Hepatocellular carcinoma (HCC) is the primary malignancy in the liver with high rate of death and recurrence. Novel prognostic model would be crucial for early diagnosis and improved clinical decision. The study aims to provide an effective lncRNA-based signature to predict survival time and tumor recurrence for HCC. Based on public database, lncRNA-based classifiers for overall survival and tumor recurrence were built with regression analysis and cross validation strategy. According to the risk-score of the classifiers, the whole cohorts were divided into groups with high and low risk. Afterwards, the efficiency of the lncRNA-based classifiers was evaluated and compared with other clinical factors. Finally, candidate small molecules for high risk groups were further screened using drug response databases to explore potential drugs for HCC treatment. Hepatocellular carcinoma (HCC) is the most common primary malignancy in the liver and the seventh most frequent neoplasm worldwide . With moIn current clinical practice, the outcome of HCC patients was mainly assessed by models based on tumor pathological characters. The Barcelona Clinic Liver Cancer (BCLC) classification is the most used and verified system for HCC with estimated median survival periods at each tumor stage . Other sNevertheless, all these fail to incorporate molecular markers as prognostic predictive factors. Generally, biological markers are viewed as a pivotal indicator for tumor diagnosis, therapeutic effectiveness, and public tumor surveillance. Not surprisingly, a considerable amount of effort has also been made in developing novel biomarkers or signatures for HCC prognosis over the last decade. For instance, Long et al has developed a four-gene  based model to predict the overall survival of HCC patients . Liu et Despite a great number of researches have done on such genomic indexes, most of them focus on protein coding region and their effect on the patients / disease. To date, the value of non-coding RNAs in HCC prognostic assessment has not been thoroughly explored. In recent years, growing evidence has indicated the crucial role of lncRNA in multiple stages of HCC development including genesis, progression, and recurrence –15. ThesTherefore, this study aims to develop a lncRNA-based tool to monitor and predict the outcomes of HCC. In detail, the HCC cohort containing lncRNA expression and clinical data was acquired from public databases. Two lncRNA-based signatures: an 8-lncRNA contained classifier for overall survival (OS) prediction and a 6-lncRNA contained classifier for relapse-free survival (RFS) prediction, were constructed by applying COX and LASSO regression with differentially expressed lncRNAs (DElncRNAs). Subsequently, the ability as prognostic predictors of both classifiers was evaluated and compared with traditional staging systems. Last but not least, as the cohort was divided into high- and low-risk groups according to the risk score determined by the signature classifiers, potential therapeutic targets and small molecules for high-risk, poor prognosis-associated patients were explored with methods described below .The comprehensive RNA expression profile containing tumor tissue (n = 369) and adjacent control (n = 50) was accessed from the TCGA database as previously described. Of the 14089 lncRNAs extracted from the RNA-seq data, 1318 lncRNAs were identified as DElncRNA under the condition of |logFC| > 1 and adj.p < 0.05 . BesidesAccording to the screening process listed above, an 8-lncRNAs-based classifier for OS prediction and a 6-lncRNAs-based classifier for RFS prediction were constructed. The information of the elemental lncRNAs was listed in detail .Subsequently, the OS cohort was further divided into two sub-groups (high-risk and low-risk) according to the median value of the risk score calculated by the OS classifier. The distribution of risk scores, the vital status of patients, and expression of element lncRNAs were compared between high-risk and low-risk subgroups of the OS cohort –3C. The The expression of the lncRNAs from both prognostic classifiers was then compared in the high-risk group, low-risk group, and non-tumor control group, to confirm the differential expression level between the high risk and low risk group. As expected, all lncRNAs for OS prediction  and RFS The predictive capacity of both OS- and RFS signatures were evaluated in all the training, validation, and whole cohorts, respectively. Kaplan-Meier log-rank tests were conducted in all 6 groups to confirm the effectiveness and consistency of the model for both OS –5C and RAfterward, the efficiency of both classifiers was checked by the time-dependent receiver operating characteristic (ROC) curve. In the OS cohort, areas under ROC curve (AUCs) of the 8-lncRNA-based classifier reached 0.798, 0.817 and 0.841 for 1, 3, and 5 years in the training group , 0.729, As described above, the lncRNA-based signatures were proved to be a prognosis indicator with high accuracy in predicting the outcomes of both OS and RFS time for HCC patients. However, whether the risk score of the novel lncRNA-based classifiers is correlated with other clinicopathologic characteristics requires further exploration.Hence, some major clinical factors were listed and compared with the risk score with Pearson chi-square test in the OS- and RFS-cohorts, separately . The anaTo compare the efficiency of lncRNA classifiers and other prognostic factors, age, gender, pT, pN, pM, tumor stage and lncRNA-based classifiers were assessed by a two-step COX regression analysis . In OS cAs the lncRNA signatures and several pathological features concordantly showed significant correlation with HCC progression, their combined efforts in predicting HCC prognosis were further check with the Normogram analysis. In specific, the risk score is the most relevant indicator in the diagram with the total points reflecting the final prognostic probability in OS of HCC, while the T status also plays a critical role . In RFS Taking together, the lncRNA-based classifiers could be considered as an independent indicator for both OS and RFS prediction of HCC with high efficiency.To identify drug candidates for high-risk patients with our LncRNA signature, two different approaches were applied using CTRP and PRISM drug response database separately. The differential drug sensitivity was identified between high- (top 20%) and low-risk (bottom 20%) patients with lower estimated AUC values in high-risk patients (Log2FC >0.05). Following this, the spearman correlation was measured to select the candidates with a negative correlation coefficient (r < -0.2) between risk scores and AUC values with both OS and RFS signatures. With the OS- signature, seven candidates from CTRP and seven candidates from the PRISM dataset were identified , 8B. AndAs HCC, the primary malignancy in the liver, remains high frequency of recurrence and mortality despite a comprehensive treatment pool and protocol including radical resection, ablation, and recently arisen targeted- or immunotherapy. For many years, physicians and scientists solely relied on models limited in pathological classification such as TNM phase and tumor stages to predict the outcomes of HCC and making clinical decisions. However, the diverse background disease and heterogenetic nature make the accurate prediction of HCC prognosis exceedingly challenging. Under the circumstance, a novel biomarker panel could be an alternative concept and beneficial for clinical surveillance and management of HCC.In recent years, numerous pieces of evidence have shown the critical role of non-coding RNAs including miRNA, circRNA and especially lncRNA in extensive biological processes. Studies have reported that lncRNAs function as master regulators of gene transcription, mRNA processing, and nucleus modification . DysreguIn this study, one large HCC cohort (TCGA-LIHC) was split into training and validation sub-groups with the cross-validation strategy to ensure the stability of the predictive ability. Moreover, the LASSO and COX regression analyses were applied to optimize the selection of candidate lncRNAs with both high expression variances and prognostic values. Last but not least, tumor recurrence is found in more than 60% of HCC patients within 5 years, which reflects the poor prognosis with the progression of the disease . TherefoAfter the establishment of signature, individual samples in overlapped OS and RFS cohorts were automatically endowed with a risk score with both OS and RFS classifier. Patients with high and low-risk scores revealed a significant difference in overall and relapse-free life expectancy according to Kaplan-Meier curves. In addition, pT, pM, tumor stage and lncRNA-based classifier are all correlated with OS in univariate COX analysis. However, the 8-lncRNA-based classifier remains relevant with remarkably high efficiency in the following multivariate COX regression analysis compared to other models. Similarly, the pT, tumor stage and 6-lncRNA-based classifier are related to RFS in univariate COX but only the lncRNA classifier remains the sole significant indicator in the multivariate COX regression model. Additionally, the classifiers exhibit superior accuracy in prognostic prediction, with AUCs exceeding 0.75 in all 1, 3, and 5 years timepoint for OS prediction, and also reaching over 0.7 for RFS prediction. In comparison, the AUCs of tumor stage as a predictor are only approximate 0.6, apparently inferior to lncRNA-based classifier in both cases .Interestingly, although the two lncRNA-based classifiers are proven to be promising predictors for HCC prognosis, lncRNAs forming the signature remain largely unstudied in tumor biology. To note, the finding that elemental lncRNAs had little mutual correlation in expression suggests they might have different or irrelevant mechanisms .Among all the lncRNAs consisting the OS classifier, LINC02580 was reported strongly down-regulated in HCC compared with normal liver—consist with our study—and low expression of LINC02580 linked with poor prognosis. Gene SRSF1 mediating genetic alternative splicing was likely the target of LNC02580 but the detailed mechanism remained unexplored . MoreoveTo further identify the potential drug targets and candidate small molecules for the high-risk patients, two drug response datasets (CTRP and PRISM) were applied for small molecules screening and the CMap database was supplied with MoA information. The top enriched drug targets are HMGCR inhibitors and topoisomerase inhibitors.Statins were widely used in patients to lower cholesterol to reduce the risk of a heart attack or stroke. As HMGCR inhibitors, statins were reported associated with reduced risk of HCC development in chronic HBV-infected patients, HCV-infected patients, and diabetes patients , 32. MorIn conclusion, this study generated paired novel lncRNA-based signatures to predict both the overall survival and recurrence of HCC. The superior effectiveness and efficiency of the model as independent prognosis indicator have been demonstrated in different manners. The application of the lncRNA-based signature, either alone or in combined efforts with other clinical factors, tend to provide novel solution for improved prognosis anticipation and clinical management of HCC, and eventually benefit both patients and doctors. But before achieving this, more efforts on validation and mechanistic exploration on these genes are still in significant need.An RNA-seq dataset of 371 HCC patients involving RNA sequencing and matched clinical characteristics were obtained from the TCGA data portal . The cohort contains 374 HCC tumor tissues and 50 adjacent liver tissue as control, and the matched clinical information was acquired from Cbioportal  .http://gencodegenes.org). Then the lncRNA was separated from gene-coding RNA and other non-coding RNAs. lncRNAs with zero counts were excluded. Differentially expressed lncRNAs (DElncRNAs) were identified by the R programming edgeR package with the criteria of |logFC| > 1 and adj.p < 0.05 between tumor and control tissues.Genome-wide all RNA expression was acquired from the TCGA dataset as described above. The data were annotated by the Gencode GTF file . Eventually, the candidate prognostic lncRNAs were determined by overlapping the DElncRNAs and univariate COX positive lncRNAs for further analysis.Next, both the OS and RFS cohorts were randomly split into training and validation groups at a 2:1 ratio. The LASSO regression was performed at 20-fold cross-validation in the two training groups to generate an 8-lncRNA-based OS classifier and a 6-lncRNA-based RFS classifier. According to the cut-off median value of the risk score, the OS and RFS cohorts were divided into high- and low-risk groups, then the expression of element lncRNAs within the classifiers in control, low-risk, and high-risk groups were compared.The predicting capability of the lncRNA classifiers in both training, validation, and whole cohort were subsequently confirmed by the Kaplan-Meier long-rank test, Time-dependent ROC curve analysis, and multivariate COX regression. All the analyses were conducted using GraphPad Prism 7 and R platform version 4.0.2 with packages ‘edgeR’, ‘carnet’, ‘survmine’, ‘glmnet’, and ‘ROCR’.https://depmap.org/repurposing/). The algorism of drug sensitivity was described in previous studies [https://clue.io/) with 2429 small molecules perturbagen types was applied for specific analysis [Drug sensitivity data of human cancer cell lines were achieved from the Cancer Therapeutics Response Portal  and PRISM repurposing dataset ( studies . Brieflyanalysis .Supplementary Figures


In major depressive disorder (MDD), self-blame-related fMRI measures have shown the potential to be used as prognostic markers for recurrence risk. Like most potential fMRI markers, however, their reliability is unclear. Here, we probed the internal reliability of self-blame-related fMRI measures, as well as the impact of different modelling approaches on reliability metrics and validity.a priori ROIs: right superior anterior temporal lobe seed region, anterior subgenual cingulate cortex, posterior subgenual cortex and right striatum / pallidum.Internal consistency  was calculated for blood oxygen level-dependent (BOLD) responses and psychophysiological interactions (PPI) related to self-blame-related biases in medication-free remitted MDD participants (n = 81) and healthy controls (n = 41). Trial-length was modelled using three durations , which was convolved with the haemodynamic response function (HRF) with and without time and dispersion derivatives. Intraclass correlation coefficients (ICCs) were calculated for simple contrasts examining activation to self-blaming emotions and other-blaming emotions and the more complex contrast of the subtraction-based difference between self- and other-blaming emotions within the following Across ROIs, we obtained fair reliability (ICC ≥ 0.40) for simple, but poor reliability (ICC < 0.40) for more complex fMRI measures related to self-blame. Despite this low internal consistency of complex measures at the individual level, we observed robust activation at the group-level, reproducing previously published results.While simple BOLD contrasts had fair reliability, previously employed PPI models had poor reliability and simple connectivity measures lacked predictive validity. This calls for the development of functional connectivity measures that strike a better balance between reliability and validity for future clinical applications, which require robust measures at the individual rather than group-level. A recenOne commonly used measure for estimating reliability is the intraclass correlation coefficient (ICC), which gives an index of similarity across repeated measurements . There aReliability metrics like ICC are dependent on the modelling approach, such as choice of haemodynamic response function (HRF), choice of regressors and definition of contrasts . For insIn addition to regional BOLD activation, functional connectivity   are of hMore and more studies are reporting on test–retest reliability, albeit with a limited focus on clinical populations and relatively small samples . Test-reEven though internal consistency places a limit on validity of a measure and on its sensitivity to detect individual difference effects , few stuHere, we investigated the internal consistency of an fMRI paradigm designed to measure self-blame-related biases in MDD, for which we have shown internal validity as predictive of recurrence risk . This paHowever, it is unknown to what extent these measures are reliable. Thus, the aim of this study was to probe the internal reliability of self-blame-related fMRI measures, using regions-of-interest based on these previous findings: RSATL seed region, anterior subgenual cingulate cortex, posterior subgenual cortex and right striatum / pallidum . Given t22.1The fMRI dataset used to assess reliability and reproducibility was collected as part of a previously published longitudinal study, which examined whether self-blame-selective alterations in anterior temporal fMRI connectivity predict subsequent recurrence of depression see . EthicalParticipants in the remitted MDD group fulfilled criteria for MDD according to Diagnostic and Statistical Manual of Mental Disorders  and wereFor the primary imaging analysis, 109 participants were included , meeting strict criteria for signal dropout  and movement (translation < 7 mm or rotation < 5 degrees). An additional thirteen participants, who did not meet the strictest quality control threshold, were included in further analysis . Please see As previously described by Green and colleagues , an fMRIAs demonstrated by measurements of the temporal signal-to-noise, i.e. “the mean of a voxel’s BOLD signal over time divided by its standard deviation over time” , overall2.3The fMRI task has been described in detail previously . In shorStimuli were presented for five seconds, followed by a jittered inter-trial interval with a mean duration of four seconds, and participants were asked to report how unpleasant they would find this  via a button press. A baseline visual fixation pattern  was pseudo-randomly interspersed across three runs, the order of which was counterbalanced across participants. For more details, please see .After the scanning session, participants rated the degree of unpleasantness associated with each stimulus on a 7-point Likert scale . Self- and other-blaming emotion trials were defined as those that were perceived as highly unpleasant  in the respective self- and other-agency conditions.2.4Pre-processed functional images were made available by the original researchers; these had been realigned, warped, co-registered to the participant’s T1-weighted images, normalised, and smoothed with a kernel of 6mm full-width-half-maximum. Each participant’s batch file was manually checked for the order of runs and the vector onsets against raw E-Prime files . Seven participants showed minor discrepancies in the order of runs and vector onsets, which were corrected before the modelling.2.4.1To keep the analysis consistent with previously published reports of the dataset , Statist2high  = 0.01, p > .05; χ2low  = 0.00, p > .05).The set-up of the fMRI paradigm, which presents a relatively large number of self- and other-blaming emotion trials  in the same testing session, lends itself to assessment of internal consistency using the split-half method. This involves splitting the trials for each subject in two halves and subsequently comparing the similarity of the neural measure between the two halves . As the For the BOLD analysis, even and odd trials of each condition ) were modelled at the first level, with 15 trials of each condition included in each split half. Movement parameters (i.e. six parameters describing movement by rotation and translation in three dimensions each) were included as covariates, and temporal and spatial derivatives of the haemodynamic response function were modelled with an event duration of 0 s. This model follows the original model parameters as reported in .In addition, alternative BOLD models were run to explore the influence of modelling without time and dispersion derivatives and with trials modelled with a varying duration on reliability. Event-related designs assume that neural activity occurs for short and discrete intervals and aim to measure transient changes in brain activity . HoweverHere, we opted to model trial-length using three different durations . Generally, events in an event-related design are assumed to have zero duration , as used as an ROI in 3.Posterior subgenual cortex (BA25), resulting from 4.Right striatum / pallidum, right hemispheric part of our a priori basal ganglia ROI used in Using the BOLD- and PPI-models, contrasts were created to examine activation to self-blaming emotions (self-agency vs fixation), other-blaming emotions (other-agency vs fixation) and the subtraction-based difference between self- and other-blaming emotions for each split half. The MarsBaR toolbox  was usedThe CONN toolbox v17.c was used to explore alternative normal seed-based connectivity and PPI measures. Pre-processed data were imported into the toolbox, and stimuli onsets and duration for the conditions  were specified. Raw BOLD signal was used as analysis unit instead of the default percent signal change to reflect the use of pre-processed data. Motion parameters and confounding temporal covariates were removed via CONNs CompCor algorithm .Reliability was assessed for both standard functional connectivity ) and gPPI models. Simple ROI-to-ROI analyses were performed to determine the functional connectivity between a seed region, i.e. the RSATL , and the previously mentioned ROIs. Within each ROI, the average BOLD time series was calculated across all voxels and bivariate temporal correlations, i.e. functional connectivity measures, were computed. For task-related changes in functional connectivity, bivariate regression coefficients were calculated.CONN’s default setting is to apply Fisher’s Z-transformation, producing ROI-to-ROI correlation matrices. For each participant, Z-transformed values were extracted from the RSATL-ROI pairs for self-blame, other-blame and fixation conditions and imported into SPSS version 26.0 . Other contrasts were created to examine connectivity in the context of self-blaming emotions (self-agency vs fixation), other-blaming emotions (other-agency vs fixation) and the subtraction-based difference between self- and other-blaming emotions for each split half.Using the imported values for the BOLD-, PPI- and CONN-models comparing response to self-blaming, other-blaming and self- vs other-blaming emotions, ICCs and their 95% confidence intervals were calculated for each ROI using SPSS. These were based on an absolute agreement, two-way random-effects model, which is equivalent to the second ICC ) as defined by Shrout and Fleiss . In the In addition to exploring reliability at a ROI-level, ICCs were calculated at a voxel-level using the MATLAB-based ICC toolbox . Even if2.4.2Using the full dataset, we investigated the trade-off between split-half reliability and reproducibility of findings between models. We therefore selected the models with optimised split-half reliability when compared against the previously published models and investigated whether these more reliable models could replicate the results of the previously published models: 1) the BOLD model without time and dispersion derivatives (duration 0 s), 2) the SPM PPI based on the first-level BOLD models without time and dispersion derivatives (duration 0 s) and 3) the CONN gPPI model at the second-level (between-subject).p = .05 and to extract cluster average regression coefficients. For further details, please see Supplementary Methods.We followed the random-effect BOLD-analysis approach outlined in , while wp = .001 (uncorrected voxel-level) and corrected for Family-Wise Error (FWE) at cluster-level or voxel-level at p = .05 over the previously used a priori SCC ROI  and the whole brain, respectively.For the gPPI model in CONN, seed-to-voxel analyses were conducted, exploring the effective connectivity between the seed ROI and all other voxels in the brain. Using random-effect analyses, connectivity measures were explored for main effect of group (stable MDD vs recurring MDD), main effect of condition (self- vs other-blame) and interaction effect of group by condition. Results were thresholded at 33.1Across ROIs, the BOLD models which included time and dispersion derivates showed poor reliability: most of the ICCs did not exceed 0.4 . Some ofIn contrast, BOLD models without time and dispersion derivates resulted in more consistent activation in both self- and other-blame conditions. With a modelled event duration of 0 s, the RSATL showed good reliability for self-blaming emotions, while subgenual BA24 and the striatum / pallidum showed good reliability for other-blaming emotions. In addition, the RSATL displayed fair reliability for other-blaming emotions, while the striatum / pallidum showed fair reliability for self-blaming emotions, and BA25 showed fair reliability for both self-blaming and other-blaming emotions. The BOLD model based on a temporal split, i.e. first and second half of the task as opposed to odd and even trials, showed fair reliability for other-blaming emotions in the RSATL and striatum / pallidum (Supplementary Table 1). Across different BOLD models and ROIs, however, the complex contrast of self- vs other-blaming emotions showed poor reliability .With regard to the internal consistency of effective connectivity, the SPM PPI model showed poor reliability across ROIs and conditions . In factAt a voxel-level, other-blaming emotions resulted in more consistent BOLD activation compared to self-blaming emotions, particularly in the frontal cortex see . In both3.23.2.1Next, we sought to replicate the findings as reported in , which sUsing the same modelling approach , but witp = .001 (uncorrected voxel-level), only the right ventrolateral putamen (extending into the claustrum) remains. We were able to reproduce these findings using the same modelling approach (again with the minor discrepancies corrected) and using the modelling approach without time and dispersion derivatives  or an interaction effect of group by condition.In recent years, reliability of fMRI results has been given more and more attention, but there is little consensus whether fMRI captures reliable measures of neural activity: some suggest fair reliability , while oHere, we probed the reliability of our blame biases paradigm in a relatively large sample of MDD and healthy control participants. Using different modelling approaches, we showed fair reliability for simple fMRI measures related to self-blame, but poor reliability for more complex measures. Our findings corroborate previous reports that reliability appears to diminish with increasing model complexity, especially when using contrasts or difference scores . InfantoMoreover, measures of functional and effective connectivity are inherently more complex than BOLD activation, which is reflected in the poor reliability estimates for our PPI models. PPI effects are relatively noisy measures, especially compared to main effects, as they contain the noise of both psychological and physiological factors in its interaction term, potentially accumulating the error term . Even thNotably, we observed fair to good reliability for both self- and other-blame conditions in our BOLD models without derivatives, while our BOLD models with time and dispersion derivatives displayed poor reliability. We expected the opposite: including derivatives has been shown to improve specificity and sensitivity of the HRF by accounting for as much variability as possible , which sThe difference in internal consistency displayed by the various modelling approaches did not lead to major discrepancies at the group-level, except for a change in lateralisation of the peak of the subgenual region BOLD activation. Our observation that it is possible to have robust activation at the group-level despite weak consistency at the individual level is in line with previous reports . It reflSomewhat counterintuitive, it is possible for a decline in reliability to result in an increase in validity , which sIt is important to recognise that many of our ICCs were negative, especially for the models with time and dispersion derivatives, pointing to inconsistent activation . HoweverEven though most of the neural measures display poor ICC values, it does not mean that our paradigm is inherently unreliable. The low values could be a reflection of low between-subject variability rather than high variability in the measure , or insuLastly, we reported on the internal consistency of our self-blame-related fMRI measures, which does not necessarily convey information about its test-retest reliability. In fact, it is possible to have good internal consistency, but poor test-retest reliability and vice versa . It is iInternal consistency of self-blame-related fMRI measures was probed using different modelling approaches, which showed that relatively simple measures had better reliability compared with more complex contrasts. While simple BOLD contrasts had fair reliability, previously employed SPM PPI models had poor reliability and simple CONN toolbox connectivity measures lacked clinical validity (i.e. predictive of recurrence risk). This calls for the development of functional connectivity measures that strike a better balance between reliability and validity for future clinical applications, for which individual, not group-level, results are paramount.Diede Fennema: Conceptualization, Methodology, Formal analysis, Writing – original draft, Visualization. Owen O'Daly: Conceptualization, Methodology, Writing – review & editing. Gareth J. Barker: Conceptualization, Methodology, Writing – review & editing. Jorge Moll: Conceptualization, Writing – review & editing. Roland Zahn: Conceptualization, Methodology, Data curation, Writing – review & editing, Supervision, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Survival for glioma patients has shown minimal improvement over the past 20 years. The ability to detect and monitor gliomas relies primarily upon imaging technologies that lack sensitivity and specificity, especially during the post-surgical treatment phase. Treatment-response monitoring with an effective liquid-biopsy paradigm may also provide the most facile clinical scenario for liquid-biopsy integration into brain-tumour care. Conceptually, liquid biopsy is advantageous when compared with both tissue sampling (less invasive) and imaging (more sensitive and specific), but is hampered by technical and biological problems. These problems predominantly relate to low concentrations of tumour-derived DNA in the bloodstream of glioma patients. In this review, we highlight methods by which the neuro-oncological scientific and clinical communities have attempted to circumvent this limitation. The use of novel biological, technological and computational approaches will be explored. The utility of alternate bio-fluids, tumour-guided sequencing, epigenomic and fragmentomic methods may eventually be leveraged to provide the biological and technological means to unlock a wide range of clinical applications for liquid biopsy in glioma. Gliomas are rare and deadly brain pathologies that possess an age-corrected incidence range of 4.67–5.73 per 100,000 persons . The curLiquid biopsy may have utility across all phases of brain-tumour investigation and management. Although screening for rare pathologies is difficult, a non-invasive, low-cost and reliable cancer- diagnostic assay could potentially benefit cancer patients and the public healthcare system. If glioma were to be included, then this may provide some benefit for certain glioma patients. Diagnosis of glioma, especially in patients for whom debulking surgery is not indicated, would benefit from minimally invasive diagnostic options such as liquid biopsy. Moreover, the ability to characterise the heterogeneous genomic landscape may be of further benefit for precision therapeutics. The expectation for disease recurrence following first-line treatment for glioma mandates the use of regular clinical follow-up associated with interval contrast-enhanced MRI. This paradigm has limitations with several radiological scenarios, such as pseudoprogression, an abnormal contrast enhancement associated with improved prognosis that affects up to one-third of patients, highlighting the need for improved detection and monitoring protocols. The frequency with which patients are currently monitored is dictated by pragmatism related to logistics and cost . MR imagMultiple analytes can be detected in the bio-fluids of patients with cancer and used as liquid biopsy for a range of clinical applications: cell-free DNA (cfDNA), cell-free RNA, mitochondrial DNA, extracellular vesicles, tumour-educated platelets, proteins and metabolites among others –9. cfDNAp = 0.037), while no difference was observed for non-progressive patients [Due to the technical limitations of the time, initial studies that recovered cfDNA focused on global DNA quantification in control and diverse malignant plasma samples . The ovepatients . Despitepatients .cfDNA is commonly detected in bio-fluids by analysing tumour-derived genomic signals, such as single- nucleotide variants (SNVs). SNVs due to their binary and potentially actionable nature, were logically the first molecular targets for cfDNA liquid-biopsy assays in the brain context. Early pan-cancer studies that evaluated cfDNA tumour fraction from various technologies have revealed that gliomas are the most challenging malignancy for liquid-biopsy applications , 20. UsiIDH1 and IDH2, ATRX, TP53, TERT, and H3F3A being suggested as having classification and prognostication potential [The blood–brain barrier protects the central nervous system and has been proposed as the main reason for low cfDNA in glioma patients. Thus, a potential strategy for improving cfDNA yield may be through collecting samples ‘closer’ to the tumour. The most direct method by which to achieve this may be via cerebrospinal fluid (CSF) sampling –30. Compotential . The repotential . Neverthotential , to targotential , low-covotential  and otheotential , 33, 34.otential . In 8 seotential . This reotential , 37, 38.otential , 40. CSFotential , and by otential . The accA practical approach to improve tumour-signal detection in cfDNA is to track multiple mutations simultaneously. Standardised panels are conceptually limited by the fraction of the genome covered, the depth of sequencing and the number of starting cfDNA molecules in the sample. By using tumour-informed  sequencing, mutations and alterations initially identified in the tumour can be sought. This approach boosts coverage across defined loci and thus increases the chances of low tumour-fraction variants being detected at modest cost. A larger number of variants, compared with standardised panels, can also be followed, therefore increasing the sensitivity of sequencing without compromising specificity. Such an approach has been validated for multiple cancer types and clinical scenarios using PCR systems , targeteMutation analysis utilises few, highly specific, mutated regions within the genome of cancer patients for signal detection and analysis. This strategy relies heavily upon the presence of loci that are present in few DNA fragments and so, inevitably, suffers from a reduction in sensitivity, especially for cancers where relatively few copies of DNA exist within a liquid-biopsy sample (e.g. brain tumours). Sequencing the cfDNA epigenome, for example, methylated (mC) DNA, is advantageous because signatures exist across multiple, less specific, regions of difference , 52. By Rather than purely detecting the presence of cancer DNA, epigenomic marks associated with the cell of origin could be also leveraged to improve detection and classification of pathologies , 56. A rEpigenomic modification of DNA can be detected through either direct or indirect means, sometimes requiring only small quantities of DNA . The mosHowever, bisulfite conversion presents limitations for cfDNA analysis. First, it necessitates harsh conditions of pH and temperature that can degrade the DNA, which when dealing with low-input quantities of cfDNA, can be limiting . MoreoveIn order to circumvent the large quantities of DNA lost through bisulfite conversion, techniques have been developed or adopted that reduce the need for large quantities of input DNA (e.g. as required for WGBS). Reduced-representation bisulfite sequencing (RRBS) enriches for CpG islands that are putatively most representative of cell methylation status. This approach reduces sequencing costs but at the cost of omitting non-CpG methylation (e.g. intergenic and enhancer regions) . It alsoBisulfite-free methods are also used and these can loosely be divided between affinity-enrichment and restriction-enzyme-mediated technologies. Affinity-enrichment methods have been developed for both methylation (MeDIP) , 74 and Direct analysis of DNA without the need for conversion or amplification is a ‘holy grail’ of methylation sequencing. Long-read sequencing may also enable the phasing of data providing contextual information that may further boost sensitivity whilst reducing epigenomic noise. Recent improvements in nanopore-sequencing analysis pipelines provide an interesting counterpoint to the short-read narrative and may deliver value for analysis where DNA molecules are limited, such as those in cfDNA for brain tumours . In addicfDNA is fragmented in plasma around 167 bp and multiple thereof, a size corresponding to the wrapping of DNA helix around the nucleosome. Plasma cfDNA from a variety of cancer cells tends to be shorter than the bulk of cfDNA derived from hematopoietic cells by 20–30 bp –83. The The methods focusing on fragmentomics, based on genome-wide paired-end sequencing, could improve existing sequencing methods, as well as mutation analysis. First, by leveraging the 20–30-bp size differences between tumour and non-tumour DNA, it is possible to either enrich for cancer signal, or filter out the false-positive signal . Indeed, clonal haematopoiesis-derived mutations should on average appear on longer fragments than mutations from tumour-derived cfDNA. Incorporating such information could reinforce the confidence in mutation calling when tumour fraction in plasma is extremely low (e.g. in gliomas). The potential of such a strategy has been demonstrated in a small cohort of glioma samples using INVAR , 48, as Leveraging other biological properties of cfDNA-fragmentation profiles could enhance the detection of tumour-derived cfDNA. By combining copy number alterations with specific fragment-size patterns using random forest classifier, cancer can be classified with a high accuracy (AUC = 0.91 for glioma cases) . RecentlThe cfDNA field has benefited from a fast-growing portfolio of technologies and from a dynamic entrepreneurial ecosystem with a desire to improve non-invasive cancer analysis. Each cfDNA technology could be well adapted to one of the clinical applications of interest for CNS cancers: screening, diagnosis/classification, treatment guidance, monitoring progression and detecting relapse earlier Table . HoweverBeyond cfDNA, multiple other tumour components (or components influenced by cancer cells) circulate within a cancer patient. Exosomes , 98, mitThe utility of liquid biopsy in glioma remains relatively underexplored at the view of its clinical potential. At every stage of the analysis paradigm, novel approaches to cfDNA have incrementally improved sensitivity and specificity. The advent of improved biobanking approaches has further improved substrate accessibility and reliability. We, therefore, hope that the past difficulties associated with translating cfDNA analysis in brain-tumour populations will not discourage further investigation amongst the liquid-biopsy community.


Key insights into emotional reactivity and regulation have been gained by studying how these dynamics evolve as older people are confronted with controlled stressors in the lab, go about their everyday routines, or develop across adulthood and old age. Yet, we are only beginning to understand how the dynamics on the different time scales observed in these study designs interact . Aiming for a comprehensive picture of the predictors, correlates, and consequences of emotional reactivity and regulation, the EMIL study integrates a lab-based study with ambulatory in-vivo assessments and a classic long-term longitudinal study. 130 young-old (65-69 years) and 59 very-old adults (83-89 years) from the ILSE study, contributing four waves of health, cognitive, and psycho-social data over almost 25 years, were tested in the lab and assessed six times a day over seven consecutive days. We provide an overview of and first across-design results from EMIL: Katzorreck et al. examined whether the frequency of exposure to daily stressors affects emotion regulation capacity as tested in the lab. Lücke et al. analyzed daily working memory performance, sleep, and its association with long-term change in cognitive functioning. Wieck et al. present differential effects of discrete negative emotions as induced in the lab and reported in daily life on social cognitive performance as indicated by empathic accuracy. Gerstorf et al. examined how long-term cognitive aging affects positive feelings and stressor reactivity in daily life. Gloria Luong will discuss the presentations, considering challenges and opportunities of integrating lab-based, ambulatory, and longitudinal study designs.


Cisplatin-based chemotherapy was previously considered as the standard adjuvant therapy for improved overall survival (OS) in patients with non-small cell lung cancer (NSCLC) after surgery. However, the benefit was limited due to high risks of recurrence and adverse events. In the present study, the efficacy of adjuvant epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) for EGFR-mutant patients after surgery was investigated using the latest updated data.This meta-analysis included a comprehensive range of relevant studies identified from database searches. Disease-free survival (DFS) and OS with hazard ratios (HRs) were calculated using random-effect or fixed-effect models. Subgroup analysis was also performed.A total of seven randomized clinical trials were included in the meta-analysis and involved 1,283 NSCLC patients harboring EGFR mutations. In resected EGFR-mutant NSCLC patients, adjuvant EGFR-TKIs were significantly better than chemotherapy in terms of DFS , without showing any benefit in OS . No significant difference in DFS was observed between patients with EGFR exon 19 deletion and those with L858R mutation. Resected EGFR-mutant NSCLC patients treated with osimertinib experienced improved DFS and a lower risk of brain recurrence than those treated with gefitinib or erlotinib. Adjuvant EGFR-TKIs reduced the risk of bone and lung relapse, without decreasing the risk of local recurrence and liver relapse.This meta-analysis shows that adjuvant EGFR-TKI therapy could significantly prolong DFS in patients with resected EGFR-mutant NSCLC. Treatment with osimertinib showed improved DFS with a lower risk of brain recurrence than treatment with gefitinib or erlotinib for resected disease. Lung cancer remains the leading cause of cancer-related mortality worldwide . Among tMolecular-targeted drugs have been successfully used as adjuvant therapy for several types of cancers, for example, imatinib for gastrointestinal stromal tumors , 7. Epid–75%, with poor postoperative morbidity  of patients treated with osimertinib was significantly longer than that with a placebo in resected EGFR-mutant NSCLC patients with stage IB to IIIA disease, consistent with the results of the CTONG 1104 and EVAN trials. However, in several previous trials, conflicting results regarding the efficacy of adjuvant EGFR-TKIs in resected NSCLC patients were reported . Furtherorbidity , 17. Undorbidity . In reseThe PubMed, Embase, and the Cochrane Library databases were used in a systematic search for studies published up to September 20, 2020 with no start date limit applied. The search terms used were “lung cancer”, “adjuvant or resected or operable”, “erlotinib or gefitinib or icotinib or afatinib or dacomitinib or osimertinib” and “randomized control trial”. We also searched meeting abstracts from the American Society of Clinical Oncology, European Society for Medical Oncology, World Conference on Lung Cancer and American Association for Cancer Research for Medical Oncology websites.versus adjuvant chemotherapy or placebo; and studies with reported hazard ratios (HRs) for survival analysis (DFS or OS) or the number of events for disease relapse patterns and adverse events (AEs) in EGFR-mutant lung cancer from the overall patient population or subgroups analyses. The exclusion criteria were as follows: studies with irretrievable or insufficient data for statistical analysis; single-arm trials, observational studies, editorials, reviews, and commentaries; duplicate studies; and abstracts and studies written in languages other than English. Two authors (L-LS and H-RC) independently searched the databases and screened articles using the titles and abstracts to find potentially relevant studies.Eligible studies that met the following criteria were included: Phase II or III randomized control trials (RCTs); and comparisons of survival in stage I–IIIA NSCLC patients treated with adjuvant EGFR-TKIs All candidate articles were independently evaluated and extracted by two investigators (R-LC and J-XZ), and all discrepancies were resolved by the consensus among all authors. From each study, the first author name, clinical trial name, trial phase, EGFR mutation status, generation of adjuvant EGFR-TKIs, other baseline clinicopathologic characteristics, planned and received treatment and toxicity, survival outcomes, and relapse patterns were extracted. The quality of the included studies was independently assessed by two authors (YC and YZ), according to the five-point Jadad scoring system .The HRs and 95% confidence intervals (CIs) for the DFS and OS of resected EGFR-mutant NSCLC patients were derived from the overall patient population and subgroups within each individual study. For dichotomous outcomes, the number of patients was used to calculate odds ratio (OR) estimates of trials with the Mantel–Haenszel method, such as disease relapse patterns and AEs.2 values , age (age ≥65 years vs. <65 years), sex , smoking status (smokers vs. non-smokers), histology (adenocarcinoma vs. non-adenocarcinoma), generation of EGFR-TKIs (gefitinib or erlotinib vs. osimertinib), and the relapse patterns.A series of subgroup analyses were conducted to explore the effects of variables on the efficacy of EGFR inhibitors for resected EGFR-mutant NSCLC. The subgroup included EGFR mutation status .EGFR-mutant NSCLC patients showed improved DFS after treatment with adjuvant EGFR-TKIs compared with the control group. Pooled HRs based on the seven RCTs indicated a lower risk of disease progression with EGFR-TKIs when compared with the control group .OS data were not available in the study by Feng et al. and not immature in the ADAURA trial. Thus, the analysis of OS was from five RCTs with available data. No significant improvement was observed between adjuvant EGFR-TKI therapy and the control group in resected EGFR-mutant NSCLC . A total of 565 patients harboring L858R mutation experienced improved DFS with EGFR-TKI therapy compared with the control group . No significant difference in DFS was observed between EGFR exon 19 deletion and L858R mutation subgroups (P for heterogeneity = 0.290).To further explore the effects of EGFR mutation status on DFS, subgroup analyses of DFS in patients with exon 19 deletion vs. gefitinib or erlotinib, HR: 0.20; 95%CI: 0.15–0.27 vs. 0.53; 0.41–0.67; P for heterogeneity < 0.001).The results of our subgroup analyses are shown in Data on disease relapse were not reported in the EVAN and RADIANT trials, or in Feng’s study. Based on the available data reported from four trials , the effects of EGFR-TKIs on disease relapse were analyzed. EGFR-TKI therapy reduced the risk of bone relapse  were investigated in our meta-analysis. Furthermore, the effects of different generations of EGFR-TKIs and the long-term tumor recurrence patterns were analyzed. The heterogeneity of DFS and OS across studies was substantial in our meta-analysis, which may be due to sex, age, smoking history, histology, stage, and generation of EGFR-TKIs. In advanced NSCLC disease, patients harboring EGFR exon 19 deletion experienced improved PFS with EGFR-TKIs compared with those harboring L858R mutation  has been considered as an excellent predictor of disease recurrence and used to identify the molecular residual disease. In a study of patients with localized lung cancer treated with curative intent, ctDNA was detected in 94% of patients with disease relapse before radiographic recurrence at a median of 5.2 months post-treatment . AdditioThe present study has several limitations. First, data for the analysis of NSCLC patients was derived from published clinical trials rather than from each individual patient. Therefore, analyzing the influence of disease stage accurately is difficult. Second, part of the data in our meta-analysis were derived from subgroup analyses of published RCTs. Thus, some important information was not collected from subgroup results, such as smoking history, stage, sex, and EGFR mutation status. Due to the lack of information on the stage of each patient, specific subgroup analyses of stage I, stage II, and stage III patients could not be performed.Despite the above limitations, the results of the current study have significant implications. This meta-analysis indicates that adjuvant EGFR-TKI therapy brought significant clinical benefit in terms of DFS in resected EGFR-mutant NSCLC patients. Osimertinib had longer DFS with lower risk of brain recurrence than gefitinib or erlotinib for resected NSCLC; however, additional studies are warranted.The original contributions presented in the study are included in the article/Conception and design: L-ZL, WH, and R-LC. Data collection or management: R-LC, L-LS, H-RC and J-XZ. Data evaluation: YC and ZY. Statistical analysis: R-LC, L-LS and C-YG. Manuscript writing and revising: R-LC, S-YW and L-ZL. All authors contributed to the article and approved the submitted version.This study was supported by grant from Traditional Chinese medicine evidence-based capacity building project (grant number 2019XZZX–ZL001), Pilot project of Integrated traditional Chinese and western medicine clinical collaboration for major and difficult diseases (lung cancer).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The present pilot study examines subjective reported symptoms of attention-deficit/hyperactivity (AD/H) in adults with Fabry disease (FD) in comparison with existing normative control data. Existing data from 69 adults with FD via the Achenbach System of Empirically Based Assessment Adult Self-Report questionnaire were analyzed. The results demonstrated a higher prevalence of AD/H symptoms in adults with FD than in the general United States population, with a roughly equal endorsement of Inattention/Attention Deficit symptoms (AD), Hyperactivity-Impulsivity (H-I) symptoms, and Combined Inattention/hyperactivity-impulsivity (C) symptoms. No gender differences were observed. While all subjects endorsing H-I symptoms fell into the symptomatic range on the AD/H scale, only two-thirds of subjects endorsing AD did so. This suggests that attention difficulties with FD are not solely explained by ADHD. Adults with FD who endorsed the AD, H-I, and C symptoms were also more likely to report mean adaptive functioning difficulties. These findings support the growing literature regarding attention difficulties in adults with FD, as well as suggesting a previously unrecognized risk of AD/H symptoms. Future research involving the objective assessment of ADHD in adults with FD is recommended. When serving adults with FD clinically, healthcare professionals should address multiple areas of care, including physical, psychological, and cognitive arenas. GLA gene, leading to a deficiency of α-galactosidase A  and resulting in the storage of globotriaosylceramide (GL3) and related lipids in the lysosome. Its incidence has historically been estimated at 1:40,000 male live births; however recent data suggests as high as 1:3000 [GLA mutations).Fabry disease (FD) is an X-linked lysosomal storage disorder (LSD) caused by mutations in the s 1:3000 ,6,7. TheHistorically, research has focused on somatic manifestations of FD, with less attention paid to neuropsychological manifestations. However, recent research suggests difficulties with cognitive functioning, particularly in the realm of attention and concentration, with implications for central nervous system (CNS) functioning in patients with FD.The initial neuropsychological screening studies of patients with FD reported contradictory results due to varying testing methods and small sample sizes. One initial study found patients with FD performed marginally better on tasks of attention than normal controls and slightly worse on tasks measuring language skills, with unimpaired performances in other cognitive domains , while aThe first study to examine neurocognitive functioning in FD using comprehensive and well-validated neuropsychological measures rather than screening tools found that males with FD demonstrated a slower information processing speed and reduced performance on measures of executive functioning compared to both females with FD and 15 age-matched normal controls . HoweverA more recent study found 29.3% of Danish patients with FD to have cognitive difficulties, with attention, psychomotor speed, and executive functioning once again being the most frequently impaired . NeitherIn comparison, subjective perceptions of cognitive impairment among Dutch subjects with FD were found to be much greater (64%) than the objective evidence of impairment 16%) . Objecti% . ObjecGiven the increasing evidence of the role of FD in aspects of attention, anecdotal patient reports regarding the use of medication for Attention Deficit Hyperactivity Disorder (ADHD) should perhaps not come as a surprise. Previously referred to as Attention Deficit Disorder (ADD) in the Diagnostic and Statistical Manual of Mental Disorders 3rd Edition (DSM III) , one of The present pilot study examines the self-reported symptoms of attention deficits/hyperactivity in adults with FD in comparison with the existing normative control data, as well as potential differences in the frequency between symptoms of attention deficits and symptoms of hyperactivity. In addition, we explored the possible association between attention-deficit/hyperactivity symptoms and poorer adaptive functioning in patients with FD. Data was derived from a subset of data in existence at the Emory Lysosomal Storage Disease Center. Specifically, data concerning Attention Deficit/Hyperactivity, Attention, Inattention, Hyperactivity-impulsivity, Somatic Symptoms, Depression, Anxiety, and Mean Adaptive Functioning were utilized from the Achenbach System of Empirically Based Assessment (ASEBA) Adult Self-Report (ASR) questionnaires completed by patients with FD between January 2005 and July 2013. Approval from the Institutional Review Board was granted through Emory University (IRB00068700).The ASEBA ASR is a reliable, validated measure of social-adaptive and psychological functioning in adults aged 18–59 and the OASR for ages 60–90+ . Norms rhttps://adm-assessment-data-manager.software.informer.com/9.0/, accessed on 1 June 2021), which produces detailed profiles on multiple aspects of psychological functioning. For this study, data from the DSM-Oriented Scale for AD/H, as well as the Attention Problem Syndrome scale, Depression scale, Somatic Complaints scale, and Mean Adaptive Functioning scale, were utilized. Subjects with T-scores in the borderline-clinical and clinical ranges were considered to have symptoms for the purposes of this study. ASEBA ASR raw data was entered into assessment data manager  ASEBA scoring software . Demographic participant characteristics were summarized using frequencies and proportions. Chi-square and Fisher’s exact tests were used to assess the associations between mean adaptive functioning and demographic variables of interest. Similarly, chi-square tests were used to assess the association between depressive symptoms and gender, AD/H symptoms, H-I symptoms, and AD symptoms. The prevalence of AD/H in our study sample was compared to the most recent estimated prevalence of AD/H among the US adult population  using FiExisting data from 69 adults with FD who completed the ASEBA ASR questionnaire was examined. The demographic information is presented in p < 0.001), using the most recently estimated prevalence (4.4%) of adult ADHD in the United States [Of the 69 subjects who completed the ASEBA ASR, twenty (29%) endorsed symptoms within the borderline-clinical-to-clinical range on the AD/H problems scale . This red States .Among the twenty subjects endorsing symptoms within the borderline-clinical-to-clinical range on the AD/H scale, the source of those scores was almost equally balanced between the symptomatic endorsement of AD items, H-I items, and combined AD/H items, with a final three subjects whose endorsement of items was evenly split such that they fell within the normal ranges on the individual subscales while still falling within the symptomatic range on the overall combined AD/H scale . All subp = 0.537). A third of the adults with FD (33%) self-reported symptoms of anxiety, with no significant differences between the male and female subjects (p = 0.0870). Almost a third of adults with FD (29%) self-reported difficulties in adaptive functioning, with no significant differences between the male and female subjects (p = 0.060). Over a third of adults with FD (38%) self-reported somatic symptoms in the borderline-clinical-to-clinical range, with no significant differences between the male and female subjects (p = 0.2468).Almost half of the adults with FD (49%) were also noted to self-report depressive symptoms in the borderline-clinical-to-clinical range on the ASEBA ASR, with no significant differences between the male and female subjects , with no significant differences between men and women. This replicates the previously reported high rates of depression among adults with FD, with prevalence estimates ranging from 15% to 62% %, with n,27,28,29While depression can have deleterious effects on attention , its intFinally, the present study found adults with FD-endorsing AD symptoms, AD/H symptoms, depressive symptoms, and anxiety were also significantly more likely to endorse adaptive functioning (AF) difficulties. An indication of the effectiveness with which individuals cope with the demands of everyday tasks and responsibilities as parents, students, employees, etc., AF is measured via evaluations such as the ASEBA focused on individuals’ relationships, jobs, education, substance use, psychological issues, and coping skills. These findings corroborate earlier research in which FD patients had a higher rate of mean AF deficits compared to population norms, with poorer AF associated with greater rates of AD/H, depression, and anxiety . All of the above findings make clear the need to pay attention to the psychological symptoms associated with FD, including the possibility of symptoms of AD/H, and expand our standard of care to include mental health treatments, if necessary. Of note, of the 20 people who self-reported AD/H symptoms in our sample, four had been prescribed medication typically used for ADHD at some point in their life, though only one had undergone a clinical diagnosis for their symptoms. As symptoms of ADHD are more heterogeneous and subtle in adults than children ,36, withThe limitations of this study include the use of self-reported symptoms at a single point in time; however, adults with ADHD have been found to be quite reliable in identifying their own symptoms via self-reported measures , and an The implications of this study include the need for greater attention to cognitive and psychological health in people with FD, particularly in the areas of attention, AD/H-like symptoms, depression, anxiety, and adaptive functioning. Genetic counselors and other healthcare providers should address such issues in their annual clinic appointments and make referrals as needed to maximize overall treatment for patients with FD. The recommendations for future research include a more objective assessment of AD/H symptoms in patients with FD, as well as further in-depth neurocognitive studying of attention/concentration in FD. Such research should utilize objective neuropsychological tests with the existing normative data with the general population.In conclusion, the present study suggests that adults with FD are at a higher risk than the general population for attention deficits, as well as symptoms of ADHD, with equal rates among men and women. When serving adults with FD clinically, genetic counselors and other healthcare professionals should address multiple areas of care, including the physical, psychological, and cognitive issues that may accompany the disease.


N-acyl glycines were higher during sleep. Regional specific changes were also detected, especially related to tyrosine analogues in the hippocampus and the cerebellum. Our findings demonstrate a strong correlation between circadian rhythms and amino acid metabolism specific for different brain regions that provide previously unknown insights in brain metabolism.Sleep is a state in which important restorative and anabolic processes occur. Understanding changes of these metabolic processes during the circadian rhythm in the brain is crucial to elucidate neurophysiological mechanisms important for sleep function. Investigation of amino acid modifications and dipeptides has recently emerged as a valuable approach in the metabolic profiling of the central nervous system. Nonetheless, very little is known about the effects of sleep on the brain levels of amino acid analogues. In the present study, we examined brain regional sleep-induced alterations selective for modified amino acids and dipeptides using Ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) based metabolomics. Our approach enabled the detection and identification of numerous amino acid-containing metabolites in the cortex, the hippocampus, the midbrain, and the cerebellum. In particular, analogues of the aromatic amino acids phenylalanine, tyrosine and tryptophan were significantly altered during sleep in the investigated brain regions. Cortical levels of medium and long chain  Sleep is a vital biological function for the survival of numerous species, and circadian rhythms play a central role in homeostatic mechanisms . The braN-Acylated amino acids are a large group of bioactive molecules involved in many physiological functions, such as molecular signaling, energy homeostasis and oxidative metabolism [N-Acylglycine, which is widely distributed in the central nervous system (CNS), was described to have several neuro-physiological properties, such as analgesic and anti-inflammatory activity [Amino acid metabolism has a key function in the central nervous system (CNS). Amino acids can regulate neuronal activity via multiple mechanisms, ranging from the synthesis of neurotransmitters, e.g., the precursor amino acids phenylalanine, tyrosine and tryptophan, to direct neurotransmission and neuromodulation, e.g., the excitatory neurotransmitters glutamate and aspartate as well as the inhibitory neurotransmitter glycine . Alterattabolism ,23,24,25activity ,26. A wiactivity ,13,18. Dactivity . One of activity ,28. ThisN-acyl glycine, glutamate, dipeptides, and N-acetylated products in the context of the sleep/wake cycle, for the first time.Mass spectrometry (MS)-based metabolomics constitutes a valuable tool for the comprehensive analysis of amino acids and analogues in brain tissue . In addiFour distinct brain regions, i.e., the cortex (CTX), the hippocampus (HC), the midbrain (MDB), and the cerebellum (CBL), were collected from two groups of mice. The mice were sacrificed either during their subjective night (referred to as sleep state/10 a.m.) or during their subjective day (referred to as wake state/10 p.m.), respectively (N = 6 per group). The samples were extracted using our standard protocols and analyzed with UPLC-MS/MS-based metabolomics . IsotopiSubsequently, the data distribution was evaluated using unsupervised principal component analysis (PCA) b. The QCt-test on sleep/wake effects, p < 0.05, A number of significantly altered features between sleep and arousal states were detected  or via MS/MS fragmentation characterization  and phenylalanine (N-AcPhe) were significantly elevated during sleep in the investigated cortical samples  occupies a large part of the cerebrum and is involved in principal executive functions a. The N- samples b,c. Isot samples b,c. The m/z 120.080 is indicative of the presence of Phe in the molecule, and its relative intensity designates the C-terminal of the dipeptide, as shown in the examples for Arg-Phe and Phe-Arg  analogues were found to be significantly elevated during sleep . Conjugaglycines .The aromatic amino acids Phe, Tyr and tryptophan (Trp) have crucial roles in CNS metabolism and neurotransmitter synthesis. Phe and Tyr serve as precursors in the synthesis of catecholamines , while Trp is the precursor of serotonin (5-hydroxytryptamine). Analogues of these amino acids were found to be significantly affected by sleep in the hippocampus (HC), the midbrain (MDB) and the cerebellum (CBL).HC is a subcortical structure of the cerebrum and a component of the limbic system which has a high involvement in learning and memory a. AcetylN-AcLeu/Ile have demonstrated sleep-induced changes, as have Tyr- and Phe-containing dipeptides  and Gly acylated with medium- to long-chain fatty acids (CTX) were found to be significantly elevated during sleep. N-Acyl amino acids have been reported as important endogenous signaling molecules [N-Acyl glycines are present in the CNS, and the N-arachidonoyl analogue has been mostly studied. A wide spectrum of neuro-physiological properties was previously attributed to this metabolite, including analgesia and neuroprotection [N-arachidonoyl Gly is mediated through modulation of glycinergic neurotransmission [N-arachidonoyl Gly and oleoyl-carnitine have demonstrated similar mechanisms of exerting palliative activity through binding to the glycine transporter GlyT2 [olecules . N-Acyl otection ,26. It wsmission . We havesmission . Indeed,er GlyT2 . Our finSleep-induced alterations were detected for multiple dipeptides that mainly contain the three aromatic amino acids Phe, Tyr and Trp, as well as non-aromatic amino acids Glu and Asp. The connection between sleep and memory consolidation has been well established ,43. In pMost of the Asp and Glu conjugates were elevated during wakefulness. Glu and Asp are excitatory neurotransmitters and therefore expected to be involved in neuronal activity performed in wakefulness. Interestingly, opposite effects were observed for several dipeptides in the HC compared to other brain regions. In particular, Tyr-containing dipeptides were significantly higher in the HC of the wake group compared to the sleep group, while the same molecules were identified to be elevated during sleep in the cerebellum. These metabolic differences reflect the distinct functionalities of these regions during sleep. HC is the major brain region involved in memory and cognitive function, while CBL is involved in motor control. However, a recent study proposed a novel neural network of cerebello-hippocampal interactions, potentially suggesting complementary metabolic needs . This fiSolvents and reagents were purchased from SigmaAldrich  or Fisher Scientific  and were used without further purification. Phe-Leu was provided by Biosynth Carbosynth . The in-house built metabolite library was obtained from MetaSci . Mass spectrometry-grade solvents were used for UPLC-ESI-MS analysis.The animal study was performed as previously described . BrieflyEvery brain region sample was weighed and transferred into bead-containing vials where methanol:water (80:20) was added (approx. 4 mL/mg brain tissue). As an internal standard (I.S.), a mixture of C-13 isotopically labeled tyrosine (5 mg/mL), phenylalanine (10 mg/mL) and valine (30 mg/mL) was used . The homm/z 50–1200. Data acquisition was performed in MSE mode. The samples were injected to the UPLC-MS system in a randomized order with QC samples injected in the beginning and end of the sample list in both ionization modes, as well as after every eight samples . Tandem MS spectra were also collected with a Maxis II ETD Q-TOF mass spectrometer equipped with an Elute UHPLC system using the same chromatographic parameters.The UPLC-MS/MS analysis was performed in a SYNAPT G2-S high-definition mass spectrometer (HDMS) using an electrospray ionization (ESI) source with an AQCUITY UPLC I-class system and equipped with a Waters ACQUITY UPLCVR HSS T3 column  ,20,51. Wwww.hmdb.ca, accessed on 21 December 2021) based on their m/z value and given the high mass accuracy provided by the mass analyzer [Significant features were primarily annotated by the human metabolome data base  for detecting consciousness state (sleep/wake) differences.The chromatograms and mass spectra were processed using the XCMS R package (version 1.4.414) for peak alignment and retention time correction ,34 in boThe UPLC-MS/MS methodology that was applied in our study enabled the detection and mapping of multiple amino acid analogues with high accuracy. Validation of the metabolite structure using tandem MS/MS analysis led to the identification of the most informative product ion spectra validating the presence of significantly altered amino acids. We also invested in the identification of the correct structure of many C-terminal dipeptides for significantly altered modified dipeptides using standards or MS/MS libraries. This approach provided a high level of confidence for the metabolite structures. Authentic standards are required to completely validate the structures of all isobaric and isomeric dipeptides and distinguish between D- and L-amino acids in future studies.In summary, our study provides, for the first time, a comprehensive analysis of the circadian impact on the levels of amino acids and their analogues in distinct brain regions. Our findings highlight the complexity of the connection between the sleep/wake cycle and the brain metabolome. We believe that these findings should be included in future animal models for the investigation of sleep disorders, such as fatal familial insomnia and sleep deprivation related to neurodegenerative diseases. Dipeptides have also demonstrated neurophysiological properties when administered exogenously . Therefo


CRC-screening promotion through e-media has great potential for increasing screening participation in PHC settings. Thus, its development should be prioritized, and it should be integrated into existing programs.Colorectal cancer (CRC)-screening reduces mortality, yet remains underutilized. The use of electronic media (e-media) decision aids improves saliency and fosters informed decision-making. This systematic review aimed to determine the effectiveness of CRC-screening promotion, using e-media decision aids in primary healthcare (PHC) settings. Three databases  were searched for eligible studies. Studies that evaluated e-media decision aids compared to usual care or other conditions were selected. Quality was assessed by using Cochrane tools. Their effectiveness was measured by CRC-screening completion rates, and meta-analysis was conducted to calculate the pooled estimates. Ten studies involving 9393 patients were included in this review. Follow-up durations spanned 3–24 months. The two types of decision-aid interventions used were videos and interactive multimedia programs, with durations of 6–15 min. Data from nine feasible studies with low or some risk of bias were synthesized for meta-analysis. A random-effects model revealed that CRC-screening promotion using e-media decision aids were almost twice as likely to have screening completion than their comparisons (OR 1.62, 95% CI: 1.03–2.62,  Colorectal cancer (CRC) remains the third most common malignancy worldwide, and it was ranked the second leading cause of cancer deaths in 2018 . The conScreening average-risk adults (age 50 to 75 years) for CRC contributes to reduced mortality ,9. NonetIt is therefore essential to invest in health-promotion programs that target the general population, focusing on increasing knowledge of the benefits of CRC screening and promoting healthy lifestyles as a matter of disease prevention. Moreover, as many patients with CRC have lower education levels ,16, it iThis systematic literature review was aimed at assessing the effectiveness of e-media decision-aid interventions used in PHC settings, from identifying to reminding patients who have not responded to CRC screening. We considered a systematic literature review the most appropriate form of review to address the research question: how effective is e-media decision aid intervention, compared with a baseline or control group, in improving CRC screening among eligible patients in PHC settings? Thus, this review helps to identify future practice and strategies for improving CRC-screening effectiveness in PHC services.The protocol for this systematic review was registered at Prospero (registration ID CRD42020220301) prior to the commencement of research to avoid duplication and minimize reporting bias.We searched the MEDLINE, Web of Science, and the Cochrane Library databases, because they were expected to contain relevant studies with numerous open-access full-text articles available online. The scope of MEDLINE is broadly defined and encompasses the areas used by health professionals engaged in basic research and clinical care, public health, health policy development, or related educational activities . Web of The literature search was performed from November 2020 to December 2020, and we searched for articles published from January 2010 up to 4 December 2020. The 10-year span was selected based on the integration of e-media and the Internet of Things, an important technical domain that has grown rapidly in the past decade and has received wide attention globally from a plethora of disciplines, including medical research . The artA preliminary search was conducted to identify the appropriate keywords and to determine whether this review was feasible. The keywords were verified and validated by two public health physicians at the Universiti Kebangsaan Malaysia. The medical subject heading (MeSH) keywords used were as follows:  AND  AND (“media” OR “mass media” OR “electronic media” OR “video”) .Potential additional studies were identified through reference tracking of systematic reviews during the database search. Given the extensive nature of the three databases, we did not search unpublished reports.n = 312). Basic information from the remaining articles was exported to a Microsoft Excel sheet. Two authors (NSR and MII) independently reviewed all titles, abstracts, and references generated by the original search to identify articles for potential inclusion. A total of 644 titles and abstracts were screened based on their relevance to the inclusion and exclusion criteria, resulting in the exclusion of 572 articles, leaving 72 articles to be assessed for eligibility.The initial search retrieved 964 articles . The search results were imported into the EndNote reference manager, and duplicates were removed  population—eligible patients in PHC settings; (b) intervention—CRC-screening promotional tools using e-media; (c) comparison—usual care or otherwise specified; and (d) outcome—CRC-screening completion, either fecal occult blood test (FOBT) or colonoscopy or sigmoidoscopy uptake. In addition, eligible articles had to be published in English, with open-access full-text articles available online. We used the modified Australian National Bowel Cancer Screening Program (NBCSP) Quality Framework  as a guiA total of 57 articles did not meet the eligibility criteria. In the next stage, two authors (NSR and AMN) assessed the full articles independently and compared the results. To increase the reliability of the study selection, all differences were reconciled by consensus. In the full assessment of the remaining 15 articles, five were excluded for falling outside the scope of this review, resulting in the selection of a final 10 articles.effective, i.e., higher CRC-screening completion rate with statistical significance; null, i.e., higher or lower CRC-screening completion rate without statistical significance; or not effective, i.e., lower CRC-screening completion rate with statistical significance, and was reported as our final outcome summary.At the final stage, NSR extracted data by using a standardized Excel spreadsheet, which was revised by another three authors . The data extracted for mapping and analysis included author, year of publication, study country, study design, participants, sample size, type of intervention including content and duration, comparison, main outcome measures, and results. The outcome measures and operational definitions used for describing CRC-screening promotion effectiveness are defined in We assessed study quality by using the Cochrane risk-of-bias tool for randomized trials (RoB 2) and the risk-of-bias tool for cluster randomized trials (RoB 2 CRT) because they are the most commonly used tool for randomized trials . Bias waI2 statistic for each pooled estimate. We used a random-effects model for heterogeneity (p < 0.05). Due to the possibility of clinical homogeneity, we performed subgroup analysis on the type of e-media decision aid used and the target population. The robustness of the results was evaluated with sensitivity analysis by excluding individual studies from each forest plot. Publication bias was assessed by using funnel plots.All statistical analyses were performed by using Review Manager 5.4.1  . We inclA total of 10 studies were included in this review. Eight studies were conducted in the United States ,36,37,38Four studies followed their patients for a minimum of 3 months ,35,38,39In total, 9393 patients were had follow-ups. The sample sizes ranged from 65  to 5271 The 10 studies yielded two types of decision aid interventions used for screening participation: videos, either with or without combination with other types of media tools ,38,39,40CRC-screening completion (or participation or adherence to either FOBT or colonoscopy or sigmoidoscopy uptake) was used as a proxy for determining the effectiveness of CRC-screening promotion intervention using e-media. The final outcome summary derived from all studies was categorized accordingly . Almost p < 0.05) , and the funnel plot generated was asymmetrical  . Nonethemetrical . The senp > 0.05); however, it did not reach statistical significance (p < 0.05) .In the subgroup analysis, two studies that used interactive programs as their e-media decision aid ,37 reporificance . On the  < 0.05) . The anaThe other main outcomes  from theThis systematic literature review highlights the effectiveness of CRC-screening promotion practiced in PHC settings and research opportunities for improving CRC-screening participation for eligible and non-adherent patients in bowel cancer screening programs. This review makes a distinction between the types of e-media decision aid used for promoting CRC screening to optimize recruitment participation and follow-up, and towards better integration of CRC screening into existing PHC prevention pathways.I2 = 93%, p < 0.05) shown may possibly reflects the variety of the population groups, sample sizes and decision aid contents including duration. In addition, the asymmetrical shape of funnel plot suggests for the existence of publication bias. This type of bias is induced by the fact that research with statistically significant results is more likely to be submitted and published than work with null or non-significant results. Thus, it poses a threat to the validity of such analyses. The implication of having publication bias may lead to an incorrect, usually over optimistic conclusion. Therefore, cautious interpretation is vital upon its existence. Finally, all secondary outcomes showed positive effects of CRC-screening promotion that uses e-media interventions when compared with their respective controls. Therefore, we conclude that e-media decision-aid interventions are effective for promoting CRC screening.Our findings demonstrate that the rates of CRC-screening completion were higher among those who received e-media decision aid interventions in the majority of studies (8/10), of which four reached statistical significance . From thperceived susceptibility, perceived severity, perceived benefits, perceived barriers, cues to action, and self-efficacy [perceived susceptibility by conveying the importance of screening, including CRC risk factors [perceived barriers [cues to action by addressing the need to change their screening behavior [self-efficacy to complete CRC screening [The effectiveness of health interventions has been linked to the use of health behavioral science theories. The health-belief model (HBM) has been applied most often for health concerns that are prevention-related and asymptomatic, such as early cancer detection, where beliefs are equally or more important than overt symptoms . The HBMefficacy . These c factors ,34,38,40barriers , providebehavior  and promcreening ,37.While the HBM focuses on the individual, it also recognizes and addresses the social context in which health behaviours take place . Thus, tperceived usefulness, perceived ease-of-use, and users’acceptance constructs towards e-media or internet use [effective-cum-statistically significant interventions in this review.Through e-media platforms, not only can decision aids such as health-promotion videos exert great influence in patient decision-making, but they can also empower the willingness of many adults to use e-media to promote cancer screening to their peers . Therefornet use . Nonethernet use . This preffective studies discussed above, two studies [self-efficacy construct at the end of the story, and the participants merely perceived the video as a form of entertainment. Hence, decision aids that only hint at why screening interventions are given are not effective in comparison with the studies that were effective.Apart from the  studies ,39 repor studies  used a vThe literature search showed that the low uptake in CRC-screening reported in many parts of the world were more prominent among underserved populations such as the uninsured, recent immigrants, and in the most ethnically diverse areas, with a striking gradient according to socioeconomic status ,51. ThisE-media decision aids were often accompanied by other types of decision aids, such as patient navigator, brochure, pamphlet, or reminder letter, as shown in studies in the present review. In some other studies, multilayer-screening interventions involving patient navigators, such as nurses and other community health workers, were also used ,53,54,55Although we have commented on the positive effect, there were also some no-effect findings on the rate of CRC-screening completion among patients in PHC following video-type intervention. Zapka and colleagues discuss such results explicitly, where their studied samples were primarily middle-class white people who had high screening rates at baseline, and the trial was conducted during a period of increased health insurance coverage for lower endoscopy procedures with wide public media attention on CRC screening . Similarcues to action for those who have never encountered this potentially deadly but preventable disease, and thus should be incorporated into health promotion activities.These findings suggest that future theory-guided trial interventions are needed, as well as the need to examine health behavior moderators and mediators. Issues that are in line with the HBM theory that could explain negative health behavior may also be considered in developing CRC-screening decision aids ; for exaReporting and publication bias may have affected our findings. The independent effect of multicomponent elements of e-media decision-aid interventions was often uncertain, as can be inferred from the high heterogeneity in meta-analysis. Despite the limitations, the findings are relevant to settings in private practice or through an organized screening program, given the role of PHC services for preventive care follow-up. To our knowledge, this is the first review that focuses on e-media decision aids in the CRC-screening pathway.Overall, this review provides effective evidence of intervention studies that used e-media platforms for promoting CRC screening. In other words, e-media decision aids have been proven to have great potential for increasing CRC-screening participation in the era of rapid electronic gadget revolution in combination with the Internet of Things. If integrated appropriately in PHC settings, these interventions could act as an effective learning interface for patients with CRC and their families. Therefore, development and application of culturally tailored e-media decision aids aimed at increasing CRC-screening completion should be prioritized. However, healthcare providers and organizations must be aware of the limitations and pitfalls of these platforms and must address them appropriately.