover, operating at low-finesse and with a tunable etalon, laser frequency drifts can be effectively handled.Several improvements can be implemented to improve the practical performances of our spectrometer. As mentioned before, the FSR of the Fabry-Perot etalon and the VIPAs should be the same to reach maximal extinction. Moreover, using the same principles demonstrated here, a reflection configuration of the 3PFP would allow to build a notch-filter rather than a bandpass filter, with similar performances of throughput and extinction but larger transmitted bandwidth, which could be useful for Brillouin measurements exhibiting large shift variations of non-homogenous sample.In conclusion, we have developed a two-stage VIPA Brillouin spectrometer with confocal sampling and a triple pass Fabry-Perot bandpass filter. We demonstrated rapid Brillouin measurement of intralipid solutions and turbid biological tissue with effective suppression of elastic scattering background. This investigation may expand the reach of Brillouin technology beyond ocular tissue to important areas such as the mechanical characterizations of tumors and atherosclerotic plaques.


Annually laminated stalagmites can be used to construct a precise chronology, and variations in laminae thickness provide an annual growth-rate record that can be used as a proxy for past climate and environmental change. Here, we present and analyse the first composite speleothem annual growth-rate record based on five stalagmites from the same cave system in northwest Scotland, where precipitation is sensitive to North Atlantic climate variability and the winter North Atlantic Oscillation (NAO). Our 3000-year record confirms persistently low growth-rates, reflective of positive NAO states, during the Medieval Climate Anomaly (MCA). Another persistently low growth period occurring at 290-550 CE coincides with the European Migration Period, and a subsequent period of sustained fast growth-rate (negative NAO) from 600-900 AD provides the climate context for the Viking Age in northern and western Europe. As a result, high (low) lamina growth rates typically reflect warm and/or dry (cool and moist) conditionsStalagmites contain a variety of potential palæoclimate proxies, and the sensitivity of any individual stalagmite proxy to climate is determined by many factors, including its location within the cave, the cave morphology, the hydrological flow path, and the overlying soil and vegetationUsing a combination of lamina counting and U-Th dating, stalagmite growth rate records from a region can be combined to produce long continuous paleoclimate records. One stalagmite from the site has been previously combined with tree-ring data in a 947-year annual resolution reconstruction of the NAO (NAOms), which suggested an unusual persistence of the positive phase of the NAO during the MCA (1050-1400 CE)67comp).Stalagmite SU963 grew on the same flowstone shelf as SU961 and SU962, and contained a continuous lamina sequence for 1802 years with no hiatuses. The growth-rate was determined from multiple manual analyses of images collected using UV microscopy  precipitation, with correlation p < 5% using CRU TS3.22For the instrumental period, SUcomp series is the temporal correlation with the winter (January-March) NAO indexcomp  that is unique within the last 1000 years . These three records do not observe a more variable NAO state in the MCA as observed in the trace element record of a Moroccan stalagmiteThe SUcomp, for example between 200 BCE – 500 CE. However, there are also periods of difference, especially before 500 BCE. Reasons for the differences could include dating uncertainties in both series  or, more likely, limitations in the strength NAO – proxy calibration in the lake sediment record. There, the NAO-like pattern is found in the third principal component, which explains only 9.4% of the variability in the data. Only further long, high-resolution proxy records can help to develop improved NAO reconstructions.Prior to the last 1000 years, we observe periods of good agreement in the reconstructed NAO between the two available records, the West Greenland lake sediment record and SUOur 3000-year record confirms persistently low growth-rates, reflective of positive NAO states, during the MCA. Another persistently low growth period occurred at 290-550 CE, and wavelet analysis demonstrates that period of slow (or fast) growth are coincident with periods of relatively low (or high) magnitude of multi-decadal variability, indicating a potential relationship between the strength of the Atlantic Multidecadal Oscillation and the NAO at these times. The phase of persistent positive NAO that occurs at ~290-550 CE coincides with a period of increased climate variability and an unusually wet growing season (April-June) in central Europe as observed in tree ringscomp record as a proxy for the NAO over the last 3000 years, this allows us to start building hypotheses relating to the impact of climate variability on environmental and societal change in NW Europe. For example, although we recognise that climatic changes alone are unlikely to be the sole driver for societal change26comp record provides a useful climatic context of colder Scandinavian climate and weaker westerly circulation for this important phase of early European history.Assuming that we can utilise the SUThe fluorescent laminae for stalagmite SU963 were counted and their thicknesses measured in duplicate using techniques detailed previouslyStalagmite SU963 was dated with U-Th mass spectrometry using methods previously reported29To further constrain the timing of deposition of SU963, the annual lamina thickness series was cross-correlated with that of stalagmites SU961, SU962 and SU967. Cross-correlations were calculated for theoretical end-of-deposition dates between 120 and 320 years before present. The strongest cross-correlations were observed when the period of divergent lamina thickness in SU963 of 150 years at ~1550 years before present was removed from the analysis: for SU961 with an end-of-deposition date of 210 year before present (r = 0.25); for SU962 with an end-of-deposition date of 229 years before present (r = 0.46); and for SU967 with an end-of-deposition date of 223 years before present (r = 0.34). The mean of these three approaches yields a date of growth cessation of 221 years before present. We have placed the SU963 lamina thickness series into the chronology with the end of deposition at 229 years before present, which is identical to the mean of the U-Th predicted date of the end of deposition and with the strongest cross-correlating stalagmite (SU962).comp. Correlation analyses were performed using Microcal Origin, SPSS, Excel and Climate Explorercomp record using a cubic smoothing spline to the approximate resolution of the lower resolution proxies .


We have recently conducted a clinical phase I study to assess the safety and spatial accuracy and precision of a newly developed dedicated MR-HIFU system for lateral breast tumor ablation . Here, wversus ablated tumor tissue. The number of sonication performed per patient (1-5) and the acoustic power (50-90 W) used for each sonication was variable. Surgery was performed at least 48 h after the MR-HIFU procedure. After surgical resection H&E and cytokeratin 8/18 staining was performed on histological sections. In order to assess the safety of the system i) the skin of the treated breast was carefully evaluated by a physician, ii) patients were asked to report pain scores according to the Numerical Rating Scale, with a score of 0 (no pain) to 10 (worst pain imaginable) and iii) the temperature evolution in the temperature imaging slice positioned on the pectoral muscle was analyzed to evaluate possible unintended heating in the far field during tumor ablation.All treatments were performed on a dedicated breast MR-HIFU system  integrated with a clinical 1.5-T MRI scanner . Ten female patients with i) pathologically proven invasive breast cancer after large-core needle biopsy and ii) tumor size ≥ 1 cm were included. The patients were under procedural sedation during the complete HIFU procedure. Fat-suppressed segmented Echo Planar Imaging was performed for PRFS-based thermometry. Online correction of the respiration-induced field disturbances was performed by a Look-Up Table (LUT)-based method . PartialSeventeen patients were initially enrolled in the study. Seven patients were excluded or withdrew from the study after a pre-treatment MRI scan.Finally, 10 patients  underwent MR-HIFU ablation. The procedural sedation protocol was improved in the course of the clinical study. This lead to less sonication related motion and involuntary patient motion. In addition, the respiration became more regular and shallower, which improved the quality of MR thermometry. The maximum temperature in the focal point increased with increasing power in each patient. Sonications performed with the same acoustic power in different patients lead to different maximum temperatures in the focal point. Finally, clear thermal damage was observed in the tumor tissue in 5 patients.The absence of thermal damage in the other patients was due to technical problems that prevented the sonication to reach temperatures inside the tumor leading to tissue damage. In none of the patients, skin redness, skin burns or other signs of skin damage were observed. Nausea and vomiting in the hours after the MR-HIFU procedure were reported as minor adverse events in one patient. Only two patients reported pain (maximum score of 5) after the MR-HIFU treatment. No temperature increases related to the tumor ablation were observed in the pectoral muscle during sonication. In conclusion, breast tumor ablation with the dedicated breast MR-HIFU system is safe and technical feasible. A good sedation protocol during HIFU ablation is essential for the success of the treatment.


In sport events like Olympic Games or World Championships competitive athletes keep pushing the boundaries of human performance. Compared to team sports, high achievements in many athletic disciplines depend solely on the individual’s performance. Contrasting previous research looking for expertise-related differences in brain anatomy at the group level, we aim to demonstrate changes in individual top athlete’s brain, which would be averaged out in a group analysis. We compared structural magnetic resonance images (MRI) of three professional track-and-field athletes to age-, gender- and education-matched control subjects. To determine brain features specific to these top athletes, we tested for significant deviations in structural grey matter density between each of the three top athletes and a carefully matched control sample. While total brain volumes were comparable between athletes and controls, we show regional grey matter differences in striatum and thalamus. The demonstrated brain anatomy patterns remained stable and were detected after 2 years with Olympic Games in between. We also found differences in the fusiform gyrus in two top long jumpers. We interpret our findings in reward-related areas as correlates of top athletes’ persistency to reach top-level skill performance over years. World class performance in athletics is thought to be a consequence of individual genetic predispositions and environmental factors such as deliberate skill practice . DeliberSkillful motor action is coordinated by subcortical and interconnected cortical brain structures such as the cortico-striatal and cortico-cerebellar pathways ,19. In mThe study was carried out in accordance with the Declaration of Helsinki and had been approved by the Ethics Committee of the University of Leipzig. Subjects gave their written informed consent prior to participation. Brain imaging and behavioral data were acquired from three track-and-field athletes . At the time of MRI scanning, these athletes performed at the highest national and intermediate international level: Two of them are long jumpers and sprinters with personal bests in long jump of >8 and >7.90 meters and each with < 11 seconds in 100 meter sprint. One of the long jumpers is a former national vice-champion. The third athlete is a javelin thrower with a personal best mark of >80 meters. The javelin thrower is a former national champion. All three athletes were active in their disciplines for at least thirteen years and conducted at least seven training session per week. Two athletes regularly participated in athletic competitions including World Championships and Olympic Games. Each of the three athletes was used for a separate case-control comparison in which deviations in brain morphology from the normal population are computed by comparing each athlete’s structural brain image (case) to an age-, sex- and education-matched control sample (control).MRI data acquisition) and control subjects were carefully selected against the following criteria for each individual top athlete . By controlling for the following factors, we aimed at controlling expertise-related brain features for age or educational background: age (±2 years), sex, education (A-level). We ensured that the control subjects had a level of current physical activity below 2 hours per week and received no former athletic or musical training .To compute significant deviations from the normal population in each athlete’s brain, T1-weighted MR images from healthy control subjects were obtained from the local MRI database at the Max-Planck-Institute for Human Cognitive and Brain Sciences. Our search yielded 46 matched control subjects for the javelin thrower, 58 matched control subjects for one long jumper and 58 control subjects for the other long jumper. We ensured that all athletes and control subjects were scanned at the same MR scanner , sex- and education-matched (A-level) control sample. The three up-and-coming athletes were used for descriptive comparison with the three top athletes. The up-and-coming athletes are active for at least nine years, conduct at least five training sessions per week and have personal bests in long jump between 6.80 and 7.20 meters.MRI data acquisition). During the behavioral tests session, a standard drop-jump test was performed to analyze general speed and jumping performance .Each of the six athletes completed a magnetic resonance imaging (MRI) scan and a behavioral test session on a different day. T1-weighted MRI scans were acquired to assess structural grey matter density  data was acquired on a 3T Magnetom Tim Trio scanner (Siemens) using a 32 channel head coil. T1-weighted images were acquired using a MPRAGE (magnetization-prepared rapid acquisition gradient echo) sequence . The acquisition time was approximately 13 minutes.http://dbm.neuro.uni-jena.de/vbm.html), implemented in the neuroimaging analysis software package SPM8  running under Matlab 7.7 (Mathworks) [Pre-processing of T1-weighted images was performed using the VBM8 toolbox (thworks) . T1-weigthworks) . High-dithworks)  was usedthworks) . Modulatthworks) . These mt test in SPM with equal variances of the two groups involving one scan in the first group (the athlete) and appropriate control subjects in the second group.To highlight characteristic features in brain structure of individual athletes, we compared each of the three top athletes to the matched control sample (see above). All control subjects were recruited from the local MPI database. Following our inclusion criteria , we obtained 46 matched control subjects for the javelin thrower and for one long jumper, and 58 control subjects for the second long jumper (note that there were more subjects matching the second long jumper’s age range). T1-weighted images from control subjects were acquired on the same MR scanner using a 12 or 32-channel head coil. We controlled for the different head coils and age (± 2 years of the athletes’ age) in each statistical model using a separate covariate of no interest. For analysis, we assumed that the athlete’s grey matter value at each voxel constitute the mean of a population with a variance equal to that of the control group ,32. Thist-value) for structural differences in regions of interests (ROIs) in the striatum and thalamus.A group of three up-and-coming long jumpers was used for descriptive comparison with the three top athletes (see above). For each of the three up-and-coming athletes we used the same procedure as for the top level athletes, including a well-matched control sample, specific to the athlete, to determine features in brain morphology deviating from the control group. The three control groups consisted of 31, 59 and 59 matched control subjects because the up-and-coming athletes differed in age and hence, the number of control subjects in the MPI database matching our inclusion criteria was different (see above). Results from each of the six case-control comparisons were plotted in a scatter plot involving maximum statistical values  in the striatum and thalamus as defined by the automated anatomical labeling atlas  relative to the reference group . Although not tested statistically due to too few degrees of freedom, both long jumpers had also larger flight times than the three up-and-coming athletes . The javelin thrower had a flight time comparable to controls (475 ms) but a low ground contact time of 124 ms relative to the two top (134 and 145 ms) and the three up-and-coming  long jumpers .Total brain volume was comparable between each of the three top athletes  and their respective control sample . The two long jumpers had a significant larger GMD in the right ventral putamen . Are these identified specific brain features of top-level athletes also observable in up-and-coming athletes? We analyzed brain imaging data from three up-and-coming long jumpers with personal bests between 6.80 and 7.20 meters. Similar to the previous case-control analyses, each up-and-coming athlete was compared against an age-, sex- and education-matched control group to determine structural features in the striatum and thalamus see . For that = 3.42, x y z -9–16 15) and fusiform gyrus  but not in the striatum (p > 0.05). This is consistent with results from separate single-case analyses where striatal effects in the two top long jumpers were spatially more dispersed . Within regions where we found significant effects in the separate single-case analyses , the combined analysis revealed more pronounced structural differences in top as compared to up-and-coming athletes in the thalamus  brain regions that have been found to be vulnerable to training-dependent structural brain plasticity and subject to morphological differences in musicians compared to controls ,35.In the top javelin thrower, we found no significant difference (increase or decrease) to the control group in the precentral gryus (PrCG) and a non-significant trend towards larger GMD in the left postcentral gyrus . Similarly, no significant difference for PrCG and a trend towards larger GMD in right PoCG was observed in one of the two top long jumpers . The other top long jumper displayed significantly larger right and left PoCG GMD  and a non-significant trend in left PrCG .t = 6.38; x y z coordinate: -32, -70, -9 and t = 5.84; x y z coordinate: -32, -70, -8; p < 0.003 and p < 0.016 FWE corrected at the voxel-level across the whole-brain; We also performed whole brain analyses to detect expertise-related brain features that were not part of our primary hypotheses. Surprisingly, we observed significantly larger cortical GMD in both top long jumpers but neither in the top javelin thrower nor in the three up-and-coming long jumpers in the left posterior fusiform gyrus  compared to age-, sex- and education-matched control subjects. While a javelin thrower showed restricted enlargements in bilateral striatal GMD that were reliably detected before and after 2 years with international tournaments in between, two long jumpers both had enlarged striatal, thalamic and visual cortical GMD compared to controls. We did not find significant differences in sensorimotor cortical regions in any of the three athletes. Our findings show significant deviations of brain structure in single healthy individuals and future studies using a similar design need to confirm these findings.Our findings are consistent with the view that individual variations in brain anatomy reflect differences in behavioral performance ,36. CorrIn both top long jumpers, we identified considerable differences in regional GMD in cortical regions of the left fusiform gyrus . The exeThe long jump is a complex athletic skill that is separated into the approach phase, take-off, flight phase and landing. Long jump performance is heavily dependent on the quality of the approach phase . The goaCortico-striatal-thalamo-cortical loops are fundamental for information processing. If we accept that performance of highly complex motor tasks involves information processing within and between widespread cortical and subcortical networks across the brain , reorganThe thalamus is implicated in the control of arousal and modulation of cortical brain activity via thalamo-cortical connections . During We did not find significant differences between each athlete and controls in areas of the sensorimotor cortex, a region that is strongly associated with motor control and learning and has been found to be enlarged in group studies of musicians ,45. AlthTherefore, it seems that the imperative for structural plasticity in motor cortical areas is modulated by motor training regimen such as musical or athletic training. Optimization of highly automatized, athletic skills may not anymore trigger expansion of neural structures while permanent acquisition of new musical pieces over the course of a musicians career causes ongoing structural plasticity in M1 . These dTherefore, we believe that the cortical motor system still has a critical role for complex skill performance in our athletes but the relevant neurobiological adaptations may be consolidated in a state that is no longer observable with our MRI methods. Long-term motor training may have caused a renormalization of initial M1 adaptations that were no longer visible after motor skill acquisition ,58. AlsoHere we compared structural MRI scans of single athletes with a group of matched control subjects to detect significant deviations from average human brain structure. These anatomical differences may be associated with their profession but single case comparisons do not permit to make inferences about the cause of these differences or potential relationships to sports behavior because differences may be mediated by factors independent of an athletes profession. We made an attempt to control for confounding effects of age, gender and education by careful selection of control subjects and multiple testing of different top athletes to show similarities in structural features. Also, differences in thalamic and visual cortical structure were prominent in the two top long jumpers but not in three up-and-coming long jumpers suggesting achievement specificity of our findings. However, we cannot exclude the possibility that other potential factors have mediated our structural differences and therefore future studies are required to confirm our findings and to shed more light into the causal relationship between brain structure and athletic training in track-and-field athletes.The behavioral underpinnings of expert performance have been subject to numerous studies in sports and cognitive science in the past decades . Here we


The same 3-fold increase in sensory nerve regeneration was found. In conclusion, side-to-side cross-bridges ameliorate poor regeneration after delayed nerve repair possibly by sustaining the growth-permissive state of denervated nerve stumps. Such autografts may be used in human repair surgery to improve outcomes after unavoidable delays.There are currently no available options to promote nerve regeneration through chronically denervated distal nerve stumps. Here we used a rat model of delayed nerve repair asking of prior insertion of side-to-side cross-bridges between a donor tibial (TIB) nerve and a recipient denervated common peroneal (CP) nerve stump ameliorates poor nerve regeneration. First, numbers of retrogradely-labelled TIB neurons that grew axons into the nerve stump within three months, increased with the size of the perineurial windows opened in the TIB and CP nerves. Equal numbers of donor TIB axons regenerated into CP stumps either side of the cross-bridges, not being affected by target neurotrophic effects, or by removing the perineurium to insert 5-9 cross-bridges. Second, CP nerve stumps were coapted three months after inserting 0-9 cross-bridges and the number of 1) CP neurons that regenerated their axons within three months or 2) CP motor nerves that reinnervated the extensor digitorum longus (EDL) muscle within five months was determined by counting and motor unit number estimation (MUNE), respectively. We found that three but not more cross-bridges promoted the regeneration of axons and reinnervation of EDL muscle by  Despite optimal management, recovery of function after peripheral nerve injury and surgical repair is rarely complete; indeed, it is frequently poor ,2. WhilsChronically denervated muscles contract more forcefully after delayed nerve repair when a prior surgery was carried out to direct sensory nerves into the denervated muscle . The expand sensory neurons contribute their axons through the side-to-side cross-bridges? 4) Does the number of cross-bridges dictate how many neurons participate in growing axons through the cross-bridges? and 5) Will optimum placement of side-to-side cross-bridges improve the regeneration of motor nerves and/or sensory nerves after delayed nerve repair? In light of there being no reliable medical or surgical options to oppose poor functional recovery when nerves regenerate through chronically denervated Schwann cells, our objective is to promote nerve regeneration and muscle reinnervation after delayed repair by ‘protecting’ chronically denervated nerve stumps with axons from a nearby donor nerve.There remain many unanswered and important questions. These are addressed in the current study: 1) Does the size of perineurial windows cut into a donor nerve and a recipient denervated nerve stump impact on the number of neurons that grow axons through side-to-side cross-bridges? 2) Do donor axons that enter the denervated distal nerve stump through side-to-side cross-bridges, continue to grow either proximal and/or distal to the cross-bridges, possibly influenced by a neurotrophic effect of the denervated targets? 3) Do both motor Thy-1-GFP transgenic rats that were developed in our laboratory. Green fluorescent protein (GFP) is expressed in neural cells under the Thy-1 promoter but is not expressed in muscle or connective tissue [All experiments were carried out on adult female Sprague Dawley rats . Of these, 26 rats were e tissue –24.Sterile surgery was carried out under inhalational anesthetic  and using an operating microscope . The rats were administered subcutaneous Metacam  for relief of post-operative pain. A warming pad was placed under the rat in order to maintain body temperature during surgery and during the brief period during which the rat regained consciousness after the surgery when the inhalational anesthetic administration ceased. The protocol was approved by the Animal Care Committee, Hospital for Sick Children, Toronto, and all perioperative care, including postoperative monitoring and analgesia, was administered according to Canadian Council for Animal Care guidelines. Post-operative care included regular daily post-operative monitoring over the first week of all surgeries followed by regular monitoring by the staff of the animal facility.Experiment #1; N = 68 rats; left CP nerve was dissected ~5–7 mm from the sciatic notch to its distal insertion into the muscles in the anterior hindlimb compartment. The CP nerve was carefully dissected free from the TIB nerve along the natural division of the two nerves as they course through the sciatic nerve. Dissection was halted at the most proximal portion of the sciatic nerve at the sciatic notch where the CP, TIB, and sural nerve fascicles were interweaved. A ~28–30 mm nerve length was harvested, cut, and kept moist in the hindlimb for later division into one to nine autologous CP side-to-side nerve cross-bridges. In the right experimental hindlimb, the CP nerve was transected and the proximal and distal nerve stumps were ligated. The distal CP nerve stump was laid parallel to the donor TIB nerve and the two CP nerve stumps were sutured to underlying innervated muscle to prevent nerve regeneration from the proximal to the distal nerve stump [left hindlimb were inserted into the sides of the donor TIB nerve and the recipient denervated CP nerve stump through equidistant perineurial windows placed over a 10 mm length , one and three 6 mm long bridges  the donor TIB motor and sensory neurons that regenerated and/or sprouted their axons into the recipient denervated CP nerve stump in the right experimental hindlimb  muscle under deep isofluorane anesthesia and at 37°C, five months after surgical coaptation of the CP nerve stumps with and without placement of three autologous CP cross-bridges : Exp. 2Ap≤0.05).The data are plotted with mean ± standard errors (SEs) as well as individual data points in regenerated their axons into the recipient CP nerve stump were backlabelled with the dye applied to the CP nerve stump but not with dye applied to the donor TIB nerve. The TIB neurons that sprouted their axons on the other hand were backlabelled from both the recipient CP nerve stump and the donor TIB nerve  nerve cross-bridges from the left hindlimb were dissected and placed in a side-to-side fashion via perineurial windows that were opened at equal distances within a 10 mm length of the intact donor tibial (TIB) nerve and the recipient denervated CP nerve stump in the right experimental hindlimb . TIB axoIB nerve .It was reported that the size of the perineurial window is an important determinant of how many axons regenerated through a denervated distal nerve stump inserted into the side of an intact donor nerve—an end-to-side coaptation –32. We fWhen wider perineurial windows were opened, significantly more donor TIB motoneurons sent their axons through three cross-bridges into the recipient denervated CP nerve stump, the mean number increasing as a linear function of the radius of the window . DespiteNote in In order to determine the direction of growth of donor TIB axons through three CP nerve autografts and into the recipient denervated CP nerve stump  and the Schwann cells in the distal nerve stumps remain chronically denervated . We demoand sensory neurons contributed axons equally across the side-to-side bridges  whether or not a perineurial window is created ,19,42. Oges Figs  and 8 ind 80–85% ,47,48. TThy-1 transgenic rats [Donor TIB neurons regenerated their axons across two perineurial windows from the donor nerve, through the cross-bridges, and into the recipient denervated CP nerve stump Figs –9. The Snic rats . During nic rats –64. The nic rats –67. Dononic rats . It is pThree months after placing the cross-bridges, the mean number of ~4500 donor axons that occupied the recipient denervated nerve stump Figs  and 9 waIt is likely that donor axons regenerate considerably more rapidly across the second as compared to the first perineurial window between each of the cross-bridges and the recipient denervated nerve stump. From the staggered time course of axon regeneration across a suture site of ~28 days, it is likely that the first axons to regenerate across the 3.2 mm long cross-bridges would reach the second window within days. In the course of the first ten days during which the extracellular matrix becomes aligned, Schwann cells cross into the suture site and are arrayed in the Bands of Bungner that in turn, guide regenerating axons into and through the endoneurial tubes of the denervated distal nerve stump . The pacAll CP motoneurons regenerated their axons through the ‘protected’ distal nerve stump when three cross-bridges were inserted through perineurial windows of a 500 μm radius as compared to the more limited 1.7-fold increase when ~50 of the TIB motoneurons sent their axons into the recipient denervated CP nerve stump through perineurial windows of half the radius, namely 250 μm (Figs The rationale of creating the side-to-side cross-bridges prior to delayed nerve repair was to prolong the permissive state of the Schwann cells by passage of donor axons into the recipient denervated nerve stump. This in turn, was to boost the capacity of the axotomized neurons to regenerate their axons within a chronically denervated nerve stump. The occupation of the distal nerve stump by donor TIB axons was increased by increasing the size of the perineurial windows cut into the donor nerve and into the recipient denervated nerve stump . As a re μm Figs . The eff μm Figs . Reinner μm Figs , and 11F μm Figs , and 11D μm Figs , and 11DIt is likely the donor axons that grew into the denervated nerve stump released agents such as neuregulin that promote the proliferation of the Schwann cells and their expression of regeneration associated genes. Those axons that made contact with Schwann cells were remyelinated by the Schwann cells Figs  and 9, cIn the current study, we allowed the babysitting donor axons to remain within the recipient denervated nerve stump after delayed nerve repair. This was done so as to model the clinical scenario of nerve regeneration of injured neurons being progressively compromised by chronic axotomy and chronic SC denervation over time and distance ,7,8,41. In summary, our animal experiments systematically investigated the optimal size of perineurial windows between a donor nerve and a recipient denervated nerve stump and revealed that windows whose radii were ~3-fold larger than that of the autologous nerve cross-bridges were optimal for promoting excellent nerve repair after delayed nerve surgery.


Viral vaccine vectors have shown to be effective in inducing a robust immune response against the vaccine antigen. Newcastle disease virus (NDV), an avian paramyxovirus, is a promising vaccine vector against human and veterinary pathogens. Avirulent NDV strains LaSota and B1 have long track records of safety and efficacy. Therefore, use of these strains as vaccine vectors is highly safe in avian and non-avian species. NDV replicates efficiently in the respiratory track of the host and induces strong local and systemic immune responses against the foreign antigen. As a vaccine vector, NDV can accommodate foreign sequences with a good degree of stability and as a RNA virus, there is limited possibility for recombination with host cell DNA. Using NDV as a vaccine vector in humans offers several advantages over other viral vaccine vectors. NDV is safe in humans due to host range restriction and there is no pre-existing antibody to NDV in the human population. NDV is antigenically distinct from common human pathogens. NDV replicates to high titer in a cell line acceptable for human vaccine development. Therefore, NDV is an attractive vaccine vector for human pathogens for which vaccines are currently not available. NDV is also an attractive vaccine vector for animal pathogens. Infectious diseases have been emerging and reemerging over millennia . Human iReplicating viral vector vaccines offer a live vaccine approach without requiring involvement of the complete pathogen or cultivation of the pathogen . ReplicaNewcastle disease virus (NDV) is a fast-replicating avian virus that is prevalent in all species of birds . In mostAvirulent NDV strains are highly safe in avian and non-avian species. NDV replicates well in vivo and induces a robust immune response. In contrast to adeno, herpes, and pox virus vectors whose genome encodes a large number of proteins, NDV encodes only seven proteins and is thus less competition for immune responses between vector proteins and the expressed foreign antigen. NDV replicates in the cytoplasm, does not integrate into the host cell DNA, and does not establish persistent infection. Recombination involving NDV is extremely rare. NDV has a modular genome that facilitates genetic manipulation. NDV infects via the intranasal route and therefore induces both mucosal and systemic immune responses. A wide range of NDV strains exists that can be used as vaccine vectors. NDV-vectored vaccine can also be used as a “differentiating infected from vaccinated animals” (DIVA) vaccine. In this review article, we have reviewed the biology of NDV, development of reverse genetic systems for generation of NDV-vectored vaccines, and use of NDV vector for development of human and veterinary vaccines.Avulavirus in the family Paramyxoviridae [NDV is a member of the genus oviridae . NDV viroviridae . The re-oviridae . In NDV, the HN and F proteins are the two integral membrane proteins. The HN protein is responsible for attachment of the virion to sialic acid containing cell surface receptors. The F protein mediates entry of the virus into the host cell by fusion of the viral envelope to the plasma membrane. The F protein is synthesized as a precursor (F0) that is cleaved by host cell protease into two biologically active F1 and F2 subunits. Cleavage of the F protein is a pre-requisite for virus entry and cell-to-cell fusion. The amino acid sequence at the F protein cleavage site has been identified as the primary determinant of virulence ,8. VirulInfectious NDV can be recovered entirely from cloned cDNA by transfecting cultured cells with plasmids encoding the viral components of a functional nucleocapsid, full-length antigenomic RNA, and the major proteins involved in replication and transcription, i.e., the N, P, and L proteins under the control of bacteriophage T7 RNA polymerase promoter  Figure . This meAvirulent NDV strains LaSota and B1 are commonly used as vaccine vectors because of their proven track records of safety. Mesogenic and velogenic NDV strains are not used as vaccine vectors because they are virulent in chickens. In an experimental study, the mesogenic strain Beaudette C (BC) was evaluated as a vaccine vector in nonhuman primates . Strain 6.5 plaque-forming units (PFU) per site of NDV did not cause any disease symptoms and its replication was restricted to the respiratory tract [10 PFU of NDV to humans was safe without causing adverse effects [9 PFU/mL) but also in Vero cells (108 PFU/mL), which is acceptable for human vaccine development. In fact, NDV has been used to express protective antigens of various human pathogens and has shown promising results in nonhuman primates. NDV-vectored vaccines for several human pathogens are discussed as follows  hemagglutinin (HA) protein . The expThe potential of NDV as a vaccine vector for use in humans was first determined in nonhuman primates. Two NDV strains LaSota and BC were evaluated as vaccine vectors in nonhuman primates by inserting the HN protein of human parainfluenza virus type 3 (HPIV3) as a protective antigen . Two dosZaireebolavirus) of infected individuals [Ebola virus (EBOV) causes severe hemorrhagic fever in humans with a fatality rate of up to 88% ). Specifically, immunization with NDV-VF vector resulted in SARS-CoV titers of a 5-fold, 61-fold, and 236-fold reduction in nasal turbinate, trachea, and lung, respectively, compared with the control animals. The NDV-BC vector was even more effective, with average reductions in viral titer of 13-fold, 276-fold, and 1102-fold in the nasal turbinate, trachea, and lung, respectively. This study demonstrated the safety and protective efficacy of NDV as a topical respiratory vaccine vector for SARS-CoV.NDV was evaluated as a vaccine vector for another important emerging pathogen, the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) . Two NDV6-fold reduction in rVac/SIVgag titers at day 5 after challenge compared to titers of control mice injected with phosphate-buffered saline (PBS). The magnitude of the protective immune response also correlated with the levels of cellular immune responses to Gag. These results suggest that NDV vector can be a suitable candidate vaccine against HIV.The use of viral vectors expressing selected HIV antigens has been a promising vaccine strategy. The potential of NDV-vectored vaccine against HIV infection was first evaluated by generating recombinant NDV expressing simian immunodeficiency virus (SIV) Gag protein (rNDV/SIVgag) . The vacThe HIV Gag and Env proteins have been expressed by NDV vector ,28,29,30Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infants and elderly . The devBorrelia burgdorferi [B. burgdorferi , immunization with vector-expressing BmpA provided a reduction of the pathogen load in the joints. This study showed the potential of NDV as a vaccine vector against bacterial pathogens. NDV has also been used as a vector to express the immunogens of a bacterial pathogen. Lyme borreliosis is a prevalent vector-borne disease in the United States, Europe and parts of Asia. NDV was used to express the basic membrane protein A (BmpA) and the outer surface protein C (OspC) of the Lyme disease pathogen gdorferi . C3H or Nipah virus (NiV) is a deadly emerging zoonotic pathogen that causes fatal encephalitis in humans and pigs . The glyNorovirus (NoV) is the most frequent cause of viral gastroenteritis in people of all ages . The inaNDV-vectored vaccines have been evaluated in several animal species  for veterinary use  Table 2Table 2. As a bivalent vaccine, the NDV strain LaSota was first used to express the host-protective immunogen VP2 of infectious bursal disease virus (IBDV), a birnavirus, which causes a highly immunosuppressive disease in chickens . The proInfectious laryngotracheitis is a major respiratory disease in chickens and caused by infectious laryngotracheitis virus (ILTV), a herpes virus . BivalenInfectious bronchitis virus (IBV), a coronavirus, is an important avian pathogen, causing respiratory disease in broilers and poor egg production in breeders and layers worldwide . The spiNDV vector has also been used for the prevention of economically important livestock diseases. Rift Valley fever virus (RVFV), a bunyavirus, causes recurrent large outbreaks in humans and in livestock . NDV expBovine herpesvirus-1 (BHV-1) is a major cause of respiratory tract diseases in cattle. Since modified live BHV-1 vaccines can cause latent infection in immunized animals, NDV expressing the glycoprotein D (gD) of BHV-1 was generated as a vectored vaccine . A singlRabies virus (RV), a rhabdovirus, causes a fatal neurologic disease in humans and in animals . To geneCanine distemper virus (CDV), a morbillivirus, infects many carnivores and cause several high-mortality disease outbreaks . The curHighly pathogenic avian influenza virus (HPAIV) is an economically important pathogen of poultry worldwide. The outbreaks involving H5N1 or H7N7 influenza viruses resulted in lethal infections in poultry and the death of a limited number of people . TherefoFor the generation of vaccines, a major protective antigen, hemagglutinin (HA) of HPAIV has been placed between the P and M genes or between the F and HN genes in lentogenic NDV strains LaSota or B1. To address a safety concern, an NDV-vectored vaccine was further generated by replacing the polybasic cleavage site in HPAIV HA with that from a low-pathogenicity strain of influenza virus . In addiAlthough use of avirulent NDV vectors has been effective in protecting chickens against clinical disease and mortality, some studies also found virus shedding in chickens after challenge with HPAIV . To enha7 PFU) induced a high titer of H5N1 HPAIV-neutralizing serum antibodies in all of the immunized monkeys. Moreover, a substantial mucosal IgA response was induced in the respiratory tract, which can potentially reduce or prevent transmission of the virus during an outbreak or a pandemic. The intranasal route of administration is also advantageous for needle-free immunization and is thus suitable for mass immunization. The protective efficacy of vaccine viruses was evaluated in African green monkeys by the intranasal/intratracheal route or by the aerosol route of administration [7 PFU of homologous HPAIV. This study demonstrated that needle-free, highly attenuated NDV-vectored vaccines were immunogenic and protective in a nonhuman primate model of HPAIV infection. For effective human vaccines against HPAIV, the immunogenicity of NDV expressing the HA of H5N1 was evaluated in African green monkeys by the intranasal route of administration . Two dosstration . Each ofNewcastle disease virus (NDV) is an attractive vaccine vector for both human and animal pathogens. The live attenuated vaccine strains used as vaccine vectors have a proven track record of safety and efficacy. NDV vectors not only induce robust humoral and cellular immune responses but also induce mucosal immune response. Therefore, NDV can be a vector of choice for mucosal immunization. The ability of NDV to infect a wide variety of non-avian species makes it a potential vector for other animals. NDV is also a promising vaccine vector for use in humans. One advantage is that most humans do not have pre-existing immunity to NDV. NDV-vectored vaccines have also become available commercially .


Clara Menéndez and colleagues conducted a randomized controlled trial among HIV-positive pregnant women in Kenya, Mozambique, and Tanzania to investigate the safety and efficacy of mefloquine as intermittent preventative therapy for malaria in women receiving cotrimoxazole prophylaxis and long-lasting insecticide treated nets.Please see later in the article for the Editors' Summary Intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) is recommended for malaria prevention in HIV-negative pregnant women, but it is contraindicated in HIV-infected women taking daily cotrimoxazole prophylaxis (CTXp) because of potential added risk of adverse effects associated with taking two antifolate drugs simultaneously. We studied the safety and efficacy of mefloquine (MQ) in women receiving CTXp and long-lasting insecticide treated nets (LLITNs).p = 0.008), placental malaria , and reduced incidence of non-obstetric hospital admissions  in the intention to treat (ITT) analysis. There were no differences in the prevalence of adverse pregnancy outcomes between groups. Drug tolerability was poorer in the MQ group compared to the control group (29.6% referred dizziness and 23.9% vomiting after the first IPTp-MQ administration). HIV viral load at delivery was higher in the MQ group compared to the control group (p = 0.048) in the ATP analysis. The frequency of perinatal mother to child transmission of HIV was increased in women who received MQ . The main limitation of the latter finding relates to the exploratory nature of this part of the analysis.A total of 1,071 HIV-infected women from Kenya, Mozambique, and Tanzania were randomized to receive either three doses of IPTp-MQ (15 mg/kg) or placebo given at least one month apart; all received CTXp and a LLITN. IPTp-MQ was associated with reduced rates of maternal parasitemia , stillbirths, and pre-term and low-birthweight babies, and is responsible for the deaths of many African women and their babies. To reduce the loss of life from malaria in pregnancy, the World Health Organization (WHO) recommends that pregnant women living in Africa receive the antimalarial drug sulfadoxine-pyrimethamine (SP) at each scheduled antenatal care visit given at least a month apart (intermittent preventive treatment in pregnancy [IPTp]). In addition, WHO advises pregnant women to sleep under insecticide-treated bed nets to protect themselves from the bites of infected mosquitoes and recommends effective case management of pregnant women with malarial illness.Pregnant women living in Africa are often infected with HIV, the virus that causes AIDS. HIV infection increases both the risk and severity of malaria infection during pregnancy, and at least one million pregnancies are complicated by co-infection with malaria and HIV in sub-Saharan Africa every year. WHO recommends that HIV-positive pregnant women take cotrimoxazole (CTX) daily to prevent opportunistic infections (CTX prophylaxis [CTXp]). Unfortunately, both CTX and SP are antifolate drugs and taking two drugs of this type increases a woman's risk of developing a severe skin reaction. Moreover, although CTXp protects children and HIV-infected adults against malaria, it is not known whether CTXp alone protects HIV-infected pregnant women adequately against malaria. Thus, evaluations of alternative drugs for use in IPTp in HIV-positive pregnant women are needed. In this randomized placebo-controlled trial, the researchers study the safety and efficacy of the antimalarial drug mefloquine (MQ) in HIV-infected women receiving CTXp. A randomized, placebo-controlled trial compares outcomes among people chosen through the play of chance to receive either the drug under investigation or a “dummy” (placebo) drug.The researchers allocated 1,071 HIV-infected pregnant women from Kenya, Mozambique, and Tanzania to receive three doses of MQ (IPTp-MQ), given at least one month apart, or three doses of placebo. All the women received CTXp and were given an insecticide-treated bed net. In an intention-to-treat analysis , the prevalence of parasitemia (parasites in the blood) at delivery among women given IPTp-MQ was 3.5% whereas the prevalence among women given the placebo was 6.9%. In other words, compared to placebo, IPTp-MQ was associated with a reduced risk of maternal parasitemia. IPTp-MQ was also associated with a reduced rate of placental malaria (parasites in the placenta) and a reduced incidence of hospital admissions for non-pregnancy related causes. There was no difference in adverse pregnancy outcomes such as stillbirth between the intervention groups but drug tolerability was poorer in the MQ group than in the placebo group. Finally, in an exploratory (unplanned) according-to-protocol analysis , women in the MQ group had a higher HIV viral load at delivery than women in the control group and were nearly twice as likely to transmit HIV to their child around the time of birth.These findings suggest that the addition of IPTp-MQ to CTXp and the use of insecticide-treated bed nets can improve malaria prevention and maternal health in HIV-infected pregnant women in Africa. However, the poor tolerability of MQ and the association of MQ treatment with both an increased HIV viral load at delivery and a higher frequency of mother-to-child-transmission of HIV when compared to placebo raise concerns about the use of MQ in IPTp. Because these last two findings came from an exploratory analysis, which is more likely to throw up a chance finding than a pre-planned analysis further studies are needed to confirm these unexpected but potentially important findings. Nevertheless, overall, the findings of this study suggest that MQ should not be recommended for IPTp in HIV-infected pregnant women in Africa and highlight the need to find alternative drugs for malaria prevention in this group of women who are particularly vulnerable to malaria.http://dx.doi.org/10.1371/journal.pmed.1001735.Please access these websites via the online version of this summary at PLOS MedicinePerspective by Richard Steketee. This study is further discussed in a PLOS MedicineResearch Article by González et al. compares the efficacy of IPTp-MQ and IPTp-SP in HIV-negative womenA related malaria  and on malaria in pregnancy; information on IPTp and the current WHO policy recommendation on IPTp with SP are available; the 2013 World Malaria Report provides details of the current global malaria situationInformation is available from the World Health Organization on malaria; a personal story about malaria in pregnancy is availableThe US Centers for Disease Control and Prevention also provides information on Roll Back Malaria Partnership on all aspects of global malaria control, including information on malaria in pregnancyInformation is available from the Malaria in Pregnancy Consortium is undertaking research into the prevention and treatment of malaria in pregnancyThe malaria (in English and Spanish)MedlinePlus provides links to additional information on trial protocol is availableMore information about the  Many African pregnant women are infected with HIV, and many of them are also exposed to falciparum malaria In Africa, intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) is recommended monthly at each antenatal care (ANC) visit, along with the use of insecticide treated nets, for malaria prevention in HIV-negative pregnant women Since SP cannot be evaluated in individuals on CTXp, other antimalarials for IPTp should be considered. Candidate antimalarials for IPTp should ideally have three main attributes: a long half-life to maximize the prophylactic effect; be administered in single dose to ensure adherence; and have a proven acceptable reproductive toxicity safety profile. We report here the results of a multicenter double-blind placebo-controlled randomized trial conducted in three sub-Saharan countries to evaluate the safety and efficacy of MQ as IPTp in HIV-infected women taking daily CTXp and in the context of long lasting insecticide treated nets (LLITNs).The study protocol and informed consent forms were reviewed and approved by the Ethics Committees from the Hospital Clínic of Barcelona (Spain), the US CDC, and the local regulatory authorities and National Ethics Review Committees from Kenya, Mozambique, and Tanzania . Study pThe study was conducted in three sub-Saharan countries: Tanzania (Makole and Chamwino), Mozambique (Manhiça and Maragra), and Kenya (Siaya). The characteristics of each site are shown in An individually randomized double-blind placebo-controlled trial was conducted to compare the efficacy of three monthly doses of MQ as IPTp with placebo-IPTp in HIV-infected pregnant women receiving daily CTXp in the context of LLITN use. The primary endpoint of the study was the prevalence of peripheral maternal malaria infection (microscopic or submicroscopic) at delivery. Based on previous estimations in the study sites Pregnant women of all gravidities attending the ANC clinic for the first time and who had not received IPTp during their current pregnancy were given the opportunity to be included in the study after providing informed consent. Enrolled women were permanent residents in the area, had a gestational age ≤28 weeks, had a positive HIV-test at recruitment, absence of history of allergy to sulfa drugs or MQ, absence of history of severe renal, hepatic, psychiatric, or neurological disease, and had not received MQ or halofantrine treatment in the preceding four weeks. In Mozambique and Tanzania, HIV-negative women were invited to participate in a randomized controlled trial evaluating the safety and efficacy of MQ IPTp compared to SP IPTp The allocation of the participants to the study arms was done centrally by block randomization (block size of 6) stratified by country to receive CTX  plus IPTp-MQ  or CTX plus IPTp-placebo . The Pharmacy Department of the Hospital Clinic in Barcelona produced and safeguarded the computer-generated randomization list for each recruiting site until unblinding, and carried out the masking, labelling, and packaging of all study interventional drugs. Study number allocation for each participant was concealed in opaque sealed envelopes that were sequentially numbered and opened only after recruitment by study health personnel. Study participants were assigned a unique study number linked to the allocated treatment group. Investigators, laboratory staff, care providers, and study participants were blinded to intervention throughout the study. All participants received a LLITN  at enrolment as part of the study intervention.Following physical examination, recruited women with gestational age ≥13 weeks received the first administration of IPTp (either placebo or MQ) under supervision. The number of tablets administered was calculated based on the participant's body weight at the first IPTp administration at a dosage of 15 mg/kg, assuming that each tablet contained 250 mg of active principle (MQ or placebo). The maximum dosage provided did not exceed 1,500 mg of MQ base. The administration of the study IPTp drugs was supervised and participants were observed for 60 minutes following IPTp administration. Women vomiting within the first 30 minutes were given a second full IPTp dose; those vomiting after 30–60 minutes were given an additional half dose. If the replacement dose was also vomited, study drug dosing was interrupted and routine ANC guidelines followed. The second and third administrations of IPTp-MQ/placebo were given at least one month apart. All women also received study CTX tablets on a monthly basis for daily prophylaxis.Drug tolerability was assessed immediately and two days after intake by home visits of field workers. Adherence to CTXp (measured by self-reported use and by counting the remaining number of tablets) and to LLITNs (assessed by self-reported use the preceding night) was evaluated at every monthly ANC visit, as well as during all unscheduled visits. Women were encouraged to attend the study health facility whenever they had any health complaint. Health care was free of charge and in general there was little availability of antimalarial drugs over the counter at all sites. A health facility-based passive surveillance system was established at each site to capture unscheduled visits of study participants during study follow-up. At each unscheduled visit, a standardized questionnaire was completed documenting signs and symptoms. Blood smears were prepared for malaria parasite examination and Hb was measured if there were current or reported symptoms and/or signs suggestive of malaria. Clinical malaria episodes were treated with oral quinine (first trimester) or artemether-lumefantrine (subsequent trimesters) for uncomplicated malaria; parenteral quinine was used for treatment of severe malaria. Solicited and unsolicited adverse events (AEs) were assessed. The former was done by directed questioning of malaria related signs and symptoms during unscheduled visits, whereas the latter were assessed through open questioning during scheduled visits. HIV/AIDS management of study participants was provided by the local government health services according to national guidelines At delivery, a sample from the mother's peripheral blood was collected for Hb, CD4+T cell count, HIV viral load, and malaria infection evaluation; cord blood and placental samples (biopsy and impression smears) were also taken, as well as blood onto filter paper for qPCR determinations. Newborns were weighed (including stillbirths) and measured and their gestational age at birth assessed using the Ballard's score www.eurotrol.com] and Hemocontrol [www.ekfdiagnostics.com]) on capillary blood samples. Plasmodium falciparum parasites were identified by microscopy on Giemsa-stained blood films according to standard, quality-controlled procedures P. falciparum-specific real time quantitative PCR (RT-qPCR) was performed on maternal peripheral and placental samples, with the inclusion in all reactions of a negative control with no template DNA Maternal HIV and syphilis serostatus were assessed at each site according to local standard procedures using rapid diagnostic tests . CD4+T cwww.openclinica.com). The main analysis was done on the Intention-to-Treat (ITT) cohort that included all recruited women that met the inclusion criteria and had data on the specific outcomes, and on the According to Protocol (ATP) cohort that included all women who had received three doses of IPTp according to the pre-specified schedule, and have results available on peripheral parasitemia at delivery. The safety cohort was defined as all recruited women who had received at least one dose of IPTp and whose data for analysis were available. The analysis of safety and tolerability was made on the Safety cohort. The ITT analyses were adjusted by country. The analyses in the ATP cohort were adjusted by country only and by baseline covariates . The proportion of women with asexual P. falciparum parasitemia (either microscopic or submicroscopic) was compared between groups using a modified binomial regression The quality of the data recorded in the study source documents and case report forms (CRFs) were monitored regularly following the principles of Good Clinical Practices by the trials' clinical monitor before their shipment to the centralized database. Data were double-entered using the OpenClinica Enterprise software for clinical data management . Heterogeneity between countries for the primary endpoint was evaluated using a Wald test. A clinical malaria episode was defined as presence of P. falciparum parasites in a blood smear plus any sign or symptom suggestive of malaria including: current fever (axillary temperature ≥37.5°C) or history of fever in the last 24 hours, and/or pallor, and/or arthromyalgias and/or headache, and/or history of convulsions P. falciparum infection.Peripheral malaria infection at delivery (primary study endpoint) was defined as the presence of asexual versus vaginal], health facility delivery, presence of moderate vomiting as AE, drug-related vomiting, maternal hospital admissions, clinical malaria during pregnancy, placental infection, RPR test result at baseline, and viral load at delivery) was performed and only those statistically significant variables (p<0.05) were included in the multivariate model.Placental infection was defined as the presence of parasites with or without pigment detected by histological examination, impression smear Immediate tolerability to study drugs was assessed as observed vomiting within one hour of drug administration. An AE was defined as any untoward medical occurrence in a study participant, to whom the study drug was administered, including occurrences, which were not necessarily caused or related to that drug. Serious adverse events (SAEs) were defined as an AE that meets any of the following criteria: (a) results in death, (b) is life-threatening, (c) requires hospitalization , (d) results in disability/incapacity, (e) is a congenital anomaly, or (f) any event of special interest  p<0.001) or in Tanzania . The OR of being in a higher category of adherence to CTXp in the MQ group compared with the placebo group was 0.86  and there was no interaction between country and intervention group .p = 0.008) . Similar results were found in the ATP analysis . The prevalence of peripheral maternal malaria infection at delivery was significantly lower in women receiving IPTp-MQ compared to those who received IPTp-placebo  0.47 [95% CI 0.27–0.82]; analysis .p = 0.021). There were no significant differences between groups in the prevalence of maternal and fetal anemia, prevalence of low birth weight, or in the prevalence of fetal and maternal parasitemia one month after delivery (p = 0.048)  compared to the control group (7.4%)  .p = 0.065) and , respectively. The incidences of clinical malaria and all-cause outpatient attendance during the study follow-up tended to be lower in women receiving MQ than in those receiving placebo, although these differences were not statistically significant    . OverallThe overall reported use of LLITNs at delivery was of 93% and of 98% one month after the end of pregnancy, with no difference between study groups.There were no differences in the prevalence of miscarriages, stillbirths, premature births, and congenital malformations between groups . The numThe overall immediate tolerability of IPTp was poorer in the MQ group (25 episodes of vomiting in the first hour following IPTp administration) as compared to placebo (one episode of vomiting) . The mosp = 0.015; with similar results in the ATP analysis) (p = 0.81), nor with the risk of MTCT of HIV (p = 0.655). In contrast, clinical malaria during pregnancy was found to be associated with an increased risk of MTCT of HIV. Higher viral loads at delivery were also associated with increased risk of MTCT of HIV  of HIV at 6 weeks of age  was performed. The proportion of infants with an HIV DNA positive PCR was higher in those born to women who had received MQ compared to those who had received placebo  . There wT of HIV .This placebo-controlled trial of IPTp with MQ (15 mg/kg dose three times at least one month apart) in HIV-infected pregnant women in the context of daily CTXp and LLITN use found significant reductions in the risk of important parameters of malaria infection in pregnancy such as maternal parasitemia at delivery and placental infection. In addition, IPTp-MQ was associated with reduction in the incidence of non-obstetric hospital admissions while the incidence of all-cause out-patient visits, all-cause hospital admissions, and clinical malaria during pregnancy also tended to be lower in women receiving IPTp-MQ. The safety profile in terms of SAEs and adverse pregnancy outcomes was similar between the two study groups, and there were no reported SAEs related to the medication. On the other hand, tolerability of MQ was poor compared to the placebo group especially for the AEs that have been commonly reported with this drug such as dizziness and vomiting. MQ IPTp was also associated with an increased risk of MTCT of HIV.Reduced frequency of placental malaria in MQ recipients was also reported in an open-label trial comparing CTXp and IPTp-MQ plus CTXp in 292 HIV-infected Beninese women The poor tolerability of MQ compared to placebo found in this study has been reported previously In an exploratory analysis, the study also found that although similar at baseline, HIV viral loads at delivery were significantly higher in the women who received MQ than in those who received placebo, and the proportion of infants who were perinatally infected with HIV was also increased if their mothers received MQ. The effect of MQ on HIV viral loads at delivery and on MTCT was not assessed in the trial evaluating MQ for IPTp in HIV-infected Beninese pregnant women, but the MTCT frequency was too low (less than 1%), given the sample size of the study population to carry out this analysis An association of presumptive clinical malaria with increased risk of MTCT of HIV has been reported Cotrimoxazole prophylaxis was administered as a trial intervention to all study participants following recommendations for HIV-infected pregnant women in poor-resource settings The majority (80%) of HIV-infected women in the world live in Africa, 75% of them being younger than 25 years of age These results show that an effective antimalarial drug given as IPTp can offer additional protection against malaria and some associated adverse outcomes when added to CTXp and a LLITN, improving malaria prevention and overall maternal health through reduction in hospital admissions. However, because of its poor tolerability at the dose of 15 mg/kg, MQ is not recommended for IPTp Figure S1Trial profile (ATP cohort).(TIF)Click here for additional data file.Figure S2Time to first episode of clinical malaria.(TIF)Click here for additional data file.Figure S3Adherence to cotrimoxazole prophylaxis by country and treatment group.(TIF)Click here for additional data file.Figure S4HIV viral load by country.(TIF)Click here for additional data file.Table S1List of regulatory authorities and national ethical review boards.(DOC)Click here for additional data file.Table S2Characteristics of study sites.(DOC)Click here for additional data file.Table S3Adverse events by treatment group and cotrimoxazole adherence.(DOC)Click here for additional data file.Table S4Placental histology results by treatment group.(DOCX)Click here for additional data file.Text S1MiPPAD study protocol.(PDF)Click here for additional data file.Text S2CONSORT checklist.(DOC)Click here for additional data file.Text S3Statistical analytical plan.(PDF)Click here for additional data file.Text S4ClinicalTrials.gov registration receipt.(PDF)Click here for additional data file.Text S5Ethics approval documentation.(PDF)Click here for additional data file.