Though the etiology of Alzheimer’s disease (AD) and many other neurodegenerative diseases are still unclear, age is known to be the biggest risk factor for neurodegeneration. And as most neurodegenerative diseases are proteopathies, protein aging is likely to be involved in the disease mechanism.2 group from the side chain extends the polypeptide backbone, disrupting the protein native structure and affecting its function. Deamidated proteins are prone to aggregation due to the loss of native conformation. In 1991 it was found that isoAsp formation in amyloid beta (Aß) peptide facilitates its aggregation [One of the major products of protein aging is isoaspartate (isoAsp), a damaging amino acid residue generated either from deamidated asparaginyl (Asn) or, less frequently, isomerized aspartatyl (Asp) residue. Both deamidation and isomerization processes occur spontaneously at physiological conditions and require no enzymes. In isoAsp, the rearrangement of a CHregation . These pL-Asp. Using S-adenosylmethionine (SAM) as a methyl donor, the enzyme protein L-isoaspartyl methyltransferase (PIMT) methylates isoAsp, which upon the spontaneous loss of methanol becomes Asp (< 25% of cases), or, more often, isoAsp again [Nature has foreseen a process of isoAsp repair, albeit not to the original Asn but to a healthy sp again . With agTo test the role of isoAsp in neurodegeneration, one needed to quantify the isoAsp levels in blood proteins of larger cohorts, which was not a trivial task due to the absence of commercial anti-isoAsp antibodies. While isoAsp is slightly immunogenic, the small chemical difference between isoAsp and Asp renders production of antibodies against isoAsp difficult. After several unsuccessful attempts we managed to develop a monoclonal antibody (mAb) with high sensitivity and specificity against isoAsp in an important domain of HSA . Using iSubsequently, via another cohort composed of a different ethnic group (Asian versus predominantly European), we validated the previous results. We also discovered the potential of isoAsp-related biomarkers in diagnostics of other neurodegenerative diseases, including frontotemporal dementia, vascular dementia and, to lesser extent, Parkinson’s disease. The most significant discovery was the best performance of isoAsp-related biomarkers (AUC = 0.92) in detection of mild cognitive impairment (MCI) compared with other current blood biomarkers  and glial fibrillary acidic protein (GFAP)). In addition, the levels of isoAsp in HSA and its antibodies were found to strongly correlate with cognitive decline, which strengthened the role of isoAsp in neurodegeneration.Being intrigued by the apparently irreversible nature of deamidation that seems to be in stark contrast with observed cell self-renewal phenomena , we investigated the possibility of true isoAsp-to-Asn repair. Besides reviewing the available theoretical arguments, we reported the first experimental evidence on the reversibility of isoAsp via protein succinimide/isoaspartate ammonia ligase (PSIAL) activity . Such acCollectively, the accumulated results provide new venues for early diagnostics, therapy and perhaps even preventions of neurodegeneration and other age-related diseases.


Electrical spinal cord neuromodulation has emerged as a leading intervention for restoring autonomic functions, such as blood pressure, lower urinary tract (LUT), bowel, and sexual functions, following spinal cord injury (SCI). While a few preliminary studies have shown the potential effect of non-invasive transcutaneous spinal cord stimulation (tSCS) on autonomic recovery following SCI, the optimal stimulation parameters, as well as real-time and long-term functional benefits of tSCS are understudied. This trial entitled “Non-invasive Neuromodulation to Treat Bladder, Bowel, and Sexual Dysfunction following Spinal Cord Injury” is a pilot trial to examine the feasibility, dosage effect and safety of tSCS on pelvic organ function for future large-scale randomized controlled trials.Forty eligible participants with chronic cervical or upper thoracic motor-complete SCI will undergo stimulation mapping and assessment batteries to determine the real-time effect of tSCS on autonomic functions. Thereafter, participants will be randomly assigned to either moderate or intensive tSCS groups to test the dosage effect of long-term stimulation on autonomic parameters. Participants in each group will receive 60 minutes of tSCS per session either twice (moderate) or five (intensive) times per week, over a period of six weeks.Outcome measures include: (a) changes in bladder capacity through urodynamic studies during real-time and after long-term tSCS, and (b) resting anorectal pressure determined via anorectal manometry during real-time tSCS. We also measure assessments of sexual function, neurological impairments, and health-related quality of life using validated questionnaires and semi-structured interviews.Ethical approval has been obtained (CREB H20-01163). All primary and secondary outcome data will be submitted to peer-reviewed journals and disseminated among the broader scientific community and stakeholders. Individuals with spinal cord injury (SCI) experience a myriad of less visible but still debilitating autonomic dysfunctions, in addition to paralysis ,2. RestoCommon impairments in sexual function following SCI include erectile and/or ejaculatory disorders in males, and reduced vaginal lubrication in females, with both sexes experiencing orgasmic difficulties or anorgasmia, as well as perceived deficits in sexual drive and sexual satisfaction ,13. CompThe LUT has two functional components: the urinary bladder (detrusor) as well as the unit of the bladder neck, urethra, and urethral sphincter, which are responsible for low pressure continent storage of urine and periodic, self-determined release of stored urine (voiding) . ImpairmNeurogenic bowel dysfunction following SCI can present as constipation, impaired colonic motility, and loss of volitional control resulting in episodes of faecal incontinence . Over 90Pelvic organs share spinal cord circuitry and rely on a low-pressure system to store urine or transport stool. Bladder and bowel elimination requires an increase in intravesical and intraabdominal pressures, with simultaneous reduction of the outlet obstruction to a minimum, by relaxing their respective external sphincters. We have extensively demonstrated that life-threatening cardiovascular dysfunctions, such as autonomic dysreflexia, are commonly associated with stimuli arising from pelvic organs or iatrogenic procedures in individuals with SCI –9. As a Electrical neuromodulation of the spinal cord has received significant attention in recent years as a promising approach to manage various sequelae of SCI ,11,27. TWe hypothesize that tSCS promotes recovery of LUT, bowel and sexual functions, without any adverse cardiovascular effects, in individuals with motor-complete SCI at T6 or above. To test this hypothesis, we plan to conduct a pilot single-blinded clinical trial of tSCS for autonomic dysfunctions with the following aims: to test the safety and feasibility of real-time tSCS in promoting recovery of LUT and bowel functions (AIM 1), and to assess the safety and dosage effect of long-term tSCS, on LUT, bowel, and sexual functions by comparing moderate (twice per week) versus intensive (five times per week) tSCS (AIM 2). The trial objectives and study design are developed in accordance with the CONSORT guideline for pilot trials ,44.An overview of the experimental schedule, intervention and outcome is illustrated in The experimental design and trial flow are illustrated in Figs Forty participants, which is justified based on the power calculation below, will be recruited. Potential participants will be identified through a variety of recruitment strategies including posters, social media advertising, letters of initial contact and upon request via email/ referral. Inclusion and exclusion criteria are outlined in As this is an open-label trial, neither the participants nor the researchers will be blinded. However, outcome assessors will be blinded.We will use an isolated spinal cord stimulator  for non-invasive tSCS , that haFollowing screening and the provision of informed consent, participants will be enrolled in the trial. Specific outcome measures for each aim at each timepoint are listed in We will conduct individualized tSCS mapping in order to elicit optimal skeletal muscle activation corresponding to specific spinal cord segments involved in LUT and bowel control. Identifying specific stimulation parameters and electrode placement are the secondary outcomes. The effect of real-time tSCS on LUT and bowel functions will be evaluated during tSCS. Bladder capacity assessed via UDS and resting anorectal sphincter pressure assessed via anorectal manometry are outcomes for LUT and bowel functions, respectively. Secondary outcomes will include the perceived desire to void, the presence of detrusor overactivity, squeezing anorectal sphincter pressure, recto-anal inhibitory reflex, anorectal sensation via anorectal manometry. Time needed for bowel management with tSCS will be reported as a secondary bowel function outcome measure.The potential efficacy of long-term tSCS for LUT and bowel functions will be evaluated before and after six weeks of tSCS. We will collect bladder capacity and resting anorectal sphincter pressure as outcomes for LUT and bowel functions, respectively. As secondary outcomes, we will assess the impact of tSCS on LUT- and bowel-related daily activities and quality of life through validated questionnaires including the Neurogenic Bladder Symptom Score (NBSS), the Incontinence-quality of life (incontinence-QoL), the Neurogenic Bowel Dysfunction Score (NBDS), the modified Wexner fecal incontinence (WFI) score and time needed for bowel management. In addition, we will use biological sex-specific validated sexual function questionnaires, including the Female Sexual Function Index (FSFI) for females and the International Index of Erectile Function (IIEF-15) for males. The aforementioned questionnaires also will also be given at the follow-up (Visits 9 and 10) to assess the sustained effects of tSCS on these autonomic functions. Upon completion of the study, we will conduct a semi-structured interview to gather additional information on sexual health-related quality of life. The ISNCSCI and Autonomic Dysfunction following Spinal Cord Injury (ADFSCI) questionnaires will be used before and after the long-term tSCS to assess the degree of neurological impairment and general autonomic dysfunction. We will also record recruitment rate, retention rate and details of adverse events as feasibility measures of this trial.UDS along with surface pelvic floor muscle EMG ,will be performed (AIM 1) in accordance with Good Urodynamic Practices recommended by the International Continence Society (ICS) . First, Anorectal manometry is a well-established methodology that provides a direct assessment of anorectal sphincter pressure and anorectal coordination during simulated defecation. The assessment is widely accepted, easy to perform and well-tolerated among participants . This asSurface EMG will be conducted  as per an established protocol to record muscle activation during mapping with and without tSCS ,55. We wThe ISNCSCI will be administered at baseline, after six weeks of tSCS and at study completion. All key muscles, light touch and pinprick sensation of the upper limb, lower limb and trunk will be assessed. In accordance with the 2019 revision, ISNCSCI examinations will assess both motor and sensory neuropathology . The comThe ADFSCI questionnaire is comprised of 24 self-reported items covering four domains, including general, medications, autonomic dysreflexia, and hypotension symptoms and severity . Ten andThe NBSS, comprises of 23 questions covering three domains, including incontinence, storage & voiding, and specific consequences, as well as one question on quality of life .The Incontinence-QoL (I-QoL) comprises ten questions covering three domains, including avoidance and limiting behaviour, psychosocial impacts, and social embarrassment, which are summarised as a total score. All scores, for each domain and a total, will be transformed into a continuous scale value . Both ofTime needed for bowel management during conventional routines will be also measured on non-consecutive days . To compare time needed for bowel management, we will record the time from ‘suppository insertion’ to ‘when bowel evacuation is completed’ at the participant’s home. Pre- and post-intervention bowel management times will be compared to determine the long-term effect of tSCS on bowel function. Our recent case study shows that epidural stimulation was able to reduce bowel management time by ~55% .Faecal continence severity will be assessed using the modified Wexner incontinence scale . The scaSelf-reported magnitude of sexual dysfunction will be assessed using sex-specific questionnaires: The International Index of Erectile Function (IIEF-15) comprises 15 questions covering five domains, including erectile function, orgasmic function, intercourse satisfaction, and overall satisfaction . The FemCardiovascular monitoring will be used as a safety measure to detect and record potential adverse cardiovascular events, such as autonomic dysreflexia, during tSCS delivery and outcome assessments (AIM 1). BP and heart rate (HR) will be measured continuously (beat-by-beat) via finger photoplethysmography and electrocardiogram  corrected to brachial pressure . An attending physician will confirm episodes of autonomic dysreflexia, which are defined according to the international standards to document remaining autonomic function after SCI as an increase in systolic BP ≥20mmHg from baseline . AutonomTo determine the real-time effects of tSCS on LUT and bowel functions, all outcomes in AIM 1 will be assessed using a non-parametric one-way repeated measures analysis of variance (ANOVA) . Long-term effects of tSCS on voiding efficiency and resting anorectal pressure without tSCS will be assessed using a non-parametric two-way repeated measures ANOVA (at baseline and the completion of tSCS) to compare changes between moderate and intensive tSCS (AIM 2). Differences in continuous variables for questionnaire data  will be assessed using two-way repeated measures ANOVA. Differences in UDS, anorectal manometry, and faecal continence severity (baseline and the completion of long-term tSCS) will be assessed using Wilcoxon signed-rank tests. All analyses will be conducted using SPSS software  at an alpha (α) threshold of p ≤ 0.05 with Bonferroni correction. A complete thematic analysis is performed using contextual qualitative data collected from the sexual health interview. All missing data will be reported with an intention to treat analysis.A sample size calculation was performed using G*power software  with power = 0.8 and α = 0.05. Data from participants that match our inclusion criteria  were used from a recent tSCS study  A minimuThis trial is focused on patient-centered outcomes that are rated as priorities for recovery by individuals with SCI. The diversity of autonomic outcomes measured in this pilot trial will provide a comprehensive/robust assessment of autonomic dysfunctions and provide insight to enhance outcome selection for future definitive trials. The techniques employed to mediate autonomic dysfunctions and measure changes in various functions are well-validated for individuals with SCI and will be performed by a collaborative, multidisciplinary team of experienced clinicians and researchers. Meanwhile, the trial limitations include the lack of a sham stimulation group to control for any potential placebo effects and the lack of participant and investigator blinding to the intervention received by participants, although outcome assessors will be blinded.All data collected (electronic or hardcopy documents) will be coded with unique identification numbers and stored centrally on the Empower database, a password-protected computer, or in a locked filing cabinet in a secure laboratory space only accessible to the study investigators. To ensure information quality and accuracy after publication, all data will be stored for 25 years, and then subsequently destroyed.Any adverse events reported during the intervention will be documented with information pertaining to their severity and anatomical location. The study coordinator will immediately report adverse events to the ethics board and Data and Safety Monitoring Committee (DSMC), which is comprised of three external, independent physician scientists with no involvement in the study, as well as the appropriate ethics board. The DSMC is responsible for safeguarding the interests of trial participants, assessing the safety and efficacy of the interventions during the trial, and monitoring the overall conduct of the clinical trial. The DSMC also provides recommendations for continuing or discontinuing the trial and outcome data use for participants who discontinue the trial. The trial progress will be biannually reported to the DSMC.This pilot study will be conducted in accordance with the Declaration of Helsinki and is consistent with the International Conference on Harmonisation Good Clinical Practice Guidelines, as well as applicable regulatory requirements. The UBC Clinical Research Ethics Board  has already approved this study protocol. The UBC CREB and DSMC will be contacted when the trial makes any important protocol modifications. The feedback base on functional outcome measure results can be provided to participants for their adherence and benefits. The results of this pilot trial will be presented at national and international conferences and will be published in peer-reviewed journals. All subsequent manuscripts will be reported in conjunction with the Consolidated Standards of Reporting Trials . AdditioS1 Checklist(DOC)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Ankle sprains occur very frequently in daily life, but people who do not pay attention to them and do not receive proper diagnosis and treatment are very prone to develop chronic ankle lateral instability (CALI) at a later stage. For CALI where conservative treatment has failed, reconstruction of the lateral collateral ligament of the ankle can achieve satisfactory results, but there are various and controversial ways of ligament reconstruction. While percutaneous reconstruction of ankle lateral ligament (PLCLR) needs to be performed repeatedly under fluoroscopy, total arthroscopic reconstruction of ankle lateral ligament (ALCLR) is increasingly recognized by experts and scholars for its minimally invasive and precise characteristics, and has achieved good clinical results. Therefore, it is imperative that a meta-analysis be performed to provide evidence as to whether there is a difference between ALCLR and PLCLR in the treatment of CALI.We will search articles in 7 electronic databases including Chinese National Knowledge Infrastructure, Wanfang Data, Chinese Scientific Journals Database, Chinese databases SinoMed, PubMed, Embase, and Cochrane Library databases. All the publications, with no time restrictions, will be searched without any restriction of language and status, the time from the establishment of the database to September 2022.We will apply the risk-of-bias tool of the Cochrane Collaboration for Randomized Controlled Trials to assess the methodological quality. Risk-of-Bias Assessment Tool for Non-randomized Studies was used to evaluate the quality of comparative studies. Statistical analysis will be conducted using RevMan 5.4 software.This systematic review will evaluate the functional outcomes and radiographic results of ALCLR in the treatment of CALI.The conclusion of this study will provide evidence for judging whether ALCLR is superior to PLCLR for treatment of CALI.CRD42022362045. The most frequent injury from sprains is to the lateral collateral ligament of the ankle, including the anterior talofibular ligament (ATFL), which has the highest rate of injury, and the calcaneofibular ligament (CFL), which has a 50% to 75% probability of injury. Acute ankle sprains that are not properly diagnosed and treated late will pose a risk of ankle instability, which can lead to chronic ankle instability, resulting in pain and limited motion and accelerated joint degeneration.–5 With increasing attention to ankle sprains, early treatment with more standardized treatment can effectively reduce the probability of late ankle instability.,7 However, due to the degree of injury to the lateral collateral ligament of the ankle, many patients still develop chronic ankle instability, and then surgery is required to restore ankle stability.,9Up to 50% to 70% of patients may develop chronic ankle lateral instability later after ankle sprains.–12 Currently, due to the prevalence of minimally invasive concepts, the main methods of ligament reconstruction are percutaneous reconstruction and arthroscopic reconstruction.–15 Percutaneous reconstruction often requires repeated x-ray fluoroscopic confirmation, which will undoubtedly increase the operative time and aggravate the damage to the medical staff and patients.,17 However, due to the rapid development of arthroscopic techniques, more and more experts and scholars have mastered the all-scopic reconstruction of ATFL and CFL ligaments.,19 Arthroscopy allows for the combined management of intra-articular injuries, such as cartilage damage and free body formation due to repeated impingement; in addition, arthroscopy allows for the precise localization of the ligament footprint area, reducing the number of x-ray fluoroscopies and avoiding increased operative time and physical damage.,21Since most of the ligaments in patients with chronic lateral ankle instability cannot be repaired, surgical treatment usually requires reconstruction of the ATFL and CFL ligaments to restore joint stability.However, due to the increasing number of relevant studies published in recent years, the lack of high-level guidelines and evidence-based medical evidence has seriously affected clinical decision making and application. In this study, we attempted to conduct a meta-analysis of relevant studies to evaluate and compare the functional outcomes and complication rates of arthroscopic reconstruction of ankle lateral ligament with percutaneous reconstruction of ankle lateral ligament for chronic ankle lateral instability, and our findings are expected to provide a reference for guiding future treatment options.We have prospectively registered this research at the international prospective register of systematic reviews-Registration number: CRD42022362045. We performed this protocol based on the Preferred Reporting Items for Systematic Review and Meta-analysis Protocols (PRISMA-P) statement guidelines.The participants diagnosed as chronic ankle lateral instability will be included regardless their country, ethnicity, sex, occupation and mechanism of injury.In the experimental group, all patients received arthroscopic reconstruction of ankle lateral ligament. In the control group, all patients received percutaneous reconstruction of ankle lateral ligament.American Orthopaedic Foot and Ankle Society midfoot score will be defined as the primary outcomes to assess the function and stability.Visual analog scale, Tegner score, complications will be defined as secondary outcomes.We will include comparative studies which published in Chinese or English, such as randomized controlled trials, retrospective studies and cohort studies. Review, case reports, experimental studies, expert experience, animal studies and conference abstracts will be excluded.CNKI, Wanfang, VIP, CBM, PubMed, Embase, and Cochrane Library databases were searched for this study. using the keywords “ankle,” “ankle instability,” “ankle ligament injury,” “arthroscopy,” “percutaneous,” “reconstruction,” and “surgery.” The search strategy in PubMed is shown in Table Different researchers (XZ and HH) will separately screen the titles and abstracts of records acquired potential eligibility which comes from the electronic databases. The obtained literature is managed by Notoexpress, irrelevant and duplicate articles are excluded by reading the title and abstract, full texts screening and data extraction will be conducted afterward independently, and finally selected according to the inclusion criteria, any disagreement will be resolved by discussion with the third author (JL) until consensus is reached or by consulting a third author. PRISMA-P flowchart Will be used to show the selection procedure Fig. .The following data were extracted: lead author; publication year; country of origin; study design; sample size; age; injury type; reconstruction technique; outcome measures and complications. Any differences of opinion will be resolved through group discussion or consultation with a third reviewer. When relevant data is not reported, we will contact the author via email or other means to obtain missing data. The PRISMA flow diagram will be filled out after the screening study is completed to provide specific information.Two independent (XZ and WG) investigators evaluated the quality of the included studies. The Cochrane Collaboration Risk of Bias Tool was used to evaluate the quality of the randomized controlled trials. The methodological quality of the non-randomized studies was assessed using the Risk-of-Bias Assessment Tool for Non-randomized Studies. The level of evidence was assessed according to the Oxford Centre for Evidence-based Medicine Levels of Evidence.I2 statistic. Cases with I2 ≤ 50% will not be considered to have significant heterogeneity, thus a fixed-effects model will be applied for meta-analysis. In cases where there is statistical heterogeneity among studies, we will further analyze the source of heterogeneity. A random-effects model will be used to pool the data, after excluding the obvious source of clinical heterogeneity, and in cases where obvious clinical heterogeneity exists, the researchers will perform subgroup, sensitivity or only descriptive analyses. Study-specific and pooled estimates will be graphically presented using forest plots, and P < .05 considered statistically significant.Statistical analysis will be conducted using RevMan 5.4 software (Cochrane Collaboration). The mean difference will be used as the effect analysis statistic for continuous variables, while the risk ratio will be used as the effect analysis statistic for categorical variables. We will also calculate 95% confidence interval for each statistic, and summarize statistical heterogeneity among summary data using the Subgroup analysis according to the age, injury type and the classifications for chronic ankle lateral instability will be performed to find the source of heterogeneity when significant clinical heterogeneity is observed.Sources of heterogeneity were assessed by sensitivity analysis, by excluding studies of low quality or small sample size, if the heterogeneity did not change significantly, the results were robust. otherwise, the excluded studies may have been source of heterogeneity.,24In this study, fewer than 10 included studies were evaluated for publication bias using funnel plot, otherwise Egger regression test would be used.No ethical approval is required because the study will be a review of literature and will not obtain data from a single patient. We will publish our findings through a peer-reviewed journal.The purpose of this study was to comparatively evaluate the functional outcomes and complications of arthroscopic reconstruction of ankle lateral ligament and percutaneous reconstruction of ankle lateral ligament in the treatment of CAIL. With the maturation of arthroscopic techniques, more and more experts and scholars are using arthroscopic minimally invasive and precise treatment of ankle instability. To the best of our knowledge, this study integrates the most recent and comprehensive clinical evidence in this field, hoping to provide patients and clinicians with useful and high-grade evidence-based medicine.Conceptualization: Xing Zhou, Jinlei Li.Data curation: Huiming Hou.Formal analysis: Ming Zhou, Hong Yin.Funding acquisition: Rujie Zhuang, Jinlei Li.Investigation: Huiming Hou, Xing Zhou, Hong Yin.Methodology: Huiming Hou, Wenxuan Guo.Resources: Ming Zhou, Xing Zhou, Wenxuan Guo.Software: Ming Zhou, Xing Zhou.Supervision: Ming Zhou, Jinlei Li.Writing – original draft: Huiming Hou, Xing Zhou.Writing – review & editing: Rujie Zhuang, Jinlei Li.


Alcohol use disorder (AUD) is one of the most common substance use disorders contributing to both behavioral and cognitive impairments in patients with AUD. Recent neuroimaging studies point out that AUD is a typical disorder featured by altered functional connectivity. However, the details about how voxel-wise functional coordination remain unknown. Here, we adopted a newly proposed method named functional connectivity density (FCD) to depict altered voxel-wise functional coordination in AUD. The novel functional imaging technique, FCD, provides a comprehensive analytical method for brain's “scale-free” networks. We applied resting-state functional MRI (rs-fMRI) toward subjects to obtain their FCD, including global FCD (gFCD), local FCD (lFCD), and long-range FCD (lrFCD). Sixty-one patients with AUD and 29 healthy controls (HC) were recruited, and patients with AUD were further divided into alcohol-related cognitive impairment group  and non-cognitive impairment group . All subjects were asked to stay stationary during the scan in order to calculate the resting-state gFCD, lFCD, and lrFCD values, and further investigate the abnormal connectivity alterations among AUD-NCI, ARCI, and HC. Compared to HC, both AUD groups exhibited significantly altered gFCD in the left inferior occipital lobe, left calcarine, altered lFCD in right lingual, and altered lrFCD in ventromedial frontal gyrus (VMPFC). It is notable that gFCD of the ARCI group was found to be significantly deviated from AUD-NCI and HC in left medial frontal gyrus, which changes probably contributed by the impairment in cognition. In addition, no significant differences in gFCD were found between ARCI and HC in left parahippocampal, while ARCI and HC were profoundly deviated from AUD-NCI, possibly reflecting a compensation of cognition impairment. Further analysis showed that within patients with AUD, gFCD values in left medial frontal gyrus are negatively correlated with MMSE scores, while lFCD values in left inferior occipital lobe are positively related to ADS scores. In conclusion, patients with AUD exhibited significantly altered functional connectivity patterns mainly in several left hemisphere brain regions, while patients with AUD with or without cognitive impairment also demonstrated intergroup FCD differences which correlated with symptom severity, and patients with AUD cognitive impairment would suffer less severe alcohol dependence. This difference in symptom severity probably served as a compensation for cognitive impairment, suggesting a difference in pathological pathways. These findings assisted future AUD studies by providing insight into possible pathological mechanisms. Alcohol use disorder (AUD) is a progressive chronic disease, and it is considered a major public health problem in the world  was proposed to provide a comprehensive analytical method for brain's “scale-free” networks by comparing differences in functional connectivity between the whole brain and the given voxel in a high spatial resolution during the past few years, and FCD mapping has proven to be instructive in diagnosing and providing supplementary evidence for symptom improvement of several psychiatric disorders such as depression and schizophrenia  or AUD with non-cognitive impairment group  groups.This present study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Zhengzhou University and complied with the declaration of Helsinki, as revised in 2008, and all participants provided informed written consent before completing the survey.Individuals with AUD were recruited from a variety of sources including inpatient ward, internet posting, and advertisements. The control participants were volunteers from the local communities.Inclusion criteria were: (1) meeting the DSM-5 criteria for AUD, based on the clinical assessment of the principal investigator; (2) drinking on average more than 14 units of alcohol per week, according to the U.K. Chief Medical Officers  were: (1) having a history of addictive , psychiatric, neurological, or physical disorder that could influence brain morphology; (2) having contraindications for magnetic resonance imaging (MRI); (3) reported currently taking centrally active medications. All subjects included in this study were men.Demographic and clinical data are shown in Patients with AUD recruited for our study were further divided into alcohol-related cognitive impairment group (ARCI) (MMSE <24 and MoCA <26) and non-cognitive impairment group (AUD-NCI) (MMSE≥24 and MoCA ≥26) by testing MMSE and MoCA scales  with a 16-channel head coil at the First Affiliated Hospital of Zhengzhou University. All subjects were requested to keep their eyes closed, and foam padding and earplugs were used to control participants' head movements and reduce noise. At the end of scanning, subjects were also asked if they had fallen asleep during scanning. The rs-fMRI data were obtained using the following parameters: TR = 1,000 ms, TE = 30 ms, field of view 220http://www.restfmri.net) , and regression of nuisance covariates including Friston 24 motion parameters . FCD maps were further transformed to z scores by subtracting the mean value and dividing by the standard deviation across gray matter voxels in the brain  corrected]. Then, we extracted mean FCD values presenting significant differences among the three groups by averaging each peak coordinate with a spherical radius of 6 mm. The mean FCD values were compared with each pair of groups by using a two-tailed two-sample t-test.One-way ANOVA was used to compare  FCD maps among the three groups, respectively. In this procedure, age and mean FD were included as covariates. Results reported in this study were corrected for multiple comparison [voxel-wise To investigate the correlation between altered FCD values and symptom severity, Pearson's correlations between these altered FCD values (extracted before) and scores of clinical scales were calculated.p < 0.001), and ADS and OCDs scores were higher in the ARCI and AUD-NCI groups than in the HCs group (p < 0.001). The details are shown in The clinical demographics of the subjects are shown in p < 0.001, cluster-level p < 0.05; GRF corrected). Specially, the main effect of global FCD was located in brain regions including left medial frontal gyrus, left parahippocampal gyrus, left inferior occipital lobe, and left calcarine  and ADS , respectively.We found a significant association between altered FCD with clinical symptoms . SpecifiIn the current study, we explored local, long-range, and global FCD maps which come from healthy controls and patients with AUD, and we extracted mean FCD values presenting significant differences among the three groups. Besides, we found that the main effect of global FCD was located in brain regions and local FCD of the three groups exhibited significant differences in brain regions. Through statistical analysis of relevant data, we found a significant association between altered FCD with clinical symptoms. Specifically, the global FCD values in left medial frontal gyrus and that in left inferior occipital lobe were correlated with MMSE and ADS, respectively, and long-range FCD values in left medial frontal gyrus were correlated with MMSE. This suggests that alternations of FCD might be associated with AUD.Our study finds that the left medial frontal gyrus has significantly deviated among HC, ARCI, and AUD-NCI in gFCD. This change in gFCD possibly stems from a significantly lower density of medial frontal gyrus in patients with AUD found in a previous finding .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.


Percutaneous coronary intervention of chronic total occlusion (CTO PCI) is a challenging procedure with high complication rates and, as not yet fully understood long-term clinical benefits. Ischemic symptom relief in patients with high ischemic burden is to date the only established clinical indication to undergo CTO PCI, supported by randomized controlled trials. In this context, current guidelines suggest attempting CTO PCI only in non-invasively assessed viable CTO correspondent myocardial territories, with large ischemic areas. Hence, besides a comprehensive coronary angiography lesion evaluation, the information derived from non-invasive cardiac imaging techniques is crucial to selecting candidates who may benefit from the revascularization of the occluded vessel. Currently, there are no clear recommendations for a non-invasive myocardial evaluation or choice of imaging modality pre-CTO PCI. Therefore, selecting among available options is left to the physician’s discretion. As CTO PCI is strongly recommended to be carried out explicitly in experienced centers, full access to non-invasive imaging for risk-benefit assessment as well as a systematic institutional evaluation process has to be encouraged. In this framework, we opted to review the current myocardial imaging tools and their use for indicating a CTO PCI. Furthermore, based on our experience, we propose a cost-effective systematic approach for myocardial assessment to help guide clinical decision-making for patients presenting with chronic total occlusions. Chronic total occlusion (CTO) of coronary arteries represents an advanced form of atherosclerotic coronary artery disease, which is currently prevalent in circa one-fifth of patients presenting for diagnostic coronary angiography . CTO is Due to the lack of randomized trials, the hard-outcome benefits of CTO PCI are not yet fully elaborated. However, the HORIZONS-AMI trial observed that the presence of a CTO in patients undergoing PCI for ST-elevation myocardial infarction was associated with worse early and late clinical outcomes .CTO PCI is a challenging procedure with increased technical complexity and a need for appropriate operator experience. Through recent advancements and the development of dedicated methods and devices in the last few years, success rates of CTO PCI have increased significantly. However, they remain lower than non-CTO PCI–#, with successful revascularization of CTO vessels ranging from 60–70% of patients in inexperienced operating centers and 90–95% in some highly experienced centers ,12,13,14The European Society of Cardiology (ESC) guidelines on myocardial revascularization suggest choosing patients for CTO PCI in a similar manner to those who need treatment for non-CTO lesions, and explain that clinical benefits are analogous among these patient groups–hence, the rationale and criteria for decision-making in the revascularization of stable CAD should apply to the CTO subset . As statMost importantly, CTO PCI is currently recommended by the ESC in selected patients with angina symptoms resistant to medical therapy   Table 2Table 2. Pre-interventional evaluation of CTO lesions has indeed to be well elaborated as the main characteristics of this specific lesion subset, such as collateral vessels and complete antegrade flow impairment, restrict diagnostic availability or alter the interpretations for clinical indication. For example, the use of the broadly recommended FFR or the novel CT-FFR measurements is not routinely possible in CTO vessels . Thus, nIn this context, suitable candidates to undergo CTO PCI should be carefully identified and selected taking into consideration diverse clinical factors and supported by appropriate cardiac imaging techniques evaluating viability and ischemia.Guided by current recommendations and clinical practice, we opted to review the available evidence on benefits of CTO PCI and shed light on pre-interventional requirements for the consideration of revascularization. Throughout our review, we used the term revascularization to reference PCI as our primary focus, if not stated otherwise. Furthermore, we summarised the available non-invasive imaging methods that support the physician to guide the patient selection process.In patients with coronary artery disease and normal ventricular function without regional wall motion abnormalities assessed by echocardiography, intact myocardial viability can be presumed . In thesn = 205) investigated the left ventricular recovery in terms of wall thickness and ejection fraction and found no differences between CTO patients who underwent revascularization and those who received optimal medical therapy alone. However, the results were limited by the low rates of ventricular dysfunction at baseline and the revascularization of diseased donor vessels in the control group [On the other hand, the clinical benefit of revascularization in patients with left ventricular dysfunction is still ambivalent. A considerable proportion of CTO patients manifest heart failure with a reduction of left ventricular function  but it iol group . Howeverol group . This stol group ,23. Howeol group . A viabiol group . On the ol group .It seems that revascularization in ventricular dysfunction has prognostic benefits, but it has been long debated if this implication depends on the revascularization method. Indeed, in the general CAD population, the recent FAME-3 trial reported a non-inferiority of functionally-guided PCI vs. CABG in 1-year follow-up. However, patients with left ventricular dysfunction were underrepresented with ca. 18% in both treatment arms .Recently, one randomized trial recently addressed the evidence gap. The REVIVED trial investigated patients with viable dysfunctional left ventricles undergoing PCI and reported no benefit in survival or cardiac events in 3 years compared to the control group which received optimal medical therapy alone. Moreover, the trial showed no improvement in left ventricular ejection fraction after PCI . HoweverOn the other hand, non-randomized data suggest PCI survival benefits in left ventricular dysfunction: Gerber et al., reported a higher 3-year survival in patients with severe left ventricular dysfunction and viable myocardium who received revascularization. Overall, the study reported similar 3-year mortality as the REVIVED trial . AlthougIn patients with ventricular dysfunction, PCI benefits may be found mostly in the presence of hibernation. An observational study on 648 patients reported that an extent of hibernating myocardium exceeding 10% was associated with the benefits of revascularization . PhysiolRevascularization of ischemic but viable myocardium aims to minimize residual ischemia and subsequently improves symptoms and prognosis. Patients with a large ischemic burden (more than 10%) are considered to benefit the most from PCI . This stNowadays, invasive functional assessment of coronary artery stenoses can derive information related to the extent of ischemia in the distal supply region. Treatment of functionally significant stenoses, as assessed by fractional flow reserve, has been proven to be superior to revascularization guided by anatomical evaluation alone. The FAME trial reported better 2-year MACE rates after revascularization of ischemic myocardium, as assessed invasively with FFR . HoweverIndeed, CTO-related myocardium can be an ischemic area even in well-developed collaterals. Werner et al., reported a sufficient collateral flow in only 5% of CTO patients with preserved left ventricular function . When paWe reviewed the currently available non-invasive diagnostic tools that can help to set the clinical indication for revascularization in CTO patients and described how they can help properly characterize this patient group by investigating quantitative and qualitative parameters . The avaResting transthoracic echocardiography (TTE) is a widely available diagnostic method in heart disease and provides an incremental value in characterizing the effects of coronary artery disease on myocardial anatomy and function. As such, it is a fast and efficient way to investigate possible myocardial conditions following a suspected or confirmed coronary artery disease ,46. TTE Dobutamine echocardiography, on the other hand, enables a dynamic assessment of the improvement in contractile function (contractile reserve) in dysfunctional areas and indicates preserved viability. A biphasic response in myocardial wall motion alterations after injection of dobutamine (motion enhancement at low doses and downgrading at high doses) indicates hibernating myocardium and preserved viability . DobutamEchocardiography represents a valuable non-invasive imaging method, but in general it can be limited by reduced image quality in patients with difficult acoustic windows, high interpreter variability, as well as low sensitivity to quantify persistent and stress-induced defects using wall motion assessment.Single Photon Emission Computer Tomography (SPECT) detects myocardial perfusion defects by deriving myocardial radiotracer uptake distribution . DiverseA classic stress myocardial perfusion imaging protocol is performed after physical or pharmacological stress using vasodilators such as dipyridamole, adenosine, or regadenoson, followed by radiotracer injection. A resting study precedes or follows  depending on the selected protocol . ReversiLimitations in SPECT perfusion imaging have been outmatched by the use of more precise positron emission tomography (PET) imaging modalities. The concept is to assess myocardial perfusion  and metabolism (using F-18-FDG) .FDG is actively transported into the inside of cells by the glucose transporters . In areaDynamic PET myocardial perfusion assessment enables quantitative information on myocardial blood flow (MBF) and myocardial perfusion reserve (MPR) ,71,72. SFunctional cardiac magnetic resonance tomography (CMR) with late gadolinium enhancement (LGE) is a highly sensitive imaging method for the evaluation of myocardial viability . GadolinWhile stress-rest SPECT may underestimate viability, the LGE stress and rest CMR can better assess scar, ischemia and hibernating myocardium . Stress CTO patients are often multimorbid patients with coexisting medical conditions that could alter diagnostics and clinical decisions, which calls for a dedicated patient evaluation in an outpatient clinical setting . For thiSupported by robust evidence, viability has a pivotal role in predicting short and long-term revascularization benefits. In patients with extended scarring in the CTO region, revascularization benefits are not warranted, while in those with smaller regional defects, the extent of viability could be interpreted along with angiographic information, to finally derive the binary labels of significant viability vs. non-viable CTO region. CMR delivers more accurate information in this context, as it can quantify both the regional extent and thickness of LGE enhancement. SPECT imaging for viability and ischemia is widely accessible and affordable; new CZT cameras and adapted protocols expose patients to less radiation. Thallium-201 SPECT rest-redistribution imaging provides better viability assessment compared to technetium-99 m stress-rest SPECT; in case of doubt, FDG-PET might be performed additionally. In the same manner, ischemia can be underestimated in some cases in the classic segmental semi-quantification . HoweverIschemia assessment is a more complex process in CTO patients, which requires careful angiography integration. Due to the development of collaterals, the CTO region and donor vessel are interconnected, thus influencing hemodynamic parameters and myocardial perfusion distribution. The collateral circulation and myocardial “steal” have been long described in the literature and recently tested in FFR studies, implicating two main effects: firstly, collaterals are rarely sufficient to provide enough flow to CTO regions; blood flow is further reduced in hyperemia due to coronary steal . SecondlSymptom improvement is the primary indication for CTO PCI with the strongest evidence to date. For this reason, ischemic symptoms and the will to accept the peri-interventional risks for improvement of life quality should stay on the plateau of the information cluster when conducting the pre-interventional evaluation. Secondly, the prognostic benefits of CTO PCI can be evaluated and taken into consideration, as stated in the European guidelines (CTO in left anterior descending or left main coronary artery, multi-vessel disease, reduced ejection fraction—Our proposed systematic workflow is presented in In conclusion, in the context of the lack of established patient evaluation algorithms including recommendations of specified cardiac imaging methods, this review has to be read as a suggestion according to the literature and the experience of the authors. Indeed, the evaluation process workflow is currently left to the physician’s discretion. However, patient evaluation should be conducted by physicians who are familiar with imaging techniques, complex coronary angiography and CTO PCI.


The adult brain can flexibly adapt behaviors to specific life-stage demands, and a classical example of this plasticity of neural circuits is the emergence of infant-rearing behavior in male mice. Both physically and mentally, the care that fathers provide is necessary for the growth of pups  on mammals  in the PVN. Then, they established a behavioral assay and applied it to wild-type (OT+/+) and OT−/− mice to assess caregiving behaviors; three unfamiliar pups from different families were put into three cages, and a father mouse that was unrelated to these pups was allowed to interact with them freely. While the OT+/+ mice displayed caring behavior, a large percentage of the OT−/− male mice ignored the young mice. This result indicates that OT neurons in the PVN are necessary to exert parental behaviors in fathers. Further examination of expectant fathers illustrated that the activity of OT neurons is essential for the first appearance of paternal caregiving behaviors.First, to explore the relationship between parenting behavior and OT in male mice, they used CRISPR-Cas9-mediated gene editing technology and Cre-expressed adeno-associated virus to conditionally knock out OT  activities in the perifornical area (peFA) were inhibited, whereas the neural activities of Calcr+ neurons  in the medial part of the MPN (MPNm) were elevated. These outcomes illustrate that OT neurons could regulate the limbic neural populations related to parental and infanticidal behaviors in virgin male mice.−/− mice, increased caregiving behaviors were displayed by the OT+/+ mice but not the OT−/− virgin male mice. Nevertheless, pup-directed attacks between these two genotypes were suppressed after the injection. Taken together, these data suggest that, compared to the significant promotion from OT, other neurotransmitters are also conducive to caregiving behaviors.Moreover, OT neurons can release non-OT neurotransmitters and neuropeptides. To identify their role in parenting behavior in the PVN, they used hM3Dq-myc driven by an OT promoter (OTp) to activate OT neurons in virgin male mice. After injection of clozapine N-oxide, which can lead to the release of neurotransmitters or neuropeptides other than OT in OTTo further investigate the change in neural connections of paternal behavior, the authors concentrated their work on PVN OT neurons. Using rabies virus-based retrograde trans-synaptic tracing, OT neurons in the fathers displayed more input from the LHA and MPNm without a change in the number of neurons compared to virgin males. Interestingly, the increased inputs observed 5 days after the birth of the pups returned to a level similar to that of virgin males after 5 weeks of isolation, revealing that the enhanced connection was temporary and reversible. Moreover, histochemical methods and further analysis of cell-type-specific marker genes demonstrated that the inputs to PVN OT neurons were mostly excitatory neurons from the LHA and MPNm, especially melanin-concentrating hormone-producing excitatory neurons in the LHA.vesicular glutamate transporter type 2 (vGluT2)-Cre mice. Consistent with the trans-synaptic tracing results, the excitatory postsynaptic currents evoked by the optogenetic stimulation of LHA and MPNm inputs were slightly, but not statistically significantly, larger in the fathers. However, the response to the DMH stimulation remained unchanged. Furthermore, optogenetic activation of excitatory neurons in the LHA evoked more spikes in PVN OT neurons, which would be even more when MPNm was activated concomitantly. These results demonstrate that the enhanced excitatory connectivity from the LHA to OT neurons is associated with life-stage transition.To examine the electrophysiological properties of excitatory inputs to OT neurons, they injected the AAV-OTp-mCherry virus into the PVN and AAV-FLEx-ChR2 (H134R) virus into the LHA, MPNm, and dorsomedial hypothalamus (DMH) of in situ staining, they found that fathers who interacted with pups expressed c-fos at a higher ratio in the LHA than those who were not exposed to pups. Notably, most c-fos+ neurons were vGluT2-positive and Pmch-expressing excitatory neurons. They focused on the functional contributions of this connection. Chemogenetic inhibition of vGluT2+ LHA neurons only slightly increased aggressive behaviors toward the pups in virgin male mice. Conversely, targeted hM3Dq-myc in the vGluT2+ LHA neurons of OT+/+ and OT−/− virgin males showed a reduction in aggression toward the pups of virgin males without evoking caregiving behaviors. More importantly, OT+/+ virgin males were significantly less aggressive than their OT−/− counterparts. Taken together, OT release mediated by excitatory LHA neurons provokes the parental behaviors of fathers by suppressing infanticide.Finally, what was the specific relationship between excitatory connections from the LHA to OT neurons and parental behavior? Through the use of Fathers play a unique role in the growth of their children  neurons can facilitate OT release from OT+ PVN neurons into central nervous system and blood, leading to parental behavior. Although OT modulation in rodents' behaviors have been extensively studied, little is known on its mechanism in the regulation of parental caregiving behaviors of virgin male mice.OT, acts in the brain as a non-canonical neurotransmitter or neuromodulator, has been long known to shape behavior in rodents  is a major clinical drug delivery route, which is a more easier, more efficient administration way and can protect the body from systemic toxicity (MacDonald et al., Intranucleus OT release from PVN neurons and into the bloodstream from the nerve terminals of this nucleus in the posterior pituitary (Eliava et al., In conclusion, Miyamichi et al. demonstrated that PVN hypothalamic oxytocin neurons and OT ligands are key regulators of parental caregiving behaviors in male mice. The plasticity of the hypothalamic neural connections is related to life stages, long distances, and specific cell types. These considerable discoveries help to provide scientific support for the mitigation of mental health conditions in children that are caused by a lack of parental caregiving behaviors and present a pattern for investigating other behavioral changes.ZC, QW, XX, ZH, and YW wrote and edited the manuscript. All authors have contributed to the manuscript and approved the submitted version.


The biomarkers era grew in the last two decades when several technical and methodological advances have improved the research in neurodegenerative diseases. All neurodegenerative conditions are progressive and debilitating disorders due to premature and irreversible neuronal loss, leading to consequent clinical deficits. The improvement in life expectancy entails a worldwide increase in these disorders, aging being the greatest risk factor for these incurable conditions. Identifying early and reliable markers for neurodegenerative diseases potentially allows the clinician to reach a timely diagnosis, provide disease staging and monitoring disease progression, increase prognostic precision, and reduce the health system burden. All these goals are essential to design clinical trials and test novel therapeutic strategies correctly.Most neurodegenerative diseases share similar pathogenic events, starting from the pathological deposition of misfolded proteins and leading to progressive neuronal damage associated with altered neuroinflammatory response. The clarification of the common pathogenic processes may open the development of effective therapeutic approaches in a field still lacking disease-modifying drugs. The current therapeutic strategies rely only on symptomatic treatment, which can stabilize or temporarily slow the clinical worsening, but they do not impact the underlying disease mechanism. For these reasons, searching for disease-specific biomarkers is a crucial step in this direction.Currently, most biomarkers are classified based on the pathogenic mechanism they can underline. Biomarkers of pathology, including markers for amyloid-β (Aβ) pathology and tau pathology, are primarily used in Alzheimer’s disease (AD) and the Frontotemporal lobar degeneration (FTLD) spectrum. They can be addressed mainly through cerebrospinal fluid (CSF), blood analysis, and positron emission tomography (PET) imaging. For Parkinson’s disease (PD) and parkinsonism, to date, the search for α-synuclein can be feasible in CSF, while no PET ligands selective for α-synuclein aggregates have been validated in humans . In addiAlzheimer’s disease (AD) is the most common neurodegenerative disease, and the biomarkers research has been primarily focused on this condition. The ATN system, based on the in vivo evidence of Aβ (A), tau pathology (T), and neurodegenerative changes (N), revolutionized the concept of the clinical diagnosis shifting to a biological definition of the AD spectrum. The AD continuum ranges from the overt dementia phase to the preclinical, asymptomatic, or subtle symptomatic phase associated with AD neuropathological changes. A precise diagnostic process along the AD spectrum is essential to provide optimal management for the patients, avoiding misdiagnosis and unsuccessful treatment .The diagnostic role of CSF biomarkers has been largely validated for dementia disorders. CSF biomarkers including phosphorylated-tau, total-tau, Aβ-42, Aβ-40, and NfL may support the in vivo identification of AD and FTLD neuropathology and neurodegenerative-related changes, facilitating differential diagnosis .Recent studies on people at risk for developing dementia pointed out this population’s heterogeneity, which can be composed of different underlying etiologies but mainly include people without signs of neurodegeneration, as detected by FDG-PET . The seaNfLs represent intriguing candidates in the prognostic workup of neurodegenerative diseases. While the turnover of NfL in healthy aging brains is slow, it is increased, in different disease-related behaviors, in several neurodegenerative conditions. To date, NfLs are largely used in Multiple Sclerosis, various dementia conditions such as AD and FTLD, and several other neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS). A study enrolling 231 patients with suspected ALS showed high diagnostic performance in discriminating patients from mimics , although CSF NfL performed slightly better than plasma NfL. In addition, higher baseline NfL levels in CSF and plasma were demonstrated in ALS patients with shorter survival. However, longitudinal plasmatic analyses of NfL showed stable levels of NfL over the disease course, questioning the role of this marker in staging the disease. This last finding can be influenced by several factors and needs further longitudinal research, but its eventual reduction in clinical trials would be a significant slowdown in the neurodegenerative process .Despite not-univocal results, the longitudinal changes in biomarker measurements may help in staging the disease progression and eventually in monitoring response to therapy. Neuroinflammation is now recognized as a critical element in the neurodegenerative process. Microglia activation can be visualized in vivo by PET imaging of neuroinflammation, specifically using tracers for the 18 kDa translocator protein (TSPO), overexpressed by activated glial cells. TSPO-PET potentially allows for visualization changes in neuroinflammatory responses over the disease course, thus revealing different phases of the neurodegenerative process. The study of microglia activation has provided intriguing results in Mild Cognitive Impairment (MCI), a transitional condition between normal aging and dementia. MCI individuals showed higher baseline TSPO-PET signals than controls, with a longitudinal decrease of microglia activation, despite an increased cerebral amyloid burden detected by amyloid-PET. In addition, AD patients showed higher baseline TSPO-PET signal than controls, but with a further increase over time. These findings supported the hypothesis of a double peak in microglia activation along the AD trajectory, with an initial, protective activation in MCI subjects, possibly contrasting neurodegenerative changes, and a late, proinflammatory activation in AD patients with deleterious consequences on neuronal functions . TSPO-PEAs shown, identifying markers able to stage the disease or monitor disease progression may be crucial in clinical trials. Identifying possible clinical trajectories along the neurodegenerative process offers the possibility of testing therapeutic strategies in a pre-symptomatic phase. For several years, many clinical trials failed; however, it is not certain whether these failed due to the actual ineffectiveness of the trial or difficulty in identifying any clinical or biological variations due to the absence of specific disease markers. Likewise, in recent clinical trial designs , biomarkers have been included among primary and secondary outcomes, highlighting the importance of biomarkers incorporation in the early phase of drug development.However, some considerations about the best biomarkers, or combination of biomarkers, to include in clinical trials are needed. Firstly, the selected biomarkers must be easily measurable and reproducible in several centers, considering the growing spread of neurodegenerative diseases. In this regard, NfL can represent a favorable biomarker for the limited costs and the recent simple technique to dose them both in CSF and plasma . SecondlIn recent decades researchers have been trying to enhance the development of reliable biomarkers for neurodegenerative diseases, albeit with conflicting and not consistently optimal results, while the medical practice has been moving toward precision medicine. However, there is still an urgent need to integrate the disease-specific biomarkers in clinical practice and develop effective disease-modifying therapies. In addition, the current identification of possible clinical trajectories along the neurodegenerative process in several subjects, such as patients with subjective cognitive complaints or patients who carried ALS mutations, offers the possibility of testing therapeutic strategies in a pre-symptomatic phase. Similarly, the investigation of neuroinflammatory responses represents a possibly invaluable tool in monitoring disease progression and predicting clinical outcomes.We are proposing a research topic focused on biomarkers in neurodegenerative diseases, aiming to gather evidence on the molecular mechanisms involved in neurodegeneration. This Special Issue represents a chance for clinicians and researchers to provide new insights into the pathogenesis of neurodegenerative diseases, encouraging the development of research for novel potential therapeutic interventions and patient management.


While the androgen receptor (AR) signalling is the mainstay therapeutic target for metastatic prostate cancers, these tumours will inevitably develop therapy resistance to AR pathway inhibitors suggesting that prostate tumour cells possess the capability to develop mechanisms to bypass their dependency on androgens and/or AR to survive and progress. In many studies, protein kinases such as Src are reported to promote prostate tumour progression. Specifically, the pro-oncogene tyrosine Src kinase regulates prostate cancer cell proliferation, adhesion, invasion, and metastasis. Not only can Src be activated under androgen depletion, low androgen, and supraphysiological androgen conditions, but also through crosstalk with other oncogenic pathways. Reciprocal activations between Src and AR proteins had also been reported. These findings rationalize Src inhibitors to be used to treat castrate-resistant prostate tumours. Although several Src inhibitors had advanced to clinical trials, the failure to observe patient benefits from these studies suggests that further evaluation of the roles of Src in prostate tumours is required. Here, we summarize the interplay between Src and AR signalling during castrate-resistant prostate cancer progression to provide insights on possible approaches to treat prostate cancer patients. Androgens and the androgen receptor (AR) play key roles during the development of prostate cancer (PCa). They are also tightly coupled with PCa progression to the castrate-resistant prostate cancer (CRPC) state. Targeting AR signalling has been a mainstay therapeutic option to manage locally advanced and metastatic PCa , 2. ThesExcept for small cell carcinoma and DNPC, most CRPC tumours express AR proteins –11. The in vitro cell models indicated that the ligand-dependent and ligand-independent AR signalling, and AR bypass mechanisms can all be possibly adopted by cancer cells. The LNCaP cell model, androgen-dependent tumour, can form xenografts only in non-castrated mice  cells, and basal cells that are believed to be sustained in a homeostasis state by stem/progenitor cells within the basal layer through differentiation . The AR The mechanisms by which the transition of AR high PCa to AR negative SCNPC or DNPC are not fully defined. Two possible mechanisms have been proposed –40. The In summary, the heterogeneity of prostate tumours is a natural challenge to AR target therapy. CRPC tumour cells have different genetic and epigenetic backgrounds, cell lineages, or evolutional stages (from being androgen-dependent to AR indifferent) within a tumour. Targeting AR signalling in the long-term is predictably ineffective since it blocks one cancer cell population and permits others to expand. Combinational therapies would be a future therapeutic option.AR can exert its transcriptional activity in the absence of androgens through its N-terminal domain (NTD) . AndrogeThe NTD is a domain with a flexible conformation that can interact with various coregulators to activate AR. It possesses the Tau5 domain (aa360-485) that mediates the ligand-independent activation of AR . InteresBlocking the LBD by ARPIs creates opportunities for NTD to interact with growth factors, cytokines, and intracellular kinases to activate AR signalling. Epidermal growth factor (EGF) acts through its membrane receptor to stimulate Src activation, which in turn activates AR Tyr534 phosphorylation . IL-6 enIntracellular kinases play critical roles in regulating ligand-independent AR signalling, among which the proto-oncogene tyrosine kinase Src had been demonstrated to contribute to CRPC progression , 60. It Src activation is mainly controlled by its intramolecular phosphorylation modifications and by Src interacting proteins. Src is inactivated when Tyr530 is phosphorylated by CSK and its homolog CHK, and recognized by the SH2 domain resulting in a closed inactive Src conformation . In contSrc was reported to play an important role in regulating cancer cell adhesion, migration, and invasion in addition to its established roles in cell proliferation. Src is required for the transition of the G2 to M phase of mammalian fibroblasts , as it fThe AR and Src can form a protein complex in PCa cells indicating that there exists crosstalk between both factors . GeneticMutual activation between AR and Src can exist not only under androgen depletion conditions but also in the presence of androgens. Under androgen deprivation conditions, growth factors  and cytokines  can mediate signalling to induce tyrosine phosphorylation of AR, and activate the transcriptional activity of AR , 58, 101In the presence of androgens, androgen-sensitive LNCaP cells were reported to stimulate Src autophosphorylation and its kinase activity by ligand-activated AR or by ligand-activated estrogen receptor beta (ERβ) , 104. Thex vivo PCa tissue, were challenged with supraphysiological doses of androgens (SPA), it induces cell cycle arrest at the G1 phase and cell senescence through cell cycling regulators such as CDK inhibitor p16, Rb1, and E2F1  all serve as the upstream regulator of Src activation, and enhanced Src activation is associated with CRPC progression. However, whether blocking Src activity will suppress CRPC remains to be defined. Src knockout mice were viable at birth but died due to impaired osteoclast function , suggestResults from preclinical studies using multiple PCa cells and xenograft models had built a reasonable rationale that targeting Src kinases would effectively suppress prostate tumour growth and progression in patients. Several phase I and II clinical trials in various tumour settings had also shown that dasatinib and saracatinib are well tolerated by patients with acceptable side effects, and the optimal dosing had been determined –115. DasThe inconsistent results from pre-clinical and clinical studies raised several questions. Is the Src activity critical for CRPC progression? Genetically engineered mouse model studies did not support that Src is essential for cell viability . The assDo all tumours/tumour cells respond to Src inhibitors? There exist inter-patient and intra-tumour heterogeneity of prostate tumours. Tumour cells carrying heterogeneous genetic and epigenetic backgrounds may respond to Src inhibitors differently. Particularly, prostate tumours in these clinical trials had progressed to the metastatic stage post castration therapies , previouAre Src activities sufficiently blocked within patient tumours? Some clinical trials had used phosphor-Src protein  or mRNAsThe Src signalling plays an important role to promote PCa development and CRPC progression. The interplay between Src and AR regulates PCa cell proliferation and metastasis under various androgen regimes, implying potential Src target therapy for CRPC. The negative results of Src inhibitors in clinical trials on PCa urge further mechanistic investigations at the molecular level on the roles of Src in PCa.Writing-original draft preparation, LG and XD; writing-review and editing, BH and XD; All authors have read and agreed to the published version of the manuscript.This work was funded by Canadian Institute of Health Research operating grants (MOP-137007 & PJT-156150) to XD.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.


Extensive research efforts have been devoted to describing yeast alcohol oxidase (AO) and its promoter region, which is vastly applied in studies of heterologous gene expression. However, little is known about basidiomycetous AO and its physiological role in wood degradation. This review describes several alcohol oxidases from both white and brown rot fungi, highlighting their physicochemical and kinetic properties. Moreover, the review presents a detailed analysis of available AO-encoding gene promoter regions in basidiomycetous fungi with a discussion of the manipulations of culture conditions in relation to the modification of alcohol oxidase gene expression and changes in enzyme production. The analysis of reactions catalyzed by lignin-modifying enzymes (LME) and certain lignin auxiliary enzymes (LDA) elucidated the possible involvement of alcohol oxidase in the degradation of derivatives of this polymer. Combined data on lignin degradation pathways suggest that basidiomycetous AO is important in secondary reactions during lignin decomposition by wood degrading fungi. With numerous alcoholic substrates, the enzyme is probably engaged in a variety of catalytic reactions leading to the detoxification of compounds produced in lignin degradation processes and their utilization as a carbon source by fungal mycelium. Drosophila melanogaster glucose dehydrogenase, Escherichia coli choline dehydrogenase, Aspergillus niger glucose oxidase, and Hansenula polymorpha methanol oxidase. Interestingly, these enzymes not only belong to different taxonomic groups but also catalyze different chemical reactions. Nevertheless, Cavener managed to identify a common pattern in their structure: a canonical ADP-binding βαβ-fold close to their amino termini, which is found in other flavoenzymes as well. Since then, a number of enzymes have been assigned to the GMC oxidoreductase family, expanding not only the number of proteins identified in many new taxa but also the number of new substrates for these oxidoreductases. The family comprises both oxidases and dehydrogenases, which are able to oxidize various sugars, alcohols, cholesterol, or choline, employing oxygen or different quinones, phenol radicals, or metal ions as the final or alternative electron acceptors . Fu. FuO-demisoforms . This maisoforms . In a siisoforms  proved ai et al.  showed tradation . Among eradation . Alcoholtrabeum) . In turnScientific data have suggested that basidiomycetous alcohol oxidase is important in secondary reactions during lignin decomposition by wood degrading fungi. With numerous alcoholic substrates, the enzyme is probably engaged in a variety of catalytic reactions leading to the detoxification of compounds produced in lignin degradation processes and their utilization as a carbon source by fungal mycelium. Hydrogen peroxide, i.e., the by-product of alcohol oxidation by AO, serves as a substrate for heme peroxidases  or is engaged in Fenton reactions, all being crucial parts of the sophisticated wood degrading machinery. Moreover, it seems that formaldehyde produced by alcohol oxidase may be used by fungi as biocontrol agents against unwanted organisms in their ecological niche.G. trabeum) in both white and brown rot fungi. As demonstrated, alcohol oxidase genes have been shown to be overexpressed in response to lignin substrates in the medium; however, it is not clear whether their expression is boosted via regulatory elements in their promoter regions or whether their genes are clustered with other wood-degrading enzymes and are thus expressed together. Future studies consisting of detailed enzyme analyses, mapping of lignin degradation pathways, and the regulation of gene expression may highlight the complete physiological role of the enzyme in wood-degrading fungi.Still, only few basidiomycetous AOs have been purified and characterized in terms of their physicochemical and kinetic properties. Little is known about their cellular localization (except for


Lymphedema is characterized by progressive and chronic tissue swelling and inflammation from local accumulation of interstitial fluid due to lymphatic injury or dysfunction. It is a debilitating condition that significantly impacts a patient’s quality of life, and has limited treatment options. With better understanding of the molecular mechanisms and pathophysiology of lymphedema and advances in tissue engineering technologies, lymphatic tissue bioengineering and regeneration have emerged as a potential therapeutic option for postsurgical lymphedema. Various strategies involving stem cells, lymphangiogenic factors, bioengineered matrices and mechanical stimuli allow more precisely controlled regeneration of lymphatic tissue at the site of lymphedema without subjecting patients to complications or iatrogenic injuries associated with surgeries. This review provides an overview of current innovative approaches of lymphatic tissue bioengineering that represent a promising treatment option for postsurgical lymphedema. The lymphatic system is present throughout the body and is an important part of circulatory, immune, and metabolic systems. The lymphatic system serves three main functions: fluid homeostasis, immune regulation, and lipid absorption ,2. The pISF enters the lymphatic system through blind-ended lymphatic capillaries in the periphery of the body and travels through lymphatic vessels before return to the blood circulation . LymphatThe lymphatic system also plays a pivotal role in immune surveillance and lipid absorption. Collecting lymphatic vessels are connected to lymph nodes, serving as a conduit for foreign antigens as well as immune cells. Dendritic cells and T cells migrate from tissues to draining lymph nodes and return to blood circulation. As such, the lymphatic system is involved in immunologic responses, inflammation, cancer metastasis, and cardiovascular and metabolic disorders . In the + cells, transcription factors, SOX18 and COUP-TFII, induce the expression of the critical lymphatic transcription regulator, PROX1. The expression of PROX1 upregulates VEGFR-3 and renders LECs more responsive to VEGF-C signaling, enhancing the LEC polarization and formation of lymph sacs lateral to the cardinal veins ). This polymer has a superior surface nanotopography and viscoelasticity than other synthetic scaffolds . The nanBiodegradable scaffolds provide a temporary scaffold that can be substituted with cellular matrix and avoid material-related side effects. Polyglycolic acid (PGA) is one of the commonly used biodegradable scaffolds with well-documented biocompatibility and ability to custom design . A preliAlthough synthetic scaffolds have demonstrated a potential to be used for tissue engineering, they did not achieve widespread acceptance for clinical uses. Instead, using biocompatible scaffolds with seed cells has gained more popularity.Fibrin and collagen are natural materials used for scaffolds. They are readily accessible and contain a spectrum of biochemical and physical cues needed for cell morphogenesis and function . They weIn the presence of appropriate biochemical stimuli such as VEGF-C and seed cells, natural proteins such as fibrin and collagen can provide adequate structural support for lymphangiogenesis. However, there are several drawbacks: the diversity of biochemical and physical cues may complicate when trying to isolate the interaction of specific cells and growth factors. Furthermore, their predefined chemical and mechanical properties can be frustrating when engineering specific lymphatic tissues.+ LYVE-1−) and lymph-like capillaries (CD31− LYVE-1+) inside the dermis. This type of dermis has some great advantages such as precise controllability of the thickness, whole and homogeneous construction, and higher biological functions.Gibot et al. described that human lymphatic endothelial cells (LECs) cocultured with dermal fibroblasts could spontaneously organize into a stable 3D lymphatic capillary network devoid of any exogenous material such as scaffolds or growth factors . In vitrVarious studies have shown the potential of organ-derived decellularized extracellular matrix (dECM) scaffolds in engineering organ-like structures. dECM scaffolds overcome the challenge associated with the design and fabrication of synthetic scaffolds. Pre-clinical and clinical application of dECM scaffolds from discarded human donor organs in tissue engineering has been explored by various studies ,115,116.Several studies have used the decellularization technique to engineer lymph nodes and lymphatic vessels ,118,119.There are challenges with dECM scaffolds including poor cell infiltration and migration due to the dense matrix that remains after decellularization, necessitating excessive times to repopulate the scaffolds. Additionally, other drawbacks of decellularized scaffolds in clinical settings are limited resources and difficulty in surgical manipulation . HoweverOne of the most recent technologies is three-dimensional (3D) bioprinting. Three-dimensional bioprinting produces a complex, high-resolution architecture that recapitulates the physiologic structure and function of human tissues. Living cells and biomaterial containing biochemical or biophysical cues can be positioned precisely into a predefined architecture ,121. TheThe feasibility of the 3D bioprinting of vascular grafts has been demonstrated ,124. KolChallenges remain in the proper selection of biomaterials and bioprinting methods to achieve desired structural, mechanical, and biological properties of the tissue . The lymThe role of interstitial fluid flow in lymphatic formation and regeneration have been extensively studied ,127,128.Given its role in lymphatic morphogenesis and functionality, interstitial flow has been utilized for lymphatic capillary engineering. Interstitial flow was incorporated into scaffolds of different compositions of fibrin and collagen and was observed to affect cellular behaviors. LECs organized best in fibrin-only matrices, which exhibit higher compliance and lower hydraulic permeability . AlthougExtracorporeal shock wave therapy (ESWT) was first introduced into clinical practice for urologic indication, to fragmentize and remove kidney stones. It is clinically used for the treatment of musculoskeletal diseases including adhesive capsulitis, calcific tendinitis, and plantar fasciitis. ESWT produces an acoustic pressure with positive and negative phases. The positive phase produces a direct mechanical force, and negative phase produces cavitation. Various mechanisms of action have been described for ESWT and one of them being its ability to amplify growth factor and protein synthesis for tissue remodeling . Low-eneThe findings of in vivo studies support its clinical application for lymphedema. Indeed, ESWT has been shown to be effective in treating lymphedema. The use of ESWT on the treatment of upper and lower extremity lymphedema was first reported in 2010. Seventy-two geriatric patients with primary or secondary lymphedema with associated fibrosis received ESWT and demonstrated a reduction in limb circumference and skin thickness . In patiLymphatic tissue bioengineering efforts are currently focused on a combination of cell-seeded scaffolds with pro-lymphangiogenic factors and external mechanical stimuli to stimulate the growth of lymphatic vessels . NaturalVarious stem cells have demonstrated potential for lymphatic regeneration including ESCs, iPSCs, HSCs, MAPCs, ECFCs, and ADSCs. Once a reliable cell source for bioengineering is seeded in the scaffold, biomolecular and mechanical stimuli are necessary to stimulate these cells to form lymphatic network. Most widely used pro-lymphangiogenic factor for lymphatic engineering is VEGF-C. Studies to date have recognized pro-lymphangiogenic effects of various factors including VEGF-C, PDGF, FGF, TGF-β, and retinoic acid. Whether these factors exert pro-lymphangiogenic effects independently or supplementary to one another are not well understood. Further studies are needed to find an ideal combination of these factors that can augment cell sprouting and support cell maturation and differentiation . ProlifeSeveral studies have highlighted the importance of supporting cells. Fibroblasts, ADSCs, and DPSCs have been explored as a co-culture model to support LEC growth. Supporting cells provide pro-lymphangiogenic growth factors and direct cell–cell contact that promote LEC growth. Fibroblasts have been most extensively used with LECs, however, several studies reported that fibroblasts form poor lymphatic vessels and take longer to form lymphatic vessels. Previous studies have shown that ADSCs support vasculogenesis and lymphangiogenesis ,137,138.Biomedical applications of bioengineered lymphatic tissues include tissue or organ implantation and reconstruction as well as disease modeling in vitro to study the pathophysiology of the disease as well as drug screening and development. Various strategies to engineer lymphatic tissues have been reported with encouraging in vitro and in vivo data. However, lymphatic tissue engineering has been relatively less studied and therefore, numerous challenges remain to be solved, including design, functionality, and long-term application before it can be implemented in a clinical management and treatment of lymphedema.Most lymphatic engineering efforts to date have been put toward regenerating lymphatic capillaries with less attempts at constructing larger collecting vessels and lymph nodes. Engineering of a larger and more complex lymphatic structure is especially important for clinical application. In secondary lymphedema, edematous areas are typically large. To effectively drain from large areas and decrease swelling, larger vessels are required for drainage . The resAnother challenge is to design an endothelialized conduit, attaching seed cells onto a scaffold and supporting the growth of these cells. Endothelialization is present in the anatomy of a native lymphatic vessel and therefore essential in engineering a successful lymphatic graft . AdditioBioengineered lymphatic structures need to be evaluated for the phenotype and functionality. To confirm the lymphatic phenotype, its identifying morphologic characteristics, such as the absence of complete basement membrane and mural cell coverage, and the expression of lymphatic specific markers, such as Prox1, Lyve1, and Podoplanin, can be evaluated. In addition, the ability of bioengineered structures to respond to pro- and anti-lymphangiogenic stimuli should be assessed . LymphatRecent advances in lymphatic tissue bioengineering have demonstrated potential for its clinical application for lymphedema. Yet, the challenges of designing larger lymphatic vessels within a complex lymphatic system hierarchy remain. With technological innovations and growing research to develop larger-sized and more complex bioengineered tissues and organs, current challenges need to be addressed in future studies.