The co-existence of wild-type and mutated mitochondrial DNA (mtDNA) molecules termed heteroplasmy becomes a research hot point of mitochondria. In this review, we listed several methods of mtDNA heteroplasmy research, including the enrichment of mtDNA and the way of calling heteroplasmic variations. At the present, while calling the novel ultra-low level heteroplasmy, high-throughput sequencing method is dominant while the detection limit of recorded mutations is accurate to 0.01% using the other quantitative approaches. In the future, the studies of mtDNA heteroplasmy may pay more attention to the single-cell level and focus on the linkage of mutations. Mitochondrial DNAs (mtDNA) form double-strand circular molecules which contain about 16,569 DNA base pairs, including a D-loop region, 13 protein encoding genes, two rRNA (12S rRNA and 16S rRNAs) and 22 tRNA encoding regions [Generally speaking, one mitochondrion can harbor 2–10 mtDNA molecules , and theAs far back as 1985, researchers had detected that more than one mitochondrial genome haplotype was present in an individual ,11. Most2− arose by electrons. After that, as the absence of effective mtDNA transcription products caused by ROS production, the descending of ETC activity leads to a higher possibility of electrons leaking [A well-known case was published in 2002 that a man with mitochondrial myopathy harbored 90% of mitochondrial genome in skeletal muscles which was the same as his father’s . Actuall leaking . In accordance with studies of mitochondrial damages, point mutations do accumulate with aging in human mtDNA ,17,18. IIn pace with developing of mtDNA heteroplasmy studies, intra-individual heteroplasmic variations were detected  and the Concurrent with the researches of heteroplasmy of mtDNA between tissues, intracellular mutations offered more detailed information. Most of these single-cell studies paid attention to the diversities of several particular variations like disease-causing genes and amplified these fragments harboring mutations before sequencing them. Some of these investigations instituted comparisons of mtDNA heteroplasmic rate between single cell and bulk cells in tissues . They coTo go deep into studies of the mitochondrial genome, more detailed and accurate mtDNA sequences should be collected. Along with the development of sequencing technologies, those requirements are responded commendably. However, due to the polymorphism of mtDNA, heteroplasmy calling especially for low-level heteroplasmic variance is still a challenge. Due to the small scale of mitochondrial genome, some larger exogenous genomes especially nuclear genome might disturb distinguishing mtDNA heteroplasmy exactly. Thus, enriching mitochondrial genome have been regarded as an essential step before heteroplasmy calling. In this review, we list several approaches to mtDNA’s heteroplasmy research, including enrichment methods and heteroplasmy calling methods.According to whether amplification means were used or not, we divided the various enrichment methods into two main categories, isolation and amplification. However, isolating mtDNA from biospecimens risks contamination from the nuclear genome, especially paralogous fragments of mtDNA. In consideration of the high copy number of mtDNA molecules compared with nuclear genome, contamination would not have a great effect on the detection of heteroplasmic variations with higher heteroplasmy frequency and the impact of nuclear DNA should be taken into account when identifying low level heteroplasmies as accurately as possible. Thus, we summarized several bioinformatics methods in a separate section which discussed some strategies to minimize the influence of contamination . Methods of isolation focus on the separation of organelles, and then obtain intact mitochondria in an early stage. Now the mitochondria extracted from biospecimens are required to maintain intact form and biological activity even the function of respiration action according to their research purposes ,27. In tIn earlier times, investigators utilized differential centrifugation to separate certain organelles from cells to extract DNA from them. As early as 1968, Bielka et al. separated mitochondria from eggs by CsCl density centrifugation . This meObviously, density gradient centrifugation greatly decreases the quality of nuclear DNA and avoids the interference of other mitochondrial homologous genes. However, this method was costly and time-consuming, and also required a large quantity of tissue samples, which limited the its widespread use. Except for extraction of mitochondria firstly, mtDNAs could directly be isolated depending on density differences. There were two kinds of dyes, ethidium bromide (EtBr) and Hoechst 33,258, which could insert between the purine and pyrimidine base pairs or bound to the large groove of mtDNA molecules. They would choose different genomes to bind according to the AT:GC ratio. The insertion of dyes also let the supercoiled DNA binds relaxed, which might cause the decrease of DNA’s density, helping separate mtDNA from nuclear DNA easier . HoweverCapture of mtDNA fragments from cracked bio-samples directly is also a frequent approach. Either used in solution or on a surface, the probes for capturing targets must be purchased from their manufacturers, which might cost a lot. Researchers would use other fragments to replace the custom probes. Briggs et al. developed a method termed primer extension capture (PEC) method where high specificity primers and a DNA polymerase was used to capture target sequences from an adaptor-ligated DNA library . MaricicDirectly capture methods often call for enough amount and freshness of the biospecimen, and display disadvantages of efficiency. As a result, amplifying mtDNA molecules can increase the content of mtDNA and cut down laborious experiments. Polymerase Chain Reaction (PCR) is one of frequently-used approaches to amplify DNA specimens, especially for enrichment of small-scale genome like mtDNA. There is a widely utilized PCR method called ‘primer-walking’ which requires two or more pairs of PCR primers with overlapping regions between each of these amplicons . The priHowever, coexisting with the convenience brought by PCR, biases cannot be ignored. For instance, if the primer-binding region harbors base alteration, PCR primers would readily coalesce homologous regions of mtDNA in the nuclear genome rather than the correct binding sites . Hence iWhole genome amplification (WGA), a method for robust amplification of an entire genome, including PCR-based and isothermal-based amplification method, is a powerful tool of amplifying DNA molecules from minute quantities to amounts that can be used for sequencing studies. PCR-based WGA includes primer-extension pre-amplification (PEP) , degenerThe methods mentioned above are all about enriching mitochondria or mtDNA directly. However, due to the feature that the majority of mitochondrial genome is gene-coding regions, some indirect approaches such as digging mtDNA sequences from exome sequencing data are also attainable. Exome data comes from all exon capture and sequencing, that is there is a huge possibility that mtDNA fragments are obtained at the same time. A research revealed that there existed some unintentionally sequenced reads including non-coding regions like introns or UTRs and mitochondrial genome discovered from exome data . As prevDespite the lower intensity of lab work this method brings, paralogous fragments turned out to be the barrier to the precision of heteroplasmy calling. Approximately 0.03% of nuclear genome is nuMTs, which can be mapped to mitochondrial reference sequence . These nConsidering the detection limit and variations type of heteroplasmy detecting approaches, we list a table  to briefIn 1977, Frederick Sanger and his colleagues developed a DNA sequencing method which based on chain-terminating dideoxynucleotides during DNA replication in vitro. This method has been the most widely used one for approximately 25 years. Nowadays, Sanger sequencing is still a frequently-used approach while studying smaller-scale project because of its speed. However, the poor quality of the first 15~40 bp sequence with the Sanger method is a common challenge due to the influence of the PCR primers as mentioned above. Naue et al. sequenced 883 samples from nine different tissues of 100 unrelated individuals by Sanger sequencing, mini-sequencing and massive parallel sequencing, then the results showed an unexpectedly high number of heteroplasmies using Sanger sequencing . A new aSanger sequencing nowadays is still a gold standard of hateroplasmic variation detection despite its comparatively lower heteroplasmy detection limit . As a coSome studies showed that heteroplasmy occurred tending to be concentrated in certain regions called mutational hotspots . Hence mAffymetrix designed a sequencing microarray named MitoChip, which was used for the detecting heteroplasmic variation position of mitochondrial genomes as a tool for early detection of cancer . This toCompared to Sanger sequencing, the microarray methods have a significant feature of high-throughput with the advantage of shorter test period and less cost, which can be applied in the field of clinical diagnosis. Capture arrays showed their convenience and efficiency in single nucleotide determination, but are unable to provide information about undiscovered abnormal loci because the design of oligonucleotide probes has to following the existing sequences. Custom capture arrays can just obtain judgments about the existence of mutations, without exactly knowing the quantified ratio of the heteroplasmic locus .Except for common amplification functions, PCR also allows some subtle changes to meet the quantitative demands with the participation of some kinds of fluorochrome or endonuclease. Quantitative real-time PCR (qPCR) is able to monitor the amount of DNA molecule being amplified in real time, so it helps determine the copy number of mtDNA in a single cell , and othAmplification Refractory Mutation System Quantitative PCR (ARMS-qPCR) is a kind of real-time PCR technology with two upstream primers and one downstream primer. Modification of one or more bases’ upstream primer makes this method possess a more sensitive heteroplasmy detection ability . By quanDuring the same time when PCR technologies are wildly utilized, as mentioned before, PCR artifacts are indistinguishable from genuine mtDNA mutations. Single-molecule PCR (smPCR), which separates the mutated and wild-type DNA molecules before amplified, has burgeoned for this reason. This approach conducting a molecule-by-molecule mode avoids too much effort being spent on nonproductive sequencing of the wild-type DNA. Due to the low abundance of template DNA, smPCR products can exclude the interferences resulting from jumping PCR or allelic preference. PCR errors can be easily identified upon direct sequencing of products as low-intensity subpeaks in the spectrum . The draAlso with the aid of emulsion PCR, the size of dPCR reaction units can be narrowed down to picoliter-forming droplets, which means a great expansion of throughput. By fastening targeted oligonucleotides on the surface of magnetic beads, Vogelstein et al. devised a beads, emulsions, amplification and magnetics (BEAMing) system improving the detection limit tremendously . This syIn terms of identifying recorded heteroplasmic variations, plenty of methods can provide diverse choices according to the sample under study. PCR-based approaches mentioned before are available on small scale species otherwise they would result in heavy expenses and labor-abundant experiments. High-throughput sequencing, which is widely known as NGS, easily obtains the whole genome of species. Working as a synthesis-by-sequencing mode, NGS offers a higher throughput than older methods, providing the conditions for detecting low-level heteroplasmy in mitochondrial genomes. Although the proportion of heteroplasmy resulting in phenotypic appearance is as high as 70~90% , the posOver the past five years, with the appearance of a barcode-based NGS technology named duplex sequencing DS, , the hetMitochondria are intergrown with eukaryotic cells, and play an important role in a considerable number of physiological processes. The behavior of mtDNA is quite different from the nuclear genome. Heteroplasmy of mtDNA hides pathogenetic mutations from regular detection which ought to be discerned as precisely as possible. Due to the mitochondrial threshold effect, phenotypic manifestation of the genetic defect occurs until the proportion of heteroplasmic variations reaches a threshold level, and threshold values of different tissues or pathologies are not identical . For exaTotally speaking, the detection of mtDNA heteroplasmy and HF determination are dominated by NGS technology at the present, supplemented by other quantitative means to verify the credibility of its results. In the future, the studies of mtDNA heteroplasmy may pay more attention to the single-cell level and focus on the linkage of mutations.


Assault is the third most common cause of traumatic brain injury (TBI), after falls and road traffic collisions. TBI can lead to multiple long-term physical, cognitive and emotional sequelae, including post-traumatic stress disorder (PTSD). Intentional violence may further compound the psychological trauma of the event, in a way that conventional outcome measures, like the Glasgow Outcome Scale (GOS), fail to capture. This study aims to examine the influence of assault on self-reported outcomes, including quality of life and symptoms of PTSD.Questionnaire were completed by 256 patients attending a TBI clinic, including Quality of Life after Brain Injury (QOLIBRI) and PTSD checklist (PCL-C). Medical records provided demographics, clinical data and aetiology of injury. Subjective outcomes were compared between assault and other causes.Of 202 patients analysed, 21% sustained TBI from assault. There was no difference in severity of injuries between assault and non-assault groups. No relationship was found between self-reported outcomes and TBI severity or GOS. The assault group scored worse in all self-reported questionnaires, with statistically significant differences for measures of PTSD and post-concussion symptoms. However, using threshold scores, the prevalence of PTSD in assaulted patients was not higher than non-assault. After adjusting for age, ethnicity and the presence of extra-cranial trauma, assault did not have a significant effect on questionnaire scores. Exploratory analysis showed that assault and road traffic accidents were associated with significantly worse outcomes compared to falls.Quality of life is significantly related to functional and psychological outcomes after TBI. Assaulted patients suffer from worse self-reported outcomes than other patients, but these differences were insignificant when adjusted for demographic factors. Intentionality behind the traumatic event is likely more important than cause alone. Differences in quality of life and other self-reported outcomes are not reflected by the Glasgow Outcome Scale. This information is useful in arranging earlier and targeted review and support. Traumatic brain injury (TBI) is a traumatically induced disruption of brain function, manifesting as loss of consciousness or memory, alteration in mental and/or focal neurological deficits. TBI is a leading cause of death and disability in both developing and developed countries . The incTBI is the third largest contributor to injury-related hospital costs in Europe , and accInterpersonal violence carries a highly significant emotive context that can compound the burden of TBI. Previous research has shown that survivors of TBI from assault suffer from worse symptoms, are more significantly disabled, less productive, a larger burden on caregivers, show less community reintegration, and gain less from inpatient rehabilitation –14. The The psychological consequences of TBI are significant, doubling the risk of developing subsequent psychiatric illness , in partTBI is generally classified by its severity, according to Glasgow Coma Scale (GCS) score: the National Institute of Health and Care Excellence (NICE) classifies a score of 13–15 as mild TBI, 9–12 as moderate, and ≤8 as severe . CT scanMost research that differentiates between aetiology of TBI focuses on socio-demographic characteristics or objective functional outcomes. This project hypothesises that rates of PTSD would be higher in patients who had suffered from assault than those who had an unintentional cause of TBI, and that this increased rate of PTSD could potentially account for some of the worse outcomes experienced by these patients. Better understanding of the influence interpersonal violence has on outcomes after TBI could lead to more targeted rehabilitation strategies, improving long-term recovery.This study aims to examine the influence of assault, compared to other aetiologies, on HRQoL, symptoms of post-traumatic stress and other subjective outcomes in victims of TBI.This is a cross-sectional study of 256 TBI patients attending the multidisciplinary TBI clinic for follow-up review at University Hospital Birmingham NHS Foundation Trust (UHBFT). The study consists of secondary analysis of an anonymised database of all patients who attended between August 2013 and February 2016. UHBFT is a major level-1 trauma centre that provides adult neurotrauma services to the Birmingham urban area and surrounding rural counties, with a catchment population of approximately 4 million. Patients are referred to the TBI clinic from hospitals within the catchment area.As part of routine care, patients who attended the clinic completed the following assessments:Quality of Life after Brain Injury Questionnaire (QOLIBRI) : a questPost-traumatic Stress Disorder checklist (civilian version) (PCL-C) : a 17-itImpact of Event Scale (IES) : a 15-quPatient Health Questionnaire (PHQ-9) : a 9-iteRivermead Post-concussion Questionnaire (RPQ) : this meHeadache Impact Test (HIT) : a 6-parAll patients completed the QOLIBRI, RPQ and either the PHQ or PHQ-9. 35 patients did not complete the PCL-C, IES and HIT, as these were introduced into the survey battery at a later date. A higher score indicates worse outcomes for all questionnaires but the QOLIBRI, where lower scores indicate worse outcomes.Patients personally completed questionnaires while attending the clinic, with a clinical team member in attendance to help with methodological or vocabulary difficulties, but not influencing responses. Patients were allowed to discuss questions with their carers but responses were only taken from the former.The responses of patients who returned questionnaires were compiled in a database. Medical records of these patients were reviewed retrospectively to collect demographic and clinical data, including:Mechanism of injuryTBI severity, according to Glasgow coma scale (GCS) on admission: severe (GCS score 3–8), moderate (9–12) and mild (13–15).Cranial CT scan, according to the Marshall classificationGlasgow outcome scale (GOS) score on dischargeHospital management Number of days hospitalisedNumber of days in an intensive care unit (ITU)Extracranial trauma or pre-existing medical conditionsAt-scene loss of consciousnessPost-traumatic amnesiaInvolvement of alcohol during eventLitigation activitiesIf a patient attended the clinic on multiple occasions, only questionnaires from their first visit were analysed. Patients were excluded from the analysis if the cause of their injury could not be ascertained; if the injury was caused in combat  or if patient had not suffered a traumatic brain injury. Incomplete questionnaires were excluded from analysis if patients did not respond to one or more questions, aside from the QOLIBRI, where scores are calculated from averages and remain reliable if two thirds of questions are answered.Ethical approval for this study was obtained from the NHS Health Research Authority–reference 17/LO/0153.Demographics, clinical data and subjective measures were compared between assaulted and non-assaulted TBI patients. Continuous variables were compared using the Mann-Whitney U Test and categorical variables using the chi-square test. Comparisons of questionnaire scores amongst severity, CT Marshall grades, and GOS score were performed using the Kruskal-Wallis test. Correlations between numerical variables were assessed using Spearman’s rank correlation coefficient. Multiple linear regression analysis was performed for each questionnaire, with cause of injury as the independent variable. Demographics that either correlated with or had an effect on each questionnaire were also included as independent variables. With all tests, pairwise was used for missing data or incomplete questionnaires to ensure maximal usage of data. A nominal significance level of p = 0.05 was used, and Bonferroni correction was used for multiple testing of items in individual questionnaires. Analysis was undertaken using the Statistical Package for the Social Sciences version 22.0 software.Of 256 patients on the database, 202 were included in analysis. Six were excluded as they did not suffer from TBI, four for injuries caused in combat and 44 for unknown aetiology of injury. 21% (n = 43) of included patients sustained their injury due to assault, with the remainder (non-assault group) sustaining injury from falls , road traffic collisions , and other causes . The median time from injury to follow-up was 5.1 months (inter-quartile range (IQR) 3.6–7.7)).Comparisons of demographics and clinical characteristics are presented in The severity of TBI ranged from mild to severe based on GCS on admission . Although the injuries tended to be more severe in the non-assault group, according to both GCS and Marshall classification, and assaulted patients tended to have better outcomes on discharge according to GOS score, these differences were not significant. Additionally, no significant effect of severity was found on any of the questionnaire scores, nor did questionnaire scores vary significantly between GOS scores. However, CT Marshall grade did have an effect on PCL-C score (p = 0.031), with more severe Marshall grades associated with more favourable PCL-C scores.All questionnaires showed a possible trend towards worse outcomes for patients in the assault groupsee . Only thWhen examining individual items in the PCL-C, more assaulted patients were symptomatic (score of 3–5) for “trouble falling or staying asleep” (p = 0.028) and “being ‘super alert’ or watchful on guard” (p = 0.001). Bonferroni correction gives a modified critical p value of p = 0.0029, so the latter result can be considered statistically significant. Diagnostic threshold scores of 36, 44 and 50 for the full checklist all showed higher prevalence of PTSD among assaulted patients, but none of these differences were significant  see .All six questionnaires correlated significantly with each other see . Age alsRegression analysis of each self-reported outcome is presented in After adjusting for confounders , assault appeared to have the greatest impact on the PCL-C, IES, and RPQ full and RPQ-13 subsection scores, but none of these changes were statistically significant . Furthermore, while assaulted patients had worse PHQ-9 and HIT scores, adjusting for confounders nullified this effect.A post-hoc analysis of observed power was performed for the comparison on our primary outcome measure (QOLIBRI) between assault and non-assault groups. This revealed a sample size of n = 166 would have 95% power to predict a difference of 15 points on the QOLIBRI between assault and non-assault groups. This difference is likely the minimum change required to be clinically significant: previous studies have suggested that changes of at least 30% are required for the change to be meaningful . Our samAn exploratory analysis was performed to identify whether, if analysed separately, assault and road traffic collisions were significantly more likely to result in poor outcomes than falls or other causes of TBI. The three groups were compared on each measure using the Kruskal Wallis test, then each group was compared separately using the Mann-Whitney U test. The comparison of assault and RTC group is not shown as all p values were greater than 0.05. The results of the other comparisons are presented in In this cohort, patients who suffered from TBI following assault were younger and less likely to be Caucasian, as expected from previous literature. Assaulted patients showed a possible trend towards worse subjective outcomes in all six questionnaires, with a significant difference in the IES, measuring symptoms of post-traumatic stress, and the first subsection of the RPQ, measuring early symptoms of post-concussion. However, there did not appear to be a significantly greater prevalence of PTSD in the assault group. Self-reported outcomes were independent of TBI severity (GCS) and of objective outcome scores (GOS). Younger patients and non-Caucasian patients scored less favourably, confounding results.Previous research suggests HRQoL after TBI is negatively impacted by the severity of post-traumatic stress, headaches, depression and extracranial trauma , 35. ThiThere is significant debate regarding the extent to which the functional outcomes measured by the Glasgow Outcome Scale influence HRQoL: some studies suggest a positive correlation and some suggest no relationship –37. The There is an unclear relationship between severity of TBI and HRQoL. Although evidence suggests that subjects with more severe injuries suffer worse cognitive and social functioning ,39, thisPrevious literature has established that younger age is protective against adverse physical outcomes after TBI . FurtherAssault is an intentional form of injury that is particularly intrusive to the victim. This study found that assaulted TBI patients reported higher IES and PCL-C scores than the non-assault group, but only the former difference was statistically significant. When considering individual components of the PCL-C, assault victims were more likely to be ‘symptomatic’ for two items measuring hyperarousal. Despite the evidence that assault leads to increased symptoms of post-traumatic stress, this did not translate to an increased prevalence of PTSD in assaulted patients, when considering PCL-C score thresholds. Furthermore, it is suggested that a change of 10–20 points in the PCL-C is required to be clinically significant ; regressExploratory analysis comparing assault and RTC separately against falls and other causes of TBI showed that assault and RTC were more likely to cause significantly worse outcomes in nearly every measure, while there was no significant difference between assault and RTC outcomes. There is increasing evidence that the feelings of anger or injustice are associated with worse outcomes following trauma ; this woThe cross-sectional, retrospective nature of this study limits the confidence with which any correlations can be drawn from the data. Additionally, the study’s reliance on questionnaires for self-reported outcomes creates a possibility for response bias, while using the PCL-C to diagnose PTSD is not as reliable as the gold standard–a clinically structured interview. Notably, the differential diagnosis of PTSD from post-TBI symptoms is particularly difficult: both conditions can cause emotional numbing and social withdrawal, as well as comorbid conditions such as depression and generalised anxiety . The IESInformation regarding potential mediators of observed relationships was highly reliant on data collected from patients’ clinical notes, which is not ideal: for example, length of coma, and socio-economic status can both influence recovery after TBI, but were not recorded in patients’ notes consistently enough to be analysed. Furthermore, many patients did not complete all questionnaires, reducing power of analysis, while the relatively small sample size and high p-values made regression analysis difficult to interpret. The study predominantly includes cases of mild TBI, and observed differences could be greater when considering moderate or severe TBI alone.These results suggest that following TBI, subjective outcomes are psychologically mediated, rather than due to organic factors like severity of injury. HRQoL correlates with symptoms of post-concussion syndrome, depression, and PTSD. There are significant potential benefits to rapidly identifying patients likely to suffer from persisting symptoms. The psychological trauma of TBI is significant and the relationship between brain injuries and the development of PTSD is complex, so all victims of TBI should be evaluated for potential adverse mental health outcomes.Future research should investigate correlation between subjective outcomes and age, potentially examining differences between patients who suffer from TBI in a larger sample, recruited from discharge rather than follow-up. Development of measures of “intentionality” attribution could help to elucidate differences seen between victims of violence and other patients, and could explain why patients undergoing litigation tend to have worse outcomes. Such information would be invaluable to inform the targeting of strategies to treat adverse outcomes from TBI.Self-reported outcomes appear independent of TBI severity, and are more sensitive to patients’ needs than objective outcome scores. Assessment of cognitive and psychological function at follow-up is as important as assessing physical function, in order to ensure improvements to HRQoL. Assaulted patients were at a higher risk of developing symptoms of post-concussion and PTSD than all other patients, but this may not correspond with a higher prevalence of PTSD. Assault and RTC were associated with worse outcomes than falls or other causes.


Spinal cord injury (SCI) can lead to severe motor, sensory and social impairments having a huge impact on patients’ lives. The complex and time-dependent SCI pathophysiology has been hampering the development of novel and effective therapies. Current treatment options include surgical interventions, to stabilize and decompress the spinal cord, and rehabilitative care, without providing a cure for these patients. Novel therapies have been developed targeting different stages during trauma. Among them, cell-based therapies hold great potential for tissue regeneration after injury. Neural stem cells (NSCs), which are multipotent cells with inherent differentiation capabilities committed to the neuronal lineage, are especially relevant to promote and reestablish the damaged neuronal spinal tracts. Several studies demonstrate the regenerative effects of NSCs in SCI after transplantation by providing neurotrophic support and restoring synaptic connectivity. Therefore, human clinical trials have already been launched to assess safety in SCI patients. Here, we review NSC-based experimental studies in a SCI context and how are they currently being translated into human clinical trials. Spinal cord injury (SCI) is one of the most disabling and devastating neurological injuries. It is recognized as a global health issue priority due to its impact on patient quality of life, complexity, and expensive medical care . In his work, Dr. Allen reported improvements in the motor function of lesioned dogs after successfully removing the inflammatory fluid . Thencef11. In hiDuring the chronic phase there is dissolution of the grey matter, white matter demyelination, deposition of connective tissue, and the formation of the glial scar. In addition to all these physiological complications, many SCI patients also experience the development of pain syndromes  and moodDespite the inhibitory environment, after the injury, the spinal cord and the brain tend to reorganize and establish new circuits in a process called neuronal plasticity. Hollis and coworkers (2016) demonstrated that after a C5 dorsal column lesion in rats the motor cortex was able to remap when continuous training was performed. Moreover, when observing SCI patients, significant alterations in the brain reorganization were shown. In comparison with the control group, SCI patients showed a reduction in the sensorimotor cortex patterning activation under functional magnetic resonance imaging (fMRI) . When loOverall, SCI is a multi-dynamic process with no effective treatment mainly due to the failure of the CNS to successfully regenerate the damaged neuronal circuits. Therefore, researchers have been exploring novel therapies that can be translated into clinical meaningful recovery.Despite the major efforts currently made to develop new therapies for SCI, there is still no effective treatment. Numerous studies have reported some positive results in preclinical SCI models, however, translation to patients is still questionable and controversial. Typically, after trauma, the patient is completely immobilized and is constantly monitored to prevent possible complications, such as respiratory dysfunctions, cardiovascular aberrations, and hypoxia . After sThe anti-inflammatory methylprednisolone sodium succinate (MPSS) was the first-line drug treatment for SCI patients . Now spaPharmacologically, new neuroprotective alternatives have been targeting secondary events such as inflammation and excitotoxicity. Among them, minocycline and riluzole are two examples that are already under clinical trials. Minocycline, a semisynthetic tetracycline antibiotic, is classified as a neuroprotective agent by improving the exacerbated inflammatory microenvironment observed during secondary SCI . After pFibroblast growth factor-2  has been implied in different biological processes, such as limb and nervous system development, wound healing, and tumor growth, due to its function in cell proliferation and survival . RegardiCytokines also exhibit interesting characteristics that can promote morphological and functional recovery of the spinal cord after trauma. The most widely studied cytokine for SCI treatment is the Glycoprotein Granulocyte Colony-Stimulating Factor (G-CSF), known to reduce inflammatory cytokine expression and to promote survival of ischemic cells . MoreoveBesides these molecular approaches, stem cell-based therapies have shown great potential over the past decades, based on the rationale that transplanted cells can differentiate and substitute the lost tissue. Moreover, stem cell-based treatments can also modify the microenvironment and regenerate the damaged circuits. However, there are also some challenges inherent to cell transplantation, including: the choice of the cellular source, the establishment of differentiation protocols, and the monitoring of cell grafts survival and integration. Regarding stem cell transplantation, encouraging results have already been reported for spinal cord repair, being particularly relevant for neuronal and glial replacement, remyelination, connectivity restoration, stimulation of precursor cells, as a bridge of cysts/cavities, and for improving the expression of beneficial neurotrophins/cytokines. A wide plethora of different cell-types has already been tested and transplanted into the injured spinal cord, both in animals and humans, including Schwann cells , olfactoThe human embryonic development of the nervous system is a complex and highly tuned-process. During the embryonic period, the primary three germ layers  are established as the basis of the various systems and organs of the body through numerous mitotic divisions. As illustrated in TBX6 and SOX2 genes in NMPs drive cells into their mesoderm or neural fate [HOX) genes  responsible for a more broad brainstem-to-rostral cervical spinal cord identity, the balance between WNT and FGF signals induces a more caudal neuroaxis spinal HOX gene expression , specifically for a cervical and thoracic spinal cord identity [At approximately the 23rd day of development, the neural plate is already observed in the embryo initiating the neurulation process. As the neural plate begins to roll-up, it develops into the neural tube and consequently to the neural crest . During ral fate . Furtheridentity ,63,64. OOver the past decades, we have been witnessing to unprecedented and groundbreaking progress in cell-based research . The potCell-based research turning point begun in the 20th-century when Stevens and Little (1954) were deciphering the complexity of teratocarcinomas. These tumors contained a relatively undifferentiated cell-type known as “Embryonal Carcinoma Cells” (ECCs), long suspected as the stem cell of the tumor . In the The ECCs therapeutic potential was compromised due to their tumorigenic potential and aneuploidy karyotype. In an attempt to overcome this drawback, in 1981 two independent laboratories reported the isolation and establishment of ESCs from early mouse embryos ,72. By rConsidering the advances in animal derived-ESCs, the isolation and culture of human pluripotent cells became an exciting challenge at the time. In 1993, Bongso et al. described for the first time the development and maintenance of ICM cultures and the following isolation of ES-like cells from its center. Cells maintained a normal karyotype and stemness-like morphology, however, a limited number of clusters differentiated into fibroblasts . In 1998Therefore, the first clinical trial using human ESC-derived OPCs was approved in 2010 (NCT01217008) ,80. The In conclusion, some convergent points stood out and common ESCs characteristics were established among the scientific community: (1) source of pluripotent cell population; (2) maintenance of a stable and diploid karyotype; (3) indefinite propagation in a primary embryonic state; (4) differentiation into any mature cell-type of the three embryonic germ layers; and (5) expression of specific nuclear and cytoplasmic markers of pluripotency . All theDespite the excitement created around the ESCs therapeutic potential some issues were hampering their translation to the clinics, mainly, ethical concerns, tumorigenic potential, and impossibility for autologous transplantation. Adult stem cells, such as mesenchymal stem cells, were a viable alternative, however, their differentiation potential is limited. Thus, further efforts were pursued to find a source of pluripotent stem cells (PSCs). Xenopus organism by transferring the nucleus from a somatic cell to an enucleated egg [Unexpectedly, in 1962 Gurdon et al. reported the full development of a complete ated egg . Althougated egg . Despiteated egg .Therefore, additional methodologies were explored to generate PSCs such as: parthenogenesis , cell fuDespite all the benefits, there are some inherent limitations of iPSCs that must be kept in mind to ensure safety , such asThe generation of iPSCs have already been done from a wide range of tissues, from adult human fibroblasts ,97 to liOn the contrary to ESCs, the use of iPSCs as a valuable source for cell transplantation was well accepted mainly due to the absence of ethical concerns. However, iPSCs also underwent some challenges; namely by the inappropriate use of animal-derived components and the use of retroviral vectors to transfect somatic cells. Thus, novel integration-free methods and xeno-free cultures were developed as well as innovative virus-free strategies ,103. TheIn 2014, Dr. Takahashi and his team  submitted the first-in-human trial using iPSCs . A patient with neovascular age-related macular degeneration (AMD), a degenerative retinal disease, received a transplant of a sheet of retinal pigment epithelial (RPE). The RPE cells were differentiated using autologous iPSCs from skin fibroblasts and no immunosuppressive agents were administrated to the patient. After transplantation, neither signs of immune rejection were observed nor aberrant genomic alterations, suggesting no tumorigenesis of the cell graft . These pHuman iPSCs are mainly obtained from skin fibroblasts however, a skin biopsy may not be the best source due to the risk of infection and scar formation ,106. ThuAdditionally, iPSCs technology has also been explored for disease modelling. The establishment of some disease-specific iPSCs lines have already allowed a better understanding of the mechanisms that trigger and are behind certain diseases . The firIn conclusion, iPSCs represent a very flexible technology that opened new and challenging opportunities to develop cell-based therapies due to its ability to differentiated into any cell-type given the right culture conditions. The numerous advantages of induced stem cells, from no immune rejection to high reproducibility, prompted the field to employ them in patient-specific diseases studies, developmental studies, cell transplantation as well as in drug screening. As a consequence, we are now moving fast towards a more personalized medicine.In the past, researchers assumed that neurons were only generated during embryogenesis, however, Altman and Das (1964) published a remarkable report describing the generation of new neurons in the hippocampal dentate gyrus of an adult rat brain . The genTherefore, the idea of transplanting NSCs is a very promising therapeutic approach for many neurodevelopmental, neurodegenerative, and neurotraumatic diseases by replacing and reestablishing damaged neuronal circuities . Thus, oNSCs from CNS primary tissues. In 1992, Reynolds and Weiss were the first to report the isolation and culture of NSCs in spherical clusters (neurospheres) from the striatal neurogenic area of an adult mouse brain [se brain . Two yease brain . Concomise brain ,116. IndDifferentiation of NSCs from pluripotent stem cells. PSCs have been used as a very attractive alternative to primary tissue isolation. Differentiation of NSCs from ESCs and iPSCs gained a lot of attention due to their therapeutic applicability potential. As previously discussed, iPSC-derived NSCs have striking advantages over ESC-derived NSCs, namely the possibility of autologous transplantation. Regarding differentiation protocols, PSCs have been cultured either as embryoid bodies (EB) or in monolayer cultures. Considering EBs, they grow in suspension and can be differentiated into neural tube-like rosettes and, subsequently, into NPCs under specific conditions [nditions . More renditions ,120. Addnditions ,122. Ovenditions ,124. On nditions ,126. Transdifferentiation of NSCs from somatic cells. Transdifferentiation is the differentiation of cells to a certain cell-type that does not follow the “normal” programmed differentiation mechanism [echanism . This teechanism . Lineageechanism  and by sechanism . Yao et echanism . The TF-echanism  and growechanism  have beeechanism . NeverthIn 1999, McDonald et al. were the first to demonstrate the potentialities of NPCs transplantation in a SCI context. After deriving neural progenitors from mouse ESCs (mESCs) and transplanting them into a rat spinal cord 9 days post-injury they observed that grafts had the capacity to survive and differentiate into neurons, oligodendrocytes, and astrocytes, and to migrate along the rostro-caudal axis from the lesion epicenter. More importantly, the transplanted experimental group showed hind limb weight support and partial walk coordination . From thAfter grafting mouse fetal striatal NS/PCs 12 wpi into a contusion T10 mice model, Kumamaru et al. (2013) observed that cells were capable of releasing multiple regenerative molecules and to differentiate into neurons/oligodendrocytes contributing for a neurogenic spinal cord environment, however, no locomotor improvements were observed . EffortsInjecting the cells in an earlier phase might be more beneficial to restore the damaged neural circuits before the formation of the glial scar barrier. Nevertheless, the current number of chronic patients is highly considerable which should also stimulate the field to get novel strategies and boost cell transplantation effects in a more chronic phase. Yet, more preclinical studies are needed to better understand the best time window of NSCs transplantation. As mention above, SCI is a two-phase injury process with multiphasic cellular and molecular responses that varies along time which makes it difficult to find the best time-window of treatment . To tack+/Mac-2+ cells at the epicenter of the injured area. Although the underlying factors remain unknown, this beneficial anti-inflammatory profile was translated into a significantly enhanced neurological function in mice [The cellular source to obtain NSCs is also an important variable to be considered. First, animal studies must be carried out to better comprehend and explore the cellular viability and to decipher some potential targets of NSCs after transplantation. Further, preclinical studies are mandatory before conducting a clinical trial mainly to assess safety. Fetal brain and spinal cord are the two main sources used to generate viable NSCs. Using mouse fetal cortex, Cheng et al. (2016) injected NSCs in an acute contusion SCI mice 7 dpi and observed a reduction in M1 macrophages activation, neutrophils and iNOS in mice . Additio in mice , to shre in mice , to the  in mice . Concerning human fetal spinal cord NSCs, positive results have also been reported regarding axonal growth at the injured site. For instance, Kadoya et al., showed that after transplantation there is an extensive regeneration of the corticospinal axons, improving synaptic connectivity and forelimb functioning . MeanwhiThe capacity of pluripotent mESCs to differentiate into the neural lineage has also been explored to reestablish the damaged circuits after a SCI. In a contusive SCI model, NSCs derived from a mESC line J1 were intrathecally transplanted 3 wpi showing the ability to differentiate into spinal GABAergic neurons and to attenuate chronic neuropathic pain . In a moFurthermore, preclinical studies have been conducted, approaching different cell sources, to better comprehend better the cellular response after transplantation namely immune rejection, tumor risk formation, and grafts survival. Using a human primary source of neural precursors from the fetal spinal cord (SPC-01) it was showed strong immunomodulatory properties through astroglial p65 NF-κB inhibition which significantly impacted the reduction of the glial scar and the cavity size . MoreoveOn the other hand, human pluripotent cells have been yielding in the generation and maintenance of NSCs. Kumamaru and coworkers demonstrated that under specific cues human ESCs (hESCs) can be derived into NSCs committed to the spinal cord improving the potential of neural cells to restore the damaged circuities. After transplantation, cellular grafts revealed to be enriched in excitatory neurons, promoting a robust corticospinal regeneration, engraftment within the host increased synaptic formation, which has culminated in hind-limb improvements .The discovery of iPSCs was one of the most remarkable innovations in regenerative medicine and biological research. The concept of convert adult somatic cells, such as blood cells or skin fibroblasts, into NSCs through iPSCs incited numerous studies in the field. Promising results have been observed after iPSC-derived NSC transplantation into SCI animal models, promoting cell survival, tissue preservation, and neuronal differentiation of cells. Moreover, functional recovery was also observed through the remyelination of axons and upregulation of supportive neurotrophic factors in the spinal cord ,152,153.Summing up, animal studies have been very useful to evaluate and decipher novel mechanistic insights after cell transplantation, yet preclinical studies are essential to target critical questions as immune rejection and tumor formation. To circumvent ethical issues and immunosuppression, iPSCs are now the most attractive human cell source, but further studies are required to ensure efficacy, feasibility and safety.After getting a valid cellular source other questions need to be addressed, namely, the following question raised is, how many cells are needed for a positive outcome? The optimal number of transplanted cells is still very debatable and few studies have been in order to elucidate the field. Yousefifard et al. (2016) have recently performed recently a systemic and meta-analysis to tackle some of these questions regarding NS/PCs transplantation. They reported that the median number of the cells per kilograms of animal’s body weight was 4.3106 (interquartile range = 1.1106–2107), but the higher the number of transplanted cells the better was the functional recovery ,155. ThiThe tumorigenicity risk associated with NSCs transplantation is also a real concern that must be addressed to ensure patients’ safety. While some studies do not report any tumor formation ,156 otheTeratomas development is mainly associated with an existent “epigenetic memory” and the lack of purification in the cellular sample used for transplantation . Some stTo avoid tumor formation during iPSCs reprogramming and differentiation there are some critical steps that must be carefully performed to avoid epigenetic and genomic instability in colonies . The choThe route of administration is also an important topic that must be considered when addressing cell transplantation. Three main injection routes have been tested to be applied in SCI context: intraspinal, intrathecal, and intravenous. Amemori et al. (2015) studied different administration routes in an acute SCI model. NSCs were either injected intraspinally into the lesion center or intrathecally into the subarachnoid space of rats with compression lesion. Both treatments facilitated functional locomotor recovery, yet the intraspinal implantation had a higher positive effect in gray and white matter sparing and axonal sprouting, and reduced astrogliosis when compared to the intrathecal injection . NeverthIntraspinal injections are the most commonly used route by researchers, however, it is important to have in consideration some positive results published regarding intravenous administration ,163. NisOn Due to the complexity of the SCI pathophysiology, different approaches have been developed combining cell-based transplantation with other target-therapies. For instance, after combining chondroitinase ABC (ChABC) treatment with iPSC-derived NSCs mice showed improvements in the forelimb grip strength and forelimb/hindlimb locomotion. The cells were injected after injury, but the ChABC treatment was only administrated 7 wpi. Interestingly, the grafts showed improved survival and differentiation potentially promoting functional synaptic connectivity . With a Moreover, biomaterials have also been currently applied as artificial extracellular matrix with the aim to increase the survival of transplanted stem cells. Using a laminin-coated hydrogel with dual porosity, iPSC-NPs were seeded and further transplanted into a rat model of chronic SCI. The cells survived over time and promoted the growth of host axons, astrocytes, and blood vessels; however, no locomotor recovery was observed . Overall, a vast number of approaches have been employed to achieve the best outcome and a significant regeneration of the damaged neurons after SCI, however, some challenges remain to be overcome. Cell-based therapy is a promising strategy since it is capable to target different events, allowing a neuroprotective environment and regenerative support in the injured spinal cord. Therefore, regeneration of the adult spinal cord cannot be thought as a one-step process, but rather as a multiple cascade-process overtime.an interdisciplinary branch of the biomedical field supported by three main pillars: benchside, bedside, and community [benchside-to-bedside. Although the encouraging results reported in preclinical studies, translation into the clinical set is still very challenging [Translational medicine has been one of the biggest challenges in science and medicine. Interestingly, the European Society for Translational Medicine (EUSTM) defined it as ommunity . Promisillenging , being tllenging ; inconsiTo date, a low number of clinical trials have been conducted for SCI patients using NSCs, nevertheless, some have already been concluded and others are still ongoing . The first clinical trial using NSCs was approved in 2005 by the Yonsei University Health System, Severance Hospital, Republic of Korea , Registration Number: KCT0000879). The phase I/ II clinical trial was based on the transplantation of NSC-derived from brain fetal tissue. Human NSCs neurospheres between P10–P20 were carefully selected and prepared for transplantation into 19 patients which were subdivided into four groups according to the time window between the injury onset and transplantation: acute (<1 week), early subacute (1–8 weeks), late subacute (9 weeks–6 months), and chronic (>6 months). A control group was also addressed to the clinical trial of 15 patients with traumatic cervical SCI. After 1 year, the study concluded that hNSCs transplantation is safe and well-tolerable by patients since no adverse evidence was observed. The patients did not exhibit evidence of cord damage, syrinx or tumor formation, neither neurological deterioration nor exacerbating neuropathic pain and spasticity. Regarding neurological outcomes, based on the American Spinal Injury Association Impairment Scale (AIS) grade, 5 out of 19 transplanted patients showed beneficial alterations, including recovery at the motor level and increased motor scores, whereas only 1 patient in the control group showed improvement . clinicaltrials.gov identifier: NCT01772810) at the University of California San Diego to intramedullary injected an FDA-approved NSI-566RSC cell line into thoracic (T2-T12) SCI subjects. NSI-566RSC line is derived from the cervical and upper thoracic regions of the spinal cord from an 8 gestational weeks human fetus. The first report was published by Curtis et al. (2018) showing that NSI-566 grafts are safe and have no detectable side effects after 18–27 months of cell transplantation [Neuralstem Inc.  began a phase I safety trial . Although some financial problems delay the initial study, a second clinical trial was initiated as a phase I/IIa dose-escalation study using the same embryonic-derived OPCs (NCT02302157). Preliminary results suggest improvements in motor function, but further data still needed [As mentioned before, Asterias Biotherapeutics Inc.  has also begun a safety trial in the implementation of human ESC-derived oligodendrocyte progenitors (GRNOPC1 cells)  in patiel needed .In 2014, a different clinical trial was also initiated to evaluate the safety and the potential of directly reprogrammed autologous NSCs (drNSCs) in subacute and chronic SCI subjects . Directly reprogrammed NSCs were generated from patients’ bone marrow cells (BMCs) using a patented reprogramming xeno-free cocktail. The grafts were directly injected into the white and grey matter of the spinal cord adjacent to the lesion after the implantation of a regeneration matrix (RMx). Although the sample size presented by the study is extremely limited (n = 5), after 6–9 months of follow-up there was no complications or side effects associated with the intervention, and all patients showed improvements in the neurologic state. Many different reports have already described the benefits of research cell lines transplantation into rodent models of thoracic contusion SCI ,187,188.In 2011, a new phase I/II clinical trial (NCT01321333) was sponsored by Stem Cells Inc. and managed at the University Hospital Balgrist . Based on previous preclinical studies, the main focus was to assess the safety and preliminary efficacy of human CNS stem cells (HuCNS-SC) transplantation. The trial included 12 thoracic (T2-T11) SCI patients who sustained an injury within 3–12 months prior to cell transplantation. At the injection time, patients received approximately a total of 20 million cells directly into the spinal cord, 2 injections rostral and 2 caudally to the injury site. After 12-months o, HuCNS-SC showed to be safe and feasible concerning the surgery and the cellular transplant. More interestingly, 7 out of the 12 patients experienced sensory improvements after neurological stability . Following these positive results, a second clinical trial was approved (NCT01725880) to perform a long-term follow-up of the transplanted HuCNS-SC subjects. However, some obstacles appeared that should be carefully considered. After 9 months of immunosuppression, the agents were removed and the sensory gains were lost. It seems that the immune system was rejecting the transplanted cells which culminate with the ceasing of the long-term follow up. Although the result observed in SCI patients was not expected, this warned the field for a real problem in transplant non-patient cells that might be challenging the host immune system. Consequently, the cell source used for clinical cell transplantation must be rethought being NSCs-derived from iPSCs one of the most attractive alternatives.Okano’s team in Japan is proposing the launch of the human clinical study using allogenic iPSC-derived NSCs for subacute SCI patients. The costs associated with quality testing, safety concerns and time-consuming for autologous transplants have led the team to an increased interest in an allogeneic alternative. Therefore, the Center for iPS Cell Research and Application (CiRA) at Kyoto University will donate “iPSCs of human leucocyte antigen (HLA) super-donors” for cell transplant intervention, which are clinical-grade allogenic immunologically match clones and no adverse outcomes are expected. Thus, the conduction of this clinical trial intends firstly to address safety and tumorigenesis issues associated with the transplantation of these clones into humans as a future viable therapeutic intervention ,191.Overall, clinical trials have been showing some modest improvements to the SCI patients. More important, these trials have not shown major safety problems, however the clinical trial perform by University Hospital Balgrist shown that after stopping immunosuppression complications may arise. For these reasons, autologous iPSC-derived NSCs are the most promising cell source to repair the injured spinal cord. This section was only based on the NSCs-based, however, several different therapeutic agents were or are being testing in clinical settings with the aim to promote SCI repair. For more information about clinical trials in a SCI context please read the following reviews ,194,195.NSCs are a promising therapeutic approach for SCI repair. NSCs can differentiate and replace the lost neural tissue as well as secrete neurotrophic factors that can protect or regenerate the tissue. Nevertheless, further studies are necessary to confirm neurological and functional benefits, safety, adjusting doses and administrations periods, and to select the most promising cellular sources to obtain NSCs. Despite all the efforts and progress, the match between preclinical models and human SCI is still poorly established but several ongoing clinical can provide an important avenue for future cell transplantation research. It is also important to highlight that cellular transplantation alone may not be a sufficient approach to completely treat the injured spinal cord, therefore, combinatory therapies may be necessary for the treatment of this devastating injury.


Numerous epidemiologic studies have examined the relation of physical activity or cardiorespiratory fitness to risk of cutaneous melanoma but the available evidence has not yet been quantified in a systematic review and meta-analysis.Following the preferred reporting items for systematic reviews and meta-analyses (PRISMA), we identified 3 cohort studies  and 5 case-control studies  of physical activity and melanoma incidence, and one cohort study (N = 49 cases) of cardiorespiratory fitness and melanoma risk.Cohort studies revealed a statistically significant positive association between high versus low physical activity and melanoma risk . In contrast, case-control studies yielded a statistically non-significant inverse risk estimate for physical activity and melanoma . The only available cohort study of cardiorespiratory fitness and melanoma risk reported a positive but statistically not significant association between the two . Potential confounding by ultraviolet (UV) radiation-related risk factors was a major concern in cohort but not case-control studies.It appears plausible that the positive relation of physical activity and cardiorespiratory fitness to melanoma observed in cohort studies is due to residual confounding by UV radiation-related risk factors.Future prospective studies need to examine the association between physical activity, cardiorespiratory fitness and melanoma after detailed adjustment for UV radiation-related skin damage. Worldwide, marked annual increases in incidence rates of invasive cutaneous melanoma have been observed across several decades, in particular among populations with low skin pigmentation –4. The mUV radiation-related DNA damage is the most relevant modifiable melanoma risk factor, and oxidative stress, chronic inflammation, and impaired immune function all represent contributing factors –9. In coAvailable evidence from epidemiologic studies of physical activity, cardiorespiratory fitness and melanoma risk has been inconsistent, which may be due to differences in study design and incomplete adjustment for potential confounding variables. We therefore conducted a systematic review and meta-analysis of physical activity, cardiorespiratory fitness and melanoma with careful consideration of the influence of study design and adjustment for potential confounding factors, including UV radiation-related skin damage, sun exposure, and sun sensitivity.Following the preferred reporting items for systematic reviews and meta-analyses (PRISMA) , two resOur systematic literature search in PubMed and Web of Knowledge was performed from inception of the databases to March 29, 2018, and it yielded 2,039 hits. Exclusion and inclusion criteria are shown in Because there was only one investigation of cardiorespiratory fitness and melanoma , we wereTwo researchers  conducted independent data extractions from the identified studies. Any disagreement was resolved by discussion. We included relative risks (RR) with 95% confidence intervals (CI) for all available physical activity domains and for any timing in life of physical activity. We used the most comprehensively adjusted risk estimates provided that those risk estimates were also adjusted for age and, if applicable, gender. If available, we extracted separate risk estimates for men and women. In a sensitivity analysis, we included only one risk estimate per study and gender. For that sensitivity analysis, we preferred recreational physical activity and recent past physical activity over all other domains and timings in life of physical activity.We classified levels of physical activity as recent past physical activity if they referred to a time period less than 10 years prior to the baseline assessment (cohort studies) or cancer diagnosis (case-control studies). If those physical activity levels referred to a period further back in time, the physical activity variable was classified as distant past physical activity. An exception was made if that variable captured more than one time period in the past and if the utmost time periods were ≥10 years apart. In that case, the physical activity variable was classified as a measure of consistent physical activity over time. We considered occupational physical activity to fall into the latter category because people usually hold occupations for more than one decade.We used the Monninkhof score  that was2-statistics to assess potential publication bias and between-study heterogeneity [We interpreted all hazard ratios, incidence rate ratios, and odds ratios as relative risks. We summarized melanoma risk estimates comparing the highest and lowest physical activity categories on the log-scale in precision-weighted random effects meta-regression models  using thogeneity –37. All ogeneity , 39.In the main analysis, we combined all available risk estimates of the association between physical activity and melanoma incidence, separately for cohort and case-control studies. In stratified analyses, we investigated the influence of the study quality score, gender, study geographic region, physical activity domain, timing in life of physical activity, physical activity measure, adjustments for UV radiation-related sun damage, sun sensitivity, sun exposure , smoking, adiposity, alcohol intake, and history of type 2 diabetes. In addition, we ran analyses stratified by adjustment for both sun sensitivity and sun exposure. To examine if any of the stratification variables explained the difference by study design, we repeated the stratified analyses for cohort and case-control studies combined. We compared summary estimates using likelihood ratio tests. In a sensitivity analysis, we included only one risk estimate per study and gender in the meta-analysis.N = 12,605 cases, of which the pooled study [N = 1,295 cases, N = 49 cases, We considered 3 cohort studies –22 .We observed a statistically significant difference between the summary melanoma risk estimates for physical activity from cohort studies and case-control studies . Similarly, the between-study heterogeneity among case-control studies was no longer apparent after adjusting for timing in life of physical activity . Case-control studies of recent and distant past physical activity observed statistically significant and non-significant inverse relations between physical activity and melanoma, respectively, whereas case-control studies of lifetime physical activity revealed a statistically non-significant positive association between the two . Similarly, using all 12 individual risk estimates rather than the pooled risk estimate from the pooled cohort study  did not The primary new finding of the current meta-analysis is that the association between physical activity and melanoma differs by study design. Specifically, we found a statistically significant positive association between physical activity and melanoma incidence among cohort studies, whereas we observed a statistically non-significant inverse relation among case-control studies. The one cohort study of cardiorespiratory fitness and melanoma reported a positive but statistically non-significant association between the two.The most important risk factor for melanoma is UV radiation-related skin damage, which includes a history of sunburns, dysplastic and common naevi, actinic keratosis, and solar lentigo –43. AddiThe presence of melanoma at sites that are rarely exposed to UV radiation and the relations of naevus patterns (site and count) to the histology and location of melanoma suggest that there are diverging pathways of melanoma incidence –46. One Moreover, various genetic factors have been related to the incidence of melanoma, including genes related to pigmentation and melanin production, the development of naevi, DNA repair, and family history of melanoma , 54, 55.Mechanistically, physical activity and cardiorespiratory fitness may prevent melanoma through enhanced immune function, increased DNA repair capacity, reduced oxidative stress, decreased chronic inflammation, and weight control , 57–61. Because none of the three cohort studies of physical activity and melanoma included in the present meta-analysis were adjusted for UV radiation-related skin damage, sun sensitivity, or sun exposure , the statistically significant positive summary risk estimate for physical activity and melanoma risk obtained from cohort studies may be a result of major confounding by UV radiation-related risk factors. The greater magnitude of the risk estimate among studies from Europe as compared to that from studies from North America indicates a greater degree of confounding of the physical activity and melanoma relation in European than North American populations, potentially due to lower levels of skin pigmentation  and greaFour of the five included case-control studies attempted to minimize the potential for UV radiation-related confounding by detailed adjustment for UV radiation-related skin damage, sun sensitivity , and sun exposure . Thus, it is conceivable that the statistically non-significant inverse summary risk estimate for physical activity and melanoma risk obtained from the included case-control studies is closer to the true association between physical activity and melanoma than that obtained from the cohort studies.Because the included studies used a large variety of physical activity assessments and because there was no single predominant type of physical activity assessment, in our main analysis we summarized risk estimates across all types of physical activity assessments to obtain a summary risk estimate of melanoma incidence for an “average” physical activity assessment. As a consequence, we included more than one risk estimate per gender in our main analysis for one  of the tThe major strength of the present systematic review and meta-analysis of physical activity and melanoma risk is the large number of cases and the inclusion of both cohort and case-control studies. Considering both study types was essential because case-control studies were methodologically superior to cohort studies in their degree of adjustment for UV radiation-related risk factors. Although case-control studies are generally considered to be prone to recall and selection biases, case-control studies of melanoma risk factors have not been subject to notable recall bias –74. AlsoIn summary, we observed significant heterogeneity between summary risk estimates from cohort and case-control studies of physical activity and melanoma incidence. It is likely that the statistically significant positive association between physical activity and melanoma risk and the positive but statistically not significant association between cardiorespiratory fitness and melanoma observed in cohort studies is the result of incomplete adjustment for UV radiation-related skin damage. Case-control studies of physical activity and melanoma generally adjusted for UV radiation-related risk factors and produced a statistically non-significant inverse relation between physical activity and melanoma incidence. To clarify the true nature of the relations of physical activity and cardiorespiratory fitness to melanoma incidence, future prospective studies are required to examine physical activity and cardiorespiratory fitness in relation to melanoma risk after careful adjustment for UV radiation-related skin damage.S1 File(DOCX)Click here for additional data file.S2 File(DOC)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Fig(EPS)Click here for additional data file.


In 2005, the Cervical Cancer Prevention Program in Zambia (CCPPZ) wasimplemented and has since provided cervical cancer screen-and-treat servicesto more than 500,000 women. By leveraging the successes and experiences ofthe CCPPZ, we intended to build capacity for the early detection andsurgical treatment of breast cancer.Our initiative sought to build capacity for breast cancer care through the(1) formation of a breast cancer advocacy alliance to raise awareness, (2)creation of resource-appropriate breast cancer care training curricula formid- and high-level providers, and (3) implementation of early detection andtreatment capacity within two major health care facilities.Six months after the completion of the initiative, the following outcomeswere documented: Breast health education and clinical breast examination(CBE) services were successfully integrated into the service platforms offour CCPPZ clinics. Two new breast diagnostic centers were opened, whichprovided access to breast ultrasound, ultrasound-guided core needle biopsy,and needle aspiration. Breast health education and CBE were provided to1,955 clients, 167 of whom were evaluated at the two diagnostic centers; 55of those evaluated underwent core-needle biopsy, of which 17 were diagnosedwith invasive cancer. Newly trained surgeons performed six sentinel lymphnode mappings, eight sentinel lymph node dissections, and 10 breastconservation surgeries (lumpectomies).This initiative successfully established clinical services in Zambia that arecritical for the early detection and surgical management of breastcancer. In this paper, we describeexperiences and initial results of a public-private demonstration project to expandaccess to breast cancer control services in Zambia. It was built on the framework ofa decade-long program that successfully integrated cervical cancer preventioninterventions within service platforms used to deliver HIV/AIDS care and treatmentin Zambia.Breast cancer in sub-Saharan Africa is characterized by low but rapidly increasingdisease incidence,13 As of February 2015, CCPPZ was officially integrated into thegovernment’s public health system with an official desk—NationalCoordinator of Cancer Prevention—now located at the Zambian MinistryHealth.Since 2005, the Zambian government, with support from the US President’sEmergency Plan for AIDS Relief, which is funded through the US Centers for DiseaseControl Prevention, and in partnership with local and US university-based publichealth oncology experts and advocates, has facilitated the implementation of apublic-sector cervical cancer prevention intervention—Cervical CancerPrevention Program in Zambia (CCPPZ). Initially focused on the provision of cervicalcancer screening and treatment services to HIV-infected women, it soon expandedservices to all women regardless of HIV status as a result of patient demand. Duringthe past decade, albeit with resources disproportionate to demand, cervical cancerprevention services have been scaled nationally to 60 government-operated publichealth clinics, at which more than 500,000 women have been screened since programinception and 50,000 are screened annually. An analysis of the first 100,000 womenenrolled in the program revealed a screen-to-detection ratio of 56 to 1 forhigh-grade cervical precancer, an overall treatment rate of 70% for women diagnosedwith precancerous lesions, and a shift in the percentage of early-stage invasivecervical cancers from 20% to 42%.Our major objectives were to leverage the successes and experiences ofZambia’s cervical cancer prevention program and to use direct participationat the point of care to build capacity for detection and surgical treatment ofbreast cancer in Zambia. We also assessed the clinical advancements that resultedfrom the capacity-building activities. The following cluster of activities wereconducted to implement the initiative:Creation of an alliance of advocacy groups: With support from the Susan G.Komen Foundation, the first step was to facilitate the formation of analliance of the five existing breast and cervical cancer advocacy groups inZambia into a consortium—CAPRAZ (Cancer Prevention AllianceZambia)—with the following goals (1) intensify breast and cervicalcancer awareness campaigns in the community to dispel myths andmisconceptions surrounding the disease and its treatment; (2) ensure thatbreast and cervical cancer health promotion messages were evidence based andcontextually relevant, and (3) coordinate and provide technical support forawareness activities of member groups.Assess capacity: After the organization of CAPRAZ into a functional nonprofitorganization, the next step was to assess breast cancer early detection anddiagnostic and surgical treatment capacities. Toward this end, breastoncology consultants from two university medical centers in the UnitedStates  were invited to Zambia to assess breast cancer care capacity at theUniversity Teaching Hospital in Lusaka Province  and Kabwe General Hospital in Central Province . Upon receipt of approval from relevant government agencies, theconsultants visited the two designated sites and worked daily alongsidelocal health professionals in their breast clinics, surgical theaters, andpostoperative wards. They also attended hospital-based educationalconferences, convened meetings with key academic stakeholders andprofessional health care providers, gave formal lectures on breast cancerdiagnosis and management to faculty and staff, and hosted informal socialgatherings. From the assessment, it was determined that the largest and mostcritical deficits in the breast cancer care pathway at the two facilitieswere lack of capacity to perform the following: (1) clinical breastexamination (CBEs), (2) breast ultrasound, (3) ultrasound-guided core needlebiopsy of palpable breast masses, and (4) conservative surgical treatment ofbreast cancer. Other weaknesses and strengths are listed in Curriculum development: The team of US consultants, in partnership withZambian health care professionals, used information from the assessment andknowledge of the local context to develop a training curriculum aimed atbuilding capacity in these four areas. Once developed, the curriculum wasvetted by senior members of the Zambian Ministry of Health’sTechnical Working Group for purposes of modification and approval.Implementation of the curriculum: Four months after their initial visit, theUS oncology consultants returned to Zambia and implemented the trainingcurriculum , in the Development of benchmarks for evaluation: The following benchmarks wereestablished for impact evaluation of the initiative:(1) Cervical cancer screening nurses working in government-operatedprimary health centers could adequately deliver breast healthawareness education and CBE.(2) Local radiographers (technicians) could accurately performultrasonographic evaluation of palpable breast masses.(3) General surgeons could safely and effectively performultrasound-guided core needle biopsy and needle aspiration of breastmasses, axillary sentinel lymph node (SLN) mapping with blue dye,SLN dissection, and breast lumpectomy.(4) Breast diagnostic centers would be established at each of the twofacilities described as assessment sites, with the capacity toperform CBE, breast ultrasound, and ultrasound-guided core needlebiopsy of palpable breast masses.(5) Data collection platforms would be established in eachclinic.(6) Referral pathways would be developed between the breastdiagnostic clinics and either the departments of general surgery atthe respective hospitals or the national cancer center  in Lusaka; referral pathways also would bedeveloped for chemotherapy and radiation at the national cancercenter in Lusaka.(7) Educational activities implemented by CAPRAZ in the catchmentareas surrounding the two hospitals would increase awareness of thesigns and symptoms of breast cancer and encourage women with breastconcerns to seek care from local health facilities. Member groupsalso would begin to increase their participation in local breastcancer initiatives, such as fundraisers, educational seminars,public lectures, and awareness campaigns.A 6-month evaluation of local activities by US consultants revealed thefollowing:Four cervical cancer screening clinics at the primary health carelevel—one in Kabwe and three in Lusaka provinces—hadsuccessfully integrated breast cancer education and CBE into their routineservice platforms.Two new breast cancer diagnostic centers were operational—one at KabweGeneral Hospital and the other at University Teaching Hospital, and eachprovided CBE, breast ultrasound, ultrasound-guided core needle biopsy, andneedle aspiration.A total of 1,955 women had undergone CBE in the four primary health centers,of which 256 (13.1%) had palpable breast masses . All werUltrasound-guided biopsies were indicated and performed in 55 of the 59 womenwho had confirmed palpable breast masses . PatholoSix SLN mappings, eight SLN dissections, and 20 breast conservation surgeries(lumpectomies) were performed independently by the Zambian general surgeons.The histologic margin status of lumpectomy surgical specimens was notreported. No surgical complications were reported .Breast cancer is projected to increase in low- and middle-income countries (LMICs)during the next two decades. The vast majority of women who live in these settingswill die from their disease primarily because of the advanced stage at which theyare diagnosed and lack of access to high-quality treatment services. In addition,attempts to develop effective breast cancer care service platforms in such settingsare made difficult by widespread myths and misconceptions about the disease as wellas by competing health care priorities. We took advantage of prior investments inthe integration of cervical cancer screening within HIV services to launch anapproach to capacity building for the early detection and surgical treatment ofbreast cancer in Zambia.14 population-based mammographic screening requires resourcesgenerally not widely available in LMICs. In addition, its benefit-to-harm ratio hasbeen questioned recently, particularly in women younger than 50 years old—theage group that produces the majority of breast cancers in LMICs.15 Although there is controversy about the benefits ofCBE,19 there is developing evidence that it has thepotential to downstage disease. In an ongoing cluster-randomized trial in India,CBE, when performed by community health care workers, detected more  early-stage cancers (stage I to IIA)in intervention versus control villages.20Cervical cancer screening nurses were trained to provide breast cancer education andperform CBE on appropriately aged asymptomatic and symptomatic women who attendedtheir clinics for cervical cancer prevention services. Although the most effectivemeans for screening is mammography,22 Ultrasound,however, is dependent on the quality and reliability of the equipment and theexperience of operators for accuracy in the diagnosis of breast lesions.23 When used effectively, it candistinguish simple cysts from solid masses, provide an estimation of the likelihoodof malignancy, and be used to aid in biopsy for pathologic diagnosis.24Two new diagnostic breast centers were established and staffed with radiographers andgeneral surgeons who were trained in ultrasound and ultrasound-guided biopsy,respectively. Ultrasound is recommended as an appropriate tool for the evaluation ofwomen with palpable breast masses in LMICs, given its relatively low cost comparedwith other imaging technologies, value as an adjunct to CBE, and ability to supportminimally invasive diagnostic procedures.25 Compared with open incisional and excisional biopsy,core-needle biopsy is less invasive, less expensive, causes minimal scarring, andcan be performed on an outpatient basis.26 Core-needle biopsy also has the benefit of provision ofadequate tissue for determination of histologic grade; vascular and lymphaticinvasion; estrogen and progesterone receptor statuses; and the presence of moleculartumor markers, such as HER2/neu.27 These prognostic variables mayhelp triage surgical interventions, such as breast-conservation therapy,28 use of SLN biopsy, or axillarylymph node dissection.27Ultrasound-guided core needle biopsies are performed to determine the etiology ofpalpable breast masses and are indicated on the basis of the sonographiccharacteristics of the mass, such as shape, orientation, margin, echo pattern,posterior acoustics, and boundary.29 It has the added benefit of better cosmetic appearance,which is important, given the impact of cultural beliefs and stigma ontreatment-seeking behavior.30 MRMs, however, will remain themainstay of treatment of breast cancer in LMICs for the foreseeable future given thelimitations imposed by the lack of radiotherapy services and inefficient resourcesfor effective follow-up programs.31 Complete level-1 and -2 axillary dissections typically areperformed during breast cancer surgeries in LMICs because of advanced-stage diseaseor the inability to perform less invasive axillary staging in early-stage disease.With proper training, however, SLN mapping and sampling can be performed, which mayincrease the accuracy of axillary staging in breast cancer and reduce morbidityassociated with complete axillary dissections.32 This will become a necessary skill as more cancers arediagnosed at an early stage.Surgery is the cornerstone of breast cancer care and is the most widely availabletherapy for breast cancer in LMICs. Modified radical mastectomy (MRM) is the mostcommonly performed surgical procedure for breast cancer in these settings, largelyas a result of late-stage presentation of disease. Although general surgeons inZambia were well versed in performance of MRMs, their exposure to breastconservation surgery (lumpectomy) and axillary SLN mapping and dissection techniqueswas limited. The number of surgical theaters was inadequate, and not enough time wasallocated to accommodate all of the surgical cases that had been scheduled forsurgical demonstrations by visiting oncologists, which resulted in aless-than-desired number for hands-on training. This ultimately limited the trainingthe surgeons received in conservative surgery techniques. Because such a largepercentage of women presented with advanced-stage disease, limited numbers ofearly-stage breast cancer cases were available on which to demonstrate breastlumpectomy and SLN mapping/dissection. Despite these challenges, the surgeons wereable to independently perform breast conservation surgery and SLN mapping anddissection techniques. Breast conservation surgery for early-stage disease is safeand has the same overall survival as mastectomy.We used direct participation at the point of care to inform the construction andimplementation of an approach to capacity building for the early detection andsurgical treatment of breast cancer in Zambia . As an oFuture initiatives will concentrate on the expansion of breast cancer early detectionand surgical treatment services throughout the country. This will require increasedtraining of nurses, radiographers, and surgeons as well as an effective system tomonitor quality, access timely and accurate pathology services, and obtain patientfollow-up information. As awareness is increased and CBE is more widely implemented,early-stage presentation will become more prevalent and the demand for breastconservation surgical skills, as well as those for breast reconstruction, willincrease.In conclusion, system-level barriers to early detection and treatment of cancercontinue to plague LMICs. We leveraged an existing cervical cancer preventionplatform to introduce breast cancer education, detection, and surgical treatmentservices by investing in the development of local clinical expertise. Overall, thisinitiative has increased breast care capacity in Zambia and, if properly scaled, canserve to improve the diagnosis and management of early-stage breast cancer at anational level, which thereby reducing breast cancer morbidity and mortality.


When retrieval occurs concomitantly with novelty detection, mismatch perception or reactivation of conflicting information, consolidated memories can enter into a labile state, and to persist, must be restabilized through a protein synthesis-dependent reconsolidation process during which their strength and content can be modified. Extensive literature implicates brain-derived neurotrophic factor (BDNF), a key regulator of synaptogenesis and synaptic plasticity, in the acquisition, consolidation and extinction of several memory types. However, the participation of BDNF in memory reconsolidation has been less studied. In this review, we discuss recent reports supporting the involvement of BDNF signaling in reactivation-induced memory updating. Memory consolidation is the time and protein synthesis-dependent stabilization process that takes place after learning to convert short-term memory into long-term memory. Consolidated memories are stable but mutable and can return to a labile state when activated during retrieval, requiring a restabilization phase known as reconsolidation to endure. Consolidation and reconsolidation share several molecular properties and result in persistent synaptic changes for memory storage. However, they are distinguishable processes that serve different biological purposes.Brain-derived neurotrophic factor (BDNF) regulates neurogenesis, neuronal differentiation, maturation and survival during development . BDNF isBDNF is one of the most commonly studied proteins in memory research. In fact, in the last two decades plenty of studies described the participation of this neurotrophin in the acquisition, consolidation and long-lasting storage of different memory types . In partde novo protein synthesis for developing new synaptic connections and/or remodeling existing ones to support lasting memory storage  that originated the conditioned fear response. Importantly, the same treatment was unable to affect retention when the reminder was omitted. The fear response also persisted in animals re-exposed to the CS alone, excluding the possibility that memory extinction could account for these results . Based oIt has been proposed that memory reconsolidation would mediate incorporation of new information into previously stored representations to support mental schema reorganization  or wouldNeurotrophins are key regulators of long-term synaptic modifications. They are synthetized and secreted in an activity-dependent manner, acting locally at active synapses to enhance neurotransmission efficacy . In partde novo hippocampal protein synthesis after retrieval, but only when novelty is perceived during reactivation, suggesting that reconsolidation recruits the hippocampus to incorporate new information into the active recognition trace  maintenance requires on trace . In neuron trace . In the on trace , and proon trace . In accoon trace . Consiston trace . PKMζ ison trace . BDNF moon trace  and mainon trace . Intereson trace . Figure on trace . This dion trace .Determining the molecular basis of retrieval-induced cognitive processes is necessary not only to understand the dynamics of the memory storage process but also to prevent memory decline and treat disorders associated with the persistent reenactment of maladaptive recollections . The stuMemories about emotionally arousing events are usually persistently stored which, in some cases, lead to intrusive and distressing recollections that may result in anxiety, phobia or other types of disarrays such as post-traumatic stress disorder PTSD; . A potenAntidepressants can influence BDNF levels bidirectionally. For example, a single dose of the serotonin re-uptake inhibitor fluoxetine decreases BDNF expression  and atteAn alternative strategy that is being study to enhance PTSD exposure therapy efficacy consist of coupling extinction sessions with physical exercise, which increases peripheral BDNF levels . In thisAlzheimer’s disease (AD) progression has also been associated with impaired reconsolidation and reduced BDNF signaling , suggestThe studies reviewed in this article suggest that BDNF mediates enduring synaptic changes required for memory strengthening and updating upon retrieval. Several pharmacological and non-pharmacological approaches to modulate BDNF signaling and expression are currently under consideration, and exposure-based psychotherapy could take advantage of those findings. However, future research should also consider BDNF signaling interaction with other mediators of memory reconsolidation. An important issue that limits the effectiveness of reconsolidation-based treatments is that retrieval does not always induce memory destabilization. However, it was recently proposed that memory destabilization can be enhanced pharmacologically , offerinMG and AR conceptualized the review, performed the literature research, interpreted the data, and wrote the manuscript. MC contributed to the data interpretation, edited parts of the manuscript, and critically revised and approved its final 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.