Action inhibition, the suppression of action impulses, is crucial for goal-directed behaviour. In order to dissociate neural mechanisms specific to motor stopping from general control processes which are also relevant for other types of conflict adjustments, we compared midfrontal oscillatory activity in human volunteers via EEG between action inhibition and two other types of motor conflicts, unexpected action activation and unexpected action change. Error rates indicated that action activation was significantly easier than the other two equally demanding tasks. Midfrontal brain oscillations were significantly stronger for inhibition than for both other conflict types. This was driven by increases in the delta range (2–3 Hz), which were higher for inhibition than activation and action change. Increases in the theta range (4–7 Hz) were equally high for inhibition and change, but lower for action activation. These findings suggest that inhibition is facilitated by neural mechanisms specific to motor-stopping, with midfrontal delta being a potentially selective marker of motor inhibition. Successful inhibition of action impulses is believed to play an important role in avoiding and overcoming harmful behavioural patterns, such as seen in addictions, overeating or obsessive-compulsive disorders3. Since inhibition implies the absence of a directly observable action, the investigation of this phenomenon is difficult at the behavioural level. Therefore, identifying neural underpinnings of inhibitory control could greatly benefit our understanding of how to stop unwanted action impulses.When changing circumstances render prevalent actions as no longer beneficial, we need to be able to stop them. The suppression of prepotent behavioural tendencies is called inhibition6. Based on such findings one might speculate that these midfrontal oscillations at least in part represent a neural marker of inhibition. For example, they might be related to a proposed fronto-basal network for motor suppression, involving the supplementary motor area (SMA), the inferior frontal cortex and the basal ganglia10.Inhibition of intended actions has regularly been found to lead to an increase in low-frequency oscillations in the delta-theta range (<7 Hz) in medial frontal brain regions as assessed via EEG11, or post-error processing13. While most studies investigated these midfrontal oscillations in the theta range (4–7 Hz), they sometimes have also been reported for the delta range (<4 Hz)15.However, increases in midfrontal low-frequency oscillations appear to be not exclusive to inhibition. Similar neural oscillatory patterns have been found in a wide variety of contexts in which a conflict arises that affords readjustments of ongoing behaviour, such as task switching18. On a neural level, they might reflect activity in the cingulate cortex, which has been related to the identification and resolution of behavioural conflicts20. Cognitive control refers to neural processes aimed at facilitating cognitive and behavioral adjustments for overcoming goal-relevant conflicts. In part, the type of adjustments necessary for successfully resolving a conflict strongly depends at the task at hand. For example, while outright inhibition of an intended motor action can represent an appropriate conflict resolution in some task contexts, other types of conflicts might afford the enactment of a new motor action instead22. Despite the task-specificity of conflict adjustments, it has been suggested that different conflict tasks partially rely on a set of overlapping neural processes for detecting conflicts and initiating cognitive control19. Such processes could be described as general cognitive control mechanisms, since they are relevant in different types of tasks. Here, midfrontal low-frequency oscillations represent one possible marker of general control processes16. However, the exact role of midfrontal low-frequency oscillations for inhibition is not yet clearly understood. Importantly, since inhibition conflicts elicit both general control and inhibition-specific neural processes, previous studies cannot clearly distinguish between oscillatory responses which are specifically related to motor stopping as compared to neural reactions related to general control processes. For understanding the neural underpinnings of successful motor inhibition, it would be important to know in how it relies on mechanisms that are specific to motor stopping compared to general control processes which are also evoked in other conflict adjustments.Since midfrontal low-frequency oscillations can be found in many different task contexts, it has been suggested that they are part of a general mechanism for facilitating cognitive control23. In order to identify inhibition-specific processes, some previous studies compared motor inhibition induced in Go-NoGo tasks with alternative types of motor conflicts (for reviews see24). For example, some previous experiments contrasted inhibition  with motor activation conflicts, where on most trials no action had to be performed, while some trials afforded a sudden button press 25. Concerning midfrontal oscillations, results of one study showed increased midfrontal theta responses during inhibition compared to activation conflicts26. This could indicate that midfrontal oscillations partly reflect an inhibition-specific neural process. However, inhibition conflict tasks compared to activation conflict tasks usually lead to higher error rates, which suggests differences in task difficulty and, thus, increased demands for cognitive control ressources27. Thus, it might be more difficult to quickly suppress a frequently performed action (inhibition conflict) compared to a situation in which one occasionally has to perform a button press, which is interleaved with longer periods of inaction (activation conflict). Accordingly, previous studies leave open the question in how far stronger midfrontal oscillatory responses for inhibition compared to activation conflicts reflect inhibition-specific processes as compared to differences in general, non-specific control demand due to the increased difficulty of action inhibition.One way to induce motor conflicts is to let participants repeatedly perform a standard action, which then unexpectedly must be replaced by another type of response. Since the frequent performance of the standard action leads to the formation of a prepotent motor tendency, the sudden change towards the infrequent response behaviour affords increased cognitive control to withstand the inclination to elicit the prepotent motor action. Thus, the need to quickly divert from the standard response pattern creates a motor conflict. For example, in the classic Go-NoGo paradigm, inhibitory conflicts are typically induced by intermixing frequent (prepotent) button presses with the infrequent (conflict-inducing) withholding of button presses28. Using EEG, this allowed for dissociation between inhibition-specific and general control aspects of midfrontal oscillatory reactions in conflict-induced motor adjustments. Additionally, we estimated the neural sources of low-frequency oscillations in midfrontal brain areas which were sensitive to the occurrence of motor conflicts across the different types of motor conflicts .The goal of the present study was to clarify the role of midfrontal oscillations in inhibition, by comparing inhibition with different types of motor conflicts. In each case we only changed the type of frequent (prepotent) and infrequent (conflict) action while keeping all other aspects of the tasks identical. As in previous studies, we employed a task inducing inhibition conflicts (infrequent stopping after periods of frequent button presses) and contrasted it with a task inducing activation conflicts (infrequent button presses after periods of frequent inactivity). Importantly, we additionally employed a task evoking conflicts due to a sudden change in motor action . Compared to  activation conflicts, a sudden change of an action has previously been found to lead to a comparable degree of control demand as motor inhibitiont(23) = 6.80, p < 0.001, d = 1.39, activation: t(23) = 3.29, p = 0.003, d = 0.67, change: t(23) = 7.39, p < 0.001, d = 1.51. This increase in error rate from prepotent to conflict trials  was significantly higher for the inhibition than the activation task, t(23) = 5.09, p < 0.001, d = 1.04, and significantly higher for the change than the activation task, t(23) = 5.54, p < 0.001, d = 1.13. In contrast, inhibition compared to change conflicts did not lead to a significant increase in errors, t(23) = −0.96, p > 0.90, d = 0.20. This indicates that the activation task was easier for participants, while the degree of difficulty did not significantly differ between motor inhibition and motor change.Mean rates of errors are shown in Table t(23) = 11.88, p < 0.001, d = 2.43, activation: t(23) = 3.68, p = 0.001, d = 0.75, change: t(23) = 4.66, p < 0.001, d = 0.95. The increase in conflict-elicited responses was significantly stronger for the inhibition than the activation task, t(23) = 3.89, p < 0.001, d = 0.79. Additionally, midfrontal responses were significantly stronger for inhibition than for change, t(23) = 3.18, p = 0.004, d = 0.65. Activation and change did not differ significantly in their conflict-induced increases, t(23) = −1.13, p = 0.80, d = 0.23. Thus, all three conflict types induced a midfrontal control signal, with inhibition leading to a significantly stronger midfrontal response than any other type of motor conflict.Analysis of baseline-corrected oscillatory activation indicated a significant main effect of TRIAL TYPE, F = 112.20, p < 0.001, t(23) = 3.81, p = 0.03, d = 0.78. Additionally, delta was significantly stronger for inhibition than for change, t(23) = 5.64, p < 0.001, d = 1.15. Activation and change did not differ in their delta increase, t(23) = 0.54, p > 0.90, d = 0.11. For theta, the conflict-related amplitude increase was significantly stronger for inhibition than for activation, t(23) = 3.53, p = 0.005, d = 0.72, but did neither differ significantly between inhibition and change, t(23) = 1.81, p = 0.25, d = 0.37, nor between change and activation, t(23) = 1.60, p = 0.37, d = 0.33.In order to test for potential differences between the frequency bands involved, we repeated our analysis separately for the delta and theta band. Adding FREQUENCY (delta/theta) as a factor to the ANOVA led to a significant FREQUENCY*TRIAL TYPE*TASK interaction, F = 4.98, p = 0.01, Figure Figure 29, as well as other cases of behavioural adjustments affording increased cognitive control31. The current study contrasted motor inhibition with two other common types of action conflicts: sudden motor activation and sudden motor change. This allowed us to distinguish between inhibition-specific and general control-related reactivity in midfrontal oscillations. For each motor task the conflict response led to increased midfrontal low-frequency activity. Importantly, inhibition compared to other motor conflicts led to significantly stronger midfrontal reactions. Midfrontal low-frequency oscillations have repeatedly been observed during conflict tasks and are commonly interpreted as part of a general (i.e. not task-specific) mechanism for enacting cognitive and behavioural control20. Thus, the finding that inhibition selectively elicited a stronger midfrontal response is of great relevance for our understanding of this frequently reported oscillatory response, as it refines our understanding of the role of midfrontal activity for inhibition and motor control. Importantly, in the current study the motor inhibition task and the motor change task showed a comparable rate of errors and obviously were equally demanding. This strongly suggests a specific midfrontal involvement in inhibition rather than differences due to mere task difficulty.Previous studies found midfrontal oscillations in the delta-theta range both during motor inhibition4. In the current study, inspection of the time-frequency data suggests that differences between inhibition and the other tasks were more likely to occur in the delta-range rather than in the theta-range. Some previous experiments suggested that delta and theta might contribute separately to motor inhibition33. For example, it has been found that delta activity but not theta activity increased during inhibition of more frequently repeated  actions32. However, these studies did not compare inhibition with other types of behavioural conflicts, and therefore cannot delineate between the contributions of inhibition-specific compared to more general control processes for such findings. The results of the present study suggest that midfrontal delta activity is a more selective marker of motor inhibition, while theta activity does not allow for a clear distinction between inhibition and motor conflicts with a similar degree of control demands.While midfrontal control signals are often reflected in both the delta and theta range, previous studies of midfrontal control signals mostly focused on theta reactivity34. Therefore, suggestions concerning a potential dissociation between the adjacent delta and theta band should be considered with caution. Nevertheless, our results indicate, that a differentiation between lower frequency bands (~delta) and higher frequencies (~theta) within the midfrontal control signal could lead to new insights concerning the neural correlates of inhibition and other types of control conflicts.It should be noted, however, that the current findings do not suggest that midfrontal oscillatory activity in the delta and theta range represent independent processes. In fact, despite the differences in delta and theta reactivity, inspection of the time-frequency maps shows a considerable overlap in their response. Increases in the delta and theta band occurred simultaneously, suggesting they are both part of one complex oscillatory signal. It must also be pointed out that the results of the time-frequency analysis inevitably represent some degree of imprecision concerning the activity in each individual frequency band. That is, estimations of the power in one frequency band are influenced by the activity in adjacent bands36. Given the overlap in their spatial and temporal occurrence, it is reasonable to assume that N2/P3 and midfrontal delta/theta at least in part an expression of the same underlying, neural mechanisms32. One approach to disentangle oscillatory activity from event-related potentials is the calculation of non-phase-locked oscillatory activity, which is assumed to lower the impact of ERPs on time-frequency results. The analysis of non-phase-locked time-frequency data in the current study showed clear evidence for midfrontal effects in the delta/theta range. This is in line with previous research showing that the removal of phase-locked activity  did not significantly diminish conflict-induced effects on midfrontal oscillatory activity39. Overall, these findings would support the view that midfrontal oscillations are not solely an expression of event-related potentials such as the N2/P3-complex. However, it should be noted that currently established analysis techniques do not allow for an unambiguous disentanglement of ERPs and oscillatory activity41. The exact relation between event-related activity and oscillations as indicators of the same cognitive phenomena is rather complex and not yet clearly understood43. Therefore, further research is needed to clarify the relation and potential dissociation of conflict-related ERPs and oscillatory activity related to the midfrontal control signal.In discussing the relevance of oscillatory measures for the study of inhibition and conflict resolution, it is worth noting that these processes have also been linked to event-related potentials (ERPs) in midfrontal regions. More specifically, conflict inhibition is known to elicit the N2/P3-complex18. We focussed on midfrontal delta/theta activity, since previous research suggests it as an essential hub for the detection and resolution of behavioural conflicts20. Source estimation of the current data suggests that the difference between inhibition and other motor conflict tasks was most strongly pronounced in anterior and posterior cingulate cortex. Previous studies identified the anterior cingulate cortex as a main source for neural activity related to conflict resolution in range of different types of conflict tasks44. Taken together with the current findings, this might suggest that inhibition and other conflict tasks share overlapping neural sources for the generation of the midfrontal control signal, but that inhibition compared to other motor conflicts leads to a stronger response in the low-frequency range. It should be noted that the precision of source estimation in the current study is necessarily limited by the fact, that we did not have access to individual brain topographies, but instead relied on a standardized brain template. Importantly, since our analysis was focused on the source of midfrontal low-frequency oscillations our findings do not rule out the existence of inhibition-specific networks which might be crucial for inhibition but are not necessarily reflected in frontal delta/theta oscillations.Our current results do not imply that low-frequency activity during conflict resolution is confined to the midfrontal brain areas. For example, some previous studies have observed increased low-frequency activity in parietal areas during behavioural conflict adjustments, which might be an indicator of frontoparietal network related to attentional and cognitive control45. Secondly, cognitive control tasks which do not necessitate an outright motor stopping in conflict situations nevertheless can evoke inhibitory tendencies. For example, surprising events as well as error-related feedback are known to evoke motor slowing, which is believed to rely on a partial activation of motor inhibition networks47. In these cases, the partial enactment of motor stopping tendencies could help to avoid automatic and potential erroneous responses and thus might facilitate goal-directed readjustment of behavioural tendencies, for example in the form of a speed-accuracy trade off49. Thus, it might be possible to characterize different cognitive control tasks with respect to their degree of inhibitory involvement. Based on the present findings, one could speculate that control conflicts that evoke a stronger tendency to withhold or delay motoric reactions  show stronger increases in the delta range than cognitive conflicts where the next motor response is less uncertain, even when these tasks are matched for their overall demand on cognitive control resources. However, future research is needed to test those assumptions, for example by comparing control conflicts that differ in their degree of motor involvement13.A precise dissociation between neural mechanisms specific to inhibition and other potentially related control functions remains a persistent problem for neuroscientists. There are two related reasons for this: First, successful inhibition relies on several cognitive functions generally necessary for behavioural readjustments such as detection of stimulus deviations  and motor planningTo conclude, previous studies observed midfrontal low-frequency oscillations both in motor inhibition and other tasks demanding behavioural adjustments due to action conflicts. The present study presents evidence suggesting that the midfrontal oscillatory response is selectively higher for inhibitory conflicts. This effect appears to be due to a differential response in the delta and theta band: Theta activity did not differentiate between inhibition and the equally demanding motor change task. This could indicate that theta waves more strongly reflect control demands that are independent of the specific type of required motor adjustment, whereas delta waves, however, are selectively increased for outright motor stopping.Participants were 27 right-handed students voluntarily taking part for either course token or financial reimbursement. Three participants were excluded from statistical analysis due to too small numbers of artefact-free EEG trials (see below). The resulting sample consisted of 24 participants  with a mean age of 25.79 years (SD = 4.55). All participants provided written informed consent before the experiment. The study was approved by the local ethical board at the Department of Psychology of the Ludwig-Maximilian-University Munich, and the procedure was in accordance with its guidelines.The experiment was presented on a 24-inch monitor at a distance of approximately 90 cm from the participants. In this study we used the three stimuli square/circle/triangle as response cues, each of which indicated one of three possible actions: Go (pressing a button), No-Go (not pressing any button), Switch-Go . The assignment between symbols and actions was randomized for each participant and remained constant throughout the experiment. Each of the three stimuli was presented in white on a grey background in the centre of the screen with a visual angle of 0.8°.EEG was recorded with a BrainVision QuickAmp amplifier using 65 active electrodes (Brain Products ActiCap), positioned according to the international 10–20 system. The FCz was used as online reference, and an additional ground electrode was mounted on the AFz-position. Impedances were kept below 15 kOhm. EEG was recorded with 500 Hz sample rate and an online filter with a bandpass between 0.016 Hz and 250 Hz.inhibition task, participants had to press the down-arrow button in prepotent trials (75% Go), but to not press any button in the conflict trials (25% No-Go). In the activation task, participants had not to press any button in the prepotent trials (75% No-Go), but to press the down-arrow button in the conflict trials (25% Go). In the change task, participants had to press the down-arrow button in the prepotent trials (75% Go), but to press the up-arrow button instead in the conflict trials (25% Change-Go). At the beginning of each task, participants were informed about the relative frequency of Go, No-Go or Change-Go trials in the upcoming sequence of trials, respectively. Participants performed a short training block consisting of 16 trials prior to each task, which was repeated in cases where instructions were obviously not followed correctly. Participants were instructed to respond by using their right index finger throughout the whole experiment. The timing of events was kept constant across all three tasks: Each trial started with the presentation of one response cue for 100 ms, which indicated the action the participant had to perform. Participants were instructed to respond as quickly and accurately as possible within a 600 ms time window starting with the onset of the cue. An error message was shown if participants incorrectly pressed a button in No-Go trials, pressed the wrong or no button in Go/Change-Go trials or gave the response too late. If participants responded correctly, no feedback was provided. Each trial was separated by a blank screen for a jittered duration of 1500 to 2000 ms.The experiment was divided in three tasks (inhibition/activation/change), each presented in separate blocks. The order of the tasks was counterbalanced between participants. Each task induced a motor conflict by contrasting one type of frequent/prepotent action, presented in 450 (75%) of all trials, with one type of infrequent/conflict action, presented in 150 (25%) of the trials, resulting in 600 trials per task. The order of prepotent and conflict trials was randomized separately for each individual task. The to-be-performed responses differed across tasks as follows: In the 50. Data was segmented into epochs from −1500 ms to +1500 ms relative to response cue onset. Components representing eye blinks were identified and removed using independent component analysis in EEGLAB, leading to a removal of 1–6 (mean = 1.6) components per participant. Trials with maximum deflections of +−80 µV were deleted to account for noise artefacts, resulting in the removal of, on average, 5.27% (SD = 4.58%) of all trials. Since the goal of the study was to investigate the implementation of successful conflict resolution, trials with incorrect responses were excluded from further analysis . On average, the number of remaining trials per participant and condition were 416.0 (SD = 29.0) for the prepotent actions and 124.9 (SD = 15.3) for the conflict actions.The data was filtered (low-pass: 30 Hz) and referenced to an average of all electrodes offline. For six participants one noisy electrode, and for one participant two noisy electrodes were replaced by spherical interpolations in EEGLAB34. This raises a challenge for a distinction between event-related potentials and oscillatory phenomena within the same time-range. Since the focus of our investigation was the role of midfrontal oscillations, we subtracted phased-locked activity from the overall neural activation prior to time-frequency calculation. More specifically, for each condition we calculated the average event-related potential by taking the mean of all trials. The condition-wise event-related potential was then subtracted from each single trial of that condition. This approach is commonly used to minimize the influence of event-related activity on time-frequency measures and, thus, provide a purer measure of non-phase locked, i.e. oscillatory activity52 (but see Discussion for a further consideration of this issue). Time-frequency power was calculated around cue onset, based on the extracted 3-second epochs to avoid edge artefacts. Frequency calculation was performed in Brainstorm using Morlet Wavelets from 2–20 Hz in 1-Hetz steps53. Basis for the wavelet generation was a template waveform  with a central frequency of 1 Hz and a temporal resolution of 2 seconds FWHW . For each individual frequency bands, target wavelets were generated by scaling the template waveform accordingly. The resulting time-frequency data was averaged for each condition. The power values at each time point were baseline-corrected via a decibel conversion relative to the frequency-specific average power between 300 and 100 ms prior to stimulus onset . The length of the baseline time window is similar to other studies concerning low-frequency oscillations54. Since in wavelet analysis each individual data point of the time-frequency results is estimated via weighted averages of surrounding time points, choosing a relatively short baseline is appropriate for time-frequency data34.Time-frequency power does not only capture purely oscillatory (non-phase-locked) activity but is also influenced by event-related (phase-locked) potentialsIn order to assess participants’ performance, the mean rate of error trials from each condition was submitted to a 3*2 ANOVA with the within factors TASK (inhibition/activation/change) and TRIAL TYPE .55. As can be seen in Fig. 6. Greenhouse Geiser corrections were applied for ANOVAs in case of violations of sphericity, with corrected p-values and original degrees of freedom being reported. Post-hoc comparisons were Bonferroni-corrected. All statistical tests were calculated in R using the packages ezANOVA and effsize.Based on previous studies of midfrontal theta, we analysed oscillatory power at the FCz electrodeprocess_test_permutation2p (using 1000 randomizations). Additionally, for each task and participant we calculated the difference map between the average activity in the conflict and prepotent trials as a measure of conflict-induced increases in oscillatory power. We then employed the same nonparametric permutation procedure to calculate the contrasts of conflict-induced increases between the three tasks . In each case, multiple comparisons were controlled for via false discovery rate correction as implemented in Brainstorm.Since the analysis of summary scores based on averaged time-frequency points might obscure information about the extent of individual effects, we also calculated statistical differences between the conditions based on individual data points. More specifically, for each condition and participant, we extracted one time-frequency map of low-frequency activity during the trial period . These maps were used to calculate statistical contrasts between the individual data points of average oscillatory activity in conflict and prepotent trials in each task. For this purpose, we employed nonparametric permutation tests as implemented in the Brainstorm function 53. Based on the MRI template ICBM152 with 15,002 dipoles, a symmetric boundary element model (BEM) of the cortex surface with a grid resolution of 5 mm was generated as forward model. For each participant a noise covariance matrix was calculated based on the −0.3 to −0.1 pre stimulus baseline. Source reconstruction was performed with the minimum norm approach with unconstrained dipole orientation based on Brainstorm’s default parameter settings56. Since the focus of our study was midfrontal oscillations, we extracted from the source-reconstructed data regions of interest (ROIs) in the central to frontal region as defined in the neuroanatomical atlas Mindboggle57. This led to the extraction of 16 ROIs : caudal anterior cingulate cortex, rostral anterior cingulate cortex, posterior cingulate cortex, superior frontal lobe, caudal middle frontal gyrus, rostral middle frontal gyrus, precentral gyrus, and paracentral cortex . For each ROI and each condition, frequency-maps were calculated for each participant for the main trial period (0–0.6 s) in the delta-theta range (2–7 Hz). Frequency maps were converted to decibel relative to the pre-stimulus baseline. The goal of this analysis was to identify potential differences in source activation between inhibition and other types of motor conflicts. Therefore, we estimated the increase in oscillatory activity in the delta-theta range due to the occurrence of a conflict within each of the three tasks and compared conflict-related increases between the tasks. More specifically, for each task we subtracted the mean activity during prepotent trials from the mean activity during conflict trials. For each ROI separately, we then contrasted this measure of conflict-induced activation between the tasks with nonparametric permutation tests across the time-frequency maps , employing false discovery rate corrections for multiple comparisons. Thus, the resulting maps indicate conflict-induced source activity which differs between the tasks.For estimating the sources of conflict-related increases we employed source reconstruction in Brainstorm


E. coli RpbL12 was affinity labeled with a tRNA analogue, resulting in the loss of activity.We have recently demonstrated that Lys-65 of the 62GANK65 motif of  In this report, we show that mutations operated at the position of Lys-65 led to an impairment in the activity of the mutant ribosomes, except the K65R or K65H bL12 mutants, suggesting that the only requirement of the reaction catalyzed or facilitated by RpbL12is the positive charge of the side chain of Lys-65. We also demonstrate that Lys-65 did not play any role in the peptidyl transferase reaction with respect to puromycin, but rather assisted the binding of the incoming aminoacyl-tRNA to the ribosomal A-site.3+ form of Lys-65 is likely to participate to the binding of aa-tRNA through ionic bonds with phosphate groups, in order to insure the accurate positioning required for the nucleophilic attack of its α-amino group on the carbonyl carbone of peptidyl-tRNA.The protonated, positively charged εNH2 group is likely to be generated by the unprotonated εNH2 form of Lys-65 which is capable of withdrawing a proton from the α-NH3+ group of aa-tRNA. This α-NH Cells were grown at 37°C in 1 liter of LB rich medium containing 50 µg.ml-1 ampicillin to 0.5 A600, after which induction with 0.2 mM isopropyl-ß-D-thiogalacto-pyranoside took place at 21°C with incubation up to 20 hours. After harvest, the cells were washed in PBS, sonicated 15 times for 10 s at 4°C in 30 ml buffer A . The supernatant obtained by centrifugation  was applied two times on 1 ml His GraviTrap affinity column  equilibrated with buffer B , the column was washed successively with buffer B containing 50 mM NaCl and 100 mM imidazole before elution of the protein with 8 ml of the same buffer containing 0.4 M imidazole. This fraction was concentrated by ultrafiltration, dialysed against buffer C  and stored at -25°C.Wild-type or mutated his-tagged proteins were expressed in 2.2.2RIML gene of Escherichia coli encodes an enzyme catalyzing the acetylation of the N-terminal serine of ribosomal protein L12, thereby converting it into L7 -N-Ac-CoA . The amount of [3H]-N-acetylated protein L12 was then determined by filtration on nitrocellulose discs that were washed twice with 3ml of ice-cold 10 mM Tris-HCl pH 7.5, 1 mM MgCl2, 1mM DTT buffer and then counted for radioactivity.180 µM L12 were incubated in 5ml 10 mM Tris-HCl pH 7.5, 1 mM MgCl2.2.4RIML acetyl-transferase and the excess of acetyl-coA which were eluted in the washing buffer B before the elution of 6His-L7 protein in the same buffer containing 0.4 M imidazole. After concentration by ultrafiltration and dialysis against buffer C, the L7 preparation was stored at -25°C.5 mM Imidazole and 0.3 M NaCl were added to the 5 ml L12 acetylation reaction mixture then loaded on 1 ml Ni-NTA superflow column (Qiagen) equilibrated with buffer B in order to eliminate the 2.2.52-terminally acetylated (L7) or non acetylated (L12) forms of bL12 were each desalted using a Ziptip C4 (Millipore Saint Quentin en Yvelines France), and the proteins were eluted with 10 µl of a solution of 39% water 1% formic acid and 60% acetonitrile and analyzed by MALDI mass spectrometry. Samples (1 μl) were loaded on a MALDI plate using a solution of 5 mg/ml of α-Cyano-4-hydroxycinnamic acid in 30% (water 0.1% TFA) 70% acetonitrile. Mass spectra were obtained on an Autoflex speed series MALDI-TOF mass spectrometer  equipped with a pulsed N2 laser (337 nm) in a positive reflectron mode. Ions formed by the laser beam were accelerated to 23 keV. The final spectra were obtained by accumulating ~1500 single laser shots.After elution from the Superdex column, the fractions of interest were pooled and solutions of the NH2.2.6Mutations were introduced with the QuickChange II XL Site-Directed Mutagenesis Kit and by using the manufacturer (Stratagen) recommended protocol. As a substrate for the mutagenesis reactions we used pET21L12C-6His plasmid. The mutagenic oligonucleotides containing the specific modified bases and their respective complementary primers for introducing the required amino acid substitutions on bL12 were synthesized and purified by Eurogentec. Following transformation of competent XL10-Gold cells, minipreps on selected transformants were analyzed by sequencing to verify the presence of desired mutations and to check the absence of secondary mutations.2.2.7E. coli elongation factors, EF-Tu, EF-Ts and EF-G were purified as described in Phenylalanine into hot trichloroacetic acid-insoluble material, as previously described Phenylalanine (5 GBq.mmol-1)) and a saturating amount of partially purified phenylalanine synthetase), 3.5 µg poly(U) and 0.3 µM EF-Tu, 0.3 µM EF-Ts, 0.2 µM EF-G and the indicated amount of 30S, 50S ribosomal subunits and bL12. After incubation, aliquots were withdrawn spotted on glass fiber filters and hot trichoroacetic acid-insoluble radioactivity was determined.Poly(Phe) synthesis was determined as incorporation of L-Phe-tRNAPhe (128GBq/mol), 20 µg poly(U), were then added to the reconstituted particles to form “Ac-[3H]Phe-tRNAPhe.poly(U).ribosome” complexes during incubation for 15 min at 30°C in a total volume of 100 µl. These complexes were then separated on superose 12 HR 10/30 column  equilibrated in the same buffer. The isolated complexes were concentrated to 200 µl using microcon 100 from Amicon and the concentration of the complexes was determined (one A260 unit of “Ac-aa-tRNA.mRNA.ribosome” complex was assumed to contain 24 pmol of ribosome complex).Reconstitution of ribosome particles was performed during incubation at 37°C for 10 min in 85 µl buffer A .reconstituted ribosome” complex, with or without 0.5 µM EF-G and 0.5 mM GTP, in the presence or absence of 1.5 mM Puromycin. After 15 min incubation at 30°C, 47 µl were withdrawn and placed on Whatman GFC filters. The filters were washed with the cold trichloroacetic acid procedure, dried and then assayed for radioactivity. The extent of puromycin reactivity (PTC activity) was determined by comparing the radioactivity of the unreacted acid-insoluble material in the presence and in the absence of the antibiotic. 100% activity corresponds to the binding of Ac-[3H]Phe-tRNAPhe to the ribosomal sites determined in the absence of puromycin.Peptidyl transferase activity was determined in 50 µl reaction mixture containing in buffer A, 0.3 µM of “Ac-[2.2.103H]Phe-tRNAPhe to the ribosomal A-site was performed as previously described PhetRNAPhe (specific activity: 128 GBq/mmol) were preincubated for 10 min at 30°C in the presence or absence of 0.4 µM preformed EF-Tu•GTP before reaction was started with the addition of 15 µl of poly(U) programmed reconstituted ribosomes with final concentrations of 0.4 µM 30S and 50S, 5 µM purified wild type bL12 or mutated bL12s, 0.4 µM tRNAPhe and 7 µg poly(U). The enzymatic binding mediated by EF-Tu•GTP and non enzymatic binding of [3H]Phe-tRNAPhe to the ribosomal A site was determined after 8 min at 30°C by the nitrocellulose binding assay.The enzymatic binding of 2) bound to one copy of protein L10 (or uL10), which anchors the pentamer to the large 50S ribosomal subunit  sample where the free NH2-terminus of the two L12 chains of rp (L7/L12)2 have been acetylated in the presence of the enzyme RIML acetyl-transferase from E. coli and of N-acetyl coenzyme A (N-Ac-CoA) as donor of acetyl groups . Analysis by chromatography on the same Superdex 75 column of the fully acetylated 6His-L7 protein eluted from the Ni-NTA superflow column  resulted in an elution profile identical to that of L12 . When the fully acetylated 6His-L7 protein was obtained by incubating the 50 kDa (L7/L12)2 protein eluted from the Superdex column with the incubation mixture of the acetylation reaction, followed by application of this mixture to the same Superdex column, the same elution profile was observed (molecular weight of 50 kDa for (L7)4) . These results suggest that acetylation of all four polypeptide chains in the (L7)4 assembly did not give rise to a different association mode for this multimeric rp. Finally, it was verified by an SDS-PAGE analysis that the (L7/L12)2 or (L7)4 samples to be used in the present study are homogenous . In addition, Fig. (1B) shows that both (L7/L12)2 and (L7)4 assemblies are made of the same polypeptide chain of about 13 kDa, as expected. We next asked whether the acetylation of all polypeptide chains in rp (L7)4 has any effect on the activity of 70S ribosomes reconstituted with this protein. We addressed this question by comparing the poly(U)-dependent poly(Phe) synthesis activities of E. coli 70S ribosomes reconstituted with rps (L7/L12)2 or (L7)4. However, a prerequisite for this study is the demonstration that all four polypeptide chains of rp (L7)4 are effectively acetylated. It should be recalled that a molecular mass of 13228 Da had been calculated for the polypeptide chain of the recombinant E. coli L12 protein used in this study, while the measured molecular mass, as determined by ESI mass spectrometry had been found equal to 13228 Da Phe-tRNAPhe positioned at the P-site, leading to the synthesis of Ac-[3H]Phe-puromycin . The N-terminally acetylated [3H]Phe-tRNAPhe is supposed to preferably bind to the P-site, because it is a mimick of peptidyl-tRNA. However, one cannot exclude that a part of the Ac-[3H]Phe-tRNAPhe binds non specifically to the A-site and competes with puromycin for binding to this site. In order to allow the N-terminally acetylated [3H]Phe-tRNAPhe to bind in majority to the ribosomal P-site, the elongation factor EF-G was added to the incubation mixture of the peptidyl transfer reaction. This translation factor is required for the translocation of the tRNA molecule from the A-site to the P-site, or from the P-site to the E-site. Therefore, in the presence of EF-G, it is most probable that Ac-[3H]Phe-tRNAPhe and puromycin are bound to the P-site and to the A-site, respectively. As shown in Fig. (5), the extent of Ac-[3H]Phe-puromycin synthesis was much lower in the absence of EF-G than in its presence, whatever the type of ribosome used in the peptidyl transferase assay. This result suggests that, as expected, independently of the presence of rp bL12 in vitro, the translation factor EF-G facilitates the binding of Ac-[3H]Phe-tRNAPhe to the ribosomal P-site, while puromycin binds freely to the A-site.Given that the activity of ribosomes in protein synthesis is composed of the peptidyl transferase reaction followed by the translocation step, the critical residue Lys-65 of rp bL12 might be involved in either of these steps at the PTC. Therefore, the uromycin , 34. As 3.65), while their poly(U)-dependent poly(Phe) synthesis activity was lost . One obvious conclusion to be drawn from these results is that the inhibition of the poly(U)-dependent poly(Phe) synthesis activity of the mutant ribosomes is most probably caused by the enzymatic binding of aminoacyl-tRNA (aa-tRNA) to the ribosomal A-site. If this is the case, the binding of [3H]Phe-tRNAPhe mediated by elongation factor EF-Tu to the A-site of poly(U)-programmed ribosomes would be restricted in some of the mutant ribosomes. As shown in Fig. (6), [3H]Phe-tRNAPhe binding to the ribosomal A-site in the presence of EF-Tu is affected in the mutant ribosomes that exhibit a reduced poly(Phe) synthesis activity. These results suggest that the defect in the occupation of the ribosomal A-site of the mutant ribosomes is directly responsible for the inhibition of the poly(U)-dependent poly(Phe) synthesis activity. Therefore, the functional role of rp bL12 is likely to consist in the binding of the incoming aa-tRNA to the ribosomal A-site at the elongation step of translation. At this stage, one can remark that the difference between the in vitro synthesis of Ac-[3H]Phe-puromycin or poly(Phe) by the ribosome is the formation of only one peptide bond (Ac-[3H]Phe-puromycin synthesis) in the former and of several consecutive repeatedly connected peptide bonds (poly(Phe) synthesis) in the latter. In accordance with this observation, we have demonstrated above that the rp bL12 did not participate to the binding of puromycin at the ribosomal A-site, in sharp contrast to the binding of the incoming aa-tRNA whose entry into the A-site was shown to be strictly dependent on the interactions with rp bL12 and the elongation factor Tu (EF-Tu). In other words, rp bL12 would be dispensable for the binding of puromycin and indispensable for the binding of the incoming aa-tRNA to the ribosomal A-site. These data are likely to reflect that the number of interactions of the tRNA molecule with the E. coli ribosome is by far larger than that of puromycin, due to the difference in size between these molecules. In conclusion, the mechanisms of the in vitro peptidyl transfer reaction with the help of puromycin and of the in vivo naturally occurring polynucleotide-dependent polypeptide synthesis are not of the same type and do not rely on the same interactions with the ribosome. This might explain why the 70S ribosomes containing the large 50SΔ subunits depleted from the original rp L7/L12 copy are fully active in peptidyl transfer, while their poly(U)-dependent poly(Phe) synthesis activity was lost.As discussed above, the 70S ribosomes containing the large 50SΔ subunits depleted from the original rp L7/L12 copy were found to be fully active in peptidyl transfer . This situation is most probable since the protonated, positively charged εNH3+ side chain of Lys-65 can be estimated to represent about 50% of this primary amine group at neutral pH , because this residue was recently shown to exhibit a pK of 7.2 [E. coli ribosomes indicated the presence at the PTC of an ionizing group of the ribosome with a pK of 7.5, which could be a catalyst of peptide bond formation by the ribosome [2 form of the Lys-65 side chain at neutral pH would be capable of performing catalysis of the formation of peptide bond. Altogether, our results make it possible to propose the following hypothetical overall mechanism for each elongation cycle of translation: (i) the first event in the course of translation elongation consists in the binding and the correct positioning of the incoming aminoacyl-tRNA at the ribosomal A-site by rp bL12 in a complex with the elongation factor Tu (EF-Tu). As discussed above, the contribution of rp bL12 to this step would consist in electrostatic interactions between the positively charged side chain of Lys-65 and phosphate groups of the CCA-arm of the tRNA molecule. It is interesting to recall that Lys-65 is located in the large globular C-terminal domain (residues 53-120) of rp bL12 that was previously shown to interact with elongation factors including EF-Tu [3+ group of the aa-tRNA positioned at the A-site by the unprotonated εNH2 group of the catalytic Lys-65 side chain to generate the nucleophilic α-NH2 group capable of attacking the ester bond of peptidyl-tRNA. These two events constitute the first step of the mechanism ; (ii) the second step is the nucleophilic attack of the α-NH2 group of the aa-tRNA on the electrophilic carbonyl group of the peptidyl-tRNA in the P-site. Subsequently, peptide bond formation would stem from this nucleophilic attack, followed by the deacylation of the P-site tRNA, as well as the protonation of its 3’-end ; (iii) finally, the third step would permit the restoration of the nucleophilic ε-NH2 group of Lys-65 following donation of a proton by ε-NH3+ to a water molecule. This last step makes it possible to initiate a next elongation cycle by abstracting a proton from the α-NH3+ group of the next incoming aa-tRNA . It should be noted that the mechanism of peptide bond formation stemming from this study is strongly supported by the following previously reported data: (i) direct visualization of A-, P-, and E-sites transfer RNAs in the E. coli ribosome by cryo-EM studies had revealed that the A-site is localized close to the L7/L12 stalk [in situ on the E. coli 70S ribosomes was shown to provoke the loss of activity [The binding and correct positioning by rp bL12 of the incoming aa-tRNA at the ribosomal A-site is likely to be insured by the critical Lys-65, because the positively charged side chain of this residue is susceptible to establish ionic bonds with phosphate groups of the polyanionic tRNA molecule, especially at its single-stranded 3’-acceptor end -dependent poly(Phe) synthesis activity of the ribosomes containing the mutant proteins. This result suggests that the only requirement of the reaction catalyzed or facilitated by rp bL12 on the ribosome is the positive charge of the side chain of the amino acid residue at the position of Lys-65. We also demonstrate that the Lys-65 residue did not play any role in the peptidyl transferase activity, but rather assists the enzymatic binding of aminoacyl-tRNA to the ribosomal A-site. The fraction of protonated, positively charged εNH3+ side chain of Lys-65 is likely to participate to the binding of the incoming aminoacyl-tRNA through ionic bonds with phosphate groups of the polyanionic tRNA molecule, especially at its single-stranded 3’-acceptor end. We propose that, in addition, the fraction of unprotonated εNH2 form of the Lys-65 side chain performs catalysis of the formation of peptide bond by withdrawing a proton from the α-NH3+ group of the aa-tRNA positioned at the A-site to generate a nucleophilic α-NH2 group. Subsequently, peptide bond formation would stem from the nucleophilic attack of this α-NH2 group on the electrophilic carbonyl group of the peptidyl-tRNA.In this report, we have used mutational analysis of the GGQ-like 62GANK65 motif of rp bL12 to demonstrate that Lys-65 of this motif is critical for the activity of


Mycobacterium tuberculosis (M.tb). Additionally, the inhibitory efficacy of PA against two critical enzymes associated with M.tb, namely, UDP-galactopyranose mutase (UGM) and arylamine-N-acetyltransferase (TBNAT), as drug targets for antituberculosis therapy were determined. PA showed a profound inhibitory effect towards all the M.tb strains tested, with minimum inhibitory concentrations (MICs) ranging between 3.2 and 4.1 µM, and selectivity indices (SIs) ranging between 18.3 and 23.4. On the other hand, the standard drug isoniazid (INH) displayed comparably high MIC values (varying from 5.4 to 5.8 µM) as well as low SI values (13.0–13.9). Interestingly, PA did not exhibit any cytotoxic effects on a human liver hepatocellular carcinoma cell line even at the highest concentration tested (75 µM). PA demonstrated remarkable suppressing propensity against UGM compared to standard uridine-5'-diphosphate (UDP), with 85.8 and 99.3% of inhibition, respectively. In addition, PA also exerted phenomenal inhibitory efficacy  value = 8.7 µM, and 77.4% inhibition) against TBNAT compared with standard INH . Furthermore, in silico analysis validated the outcomes of in vitro assays, as the molecular interactions of PA with the active sites of UGM and TBNAT were unveiled using molecular docking and structure–activity relationship studies. Concomitantly, our findings present PA as an effective and safe natural drug plausible for use in controlling tuberculosis infections.The current study explores the antimycobacterial efficacy of lichen-derived psoromic acid (PA) against clinical strains of  Globally, tuberculosis (TB) has been placed next to HIV at the top of the list of communicable infectious diseases causing significant mortality and morbidity, in the long run posing a severe threat to public health . An estiStreptomyces griseus) followed by rifamycin B (an ansamycin-type macrolactom), rifampicin (a potent inhibitor of RNA polymerase and a semisynthetic derivative of rifamycin B), and amikacin, kanamycin (aminoglycosides) and cycloserine in combination with capreomycin (second-line drug combinations) [For the past four decades, efforts have been intensified for potent TB drug development. This eventually culminated in the discovery of the new ATP synthase inhibitors bedaquiline and delamanid  . In TB dnations) ,5. RecenMycobacterium tuberculosis (M.tb), the obligate slow growing pathogenic bacterium, causes TB by targeting alveolar macrophages. Based on its replication behaviour, the disease is characterized by replicating mycobacteria and non-replicating mycobacteria, wherein the former lead to disease and the latter to asymptomatic infection [d-galactofuranose  residues. The flavoenzyme uridine 5’-phosphate (UDP)-galactopyranose mutase (UGM) inevitably catalyses the biosynthesis of β-d-Galf residues from UDP-galactopyranose  using the precursor-UDP-galactofuranose  [f and UGM are vital for the viability and growth of most human pathogens including M.tb, Klebsiella pneumoniae, Trypanosoma cruzi, Leishmania major, and Aspergillus fumigaus [f and UGM are absent in mammalians and hence, targeting these enzymes involved in cell wall biosynthesis of mycobacteria could plausibly be an innovative strategy to develop potent and safe TB drugs. The enzyme inhibitors targeting UGM are now receiving much attention among natural product chemists, and numerous reports are being published [nfection ,7. UnlikDP-Galf) . Galf anfumigaus . Of noteublished ,10,11,12Through the Ping-Pong Bi–Bi mechanism, arylamine-N-acetyltransferase (NAT) catalyzes the hydrolysis of an acyl or acetyl group to arylamine via a conserved cysteine residue. This enzyme has been discovered in many microorganisms including M.tb as well as other microorganisms that infect humans . Drugs sUsnea, Psoroma, and Alectoria. Although PA was first isolated in the year 1880, its structure was resolved in 1958 [Lichens are symbiotic associations of two different organisms usually fungus and one or several algae or cyanobacteria (a photosynthetic partner). Lichens can produce more than 1000 different bioactive metabolites  with prominent pharmacological efficacies . Hithert in 1958 . Of late in 1958 ,23,24,25Although several natural products and its derivatives have been reported as having an antimycobacterial effect , the cytotoxicity and/or selectivity index associated with reported compounds have been little  reported. Hence, the present study was deliberately focused on determining the antimycobacterial effect of lichen-derived PA against eight M.tb clinical strains in addition to the reference strain, along with the inhibitory properties against UGM and TBNAT enzymes of M.tb. Attempts were also made to understand the mechanism of action of PA against M.tb, and structure–activity relationships through in silico analysis of PA with UGM and TBNAT. Furthermore, the toxicity and drug-likeness of PA were also evaluated through cytotoxicity and lipophilicity studies. To the best of authors’ knowledge, the current study is the first of its kind to decipher the underlying mechanism of action of PA, along with the inhibitory properties against two critical enzymes of M.tb, UGM and TBNAT. 37Rv  was received from the Czech National Collection of Type Cultures (CNCTC), National Institute of Public Health , as well as eight clinical isolates of M.tb , were kindly obtained from Motol University Hospital in Prague, Czech Republic. All clinical isolates were identified using biochemical and molecular protocols following the approved guidelines of Clinical and Laboratory Standards Institute (CLSI) [Standard PA (purity > 98%) was kindly obtained from University of Chemistry and Technology , whereas the standard antitubercular drug isoniazid (INH) was acquired from Sigma Aldrich . The reference pathogenic strain of M.tb. He (CLSI) . All M.tFor antimycobacterial activity, a microdilution assay was performed according to the process recommended by the CLSI . INH was50) was performed by a nonlinear regression analysis of the inhibitory curves. For cytotoxicity evaluation of PA and INH, the human liver hepatocellular carcinoma cell line HepG2-P9; purchased from Health Protection Agency Culture Collections , was cultured and prepared in recommended conditions, and the cytotoxic effect of PA and INH was evaluated spectrophotometrically as described earlier . The staThe lipophilicity parameters (Log P) for PA and INH were determined using the software CS ChemBioDraw Ultra 14.0 .UGM was expressed and purified from M.tb  as previously described . The con2S2O3 on ice for 1 min. Further, PA and UDP at concentrations of 20 mM were incubated with the solution mixture for 1 min. The UGM-catalyzed reaction took place by adding UDP-Galf at a concentration of 63 µM, at 25 °C. The reactions were terminated at various times by adding ice-cold HCl and exposed to quick freezing in liquid nitrogen. The activity of UGM in the presence of 5% (v/v) DMSO was assayed as a control. UGM activity was analyzed by HPLC (Agilent 1100 series), following the procedures of the methods as mentioned above for the instrumental setup, operational conditions, and the degree of conversion was quantified through the comparison of the integration of substrate as well as product peaks.Enzymatic activity was carried out as described earlier ,31. PA aTBNAT was prepared and purified as recombinant protein following the previously reported method . The pur50 values of test inhibitors by preparing the inhibition curves which were acquired by non-linear fitting of the percentage inhibition and the logarithmic concentration of the inhibitor versus the response.The catalytic action of TBNAT was assayed based on measuring the rate of hydrolysis of acetyl CoA by detection with 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), and the absorbance was detected at 405 nm  as reported earlier . BrieflyM. tuberculosis UDP-galactopyranose mutase in complex with UDP , the 3D-crystal structure of arylamine-N-acetyltransferase from M.tb , and the 3D-structure of PA (SDF format code: PJA) were recovered from the RCSB Protein Data Bank. To verify the docking procedure, the co-crystallized ligand (UDP) from the PDB (PDB code: 4RPJ) structure was removed and re-docked using PyRx docking tool via Autodock VINA software. UGM and TBNAT were inserted in the PyRx tool. The solvent molecules were extracted, and hydrogen and Gasteiger charge calculations were computed. The SDF file of PA was inserted in Autodock VINA, outfitted for docking, and was energy minimized. The UGM and TBNAT receptors and the SDF file of PA were transformed into the pdbqt format, and the grid center was placed on the active sites of UGM and TBNAT. The exhaustiveness values were set to increase the binding conformational analyses. The docking analyses were inspected based on binding affinity values  as well as hydrogen, hydrophobic, and electrostatic bonding interactions. The docking calculations, the protonation condition of the UGM-PA and the TBNAT-PA complexes, and the overall charge were determined as described earlier [Based on the results of the anti-UGM activity and anti-TBNAT activity, and to understand the interaction of PA with UGM and TBNAT active sites, molecular docking studies on the interaction of PA with UGM and NAT were achieved using the PyRx docking tool via Autodock VINA software . The thr earlier . The gra earlier .Challenged with the global requirement to develop novel antimycobacterial drugs with activity against drug-resistant bacteria, we focused on PA as a natural lichen-derived product. In the present study, the in vitro inhibitory effect of the test compound PA and the standard drug INH was assessed against nine strains of M.tb including a reference strain. The MIC determination assay revealed that the effectiveness of PA was more highly significant than that of the standard drug INH in inhibiting the growth of mycobacterial cells . 37Rv) with an MIC value of 44 µg/mL (122.8 µM) at pH = 5.8. It has been stated that the antimycobacterial activity of an antibiotic or antibacterial agent is dependent on the conditions of biological assays used, such as the pH value [37Rv) was significantly enhanced at pH = 6.6 (as recommended by CLSI), with an MIC value of 3.2 µM, in comparison with the results obtained by Tasdemir and Franzblau. This indicates that the used pH in the antimycobacterial assay plays an essential role in the inhibition activity of compounds screened against M.tb. Notably, PA showed the MIC range of 3.2 to 4.1 µM against the tested mycobacterial cells. On the other hand, the concentrations required for INH to kill the test pathogens were comparatively higher, and in turn, the antimycobacterial efficacy was statistically insignificant, with MICs ranging from 5.4 to 5.8 µM. The obtained MICs for INH are in accordance with the MIC breakpoints for INH (for susceptible M.tb strains) as previously reported by CLSI . FurtherpH value ,42. Base50 value required to reduce the viability of cell population to 50% compared to a control (100% cell viability). In the human liver hepatocellular carcinoma cell line HepG2, the test compounds did not show any cytotoxic effect, even at a high concentration (75 µM). It is recognized that lipophilicity is an essential physicochemical characteristic of any drug which outlines the permeation within biological membranes via passive diffusion [It is known that the use of antibiotics is the first-line treatment strategy for M.tb is associated with undesirable side effects such as a high risk of hepatotoxicity . Therefoiffusion . Thus, aiffusion . AlthougGiven the prominence of PA in posing a phenomenal inhibitory efficacy against a stretch of clinical isolates of mycobacterial cells, it would be worthwhile to investigate its inhibitory efficacy towards UGM, an essential enzyme required for the biosynthesis of the cell wall in M.tb. Data of the assay revealed that PA showed a profound inhibition of UGM . The untreated cells were found to synthesis 58.4% of UGM, whereas the PA and UDP treatment showed dramatically reduced levels of production to 8.3 (±2.6) and 0.4 (±2.3)%, respectively. There is mounting of scientific evidence showing that any molecule with proficiency as an UGM inhibitor could block mycobacterial growth significantly ,45,46. TIn recent years, TBNAT has emerged as a potential drug target for tuberculosis treatment. Therefore, we performed an enzymatic activity assay to evaluate the inhibition properties of PA towards TBNAT, and results are presented in 50 value of 8.7 µM, 77.4% inhibition) as compared with INH . Although this is the first report on the inhibition activity of PA against TBNAT, the obtained results agree with several investigations that have reported natural products with inactivation properties against various types of NAT enzymes from microorganisms and of human origin [The results declared that PA exerted great inhibitory activities against TBNAT  (The molecular docking studies were performed with PA on UGM of M.tb He: 4RPJ) . The enze: 4RPJ) , whereas37Rv (PDB code: 4BGF) was performed (The binding of PA in the active pocket of TBNAT from M.tb Herformed , and TBNerformed , whereasIn the drug discovery process, molecular docking is a valuable tool performing a critical role in the basic design of drugs, and convincingly envisages the best orientation of specific active molecules in the supposed target . It has In recent years, treatment of tuberculosis infections has globally been the focus of attention of many researchers and healthcare providers. This is due to the problem of drug resistance caused by the extensive use of antibiotics. Therefore, there is an urgent need to find new antitubercular drugs. Our findings revealed that M.tb strains chemically treated with PA were potently inhibited when compared to strains chemically treated with INH. Besides, PA showed a high safety profile along with prominent inactivation properties against two critical enzymes associated with M.tb, UGM and TBNAT. Based on the findings mentioned above, we can conclude that PA might be a future drug for possible treatment of tuberculosis infections. Further investigations should be performed in vivo to evaluate efficacy as well as pharmacokinetic and pharmacodynamic properties, leading to effective optimization of PA as a natural antitubercular agent.


Infectious bronchitis virus (IBV) causes a highly contagious respiratory, reproductive and urogenital tract disease in chickens worldwide, resulting in substantial economic losses for the poultry industry. Currently, live-attenuated IBV vaccines are used to control the disease. However, safety, attenuation and immunization outcomes of current vaccines are not guaranteed. Several studies indicate that attenuated IBV vaccine strains contribute to the emergence of variant viruses in the field due to mutations and recombination. Therefore, there is a need to develop a stable and safe IBV vaccine that will not create variant viruses. In this study, we generated recombinant Newcastle disease viruses (rNDVs) expressing the S1, S2 and S proteins of IBV using reverse genetics technology. Our results showed that the rNDV expressing the S protein of IBV provided better protection than the rNDV expressing S1 or S2 protein of IBV, indicating that the S protein is the best protective antigen of IBV. Immunization of 4-week-old SPF chickens with the rNDV expressing S protein elicited IBV-specific neutralizing antibodies and provided complete protection against virulent IBV and virulent NDV challenges. These results suggest that the rNDV expressing the S protein of IBV is a safe and effective bivalent vaccine candidate for both IBV and NDV. IB causes major economic losses in commercial chickens throughout the world3. It is one of the most prevalent diseases in the poultry industry. The disease is usually characterized by respiratory signs including ocular and nasal discharges, gasping, coughing and sneezing. However, the virus can also infect urogenital and reproductive tracts causing renal dysfunction and decreased egg production4.Infectious Bronchitis (IB) is an acute and highly contagious viral respiratory disease of chickensCoronaviridae. The genome of IBV is a single stranded, positive-sense RNA of approximately 27.6 kb in length. It encodes five major structural proteins: spike (S), small envelope (E), membrane (M), nucleocapsid (N) and fifteen non-structural proteins. The S glycoprotein is the major antigen against which neutralizing and protective antibodies are produced. The S protein is partially or completely cleaved into the amino-terminal S1 and to the carboxy-terminal S2 subunits post translationally by a furine-like protease of host5. The S1 subunit is highly variable among different isolates of IBV. It is responsible for viral attachment to host cell and contains major neutralizing epitopes. The S2 subunit is highly conserved among IBV strains and contributes to viral fusion activity and elicits some minor but broadly reactive neutralizing antibodies10.IB is caused by infectious bronchitis virus (IBV), a member of the family 11. Live vaccines have been found as one of the sources of IB outbreaks12. Live-attenuated vaccine strains also are not genetically stable and have a tendency to revert back to virulence14. Furthermore, circulation of live-attenuated viruses in the environment provides a setting in which the viral population may undergo mutations and recombination leading to creation of variant viruses16. Moreover, live IBV vaccines may cause pathological lesions or secondary bacterial infections in young vaccinated chicks18. Inactivated IBV vaccines, which usually are administered by injection to layers and breeders are not an alternative for live-attenuated IBV vaccines. Inactivated vaccines of IBV do not elicit strong immune responses in chickens against circulating virulent IBV strains20. The production and administration of inactivated vaccines are also time consuming and costly14. Therefore, there is a great need to develop an alternative IBV vaccine.Currently IB in commercial chickens is controlled by the use of live-attenuated and inactivated IBV vaccines. However, safety, attenuation and immunization outcomes of current live-attenuated vaccines which, were developed by serial passaging of field isolates of IBV in chicken embryonated eggs, are not guaranteed, particularly with regards to the age at immunization, vaccine dose, type of bird and route of vaccination25. Among these viral vectors NDV holds great promise, because it induces effective local and systemic immune responses, it is a safe vaccine vector, and it will serve as a bivalent vaccine against IBV and NDV.Development of viral vectored vaccines to control IB is an alternative approach. One of the important advantages of vectored IBV vaccines is that it will not create variant viruses, which is a major drawback of the current live-attenuated IBV vaccines. Several viruses, such as, fowl pox virus, adenovirus and Newcastle disease virus (NDV) have been evaluated as vaccine vectors for IBVAvulavirus in the family Paramyxoviridae. Virulent NDV strains cause a fatal neurological disease in chickens. NDV strain LaSota has been used as a safe and effective live vaccine for more than 60 years27. Recombinant NDV strain LaSota has been evaluated as a vaccine vector against several avian pathogens including IBV32. In one study, rNDV expressing S2 protein of IBV was found to induce partial protection against a virulent IBV challenge23. In another study, it was shown that rNDV expressing S1 protein of IBV resulted in inducing protective immunity against virulent IBV challenge24. However, the protective efficacy of the whole S protein of IBV expressed by rNDV vector has not been evaluated and it is also not known among S1, S2 and S proteins of IBV, which is the best protective antigen for the development of viral vectored IBV vaccines. In this study, we have used reverse genetics to generate rNDVs expressing S1, S2 and S proteins of IBV strain Mass-41. The protective efficacies of rNDVs expressing S1, S2 or S protein of IBV were compared in chickens against a virulent IBV strain Mass-41 challenge. Our results showed that the whole S protein is the best protective antigen of IBV. We also showed that rNDV strain LaSota expressing the S protein of IBV strain Mass-41 protects chickens against both virulent IBV and virulent NDV challenges.NDV belongs to the genus PmeI site, between P and M genes . rNDV/S1, rNDV/S1(cs+) + NDV-F-TM&CT, rNDV/S2, rNDV/codon optimized-S and rNDV were evaluated in the presence of exogenous protease in DF-1 cells  route. At three weeks post-immunization, chickens were challenged with virulent IBV strain Mass-41. The severity scores of IBV clinical signs were recorded twice a day for 10 days post-challenge Fig. . Compare3.1 EID50) of virulent IBV strain Mass-41 at 3 week post-immunization1. The severity scores of IBV clinical signs were recorded twice a day for 10 days post-challenge  recommended dose   Fig. . In orde05) Fig. .Figure 8To evaluate the protective efficacy of rNDV expressing S gene of IBV against a virulent NDV strain, groups of five 1-day-old chicks were immunized with rNDV, rNDV expressing codon optimized S protein and PBS. Three weeks after immunization, chickens were challenged with virulent NDV strain Texas GB in our BSL-3 plus facility. Our results showed that all chickens immunized with the rNDV and rNDV expressing codon optimized S gene of IBV survived after highly virulent NDV challenge, while all chickens in PBS group died at day 5 and 6 post-challenge Fig. .Figure 9Hemagglutination inhibition (HI) assay using a standard protocol of OIE was used to assess the level of antibodies mounted against NDV in serum samples of chickens 21 days after immunization. The results showed that HI titers of NDV was detected in serum samples of all chickens immunized with rNDV and rNDV expressing codon optimized S protein. There was no significant differences observed among HI titers against NDV in serum samples of chickens from groups immunized with rNDV and rNDV expressing S protein Fig. . Virus n37. Four different versions of IBV S1 gene were used to identify the version that is expressed at the highest level and incorporated into NDV particles. We were able to recover all the recombinant viruses and their growth characteristics were similar to rLaSota. However, the recombinant viruses containing IBV S gene grew slightly slowly than the parental virus. The viruses were stable after passages in SPF chicken embryos. Western blot analysis showed that chicken codon optimized S2 and S proteins were expressed at much higher levels and were incorporated into NDV particles. Whereas, all the four versions of S1 protein were detected at very low levels by Western blot analysis. It is noteworthy that the unmodified S1 protein was detected in the infected cell culture supernatant, indicating that the modification of S1 protein probably caused retention of the protein in the cell. These results suggest that the S2 protein acts as a chaperone to assist in the folding of the S1 protein. The S1 protein is folded incorrectly in the absence of S2 protein and the new structure probably causes loss of some conformational epitopes for IBV antibodies.This study was conducted to compare the protective efficacies of S1, S2, and S proteins of IBV using rNDV as a vaccine vector. The S1, S2, and S genes of IBV strain Mass-41 were individually inserted between the P and M genes of rNDV strain LaSota. This site was chosen because it has been identified as the optimal site for insertion of foreign genes into NDV genome24. These results also support the recent report that a spike ectodomain subunit vaccine protects chickens against IBV38.In the first IBV protection experiment, we found that 1-day-old chicks immunized with rNDV expressing the S protein of IBV conferred better protection from disease compared to 1-day-old chicks immunized with rNDVs expressing either S1 or S2 protein of IBV. Our results showed that the S protein, which contains both S1 and S2 proteins, is the best protective antigen of IBV. The S2 protein lacks major neutralizing epitopes which are present in the S1 protein, hence it is not an effective antigen. The S1 protein contains major neutralizing epitopes, but it losses some conformational epitopes when expressed separately. Although in the first study we showed that rNDV expressing S protein provided enhanced protection, it could not reduce virus shedding, indicating that we needed to determine whether the elimination of virus shedding would probably require a much higher level of immune response than that is induced by rNDV expressing S protein or would require the optimization of the IBV protection study. In this study, our results support previous reports that rNDV vectored IBV vaccines prevent disease but do not stop virus sheddingIn the second IBV protection experiment, we investigated whether age at immunization influences the outcome of IBV challenge. Our results showed that a single immunization of 4-week-old chickens with rNDV expressing S protein completely protected chickens against IBV challenge based on disease and viral load in tracheas. Indeed, the level of protection conferred by rNDV expressing S protein was similar to that of a commercial IBV vaccine. However chickens immunized with either commercial live attenuated IBV vaccine or rNDV expressing IBV S protein showed very low levels of tracheal viral load. This showed that protection was greater when the chickens were immunized at an age when their immune system is relatively well developed.In the third IBV protection experiment, we showed that rNDV expressing IBV S protein protects adult chickens against a higher dose of virulent IBV challenge. However, compared to standard challenge dose of virulent IBV, a higher challenge dose of virulent IBV caused higher levels of tracheal viral load in adult chickens immunized with rNDV expressing IBV S protein and low levels of tracheal viral load in chickens immunized with commercial live attenuated IBV vaccine. Our result showed that although both the age of immunization and dose of challenge virus affect the results of IBV challenge, the influence of the age of immunization is greater than the effect of the dose of challenge virus.Our results also showed that when we challenged adult immunized chickens with standard challenge dose of virulent IBV, rNDV expressing S protein showed slightly better protection than a commercial live IBV vaccine, based on disease and viral shedding in trachea, but when the adult immunized chickens were challenged with a higher dose of virulent IBV, commercial live IBV vaccine showed slightly better protection than rNDV expressing S protein. Hence to compare the efficacy of rNDV expressing S protein of IBV with the efficacy of live attenuated IBV vaccine, a large IBV protection study using commercial chickens is needed.In the fourth IBV protection experiment, we showed that rNDV expressing IBV S protein protected young chickens against virulent IBV challenge by the intraocular route. The route of challenge has been recommended by USDA-CFR-9. Our results showed that compared to infection of chickens with virulent IBV by the oculanasal route, infection of chickens with virulent IBV by the intraocular route, caused much lower levels of tracheal viral load in young chickens immunized with rNDV expressing IBV S protein and low levels of tracheal viral load in chickens immunized with commercial live attenuated IBV vaccine. Our results showed that the route of the challenge virus inoculation affected the results of the tracheal virus shedding in young chickens immunized by rNDV expressing S protein following IBV challenge; however, it did not affect the outcomes of the severity of clinical signs.Although our studies showed that the rNDV expressing the S protein and commercial live IBV vaccine provided comparable protection, rNDV expressing the S protein has several advantages over live IBV vaccines in controlling IB in the field. (i) NDV vectored IBV vaccine is highly safe in 1-day-old chicks, (ii) it will not create new vaccine derived variant viruses, which is a major concern in using live modified IBV vaccines, (iii) a single vaccine can be used to control both NDV and IBV, (iv) we believe that the level of immunity induced by the NDV vectored vaccine against IBV is probably sufficient to completely stop IBV infection in field condition, and (v) the immune response of NDV vectored vaccine can be enhanced by prime-boost vaccination strategy.In summary, we have shown that although the S1 and S2 proteins of IBV are known to contain virus neutralizing epitopes, the presence of the whole S protein is necessary for eliciting a strong protective immune response. The S protein is the antigen of choice for any vectored IBV vaccine. NDV is an attractive vaccine vector for IBV, because it can be used as a bivalent vaccine. Our results suggest that a recombinant NDV vectored IBV vaccine is the vaccine of choice for controlling IBV infection in the field.39. The rNDV and rNDVs expressing chicken codon optimized S1, S2 and S genes and non-codon optimized S gene of IBV strain Mass-41 were grown in 9-day-old embryonated SPF chicken eggs at 37 °C. The virulent IBV strain Mass-41 was propagated in 10-day-old SPF embryonated chicken eggs and harvested five days after infection. The titer of virus in harvested allantoic fluid was determined by 50% embryo infectious dose (EID50) method. Briefly, ten-fold serial dilutions of IBV strain Mass-41 was inoculated into 10-day-old embryonated SPF chicken eggs. Seven days after inoculation, infected embryos were examined for IBV specific lesions such as stunting or curling. The titer of virus was calculated using Reed and Muench method40.The modified vaccinia virus strain Ankara expressing T7 RNA polymerase (MVA-T7) was propagatd in monolayer primary chicken embryo fibroblast cells.Chicken embryo fibroblast (DF-1) cells and human epidermoid carcinoma (HEp-2) cells were obtained from the American Type Culture Collection . They were grown in Dulbecco’s minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS). The recombinant avirulent NDV strain LaSota was generated previously in our laboratory using reverse genetics39. In order to develop an effective IBV vaccine the maximum neutralizing epitopes with correct conformation are needed to be displayed. Most neutralizing epitopes are located in the S protein. In this study seven transcription cassettes containing S, S1 or S2 genes of IBV were constructed to identify the best protective antigen for the development of NDV vectored IBV vaccines. The S, S1 and S2 genes were chicken codon optimized for higher level of expression in chickens. The following transcription cassettes were designed: (i) a transcription cassette containing the S gene of IBV strain Mass-41 (3489 nt) was designed to determine whether the expression of the whole S gene from NDV will lead to display the maximum neutralizing epitopes in correct conformation, (ii) a transcription cassette containing the S2 subunit of S gene (1878 nt) of IBV fused with C-terminus of signal peptide sequence of S gene (69 nt) was constructed for transport of the protein from the cell (iii) a transcription cassette containing the S1 subunit of S gene (1614 nt) was designed to determine the protective efficacy of S1 protein, (iv) a transcription cassette containing the S1 gene (1611 nt) fused with N-terminus of transmembrane and cytoplasmic tail of S gene (255 nt) was designed for incorporation into NDV envelop, (v) a transcription cassette containing the S1 subunit of S gene without S1 protein cleavage site residues (1593 nt) fused with N-terminus of transmembrane and cytoplasmic tail of NDV F gene (171nt) was designed for incorporation of the S1 protein into envelope of NDV. (vi) a transcription cassette containing the S1 subunit of S gene containing S1 protein cleavage site residues (1611 nt) fused with N-terminus of transmembrane and cytoplasmic tail of NDV F gene (171 nt) was designed to incorporate the S1 protein into NDV envelope and also to know whether adding the cleavage site residues has any effect on the fusion of two proteins, and (vii) a transcription cassette containing the non-codon optimized S gene (3489 nt) was constructed to compare the level of protein expression between the codon optimized and non-codon optimized S genes.A plasmid containing full-length antigenomic cDNA of NDV strain LaSota has been constructed previously26. Any of the gene junctions is a potential insertion site for the transcription cassette of a foreign gene. However, we and others have found that the intergenic region between the P and M genes is a good site for expression of most foreign genes37. The transcription cassettes containing IBV genes contained PmeI restriction enzyme sequence, 15 nt of untranslated region (UTR) of NDV, NDV GE signal, one T nucleotide as intergenic sequence, NDV GS signal, extra nucleotides to maintain the rule of six41, Kozak sequence at the upstream of foreign gene ORFs and PmeI restriction enzyme sequence at downstream of foreign gene ORF. The transcription cassettes of codon optimized and non-codon optimized S gene were digested from two commercially synthesized  plasmids containing codon optimized  and non-codon optimized S gene of IBV strain Mass-41(GenBank Accession no. AY851295. 1), respectively. The transcription cassettes of codon optimized S1 and S2 genes were amplified from the commercially synthesized plasmid containing codon optimized S gene of IBV strain Mass-41 and cloned into individual shuttle vectors . Then the flanking DNA of transcription cassettes were digested from shuttle vectors. The transcription cassettes derived from shuttle vectors were cloned into complete individual plasmids containing cDNA of rLaSota at P and M gene junction using PmeI site  + NDV-F-TM&CT, rNDV/S1(cs−) + NDV-F-TM&CT, rNDV/S2, rNDV/codon optimized-S and rNDV/non-codon optimized-S, respectively. The IBV genes were amplified from the rNDV constructs by RT-PCR.NDV genome contains six genes: nucleocapsid(N), phosphoprotein(P), matrix(M), fusion(F), hemagglutinin-neuraminidase(HN) and large(L). The genes are ordered 3′-N-P-M-F-HN-L-5′. The beginning and the end of each gene contain conserved transcriptional sequences known as the gene-start (GS) and gene-end (GE), respectively. Between the genes, there are gene junctionsConfluent monolayers of DF-1 cells were infected at a multiplicity of infection (MOI) of 0.01–0.1 with rNDV strain LaSota, rNDV/S1, rNDV/S1 + IBV-S-TM&CT, rNDV/S1(cs+) + NDV-F-TM&CT, rNDV/S1(cs−) + NDV-F-TM&CT, rNDV/S2, rNDV/codon optimized-S or rNDV/non-codon optimized-S. DF-1 cells were harvested 30 hours post-infection, lysed and analyzed by Western blot. A polyclonal chicken anti-IBV strain Mass-41 was used to detect the expression of S1, S2 and S proteins of IBV. To determine the incorporation of IBV proteins into NDV envelope, rNDV, rNDV/S2, rNDV/codon optimized-S and rNDV/non-codon optimized-S were inoculated into 9-day-old embryonated SPF chicken eggs. Three days after incubation, recombinant viral particles from infected allantoic fluids were partially purified by sucrose density gradient centrifugation and analyzed by Western blot analysis. A monoclonal anti-NDV/HN antibody also was used to detect HN protein of NDV in lysates and purified virions by one more Western blot analysis.2. Aliquots of 200 µL of supernatant from infected cells were collected and replaced with fresh DMEM including FBS at intervals of 8 hours until 64 hours post-infection. The titer of virus in the harvested samples was determined by TCID50 method in DF-1 cells in 96-well tissue culture plates.In order to determine the growth kinetics of rNDVs expressing S1, S2 or S protein of IBV, confluent monolayers of DF-1 cells in 6-well tissue culture plates were infected at a MOI of 0.1 with rNDV, rNDV/S1(cs+) + NDV-F-TM&CT, rNDV/S2 and rNDV/codon optimized-S and adsorbed for 90 minutes at 37 °C. After adsorption, cells were washed with PBS, then incubated with DMEM containing 2% FBS and 10% fresh SPF chicken egg allantoic fluid at 37 °C in presence of 5% COBased on the level of expression of S1, S2 and S proteins of IBV from rNDVs, rNDV/S1(cs+) + NDV-F-TM&CT, rNDV/S2, and rNDV/codon optimized-S viruses were selected for animal study to evaluate their protective efficacy against virulent IBV challenge.7 EID50 of rNDV, rNDV/S1(cs+) + NDV-F-TM&CT, rNDV/S2 and rNDV/codon optimized-S strains via oculonasal route. The fifteen chicks of group five and five chicks of group six were inoculated with PBS. Three weeks after immunization, all immunized chickens, were challenged with 103.1 EID50 of virulent IBV strain Mass-41. This challenge virus dose was determined by an experimental chicken infection study. The severity scores of clinical signs of IBV including, nasal discharge, ocular discharge and difficulty in breathing  were recorded twice a day for 10 days post-challenge. In order to evaluate protective efficacy of rNDVs expressing S1, S2 and S genes of IBV in preventing shedding of virulent IBV in immunized chickens, at day five post-challenge, tracheal swab samples were collected from fifteen birds of each group and placed in 1.5 mL serum free DMEM with 10 X antibiotics. The swab samples were analyzed for quantification of viral RNA using an IBV-N gene-specific RT-qPCR.In this study, the protective efficacy of rNDVs expressing S1, S2 or S protein of IBV strain Mass-41 were evaluated in 1-day-old SPF chicks. Briefly, a total of eighty 1-day-old chicks were divided into five groups of fifteen each and one group of five. Chicks of the first four groups were inoculated with 101. A total of twenty 4-week-old SPF chickens were divided into four groups of five each. Five chickens of groups one and two were inoculated with 107 EID50 of rNDV and rNDV/codon optimized-S, respectively, via oculanasal route. Five chickens of group three were inoculated with 10 recommended doses of a commercial live attenuated Mass-type IBV vaccine via oculanasal route and chickens of group four were inoculated with PBS. Three weeks after immunization, chickens of all groups were challenged with 103.1 EID50 of virulent IBV strain Mass-41 by the oculonasal route. The severity scores of clinical signs of IBV, described in IBV protection experiment 1, were recorded for 10 days post-challenge. In order to evaluate the efficacy of rNDV expressing S protein of IBV in preventing shedding of virulent IBV in immunized chickens, at day 5 post-challenge, tracheal swab samples were collected from twenty chickens and placed in 1.5 ml serum free DMEM with 10 X antibiotic. The swab samples were analyzed for quantification of viral RNA using an IBV-N gene-specific RT-qPCR.In this study, the protective efficacy of rNDV expressing codon optimized S protein of IBV was evaluated in 4-week-old SPF chickens against the OIE recommended dose of virulent IBV challenge7 EID50 of rNDV and rNDV/codon optimized-S, respectively, via oculanasal route. Eight chickens of group three were inoculated with 10 recommended doses of a commercial live attenuated Mass-type IBV vaccine via oculanasal route and chickens of group four were inoculated with PBS. Three weeks after immunization, chickens of all groups were challenged with 104.7 EID50 of virulent IBV strain Mass-41 by the oculonasal route. The severity scores of clinical signs of IBV, described in IBV protection experiment 1, were recorded for 8 days post-challenge. At day 4 post-challenge, three chickens from each group were euthanized for tracheal ciliostasis analysis (data not shown). In order to evaluate the efficacy of rNDV expressing S protein of IBV in preventing shedding of virulent IBV in immunized chickens, tracheal swab samples were collected from five chickens from each group and placed in 1.5 mL serum free DMEM with 10 X antibiotic. Each fluid was tested for IBV specific lesions on chicken embryo by inoculation (0.1 ml) of one 10-day-old embryonated SPF chicken egg. The swab samples were also analyzed for quantification of viral RNA using an IBV-N gene-specific RT-qPCR. The swab samples collected from two non-vaccinated SPF chickens involved in another IBV protection study also were used as control.In this study, the protective efficacy of rNDV expressing codon optimized S protein of IBV was evaluated in 4-week-old SPF chickens against a higher dose of virulent IBV challenge. A total of thirty two 4-week-old SPF chickens were divided into four groups of eight each. Eight chickens of group one and two were inoculated with 1033. A total of forty five 1-day-old SPF chickens were divided into three groups of ten each and three groups of five each. Ten chickens of group one and two were inoculated with 107 EID50 of rNDV, rNDV/codon optimized-S, respectively, via oculanasal route. Ten chickens of group three were inoculated with one recommended dose of a commercial live attenuated Mass-type IBV vaccine via oculanasal route and chickens of groups four to six were left non-vaccinated. Three weeks after immunization, chickens of all groups one to four were challenged with 104 EID50 of virulent IBV strain Mass-41 by the intraocular route, chickens of group five were challenged with 104 EID50 of virulent IBV strain Mass-41 by the oculanasal route, and chickens of group six were left non-infected. The severity scores of clinical signs of IBV, described in IBV protection experiment 1, were recorded for 10 days post-challenge. In order to evaluate the efficacy of rNDV expressing S protein of IBV in preventing shedding of virulent IBV in immunized chickens, at day 5 post-challenge, tracheal swab samples were collected from all chickens of each group and placed in 3 mL serum free DMEM with 10 X antibiotic. Each fluid was tested for IBV specific lesions on chicken embryo by inoculation with 0.2 ml to each of five 10-day-old embryonated SPF chicken egg. The sample was considered positive for virus shedding, if any of the five embryos showed IBV lesions.In this study, the protective efficacy of rNDV expressing codon optimized S protein of IBV was evaluated in 1-day-old SPF chicks against virulent IB challenge virus infected by the intraocular route. Intraocular route was used, because this route of IBV challenge has been specified by the USDA-CFR-9We performed all experiments involving virulent IBV in our USDA approved Biosafety level-2 and Biosafety level-2 plus facilities following the guidelines and approval of the Animal Care and Use Committee (IACUC), University of Maryland.7 EID50 of rNDV and rNDV/IBV-codon optimized-S via oculonasal route. The five chickens of group three were inoculated with PBS. Three weeks after immunization, blood samples of all birds were collected for NDV antibody response analysis and challenged with one hundred 50% chicken lethal dose (CLD50) of the highly virulent NDV strain GB Texas via oculonasal route. The chickens were observed daily for 10 days after challenge for mortality with clinical signs of disease . We performed the experiment involving virulent NDV in our USDA approved Biosafety level-3 plus facility following the guidelines and approval of the Animal Care and Use Committee (IACUC), University of Maryland.The protective efficacy of rNDV expressing S protein of IBV strain Mass-41 was evaluated against a virulent NDV strain GB Texas challenge in our biosafety level 3 (BSL-3) plus facility. Briefly, a total of fifteen 1-day-old chicks were divided into three groups of five each. Chicks of two groups were inoculated with 1027. The virus neutralization assay according to OIE was used to measure the level of neutralizing antibodies mounted against IBV1. Briefly, serum samples of three birds from the group immunized with rNDV expressing codon optimized S protein of IBV and serum samples of three birds from the group immunized with commercial IBV vaccine group were incubated at 56 °C for 30 minutes. One hundred EID50 of IBV strain Mass-41 was mixed with 2 fold dilutions of antiserum and incubated for 1 hour at 37 °C. One hundred µL of each serum and virus mixture was inoculated into three 10-day-old embryonated SPF chicken eggs. To confirm that at least 100 EID50 of virus was inoculated into each egg, three eggs were inoculated with 100 µl of PBS containing 100 EID50 of IBV. Three eggs were inoculated with 100 µL of PBS as negative control. Three eggs were inoculated with a mixture of 100 EID50 of IBV and a dilution of 1:8 of a randomly selected serum sample collected from a bird immunized with rNDV strain LaSota as vector control. The eggs were incubated at 37 °C and were observed daily for dead chicken embryos for 7 days post inoculation. The serum titers were calculated according to the method of Reed and Muench40, based on mortality and IBV specific lesions on chicken embryos.The level of antibodies induced against NDV and IBV were evaluated. The serum samples were collected three weeks post-immunization. Hemagglutination inhibition (HI) assay using a standard protocol OIE was used to assess the level of antibody titer mounted against NDV in chickens immunized by rNDVs7.5 EID50/ml was used to establish the standard curve. The cDNA synthesized from extracted RNA of allantoic fluid stock of a virulent IBV strain Mass-41 and the cDNA synthesized from extracted RNA of swab sample solution were served as positive and negative controls, respectively. Melting point analysis was used to confirm the specificity of the test.RNA was extracted using TRIzol Reagent (Invitrogen) from tracheal swab samples collected from chickens. The first strand cDNA was synthesized using Thermo Scientific RevertAid Reverse Transcriptase (RT). SYBR green RT-qPCR was performed using a specific primer pair set: (a) N gene - 296 forward primer: 5′ GACCAGCCGCTAACCTGAAT 3′ and (b) N gene - 445 reverse primer: 5′ GTCCTCCGTCTGAAAACCGT 3′ amplifying 150 nt of N gene of IBV strain Mass-41. PCRs were performed using a Bio-Rad CFX96 Cycler. Each 20 µl reaction was carried out using 5 µl of cDNA, 10 µl of iTaq Universal SYBR Green Supermix (Bio-Rad), 2 µl of forward and reverse primers and 3 µl of nuclease free water. Forty cycles of PCR at 95 °C for 10 s (denaturation), 58 °C for 20 s , and 72 °C for 30 s (elongation) followed by melting curve analysis that consisted of 95 °C for 5 s and 65 °C for 60 s. A serial 10 fold dilution of cDNA synthesized from extracted RNA of allantoic fluid stock of a virulent IBV strain Mass-41 with 10t-test was used to compare two groups. To avoid bias, all animal experiments were designed as blinded studies.Data were analyzed among groups by One-Way-ANOVA test. The student Supplementary data