Sexual behaviour is the result of an interplay between distinct neural inhibitory and excitatory mechanisms. Individual differences in sexual excitation and sexual inhibition are proposed to play an important role in the processes sustaining the regulation of sexual behaviour. While much research has focused on the neural correlates of response inhibition, highlighting a prominent role of the inferior frontal gyrus (IFG), very little is known regarding the neural mechanisms underlying different aspects of sexual inhibition. Here, we experimentally combined functional magnetic resonance imaging (fMRI) and transcranial magnetic stimulation (TMS) to: (i) test the functional role of IFG during motivational and cognitive sexual inhibition; and (ii) reveal whether this IFG involvement in sexual inhibitory processes depends on sexual excitation and sexual inhibition as traits. Twenty-two participants performed an Approach-Avoidance (AA) and a Negative Affective Priming (NAP) paradigm to assess motivational and cognitive sexual inhibition respectively. Our fMRI study showed IFG being selectively activated during cognitive but not motivational sexual inhibition. Importantly, the level of this neural activity was modulated by individual sexual excitation scores. Interestingly, a transient disruption of IFG activity using TMS led to an improvement in cognitive, not motivational, sexual inhibition, but only when accounting for individual sexual excitation scores. These findings clearly document that sexual excitation modulates IFG activity levels during cognitive sexual inhibition, and at the same time determines the effects of TMS on IFG by improving cognitive control exclusively for individuals with high sexual excitation scores. These findings provide new insights regarding the functional role of IFG, and underscore the relevance of individual psychological differences in understanding the brain mechanisms underlying socioaffective processes. Sexuality is evolutionary relevant and individually rewarding and thus constitutes one of the leading motivating forces in human behaviour. Since sexual arousal can arise fairly automatically in response to sexual stimuli that are omnipresent in everyday life, adequate social behaviour requires the ability to inhibit sexual responses that potentially harm social norms and individual well-being.The ability to inhibit unfolding sexual responses comprises different psychological and physiological mechanisms that occur in parallel with those processes that elicit sexual arousal. According to the dual control model of sexual response, any form of sexual behaviour is the consequence of the interplay and balance between mechanisms that activate and facilitate the unfolding of the sexual response  and mechanisms that diminish or avoid this response . Sexual inhibition and sexual excitation cannot only be studied as a process but also as traits, as individuals differ in their propensity to become sexually aroused or inhibited  paradigm assessing the cognitive component of inhibitory control, and the Approach-Avoidance (AA) paradigm designed to involve a motivational-motor driven component of sexual inhibition. Previous research has shown that both paradigms were sensitive to predict the frequency of sexual thoughts and pornography watching respectively, and represent two independent inhibitory mechanisms . All participants received a written description of the experiment and the relevant practicalities about the use of MRI prior to accepting to participate. They gave written informed consent and were financially reimbursed for their participation. The study was approved by the local Ethical Committee of the Faculty of Psychology and Neuroscience at Maastricht University.Twenty-four healthy male participants (18–34 years old) with no neurological or psychiatric disorders took part in this study. One participant was excluded due to extensive head movements and a second participant due to technical difficulties leading to an incomplete data set  and one non-sexual (Go/No-go) inhibition paradigm inside the MRI scanner. The order in which the two sexual tasks were presented was counterbalanced. The Go/No-go task was taking place between the sexual tasks to prevent habituation to sexual stimuli. At the end of the session participants filled out the computerised self-reports .motivational sexual inhibition, we adapted the AA task using sexual and neutral stimuli  Sex Priming; (b) Sex No Priming; (c) Non-Sex Priming; and (d) Non-Sex No Priming. A trial-sequence consisted of a prime trial and a probe trial; during each, two pictures were presented simultaneously. The pictures were displayed one above the other, and one was surrounded by a black frame and the other by a grey frame. The instruction was to attend only to the black frame picture (target), therefore ignoring the grey frame one (distractor) and to indicate whether the target displayed sexual or non-sexual content through button pressing. During the priming trial-sequences, the content type of the distractor in the prime trial matched the content of the target picture in the probe trial. In the control trial-sequences (No Priming) the content type of the distractor in the prime trial was different from the target in the probe trial. The target of the probe trial could be sexual or non-sexual. Figure The four types of trial-sequences were presented randomly in equal proportions throughout each of three blocks. Each block contained 32 trial-sequences. The prime and probe trials were presented for 1700 ms each and were separated by a fixation cross displayed for the same duration. The probe and prime trials from different sequences were separated by the same fixation cross which lasted for 3400, 4250, or 5100 ms Figure . Sexual general inhibition. Participants were instructed to respond to a frequent Go stimulus and to not respond to an infrequent No-go stimulus. They responded with the right index finger on a button-box . All tasks described in this manuscript were programmed and presented with PsychoPy software Peirce, .This 45 items-scale measures the individual propensity for sexual inhibition and excitation. It contains one factor quantifying sexual excitation and two factors quantifying sexual inhibition: (a) SIS1—inhibition derived from threat of sexual performance failure, distraction or lack of physical stimulation (14 items); and (b) SIS2—inhibition due to the threat of performance consequences, such as risk of being caught, unwanted pregnancy, sexually transmitted diseases, feeling or causing pain, and partner’s too young age (11 items). Answers were registered on a four-point Likert scale . Online-scanner reconstruction was performed using the slice-GRAPPA algorithm . The echo images were combined using an optimised echo weighting method as described in Poser et al. .High-resolution anatomical images were acquired with an MPRAGE sequence . The images were motion-corrected  and corrected for slice timing skew using temporal sinc interpolation. A temporal high pass filter (three cycles) was applied. Images were co-registered to the individual T1 weighted images and normalised to Talairach stereotaxic space. Volume time courses were spatially smoothed using a 6 mm full width half maximum Gaussian kernel.We conducted a random-effects general linear model (GLM) analysis for every task. An event-related approach was considered for every task and each condition was entered as a regressor in the design matrix . For each sexual task we contrasted the sexual inhibitory condition  against their respective control inhibitory condition . Motion correction parameters were included as confound variables in the GLM. In separate models, we performed the same analyses including the SES and SIS scores as covariates. The resulting maps were corrected for multiple comparisons by means of cluster threshold level estimation .The motivational sexual inhibition condition (Sex Avoid > Dance Avoid) led to decreased activation in the middle frontal gyrus, middle temporal gyrus, inferior parietal lobule and cuneus .During cognitive sexual inhibition (Sex Priming > Non-Sex Priming), increased activation was observed in the right inferior and middle frontal gyri, the posterior cingulate, the inferior and middle temporal gyri and the fusiform gyrus  an increased activation was observed in the right superior frontal gyrus and bilaterally in the insula, extending to the IFG on the right side (CLTC p < 0.005). The latter cluster overlapped with the cluster engaged during the cognitive sexual inhibitory condition  scores did not modulate the IFG neural response during the same condition (CLTC p = 0.01). The SES, SIS1 and SIS2 scores did not hold significant correlation with the IFG neural response during the sexual no priming trials of the NAP task , nor during the sexual avoiding trials of the AA task . Unexpectedily, SIS1 scores—but not SES or SIS2—modulated the IFG  neural response during the non-sexual inhibitory trials : 1823; CLTC p = 0.001). This cluster extended to the insula, where it showed an overlap with the non-modulated neural activation during the No-go trials  within the IFG and in the middle frontal gyrus extending to the IFG  but not during the execution of the motivational sexual inhibition (AA) task. As expected, the IFG was also recruited during the non-sexual inhibitory Go/NoGo task. However, whereas the portion of the IFG recruited by cognitive sexual inhibition was located dorsally in the pars triangularis, the region engaged during the non-sexual inhibition was located ventrally in the opercular subdivision, in concordance with previous evidence  indeed significantly modulated the neural activity of the IFG during cognitive sexual inhibition but not during motivational sexual inhibition. This finding supports that individuals who are more prone to be sexually aroused have more inhibitory demands during cognitive sexual inhibition, resulting in an increased IFG activity during sexual inhibition which is modulated by sexual arousal proneness.Sexual inhibition traits did not predict the IFG neural response during sexual inhibitory processes. Whereas the sexual inhibition traits assess the proneness to inhibit the sexual response and sexual arousal due to the threat of sexual performance, our paradigms targeted sexual inhibition at a cognitive and at motor-motivational levels. These processes may not be related at all or they may relate in other components of socioaffective cognition networks.Although, we did not have specific predictions regarding the modulation of sexual individuals traits over the IFG neural response during general response inhibition, we observed that individuals higher in the first factor of sexual inhibition  recruited to a higher extent the IFG pars orbitalis during the No-go trials. This factor of sexual inhibition has been related to general anxiety and also proposed to be related to inhibitory tone  showed to be specifically activated during the execution of cognitive sexual inhibition and its activity correlated with sexual excitation scores. Although these results seem to support the idea of IFGpt being an inhibitory node during cognitive sexual inhibition, the findings do not provide conclusive evidence for its causal involvement. From our correlational data we still cannot exclude alternative explanations, such as that the IFGpt processes the saliency of incoming information, increasing activity in individuals who are more easily sexually aroused when presented with sexual primed stimuli. In order to investigate whether the engagement of the IFGpt was indeed of inhibitory nature, we performed a subsequent brain stimulation experiment.In our previous neuroimaging study, we could demonstrate that the right IFGpt is engaged during cognitive but not motivational sexual inhibition. Moreover, the activation during cognitive sexual inhibition correlated with sexual excitation as a trait.Although this region has been typically associated with inhibitory and self-regulation processes .To further increase the specificity of potential findings, we also targeted the precuneus, as this region was active during the sexual trials of both sexual inhibition tasks and did not show activation specificity for the inhibitory conditions. Thus, this region seems to play a role in sexual cognition that is not specific to the inhibitory component. Therefore, disrupting this area could modulate sexual inhibition by generally dysregulating sexual cognition in a non-specific way. To pursue our goals, we used a repeated-measures design in which participants performed the same tasks as described in Experiment 1 to target cognitive sexual and motivational sexual inhibition (NAP and AA) as well as a motor-response general inhibition task (Go/No-go) to additionally control for possible effects on a non-sexual inhibitory process.n = 22; mean age = 21.8, SD = 6.25). The study was approved by the same ethical committee as in Experiment 1.Twenty-five healthy and self-reported heterosexual males were recruited for this study. One participant did not attend the third session and two participants stopped during the first session as they experienced the stimulation as uncomfortable. All participants were informed about the risks and effects of the TMS and signed an informed consent. Neither of the participants had a history of neurological disorders and all of them were right-handed . During the TMS sessions, participants received continuous Theta burst stimulation , and misses  were recorded.Participants filled in the same questionnaires as in Experiment 1.n = 19) were recruited for the present TMS study and since TMS effects are highly dependent on spatial precision, we used cortex based-alignment (CBA) for individualised TMS coil positioning. Unlike Talairach coordinates, the CBA method takes the macroanatomical differences of participants into account by aligning the individual anatomical data with pre-existing functional data in a surface space. Therefore, this method preserves a functional-anatomical correspondence, resulting in a higher spatial stimulation specificity over the region of interest and consequently stronger TMS effects  from which a curvature map was extracted and transformed into a spherical space. The individual spherical space of each participant in this study was aligned to the group average spherical space from the fMRI sample proceeding from most prominent to most fine anatomical landmarks. After the alignment, the individual space was back-transformed to the individual brain anatomy and the target regions in the average surface map from fMRI were copied to the individual surface maps.Anatomical images from the participants were collected with the same methodology as in Experiment 1.x, y, z = 40, 28, 20). We also executed a conjunction analysis of the sexual conditions of both sexual inhibition tasks (Sex Approach ∧ Sex Avoid ∧ Sex Priming ∧ Sex No Priming); from the resulting map we extracted a patch of interest in the precuneus .As a target area, we selected the IFG cluster that was active during the cognitive sexual inhibition paradigm (NAP) in Experiment 1 and whose activity was shown to be modulated by the sexual excitation scores. The selection of the area was based on the surface map GLM for the Sex Priming > Non-Sex Priming contrast  and a figure-of-eight coil (MC-B70). The co-registration of the participant’s head with their MRI anatomical data was done with the Brain Voyager TMS Neuronavigator , which allowed the localisation and targeting of the regions of interest. The coil was manually held tangentially to the scalp and oriented at 45°, 90° and 45° to the central sulcus for the IFG, pre-supplementary motor area (sham), and precuneus respectively. cTBS was applied at 100% of the individual resting motor threshold . For the sham stimulation a placebo coil was used (MC-P-B70).F = 1.53, p = 0.22) or the NAP  = 2.47, p = 0.09) main indexes across the three sessions. Similarly, a Friedman’s two-way ANOVA by ranks showed no differences in the number of Misses and False Alarms in the Go/No-go task . Table A repeated measures analysis of variance (ANOVA) revealed no main stimulation effect on sexual inhibition as there were no significant differences on the AA  = 6.24, p = 0.004) was observed with participants showing a higher sexual priming effect after TMS over IFGpt as compared to the precuneus and the sham stimulation sessions  = 0.72, p = 0.49). After adjusting for multiple comparisons, the difference between the two experimental conditions (IFGpt and precuneus stimulation) remained significant (p = 0.05) while the difference between the sham and the IFGpt stimulation conditions did not (p = 0.16). Stimulation had different effects on participants depending on their sexual excitation scores  = 5.73, p = 0.01). Regression analyses showed that higher sexual excitation scores predicted a smaller sexual priming effect during the sham session  IFGpt disruption to induce changes specifically in cognitive sexual inhibition -not in motivational sexual inhibition or general motor-response inhibition; and (b) sexual excitation as a trait to modulate these behavioural effects induced by the IFGpt disruption.We observed that disrupting the IFGpt did modulate sexual inhibition in a specific way. Whereas brain stimulation affected cognitive sexual inhibition, it did not produce significant changes in motivational sexual inhibition or general motor-response inhibition. Importantly, this effect was significant only when taking into account sexual excitation proneness.The present findings are relevant in two crucial ways. First, the effect was specific to cognitive sexual inhibition and did not affect other forms of inhibition. In our previous study, the different sexual inhibitory mechanisms were not related with each other behaviourally, and they distinctively predicted the frequency of different aspects of sexuality  has previously been engaged in inhibitory paradigms  were consistent in showing the modulatory effects of sexual excitation over the IFGpt during sexual cognitive inhibition. If the IFGpt is sensitive to detecting salient information, we would expect that the underlying mechanism is more prominent in individuals who are more sensitive to sexual cues during a cognitive sexual inhibition paradigm. In line with this, disrupting this process would cause a lower sensitivity to affective salient information resulting in a better affective cognitive inhibition. This reasoning is in concordance with the current results. The TMS effects were dependent on sexual excitation with individuals scoring higher in this trait showing larger benefits in cognitive sexual inhibition as a consequence of the experimentally-induced IFGpt disruption. This is, whereas individuals that scored high in sexual excitation showed less sexually cognitive inhibition during the baseline condition, this relationship was reversed when IFGpt activity was disrupted with cTBS. It can be expected that individuals who are more easily sexually aroused have a lower threshold to detect sexual cues. This salience detection mechanism interferes with their inhibitory demands. However, when this mechanism is disrupted, they can more easily ignore sexual cues facilitating their inhibitory processing.In conclusion, this pair of studies shows that the right IFGpt is a relevant node during sexual inhibition . However, the current evidence did not support that this region sustains the inhibitory processing, as the temporal disruption of the IFGpt produced an increase in cognitive sexual inhibition. The present evidence supports the role of the right IFGpt as a reorienting system driven by salient stimuli detection. Importantly, our study highlights the role of individual differences in psychological processes and their underlying neural mechanisms. The IFGpt activity during cognitive sexual inhibition was modulated by sexual excitation and, in line with this, the effect of TMS on the participants’ performance was also dependent on the levels of sexual excitation. These findings showed that sexual excitation proneness influence basic sexual inhibition processing and the underlying neural mechanisms. This was specific to cognitive sexual inhibition and presumably due to a higher sensitivity to sexual cues. Finally, the modulation of sexual excitation trait over the rIFG during sexual inhibition provides empirical support to the dual control model of male sexual response which states that the interplay of excitatory and inhibitory mechanisms determine the occurrence of sexual responses and associated behaviours  or are contra-indicated because of the use of other pharmaceuticals. Although more research is needed, the rIFG activity could be a potential biomarker for detecting the suitability of TMS treatment. Alternatively, the modulation of cognitive sexual processes can be addressed by targeting different sub-processes such as reward processing (Prause et al., GR, AS, MD and TS made substantial contributions in the design of the study, the data interpretation and the final manuscript. GR performed the experiments and data analyses.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To compare the clinical effect of 3D-printed template technology with X-ray fluoroscopy in assisting surgery for sacroiliac screws placement.Institutional review board-approved retrospective analysis.The clinical data of 31 cases of sacroiliac complex injury between January 2015 and December 2016 were analyzed. There were 16 patients, males 11 and females 5, who underwent surgery assisted by 3D-printed template in template group, and that of contemporaneous 15 patients, males 11 and females 4, who underwent traditional surgery were gathered as fluoroscopy group. All those patients were followed up for more than 6 months.The operation time and X-ray fluoroscopy times for each screw placement, and the Matta and Majeed score were analyzed and the difference between the two group was tested.All cases were followed up for 6–20 months, average 11.4 ± 0.6 months. In template group, 19 screws were implanted. Each screw spent 25–38 min, average 27.2 ± 5.3 min, and need 2–5 times fluoroscopy, average 2.7 ± 0.5. The fracture reduction quality was evaluated by Matta score scale: excellent 10, well 4, fair 2, good rate 87.5%; and pelvic function were evaluated by Majeed score scale: excellent 11, well 3, fair 2, and good rate 87.5%. In fluoroscopy group, 17 screws were implanted. Each screw spent 45–70 min, average 60.3 ± 5.8 min, and needs 11–23 times fluoroscopy, average 15.4 ± 3.5. The fracture reduction quality was evaluated by Matta score scale: excellent 7, well 6, fair 2, and good rate 86.7%; and pelvic function was evaluated by Majeed score scale: excellent 6, well 6, fair 3, and good rate 80.0%. The difference in operation time, X-ray fluoroscopy times between template group and fluoroscopy group had statistical significance. But the Matta and Majeed score had no difference between two groups.Compared with traditional surgery, 3D-printed template technology-assisted surgery for sacroiliac screws placement in sacroiliac complex injury patients possesses advantage such as shortened operation time and reduced X-ray exposure times. This technology improves the safety profile of this operation and should be further studied in future clinical applications. Since the development of transportation and acceleration of urbanization, the sacroiliac joint complex injury caused by high-energy trauma is more and more common . The sacInclusion criterion: (1) fresh and closed pelvic fracture, (2) type Tile C1 and C2 pelvic fracture, (3) the fracture dislocation could be corrected by traction, (4) underwent sacroiliac screw fixation, (5) the clinical data were intact and follow-up time was no less than 6 months.Exclusion criterion: (1) old or open pelvic fracture, (2) severe lumbosacral trunk nerve injury, (3) non-screw internal fixation, (4) the clinical data were incomplete, or follow-up time was less than 6 months.There were altogether 31 pelvic fracture patients brought into this study . The pre-operation time sustained to 8–13 days. In template group, the CT data were saved as DICOM format in mobile hard disk, and imported to a computer for further disposition. With the help of software Mimics 14.0 , the 3D pelvic model was easy to be reconstructed, and the surgeon could simulate pelvic fracture reduction. On the basis of injury side ilium after reduction, the sacroiliac screw template was designed and printed, meanwhile the screw diameter and length were also confirmed. The process of template designation was as follows [ kg. The The DICOM data were imported to Mimics, to reconstruct 3D pelvic model. Draw two lines: one to pass posterior and anterior superior iliac spine, another along with the longitudinal axis of femur. Choose the crossing point as initial point, the central point of terminal plate of sacral vertebrae 1 as end point. The distance between the two points is the screw channel. Simulate nailing process, observe and measure the breadth in three-dimensional form to make sure of the length and diameter of screw. When screw channel is confirmed, the sleeve of template could be designed according to its axis, with a 2-mm inner diameter and a 10-mm outer diameter. On the other hand, the anatomical character of local iliac crest is collected, and based on which an anastrophic basic template is established. Then combine the screw sleeve and basic template to produce template rudiment. Get through the sleeve channel, add two 1.5-mm pilot holes on both sides of the sleeve, and the individualized sacroiliac screw template is ready.The surgical procedure was conducted by the same team who had rich experience in pelvic surgery. Prone position was recommended after general anesthesia. Pelvic anterior–posterior and lateral position, inlet and outlet view were observed by fluoroscopy, to evaluate the fracture position. When vertical displacement persisted, large dose of traction of the ipsilateral lower limb was performed to reduce the dislocation. In template group, in accordance with pre-operation plan, a 4 cm curved incision along the iliac crest was made to expose segmental posterior superior iliac spine and proximal iliac crest. Soft tissue on the outer table iliac was peeled off, the template was attached onto outer table iliac, which was fastened temporarily by two 1.5-mm Kirschner wires (K-wire). Along with the sleeve, place a 2.0-mm K-wire into the screw channel as guiding wire. Remove the template and insert 7.3-mm cannulated screw (Beijing LiBeiEr Bioengineering Institute Limited Company). In fluoroscopy group, in accordance with traditional experience, the guiding K-wire was placed through the mid-lower third intersection of connecting line of posterior and anterior superior iliac spine. Then by means of repetitious X-ray fluoroscopy, adjust the orientation and depth step by step. After confirmation under K-wire, measure the length and insert suitable cannulated screw. In both groups, inlet and outlet view were observed by fluoroscopy once more when the operation was complete.Routinely preventive antibiotic application should sustain 48 h post operation. Both X-ray and CT scanning and 3D reconstruction of pelvic anterior–posterior and lateral position were captured again on the day after operation. The exercise of lumbodorsal muscles and lower limb muscles strength was also carried out the first day after operation. The patients were told to come to the hospital and take pelvic X-ray at 1 month, 3 months, 6 months, and 12 months after operation, and the time for weight bearing depended on the fracture union status.Observe and record the time for inserting single screw, fluoroscopy times for each screw and follow-up time. Fracture reduction effectiveness was evaluated by Matta scale : excellet test for difference between the two groups. Enumeration data are showed as ratio, and compared by χ2 test for difference between the two groups. P < 0.05 was regarded as statistically difference.All data were assessed by software SPSS . Measurement data are shown as t = 0.493, P > 0.05). The screw number was 19 for template group and 17 for fluoroscopy group. The time for each screw insertion was (27.2 ± 5.3) min in template group, and (60.3 ± 5.8) min in fluoroscopy group, and there was statistical difference between two groups . The fluoroscopy times for each screw were (2.7 ± 0.5) in template group, and (15.4 ± 3.5) in fluoroscopy group, and there was statistical difference between two groups  . Assessed by Majeed scale, the excellent and good rate of pelvic function was 87.5% (14/16) in template group and 80.0% (12/15) in fluoroscopy group, and there was no statistical difference . Typical case as in Fig. Assessed by Matta scale, the excellent and good rate of fracture reduction was 87.5% 14/16) in template group and 86.7% (13/15) in fluoroscopy group, and there was no statistical difference  improving the accuracy and security in screw insertion 2) shortening the time for surgery procedure (3) decreasing the radiation exposure for both doctor and patient (4) enhancing the communication between doctor and patient. The sacroiliac joint complex is featured with complicated anatomy and surrounded by dense vessels and nerves  shorteni. So it iIn our retrospective study, each screw insertion in fluoroscopy group spent an average of 60 min, and needed an average of 15 times fluoroscopy, which was similar to other scholars . HoweverAlthough has its advantage in assisting surgery obviously, but 3D printing technology also has its limitation. Zhang et al.  pointed In our experience, the pelvic anterior–posterior and lateral position, inlet and outlet view should be observed by fluoroscopy before operation to assess the reduction effect of traction. When the operation begins, try to reduct the fracture by pry-poking or reverse-rotation via Schanz screw. Open reduction internal fixation is the alternative scheme in case of poor reduction. For another thing, the 3D-printed template needs to rely on the anatomic landmark of iliac bone, so it is important to peel off the attached soft tissue thoroughly on the iliac crest; and in this process, it may injure the local soft tissue and damage the bone blood supply for fully stripping, or cause a deviation and decrease the security of operation for deficiently stripping. In our study, two cases were excluded for the changed operation plan for poor reduction, and another two cases showed a transient worsening of the neurological symptoms, which may be due to the severe displacement and nerve drag injury after reduction, or the nerve irritability during the screw insertion. Yang et al.  also poiThere are still several shortcomings about the study as follows: it is a retrospective study; the sample size is not big enough; there is systematic error for analyzing the short-term and long-term complications. Furthermore, the application of 3D printing technology in clinical practice is still at its early stage, and how to improve the fracture reduction skill, and how to enhance the template accuracy both need to be further studied. At last, the comparison between 3D printing technology and 3D navigation technology also needs to be studied.In conclusion, 3D-printed template technology-assisted surgery for sacroiliac screws placement in sacroiliac complex injury patients possesses advantage such as shortened operation time and reduced X-ray exposure times. This technology improves the safety profile of this operation and should be further studied in future clinical applications.


Xenopus oocytes, an odorant receptor from the southern house mosquito, CquiOR32, generated inward (regular) currents when challenged with cyclohexanone and methyl salicylate, whereas eucalyptol and fenchone elicited inhibitory (upward) currents. Responses of CquiOR32-CquiOrco-expressing oocytes to odorants were reduced in a dose-dependent fashion by coapplication of inhibitors. This intrareceptor inhibition was also manifested in vivo in fruit flies expressing the mosquito receptor CquiOR32, as well in neurons on the antennae of the southern house mosquito. Likewise, an orthologue from the yellow fever mosquito, AaegOR71, showed intrareceptor inhibition in the Xenopus oocyte recording system and corresponding inhibition in antennal neurons. Inhibition was also manifested in mosquito behavior. Blood-seeking females were repelled by methyl salicylate, but repellence was significantly reduced when methyl salicylate was coapplied with eucalyptol.How chemical signals are integrated at the peripheral sensory system of insects is still an enigma. Here we show that when coexpressed with Orco in   •We found dual inhibitory/excitatory odorant receptors (ORs) in mosquitoes•Inhibitory and endogenous ORs coexpressed in flies showed lateral inhibition•The bipolar nature of these inhibitory ORs was displayed in electrophysiology•The duality excitation/inhibition was also manifested in mosquito behavior Biological Sciences; Neuroscience; Sensory Neuroscience Cx. quinquefasciatus displayed excitatory responses (increased spike frequency upon stimulus), but evidence for inhibitory responses (reduction in spontaneous activity upon stimulus), already known for Ae. aegypti  stimulation of an ORN, the  potential (of the sensillum lymph surrounding dendrites) declines. Consequently, per channel current generated by a cocompartmentalized neuron (when stimulated by its cognate ligand) is reduced  remains one of the least understood mechanisms of insect olfaction, particularly in mosquitoes. Despite the great progress made in the last 2 decades in understanding how receptors form the basis of chemosensory perception in insects, how olfactory (as well as taste) signals integrate at the periphery remains an enigma . “It is  aegypti , is now asciatus . It has asciatus , beetlesasciatus , the fruasciatus , and mosasciatus  that actasciatus . Althougasciatus , the com reduced . This scCx. quinquefasciatus, we challenged Xenopus oocytes coexpressing CquiOR32 along with the obligatory coreceptor Orco with a panel of more than 200 compounds, including mostly physiologically and behaviorally relevant compounds  as a source of bulky, impermeable mono cation (+ by less permeable NMG (+ with NMG had a minor effect on the I-V curves (− with gluconate seems to suggest that Cl− is implicated in this inhibition (To gain better insights into possible mechanism(s) of these currents in reverse direction, we obtained current-voltage (I-V) curves from oocytes expressing wild-type (WT) CquiOR32-CquiOrco with voltage clamped at −80, −60, −40, −20, 0, +20, and +40 mV and using methyl salicylate and eucalyptol as stimuli  and then eucalyptol (1 mM) and recorded regular and reverse currents, respectively . Then wenogaster .in vivo at the periphery of the olfactory system, we generated transgenic flies, with CquiOR32 expression driven by DmelOrco promoter, and recorded electroantennogram (EAG) responses by using a standard method (1118) gave strong response to (E)-2-hexenal and weak response to methyl salicylate (E)-2-hexenal unchanged -2-hexenal when it was delivered alone or in combination with eucalyptol. EAG responses from w1118 flies to 0.1% (E)-2-hexenal alone or in combination with 10% eucalyptol did not differ significantly (E)-2-hexenal plus 10% eucalyptol were significantly lower than those elicited by 0.1% (E)-2-hexenal alone (E)-2-hexenal were reduced when coapplied with eucalyptol  (E)-2-hexenal does not activate CquiOR32 (E)-2-hexenal. In these continuous experiments, a small difference between EAG responses before and after costimulus tests may be due to loss of this volatile semiochemical from the cartridge rather than adaptation. Similar inhibition was observed when 2-heptanone was applied alone or coapplied with eucalyptol -2-hexenal suggests that intraneuronal inhibition occurred. A few lines of evidence support this hypothesis. First and foremost, eucalyptol does not cause inhibition in control flies (E)-2-hexenal does not activate CquiOR32 (E)-2-hexenal. In short, inhibitor and agonist are likely to be acting on different receptors in the same neurons, thus an intraneuron inhibition. To further test the notion of intraneuronal inhibition, we turned to single sensillum recordings (SSRs).Next, we recorded EAG responses when flies were challenged with odorants and an inhibitor. First, we compared the response of wficantly A. By conal alone B. We theal alone C but remand 10%) D. Of notcalyptol . Taken tol flies A and (E)CquiOR32 . The simE)-2-hexenal , mosquitoes (and possibly other insect species) can coexpress multiple ORs in the same neuron  showed that this neuron responded to cyclohexanone with dose-dependent excitatory responses A and to g system . Additiog system D and feng system E in a doXenopus oocyte recording system  and challenged the oocytes with compounds that elicited inward and inhibitory currents in CquiOR32. Cyclohexanone elicited inward currents, but the compounds generating the largest inward currents were 4,5-dimethylthiazole (DMT) and 2-methyl-2-thiazoline (2MT) , with 55.5% identity. We sequenced 20 clones and obtained 19 AaegOR71 sequences. Five clones showed sequences identical to the sequence in VectorBase and were, therefore, considered the WT. We expressed AaegOR71-WT in the ne (2MT) ; no respne (2MT) .Xenopus oocyte recording system. The other 7 clones differed from the WT in 6–12 amino acid residues. AaegOR71-V5 (MG593071), AaegOR71-V14 (MG593074), and AaegOR71-V15 (MG593075) differed in 12, 9, and 11 amino acid residues, respectively, and none of them responded to odorants. AaegOR71-V8 (MG593072) differed in 10 amino acid residues and showed very weak response only to the Orco agonist VUAA-1. AaegOR71-V4 (MG593070), AaegOR71-V9 (MG593073), AaegOR71-V17 (MG593076) differed in 8, 10, and 6 amino acid residues but gave weak to moderate responses to odorants. None of these variants elicited detectable inhibitory currents when challenged with eucalyptol or fenchone.Four clones  differed from WT in 7 amino acid residues, and 3 clones  differed from WT in 11 amino acid residues. They both showed weak responses to odorants when tested in the in vivo in the antennae of the yellow fever mosquito. With 350 contacts, 69 recordings were made from SST-2 sensilla. SSR showed that cyclohexanone, 2-methyl-2-thiazoline, and 2,4-dimethylthiazole elicited dose-dependent excitatory responses in neuron-A in SST-2, whereas eucalyptol and fenchone showed inhibitory responses . With a higher dose (1%), 44.3 ± 5.7% and 55.7 ± 5.7% mosquitoes responded to treatment and control, respectively , thus showing that eucalyptol per se is not a potent spatial repellent. Likewise, cyclohexanone did not show repellence activity at 0.1%  or 1% .Evidence in the literature suggests that eucalyptol is an oviposition deterrent . We thenContrary to eucalyptol, methyl salicylate showed repellence activity. At a dose of 0.1%, the number of mosquitoes responding to the treatment side of the arena was significantly lower than the number responding to control . At a higher dose (1%), the difference was highly significant .Because eucalyptol per se is not a repellent, we then tested the effect of eucalyptol on repellence elicited by methyl salicylate. A mixture of 1% methyl salicylate and 1% eucalyptol had significantly lower protection than methyl salicylate alone . We repein vitro and in vivo evidence suggesting that negative allosteric modulation may occur. It is known that insect ORx-Orco complexes form agonist-activated cation channels  depolarizes the membrane further (− currents are involved.We provided evidence showing that the same receptor (CquiOR32) is activated by some ligands and inhibited by others (intrareceptor inhibition). When this “inhibitory receptor” was coexpressed with another OR we found evidence of intraneuron inhibition. Are these inhibitory compounds merely inverse agonists? If receptors are constitutively open as previously suggested for a case in the vinegar fly , bindingchannels . Upon bi− influx : sensill balance . In the  further , thus pr further . In summOur dataset provided strong evidence for intrareceptor and intraneuron inhibition, but to unambiguously elucidate the mode of action of these inhibitory responses that lead to (outward) reverse currents in oocytes and “inverse” EAG responses we must wait for the elucidation of the structures of CquiOR-CquiOrco or other OR-Orco complexes with similar features.All methods can be found in the accompanying


B. canis-caused babesiosis with no organ dysfunctions by employing gel-free LC-MS/MS analysis of individual samples and tandem mass tag (TMT) label-based quantitative analyses of pools, both supported by phosphopeptide enrichment. Results showed a moderate number of phosphorylated proteins (50–55), with 89 phosphorylation sites not previously published for dogs although a number of them matched phosphorylation sites found in mammalian orthologs. Three phosphopeptides showed significant variation in babesiosis-affected dog sera compared to controls: Serum amyloid A (SAA) phosphorylated at serine 101 (up-regulation), kininogen 1 phosphorylated at threonine 326, and fibrinogen α phosphorylated at both threonine 20 and serine 22 (down-regulation). 71.9% of the detected phosphorylated sites were phosphoserine, 16.8% phosphothreonine and only 11.2% phosphotyrosine residues. TMT label-based quantitative analysis showed α-2-HS-glycoprotein / Fetuin A to be the most abundant phosphoprotein  followed by kininogen-1 (10–20%). The alterations of phosphorylated proteins observed in canine babesiosis caused by Babesia canis suggest new insights into the largely neglected role of extracellular protein phosphorylation in health and disease, encouraging urgent further research on this area. To the best of our knowledge the present study represents the first attempt to characterize canine serum phosphoproteome.Phosphorylation is the most commonly studied protein post-translational modification (PTM) in biological systems due to its importance in controlling cell division, survival, growth, etc. Despite the thorough research in phosphoproteomics of cells and tissues there is little information on circulating phosphoproteins. We compared serum from 10 healthy dogs and 10 dogs affected by  Protein phosphorylation is the most widespread known post-translational modification involved in almost all cellular events, controlling the most diverse cellular pathways . At leasThe increase in sensitivity of high-throughput proteomic techniques has allowed the study of protein phosphorylation in an extensive way and favored the opening of new horizons in biomedical research. Limited biomarker discovery studies for differential protein levels has resulted in a disappointingly low rate of application of biomarkers in clinical practice . A revisQualitative differences can be inferred from the presence or absence of detected sites calculated from the relative number of times a site is identified in different samples  but this method is considered rather semi-quantitative due to lack of accuracy . Unlike . Furthermore, the phosphorylation state of abundant phosphorylated serum proteins has been used as a marker of aging [Along with MS/MS, the optimization of phosphoprotein/phosphopeptide enrichment procedures has greatly contributed to the expansion of phosphoproteomics . Recent of aging , schizopof aging , etc.Despite emerging interest, there is little knowledge about serum/plasma phosphoproteins as markers for infectious diseases. Some phospho-proteins/-peptides are components of the pathogens in the host and can be converted into circulating or membrane associated antigens . On the Babesia canis, present in North America, Southern Europe, parts of Africa and Asia. Several markers have been identified to be useful for prognosis and for monitoring after antibabesial therapy [32).[Canine babesiosis is a significant tick-borne disease caused by several intraerythrocytic protozoan species of the genus Babesia. This pathology is most frequently caused by apy [32)., 34. Cytapy [32)., 36.Babesia Rossi- infected erythrocytes that is suspected to constitute a major virulence factor [Babesia canis-caused infection could potentially contribute with the parasite’s phosphoproteins to the canine serum proteome, acting as biomarkers of the infection. Some other phosphoproteins such as Trypanosoma cruzi’s calcineurin B, a phosphatase, are involved in host cell invasion [Leishmania major [Interestingly, a polymorphic membrane phosphoprotein (BrEMA1) has been described on the plasma membrane of e factor . Babesiainvasion  and ectoia major . Moreoveia major , 6, makiia major .The present study shows an approach to characterize qualitatively individual serum samples and quantitatively pools containing the same 10 control samples and 10 samples from dogs suffering from babesiosis before the onset of organic dysfunction . In ordeBabesia canis (B. canis), admitted to the Clinic for Internal Diseases, Faculty of Veterinary Medicine, University of Zagreb, Croatia, with clinical signs of acute babesiosis. Dogs in this group were of various breeds, between 1 and 14 years of age and 6 of them were males. The clinical manifestations were variable and included anorexia, lethargy and fever, ticks found by the owner or veterinarian, pale mucous membranes, anaemia, jaundice, hemoglobinuria or hematuria, splenomegaly, tachycardia and vomiting. The main complication was the development of an excessive inflammatory response called “systemic inflammatory response syndrome” or SIRS61. The diagnosis was confirmed by demonstration of the parasites within infected erythrocytes in Romanowsky-stained thin blood smears. One dose (6 mg/kg of body weight) of imidocarb dipropionate  was administered to all dogs subcutaneously on the day of admission. Additional treatment consisted of various fluids , and whole blood transfusion when this was indicated. Subspecies were confirmed using PCR (polymerase chain reaction) [The study was performed on two groups of animals. Group 1 consisted of 10 dogs naturally infected by eaction) .9/L or more than 12 x 109/L or more than 10 percent band neutrophils. None of the dogs selected for this study presented organ dysfunction at the time of blood extraction.On the basis of clinical manifestations and laboratory data, the affected dogs were classified as SIRS positive. The SIRS criteria used in the study were established on the basis of the criteria proposed by Purvis and Kirby (1994)  and WelzGroup 2  consisted of 10 dogs of different breeds and sexes, aged from 1 to 10 years. These dogs were considered healthy based on physical examination as well as hematological and biochemical data, and they attended the hospital to receive a prophylactic dose of imidocarb dipropionate (6 mg/kg) as a preventive measure against babesiosis upon their owners’ request. The protocol was approved by the Ethics Committee for Animal Experimentation, Faculty of Veterinary Medicine, University of Zagreb, Croatia (Permit No: 251–61–01/139–12–2). All dogs were admitted to the Clinic for Internal Diseases, Faculty of Veterinary Medicine, University of Zagreb, Croatia and written owner consent was obtained for all dogs included in this study as part of a routine clinical protocol. Owners were informed about the experimental use of the specimens.D. immitis antigen and antibodies, both immunoglobulin G and M, to E. canis, B. burgdorferi sensu lato and A. phagocytophilum with the SNAP 4Dx test . The same samples were further qualitatively tested for antibodies to L. infantum with SNAP Leishmania (IDEXX Laboratories). Both tests were performed according to the manufacturer’s instructions. All samples were negative for the aforementioned vector-borne pathogens.All serum samples from dogs were screened for simultaneous qualitative detection of circulating Blood samples for analysis from groups 1 and 2 were collected from the cephalic vein on the day of admission, and before the administration of imidocarb dipropionate (Imizol). The samples were placed in tubes with ethylenediaminetetraacetic acid (EDTA) for haematological analysis and for establishing the diagnosis. Blood smears and PCR were performed from samples taken at admission. The samples for biochemical analysis were collected in tubes with no anticoagulant and were centrifuged at 1200 × g. A portion of the obtained serum was used for establishing biochemical profiles while the remainder was stored at –80°C until used for proteomic analysis. Complete blood count was analyzed using an automatic haematology analyzer, Horiba ABX . Biochemical profiles were generated according to standard methods using an automated biochemistry analyzer .We performed filter aided sample preparation (FASP) using 10kDa cut-off filters. 10 control serum samples and 10 serum samples from dogs with babesiosis (7 μl per sample) were incubated for 30 min in 20% acetonitrile / 80% 50mM ammonium bicarbonate (18μl) at room temperature to allow the release of peptides bound to albumin in the presence of phosphatase inhibitor cocktail set V 50x  diluted 50-fold. 5μl of 200mM DTT was added to each sample and reduction was allowed to proceed for 30 min at room temperature. After reduction, 20μl of 200mM IAA was added and samples were incubated for 30 min at room temperature.After alkylation, 20μl of 200 mM DTT was added to reduce any excess IAA and samples were incubated for 30 min at RT. Digestion was performed by adding 1μg of Trypsin Gold to each sample . Digestion was allowed to proceed overnight at 37°C and peptides were dissolved in 80% acetonitrile and 2% formic acid. Peptides were kept at -20°C prior to phosphopeptide analysis.For identification, prior to LC-MS/MS analysis, enriched tryptic peptides were desalted using ZipTip C18  according to the manufacturer’s procedure and vacuum-dried.Babesia-infected dogs were pooled and both pools were mixed with protease inhibitors. The protein concentration of healthy and Babesia-infected pools was determined by BCA assay. Peptides were labelled using two different tags from a TMTsixplex isobaric label reagent set (m/z 128 and m/z 129 isobaric tags) according to the manufacturer’s procedure  with some modifications. In short, 35 μg of total protein was diluted to a final volume of 50 μl using 0.1 M triethyl ammonium bicarbonate . A volume of 2.5μl of 200 mM DTT was added to each sample followed by incubation for 1 h at 55°C. Alkylation was performed by adding 2.5 μl of 375 mM IAA and incubating for 30 min at room temperature in the dark. Proteins were acetone-precipitated (overnight at -20°C) and centrifuged at 8000 xg for 10 min at 4°C. Protein pellets were reconstituted in 50 μl of 0.1 M TEAB and trypsin digested  at 37°C overnight. TMT reagents were dissolved according to manufacturer recommendations. For the labelling reaction, 18 μl of freshly prepared TMT reagent was added to each sample and incubated for 1h at room temperature. The reaction was quenched by adding 8 μl of 5% (w/v) hydroxylamine HCl solution (incubation for 15 min). Finally, equal amounts of differentially TMT-labelled peptides were combined into the final sample. 10 μg of the final sample was vacuum-dried and stored at -80°C for LC-MS/MS analysis. The remaining sample was used for phosphopeptide enrichment.Sera from 10 healthy dogs were pooled. Separately, sera from 10 TM phosphopeptide enrichment containing TiO2 magnetic microbeads  was used to enrich phosphopeptides. Briefly, 30 μl of bead mixture for every sample was equilibrated with binding buffer by washing the microbeads three times on a magnetic support. Serum peptides obtained after digestion were added to the microbeads and resuspended by repeated pipetting. After a short incubation (2 min) at room temperature, non-bound peptides were washed off from the microbeads by adding binding buffer and washing on a magnetic support (3 times). After binding, washing buffer was added quickly and then removed completely. Elution buffer (30 μl) was added, the sample resuspended repeatedly and allowed to stand for 10 min at room temperature after which eluted phosphopeptides were collected, quantified using nanodrop and kept at -20°C for further LC-MS/MS analysis.PiercePeptides were reconstituted in 0.1% (v/v) FA and 1μg was separated on an Ultimate 3500 RSLS nanoflow system (Dionex) before online ESI-MS/MS analysis with a Q Exactive Plus mass spectrometer (Thermo Scientific). For phosphoprotein ID, direct injection onto a PepMap RSLC C18 analytical column (15 cm x 75 μm) with a linear gradient 5–35% mobile phase B (0.1% FA in 80% ACN) over 180 min at a flow rate of 300 nL /min was used to separate peptides. For TMT-based relative quantification, peptides were loaded onto a cartridge trapping column  using a 15 μl/min flow rate and desalted for 12 minutes by mobile phase C (1% ACN in 0.1% FA) before separation on a PepMap RSLC C18 (50 cm x 75 μm) analytical column.m/z 350 to m/z 1900 with a resolution of 70000. The maximum injection time for the FTMS full scan was set as 110 ms reaching an AGC target value of 3x106. The eight most intense precursor ions were chosen for HCD fragmentation with a resolution of 17500 using an AGC target of 2x105. The step collision energy was set to 28% and 35% NCE, respectively. For relative quantification, as a third step 40% NCE was used. A ± 1.6 Da isolation window was applied to isolate precursor ions with a dynamic exclusion of 30 s. Minimal PSM number to confirm the presence of a phosphopeptide was set at 2. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD010894.The MS instrument was operated using a Nanospray Flex ion source with a SilicaTipemitter (New Objective). The ionisation voltage was set to 1.9 kV and the ion transfer capillary temperature to 275°C. MS was operated in positive ion mode using FT HCD MS2. Full scan FTMS spectra were acquired in the range from Canis lupus familiaris fasta files (downloaded from NCBInr database 2016/10/13) was employed for data analysis. Static peptide modification included carbamidomethylation (C), dynamic oxidation (M) and phosphorylation . One trypsin missed cleavage was allowed. Precursor tolerance and ion fragment tolerance were set at 10 ppm and 0.05 Da, respectively. Confidence levels were set to 1% FDR (high confidence) and 5% FDR (middle confidence). On average 90% of the identified phosphopeptides showed high confidence (FDR 1% or lower).Proteome Discoverer (version 2.0.0.802) software performing SEQUEST search of RS site probabilities above 75% indicate that a site is truly modified. In multiply phosphorylated peptides ptmRS site probabilities often drop below 50% although the same phosphorylation site was observed in the same monophosphorylated peptide at higher ptmRS probabilities. In the present study only phosphorylation sites reaching 95% or higher ptmRS probabilities were considered.Proteome Discoverer node ptmRS was used for analysis and mapping of peptide/protein phosphorylation sites. ptmRS Best Site Probabilities displays the most likely positions of the modifications and their site score for each PSM. For each modification site, this value is an estimate of the probability (0–100%) that the site is truly modified. Any ptmModification Site Probabilities: Displays the modification site probabilities for all possible modification positions for a single modification. In the column title, Modification is replaced by the name of the actual modification, for example, Phosphorylation Site Probabilities [https://www.phosphosite.org/homeAction) [http://www.cbs.dtu.dk/services/NetPhos/)[https://weblogo.berkeley.edu/logo.cgi [For peptide groups, this column shows the best site probabilities of the first PSM. ptmRS bilities . PhosphoeAction) . AssignmNetPhos/). Weblogologo.cgi .2 magnetic microbeads. Canine serum phosphoproteins were analyzed qualitatively and quantitatively after phosphopeptide enrichment by means of metal oxide affinity using TiO2 after several binding steps followed by extensive washing and a final elution step. After optimizing sample preparation and LC-MS/MS protocols as well as data processing, we observed qualitative changes in phosphoprotein content of individual serum samples. Additionally, we performed a preliminary quantitative analysis using TMT- labeling, along with LC-MS/MS, of a serum pool composed of 10 control samples and a babesiosis serum pool composed of 10 serum samples from Babesia-infected dogs.Phosphopeptides were enriched according to their affinity for TiOAfter LC-MS/MS analysis of the enriched phosphopeptides, we found approximately 50 proteins with detected phosphorylation sites and a variable number of non-phosphorylated proteins . The numQualitative results showed approximately 89 phosphorylation sites in about 50 proteins: 64 phosphoserines (71.9%), 15 phosphothreonines (16.8%) and 10 phosphotyrosines (11.2%) . The preThere was an important difference in the presence of serum amyloid A (SAA), an acute phase protein, in samples from babesiotic dogs in comparison to controls. A considerable number of phosphosites was detected for SAA in diseased samples with serine (S) 101 phosphorylated in all controls but in none of the sera from babesiotic dogs. Fibrinogen β was found to be phosphorylated at tyrosine (Y) 32 only in 2 samples from infected animals and in none of the controls. Kininogen-1, an important protein in coagulation, was phosphorylated at threonine (T) 326 and serine (S) 332 in all samples. Another protein with a relatively high number of phosphorylation sites was α-2-antiplasmin, which was phosphorylated at S514 in 10 samples from diseased dogs vs. 5 control samples.On the other hand, a higher number of phosphorylated proteins was found in control samples in case of ApoA1 at S220 (5 controls vs. none in babesiosis samples), Chromogranin A at S190 (4 controls vs. 1 babesiosis) and Fibrinogen α at S22 (9 controls vs. 6 babesiosis). Finally, multiple phosphofibronectin isoforms were detected in this analysis in all of the samples .Out of all phosphopeptides detected in individual samples, 3 showed significantly different spectral intensities in babesiosis-affected dogs compared to controls. Serum amyloid A phosphorylated at S101 was the most significant increase (p = 0.00018) in phosphopeptide levels according to Mann-Whitney test , since iKininogen1 phosphorylated at T92 (p = 0.0257) and fibrinogen α phosphorylated at T20 and S22  showed significant decreases in babesiosis samples. Sequence alignment of canine SAA and α2-HS-glycoprotein with human orthologs revealed some conserved phosphorylation sites according to PhosphoSitePlus database search .Phosphorylation sites detected in the present analysis were compared with the phosphosite plus database data for human, mouse, rat and cow and conserved sites are listed in TMT-labeling-based quantitative analysis of the control and babesiosis pools revealed often, most notably in the case of SAA, that the global level of a protein (phosphorylated and non-phosphorylated) was several fold higher in babesiosis pool (pool B) . SulfhydAfter summarizing quantitative data for the top TMT-labelled phosphoproteins detected, our data show that alpha-2-HS-glycoprotein / fetuin-A is the most abundant phosphoprotein present in dog serum both in enriched and non-enriched pools  with aroSecond in abundance was kininogen-1 with 19.9% of total intensity of TMT-labelled phosphopeptides in the control pool vs. 16.5% in the babesiosis pool. Other remarkable changes observed were serum amyloid A (0.4% in the control pool vs. 1.1% in the babesiosis pool), sulfhydryl oxidase (1.3% to 3.3% increase), fibrinopeptide B (1.5% to 2.3%), fibrinogen gamma (0.6% to 1.02%) and coagulation factor V (0.99% to 1.06%), while most other proteins remained at similar levels . Data beAmino acid sequences of selected phosphopeptides observed in the present study were submitted to weblogo analysis in order to determine sequence conservation and to allow comparison with previously reported phosphorylation sites .We have focused our study on the relatively new field of serum phosphoproteomics combining individual sample qualitative/quantitative label-free and TMT label-based quantitative pool proteomic analysis. The present analysis is to our knowledge the first to accomplish a preliminary characterization of phosphorylation sites of major serum proteins in dogs to yield an initial characterization of the canine serum phosphoproteome. Additionally, our quantitative study aimed to perform an initial assessment of the detectable modifications of serum phosphoprotein abundancies induced by babesiosis.2 magnetic beads due to their apolarity as preciously observed [Our data suggest that the increase in concentration of acute phase proteins, proteins released from lysed erythrocytes due to hemolysis, and apolipoproteins could lead to an increase of unspecific binding of these proteins to TiOobserved , 49. Desobserved Homo sapiens, Mus musculus, Rattus norvegicus, Drosophila melanogaster, Caenorhabditis elegans, Sacchamomyces cerevisiae and Schizosaccharomyces pombe [Canis lupus familiaris. Computational analysis of phosphorylation sites in protein databases has shown that kinases hold the highest degree of conservation among the hundreds of organisms studied [canis lupus familiaris and homo sapiens proteins compared to mus musculus and rattus norvegicus proteins. In some cases, as for SAA, phosphorylation was observed to occur in the extremes of α- helical motifs, suggesting that solvent-exposed residues could be more prone to phosphorylation by ecto-kinases. The action of ecto- kinases and ecto-phosphatases is still very poorly characterized but there are evidences of extensive cell surface and serum protein phosphorylation in cell culture medium [Our analysis found several phosphorylation sites not included in current protein phosphorylation databases such as PhosphoSitePlus, PhosphoElm 9.0, PHOSIDA, dbPAF, dbPTM 3.0 Uniprot and others. These databases contain a maximum of a half million phosphorylation sites found in around 50,000 proteins from different species, including es pombe , but ver studied . Phosphoe medium  and in ee medium .Many of the observed phosphorylation sites show a high probability to be substrate of protein kinases CKI and CKII in agreement with recent findings suggesting a major role of atypical protein kinases that localize within the Golgi apparatus (Fam20C kinase family) that phosphorylates secretory pathway proteins within S-x-E motifs , 54 Our Quantitative data showed that α-2-HS-glycoprotein / Fetuin-A is the most abundant phosphoprotein in dog serum , with kininogen-1 second in abundance (with 16–20% of intensity attributed to phosphopeptides). Previous studies have indicated fetuin-A to be an abundant phosphoprotein in human serum  based onSerum amyloid A (SAA), displays a very significant increase in phosphorylation at S 101 (p = 0.00018) in individual babesiosis samples and the highest increase in the TMT-labeled babesiosis pool both in phosphorylated and non-phosphorylated forms of all proteins observed. Some ELISA-based quantification studies have shown an 800-fold increase of SAA levels in canine babesiosis  and signi.e., calcium/calmodulin-dependent serine protein kinases secreted by platelets [SAA has been reported to promote chemotaxis, migration, and adhesion of monocytes/macrophages . Recent latelets ) may plalatelets  stronglyPlasmodium Falciparum and Plasmodium vivax) and in SARS [B. canis, the reported diagnostic sensitivity of CRP, SAA and platelet counts is significantly higher than haptoglobin, erythrocyte sedimentation rate, hematocrit and white blood cell count [It is known that, as in many infectious diseases affecting humans, canine babesiosis elicits the exacerbated secretion of acute phase proteins (APP) . A recen in SARS . Moreovell count .Babesia canis have been reported to be insufficient to activate the kallikrein system in dogs infected with Babesia canis [We obserbed as well a significant level reduction in Kininogen 1 phosphorylated at T326 (p = 0.0257). Phosphorylated kininogen1 has been previously reported in human serum . High moia canis . There iia canis  could exSimilarly, fibrinogen α /fibrinopeptide A phosphorylated at both T20 (p = 0.0257) and S22 (p = 0.0193) was found to be significantly down-phosphorylated in individual serum samples from babesiotic dogs compared to healthy controls. Another phosphoprotein is involved in coagulation, fibrinogen beta/ fibrinopeptide B (phosphorylated at T35), suggesting an influence of modifications on hemostasis. Phosphorylation has been previously observed as well in coagulation factor V, FVIII, FIX and prothrombin . During B.canis-caused babesiosis [Lower levels of phosphorylated coagulation factors observed in our data might be related to thrombocytopenia observed in besiosis , 78 as wbesiosis . Moreovebesiosis .in vivo experiments are needed to elucidate the role of phosphorylation of many serum proteins as well as to characterize the role of ecto- kinases and ecto-phosphatases in modulating extracellular protein phosphorylation.In summary, our qualitative individual sample data showed novel and conserved phosphorylation sites in canine serum phosphoproteins and significant variations in a number of phosphoprotein levels upon babesiosis. In addition, quantitative data are valuable to establish relative abundances of serum phosphoproteins jointly offering an initial characterization of variations occurring in babesiosis. Further investigation with a larger number of samples and more extensive enrichment is required to increase the number of detected phosphoproteins. Further The present analysis represents to the best of our knowledge the first characterization of canine serum phosphoproteins and the first approach to map modifications of serum protein phosphorylation status elicited by babesiosis. Phosphorylation sites detected in canine serum proteins showed a higher degree of conservation when compared with human than with other mammalian species present in phosphorylation databases. Additionally, the results show that the canine serum phosphoproteome is characterized by a wide dynamic range, with fetuin-A and kininogen-1 as major phosphoproteins. Moreover, significant differences in SAA, kininogen 1 and fibrinogen α phosphorylation levels were detected in canine babesiosis that could be useful as candidate biomarkers. These findings suggest that extracellular phosphorylation / de-phosphorylation catalyzed by extracellular kinases and phosphatases, a field which is gaining much attention in recent years, could be involved in relevant physiological processes related with infectious diseases.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.


O-deethylation reaction of phenacetin was used for monitoring CYP1A2 enzyme activity, coumarin 7-hydroxylation for CYP2A6 enzyme activity, 6-α-hydroxylation of paclitaxel for CYP2C8 enzyme activity, and dextromethorphan O-demethylation for CYP2D6 enzyme activity. The generated metabolites were monitored by high-performance liquid chromatography coupled with diode array detection. Hesperetin, pinocembrin, chrysin, isorhamnetin, and morin inhibited CYP1A2 activity; apigenin, tangeretin, galangin, and isorhamnetin inhibited CYP2A6 activity; and chrysin, chrysin-dimethylether, and galangin inhibited CYP2C8. None of the analyzed flavonoids showed inhibition of CYP2D6. The flavonoids in this study were mainly reversible inhibitors of CYP1A2 and CYP2A6, while the inhibition of CYP2C8 was of mixed type (reversible and irreversible). The most prominent reversible inhibitor of CYP1A2 was chrysin, and this was confirmed by the docking study.Cytochromes P450 are major metabolic enzymes involved in the biotransformation of xenobiotics. The majority of xenobiotics are metabolized in the liver, in which the highest levels of cytochromes P450 are expressed. Flavonoids are natural compounds to which humans are exposed through everyday diet. In the previous study, selected flavonoid aglycones showed inhibition of CYP3A4 enzyme. Thus, the objective of this study was to determine if these flavonoids inhibit metabolic activity of CYP1A2, CYP2A6, CYP2C8, and CYP2D6 enzymes. For this purpose, the  Cytochrome P450 (CYP) enzymes are the most significant enzymes for the metabolism of substances foreign to the human body, including drugs . These eThe reactions catalyzed by cytochromes P450 are not limited to one type of reaction or one substrate. Instead, each cytochrome P450 enzyme has numerous substrates and catalyzes chemically diverse reactions. Beside oxidation reactions , cytochromes catalyze reduction, desaturation, ester cleavage, ring expansion, ring formation, aldehyde scission, dehydration, ipso attack, one-electron oxidation, coupling reactions, rearrangement of fatty acid, and prostaglandin hydroperoxides .The major site of cytochromes P450 activity within the human organism is the liver. The liver cytochromes P450 involved in the metabolism of most drugs are: CYP3A4/5 (30%), CYP2D6 (20%), CYP2C9 (13%), CYP1A2 (9%), and others [Drugs and other xenobiotics can be substrates of cytochromes P450, but they can also act as inhibitors. Two substrates competing for the same active site of the cytochrome P450 enzyme present the most common observed interaction, e.g., ketoconazole is a competitive CYP3A4 inhibitor that can reduce terfenadine metabolism, which is almost exclusively metabolized by CYP3A4 [Inhibitions that can have more significant consequences are irreversible. These are usually caused by inactivation of cytochromes P450 by a reactive intermediar that covalently binds to the apoprotein or the heme part of cytochrome P450. A good example is the antihypertensive drug mibefradil, which irreversibly inhibits CYP3A4. Mibefradil is converted to a reactive intermediar in the catalytic cycle of CYP3A4. The generated intermediar covalently binds to the heme part of CYP3A4, causing the inactivation of the enzyme .Except drugs, other xenobiotics like flavonoids can interact with cytochrome P450. Flavonoids are natural polyphenols abundantly present in higher plants, including fruits, vegetables, and plant-derived products, such as wine and propolis ,8,9. FlaO-dealkylations, which are catalyzed by cytochromes P450 [Flavonoids, in nature, mainly come bound to a sugar moiety as glycosides. Glycosides are susceptible to hydrolysis, and aglycones are liberated and absorbed in the gut . In the mes P450 ,15. The mes P450 ,15. Flavmes P450 ,19,20,21p < 0.1)  . Thus, tO-deethylation for monitoring CYP1A2 enzyme activity, coumarin 7-hydroxylation for monitoring CYP2A6 enzyme activity, paclitaxel 6-α-hydroxylation for monitoring CYP2C8 enzyme activity, and dextromethorphan O-demethylation for monitoring CYP2D6 enzyme activity (vide infra).To test the enzyme activity of each individual cytochrome P450, marker substrates/reactions were used, namely phenacetin activity . All genCYP1A2 activity was inhibited by five of the tested flavonoids, namely chrysin, hesperetin, isorhamnetin, morin, and pinocembrin .p = 0.001) and 18 ± 4% (p = 0.004), respectively), it can be concluded that inhibition is reversible. This is in accordance with previously published data. Lee et al. [N3-demethylation of caffeine as a marker reaction, and chrysin showed reversible, competitive inhibition. On rat liver microsomes, Siess et al. [O-deethylation and caffeine N3-demethylation are marker reactions of CYP1A2, ethoxy- and pentoxy-resorufin, while dealkylation was used to characterize the overall contribution of all cytochromes. The inhibition observed in the study performed by Siess et al. [O-deethylation reaction on a recombinant cytochrome P450 system. Mixed inhibition is in accordance with observed results of the TDI assay that showed complete inhibition of CYP1A2  and time dependent (TDI), as well as in the direct inhibition (DI) assay. As direct inhibition and metabolism inhibition assay are of comparable residual activities .Chang et al.  showed cOn rat liver microsomes, Siess et al.  showed tPinocembrin inhibited CYP1A2 enzyme activity. The observation that pinocembrin acts as a metabolism dependent, time dependent, and direct inhibitor of CYP1A2 has not been previously reported. As in the case of the most of aforementioned flavonoid inhibitors of CYP1A2, time dependent inhibition is the most prominent compared to other types. This indicates that these flavonoids require time to interact with CYP1A2 for the inhibition to be observed, and could be attributed to allosteric, nonspecific binding to the enzyme.Out of 11 analyzed flavonoids, apigenin, galangin, isorhamnetin, and tangeretin inhibited CYP2A6 enzyme activity . AlthougIC50 values in metabolism dependent and direct inhibition assays of 0.77 ± 0.16 µM and 0.90 ± 0.07 µM, respectively. Likewise, under the similar experimental conditions and 1 µM concentration of apigenin, we observed comparable values of residual CYP2A6 enzyme activity: 47 ± 7% (p = 0.037) and 48 ± 1% (p = 0.018). This indicates that apigenin is a direct, reversible inhibitor of the CYP1A2 enzyme.Apigenin was reported to inhibit CYP2A6 activity in the metabolism dependent inhibition assay, as well as the direct inhibition assay of CYP2A6. Boonruanga et al.  reportedGalangin showed similar values of residual CYP2A6 activity in MDI and DI assays, indicating that it is a reversible, direct inhibitor of CYP1A2. This is in accordance with the results of Tiong et al. , who conp = 0.025). . . This ina et al. .Although it has been reported that tangeretin is metabolized by cytochromes P450, namely CYP1A2, CYP3A4, and CYP2D6 ,15, it iAlthough the inhibition of CYP2C8 was reported for some flavonoids ,35, thisInterestingly, no inhibition of CYP2D6 was observed on the set of analyzed flavonoids, neither reversible nor irreversible. Flavonoids are weak acids, having mainly hydroxyl groups, while typical CYP2D6 substrates contain a nitrogen atom, which can be protonated at physiological pH . This coIn our previous research, a significant number of irreversible flavonoid inhibitors of CYP3A4 was reported . HoweverTo assess the reversible binding of flavonoids to cytochromes P450, a docking study of the most potent reversible inhibitor, i.e., chrysin, to the CYP1A2 enzyme was conducted.The redocking of alpha-naphthoflavone, both in presence and in absence of HOH 733, was performed and compared with crystallographic data . The docFor both chrysin species, docking with and without the HOH 733 water molecule was done and their positions were compared , Table 1The anion species at position 7 binds in the same position both in presence and in absence of the HOH 733 water molecule, with the 7-O- group closest to the heme iron. For the docked molecule species, the HOH 733 water molecule does not play a significant role. The top-ranked docking pose in both cases was identical, with the 5-OH and 4-keto group being oriented toward the heme iron. Meanwhile, in the less populated clusters, the group closest to the heme iron was the 7-OH group. The second-ranked cluster of docked molecule species in presence of HOH 733 is not shown, but it was orientated with the B ring facing the heme iron. The binding energy for the molecule species  was muchIn conclusion, out of 11 analyzed flavonoids, hesperetin, pinocembrin, chrysin, isorhamnetin, and morin inhibited CYP1A2 activity; apigenin, tangeretin, galangin, and isorhamnetin inhibited CYP2A6 activity; and chrysin, chrysin-dimethylether, and galangin inhibited CYP2C8. None of the analyzed flavonoids showed inhibition of CYP2D6. The flavonoids in this study were mainly reversible inhibitors of CYP1A2 and CYP2A6, while the inhibition of CYP2C8 was of mixed type (reversible and irreversible). The determined types of inhibition are important for the further assessment of flavonoid-drug interactions. If flavonoid is a reversible inhibitor, flavonoid-rich foods or dietary supplements could inhibit drug metabolism mediated by cytochromes P450 and dose adjustment if needed. If flavonoid is an irreversible inhibitor of cytochrome P450 enzyme, combinations with drugs that it can interact with should be avoided.Eleven flavonoids were used in this study. Acacetin, hesperetin, pinocembrin, chrysin, flavone, tangeretin, galangin, isorhamnetin, and morin were acquired commercially from TransMIT , while chrysin-dimethylether and tamarixetin were obtained from Extrasynthèse . The marker substrates and their corresponding metabolites were purchased from Sigma-Aldrich , except 7-hydroxycoumarin, which was purchased from Extrasynthèse .5 were used as a source of enzyme. Baculosomes were commercially obtained from Thermo Fisher Scientific , and all other chemicals from Sigma-Aldrich if not otherwise indicated. A 1.0 M solution phosphate buffer of pH 7.4 was prepared in house. NADPH generating system was prepared of 0.1 M glucose-6-phosphate, 10 mg mL−1 NADP+, and 1000 IU mL−1 glucose-6-phosphate dehydrogenase in ratio 100:50:2 (v/v/v) [The recombinant baculosomes with hyperexpressed cytochromes P450  and coexpressed NADPH cytochrome P450 reductase and cytochrome b (v/v/v) .g), the clear solution was transferred to a vial for HPLC analysis [Phenacetine O-deethylation was used as a marker reaction for monitoring CYP1A2 enzyme activity . Incubatanalysis .t/min, %B): , , , , . The flow rate was 1 mL/min, and chromatograms were recorded at 254 nm. Volume of incubation injected to the column was 15 µL. Retention time of substrate was 12.2 min, while product was detected at 5.7 min. The amount of generated product (acetaminophen) was determined as the area under the curve based on the calibration curve of the standard.HPLC analysis was performed on Agilent 1100 instrument  coupled with a diode array detector (DAD) on Luna C18 column . The gradient method was used for separation. Mobile phase A consisted of water, acetonitrile, and glacial acetic acid in the volume ratio of 90:10:0.1, while mobile phase B contained the same solvents in the ratio 10:90:0.1. The following gradient timetable was used (The aromatic hydroxylation of coumarin at the position 7 was used as a marker reaction for CYP2A6. The incubation conditions were the same as for CYP1A2 enzyme (vide supra) unless otherwise stated. The final concentration of coumarin was 10 µM, while the incubation time was extended to 30 min.HPLC-DAD analysis was conducted on the same system as the CYP1A2 enzyme with the following modifications: The gradient timetable  was , , , , , and the detection wavelength was 330 nm. The retention time of the substrate (coumarin) was 7.5 min, while the product (7-hydroxycoumarin) was detected at 4.8 min. The amount of generated product was determined as the area under the curve based on the calibration curve of the standard .Paclitaxel 6α-hydroxylation was used as a marker reaction for monitoring CYP2C8 enzyme activity . The incHPLC-DAD analysis was conducted on the same system as the CYP1A2 enzyme with the following modifications: The gradient timetable  was , , , , , and the detection wavelength was 227 nm. The retention time of the substrate (paclitaxel) was 6.1 min, while the product (6α-hydroxypaclitaxel) was detected at 3.4 min. The amount of generated product was determined as the area under the curve based on the calibration curve of the standard [O-demethylation of dextromethorphan to dextrorphan was used as a marker reaction for monitoring CYP2D6 enzyme activity . The incHPLC-DAD analysis was conducted on the same system as for CYP1A2 enzyme with the following modifications: The gradient timetable  was , , , , and the detection wavelength was set to 224 nm. The retention time of the substrate (dextromethorphan) was 7.1 min, while the product (dextrorphan) was detected at 4.1 min. The amount of generated product was determined as the area under the curve based on the calibration curve of the standard [Three types of experiments were conducted to determine metabolism dependent inhibition, time dependent inhibition, and direct inhibition of CYP1A2, CYP2A6, CYP2C8, and CYP2D6 enzymes .To determine metabolism dependent inhibition (MDI assay), the flavonoid was first preincubated with the enzyme with the addition of the generating system for 30 min, after which the marker substrate was added to determine residual enzyme activity (as described above for each enzyme).If metabolism dependent inhibition was determined, time dependent inhibition and direct inhibition were tested. Time dependent inhibition (TDI assay) was assessed by preincubating the flavonoid and enzyme, without the generating system, after which residual activity was determined by adding the NADPH generating system along with the substrate (as described above for each enzyme).The direct inhibition assay (DI assay) was conducted without preincubation, i.e., the NADPH generating system was added to the incubation mixture containing flavonoid and substrate following the experimental set-up described above for each individual cytochrome P450 enzyme .If the flavonoid is an irreversible inhibitor of the cytochrome P450, it will decrease enzyme activity in the MDI assay, while no reduction of enzyme activity will be observed in TDI and DI assays. Pure time dependent inhibitors require time to interact with the enzyme. Thus, TDI inhibitors will show a reduction of enzyme activity in assays with preincubations, i.e., the MDI and TDI assays, but not in the DI assay (no preincubation). Pure direct inhibitors are reversible inhibitors that usually compete with the substrate binding to the active site and will show a decrease of enzyme activity with or without preincubation. DI inhibitors will reduce enzyme activity in all three assays .To examine the binding of chrysin to the active site of cytochrome P450 1A2, a docking study was performed using AutoDock 4.2.6.  , which uAll incubations were conducted in triplicate. The results are expressed as the residual activity of the enzyme, i.e., the percentage of product generated in incubation with the addition of flavonoid in ratio to the control without flavonoid. The statistical significance was tested with Student’s t-test in the program R .


E. coli and purified. MSTNpro regions consisting of residues 45–68, -69, and -70 with MBP fusion suppressed MSTN activity with a potency comparable to that of full-sequence flatfish MSTNpro in a pGL3-(CAGA)12-luciferase reporter assay. Even though the MSTN-inhibitory potency was about 1,000-fold lower, the flatfish MSTNpro region containing residues 45–65 (MBP-Pro45-65) showed MSTN-inhibitory capacity but not the MBP-Pro45-64, indicating that the region 45–65 is the minimum domain required for MSTN binding and suppression of its activity. To examine the in vivo effect of MBP-fused, truncated flatfish MSTNpro, MBP-Pro45-70-His6 (20 mg/kg body wt) was subcutaneously injected 5 times for 14 days in mice. Body wt gain and bone mass were not affected by the administration. Grip strength and swimming time were significantly enhanced at 7 d after the administration. At 14 d, the effect on grip strength disappeared, and the extent of the effect on swimming time significantly diminished. The presence of antibody against MBP-Pro45-70-His6 was observed at both 7 and 14 d after the administration with the titer value at 14 d being much greater than that at 7 d, suggesting that antibodies against MBP-Pro45-70-His6 neutralized the MSTN-inhibitory effect of MBP-Pro45-70-His6. We, thus, examined the MSTN-inhibitory capacity and in vivo effect of flatfish MSTNpro region 45–65 peptide (Pep45-65-NH2), which was predicted to have no immunogenicity in silico analysis. Pep45-65-NH2 suppressed MSTN activity with a potency similar to that of MBP-Pro45-65 but did not suppress GDF11, or activin A. Pep45-65-NH2 blocked MSTN-induced Smad2 phosphorylation in HepG2 cells. The administration of Pep45-65  increased the body wt gain with a greater gain at 14 d than at 7 d and muscle wt. Grip strength and swimming time were also significantly enhanced by the administration. Antibody titer against Pep45-65 was not detected. In conclusion, current results indicate that MSTN-inhibitory proteins with heterologous fusion partner may not be effective in suppressing MSTN activity in vivo due to an immune response against the proteins. Current results also show that the region of flatfish MSTNpro consisting of 45–65 (Pep45-65) can suppress mouse MSTN activity and increase muscle mass and function without invoking an immune response, implying that Pep45-65 would be a potential agent to enhance skeletal muscle growth and function in animals or to treat muscle atrophy caused by various clinical conditions.Myostatin (MSTN) negatively regulates skeletal muscle growth, and its activity is inhibited by the binding of MSTN propeptide (MSTNpro), the N-terminal domain of proMSTN that is proteolytically cleaved from the proMSTN. Partial sequences from the N-terminal side of MSTNpro have shown to be sufficient to inhibit MSTN activity. In this study, to determine the minimum size of flatfish MSTNpro for MSTN inhibition, various truncated forms of flatfish MSTNpro with N-terminal maltose binding protein (MBP) fusion were expressed in  Myostatin (MSTN), a member of the TGF-β superfamily, is a negative regulator of skeletal muscle development and growth. Knockout of the MSTN gene resulted in a dramatic increase in skeletal muscle mass in mice with little effect on other organs , while sin vivo by various approaches, including plasmid-mediated delivery , where Y is % inhibition, Bottom is the lowest value of % inhibition set at 0%, Top is the highest value of % inhibition set at 100%, and X is Log ligand concentration. IC50 values were analyzed by ANOVA using the same program.The anti-MSTN activity was measured using a pGL3-(CAGA)onstruct . HEK293 eviously  using a 5 cells per well and grown in DMEM containing 10% FBS, 1% penicillin/streptomycin at 37 °C with 5% CO2 for 24 h. Following serum-free DMEM containing 10% FBS, 1% penicillin/streptomycin for 4 h, cells were treated with or without 10 nM MSTN , 10 μM SB431542, an inhibitor of activin receptor-like kinase (ALK)4/5/7  and 600 nM MBP-Pro45-70-His6 for 30 min. Then, cells were lysed in RIPA buffer  containing protease inhibitor cocktail and phosphatase inhibitor cocktail . The lysates were disrupted by sonication for 15 s on the ice and centrifuged at 12,000 rpm for 20 min at 4 °C. Protein lysates were quantified by BCA assay . Fifty μg of protein were separated by 10% polyacrylamide SDS-PAGE and transferred to polyvinylidene fluoride membranes . The membranes were blocked with 5% nonfat or 5% BSA in Tris-buffered saline (TBS) containing 0.1% tween 20 (TTBS) for 3 h at room temperature. To assess phosphorylated and total protein of signaling molecules, the membranes were incubated with antibodies against respective phospho- or total protein for 3 h at room temperature. The following primary monoclonal antibodies and polyclonal antibody (β-actin) were used and purchased from Cell Signaling Technology : Smad2 (Cat#31221:1000), phospho-Smad2 , Smad3 , phospho-Smad3 , β-actin . The membranes were washed with TTBS for 10 min four times and then incubated for 3 h at room temperature with horseradish peroxidase-conjugated matching secondary antibodies (1:5000): anti-mouse IgG for Smad2, p-Smad2, Smad3, and p-Smad3; anti-Rabbit IgG for β-actin. The membranes were washed 3 times with TTBS for 10 min. The immuno-reactive bands were detected by the SuperSignal West Femto Maximum Sensitivity Substrate  and visualized on Kodak Omat X-ray films. Densitometry analysis of the images obtained from X-ray films was performed using the Image J software (NIH).The effect of MBP-Pro45-70-His6 on MSTN-induced phosphorylation of Smad2 and Smad3 was examined in HepG2 cells that induce Smad2 phosphorylation in the presence of MSTN . HepG2 cad libitum. For the MBP-Pro45-70-His6 administration study, mice (5~6 week old weighing 24–26 g) were randomly divided into two groups (n = 12 per group). Animals were injected subcutaneously with 0 or 24 mg/kg body wt of MBP-Pro45-70-His6 five times (3 days apart) for 14 days. MBP-Pro45-70-His6 was dissolved in PBS (2 μg/μL) for mice in the treatment group, and PBS was injected for mice in the control group. Body wt was measured at the time of injection.ICR male mice were obtained from Hyochang Science . We followed the current regulations for the care and use of laboratory animals under the guidance of the Animal Ethics Committee of Pukyong National University, Busan, Korea. The ethics committee approved this study under protocol AEC-201405. The mice were maintained in a room at a constant temperature of 24±1°C under a 12 h light/12 h dark cycle at 65% humidity. Animal pellet  and water were provided For the Pep45-65-NH2 administration study, mice (6~8 week old weighing 27–30 g) were randomly divided into two groups (n = 12 per group). Pep45-65-NH2 was dissolved in PBS (2 μg/μL), and subcutaneously injected (10 μL/g body wt) five times (3 days apart) for 14 days for mice in the treatment group. Mice in the control group were injected with PBS. Body wt was measured at the time of injection.2 metal net . Briefly, the mouse was slowly pulled by the tail in the opposite direction while its two forelimbs gripped the metal net. The grip strength meter was recorded in Newton (N). Each mouse was subjected to three times grip test with at least a 1 min rest between trials. The maximum grip force was recorded as the forelimb grip strength. Grip strength (N) was measured on day 0 (before the first injection), day 7, and day 14.Forelimb grip strength was measured 1 h after each injection. The tensile force generated by each mouse was measured using a force transducer with a rectangular 4×5 cmst measurement.The forced swimming test was performed 1 h after the grip strength measurement. For the forced swimming test, mice were dropped individually in a 1,000 mL beaker  filled with water at 24±1 °C. After a 2 min adaptation period, the swimming time (s) was measured following the procedure described previously . AnotherOn day 14, the mice were anesthetized with Zoletil 50 , and blood was collected by cardiac puncture . The bloAfter peeling the skin off the euthanized mice, forelimbs and hindlimbs were disarticulated from scapula to carpus and from ilium to medial malleolus, respectively. The wt of forelimbs and hindlimbs were immediately measured, as well as the bone wt. The skeletal muscle mass was calculated by subtracting the wt of the bone from those of the forelimbs and hindlimbs, respectively.Blood for ELISA was collected on day 7 using the facial vein technique  and on d12-luciferase reporter assay, and IC50 values for 1 nM MSTN inhibition were estimated . Removal of 47 and 48 from the N-terminal side of Pro45-70 drastically diminished the MSTN-inhibitory capacity with IC50 values of 1.03 μM.Fifteen truncated, MBP-fused MSTNpro proteins, including MBP-Pro45-70, MBP-Pro45-69, MBP-Pro45-68, MBP-Pro45-67, MBP-Pro45-66, MBP-Pro45-65, MBP-Pro45-64, MBP-Pro45-63, MBP-Pro45-62, MBP-Pro45-61, MBP-Pro45-60, MBP-Pro46-70, MBP-Pro46-69, MBP-Pro47-70, and MBP-Pro49-70 were expressed and purified by amylose affinity chromatography. Sequence analysis confirmed the amino acid sequences of each MSTN prodomain. The anti-MSTN activities were measured using the pGL3-(CAGA)stimated . MBP-Proectively . Removin50 value of MBP-Pro45-70-His6 was 1.43 nM for 1 nM MSTN inhibition  and Activin A (52.8 nM) were about 8-fold and 37-fold higher, respectively than for 1 nM MSTN  and induces phosphorylation of activin receptor-like kinase4 (ALK4) or activin receptor-like kinase5 (ALK5) type I receptor . The MSTThe administration of MBP-Pro45-70-His6 tended to increase the body wt gain during 7 days administration period as compared to the control, but no difference in body wt gain was observed after 14 days administration . The musThe effect of MBP-pro45-70-His6 on swimming time was also examined by measuring the change in swimming time from day 0 to day 7 and from day 0 to day 14 . The micThe concentrations of serum cholesterol, triglyceride, and free fatty acid of mice administered with MBP-Pro45-65-His6 were significantly lower than those of control mice, while no difference in serum glucose concentration was observed between the two groups .MBP-Pro45-70-His6 is a heterologous protein to mice, thus the generation of antibody against MBP-Pro45-70-His6 was examined using ELISA . The pre50 value of 2.66 μM , as well as from day 0 to day 14 (0.0800 N vs 0.0325 N) . The micThe concentrations of serum glucose, triglyceride, and free fatty acid of mice administered with Pep45-65-NH2 were significantly lower than those of control mice, while no difference in serum cholesterol concentration was observed between the two groups .Myostatin propeptide (MSTNpro) is a well-known MSTN suppressor, and the administration of recombinant MSTNpro enhanced skeletal growth or muscle repair in mice , 27, 32,50, 1.18 nM) of flatfish MSTNpro region consisting of residues 45–70 with MBP fusion (MBP-MSTNpro45-70) is not different from that of MBP-MSTNpro-45-80  reported previously [The current study shows that MSTN-inhibitory capacity , which suppressed MSTN activity, was predicted to induce little immunogenicity in silico assay. Pep45-65 suppressed the MSTN-induced Smad2/3 phosphorylation dose-dependently in HepG2 cells, suggesting its potential to suppress MSTN activity Pep45-65 , suggestIn addition to its effect on muscle mass, MSTN also regulates fat mass and energy metabolism. Studies have shown that the inhibition of MSTN decreases fat mass, improves insulin sensitivity and whole-body metabolism in mice –52, and 50 value of Pep45-65 for MSTN inhibition (12.5 ng/mL) was about 3 μM in our current and a previous study [50 value of p23 for MSTN inhibition (10 ng/mL) was reported to be 4.1 μM in a (CAGA)12 reporter gene assay [The size of Pep45-65 is smaller than that of mouse MSTNpro peptide fragment consisting of residues from 44 to 66 (p23) that was reported to be the minimum peptide for MSTN inhibition . In spitus study , while tne assay . The remne assay , suggestin vivo. These results, together, imply that Pep45-65-NH2 would be a potential agent to enhance skeletal muscle growth and function in animals or to treat muscle atrophy caused by various clinical conditions.In conclusion, current results indicate that MSTN-inhibitory proteins with heterologous fusion partner may not be effective in suppressing MSTN activity in vivo due to an immune response against the proteins. Current results also show that the region of flatfish MSTNpro consisting of 45–65 (Pep45-65-NH2) can suppress MSTN activity and increase muscle mass and function without invoking an immune response in the mouse. The Pep45-65-NH2 with its smaller size than the mouse propeptide  showed a comparable to or better MSTN-inhibitory potency than the mouse propeptide. Also, Pep45-65-NH2 did not interfere with the GDF11 and Activin A signaling, suggesting minimal off-target effect S1 TableUnderline indicates EcoRI; site used in the construction of expression vectors.(PDF)Click here for additional data file.S1 FigM, protein ladder; T, total protein; S, soluble fraction; P: pellet (insoluble fraction), FT1: flow-through fraction of Ni-NTA agarose, W1: washing fraction of Ni-NTA agarose, E1: elution fraction of Ni-NTA agarose, FT2: flow-through fraction of amylose resin, W2: washing fraction of amylose resin, E2: elution fraction of amylose resin.Ni-NTA agarose affinity chromatography was first used, followed by amylose resin affinity chromatography for purification. Proteins were visualized with Coomassie blue staining. (TIF)Click here for additional data file.S2 Fig1000-fold diluted sera were used for titer measurement. Open circle (○) and closed square (■) indicate 0 and 20 mg/kg body wt administration of Pro45-65-NH2, respectively. Student`s t-test was used to compare the mean difference.(TIF)Click here for additional data file.