Determining readiness for duty after mild traumatic brain injury (mTBI) is essential for the safety of service members and their unit. Currently, these decisions are primarily based on self-reported symptoms, objective measures that assess a single system, or standardized physical or cognitive tests that may be insensitive or lack ecological validity for warrior tasks. While significant technological advancements have been made in a variety of assessments of these individual systems, assessments of isolated tasks are neither diagnostically accurate nor representative of the demands imposed by daily life and military activities. Emerging evidence suggests that complex tasks, such as dual-task paradigms or turning, have utility in probing functional deficits after mTBI. Objective measures from turning tasks in single- or dual-task conditions, therefore, may be highly valuable for clinical assessments and return-to-duty decisions after mTBI. The goals of this study are to assess the diagnostic accuracy, predictive capacity, and responsiveness to rehabilitation of objective, dual-task turning measures within an mTBI population. These goals will be accomplished over two phases. Phase 1 will enroll civilians at three sites and active-duty service members at one site to examine the diagnostic accuracy and predictive capacity of dual-task turning outcomes. Phase 1 participants will complete a series of turning tasks while wearing inertial sensors and a battery of clinical questionnaires, neurocognitive testing, and standard clinical assessments of function. Phase 2 will enroll active-duty service members referred for rehabilitation from two military medical treatment facilities to investigate the responsiveness to rehabilitation of objective dual-task turning measures. Phase 2 participants will complete two assessments of turning while wearing inertial sensors: a baseline assessment prior to the first rehabilitation session and a post-rehabilitation assessment after the physical therapist determines the participant has completed his/her rehabilitation course. A variable selection procedure will then be implemented to determine the best task and outcome measure for return-to-duty decisions based on diagnostic accuracy, predictive capacity, and responsiveness to rehabilitation. Overall, the results of this study will provide guidance and potential new tools for clinical decisions in individuals with mTBI.Clinical Trial Registration: clinicaltrials.gov, Identifier NCT03892291. Determining readiness for duty after mild traumatic brain injury (mTBI) is essential for the safety of service members (SMs) and their units. Sensory, motor, and cognitive deficits such as delayed reaction time , imbalanCurrently, RTD decisions are based on self-reported symptoms, objective measures that assess a single system, or standardized physical tests that lack ecological validity for warrior tasks or battle drills. Duty-readiness is primarily assessed using patient-reported symptoms –16 and tWhile understudied in mTBI populations , ambulatTurning velocity and head–body coordination during activities with cognitive demands are critical to an SM's ability to safely and effectively scan and maneuver through operational environments. The ability to successfully acquire, aim, and hit a target is a critical warrior task that requires rapid and coordinated reorientation of the head and body . FurtherQuantifying turning performance has traditionally been difficult in clinical settings, but recent advances in wearable sensors have enabled objective, robust, reliable, and sensitive assessments of turning performance in other clinical populations , 55–59. ClinicalTrials.gov (NCT03892291).This study has two phases: for Phase 1, participants will be recruited from the general civilian populations surrounding three sites  and active-duty SMs at one site [Fort Sam Houston (FSH)] to address the first two goals of the study (diagnostic accuracy and predictive capacity). For Phase 2, participants will be recruited from active-duty SMs referred to military medical treatment facilities  for physical therapy following mTBI to address the third goal of the study (responsiveness to rehabilitation). This study has been registered on Phase 1 will include approximately 50 civilians with persistent symptoms from mTBI, 50 civilian healthy controls, and 40 active-duty SM healthy controls. Three non-military sites  will test civilians. A fourth military site (FSH) will enroll and test 40 active-duty SM healthy controls across a range of military experience and ability levels.Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (Participants may be active duty (at FSH), veterans, or nonveterans and must (1) have a diagnosis of mTBI based on Veterans Affairs/Department of Defense (VA/DoD) criteria , 62, (2) Edition ; (3) disAll assessment procedures for Phase 1 will occur in a single session. Potential participants will be screened for eligibility prior to scheduling their assessment and their arrival at the clinic. Upon arrival at the clinic, study team personnel will review the study procedures and obtain informed written consent from the participant. All participants will be assessed using a battery of assessments including self-reported questionnaires, computerized neurocognition, single-task and DT turning, ecologically relevant mobility tests, clinical mobility tests, and vestibular–ocular motor screening. All procedures have been approved by local institutional review boards and the US Army Medical Research and Materiel Command, Office of Research Protections, Human Research Protection Office.The primary outcome measures will be obtained through a series of clinical turning tasks . Each paTo assess the capacity of DT turning measures to predict performance in a civilian ambulatory task (CAT), participants will navigate through an uncontrolled pedestrian environment while wearing inertial sensors as shown in To assess the capacity of DT turning measures to predict performance in a military-relevant task, participants will complete a simulated urban patrol task SUP, . During Participants will be instructed to enter the environment and secure the area as quickly as possible by clearing all hostile targets without harming friendly targets. Participants will use a mock weapon with a trigger-activated laser  to clear targets. To clear hostile targets, participants will need to accurately scan each room and tag the hostile target with their laser. Each target will contain infrared-sensing diodes that will identify when the target was tagged with the laser. Upon tagging each target with a laser, the LEDs surrounding the hostile targets will turn off, while the LEDs surrounding the friendly targets will turn white (indicating an incorrect shot). Following the task, participants will also be asked to recount the number of hostile targets and number of friendly targets in each room. The primary outcome of functional performance for the simulated SUP task will be a throughput score calculated using the Comstock method , which ag effect sizes determined through preliminary data on turning in people with mTBI . For the second goal, the power analysis was based on the correlation between clinic-based peak turning velocity and the duration of walking bouts, a common measure of daily ambulatory function, exhibited during daily, unmonitored activities at home . With 140 planned participants (100 civilian participants and 40 active-duty participants), we will have 97% power to detect a correlation between the lumbar turning velocity measured in the clinic and daily function with a two-tailed 0.01 significance level if we observe the same effect size. Power calculations were performed in G*Power 3.1.9.2  curves using each objective turning outcome and clinical assessment of activity. The area under the ROC curve (AUC) will be calculated, and AUC confidence intervals will be generated through a bootstrapping procedure. We will compare the AUCs and confidence intervals between each objective turning measure and clinical assessment of activity using tests of equality and a 0.05 significance level with Holm-Bonferroni corrections to adjust for multiple comparisons.To assess the capacity of DT turning measures to predict performance in civilian or military-relevant tasks, we will use Pearson correlation coefficients and linear regression models. Pearson correlation coefficients will compare the linear relationship between objective turning outcomes and the primary outcome of task completion time during the CAT and throughput during the SUP tasks within participants with mTBI, healthy control participants, and all participants together. To determine how changes in objective turning measures correspond to changes in functional performance, linear regression models will be built for each objective turning measure that was significantly associated with functional performance. The primary outcomes for the civilian and military-relevant tasks will be modeled as the dependent variable. To account for potential differences in performance between civilians and active-duty SMs, a group effect will be included as a covariate. Beta coefficients will be interpreted to identify the relative change in objective turning measures that are associated with changes equal in magnitude to 1 standard deviation of the healthy control participants' performance.n” model until the addition of outcomes does not significantly change the AUC.To evaluate if turning outcomes have added value over standard clinical assessments of physical function, base multivariate logistic regression models will be constructed for each clinical assessment of physical function . A “base + 1” logistic regression model will be built by adding the objective turning measure with the highest AUC as described above. ROC curves will be constructed for the base and “base + 1” models, and the AUC calculated and compared using likelihood ratio tests. If the AUC from the “base + 1” model is greater than the AUC of the base model, then the process will be repeated iteratively—the outcome measure with the next largest univariate AUC will be added to the “base + 1” logistic regression model to create a “base + * turning outcome interaction will assess whether the association between objective turning outcomes and ICF function varies by group through the interaction term. A 0.05 significance level will be used with stepwise Holm-Bonferroni corrections to adjust for multiple comparisons.To evaluate if objective turning measures will be associated with impairments in ICF model components, including body functions and structures, activities, and participation domains, Pearson and Spearman correlation coefficients will be implemented separately within healthy civilian control participants, healthy active-duty control participants, and civilians with mTBI. Linear regression models with ICF impairments  as depenTo determine clinically viable measures of turning based on clinometric properties, we will calculate the repeatability and minimum detectable difference using a Bland-Altman framework on the subset of participants who complete two blocks of turning tasks , 81. Linramework , 81.To determine whether healthy active-duty SMs perform turning tasks differently than healthy civilians, we will fit linear regression models for each turning outcome with main effects of group (active-duty SMs vs. civilians) and adjusted for age, body mass index, and other potential covariates. Inferences will be made using a significance level of 0.05 and a Holm-Bonferroni adjustment for multiple comparisons. If we find outcomes significantly differ between active-duty populations and control populations, we can conclude that population-specific normative values are needed. Given the large sample size of active-duty SMs and controls, we will then separate active-duty and civilian control groups for future analyses using those outcomes with significant differences between active-duty and civilian control participants. If we fail to find differences between active-duty and civilian control groups, it suggests performance does not vary by service status, and normative means, standard deviations, and minimum detectable changes will be calculated by combining data from all healthy control participants. We will also compare the distributions of performance using interval estimators to determine the extent to which the range of performance overlaps between active-duty SMs and civilians.Phase 2 will include 40 active-duty SMs with persistent symptoms from mTBI who are referred for physical therapy because of their symptoms. Participants will be recruited from two military medical centers specializing in the rehabilitation of active-duty personnel after mTBI, the Evans Army Community Hospital (EACH) at Fort Carson, CO; and Madigan Army Medical Center (MAMC) at Joint Base Lewis-McChord, WA. Each site will recruit 20 participants for Phase 2. Participants will be tested before and after their rehabilitation; this study will have no influence or input on the rehabilitation prescribed or delivered to the participants.All participants must be active-duty SMs who are referred to either the EACH or MAMC for physical therapy following mTBI. All other inclusion and exclusion criteria will be identical to those in Phase 1.Participants will complete two assessments: a baseline assessment prior to the first rehabilitation session and a postrehabilitation assessment after the physical therapist determines the participant has completed his/her rehabilitation course. At each assessment, patients will complete symptom inventories  and will be outfitted with inertial sensors. Participants will then complete the three turning tasks described in Phase I while wearing the sensors. One block will be performed, consisting of one 1MW, one mIAT, and one CCTC performed with no additional cognitive task, and one 1MW, one mIAT, and one CCTC performed while simultaneously performing a cognitively demanding task. At the postrehabilitation assessment, participants will complete the Patients' Global Impression of Change Scale (PGIC) . ParticiTo determine the responsiveness to rehabilitation, we will (1) determine the clinically important difference for objective turning outcomes and (2) compare the standardized response mean of each to rehabilitation to the minimum detectable change calculated in Phase I.To determine the clinically important difference of turning outcomes, changes in objective turning outcomes will be anchored to the PGIC. Linear regression models will be fit to determine the differences in objective turning outcomes that are associated with a unit improvement in the patients' activity, symptoms, emotions, and overall quality of life, based on the PGIC.To compare the effect of rehabilitation to the minimum detectable change, calculated from Phase 1, for each outcome, we will compute the mean difference between pre-rehabilitation and post-rehabilitation for each objective turning outcome. For each outcome, the ratio of the mean difference over minimum detectable change will be calculated. Ratios >1 will indicate that the mean difference due to rehabilitation was greater than normal variation between measurements, with larger ratios indicating larger effects due to rehabilitation.To reduce the number of tasks and outcomes, a variable selection procedure will be implemented. First, objective turning outcomes for each task will be ranked according to their (1) AUC in differentiating people with mTBI from healthy control subjects, (2) correlation with functional performance in the civilian-relevant task, (3) correlation with functional performance in the military-relevant task, and (4) ratio of mean effect of rehabilitation over minimum detectable change. Outcome–task combinations will then be ordered according to the sum of their ranks across these four categories, where a lower total is better. The outcome, and associated task, with the lowest total sum will be identified and put forward in future applications and clinical training tools. Additionally, a civilian-specific task/outcome combination will be identified by summing the ranks, but excluding the rank corresponding to the correlation with functional performance in the military-relevant task. A military-specific task/outcome combination will be identified similarly, but summing all ranks except the correlation with functional performance during the civilian-specific task.The functional deficits after mTBI can be subtle and are often overlooked in routine clinical assessments. Overt balance and mobility deficits during clinical tests typically resolve within 3 to 5 days after mTBI , but perClinical feasibility and ceiling effects in high-performing individuals are two main challenges in designing ecologically relevant assessments for RTD decisions. In tackling these challenges, we considered the following principles—the RTD assessment must be limited in time and equipment and easily administered; the clinically feasible assessment should, on its face, be ecologically relevant; performance on the assessment should be directly associated with performance in real-world tasks; and there should be no ceiling caused by the measurement technique . These pIf we find the DT turning performance can improve diagnostic accuracy of standard clinical assessments, is associated with functional performance in ecologically relevant tasks, and is responsive to rehabilitation, the results of this study will motivate rapid adoption of DT turning assessments in mTBI rehabilitation and RTD decisions. However, we anticipate that a single outcome measure may not satisfy all of these criteria. By instrumenting various body segments with wearable inertial sensors, we will simultaneously capture multiple outcome measures, each quantifying distinct aspects of movement , for eacOccupational demands and physical capabilities vary widely between and within civilians and military populations. Thus, adequate performance on the proposed turning tasks may be viewed as the bare minimum standard for RTD decisions. Because of the vast heterogeneity in occupational demands and physical capabilities both within and outside of the military, the current study is not designed to determine how performance varies by these factors. However, we anticipate addressing the extent to which performance differs between general civilian and military populations. If military populations perform differently , it supports the use of population-adjusted normative ranges. Yet, even population adjusted normative ranges fail to address the variability within each subset due to occupation or physical health. Accordingly, the results of this study may be seen as a stepping stone for more individualized, and perhaps more complex, RTD assessments based on specific occupational duties.Similar heterogeneity exists in the timeline of care and symptomology for individuals with mTBI. Based on recent evidence that active rehabilitation can be initiated within several weeks after mTBI and may yield greater gains than a traditional wait-and-see approach –99, the Because of the large sample size, multisite design, and inclusion of both civilian and active duty military populations, the results of this study are generalizable and can be rapidly implemented into clinical practice. To translate the results of this study into clinical environments, we will (1) develop standardized administration manuals and scoring sheets for each turning task/outcome selected for clinical use and (2) make recommendations to integrate these training and administration materials into DoD professional development and training courses for rehabilitation specialists. To facilitate widespread implementation, we will publish all analysis algorithms and clinical protocols on freely available, open-access repositories. Finally, we plan to seek future funds to translate the analysis algorithms and protocols to ubiquitous mobile technologies  that do not rely on relatively expensive and specialized commercial technologies.The studies involving human participants were reviewed and approved by Oregon Health and Science University IRB, University of Utah IRB, Allina Health IRB, U.S. Army Medical Research and Materiel Command (USAMRMC), Office of Research Protections (ORP), Human Research Protection Office (HRPO). The patients/participants provided their written informed consent to participate in this study.PF, MW, LD, ML, JA, AS, HR, and LK contributed to the study design and procurement of funding. CH and LP contributed to the implementation of the protocol across sites. PF wrote the first draft of the manuscript. All authors contributed to editing and revising the manuscript.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.


Buried penis is a condition where the penis is hidden by abdominal or suprapubic skin or fat. In adult men, buried penis occurs as an acquired condition most commonly caused by morbid obesity. The clinical characteristics of three obese men with adult acquired buried penis are described and the associated features of the buried penis are reviewed. In addition to morbid obesity, a buried penis can result from other etiologies, such as hidradenitis suppurativa, iatrogenic causes such as elective surgeries, infections, lichen sclerosus, penoscrotal lymphedema, and traumatic events. Lower urinary tract symptoms, such as voiding, and post-voiding problems are the most common presenting complaints; however, bacterial and fungal infections, phimosis, psychological issues, and sexual dysfunction, are also buried penis-related symptoms. The evaluation of a man with adult acquired buried penis begins with a detailed history for condition-related symptoms. Examination of the patient, both standing and supine with an attempt to demonstrate the penis using digital compression of the surrounding skin and fat, should be performed to determine the extent of the problem and whether comorbid conditions-such as infection and lichen sclerosus-are present. Both buried penis and lichen sclerosus can predispose to the development of penile squamous cell carcinoma; the diagnosis of this tumor can be delayed in men with adult acquired buried penis since an adequate penile examination is difficult or impossible. A multidisciplinary approach-including surgeons, primary care physician, registered dietitian nutritionist, and psychiatrist-should be considered for a patient with a buried penis. The surgical management is individualized and based on not only the extent of the problem but also whether an associated condition, such as urethral stricture, is present. Most patients are pleased with the functional and aesthetic outcome following surgery. Obesity is a prevalent health problem of epidemic proportion. Indeed, a large portion of the population is overweight or obese. There are several obesity-associated medical conditions, such as diabetes mellitus and hypertension; in addition, adult acquired buried penis is a comorbid problem that may develop in obese men -4.Buried penis describes a condition in which either skin and/or fat in the prepubic area encompasses a penis of normal size; clinically, the penis is not visible or appears short and buried in the prepubic tissue when the patient is examined. Buried penis can be a congenital condition or an acquired disorder; it occurs in children and adults who have either a normal or an elevated body mass index. There are multiple potential causes of acquired buried penis; however, obesity is the most common etiology in adult men -16.The features of buried penis in three obese adults are described. One of the men also had biopsy-confirmed penile lichen sclerosus. The characteristics of adult acquired buried penis-a hidden problem in obese men-are reviewed.Case 1An obese 62-year-old man presented for a total body skin examination. His past medical history was significant for diabetes mellitus, heart disease, hyperlipidemia and hypertension. His cutaneous history had been remarkable for benign pigmented purpuric eruption of his legs, intertrigo of his groin region, onychomycosis, actinic keratoses, and squamous cell carcinoma on his right forearm.Cutaneous examination showed new actinic keratoses presenting as keratotic plaques on his arms, face, and scalp. He had recurrence of his intertrigo; the candidiasis appeared as confluent erythematous patches with minimal scaling on his bilateral inguinal grooves, the skin fold beneath his lower abdomen, and his groin. His pubic region demonstrated scrotal alopecia and scrotal laxity; his circumcised penis was buried and only the tip of the glans was visible Figure .His actinic keratoses were treated with cryotherapy using liquid nitrogen. His recurrent fungal intertrigo, a buried penis-related comorbidity, was successfully managed by applying ketoconazole 2% cream twice daily. He did not want to pursue any treatment for his adult acquired buried penis.Case 2An obese 71-year-old man presented for a total body skin examination. His past medical history was significant for diabetes mellitus, gastroesophageal reflux disease, hyperlipidemia, hypertension, and low grade papillary urothelial bladder carcinoma. His cutaneous history had been remarkable for stasis dermatitis of his legs, onychogryphosis and pincer nails of the toenails on both feet, and a basal cell carcinoma on his right proximal nasal bridge near the medial canthus.Cutaneous examination of his pubic region demonstrated not only scrotal alopecia, but also that his circumcised penis was buried and only the tip of the glans penis was visible Figure . His oriCorrelation of his history and examination established diagnoses of obesity-associated adult acquired buried penis and lichen sclerosus. He had been applying ketoconazole 2% cream to his glans penis without improvement. Halobetasol 0.05% ointment twice daily was prescribed to be applied to his penile shaft and glans penis. Treatment for his adult acquired buried penis was not desired.Case 3An obese 87-year-old man presented for a total body skin examination. His past medical history was significant for arthritis, diabetes mellitus, hypertension, and seasonal allergies. His cutaneous history had been remarkable for actinic keratoses, dermatitis on his lower back and leg, basal cell carcinomas on his nasal bridge and chest, and a squamous cell carcinoma on his right cheek.Cutaneous examination showed keratotic plaques on his arms and scalp and xerosis on his lower back and legs; the actinic keratoses were treated with cryotherapy using liquid nitrogen. He has scrotal alopecia and his uncircumcised penis was completely hidden Figure . After pThe term buried penis was introduced by Edward Loughborough Keyes, MD in 1919 ,5,15. ItSeveral etiologies have been associated with the development of buried penis. In children, buried penis has primarily resulted from two causes. These include poor penile skin suspension secondary to abnormal mobility of the dartos fascia, or inadequate attachment to Buck facia, or both ,10,11,15In adults, there are several possible causes of acquired buried penis. In addition to morbid obesity, some of the causes of buried penis are hidradenitis suppurativa, iatrogenic etiologies , infections , lichen sclerosus, penoscrotal lymphedema , and traumatic events. Wearing tight underwear can also potentially result in bacterial or fungal infection of the glans penis; the balanitis can promote the development of buried penis ,3,5,6.Buried penis in adults is a common problem that is increasingly being seen by reconstructive urologists. Indeed, 87% of the men who underwent surgical treatment for buried penis were obese. However, based on the increased prevalence of obesity, there seems to be a paucity of the literature on buried penis ,5,6.Originally, a classification of buried penis in children was established ,11. HoweTausch et al. introduced a classification system for adult acquired buried penis in 2016. Grade 1 patients had viable penis skin, whereas grade 2 patients had nonviable penile skin and grade 3 patients had genital lymphedema. Treatment included phalloplasty with or without a scrotal flap for grade 1 patients, replacement of the denuded skin with a split-thickness skin graft for grade 2 patients, and excision of involved tissues and any appropriate adjunctive procedures followed by split-thickness skin grafting [Subsequently, in 2018, three additional classification systems were proposed. Mirastschijski's novel classification system of adult acquired buried penis included type 1 (pseudo-buried penis in which excessive prepubic soft tissue covers the penis without invagination), type 2 , and type 3 . Type 1 required apronectomy and a prepubic lift; types 2 and 3 both required apronectomy and penile anchoring sutures-however, type 3 also needed skin grafts or local flaps for penile shaft reconstruction [In contrast, Pariser et al. proposed a classification system based on the complexity of the surgical repair. There are five categories; patients requiring more than one procedure were assigned to the highest category. The surgical complexity ranged from penile unburying with a local skin flap (category I) to abdominal panniculectomy (category V). In between, the procedures included penile skin grafting (category II), scrotectomy or scrotoplasty (category III), or excutcheonectomy (category IV) .Finally, the Wisconsin classification  stratified patients with adult acquired buried penis according to preoperative examination findings. Type 1 men had a buried penis secondary to skin deficiency, iatrogenic scarring, and/or diseased penile skin; treatment included peri-penile skin excision and split-thickness skin grafting. Type II men with buried penis had excessive abdominal and/or suprapubic fat with lymphedema but no penile skin deterioration; they were treated with panniculectomy, or escutcheonectomy, and/or suprapubic debulking followed by suprapubic suspension. Type III buried penis patients also had excessive abdominal and/or suprapubic fat with lymphedema and penile skin deterioration; in addition to penile exhumation and peri-penile skin excision, they were treated with panniculectomy, or escutcheonectomy, and/or suprapubic debulking followed by split-thickness skin grafting. And type IV patients had excessive abdominal and/or suprapubic fat with penile skin deterioration and severe scrotal edema; their treatment not only included panniculectomy and/or escutcheonectomy, but also penis exhumation and peri-penile skin excision with split-thickness skin grafting in addition to scrotectomy and translocation of the testes .The most common presenting complaints of patients with adult acquired buried penis are lower urinary tract symptoms. These predominantly include both voiding difficulties such as inability to control urination (secondary to hesitancy and poor flow) and post-voiding problems such as dribbling and incontinence. In addition, other symptoms include bacterial and fungal infection of the soft tissues and urinary tract, phimosis, psychological issues (such as depression and low self-esteem) and sexual function issues  ,7.The evaluation of a man with adult acquired buried penis should include a detailed history focused on the duration of time that the penis has been buried and whether the occurrence of the vanishing penis was associated with either gaining or losing weight. Inquiry should be made regarding not only symptoms associated with voiding problems or sexual dysfunction, but also the presence of absence of benign prostatic hypertrophy, injections for genital enlargement, prior surgeries , and urethral stricture. In addition, evaluation for potentially associated conditions, such as diabetes mellitus, hidradenitis suppurativa, hypertension, obesity, pulmonary issues (related to obesity), and urologic problems, should be conducted ,4,6,8.A man who is being evaluated for adult acquired buried penis should be examined both standing and supine; either the abdominal panniculus or the suprapubic fat pad (escutcheon) or both may be covering the penis. The penis should be examined to determine whether it has been circumcised and the penile meatus should be inspected for stenosis; in addition, the penis and scrotum and suprapubic region should be assessed for lymphedema, thickening of the skin, and whitening of the skin associated with lichen sclerosus ,8.The skin and fat surrounding the penis should be compressed by the evaluator’s fingers to determine if the penis can be demonstrated. Overabundance of escutcheon or phimosis may prevent deliverance of the penis. Also, the presence of scrotal enlargement may be a clue to associated hidradenitis suppurativa ,6,7.Voiding symptoms in men with buried penis may be secondary only to the hidden penis. However, lichen sclerosus or urethral stricture or both may result in voiding symptoms. Although urethral strictures are frequently observed in men with adult acquired buried penis, an etiologic association between these conditions has not been established; however, the presence of lichen sclerosus in adult acquired buried penis patients who also have distal penis (fossa navicularis and/or meatus) urethral strictures may share a common pathogenesis related to the lichen sclerosus that predisposes to both buried penis and urethral strictures. Cystoscopy, with or without imaging studies (such as a retrograde urethrogram), may be necessary to establish the diagnosis of urethral stricture ,6,8,9.There are several comorbid conditions associated with buried penis; the disorders can have an etiologic role in the development of a hidden penis and/or be a sequelae of the buried penis. Poor hygiene and trapped moisture results in bacterial and fungal colonization and infection with subsequent maceration of the penile shaft tissue. Lichen sclerosus can also promote or result from a buried penis ,9,16-20.Lichen sclerosus  presents as white fibrotic induration of the glans penis, penile shaft and/or foreskin (if present) with possible phimosis; it is characterized microscopically by a band-like lichenoid lymphocytic infiltrate in the upper dermis, often with vacuolar alteration of the basal layer of the atrophic epidermis with overlying hyperkeratosis. Medical management predominantly involves the topical application of ultra-high potency corticosteroids; however, recently a potential role for intralesional platelet-rich plasma has been proposed. Exacerbation or recurrence of buried penis can be a postoperative complication of circumcision of the lichen sclerosus-affected tissue -20.One of the reported patients with adult acquired buried penis had lichen sclerosus. The diagnosis of lichen sclerosus was delayed as a result of the difficulty his clinicians had in examining his penis. Although he had previously been circumcised, when he eventually presented to a urologist the affected tissue was surgically removed; subsequently, the residual disease on his glans penis was managed medically with twice daily application of halobetasol 0.05% ointment.Squamous cell carcinoma can develop in the setting of either buried penis or lichen sclerosus -14; a syThe management of adult acquired buried penis is based upon the etiology and the extent of disease involvement as determined by the clinical presentation of the patient. Ideally, there should be a multidisciplinary approach to the patient. This would include not only the surgeons  but also the primary care physician, registered dietitian nutritionist, and psychiatrist .The specific surgical intervention must be determined for each individual patient. However, general concepts for the management of adult acquired buried penis include mobilization of the penis (by the release of tethering bands and scars), fat removal , appropriately suspending the base of the penis (to prevent retraction), and restoring penile skin if the penis has been denuded; when replacement penile tissue is necessary, recommendations vary with regards to using split-thickness skin grafts, thick split-thickness skin grafts and/or full-thickness skin grafts. Scrotectomy may also be necessary for some of the men with adult acquired buried penis -9,15,16.Postoperative complications may occur following surgical treatment of buried penis. Some of these include abnormal scarring, granuloma formation, paradoxical penile shortening, penile deformity, psychological sequelae, and sexual dysfunction. However, most of the men are pleased with the functional and aesthetic outcome following surgical management of their adult acquired buried penis ,9.Obesity is the major cause for adult acquired buried penis. A buried penis is a normal sized penis that either appears short or is hidden from view by abdominal or suprapubic skin or fat. Three older men who had adult acquired buried penis are described; their buried penis was an incidental finding during a total body skin examination. Prior to their cutaneous evaluation, the men did not realize that there were surgical interventions that might be useful to remedy their buried penis; however, perhaps because of their age or the chronicity of their problem or both, none of the patients desired to have their buried penis further evaluated or treated. There are several condition-related symptoms  and disorders (such as lichen sclerosus) that can be present in men who have a hidden penis; urethral strictures are also commonly present in patients with adult acquired buried penis. In addition, both buried penis and lichen sclerosus can be associated with the development of penile squamous cell carcinoma. The optimal management of adult acquired buried penis is multidisciplinary; surgical intervention is often successful in providing a functional and aesthetic outcome.


Endocrine-disrupting chemicals (EDCs) are exogenous substances able to mimic or to interfere with the endocrine system, thus altering key biological processes such as organ development, reproduction, immunity, metabolism and behavior. High concentrations of EDCs are found in several everyday products including plastic bottles and food containers and they could be easily absorbed by dietary intake. In recent years, considerable interest has been raised regarding the biological effects of EDCs, particularly Bisphenol A (BPA) and phthalates, on human pregnancy and fetal development. Several evidence obtained on in vitro and animal models as well as by epidemiologic and population studies strongly indicated that endocrine disruptors could negatively impact fetal and placental health by interfering with the embryonic developing epigenome, thus establishing disease paths into adulthood. Moreover, EDCs could cause and/or contribute to the onset of severe gestational conditions as Preeclampsia (PE), Fetal Growth Restriction (FGR) and gestational diabetes in pregnancy, as well as obesity, diabetes and cardiovascular complications in reproductive age. Therefore, despite contrasting data being present in the literature, endocrine disruptors must be considered as a therapeutic target. Future actions aimed at reducing or eliminating EDC exposure during the perinatal period are mandatory to guarantee pregnancy success and preserve fetal and adult health. Natural and man-made chemicals may mimic or interfere with the endocrine system, a complex communication network among the nervous system and key biological functions such as reproduction, immunity, metabolism and behavior . These cTherefore, the objective of this paper is to review the most recent findings on fetal–maternal exposure to endocrine disruptors, particularly focusing on Bisphenol A (BPA) and phthalates, pregnancy outcomes, the in-utero setting of post-natal and adult conditions as well as up-to-date guidelines to reduce dietary EDCs exposure during the perinatal period.According to the World Health Organization (WHO), an endocrine disruptor is “an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub) populations” .EDCs disrupt endocrine functions mainly by mimicking natural hormones like estrogens, androgens or thyroid hormones. Moreover, they could alter hormones’ metabolism, thus blocking and antagonizing their interaction with the specific membrane and/or intra-cellular receptors . Almost BPA is an industrial chemical obtained from condensation between phenol and acetone that has been used to produce plastics and resins since the 1960s. It is considered the first synthetic estrogen produced, although it does not possess a steroid structure since it does not include the phenanthrene nucleus . BPA is BPA is well known for its estrogenic activity exerted through the activation of the Estrogen Receptor (ER), with a stronger affinity for ERβ than for ERα, whereas BPA-G did not show ER-mediated estrogenic activity , but it Phthalates are diesters of phthalic acid classified into high- and low-molecular-weight ones. The first category includes several compounds that are largely adopted to increase plastic flexibility and durability. Among them, the most commonly employed additive is di (2-ethylhexyl) phthalate (DEHP). Low-molecular-weight phthalates, like diethyl phthalate (DEP), are mainly used in personal care products and cosmetics, but they could be also found in pesticides and in food packaging . ExposurBPA and phthalates are therefore widely present in everyday life due to the considerable volume of plastic produced . They coWorldwide estimations of ECD daily intake based on urinary exposures revealed different exposure levels across continents, countries, regions, and towns .The globally estimated BPA daily intake in adults is 30.76 ng/kg bw/day, whereas a notably higher daily intake, corresponding to 42.03 ng/kg bw/day, was found for pregnant women . Both exAs regards phthalate daily intake, China’s adult population seems to be the most exposed , with peAlthough providing interesting data to identify populations at a greater exposure risk and to assist policy makers in making regulations to reduce EDC exposure, one has to be aware of several pitfalls when interpreting exposure to EDC based on urinary concentrations. First, the use of an equal body weight for all populations risks overestimating exposure for some countries and underestimating it for others. Second, BPA has been shown to accumulate in adipose tissue, affecting urinary excretion ,43,44,45All of this should be taken into consideration when estimating and interpreting the daily intake of BPA and phthalates.Human reproduction and pregnancy success depend on both female and male reproductive health and EDCs, as BPA and phthalates could affect fertility of both genders.The ovaries are responsible for female gametogenesis and endocrine functions during reproductive life. It has been demonstrated that prenatal BPA exposure inhibited germ cell nest breakdown in F1 generation ovaries in mice, decreased the numbers of primordial, primary, preantral and total healthy follicles at post-natal day 21 and decreased estradiol levels in female rats dosed for 1 year ,47. Hu aPhthalates were shown to disrupt female fertility by altering oocytes development and maturation. Data are available from animal models. Adult CD-1 mice orally daily dosed with DEHP (20–750 mg/kg/day) for 10 and 30 days showed a decreased percentage of primordial follicles and an increased percentage of primary follicles, thus reducing reproductive lifespan. The mechanism by which DEHP accelerates primordial follicle recruitment is likely via over-activation of the phosphatidylinositol 3-kinase (PI3K) signaling, a pathway that regulates primordial follicle survival, quiescence and recruitment. DEHP exposure increased ovarian mRNA levels of 3-phosphoinositide-dependent protein kinase-1 (Pdpk1), mammalian target of rapamycin complex 1 (Mtorc1), factors that drive primordial follicle recruitment, and decreased Phosphatase and tensin homolog (Pten) and Tuberous sclerosis 1 (Tsc1) mRNA levels, promoters of primordial follicle quiescence . MEHP, tIn males, EDCs exposure was associated with declined semen quality, increased sperm DNA damage, alterations in testis morphology and endocrine function ,53,54,55Even phthalates could negatively impact on male fertility. DEHP exposure (750 mg/kg/day) in male CD-1 mice induced lower serum testosterone levels, accompanied by higher serum estradiol and LH levels. Moreover, histological mice evaluations showed that male mice prenatally exposed to DEHP were characterized by increased germ cell apoptosis, and the degeneration of seminiferous tubules, oligozoospermia, asthenozoospermia, and teratozoospermia, leading to premature reproductive senescence . Yuan anTherefore, EDCs as BPA and phthalates could have severe detrimental effects on human reproduction, impairing both female and male fertility before pregnancy onset and causing gametes anomalies during fetal development that could be inherited by the following generations.The reproductive age is a critical phase in the life of a woman, as the development of certain pathological conditions during this phase may influence the outcomes of future pregnancies. The presence of obesity , diabeteNevertheless, the prevalence of obesity, diabetes and metabolic syndrome continuing to increase worldwide . UnhealtA small cross-sectional study involving 103 women aged 19–50 found that urinary BPA was associated with BMI and, marginally, with waist circumference, after adjustment for energy intake and energy requirement . SimilarAt this stage, it is clear that it is not possible to draw conclusions on the relationship between exposure to EDC during reproductive age and the risk of developing pathological conditions potentially affecting pregnancy outcome. However, given the effect that obesity and metabolic complications can have on the health of both mother and child, it is urgent to conduct epidemiological studies specifically designed for this issue.The Developmental Origins of Health and Disease theory (DOHaD) introduced the concept that maternal diet, environmental insults and lifestyle could significantly impact on fetal–placental, development, thus establishing disease paths into adulthood ,75. In tEpigenetics defines heritable phenotype changes that involve alterations in gene expression and not in DNA sequence. Epigenetic mechanisms include DNA methylation, acetylation, genomic imprinting, as well as the expression of microRNAs (miRNAs) and non-coding RNAs. These modifications usually have an inhibitory effect on gene expression since they modulate DNA accessibility to transcription factors and regulatory proteins by altering chromatin structure and/or by the recruitment of histone modifiers. Early fetal development is particularly vulnerable to epigenetic insults since it is characterized by a high DNA synthesis rate and because the complex machinery modulating DNA methylation and chromatin organization is established at this time .Studies investigating EDCs’ impact on fetal–placental epigenetics in early human pregnancy are few due to the difficulties of collecting samples during the first trimester of pregnancy. Therefore, most of the data available came from in-vitro and animal studies.Data obtained on human fetal liver biopsies revealed that even low BPA doses could significantly influence in-utero epigenetic regulation of xenobiotic metabolizing enzymes (XMEs), pivotal for compounds metabolism and excretion. In particular, they described increased methylation at COMT and SULT2A1 promoters related to higher BPA levels, anomalies that could alter disease susceptibility later in life .A recent cohort study reported the negative association among first-trimester maternal exposure to BPA and phthalates and term cord blood methylation of imprinted  and non-imprinted  genes and LINE-1 repetitive elements , epigeneMoreover, early life EDC exposure could affect obesity epigenetic programming through endocrine disruptors’ ability to bind nuclear receptors as the Peroxisome Proliferator-Activated Receptor (PPAR)γ, master regulator of adipogenesis modulating the expression of metabolic genes during differentiation . EDC-indAnother EDC target with important consequences for epigenetic regulation is the Estrogen Receptor (ERα) ,87. ERα Even the placenta is directly affected by EDC activity. Studies conducted in mice demonstrated that BPA intrauterine exposure markedly altered the placental methylation of imprinted genes  affectinIndeed, different EDCs types such as BPA and phthalates have specific gene targets and their actions could be influenced by fetal sex. Importantly, so-called safer alternatives to BPA as BPS could have detrimental effects on fetal programming too. These data open new perspectives into the understanding of EDCs’ influence on the prenatal development of adult health susceptibilities and pregnancy-related disorders.Beside impacting on epigenetic switches and fetal programming, BPA and phthalates could directly alter pregnancy physiology, undermining its success. As reported for EDC-induced epigenetic modifications, most of the mechanistic data present in the literature about endocrine disrupting chemicals and human pregnancy disorders derive from animal and in vitro data.Starting from the very beginning of gestation, EDCs were recognized as potent perturbators of human Chorionic Gonadotropin (hCG) production and secretion . hCG is PE is a major cause of mortality and morbidity worldwide, complicating 3 to 8% of all pregnancies and characterized by maternal hypertension and multiorgan damage. It originates during early gestation and, despite its etiopathogenesis remaining unclear, it is widely accepted that a placental/systemic vascular dysfunction characterized by an imbalance between pro- and anti-angiogenic factors such as placental growth factor (PlGF) and soluble fms-tyrosine kinase (sFlt)−1 underlie PE onset.In a recent population-based prospective cohort study including 1233 women, a positive association was reported between increased first-trimester urine high molecular weight phthalates metabolites and a higher sFlt-1/PlGF ratio, parameter used for PE screening . HoweverAmong the proposed mechanisms by which EDCs could lead to PE, the disruption of normal placental development plays a major role. A pregnant mouse model exposed to a BPA minimum effective dose of 4 μmol/L developed preeclampsia-like features such as hypertension, abnormal circulating and placental sFlt-1/PlGF levels and kidney damage . The BPAPreeclampsia is often associated with Fetal Growth Restriction (FGR) defined as failure of the fetus to achieve its genetically determined growth potential . DEHP exEven BPA exposure was linked to FGR development. In a recent work, a considered safe dose of BPA (50 μg/kg BPA/day) was administered to pregnant mice during the implantation window (day 1 to 7 of gestation), thus inducing defective remodeling of maternal spiral arteries by placental trophoblast with consequent intrauterine growth restriction .The deleterious EDCs effects on physiological pregnancy development were also demonstrated in in vitro human placental models and population studies, even though contrasting data are present in the literature. MEHP inhibited trophoblast invasion by activating the PPARγ pathway in human HTR-8/SVneo extravillous trophoblast cell lines, mechanisms associated with early pregnancy loss . A prospIn conclusion, despite controversial evidence in epidemiologic, in vitro and animal studies, there is a general consensus on the harmful effects of EDC exposure during fetal life. Several molecular mechanisms have been proposed to explain the role of BPA and phthalates in the onset of severe pregnancy-related conditions such as PE and FGR, suggesting as a main outcome an aberrant placental development. Most of the differences in the literature results and interpretations are probably due to an extreme variability in EDC types and concentrations used, time of administration, for animal or in vitro models, or sampling windows for human gestation studies.It is well known that even during a healthy pregnancy, the release of numerous placental hormones promotes insulin-resistance, which is addressed through a compensatory insulin secretion by the pancreatic β-cells . Gestatist-trimester urinary phthalate metabolite concentrations were related to early gestational weight gain . The association between MEP and gestational weight gain followed a U-shape with increasing gestational weight gain in the second quartile compared to the lowest one . Differently, the dose–response association between DEHP and gestational weight gain was described by an inverse U-shape [Excessive weight gain during pregnancy is a risk factor for GDM. Some recent studies suggest a possible link between the gestational exposure to EDCs and weight gain in pregnancy. In a population-based prospective cohort study among 1213 pregnant women, each log unit increase in early pregnancy BPA and di-n-octylphthalate urine concentrations were associated with lower mid-to late pregnancy gestational weight gain  . Differe U-shape .In a Chinese cohort prospective study including 620 pregnant women, plasma glucose at 2 h in the 75-g OGTT was 0.36 mmol/L lower  for women with urine BPA in the high versus the low tertile, and for each log unit increase, the odds of GDM was reduced by 27%  . On the In conclusion, the limited evidence and conflicting results do not allow a definitive conclusion. The discrepancies in the literature results are presumably due to the different time windows considered for the exposure assessment, the use of a single-spot urine sampling to assess exposure, the different criteria used to diagnose GDM, and not considering pre-gestational exposure.Although there is an urgent need to have data on the association between the level of EDCs in reproductive age and pregnancy outcomes, it is well known that dietary exposure is relevant. There can be substantial variability in phthalate concentrations within food groups based on the region of food production, processing practices, presence and type of packaging and lipid content ,120. RecAccording to the EFSA Panel on Contaminants in the Food Chain, some foods  are an important source of exposure to PFCs. Moreover, PFOA has been used in the past in the production of non-stick coatings. Currently, Italian manufacturers of cookware coatings do not use products with PFOA. Therefore, consumers have to turn their attention to products coming from non-European countries, especially those without the CE mark .Food contact materials  are the main dietary sources of DEHP. However, DEHP use in Europe has dramatically dropped; for some usages, such as flooring and food contact film, European manufacturers have almost completely substituted and phased-out DEHP .PAHs are a group of compounds that are formed from combustion processes, both industrial and household. Nutrition plays a key role in the prevention of exposure: PAHs form at high temperatures within overheated parts of foods, especially with some cooking methods, such as grilling or charring, smoking, or barbecuing .As mentioned above, BPA is a chemical «building block» for the manufacture of polycarbonate plastic used in food contact materials and epoxy resins (lining protective of most cans and food recipients). Canned foods and beverages are the main source of dietary exposure for all age groups, and consumers may be exposed to either the residual monomer that migrates from cans to beverages and foods, or the products that result from polymer hydrolysis .Endocrine-disrupting chemicals represent a health threat that should not be underestimated, especially when dealing with human pregnancy. Pregnant women can be easily exposed to a large number of EDCs by dietary intake. Because of their biochemical features, EDCs pass the placental barrier and reach the developing fetus, causing aberrant genetic/epigenetic regulation with sex-specific modifications and contributing to the onset of placenta- and pregnancy-related disorders. The medical-scientific community has still a lot of work to do in order to clarify the pathways involved in EDCs’ effects on pregnancy physiology. The main limitation that explains the contrasting results present in the literature is the high variability in experimental conditions and models used to investigate EDC activity in the perinatal period. Therefore, a great effort must be made by researchers in the field to set up common protocols and guidelines in order to improve data reliability.


Gene expression in the brain is dramatically regulated by a variety of stimuli. While the role of neural activity has been extensively studied, less is known about the effects of metabolism and nutrition on transcriptional control mechanisms in the brain. Extracellular signals are integrated at the chromatin level through dynamic modifications of epigenetic marks, which in turn fine-tune gene transcription. In the last twenty years, it has become clear that epigenetics plays a crucial role in modulating central nervous system functions and finally behavior. Here, we will focus on the effect of metabolic signals in shaping brain DNA methylation, both during development and adulthood. We will provide an overview of maternal nutrition effects on brain methylation and behavior in offspring. In addition, the impact of different diet challenges on cytosine methylation dynamics in the adult brain will be discussed. Finally, the possible role played by the metabolic status in modulating DNA hydroxymethylation, which is particularly abundant in neural tissue, will be considered.  The “nature versus nurture” issue is one of the most ancient debates in neuroscience and psychology. The word “nature” refers mainly to our genes and, thus, to our genetic heritage. On the other hand, “nurture” refers to all the environmental influences, experiences and stimuli which we have been in contact with from our infancy and even during the fetal stage. The ultimate question is: how do these factors interact in shaping neural circuits and eventually our behavior? Nowadays, it is clear that the problem to address consists of understanding how environmental factors affect gene expression and how gene products can alter neural function.Epigenetics (the study of heritable changes in gene expression that do not involve changes to the underlying DNA sequence ) can be The last twenty years have witnessed challenging efforts to identify epigenetic mechanisms both in health and disease: histone post-translational modifications (PTMS), DNA methylation, non-coding RNA and nucleosome remodeling. Among several epigenetic mechanisms used by the cell, DNA methylation, catalyzed by DNA methyltransferases (DNMTs), has been deeply investigated as a key regulator of transcriptional silencing. For instance, it plays a paramount role in several cellular processes such as regulation of gene expression, silencing of transposable elements, genomic imprinting and X chromosome inactivation . MethylaThis review will describe how metabolic signals, prevalently derived by nutritional changes, affect DNA methylation levels in the brain during development and adulthood. The possibility that metabolism could modulate brain 5-hydroxymethylcytosine (5-hmC) will be also discussed. Finally, open questions and future approaches to potentiate the causal connection between neuroepigenetics and metabolism will be examined.DNA methylation was long considered a stable epigenetic mark in post-mitotic cells, such as neurons. However, despite their necessity to maintain genomic stability, neurons continuously integrate endogenous and exogenous signals, often resulting in plastic mechanisms at different levels . The epigenome represents a platform able to process environmental stimuli, translating them into plasticity, and giving the neuronal cells the opportunity to switch from a poised/quiescent state to an active state toward circuit modifications.In the last decade, a plethora of studies demonstrated how brain DNA methylation is highly modulated by activity and experience during prenatal and postnatal development, and adulthood, challenging the traditional view of a stable epigenetic mark . For insIGF2 gene still present six decades later [vyA) mouse model, in which coat color variation is correlated to epigenetic marks established early in development, has been used to investigate the impact of nutritional and environmental influences on the fetal epigenome. This mouse results from the insertion of an intracisternal A particle (IAP) murine retrotransposon upstream of the transcription start site of the Agouti gene. DNA methylation of the IAP inversely correlates with the level of ectopic Agouti expression and causes a wide variation in coat color ranging from yellow (unmethylated) to light brown (methylated) [Environmental stimuli including diet, physical exercise and stress often converge into changes in the metabolic status of the individual. Metabolism impinges on whole-body physiology and health, including the central nervous system (CNS). This happens in part through the modulation of epigenetic processes. A historical example is represented by periconceptual exposure to the Dutch Hunger Winter (1944–45), which caused an increase in DNA methylation of the imprinted es later . The agohylated) . In the next sections we will focus on nutritional challenge-driven changes in DNA methylation during brain development and in adulthood and the subsequent effect on neural function and cognitive and emotional outcomes. In the last decade, a growing body of studies has linked nutrition to epigenetics since the chemical moieties used for PTMS are often donated by metabolites . Indeed,One-carbon metabolism integrates information coming from the metabolic status of the body into three different biochemical pathways: the folate cycle, the methionine cycle and the transsulfuration pathway . The metAs methionine is introduced with the diet, it is easy to theorize that alterations in the nutritional level of methionine could impact on SAM production and, as a consequence of that, on the level of DNA methylation. However, other micronutrients present in the food can contribute to methyl group production, such as folate and choline, as they participate in one-carbon metabolism as precursors. Notably, choline is also required for the synthesis of phospholipids, transmembrane signaling, lipid-cholesterol transport and cholinergic neurotransmission . This meDnmt3a expression was lower in both the cortex and hippocampus, suggesting the possibility of epigenetic alterations underlying the observed phenotype [It is worth noting that during gestation the nutrition of the fetus relies completely on maternal diet. DNA methylation in the fetal brain has been shown to be present and localized on specific genomic regions in both humans and mice ,38. Thushenotype . High fohenotype . Kcnmb2 and hypermethylation of the same gene. Overexpression of Kcnmb2 in the offspring was able to rescue the phenotype and hippocampal dysfunction [It is worth noting that paternal dietary methyl donor intake also influences behavioral and cognitive functions in the next generation. The offspring of fathers fed a methyl donor-enriched diet  displayed deficits in contextual fear conditioning and spatial learning associated with impaired hippocampal long-term potentiation (LTP) and electroencephalogram oscillatory pattern in the 5–10 Hz range (theta oscillations). Gene expression analysis revealed a downregulation of the BK channel subunit function ,46. On tfunction . In addifunction ,49. ThosEpidemiological and preclinical studies report that exposure to maternal obesity or high fat diet (HFD) during gestation increases the risk of neurodevelopmental disorders ,51,52. IAll together these findings confirmed that micronutrient availability fine tunes DNA methylation in the offspring brain and that subtle alterations in the balance of specific metabolites can have dramatic effects on the chromatin landscape of neural tissue, with severe behavioural outcomes. Lastly, aberrant epigenetic remodeling driven by a food challenge could be normalized by specific micronutrient supplementation. These observations open questions about how epigenetic enzymes sense metabolic cofactor availability and how plastic 5-mC acquired during gestational epigenetic programming could be. Moreover, further studies are needed to dissect alterations in the whole brain methylome in different CNS regions and even at the level of specific neuronal cell types.Pomc gene locus has been shown to be a target of the epigenetic remodeling driven by maternal diet. POMC-expressing neurons are enriched in the arcuate nucleus of the hypothalamus, and they are critical regulators of metabolism, energy balance (reducing food intake and increasing energy expenditure) and reproduction [Pomc promoter methylation and body weight, even upon consumption of a balanced/standard diet , after weaning [Pomc promoter and enhancer regions causing hypermethylation in the arcuate nucleus of rats weaned on a low-fat diet [Pomc transcription in the arcuate nucleus. The Pomc promoter of balanced diet-fed rats exposed to maternal HFD had increased 5-mC, while one of the offspring from balanced chow-fed mothers displayed enrichment in 5-hmC. Importantly, there were negative correlations in the body weight of individual rats and their 5-hmC levels. In contrast, 5-mC levels were positively correlated with body weight [Pomc promoter hypomethylation and increased Pomc expression in the hypothalamus, together with increased body weight and decreased glucose tolerance and insulin sensitivity [Pomc or other gene loci in response to maternal HFD feeding. Although we lack a profound overview of the maternal diet-dependent neural tissue DNA methylome, some genes seem to be exquisitely impacted by changes in 5-mC. For instance, the oduction . It is koduction . Offspri weaning . In a sifat diet . It seemy weight . On the sitivity . The disPomc gene expression and attenuated stress hyperresponsiveness during adulthood [As previously mentioned, choline is an important donor of methyl groups. This metabolite is oxidized to betaine, which is a substrate in the betaine-homocysteine methyltransferase reaction, which links choline and betaine to folate-dependent one-carbon metabolism . Remarkadulthood . Siah1b, Prkx) displayed genomic loci hypomethylation and subsequent upregulation in their levels. Those genes might be relevant players in this pathology, as the Wnt pathway is an important regulator of neural tube development [Lastly, it is well known that inadequate levels of folate during pregnancy could increase the risk of developing neural tube defects (NTD) in the baby. How folate supplementation could avoid NTD is not known; nonetheless, some studies have suggested that altered patterns of DNA methylation in humans could be involved . HPLC anelopment . DespiteIn summary, these lines of evidence indicate that DNA methylation may respond to the supply of methyl groups in a complex fashion that includes alterations in the activities of DNA methylating and/or demethylating enzymes. For example, diet can influence the expression of DNMTs ,73,74. IDespite the decrease in plasticity observed after sensitive periods of postnatal development, the adult brain still experiences plastic processes at molecular, anatomical and functional levels. Epigenetic marks dynamically react to external signals and can be modified based on the availability of cofactors and metabolites all life long. Nutritional challenges can modulate DNA methylation in the adult CNS with relevant consequences in gene expression, physiology and behavior. Netrin1 promoter and downregulation of Netrin1 gene expression. Importantly, intracerebral administration of Netrin1 considerably restored long-term fear-motivated memory [Feeding post-weaning mice a folate, methionine and choline deficient chow impaired hippocampus-dependent memory and caused promoter hypermethylation and low expression of specific subunits of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors . A diet d memory . Methyl d memory . As maternal HFD was shown to impact offspring brain DNA methylation, feeding a Western diet to adult mice for 12 weeks caused a decrease in global DNA methylation in the frontal cortex. Genome-wide bisulfite sequencing identified differentially methylated loci belonging to genes related to neurological function and disease, neuroactive ligand-receptor interaction, axon guidance, regulation of metabolism, cell adhesion, leukocyte transendothelial migration and inflammation . This isPpp1cb, Reln, Sirt1) in the hippocampus. Intriguingly, all the promoters of the memory-related genes displayed hypermethylation. Neuron-specific Sirt1 knockout (KO) mice recapitulated deficits in hippocampal memory, while resveratrol treatment rescued them in obese mice [Npy and Pomc promoter regions in the paraventricular nucleus and the arcuate nucleus, respectively. The epigenetic regulation of gene expression was correlated with changes in energy intake, body weight and fat depots, suggesting that cafeteria diet alteration of 5-mC hypothalamic nuclei could be responsible for long-lasting modifications in metabolic homeostasis [Diet-induced obesity impaired hippocampal performance in spatial memory, synaptic plasticity tested through LTP and decreased the levels of memory related genes (ese mice . Cafetereostasis . Mat2a and S-adenosyl homocysteine hydrolase (Ahyc) in rat cerebellum. 5-mC was not analyzed, although the authors expected brain hypermethylation based on their data [Alcohol, an abuse substance commonly drunk in a social context, is often part of the diet in adult individuals. Its moderate to heavy consumption is known to deteriorate cognitive abilities and to induce neural damage over time. The specific mechanisms are still under exploration; however, metabolites generated upon alcohol metabolism might contribute through epigenetic remodeling and long-term modulation of transcription . As sucheir data . With increased life expectancy, healthy aging has become an important matter in the scientific field, and the CNS has definitely been in the spotlight, since brain aging is often accompanied by an increased risk of cognitive decline and neurodegenerative diseases . IntriguThe investigations performed both during development and adulthood convincingly indicate the existence of a tight link between metabolic changes and DNA methylation. However, more exploration is needed to identify, on a genome-wide scale, which chromatin regions are affected, which specific genes are modulated and, on top of that, how those epigenetic/molecular modifications are causally related to alterations in neural function and finally behavior. Reprogramming of 5-mC driven by HFD has been observed in different tissues, thus suggesting that methylation plays an important role in regulating the response to diets rich in fat. Changes in the level of one-carbon metabolism components, such as SAM and SAHA, could be taken into account. For example, hepatic SAM and SAHA gained diurnal oscillation in their level in adult HFD-fed mice together with some transcripts coding for the enzymes related to the same biochemical pathway . In the CR effects on DNA methylation in different tissues is of much interest in light of the new concept of epigenetic clocks, which measures changes in CpG sites to predict chronological age . CR has It is worth noting that tissue- and cell-specific analysis is required to discover how nutrition affects DNA methylation and other epigenetic marks, especially in the brain. It could be likely that, based on their function, brain regions will not be equally affected by metabolic alterations. Importantly, as the neural tissue is so heterogeneous, the distinct cell types might respond in different ways and their chromatin landscape could be more or less resilient to the surrounding metabolic milieu. For example, inhibitory interneurons in the human prefrontal cortex displayed high levels of 5-hmC at specific enhancers as compared with glutamatergic neurons. In gene bodies, the range of 5-hmC levels between genes with low and high expression was also significantly higher in inhibitory than in excitatory neurons, with better correlation of DNA hydroxymethylation with transcription in GABAergic neurons . Thus, cFinally, the gut microbiota is strongly intertwined to the host metabolism and might be another, so far underestimated, player in the topic of metabolism-driven neuroepigenetics. The gut microbiota are involved in immune system function , metabolMicrobial-derived short-chain fatty acids , obtained through the fermentation of indigestible polysaccharide, work as histone deacetylase inhibitors (HDAC) and have been shown to cross the blood‒brain barrier. SCFA contribute to maturation and maintenance of microglial cells function  and may At the moment, there is no direct evidence demonstrating that the intestinal commensals could influence epigenetics in the CNS. Notwithstanding, the gut-microbiota-brain axis has been involved in neurophysiology and pathology at different levels; therefore, we cannot exclude that microbes-derived metabolites could reach the brain and impact on the epigenetic landscape of its cells. The above-mentioned studies and many more demonstrate the dynamic nature of cytosine methylation and its reversibility in the neural tissue in response to metabolic challenges. How do 5-mC dynamic changes occur? A single enzyme capable of breaking the strong carbon‒carbon bond in order to directly demethylate cytosine has not been found yet. However, cytosine demethylation can occur through a series of chemical reactions of deamination and/or oxidation catalyzed by specific enzymes. Among the enzymes, growth arrest and DNA damage-inducible 45 proteins (GADD45A and GADD45B) have been shown to take part in the demethylation process in the brain ,115. A m5-hmC immediately raised neuroscientists’ curiosity as quantitation experiments have revealed this mark to be remarkably abundant in the CNS with respect to other organs , perhaps2+, 2-oxoglutarate/α-ketoglutarate (AKG) and reducing agents, such as ascorbic acid , to catalyze chemical reactions [As TET enzymes belong to the family of 2-oxoglutarate-dependent dioxygenases, which needs Feeactions , the metPomc in the hypothalamus [Sirt1 promoter with consequences in gene expression and memory performance [A recent article reported that CR could prevent alterations in 5-hmC levels observed in the hippocampus in old mice ; a similthalamus , and dieformance . Despite sparse information in a few studies, there is no direct evidence connecting 5-hmC to the metabolic status of the individual. However, it is possible to speculate that changes in the level of ascorbic acid in the diet could affect TET activity and finally 5-hmC. Strikingly, vitamin C has the highest concentration of the whole body in the brain, where it is actively transported via the sodium-dependent vitamin C transporter SVCT-2 . Moreove+/NADH) generation, which in turn could influence reactive oxygen species levels [AKG is a Krebs cycle intermediate synthesized by IDH1/2 enzymes. As such, it regulates anabolic and catabolic Krebs cycle products and substrates, thereby regulating amino acid synthesis, ATP production and reducing equivalent (NADs levels . Indeed,s levels  and evens levels . Recentls levels . Notablys levels . Although the possible relationship between AKG levels and neural DNA hydroxymethylation has not been explored yet, dietary AKG supplementation might contribute to modulate 5-hmC and in turn dynamic DNA demethylation in neuronal cells. In summary, there are no systematic studies demonstrating that nutrition and/or metabolism can affect DNA hydroxymethylation in the brain. The literature might suggest this possibility, however, future investigations are necessary to understand how diet and/or specific supplements could influence 5-hmC with subsequent impact on neural physiology. This might be relevant since vitamin C is commonly used and often recommended to children, adults and the elderly and AKG is commercially available to ameliorate sport performance. The intricate relationship between epigenetics and metabolism has been emerging in several fields, including neuroscience. The brain is sensitive to the metabolic status, as shown by behavioral and functional alterations after malnourishment , differeThus, several questions still need an answer: what are the tissue-specific mechanisms responsible for diet-driven alterations in DNA methylation? In which brain areas is DNA methylation more sensitive to micronutrient supplementation/deficiency and how this can be connected to changes in brain function and behavior? Is DNA methylation in distinct brain cell types impacted in the same way by diet and micronutrient availability? Can we use the cell-specific or area-specific diet/micronutrient-driven DNA methylome to predict neurological disease or behavioral outcomes during development, adulthood or aging? Could the gut microbiota play a role in shaping CNS DNA methylation through specific, still unknown signaling molecules or microbe-derived metabolites?A deeper exploration at the level of distinct areas and/or cells will help to comprehend the specific molecular/epigenetic mechanisms, which could be clinically relevant for the treatment and prevention of neuropsychiatric disorders or metabolic diseases.


Central nervous system (CNS) injury, including stroke, spinal cord injury, and traumatic brain injury, causes severe neurological symptoms such as sensory and motor deficits. Currently, there is no effective therapeutic method to restore neurological function because the adult CNS has limited capacity to regenerate after injury. Many efforts have been made to understand the molecular and cellular mechanisms underlying CNS regeneration and to establish novel therapeutic methods based on these mechanisms, with a variety of strategies including cell transplantation, modulation of cell intrinsic molecular mechanisms, and therapeutic targeting of the pathological nature of the extracellular environment in CNS injury. In this review, we will focus on the mechanisms that regulate CNS regeneration, highlighting the history, recent efforts, and questions left unanswered in this field. Damage to the central nervous system (CNS), such as in stroke, spinal cord injury (SCI), and traumatic brain injury, leads to severe neurological dysfunction due to neuronal cell death and axonal degeneration. Although neurons have the potential to undergo dynamic differentiation and development processes, their ability to regenerate after injury is very low in adults. Early studies have established that the pathogenic nature of the extracellular environment in CNS injury, such as myelin debris and glial scars, have inhibitory effects on axon regeneration ,2. In adRecent advances in high-throughput technologies utilized in transcriptomics, proteomics, genomics, and cellular imaging have accelerated the identification of novel molecular mechanisms as a therapeutic target that effectively regulates axon growth, regeneration, and remyelination, and promotes functional recovery after CNS injury. Here, we review recent reports mainly focusing on the adaptation of novel techniques to investigate the molecular mechanisms regulating axonal regeneration and remyelination in the CNS.NTR and Lingo-1. p75NTR mediates the activation of Ras homologous member A (RhoA), a Rho family small GTP-binding protein, by releasing Rho guanosine nucleotide dissociation inhibitors (Rho-GDIs) from GDP-bound inactive form RhoA. Then GTP-bound active form RhoA activates Rho kinase (ROCK), which leads to axon growth inhibition thorough phosphorylation of various molecules related to formation of cytoskeleton [In the mature CNS, neurons have little ability to regenerate their axons and reconstruct lost neural circuits after injury. It has been widely accepted that both extrinsic factors derived from the external environment of damaged axons and poor intrinsic potential for the regeneration of adult CNS neurons limit axonal regeneration and sprouting a. Growthskeleton ,12,13  are a class of epigenetic regulation factors. It has been reported that a large number of miRNAs are differentially expressed after nervous system injury, suggesting that miRNAs play important roles in axon regeneration. A phenotypic screen using an miRNA library directly identified functional miRNAs in axon regeneration . In thisScreening using small molecules has also been utilized to investigate novel mechanisms underlying axonal regeneration ,40,41. ADemyelination in the CNS is one of the hallmarks of neurological disorders such as MS. Remyelination is a regenerative process that spontaneously occurs but is often insufficient to prevent axonal loss, restore lost axonal function, and improve neurological deficits after demyelination. Remyelination in the CNS is mainly mediated by adult oligodendrocyte progenitor cells (aOPCs), which are distributed throughout the mammalian CNS  Figure a. aOPCs A lot of high-throughput screens using these models have provided several potential small molecules for remyelination, which have become a focus of clinical attention  52,53,5,57,58.A small molecule screening using OPCs derived from rat optic nerve identified Benztropine, a muscarinic antagonist, as an inducer of OPC differentiation . BenztroThe next step after OPC differentiation is the formation of myelin sheets, but the cell-autonomous mechanisms regulating myelination in vitro have not been sufficiently investigated. Interestingly, oligodendrocytes can myelinate synthetic axon models, such as nanofibers. A recent study developed a platform with conical micropillar arrays to examine myelin formation around the 3D structure . When OPIt has been reported that disruption of the vascular barrier often occurs in demyelinating diseases and related experimental animal models. Several circulating molecules in leaked blood have been shown to promote the development of OPCs during remyelination ,65 Figub. In a p9-cis-retinoic acid (9cRA) promotes the differentiation of cultured oligodendrocytes. Intraperitoneally administration of 9cRA was found to promote remyelination after toxin-induced focal demyelination in rats. A recent study using mice expressing GFP in OPCs has provided more precise insight into the molecular mechanism regulating OPC differentiation in response to demyelinating injury [A set of studies using comprehensive transcriptional analysis identified several key gene networks regulating remyelination. An earlier study identified a role for retinoid acid receptor RXR signaling in remyelination by transcriptome-profiling of rat cerebellar tissues, which were isolated at several time points after toxin-induced demyelination . siRNA-mg injury . In thisg injury . Anotherg injury . Olig1-cg injury  to generg injury , and it g injury . These sThe ultimate goal of research on neural circuit repair is to reestablish functional neural circuits and restore lost neurological function. Recent advances in omics analysis and other high-content techniques have accelerated the understanding of the molecular mechanisms of axonal regeneration and remyelination, which is the first step toward that goal. Notably, there is a large amount of web-accessible resources and a number of data-mining software programs based on such studies, which allow us to effectively identify new therapeutic targets by reanalyzing data from different perspectives. However, it is still unclear how reconstruction of the circuitry restores motor and cognitive functions. The development of imaging and genetic modification techniques has made it possible to observe damaged neural circuits and analyze the function of specific genes. Continued investigation using these techniques will provide us with consistent knowledge from molecules to cells and neurological functions, which will contribute to the establishment of methods to re-acquire functional neural circuits.


Ezrin, as encoded by the EZR gene, is a member of the Ezrin/Radixin/Moesin (ERM) family. The ERM family includes three highly related actin filament binding proteins, Ezrin, Radixin, and Moesin. These three members share similar structural properties containing an N-terminal domain named FERM, a central helical linker region, and a C-terminal domain that mediates the interaction with F-actin. Ezrin protein is highly regulated through the conformational change between a closed, inactivate form and an open, active form. As a membrane-cytoskeleton linker protein, Ezrin facilitates numerous signal transductions in tumorigenesis and mediates diverse essential functions through interactions with a variety of growth factor receptors and adhesion molecules. Emerging evidence has demonstrated that Ezrin is an oncogene protein, as high levels of Ezrin are associated with metastatic behavior in various types of cancer. The diverse functions attributed to Ezrin and the understanding of how Ezrin drives the deadly process of metastasis are complex and often controversial. Here by reviewing recent findings across a wide spectrum of cancer types we will highlight the structures, protein interactions and oncogenic roles of Ezrin as well as the emerging therapeutic agents targeting Ezrin. This review provides a comprehensive framework to guide future studies of Ezrin and other ERM proteins in basic and clinical studies. Cancer is one of the most debilitating diseases worldwide. The molecular mechanisms of carcinogenesis provide essential implications for potential prevention and treatment of cancers. Extensive studies have been conducted on tumor invasion and metastasis, and multi-step processes have been described. Previous research has shown that cell adhesion, migration, and morphogenesis regulate tumor invasion and metastasis . HoweverThe Ezrin/Radixin/Moesin (ERM) family proteins regulate cell networks through linking actin cytoskeleton to the cell membranes . ERM famEzrin, a member of ERM family, is phosphorylated by threonine and tyrosine kinases . Ezrin iEncoded by the EZR gene that locates at chromosome 6q25.2-q26, the Ezrin protein is the most studied member of the Ezrin/Radixin/Moesin (ERM) family, containing an FERM domain , a central helical linker region and an ERM-associated domain .2- and COOH-terminal bind to each other, full length Ezrin is in a closed inactive form. The abolition of the intramolecular head-to-tail interaction is required to expose the actin binding sites, since the F-actin binding site at the C-terminal domain is normally masked in the full length Ezrin   . Ezrin ie (PIP2) . Additioe (PIP2) . Interese (PIP2) . Althouge (PIP2) .As discussed earlier, unphosphorylated/inactive Ezrin remains in a folded conformation through head-to-tail interaction, masking binding sites for other molecules. Phosphorylation on the conserved threonine residue T567 causes conformational changes, unmasking binding sites . T567 keIn normal cells, Ezrin protein is known to contribute to epithelial morphogenesis, adhesion, and migration . Under pDuring cancer development, the relative membrane localization of Ezrin proteins is increased and cell–cell contact is disrupted. Therefore, the activation of Ezrin proteins facilitates the process of tumor progression and invasion. The effect of enhanced Ezrin proteins in cancer metastasis takes different forms in various cancers. For example, in osteosarcoma, Ezrin allows metastatic tumor cells to overcome a number of stresses as cells from the primary lesion are able to break loose and effectively initiate the growth of secondary lesions by generating additional ATP from a variety of sources . In pancActivated Ezrin regulates key events and interacts with different proteins in a variety of cancer types. The precise spatial and temporal activation of Rho GTPases establishes cell polarity and morphology . The antThe E-cadherin-catenin adhesion complex maintains tissue architecture and is critical for intercellular adhesiveness. Ezrin suppression promotes the expression of E-cadherin and β-catenin. Both E-cadherin and β-catenin play a key role in epithelial cell adhesion. Co-precipitation experiments suggests Ezrin associates with E-cadherin and β-catenin . The modEzrin mediates cell growth and survival through Akt signaling, but not the mitogen-activated protein kinase (MAPK) pathway in certain cancers, which is essential for cancer proliferation, invasion, migration and survival . Ezrin iAs an important member of the ERM family of proteins, Ezrin has been well studied in many cellular events. As summarized earlier, Ezrin plays a vital role in molecular signaling, including cell proliferation, cell polarity establishment, cell motility, and cell adhesion . Since tHighly expressed Ezrin has been detected in lung cancer cell lines and primary lung cancer tissues. Ezrin has been found to be primarily distributed in the cytoplasm of lung cancerous tissue and metastatic foci . Ezrin ein vivo in real time  . As a miEzrin plays a key role in cervical cancer invasion and is a potential prognostic immunomarker. Interestingly, Ezrin expression is correlated with HPV associated lesions , suggestGastric cancer is the second most prevalent cause of cancer death. The Ezrin protein is up-regulated in gastric cancer lesions. Ezrin expression is correlated with tumor size, tumor location, lymph node invasion and metastasis, and shortened survival in stages I, II, and III . SpecifiDysregulation of miR-183 through Ezrin targeting promotes osteosarcoma tumor metastasis. Ezrin is required for metastasis in osteosarcoma and its high expression is associated with poor outcomes in pediatric osteosarcoma patients. As discussed earlier, Ezrin phosphorylation regulation contributes to Ezrin’s molecular plasticity. Hyperphosphorylation at the C-terminal threonine residue (T567) is significantly correlated with an invasive clinical hepatocellular carcinoma (HCC) . TherefoInconsistent with most of cancers mentioned above, membranous expression of Ezrin is significantly lower in high grade bladder cancer and significantly associated disease-specific overall survival . Ezrin iEzrin expression negatively correlated with renal cell carcinoma (RCC) metastasis, and the inhibition of Ezrin expression suppressed the invasive abilities of RCC cells . Using iIn colorectal cancer (CRC), Ezrin binds with a cell-neural adhesion molecule (L1CAM) and mediates the phosphorylation of NF-κB as well as the activation of NF-κB signaling . It has in vitro invasion in medulloblastoma , which carries out opposite activities in tumor growth . Notabilblastoma .In primary melanomas of the skin and metastatic tumors, Ezrin expression correlates with tumor progression and suggests worsening clinical disease behaviors. The molecular mechanism involves molecules related to metastatic functions such as CD44, merlin, and Ras signaling . ConsistThe above sections are not meant to recap all the latest important findings in Ezrin research but rather to provide an overview of the evidence showing the oncogenic roles and prognostic value of Ezrin in a wide range of cancer types. One of the questions that remain to be answered is what the clinical implication of Ezrin is. As described above, high levels of Ezrin are observed in many cancers with lung metastasis, indicating poor survival and bad prognoses. Ezrin as an essential prognosis predictor of various cancers has been demonstrated to be a key modulator of tumor metastasis. All the existing studies, taken together, highlighted the fact that Ezrin may serve as a potential therapeutic target in cancer . This prOver the past decade, many studies have attempted to develop targeted cancer treatment strategies using small molecule inhibitors of Ezrin . For theBecause of the oncogenic role of Ezrin, studies investigating Ezrin activator are rare. Ezrin activation has been linked to CDK5 in the senescent phenotype as CDK5 is able to activate Ezrin by phosphorylating T235 of Ezrin . InteresIn the literature, the oncogenic roles of Ezrin were intensively studied but there are a limited number of studies investigating the predictive performance of Ezrin expression level. In this review, we summarized not only the oncogenic roles of Ezrin but also its pathophysiological roles and potential pharmacological regulators in a wide range of cancer types. Our understanding of Ezrin as a potential drug target is strongly influenced by the idea that Ezrin is commonly proved to promote tumor metastasis and predicts poor prognosis in different types of cancers. Therefore, direct inactivation of Ezrin by the small molecule inhibitors should provide a new strategy for metastatic treatment in many cancers. While this hypothesis is indeed supported by a few lines of evidence in a couple of cancer types such as lung cancer, this rule seems failed to expand in many other cancer types.in vivo activity of the existing small molecule inhibitors as potential tools in cancer therapeutics.Many fundamental questions in the roles of Ezrin remain to be answered. From this work, some basic understanding of Ezrin protein may be challenged. For example, the expression level of Ezrin in bladder cancer is reduced while it is commonly up-regulated in many other cancer types. The predictive value of Ezrin in bladder cancer is also found opposite to the other cancer types. Given metastasis is a complicated process that involves many steps that are poorly understood at this time, some of which may include tissue type-specific mechanism involving Ezrin. This mechanism may not be shared within ERM family proteins as the other ERM protein Mosin was found enriched in bladder cancer and consistent with its oncogenic role in invasion process. Future work is needed to uncover new pharmacological inhibitors and to explore the WX and YY designed the manuscript. YS and XM wrote and revised the manuscript. MZ searched the references. MW and GW drafted the figures. All authors contributed to the article and approved the submitted version.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.


Determination of the relative copy numbers of mixed molecular species in nucleic acid samples is often the objective of biological experiments, including Single-Nucleotide Polymorphism (SNP), indel and gene copy-number characterization, and quantification of CRISPR-Cas9 base editing, cytosine methylation, and RNA editing. Standard dye-terminator chromatograms are a widely accessible, cost-effective information source from which copy-number proportions can be inferred. However, the rate of incorporation of dye terminators is dependent on the dye type, the adjacent sequence string, and the secondary structure of the sequenced strand. These variable rates complicate inferences and have driven scientists to resort to complex and costly quantification methods. Because these complex methods introduce their own biases, researchers are rethinking whether rectifying distortions in sequencing trace files and using direct sequencing for quantification will enable comparable accurate assessment. Indeed, recent developments in software tools  indicate that quantification based on direct Sanger sequencing is gaining in scientific acceptance. This commentary reviews the common obstacles in quantification and the latest insights and developments relevant to estimating copy-number proportions based on direct Sanger sequencing, concluding that bidirectional sequencing and sophisticated base calling are the keys to identifying and avoiding sequence distortions. No. NoFCGR2R2 CD32, . 2+ [2+ . The rec2+ . However2+ . The dem2+ . Thus, nRecent developments in estimating copy-number proportions based on direct Sanger sequencing indicate that such quantification is gaining in scientific acceptance. Best quantification is achieved when the mixed DNA molecular species involve microindels for which superimposed trace files of the indel site allow peak-height ratio analysis. Frequent indels are associated with outcomes of CRISPR-Cas9 base editing and with analyses of mitochondrial heteroplasmy; and the need to quantify these sparked the development of new software tools to infer copy-number proportions from Sanger trace files. As the rate of incorporation of dye terminators is dependent on dye type, the adjacent sequence string and the secondary structure of the sequenced strand, such inference is less acceptable in cases of single-base variations. Frequent observation of overscaled C signals renders direct bisulfite sequencing unreliable for quantification of CpG methylation. However, use of peak-height analysis of a single site has been reported to successfully determine gene copy-number proportions in CNVs and accurately quantify RNA editing. Thus, identifying and rectifying distortions in sequencing trace files may further promote the use of direct Sanger sequencing for quantification. Aside from consulting the available literature presenting characterized base strings associated with distortions, bidirectional sequencing is an easy way to identify and avoid these local sequence effects. Commercial companies have not disclosed the algorithms of their basecallers, which are mostly marketed as part of the software that operates capillary sequencers. However, it is apparent that despite high predictability of distorting effects on peak intensities, current basecallers focus on the identification of bases and not on reporting their relative proportions in mixed DNA templates. This emphasizes the need for base-calling algorithms that can take into account the effects of base terminators on incorporation rates. As different rates are generated by the ever-changing microenvironments of different salt and DNA concentrations, using sophisticated methods, such as neural networks, for base calling  may prov