The conductive polymeric composites incorporating carbon nanotube (CNT) and carbonyl iron powder (CIP) have attracted much attention for various sensor applications. In this paper, a comprehensive study of the magneto-sensing property of a CNT-CIP embedded polymer composite is conducted to implement the composite as magneto-sensors. Thus, this study experimentally investigated the magneto-sensing performances of CNT-doped polymeric composites with the addition of CIP in terms of electrical conductivity, sensitivity, repeatability, and response time. First, the CNT-CIP clusters were manufactured and their interactions were analyzed with the zeta potential measurement and SEM observation. Then, the CNT-CIP clusters were embedded into the polymeric composites for the magneto-sensing evaluations. Experiments showed that the CNT contents in the range of percolation threshold  are optimal values for sensor applications. The addition of CNT 0.5% and 0.75% resulted in a high sensitivity of 7% and a faster response time within 400 ms. Experiment evaluation confirmed a high potential of implementing CNT-CIP composite as magneto-sensors. Expeditious advancements of conductive polymeric composites (CPCs) have been gaining attention due to their high applicability, such as multi-functional sensors ,2,3, ele® 0010 of poly-butylene adipate-co-terphthalate (PBAT) is utilized as the polymeric matrix. It is a flexible polymer matrix, widely used in related research [® 0010 is explained in the study by Wei et al. [® was then added to the sonicated mixtures, and they were heated for 12 h at 120 °C with stirred at 200 rpm to both improve the dispersion of CNTs and evaporate the IPA solvent in the Ecoflex® matrix. After 12 h, CIP 50% by polymer weight was added to the mixtures, and they were hand-mixed for an additional 3 min. The mixtures were cured in a 20 3 mold at an oven with a temperature of 60 °C for 24 h  was used to measure the potential differences of the used materials in IPA solvent. A micro-structural image of the sample was taken through field emission scanning electron microscopy , and they were discussed with the formation of electrical conductivity networks and the overall magneto-resistive sensing test results. The electrical resistance of the samples was measured using a portable digital multi-meter with a two-probe method, and they were converted to the electrical resistivity. A total of three samples in each mix proportion were chosen to calculate the average values. Input voltage (1 V) was applied to the samples and their electrical resistance variations were measured to observe the electrical stability of the samples under the static conditions for 4 h. The magneto-resistive sensing performances of the samples were evaluated with and without a magnetic field. Magnetic field levels (0 and 200 mT) were applied to the samples for 30 s, respectively, and the corresponding electrical resistances of the samples were measured simultaneously. The identical process was repeated for 3 cycles for every sample. The magneto-sensing performances of the samples including sensitivity and response time were evaluated in terms of the resistance change over time.First, the CIP-based clusters coated with CNT were developed. Then, their formation was investigated using the zeta potential analysis and SEM observation. In The electrical resistivity values of the samples are shown in The magneto-resistive sensing performances of the samples under the different levels of the applied magnetic field are displayed in ®-based composites around 332 ms due to the ultra-high viscoelastic properties [The process as described in operties ,23.In the present study, the experimental investigations about the electrical characteristics and magneto-resistive sensing performances of CIP@CNT clusters-embedded polymeric composites for sensor applications were evaluated. First, the CIP coated with CNT (CIP@CNT) clusters were fabricated, and their formation was investigated with the zeta potential measurement and SEM images. In observation of electrical characterization, the effects of included CNT contents on the electrical conductivity of samples were observed, and the percolation threshold (0.5% to 0.75%) was found. Through the magneto-resistive sensing test, the samples with CNT contents in the percolation threshold showed higher sensitivity, i.e., approximately 7%, and faster response within 400 ms compared to that found in the previous studies. Thus, the present experimental results exhibited that CIP@CNT clusters-embedded polymeric composites have potential applications as magneto-sensors. For the further work, the optimization of polymer matrix selection, proportions of CIP and CNT, and addition of other fillers are recommended to be conducted to maximize the overall magneto-resistive sensing performance. After the aforementioned research, the optimal magneto-resistive sensors based on the CIP@CNT-embedded polymeric composites are expected to be manufactured for practical and commercial applications.


Our meta-regression analysis with pandemic prevalence as the predictor explains almost half the variance in risk ratios across trials . This study finds that efficacy of SARS-CoV-2 vaccines reported in Phase 3 trial declines as pandemic prevalence at trial sites increases. Trials conducted in locations with low pandemic prevalence reported higher efficacies as compared to trials conducted in high pandemic prevalence locations.The efficacy of SARS-CoV-2 vaccines reported in Phase 3 trials varies from ~45% to ~95%. This study tests the hypothesis that the observed variation in efficacy of SARS-CoV-2 vaccine candidates can be explained by the prevalence of the COVID-19 pandemic at trial sites. To test the proposed hypothesis, we conducted a systematic search following PRISMA guidelines. Our search resulted in 8 vaccine candidates that had reported efficacy data from a total of 20 Phase 3 trials, representing a total of 221,968 subjects, 453 infections across the vaccinated groups and 1,554 infections across the placebo groups. We use meta-regression models to analyse the potential associations between prevalence of COVID-19 pandemic at trial sites and efficacy of the reported SARS-CoV2 vaccines. The overall estimate of the risk-ratio is 0.24 , with a high degree of heterogeneity (τ The SARS-CoV-2 pandemic has generated an unprecedented effort towards developing vaccines to halt the pandemic. A number of vaccines are under development, many have reported results of Phase 3 trials, and many have received emergency approval too. Vaccine efficacies reported across Phase 3 trials range from 45% to 96% –5, see TDifferences in vaccine efficacies reported across trials could be due to various sources of heterogeneity between trials , 22, 23.E, Kaslow [E malaria vaccine, observed that vaccine efficacy “tends to be lower in high transmission areas” [Variations in vaccine efficacy under different prevalence conditions have been reported previously in Phase 3 trials of other vaccines too . For ins, Kaslow  reports , Kaslow . Similarn areas” , 27. Kasn areas”  further n areas” .ceteris paribus, when there are more breakthrough infections, i.e. a greater number of subjects in the vaccinated group of a trial getting infected. Consequently, as mutant strains generate a greater number of breakthrough infections, vaccine efficacy is likely to decrease in the presence of mutant strains. Genetic mutations and antigenic drift in a focal virus can also lead to higher prevalence. Mutant strains often possess additional paths, as well as a stronger ability, to infect human cells [Variations in vaccine efficacy have been attributed to the presence of mutant strains of a virus, among other factors , 30. Genan cells , 30. Thoan cells , 30. Thean cells  reports Phase 3 trials of SARS-CoV-2 vaccines carried out in high prevalence areas are likely to report lower efficacies.As mutations and antigenic drifts are responsible for both higher prevalence as well as lower efficacy, we hypothesize that This study is a systematic review and meta-analysis of the efficacy data reported in Phase 3 trials of SARS-CoV-2 vaccine candidates. The meta-analysis reports an overall summary estimate of the risk-ratio and conducts a meta-regression to test the effect of between-trial differences in prevalence of the SARS-CoV-2 pandemic on efficacies reported across trial sites. The study was registered at PROSPERO (Registration Number CRD42021243121).To ensure the latest information is available, the landscape will be updated twice a week  by searching, gathering and cross-checking data from multiple sources such as the Cochrane vaccine mapping tool, PubMed, ClinicalTrials.gov, WHO ICTRP and from a network of researchers and industry for new candidate vaccines by screening registered trials for clinical information. Where data is missing, … we supplement information gathered from press or public releases” [PubMed and ClinicalTrials.gov, we relied on those sources to identify vaccine candidates that had progressed to Phase 3 trials.Vaccine candidates were included in this study if they reported efficacy results from Phase 3 randomised controlled trials. Results of Phase 3 trials reported till March 31, 2021 were included in the analysis. Phase 3 clinical trials of vaccine candidates are being conducted by the vaccine manufacturers only. Accordingly, we started our search by identifying vaccine candidates that had registered Phase 3 trials, following PRISMA guidelines. Given the high level of interest in the development of SARS-CoV-2 vaccines, such information is widely shared by the manufacturers and tracked by multiple reliable sources [ClinicalTrials.gov to identify the start dates of the Phase 3 trials for each vaccine candidate. Given the time lag between the start of Phase 3 trials and the reporting of efficacy results, trials that had not started by November 1, 2020 were excluded from a further search of results of Phase 3 trials. That left a total of 11 vaccine candidates to be searched for the results of efficacy data from Phase 3 trials. AA independently validated every step of the search and the master list of vaccine candidates through reference to additional sources  based on the number of infections and participants in the experimental and control arms of the trials. A random-effects model with restricted maximum likelihood estimator was employed in the meta-analysis. Heterogeneity between studies was assessed using the τatistics . The effClinicalTrials.gov and corroborated with the information reported in the peer reviewed documents, company reports and company press releases.Pandemic prevalence associated with a trial is estimated from the country-wise data on the SARS-CoV-2 positivity rates reported on Oxford University’s ‘Our World in Data’ portal . The porThe SARS-CoV-2 prevalence data across countries are not directly comparable due to the different protocols and practices across the different national SARS-CoV-2 testing programs . Some of2 statistic is 0.50 and the I2 statistic is 88.73%, suggesting that the extent of heterogeneity is considerable, and that a high proportion of the total variance is due to between-study heterogeneity [The overall estimate of the risk-ratio   , Fig 4. The funnel plot See  shows noSensitivity analyses were conducted to analyse if the result of the meta-regression is sensitive to the inclusion of multiple trials for the Janssen vaccine  or the AstraZeneca vaccine 4 trials). Two meta-regressions were run, one with an additional dummy variable for the Janssen vaccine, and the other with an additional dummy variable for the AstraZeneca vaccine. In both cases, the regression coefficient for pandemic prevalence was significant, while the coefficients for the dummy variable (Janssen/AstraZeneca) were non-significant   .Risk of bias was assessed as low using the Cochrane tool, while certainty of evidence was assessed as high using GRADE , attributable to between-study differences [The meta-analysis . Support for the hypothesis is robust, and not sensitive to potential sources of validity threats. The inclusion of multiple trials for the Janssen and AstraZeneca vaccine candidates does not affect the results of the meta-regression reported in The results support our hypothesis that increasing pandemic prevalence is associated with lower efficacy of SARS-CoV-2 vaccine candidates  is 25% and the I2 statistic for the residual heterogeneity is 79.3%. This suggests that it is quite likely that the residual heterogeneity could be explained by additional between-trial differences. There are a number of other sources of heterogeneity that occur across Phase 3 trials of the SARS-CoV-2 vaccine candidates as well, including, but not limited to dosing protocols, interval between doses, and the definition of primary endpoints [ndpoints , 23, 29.The meta-regression analysis  reports The finding that pandemic prevalence affects vaccine efficacy suggests three important implications for the conduct of Phase 3 trials of SARS-CoV-2 vaccine candidates. First, when efficacy results of Phase 3 trials are reported, they should be reported at the level of each trial site and include the level of pandemic prevalence associated with each trial site. Given the findings of this meta-analysis, a meaningful interpretation of observed efficacy in Phase 3 trials can only be done in conjunction with the associated data on pandemic prevalence across trial sites.Second, it suggests that protocols for the design and conduct of Phase 3 trials need to take pandemic prevalence into account. The design of trials should include randomization across locations that are likely to vary in the prevalence of the pandemic. Further, for the duration of the Phase 3 trials, manufacturers should monitor pandemic prevalence at all locations where the trials are conducted. Results of Phase 3 trials should include an analysis of the effect of prevalence on vaccine efficacy.Finally, the findings of this study suggests that Phase 3 trials should also analyse participant samples for the presence of mutant strains. Following prior research on the rotavirus and malaria vaccines, this study argues that the presence of mutant strains of the SARS-CoV-2 virus leads to both higher pandemic prevalence as well as a higher number of breakthrough infections. However, we are unable to directly test that mechanism as data on prevalent mutant strains at trial sites is not reported as part of the reporting protocols for Phase 3 trials. It is inevitable that there is a time lag between the sequencing of a mutant strain and assays to test for the presence of that strain. Hence, it is not always possible to report a mutant-specific analysis of the results of Phase 3 trials. However, the findings of this study suggest that this is an important relationship that has implications both for the development of vaccines as well as strategies for managing the pandemic. Accordingly, we suggest that protocols for Phase 3 trials should include the preservation of samples for the identification of mutant strains at various trial sites once the assays are available.Overall, a key strength of this study is that it summarizes the entire current publicly available global evidence on the efficacy of SARS-CoV-2 vaccine candidates. It is also a strength of this study that it can explain almost half the variance in between-trial efficacies with one moderator variable only, pandemic prevalence. Another important strength of this meta-analysis is that data on pandemic prevalence is obtained from sources independent of the trials.While this meta-analysis finds that, as hypothesized, higher pandemic prevalence is associated with lower vaccine efficacy, causal inferences need to be drawn with caution. As is the case with any meta-analysis, this is a post-hoc observational study. However, given the importance of the observed association, we suggest that future Phase 3 trials should employ protocols that allow this relationship to be tested via randomized controlled trials .The overall sample is small, 20 trials across 8 vaccine candidates. Efficacy data on three other vaccine candidates that have completed Phase 3 trials and are currently approved for use are not publicly available and could not be included in this meta-analysis. All trials are multi-location, and a few are multi-country as well, e.g., Pfizer/BioNTech’s trial conducted in the US, Chile and Peru see , Note a.Pandemic prevalence has a significant negative effect on efficacies observed across Phase 3 trials of SARS-CoV-2 vaccine candidates; trials conducted in locations with low pandemic prevalence reported higher efficacies as compared to trials conducted in high pandemic prevalence locations.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


Using 180 normal adults’ rsfMRI data from the human connectome project database, we visualized inter-voxel relations by embedding voxels on the hyperbolic space using the k-core percolation on 30 participants to investigate core voxels for each individual. It recursively peels the layer off, and this procedure leaves voxels embedded in the center of the hyperbolic disc. We used independent components to classify core voxels, and it revealed stereotypes of individuals such as visual network dominant, default mode network dominant, and distributed patterns. Characteristic core structures of resting-state brain connectivity of normal subjects disclosed the distributed or asymmetric contribution of voxels to the kmax-core, which suggests the hierarchical dominance of certain IC subnetworks characteristic of subgroups of individuals at rest.Hyperbolic disc embedding and  The conundrum of how these modules are aggregated to form a single coherent network with preserved functionality remains a fundamental question for unveiling the functional architecture of the human brain in the resting state and upon activation7. Individual differences add complexity to a succinct understanding of this question.The brain is a high-dimensional complex and integrated network that is composed of multiple modular and specialized networks, distributed spatially, and combined to form a multimodular structure9. Essential nodes were initially supposed to be hub nodes with high degrees or high centrality on brain graphs but were soon suggested to be core influencer nodes with a wide range of initial degrees on decomposition8. In physical networks, disruption in these core nodes leads to abrupt disintegration, which is called network dismantling or targeted damage13. Disruption in the brain graph is associated with serious neuropsychiatric diseases with disrupted associative functionality15.Recent works in network science suggest that the aggregation of these modules is facilitated by a set of essential voxels that integrate intramodular and intermodular information throughout the network16, implementing hubness, centrality measures such as degree, betweenness, eigenvector, and leverage centrality20, or k-core25. The k-core percolation describes the architecture of the backbones of the network by filtering out peripheral nodes and searching for remaining central nodes, where the coreness, k, acts as the threshold for sustaining node connectivity along the filtration. The k-core percolation was used to understand the forward (phase transition) and backward (k-decomposition) behavior of networks, and brain networks can be dissected in a similar way as was done for graph filtration thresholding30.Therefore, it is important to identify which nodes compose the core structure, and if ever the resting state core is individually unique, then their individual differences in the core composition should be disclosed using voxel-based representation of brain graphs. Recent studies of mathematics and neurosciences have accomplished this job successfully with physical networks and probably with the brainIn our previous work, we addressed the problem of difficulty in visualization and thus formed mental imagery of the object brain graphs by considering their geometric characteristics. We adopted an analytical framework for visualizing a complex, multimodular network for functional brain networks with scale-freeness by embedding the networks into the latent geometric model of 31, the geometric meaning of Based on the Popularity 33. The embedding of the network into the geometric model was performed by using the software named Mercator, introduced by García-Pérez et al.33, which makes use of Laplace eigenmaps (LE) for the reduction of dimension and maximum likelihood estimation (MLE) techniques for acquiring the most appropriate geometric object on hyperbolic discs representing the original network with fidelity34. This solved two issues: (1) 2-dimensional representation of complex brain graphs with flexible annotation of functionally cooperating voxel groups and (2) thresholding to sort out necessary edges to make the complex brain graphs obey power law and thus scale-freeness34, which implies the self-similarity and heterogeneity of degree distribution. This shares some topological features with functional brain network, and compatible with hyperbolic embedding of the network34.The model has been successful in revealing the hidden geometry of many other real complex networks of non-Euclidean naturek-core percolation as a measure of centrality. We characterized the kmax-core voxels for their degree distribution belonging to each IC, showing the plausible influencer behavior of these IC subnetwork voxels upon k-core percolation eventually to find which subnetworks are the dominant by counting the voxels belonging to them at rest in normal individuals. We asked whether the individuals had common or characteristic core structures in terms of their kmax-core IC-voxel compositions.In this work, we analyzed functional brain networks from healthy human young adults by analyzing rsfMRI data and visualized functional subnetworks, i.e., independent components (ICs), using hyperbolic embedding. Then, we investigated how each functional subnetwork was composed of the subset of voxels with high core-ness, revealed by 34 that looked for an optimal non-Euclidean space for embedding the intervoxel correlation structure, we simply chose 2-dimensional hyperbolic disc embedding. Hyperbolic disc representation reflected the original high-dimensional edge information similarly well to the high-dimensional Euclidean embedding alternatives34. To the best of our knowledge, the high-dimensional correlation structure rendered by hyperbolic disc embedding serves an easy-to-recognize visibility, which is very difficult to achieve via Euclidean 2-dimensional representation  information to the hyperbolic disc space. According to our previous investigationion Fig. a,b.Figur34, we used voxel correlation to visualize intervoxel relationships for hyperbolic disc embedding using the 33. The output easily disclosed the belonging characteristics of the voxels to ICs on a hyperbolic polar coordinate. Edge weight on the voxel-voxel correlation matrix was thresholded to yield the adjacency matrix after confirming the scale freeness of the resultant degree distribution of the voxels while preserving the size of the largest component as large. This allowed us to confirm the power law of its degree distribution and scale freeness to be fit for the hyperbolic model. Reproducibility on repeated embedding was tested in an exemplary case with repeated embedding with the Mercator algorithm34 , with edges ranging from 274,634 to 3,894,033 (1.6–22% of possible edges). A randomly sampled case was repeatedly embedded in this The adjacency matrix was then converted to fit into the mentclass2pt{minimmentclass2pt{minim33. This enabled us to assume that the angular coherence on the hyperbolic disc reveals similarity of the group of voxels and that the closer distance of a voxel to the disc center represents a higher degree with greater popularity34. We identified the voxels belonging to specific ICs (fifteen ICs) obtained from conventional group ICA performed in all 180 subjects36.This embedding provided clearer visibility of intervoxel relations on 2-dimensional space (disc) than any conventional method of visualization Fig. a,b. Insp37  ) to 443  2) . We counted how many kmax-core voxels belonged to specific resting-state IC/lobe subnetworks  had the smallest . Interestingly, the distribution of the degrees of the kmax-core voxels varied between individuals who showed a spectrum in the distribution of the dominance  of the ICs to which the kmax-core voxels belonged  than any other resting-state IC subnetwork, and more than half of the kmax-core voxels belonged to VN in 40% of subjects (12 among 30). The degrees of kmax-core voxels belonging to VN ranged from high to low values, similar to the degrees of the voxels belonging to the other ICs, such as DAN, DMN, and SN , and VN occupied the largest number of these common kmax-core voxels   mainly belonged to occipital and parietal regions. In more detail, 17 voxels in bilateral lateral superior occipital regions, 13 voxels in the bilateral ventromedial occipital regions, three voxels in the parietal regions , and one in the frontal region participated in the kmax-core. We suggest that posterior area voxels, including VN/PCN or occipitoparietal regions, are candidates for the common core in more than half of normal subjects to preside over their hierarchically lower voxels in the same ICs and the voxels belonging to the other ICs or lobes.More specifically, VN included the largest number of kmax-core voxel-IC composition among individuals using functional labels: DMN-dominant, VN-dominant, and uneven but distributed  voxel-IC compositions was named as having a distributed pattern . Stacked histograms of the degree distribution of kmax-core voxels are presented in Fig. kmax-core voxel-IC compositions. An exemplary case showed the changes of k-cores in terms of IC-voxel rendering on brain template along k-core percolation  or distributed patterns on the kmax-core voxels-ICs composition analysis.In this study, we applied hyperbolic embedding and 4. Node degree and betweenness centrality are well-known measures; however, they only show a local or global property5. Node degree measures local influence since they include only neighbor nodes in calculation. The betweenness centrality of a node is calculated based on shortest path length, which considers whole network. However, k-core percolation provides hierarchy in their structures considering both the local and the global connections at each step of k-core decomposition41 along with dynamic re-checking the degree within k-core of the networks while removing the nodes of k-1 shell29. The nodes of outer k-shells are locally central, while ones in the central shells are globally central. Hence, the k-core percolation reveals topological importance of the network. It peels the network layer by layer to reveal the most internal one and provides hierarchical fingerprints29. In this study, we aimed to discover one unique central core which has the densest connection and pivotal hub of individual’s brain network.Previous studies reported hierarchical organization of human brain network. Centrality measure is one of the most commonly used methods to investigate hierarchical nature of human braink-core percolation has recently been interpreted as one part of explosive percolation42, which was once considered to be discontinuous but later continuous43 and finally proposed to be hybrid44 in its configuration dynamics according to the percolation process. In explosive percolation in complex networks, dynamic changes in network configuration were observed/simulated in a forward way, meaning how global connectivity was formed with the addition of new edges to the networks. The formation of the largest component could have been discontinuous, continuous or hybrid44. In contrast, optimal percolation was also introduced to find the vulnerable nodes for targeted attack and trial of dismantling12 eventually to have defined influencer nodes10. This idea has become more popular for understanding epidemic spread, power grid failure15 and social message propagation25, either viral or fake, and has introduced algorithms of collective influence or belief propagation13. Optimal percolation tried to reveal a minimal set of nodes and thus kept itself different from k-core percolation decomposition, saying that k-core percolation finds a group of nodes not pinpointing the nodes with the highest collective influencer score . In the optimal percolation or collective influence algorithm, of course, they proceed backward from the largest component to fragments. We followed the idea of k-core percolation; the largest component with the initial input and the adjacent matrix was reconfigured while removing the k-shell in this investigation.The k-core percolation functioned as the tool to find the survivors of this endeavor. Adoption of the k-core percolation algorithm from the literature45 easily yielded the voxels of kmax-core. We applied k-core percolation to each participant’s brain network which consisted of about 5000 voxels to reveal the innermost central core of the hierarchical structure. After reaching kmax, if one goes a step further , the largest component was disintegrated into many smaller pieces. As expected, the degree distribution of voxels belonging to the ICs of these kmax-cores ranged from the highest to the mid-level  and its contribution to the multilayered duplex interdependent brain network remain to be studied. Such is also the case with the temporal variation in kmax-core voxels and their IC-voxel composition.In the resting state, fMRI renders information on the fluctuation of BOLD signals per voxel, and evidently, the individuals are conscious, although sometimes their minds are drifting from introspection/imagination to paying attention to the milieu, full of MRI radiofrequency-derived noise or the scenes within the gantry. Individual differences in this mindset are expected, and temporal fluctuations will add up to make the stationary setting. In this study, we assumed perfect stationarity, meaning we calculated a single-digit correlation between every voxel k-core percolation was presented using flag plots , or central executive network (CEN), to vanish. Another point of merit is that voxels belonging to the SN disappear completely in a few individuals but remain in the others. Regarding DAN, CEN, AN, voxels seemed to vanish, but a small number of voxels belonging to these ICs remained definitely and joined the group of voxels of kmax-core. This interesting phenomenon on k-core percolation is just reported here and will be the subject of further study to understand the conscious resting-state of mind in normal individuals and its correlates on rsfMRI.Using the adjacency matrix and its consequent network, which defined the stationary voxel correlations at rest on rsfMRI, we proceeded to visualize the configuration in 2 dimensions with the hyperbolic disc. All the voxels were designated to belong to one (rarely more than one) IC, and the behavior of dynamic change of surviving voxels at each step of ts Figs. c, 4c,d ats Figs. a,b. Flag51. Global neuronal workplace theory50 advocated distributed subnetworks interconnected onto each other in conscious states. Thus, the isolated subnetworks are not the correlates for consciousness, and instead, once connected in a network of subnetworks, consciousness is achieved, emphasizing input–output processing. In contrast, integrated information theory49 measures cause-effect power with maximally irreducible integrated information in some areas, most likely suggesting the posterior area of the brain. Irrespective of which theory suits the data better, the subnetworks participating in the maintenance of consciousness should be discovered on each modality . If it is true that the posterior area contributes to the conscious resting state, as indicated by IIT, we need to investigate whether the VN we found in this study would be in charge. A perturbation study and/or calculation of integrated information, ϕ, of IIT will also be necessary51. The method we introduced in this study will be a good platform for visualization of intervoxel correlations and for elucidation of the changes in IC-voxel composition upon k-core percolation using a flag plot for these studies.Conscious individuals, evidently in an awake resting state on examinations, and their electrophysiological or perfusion correlates on electroencephalography (EEG), magnetoencephalography (MEG) or rsfMRI were studied by various investigators to yield a representative theory of consciousness, such as global neuronal workplace theory and integrated information theory (IIT)kmax-core voxels of the parietooccipital area are dominant in more than half of the studied individuals and that VNs obviously participate in the kmax-core in the remaining distributed or DMN-dominant individuals would mean that posterior or parietooccipital areas are one of the important correlates of resting-state consciousness. This indicates that everyone had VN in their kmax-core voxels  mainly DMN and several minor ICs or (2) VN and CEN and few minors in the resting state, respectively. This might imply that dominant IC subnetworks are sustained as characteristics of the individuals and/or that in every individual, there is a fluctuation of mental states, which drift between the distributed pattern and, in some instances, enter the DMN major or the VN major states, which would also be observed by rsfMRI. Former or latter, whichever interpretation might be the fact/truth, further study with sliding window segmentation and phase-shift observation of rsfMRI is warranted.The core of the individuals showed diversity in size and IC voxel composition. This IC-voxel composition pattern was arbitrarily classified among the individuals: DMN-dominant, VN-dominant, and uneven but distributed types. Individuals with DMN or VN dominance revealed that their kmax-core, and more than half of all had voxels belonging to VNs or DMN in their kmax-core. It is interesting to know whether DMN- or VN-dominant patterns might be a drifting accentuation of these two subnetworks, while evenly distributed IC voxels are the background default state at rest in humans. Then, DMN and VN-dominant patterns are the two extremes of the spectrum, and the distributed pattern resides between them. Regarding hubs, there was a controversy that in some reports57, hub regions were composed of voxels belonging to various subnetworks and not confined to a specific system in one report. In other reports58, there was a predominance of the visual system and precuneus among hub nodes. We suggest that this kmax core IC-voxel composition be used as a fingerprint to identify and describe an individual’s physiological or pathological characteristics of their resting IC compositions of the cores.Individuals with the distributed pattern tended to have a larger size of voxels in their kmax core voxels using an established visualization method on hyperbolically embedded discs accompanying k-core percolation raised the possibility of studying stationary and dynamic functional connectivity of voxels and their hierarchy upon filtration/percolation. The k-core percolation disintegrated the initial largest component gradually but sometimes abruptly; thus, this descent pattern seemed to represent the core and subcore configuration of voxels hidden under just the simple-looking scale-free functional brain network. The hidden relation between areas/regions/voxels on rsfMRI was recently investigated with either a coactivation pattern (CAP)59 or hidden Markov model (HMM)61, both of which followed the success of elucidation of dynamic changes of various CAPs on the analysis of MEG data62 or of discovering variable HMM states and finding the transition between HMM states at rest using MEG63, respectively. On the temporal scale and/or cross-modal investigations, both CAP and HMM methods were used to understand the twitches and other trivial movement/activities of humans during imaging64 and eventually consciousness. Our method of kmax-core detection and the annotation of the kmax-core voxels to ICs upon filtration will lead us to define the hierarchical structure of core-periphery coherent gathering of voxels. The 2-dimensional display of hyperbolic discs allowed us to visualize how the kmax-core was formed by the simple rule of k-shell peeling or decomposition. This is the new platform to understand finally the awake, twitching intermittently, mentally drifting with various attention to milieu or his/her mind in the MRI gantry in a conscious resting-state of human individuals. Temporal variation65 with rsfMRI and cross-modal investigation with MEG or EEG66 will be the next step of investigation using the current method of hyperbolic disc embedding and k-core percolation.Finally, finding 67. It implies self-similarity and heterogeneity of degree distribution. To test the results of kmax-core depending on thresholding, we selected one representative participant of each three pattern (VN/DMN/Distributed pattern) and applied various thresholds . Despite the arbitrariness of threshold, patterns of kmax-core voxels were consistent . This open access data is available to those who register an account at ConnectomeDB and agree to the open access data use terms. The acquisition parameters and preprocessing steps were described in Ref.71. All methods were carried out in accordance with relevant guidelines and regulations. Participants were free of neurological diseases and psychiatric disorders . We included all 180 subjects in angular coherence analysis and 30 subjects in k-core percolation analysis .We included 180 participants from the Human Connectome Project (HCP) S1200 release, which is available with open access (www.fil.ion.ucl.ac.uk/spm/) and FMRIB Software Library 71. EPI images were corrected gradient and motion-induced distortion, and a field map-based nonlinear transform was also used to correct distortion. After images were coregistered and normalized into standard space, intensity normalization was performed. These minimal preprocessing results in 2 × 2 × 2 mm sized voxel images71. We additionally conducted smoothing using 6 mm full-width at half maximum (FWHM) of the Gaussian kernel, and bandpass filtering (0.01–0.1 Hz). Finally, we downsampled the data to reduce the computational load of voxel-based whole-brain network analysis , and 5937 voxels that consisted of the gray matter were used.rsfMRI was obtained with a 3 T scanner with the following parameters: TR = 720 ms; TE = 33.1 ms; flip angle = 52°; FOV = 208 × 180 mm, 2 mm isotropic voxels. The minimally preprocessed data from HCP were further preprocessed using Statistical Parametric Mapping , SN, DAN, left CEN (L CEN), right CEN (R CEN), SMN1, SMN2, AN, VN1, VN2, VN3, VN4, and VAN. The spatial maps were manually inspected and classified based on previous studies73. To present our results in a more comprehensible summary, we also combined 15 resting-state networks into seven categories: DMN, SN, DAN, CEN, SMN, AN, and VN. Both schemes were used for functional label. The Brainnetome atlas was used to generate 15 left/right anatomical lobes, and seven lobes consisted of bilateral brain regions for anatomical labeling40.We performed independent component analysis (ICA) using MELODIC (multivariate exploratory linear optimized decomposition into independent components) to extract resting-state networks1…, XN) of a given voxel, the variance was computed by the sample variance 1…, XN) and Y = , the functional connectivity was estimated by the sample Pearson correlation coefficient For the scale of the 5937 cubic isotropic voxels, we measured blood oxygen level-dependent (BOLD) signals from each voxel of fMRI data and characterized the spontaneous fluctuations over the time series, From the sample Pearson correlation coefficient 34. Connectomes were assumed to exist in underlying geometric space, linked with the observed topologic properties through a law of connection probability that defined the likelihood if the two regions of the brain were linked. In the model, the connection probability between two voxels  k is the curvature of the plane, Mercator (available at https://github.com/networkgeometry/mercator), introduced by García-Pérez et al.31, which applied maximum likelihood techniques and a machine learning approach for embedding the network. By this means, by computing the most appropriate set of polar coordinates for The resulting binary graph from each subject was mapped onto a hyperbolic disc using the 31. The angular coherence For the assessment of narrow or widespread aggregation of voxels in the hyperbolic disc space, we used a metric called angular coherence, which was previously devised to investigate how points were angularly similar on the hyperbolic disck-core percolation to investigate individual-specific core subnetworks of brain connectivity. The k-core of a network is the maximal subgraph of the network in which all vertices have a degree of at least 29. The voxels that belonged to the k-core percolation, the changes in the size of the subnetwork at the k-core were well visualized in these flag plots over increasing k, revealing the association between the coreness of voxels and subnetworks of ICs or lobes to which the voxels belonged. Voxels and their belonging to ICs or lobes were drawn as flag plots to describe how the number of voxels consisting of each IC/lobe decreased with increasing coreness k.We performed  k increased, the step maximum was defined as the one where on step further, at kmax-core. We evaluated the size and degree distribution of this kmax core for ICs and lobes.As the corenessSupplementary Information.


Repetitive transcranial magnetic stimulation (rTMS) has shown promising efficacy in improving the language functions in poststroke aphasia. However, randomized controlled trials were lacking to investigate the rTMS-related neuroimaging changes underlying the therapeutic effects on language improvement in chronic aphasia.In this study, we aimed to evaluate the effects of low-frequency rTMS (LF-rTMS) on chronic poststroke aphasia. We hypothesized that the deactivation of the right pars triangularis could restore the balance of interhemispheric inhibition and, hence, facilitated the functional remodeling of language networks in both the hemispheres. Furthermore, the rTMS-induced functional reorganization should underpin the language recovery after rTMS.A total of 33 patients  with chronic stroke in the left hemisphere and nonfluent aphasia were recruited in this randomized double-blinded study. The ratio of randomization between the rTMS and sham groups is 17:16. All the patients received real 1-Hz rTMS or sham stimulation (placebo coil delivered < 5% of magnetic output with similar audible click-on discharge) at the right posterior pars triangularis for 10 consecutive weekdays (stroke onset to the first stimulation: 10.97 ± 10.35 months). Functional connectivity of language networks measured by resting-state fMRI was calculated and correlated to the scores of the Concise Chinese Aphasia Test by using the stepwise regression analysis.After LF-rTMS intervention, significant improvement in language functions in terms of comprehension and expression abilities was observed compared with the sham group. The rTMS group showed a significant decrease of coupling strength between right pars triangularis and pars opercularis with a strengthened connection between right pars orbitalis and angular gyrus. Furthermore, the LF-rTMS significantly enhanced the coupling strength associated with left Wernicke area. Results of regression analysis showed that the identified functional remodeling involving both the hemispheres could support and predict the language recovery after LF-rTMS treatment.We reported the therapeutic effects of LF-rTMS and corresponding functional remodeling in chronic poststroke aphasia. Our results provided neuroimage evidence reflecting the rebalance of interhemispheric inhibition induced by LF-rTMS, which could facilitate future research in the refinement of rTMS protocol to optimize the neuromodulation efficacy and benefit the clinical management of patients with stroke. Poststroke nonfluent aphasia is characterized by a disabling linguistic output resulting from the damage of language-related circuits in the brain. Patients with this condition have difficulty in performing basic communication tasks and recovering previous daily status . StudiesNeuroplasticity associated with language recovery after aphasic stroke involves both the left and right hemispheres , 13. HowResting-state fMRI (rs-fMRI) has been widely used to provide evidence of functional changes after stroke. Unlike task-based fMRI methods, rs-fMRI measures the intrinsic fluctuation of brain activities and provides a more flexible approach to investigate functional networks . Recent In this RCT, we evaluated the therapeutic effects of LF-rTMS and associated functional remodeling in patients with chronic poststroke aphasia. Patients were randomly allocated to the rTMS or sham groups. We hypothesized that the deactivation of the right pars triangularis would restore the balance of interhemispheric inhibition, thus facilitating the functional remodeling of language networks in both the brain hemispheres. Furthermore, we anticipated that the rTMS-induced functional reorganization, which involves FC enhancement in the left hemisphere and corresponding modulation in the right hemisphere, could support language recovery after rTMS. Considering the predominant lateralization of language functions and aphasia as a network disorder, we investigated the modulation of the bilateral networks and effects on language improvement relevant to treatment-induced recovery.https://clinicaltrials.gov/ct2/show/NCT03059225). All the patients were right-handed and had scores of > 90 in the Edinburgh Inventory  a first ischemic stroke affecting the left territory of the middle cerebral artery as confirmed through MRI; (2) at least 3 months after stroke onset; (3) no history of dementia, affective disorders, or other neurodegenerative diseases; and (4) absence of rTMS contraindications. Nonfluent aphasia is characterized by anomia, short phrase length (0–5 words per breath unit), with or without auditory comprehension, or repetition impairment . Phrase nventory . Figure n = 17) for 10 daily sessions or untreated . p = 0.086, two sample t-test), sex , and pretreatment language performance measured by the CCAT  were observed between the rTMS and sham groups. All the statistical analyses were performed by using the statistics toolbox in MATLAB R2020 environment.The enrolled patients were randomly allocated into one of the study groups  and eith2  with a 70-mm figure-of-eight coil. A Dantec keypoint electromyograph  was connected to the stimulator to record the motor-evoked potentials (MEPs) of the left first dorsal interosseous muscle. We recorded MEPs by using surface Ag/AgCl electrodes, probing the resting motor threshold (rMT) on a 9 cm × 9 cm grid over the motor cortex in the frontal area. The amplified (100 μV−1 mV/div) and bandpass-filtered  signals were digitized at a sampling rate of 20 kHz. Subsequently, rMT was defined as the minimum intensity at which MEPs with amplitudes of at least 50 μV could be elicited in half of the 10 consecutive trials. 3T-MRI revealed that the target area of the right posterior pars triangularis was located on the rostral part of the vertical ascending ramus of the Sylvian fissure and on the caudal part of the triangular gyrus  was used for the sham stimulation; in this approach, < 5% of the magnetic output was delivered and minimal scalp current sensation was induced. The placebo coil was identical in shape and size to the real stimulation coil and produced the same audible click on discharge as the real coil. Because none of the patients had ever undergone rTMS, they could not identify whether the stimulation was real or sham. During the initial assessment for rMT, we also used the sham coil to provide a consistent sensation as during the treatment sessions. Each patient alternately received intervention in a separate room at an appointed time to avoid conversation. Intervention for both the groups consisted of 10 daily sessions (from Monday to Friday for 2 weeks) by another TMS operator different from the one who assessed the rMT. The TMS operator was blinded to patient grouping. At the end of the first and final treatment, patients were asked to state whether they could tell if the treatment they were receiving was a real or sham stimulation. All the patients reported that they could not differentiate the type of stimulation at both the time points.No accompanying language rehabilitation training was performed during the rTMS intervention in either the rTMS or sham groups. All the participants underwent a 40-min training program by a speech-language pathologist, who was blinded to patient grouping. The training program was performed 10 min after the rTMS intervention on Monday (once a week). The training program emphasized verbal expressive skills, including repetition, phonemic training, semantic training, naming, conversation, picture description tasks, and phrase-generation tasks.A blinded speech and language therapist determined the language performance before and after rTMS intervention by using the CCAT , which wt-test was used to assess the time effects on the 12 items of the CCAT  within each group and a two-sample t-test was used to identify the significant difference of change scores (post- and pre- assessment) between the rTMS and sham groups. Cohen's d, i.e., the standardized mean difference, was further calculated to estimate the effect size  of this study was relatively small, we applied the last two terms of the equation to adjust the Cohen's d (N = 33 in this study) (where s study) .Magnetic resonance imaging data, including three-dimensional fast spoiled gradient recalled acquisition in the steady state (3D-FSPGR) T1-weighted images [repetition time/echo time (TR/TE): 9.4/4.0 ms and voxel size: 1.0 mm × 1.0 mm × 1.0 mm] and blood oxygenation-level dependent (BOLD) rs-fMRI images  were acquired on a 3T MRI scanner (GE Discovery MR750) by using an 8-channel head coil. Each patient received two MRI scans before and after the treatment to evaluate the changes in brain functional networks. The average duration between pre- and post- treatment MRI was 25.67 ± 20.89 days based on the scanner and patient's schedule (the rTMS treatment took 2 weeks). The fMRI data were preprocessed based on the standard procedures . The avet-test was conducted to examine the time effect. The significance of FC changes with control of family-wise errors was examined by using the following procedures  as p0. Finally, the permutation p-value was calculated by dividing the number of pt that was smaller than p0 by the total shuffling time  and permutation p = #(pt < p0)/T. FC changes were considered significant if permutation p < 0.001.For comparing posttreatment and pretreatment FCs for each group (intragroup test), a paired ocedures . First, s method . We denot-test (p < 0.05) was first applied to identify the significant difference of coupling strength changes (post-FCs and pre-FCs). We further performed a one-way multivariate ANOVA (MANOVA) (p < 0.05) to investigate whether the significant difference of coupling strength changes could be identified between the two groups by considering the brain as an integrated network  and F statistic vs. a constant model (with p < 0.05 as significance). The influence of each dependent variable  on the prediction of language improvement was considered significant if p < 0.05.We further performed stepwise linear regression analysis to investigate the relationship between the altered functional networks and language improvement in the rTMS group . Considet-test revealed that the improvements in total CCAT , auditory comprehension , naming , imitation writing , 4-expression , and 3-comprehension  were significantly greater in the rTMS group than that in the sham group  of functional remodeling between the rTMS and sham groups  with at least one CCAT item were within left or right hemispheres , which were correlated with the language improvement after rTMS treatment. Our findings provide insights into the neuroplastic mechanisms of functional recovery in chronic poststroke aphasia. The discussion of therapeutic mechanisms of rTMS, LF-rTMS-related FC changes, and the association between functional remodeling and language recovery are addressed in the following sections.Both the left and right hemispheres contribute to language recovery to different extents under varied hypotheses following a stroke. Evidence from studies employing neuroimaging suggested that language recovery is primarily attributed to left hemisphere reorganization, whereas the right hemisphere assists in relevant tasks , 39. In Brain stimulation methods such as rTMS might alter neural plasticity and promote language restoration through long-term potentiation and long-term depression mechanisms , 15, 41.In this study, the rTMS group exhibited favorable outcomes in terms of the total CCAT score, reflected in auditory comprehension, naming, expression, and imitation writing compared with the sham group. We demonstrated the substantial effect of rTMS in multiple domains of language skills for treating chronic poststroke aphasia.Harvey et al. applied LF-rTMS to the contralesional par triangularis in nine patients with chronic aphasia following stroke and found a long-lasting improvement in naming coupled with activation changes in the right frontal region and left hemisphere in fMRI . This reIn addition to the changes in local structure and functional activation after stroke, recent articles have addressed the alteration of interregional coupling measured by using FC and its association with the deficit in language functions , 46. AnoOur results shown in Because few studies have focused on LF-rTMS effects on patients with chronic stroke, rTMS-related neuroimaging evidence to support and elucidate language improvement remains unclear. In this study, we applied stepwise linear regression, in which we considered multiple independent variables (multiple FC changes) for investigating the relationship between functional remodeling and language improvement after rTMS treatment. Compared with the univariate correlation analysis between each FC change and language performance, the multivariate linear regression analysis is more flexible for considering the interactions between FCs and assess the comprehensive effect of network remodeling on language improvement . Among tThe FCs selected in the regression models were associated with the Broca area, Wernicke area, Geschwind area, striatum, and cerebellum in both the hemispheres. Comprehension of spoken or written words requires the engagement of the superior temporal gyrus, whereas only the left hemisphere is involved in nonlexical phonemic recording . The lefIt is worth to note that in a tonal language, such as Chinese, pitch can distinguish word meanings, while in a nontonal language, such as English, pitch is used to convey the lexical and intonation in phonetic . PreviouWe summarized the neuroplastic mechanism underlying the LF-rTMS treatment based on our findings in per session during the rTMS treatment instead of 1,200 pulses per session, as used in some other studies. This discrepancy in conditioning pulses may influence the modulation to the functional remodeling measured by the FC changes. Third, the recruited patients received language therapy once a week after the rTMS intervention. The rTMS might boost receptiveness to the language therapy session. To reduce this synergy effect, we have already reduced the frequency of language therapy to once a week. However, we were unable to completely eliminate this potential confound factor considering the patients' right to receive the language therapy. Fourth, a larger patient cohort may be required in the further study to confirm our findings. Finally, heterogeneity of lesions can be a critical factor in most stroke studies. The effects of lesion variability should be carefully addressed in the future studies.Several issues of this study should be further recognized. First, this study had a relatively high exclusion rate. We excluded 21 patients because of significant head motions during the fMRI scan. Some of the patients with stroke had difficulty in fixing the head position. Despite the high exclusion rate, the final number of included participants (33 patients) was still higher than those of most prospective rTMS studies, which have enrolled fewer than 10 participants. Second, we applied a protocol with 900 pulses Further considerations for the future studies might be proposed. In addition to nonfluent aphasia, LF-rTMS modulation on the right pars triangularis also resulted in concomitant improvement in language function in patients with Wernicke's aphasia in our experience. This observation suggests that fluent aphasia might benefit from this approach through the strengthening of the Wernicke-related circuits, even though the LF-rTMS target is not directly on the perceptive pathway .In this study, we demonstrated the promising therapeutic effects of LF-rTMS on patients with chronic aphasia following stroke. The rTMS group exhibited significant improvement in the comprehension and expression of language functions compared with the sham group. Our data further indicated that the rTMS-induced functional remodeling, involving the core language cortices and supplemental modulating areas in both the hemispheres, supported the language recovery. The engagement of the identified circuits may inspire future research on refining the rTMS protocol to improve the therapeutic effects on chronic poststroke aphasia.The raw data cannot be made publicly available for ethical and legal reasons. However, researchers can submit inquiries for analyzed data to the corresponding authors upon reasonable request.The studies involving human participants were reviewed and approved by the local Institutional Review Board in Taipei Veterans General Hospital, Taiwan. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.C-FL and P-YT: conceptualization, funding acquisition, resources, and supervision. B-FL and C-FL: methodology and writing—original draft preparation. B-FL, C-FL, and P-YT: formal analysis and investigation. B-FL, S-CY, Y-CK, C-FL, and P-YT: writing—review and editing. All authors contributed to the article and approved the submitted version.This study was funded by Ministry of Science and Technology, Taiwan  and Veterans General Hospitals and University System of Taiwan Joint Research Program (VGHUST110-G7-2-2).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Since the emergence of the COVID-19 pandemic, there have been lots of published work examining the association between COVID-19 and mental health, particularly, anxiety and depression in the general populations and disease subpopulations globally. Depression is a debilitating disorder affecting individuals' level of bio-psychological-social functioning across different age groups. Since almost all studies were cross-sectional studies, there seems to be a lack of robust, large-scale, and technological-based interventional studies to restore the general public's optimal psychosocial wellbeing amidst the COVID-19 pandemic. Transcranial pulse stimulation (TPS) is a relatively new non-intrusive brain stimulation (NIBS) technology, and only a paucity of studies was conducted related to the TPS treatment on older adults with mild neurocognitive disorders. However, there is by far no study conducted on young adults with major depressive disorder nationwide. This gives us the impetus to execute the first nationwide study evaluating the efficacy of TPS on the treatment of depression among young adults in Hong Kong.This study proposes a two-armed single-blinded randomised controlled trial including TPS as an intervention group and a waitlist control group. Both groups will be measured at baseline (T1), immediately after the intervention (T2), and at the 3- month follow-up (T3).A total of 30 community-dwelling subjects who are aged 18 and above and diagnosed with major depressive disorder (MDD) will be recruited in this study. All subjects will be computer randomised into either the intervention group or the waitlist control group, balanced by gender and age on a 1:1 ratio.All subjects in each group will have to undertake functional MRI (fMRI) before and after six 30-min TPS sessions, which will be completed in 2 weeks' time.Baseline measurements and post-TPS evaluation of the psychological outcomes  will also be conducted on all participants. A 3-month follow-up period will be usedp < 0.05.to assess the long-term sustainability of the TPS intervention. For statistical analysis, ANOVA with repeated measures will be used to analyse data. Missing data were managed by multiple mutations. The level of significance will be set to Results of this study will be used to inform health policy to determine whether TPS could be considered as a top treatment option for MDD.ClinicalTrials.gov, identifier: NCT05006365. Since the emergence of the COVID-19 pandemic, there have been lots of published work examining the association between COVID-19 and depression in the global general populations  and vulnN = 25,053) between March 24 and April 30, 2020 amidst the COVID-19 pandemic , among whom 22% exhibited SI. In addition, Hong Kong had the highest depression prevalence among five Asian collaborative countries in this multi-country study. Prolonged social isolation, loneliness, and social distancing may inevitably increase the likelihood of developing depression during the pandemic and beyond.Our project team has conducted a multi-country cross-sectional study on 10 countries  score ≥ 10)   during tre ≥ 10) .n = 584) reported psychological problems, and 14.4% reported posttraumatic stress symptoms. These pieces of evidence suggest that COVID-19 has a significant impact on youth mental health. Nonetheless, it is not uncommon for young adults with probable depression to avoid seeking professional help. Common reasons for refusal include lack of trust in the treatment and fear of social consequences of help-seeking  such as transcranial direct current stimulation (tDCS)  and tranTranscranial pulse stimulation uses repetitive single ultrashort pulses to stimulate the human brain. With a neuro-navigation device, TPS can target the human brain in a highly focal and precise manner . TPS difThe basic mechanism of TPS is mechanotransduction. TPS can promote the formation of new blood vessels (angiogenesis) and nerve regeneration, stimulate vascular growth factors (VEGF) , 35 and Since TPS is the latest NIBS device, the underlying mechanism of TPS on how ultrashort ultrasound shock waves may affect neurons and generate neuroplastic effects remain elusive, despite recent TPS studies providing further insight , 43. NonA receptors, wherein affected GABAergic transmission is believed as a mechanism of MDD (Dopamine (DA) is a neurotransmitter that plays an important role in learning, reward, motor control, emotion, and executive functions. Dysfunction of the frontal-striatal dopamine circuit is thought to be involved in depression . Thus, fm of MDD .Focused ultrasound demonstrated the neuromodulation effect in the human brain. Focused ultrasound can modulate the amplitude of somatosensory evoked potentials (SEPs) when targeted at the cortical regions  and the Past research showed that TPS was effective in treating patients with unresponsive wakefulness syndrome. Five patients received 4-week (3 times per week) TPS treatment, having 4,000 pulses each, every 6 months for an average of 2–4 years. Patients showed significant improvement in vigilance, of which three patients' percutaneous endoscopic gastrostomy (PEG) tube could be removed due to improved oropharyngeal motor function .p = 0.005) and Beck Depression Inventory (BDI) (p < 0.0001) at 1- and 3-months post-stimulation follow-up compared with the baseline scores  score (immediately after intervention and at 1- and 3-month after intervention). Functional MRI (fMRI) also showed significantly increased connectivity within the memory network . Beisteie scores . Increase scores  in the 3The aims of this study are to (1) evaluate the effects of TPS on symptoms of depression among young adults in Hong Kong; (2) improve young adults' anhedonia symptoms after the 2-week TPS intervention and will be maintained at 3-month post-intervention follow-up; (3) improve young adults' instrumental activities of daily living (IADL) after the 2-week TPS intervention and will be maintained at 3-month post-intervention follow-up; (4) improve young adults' cognition after the 2-week TPS intervention and will be maintained at 3-month post-intervention follow-up; (5) evaluate if there are any structural and functional connectivity in participants' brain after the 2-week TPS intervention compared to pre-TPS MRI scan.Primary objective: To evaluate the effects of TPS on symptoms of depression among young adults in Hong Kong.Secondary objectives:To improve young adults' anhedonia symptoms after the 2-week TPS intervention and will be maintained at 3-month post-intervention follow-upTo improve young adults' instrumental activities of daily living (IADL) after the 2-week TPS intervention and will be maintained at 3-month post-intervention follow-up.To improve young adults' cognition after the 2-week TPS intervention and will be maintained at 3-month post-intervention follow-up.Post-MRI scan will show a remarkable structural and functional connectivity in participants' brains after the 2-week TPS intervention compared to pre-MRI scan.1), immediately after the intervention (T2), and at the 3-month follow-up (T3). A total of 30 community-dwelling subjects, aged 18 and above, will be recruited. All subjects will be computer randomised into either the intervention group or the waitlist control group, balanced by gender and age on a 1:1 ratio. All subjects in each group will have to undertake fMRI before and after six 30-min TPS sessions, which will be completed in 2 weeks' time. Baseline measurements and post-TPS evaluation of the psychological outcomes  will also be conducted on all participants. Based on the previous studies, a 3-month follow-up is sufficient to assess the long-term sustainability of the TPS intervention  statement . In our rvention .2 and pulse frequencies of 4–5 Hz (pulses per second). During the TPS session, participants will sit in a comfortable chair in the treatment venue. Participants will wear a BodyTrack system consisting of a 3D camera, tracking glasses with markers, and a TPS handpiece with markers -licenced medical staff/other trained medical professionals will deliver the intervention. The TPS system consists of a mobile single transducer and an infrared camera system for MR-based neuro-navigation . TPS generated single ultrashort (3 μs) ultrasound pulses with typical energy levels of 0.2–0.25 mJ/mm markers . This BoWe will target the left DLPFC while guided by real-time MRI brain imaging. The selected brain region is based on previous research  in deprevia variable stand-offs at the handpiece for depth regulation and manual movement of the handpiece over the skull with real-time visualisation of the individual pulses on the patients' MR brain images. The treatment will be done by a trained staff holding the applicator in their hand. The whole treatment session will be recorded for post-hoc evaluation of the individual intracerebral pulse localisation  age ≥ 18 years; (2) able to understand/read Chinese; (3) obtain a Hamilton Depression Rating Scale (HAM-D-17) score of ≥ 8.Patients will be excluded if they corresponded to the following characteristics: (1) individuals being prescribed a DSM-5 diagnosis other than a major depressive disorder ; (2) alcohol or substance dependence; (3) concomitant unstable major medical conditions or major neurological conditions such as brain tumour and brain aneurysm; (4) haemophilia or other blood clotting disorders or thrombosis; (5) significant communicative impairments; (6) having a metal implant in the brain or treated area of the head; (7) those who undertook corticosteroid treatment within the last 6 weeks before the first TPS treatment; (8) pregnant or breastfeeding women.The primary investigator will obtain written informed consent from all subjects in this trial.The original written consent will cover the approval of receiving neuroimaging (MRI) scan. No biological specimens or ancillary studies will be involved for additional consents.All participants involved will receive TPS treatment. The comparing groups were referred to as the waitlist control group. At the same timepoints, we will appropriately compare the effect of the TPS treatment between those who received it in the intervention group and those who did not receive it in the waitlist control group .Purpose of the intervention: The key tenets of the TPS intervention is neuromodulation, i.e., using ultrasound-based brain stimulation techniques to modulate the human brain in a focal and targeted manner  will receive six 30-min TPS sessions  in 2 weeks' time  per week, a total of 3 h). Participants will be followed up at a 3-month period after the intervention  will be delivered to participants to monitor their progress and adherence throughout the TPS study.Participants will be instructed continue to take their prescribed medications for other conditions as normal. Self-prescription of any psychotropic medications to mitigate depressive symptoms is strongly discouraged to avoid contamination of data. Throughout the intervention period, participants will be advised to avoid strenuous exercise and adhere to healthy lifestyles and regular resting periods.Participants who are finished receiving the TPS treatment will have a follow-up assessment at a 3-month period. They could contact the PI during the post-trial for any potential physical or mental health issues related to the TPS treatment.The diagnosis of MDD will be confirmed using the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders (SCID-5).We will solicit the demographic data of the patients, including age, gender, educational attainment, place of birth, marital status, number of children, financial condition, family history of depression/mood disorders, and family household income. Medical comorbidities will be assessed with the Cumulative Illness Rating Scale (CIRS). The handedness of the participants will be evaluated using the Edinburgh Handedness Inventory—Short Form. Participants' medical and psychiatric history, including the age of onset of major physical illnesses, depressive symptoms (with or without current medication), and treatment /drug dosage, will also be recorded at baseline.Hamilton Depression Rating Scale-17  will be The core symptom of depression will be assessed by the Chinese version of the Snaith-Hamilton Pleasure Scale . SHAPS is a 14-item scale assessing participants' hedonic experience in the last few days. SHAPS includes four domains , rated on a 4-point Likert-type scale . Total score will range from 14 to 56, with higher scores representing more pronounced anhedonia. It is one of the most widely used self-report questionnaires in clinical research for evaluating anhedonia with good psychometric properties .Instrumental activities of daily living will be assessed by the Hong Kong Chinese version of the Lawton Instrumental Activities of Daily Living Scale. This IADL is a valid and reliable tool to assess the daily functioning of Hong Kong adults . The scaGlobal cognition will be measured using the Hong Kong Chinese version of the Montreal Cognitive Assessment (MoCA) . The MoC2 and two b = 0 images. The imaging parameters were the following: TR/TE = 1,200/82 ms, matrix size = 128 × 128, field of view (FOV) = 240 mm, slice thickness = 3 mm with no intersection gap, number of excitations = 2, and number of slices = 67. Finally, resting-state fMRI of 150 T2-weighted gradient echo-planar imaging (EPI) will be acquired with TR = 2 s and TE = 32 ms, with 32 slices, and a resolution of 3 × 3 × 4 mm, during which subjects will view a fixation cross (“+”) passively at the centre of the screen. Images processing and analysis will be performed using software packages including FSL (http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/), Freesurfer, and SPM8 . Total brain and its total grey and white matter volumes will be extracted from the T1 structural scan. Grey matter and white matter tissue maps will be segmented and compared for regional tissue density differences using voxel-based morphometry (VBM) , and resting state-fMRI (rs-fMRI) will be performed using a 3T scanner at the University Research Facility in Behavioural and Systems Neuroscience, The Hong Kong Polytechnic University. The subjects will be closely monitored by the research assistant and the radiographer during scanning. The whole scan will last around 45 min including preparation. Structural MRI scans, including T1- and T2-weighted fluid attenuation inversion recovery (T2-FLAIR) sequences, will be used for assessing regional volume differences across the whole brain. High resolution sagittal 3D T1-weighted [spoiled gradient recalled echo (SPGR)/magnetisation-prepared rapid gradient echo (MPRAGE)] images of 1 mm x 1 mm x 1 mm were acquired with repetition time (TR) = 1,820 ms, echo time (TE) = 3.75 ms, inversion time (TI) = 1,100 ms, and flip angle = 70°. This will be followed with axial 2D-T2-weighted FLAIR images of 0.9 × 0.9 × 3 mm, which will be acquired with TR = 9,160 ms, TE = 90 ms, TI = 2,500 ms, and flip angle = 150°, for the quantification of white matter hyperintensities (WMH). DTI sequence will be conducted using a single-shot spin-echo echo-planar imaging, with diffusion-sensitising gradients applied along 16 non-collinear directions with diffusion weighting factor b = 1,000 s/mmry (VBM) . Structuhttps://sites.google.com/site/bctnet/). A dual regression approach will be used to investigate between-group differences in the individual functional networks. The significance threshold of the voxel-wise differences will be set at p < 0.05 (family-wise error corrected).For functional connectivity, all resting state-fMRI (rs-fMRI) volumes will be pre-processed, with motion correction and slice timing correction, and then linearly registered to the Montreal Neurological Institute (MNI) standard space. A data-driven approach will be used for the analysis of rs-fMRI data. Independent component analysis will be done with Multivariate Exploratory Linear Decomposition in FSL. A set of independent components will be identified as the common resting-state functional networks. The global and local efficiency, modularity, and hubs will be computed using the Brain Connectivity Toolbox  to ensure concealment of randomisation. To avoid information flow, participants in both groups will be advised not to reveal their study participants to the other group, to minimise potential contamination of the effects of TPS or subject bias. The participant list will be concealed from the recruitment venues throughout the study.An independent statistician will use a computer-generated list of random numbers , immediately after the intervention (T2), and at the 3-month follow-up  will be delivered to participants to monitor their progress and adherence throughout the TPS study.The DMP is enforced according to the Personal Data (Privacy) Ordinance, Code of Ethics for Research Involving Human Subjects, and the Policy on Ownership of Intellectual Property issued by the PolyU. All the anonymized data including fMRI scans, laboratory results and documentation will be converted into.csv, and.pdf formats for long-term preservation and access and stored in a Figshare and PolyU home drive for 5 years with a DataCite DOI as an identifier.All consenting participants will be listed in alphabetical order according to their surnames, and each participant will be assigned a unique study number. All personal information collected for potential and enrolled participants will only be shared under the supervision of the principal investigator. All confidential data will be kept and maintained by the principal investigator after the trial ends in case any emergencies will be raised from participants. The confidential data will be destroyed once publications have been made.Not applicable.p-values of < 0.05 (two-tailed) will be considered significant. For Objective 5, the neurological rs-fMRI data, longitudinal VBM will be used to examine whether TPS produces local changes in grey matter. Specifically, relative local increases and decreases between pre- and post-intervention scans will be compared within and between study groups. The diffusion MR data will be analysed using the diffusion tensor model. After a mathematical diagonalization process, the eigenvectors and eigenvalues describing the tensor ellipsoid will be determined. Then, two standard diffusion indices will be obtained which are the following: (1) the apparent diffusion coefficient and (2) the fractional anisotropy. DTI scans pre- and post-treatment will permit the assessment of changes in axial diffusivity, with lower values being interpreted as the structural enhancement of white matter. For the cognitive data analysis, general linear modelling (GLM) will be used to compare the difference in performance in the forward and backward digit span, the Stroop test, the category verbal fluency test, and the TMT Parts A and B between the TPS group and the waitlist control group. Performance on the TMT between the TPS group and the waitlist control group will be analysed by one-way repeated-measures ANOVA. Participants' narrative feedback on TPS will be analysed with descriptive content analysis.Data will be analysed by the SPSS for Windows, version 27. The normality of the primary outcome (depression) scores will be tested by the Kolmogorov-Smirnov test. If significant differences between sociodemographic variables are found, covariates will be used as a control or confounding variables in the main analysis. For Objective 1, multilevel modelling analysis will be used to investigate changes in the outcome measures (depression) at each time interval (2 weeks and 3 months) with respect to their baseline measurements to examine the effects of TPS. A two-level model will be built, with repeated time points at level 1 and respondents at level 2. The baseline difference between groups will be tested using the Wald test based on the regression coefficient estimates of the indicator. The changes in the outcome measures over time will be included in the fixed part of the model to estimate change from baseline to 2 weeks and from baseline to 3 months. To assess the effects of TPS over time, interaction terms will be included in the fixed part of the model. The significantly different demographics between groups identified in step one of the analysis and other possible confounders will be adjusted by including them in the main-effect model. The within-respondent random effects of the outcome measurements will be taken into account by the level-2 variance in the model. For Objectives 2, 3, and 4, pairwise comparison and other hypotheses on the intervention effects will be tested using a generalised Wald test with an adequate degree of freedom from the modelling analysis . The samOnly the PI will have access to all participants' data. Should any participants suffer from any adverse effect during or after the TPS treatment, they will be duly assessed by the PI and TPS treatment will be terminated as deemed necessary.Subgroup analyses will be conducted between the TPS group and the waitlist control group in terms of the primary and secondary outcomes.Multiple imputations will be used to manage missing data in any treatment phrases.Anonymous data is available on request.Since this is a small-scale study comprising 30 subjects, the PI will closely monitor the progress and intervention of this trial. In addition, the PI will organise regular group meetings with the project team members to update the progress of the study until the completion of this trial.Data monitoring committee is not required in this trial as there is no competing interest between the sponsor and the project team members. The sponsor is not involved in the research design, data collection, and execution of this study.In vivo animal TPS study did not cause any tissue damage despite using 6- to 7-fold higher energy levels compared with those in human studies. Furthermore, the intervention did not cause any serious adverse effects such as intracranial bleeding, oedema or other intracranial pathology, as confirmed with MRI in a previous AD study. Few subjects reported headache (4%), pain or pressure (1%), and mood deterioration (3%)  . The CliA checklist of potential adverse effects associated with TPS administration will be generated from existing literature . The cheThe PI will be responsible to conduct random and non-random audit throughout the trial period, ensuring that the protocol and TPS intervention has strictly adhered to protocol.Should there be any changes in the eligibility criteria or outcome measurements, IRB and research ethics committee in The Hong Kong Polytechnic University will be immediately notified.via international journal publications, international conferences, and press conferences.Investigators will disseminate the study findings Our results will highlight the efficacy and long-term sustainability of TPS in the treatment of depressive symptoms among young adults. Our findings may add new knowledge on the efficacy of using TPS as a non-intrusive, and non-pharmacological treatment of depression in Psychiatry. It will also lay down the groundwork for a larger clinical trial in the near future in order to evaluate if this new treatment modality in psychiatry can be used to treat patients suffering from other types of neuropsychiatric disorders.Using NIBS in the treatment of neuropsychiatric disorders is the future trend in the field of psychiatry and neuroscience. Existing findings have already proven that TPS is an effective treatment modality in Alzheimer's Disease. Our study findings will further support the notion that TPS can also be used in the treatment of MDD. Study findings may have a significant impact on researchers/clinicians, individuals who are treatment-resistant to all sorts of antidepressants, and/or those who have poor drug compliance and insight into their neuropsychiatric disorders.Since TPS is a relatively new NIBS technology, only a paucity of studies was conducted on older adults with mild NCD . There iTo the best of our knowledge, our proposed study is the first pilot study to use TPS to treat young adults with depression in Europe, Asia, and Hong Kong.Health policy-makers and researchers are working to ameliorate hard to formulate effective mental health interventions to curb the depression epidemic brought by the pandemic situations , 72 and There are some limitations that need to be addressed in this protocol. First, since we will execute the first pilot RCT nationwide interrogating the effects of TPS on MDD, we can only use existing NIBS studies like TMS and tDCS to act as a comparison for our findings. Since both TMS and tDCS use non-invasive neuromodulation technology by electrical stimulation, the mechanism will be somehow different from TPS which uses ultrashort ultrasound waves in the stimulation of the human brain. Thus, our findings will need to be interpreted with caution. Second, participants in the waitlist control group will be given the identical TPS intervention, albeit in different timepoints, but they will all receive the same TPS pulse/frequency/energy in this TPS trial. Third, the TPS machine itself may have a potential placebo effect which may bias the results. Fourth, there could be a potential risk of contamination if our waitlist control group utilised self-prescribed pharmaceutical input or other TCM modalities  to treat their symptoms of depression while waiting the TPS treatment. Such contamination may impact the receptivity and efficacy of our TPS intervention. Fifth, participants will be administered the same sets of psychological instruments at three different time points so there is a potential risk that some participants may recall some contents of these instruments. To prevent this, our research personnel will not provide accurate answers to these tests throughout each study phase to minimise recall bias. Last but not least, individuals with severe depression with fleeting suicidal ideas will be excluded in this study, and thus, the efficacy of TPS on these severely depressive and suicidal community-dwelling individuals will remain unknown. Future replication of a multi-centre study may adopt a double-blinded, sham-controlled RCT to philtre out the placebo effects on a larger sample.ClinicalTrials.gov on August 16, 2021 (protocol version). Recruitment commenced on 1 August 2021 and was completed on 30 October 2021.This trial was registered with The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.All participants are required to provide written informed consent to participate in this study. Ethical approval will be sought from the Human Subjects Ethics Sub-committee, The Hong Kong Polytechnic University. Research Safety Approval will also be obtained prior to the commencement of the study. This study adheres strictly to the Declaration of Helsinki ethical principles developed by the World Medical Association. All participants will be covered by trial insurance. Potential risks involved in fMRI will be clearly indicated in the Information Sheet. Voluntary participation, anonymity and confidentiality, and the right to withdraw are respected. All participants will be given a HK$50 supermarket cash coupon for their time and travel-related expenses for the pre-and post-treatment fMRI.TC, YH, KF, RB, CC, and GK conceived the study. TC and CC designed, executed the study, administered the TPS, and arranged neuroimaging logistics. YH assisted in the administration of TPS and baseline and post-TPS measurements. GK, CC, and TF assisted in data entry and administration of baseline and post-TPS measurements. RB, KF, and CC offered expert advice in fMRI data analysis and manuscript writing. All authors contributed to the article and approved the submitted version.This work was supported by the Departmental General Research Funds, The Hong Kong Polytechnic University, Hong Kong (Grant Number P0036773). RB is supported by the Herzfelder Stiftung, Austria.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


PMAP-23, a cathelicidin-derived host defense peptide, does not cause severe membrane permeabilization, but exerts strong and broad-spectrum bactericidal activity. We have previously shown that it forms an amphipathic α-helical structure with a central hinge induced by the PXXP motif, which is implicated in the interaction of PMAP-23 with negatively charged bacterial membranes. Here, we studied the potential roles of the PXXP motif in PMAP-23 translocation across the lipid bilayer by replacing Pro residues with either α-helix former Ala (PMAP-PA) or α-helix breaker Gly (PMAP-PG). Although both PMAP-PA and PMAP-PG led to effective membrane depolarization and permeabilization, they showed less antimicrobial activity than wild-type PMAP-23. Interestingly, we observed that PMAP-23 crossed lipid bilayers much more efficiently than its Pro-substituted derivatives. The fact that the Gly-induced hinge was unable to replace the PXXP motif in PMAP-23 translocation suggests that the PXXP motif has unique structural properties other than the central hinge. Surface plasmon resonance sensorgrams showed that the running buffer almost entirely dissociated PMAP-23 from the membrane surface, while its Pro-substituted derivatives remained significantly bound to the membrane. In addition, kinetic analysis of the sensorgrams revealed that the central PXXP motif allows PMAP-23 to rapidly translocate at the interface between the hydrophilic and hydrophobic phases. Taken together, we propose that the structural and kinetic understanding of the PXXP motif in peptide translocation could greatly aid the development of novel antimicrobial peptides with intracellular targets by promoting peptide entry into bacterial cells. Antimicrobial peptides (AMPs) provide the first line of host defense and are capable of killing or inhibiting a wide variety of bacterial pathogens ,2,3. AMPMany studies on peptide-lipid interactions using several lipid model systems have been performed to elucidate key aspects of the mode of action of AMP. For example, previous studies have revealed how the hydrophobicity and secondary structure play a crucial role in the process of interaction with lipid bilayers other than the overall net charge for magainin 2, an important class of AMPs ,22,23. RCathelicidins are the largest family of AMPs found in invertebrates. Porcine cathelicidin PMAP-23 exerts rapid and potent microbicidal activity against a broad spectrum of pathogens ,27,28. PIn this study, we investigated whether PMAP-23 translocation across lipid membranes was associated with antimicrobial activity. To examine the roles of the central PXXP motif in the peptide translocation at the molecular and structural level, two Pro residues of PMAP-23 were replaced with either α-helix stabilizing Ala or α-helix destabilizing Gly. We found that Pro to Ala or Gly substitution increased membrane depolarization and permeabilization but decreased antimicrobial activity. Interestingly, we showed that the PXXP motif enables efficient PMAP-23 translocation across the lipid bilayer. Analysis of surface plasmon resonance (SPR) sensorgrams using a two-state reaction model revealed that the PXXP motif could readily dissociate PMAP-23 from the lipid bilayer. The possible role of the central PXXP motif in the antimicrobial mechanism of PAMP-23, including primary permeabilization of bacterial membranes and secondary intracellular targets, is discussed.PMAP-23 has an N-terminal helix, a central hinge induced by the PXXP motif, and a C-terminal helix. We have previously shown that the central hinge plays an important role in maintaining the amphipathic structure and exerting strong antimicrobial activity against PMAP-23 ,13. To fS. aureus and gram-negative E. coli . When diSC3(5) was added to a suspension of S. aureus, the fluorescence intensity was strongly extinguished and stabilized after approximately 300 s, indicating the accumulation of diSC3(5) within the bacterial cytoplasmic membrane (3(5) fluorescence, reflecting membrane depolarization, followed by the addition of gramicidin D to fully dissipate the membrane potential. We found that PMAP-PA with a high degree of α-helicity caused an immediate increase in diSC3(5) fluorescence, indicating the rapid collapse of the transmembrane electrochemical gradients of S. aureus. PMAP-23 and PMAP-PG exhibited similar abilities to depolarize the bacterial membrane, but were much lower than PMAP-PA. These results suggest that the α-helicity of peptides influences their membrane depolarization activity. However, the lack of correlation between membrane depolarization and antimicrobial activity indicates that loss of membrane potential is not the main bactericidal effect of PMAP-23, and mechanisms may exist.A common feature observed for antimicrobial peptides is their ability to disrupt membrane integrity, leading to the collapse of the transmembrane potential. To determine whether membrane depolarization induced by PMAP-23 and its analogs is related to bacterial death, we assessed their ability to depolarize bacterial cytoplasmic membranes using a potential-sensitive fluorescent probe diSCmembrane . SubsequKSV values were calculated using a fluorescence quenching experiment with a water-soluble fluorescence quenching agent, acrylamide  than in PC vesicles, suggesting that Trp residues of the peptides insert more strongly and deeply into the hydrophobic core of the bacterial membrane. The lower KSV value of PMAP-PA over PMAP-23 and PMAP-PG in PC vesicles further explains the direct correlation between its binding to zwitterionic membranes and its cytotoxicity against mammalian cells.Lipid composition of the cytoplasmic membrane is very different in eukaryotic and prokaryotic cells. Eukaryotic cell membranes are predominantly composed of zwitterionic phospholipids, such as phosphatidylcholine (PC), whereas bacterial cell membranes are composed largely of negatively charged phospholipids, such as phosphatidylglycerol (PG) . We examrylamide . If the We next investigated the ability of PMAP-23 and its analogs to disrupt membranes by assessing the release of entrapped calcein from vesicles . ConsistTo determine whether the antimicrobial activity of peptides was related to their intracellular targets, we investigated the translocation of peptides through the lipid bilayer. The ability of the peptide to cross lipid bilayers was assessed by the resonance energy transfer from the Trp residue of the peptides to the dansyl group of phosphatidylethanolamine (PE) incorporated into the vesicles. When peptides translocate across the lipid bilayer, they are broken down by trypsin confined in vesicles, resulting in a loss of fluorescence intensity . We obseka1, ka2) and dissociation rate , as shown in ka1 and kd1 values involved in the first step were similar for all peptides. However, PMAP-23 showed significantly different ka2 and kd2 values in the second step compared to the other peptides. This result suggests that after binding to the membrane surface, PMAP-23 is more readily inserted into the lipid bilayer and separates from the membrane faster than other peptides. In the second step, the low association and dissociation rate of PMAP-PG suggests that the Gly induced hinge cannot interact with the membrane as fast as the PXXP motif.AMPs bind electrostatically to the membrane surface first, then insert themselves into the lipid bilayer via hydrophobic interactions. Finally, SPR was used to investigate how the kinetic interactions of peptides with membranes are related to peptide translocation, since SPR is useful for understanding the kinetic basis for the two-step process. The first step is the binding of peptides to the membrane surface, while the second step corresponds to the insertion of peptides into the hydrophobic core of the lipid bilayer. The SPR sensorgrams consisting of the association and dissociation phases are shown in A progression model of PMAP-23 translocation across lipid bilayers is illustrated in In summary, PMAP23 was bactericidal without causing significant membrane depolarization and permeabilization. Here, we found a correlation between antimicrobial activity and translocation in PMAP-23 and its analogs, and determined that the PXXP motif is a critical structural factor for the cell-penetrating property of PMAP-23. Furthermore, SPR sensorgrams demonstrated that PMAP-23 was rapidly inserted into the hydrophobic lipid core and readily separated from the membrane for translocation through the lipid bilayer. We propose that the central PXXP motif promotes dynamic association and dissociation of PMAP-23 to the lipid bilayer, allowing PMAP-23 to enter bacterial cells. From an experimental point of view, possible strategies that can be adopted to demonstrate the specific mechanism of action and the precise role of the PXXP motif may include optical super-resolution techniques or scanning probe techniques, such as atomic force microscopy. Most importantly, we believe that the PXXP motif can be applied to other AMPs with an amphipathic α-helical structure, which can be of great help in the further development of AMPs with intracellular targets and can generate multifunctional AMPs.3(5)], were purchased from Invitrogen corporation . Microorganisms were obtained from the Korean Collection for Type Cultures (KCTC). All other reagents were of analytical grade.Phospholipids and peptides were purchased from Avanti Polar Lipids Inc.  and Anygen Co. Ltd. , respectively. Fluorescent probes, including calcein and 3,3-dipropylthiacarbocyanine  = F0/F − 1, where [Q] is the concentration of acrylamide, F0 is the Trp fluorescence in the absence of acrylamide, and F is the Trp fluorescence in the presence of acrylamide. The measurements were repeated in triplicate at a given condition to ensure reproducibility.Peptide binding to membranes was estimated using Trp fluorescence measurements, as described previously . BrieflyPeptide translocation across a phospholipid bilayer was evaluated by fluorescence resonance energy transfer (FRET) from the Trp of peptides to a 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl or DNS) group of phosphatidylethanolamine (PE), as described previously ,38. BrieThe kinetics of peptide binding to and dissociation from the lipid bilayer were assessed by SPR using BIACORE 2000, as described previously . We inje