Eleven healthy subjects executed or inhibited previous learned finger and foot movements indicated by a visual cue. The execution of finger/foot movements caused a typical activation pattern namely an increase of [oxy-Hb] and a decrease of [deoxy-Hb] whereas the inhibition of finger/foot movements caused a decrease of [oxy-Hb] and an increase of [deoxy-Hb] in the hand or foot representation area . Additionally an increase of [oxy-Hb] and a decrease of [deoxy-Hb] in the medial area of the anterior prefrontal cortex (APFC) during the inhibition of finger/foot movements were found. The results showed, that inhibition/execution of learned motor programs depends on an interplay of focal increases and decreases of neural activity in prefrontal and sensorimotor areas regardless of the effector. As far as we know, this is the first study investigating inhibitory processes of finger/foot movements by means of multi-channel fNIRS.In the present study inhibitory cortical mechanisms have been investigated during execution and inhibition of learned motor programs by means of multi-channel functional near infrared spectroscopy (fNIRS). fNIRS is an emerging non-invasive optical technique for the  In daily life successful human behavior strongly depends on learning and inhibition of inappropriate behavior. Specifically inhibitory control is an essential function to provide appropriate preparation and online control of required motor programs. Furthermore a fine balance between activation and inhibition is necessary for preparation of movement, initiation, motor control, and timely inhibition of the act. There are a lot of studies focusing on the question about the neural correlate of effective inhibition or suppression of behavior. Most of them used experimental paradigms like GO/NOGO  aged from 22 to 37 years . All subjects were right-handed and had normal or corrected to normal vision. Hand performance was assessed with the “Hand Dominance Test” (HDT) by Steingrüber and Lienert . This teThree weeks prior to the experiment, subjects were instructed to train themselves on six sequences of right hand finger and right foot movements of two different task complexities at home. In Table During the experimental sessions all subjects were seated in a comfortable arm-chair in front of a TFT monitor. The distance between the participants and the screen was about 120 cm. To avoid artifacts, the participants were instructed to relax as much as possible during the measurement. The study consisted of three sessions (Table During the finger movement session subjects had to execute or inhibit 48 sequences of right hand finger movements presented on the monitor. In order to indicate if execution or an inhibition task was required a green (execution) or a red (inhibition) frame was shown around the picture of the hand one second after sequence presentation. One sequence consisted of eight movements [presented on the screen as a sequence of eight numbers digits 1–4)] and lasted 10 s. The fingers were labeled corresponding to these digits as follows: the index finger “1,” the middle finger “2,” the ring finger “3,” the little finger “4.” During the execution task the subjects were instructed to perform the requested sequence  on a modified keyboard until the screen turns black. The average frequency of finger Figure  tapping –4] and lDuring the foot movement session subjects had to execute or inhibit foot movement sequences on a custom made console Figure . The aveFinally, in the session with randomized finger and foot movements the subjects had to execute or inhibit 24 fingers and foot movement sequences in random order. In contrast to the finger and foot block, four new sequences occurred, so the block consisted of 52 trials. Between the blocks the subjects had short breaks of about 5 min  with an error estimated (SD) was calculated , which is based on the continuous wave principle was used. The sampling rate was set to 10 Hz. The multi-channel system measures the change of [oxy-Hb] and [deoxy-Hb]After a visual inspection of the raw fNIRS data by a trained expert, trials containing motion artifacts were removed manually. Additionally, channels with poor signal quality, e.g. containing noise (on average less than 7% of the channels), were excluded. Baseline drifts were reduced by using a 0.01 Hz Butterworth high pass filter of order 6 with 30 dB attenuation in the stop band. Afterwards a common average reference (CAR) spatial filter was used to remove global influences like respiratory or blood pressure rhythms. As a result, for every time point, the mean of all non-excluded channels was calculated and subtracted from each channel  were calculated. For the excluded channels (at the maximum 6 out of 46 channels) the changes were recalculated by interpolation of the surrounding channels. In all subjects not more than one channel was interpolated in each ROI. Furthermore no interpolation was performed in frontal ROIs (FP1 and FP2). As the fNIRS data was checked for artifacts, such interpolation of channels will only cause a spatiotemporal smoothing of the hemodynamic pattern. The topographic distributions during the tasks are further visualized by plotting the [oxy-Hb] and [deoxy-Hb] values at their corresponding spatial position. A 2-D interpolation on a fine Cartesian grid was used to generate a scalp distribution. The average over two different time windows are calculated. The first time window between 0 and 4 s corresponds to the cue presentation and start of the task. The second time window between 10 and 12 s corresponds to the end of the task. The mean concentration changes of oxy-Hb and deoxy-Hb are visualized in different plots with the same scale. Increases are plotted in blue and decreases in red (according to the toolbox “EEG-Lab” from Matlab). Only well trained sequences run into analyses, concretely the mean task related concentration changes of 48 trials for each condition are plotted. The new sequences during the experimental trials were only used to keep attention.Before running statistical analyses the following pre-processing steps were performed:First, five regions of interest  covering the frontal and motor cortex of both hemispheres were defined: Frontal cortex: FP1 ; FP2 ; Motor cortex: C3 ; Cz ; C4 . The MNI coordinates and anatomical locations of the included channels are given in Table In general all subjects showed strong changes of [oxy-Hb]F-values for [oxy-Hb] and [deoxy-Hb]. F-values at 5% level are marked with one asterisk (*), at 1% level with two asterisks (**). All repeated measures tests are Huynh–Feldt corrected.In the following paragraphs significant results of the 2 × 2 × 2 univariate ANOVA are reported for finger and foot condition separately. Table * FRONTAL/CENTRAL . This interaction indicated that the type of task leads to different hemodynamic responses at frontal and central brain regions. Post hoc-tests (Bonferroni) showed a stronger increase in [oxy-Hb] during motor inhibition compared to active movement at frontal brain regions . At central sites  no significant difference in [oxy-Hb] between motor inhibition and active movement was found. Additionally [oxy-Hb] increased over central compared to frontal sites in the active movement condition. Furthermore the three-way interaction effect EXEC/INHIB * FRONTAL/CENTRAL * HEMI  was significant. Post hoc-tests (Bonferroni) showed stronger increases in [oxy-Hb] during motor inhibition compared to active movement at frontal left brain regions (FP1) and no significant difference at frontal right areas (FP2). At central left areas (C3) [oxy-Hb] was higher during active movement than during motor inhibition. Like at frontal right sites, these two conditions showed no significant difference in [oxy-Hb] at central right sites (C4). In the inhibition condition, [oxy-Hb] was significantly increased at frontal left and right compared to central left sites. In the active movement condition, [oxy-Hb] was significantly higher at central left sites than at frontal left and right sites. Summarizing, the results showed significant differences in oxy-Hb concentration changes between execution and inhibition at central and frontal sites. There is no difference in [oxy-Hb] between left and right hemisphere in the inhibition condition, leading to a more bilateral activation. The mulit-channel maps in Figure For [oxy-Hb], the ANOVA revealed a significant two-way interaction effect of EXEC/INHIB l sites C, Cz, C4 * FRONTAL/CENTRAL * HEMI  was significant. Post hoc-tests (Bonferroni) indicated a stronger decrease of [deoxy-Hb] during active movement than during motor inhibition at central left brain regions (C3).For [deoxy-Hb] in the finger condition, the three-way interaction effect of EXEC/INHIB F = 10.79, p < 0.01; η2 = 0.52]. The significant interaction effect of EXEC/INHIB * FRONTAL/CENTRAL  confirmed a substantial frontal increase of [oxy-Hb] during motor inhibition compared to active movement. Additionally, [oxy-Hb] was higher at frontal sites  than at central sites (Cz) in the motor inhibition condition, whereas in the active movement condition [oxy-Hb] was higher at central sites (Cz) compared to frontal areas . This effect is clearly visible in the following multi-channel map of foot movement execution  = 7.42, p < 0.05; η2 = 0.43] was significant, too. At central sites (Cz) no significant differences in [oxy-Hb] between active movement and motor inhibition could be found. At frontal sites , [oxy-Hb] was higher in the inhibition condition than during active movement in both hemispheres. During inhibition [oxy-Hb] was lower at central sites (left and right) compared to frontal sites (left and right). During active movement [oxy-Hb] was higher at central sites (left and right) compared to frontal sites (left and right). No significant differences in [oxy-Hb] between frontal left and right sites during inhibition were found. Again during inhibition, activation was bilateral at frontal sites like in the finger movement condition.Furthermore, the three-way interaction effect EXEC/INHIB * FRONTAL/CENTRAL . Like for [oxy-Hb] the type of task evokes different hemodynamic responses at frontal and central sites. Additionally, the interaction effect of FRONTAL/CENTRAL * HEMI  was significant, due to a stronger decrease in [deoxy-Hb] over central right sites compared to frontal right sites.For [deoxy-Hb] the ANOVA revealed a significant interaction effect of EXEC/INHIB The overall results clearly show differences in the frontal and central brain regions depending on the type of task. In Figure Particularly during the finger movement execution an [oxy-Hb] increase was found centrally compared to the inhibition condition, where frontal regions showed [oxy-Hb] increase and [deoxy-Hb] decrease. This effect is clearly visible in the topographic maps of Figures The grand average hemodynamic response during finger movement execution showed a typical activation pattern, namely an increase in the [oxy-Hb] and a decrease of [deoxy-Hb] in the hand representation area (left sensorimotor cortex). In parallel with this activation pattern an [oxy-Hb] decrease and an increase of [deoxy-Hb] in the medial area of the anterior prefrontal cortex  was also observed. Furthermore, the responses during finger movement inhibition showed a decrease in the [oxy-Hb] and an increase of [deoxy-Hb] in the hand representation area (left sensorimotor cortex) whereas in the medial area of the APFC [oxy-Hb] increased and [deoxy-Hb] decreased. These findings are in line with previous fMRI studies , insula, basal ganglia, pre-SMA, and dorsolateral prefrontal cortex (DLPFC)  were missing.During finger movement execution of right handed subiects, we found an increase of [oxy-Hb] and a decrease of [deoxy-Hb] in the hand representation area (left sensorimotor cortex). Additionally a [oxy-Hb] decrease and an increase of [deoxy-Hb] in the medial area of the APFC were observed, more prominently in the left hemisphere. During finger movement inhibition a decrease in the [oxy-Hb] and an increase of [deoxy-Hb] in the hand representation area was found. Furthermore, an [oxy-Hb] increase and a [deoxy-Hb] decrease in the medial area of the APFC bilaterally and the supplementary sensorimotor regions was observed. These bidirectional neuronal control which is represented by increase/decrease of oxy-Hb and deoxy-Hb concentration are in line with the results by Hummel et al.  suggestiFurthermore the same interpretation is valid for foot movements, where we found an increase of [oxy-Hb] over APFC during the inhibition condition. This novel finding reinforces the claim that the PFC plays an important role during inhibitory control of motor responses (Hummel et al., 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.


A neuroimaging study reveals how coupled brain oscillations at different frequencies align with quasi-rhythmic features of continuous speech such as prosody, syllables, and phonemes. Cortical oscillations are likely candidates for segmentation and coding of continuous speech. Here, we monitored continuous speech processing with magnetoencephalography (MEG) to unravel the principles of speech segmentation and coding. We demonstrate that speech entrains the phase of low-frequency  and the amplitude of high-frequency (gamma) oscillations in the auditory cortex. Phase entrainment is stronger in the right and amplitude entrainment is stronger in the left auditory cortex. Furthermore, edges in the speech envelope phase reset auditory cortex oscillations thereby enhancing their entrainment to speech. This mechanism adapts to the changing physical features of the speech envelope and enables efficient, stimulus-specific speech sampling. Finally, we show that within the auditory cortex, coupling between delta, theta, and gamma oscillations increases following speech edges. Importantly, all couplings  attenuate for backward-presented speech, suggesting top-down control. We conclude that segmentation and coding of speech relies on a nested hierarchy of entrained cortical oscillations. Continuous speech is organized into a nested hierarchy of quasi-rhythmic components  with different time scales. Interestingly, neural activity in the human auditory cortex shows rhythmic modulations with frequencies that match these speech rhythms. Here, we use magnetoencephalography and information theory to study brain oscillations in participants as they process continuous speech. We show that auditory brain oscillations at different frequencies align with the rhythmic structure of speech. This alignment is more precise when participants listen to intelligible rather than unintelligible speech. The onset of speech resets brain oscillations and improves their alignment to speech rhythms; it also improves the alignment between the different frequencies of nested brain oscillations in the auditory cortex. Since these brain oscillations reflect rhythmic changes in neural excitability, they are strong candidates for mediating the segmentation of continuous speech at different time scales corresponding to key speech components such as syllables and phonemes. A large number of invasive and non-invasive neurophysiological studies provide converging evidence that cortical oscillations play an important role in gating information flow in the human brain, thereby supporting a variety of cognitive processes including attention, working memory, and decision-making Interestingly, speech comprises a remarkably similar hierarchy of rhythmic components representing prosody (delta band), syllables (theta band), and phonemes (gamma band) Previous studies have demonstrated amplitude and phase modulation in response to speech stimuli in the delta, theta, and gamma bands using electroencephalography (EEG)/magnetoencephalography (MEG) This attractive model leaves three important points largely unresolved: First, a comprehensive account of how rhythmic components in speech interact with brain oscillations is still missing and it is uncertain if the previously reported hemispheric asymmetry during speech perception is also evident in a lateralized alignment of brain oscillations to continuous speech. Behavioural, electrophysiological, and neuroimaging studies Here, we addressed these three points using continuous speech and analysis based on information theory. Importantly, all three points were investigated for intelligible and unintelligible (backward played) speech. We analysed the frequency-specific dependencies between the speech envelope and brain activity. We also analysed the dependencies between cortical oscillations across different frequencies. We first hypothesised that a multi-scale hierarchy of oscillations in the listener's brain tracks the dynamics of the speaker's speech envelope—specifically, preferential theta band tracking in the right auditory cortex and gamma band tracking in the left auditory cortex. Second, we asked whether speech-entrained brain oscillations are hierarchically coupled and if so how that coupling is modulated by the stimulus. Third, we asked whether phase of low-frequency brain oscillations  in the auditory cortex coincide with and adapt to salient events in speech stimuli.We presented a 7-min long continuous story binaurally to 22 participants while recording neural activity with MEG (“story” condition). As a control condition the same story was played backwards (“back” condition). We used mutual information (MI) to measure all dependencies (linear and nonlinear) between the speech signal and its encoding in brain oscillations Our results reveal hierarchically coupled oscillations in speech-related brain areas and their alignment to quasi-rhythmic components in continuous speech , with pronounced asymmetries between left and right hemispheres. Edges in the speech envelope reset oscillatory low-frequency phase in left and right auditory cortices. Phase resets in cortical oscillations code features of the speech edges and help to align temporal windows of high neural excitability to optimise processing of important speech events. Importantly, we demonstrate that oscillatory speech tracking and hierarchical couplings significantly reduce for backward-presented speech and so are not only stimulus driven.p<0.05, false discovery rate [FDR] corrected, see We first asked whether there is phase-locking between rhythmic changes in the speech envelope and corresponding oscillatory brain activity. Whereas most previous studies quantify phase-locking to stimulus onset across repeated presentations of the same stimulus, here we studied phase-locking over time directly between speech envelope and brain oscillations. To do this, we compared the phase coupling between the speech and oscillatory brain activity (in 1 Hz steps between 1 and 60 Hz) in two conditions: story and back. To test for other couplings between the speech and cortical oscillations, we also computed MI between the amplitude of the speech and the amplitude of cortical oscillations and between the amplitude of the speech and the phase of cortical oscillations for each frequency between 1 and 60 Hz. These computations revealed no significant dependencies. Finally, we flipped the computations around, to test whether the phase of the speech envelope modulated the amplitude of cortical oscillations. Again, we carried out this computation across frequencies, for all combinations between 1 and 60 Hz and found one significant phase-amplitude coupling. In sum, this comprehensive analysis revealed two distinct speech tracking mechanisms in the brain. First, low-frequency speech modulations entrain  delta and theta oscillations in the auditory cortex. Second, low-frequency speech modulations also entrain the amplitude dynamics of gamma oscillations. Both tracking mechanisms are especially sensitive to intelligible speech because the effects are stronger for the story than the back condition. Since the theta phase of the speech envelope is coupled to both, the theta phase  and gammt-tests of MI values for corresponding voxels in the left and the right hemisphere for the story condition. Interestingly, although present in both left and right hemisphere  increase of phase-locking in an early (100–300 ms) and late (400–600 ms) time window compared to baseline (−200 to 0 ms).We used a thresholding algorithm to identify 254 separate temporal edges in the continuous stimulus . This demonstrates that speech continuously entrains brain rhythms beyond a stereotypical short-lived phase reset evoked by edges.To measure the extent to which this increase can be explained by a stereotypical edge-evoked response we computed phase-locking of auditory theta activity across trials time-locked to edge onset (dashed lines). This measure captures the evoked response to edge onset. As expected, this evoked response (dashed lines) increased following edge onset with a similar dynamics as the phase-locking to speech (solid lines). But importantly, phase-locking to speech (solid lines) is significantly stronger in the late time window than phase-locking to edge onset (dashed lines) . This could indicate that phase resetting in the left hemisphere is partly driven by the right auditory cortex.Finally, we computed the phase-locking between left and right auditory theta activity  between the speech envelope and low-frequency oscillations in the auditory cortex implements a mechanism for efficient sampling and segmentation of speech t-tests, all p<0.05). Speech sampling by amplitude in the gamma band is significantly higher for the story compared to the back condition in the left auditory cortex . Although the pattern of lateralisation was overall consistent with These results indicate that temporal edges in speech amplitude induce modulations in low-frequency phase and high-frequency amplitude dynamics of brain oscillations that align windows of high neural excitability to salient speech events. Importantly, this alignment is not caused by an identical phase resetting for all edges because shuffling the speech trials reduces the correlation. We predicted that edge-specific phase resets coding stimulus features  cause this trial-specific alignment. We tested this hypothesis by sorting our previously identified 254 trials by maximum amplitude of speech envelope in the 200 ms window after onset. For each participant we computed in the left and right auditory cortex the theta phase at 100 ms after onset and correlated both quantities using circular correlation Together, these results demonstrate that the phase of low-frequency cortical oscillations and the amplitude of high-frequency oscillations align to trial-specific speech dynamics, adapting to variations of speech over time. This trial-specific alignment suggests that oscillatory windows of high excitability sample salient speech components. Our analysis on the continuous data  and S4 hWe first characterised the spatial distribution of edge-induced changes in cross-frequency coupling by computing coupling of gamma amplitude to theta phase in all brain voxels. We then computed the full cross-frequency coupling matrix separately for the left and the right auditory cortex.t-statistic of MI between theta phase and gamma amplitude for the two 500 ms windows preceding and following speech onset. Since this computation is based on the difference between post-stimulus and pre-stimulus data it captures the edge-induced changes of cross-frequency coupling. We performed the computation for both the story and back condition. As in t-test (story versus back condition) with randomisation-based FDR correction. Group t-maps are displayed with thresholds corresponding to p<0.05 (FDR-corrected). As before, we used MI to analyze cross-frequency oscillatory coupling as in  but now t-statistics for the difference between the story condition and surrogate data  in the story condition compared to the back condition, demonstrating a more precise hierarchical nesting of cortical oscillations for intelligible than unintelligible speech.The second analysis used the left and right auditory cortex as regions of interest to compute the full cross-frequency coupling matrix. Here, we computed MI as before but now for all combinations of phase (1–10 Hz) and amplitude (4–80 Hz). We computed group Finally, we studied lateralisation of the cross-frequency coupling shown in Our results provide direct evidence for the hypothesis that a listener's brain oscillations segment and encode continuous speech in a frequency-specific manner. This suggests that these oscillations play a functional role in efficient sensory sampling. MI analysis reveals alignment of low-frequency phase and high-frequency amplitude to the speech envelope that is frequency specific, shows hemispheric asymmetry, and is modulated by intelligibility . The low-frequency phase alignment is preserved over time by transient events in the stimulus (edges) that lead to phase adjustments. These phase adjustments are stimulus specific and depend on the amplitude of transient events (and likely other features of the stimulus). Interestingly, brain activity in the three observed frequency bands is hierarchically coupled. This cross-frequency coupling is increased following edge onset and the increase is stronger for speech than for reverse speech.We observed phase alignment between low-frequency components of the speech envelope and brain activity in the delta and theta band. No consistent phase-phase coupling was observed for frequencies higher than 10 Hz. Previous studies have shown that speech envelope frequencies below 10 Hz are important for intelligibility We reported two different mechanisms. First, the low-frequency phase in the speech envelope entrains the low-frequency phase of brain oscillations in delta and theta frequency bands. The specific entrainment patterns support the idea that delta and theta bands are qualitatively different The second mechanism revealed in our analysis is the alignment of gamma-amplitude modulations to the theta phase of the speech envelope in bilateral temporal, frontal, and parietal areas with lateralisation to the left hemisphere. Taken together, the auditory cortex showed right-lateralisation for theta phase entrainment and left-lateralisation for gamma amplitude entrainment. These results support the asymmetric sampling in time (AST) model All entrainment effects were identified in a statistical contrast between the story and the back condition. This is important because it demonstrates that these entrainments are not just unspecific stimulus-driven effects but that they are modulated by intelligibility of the stimulus. A previous study Our study supports emerging models of speech perception that emphasise the role of brain oscillations Our results indicate that sharp large-amplitude transients (edges) in speech reset oscillations in the auditory cortex with important consequences. First, these resets increase the alignment between auditory oscillations and the speech envelope . This isIn the rat auditory cortex, increases in sound power in the frequency band matching the tonotopy of the considered location lead to large depolarizing currents in the input layers that reset intrinsic oscillations to an “excitable” phase In summary, we report a nested hierarchy of auditory oscillations at multiple frequencies that match the frequency of relevant linguistic components in continuous speech. These oscillations entrain to speech with differential hemispheric preference for high (left) and low (right) frequencies. Our results indicate that temporal edges in speech increase first the coupling between auditory oscillations across frequency bands and, second, their coupling to the speech envelope.We can only speculate about the nature of the observed phase/amplitude alignments. Most likely the alignments are caused by a combination of modulatory and evoked effects Finally, going beyond speech perception, the entrainment of hierarchically organized oscillations between speaker and listener may well have a more general role in interpersonal communication 22 healthy, right-handed volunteers participated in the study . All participants provided informed written consent and received monetary compensation for their participation. The study was approved by the local ethics committee  and conducted in conformity with the Declaration of Helsinki.MEG recordings were obtained with a 248-magnetometers whole-head MEG system  at 1,017 Hz sampling rate.The analysis of the MEG signal was performed using the FieldTrip toolbox Stimuli have been previously used in an fMRI study We computed the amplitude envelope of auditory signals following Chandrasekaran et al. MEG signals were denoised with information from the reference sensors using the denoise_pca function in FieldTrip. Bad channels were excluded by visual inspection.T1-weighted structural magnetic resonance images (MRIs) of each participant were co-registered to the MEG coordinate system using a semi-automatic procedure. Anatomical landmarks  were manually identified in the individual's MRI. Initial alignment of both coordinate systems was based on these three points. Subsequently, numerical optimisation was achieved by using the ICP algorithm Individual head models were created from anatomical MRIs using segmentation routines in FieldTrip/SPM5. Leadfield computation was based on a single shell volume conductor model Cross-spectral density was computed using Fast Fourier Transform on 1-s segments of data after applying Hanning window. For frequencies above 40 Hz spectral analysis was performed using multitaper  beamformer coefficients to the MEG data filtered in the same frequency band . The broadband speech envelope was processed identically. Second, Hilbert transform was applied to the bandpass filtered data to compute phase or amplitude dynamics. Finally, MI was computed between the speech signal and brain signal for each voxel, frequency band, and for all combinations of signals . MI computation was performed using the direct method with quadratic extrapolation for bias correction in the Information-Theory Toolbox p<0.05 corrected) are reported. Group statistics were computed to compare the story condition to back condition and surrogate analysis. Final statistical maps (thresholded at p<0.05 corrected) are rendered on the MNI template brain. To confirm that MI for phase-phase interaction is due to phase-locking of speech and brain signals we computed PLV Group statistical analysis was performed on the data of all 22 participants using non-parametric randomisation statistics in FieldTrip (Monte Carlo randomisation). Specifically, individual volumetric maps were smoothed with a 10 mm Gaussian kernel and subjected to dependent-samples T-test. The null distribution was estimated using 500 randomisations and multiple comparison correction was performed using FDR t-test against 0) following the approach described in the previous paragraph with FDR correction for multiple comparisons. For Statistical analysis of lateralisation was performed in three steps. First, corresponding voxels in both hemispheres were identified on the basis of their coordinates. Second, the lateralisation index (LI = [right−left]/[right+left]) was computed for each voxel. Third, significance of lateralisation index was tested (theta) and theta brain phase (Btheta) is significantly increased by including gamma amplitude in the computation  and I  using bias-corrected mutual information estimates . The significance of the difference is tested by computing a null distribution without bias correction for I  where Bgamma is shuffled for fixed values of the binned signal Btheta. The null distribution is then compared to I  computed without bias correction. These separate computations are motivated by the fact that bias correction decreases statistical power but increases accuracy of magnitude estimation To address the question whether MI I of theta speech phase . Time-locked to these onsets we have extracted trials from −500 ms to 1,000 ms.A thresholding algorithm was used to identify temporal edges in speech. The speech envelope was normalised to a maximum amplitude of 1. Speech edges were defined using the following criteria: (1) Mean amplitude in 400 ms before onset is less than 0.05. (2) Mean amplitude in 1 s after onset is larger than 0.05. (3) The difference between the mean amplitude 20 ms before and 20 ms after onset is larger than 0.05. For our particular speech stimulus this resulted in 254 time points characterised by a short period of low speech envelope amplitude followed by a sharp increase in amplitude. Onsets were confirmed by visual inspection of the speech envelope. Speech onset results were robust against small changes of these criteria. The same algorithm was applied to identify speech edges in the back condition. Mean and maximum amplitude and mean and maximum slope in the 100 ms following edge onsets were compared for the story and back condition and showed no significant difference (n|∑ exp(i * ph)| where n is the number of trials and ph the phase of auditory theta signal). Second, the phase-locking of the phase difference between auditory theta signal and the theta speech envelope was computed (PLVsp = 1/n |∑ (exp(i * (ph−phs))| where n is the number of trials and ph the phase of auditory theta signal and phs the theta phase of speech envelope). Third, the phase-locking between left and right auditory theta activity (PLVsp = 1/n |∑ (exp(i * (phl−phr))| where n is the number of trials and phl and phr the phase of left and right auditory theta signal, respectively). Time-resolved PLV data were averaged in three time windows  and subjected to Anova analysis with factors time window and PLV measure. Both factors and their interactions were highly significant .PLVs For each voxel the instantaneous amplitude A and phase ph for each speech trial was computed . For eacWe performed two separate analyses to investigate the spatio-spectral distribution of cross-frequency coupling . First, t-values of contrast post-onset versus pre-onset were computed across trials. The computation was performed for the story and back condition. As in t-test with randomisation-based FDR correction. Group t-maps are displayed with thresholds corresponding to p<0.05 (FDR corrected). The second analysis was performed only in the left and right auditory cortex. Here, we computed MI as before but now for all combinations of phase (range 1–10 Hz) and amplitude (range 4–80 Hz). Group t-statistic was computed for the difference between story condition and surrogate data .The first analysis was motivated by For each frequency-frequency pair we computed a bootstrap confidence level by randomly drawing 22 participants with replacement in each of 500 bootstrap iterations and computing the 95th percentile.The lateralisation analysis in Figure S1(A) Mutual information group statistics for surrogate data. Group statistical map of phase-phase MI dependencies in the theta frequency band. This figure corresponds to (PDF)Click here for additional data file.Figure S2Bar plot of individual lateralisation indices. For each participant the lateralisation index for theta-phase lateralisation (red) and theta-gamma lateralisation (blue) in Heschl's gyrus (left panel) and superior temporal gyrus  is shown. Each pair of red/blue bars corresponds to an individual.(PDF)Click here for additional data file.Figure S3Bar plot of mutual information in the auditory cortex. For each panel mean and SEM is shown for the left and right auditory cortex for all conditions. An asterisk indicates relevant significant differences (t-test with p<0.05). Control condition is computed from surrogate data where brain activity from story condition is used together with speech envelope from back condition. (A) Bar plot for delta phase. (B) Bar plot for theta phase. (C) Bar plot for mutual information between theta phase in speech and gamma amplitude in the auditory cortex. (D) Bar plot for mutual information between theta phase and gamma amplitude in the auditory cortex. Here, control condition was obtained from mutual information with gamma time series reversed.(PDF)Click here for additional data file.Figure S4Group statistics of cross-frequency coupling. (A) Statistical map of difference between story and back condition for mutual information between delta phase and theta amplitude. (B) Statistical map of lateralisation of mutual information between delta phase and theta amplitude for the story condition. (C) Statistical map of difference between story and back condition for mutual information between theta phase and gamma amplitude. This map corresponds to (PDF)Click here for additional data file.Figure S5Phase coding of speech amplitude. The phase of theta oscillations at 100 ms after speech onset in the left (black) and right (red) auditory cortex codes the maximum amplitude of speech envelope in the first 200 ms following onset. The area signifies the 95% confidence interval around the median obtained from bootstrap analysis.(PDF)Click here for additional data file.


The case of a patient with narrow QRS-complex supraventricular tachycardia and atrial flutter is described. The 12-lead surface electrocardiogram (ECG) revealed sinus rhythm with ventricular pre-excitation and negative δ waves in leads II, III and aVF, indicating Wolff-Parkinson-White syndrome with a posteroseptal accessory pathway (AP). Coronary sinus angiography revealed the presence of a diverticulum near the coronary sinus ostium. The AP was successfully ablated using radiofrequency energy applied in the neck of the diverticulum, following several failed attempts at catheter ablation from the endocardial surface of the posteroseptal space. Radiofrequency catheter ablation (RFCA) has become the curative treatment of choice for supraventricular tachycardia (SVT) in patients with Wolff-Parkinson-White (WPW) syndrome . The posA 46-year-old male presented with recurrent SVT associated with symptoms of palpitations, dizziness and diaphoresis. The termination of tachycardia with drugs is not always successful and electrical cardioversion may be required. The patient in this study used antiarrhythmic drugs prior to radio-frequency catheter ablation (RFCA) and drugs alone were used during the attacks. A sinus rhythm electrocardiogram (ECG) showed ventricular pre-excitation with a positive δ wave in leads I, V1, V2 and a negative δ wave in leads II, III and aVF , indicatAn EPS was performed to identify the mechanism of tachycardia and RFCA. A deflectable decapolar catheter  was placed in the coronary sinus via the left subclavian vein. Quadripolar catheters (Cordis-Webster) were introduced through the right femoral vein and positioned in the His-bundle region and the right ventricular apex (RVA). The intracardiac electrogram revealed the earliest atrial activation was at the CS ostium and RVA pacing initiated orthodromic AVRT. The AP was located in the left posteroseptal region. An 8-French ablation catheter with a 4-mm tip electrode (Cordis-Webster) was introduced via the right femoral artery and placed under the mitral valve close to the annulus. The ventricular wave could not be significantly ahead of the atrial wave and the AV wave was not always merged. The applications of radiofrequency energy  delivered in the posteroseptal region of the mitral valve annulus failed to ablate the AP. Due to the consideration that the AP may have been located at the epicardium, CS angiography was implemented and a CS diverticulum was identified near the ostium . The ablA CS diverticulum is a rare congenital abnormality of the intracardiac veins and is shown to contain myocardial fibers which connect to the ventricle and the CS myocardial coat. The connections between the CS myocardial coat and the ventricle may serve as an AP ,3. CS diA CS diverticulum is usually associated with manifest posteroseptal APs and the manifest APs have bidirectional atrioventricular conduction properties . These AThis case report illustrates a patient who had SVT and atrial flutter with a left posteroseptal AP associated with a CS diverticulum. RFCA at the neck of the CS diverticulum effectively interrupted the AP conduction. The current case highlights the potential importance of CS angiography and identification of AP potentials in patients with a posteroseptal AP which is difficult to ablate by the endocardial approach.


Brain metastasis is a significant cause of morbidity and mortality and a critical complication of non-central nervous system primary carcinoma. The present study describes the clinical case of a 46-year-old male with lung cancer and life-threatening brain metastases. The patient was diagnosed with lung cancer with a clinical stage of T2N0M1 (stage IV). Six months after the initial diagnosis and administration of conformal radiotherapy combined with three cycles of chemotherapy, an enhanced computed tomography (CT) scan of the brain revealed abnormalities with double-dosing of intravenous contrast. The CT scan identified >24 lesions scattered in the whole brain. The patient was treated with three-fraction Cyberknife radiotherapy at 22 Gy, delivered to the brain metastases at the Center for Tumor Treatment of People’s Liberation Army 107th Hospital. Following CyberKnife therapy, a CT scan of the brain revealed that most of the tumors had disappeared with almost no residual traces. The stereotactic radiosurgery (SRS) conducted using CyberKnife, an image-guided frameless robotic technology for whole-body radiosurgery, had produced a marked response. The present case report demonstrates that CyberKnife therapy plays a significant role in the management of multiple meta-static brain tumors. Brain metastases are a common type of intracranial malignancy derived from the transfer of tumor cells outside the central nervous system to the brain tissue. In a study by Lu-Emerson and Eichler, brain metastases were reported to occur in ∼20–40% of cancer patients, with an onset age of 50 to 70-years-old. The study reported that 1/3–1/2 of cancer patients died as a direct result of brain metastases  in April 2010. The patient was suffering with a long-term cough and pain in the chest and back. Using X-ray, a mass was detected in the right lobe of the lung. Enhanced computed tomography (CT) scans confirmed the suspicion of lung cancer and also identified a right adrenal metastasis. The tumor was clinically staged as T2N0M1 (stage IV). The patient underwent conformal radiotherapy with 66 Gy/33 F combined with three cycles of cisplatin and vinorelbine chemotherapy. A good partial remission was achieved after the administration.Six months after the initial diagnosis, the patient experienced severe headaches with frequent vomiting and blurred vision. An enhanced CT scan of the brain revealed abnormalities with a double-dosing of intravenous contrast, which demonstrated that there were no less than 24 lesions scattered in the whole brain, including the frontal, parietal and temporal lobes and the brainstem . A discuBrain metastases from systemic cancer are the most common type of intracranial neoplasm in adults, being almost 10 times more common than primary malignant brain tumors, which cause a significant burden on the management of patients with advanced cancer . Symptomet al , which is coupled with real-time imaging to track and compensate for the patient’s or target’s motion. As a relatively non-invasive treatment modality, CyberKnife demonstrates certain benefits, including a more accurate target localization and improved dose delivery for the management of metastatic brain tumors that allows higher biologically effective dose delivery without increased incidence of toxicity. Nishizaki et al  producedet al , which were scattered throughout the whole of the brain, were completely resolved within 3 months following CyberKnife radiosurgery. No opportunistic recurrence or metastasis occurred during the follow-up, as revealed by CT imaging studies. Although the patient succumbed to liver metastasis, the survival results were comparable to those of published studies. Kim et al  conductel (et al  showed tberKnife .In the present case, the results for the treatment of multiple brain metastases after CyberKnife surgery with a 7–8 Gy marginal dose was promising. CyberKnife for metastatic brain tumors is an effective and safe method for reducing the marginal dose prescribed for multiple brain metastases and for minimizing the radiation-related neurotoxicities. In conclusion, CyberKnife, a focused, highly-targeted radiosurgery and fractionated radiotherapy is particularly useful for multiple brain metastases. CyberKnife provides the advantage of the management of local recurrence and a tolerable complication rate. Although the treatment of brain metastases has been performed with CyberKnife, the clinical significance and optimal dose fractionation scheme require further investigation.


Paramyxoviridae as proposed for some other viral families. Additionally, the high rate of bat–human contact at the study site calls for the zoonotic potential of the detected viruses to be investigated further.Bats constitute a reservoir of zoonotic infections and some bat paramyxoviruses are capable of cross-species transmission, often with fatal consequences. Determining the level of viral diversity in reservoir populations is fundamental to understanding and predicting viral emergence. This is particularly relevant for RNA viruses where the adaptive mutations required for cross-species transmission can be present in the reservoir host. We report the use of non-invasively collected, pooled, neat urine samples as a robust sample type for investigating paramyxoviruses in bat populations. Using consensus PCR assays we have detected a high incidence and genetic diversity of novel paramyxoviruses in an urban fruit bat population over a short period of time. This may suggest a similarly unique relationship between bats and the members of the family  Bats are an increasingly recognized source of emerging zoonoses and are known to harbour a wide range of viruses, including highly virulent zoonoses such as henipa-, filo- and lyssa- viruses (Eidolon helvum) sampled in Ghana have been shown to have neutralizing antibodies against henipaviruses  from E. helvum were collected over 12 sampling intervals between September and November 2010 (E. helvum (co-roosting with other species has not been observed at this site) that comprise part of a much larger colony of up to 1 000 000 bats in Accra (Pooled urine samples (ber 2010 . Samplesin Accra . On retuParamyxovirinae PCR (PMV-PCR) was used to amplify a 531 bp (excluding primers PAR-F2 and PAR-R) fragment of polymerase genes of viruses belonging to the subfamily Paramyxovirinae, that spanned positions 13 898–14 428 of the hendra virus genome (GenBank accession no. NC_001906). A PCR targeting an upstream portion of the polymerase gene  of respiro-, morbilli- and henipa- viruses (RMH-PCR) was also used.Extracted RNA was tested for the presence of paramyxovirus polymerase gene RNA using two heminested RT-PCRs  and one or more clones from each sample were sequenced . Obtained sequences were aligned with polymerase genes from the subfamily g muscle  and cluslustal_x  under th from the subfamily Paramyxovirinae, but also allowed for comparison of the assays’ performance on field samples (The urine samples were frequently found to contain paramyxovirus sequences with PCR-positive samples being collected on 8 of the 12 sampling dates (over a 2 month period) and 31 of 72 samples (43 %) being positive. Fifteen of the samples were positive by both PMV-PCR and RMH-PCR, with each PCR detecting a further 10 and 7 (respectively) positive samples uniquely  and 2. P samples  and 2. GE. helvum population in Accra, with sequences having pairwise nucleotide identities of 57–89 % with known paramyxoviruses. These sequences expanded the Paramyxovirinae phylogeny, with the PMV-PCR detecting sequences in five clusters throughout the phylogeny  within the clade containing known rubulaviruses  and the RMH-PCR being restricted to respiro-, morbilli- and henipa- viruses. This is likely caused by the greater redundancy of the PMV-PCR primers and is further evidenced by the results from 10 samples in which sequence was detected using PMV-PCR but not using RMH-PCR. The sequences derived from these 10 samples showed phylogenetic clustering , the RMH-PCR detected seven samples as positive that were negative on PMV-PCR. However, the phylogenetic clustering of these samples’ sequences was not as marked as in the previous case . Thus, iE. helvum in Accra, Ghana. This demonstrates that neat urine pools are a robust sample type for investigation into paramyxoviruses in bat populations. Indeed, novel paramyxoviruses have since been isolated and characterized from this population . Not only is collecting urine from underneath roosts highly efficacious for qualitative virus population studies, but the non-invasive nature of the collection method would facilitate the study of infection dynamics free from sampling-induced stress. However, one disadvantage is that the viral sequences detected here cannot be absolutely confirmed as having been derived from E. helvum. Although co-roosting with other species has not previously been observed, and sheeting was supervised throughout the sampling period, detection of these sequences in samples directly collected from individual bats would be required to confirm their origin.Irrespective of assay differences, novel paramyxovirus sequences were detected at a high incidence in pooled urine samples collected from underneath a single roost-tree of E. helvum  supports these findings and increases our understanding of the diversity of paramyxoviruses in Africa. Indeed, the sequences described here support the further expansion of the genus Rubulavirus, which appears to have commenced with the detection of other bat-derived paramyxoviruses namely menangle, tioman and tuhoko viruses. Also, our data give rise to numerous phylogenetically distinct, but poorly placed clusters of viral sequences, perhaps indicating that viruses belonging to novel genera may be circulating in the study population. Such diversity and expansion of the paramyxovirus phylogeny within a single population implies that adaptive mutations required for emergence may be present in the reservoir host, and also may suggest that a co-evolutionary relationship between Chiroptera and Paramyxoviridae exists, as has been proposed for other viral families (The detected sequences are highly novel and demonstrate significant genetic diversity of paramyxoviruses within a single bat population over a short period of time. Furthermore, their close relationships with previously detected paramyxovirus sequences in Ghanaian E. helvum is widely hunted in Ghana for bush meat (Sequences were detected that had close relationships with known human pathogens.


Yersinia pestis, the secretion of effector proteins, termed Yersinia outer proteins (Yops), is regulated by the activity of the YopN/SycN/YscB/TyeA complex. YopN is a secreted protein that interacts with the SycN/YscB chaperone via an N-terminal chaperone-binding domain (CBD) and with TyeA via a C-terminal TyeA-binding domain (TBD). Efficient YopN secretion is dependent upon its N-terminal secretion signal (SS), CBD, and the SycN/YscB chaperone. In this study, we investigate the role of the YopN CBD in the regulation of Yop secretion. Analysis of YopE/YopN hybrid proteins in which the YopN SS or SS and CBD were replaced with the analogous regions of YopE indicated that the YopN CBD or SycN/YscB chaperone play a role in the regulation of Yop secretion that is independent of their established roles in YopN secretion. To further analyze the role of the YopN CBD in the regulation of Yop secretion a series of tetra-alanine substitution mutants were generated throughout the YopN CBD. A number of these mutants exhibited a defect in the regulation of Yop secretion but showed no defect in YopN secretion or in the interaction of YopN with the SycN/YscB chaperone. Finally, conditions were established that enabled YopN and TyeA to regulate Yop secretion in the absence of the SycN/YscB chaperone. Importantly, a number of the YopN CBD mutants maintained their defect in the regulation of Yop secretion even under the established SycN/YscB chaperone-independent conditions. These studies establish a role for the CBD region of YopN in the regulation of Yop secretion that is independent from its role in YopN secretion or in the binding of the SycN/YscB chaperone.Numerous Gram-negative bacterial pathogens employ type III secretion systems (T3SSs) to inject effector proteins into eukaryotic cells. The activation of the type III secretion (T3S) process is tightly controlled in all T3SSs. In  Numerous Gram-negative bacterial pathogens use type III secretion systems (T3SSs) to inject effector proteins into eukaryotic cells Ghosh, . The injYersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica) carry a ca. 70 kb plasmid encoding a T3SS that is required for virulence  into targeted eukaryotic cells  proteins  regained some ability to regulate Yop secretion under the SycN/YscB-independent conditions, indicating that the earlier defect in regulation was chaperone-dependent and likely the result of an improper interaction between the mutant YopN proteins and the SycN/YscB chaperone. Indeed, both SycN and YscB failed to immunoprecipitate with the YopN(S48-D51A) mutant. In contrast, both SycN and YscB immunoprecipitated with YopN(F60-R63A), suggesting that the chaperone-dependent defect in YopN function in this mutant was more complex and not due to a simple loss of chaperone binding. Importantly, these types of mutations are expected and support the previously identified role of the SycN/YscB chaperone in efficiently targeting YopN for secretion. In contrast, to chaperone-dependent mutants discussed above, two of the YopN CBD mutants [YopN(M52-E55A) and YopN(V56-V59A)] secreted Yops constitutively under chaperone-independent conditions, indicating that these mutants were likely defective in regulation due to a unique (SycN/YscB-independent) role of the YopN CBD in the regulation of Yop secretion.Shigella flexneri MxiC protein, a homolog of YopN and TyeA, interacts with Spa47, the (T3S) ATPase (YscN in the Yersinia T3SS)  is required for the efficient targeting of the wild-type YopN/TyeA complex to the T3S apparatus. At the T3S apparatus, the SycN/YscB chaperone may be removed, revealing the CBD. Alternatively, it is possible that the SycN/YscB chaperone remains bound until the secretion of YopN is triggered. We hypothesize that an early interaction mediated via the CBD region is required for the C-terminal region of YopN complexed with TyeA to establish its unique interaction with the T3S apparatus that blocks Yop secretion. This CBD-dependent interaction with the secretion apparatus may be required to induce a conformational change in the YopN/TyeA complex that is required for it to establish its block in Yop secretion. This mechanism would ensure that the YopN/SycN/YscB/TyeA complex would only block the T3S process at the proper time, when the needle assembly is complete and translocator- and effector-type substrates are targeted to the T3S apparatus. We have previously isolated mutations in YopN that allow the YopN/TyeA complex to constitutively block secretion regardless of the level of extracellular calcium (Ferracci et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The target tissue was suctioned by a device made of polydimethylsiloxane (PDMS) following the intravenous injection of naked pDNA or siRNA. Transfection of pDNA encoding luciferase was achieved by the suction of the kidney, liver, spleen, and heart, but not the duodenum, skeletal muscle, or stomach. Luciferase expression was specifically observed at the suctioned region of the tissue, and the highest luciferase expression was detected at the surface of the tissue (0.12±0.03 ng/mg protein in mice liver). Luciferase expression levels in the whole liver increased linearly with an increase in the number of times the liver was suctioned. Transfection of siRNA targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene significantly suppressed the expression of GAPDH mRNA in the liver. Histological analysis shows that severe damage was not observed in the suctioned livers. Since the suction device can be mounted onto the head of the endoscope, this method is a minimally invasive. These results indicate that the in vivo transfection method developed in this study will be a viable approach for biological research and therapies using nucleic acids.We have developed an  Many in vivo transfection methods have been developed using recombinant viral vectors or non-viral carriers such as cationic liposomes and polymers In the post-genomic era, increased importance has been placed on the development of et al. reported that non-invasive gene delivery to the liver was performed by a mechanical massage around the abdomen after intravenous injection of naked pDNA in mice Liu in vivo experiments revealed that the damage was negligible In tissue pressure-mediated transfection, 2 objects were used to apply direct pressure effectively to the target tissue; the first is used to directly press the target tissue, and the other is used to support the pressed tissue. Examples include the index finger and the thumb in vivo site-specific transfection of naked pDNA or siRNA in mice. In this study, transfection of mouse kidney, liver, spleen, heart, duodenum, skeletal muscle, and stomach were evaluated after tissue suction by a PDMS device following intravenous injection of naked pDNA or siRNA. This is our initial study concerning in vivo site-specific transfection using a tissue suction device.Our previous results using the pneumatic balloon actuator and the renal case suggested that tissue deformation following the tissue pressure would be a key factor for the transfection efficiency of naked pDNA in vivo imaging of luciferase activity was performed 6 h after the suction. As shown in Ex vivo imaging of the suctioned liver clearly shows that the luciferase was expressed at the site of tissue suction , with an increase in the suction number and reached approximately 0.02 ng/mg protein.We investigated the effects of the number of tissue suctions on pDNA expression. First, 2 different parts of the tissue were simultaneously suctioned by using 2 tissue suction devices just after intravenous injection of pCMV-Luc. To evaluate the potential for naked oligonucleotide delivery of the transfection by tissue suction, we demonstrated silencing of an endogenous gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in the serum were examined to determine the occurrence of liver toxicity after treatment with the tissue suction device . pCMV-Luin vivo transfection of naked nucleic acids such as pDNA and siRNA can be achieved by tissue suction. Previously, positive pressure had been used to directly press the target tissue to induce transfection in vivo transfection with greater ease than previously reported. Moreover, since the tissue suction devices are small enough to be mounted to the head of the endoscope, it is possible to perform transfections less invasively than before. Thus, we have successfully innovated a useful procedure for tissue pressure-mediated transfection employing tissue suction.In the present study, it was shown that We made the tissue suction devices in several sizes  and suctIn vivo transfection by tissue suction achieved site-specific gene transfection in the applied organs  A tissue suction device was fabricated as described previously, with some modifications In a typical case of pDNA transfection using tissue suction devices, the mice were anesthetized with isoflurane, and the target tissue was minimally exposed. pCMV-Luc  was intravenously injected into the mice, and the target tissue was suctioned by the device with small amounts of negative pressure . The tisIn siRNA transfection experiments, anesthetized mice were intravenously injected with GAPDH siRNA or scramble siRNA , immediately followed by small negative pressure-mediated suctioning of the liver by the suction device.The hydrodynamic method was performed as described previously 2 by using the pressure control devices, namely, a syringe-like device and a spatula.The liver pressure-mediated transfection method was performed as previously described Gene expression levels were determined by luciferase assay after 6 h of tissue suction as described previously pCMV-GFP  was intravenously injected into the mice and the livers were suctioned using the type III device. After 6 hours, the mice were sacrificed and the surfaces of the target liver parts were observed under an inverted fluorescence microscope .5′-GGC AGT GAT GGC ATG GAC TGT-3′ (reverse); β-actin, 5′-GTT CTA CAA ATG TGG CTG AGG ACT T-3′ (forward) and 5′-TTG GGA GGG TGA GGG ACT T-3′ (reverse). mRNA copy number was calculated for each sample from the standard curve using the thermal-cycler software . Results were expressed as relative copy number calculated relative to β-actin mRNA (GAPDH mRNA copy number/β-actin mRNA copy number).Total RNA was isolated from the cells and organs using a GenElute Mammalian Total RNA Miniprep Kit (Sigma- Aldrich). Reverse transcription of mRNA was carried out using PrimeScript® RT reagent Kit . The detection of complementary DNA (GAPDH and β-actin) was conducted using real-time PCR using SYBR® Premix Ex Taq (Takara Bio) and a Lightcycler Quick System 350S . Primers for GAPDH and β-actin cDNA were synthesized by Invitrogen as follows: GAPDH, 5′-CTC ACT CAA GAT TGT CAG CAA TG-3′ (forward) and Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in the serum were determined as described previously The mice livers were harvested at 0 and 7 days after the suction and fixed in 10% neutral buffered formalin. Four-micrometer thick paraffin sections were stained with hematoxylin and eosin (HE). The histology of the liver sections was microscopically examined.Prism 5 software  was used. Statistical significance was determined by two-tailed t-test or one-way analysis of variance (ANOVA), followed by Bonferroni’s test.Figure S1Effects of the size of the tissue suction devices on luciferase levels.In vivo transfection by tissue suction was applied to the right kidney by using type II and IV device. *p<0.05 versus type IV device . All mice were alive at the end of the experiment.(TIF)Click here for additional data file.


Smac/DIABLO promotes apoptosis by antagonizing inhibitor of apoptosis proteins. The expression of Smac/DIABLO in tissues has been reported in various cancers; however, little is known about circulating levels of Smac/DIABLO. The present study was designed to first determine if Smac/DIABLO can be detected in the serum and then assess whether the circulating levels of Smac/DIABLO are of prognostic significance in patients with bladder cancer. The levels of Smac/DIABLO in the sera of 173 patients with bladder cancer and 36 normal donors were determined by using an enzyme-linked immunosorbent assay. The mean serum level of Smac/DIABLO in patients with bladder cancer was approximately 2-fold lower than that in normal donors. The mean level of serum Smac/DIABLO in patients with muscle-invasive bladder cancer was lower than that in patients with non-muscle invasive cancer. In addition, the mean serum Smac/DIABLO level in patients with T4 muscle-invasive bladder cancer was lower than that in patients with T2 and T3 cancers. The mean serum level of Smac/DIABLO in patients with Grade 3 bladder cancer was lower than that in patients with Grade 1 and Grade 2 cancers. Analysis by Kaplan-Meier revealed that patients with Ta and T1 non-muscle invasive bladder cancer with high level of serum Smac/DIABLO  had a longer postoperative tumor-free interval than those with low level  in the 3-year follow-up. Furthermore, patients with T2–T4 muscle-invasive bladder cancer with high serum Smac/DIABLO level  had a higher postoperative disease-free rate when compared with patients with low level  in the 5-year follow-up. The present study is the first to analyze circulating levels of Smac/DIABLO in the serum. The findings demonstrate that the mean serum level of Smac/DIABLO was downregulated in patients with bladder cancer compared to control healthy individuals, especially high grade muscle-invasive bladder cancer. Noteworthy, lower serum level of Smac/DIABLO predicted early recurrence in patients with bladder cancer. Overall, the findings suggest that measuring the levels of Smac/DIABLO in the serum may be considered a prognostic parameter in patients with bladder cancer. Furthermore, Smac/DIABLO may be a molecular therapeutic target in bladder cancer. Bladder cancer accounts for ~4% of all cancers worldwide. Clinical factors have been used historically as prognostic or predictive markers such as stage and grade of bladder cancer. Recent advances have been achieved to unravel both the pathogenesis and molecular biology of bladder cancer with the objective of developing new methods for early diagnosis and better prognosis for patients with bladder cancer . AlthougCancer cells respond to cytotoxic therapies by activation of the type II mitochondrial apoptotic pathway. The activation of this pathway results in the depolarization of the mitochondrial membrane potential and the release from the mitochondria into the cytosol both cytochrome c and second mitochondria-derived activator of caspase/direct inhibitor of apoptosis protein (IAP)-binding protein with low pl (Smac/DIABLO). These lead to the activation of caspases 3, 8 and 9 and apoptosis . DIABLO Previous studies have reported that overexpression of Smac/DIABLO sensitizes the resistant cancer cells to death receptor- or anticancer cytotoxic agent-induced apoptosis ,7. ThesePeripheral blood was obtained from 173 patients with initial primary bladder cancer before surgery or anticancer therapy. They included 140 male and 33 female patients, ranging in age from 24 to 89 years. Histologic diagnosis revealed that all patients had urothelial carcinoma of bladder cancer. Their histologic classification and staging according to the TNM classification were: Tis (n=8), Ta (n=89), T1 (n=45), T2 (n=9), T3 (n=16), T4 (n=6), and G1 (n=51), G2 (n=63), G3 (n=59), respectively. The cases had no metastasis.The patients had no treatments for bladder cancer before surgery. Patients with Ta and T1 non-muscle invasive bladder cancer were not treated after transurethral resection of bladder tumor (TUR-Bt) until recurrence. When patients with muscle-invasive bladder cancer had recurrence or metastasis after radical cystectomy, they were treated with cisplatin-based chemotherapy.Blood samples were also collected from 36 healthy donors without malignancy based on the medical history. This study was performed after approval by a local Human Investigations Committee. Informed consent was obtained from each normal and cancer patient.The sera were separated by centrifugation of the blood after collection, and stored frozen at −80°C until future use for an enzyme-linked immunosorbent assay (ELISA).The levels of Smac/DIABLO in the sera were quantitated by the sandwich ELISA according to the manufacturer’s protocol . The serum concentrations of Smac/DIABLO were calibrated from a dose response curve based on reference standards. This method made it possible to estimate serum Smac/DIABLO levels >7.81 pg/ml. Repeated measurements yielded the same results.Patients were divided into two groups according to the level of Smac/DIABLO in the serum. The level of serum Smac/DIABLO greater than the mean value was regarded as ‘high level’ and the level less than the mean value was regarded as ‘low level’.All determinations were made in triplicate. For statistical analysis, Student’s t-test was used. Postoperative tumor-free interval and postoperative disease-specific survival rate was determined by the Kaplan-Meier method. The Cox-Mantel test was used to establish the statistical difference in tumor-free period and disease-specific survival rate between the patients with high and low serum Smac/DIABLO levels. P<0.05 was considered significant.The serum levels of Smac/DIABLO in samples derived from normal healthy controls and patients with all histological stages of bladder cancer were determined by ELISA. The mean serum levels of Smac/DIABLO in normal individuals and patients were 257 and 136 pg/ml, respectively . In compWe examined the serum levels of Smac/DIABLO in patients with bladder cancer as a function of their histologic stages and grades of the disease. The mean serum level of Smac/DIABLO was significantly lower (P<0.05) in patients with muscle-invasive bladder cancer (T2–T4N0M0) than that in patients with non-muscle invasive cancer  . FurtherThese findings demonstrate that analyses based on the histological stage and grade of bladder cancer revealed a tendency for decrease in the serum level of Smac/DIABLO as a function of disease progression and a higher grade.Ta and T1 non-muscle invasive bladder cancer patients undergoing TUR-Bt were retrospectively evaluated for the postoperative clinical course. The postoperative tumor-free period was estimated by Kaplan-Meier analysis. Based on the analysis, patients with non-muscle invasive bladder cancer were divided into two groups, namely, those with high level of serum Smac/DIABLO  and those with low level of serum Smac/DIABLO . Patients with Ta and T1 bladder cancer with high level of serum Smac/DIABLO showed a significantly longer postoperative tumor-free period as compared to those with low level in the 3-year follow-up (P<0.05) . These rT2–T4 (N0M0) muscle-invasive bladder cancer patients undergoing radical cystectomy were also retrospectively evaluated for the postoperative clinical course. Based on the analysis, patients with muscle-invasive bladder cancer were divided into two groups, namely, those with high level of serum Smac/DIABLO  and those with low level of serum Smac/DIABLO . Patients with muscle-invasive bladder cancer with high level of serum Smac/DIABLO showed a higher disease-specific survival rate as compared to those with low level in the 5-year follow-up (P<0.05) . These rThe present study demonstrates for the first time that Smac/DIABLO can be detected in the serum. Further, we investigated whether the serum level of Smac/DIABLO in patients with bladder cancer was modified as a function of both the stage and the grade of the disease. We report that the mean level of serum Smac/DIABLO in patients with bladder cancer was significantly lower than that in normal volunteers, and that the mean serum level of Smac/DIABLO inversely correlated both with the progression of the stage and the increase of the grade of bladder cancer. To our knowledge, this study is the first to show that patients with Ta and T1 non-muscle invasive bladder cancer patients with high serum level of Smac/DIABLO had a longer tumor-free interval than those with low level in the 3-year follow-up. In addition, patients with muscle-invasive bladder cancer with high serum level of Smac/DIABLO had a higher disease-specific survival rate than those with low level in the 5-year follow-up. Although we report a small number of patients during a short-term follow-up in this study, our preliminary data indicate that the level of serum Smac/DIABLO may be one of the significant prognostic parameters in patients with bladder cancer.There are many reported studies on the expression of Smac/DIABLO in both normal and cancer tissues ,9. HowevThe current study demonstrates for the first time that the serum level of Smac/DIABLO predicted the clinical outcome and that the high level in the serum was a good prognostic sign in patients with bladder cancer. The precise underlying mechanism responsible for this relationship remains unclear. Since Smac/DIABLO is a proapoptotic regulatory molecule, it is reasonable to assume that the elevated levels of Smac/DIABLO in the circulation might have resulted from its release following apoptosis of cells. In contrast, the low serum levels of Smac/DIABLO in the serum may reflect tumor escape from apoptosis.The precise cellular origin of Smac/DIABLO has not been elucidated. We speculate that Smac/DIABLO may be derived from cancer cells and/or normal tissues. Previous studies demonstrated that Smac/DIABLO was expressed by various cancers –12. NormSmac/DIABLO is not a secretory molecule. It is possible that its presence in the circulation may be due to the physiological cell death of normal tissues and cell death of cancer cells. Based on our findings, the circulating levels of Smac/DIABLO in bladder cancer patients on the average are lower than those detected in normal healthy individuals presumably due to a disturbance in the turnover rate of Smac/DIABLO in cancer patients. However, our findings in normal individuals revealed that there was a significant subpopulation of individuals who had levels of Smac/DIABLO in the same range as bladder cancer patients. This may be interpreted that such individuals might have a distinct turnover rate and/or possibly that Smac/DIABLO is degraded and modified and not detected by the ELISA assay used.Preliminary experiments demonstrate that the level of Smac/DIABLO in the serum derived prior to surgery increased after curative surgery in patients with muscle-invasive bladder cancer (data not shown). However, in patients with non-muscle invasive bladder cancer, the mean serum level of Smac/DIABLO derived before surgery did not change after TUR-Bt. Since Smac/DIABLO is a proapoptotic regulatory molecule, it is reasonable to assume that elevated level of Smac/DIABLO in the circulation may contribute to induce apoptosis in muscle-invasive bladder cancer through its ability to inhibit IAP family members. Therefore, low level of Smac/DIABLO in the serum may be a novel mechanism of tumor cells’ escape from apoptosis in muscle-invasive bladder cancer. If these were the case, the present findings suggest that Smac/DIABLO mimetics may provide a therapeutic means of preventing the growth of muscle-invasive bladder cancer.IAPs such as XIAP are highly expressed in various cancers and are associated with poor prognosis and resistance to apoptosis ,14. Our Our findings correlating the levels of Smac/DIABLO decreasing as a function of stage and grade of disease might be interpreted by at least two possibilities. The first is that the more aggressive the tumor, the less apoptotic cell death takes place and less Smac/DIABLO is released in the circulation. Alternatively, we have reported that the level of XIAP varies as a function of disease progression, since XIAP induces ubiquitation and degradation of Smac/DIABLO, the more XIAP level in the cell the less Smac/DIABLO will be available and detected in the serum. Furthermore, Smac 3, a Smac/DIABLO isoform, was recently identified . Smac 3 In conclusion, the data presented in this communication have demonstrated that Smac/DIABLO can be detected in the sera of both normal and cancer patients. The levels of Smac/DIABLO in patients with bladder cancer inversely correlated with histologic stage and grade of the disease; and low serum levels of Smac/DIABLO were associated with early recurrence in patients with bladder cancer. The correlation between the level of serum Smac/DIABLO and postoperative prognosis suggests that the serum level Smac/DIABLO could be used as a prognostic marker in patients with bladder cancer. The accurate prediction of prognosis may help select patients for more intensive surgical or chemotherapeutic approaches in combination with Smac/DIABLO agonists.


MicroRNAs (miRs) have an important role in lung carcinogenesis and progression. Single-nucleotide polymorphisms (SNPs) in genes involved in miR biogenesis may affect miR expression in lung tissue and be associated with lung carcinogenesis and progression.POLR2A, RNASEN and DICER1 genes in 1984 cases and 2073 controls from the Environment And Genetics in Lung cancer Etiology (EAGLE) study. We investigated miR expression profiles in 165 lung adenocarcinoma (AD) and 125 squamous cell carcinoma tissue samples from the same population. We used logistic and Cox regression models to examine the association of individual genotypes and haplotypes with lung cancer risk and with lung cancer-specific survival, respectively. SNPs-miR expression associations in cases were assessed using two-sample t-tests and global permutation tests.We analysed 12 SNPs in RNASEN (Drosha) was significantly associated with shorter lung cancer survival . In AD cases, a SNP within the same haplotype was associated with reduced RNASEN mRNA expression (P=0.013) and with miR expression changes  of miRs known to be associated with cancer .A haplotype in Inherited variation in the miR-processing machinery can affect miR expression levels and lung cancer-specific survival. PolR2A); excision of a stem-loop structure by the nuclear RNaseIII enzyme (Drosha) to generate the pre-miR; transportation of the pre-miR to the cytoplasm and processing by another RNaseIII enzyme (Dicer) into a ∼22-base mature duplex RNA (MicroRNAs (miRs) are small non-coding RNAs that bind to the target transcript in the 3′-UTR and can inhibit the translation of proteins and destabilise their target mRNA . Polymorplex RNA . An alteWe performed SNP genotyping and miR expression profiling using blood and tumour tissue samples from the Environment And Genetics in Lung cancer Etiology (EAGLE) study  and DICER1 (http://snp500cancer.nci.nih.gov). Duplicate quality-control samples (2%) showed 100% agreement in all assays. Subjects with at least a 90% genotype call-rate  were included in the final analyses. All SNPs passed the Hardy–Weinberg equilibrium test among controls (P⩾0.26).Genomic DNA was isolated from blood samples from 1984 cases and 2073 controls and used to genotype 12 SNPs  coveringd DICER1 . GenotypRNASEN/rs640831 and RNASEN gene expression in non-involved lung tissue from 45 AD patients from EAGLE using data from an Affymetrix Chip HG U133A .The miR expression data were derived from formalin-fixed paraffin-embedded (FFPE) tissue samples in 165 lung adenocarcinoma (AD) and 125 squamous cell carcinoma (SQ) cases from EAGLE, who had not undergone chemotherapy or radiation therapy before tissue collection. Lung cancer histology and the presence of malignant cells in the FFPE tissue blocks were ascertained by the EAGLE local pathologists and were reviewed by a pathologist from the NCI. We excluded tissue blocks with mixed histologies or low frequency of malignant cells. miR expression profiles were obtained using a custom-made two-channel oligo array. The miRs represented on the array, data pre-processing, quality control procedures and selection of the analysed 199 human miRs were described previously (n=439) or died for causes unrelated to lung cancer (n=98) were censored in the analyses. The association of SNPs and haplotypes with survival-time adjusted for age, sex, stage, and smoking status was estimated by fitting Cox proportional hazards model , smoking intensity (cigarettes per day), and years-since-quitting smoking. We also performed subgroup analyses by smoking status (never/ever) and major histology types and analyses restricted to patients with resectable tumours . Lung cancer-specific survival was defined as time from diagnosis of lung cancer to time of death due to lung cancer. Patients who were still alive at the time of last follow-up (ds model  in patiet-test statistic for each miR-SNP combination. For each SNP we counted the number of significant (P⩽0.05) miR-SNP associations (ns) and then computed a global permutation P-value to evaluate the significance of the association between the SNP and the global miR expression profile. The permutation test for each SNPi  was performed by repeating 9999 times the 199 miRj−SNPip  association t-tests, where each time SNPip was a random permutation across subjects of the original SNPi  data and the number of significant miRj−SNPip associations (nsp) was recorded for each permutation. The global P-value was then defined as one plus the number of times in which nsp was at least as large as ns (numerator) divided by the total number of permutations plus one . Finally, we evaluated the association between RNASEN mRNA expression and the rs640831 SNP with a dominant model in 45 AD cases using the t-test statistic.We evaluated the association between miR expression and SNPs within a dominant model in 290 cases using the P-value threshold for statistical significance. All analyses were implemented and performed using the R-project (v2.10) statistical package (http://www.r-project.org/index.html) with the exception of the haplotype analysis, conducted using the THESIAS program , was significantly associated with lung cancer-specific reduced survival compared with the most common haplotype GTACCT (frequency=30%) among all cases with hazard ratio (HR)=1.86, 95% CI=1.19–2.92 and P=0.007. Similar results were obtained in AD and SQ histological subtypes  and among ever smoker patients . This haplotype-survival association was not observed among the 133 never smoker patients . When we repeated this analysis among the 821 patients with early stage resectable tumours , the association between RNASEN haplotype GTAATC and reduced lung cancer-specific survival was even stronger . Results were also significant in patients with resectable AD and resectable SQ separately .None of the investigated polymorphisms in  results . HoweverRNASEN/rs640831, included in the GTACCT haplotype, was associated with the expression of 56 miRs . As expected, the correlation between the microarray and qRT–PCR data was inverse as qRT–PCR values are measured in terms of number of measurement cycles needed to reach a certain expression level: the lower the number of cycles the higher the detected expression level. In addition, the association between the expressions as measured by qRT–PCR and RNASEN/rs640831 was qualitatively concordant with the microarray-based results (inverse association in the 24 AD but not in the 23 SQ cases).We validated the microarray results by qRT–PCR for 4 of the 56 miRs significantly associated with RNASEN/rs640831 and the miR expression profile among AD cases, we tested the association between RNASEN gene expression and the rs640831 polymorphism in non-involved lung tissue of 45 AD patients from EAGLE. The 25 AD patients carrying one or two rs640831 minor variants showed a significantly lower mRNA expression than the 20 AD patients homozygous with the more frequent allele .Finally, to further elucidate our finding of a correlation between the RNASEN, particularly, among early stage patients and (ii) a differentially expressed miR profile and RNASEN gene expression by RNASEN/rs640831 status in lung tissue. Carrying the minor variant A vs the common variant C in RNASEN/rs640831 contributed to the survival association for the RNASEN haplotype AGTATC compared with the haplotype CGTACT. Our results are consistent with the combined effect of multiple genetic markers within a haplotype as better representing the impact of the genetic locus on disease progression than individual markers  an association between lung cancer survival and a haplotype in  markers . This isn∼2000), allowing for exploration of several models, including haplotypes, the miR expression analysis was based on a smaller sample size . Consequently, only the most informative SNP (MAF=34%) could be adequately tested with the miR profile. In contrast, we were not able to detect possible effects of the full haplotype in association with the miR profile, because this test would have required hundreds of samples with miR expression data. Further functional studies and/or larger miR profiling studies are necessary to confirm our findings.In contrast to the survival analysis that was based on a large sample (RNASEN gene, coding for the Drosha enzyme, was associated with reduced expression of the Drosha gene and changes in the expression of miRs involved in many cancer-related processes. A haplotype in Drosha that included the inherited RNASEN variant and five additional variants was associated with poor lung cancer survival, particularly, among early stage patients. Drosha gene variants may affect miR-processing machinery including miR maturation and expression level, which may consequently affect the function of miRs in transcript and protein translation regulation. These variants in Drosha may have potential prognostic implications in lung cancer.In conclusion, in the largest study of SNPs in genes involved in miR biogenesis and lung cancer to date, an inherited variant in the


Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infection on host gene expression in Spodoptera exigua 4th instar larvae was investigated through the use of 454 sequencing-based RNA-seq of cDNA libraries developed from insects challenged with active AcMNPV or heat-inactivated AcMNPV.The impact of By comparing the two cDNA libraries, we show that 201 host genes are significantly up-regulated and 234 genes are significantly down-regulated by active AcMNPV infection. Down-regulated host genes included genes encoding antimicrobial peptides, namely three gloverin isoforms and an attacin, indicating that the viral infection actively repressed the expression of a portion of the host immune gene repertoire. Another interesting group of down-regulated host genes included genes encoding two juvenile hormone binding proteins and a hexamerin, all of which are involved in juvenile hormone regulation. The expression of these genes was enhanced by the topical application of Juvenile Hormone III (JHIII) in the insects challenged with heat-inactivated AcMNPV. However, infection with the active virus strongly suppresses the expression of these three genes, regardless of the absence or presence of JHIII.Using RNA-seq, we have identified groups of immune-regulated and juvenile hormone-regulated genes that are suppressed by infection with active AcMNPV. This information and further studies on the regulation of host gene expression by AcMNPV will provide the tools needed to enhance the utility of the virus as an effective protein expression system and as an insecticide. Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), which can infect a wide range of lepidopteran insects Baculoviruses are large DNA viruses that primarily infect insects. Most baculoviruses are quite host-specific, infecting only a single species or a few closely related species, except for Cydia Pomonella) Anticarsia gemmatalis) Helicoverpa armigera) Baculoviruses have long been used for two major purposes: as viral insecticides to control insect pests in agriculture and forestry, and as the basis of a popular eukaryotic protein expression system The remaining challenges for baculovirus expression systems include the need to improve protein quality by combining various post-translational modifications  and the need to stabilise the viral genome and the expression of the heterogeneous genes over longer periods of times. In addition, the slow speed of host killing by baculovirus, host adaptations and the complexity of producing standardised viral preparations limit the usage of this virus for insect control. Understanding how baculoviruses interact with their host cells at a molecular level will make it possible to engineer these viruses in a way that will enhance their usefulness as effective insecticides and protein expression systems.S. frugiperda cells Many viral proteins have been reported or predicted to be involved in host-virus interactions, resulting in host morphological changes after viral infection, inhibition of host apoptosis or molting, regulation of host stress, and host disintegration. During the viral replication cycle, an electron-dense, chromatin-like structure, the virogenic stroma, can be found near the centre of the nuclei of infected cells Bombyx mori NPV (BmNPV) infection triggered a global down-regulation of host gene expression in insect cells beginning at approximately 12–18 h post infection (hpi) Spodoptera frugiperda (Sf9) cells has been reported in multiple studies Baculovirus infection is also reported to affect the expression of host genes. Spodoptera exigua larvae, we used 454 sequencing to analyse the transcriptome. This is a newer alternative to traditional EST sequencing and is a much more cost effective means of sequencing transcriptomes. In addition, this method allows de novo sequencing, assembly and annotation of expressed genes in a non-model organism for which genome sequences are currently unavailable. The 454 sequencing technique can also be used to investigate transcriptome-wide differential gene expression between differently treated samples. For this study, we sequenced the cDNA libraries from insects treated with active AcMNPV and heat-inactivated AcMNPV 12 h after treatment. The combined read sequences from the two transcriptomes were then used to construct a pool of contigs. The read numbers from the two transcriptomes were compared to identify host genes that were up- or down-regulated after viral infection. Out of 5,945 total contigs, 201 genes are significantly up-regulated and 234 genes significantly down-regulated by active AcMNPV infection, as compared to heat-inactivated AcMNPV infection. Two small groups of host genes, a group of genes encoding antimicrobial peptides/proteins (AMPs) (gloverins and an attacin) and a group of three juvenile hormone-related genes, were down-regulated. The genes encoding the AMPs were strongly induced by challenge with the heat-inactivated AcMNPV, but this induction was suppressed by active AcMNPV. The genes encoding the two juvenile hormone binding proteins and a hexamerin were induced by challenge with the heat-inactivated AcMNPV and additionally enhanced by the application of Juvenile Hormone III (JHIII). This up-regulation was not observed in insects infected with active AcMNPV. These results strongly suggest that the active virus can suppress the expression of specific host genes.To identify the effect of AcMNPV infection on the expression of host transcripts in Spodoptera exigua larvae challenged with active AcMNPV or heat-inactivated AcMNPV were subjected to a 1/8-plate production run on the 454 GS-FLX sequencing instrument, resulting in 77,616 and 74,928 reads, respectively. Files containing these data were deposited in the Short Read Archive of the National Center for Biotechnology Information (NCBI) with accession numbers of SRX110132  and SRX110248 , respectively. After the sequence reads of viral origin were removed, a total of 130,335 total reads were obtained from the two cDNA libraries, which were assembled to create 5,945 contigs   . A totalS. exigua transcriptome, we compared the 5,945 contigs with a Drosophila database using BLASTX. A Drosophila-based gene ontology search categorised 2,699 hits into 15 functional groups  differences in the expression of different contigs were determined by comparing the read number of each contig between the active AcMNPV-infected sample (A-read) and the heat-inactivated AcMNPV-treated sample (I-read). Using this method, we identified 201 host genes that are significantly up-regulated and 234 genes that are significantly down-regulated by infection with active AcMNPV  & S3. ThWe characterised the sequences of viral origin from both the A-read and the I-read by performing a BLASTN homology search to AcMNPV open reading frame (ORF) sequences. The A-read from the active AcMNPV challenged-cDNA library included 614 read sequences that originated from AcMNPV, whereas the I-read from the heat-inactivated AcMNPV sample only produced one sequence . Out of per os infectivity factors, which are involved in the initiation of midgut infections To obtain an overview of AcMNPV gene expression 12 hpi, we grouped the AcMNPV ORFs based on their known functions and on their abundance in the A-read . Here, wThe number of I-reads and A-reads for each contig were graphed on an x,y plot, showing that the expression of the majority of host genes is not significantly affected (p>0.1) . Only apA global down-regulation of host transcription at late time points of infection has been reported in several studies in vitro system using Sf9 cells, which were infected with the virus at an MOI (multiplicities of infection) of 10. Because most cultured cells (over 99%) were infected with the virus simultaneously under these infection conditions, transcripts for the majority of host genes in Sf9 cells were shown to decline substantially between 12–24 hpi. In contrast, in this study, in vivo S. exigua larvae were used as host system, in which only several parts of tissues including hemocyte and fatbody could be actually infected with injected viruses and most of tissues were remained as non-infected at 12 hpi, at which time point total RNA was isolated from whole body of the infected larvae. This was further supported by the viral gene expression at 12 hpi  acts as an antimicrobial peptide (AMP) against Bacillus thuringiensisHyalophora cecropiaS. exiguaWhen the number of I-reads and A-reads for the contigs encoding immune-related peptides/proteins were graphed, we observed that the expression of several genes involved in the host immune response was significantly down-regulated in larvae infected with active AcMNPV . These dS. exigua larvae upon hemocoelic injection of AcMNPV (regardless of active and heat-inactivated), but the further expression of them was suppressed by viral modulation of host immune mechanisms in S. exigua larvae injected with the active AcMNPV at 12 hpi. This result may also suggest a possible antiviral role for these glycine-rich AMPs, a hypothesis that will be addressed by further research.We analysed the expression profiles of GLV1 and attacin by qPCR. Gene expression was slightly up-regulated at 6 hpi in the control insects, which were mock-injected with Sf9 growth medium. We observed 5–7 times greater expression of both AMP genes 6 hpi in the insects infected with active or heat-inactivated AcMNPV , as compbona fide JH-binding proteins The group of DOWN contigs included three genes encoding proteins that belong to the juvenile hormone binding protein (JHBP) family and three genes encoding hexamerin proteins . Low molWe analysed the expression profile of two JHBP genes (JHBP1 and JHBP2) and one hexamerin gene (contig00429) by qPCR. The expression was strongly induced at 12 hpi with heat-inactivated AcMNPV . This inWe propose that JH may be involved in the host defence against baculovirus infection by interfering in the viral life cycle. In order for successful viral replication to occur, the baculovirus needs to control host molting and pupation in infected larvae. Host metamorphosis is regulated by two hormones: ecdysone, which causes molting, and JH, which allows larval molting but prevents pupation. Host insects may respond to AcMNPV infection by increasing JH trafficking, which could explain the activation of JH carrier genes by heat-inactivated AcMNPV. Live AcMNPV counteracts this measure by suppressing gene activation, thereby controlling host molting and pupation to be more favourable for the viral replication cycle.Host ecdysone levels have been reported to be controlled by viral ecdysteroid UDP-glucosyltransferase (EGT). Viral EGT functions to block molting and pupation in infected insect larvae by inactivating ecdysone hormone In this paper, we describe the suppression of host JH-related genes by AcMNPV. We hypothesise that the host insect increases the level of available juvenile hormone after the viral infection, but that active AcMNPV counteracts this measure through an unknown mechanism that does not involve viral EGT regulation of host ecdysone levels.S. frugiperda cell line, Sf9, was maintained in TC-100 medium  supplemented with 10% heat-inactivated  fetal bovine serum  at 27°C. The S. exigua larvae were obtained from a laboratory colony and raised at 25°C under a 16 h∶8 h light∶dark cycle with an artificial diet The 7 PFU/ml) (containing active or heat-inactivated virus) with 6 mg/ml kanamycin was injected just underneath the dorsal cuticle of each S. exigua 5th instar larva using a Hamilton digital syringe  fitted with a 22s-gauge needle. JHIII  was applied topically  to S. exigua larvae. After incubation at 25°C for 12 h, total RNA was isolated from infected S. exigua larvae using TRIZOL Reagent  according to the manufacturer's instructions. The extracted total RNA was first treated with DNaseI  to remove contaminating genomic DNA.Inoculation of the insect larvae was carried out as described previously S. exigua 5th instar larvae, mRNAs were purified using the Oligotex mRNA Midi Kit . Next, double-stranded cDNA was synthesised using the SMART cDNA Library Construction Kit . The cDNA was further purified using the QIAquick PCR Purification Kit  and checked for quality using an Agilent 2100 Bioanalyzer. Approximately 5 µg of cDNA was used for sequencing on a Roche/454 GS-FLX Titanium sequencer . A 1/8 plate sequencing run was performed at MACROGEN Co. (Korea) according to the manufacturer's instructions. The raw 454 sequence files were processed to remove low quality regions and adaptor sequences using the SeqClean program (http://compbio.dfci.harvard.edu/tgi/software). The resulting sequences were then screened against the NCBI UniVec database and E. coli genome sequences to remove contaminating sequences. Sequences shorter than 100 bp were discarded. The processed sequences were assembled into contigs using the iAssembler program (http://bioinfo.bti.cornell.edu/tool/iAssembler).From the total RNA extracted from whole body of −ΔCt method The 28S rRNA gene was used as a reference gene Drosophila proteins using BLASTX . The resulting data set was used to reconstruct functional class profiles and pathways using the KEGG (Kyoto Encyclopedia of Genes and Genomes), except for the Immune response category, which was directly defined by comparing to Drosophila immune genes using BLASTN. The annotation results of S. exigua contigs derived from BLASTX were subsequently analysed in PROSITE, SMART, PFAM to confirm conserved domain structures.The contigs were analysed by comparing to Table S1List of assembled contigs with total reads from the cDNA libraries from both the AcMNPV-challenged and the heat-inactivated AcMNPV-challenged S. exigua.(XLS)Click here for additional data file.Table S2List of S. exigua genes significantly up-regulated by active AcMNPV infection.(DOC)Click here for additional data file.Table S3List of S. exigua genes significantly down-regulated by active AcMNPV infection.(DOC)Click here for additional data file.Table S4Number of read sequences originating from AcMNPV.(DOC)Click here for additional data file.Table S5Fasta sequence of the 93 AcMNPV unigenes assembled from the RNA-seq.(DOC)Click here for additional data file.Table S6Primer sequences used in quantitative real-time PCR analysis.(DOC)Click here for additional data file.